study of the ultra high pressure homogenization (uhph ... · su dirección, en el Área de ciencia...

Facultat de Veterinària

Departament de Ciència Animal i dels Aliments

Study of the Ultra High Pressure Homogenization (UHPH) technology for producing high quality soymilk

Memòria presentada per optar al grau de Doctor en

Ciència i Tecnologia dels Aliments

Fábio Henrique Poliseli Scopel

Bellaterra, 2012

VICTORIA FERRAGUT PÉREZ y MANUELA HERNÁNDEZ HERRERO, profesoras

titulares de Tecnología de los alimentos de la Universitat Autònoma de Barcelona,

HACEN CONSTAR: que D. FÁBIO HENRIQUE POLISELI SCOPEL ha realizado, bajo

su dirección, en el Área de Ciencia y Tecnologia de los Alimentos de la Universitat

Autònoma de Barcelona, el trabajo titulado “Study of the Ultra High Pressure

Homogenization (UHPH) technology for producing high quality soymilk” que presenta

para optar al grado de Doctor.

Y para que así conste firmamos el presente documentos en:

Bellaterra (Cerdanyola del Vallès), 27 de septiembre del 2012.

Dra. Victoria Ferragut Pérez Dra. Manuela Hernández Herrero

Esta tesis doctoral fue realizada con la financiación aportada por el Plan Nacional de

Investigación, Desarrollo e Innovación (AGL 2008-05430), por la comunidad europea a

través del proyecto FUNENTECH (FP7/2007-2013-SME-232603), por el programa de

becas (ALBAN) y por el Centro de Recerca Planta de Tecnologia dels Aliments

(CERPTA).

i

Abstract

Soymilk consumption is experiencing a noticeable increase due to it being considered as a healthy product. Soymilk has often been used as an alternative to dairy milk for people who have intolerance to dairy products. Nowadays, it is known for its important health benefits that can contribute to the reduction of chronic illness commonly prevalent in the modern style life. This is due, primarily, to characteristics of protein fraction and minor components rich in antioxidant activity (flavonoids, tocopherols and poliamines) taking into account the excellent nutritional profile of soymilk.

This thesis project was focused on the application of an emerging technology, Ultra High Pressure Homogenization (UHPH), in the production of soy vegetable milk. This non-thermal technology consists of a high pressure machine capable of applying pressures of up to 400 MPa using a special homogenizing system designed to produce a conserving effect, improving the colloidal stability while maintaining good nutritional and sensory qualities. Considering this hypothesis, UHPH could be an alternative technology to those commonly applied in the food industries. For that, a comparative study of UHPH with thermal treatments (pasteurization and UHT) was carried out in this work.

In the first part of this thesis, different UHPH conditions (200 and 300 MPa at 55, 65 and 75ºC of inlet temperature) were performed on soymilk in order to select optimal treatment conditions for producing a good quality product whether intended for refrigeration or long-term storage at room temperature. In this first step, two independent evaluations were performed. On one hand, quality parameters related to chemical, enzymatic, microbiological and colloidal characteristics were evaluated and on the other hand, an inoculation study with different strain spores was carried out in order to determine the inactivation kinetic of the UHPH treatment. Results indicated that treatments at 300 MPa were able to produce soymilk with high chemical and colloidal stability. It is also worth noting that an excellent reduction of bacterial spores was reached applying inlet temperature of 85ºC at the same pressure.

The second part consisted in the shelf-life evaluation of soymilk treated by UHPH using the selected optimal conditions determined in the previous step. As a result, soymilk was obtained with similar characteristics to those produced by pasteurization and with extended shelf-life similar to those obtained by UHT treatments. To achieve this purpose, microbiological aspects, colloidal stability, color changes, chemical parameters and sensory quality were applied to evaluate the overall quality of soymilk and its acceptance by the consumers. Refrigerated soymilk and that produced for an extended shelf-life respectively reached 1 and 6 months of storage in good conditions for consumption and with better quality than those obtained by thermal treatments.

iii

Resumen

El consumo de licuado de soja está experimentado un notable incremento debido a su consideración de producto saludable. El licuado de soja, además de ser una alternativa a la leche de vaca, sobre todo para las personas que poseen alguna intolerancia a los productos derivado de la leche, tiene componentes bioactivos (flavonoides, vitamina E y poliaminas) que pueden contribuir a prevenir algunas dolencias crónicas prevalentes en la sociedad actual.

En este estudio se planteó la utilización de una tecnología emergente, la ultra alta presión de homogenización (UHPH) para la obtención de licuado de soja. Esta tecnología no térmica consiste en la aplicación de presiones de hasta 400 MPa utilizando un sistema de homogenización, especialmente diseñado para producir un efecto conservador, al mismo tiempo que se mejora la estabilidad coloidal y se mantiene la calidad nutricional y sensorial. Con esta hipótesis de partida, la UHPH podría ser una tecnología alternativa a las comúnmente aplicadas a nivel industrial. Por ello, en el planteamiento de este trabajo se incluyó el estudio comparativo de la UHPH con los tratamientos térmicos de pasteurización y UHT.

En la primera parte de esta tesis se llevaron a cabo diferentes tratamientos UHPH (200 y 300 MPa con temperaturas de entrada de 55, 65 y 75ºC) con la finalidad de seleccionar las condiciones óptimas para obtener productos de buena calidad, tanto de almacenamiento en refrigeración, como de larga duración de almacenamiento a temperatura ambiente. El estudio se realizó a dos niveles independientes. Por una parte se evaluaron parámetros característicos de la calidad química, coloidal, enzimática y microbiológica de licuados de consumo habitual, y por la otra, se realizó un estudio con licuados inoculados con diversas cepas microbianas para conocer su cinética de destrucción frente a tratamientos UHPH. De estos estudios se concluyó que los tratamientos a 300 MPa produjeron licuado de soja con muy buena estabilidad coloidal y química y que, aplicando una temperatura de entrada de 85ºC en combinación con dicha presión, se alcanzó una excelente reducción de las esporas bacterianas.

La segunda parte del trabajo consistió en el estudio de la evolución durante el almacenamiento de los licuados UHPH tratados en las condiciones óptimas seleccionadas del estudio previo. De este modo, se obtuvieron tanto licuados frescos similares a los pasteurizados, como licuados de larga duración, similares a los tratados por UHT. Para lograr este propósito, se evaluaron una serie de aspectos, tales como microbiológicos, estabilidad coloidal, cambios de color, parámetros químicos y sensoriales que permitieron evaluar la calidad global de los productos de soja, así como su aceptación por los consumidores. Los licuados de soja fresco y de larga duración alcanzaron respectivamente 1 y 6 meses de caducidad con mejor calidad que aquellos tratados térmicamente.

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Table of contents

CHAPTER 1. BACKGROUND, OBJECTIVES AND WORKING PLAN ................... 1

1.1 Background .................................................................................................................. 3

1.2 Objectives .................................................................................................................... 7

1.3 Working plan ............................................................................................................... 8

1.4 References ................................................................................................................. 11

CHAPTER 2. INTRODUCTION ..................................................................................... 15

2.1 Soybeans .................................................................................................................... 17

2.1.1 Definition ............................................................................................................ 17

2.1.2 Proximate composition ....................................................................................... 17

2.2 Soymilk ...................................................................................................................... 22

2.2.1 Definition and composition ................................................................................ 22

2.2.2 Soymilk processing ............................................................................................ 23

2.3 Ultra high pressure homogenization .......................................................................... 26

2.3.1 Concept and history ............................................................................................ 26

2.3.2 High-pressure homogenization equipments ....................................................... 28

2.3.3 Temperature increase during the UHPH treatment ............................................ 31

2.3.4 UHPH applications ............................................................................................. 32

2.3.5 UHPH effect on microbial inactivation .............................................................. 34

2.3.6 UHPH effects on physico-chemical properties .................................................. 36

2.3.7 UHPH effects on proteins and enzymes ............................................................. 38

2.4 References ................................................................................................................. 41

CHAPTER 3. MATERIAL AND METHODS ............................................................... 55

3.1 Soymilk elaboration ................................................................................................... 57

3.2 Soymilk treatments: UHPH, pasteurization and UHT .............................................. 58

3.3 Production yield ......................................................................................................... 58

3.4 Storage of soymilk ..................................................................................................... 59

3.5 Soymilk and soybeans physico-chemical analysis .................................................... 60

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3.6 Microbiological analysis ........................................................................................... 60

3.6.1 Microbiological quality ...................................................................................... 61

3.6.2 Isolate collection and selection ........................................................................... 61

3.6.3 Sporulation conditions ........................................................................................ 62

3.6.4 Spores recovery .................................................................................................. 62

3.7 Lipid oxidation .......................................................................................................... 63

3.7.1 Lipoxygenase activity ......................................................................................... 63

3.7.2 Hydroperoxide index .......................................................................................... 63

3.8 Trypsin inhibitor activity ........................................................................................... 64

3.9 Particle size determination ........................................................................................ 65

3.10 Particle sedimentation ............................................................................................. 65

3.11 Transmission electron microscopy .......................................................................... 66

3.12 Surface hydrophobicity ........................................................................................... 67

3.13 Color measurements ................................................................................................ 67

3.14 Headspace analysis of volatile compounds ............................................................. 67

3.14.1 SPME – gas chromatography mass spectrometry ............................................ 67

3.15 Sensory analysis ...................................................................................................... 68

3.16 Statistical analysis ................................................................................................... 69

3.17 References ............................................................................................................... 70

CHAPTER 4. OPTIMIZATION OF SOYMILK ELABORATION AT PILOT

PLANT SCALE ................................................................................................................. 73

4.1 Introduction ............................................................................................................... 75

4.2 Results and discussion ............................................................................................... 76

4.2.1 Chemical composition ........................................................................................ 76

4.2.2 Production yield .................................................................................................. 77

4.2.3 Lipoxygenase activity ......................................................................................... 78

4.2.4 Trypsin inhibitor activity .................................................................................... 79

4.3 Conclusions ............................................................................................................... 80

4.4 References ................................................................................................................. 80

vii

CHAPTER 5. COMPARISON OF UHPH AND CONVENTIONAL THERMAL

TREATMENTS ON THE MICROBIOLOGICAL, PHYSICAL AND CHEMICAL

QUALITY OF SOYMILK ................................................................................................ 83

5.1 Introduction ............................................................................................................... 85

5.2 Results and discussion ............................................................................................... 86

5.2.1 Soymilk chemical composition .......................................................................... 86

5.2.2 Temperature changes in UHPH processing ........................................................ 86

5.2.3 Microbiological quality ...................................................................................... 87

5.2.4 Colloidal stability: particle size and sedimentation ............................................ 89

5.2.5 Chemical stability: oxidation and trypsin inhibitor activity ............................... 93

5.3 Conclusions ............................................................................................................... 96

5.4 References ................................................................................................................. 96

CHAPTER 6. STUDY OF THE POTENTIAL INACTIVATION OF UHPH

TREATMENT ON SELECTED BACILLUS SPORES ISOLATED FROM

SOYMILK ....................................................................................................................... 101

6.1 Introduction ............................................................................................................. 103

6.2 Results and discussion ............................................................................................. 104

6.2.1 Spore-former occurrence .................................................................................. 104

6.2.2 UHPH effect on spores survive ........................................................................ 105

6.3 Conclusions ............................................................................................................. 109

6.4 References ............................................................................................................... 110

CHAPTER 7. CHARACTERIZATION OF VOLATILE PROFILE IN SOYMILK

TREATED BY UHPH ..................................................................................................... 113

7.1 Introduction ............................................................................................................. 115


7.2.1 Volatile compounds .......................................................................................... 115

7.2.2 Aldehydes ......................................................................................................... 117

7.2.3 Ketones ............................................................................................................. 119

7.2.4 Alcohols ............................................................................................................ 121

7.2.5 Furans ............................................................................................................... 123

viii

7.2.6 Esters and Acids ............................................................................................... 124

7.2.7 Principal component analysis (PCA) ................................................................ 125

7.3 Conclusions ............................................................................................................. 129

7.4 References ............................................................................................................... 129

CHAPTER 8. CHARACTERISTICS OF REFRIGERATED UHPH-TREATED

SOYMILK AND SHELF-LIFE ESTIMATION .......................................................... 133

8.1 Introduction ............................................................................................................. 135


8.2.1 Microbiological quality and pH measurements ................................................ 136

8.2.2 Microstructure and hydrophobicity characterization ........................................ 138

8.2.3 Physical stability ............................................................................................... 143

8.2.4 Chemical stability ............................................................................................. 148

8.2.5 Sensory analysis ............................................................................................... 151

8.3. Conclusions ............................................................................................................ 156

8.4 References ............................................................................................................... 157

CHAPTER 9. SHELF-LIFE EVALUATION OF ASEPTICALLY PACKAGED

UHPH-TREATED SOYMILK ....................................................................................... 161

9.1 Introduction ............................................................................................................. 163


9.2.1 Microbiological quality and pH measurements ................................................ 164

9.2.2 Colloidal stability ............................................................................................. 166

9.2.4 Chemical stability ............................................................................................. 171

9.2.5 Sensory analysis and color quality ................................................................... 178

9.3 Conclusions ............................................................................................................. 184

9.4 References ............................................................................................................... 185

CHAPTER 10. CONCLUSIONS ................................................................................... 193

CHAPTER 11. APPENDIX ............................................................................................ 195

ix

Abbreviations key

ANS 8-Anilino-1-naphtalene sulfonic acid

ANOVA Analysis of variance

cm centimeters

cfu Colony forming unit

et al. et alter (and others)

GLM Generalized linear model

g gram

g Centrifugal force

GC Gas chromatography

h hour

kV kilovolts

L* Luminosity

L Litter

LOX Lipoxygenase

M Molarity

m metter

meq milliequivalent

min minute

mg milligram

mL milliliter

mm millimeter

mM millimolar

MPa Megapascal

MS Mass spectra

nm nanometer

o/w oil-in-water

Pa Pascal

PCA Principal component analysis

PCR Polymerase chain reaction

psi Pound per square inch

RHHTC Rapid hydration hydrothermal cooking

s second

x

SD Standard deviation

SPME Solid phase microextraction

TEM Transmission electron microscopy

TI Trypsin Inhibitor

UHPH Ultra high-pressure homogenization

UHT Ultra high temperature

w/w weight/weight

ΔB Backscattering difference

ΔE Color difference

μg microgram

μL microlliter

μm micrometer

xi

List of tables

Table Page

Table 1-1. Soybeans trade and production. ........................................................................... 4

Table 1-2. Functional properties of soybeans in food systems. ............................................ 5

Table 2-1. Soybean amino acid composition....................................................................... 18

Table 2-2. Main soybean fatty acids composition. .............................................................. 19

Table 2-3. Chemical composition of soymilk ..................................................................... 22

Table 2-4. UHPH effect on different food applications ...................................................... 33

Table 4-1. Chemical composition of soymilk extracted at 60ºC and 80ºC ......................... 77

Table 4-2. Production yield of soymilk extracted at 60ºC and 80ºC ................................... 78

Table 5-1. Temperature changes of UHPH soymilks during processing ............................ 87

Table 5-2. Microbial populations of untreated and treated soymilks .................................. 88

Table 5-3. Solids sedimentation and particle size untreated and treated soymilks ............. 90

Table 6-1. Counts of bacterial spores inoculated in soymilks ........................................... 106

Table 6-2. Temperature and pressure changes of UHPH soymilk during processing ....... 108

Table 7-1. Total of volatile compounds by chemical family in BP and treated soymilks. 117

Table 7-2. Abundance of aldehydes detected in the volatile fraction of soymilks ............ 118

Table 7-3. Abundance of ketones detected in the volatile fraction of soymilks ............... 120

Table 7-4. Abundance of alcohols detected in the volatile fraction of soymilks .............. 122

Table 7-5. Abundance of alcohols detected in the volatile fraction of soymilks .............. 123

Table 7-6. Abundance of esters and acids detected in the volatile fraction of soymilks ... 125

Table 7-7. Variance accounted by PC1 and PC2 of soymilk volatile profile .................... 127

Table 8-1. Solids sedimentation of treated soymilks during refrigerated storage ............. 144

Table 8-2. Hydroperoxide index values of untreated and treated soymilks ...................... 148

Table 8-3. Main volatile compounds detected in untreated and treated soymilks ............. 153

Table 8-4. Color parameters of untreated and treated soymilks ........................................ 155

Table 9-1. Changes in particle size distribution of UHT and UHPH soymilks................. 168

Table 9-2. Solids sedimentation of treated soymilks during storage ................................. 169

Table 9-3. Hydroperoxide values of untreated and treated soymilks ................................ 172

Table 9-4. Main volatile compounds detected in soymilk treated by UHT and UHPH .... 174

Table 9-5. Changes in color parameters during storage in Tetra Brik containers ............. 180

Table 9-6. Preference results of panel evaluation.............................................................. 184

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List of figures

Figure Page

Figure 1-1. Working plan: previous study and first step ....................................................... 9

Figure 1-2. Working plan: fresh and sterile soymilk and fourth step: inoculation study .... 10

Figure 2-1. Soymilk processes. ........................................................................................... 24

Figure 2-2. Valve design of a conventional homogenizer (APV-Gaulin) ........................... 27

Figure 2-3. Schematic view of the Microfluidics International Corporation ...................... 28

Figure 2-4. Schematic view of the Stansted High Pressure Homogenization valve ........... 29

Figure 2-5. Scheme of the UHPH equipment ...................................................................... 31

Figure 5-1. Particle size distribution of untreated and treated soymilks ............................. 92

Figure 5-2. Hydroperoxides values of untreated and treated soymilks ............................... 94

Figure 5-3. Trypsin inhibitor values of untreated and treated soymilks .............................. 95

Figure 6-1. Lethality of bacterial spores inoculated in soymilk UHPH treated ................ 107

Figure 7-1. Principal component analysis defined by PC1 and PC2 ................................. 126

Figure 8-1. Microbialogical pH quality of pasteurized and UHPH soymilks ................... 137

Figure 8-2. Protein surface hydrophobicity of untreated and treated soymilk .................. 139

Figure 8-3. Transmission electron micrographs untreated and treated soymilk ............... 142

Figure 8-4. Backscattering of untreated and treated soymilk ............................................ 145

Figure 8-5. ΔB untreated and treated soymilk for 28 days of storage ............................... 147

Figure 8-6. Total volatiles compounds and percent ratio of hexanal ................................ 149

Figure 8-7. Sensory attributes of pasteurized and UHPH soymilks .................................. 152

Figure 9-1. pH measurements of UHT and UHPH soymilks ............................................ 165

Figure 9-2. Height of solids settled of untreated and treated soymilks ............................. 167

Figure 9-3. Protein surface hydrophobicity of untreated and treated soymilks................. 170

Figure 9-4. Ratio of hexanal to total volatiles UHT and UHPH soymilks ........................ 177

Figure 9-5. Sensory attributes of UHT and UHPH soymilks ............................................ 182

Chapter 1

Background, objectives and working plan

3

1


1.1 Background

For more than 2000 years people throughout East Asia have consumed soybeans in the

form of traditional foods, such as nimame (cooked whole soy), soy sauce, tofu and soymilk

(Fukushima, 2001). Historical and geographical evidence indicates that the soybean first

emerged as a domestic crop in the eastern half of North China (Hymowitz, 1970; Liu,

1999).

The soybean (Glicine max L.) remained exclusive to the Orient for many centuries. Its first

introduction into Europe was about 1712. However, due to poor climate and soil

conditions, soybean production has been limited in Europe (Liu, 1999). In 2008, the

European Union produced less than 1% of total world production (USDA, 2012).

In western countries, the soybean first drew attention in the 1960s as an economical and

high quality vegetable protein source for humans. The part of the seed with greatest

interest is formed by cotyledons (90%) with around 20% of lipids and 40% of protein in

dry matter (Liu, 1999).

World production of soybeans has increased substantially in the last 50 years; from 13

million tons in 1939 to 265 million tons in 2011 (Kwok & Niranjan 1995; USDA, 2012).

Nowadays, western countries have become the main soybean producer and exporter,

whereas eastern countries have become the main soybean importer. Data on world

production and trade are shown in Table 1-1.

Chapter 1

4

Table 1-1. Soybeans trade and production (millions tons), 2010 – 20111. Production Export Import

World 264.69 88.85 92.64

United States 90.61 40.86 0.39

Brazil 75.50 29.95 0.04

Argentina 49.00 9.21 0.01

China 15.10 0.19 52.34

European Union 1.04 0.06 12.48

Japan 0.22 0.00 2.92 1 World imports and exports may not balance due to differences in local marketing years and to time lags

between reported exports and imports (USDA, 2012).

Of the total soybean importations of European Union in 2010, 12.7% correspond to France,

13.0% to Spain, 14.2% to the United Kingdom, 21.0% to the Netherland and 35.4% to

Portugal. Total production of Spain in 2009 was 2767 tons, with Extremadura as the

greatest producer with 68% of the total and Catalonia was responsible for 1.5% (MAPA,

2010).

In 2009, consumption of soy foods was around 30.1 million tons and predicted to reach

around 33.3 million tons in 2012 (USDA, 2012). The increased soy consumption in the

world and in Europe is probably related to the high content of lipids and proteins. These

components in combination with others of minor content (minerals and lecithin for

example) give soybeans a great applicability in the food industry. The interest in soybeans

is not only based on nutritional and biological quality but also on the functional properties

of components such as gel formation, emulsification, thermal stability, water and fat

absorption and sparkling (Utsumi et al., 1997; Fukushima, 2001). Table 1-2 shows a

complete list of these and other properties.

In addition to the industrial applications, there is an extensive bibliography about nutritive

or health properties of soy components. The most relevant effects are, reduction in

cholesterol levels (Potter et al., 1993; Carroll & Kurowska 1995), mitigation of menopause

and osteoporosis symptoms, and reduction in risk of heart diseases and cancer (Khatib et


5

al., 2002; Huang et al., 2006; Rochfort & Panozzo, 2007). However, there is a

controversial opinion in the scientific community. The possible health benefits have not

been evidenced according to reports published by EFSA (2010) and EFSA (2011).

Table 1-2. Functional properties of soybeans in food systems (Wolf, 1970).

Functional property Food systems

Emulsification

Formation Sausages, frankfurters, bologna, breads, cakes and soups

Stabilization Frozen desserts, sausages, frankfurter, bologna and soups

Fat absorption

Promotion Sausages, frankfurter, bologna and meat patties

Prevention Doughnuts, pancakes

Water absorption

Uptake Breads, cakes, macaroni and confections

Retention Breads and cakes

Texture

Viscosity and gelation Soups, gravies, chili and simulated ground meats

Shred and fiber formation Simulated meats

Dough and film formation Baked goods, frankfurter and bologna

Adhesion Sausages, lunch meats, meat loaves, hams, meats dehydrated

Cohesion Baked goods, macaroni and simulated meats

Elasticity Baked goods and simulated meats

Color control

Bleaching Brads

Browning Breads, pancakes, waffles

Aeration Confections

Chapter 1

6

According to FAO, of all soy products soymilk was the product which has most grown in

consumption (FAO, 2002). Despite the consumption rise in western countries in the last

decade, soymilk has been somewhat restricted mainly because of its typical beany flavor

(Yuan & Chang, 2007; Achouri et al., 2008), and finding alternative processing methods

that can reduce soymilk off-flavors has become a challenge to the food industry.

Consumer opinion is a key element for the development and modernization of the

industrial process, and taste is fundamental for the acceptance of products introduced into

market, especially culturally different products, such as soymilk.

Consumers demand for safe food products, which are environmental friendly and which

exhibit high nutritional quality has abruptly increased in recent decades. Thus, it is a

challenge for the food industry to adapt the industrial processes, and to search and test for

alternative technologies which improve the organoleptic quality of the products while

preserving nutritional properties and reducing losses and energy costs.

This tendency impacts directly on traditional technologies, such as heat treatments, so that

lately non-thermal technologies, such as pulsed electric field and oscillating magnetic field

(Deeth & Datta, 2002), irradiation, ultrasonication, centrifugation, microfiltration and

hydrostatic high pressure (HHP) (Datta & Deeth, 2002ab; Diels et al., 2005) have been

investigated and developed.

In the case of soymilk, heat treatments cause undesirable chemical changes which may

lead to the destruction of amino acids and vitamins, browning reaction, development of

cooked flavor (Kwok & Niranjan, 1995) and negative effects on solubility and water

absorption (Zhang et al., 2005).

Several studies applying non-thermal technologies, such as hydrostatic high pressure

(HHP) were carried out on soymilk. For instance, Zhang et al. (2005) studied HHP effects

on soymilk proteins and Jung et al. (2008) investigated on isoflavone profiles. However,

HHP technology is a discontinuous process which is not fully adapted to the needs of

soymilk processing. In the last decade, ultra-high pressure homogenization (UHPH) has

been atracting certain interest for application in liquid foods since it is a non-thermal

continuous process which may improve several aspects of the overall quality of such

products.

The Centre Especial de Recerca Planta de Tecnologia dels Aliments (CERPTA) of the

Universitat Autònoma de Barcelona, has been working with UHPH technology since 2000

and has participated in several Spanish and European projects. Some results for soymilk

showed that UHPH treatment was efficient in reducing microbial populations, improved


7

physical stability and presented high digestibility, similar to heat treatment (Cruz, 2008).

These preliminary results raised expectations for UHPH as a possible alternative to the

conventional heating processing in soymilk production. UHT has some detrimental effects

on the nutritive and sensory quality of soymilk, in addition to loss of colloidal stability

during storage, and UHPH has a great potential to produce commercial soymilk with

improved overall characteristics. According to this hypothesis, the following objectives

were proposed.

1.2 Objectives

This thesis is framed into the national project (AGL2008-05430) named “Application of

ultra high pressure homogenization on the elaboration of high quality vegetables milks

(soymilk and almond milk)”. In addition, part of this study was included in the European

project (FP7/2007-2013-SME-232603) called “Study of functionality, nutritional and

safety aspects of liquid foods, liquid food preparations and cosmetics processed by ultra

high pressure homogenization”.

General objective

The general objective of this thesis was to study the effects of applying UHPH technology

to soymilk production as an alternative to conventional heat treatments such as

pasteurization and UHT, in order to obtain a high quality soymilk with an extended shelf-

life.

Specific objectives

• To develop an optimized method at pilot plant level for soymilk elaboration with

high standards of chemical composition and good production yield.

• To study the influence of combining temperature and pressure at different levels for

UHPH treatments and their influence on soymilk quality parameters. Then,

selecting the best conditions for obtaining fresh soymilk (to be stored in

refrigeration) and long storage soymilk (room temperature).

• To identify the potential spoilage-related microbiota in soymilk that may be

resistant to UHPH treatments and to study the influence of combining temperature

and pressure in the kinetic of inactivation.

Chapter 1

8

• To study the evolution during storage of soymilks selected previously: stored at

cold temperatures and long term aseptically packaged stored at ambient

temperature.

• To study the aroma profile after processing and during storage of fresh soymilk and

treated soymilk (pasteurized, UHT and UHPH).

1.3 Working plan

A preliminary study was carried out in order to obtain a standard method of soymilk

elaboration with high quality characteristics and yield, using the infrastructure at the UAB

pilot plant (Figure 1-1).

The next step was to apply UHPH treatments on soymilk base product (BP). Samples were

subject to 200 MPa and 300 MPa of pressure combined with 55ºC, 65ºC and 75ºC of inlet

temperature (Figure 1-1). Microbiological, physico-chemical and biochemical quality

parameters were evaluated.

The third step included the study to evaluate the inactivation of spores of Bacillus. After

isolation and identification of some spore-forming bacteria from original soymilk treated at

300 MPa and 65ºC of inlet temperature, two bacterial strains were selected (Bacillus

cereus and Paenibacillus taichungensis) to study the kinetic of inactivation of spores in

inoculated soymilk. UHPH conditions applied were 300 MPa at 55ºC, 65ºC, 75ºC and

85ºC inlet temperature (Figure 1-2).

For the fourth step, and taking into account the previous results, UHPH conditions of 200

MPa at 55ºC and 75ºC of inlet temperature were selected to produce a fresh soymilk for

achieving similar or better quality characteristics than pasteurized soymilk.

Microbiological, chemical and physical changes were studied for 28 days of storage at 4ºC

(Figure 1-2). Moreover, volatile profile at day 1 and day 28 and sensory analysis at day 15

were also studied.

Finally, the last step was performed in order to obtain a product with similar or better

characteristics than UHT soymilk. Soymilk BP was treated by UHPH (300 MPa, 80ºC) and

UHT with subsequent aseptic packaging in both treatments. Microbiological, chemical and

physical changes were studied for 180 days of storage at room temperature (Figure 1-2).

Figure 1-1. Working plan: previous study and second step.

Soymilk base

Pasteurization 90ºC 30 s

UHT 142ºC 6 s

UHPH 200 MPa 55ºC-65ºC-75ºC 300 MPa 55ºC-65ºC-75ºC

• Mesophilic aerobic counts • Mesophilic spores counts • Enterobacteria counts • Yeasts and molds • Staphylococcus aureus • Bacillus cereus • Salmonella spp. • Sterility test •Isolation, purification and identification

Grinding 80ºC, 20 min

Soybeans

Soaking

Grinding 60ºC, 20 min

Filtration

Soymilk base product

Chemical composition

Production yield

Filtration


Lipoxygenase activity Trypsin inhibitor activity

Optimization of soymilk elaboration

Second step: Initial UHPH processing

Microbiological analysis Physico-chemical Chemical stability

• Lipoxygenase activity • Hydroperoxide index • Trypsin inhibitor activity

• Chemical composition • Particle sedimentation • Particle size • pH

Figure 1-2. Working plan: third, fourth and fifth steps.


UHT 142ºC 6 s

UHPH 300 MPa 80ºC

Pasteurization 90ºC 30 s

UHPH 200 MPa 55ºC – 75ºC

Storage condition: room temperature

Aseptic packaging (Tetra Brick)

Analysis: 1, 20, 40, 60, 90, 120, 150 and 180 days

Storage condition: 4ºC

Analysis: 1, 7, 14, 21 and 28 days

Microbiological analysis

• Mesophilic aerobic counts • Mesophilic spores counts • Enterobacteria counts • Bacillus cereus • Thermophilic bacteria

Physico-chemical analysis

• pH • Color (L* a* b*) • Particle sedimentation • Particle size • Microstructure (TEM) • Hydrophobicity • Hydroperoxide index • Volatile compounds profile

Sensory analysis

• Selection of panelist • Training of panelist • Triangular test • Descriptive test • Preference test

Incubation: 30ºC, 20 days


Sterilization 121ºC 15 min

Inoculation Bacillus cereus

Paenibacillus taichunengis

UHPH 300 MPa 55ºC-65ºC-75ºC-85ºC

Microbiological analysis

Incubation: 30ºC, 20 days

Total bacteria counts

Third step: Microbial inoculation

Four and Fifith steps: shelf-stable soymilk to refrigeration or room temperature


11

1.4 References

Achouri, A., Boye, J.I. & Zamani, Y. (2007). Changes in soymilk quality as a function of

composition and storage. Journal of Food Quality. 30, 731-744.

Achouri, A., Boye, J.I. & Zamani, Y. (2008). Soybean variety and storage effects on

soymilk flavour and quality. International Journal of Food Science & Technology.

43, 82-90.

Carroll, K.K. & Kurowska, E.M. (1995). Soy Consumption and cholesterol reduction -

Review of animal and human studies. Journal of Nutrition. 125, S594-S597.

Cruz N. (2008). Efecto de la alta presión de homogeneización en licuado de soja y su

comportamiento en el desarrollo de un producto fermentado [dissertation]. Universitat

Autònoma de Barcelona, Spain.

Datta, N. & Deeth, H.C. (2002a). Heat treatment, alternatives to: high-pressure processing.

In Encyclopedia of Dairy Sciences (Edited by, Hubert Roginski), Elsevier, UK. pp.

1327.

Datta, N. & Deeth, H. C. (2002b). Heat treatment, alternatives to: Other nonthermal

technologies. In Encyclopedia of Dairy Sciences (Edited by, Hubert Roginski),

Elsevier, UK. pp. 1339.

Deeth, H.C. & Datta, N. (2002). Heat treatment, alternatives to: Pulsed energy

technologies. In Encyclopedia of Dairy Sciences (Edited by, Hubert Roginski),

Elsevier, UK. pp. 1333

Diels, A.J., Callewaert, L., Wuytack, E.Y., Masschalck, B. & Michiels, C.W. (2005).

Inactivation of Escherichia coli by high-pressure homogenisation is influenced by

fluid viscosity but not by water activity and product composition. International

Journal of Food Microbiology. 101, 281-291.

EFSA. (2011). Scientific opinion on the substantiation of health claims related to soy

isoflavones and protection of DNA, proteins and lipids from oxidative damage,

maintenance of normal blood LDL-cholesterol concentrations, reduction of

Chapter 1

12

vasomotor symptoms associated with menopause, maintenance of normal skin

tonicity, contribution to normal hair growth, “cardiovascular health”, treatment of

prostate cancer and “upper respiratory tract” pursuant to Article 13(1) of regulation

(EC), No 1924/20061. European Food Safety Authority. 1-44.

EFSA. (2010). Scientific opinion on the substantiation of a health claim related to soy

protein and reduction of blood cholesterol concentrations pursuant to article 14 of the

regulation (EC), No 1924/20061. European Food Safety Authority. 1-14.

FAO. (2002). Chapter XIX soybeans: Post-harvest operations. Acessed January/2012.

http://www.fao.org/inpho/content/compend/text/Ch19sec1.htm#

Fukushima, D. (2001). Recent progress in research and technology on soybeans. Food

Science and Technology Research. 7, 8-16.

Huang, H., Liang, H. & Kwok, K.C. (2006a). Effect of thermal processing on genistein,

daidzein and glycitein content in soymilk. Journal of the Science of Food and

Agriculture. 86, 1110-1114.

Hymowitz, T. (1970). Domestication of Soybean. Economic Botany. 24, 408-421.

Jung, S., Murphy, P.A. & Sala, I. (2008). Isoflavone profiles of soymilk as affected by

high-pressure treatments of soymilk and soybeans. Food Chemistry. 111, 592-598.

Khatib, K.A., Aramouni, F.M., Herald, T.J. & Boyer, J.E. (2002). Physicochemical

characteristics of soft tofu formulated from selected soybean varieties. Journal of

Food Quality. 25, 289-303.

Kwok, K.C. & Niranjan, K. (1995). Review: Effect of thermal processing on soymilk.

International Journal of Food Science & Technology. 30, 263-295.

Liu, K. (1999). Soybeans: Chemistry, technology and utilization. Aspen publisher, Inc.

Gaitherburg, USA. pp. 532.

MAPA. (2010). Anuario de estadística agroalimentario. Ministerio de Agricultura, Pesca

y Alimentación. Accessed: January/2012. Available from:

http://www.magrama.gob.es/estadistica/pags/anuario/2010/AE_2010_13.pdf.


13

Potter, S.M., Bakhit, R.M., Essexsorlie, D.L., Weingartner, K.E., Chapman, K.M., Nelson,

R.A., Prabhudesai, M., Savage, W.D., Nelson, A.I., Winter, L.W. & Erdman, J.W.

(1993). Depression of plasma-cholesterol in men by consumption of baked products

containing soy protein. American Journal of Clinical Nutrition. 58, 501-506.

Rochfort, S. & Panozzo, J. (2007). Phytochemicals for health, the role of pulses. Journal of

Agricultural and Food Chemistry. 55, 7981-7994.

USDA. (2012). Soybeans: World Supply and Distribution. Accessed:January/2012.

Avilable from:

http://www.usda.gov/wps/portal/usda/usdahome?navid=HOME&navtype=MA

Utsumi, S., Matsumura, Y. & Mori, T. (1997). Structure-function relationships of soy

proteins. In S Damodaran (Edited by A Paraf), Food Proteins and Their Applications.

Marcel Dekker, USA. pp 257–291.

Wolf, W.J. (1970). Soybean proteins: their functional, chemical, and physical properties.

Journal of Agricultural and Food Chemistry. 18, 969-976.

Yuan, S. & Chang, K.C. (2007). Selected odor compounds in soymilk as affected by

chemical composition and lipoxygenases in five soybean materials. Journal of


Zhang, H., Li, L., Tatsumi, E. & Isobe, S. (2005). High-pressure treatment effects on

proteins in soy milk. Lebensmittel-Wissenschaft Und-Technologie,. 38, 7-14.

Chapter 2

Introduction

17

2 Introduction

2.1 Soybeans

2.1.1 Definition

Soy plant is botanically denominated as Glicyne max L. Glicyne is a greek word meaning

“sweet” and it applies to all the groundnut species of legumes. The word max means

“large”, referring to the large nodules of the soybean plant (Liu, 1999). Mature soybeans

are nearly spherical in shape, but may change considerably according to cultivar and

growing conditions. Soybean seed consists in three major parts: seed coat or hull,

cotyledons, and germ or hypocotyl. The seed coat contains hilum, which is the point of

attachment to the pod (Perkins, 1995).

2.1.2 Proximate composition

Protein and fat content of whole soybeans are about 40% and 20% (dry basis),

respectively. Remaining dry matter is composed mainly of carbohydrates (35%) and ash

(about 5%). Water content of stored mature soybean is usually about 13% (Liu, 1999). In

addition, important components such as isoflavones and saponins that have apparently

valuable health effects are present in the seed (Perkins, 1995; Huang et al., 2006a;

Rochfort & Panozzo, 2007; Jung et al., 2008).

The most abundant components in soybeans are proteins. About 40 to 90% of them are

present as storage proteins. Because of this, some researchers have suggested that soybeans

should be called protein seeds rather than oilseeds (Liu, 1999). Due to the great content of

proteins, soybean is used in many industrial processes that include protein isolation and

concentration, such as infant formulas and soy foods (tofu and soymilk for example)

(Friedman & Brandon, 2001).

According to biological functions, soy proteins can be characterized as metabolic proteins

or storage proteins. Based on solubility, proteins are divided into albumins and globulins

(Liu, 1999). Globulins are the major and the most important soy protein, being soluble in

water or diluted salt solutions at pH values above or below isoelectric point (Wolf, 1970).

Chapter 2

18

A precise technique to identify protein is based in approximate sedimentation coefficients

by using ultracentrifugation to separate proteins of the seed (Thanh et al., 1975; Howard et

al., 1983). After ultracentrifugation and under appropriate conditions, soy globulin exhibits

four fractions, named 2S, 7S, 11S and 15S. Analysis of these fractions has shown that 11S

and 15S fractions are pure proteins. In particular, 11S fraction corresponds to soybean

glycinin and accounts for at least 33% of extractable protein, whereas 15S fraction is

thought to be a polymer of glycinin and accounts for 10%. 2S fraction represents 20% of

the extractable protein and includes trypsin inhibitor and cytochrome C, whereas 7S

fraction represents an additional third of the extractable protein and consists of β-

conglycinin, α-amylase, lipoxygenase, and hemagglutinin (Nielsen, 1985).

An important chemical property of soy protein which determines protein nutritional value

is its amino acid composition. Table 2-1 lists essential amino acid composition of soybean.

Table 2-1. Soybean amino acid composition. Amino acid mg/g protein (dry matter basis)

(Fukushima, 1991) (Friedman, 1996) (USDA, 2003)

histidine 27.0 25.4 27.51

isoleucine 48.0 47.1 49.42

leucine 78.0 85.1 82.97

lysine 61.0 63.4 67.85

methionine + cysteine 65.0 68.1 -

phenylalanine +

tyrosine 90.0 96.6 91.80

tryptophan 13.0 11.4 14.82

threonine 35.0 38.4 44.28

valine 48.0 49.1 50.88

The amino acids above mentioned are considered essential amino acids, and cysteine and

tyrosine are considered conditionally essentials. These amino acids are responsible for

promoting growth in young people, preventing diseases and maintaining a positive

nitrogen balance during youth and old age (Laidlaw & Kopple, 1987). In general, soybeans

Introduction

19

contain all essential amino acids required for human or animal nutrition, paying attention

to lysine by its valuable content compared to other cereal proteins and by its important role

in the growth of young children (Liu, 1999). Accoding to Carrol and Kurowska (1995), soy

amino acids are less hypercholesterolemic than milk amino acids, making soy proteins a

good substitution of animal protein in the diet contain cholesterol by the reductions of the

serum cholesterol concentration.

Lipid composition of soybean is formed by 90% of triglycerides, 7% of phospholipids and

3% of glico-lipids. Fatty acids are distributed in saturated (15%) and unsaturated (85%)

(USDA, 2003). Linoleic acid is the most abundant fatty acid and represents about half of

the total amount, although oleic and linolenic acids are also in a considerable quantity

(Sangwan et al., 1986; Liu, 1999). Table 2-2 shows fatty acid composition of soybeans

from three different sources. Among of soy lipids, phospholipids, commonly known as

“lecithin” have significant commercial value as emulsifying agent in food industry.

Additionally, wetting and colloidal and antioxidant are also important properties of soy

lecithin.

Table 2-2. Main soybean fatty acids composition.

Fatty acid Percent (%)

(Sangwan et al., 1986) (Liu, 1999) (USDA, 2003)

Saturated (Total)

Palmitic (C16:0) 9.3–17.4 8–17 11.5

Stearic (C18:0) 2.2–7.0 3–30 3.90

Unsaturated (total)

Oleic (C18:1) 15.2–29.6 25–60 23.6

Linoleic (C18:2) 33.8–59.6 25–60 53.9

Linolenic (C18:3) 4.3–15.0 2–15 7.20

Carbohydrate content in soybeans is about 35% on dry basis, making them the second

largest component in the seeds. However, the economical value of soy carbohydrates is

considered much less important than soy protein and oil (Liu, 1999). They are divided in

monosaccharides, such as glucose and arabinose, and oligosaccharides known also as α-

Chapter 2

20

galactosides of sucrose, including raffinose and stachyose. These saccharides are included

in the category of soluble carbohydrates and the composition of some of them ranges from

2.5 to 8.2% for sucrose, from 1.4 to 4.1% for stachyose and from 0.1 to 0.9% for raffinose.

In the category of insoluble carbohydrates include hemicelluloses, pectin and cellulose,

containing respectively 50, 30 and 20% in the soy cell walls (Liu, 1999). Among of soluble

carbohydrates, raffinose and stachyose receive more attention because of their flatulence

and abdominal discomfort associated with consumption of soybeans and soy products. The

lack of enzymes in human intestinal tract to hydrolyze galactosidic linkages of raffinose

and stachyose into simple sugars, allows natural bacteria to metabolize intact glucide

molecules and thus generate unpleasant flatus feeling (Calloway et al., 1971; Hymowitz et

al., 1972; Knudsen & Li, 1991; Perkins, 1995; Wilcox & Shibles, 2001).

Despite the presence of oligosaccharides in soybeans and soy products, generally

considered undesirable in terms of flatus activity, some studies have shown beneficial

effects of oligosaccharides inclusion in the human diet. The main benefit reported was the

growth stimulation of Bifidobacteria population in the intestinal tract preventing

constipation problems and helping in the production of vitamins (Gibson & Roberfroid,

1995; Martínez et al., 2005).

Major minerals present in total ash (about 5%) of soybeans are potassium (1800 mg/100g),

phosphorus (700 mg/100g), magnesium (280 mg/100g) and calcium (275 mg/100g). Minor

minerals include iron, zinc, arsenic and selenium. Water-soluble vitamins content include

thiamin, riboflavin, niacin, pantothenic acid and folic acid and on the other hand, oil-

soluble vitamins are A, E and K (USDA, 2003).

Another minor component, ranged from 0.3 to 0.8% (dry matter basis) and has converted

soybean extensively known are isoflavones (Jung et al., 2008). These components are a

subclass of flavonoids and belong to the group of phytochemicals (Craig, 1997) and seem

to act as phytoestrogens in human metabolism. According to data from several studies,

isoflavones are believed to potentiate the decrease of cholesterol levels, to prevent both

prostate and breast cancers, to attenuate bone loss in postmenopausal women and to

alleviate menopausal symptoms (Jenkins et al., 2002; Achouri et al., 2005; Huang et al.,

2006a; Jung et al., 2008; Aparicio et al., 2008). On the other hand, these health benefits

have not been established by the scientific community of European Union (EFSA, 2010;

EFSA, 2011).

Introduction

21

In spite of apparent health benefits of soybeans consume, substances with antinutritional

properties known as trypsin inhibitors are included in the composition. These substances

have a proteolytic activity which reduces the availability of trypsin, an important protease

in the animal digestive function (Friedman & Brandon, 2001). Two types of trypsin

inhibitors are present in soybeans: Kunitz trypsin inhibitor and Bowman-Birk (BB)

inhibitor. These protein inhibitors (TI) have strong affinity for human digestive enzymes;

interfere in the digestion and absorption of proteins and may cause pancreatic enlargement

involving hypertrophy followed by hyperplasia of the exocrine cells by the production of

trypsin, chymotrypsin, elastase, amylase and serine proteases (Gallaher & Schneeman,

1986; Weder, 1986; Liener et al., 1988; Kwok & Niranjan, 1995). On the other hand, some

authors have reported that soybean BB inhibitor may contribute to the prevention of cancer

because of its anticarcinogenic and cancer chemopreventive properties (Kennedy, 1998;

Sessa & Wolf, 2001; Akoum et al., 2006).

To avoid potential deficiency in nutrient absorption, both inhibitors should reach high

levels of inactivation. However they are rather heat stable due to the presence of disulphide

bonds in their molecular structure. Heat treatment during long time, such as 60 to 70 min at

93ºC or 5 to 10 min at 121ºC are required to reach 90% of inactivation. However,

overheating in order to remove completely trypsin inhibitor activity reduces nutritive value

of soybeans and results in amino acid degradation and other deteriorative reactions (Kwok

et al., 2002).

Additionally to the inactivation of trypsin inhibitors, lipoxygenase (LOX), an important

enzyme which act as catalyst in lipid oxidation, should be inactivated as much as possible

in all soy foods (Van der Ven et al., 2005; Min et al., 2005). LOX (linoleate oxygen

redutase; EC 1.13.11.12) is a non-heme and non-sulfur iron containing dioxygenase which

catalyses the oxidation of 1,4-cis,cis-pentadiene to pentadienyl, which upon abstraction of

hydrogen, results in a pentadienyl radical intermediate. Pentadienyl radical may react with

oxygen to form peroxyl radical isomers and fatty acid hydroperoxides. Hydroperoxides as

well as decomposed products are potentially reactive substances that may cause

deterioration of food proteins and formation of volatile compounds, such as aldehydes and

ketones in presence of oxygen, light, enzymes and high temperatures (Kumar et al., 2003;

Huang et al., 2006b; Ying-Qiu et al., 2008). For soy food applications, soymilk for

example, LOX inactivation is commonly achieved by thermal treatments. Studies showed

that heat treatment during 10 min from 80 to 100ºC were effective in its inactivation

(Kwok & Niranjan,1995; Wang et al., 2008).

Chapter 2

22

2.2 Soymilk

2.2.1 Definition and composition

Soymilk has been consumed in China since 2000 years ago. Since then, the soymilk

consume was higher in China and Asian countries than milk in western countries.

Furthermore, soymilk is an intermediate for preparing soy foods, such as tofu (Kwok et al.,

1993; Liu, 1999; Wang et al., 2001).

Soymilk is an aqueous extract of soybeans. It is a fine emulsion closely resembling to milk

in appearance and composition, in addition to be lactose free. Table 2-3 shows chemical

composition of typical soymilk from different authors.

Table 2-3. Chemical composition of soymilk1

Reference Moisture Protein Fat Carbohydrate Ash

Iwuoha & Umunnakwe (1997)

Liu (1999)

Wang et al. (2001)2

Cruz et al. (2007)

91.6

90.8

85.7

91.7

3.1

3.6

2.2

3.8

1.82

2.00

1.60

1.86

2.33 0.94

2.90 0.50

10.50

0.68 0.68 1 Mean values expressed as g/100g (w/w). 2 Carbohydrates + ash.

In addition to chemical composition, quality parameters of fresh soymilk are: pH 6.60,

titratable acidity about 2.7%, viscosity 38.0 Pa·s and specific gravity of 1.055 according to

Iwuoha and Umunnakwe (1997).

Soymilk is a good source of vitamins. Contains about 7.36 mg/100mL of riboflavin (B2),

0.33 mg/100mL of thiamin (B1) (dry basis) (Hou et al., 2000) and pyridoxide (B6) and folic

acid (Kwok & Niranjan, 1995). Soymilk minerals are potassium (131.32 mg/100mL),

magnesium (22.43 mg/100mL), sodium (2.86 mg/100mL) (Achouri et al., 2007),

phosphorus (38.87 mg/100mL) and calcium (18.34 mg/100mL) (Achouri et al., 2008).

Thanks to rich composition, soymilk compares favorably to human milk (Liu, 1999).

Introduction

23

2.2.2 Soymilk processing

Traditional method of soymilk processing consists basically of the following steps: whole soybeans are soaked in water for 8-12 hours or overnight, then are washed and ground with water at a water:bean ratio between 8:1 and 10:1, depending on the desired final concentration. Slurry is then filtered through a cloth to remove the insoluble residue, known as “soy pulp” or “okara”, and then the filtrate is cooked for about 30 minutes (Johnson et al., 1983; Liu, 1999; Prawiradjaja, 2003). There are different industrial methods for soymilk production. The following processes showed in Figure 2-1, represent the traditional method and important developments and improvements in soymilk processing. Examples of these methods are Cornell, Illinois, Rapid Hydration Hydrothermal Cooking (RHHTC), cold-grind under vacuum (Prosoya), deodorization, and antioxidant and alkali treatment methods. Soymilk process can use a combination of these methods to produce a nutritive soymilk with high yields in solids and protein recovery (Golbitz, 1995; Liu, 1999; Prawiradjaja, 2003). Both Illinois and RHHTC methods incorporate all of the soybean parts into the soymilk due to high shear generated in the process. This particularity results in a soymilk with high percentage of solids (86-89% w/w) and protein recovery (90-93% w/w). Prosoya and Cornell methods are modifications of the traditional methods. Both methods reach low percentage of solids (55-65% w/w) and protein recovery (70-80% w/w) (Golbitz, 1995; Kwok & Niranjan, 1995). Even though some process modifications have improved soymilk quality, each method has its own advantages and disadvantages. RHHTC for example, applies temperature around 154ºC during soymilk processing (Figure 2-1). At this temperature chemical changes may occurs favoring formation of volatile compounds, which are responsible for undesirable off-flavors and color changes. The rest of the methods, applies moderate temperatures (about 100ºC), which may ensure microbial quality but at short shelf-life. In addition, controlling process parameters such as time, temperature and water-to-bean ratio have been suggested to improve off-flavors of soymilk (Achouri et al., 2008). On the other hand, choosing the right genetic variety of soybeans may have an important role affecting quality of finished product (Deman et al., 1975). Three basic heat treatments are carried out to extend shelf-life of soymilk: pasteurization, sterilization and ultra high temperature (UHT). Pasteurization refers to heat treatment, normally below 100ºC, which is adequate to destroy pathogenic microorganisms. This process also inactivates some enzymes and reduces total microbial counts. Sterilization is a severe heat treatment that requires heating at 121ºC for 15-20 min to achieve sterility. UHT process involves high temperatures (135-150ºC) for short time (a few seconds) to achieve a commercially sterile product (Kwok & Niranjan, 1995).

Figure 2-1. Soymilk processes.

Traditional method

Cornell method

Illinois method

The Rapid Hydration Hydrothermal Cooking

Prosoya method

Cold water soaking

(8-12 Hrs)

Wash

Water

Grinding

Okara Extract Cook

Cook Extract Okara

Soymilk

Soybean

Grinding with hot water

Okara Extraction

Cooking

Homogenization

(13.8 - 24.1 MPa)

Soymilk

Soak overnight in 0,5%

NaHCO3

Blanching at 100ºC

10-20 min

Grinding

Heat slurry to 82ºC

Homogenization at 3.45 and 24.1 MPa

Add water to give 12% solid

Neutralization

Add sugar, salt and flavoring

Pasteurization

Homogenization (24.1 MPa)

Soymilk

Soybean

Grind to flour

Slurry in water

Cook

(154ºC, 20 seconds)

Cooled

Centrifugation

Soymilk Okara

Soybean

Soak overnight

Cold grind in water

without oxygen

Cooking

(100ºC, 10-20 min)

Filtration

Soymilk Okara

Source. (Johnson et al., 1983; Gupta & Gupta, 1988; Golbitz ,1995; Kwok & Niranjan, 1995; Liu, 1999; Prawiradjaja, 2003).

Chinese method

Japanese method

Introduction

25

Consumption of soymilk in western countries has been limited partially by natural soymilk

off-flavors. These unpleasant soymilk characteristics are primarily derived from enzymatic

action which catalyzes the oxidation of polyunsaturated fatty acids, such as linoleic and

linolenic acids. Oxidation reactions initiate mainly in the soaking and grinding steps of

soymilk processing, when lipoxygenase is still active (Mizutani & Hashimoto, 2004). To

avoid potential deterioration problems in soymilk as a consequence of oxidation reactions,

studies applying heat treatments have reached great levels of lipoxygenase inactivation.

For instance, Gupta and Gupta (1988) achieved complete LOX inactivation applying 80ºC

for 10 min in the grinding step of soymilk elaboration. Other studies suggested that adding

extra ingredients such as antioxidants, sodium bicarbonate or masking agents would

improve soymilk flavor (Prawiradjaja, 2003).

As described above, oxidation reactions that take place during soymilk elaboration involve

the formation of hydroperoxides as primary reaction. These reactive substances originate a

high variety of both non-volatile and volatile final products, such as aldehydes, ketones,

alcohols, furans and acids. Most compounds of these chemical families are responsible for

off-flavors development, such as beany, grassy and rancid flavors, which adversely affect

organoleptic quality of soymilk (Torres-Penaranda & Reitmeier, 2001; N'Kouka et al.,

2004). In addition, oxidation products can be related to heart diseases, cancer and aging

problems. Although thermal treatments inactivate effectively LOX and high inactivation

levels of antinutritional factors (trypsin inhibitor activity), they also denature soy proteins

resulting in loss of stability and amino acid degradation. Other deteriorative reactions may

take place during treatment at high time-temperature, such as color changes and

development of cooked flavor. Moreover, nutrients such as vitamins may also be affected

by heat treatment (Kwok & Niranjan, 1995; Ying-Qiu et al., 2008). Generally, solids

recovery in soymilk extracts decreases as a function of extent and intensity of heat

treatment (Johnson et al., 1983).

Soymilk is an excellent growth medium for many microorganisms. High moisture, neutral

pH, high amount of nitrogenous compounds, fat, carbohydrates, minerals and vitamins,

make soymilk similar to microbial milk spoilage patterns. Microbial patterns of untreated

soymilk include mesophilic spores, enterobacteria, total mesophilic bacteria and Bacillus

gender (Kwok & Niranjan, 1995). At room temperature, untreated soymilk undergoes acid

curdling with a rapid drop in pH accompanied by separation of curds and whey. This

spoilage usually occurs after standing for 24h out of refrigeration. Proteolytic spoilage may

take place within week at refrigeration temperatures of 1ºC (Kwok & Niranjan, 1995; Bai

Chapter 2

26

et al., 1998). Soymilk treated by heat treatments, normally presents great reduction of

microbial counts. In general counts are below the detection limit (< 10 cfu/mL) and no

coliforms or Escherichia coli are found in soymilk products (Bai et al., 1998; Achouri et

al., 2007). Bouno et al. (1989) investigated the effect of different heat treatments on the

destruction of indigenous Bacillus spores in soymilk. Results showed that microbial load

was reduced from 3.34 to 1.52 log cfu/mL for boiling soymilk (1 min) in microwave oven

and to 1.40 log cfu/mL for steam heated soymilk at 110°C for 20 min. No microorganisms

were detected in autoclaved soymilk (121ºC, 15 min). Evidently, sterilization requires a

severe heat treatment which practically destroys all microorganisms, including spores.

In addition to mesophilic spore bacteria, thermophilic spore bacteria are extremely heat

resistant and could compromises soymilk quality. Processes designed to destroy

thermophilic spore bacteria may result in a product overheated obtaining sensory and

nutritive characteristics degraded. Thermophilic spores may survive conventional

treatments, such as UHT, but their growth and spoilage are affected under room

temperature storage conditions (Kwok & Niranjan, 1995).

2.3 Ultra high pressure homogenization

2.3.1 Concept and history

Emulsions are dispersions of phases that consist of two or more liquids largely immiscible.

In order to produce emulsions, disperse phase should be distributed in fine divided droplets

through the continuous phase, but a surface active of molecules should also be in the

interface of the droplet to prevent instantaneous coalescence (Floury et al., 2000).

Homogenization was first presented by August Gaulin at Paris in 1900 and was since then

largely used in the industries to disperse non-miscible phases, stabilize emulsion, or

prepare products with appropriate rheological properties (Thiebaud et al., 2003; Hayes &

Kelly, 2003b). The basic homogenizer design consists of positive-displacement pump

coupled to a pressure intensifier forcing the fluid to pass through the homogenization valve

(Middelberg, 1995). In any type of homogenizer valve, the fluid flow under pressure

through a convergent section called the homogenization gap and then expands (Figure 2-

2). As a result, a combination of mechanical forces takes place producing disruption of the

droplets. The operating pressure is controlled adjusting the distance between valve and seat

(Floury et al., 2004b).

Introduction

27

The best known food application is probably the homogenization of milk. Usually, the

process is performed between 60 and 70ºC and involves breaking milk fat globules into

fine fat droplets, preventing cream separation, thereby increasing stability and shelf-life of

milk (Diels et al., 2005; Zamora et al., 2007). In the classical design (Figure 2-2), the fluid

is fed axially into the valve seat, and then accelerated radially into the small gap between

valve and seat. Once the fluid leaves the gap, it becomes a radial jet that stagnates on an

impact ring before leaving the homogenizer at atmospheric pressure (Kleinig &

Middelberg, 1996; Kelly & Muske, 2004). APV-Gaulin design is often used in dairy

industry, where at large-scale processes two homogenization valves (two-stage) are used

under moderate pressures (70-100 MPa) (Thiebaud et al., 2003; Pereda et al., 2007).

Figure 2-2. Valve design of a conventional homogenizer (APV-Gaulin) obtained from Floury et al. (2004b).

In the last 10 years, studies about homogenization technology have evolved primarily by

the increase demand of products with nutritive quality, long shelf-life and high colloidal

stability. Thanks to the advances in material science, improvements in the homogenizer

designs allowed increasing the pressure level of working, leading to products of high

quality. Especial designs, very small dimensions and material changes are required to

withstand very high stresses during treatment (Pandolf & Kinney, 1998). Since then,

UHPH is largely used in the chemical, pharmaceutical and biotechnological industries to

emulsify, disperse, mix and process products.

Chapter 2

28

2.3.2 High-pressure homogenization equipments

There are different types of high pressure homogenization equipments, from prototypes to

industrial scale: MICROFLUIDIZER (Microfluidics International Corporation, USA),

NANOJET (Haskel, USA), EMULSIFLEX (Avestin, Canada) and STANSTED (Stansted

Fluid Power Ltd., UK) are equipments which achieve pressures higher than 200 MPa,

being known as UHPH equipments.

MicrofluidizerTM (Microfluidics International Corporation)

This technology reported by (Paquin & Giasson, 1989) was introduced in the food industry

in 1987. The system is divided into two micro-channels and then recombined in a reacting

chamber where jets of fluid collide at high velocity (up to 400 m/s), dissipating energy

instantaneously at the point of impact (Figure 2-3). The reaction chamber is static and

contains no moving parts. The limited aspect of this technology is the pressure delivered

by the equipment, which is linked to the flow and equipment design (Middelberg, 1995;

Paquin, 1999; Geciova et al., 2002). There are industrial scale equipments (2000 L/h) using

this technology able to work at 270 MPa pressure.

Figure 2-3. Schematic view of the Microfluidics International Corporation, obtained from

http://www.iesmat.com/Productos-MFL-LAB-M-110P.htm

EmulsiflexTM (Avestin)

This technology has been applied for pharmaceutical emulsions, liposomes and

dispersions. The reaction chambers are coated with ceramic material reaching pressures up

to 220 MPa. Valve adjustment is achieved using a micrometer screw allowing a fine

adjustment of the gap between valve seat and head. This mechanism is very critical at high

Introduction

29

pressures (Paquin, 1999). The process has a constant flow rate of 3 L/h and is able to work

at small sample volume (10 mL).

Stansted technology (Stansted Fluid Power Ltd.)

The design of Stansted homogenization valve is made from ceramic material that allows to

withstand ultra high pressure levels. Moreover, geometry of the valve has been modified

compared to classical design of APV-Gaulin (Figure 2-2). In the Stansted valve (Figure 2-

4) the flow directions through the valve are reversed, the fluid is first fed axially at high

pressure along the mobile part of the valve and flows with high velocity through the

narrow gap between the valve and valve seat. The size of the gap and the resulting velocity

of the fluid generated by the high pressure depend on the force acting in the valve piston,

which can be adjusted to regulate the homogenizing intensity. The pressure drop of the

fluid in the valve is called the homogenization pressure (Floury et al., 2004ab). The

maximum pressures reached by Stansted valve (400 MPa) are due to the narrow gap (2-5

μm vs 10-30 μm) (Floury et al., 2004a).

Figure 2-4. Schematic view of the Stansted high pressure homogenization valve obtained from Donsì et al. (2009a).

Although this configuration may look simple, the fluid dynamics involved is quite

complex. According to Floury et al. (2000), intense energy changes occur in the

homogenization valve, causing strong turbulent flow, cavitation and shear phenomena.

Chapter 2

30

Studies provide some evidence supporting that shear phenomenon is one of the primary

mechanisms of cell or particle disruption during UHPH treatment (Middelberg, 1995;

Kleinig & Middelberg, 1998). Disruption is in fact obtained in the narrow valve gap when

deformation beyond a critical level is induced by the intense shear forces and elongational

flow caused by the restriction between the piston and the seat of the valve (Figure 2-4).

Cavitation is the formation of cavities due to the local vaporization of the fluid under

conditions of pressure lower than its vapor pressure. When cavities flowing within the

liquid through the system find a region of high pressure, they collapse violently, causing

vibrations with a disruption effect (Diels et al., 2005). Turbulence occurs when a fluid

flows at high speed over a surface. Due to surface roughness, above a certain velocity the

fluid streamlines no longer follow the shape of the surface, but deviate from the surface,

resulting in the formation of vortices which interfere one another, causing a disorderly

movement of the fluid particles (Doulah et al., 1975) This fluid movement leads to the

break-up of the dispersed phase into small droplets which can collide among then, leading

sometimes to coalescence. Usually a dynamic equilibrium between breakage and

coalescence is achieved.

A schematic view of UHPH equipment (FPG11300) used in the present study is shown in

Figure 2-5.

Introduction

31

Figure 2-5. Scheme of the UHPH equipment (FPG11300:400) used in the present study, Stansted Fluid

Power Ltda., Essex, UK.

2.3.3 Temperature increase during the UHPH treatment

Generally UHPH technology is considered as an alternative to heat treatments. However

during high pressure process, a linear temperature increase occurs with the increase of

homogenization pressure at constant inlet temperature (Floury et al., 2003; Sandra &

Dalgleish 2005; Serra et al., 2008b). Temperature increase is due to the adiabatic heating

generated by the viscous stress when the fluid is impinged at high velocity on the

homogenization valve during treatment (Hayes & Kelly, 2003b; Bouaouina et al., 2006).

Thus, kinetic energy is converted into thermal dissipation by the phenomena described

above, generating fluid heating (Thiebaud et al., 2003; Floury et al., 2004b). Pereda et al.

(2007) described an increase of 19.5ºC per 100 MPa from 100 to 300 MPa at 40ºC of inlet

temperature, using Stansted FPG11300 equipment. These results are in accordance with

Thiebaud et al. (2003) who found an increase of 18.5ºC in the same range of pressure using

4, 14 and 24ºC of inlet temperatures. Donsì et al. (2009b) reported a linear increase of 18ºC

per 100 MPa, applying pressures of 250 MPa and inlet temperature of 2ºC, whereas Serra

(2008c) described an increase of 19.5ºC per 100 MPa applying pressures of 100, 200 and

300 MPa at 30ºC of inlet temperature. All these groups carried out their studies using

Stansted Fluid Power equipment. The resultant temperature increase in the high pressure

Chapter 2

32

valve according to inlet temperature and pressure applied, may reach temperatures higher

than 120ºC. The holding time at high temperature was estimated being lower than 1 second

(Picart et al., 2006). Therefore, product treated by UHPH technology undergoes a

combined effect between pressure and temperature at low holding time, minimizing the

thermal damage in the overall quality of the product.

2.3.4 UHPH applications

The first high-pressure homogenization application in industry was intended to prepare or

stabilize, disperse and mix emulsions. To reach these objectives pressure around 20 MPa

was applied in different types of processes, such as pharmaceutical, chemical, cosmetics

and ceramic industry (Kielczewska et al., 2003; Floury et al., 2004a; Diels et al., 2005).

During the last decade, UHPH has been investigated for its potential application in the

food industry. Several research groups have published studies focused on UHPH food

applications using different pressures and inlet temperature combinations, either single or

double homogenization stage. Table 2-4 lists the main UHPH applications.

Nowadays, it have been designed equipments able to reach pressures of 400 MPa at 500

L/h flow rate, however these equipments are still prototypes.

Table 2-4. UHPH effect on different food applications

Food Pressure (MPa) Inlet temperature (ºC)

Main UHPH effect Reference

Oil-in-water emulsions

20, 150 and 300 4 Results showed significant modifications in the structure and texture with increasing pressure. Drolpet size was reduced with increasing pressure

Floury et al. (2000)

Soy protein emulsions

20 to 350 5 and 20 Droplet sizes of emulsions were greatly reduced and treatments. Pressure of 350 MPa produced highly viscous and stable emulsions.

Floury et al. (2002)

Milk 100 and 300 4, 14 and 24 At high pressure and high inlet temperature, the inactivation of endogenous flora increases. High reduction of fat globule size was reached.

Thiebaud et al. (2003)

Milk 150, 200 and 250 45 Great reduction of fat globule size and complete inactivation of psychrotropic bacteria. Reductions of mesophilic bacteria were 1.3, 1.83 and 3.06 log cfu/mL at 150, 200 and 300 MPa, respectively.

Hayes, et al. (2005)

Milk for cheese making

179 and 100 to 300+30 (first and

double stage)

25 and 30 Improvement of coagulation properties and aggregation of casein micelles. Increasing of the wet yield of curd and moisture content.

Sandra and Dalgleish (2007) and Zamora et al. (2007).

Soymilk for producing soy yogurt

200 and 300 40 and 50 Considerably reduction of microbial load and highly physical stability of soymilk. Disruption of colloidal particles and aggregates at 300 MPa. High homogeneity and compact network structure of soy yogurt.

Cruz et al. (2007) and Ferragut et al. (2009)

Milk for producing yogurt

100 to 300 30 UHPH was capable of reducing particle size leading to the formation of fine dispersions. Desnsity of gel, aggregation rate and water retention are improved as increased pressure conditions.

Serra et al. (2007) and Serra et al. (2008a)

Apple juice 100, 200 and 300 4 and 20 Successful reduction of microbial load at 200 and 300 MPa (comparable to pasteurization). During 60 days of storage (4ºC) mesophilic counts did not change.

Suárez-Jacobo et al. (2010) and Suárez-Jacobo (2011)

Chapter 2

34

2.3.5 UHPH effect on microbial inactivation

Traditionally UHPH has been used in the pharmaceutical and biotechnological industry for

the disruption of microbial cells for obtaining intracellular extracts for several applications

(Bury et al., 2001; Geciova et al., 2002). However, for the UHPH application in liquid

food, the challenge relies on finding the right conditions for microbial inactivation without

compromising food characteristics.

Several studies have been published in relation to the UHPH effect in the native microbiota

inactivation of different food products, such as Hayes et al. (2005) in milk samples, Cruz et

al. (2007) in soymilk and Suárez-Jacobo et al. (2010) in apple juice (Table 2-4). On the

other hand, the UHPH effect on specific pathogen bacteria has been published by several

groups. Briñez et al. (2007) applied pressure of 300+30 MPa (first and second valve) at 6

and 20ºC of inlet temperature in milk and orange juice inoculated with Staphylococcus

aureus and Staphylococcus carnosus. They found that inlet temperature, food matrix and

bacterial strain influenced the lethality level, being higher for S. aureus in whole milk at

20ºC of inlet temperature. Velazquez-Estrada et al. (2008) treated whole egg inoculated

with Salmonella enteric serovar Senftenberg 775W at 150, 200 and 250 MPa and 6ºC of

inlet temperature. They found that level of pressure influenced significantly the lethality,

obtaining a reduction of 3.8 log units at 250 MPa. Vachon et al. (2002) who applied

pressures of 100, 200 and 300 MPa (1, 2 and 3 passes) at 25ºC of inlet temperature on milk

inoculated with Listeria monocytogenes and E. coli O157:H7, found significant reduction

of both microorganisms as increased pressure and number of passes. However, E. coli was

much more sensitive to the treatment than L. monocytogenes. Other groups have been

shown that increasing the passes number or cycles increase microbial reduction (Hayes &

Kelly 2003b; Picart et al., 2006; Smiddy et al., 2007). Donsì et al. (2009b) who worked

with Escherichia coli and Saccharomyces cerevisae inactivation at 250 MPa, observed that

microbial inactivation increased as the pressure level increased, reaching in some cases, a

complete inactivation. Similar conclusions were found by Thiebaud et al. (2003), who

reported that microbial inactivation was a function of homogenization pressure and inlet

temperature. They concluded that high inlet temperature combined to high pressure

produced an increase of microbial inactivation. Therefore, parameters of UHPH treatment

such as pressure level, temperature, multi-pass homogenization play an important role in

the microbial inactivation.

Introduction

35

Studies have shown that cellular membrane is the main site affected by pressure

(Earnshaw, 1992). The exact cause of cell disruption is controversial in the literature. In

fact, it is not possible to specify a single overall disruption mechanism, without taking into

account the product parameters (e.g. viscosity), operating parameters (e.g. flow rate,

temperature), and device parameters (e.g. valve geometry) (Donsì et al., 2009a). Therefore,

homogenization valve and machine design could be one of the most important factors,

although temperature increase produced by UHPH valve can affect microbial inactivation

(Pereda et al., 2007). In this way, temperature noticeably affects membrane lipid

composition and physical state of bacterial cell. At low inlet temperature (2ºC to 10ºC),

crystallization of phospholipids occurs and cell membranes become more rigid and

consequently more sensitive to pressure (Vachon et al., 2002). Microbial cells experience a

nonspecific tearing apart of the cell wall (Middelberg, 1995), which is determined by the

physical interaction of the cells with the small-gap homogenization valve, in a co-operative

action between the destructive stresses originated from the fluid condition and physical

strength of the cells (Shamlou et al., 1995).

Regarding bacterial spores, little information is available. However, using mild

temperatures and repeated treatment cycles, may increase significantly the inactivation of

Bacillus spores, although high resistance to UHPH treatment is expected (Feijoo et al.,

1997; Chaves-López et al., 2009).

Microbial strain and cell concentration

Some studies have shown changes in cell morphology as well as splits in the cytoplasmic

membrane of bacteria submitted to UHPH treatments (Kheadr et al., 2002). Several studies

indicated that Gram-negative bacteria, characterized by a thinner cell wall membrane, are

more sensitive to high pressure homogenization than Gram-positive bacteria. This suggests

a correlation between cell wall structure and UHPH resistance, which indicates that high

pressure homogenization, destroys vegetative bacteria mainly through mechanical

disruption of the cell integrity during the pass through UHPH equipment (Vachon et al.,

2002; Wuytack et al., 2002).

On the other hand, initial cell concentration can have an important role in the UHPH

efficacy. Studies carried out with this purpose indicated a correlation between the initial

cell concentration and high pressure effect. For instance, Moroni et al. (2002) observed that

UHPH treatment became less effective at greater initial load of lactococcal bacteriophages.

Chapter 2

36

Similar results were found by Tahiri et al. (2006) and Donsì et al. (2006) who worked with

different microbial strains and demonstrated that UHPH effectiveness increased at low

initial bacterial concentration.

2.3.6 UHPH effects on physico-chemical properties

Additionally to UHPH effects on microbial inactivation, fat globules, particle size,

proteins, enzymes and other colloidal components are affected by high pressure

homogenization. Due to the high number of publications applying UHPH treatments in

milk and milk products, most of the effects reported up to now have been on dairy

products. Some of them are reported in this section.

Effect on particle and fat globule size

The different phenomena that a fluid experience thought the pass of high-pressure valve

impact strongly in the particle, protein and fat globule distribution in the continuous phase

of an emulsion. For the case of whole milk, the fat droplets diameter in non-homogenized

sample is usually between 0.1 and 20 μm, having an average between 3 and 5 μm (Pereda,

2008). Generally, milk homogenized by conventional Gaulin valve achieved particle

diameter of 1 μm compared to non-homogenized milk (Dalgleih et al., 1996).

Several studies carried out in milk (Hayes & Kelly 2003b; Thiebaud et al., 2003; Hayes et

al., 2005; Zamora et al., 2007; Pereda et al., 2007) demonstrated that fat globule size was

drastically reduced applying pressures of 200 MPa, compared to conventional

homogenized milk. Picart et al. (2006) obtained fat globule diameter lower than 0.36 μm

representing around 78 and 93% of the total fat volume in UHPH-treated milk at 200 and

300 MPa respectively. For soymilk samples, few studies have investigated soymilk treated

by UHPH technology at the present moment. For instance, Cruz et al. (2007) obtained

evidences that fat droplets size of soymilk decreased after UHPH treatment, although no

apparent differences were observed between 200 and 300 MPa conditions. Nevertheless,

they observed an increasing of aggregates as pressure increased. Authors such as Thiebaud

et al. (2003), Hayes et al. (2005), Pereda et al. (2007) and Zamora et al. (2007) also

observed an increase in fat globule aggregates of milk treated at 250 and 300 MPa. The

formation of aggregates could take place by different reasons. According to Floury et al.

(2002), aggregates are formed by coalescence phenomenon of freshly-disrupted oil

droplets that occurs directly after the homogenization valve. This phenomenon depends on

Introduction

37

the flow rate in the homogenizing device, where turbulence zones and velocity gradients

take place at the exit of the valve gap. In this sense, Desrumaux and Marcand (2002)

suggested that, flow rate, high shear rates and heat dissipation by the pressure drop inside

the high pressure valve increase the probability of collision between fat droplets and

coalescence. On the other hand, some authors observed that proteins, such as milk-caseins

may affect the fat globule size. Dalgleish et al. (1996) suggested that the reduction of fat

globule size by HPH treatment, cause a strong increase of surface-active of the globule in

which a great proportion of casein may be adsorbed into exposed interface. However, at

higher pressures, the amount of available casein may become limited, which would

account an increase in fat globule size after treatment by partial agglomeration of very

small globules insufficiently covered by surface-active material (Datta et al., 2005; Hayes

et al., 2005).

Inlet temperature used during UHPH treatment of milk also affects the fat globule size.

Datta et al. (2005) reported that the fat state (liquid or solid, or part-liquid/part-solid) has a

significant influence in the extent of globule size reduction. They concluded that most of

the milk fat needs to be in liquid state prior to homogenization valve to ensure the

treatment effectiveness (Hayes & Kelly, 2003b; Thiebaud et al., 2003).

Color

Due to the new state of particle distribution caused by the UHPH treatment on the food

fluid, the reflection and transmission of the light may change and thus affecting color

parameters (L*, a* and b* in the CIELAb scale). For milk samples for example, Pereda et

al. (2007) and Hayes and Kelly (2003b) observed an increase of lightness (L*) compared

to untreated milk, due to the increase of light reflected produced by the fat globules in

homogenized milks. However, (Serra et al., 2008a) found a decrease of lightness in skim

milk treated at pressures between 200 and 300 MPa. They attribute the results to the casein

micelles aggregation decreasing the surface of reflection. For soymilk samples, Cruz et al.

(2007) observed a decrease of lightness which was related to the protein and lipid-protein

aggregates causing a decrease of light reflection. For apple juice samples, Saldo et al.

(2009) observed values of lightness slightly lower for treatments at 100 and 300 MPa

compared to untreated and pasteurized samples. Therefore, particle distribution, state of

aggregation and nature of chomophores of food matrix and the intensity of pressure

applied, define the response of quality color.

Chapter 2

38

Viscosity

The high homogenization obtained after UHPH treatment may decrease the viscosity of the

food fluid by the reduction of particle size and better protein dispersion in the interface o/w

(oil-water). Some studies carried out viscosity analysis in order to determine the effect of

UHPH on raw material products. For instance, Cruz et al. (2007) reported that soymilk

treated at 200 and 300 MPa present lower viscosity values than untreated soymilk. In that

case, untreated soymilk was submitted to the action of a colloidal mill to obtain a coarse

emulsion. However UHT-treated soymilk showed lower viscosity than those UHPH

soymilks. This result was attributed to the increase in effective/volume of disperse phase

due to the decrease of particle size in UHPH treatment that increased the internal friction

leading to the detection of high viscosity. On the contrary, Floury et al. (2002) studied

UHPH treatment on of globulin 11S fraction. They reported an increase of viscosity as

homogenization pressure increases in protein emulsions treated by UHPH. This result was

attributed to the formation of large aggregates by intermolecular interactions among

denatured protein molecules. They concluded that soy-proteins emulsion treated at 350

MPa produces highly viscous and stable emulsion.

For milk samples, Serra et al. (2008a) did not observe differences between milks treated at

100 and 300 MPa. On the other hand, Pereda et al. (2007) reported lower viscosity values

of milks treated at 200 MPa instead of 300 MPa. They attributed these differences to large

particles or fat aggregates formed in those samples treated at 300 MPa.

2.3.7 UHPH effects on proteins and enzymes

The UHPH effect observed in the particle distribution as well as fat globules, protein being

macromolecular components may undergoes important changes in the structure which

affects its solubility and functionality. The most studied proteins and enzymes are reported

in this section.

Whey protein and casein micelles

High pressure homogenizer treatment causes different effects on different types of whey

protein. According to Paulsson and Dejmek (1990), the major whey proteins present in the

milk are β-lactoglobulin and α-lactoalbumin. Because of this, they may experience the

most UHPH effects. Protein denaturation is one of the effects that take place in UHPH

Introduction

39

treatments. For instance, Hayes et al. (2005), Datta et al. (2005), Zamora et al. (2007) and

Pereda et al. (2008) reported some degree of whey protein denaturation induced by UHPH

treatment. Similar conclusions were obtained by Serra et al. (2008a) who found an increase

of β-lactoglobulin denaturation degree (from 14 to 26%) from 100 to 300 MPa. β-

lactoglobulin was more sensible than α-lactoalbumin in both heat and UHPH treatments.

Denaturation process of α-lactoalbumin is around 80-90% reversible after short-time

heating (Ruegg et al., 1977), converting α-lactoalbumin more resistant than β-lactoglobulin

to thermal denaturation. On the contrary, Hayes and Kelly (2003b) did not observe whey

protein denaturation at pressures between 50 and 200 MPa (single stage or double stage)

using Stansted equipment. This result is in agreement with those obtained by Sandra and

Dalgleish (2005) and Bouaouina et al. (2006).

Hayes and Kelly (2003b) reported that casein was not affected in milk treated at pressures

lower than 150 MPa. On the other hand, at 200 MPa a decrease of 5% was found in the

size of casein micelle (from 180.75 nm to 170.65 nm). In this way, Roach and Hart (2008)

observed a linear decrease of 30% of casein micelle size at pressures from 0 to 200 MPa

reducing from 278 nm to 171 nm. These results suggested that UHPH partially remove

parts of the casein micelle surface, leaving it still active (Sandra & Dalgleish 2005, 2007).

Soy protein

As reported in the section 2.1.2, the major soy protein is formed by globulins. The UHPH

effect on globulins solubility was carried out by Floury et al. (2002). In that study an

aqueous solution of soy globulin 11S was treated at pressures between 20 and 350 MPa.

They observed that protein solubility was preserved at relative moderate pressures (≤150

MPa). However, increasing the homogenization pressure above 200 MPa, led to a quite

strong decrease in the globulin solubility with a wide variation of 40%. The loss of

globulin solubility (above 200 MPa) was caused by the protein denaturation due to the

homogenization process. The mechanical forces that take place during UHPH process

could have affected the macromolecular conformation of soy globulin. Changes in

macromolecular structures lead to interactions among proteins which may induce

denaturation and aggregation. The strength increase of hydrophobic effect causes

improvement of protein-protein interactions at high pressure of homogenization. These

effects produce highly viscous and stable emulsions. Cruz (2008) achieved also high

Chapter 2

40

colloidal stability of UHPH-treated soymilk, despite of complete globulin denaturation

reached at 300 MPa.

Trypsin inhibitor activity

Trypsin inhibitors are substances that adversely affect nutritional properties of soy

products, including soymilk (see 2.1.2). Therefore, a maximum inactivation should be

achieved as much as possible. Few studies have reported the UHPH effect on TI

inactivation. For instance, Cruz (2008) observed that the increase of inactivation was

linked to the pressure increase in UHPH-treated soymilk. Poliseli-Scopel et al. (2012)

reached for UHPH-treated soymilk at 300 MPa similar degree of inactivation to

pasteurization treatment (37% of initial activity). On the other hand, nowadays there is not

a European regulation or recommendation about TI inactivation. Similarly, at the present

time it has not reported health problems due to the consumption of soymilk in humans.

Enzymes

Regarding to enzymes, several studies have published the UHPH effect on native enzymes

of different foodstuffs. Hayes and Kelly (2003a) studied the high-pressure homogenization

on alkaline phosphatase and plasmin activities in raw whole milk. They observed that

inactivation of plasmin increased as pressure increased. Two stage treatments were more

effective than single stage treatment. However, UHPH treatment was not effective in the

inactivation of alkaline phosphatase. On the other hand, Datta et al. (2005) observed

important reduction of alkaline phosphatase, plasmin and lactoperoxidase activity in milk

treated at 200 MPa and 45ºC of inlet temperature. Complete lactoperoxidase inactivation

was achieved at 200 and 300 MPa by Pereda et al. (2007). However, they observed that

psychrotrophic counts remained below the legal limit established for 21 days in samples at

200 MPa at 30ºC, since the lactoperoxidase was only partially inactivated. At pressures of

150, 200 and 250 MPa, Hayes et al. (2005) observed residual activity of 91 and 34% for

150 and 200 MPa respectively and complete inactivation at 250 MPa.

Pectin methylesterase is an enzyme heat resistant and responsible for the loss of turbidity

in orange juice during storage. Lacroix et al. (2005) obtained a reduction of 20% in UHPH-

treated orange juice without pre-warming. Nevertheless, a combination of UHPH and pre-

Introduction

41

warming (50ºC, 10 min) increased significantly the effectiveness of pectin methylesterase

inactivation compared to only pre-warming. On the other hand, Velazquez-Estrada et al.

(2008) observed a drastic reduction (> 90%) under 200 and 300 MPa of pressure at 10 and

20ºC of inlet temperatures in orange juice compared with conventional pasteurization.

Another enzyme which plays an important role in the quality of fruit juice is

polyphenoloxidase. This enzyme often catalyzes reactions of color degradation,

undesirable flavor formation and lost of nutrient impacting negatively in the product

quality. Suárez-Jacobo, (2011) reported complete inactivation of polyphenoloxidase in

apple juice treated at 300 MPa and 4ºC of inlet temperature.

Finally, lipoxygenase is commonly present in cereal foodstuffs and primarily in soy

products. In soymilk it has a particular interest by its implication in flavor quality (see

2.1.2). There are few studies about the effect of UHPH on lipoxygenase activity. Cruz

(2008) obtained complete inactivation at pressures of 200 and 300 MPa and inlet

temperature of 40 and 50ºC for soymilk samples. Probably, physical phenomena produced

by homogenization valve in combination with rising temperature, induced structural

modifications on enzyme conformation causing loss of activity.

2.4 References



Achouri, A., Boye, J.I. & Belanger, D. (2005). Soybean isoflavones: Efficacy of extraction

conditions and effect of food type on extractability. Food Research International. 38,

1199-1204.


soymilk flavour and quality. International Journal of Food Science & Technology. 43,

82-90.

Akoum, O., Richfield, D., Jaffrin, M.Y., Ding, L.H. & Swart, P. (2006). Recovery of

trypsin inhibitor and soy milk protein concentration by dynamic filtration. Journal of

Membrane Science. 279, 291-300.

Chapter 2

42

Aparicio, M.I., Cuenca, A.R., Villanueva, S.J. & Zapata, R.A. (2008). Soybean, a

promising health source. Nutricion Hospitalaria. 23, 305-312.

Bai, Y., Wilson, L.A. & Glatz, B.A. (1998). Quality of commercial shelf-stable soymilk

products. Journal of Food Protection. 61, 1161-1164.

Bouaouina, H., Desrumaux, A., Loisel, C. & Legrand, J. (2006). Functional properties of

whey proteins as affected by dynamic high-pressure treatment. International Dairy

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Bouno, M.A., Niroomand, F., Fung, D.Y.C. & Erickson, L.E. (1989). Destruction of

indigenous Bacillus spores in soymilk by heat. Journal of Food Protection. 52, 825-826.

Briñez, W.J., Roig-Sagués, A.X., Hernández-Herrero, M. & Guamis, B. (2007)

Inactivation of Staphylococcus spp. strains in whole milk and orange juice using ultra

high pressure homogenization at inlet temperature of 6 and 20ºC. Food Control. 18,

1282-1288.

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Chapter 3

Material and methods

57

3


This chapter includes description of all analyses and methodologies used to evaluate the

soymilk produced in pilot scale. Some methods applied, such as soymilk elaboration,

treatments and trypsin inhibitor activity were performed by two similar methods, being the

second one an alternative of the first one. Soymilk elaboration, treatments and some

methods of analysis, were used in more than one study. The methods used in each study

are reported in the specific chapter.

3.1 Soymilk elaboration

Method A. Soybean (Majesta variety) used in this study was provided by Liquats Vegetals,

S.A. (Girona, Spain). The methodology used to produce soymilk was based in the method

described by Liu (1999) and Yuan and Chang (2007). Whole soybeans were soaked (3:1

water-to-soybean ratio) during 15 h at room temperature. The volume increase was

expressed as: (W2/W1)x100, where W2 is the mass of wet soybeans and W1 is the mass of

dry soybeans.

Soaked soybeans immersed in 75% of the total water used in the soymilk elaboration and

then ground in a crushing machine (9:1 water-to- hydrated soybean ratio) with heating

control (adapted from Frigomat, Italy) for 20 minutes at 60ºC and 80ºC with recirculation

in a colloidal mill (E. Bachiller B. S.A., Spain). After that, pulp was separated by filtration

using a 0.2 mm steel sieve (model: CE98, Mejisa – Mectufry, Spain). For the second

extraction, the pulp was immersed in the 25% remaining water and then the mixture was

heated at 60ºC or 80ºC at continuous agitation. After that, the mixture was filtered and then

mixed with the first fraction of soymilk. Soymilk and pulp were finally weighted. This

mixture was considered the soymilk BP.

Method B. Using the same soybeans variety described in method A, soymilk elaboration

was the same with the following modifications. After soaking soybeans, only an extraction

Chapter 3

58

at 80ºC for 20 min was used to elaborate soymilk. Weighting control in each step of

elaboration was not performed. This method was applied in all chapters, except chapter 4.

3.2 Soymilk treatments: UHPH, pasteurization and UHT

UHPH A. UHPH treatments were conducted with an ultra high-pressure homogenizer

(model: FPG11300, Stansted Fluid Power Ltd., UK). This device (flow rate of 120 L/h) is

provided with two intensifiers, driven by a hydraulic pump and a high-pressure ceramic

valve able to support 400 MPa. Inlet and outlet temperatures of soymilk were controlled by

two heat exchangers (Garvía, Spain) located before the machine entrance and after the

high-pressure valve, respectively. During treatments inlet temperature and temperature

after homogenization valve was monitored.

UHPH B. Benchtop ultra high pressure homogenizer (model: FPG12500 Stansted Fluid

Power Ltd., UK) was used for the assays. This device (flow rate of 15 L/h) is provided

with two intensifiers, driven by a hydraulic pump and a high pressure ceramic valve able to

support 400 MPa. Inlet and outlet temperature of soymilk were controlled and monitored

by hot bath located before the machine entrance and by cold interchange located after the

high-pressure valve. This UHPH system was applied to carry out the third step described in

the working plan (Figure 1-2). The spores (105–106 spores/mL) suspended in 500 mL of

sterilized soymilk (sterilization at 121ºC for 10 min) were subjected to UHPH treatment of

300 MPa and 55, 65, 75 and 85 ºC of inlet temperature.

Pasteurization. Soymilk BP was homogenized at 18 MPa (LAB type: 22.51, Rannie,

Denmark) and subsequently pasteurized using a tubular heat exchanger (ATI, Spain) at

95ºC for 30 s.

UHT. BP samples were subjected to homogenization of two stages (18 and 4 MPa)

performed in a homogenizer (X68IE+X68P, Niro Soavi, Italy) previous to UHT treatment

(142ºC for 6 s) with indirect system equipment (6500/010, GEA Finnah GmbH Ahaus,

Germany).

3.3 Production yield

To determine production yield, mass balance was applied to 4 independent elaborations

taking into account the weight of dry seeds, soaked seeds, and ground seeds in water,

soymilk and pulp. Values were calculated according to law of mass conservation for


59

which, in a determined volume, the sum of ingredients mass is equal to the sum of products

mass:

. .

i e

d mc mi medt

= −∑ ∑

Assuming steady state, the equation is reduced at:

. .

0vcdmdt

mi me

=

=∑ ∑

Equation indexes, “mi” and “me” are, respectively, input mass and output mass.

To calculate global mass balance or component mass balance, input mass should be the

same of output mass in the control volume. In this process there is not continuous flow

neither time variation. Each production was performed by bath in a close system.

Therefore, it is convenient to considerate no consumption neither generation of mass or

energy from chemical reaction or mass transference. Productivity of soybeans during

soaking, solids recovered, loss of water and overall yield expressed as g/100g was

calculated by this method.

Another way to express yield is based in the ratio of soymilk produced by the soybeans

seed used:

Yield = litters of soymilk produced kilograms of seeds

3.4 Storage of soymilk

Condition A. To perform the third step described in the working (Figure 1-2), UHPH

treated and pasteurized soymilks were stored at 4ºC for 28 days. Samples were transferred

aseptically to sterile bottles of 100 mL and divided for each day of analysis: 1, 7, 14, 21

and 28 days of storage. Each bottle was aseptically opened in the correspondent day of

analysis.

Chapter 3

60

Condition B. To achieve the forth step described in the working plan (Figure 1-2), UHPH

and UHT soymilks were packaged in a coater paperboard cartons (200 mL Tetra Brick

containers) by using a Tetra Pak (TBA9 slim line, Switzerland) aseptic technology. The

tetra brick containers were stored at room temperature during 180 days. Samples were

analyzed in triplicate at day 1, 20, 40, 60, 90, 120, 150 and 180 days of storage. For each

day of analysis, 3 bricks were randomly selected and then mixed in a glass prepared for

this purpose. This procedure was carried out for each treatment.

3.5 Soymilk and soybeans physico-chemical analysis

Soybeans were ground in a crushing machine (Morphy Richards, S., UK) prior to the

analysis. Dry matter and ash content were analyzed by reference AOAC method (AOAC,

2000); total nitrogen content was analyzed by Dumas method (FIL - IDF, 2002) in both

soybeans and soymilk samples. The pH of soymilk was measured with a pH meter (model

GLP 21+ Crison, Spain). Soybeans and soymilk fat content was determined by ASE 200

(Accelerated Solvent Extraction, USA). For soybeans, 2 g of sample were mixed with sea

sand (~ 2:1) and then transferring the mix to 11 mL cell extraction. 3 cycles of 10 min at

105ºC and 1500 psi were applied according to the method proposed by the manufacturer.

Petroleum ether was used as extracting solvent which were further evaporated with

nitrogen and the residue weighted. For soymilk, 1 g of sample was weighted and mixed

with 1.1 g of sea sand and 0.9 g of celite and then transferring the mix to 11 mL cell

extraction. The fat extraction method was the same described above with the following

modifications: 3 cycles of 1 min at 120ºC and 1500 psi were applied. Petroleum ether and

2-propanol (60:40) were used as extracting solvents. The fat results were contrasted with

Soxhlet reference method (AOAC, 2000). These analyses were performed in soymilk in all

study steps described in the working plan, except inoculation study.

3.6 Microbiological analysis

This topic are related to the microbial quality of BP and treated soymilks and on the other

hand related to the inactivation study of spores inoculated in sterile soymilk BP.


61

3.6.1 Microbiological quality

Microbiological quality of soymilk samples was assessed by enumerating the following

microorganisms: mesophilic aerobic bacteria were counted on PCA medium (Oxoid Ltd,

UK) incubated for 48 h at 30ºC. Mesophilic aerobic spore counts were assessed by heat

shock at 80ºC for 10 min, quickly cooled in ice and plated on PCA medium (Oxoid) and

incubated for 48 h at 30ºC. Enterobacteria counts were determined in violet red bile

glucose agar (VRBG, Oxoid) incubated at 37ºC for 24 h. Yeasts and moulds were detected

in rose-bengal chlorampenicol medium (Oxoid), incubated at 25ºC for 5 days.

Staphylococcus aureus was determined in Baird-Parker RPF (bioMérieux) and incubated at

37ºC for 48h. Bacillus cereus was determined in brilliance Bacillus cereus medium

(Oxoid), supplemented with Bacillus cereus selective supplement (Oxoid) and incubated at

30ºC for 48 h. Salmonella sp. was investigated through sample pre-enrichment at 37ºC for

24 h and then enriched in Muller Kauffman broth (bioMérieux S.A.) and Rappaport

Vassiliadis broth (bioMérieux S.A.) at 37ºC and 42ºC for 24h, respectively. Enrichments

were streaked onto XLD (Oxoid) and SM2 agar media (bioMérieux S.A.) and incubated at

37ºC for 24 h.

Sterility test. Soymilk samples were incubated at 30ºC for 20 days. During this period,

coagulation and phase separation were checked visually. Mesophilic aerobic bacteria was

determined on PCA medium (Oxoid) in all incubated bottles.

3.6.2 Isolate collection and selection

Isolates were obtained from soymilk samples treated by UHPH at 300 MPa and 55 and

65ºC of inlet temperature. Spore counts were assessed by heat shock at 80ºC for 10 min,

quickly cooled in ice and plated on PCA medium (Oxoid) and incubated for 48-72 h at

30ºC. Typically, colonies representing visually distinct morphology (ranging from 1 to 5

colonies per sample) were selected and streaked for purity on TSA (TSA, Oxoid).

Biochemical testing on cultures using the API 50 CHB kit (bioMérieux, France) was

performed and thereafter, genetic identification was established based on comparative 16S

rRNA sequences. Briefly, isolates were cultured on TSA. Bacterial genomic DNA was

prepared by using DNeasy tissue kit (Qiaagen, Valencia, USA). 16S rRNA genes were

amplified by conventional PCR. Amplicons were analyzed on 1% agarose gels containing

ethidium bromide. The sequencing was performed at Macrogen Inc (Korea). DNA

sequences of the 16S rRNA were aligned with the Clustal method from MegAlign

Chapter 3

62

(DNAStar Inc., USA) assembling with 95% minimum match. The obtained nucleic acid

sequences were analyzed with the algorithm Blastn at the National Center for

Biotechnology Information (NCBI).

3.6.3 Sporulation conditions

Sporulation of all Bacillus strains was performed following the method UNE-EN-13704

standard (Anonymous, 2002). A suspension of Bacillus spores was prepared from an

exponential phase culture of vegetative bacteria in tryptone glucose broth (0.25% yeast

extract; 0.5% tryptone; 0.1% glucose and pH was adjusted to 7.2). Approximately 2–3 mL

of this culture were transferred to Roux flasks containing meat yeast extract agar (1% meat

extract; 0.2% yeast extract, 0.004% MnSO4·H2O; 1.5% agar and pH was adjusted to 7.2)

and was incubated for 8–10 days at 30°C. The culture was harvested and purified by

repeated centrifugation (10000 g for 20 min) and washed with sterile distilled water. The

suspensions were heat-shocked for 30 min at 75°C in order to kill vegetative cells. All the

spore preparations were free (> 97%) of sporulating cells, cell debris and germinated

spores, as determined by phase-contrast microscopy. Finally, the spores were stored in

double distilled sterile water at 4ºC until use.

3.6.4 Spores recovery

Between 80 and 100 ml of the processed samples were taken for analysis. Spore counts

were quantified in glucose yeast agar (0.1% casamino acids without vitamins; 0.1% soluble

starch; 0.25% glucose; 0.5% yeast extract; 0.01% FeSO4; 0.00001% MnSO4·H2O; 1.5%

agar and pH was adjusted to 6.8), incubating at 30°C for 72 h. Lethality was calculated as

the difference between the logarithms of colony counts of the untreated and treated

samples (log No- log N). Further investigation was carried out to confirm if complete

inactivation had been achieved. For this purpose, 50 mL of beverage samples were

incubated at 30ºC for 10 days. During this period, coagulation and phase separation were

checked visually. Moreover, a loopful of incubated samples was streaked out on glucose

yeast agar, which was incubated at 30°C for 48-72 h.


63

3.7 Lipid oxidation

3.7.1 Lipoxygenase activity

LOX extraction.The extraction was performed following the method described by Van der

Ven et al. (2005) with some modifications. Soymilk (30 mL) were placed in polypropylene

tubs (32 mm diameter and 115 mm length) and centrifuged for 60 minutes at 12000 g at

4ºC. Supernatant was used for enzyme activity assay.

LOX assay. The assay was performed following the method described by Axelrod et al.

(1981). The reaction was carried out at 25ºC in quartz cuvette of 1.0 cm light path in

spectrophotometer (Cecil 9000, UK) at 234 nm. The assay mixture contained 2.975 mL of

borate buffer, pH 9.0, 0.025 mL of sodium linoleate substrate (10 mM), and 0.030 mL of

LOX extract (enzyme). After each addition the mixture was stirred. The blank cuvette

contained no enzyme.

The activity was expressed as absorbance per minute read (abs/min) in the

spectrophotometer and transformed into units of lipoxygenase activity (ULA). One ULA

was defined as a change of 0.1 units of absorbance per minute of enzyme extract.

3.7.2 Hydroperoxide index

Lipid hydroperoxides in soymilk were determined by using the method described by

Ostdal et al. (2000) with some modifications. The reaction consists of the oxidation of

ferrous to ferric ion by hydroperoxides in the presence of ammonium thiocyanate to

produce ferric thiocyanate whose absorbance can be measured at 500 nm. Soymilk (0.4

mL) was mixed with water (1.6 mL). Then 2 mL of methanol were added and stirred.

Chloroform (4 mL) was added and vortexed for approximately 30 s. After centrifugation

for 10 min at 12000 g, 1 mL of the chloroform phase was transferred to a test tube and

mixed with 1 mL of Fe (II)/thiocyanate in methanol/chloroform (1:1). The mixture was

allowed to react for 5 min at room temperature before the absorbance at 500 nm was read.

Soymilk samples were analyzed in triplicate, and data were expressed as meq peroxide/L

of sample as described by (Hornero-Méndez et al., 2001). The calibration curve was

prepared according to the methodology described by FIL - IDF (1991). Measure is based

on spectrophotometric reading at 500 nm of a series of dilutions that contain Fe3+ in

chloroform/methanol (70:30).

Chapter 3

64

( )/55.84 x 2 x x

As Abm eq Lm Vs

−=

Where As is the absorbance of the sample, Ab is the absorbance of the blank; 55.84 is the

atomic weight of Fe; 2 is the factor to convert miliequivalents (meq) of Fe to meq of

peroxide; m is the slope of Fe3+ calibration plot; Vs is the sample volume in litters.

3.8 Trypsin inhibitor activity

Method A. Trypsin inhibitor (TI) extraction was prepared following the method described

by Van der Ven et al. (2005). Soymilk samples were diluted (1:5) with 0.015M of NaOH

and 0.5M of NaCl and stirred for 2 h at room temperature. After mixing, the mixture was

ultra-centrifuged (Beckman, model L8 60M, USA) at 30000 g for 20 min at 20ºC.

Supernatant was used for TI assay.

TI determination was based in the method described by Hamerstrand et al. (1981).

Dissolutions of Tris Buffer: hydroxymethyl aminomethane (1.21 g) and 0.59 g of CaCl2

dihydrated were dissolved in 180 mL of distilled water with adjusted pH 8.2 and then made

up to 200 mL with distilled water. BAPA solution: Nα-Benzoyl-L-arginine 4-nitroanilide

hydrochloride (0.080 g) was dissolved in 2 mL of dimethyl sulfoxide and diluted to 200

mL with Tris Buffer. Trypsin solution: trypsin (0.0040 g) was diluted to 200 mL with

0.001N HCl.

Soymilk TI extract (2 mL) was added in four tubes. 2 mL of trypsin solution were added in

the first 3 tubes and then placed in a constant temperature bath of 37ºC for 10 min. Five

milliliters of BAPA solution (prewarmed to 37°C) were rapidly added into each tube. The

mixture was stirred immediately and the tubes were replaced in the bath. The reaction was

stopped exactly 10 min later by adding of 1 mL of 30% acetic acid. In the fourth tube was

then added 2 mL of trypsin solution. Two extras tubes (trypsin standard and blank) were

prepared applying the same procedure without soymilk. The absorbance was determined at

410 nm (Cecil 9000, UK) and inhibition percentage was calculated according to the

relation described by Kumar et al. (2003) with some modifications:

Percentage of inhibition = 100 x Absorbance of the sample Absorbance of the standard trypsin


65

Method B. Due to infrastructural conditions of the laboratory and for accurate results, some

modifications were applied in the analytical method described above, respecting the

sequence proposed by the authors. Soymilk samples were diluted twice with 0.02 N NaOH

(the pH was adjusted to 8.4-10.0) and stirred for 2 hours. The extract was then centrifuged

at 12000 g for 35 min and filtered through Whatman paper No. 42. The TI extract was

diluted so that 2 mL of the extract dilution inhibited 40% - 60% of the trypsin used as

standard in the analysis. Values of inhibition superior to 60% cause loss of linearity of the

trypsin inhibitor reaction, causing turbidity of the sample extract. Percentage of inhibition

was previously determined in untreated soymilk extract in order to find out the optimal

volume of treated soymilk extract needed to be used in the assay.

TIA assay was assessed following the modifications of method A: 3 tubes of TI soymilk

extract were prepared and blank sample consisted of distilled water without sample extract.

To define TI activity, one trypsin unit (TU) was arbitrarily described as the increase of

0.01 absorbance units per 10 mL reaction mixture at 410 nm (Kakade et al., 1969).

Absorbance values obtained from soymilk TI extracts were subtracted from the values of

trypsin standard and the TI activity was expressed as mg of TI/mL of sample extract by

assuming that 1 µg of pure trypsin was equivalent to 0.019 absorbance units.

3.9 Particle size determination

Particle size distribution was performed by laser light-scattering, supported by the patented

multi-wavelength system (PIDS) which provides accurate results in the 0.04 and 2000 µm

regions. An LSTM 13 320 Series Particle Size analyzer (Beckman Coulter, USA) using

soymilk optical model with a refractive index of 1.46 (Malaki-Nik et al., 2008) and

dispersant phase (water) of 1.332 was used for this purpose. Soymilk samples were diluted

until obscuration of 2% or 7%, depending on the sample. Particle size distribution was

characterized by the Sauter mean diameter, d3.2 (particle diameter that has the same

specific surface as that of the full distribution) and by the d4.3 (diameter of the sphere of

equivalent volume to measured particles).

3.10 Particle sedimentation

Centrifugation method. Approximately 30 g of soymilk were poured into flexible plastic

tubes (32 mm diameter, 115 mm length) and centrifuged at 1046 g for 45 min at 20ºC.

Chapter 3

66

Values were expressed as percentage (w/w) of solid deposition obtained after

centrifugation.

Particle migration method. Soymilk samples were transferred into borosilicate glass tubes

of 27.5 x 70.0 mm up to 40 mm of height and sodium azide (0.04% NaN3) was added.

Three tubes of each treatment and untreated soymilk were prepared and stored at 4ºC. On

each day of analysis samples were carefully placed in the Turbiscan equipment (LAB

expert Formulaction, France) and a near infrared light from top to bottom measured the

percentage of light backscattered through the sample at 25ºC. Results were expressed in

base to changes of backscattering (ΔB) in the bottom of the tube (chapter 8) and by the

determination of the height of solids layer settled during the period of storage (chapter 9).

The latter was calculated using “sediment phase thickness” of the manufacturer software.

3.11 Transmission electron microscopy

Transmission electron microscopy (TEM) of soymilk was determined following the

method described by Cruz et al. (2007). Soymilk was mixed with 3% glutaraldehyde in a

bijou bottle and then mixed with warm 2% low-temperature gelling agar at 1:1 ratio. The

mixture was allowed to gel and was chopped into 1 mm3 cubes. Then cubes were washed

with 0.1 M sodium cacodylate buffer, pH 7.2 for 30 min and again for 1 h, and then left for

a further 1 h prior to replacement with 1 ml of a solution containing equal amounts of 2%

osmium tetroxide and 50% of 0.1 M cacodylate/HCL buffer. This was left to stand for 2 h

before being replaced with 1 ml of 1% uranium acetate for 30 min. Product cubes were

washed with water before dehydration. Dehydration included washing with 50, 70 and

90% of ethanol for 5, 30 and 180 min, respectively. 100% ethanol was changed after 30

and 60 min. Ethanol was poured off and the bottle was filled with incomplete resin [20 ml

epoxy resin, 20 ml dodecylsuccinic anhydride (DDSA) and 1 drop of dibutyl phthalate]

and placed on a rotator overnight before replacement with complete resin [incomplete

formulation with addition of 0.6 ml of the plasticiser benzyldimethylamine (BDMA)] and

then placed on the rotator for a further 4 h. One cube of sample was added to each of three

moulds containing fresh complete resin which were then baked overnight at 60ºC. Samples

were cut (0.03–0.05 µm) using a Reichert Ultracut microtome and mounted in 3 mm

copper grids and stained using uranyl acetate and lead citrate before examination in a

Philips 201 transmission electron microscope at an accelerating voltage of 60 kV (NL-

5600 MD Philips, The Netherlands).


67

3.12 Surface hydrophobicity

Surface hydrophobicity of soymilk protein was measured using 8-Anilino-1-naphtalene

sulfonic acid (ANS) as reactive (Shimoyamada et al., 2008). Soymilk samples (50 µL)

were diluted with 50 µL of 0.01 M phosphate buffer (pH 7.0) and mixed with 20 µL of 8

mM ANS solution and 4 mL of the same buffer. The resulting mixtures were subjected to

fluorescence spectrometry (Eclipse Spectrophotometer, Varian Inc., USA) and

fluorescence was measured (excitation, 390 nm; emission, 470 nm). This analysis was only

carried out at day 1 (chapter 8 and 9).

3.13 Color measurements

Soymilk color was measured using a Hunter Lab colorimeter (MiniScan XE Hunter

Associates Laboratory Inc., USA). D65 was used as illuminant with an observation angle

of 10º using the ring and disk set for translucent liquids (HunterLab, 2008). Data was

acquired in the CIELab color space, L* (luminosity), a* (red-green) and b* (blue-yellow)

were then used to calculate the total color difference ΔE by means ΔE = [(ΔL)2 + (Δa)2 +

(Δb)2)]1/2 equation. ΔL, Δa and Δb are the differences in the tristimulus coordinates

between reference sample and treated soymilks.

3.14 Headspace analysis of volatile compounds

Changes in volatile compounds produced by thermal and high pressure treatments could

strictly modify odor and flavor of soymilk. Solid-phase microextraction (SPME) is a

simple and sensitive technique which allows a direct extraction of the volatile compounds

present in soymilk. In addition, this method is economic and ecologic because it does not

consume large quantities of solvent. The analyses were carried out after UHPH and

thermal treatments.

3.14.1 SPME – gas chromatography mass spectrometry

Volatile compounds analysis was performed following the method described by Achouri et

al. (2006) with some modifications. The SPME fiber used was 85 µm CAR-PDMS

(Supelco, USA). 1.5 mL of soymilk sample (previously homogenized using ultrasound

Chapter 3

68

equipment with temperature control for 10 min) were placed in tubes (4 mL) and incubated

for 30 min at 40ºC. The adsorbed volatiles were desorbed in the injector port in splitless at

300ºC for 1 min. Headspace of the volatile compounds was analyzed using an automated

gas chromatography (model: HP 6890 Series II, Agilent, USA). The column model used

was 0.25 µm in a 60 m x 0.25 mm (TRB-Was, Agilent technologies). The mass

spectrometry (MS) detector was used in the electron impact ionization (model: HP 5972

Agilent, USA) with a mass range of 30 – 250 m/z. Before each analysis, the fiber was

preconditioned for 1 h at 300ºC. The temperature was programmed in 2 stages. The initial

temperature was kept at 35ºC for 9 min, and then increased at 5 ºC/min to 110ºC for 10

min. In the second stage, the temperature increased at 10 ºC/min to 250ºC for 10 min.

Retention indices, relative to C8-C26 n-alkanes were determined injecting 1 µl of each

standard solution (Alkane standard solution C8-C20 from Sigma-Aldrich, and Connecticut

ETPH calibration mixture C9-C36 were used as standards) in triplicate with a split ratio of

1:200. Signals were processed using Agilent MSD Productivity ChemStation Enhanced

Data Analysis software (Agilent technology, USA). The identification of some volatile

compounds (hexanal, pentanal, 1-octen-3-one, 2,3-pentanodione, 1-hexanol, 1-pentanol, 1-

octen-3-ol and 2-penthyl furan) were confirmed by comparing their retention times and

mass spectra with those of authentic reference compounds injected under the same

operating conditions Otherwise, tentative identifications were made based on comparisons

with the mass spectra data of NIST08 and Wiley 7n1 libraries and retention index of the

literature. Main, molecular, and qualifier ions were selected for each compound

indentified.

3.15 Sensory analysis

The attributes required for the training as well as for the sensory analysis were based on the

study described by Torres-Penaranda et al. (1998) and Torres-Penaranda and Reitmeier

(2001).

Selection and training of panelists. Twelve judges from students and staff at the

Universitat Autònoma de Barcelona with a previous experience evaluating different

products were pre-selected based on availability, interest and habitual consumption or not

of soymilk. Panelists were trained in 3 sessions for approximately 30 minutes on different

days. In each session, judges were exposed to different tastes: sweet (10 g/L of

saccharose), salt (5 g/L of sodium chloride), bitter (0.3 g/L caffeine) and acid (0.1 g/L


69

citric acid) in order to check if judges were able to identify differences. Moreover,

attributes of flavor and aroma such as astringency (alum in water 0.3 g/L), green or beany

flavor (50 g/L of fresh soybeans in water), grassy aroma (0.02 g/L cis-3-hexen-1-ol) and 2

solutions of oxidized aroma (1 mg/L 2-nonenal and 10 mg/L 2-heptenal), were prepared

and exposed to the panelist. At the end of each meeting, judges evaluated a commercial

soymilk sample according to the trained attributes in the session.

Sensory tests applied. Sensory tests were divided into 3 parts. The first one included a

triangular test, where panelists were asked to identify possible differences between UHPH-

treated soymilk and UHT-treated soymilk, both presented in random order. The second

part included a descriptive test, where the following seven attributes were evaluated: beany

flavor, grassy aroma, oxidized aroma, astringent mouthfeel, thickness, darkness and

yellowness. In addition, judges were instructed to describe uncommon flavor perceived.

Responses were recorded on an intensity scale from 0 to 5 points, where 0 = not intense

and 5 = extremely intense. In the last part a preference test was applied using a hedonic

scale with 9 categories ranging from “dislike extremely” to “like extremely”. Soymilk

samples (~ 30 mL) at room temperature was presented in white plastic cups with a 3-digit

random code

3.16 Statistical analysis

To carry out the preliminary study of soymilk elaboration, two individual soymilk

extractions were performed. For the initial screening and shelf-life evaluation of

refrigerated UHPH-treated soymilk, three individual treatments were carried out and

analyses of the treated samples were performed in triplicate. Results were analyzed by

ANOVA using GLM procedure of SAS (SAS Institute, 2004) to determine differences.

SNK (Student Newnan Keuls) test was used for comparing sample data in chapter 4, 5 and

6 and Tukey test was used in chapters 7, 8 and 9. Principal component analysis (PCA) was

performed to reduce the data in two dimensions and identify patterns of variation in the

results of volatile profile. R software (R software, New Zealand) was used for this purpose.

Results of triangular test were analyzed using Chi-square test of SAS to accept or reject the

null hypothesis. Hedonic test data were analyzed by two-sample T-test of SAS. Data

analyses were based on a significant level of P < 0.05.

Chapter 3

70

3.17 References

Achouri, A.Boye, J.I. & Zamani, Y. (2006). Identification of volatile compounds in

soymilk using solid-phase microextraction-gas chromatography. Food Chemistry. 99,

759-766.

Anonymous (2002). UNE-EN-13704. Chemical disinfectants. Quantitative suspension test

for the evaluation of sporicidal activity of chemical disinfectants used in food,

industrial, domestic, and institutional areas. Test methods and requirements (phase 2,

step 1).

AOAC, (2000). Official methods of analysis, Association of Official Analytical Chemists,

Washington, DC: Association of Official Analytical Chemists.

Axelrod, B., Cheesbrough, T. M., & Laasko, S. (1981). Lipoxygenase from soybeans. In

Methods in Enzimology (Edited by, J. M. Lowenstein), USa. pp. 441-451.


Ultra high pressure homogenization of soymilk: Microbiological, physicochemical

and microstructural characteristics. Food Research International. 40, 725-732.

FIL - IDF. (2002.). Milk and milk products. Determination of nitrogen content. Routine

method using combustion according to the Dumas principle. Standard 185. Brussels:

International Dairy Federation.

FIL - IDF. (1991). Peroxide index determination. Standard 74A. Brussels: International

Dairy Federation.

Hamerstrand, G.E., Black, L.T. & Glover, J.D. (1981). Trypsin-inhibitors in soy products -

modification of the standard analytical procedure. Cereal Chemistry. 58, 42-45.

Hornero-Méndez, D., Pérez-Gálvez, A. & Mínguez-Mosquera, M.I. (2001). A rapid

spectrophotometric method for the determination of peroxide value in food lipids with

high carotenoid content. Journal of the American Oil Chemists' Society. 78, 1151-

1155.


71

Kakade, M.L., Simons, N. & Liener, I.E. (1969). An evaluation of natural vs synthetic

substrates for measuring antitryptic activity of soybean samples. Cereal Chemistry.

46, 518-526.

Kumar, V., Rani, A., Tindwani, C. & Jain, M. (2003). Lipoxygenase isozymes and trypsin

inhibitor activities in soybean as influenced by growing location. Food Chemistry. 83,

79-83.



Malaki-Nik, A.M., Tosh, S., Poysa, V., Woodrow, L. & Corredig, M. (2008).

Physicochemical characterization of soymilk after step-wise centrifugation. Food

Research International. 41, 286-294.

Ostdal, H., Andersen, H.J. & Nielsen, J.H. (2000). Antioxidative activity of urate in bovine

milk. Journal of Agricultural and Food Chemistry. 48, 5588-5592.

SAS (2004). SAS procedures, version 9.2. Cary, NC, USA: SAS Institute, Inc.

Shimoyamada, M., Tsushima, N., Tsuzuki, K., Asao, H. & Yamauchi, R. (2008). Effect of

heat treatment on dispersion stability of soymilk and heat denaturation of soymilk

protein. Food Science and Technology Research. 14, 32-38.



Torres-Penaranda, A.V., Reitmeier, C.A., Wilson, L.A., Fehr, W.R. & Narvel, J.M. (1998).

Sensory characteristics of soymilk and tofu made from lipoxygenase-free and normal

soybeans. Journal of Food Science. 63, 1084-1087.


trypsin inhibitors and lipoxygenase by high-pressure processing. Journal of


Chapter 3

72




Chapter 4

Optimization of soymilk elaboration at pilot plant scale

75

4 Optimization of soymilk elaboration at pilot plant scale

4.1 Introduction

Soybean variety, geographic area of cultive and climate are the main factors influencing

soymilk elaboration (Liu, 1999). Some soybean varieties have become well known for

their superior processing properties, but storage and environmental conditions during the

growing season affects soybean seed size and composition and consequently soymilk yield

and quality (Mullin et al., 2001; Alpaslan & Hayta, 2002).

Main steps of soymilk elaboration include: soaking, grinding, filtration, heat treatment and

packaging. Soaking reduces the power requirement for grinding, breaks out some

oligosaccharides resulting in a better dispersion of solids during extraction, improving

production yield.

The best known methods and extensively applied in industries 40 years ago are Cornell and

Illinois methods. Cornell method apply heat grinding between 80ºC and 100ºC and Illinois

method apply blanching for 10-20 min at 100ºC before grinding with cold water (Liu,

1999). Due to technology improvement occurred in the last decade, nowadays industries

commonly apply modern and automatic methods in the soymilk production. Heat

treatments using steam injection at high temperatures is a continuous flow method

extensively used nowadays.

Production yield is an important factor for all industrial process. There are several ways to

express this parameter. Some of them include expressing by weight or volume of soymilk

from original soybeans quantity and by total solids calculated as percentage of protein or

solids recovery in the final product (Liu, 1999). Additionally to production yield, trypsin

inhibitor activity and lipoxygenase activity play an important role in the soymilk quality.

The presence of trypsin inhibitors affects trypsin hydrolytic action causing reduction of

nutrients absorption (Liu, 1999). In animal feed, for example it has been associated with

growth suppression and pancreatic hypertrophy. On the other hand, the effects of trypsin

inhibitors in humans are not fully clear, however its reduction is highly recommended to

soy products and generally is achieved by heat treatments (Friedman & Brandon, 2001).

Chapter 4

76

Lipoxygenase is an enzyme which catalyzes the hydrolysis of polyunsaturated fatty acids

(Vijayvaragiya & Pai, 1991), favoring lipid oxidation which result in the formation of off-

flavors (Yuan & Chang, 2007).

Soymilk used in previous studies in our research group was provided by a commercial

company. However, in order to control the influence of the elaboration process of the

soymilk, the first goal of this study was to establish and standardize the conditions of

soymilk base product production. For this purpose, two temperatures of soymilk extraction,

60 and 80ºC (see 3.1 method A) were used and analytical techniques such as chemical

composition of soybeans and soymilk (see 3.5), production yield (see 3.3), lipoxygenase

activity (see 3.7.1) as well as trypsin inhibitor activity (see 3.8 method A), were evaluated

as quality parameters to select the best condition of soymilk base product elaboration.

4.2 Results and discussion

4.2.1 Chemical composition

Composition of Majesta variety soybeans was the following: 9.94 ± 0.04 g/100g dry

matter; 39.67 ± 0.32 g/100g protein; 21.20 ± 0.29 g/100g fat; 23.29 ± 0.24 g/100g

carbohydrate and 5.72 ± 0.11 g/100g ash. Similar results were reported by (Cai et al.,

1997) and (Mullin et al., 2001) for different soybean varieties. Composition of soymilk

extracted at 60ºC and 80ºC are shown in Table 4-1. Results indicated that the temperature

used in soymilk extraction did not influence dry mater and fat composition. However,

soymilk extracted at 60ºC presented values of protein slightly higher than those observed at

80ºC (P < 0.05). This difference could be attributed to the protein solubility. At 80ºC

soymilk protein may have partially reduced its solubility, as a consequence of denaturation

process that takes place at high temperatures. The filtration step could therefore be

essential for the protein recovery, because part of protein precipitated could form

complexes with particles and macromolecules in the pulp, reducing then its availability.

Kwok & Niranjan (1995) observed that combination of temperature and time applied

during soymilk extraction may increase solid and protein recovery from the seeds or, on

the contrary, insolubilized proteins and decrease yield. Results of soymilk composition

were similar to those reported in commercial soymilks (Liu, 1999; Cruz et al., 2007; Liu &

Chan, 2012).


77

Table 4-1. Dry matter, fat and protein content1 of soymilk extracted at 60ºC and 80ºC.

Extraction at 60ºC Extraction at 80ºC

Dry matter 6.28 ± 0.11a 5.78 ± 0.24a

Fat content 1.53 ± 0.08a 1.36 ± 0.17a

Protein content 3.21 ± 0.04a 3.10 ± 0.04b

a-b Different superscripts in the same raw are significantly different (P < 0.05). 1 Mean values (g/100g) of chemical composition of soymilk base product.

4.2.2 Production yield

Productivity of soybeans during soaking step was 228.06 ± 3.49 (% w/w). This result

indicates an increase of 2.28 times the original weight of the seeds. Soaking step would be

completed when seeds reach 2.4 times the initial weight (Wang, 1986). However, a partial

hydration of twice initial weight of the seeds is very common in soymilk extraction (Liu,

1999).

Yield production of soymilk elaborated at 60ºC and 80ºC is shown in Table 4-2. No

significant differences were observed for all parameters studied. Total solids recovery from

soybeans is decisive for the content of lipids and proteins of soymilk as well as its

nutritional value. Amount of 56.68 and 52.09% obtained for soymilk extracted at 60ºC and

80ºC, respectively, are in agreement with the typical soymilk production, commonly

known as Cornell method as described by Prawiradjaja (2003). The same author reported

values of 55-65% (w/w) of total solids recovery using Cornell method, whereas Illinois

method values were about 86-89% (w/w). In the Illinois method, all parts of the soybeans

including the pulp are incorporated into the soymilk, favoring high percentage of solid

extraction (Golbitz, 1995; Kwok & Niranjan, 1995).

The relation soybean:soymilk produced is another way to express production yield. A

relation of 8.17 means that total volume of soymilk produced was 8.17 times the total

seeds used. Traditional method of soymilk extraction reaches values between 8-10 times

the original values of seeds weighted before the soaking step (Liu, 1999). Total loss of

water was about 13% for the whole process with an overall yield about 81%. According to

conventional industrial process, 19% of losses are higher than desired. Grinding and

filtration are the crucial steps to avoid or to reduce losses during soymilk elaboration (Liu,

Chapter 4

78

1999). In this study, grinding step produced the major losses compared to the rest of

elaboration steps, reaching values around of 68%. Infrastructure conditions of production

used in this study did not allow elaborating soymilk with better overall productivity. On

the other hand, results of soybean:soymilk obtained in this study as well as results of total

solids recovery are in agreement with different methods of soymilk production, traditional

or industrial. However, some studies published have reported high overall productivity by

adding sugar in the soymilk formulation using conventional method of extraction

(Prawiradjaja, 2003).

Table 4-2. Production yield1 of soymilk extracted at 60ºC and 80ºC.

Extraction at 60ºC Extraction at 80ºC

Solids recovered 56.68 ± 6.16a 52.09 ± 3.30a

Soybean:soymilk2 8.17 ± 0.74a 8.19 ± 0.01a

Loss of water 13.03 ± 9.67a 13.46 ± 0.04a

Overall yield 81.68 ± 7.39a 81.81 ± 0.14a

a Different superscripts in the same raw are significantly different (P < 0.05). 1 Mean values (g/100g) of production yield parameters of soymilk base product. 2 The relation was based in volume of soymilk (L) per kilograms of soybeans.

4.2.3 Lipoxygenase activity

Soybeans contain about 20% of lipids, being 80% of them composed by unsaturated fatty

acids. Linoleic acid (51%) is the most predominant unsaturated fatty acid, followed by

oleic acid (23%) and linolenic acid (7%). Most of theses fatty acids are present in soymilk

and makes a perfect substrate of lipoxygenase enzyme (Liu, 1999; Prawiradjaja, 2003).

Lipoxygenase (LOX), an iron-containing dioxygenase, catalyse the oxidation of

polyunsaturated fatty acids containing cis,cis-1,4-pentadiene units to the corresponding

conjugated hydroperoxydiene derivatives by the addition of molecular oxygen (Wang et

al., 2008). In advanced stages of oxidation reaction, secondary products are formed such as

aldehydes and ketones. These volatile compounds are part of the off-flavors which limit

the acceptance of soymilk. According to Min et al. (2005), off-flavor compounds are often

formed during soaking and grinding steps in soymilk elaboration. For this reason,


79

controlling parameters such as time and temperature in the soaking and grinding steps may

play an important role in the sensory perception and overall quality of soymilk.

Results showed that lipoxygenase activity was influenced by the temperature. Soymilk

extracted at 60ºC presented a residual activity of 6.31 ULA (units of lipoxygenase

activity), while no activity in soymilk extracted at 80ºC was detected. According to Yuan

and Chang (2007), lipoxygenase is denatured approximately at 80ºC, which causes

inactivation of its catalytic function. Thus, the heating at 80ºC applied in the grinding step

was the reason of the LOX inactivation. In addition, soybeans cultivars, year of production

and geographic location have been reported to affect the content and activity of soybean

lipoxygenase. Inactivation of lipoxygenase can be achieved by blanching soaked soybeans

in boiling water for 10 min or by dropping dry seeds directly into boiling water for 20 min

(Kwok & Niranjan, 1995).

4.2.4 Trypsin inhibitor activity

Trypsin inhibitors (TI) are substance with antinutritional properties. They affect protein

digestibility, may cause pancreas hyperactivity and their presence in animal feed has been

associated with growth suppression and pancreatic hypertrophy (Van der Ven et al., 2005).

These substances have a proteolytic activity which reduces the availability of trypsin, an

important protease in the animal digestive function (Friedman & Brandon, 2001). Two

types of trypsin inhibitors are present in soybeans: Kunitz trypsin inhibitor (KT) and

Bowman-Birk (BB) inhibitor. BB is considered more heat stable than KT due to higher

proportion of disulfide bonds which stabilize the molecular composition required for

biological activity (Kwok & Niranjan, 1995). Therefore the extent of destruction of TI (KT

and BB fractions) in soymilk should be achieved for maximum nutritive value.

Values obtained were expressed as residual acitivity calculated according to the trypsin

used as standard. As a result, soymilk extracted at 80ºC showed higher values of trypsin

inhibitor inactivation (76.3%) compared to soymilk extracted at 60ºC (65.0%). Using

different conditions of temperature in soymilk processing, Kwok et al. (1993) obtained

70% of TI inactivation from initial activity of untreated sample applying 93ºC for 20 min.

Lei et al. (1981) found 30% and 34% of trypsin inhibitor inactivation for soybeans water-

extract heated at 70ºC and 80ºC, respectively, during 30 min. To reach an 80% of TI

inactivation requires heating of 100ºC for 14 min and 30 min for a 90% of inactivation at

same temperature (Liu, 1999). Other methods to reach 90% of TI inactivation were

Chapter 4

80

reported by using high temperatures and/or high holding time. For instance, Kwok et al.

(2002) obtained 90% of TI inactivation from initial activity of untreated sample applying

93ºC during 60-70 min or 121ºC for 5-10 min. Ultra high temperature (UHT) treatments of

154ºC also reach 90% of TI inactivation, but it is necessary to apply heating during 60

seconds (Kwok et al., 1993). However, overheating reduce nutritive value and cause

destruction of important amino acids, such as cysteine, arginine and lysine (Skrede &

Krogdahl, 1985). The results of TI inactivation obtained in this study are in agreement with

the authors cited, indicating that time and temperature plays a fundamental role in the TI

inactivation.

4.3 Conclusions

Soymilk samples extracted at 60ºC and 80ºC did not present significant differences in the

values of dry matter and lipids in chemical composition results. However values obtained

of protein content showed that proportion water:soybeans in the soymilk extraction as well

as parameters such as temperature and time, affected total protein content. On the other

hand, overall yield were not affected by these conditions (P < 0.05).

Soymilk base product elaborated at 80ºC was efficient in the lipoxygenase inactivation and

caused a partial inactivation of the trypsin inhibitor activity. The method of soymilk

elaboration applied was able to produce a product with good quality and feasible to

available infrastructure of UAB pilot plant.

4.4 References

Alpaslan, M. & Hayta, M. (2002). Hydration properties, soymilk and okara yield of

soybean affected by agronomic factors. Molecular Nutrition & Food Research. 46,

141-143.

Cai, T.D., Chang, K.C., Shih, M.C., Hou, H.J. & Ji, M. (1997). Comparison of bench and

production scale methods for making soymilk and tofu from 13 soybean varieties.

Food Research International. 30, 659-668.


81




Friedman, M. & Brandon, D. L. (2001). Nutritional and health benefits of soy proteins.


Golbitz, P. (1995). Traditional soyfoods - processing and products. Journal of Nutrition.

125, S570-S572.



Kwok, K.C., Liang, H.H., Niranjan, K. (2002). Mathematical modelling of the heat

inactivation of trypsin inhibitors in soymilk at 121-154°C. Journal of the Science of

Food and Agriculture, 82, 243-247.



Lei, M.G., Bassette, R. & Reeck, G.R. (1981). Effect of cysteine on heat inactivation of

soybean trypsin-inhibitors. Journal of Agricultural and Food Chemistry. 29, 1196-

1199.


Gaitherburg, USA . pp. 532.

Liu, Z.S. & Chang, S.C. (2012). Nutritional profile and physicochemical properties of

commercial soymilk. Journal of Food Processing and Preservation. 1-11.

Min, S., Yu, Y. & Martin, S. S. (2005). Effect of soybean varieties and growing locations

on the physical and chemical properties of soymilk and tofu. Journal of Food Science.

70, C8-C21.

Mullin, W.J., Fregeau-Reid, J.A., Butler, M., Poysa, V., Woodrow, L., Jessop, D.B. &

Raymond, D. (2001). An interlaboratory test of a procedure to assess soybean quality

for soymilk and tofu production. Food Research International,. 34, 669-677.

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82

Prawiradjaja, S. (2003). Process optimization for solid extraction, flavor improvement and

fat removal in the production of soymilk from full fat soy flakes [dissertation]. Iowa

State University, USA.

Skrede, A. & Krogdahl, A. (1985). Heat affects nutritional characteristics of soybean-meal

and excretion of proteinases in mink and chicks. Nutrition Reports International. 32,

479-489.


trypsin inhibitors and lipoxygenase by high-pressure processing. Journal of


Vijayvaragiya, R.R. & Pai, J.S. (1991). Lowering of lipoxygenase activity in soy milk

preparation by propyl gallate. Food Chemistry. 41, 63-67.

Wang, H.L. (1986). Production of soymilk and tofu. French Soyfood Industry Publisher.

France. pp. 27.

Wang, R., Zhou, X. & Chen, Z. (2008). High pressure inactivation of lipoxygenase in soy

milk and crude soybean extract. Food Chemistry, 106, 603-611.




Chapter 5

Comparison of ultra-high pressure homogenization and conventional

thermal treatments on the microbiological, physical and chemical quality of

soymilk

85

5 Comparison of ultra-high pressure homogenization and conventional

thermal treatments on the microbiological, physical and chemical quality

of soymilk.

5.1 Introduction

Commercial soymilk is produced mainly by conventional technologies, such as UHT (ultra

high temperature); high temperatures, however, cause undesirable chemical changes which

include destruction of amino acids and vitamins, browning reactions, and development of

cooked flavors (Kwok & Niranjan 1995). In addition, working at high temperatures can

accelerate the oxidation process, which is related with the formation of off-flavors and

overall quality of soymilk.

Consumers demand high quality food products, which mean that they have to guaranty

good nutritional quality, long shelf-life and high physical stability. To define the overall

quality of a product a number of parameters have to be studied. In addition to good

hygienic quality, soymilk should have low oxidation values, low antitrypsin activity, high

emulsion stability and high nutritional values.

A previous study dealing with UHPH treated soymilk (Cruz et al., 2007) resulted in

microbial reduction similar to pasteurization and an excellent physical stability. The

objective of the present work was to extend UHPH conditions to obtain high quality

soymilks paying special attention to finding the right conditions for commercial sterility of

the products. For this purpose six UHPH treatments were performed by combining, 200

MPa and 300 MPa with different inlet temperatures (55, 65 and 75ºC) and compared with

pasteurized and UHT soymilks. Microbiological quality (see 3.6.1), particle size

determination (see 3.9), particle sedimentation (see 3.10 centrifugation method), lipid

oxidation (see 3.7) and trypsin inhibitor activity (see 3.8 method B) were applied as quality

parameters.

Chapter 5

86


5.2.1 Soymilk chemical composition

Soymilk basic composition was not affected by the treatment applied. Data are given as

mean values regardless of the treatment applied to soymilk (heat treatments or UHPH):

2.68 ± 0.17 g/100g protein; 1.92 ± 0.17 g/100g fat; 5.53 ± 0.39 g/100g dry matter; 1.35 ±

0.05 g/100g carbohydrate; 0.18 ± 0.05 g/100g ash. Mean pH value was 6.65 ± 0.05.

Authors such as Liu (1999), Wang et al. (2001) and Cruz et al. (2007) presented similar

composition of soymilk in their studies.

5.2.2 Temperature changes in UHPH processing

Temperature of soymilk reached in the high pressure valve in combination with very short

time (approximately 0.7 s in this UHPH machine) at this temperature determined the

quality characteristics of UHPH-treated samples. Temperature increase during UHPH

treatment (Table 5-1) was about 20ºC between 200 and 300 MPa at the different inlet

temperatures used in this study. This temperature increase was close to those observed by

other authors. Pereda et al. (2007), who pressurized milk in the same UHPH equipment at

inlet temperatures of 30 and 40ºC and pressures from 100 to 300 MPa, found the same

temperature change. Thiebaud et al. (2003) reported a temperature increase of 18.5ºC per

100 MPa, in the pressure range 100 to 300 MPa for milk samples. The increase of

temperature during UHPH treatment is the consequence of the adiabatic heating generated

in the machine in addition to the turbulence, shear, and cavitation forces that the food

experiments when passing through the ultra high pressure valve (Thiebaud et al., 2003;

Hayes & Kelly, 2003).

Comparison of UHPH and thermal treatments

87

Table 5-1. Temperature changes1 of UHPH-treated soymilks during processing.

Ti (ºC) Treatment (MPa) T1 (ºC) Tf (ºC)

55 200 105.7 ± 0.58 27.1 ± 1.0 300 128.3 ± 1.53 27.3 ± 1.1

65 200 111.7 ± 1.15 27.0 ± 1.0 300 130.7 ± 1.15 26.2 ± 0.8

75 200 117.0 ± 2.00 25.6 ± 2.7 300 135.7 ± 1.53 26.2 ± 2.2

1 Ti = inlet temperature; T1 = temperature after the homogenization valve; Tf = temperature after final heat

exchanger.

5.2.3 Microbiological quality

Table 5-2 shows the microbiological counts (log cfu/mL) of BP, heat treated (pasteurized

and UHT) and UHPH samples. In general, UHPH treatments at 200 and 300 MPa were

more effective than pasteurization against almost all the microorganisms. However,

significant differences (P < 0.05) were detected between UHPH samples at 200 MPa (55

and 65ºC inlet temperature) and pressurized samples at 300 MPa. According to some

authors, the reduction in microbiological counts was pressure dependent, showing better

inactivation as homogenization pressure increased (Thiebaud et al., 2003; Hayes et al.,

2005). In contrast, Pereda et al. (2007) reported that no significant differences were

detected between UHPH milk samples at 200 and 300 MPa at 30 and 40ºC inlet

temperature, immediately after treatment. However, they concluded that milk treated at

300 MPa, 30ºC of inlet temperature had similar or better microbial shelf life than

pasteurized milk.

Additionally to the pressure applied in the UHPH treatment, inlet temperature could also

be considered as a factor affecting microbial lethality, especially when samples were

treated at 200 MPa. In this study, the highest temperature reached was 135°C at 300 MPa,

75ºC inlet temperature. All treatments at 200 MPa were more effective than pasteurization

in reducing aerobic spore counts. UHPH samples treated at 200 MPa, 55ºC inlet

temperature showed no significant differences (P ≥ 0.05) in the total counts of mesophilic

aerobic bacteria and B. cereus counts with pasteurized samples, while UHPH treatment at

200 MPa, 65ºC significantly reduced B. cereus and total mesophilic bacteria and spore

counts compared with BP, pasteurized and 200 MPa, 55ºC samples. Cruz et al. (2007) who

worked with soymilk at 200 MPa, 40ºC inlet temperature, obtained a reduction in the total

Chapter 5

88

mesophilic bacteria counts of 2.42 log units, which was slightly higher than the reduction

obtained in this study at inlet temperature of 55ºC and similar to those obtained at 65ºC

inlet temperature, where reduction reached was of 1.6 and 2.58 log units, respectively.

Counts below the detection level for B. cereus (< 5 cfu/mL) and mesophilic aerobic

bacteria and mesophilic aerobic spore (< 0.5 cfu/mL) were obtained for 200 MPa, 75ºC

and 300 MPa at 55, 65 and 75ºC inlet temperatures. Although Cruz et al. (2007) obtained

in soymilk samples a significant reduction of total mesophilic bacteria and spore counts (4

and 2 log units, respectively) after UHPH treatment at 300 MPa, 40ºC, it was not achieved

a complete inactivation. Picart et al. (2006) reported a reduction on the total bacteria in

milk samples of 2.9 log units at 300 MPa, 24ºC inlet temperature, while Pereda et al.

(2007) reported in milk samples reductions about 3.3 log units at 300 MPa, 30 or 40ºC

inlet temperatures. However, Smiddy et al. (2007) found a reduction of about 5 log units

for UHPH-treated milk at 250 MPa, 55 and 70°C. As it was above mentioned, differences

observed in microbial counts between this study and others could be explained by the

effect caused of the maximum temperature reached in the UHPH treatment.

Table 5-2. Microbial populations (log cfu/mL ± SD) of BP and treated soymilks.

Treatment Total bacteria Total spores B. cereus BP 5.02 ± 2.24a 3.46 ± 1.21a 3.55 ± 2.37a Pasteurized 3.46 ± 1.23b 2.85 ± 0.95a 2.56 ± 1.91b UHT ND1 ND ND 200MPa 55ºC 3.39 ± 1.55b 1.75 ± 0.69b 2.31 ± 1.38b 200MPa 65ºC 2.44 ± 1.59c 0.85 ± 0.57c ND2 200MPa 75ºC ND ND ND 300MPa 55ºC ND ND ND 300MPa 65ºC ND ND ND 300MPa 75ºC ND ND ND

a-c Values in the same column with different superscripts are significantly different (P < 0.05). 1 ND = no detected. 2 Bacillus cereus growth was detected only in one production (3.40 ± 0.12 log cfu/mL).

Despite the fact that in some UHPH treatments, apparently were achieved a complete

microbial inactivation, after incubation at 30ºC for 20 days to test the sterility of treated

samples, it was observed that only the UHT samples and those treated at 300 MPa, 75ºC


89

did not show bacterial growth. Samples treated at 200 MPa, 65 and 75ºC inlet temperature

coagulated after 2 days of incubation and those treated at 300 MPa, 65ºC showed microbial

growth of 3.97 ± 0.08 cfu/mL after one week of incubation and coagulated on day 20.

Spores showed a high resistance. According with Suárez-Jacobo et al. (2010), who worked

with fruit juice, all vegetative cells were already destroyed at 200 MPa, and the differences

between 200 and 300 MPa were only accounting for the destruction of spores. Cruz et al.

(2007) and Pereda et al. (2007), also detected that bacterial spores were not completely

eliminated by UHPH treatments with inlet temperatures of 30 or 40ºC at 300 MPa.

Coliform counts (data not shown) were only detected in one production of BP (2.57 ± 0.02

log cfu/mL). However, coliforms counts were below the detection level after

pasteurization, UHT and UHPH treatments (< 0.5 cfu/mL). Cruz et al. (2007) found

enterobacteria counts below detection level (initial count of 2.3 log cfu/mL) in soymilk

samples when homogenized at 200 and 300 MPa, and Hayes et al. (2005) and Pereda et al.

(2007), did not detect coliforms in milk samples, after treatment at 200 MPa and above.

Yeasts and moulds, Salmonella spp. and Staphylococcus aureus (data not shown) were not

detected in both BP and treated soymilks Tahiri et al. (2006) obtained inactivation of

Saccharomyces cerevisiae and Penicillum spp. in orange juice by 2.5 and 4.0 log units,

respectively, working at 200 MPa, 25ºC. Suárez-Jacobo et al. (2010) obtained reductions

of yeast and mould counts around 5 log units in apple juice treated at 200 MPa and 300

MPa and Donsì et al. (2009) achieved a reduction of 5 log units for S. cerevisae inoculated

in sterile water at 250 MPa, showing high susceptibility of some yeasts to UHPH

treatment.

5.2.4 Colloidal stability: particle size and sedimentation

In soymilk, particles dispersed in the aqueous phase are of different characteristics such as

oil droplets, native protein aggregates (protein bodies), and other aggregates formed from

oil droplets and proteins and/or polysaccharides (Cruz et al., 2007; Malaki-Nik et al.,

2008). The most common stability problems of vegetable beverages are the creaming of oil

droplets and the sedimentation of solid particles; both phenomena depending to a great

extent on particle size distribution. Colloidal stability was assessed by measuring solids

sedimentation after centrifugation of soymilk and by means of particle size distribution

(Table 5-3). It was observed that UHPH treatments reduced solids sedimentation induced

by centrifugation compared with conventional thermal treatments applied in this study.

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90

Samples treated at 200 and 300 MPa at any inlet temperature did not show significant

differences (P ≥ 0.05) in solids sedimentation values. In the same way, samples did not

exhibit additional sedimentation increase after 15 days of storage (data not shown).

Another test to qualitatively evaluate the physical stability of samples consisted of

detecting any destabilizing phenomena in glass bottles of treated soymilks for one week

while maintained at rest. UHPH-treated soymilks remained in perfect dispersion state

without any visible phase separation, while UHT and pasteurized soymilk exhibited a layer

on the bottom of the bottles easily observable. All these results indicated that applying

pressure in such a magnitude compared with those conventional applied at the food

industry (in this case 18 MPa in a single or double step) is very effective for dispersing

solid particles, even during storage periods. However this latter aspect should further be

study.

Table 5-3. Solids sedimentation and particle size parameters of BP and treated soymilks.

Treatment Stability index1 d3.2 (µm)2 d4.3 (µm)3

BP 7.03 ± 0.55a 0.51 ± 0.05a 12.98 ± 3.82a Pasteurized 3.32 ± 0.30b 0.68 ± 0.04b 48.00 ± 10.2b UHT 3.08 ± 0.23b 0.39 ± 0.03c 15.65 ± 1.83a 200MPa 55ºC 1.32 ± 0.11c 0.13 ± 0.02d 4.82 ± 2.27c 200MPa 65ºC 1.34 ± 0.10c 0.14 ± 0.02d 0.14 ± 0.01d 200MPa 75ºC 1.38 ± 0.12c 0.14 ± 0.01d 3.09 ± 0.37e 300MPa 55ºC 1.55 ± 0.40c 0.12 ± 0.01d 4.39 ± 1.16ce 300MPa 65ºC 1.61 ± 0.36c 0.11 ± 0.01d 1.32 ± 0.55d 300MPa 75ºC 1.38 ± 0.20c 0.11 ± 0.01d 3.39 ± 1.83ce

a-e Different superscripts in the same column are significantly different (P < 0.05). 1 Mean values ± SD (g/100g w/w) of solids sedimentation after centrifugation. 2 Mean values ± SD (µm) of average diameter (surface weighted mean diameter). 3 Mean values ± SD (µm) of average diameter (volume weighted mean diameter).

Analyzing particle size distribution in soymilk is useful not only to determine the

individual particle size but also to detect aggregates, which behave as big particles.

Analysis of particle size was performed by examining the distribution curves and through

the parameters d3.2 and d4.3 which are the average diameter of particles based on surface


91

area and that based on volume of particles, respectively. The latter is especially sensitive to

the presence of aggregates. All samples exhibited a bimodal curve of particle size

distribution, which was characterized by the presence of two peaks (Figure 5-1). UHPH-

treated soymilks were characterized by a big peak in the range of 0 - 0.6 µm which

represented 80 - 95% of the total volume of particles, and a second small peak (> 3 µm)

which represented 5 - 20% of the total volume of particles, depending on pressure applied.

UHT and pasteurization treatments were characterized by a peak in the range of 0.1 - 1.6

µm which represented approximately 65 and 20% respectively of the total volume of

particles, and a second large peak (> 3 µm) representing about 35% of total volume of

particles for UHT and 80% for pasteurized soymilk.

Particle size parameters exhibited a significant (P < 0.05) decrease in UHPH-treated

soymilks compared to BP and to those soymilks processed by heat treatment (Table 5-3).

However, pasteurized soymilk showed unexpected high values of particle size parameters

compared to BP. This might be due to the aggregation of particles caused by using a single

effect homogenizer as part of the pasteurization line in the present study. This

phenomenon, well known in dairy industry (Walstra et al., 1999), makes using double

effect homogenizers to be preferred. The second valve in double effect homogenizers, such

as this used in UHT-treated soymilk in this study, have the objective of dispersing

aggregates formed in the first valve, into smaller particles. Another data that supports this

explanation is the fact that particle sedimentation was similar in both heat treated soymilks,

pasteurized and UHT, since oil droplets aggregates, experienced creaming in the

centrifugation of samples.

UHPH technology produced a considerably reduction of the mean particle size and also

caused the reduction of aggregates volume fraction. On the other hand, in UHPH

treatments the combination of pressure and inlet temperature did not produce significant

variations in the mean diameter of particles, while it affected the production of aggregates.

However, no specific relation was observed between UHPH treatment applied and

aggregates formation. Despite the presence of small quantity of aggregates in the UHPH-

treated soymilks, physical stability of these samples was not affected as demonstrated by

the observed values of particles sedimentation. Thus UHPH dramatically increased

colloidal stability of soymilk. The presence of aggregates in UHPH-treated soy-based food

products has also been described by Floury et al. (2002) in emulsions stabilized with soy

proteins, and Cruz et al. (2007) in soymilk. The formation of large particles was observed

at 300 MPa for milk samples (Thiebaud et al., 2003) and for soymilk (Cruz et al., 2007).

Chapter 5

92

They attributed the aggregates presence to unfolding and denaturation of whey proteins,

which in this condition may interact with other proteins and/or fat globules. In the UHPH

treatment, phenomena of cavitation, shear and turbulence are involved simultaneously,

affecting the macromolecular conformation of proteins. In the case of soybean, globulins

are strongly affected by the decrease in solubility, due to the denaturation produced by

UHPH treatment above 200 MPa, with the consequent aggregation (Floury et al., 2002).

Figure 5-1. Particle size distribution determined by light-scattering of BP (—), pasteurized (--), UHT (…),

200 MPa, 75ºC(--) and 300 MPa, 75ºC (-Δ-).

Malaki-Nik et al. (2008) have study the colloidal stability of soymilk in terms of particle

size distribution as influenced by heat treatment and homogenization. They concluded that

heating of soymilk disrupts large aggregates of soy proteins and causes a decrease in the

particle size. Their conclusions may be partially in agreement to those observed in this

study. However, results have to be interpreted in their specific context. They applied a mild

thermal treatment (60ºC) to obtain the BP sample which was further heat treated (95–

100ºC for 7 min) or heated and subsequently homogenized (69 MPa). However, in the


93

present study, soymilk BP was obtained by a method which included heating at 80ºC,

causing therefore a partial denaturation of soy proteins. Subsequent heat treatments and

homogenization of soymilks were made in different conditions and sequence as those

performed by Malaki-Nik et al. (2008). The same authors, Malaki-Nik et al. (2009) have

further investigated the influence soy protein subunits by studying different soy varieties

and confirmed the influence of protein subunit composition in the colloidal stability of

soymilks. However, it is important to note that changing the elaboration process of soymilk

might produce different results.

5.2.5 Chemical stability: oxidation and trypsin inhibitor activity

Lipid oxidation involves the formation of hydroperoxides as primary products in reactions

which are developed through free radicals. There are many factors which favor lipid

oxidation: presence of oxygen, light and enzymes such as lipoxygenase, and high

temperatures. In this study, the oxidation process was evaluated by measuring the

lipoxygenase activity and the hydroperoxide concentration at days 1 and 15 after

processing of soymilks. As primary reaction, lipoxygenase catalyzes the hydroperoxidation

by molecular oxygen of linoleic acid and other polyunsaturated lipids (Axelrod et al.,

1981). Lipoxygenase activity was determined because it is an initiator of hydroperoxides

formation and it may give some information about raw material manipulation previously or

during processing. In this study, lipoxygenase was completely inactivated during the

elaboration of soymilk BP and consequently, all subsequent treatments applied to soymilk

did not present any lipoxygenase activity (dates not shown). During the elaboration of the

BP sample, soybeans were ground at 80ºC for 20 minutes which explains total inactivation

of lipoxygenase. Kwok and Niranjan (1995) reported different ways of lipoxygenase

inactivation: grinding with water between 80ºC and 100ºC for 10 minutes, blanching

soaked soybeans in boiling water for 10 minutes, and boiling dry whole beans in water for

20 minutes.

Hydroperoxide determination was carried out on the first day and 15 days after soymilk

storage at 4ºC (Figure 5-2). On the first day, soymilk samples presented significantly lower

hydroperoxide values than at day 15 at cold storage as expected (P < 0.05). The initial

hydroperoxide values of samples did not exhibit a tendency when comparing UHPH and

heat treatments, although the index ranged in a narrow interval between 0.2 and 0.4 meq/L

for all samples. After 15 days of cold storage all treated soymilks presented higher

Chapter 5

94

hydroperoxide index and the tendency was defined: heat treated soymilks had higher

values of oxidation index than UHPH treatments, 300 MPa at any inlet temperature

showed the lowest hydroperoxide index. Pereda et al. (2008) who applied UHPH to milk

reported that 300 MPa produced samples with less hydroperoxides index compared to 200

MPa. However, the secondary oxidation, studied through malondialdehyde formation, was

higher at this UHPH condition, which indicated an evolution in the oxidation process to

final products as also confirmed by the higher hexanal content of these samples.

Figure 5-2. Hydroperoxides values (mean ± SD) of BP, heat treated (pasteurized and UHT) and UHPH

soymilks at day 1 ( ) and day 15 ( ). a-e Different letters above bars of day 1 indicate that samples are significantly different (P < 0.05). g-l Different letters above bars of day 15 indicate that samples are significantly different (P < 0.05).

Trypsin inhibitors are substances with anti nutritional properties which are present in

animal and vegetal tissues, such as soybeans. They reduce the availability of trypsin, an

important protease in the animal digestive function for splitting proteins to render

dipeptides and tripeptides (Guerrero-Beltrán et al., 2009). Consumption of raw or

inadequately cooked soy products may cause a decrease in protein digestibility and

nutritive value (Yuan et al., 2008). For this reason, reaching the maximum inactivation of

trypsin inhibitors is an objective in the production of soy products. However, trypsin

0

0.2

0.4

0.6

0.8

1

1.2

1.4

BP Past UHT 200MPa 55ºC

200MPa 65ºC

200MPa 75ºC

300MPa 55ºC

300MPa 65ºC

300MPa 75ºC

Lip

id h

ydro

pero

xide

s (m

eq p

erox

ide/

L sa

mpl

e)

h

b

ga

i

cd

c

k

dce

de

k

kjhj

a

l

ce


95

inhibitors (TI) are very stable due to the presence of disulfide bonds in their molecular

structure (Wolf, 1977). In the present study, original soybeans used to prepare soymilk

presented a total content of 9.25 g of TI/L of sample extract.

Figure 5-3. Trypsin inhibitor values (mean ± SD) of BP, heat-treated and UHPH soymilks. a-c Different letters above bars of each treatment indicate that samples are significantly different (P < 0.05).

Figure 5-3 shows results of residual TI activity after treatment of soymilks. According to

these results, UHPH treatments caused similar inactivation compared to the pasteurized

sample (about 37% of initial activity in BP sample). However, to reach the same

inactivation effect on soymilks, it was necessary that pasteurization acted during 30 s at

90ºC, whereas in UHPH treatment the holding time at high temperature (from 105 to

135ºC depending on inlet temperature and pressure applied) was about 0.7 s. So, to reach a

significantly higher reduction of TI, it was necessary to combine higher temperatures and

longer holding time such as in UHT treatment applied in this work (142ºC for 6 s). Under

these conditions, TI inactivation achieved 60.8% of the initial TI activity in the BP sample.

This result is in accordance with values observed in commercial UHT soymilks (data not

shown). Taking into account the total TI values of soybeans, UHPH and pasteurized

soymilks achieved total inactivation about 80%, whereas UHT-treated soymilk achieved

90%.

0

0.5

1

1.5

2

2.5

3

BP Past UHT 200MPa 55ºC

200MPa 65ºC

200MPa 75ºC

300MPa 55ºC

300MPa 65ºC

300MPa 75ºC

g of

TI/

L o

f sam

ple

a

b b b b bb b

c

Chapter 5

96

Considering TI values of untreated soymilk, Kwok et al. (1993) reached about 90% of

inactivation in soymilk at 93ºC with holding time of 60 min or 154ºC using UHT treatment

for about 60 s. Miyagi et al. (1997) boiled soymilk by traditional cooking method at 100ºC

for 30 min and reduced TI to about 10% of the initial activity (whole soybeans). All the

above-mentioned studies applied higher temperature during longer holding times compared

to UHPH treatment applied in this work.

5.3 Conclusions

The present study demonstrates that UHPH was effective in the reduction of microbial

populations. Inlet temperature was decisive to achieve complete reduction in combination

with pressure. UHPH at 300 MPa and 75ºC inlet temperature was able to produce sterile

soymilks. In terms of physical stability, UHPH conditions applied produced high stability

products, with a considerable reduction of particle size and no deposition layer of particles

observed during the first week compared to that produced in heat treated samples. The

hydroperoxides index also showed lower values in UHPH-treated samples compared to

heat-treated samples, although in order to assess the complete oxidation caused by

treatments, further analysis must be completed by means of determination of the secondary

oxidation products, as well as profile of volatile compounds and finally sensorial analysis.

UHPH treatments did not achieve the same level of TI inactivation as UHT treatment.

However, a further study of digestibility should be performed to assess the real importance

of the residual activity of TI after UHPH selected treatment.

5.4 References

Axelrod, B., Cheesbrough, T. M., & Laasko, S. 1981. Lipoxygenase from soybeans. In

Methods in Enzimology (Edited by, J. M. Lowenstein), USA. pp. 441-451.




Donsì, F., Ferrari, G., & Maresca, P. (2009). High-pressure homogenization for food



97

Barbosa-Cánovas, Alan Mortimer, David Lineback, Walter Spiess, Ken Buckle, &

Paul Colonna), Academic Press, USA. pp. 309-352.




Guerrero-Beltrán, J.A., Estrada-Girón, Y., Swanson, B.G. & Barbosa-Cánovas, G.V.

(2009). Pressure and temperature combination for inactivation of soymilk trypsin

inhibitors. Food Chemistry. 116, 676-679.

Hayes, M.G., Fox, P.F. & Kelly, A.L. (2005). Potential applications of high pressure


Hayes, M.G. & Kelly, A.L. (2003). High pressure homogenisation of raw whole bovine

milk (a) effects on fat globule size and other properties. Journal of Dairy Research.

70, 297-305.







Malaki-Nik, A., Tosh, S., Poysa, V., Woodrow, L. & Corredig, M. (2008).



Malaki-Nik, A., Tosh, S.M., Woodrow, L., Poysa, V. & Corredig, M. (2009). Effect of soy

protein subunit composition and processing conditions on stability and particle size

distribution of soymilk. LWT - Food Science and Technology. 42, 1245-1252.

Chapter 5

98

Miyagi, Y., Shinjo, S., Nishida, R., Miyagi, C., Takamatsu, K., Yamamoto, T. &

Yamamoto, S. (1997). Trypsin inhibitor activity in commercial soybean products in

Japan. Journal of Nutritional Science and Vitaminology. 43, 575-580.


ultra-high pressure homogenization on microbial and physicochemical shelf life of

milk. Journal of Dairy Science. 90, 1081-1093.


ultra-high-pressure homogenization treatment on the lipolysis and lipid oxidation of

milk during refrigerated storage. Journal of Agricultural and Food Chemistry. 56,

7125-7130.

Picart, L., Thiebaud, M., René, M., Pierre Guiraud, J., Cheftel, J.C. & Dumay, E. (2006).

Effects of high pressure homogenisation of raw bovine milk on alkaline phosphatase

and microbial inactivation. A comparison with continuous short-time thermal

treatments. Journal of Dairy Research. 73, 454-463.

Smiddy, M.A., Martin, J.E., Huppertz, T. & Kelly, A.L. (2007). Microbial shelf-life of

high-pressure-homogenised milk. International Dairy Journal. 17, 29-32.

Suárez-Jacobo, Á., Gervilla, R., Guamis, B., Roig-Sagués, A.X. & Saldo, J. (2010). Effect

of UHPH on indigenous microbiota of apple juice: A preliminary study of microbial

shelf-life. International Journal of Food Microbiology. 136, 261-267.

Tahiri, I., Makhlouf, J., Paquin, P. & Fliss, I. (2006). Inactivation of food spoilage bacteria

and Escherichia coli O157:H7 in phosphate buffer and orange juice using dynamic

high pressure. Food Research International. 39, 98-105.




Walstra, P., Geurts, T. J., Noomen, A., Jellema, A., & van Boekel, M. A. J. S. (1999).

Homogenization. In Dairy technology: Principles of milk properties and processes

(Edited by, Anonymous), Marcel Dekker, Inc., USA. pp. 245-264.


99



Wolf, W.J. (1977). Physical and chemical properties of soybean proteins. Journal of the

American Oil Chemists Society. 54, A112-A117.

Yuan, S., Chang, S.C., Liu, Z. & Xu, B. (2008). Elimination of trypsin inhibitor activity

and beany flavor in soy milk by consecutive blanching and ultrahigh-temperature

(UHT) processing. Journal of Agricultural and Food Chemistry. 56, 7957-7963.

Chapter 6

Study of the potential inactivation of UHPH treatment on selected Bacillus

spores isolated from soymilk

103

6

Study of the potential inactivation of UHPH treatment on selected

Bacillus spores isolated from soymilk

6.1 Introduction

The ability of UHPH to inactivate microorganisms has been already recognized by some

authors, Wuytack et al. (2002), Briñez et al. (2006) and Donsì et al. (2009). Several

microbial strains were exposed to UHPH in order to evaluate the sensitivity of the

microorganisms to the treatment and the efficiency in sanitization process. Investigations

reported that microbial inactivation was influenced by microbial strains, medium of

suspension, pressure and temperature in the homogenization valve.

Sporogenesis is a characteristic of some bacteria that permit organisms to withstand

environmentally harsh conditions, allowing long-term survival and favoring contamination

of many foods. Bacillus genus is a class of bacteria able to form spores and usually

detected in soy foods, such as soymilk. The characteristic of endospores to be able to

survive traditional heat treatments (pasteurization and UHT) widely used in dairy industry,

allows, at good conditions, growth of vegetative bacteria at refrigeration temperatures

(Feijoo et al., 1997; Van Opstal et al., 2004).

The few data available on the effects of high-pressure homogenization on spore

inactivation indicate a resistance of spores to this treatment. Therefore, the purpose of this

study was to evaluate the kinetics of inactivation of Bacillus cereus and Paenibacillus

taichungensis previously isolated and purified from original soymilk. The spores of these

microorganisms were then inoculated in sterile soymilk followed by UHPH treatments at

300 MPa and 55, 65, 75 and 85ºC of inlet temperature. For this purpose, a Benchtop ultra

high pressure homogenizer (UHPH B) was used for soymilk treatment. The methodology

applied was the follow: isolation and selection of the strains (see 3.6.2), sporulation

conditions (see 3.6.3) and spores recovery (see 3.6.4).

Chapter 6

104


6.2.1 Spore-former occurrence

Spore-forming bacteria prevalence in soymilk treated at 300 MPa, 65ºC of inlet

temperature was investigated. Several genus and species of Bacillus were identified:

Lysinibacillus and Paenibacillus (P. taichunengis and P. glucanolyticus). Of these Bacillus

species, B. cereus and Paenibacillus taichungensis were the most frequently encountered

on UHPH soymilk. Precisely, these indigenous spore-forming bacteria were selected to

evaluate the UHPH effect on their lethality at 300 MPa and 55, 65, 75 and 85ºC inlet

temperatures.

The contamination of foods with bacterial spores is well documented in several foodstuffs.

The multiplication of the vegetative cells formed after spore germination and outgrowth

can occur at a wide range of temperature (spore-forming bacterial species which include

psychrotrophic, mesophilic or thermophilic). Water activity and pH can be the cause of

foodborne poisoning or food spoilage (Carlin, 2011). In the present study, the more

resistant aerobic mesophilic spore-forming bacteria found in soymilk treated by UHPH at

300 MPa and 65ºC of inlet temperature coincide partially with those found by Postollec et

al. (2012). They investigated the occurrence of spore-forming bacteria in 90 foodstuffs

(raw materials, dehydrated ingredients and processed foods) and reported that the most

frequently encountered Bacillus species were B. cereus, B. subtilis and B. licheniformis,

isolated from dehydrated vegetables. In food industry, hygiene management and

processing largely contribute to lower spore-forming bacterial contamination ensuring

quality and safety of final products (Postollec et al., 2012). Bacillus cereus, for instance, is

widely distributed in natural and commercial products due to the strong resistance of its

spores to physical and chemical disinfectant agents. The organism is associated with two

types of gastrointestinal syndromes, denominated as emetic type and diarrhoeal type (Ju et

al., 2008; De Jonghe et al., 2010). Food spoilage can be produced by two different ways:

by survival of viable spores to the treatment (pasteurization/UHT) with subsequent growth,

causing therefore the characteristic spoilage; or by extracellular enzymes (proteases,

lipases and lecithinases) that are synthesized prior to heat treatment. According to

Rodríguez-Lozan et al. (2010), conventional treatments are able to eliminate viable

organisms, but do not inactivate the preformed heat-stable enzymes.

Inoculation study

105

Paenibacillus has recently been recognized as a separate genus from Bacillus and many

new species of Paenibacillus continue to be identified (Ivy et al., 2012). They have been

isolated from various different pasteurized foodstuffs (Guinebretiere et al., 2001) and even

by ultra high temperature (UHT) treatment applied in milk (Scheldeman et al., 2004).

Recently, Paenibacillus spp. has described as spoilage organisms. Some species appear to

be predominantly psychrotolerant with an ability to grow in milk and possibly other foods

at temperatures as low as 6ºC (Ivy et al., 2012).

6.2.2 UHPH effect on spores survive

Spores of Bacillus genus are greatly resistant to conventional treatments, including

homogenization (Popper & Knorr, 1990). In this study, the effect of UHPH on spores of B.

cereus and P. taichungensis suspended in sterilized soymilk has been evaluated by

measuring the capability of spores to survive and proliferate in solid medium after

treatment. Among of 7-8 log cfu/mL of each bacterial strain were inoculated in sterilized

soymilk before treatment. In order to evaluate UHPH efficiency in the total elimination of

inoculated strains and the possible recovery of sub-lethally injured cells, samples of drastic

UHPH conditions (75ºC and 85ºC of inlet temperature) were incubated for 10 days at

30ºC. Table 6-1 shows UHPH effect in the reduction of B. cereus and P. taichungensis in

sterile soymilk. All treatments applied caused significant reduction in the colony counts of

the two strains inoculated (P < 0.05).

In general, B. cereus achieved great log reductions for all UHPH treatments. P.

taichungensis obtained higher log reductions compared to B. cereus, reaching complete

inactivation at 300 MPa, 85ºC. Despite samples of P. taichungensis showed higher initial

load compared to B. cereus, the resistance to the treatment was observed being higher for

B. cereus. These results indicated that inlet temperature played an important role in the

strains resistance against treatment. In addition, as inlet temperature increased, high

microbial inactivation was reached. During the period of incubation, no coagulation and no

microbial growth were observed for P. taichungensis treated at 300 MPa, 85ºC. For the

UHPH condition of 300 MPa, 85ºC, B. cereus strain reached reduction about 5 log cfu/mL,

whereas 55, 65 and 75ºC reached, respectively, values around of 1, 2.5 and 3.5 log cfu/mL.

Chapter 6

106

Table 6-1. Counts (log cfu/mL ± SD) of two bacterial strains inoculated in sterilized

soymilk before and after treatment.

Treatments B. cereus P. taichungensis

Before treatment 5.28 ± 0.55a 6.01 ± 0.24a

300 MPa at 55ºC 4.08 ± 0.22b 3.19 ± 0.57b

300 MPa at 65ºC 2.90 ± 0.99bc 1.98 ± 0.93c

300 MPa at 75ºC 1.78 ± 1.55c 0.20 ± 0.35d

300 MPa at 85ºC 0.54 ± 0.47d ND1

a-d Different superscripts in the same column are significantly different (P < 0.05). 1 No coagulation was detected after sample incubation at 30ºC for 10 days.

Lethality strains of each UHPH condition are shown in Figure 6-1. Significant differences

were observed for B. cereus strain between inlet temperatures (P < 0.05), except for 65 and

75ºC. For P. taichungensis the highest reduction was detected at 75 and 85ºC of inlet

temperature (around 6 log units), whereas 65 and 55ºC achieved values between 3.5 and

2.0 log units, respectively. No significant differences were observed between 55 and 65ºC

and on the other hand between 75 and 85ºC (P ≥ 0.05). Taking into account inlet

temperature for each bacterial strain, only significant differences were observed for 75ºC

and 85ºC (P < 0.05).

Feijoo et al. (1997) investigated spores inactivation of Bacillus licheniformis in ice cream

high-pressure homogenized. They reached reductions of 0.5 log units applying pressures

from 50 to 200 MPa at 33ºC of inlet temperature. Chaves-López et al. (2009) evaluated the

influence of high-pressure homogenization treatment at 150 MPa (one, two or three stages)

in the inactivation of Bacillus cereus spore and Bacillus subtilis spore suspended in

sterilized distilled water. When single stage was applied, a negligible reduction of colony

counts at 150 MPa was observed. In fact, they obtained inactivation values of 0.5 log

cfu/mL for B. subtilis and 0.3 log ufc/mL for B. cereus. However, applying three

consecutive treatment stages, high reduction of colony counts (about 5 log units) were

obtained. In the present study, UHPH treatment at single stage was enough to reach about

6 log units, indicating that pressure of 300 MPa was more effective than number of stages

at 150 MPa.

Inoculation study

107

Figure 6-1. Lethality of Bacillus cereus and Paenibacillus taichungensis inoculated in soymilk UHPH

treated at ( ) 300 MPa, 55ºC, ( ), 300 MPa, 65ºC, ( ) 300 MPa, 75ºC and ( ) 300 MPa, 85ºC. a-b Different letters above bars of each strains indicate that samples are significantly different (P < 0.05). x-y Different letters above bars of each inlet temperature indicate that samples are significantly different (P <

0.05).

Treatment parameters such as inlet temperature, temperature after homogenization vale,

outlet temperature and pressure were monitored during UHPH treatment. Table 6-2 shows

changes of temperature and pressure during soymilk processing. Treatments at 55ºC, 65ºC,

75ºC and 85ºC of inlet temperature achieved maximum temperature after high-pressure

valve near to 109ºC, 118ºC, 128ºC and 137ºC respectively. These values may explain why

spores lethality reached high values in samples treated at 85ºC, as expected. In the UHPH

processing, the food fluid is brought to high pressure in few seconds and then forced

through a very small orifice, the valve gap of few micrometres in width. The resulting

pressure drop generates intense mechanical forces and elongational stress in laminar flow

at the valve entrance and in the valve gap. Turbulence, cavitation and impacts with solid

surfaces occur at the gap outlet. Due to these phenomena, the food fluid experiences a

short-heating phenomenon that increases as pressure increase (Dumay et al., 2012).

According to Sharma et al. (2009), spore inactivation requires a combination of shear and

0

1

2

3

4

5

6

7

B. cereus P. taichungensis

Leth

ality

log

(No/

N)

ax

bx

bxcx

ax

ax

by by

Chapter 6

108

temperature. Phenomena of turbulence, high shear, and cavitation forces increase as

pressure increase, resulting in thermal dissipation on the product. The resultant temperature

elevation has a positive effect in the destruction of bacterial spores. Some studies have

reported germination stimulation of spore accompanied by sub-lethal heating, rendering

germinated spore more sensitive to destructive action of temperature and mechanical

forces as were vegetative cells (Hyung-Yong et al., 1999; Ananta et al., 2001). Therefore,

the flow of inoculated soymilk through the homogenizing valve may have stimulated

spores germination in the food fluid, rendering them sensitive to heat and mechanical

forces (Diels & Michiels, 2006; Chaves-López et al., 2009). Nevertheless, it is worth to

point that the holding time at high temperature was lower than 0.3 s, resulting in a minimal

thermal damage respect to usual heat treatments applied in food industries. Although the

residence time was very low, it was essential to reach high levels of spore inactivation.

Table 6-2. Temperature and pressure changes1 of UHPH-treated soymilk during

processing.

Pressure Ti (ºC) T1 (ºC) Tf (ºC)

305.0 ± 1.73 55.6 ± 0.55 108.8 ± 0.84 4.80 ± 1.10

302.3 ± 2.52 65.2 ± 0.45 117.8 ± 1.10 7.60 ± 2.88

307.7 ± 3.21 74.6 ± 1.14 127.6 ± 1.52 7.60 ± 1.95

302.0 ± 2.65 84.0 ± 1.41 137.2 ± 1.30 8.20 ± 2.86

1 Ti = inlet temperature; T1 = temperature after the homogenization valve; Tf = temperature after final heat

exchanger. Mean values ± SD from 3 individual productions.

Additionally to processing parameters, the type of foodstuff may influence the efficacy of

the treatment. Available results indicated that UHPH treatments were more effective

against microorganisms in saline buffered solutions than complex matrices (Vachon et al.,

2002; Diels et al., 2005). Foodstuff is a complex chemical system in which most of

components, such as protein, lipids and carbohydrates may affect microbial tolerance to

heat or pressure, making difficult to study the possible protective mechanism of the

interactions among those components (Roig-Sagués et al., 2009). Therefore, the possible

reason of higher resistance of B. cereus to the UHPH treatment than P. taichungensis could

Inoculation study

109

be due to soymilk composition and to the components interaction in addition to cellular

structure of the spore strain. Structurally, an endospore consists of a core, surrounded by a

cortex of peptidoglycan, a spore coat of protein and in some species a delicate thin layer

called exosporium. Mature core of endospore differs greatly of cytoplasm of vegetative

cell from which it is derived. In addition, it is rich in calcium dipicolinate, contains small

water content (10-30%) from the vegetative cell, becoming gel consistency the cytoplasm

core (Diels & Michiels, 2006). These characteristics of spore confer high resistance to the

treatment in addition to the matrix characteristics. On the other hand, when cell water

content is about 10% the resistance to pressure and temperature remained unaffected.

However, beyond 10% moisture, the resistance of spores is gradually reduced, enabling a

particular pressure-temperature combination to achieve an adequate level of spore

reduction (Ananta et al., 2001). In addition, water content from the matrix, in this case

soymilk, could be transferred to the spore cells increasing its sensibility to pressure-

temperature effect (Chaves-López et al., 2009). This approach was first elucidated from

heat inactivation studies of spores in fat systems (Molin & Snygg 1967; Harnulv et al.,

1977).

6.3 Conclusions

Bacterial endospores inactivation is the main objective in food sterilization, due to high

resistance to several chemical and physical agents as well as heat treatments. The main

potential spores identified and isolated from soymilk bellowed to Bacillus genus, in

particular Bacillus cereus and Paenibacillus taichungensis. UHPH treatment was able to

reduce noticeably these bacterial strain inoculated in sterilized soymilk. It was observed

that as the inlet temperature increased, the levels of inactivation increased, especially at

85ºC. Inactivation of Paenibacillus taichungensis was more effective instead of Bacillus

cereus at any inlet temperature, reaching complete inactivation at 85ºC. The maximum

temperature achieved in the high-pressure valve was 137ºC when inlet temperature of 85ºC

was applied. The combined effect of high-pressure and temperature was determinant in the

bacillus strains inactivation, taking into account that the holding time at high temperature

was about 0.3 seconds. Therefore, the use of UHPH system could be an alternative to

increase shelf-life and microbial safety in soymilk processing, avoiding the typical thermal

damage caused by conventional technologies.

Chapter 6

110

6.4 References

Ananta, E., Heinz, V., Schlüter, O. & Knorr, D. (2001). Kinetic studies on high-pressure

inactivation of Bacillus stearothermophilus spores suspended in food matrices.

Innovative Food Science & Emerging Technologies. 2, 261-272.

Briñez, W.J., Roig-Sagués, A.X., Hernández-Herrero, M. & Guamis, B. (2006).

Inactivation of two strains of Escherichia coli inoculated into whole and skim milk by

ultrahigh-pressure homogenisation. Lait. 86, 241-249.

Carlin, F. (2011). Origin of bacterial spores contaminating foods. Food Microbiology. 28,

177-182.

Chaves-López, C., Lanciotti, R., Serio, A., Paparella, A., Guerzoni, E. & Suzzi, G. (2009).

Effect of high pressure homogenization applied individually or in combination with

other mild physical or chemical stresses on Bacillus cereus and Bacillus subtilis spore

viability. Food Control. 20, 691-695.

De Jonghe, V., Coorevits, A., De Block, J., Van Coillie, E., Grijspeerdt, K., Herman, L.,

De Vos, P. & Heyndrickx, M. (2010). Toxinogenic and spoilage potential of aerobic

spore-formers isolated from raw milk. International Journal of Food Microbiology.

136, 318-325.


Inactivation of Escherichia coli by high-pressure homogenisation is influenced by

fluid viscosity but not by water activity and product composition. International

Journal of Food Microbiology. 101, 281-291.

Diels, A.M.J. & Michiels, C.W. (2006). High-pressure homogenization as a non-thermal

technique for the inactivation of microorganisms. Critical Reviews in Microbiology.

32, 201-216.

Donsì, F., Ferrari, G., Lenza, E. & Maresca, P. (2009). Main factors regulating microbial

inactivation by high-pressure homogenization: Operating parameters and scale of

operation. Chemical Engineering Science. 64, 520-532.

Inoculation study

111

Dumay, E., Chevalier-Lucia, D., Picart-Palmade, L.T., Benzaria, A., Gràcia-Julià, A. &

Blayo, C. (2012). Technological aspects and potential applications of (ultra) high-

pressure homogenisation. Trends in Food Science & Technology. 0, 1-14.

Feijoo, S.C., Hayes, W.W., Watson, C.E. & Martin, J.H. (1997). Effects of

microfluidizer® technology on Bacillus licheniformis spores in ice cream mix.

Journal of Dairy Science. 80, 2184-2187.

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Chapter 7

Characterization of volatile profile in soymilk treated by ultra high pressure

homogenization

115

7 Characterization of volatile profile in soymilk treated by ultra high

pressure homogenization

7.1 Introduction

The main off-flavors that could be associated to the limited soymilk acceptation by

consumers have been described as green, grassy, paint, rancid, astringent, and bitter

(Torres-Penaranda, & Reitmeier, 2001; N'Kouka, et al., 2004; Lozano, et al., 2007). Soy

odors in soymilk are primarily derived from enzymatic oxidation. Moreover, auto-

oxidation of linoleic and linolenic acids also plays an important role in the off-flavor

generated by degradation products in the last steps of oxidation reaction (Min, et al., 2005).

Thus, the profile of volatile compounds in soymilk is strongly related to its quality and

acceptability.

Identification of volatile compounds in soymilk treated by UHPH and comparing them

with those identified by conventional thermal treatments could provide information related

to sensory quality and better acceptation of soymilk. The aim of this study was to

characterize the volatile profile of soymilk treated at 200 MPa at two inlet temperatures (55

and 75ºC) and 300 MPa at 80ºC of inlet temperature. UHPH soymilks were then compared

to UHT and pasteurized treated samples. SPME analysis (see 3.14) was applied in order to

characterize soymilk volatile profile.


7.2.1 Volatile compounds

In addition to those natural compounds from soy seeds, responsible for the characteristic

volatile profile, other compounds may be generated and the amount of the original

compounds may be changed during processing of soymilk. An important part of those

compounds is consequence of lipid auto-oxidation. This important lipid degradation in soy

and derived products depends on soymilk processing conditions such as light incidence,

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116

partial pressure of oxygen and processing temperature. Oxidation may also take place by

the enzymatic pathway through the lipoxygenase action. In both, enzymatic and no

enzymatic auto-oxidation, hydroperoxidation of polyunsaturated fatty acids such as linoleic

and linolenic acids by molecular oxygen is the primary reaction (Boatright & Lei, 1999;

Kakumyan, et al., 2009). The hydroperoxides formed are very unstable and they can easily

be transformed to final products such us aldehydes, ketones, furans, alcohols, polymers,

etc. Maillard reaction and Strecker degradation might also contribute to the formation of

volatiles compounds through saccharides and amino acids reaction in thermally processed

soymilk (Lozano et al., 2007; Plutowska & Wardencki, 2007).

Fifty-seven compounds were identified in the headspace of soymilks studied in the present

work, which consisted of untreated, pasteurized, UHT and UHPH treated at 3 different

combinations of temperature and pressure. Table 7-1 shows total abundance of volatile

compounds grouped by chemical families in BP, pasteurized, UHT and UHPH soymilks.

Chemical families included aldehydes, ketones, alcohols, furans, esters and acids and were

listed in Tables 7-2 to 7-6.

Adehydes and alcohols were the most representative compounds in all chromatographic

profiles studied for all treatments (Table 7-1) in agreement with the study reported by

Achouri et al. (2006). In general and considering the total of volatiles for each family of

compounds, soymilk treated at 200 MPa, 55 and 75ºC inlet temperature respectively,

caused similar effects to the pasteurized and unprocessed soymilks. Only some differences

were found between specific treatments and families which are later discussed without

altering general results (Table 7-1). At 300 MPa, the abundance of compounds of all

chemical families increased with the increase of pressure and inlet temperature, reaching

similar results to UHT soymilk for all groups except for furans for which UHT obtained

the highest abundance values (P < 0.05). These results indicated that the adiabatic

temperature increase caused by high pressure could be the main reason for the formation of

volatile compounds. Temperature after homogenization valve in UHPH treatments at 200

MPa reached values between 106ºC and 117ºC when inlet temperatures of 55ºC and 75ºC,

respectively, were applied and 144ºC were reached for soymilk treated at 300 MPa, 80ºC

inlet temperature. The residence time at these temperatures in the UHPH treatment was

only 0.7 seconds as described in chapter 5, while pasteurization reached 95ºC for 30

seconds and UHT 142ºC for 6 seconds. The differences of temperatures after

homogenization valve between 200 and 300 MPa were the main cause of the changes

observed in the volatile profile among UHPH treatments. On the other hand, differences

Volatile profile characterization

117

observed between UHT and 300 MPa, 80ºC indicated that high pressure additionally to the

temperature could have an important effect on formation of the volatile profile, especially

in the oxidative process during treatment application, as discussed below.

Table 7-1. Total of volatile compounds1 by chemical family in BP and treated soymilks.

BP Pasteurized UHT 200 MPa 55ºC 200 MPa 75ºC 300 MPa 80ºC

Aldehydes 150.64 ± 19.45a 188.14 ± 11.28a 252.42 ± 44.02b 158.19 ± 16.05a 184.64 ± 9.03a 244.47 ± 7.40b

Alcohols 248.92 ± 60.31ab 244.61 ± 30.46ab 207.47 ± 8.02ab 171.26 ± 20.92a 174.46 ± 17.40a 258.62 ± 38.48b

Ketones 28.20 ± 3.97a 36.19 ± 2.71a 47.12 ± 1.82b 35.28 ± 2.55a 34.06 ± 5.41a 46.45 ± 5.12b

Furans 19.84 ± 3.57a 28.87 ±1.51ab 104.83 ± 9.43c 20.36 ± 0.76a 21.77 ± 0.70ab 32.56 ± 2.05b

Esters 9.95 ± 0.67ab 8.96 ± 0.77a 12.22 ± 1.37b 6.48 ± 1.38c 6.01 ± 0.77c 9.79 ± 0.67ab

Acids 26.50 ± 3.96a 39.39 ± 2.27b 44.30 ± 8.95bc 33.83 ± 3.58ab 40.73 ± 6.05bc 49.18 ± 4.64c a-c Different superscript in the same row are significantly different (P < 0.05). 1 Integrated area counts. Mean values x 105 ± SD.

7.2.2 Aldehydes

UHT treatment and 300 MPa, 80ºC UHPH condition caused the most significant effect in

the total aldehydes compounds. The abundance of this chemical family influenced by

treatment is shown in Table 7-2. Hexanal was the most abundant compound identified

followed by pentanal. Other compounds were also detected in low levels such as

acetaldehyde, propanal, heptanal, benzaldehyde and 2-hexanal. Except acetaldehyde, all

other aldehyde levels changed after treatment (P < 0.05).

Of the aldehydes identified in soymilk, hexanal is the most studied because it is indicative

of the oxidation degree of product and, as reported, it plays the most important role in the

sensory off-flavors. Commonly it is related to beany, grassy and green flavors in soymilk

(Yuan & Chang, 2007a). According to Hashim and Chaveron (1995), hexanal has a very

low sensory threshold, so content above 25 µg/kg allows its detection. Therefore, hexanal

content could be decisive in the sensory quality and as a result soymilk with a low hexanal

content would receive a better acceptance by the consumers. Table 7-2 shows that UHPH

treatments at 200 MPa achieved similar levels of hexanal compared to pasteurized and BP

soymilks. However, soymilk treated at 200 MPa, 75ºC inlet temperature reached values

slightly higher than those observed at 200 MPa, 55ºC, although this difference was not

Chapter 7

118

significant (P ≥ 0.05). These results are in accordance to the temperature increase during

UHPH application. High temperatures combined with oxygen have an important role in the

oxidative processes that take place during and after treatment. Alternatively, 300 MPa,

80ºC and UHT treatments reached the highest levels of hexanal. Similar results were

observed by Pereda et al. (2008), who analyzed the effect of UHPH in the volatile profile

in milk. They found that 300 MPa, 30 and 40ºC inlet temperatures showed higher content

of hexanal compared to 200 MPa at the same inlet temperatures. Several authors (Min et

al., 2005; Yuan & Chang, 2007ab; Achouri et al., 2007; Achouri et al., 2008) found also

hexanal as the main compound in heat-treated soymilk.

Table 7-2. Abundance1 of aldehydes detected in the volatile fraction of soymilks Treatments4

Name ID2 KI3 BP Past UHT P1 P2 P3 Hexanal MS, RI, P 1092 122.20 a 143.14 a 200.03 b 123.35 a 141.37 a 192.48 b Pentanal MS, RI, P 990 9.88 a 14.77 a 22.22 b 12.34 a 14.54 a 20.23 b Acetaldeyde MS 4.29 a 4.63 a 4.77 a 2.74 a 4.23 a 4.66 a Benzaldehyde MS, RI 1563 1.86 a 6.36 c 3.10 ab 3.66 b 4.28 b 4.66 bc Propanal MS, RI 804 2.78 a 5.53 b 4.22 ab 4.05 ab 4.00 ab 4.41 ab 2-Butenal MS, RI 1056 1.16 ab 2.01 bc 0.62 a 3.15 c 5.65 d 2.22 bc 3-Methylbutanal MS, RI 929 0.91 a 1.52 ab 1.36 ab 0.93 a 1.23 ab 2.15 b Heptanal MS, RI 1192 2.44 ab 2.64 a 4.37 d 1.43 c 1.59 ab 3.80 d 2-Hexenal MS, RI 1219 1.48 a 2.03 ab 3.87 c 2.14 ab 2.49 ab 2.73 bc 2-Heptanal MS, RI 1345 1.00 a 1.55 ab 2.67 bc 1.34 ab 1.63 abc 2.77 c Nonanal MS, RI 1407 0.91 a 1.21 ab 1.69 b 0.96 ab 1.17 ab 1.54 ab 2,4-Hexadienal MS, RI 1430 0.48 a 0.65 ab 1.02 c 0.63 a 0.73 abc 0.95 bc Octenal MS 0.22 a 0.37 abc 0.72 b 0.31 ac 0.37 abc 0.61 bc 2,4-Heptadienal MS, RI 1520 0.13 a 0.32 bc 0.96 d 0.31 b 0.37 b 0.68 c 2-Pentenal MS, RI 1145 0.90 ab 1.41 c 0.80 ab 0.84 ab 0.97 a 0.59 b Total 150.64 a 188.14 a 252.42 b 158.19 a 184.64 a 244.47 b

a-d Different letters in the same row are significantly different (P < 0.05). 1 Integrated area counts. Mean values x 105. 2 Identification: MS = Mass spectra, RI = Retention index compared to Pherobase database, P = Positively

identified by comparison with authentic standard. 3 Kovats retention index calculated. 4 BP = Base product; Past = Pasteurization; P1 = 200 MPa, 55ºC; P2 = 200 MPa, 75ºC; P3 = 300 MPa, 80ºC.


119

Percentage of hexanal detected related to total volatile compounds in all treated soymilks

varied between 25 and 30%. UHT, 200 MPa, 75ºC and 300 MPa, 80ºC were those

treatments which reached the highest hexanal percentage with values around 30%

compared to 25% of BP. Wilkens and Lin (1970) reported that hexanal comprises about

25% of the total volatile profile in soymilk. On the other hand, Suratman et al. (2004)

found 34%-49% of hexanal related to total volatiles in soymilk added of cyclodextrins and

heated at 95ºC for 15 min. Reported hexanal percentage in soymilk by Achouri et al.

(2008) was between 50 and 66% taking into account a total of 14 volatile compounds

identified in soymilk made from soybeans stored for 10 months. They concluded that

making soymilk from stored soybeans for 3 months, resulted in a product with lower

volatile compounds formed. Thus, controlling process conditions, method of treatment as

well as soybean quality, could help to minimize the hexanal formation and improve

soymilk sensory quality.

Pentanal was the second most detected compound in the aldehydes group (Table 7-2). It

was formed from oxidation of linoleic acid by the catalytic action of lipoxygenase, mainly

in the grinding step of soybeans, when lipoxygenase was still active (Min et al., 2005;

Mizutani & Hashimoto, 2004). The two treatments performed at 200 MPa did not produce

pentanal level differences from untreated soymilk. On the other hand, UHT and 300 MPa

soymilks showed higher values (P < 0.05) than those observed at 200 MPa (Table 7-2).

Minor detected compounds such as heptanal, nonanal and 2-hexenal were also identified

by Wilkens and Lin (1970), Suratman et al. (2004) and Min et al. (2005). These

compounds in soymilk may be related to grassy flavor and oxidized aroma. In general,

results indicated that temperature reached during UHPH treatment, in addition to the

pressure, was decisive in aldehydes formation.

7.2.3 Ketones

Ketone compounds are derived mainly from linoleic acid (Wilkens & Lin, 1970). Table 7-

3 shows the chromatographic areas of ketones in soymilk studied samples. UHT and 300

MPa, 80ºC treatments significantly (P < 0.05) increased total ketones composition while

pasteurization and UHPH treatments at 200 MPa reached equivalent values to BP sample.

The main compound detected was ketone, followed by 2,3-octanedione, 2,3-pentanedione,

2-butanone, 2-heptanone and 1-octen-3-one. Some of these compounds were also

identified by Wilkens and Lin (1970), Boatright and Lei (1999) and Lozano et al. (2007)

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120

on soymilk. As observed in Table 7-3, 2-pentanone, 1-octe-3-one, 2-octanone and 3-

ocatanone, were not affected by treatment applied compared to BP (P ≥ 0.05). UHT

treatment reached higher values of ketone and 2-heptanone and 300 MPa, 80ºC had higher

levels of 2-butanone and 2,3-octanedione compared to the rest of the treatments. UHPH

treatments at 200 MPa did not show significant differences between them and BP sample,

except for acetophenone, 2,3-octanedione and 2,3-pentanedione. The most interesting

compound of the ketones group is 1-octen-3-one. It has been described by many authors to

cause an undesirable flavor in soymilk. It was related to green-beany odor (Wilkens & Lin,

1970) and mushroom aroma (Boatright & Lei, 1999; Lozano et al., 2007). In addition, its

sensory threshold was described to be about 7 µg/mL in heat-treated soymilk (Yuan &

Chang, 2007a).

Table 7-3. Abundance1 of ketones detected in the volatile fraction of soymilks

Treatments4

Name ID2 KI3 BP Past UHT P1 P2 P3 Ketone MS, RI 826 12.05 abc 12.99 bc 25.10 d 9.87 ab 6.95 a 15.91 c2,3-Octanedione MS, RI 1333 3.72 a 5.46 ab 4.36 a 7.34 bc 8.00 c 8.47 c2,3-Pentanedione MS, RI, P 1072 3.77 a 5.17 b 3.30 a 6.39 bc 7.24 c 6.14 bc2-Butanone MS, RI 910 2.30 a 2.83 a 2.68 a 3.57 a 3.05 a 6.14 b2-Heptanone MS, RI 1190 2.07 a 3.85 b 5.63 c 2.59 ab 2.76 ab 3.36 ab1-Octen-3-one MS, RI, P 1314 1.49 a 1.79 a 1.64 a 1.94 a 1.90 a 1.94 a2-Octanone MS, RI 1295 1.02 a 1.51 a 1.30 a 1.25 a 1.27 a 0.99 a2-Pentanone MS, RI 1070 0.62 a 0.87 a 0.95 a 0.78 a 1.22 a 1.00 a3-Octen-2-one MS, RI 1427A 0.54 a 0.64 a 1.20 b 0.64 a 0.67 a 1.30 b3-Octanone MS 0.46 a 0.62 a 0.64 a 0.52 a 0.50 a 0.55 aAcetophenone MS, RI 1693 0.16 a 0.47 bc 0.32 ab 0.41 bc 0.49 bc 0.63 cTotal 28.20 a 36.19 a 47.12 b 35.28 a 34.06 a 46.45 b a-d Different letters in the same row are significantly different (P < 0.05). 1 Integrated area counts. Mean values x 105. 2 Identification: MS = Mass spectra, RI = Retention index compared to Pherobase database and (A)

Kobayashi et al. (1995), P = Positively identified by comparison with authentic standard. 3 Kovats retention index calculated. 4 BP = Base product; Past = Pasteurization; P1 = 200 MPa, 55ºC; P2 = 200 MPa, 75ºC; P3 = 300 MPa, 80ºC.


121

Due to low sensory threshold, hexanal and 1-octen-3-one may compromise sensory quality

of soymilk as described by Yuan and Chang (2007a). Authors reported that a 1-octen-3-

one content was affected by soybean material, heating method, and heating time. They

achieved a drastic reduction of 1-octen-3-one compared to unprocessed soymilk according

to the combination of temperature and time parameters during treatment.

In addition to 1-octen-3-one, acetophenone and 2,3-pentanedione respectively possess

penetrating green and buttery unpleasant odors which may compromise the acceptance of

soymilk (Boatright & Lei, 1999; Lozano et al., 2007). However, in our working conditions,

the levels of this compound were not affected by any treatment applied.

7.2.4 Alcohols

Volatile alcohols were the second most detected group in all treated soymilks, even in

untreated soymilk. Table 7-4 shows the relative abundance area of alcohols for all

soymilks studied. Most of them were associated with green and beany aromas which are

characteristic off-flavors of soymilk. No changes were observed in the total alcohols due to

heat and UHPH treatment taking into account BP sample as reference level, although a

slight decrease was observed in treatments at 200 MPa (P ≥ 0.05). Some compounds were

not affected significantly by the type of treatment: 1-octen-3-ol, (Z)-2-penten-1-ol, 2-

heptanol, 1-octanol, 2-octanol, (Z) 3-hexen-1ol, 3-octanol and 2-ethylhexanol. In general,

treatments, except pasteurization, presented only one significantly different compound (P <

0.05) to the untreated sample. Ethanol was one of them and moreover, it was the most

abundant among this family of compounds. Its origin was reported to be from oxidation of

linolenic acid (Wilkens & Lin, 1970). As shown in Table 7-4, ethanol levels decreased in

all treatments compared to BP and showed specially a stronger reduction in 200 MPa

UHPH treatments. Min et al. (2005) identified ethanol in soymilk samples as the second

most abundant compound detected. On the other hand, Wilkens and Lin (1970) found

ethanol as the least abundant alcohol compound in soymilk. 1-Hexanol and 1-pentanol

were also identified as relevant compounds in the present study in terms of high volatile

contents. Both compounds may be originated from linoleic acid as reported by Wilkens

and Lin (1970) and Kobayashi et al. (1995), although a possible alternative of 1-hexanol

formation could be through hexanal reduction by alcohol dehydrogenase before treatment

(Kakumyan et al., 2009). 1-Hexanol and 1-pentanol were associated to beany and green

odors (Achouri et al., 2008) and 1-hexanol may also be related to harsh grassy and painty

Chapter 7

122

odors (Wang et al., 2001). Therefore, these compounds in soymilk samples may play an

important role in the overall quality aroma of soymilk. UHPH treatment did not affect 1-

hexanol levels as shown in Table 7.4, but pasteurization treatment caused a significant

effect with higher values detected. For 1-pentanol, no significant effect of treatment was

found. Relevant levels of 1-octen-3-ol and 1-penten-3-ol were also identified in the

alcohols group. These compounds were associated respectively to mushroom and pungent

aroma (Lozano et al., 2007) and their formations were linked to the oxidation of linoleic

acid and linolenic acid, respectively (Wilkens & Lin, 1970). All treatments applied did not

affect levels of 1-octen-3-ol, but slight differences, although not significant, were found

between 200 MPa, 55ºC (low values) and 300 MPa, 80ºC (high levels).

Table 7-4. Abundance1 of alcohols detected in the volatile fraction of soymilks

Treatments4

Name ID2 KI3 BP Past UHT P1 P2 P3 Ethanol MS, RI 944 143.76 a 102.99 ab 103.54 ab 65.93 b 63.84 b 133.41 a1-Pentanol MS, RI, P 1260 32.58 a 33.36 a 27.53 a 31.20 a 32.90 a 43.14 b 1-Hexanol MS, RI, P 1359 31.76 a 62.91 b 34.15 a 39.28 a 41.70 a 37.47 a 1-Octen-3-ol MS, RI, P 1454 15.88 a 16.94 a 17.54 a 11.82 a 12.36 a 15.63 a 1-Penten-3-ol MS, RI 1175A 12.72 ab 12.07 ab 13.10 ab 10.64 a 10.86 ab 14.14 b 3-Methyl-1-butanol MS, RI 1260 2.00 ab 4.45 c 1.40 b 2.59 a 2.63 a 3.09 a (Z)-2-penten-1-ol MS, RI 1328 2.52 a 2.77 a 1.72 a 1.88 a 1.93 a 2.44 a 2-Methyl-1-butanol MS, RI 1217 1.88 ab 2.34 b 1.32 a 2.01 ab 2.07 b 2.11 b 1-Butanol MS - 1.05 ab 1.20 ab 1.56 b 0.93 a 1.10 ab 1.42 ab 1-Heptanol MS, RI 1460 0.94 a 1.13 ab 1.34 b 0.93 a 1.01 ab 1.24 ab 2-Heptanol MS, RI 1323 0.73 a 0.99 a 0.83 a 0.77 a 0.80 a 0.78 a 1-Octanol MS, RI 1562 0.73 a 0.71 a 0.69 a 0.74 a 0.63 a 0.71 a 2-Octanol MS, RI 1421 0.66 a 0.86 a 0.73 a 0.71 a 0.73 a 0.63 a (Z)-3-hexen-1-ol MS, RI 1466 0.59 a 0.78 a 0.54 a 0.57 a 0.62 a 0.67 a 3-Octanol MS, RI 1395 0.40 a 0.48 a 0.40 a 0.38 a 0.38 a 0.41 a 2-Ethylhexanol MS, RI 1492 0.40 a 0.27 a 0.66 a 0.59 a 0.61 a 0.88 a (E )-2-penten-1-ol MS - 0.33 ab 0.37 ab 0.42 ab 0.29 a 0.28 a 0.47 b Total 248.92 ab 244.61 ab 207.47 ab 171.26 a 174.46 a 258.62 b

a-c Different letters in the same row are significantly different (P < 0.05). 1 Integrated area counts. Mean values x 105. 2 Identification: MS = Mass spectra, RI = Retention index compared to Pherobase database and (A) Vichi, et

al. (2003), P = Positively identified by comparison with authentic standard. 3 Kovats retention index calculated. 4 BP = Base product; Past = Pasteurization; P1 = 200 MPa, 55ºC; P2 = 200 MPa, 75ºC; P3 = 300 MPa, 80ºC.


123

1-Hexanol, 1-pentanol and 1-octen-3-ol were also identified by Suratman et al. (2004) and

Achouri et al. (2008) and 1-penten-3-ol by Wilkens and Lin (1970) and Lozano et al.

(2007). Volatile compounds identification reported by these authors was carried out in

heat-treated soymilk.

7.2.5 Furans

Generally, furans in soymilk are associated with unpleasant flavor besides being an

indication of color changes due to treatment applied. Furans may be formed by the

oxidation of unsaturated fatty acids or by Maillard reaction products (Achouri et al., 2007;

Yuan & Chang, 2007a). UHT treatment (Table 7-5) caused the most significant effects in

furans composition (P < 0.05). Levels of all compounds increased substantially comparing

those of UHPH treatments, pasteurization and untreated soymilk. The Maillard reaction

which favors furans formation take place when processing at high temperatures, for

instance 121ºC, 143ºC and 154ºC (Kwok & Niranjan, 1995). Processing conditions (time

and temperature) of UHT treatment used in the present study were probably the reason for

the occurrence of browning reactions, contributing to the increase of furan compounds.

Table 7-5. Abundance1 of furans detected in the volatile fraction of soymilks

Treatments4

Name ID2 KI3 BP Past UHT P1 P2 P3 2-Penthyl furan MS, RI, P 1235A 10.61 a 13.96 a 40.89 b 10.97 a 11.96 a 16.36 a2-Ethyl furan MS, RI 965A 6.56 a 9.70 a 35.00 b 6.42 a 6.65 a 11.03 a2-Propyl furan MS, RI 1044 1.22 a 1.58 a 22.11 b 1.42 a 1.54 a 3.17 a 2-Vinyl furan MS, RI 1085 0.74 a 0.75 a 2.98 b 0.71 a 0.68 a 0.98 a 2-n-Butyl furan MS, RI 1135 0.46 a 0.53 a 2.61 b 0.59 a 0.65 a 0.57 a 2-Methyl furan MS, RI 881 0.24 a 0.35 ab 1.25 c 0.26 a 0.28 a 0.46 b Total 19.84 a 26.87 ab 104.83 c 20.36 a 21.77 ab 35.56 b a-c Different letters in the same row are significantly different (P < 0.05). 1 Integrated area counts. Mean values x 105. 2 Identification: MS = Mass spectra, RI = Retention index compared to Pherobase database and (A) Vichi et

al. (2003), P = Positively identified by comparison with authentic standard. 3 Kovats retention index calculated. 4 BP = Base product; Past = Pasteurization; P1 = 200 MPa, 55ºC; P2 = 200 MPa, 75ºC; P3 = 300 MPa, 80ºC.

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Treatments at 200 MPa (Table 7-5) and that processed by pasteurization did not affect

furan composition compared to BP sample. UHT and 300 MPa, 80ºC produced a

significant increase in the levels of 2-methyl furan compared to BP, noticeably reaching

UHT soymilk levels. The most abundant compound detected was 2- penthyl furan which is

the main compound related with grassy and beany off-flavors (Boatright & Lei, 1999; Min

et al., 2005; Achouri et al., 2006). 2-Penthyl furan is formed from linoleic acid by singlet

oxygen that could be obtained by soy riboflavin or by atmospheric air under light (Min et

al., 2005). Moreover, it has a low perception threshold (Yuan & Chang, 2007a). Results

showed that UHPH treatment did not affect levels of 2-penthyl furan which, on the

contrary, occurred in UHT soymilk (P < 0.05).

7.2.6 Esters and Acids

Ester and acid were the minority compounds in the whole volatile profile of soymilks in

this study. In Table 7-6, a total of 3 acids were identified in soymilk: butanoic acid,

pentanoic acid and hexanoic acid. Prevalence of all acids detected increased significantly

due to treatment applied, either heat or UHPH. However, no changes were observed in 200

MPa, 55ºC compared to BP sample. The predominant compound detected was hexanoic

acid. In general, its levels increased in all treatments compared to soymilk BP, with a more

important increase in 300 MPa, 80ºC (P < 0.05). Lozano et al. (2007) found butanoic acid

and hexanoic acid in heat-treated soymilk. These compounds were related to cheese aroma

and sweaty odor, respectively. According to Wilkens and Lin (1970), hexanoic acid is

formed by hexanal oxidation in presence of oxygen which in turn causes a fetid odor.

In the esters group, treatments at 200 MPa caused a significant decrease in the total esters

composition, while esters remained stable in heat treatments and 300 MP, 80ºC compared

to untreated soymilk. Methyl acetate and ethyl acetate were the most abundant compounds

detected (Table 7-6). For methyl acetate, only UHT treatment caused a significant increase

in the levels detected (P < 0.05) and, on the other hand, ethyl acetate decreased

significantly in all treatments applied. Achouri et al. (2006) found ethyl heptanoate, ethyl

octanoate and isoamyl acetate in 6 different commercial soymilks, and Kato, et al. (1981)

found ethyl methanoate and ethyl acetate in roasted soybeans, but in this study only ethyl

acetate was identified. There is no information in the literature about esters formation or

about sensory effect on soymilk, however, they have been related with floral and fruity

flavors in other products.


125

Table 7-6. Abundance1 of esters, acids and others compounds detected in the volatile

fraction of soymilks

Treatments4

Name ID2 KI3 BP Past UHT P1 P2 P3 Esters Ethyl acetate MS, RI 897 6.02 a 4.45 b 3.70 bc 2.84 bc 2.89 bc 4.84 abMethyl acetate MS, RI 839 3.02 abc 3.69 ab 7.33 d 2.33 bc 2.18 c 3.85 an-Hexyl acetate MS, RI 1278 0.41 a 0.38 a 0.48 a 0.46 a 0.46 a 0.45 an-Amyl acetate MS, RI 1178 0.32 a 0.44 ab 0.55 ab 0.70 b 0.33 a 0.42 abTotal 9.95 ab 8.96 a 12.22 b 6.48 c 6.01 c 9.79 abAcids Hexanoic acid MS, RI 1946 24.52 a 35.44 bc 39.38 bc 30.65 ab 36.72 bc 44.46 cPentanoic acid MS, RI 1747 1.84 a 3.63 bc 4.64 c 2.92 ab 3.66 bc 4.39 bcButanoic acid MS, RI 1638 0.14 a 0.32 ab 0.28 ab 0.27 ab 0.35 b 0.33 bTotal 26.50 a 39.39 b 44.30 bc 33.83 ab 40.73 bc 49.18 cOthers Carbon dissulphide MS 1.48 a 1.42 a 0.43 b 1.22 a 1.32 a 1.38 a a-d Different letters in the same row are significantly different (P < 0.05). 1 Integrated area counts. Mean values x 105. 2 Identification: MS = Mass spectra, RI = Retention index compared to Pherobase database, P = Positively

identified by comparison with authentic standard. 3 Kovats retention index calculated. 4 BP = Base product; Past = Pasteurization; P1 = 200 MPa, 55ºC; P2 = 200 MPa, 75ºC; P3 = 300 MPa, 80ºC.

7.2.7 Principal component analysis (PCA)

PCA is a statistical analysis for resolving sets of data into orthogonal components, whose

linear combinations (principal components, PC) approximate the original data to any

desired degree of accuracy. In most cases, two components are sufficient to explain a great

proportion of the variation in the original parameters. Figure 7-1 shows the distribution of

the samples in the PC1 and PC2 according to volatile composition. 38% and 19% of the

variability was explained by PC1 and PC2, respectively. As observed in PC1 there is a

clear separation between drastic treatment conditions and mild treatments. UHT and 300

MPa, 80ºC are located on the negative side of PC1 and treatments at 200 MPa, pasteurized

and untreated soymilks are located on the positive side.

Chap

126

Figur

first p

Rega

two

furan

comp

therm

samp

addit

MPa

value

one

value

relate

aldeh

contr

pter 7

re 7-1. Loadin

principal comp

arding PC1

alcohols (1

ns achieved

pounds com

mal damage

ples are dis

tion, 300 M

a treatments

es in the ald

and 2,3-oc

es in the PC

ed to UHT

hydes, som

rary, benza

ngs plot after p

ponents (PC1

model, sev

-butanol an

d very high

mpared to 30

e was high

stributed al

MPa, 80ºC s

s. Concerni

dehydes gro

tanedione),

C2 (Table

T soymilk.

me ketones

aldehyde, w

principal comp

and PC2).

veral aldehy

nd 1-heptan

h loadings

00 MPa, 80

er in UHT

long the PC

soymilk sho

ing PC2, pr

oup. Some k

some alco

7-7). More

As mentio

and alcoho

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ydes, 3 keton

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(Table 7-7

0ºC. Due to

soymilk th

C1 compon

ows a subtl

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ketones (2,3

ohols and a

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oned above

ols, are rel

reported

sis of the indiv

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han 300 M

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3-pentanedi

all furan co

higher value

e, this clas

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as having

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shows high

parameters

MPa, 80ºC s

ding to trea

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y benzaldeh

ione, acetop

ompounds o

es of all fur

ss of comp

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plane defined

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anoic acids)

her values o

s of UHT tr

soymilk. Th

atment inten

pasteurized

hyde achiev

phenone, 1-

obtained ve

ran compou

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ptanone),

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of these

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unds are

well as

On the

flavour


127

(Boatright & Lei, 1999), shows negative loading in the PC2. This compound is more

representative in 300 MPa, 80ºC treatment.

Differences in the holding time at high temperature and pressure applied during the process

played an important role on the type of compound affected. To support this affirmation,

treatments at low UHPH pressures and temperatures had similar results to pasteurized and

untreated soymilk. Therefore, the real impact of pressure on volatile formation was

associated to the temperature generated during UHPH process as a consequence of inlet

temperature. High holding times at high temperature was more beneficial to volatile

formation than combination of pressure and middle and high temperature at low holding

time.

Table 7-7. Loading and percentage variance accounted by the first two principal

components of soymilk volatile profile.

Principal Component PC1 PC2 Aldehydes 2-Butenal 0.093 -0.149 2-Pentenal 0.062 -0.056 Benzaldehyde -0.038 -0.246 Acetaldeyde -0.072 0.086 Propanal -0.078 -0.195 3-Methylbutanal -0.093 -0.148 Nonanal -0.180 -0.038 2-Heptanal -0.187 -0.078 Heptanal -0.188 0.061 2,4-Hexadienal -0.190 -0.087 Octenal -0.193 -0.045 2-Hexenal -0.194 -0.044 Hexanal -0.197 -0.021 Pentanal -0.198 -0.056 2,4-Heptadienal -0.204 -0.010 Ketones 2,3-Pentanedione 0.065 -0.249 2,3-Octanedione 0.004 -0.255 1-Octen-3-one -0.033 -0.243 2-Octanone -0.039 -0.115 2-Butanone -0.052 -0.200 Acetophenone -0.053 -0.273 2-Pentanone -0.069 -0.178 3-Octanone -0.122 -0.029

Chapter 7

128

Principal Component PC1 PC2 Ketones Ketone -0.176 0.106 3-Octen-2-one -0.180 -0.045 2-Heptanone -0.198 0.009 Alcohols 2-Methyl-1-butanol 0.113 -0.164 3-Methyl-1-butanol 0.069 -0.175 (Z)-3-hexen-1-ol 0.032 -0.078 1-Hexanol 0.017 -0.196 (Z)-2-penten-1-ol -0.002 -0.076 1-Pentanol -0.025 -0.177 Ethanol -0.029 0.128 2-Octanol -0.033 -0.093 3-Octanol -0.042 -0.103 1-Octanol -0.044 -0.017 2-Heptanol -0.061 -0.122 2-Ethylhexanol -0.080 -0.121 1-Octen-3-ol -0.110 0.045 1-Penten-3-ol -0.127 -0.006 (E )-2-penten-1-ol -0.150 -0.053 1-Butanol -0.175 0.019 1-Heptanol -0.189 -0.037 Furans 2-n-Butyl furan -0.178 0.102 2-Ethyl furan -0.181 0.119 2-Propyl furan -0.182 0.122 2-Vinyl furan -0.185 0.116 2-Methyl furan -0.190 0.102 2-Penthyl furan -0.195 0.094 Esters Ethyl acetat 0.027 0.129 n-Hexyl acetat -0.050 -0.044 n-Amyl acetat -0.053 0.009 Methyl acetat -0.169 0.145 Acids Butanoic acid -0.078 -0.245 Hexanoic acid -0.156 -0.186 Pentanoic acid -0.172 -0.145 Ohters Carbon dissulphide 0.139 -0.054 Porcentage of variance explained 38 19


129

7.3 Conclusions

The soymilk aroma profile was characterized primarily by aldehyde and alcohol.

Compounds of these chemical families were the most detected in all treatments applied as

well as untreated soymilk. Furan and ketone compounds were identified in low levels, but

they are not less relevant. In general, the main compounds detected in soymilk by other

authors were also identified in the present study, primarily compounds responsible for

generating off-flavors. On the other hand, not all compounds identified possess a

disagreeable aroma. For example, it is well recognized that benzaldehyde has an almond

aroma, octanol has an odor slightly reminiscent of roses and 2-heptanone has a flowery

odor. Hence, the characteristic aroma of soymilk is not formed by an individual compound,

but by a mixture of them which can be formed in higher or lower levels according to the

treatment applied. Pasteurization and UHPH treatments at 200 MPa produced slight

changes in the volatile profile compared to untreated soymilk. Although UHPH treatment

at 300 MPa, 80ºC inlet temperature achieved similar results to UHT treatment, PCA

analysis indicated higher levels of furans in UHT soymilk. High temperature and high

holding time were the main processing parameters responsible for affecting changes in

volatile profile. Therefore, UHT could be the treatment that produces soymilk with lower

sensory acceptance due to results of furan compounds combined with high levels of

aldehydes, ketones and alcohols chemicals group. On the other hand, UHPH treatment at

300 MPa, 80ºC with low levels of furan compounds could have good sensorial acceptation.

7.4 References




soymilk flavour and quality. International Journal of Food Science & Technology. 43,

82-90.

Achouri, A., Boye, J.I. & Zamani, Y. (2006). Identification of volatile compounds in


759-766.

Chapter 7

130

Boatright, W.L. & Lei, Q. (1999). Compounds contributing to the "beany" odor of aqueous

solutions of soy protein isolates. Journal of Food Science. 64, 667-670.

Hashim, L., & Chaveron, H. (1995). Isolation and identification of off-flavor components

from soy milk. In Developments in Food Science (Edited by, Anonymous), Elsevier,

France. pp. 1007-1019.

Kakumyan, P., Kato, M., Hajika, M. & Matsui, K. (2009). Development of a screening

system for the evaluation of soybean volatiles. Bioscience, Biotechnology, and

Biochemistry. 73, 1844-1848.

Kobayashi, A., Tsuda, Y., Hirata, N., Kubota, K. & Kitamura, K. (1995). Aroma

constituents of soybean [Glycine max (L.) Merril] milk lacking lipoxygenase

isoenzymes. Journal of Agricultural and Food Chemistry. 43, 2449-2452.



Lozano, P.R., Drake, M., Benitez, D. & Cadwallader, K.R. (2007). Instrumental and

sensory characterization of heat-induced odorants in aseptically packaged soy milk.


Min, S., Yu, Y., Yoo, S. & Martin, S.S. (2005). Effect of soybean varieties and growing

locations on the flavor of soymilk. Journal of Food Science. 70, C1-C11.

Mizutani, T. & Hashimoto, H. (2004). Effect of grinding temperature on hydroperoxide

and off-flavor contents during soymilk manufacturing process. Journal of Food

Science. 69, 112-116.

N'Kouka, K.D., Klein, B.P., & Lee, S. (2004). Developing a lexicon for descriptive

analysis of soymilks. Journal of Food Science, 69, 259-263.

Pereda, J., Jaramillo, D.P., Quevedo, J.M., Ferragut, V., Guamis, B. & Trujillo, A.J.

(2008). Characterization of volatile compounds in ultra-high-pressure homogenized

milk. International Dairy Journal. 18, 826-834.


131

Plutowska, B. & Wardencki, W. (2007). Aromagrams – Aromatic profiles in the

appreciation of food quality. Food Chemistry. 101, 845-872.

Suratman, L.L.I., Jeon, I.J. & Schmidt, K.A. (2004). Ability of cyclodextrins to entrap

volatile beany flavor compounds in soymilk. Journal of Food Science. 69, 109-113.

Torres-Penaranda, A.V., & Reitmeier, C.A. (2001). Sensory descriptive analysis of

soymilk. Journal of Food Science, 66, 352-356.

The Pherobase. Database (Kovats-Index) of insect pheromones and semiochemicals.

Accessed June/2012. Available from:

<http://www.pherobase.com/database/kovats/kovats-index.php>.



Wang, Z.H., Dou, J., Macura, D., Durance, T.D. & Nakai, S. (1997). Solid phase extraction

for GC analysis of beany flavours in soymilk. Food Research International. 30, 503-

511.

Wilkens, W.F. & Lin, F.M. (1970). Gas chromatographic and mass spectral analyses of

soybean milk volatiles. Journal of Agricultural and Food Chemistry. 18, 333-336.

Vichi, S., Pizzale, L., Conte, L.S., Buxaderas, S. & lópez-Tamames, E. (2003). Solid-phase

microextraction in the analysis of virgin olive oil volatile fraction: modifications

induced by oxidation and suitable markers of oxidative status. Journal of Agricultural

and Food Chemistry, 51, 6564-6571.

Yuan, S.H. & Chang, K.C. (2007a). Selected odor compounds in cooked soymilk as

affected by soybean materials and direct steam injection. Journal of Food Science. 72,

S481-S486.

Yuan, S.H. & Chang, K.C. (2007b). Selected odor compounds in soymilk as affected by



Chapter 8

Characteristics of refrigerated UHPH-treated soymilks and shelf-life

estimation

135

8

Characteristics of refrigerated UHPH-treated soymilks and shelf-life estimation

8.1 Introduction

The consumption increase experimented by soymilk in the last years is accompanied by a specific segment of consumers due to the healthy characteristics. In fact, in North American markets, in addition to UHT treatment, soymilk is also commercialzed under different conditions of pasteurization to ensure that consumers receive a product with no threat to public health (The Soyfoods Association of America, 1996). Generally, heat-treated soymilks are packaged in bottles that require refrigeration at 4ºC to maintain a shelf-life of about one week (Kwok & Niranjan, 1995). Despite the benefits of the heating process, such as microbial safety and extended shelf-life, UHT processing induces important modifications in sensory characteristics, resulting in changes in color, and loss of nutritive value (Achouri et al., 2007; Lozano et al., 2007). Pasteurization is a thermal processes applied at food industry considered less aggressive to the overall quality parameters of the product. The low temperature applied in pasteurization, compared to UHT and sterilization treatments, is probably the main factor responsible for this advantage. However, the holding time used in the pasteurization process can be decisive in the detrimental effects of nutritional and quality attributes of soymilk. Therefore, the present study aims to evaluate the UHPH effect on changes in quality parameters of soymilk in order to produce a fresh product to be stored at refrigeration. For this purpose, pressure of 200 MPa at 55 and 75ºC were applied on soymilk BP (untreated sample) and compared with pasteurized soymilk (95ºC, 30 s). Samples were kept in bottles at 4ºC to determine shelf-life (see 3.4 condition A). UHPH conditions (200 MPa, 55 and 75ºC) were selected based on microbial and chemical stability of soymilk from a previous study (chapter 5). Microbiological analysis applied included mesophilic aerobic bacteria and spore and enterobacteria counts (see 3.6.1). The determinations applied were: particle size (see 3.9), particle sedimentation (see 3.10 centrifugation and particle migration methods), hydroperoxide index (see 3.7.2), TEM (see 3.11), color (see 3.13), pH (see 3.5), surface hydrophobicity (see 3.12), volatile profile evolution (see 3.14) and sensory analysis (see 3.15).

Chapter 8

136


8.2.1 Microbiological quality and pH measurements

Figure 8-1 shows microbiological results and pH measurements of untreated, UHPH and

pasteurized soymilk during 28 days of storage at 4ºC. Initial counts of mesophilic bacteria

and spores of untreated soymilk were respectively 2.13 log cfu/mL and 1.54 log cfu/mL.

Enterobacteria counts were not detected in any sample (limit detection 1 cfu/mL ) (data not

shown). UHPH treatments at 200 MPa were more effective in the microbial inactivation

than pasteurization treatment, being 75ºC of inlet temperature the most efficient UHPH

condition. Pasteurized soymilk showed an increasing in mesophilic bacteria counts of 4.2

log units from day 1 to day 28, whereas soymilk treated at 200 MPa of pressure and 55 and

75ºC of inlet temperature of just 3.2 and 0.3 log units respectively. During storage,

mesophilic spore counts increased of 2.0 and 0.8 log units respectively for pasteurized and

200 MPa, 55ºC soymilks. In general terms, pasteurized soymilk presented higher bacterial

counts compared to UHPH soymilks. From day 1 to day 14, mesophilic bacteria increased

progressively in pasteurized and 200 MPa, 55ºC soymilks, but beyond this point an

accelerated increase occurred mainly in pasteurized soymilk. In parallel with bacterial

growth, pH values of pasteurized sample decreased considerably after 14 days of storage.

On the last day of measurements, pH reached values about 6.49, indicating a slight

acidification produced by this microbial gowth. UHPH-treated soymilks revealed similar

pH evolution during the period of storage. A decreasing in the pH values was observed

after 21 days of storage, with values on the last day of analysis of 6.73 and 6.77 for 200

MPa, 55 and 75ºC of inlet temperature, respectively.

The marked microbiological growth observed in pasteurized soymilk, was related to the

recovery of health and sub-lethally injured cells during storage or by spore germination

that remained after treatement, while this recovery was not observed in soymilk treated at

200 MPa and 75ºC of inlet temperature. Phenomena, such as cavitation, shear forces and

turbulence take place at high pressure causing destructive stresses of the bacterial cell

(Middelberg, 1995; Donsì et al., 2009).

Shelf-life estimation under refrigeration conditions

137

A

B

C

Figure 8-1. Development of (A) mesophilic aerobic bacteria counts, (B) mesophilic aerobic spores counts

and (C) pH measurements of pasteurized soymilk (-♦-), 200 MPa, 55ºC (--) and 200 MPa, 75ºC soymilks (-

-).

0

1

2

3

4

5

6

7

0 7 14 21 28

Log

coun

ts (c

fu/m

L)

Days

0

1

2

3

4

0 7 14 21 28

Log

coun

ts (c

fu/m

L)

Days

6.5

6.6

6.7

6.8

6.9

0 7 14 21 28

pH

Days

Chapter 8

138

Similar increase of spores counts were observed in milk samples treated in the same UHPH

equipment at 200 MPa, 30ºC and 40ºC (Pereda et al. 2007). However, mesophilic bacteria

growth of that study presented higher counts than the present study with values of 5.4 and

2.3 log cfu/mL, respectively, after 21 days of storage at 4ºC. These differences may be

attributed especially to the inlet temperature applied in each study and to the characteristics

of indigenous microbiota of milk.

8.2.2 Microstructure and hydrophobicity characterization

Native protein organization in untreated soymilk is partially dispersed along of the

interface between fat globules and continuous phase and is also structured in form of

protein bodies (high density particles from seed grinding during soymilk processing).

Depending on the intensity of the homogenization process, better protein dispersion is

obtained in the interface o/w (oil-in-water) and great coverage of the surface of small

particles finely distributed in the continuous phase is obtained. As a result, the soymilk

stability can be improved dramatically.

Surface hydrophobicity measurements are related to the exposure or unmasking of buried

hydrophobic zones of the protein and is measured through fluorescence derived from the

interaction between ANS (8-Anilino-1-naphtalene sulfonic acid) and protein (Bouaouina et

al., 2006; Miriani et al., 2011). This analysis gives information about protein structural

organization from native to unfolding state. The major soy globulin proteins are glycinin

and β-conglycinin and probably are the main fractions affected by the treatment conditions.

During processing, soy proteins can undergo structural changes which may affect the

functional properties, such as solubility and stability. Exposure of the inner regions leads to

protein becoming more active to hydrophobic interactions between protein molecules,

protein-lipids molecules and complexes among protein, micro particles and fat globules by

means of phospholipids (Ono et al., 1996). Results of surface hydrophobicity are shown in

Figure 8-2.


139

Figure 8-2. Protein surface hydrophobicity of BP (―), soymilk pasteurized (―), UHPH-treated soymilk at

200 MPa, 55ºC (―) and 200 MPa, 75ºC (―).

Due to the protein dispersion in the BP sample, most of the protein hydrophobic zones

could be masked for binding with ANS, resulting in lowest fluorescence values. The low

homogenization intensity and the subsequent heat treatment (95ºC, 30 s) of the pasteurized

soymilk, favored the exposition of the inner hydrophobic regions of the soy globulins for

binding to ANS. UHPH treatments resulted in higher fluorescence values compared to

pasteurization treatment and BP samples, indicating high hydrophobicity for UHPH

samples. No relevant differences were observed between UHPH conditions, so a difference

of 20ºC in the inlet temperature was not enough to produce higher fluorescence. However,

the large aggregates and/or native large particles detected by particle size determination

(see chapter 5, Table 5-3) could partially have masked some of these hydrophobic regions,

making them unavailable for hydrophobicity determination. Since for UHPH samples, the

drastic intensity of homogenization linked to the temperature in the high pressure valve

experienced by soymilk, produced strong disruption of large particles, fat globules and

protein aggregates. As a consequence, an efficient exposition of hydrophobic regions of the

protein was obtained, allowing the complex protein-ANS.

Hydrophobic interactions and disulfide (S-S) binding are the main way of complexes

between soy globulins, oil droplets and micro particles (Fukushima, 2001; Floury et al.,

2002; Bouaouina et al., 2005). Surface hydrophobicity of UHPH-treated whey protein

0

50

100

150

200

250

300

350

400 450 500 550

Rel

ativ

e flu

ores

cenc

e

Wavelength (nm)

Chapter 8

140

increased gradually as pressure increased (Bouaouina et al. 2006), showing better

stabilizing properties by the strong increase of hydrophobicity attributed to increased

exposure of buried hydrophobic zones of the protein aggregates. An increasing in the

hydrophobicity of soymilk was also observed by Shimoyamada et al. (2008) during heating

process. Their results were related to the improvement of soymilk stability by the

aggregates formed due to the soy globulins denatured.

Protein solubility is controlled by a delicate balance between repulsive and attractive

intermolecular forces. Although each protein possesses a unique, well-defined structure in

the native state, after drastic treatment such as UHPH or heat treatment, protein changes its

conformation, creating several non specific structures according to the type and extent of

treatment. For instance, β-conglycinin fraction undergoes a supra-molecular rearrangement

involving a re-organization of its quaternary without affecting its tertiary structure (Floury

et al., 2002; Miriani et al., 2011). Disulfide bonds were reported as being responsible for

the preservation of the soy globulin tertiary structure due to high resistance to UHPH

treatment of aqueous soy protein solution as reported by Floury et al. (2002). Therefore,

the results of surface hydrophobicity of UHPH soymilks are indicative that protein

denaturation occurred during UHPH treatment could not be related to loss of solubility, but

on the contrary with better soymilk stability. Evidently, extreme values could indicate

complete protein denaturation which would result in the formation of precipitates. Possible

changes occurred during storage at 4ºC were evaluated by solids sedimentation

(centrifugation and particle migration methods).

Micrographs of BP, pasteurized and UHPH-treated soymilk at 200 MPa, 75ºC are shown in

Figure 8-3. In BP samples (Figure 8-3A), several groups of protein-fat globule aggregates

and fat globules are observed distributed in the continuous phase. Most of fat globules are

located far away to each other and they did not have spherical format as expected. In

addition, a large aggregate of protein-fat globule is observed, masking hydrophobic zones

of the soy protein. The distorted shapes of the oil droplets suggest that interactions between

the aggregated proteins are stronger than surface tension forces (Malaki et al., 2008). For

pasteurized sample (Figure 8-3B) a better distribution of oil droplets and small protein-fat

aggregates could be seen. In this case, the homogenization process and heat treatment

dispersed partially protein in the surface of fat globule favoring the formation of spherical

droplets. On the other hand, large protein aggregates, possibly glycinin and β-conglycinin

fractions, could be seen. The rearrangement of the protein structure caused by the heating


141

process, has unmasked some hydrophobic zones allowing interactions between proteins.

These results are in accordance to that obtained in particle size determination (Table 5-3),

which indicated particles of large volume of pasteurized soymilk. Great spherical fat

globules dispersion, some protein macromolecules and small protein-fat globule aggregate

are observed in UHPH-treated soymilk (Figure 8-3C). Partially unfolded proteins and their

molecular interactions in UHPH-treated soymilk could have resulted in the protrusion of

very small fat globules into a protein aggregate. Therefore, great dispersion of the soy

protein in the surface of oil droplets and micro particles finely distributed in the aqueous

phase was obtained in UHPH treatment. In addition to the reduced particle size (Table 5-

3), a high number of micro particles solubilized in the continuous phase were produced.

Because of this, several remained protein molecules are available for binding with ANS.

Cruz et al. (2007) and Malaki-Nik et al. (2008) also observed small aggregates of protein

and fat globules in homogenized soymilk.

Chapter 8

142

A

B

C

Figure 8-3. Transmission electron micrographs of (A) BP, (B) pasteurized soymilk and (C) UHPH-treated

soymilk at 200 MPa, 75ºC. Different colloidal structures are indicated: (FG) fat globule, (PFA) protein-fat

globule aggregates, (PA) protein aggregates, (P) protein and (AFP) aggregate of fat droplets protruded into

protein.

2 µm

2 µm

2 µm

0.2 µm

PFA

PFA

FG

PFA

FG

PA

0.2 µm

AFP

PFA

0.2 µm

PFA P

FG


143

8.2.3 Physical stability

Soymilk is a water extract of soybeans in form of oil-in-water emulsion. The most relevant

functional property of proteins in this system is to cover oil droplets of the lipid fraction for

maintaining a good dispersibility of those in the continuous phase during storage. Thus,

soy proteins make soymilk to be a very stable emulsion. In spite of this, creaming of oil

droplets and sedimentation of solid particles are the primary mode of destabilization of

vegetable beverages. Both phenomena are dependent to a great extent on particle size

distribution (Durand et al., 2003; Poliseli-Scopel et al., 2012).

Particles in soymilk include not only fat globules, but also small particles, such as protein

bodies, protein aggregates and even protein-fat globule and globule aggregates (Cruz et al.,

2007). Untreated and pasteurized soymilk presented highest values of particle size

parameters (Table 5-3, chapter 5), indicating that conventional homogenization at low

pressure (18 MPa) applied before heat treatment did not produce an additional decrease

compared to the colloidal mill in the grinding step of soymilk elaboration. On the other

hand, pasteurization with single effect homogenization used in this study was not enough

to disperse aggregates formed into small particles. The dairy industries commonly apply

double effect homogenization to avoid this phenomenon (Poliseli-Scopel et al., 2012).

Depending on the heat conditions, glycinin and β-conglycinin, major globulin fractions of

soy protein are able to form large aggregates (Tay et al., 2005). As expected, UHPH-

treated soymilk showed higher reduction in the particle size parameters compared to

pasteurized soymilk. According to the parameters evaluated, no significant differences

were observed in particle size between UHPH soymilks (Table 5-3) in spite of inlet

temperature and pressure. Previous results of particle size were supported by particle

sedimentation through low-speed centrifugation during 28 days of storage (Table 8-1).

Particle sedimentation measured by centrifugation is indirectly related to the stability of the

system. Under the same conditions, centrifugation was applied to all samples, forcing big

particles and aggregates to separate from the bulk, either to the top, in the case of fat

globules, or to the bottom, in the case of solid particles. This analysis can be considered as

indicative of the sedimentation potential of soymilk during long storage periods and

especially as a comparative measurement among treatments applied. Pasteurized soymilk

presented higher amount of solids settled by centrifugation than UHPH soymilks

throughout 28 days of storage (Table 8-1). As storage time increased, a slight increase in

Chapter 8

144

the percentage of sediments was observed, and differences (P < 0.05) were especially

marked from 14 days of storage. Solids sedimentation values in UHPH samples were very

reduced and only slight increase was (P < 0.05) observed at day 28.

Single effect homogenization and intensity of the pressure (18 MPa) before heating was

not able to reduce solid particles size for providing enough stability of the product during

28 days, revealed by solids accumulation in the bottom of the bottle. On the contrary, the

state of particle dispersion in the UHPH-treated soymilks was sufficiently intense to resist

the centrifugal force producing low solids sedimentation (Table 8-1). Similar results were

previously described in chapter 5. Applying similar centrifugation method of particle

sedimentation in soymilk heated (70 to 115ºC), Shimoyamada et al. (2008) observed that

the interaction between soy globulin fractions played an important role in the colloidal

stability, mainly at high temperatures. They reported that the combination of denatured β-

conglycinin and native glycinin caused lower dispersion stability of soymilk, whereas the

combination of denatured β-conglycinin and denatured glycinin increased the stability.

Table 8-1. Solids sedimentation1 of treated soymilks during refrigerated storage.

Treatment Storage day 1 7 14 21 28 BP 5.97 ± 0.47a Pasteurized 3.68 ± 0.17bx 3.83 ± 0.12axy 4.01 ± 0.09ay 3.71 ± 0.08ax 3.96 ± 0.08ay

200 MPa 55ºC 1.34 ± 0.10cx 1.36 ± 0.13bx 1.53 ± 0.16bx 1.48 ± 0.19bx 1.86 ± 0.41by

200 MPa 75ºC 1.19 ± 0.05dx 1.18 ± 0.11cx 1.34 ± 0.06cx 1.28 ± 0.04cx 1.95 ± 0.41by

a-d Different superscript in the same column are significantly different (P < 0.05). x-y Different superscript in the same row are significantly different (P < 0.05). 1 Mean values ± SD (g/100g w/w) of solids sedimentation after low-speed centrifugation.

To evaluate the real impact of the treatment and its influence on the colloidal system

stability, particles migration along the bottle containing the soymilk sample was performed

in a TurbiscanR equipment. This system measure the percentage of backscattered light

which depend on the particle size and particle concentration. In such a way, it can be

detected destabilization phenomena such as aggregation, sedimentation and creaming;

depending on what part of the bottle the backscattered light is produced. Results of

percentage of light backscattered in the bottom of the analysis tube (from 1.1 to 2.0 mm)


145

are shown in Figure 8-4. An increase in backscattering intensity indicates that particle size

and/or particle concentration has increased, while a decrease in backscattering intensity

indicates a decrease in particle size and/or particle concentration.

All samples including untreated soymilk (BP) increased the percentage of backscattering

over time. BP and pasteurized soymilks showed the most pronounced increase, mainly in

the first 7 days of storage when an accelerated rate was observed. Beyond this point, % of

backscattering of these samples increased at a slow rate.

Figure 8-4. Percentage of backscattering in the bottom of the tube of soymilk BP (-♦-), pasteurized soymilk

(--), soymilk UHPH treated at 200 MPa, 55ºC (--) and 200 MPa, 75ºC (-♦-) during storage at 4ºC.

The difference in backscattering from day 1 to day 28 for the BP and pasteurized soymilks

was 18 and 13% respectively, indicating, as expected high concentration of particles in the

bottom of BP samples. In the same way, backscattering values of pasteurized soymilk was,

respectively 10 and 15% higher than UHPH soymilk in the first and last day of analysis.

On the other hand, UHPH soymilks showed a difference between first and last day of

analysis of just 7.6%, indicating low particle concentration at the bottom of the analysis

tube which resulted in higher stability of the soymilk during storage than pasteurized and

BP samples. UHPH conditions presented a similar increase of backscattering at slow rate

on all days analyzed. These results were supported by centrifugation and particle size

parameters. Durand et al. (2003) investigated stability of milk of different vegetable

beverages, including soymilk. All samples were treated by commercial UHT process and

0

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60

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0 7 14 21 28

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Chapter 8

146

changes in backscattering intensity were analyzed during 12 h of measurements. They

observed that milk and soymilk samples did not exhibit sedimentation phenomenon, but

slight amount of creaming in the surface of the sample. Creaming phenomenon was

attributed to the tendency of oil droplets to coalesce by the large particles in the floating

portion at the top of the tube. On the contrary, the present study revealed different levels of

sedimentation of untreated and treated soymilks analyzed for 28 days as mentioned above.

Figure 8-5 shows ∆B (backscattering difference calculated of each measurement taking

into account the first one as reference) versus sample length of the tube for BP, pasteurized

and UHPH-treated soymilks during the different storage periods. The increase in ∆B at the

bottom of the tube (between 0 mm and 5 mm) indicates an increasing in particle

concentration and thus, indicative of the sedimentation phenomenon. The strong decrease

of particle size as a consequence of UHPH treatment (chapter 5), have allowed particle

migration along the tube with discrete particle concentration at the bottom, which was not

visually perceptible. Creaming phenomenon of emulsion destabilization is identified by an

increasing in ∆B at the top of the tube (near 40 mm), indicating the migration of the oil

droplets due to the density difference between continuous and disperse phases. As

observed in the figure no creaming was detected in any of the treated soymilks. Straight

line at zero ∆B across the sample length indicates stable sample in which no changes

occurred. UHPH-treated soymilk exhibited lower variation in the straight line compared to

pasteurized treatment, indicating higher stability of the sample.

A

B

C

Figure 8-5. Delta backscattering for (A) BP, (B) pasteurized soymilk and (C) UHPH-treated soymilk at 200 MPa, 75ºC for 28 days of storage.

Chapter 8

148

8.2.4 Chemical stability

Hidroperoxide formation in processed foods causes several deterioration reactions which

negatively affect quality and storage life of food products. Stability reduction, changes in

color parameters and formation of off-flavors are the main undesirable modifications

which affect soymilk and its consumer acceptance (Gray & Monahan, 1992; Hornero-

Méndez et al., 2001). Determination of hydroperoxide index in soymilk allows the

evaluation of the initial stages of oxidation and its evolution through time. As a

consequence, high number of non-volatile and volatile secondary compounds are

originated, affecting the quality of soymilk. Results of hydroperoxides formation are

shown in Table 8-2.

Table 8-2. Hydroperoxide index values1 of untreated and treated soymilks

Treatment Storage day 1 7 14 21 28 BP 0.38 ± 0.02a Pasteurized 0.48 ± 0.02xb 0.77 ± 0.03ya 0.82 ± 0.03yza 0.79 ± 0.07ya 0.92 ± 0.10za

200 MPa 55ºC 0.43 ± 0.01xc 0.51 ± 0.02yb 0.54 ± 0.03yb 0.56 ± 0.03yzb 0.63 ± 0.09zb

200 MPa 75ºC 0.44 ± 0.01xc 0.44 ± 0.01xc 0.47 ± 0.02xc 0.66 ± 0.03yc 0.66 ± 0.04yb

a-c Different superscript in the same column are significantly different (P < 0.05). x-z Different superscript in the same row are significantly different (P < 0.05). 1 Mean values ± SD (meq peroxide/L sample) of hydroperoxides.

Results showed slight increases of the hydroperoxide index for all treated soymilks during

storage (P < 0.05), being pasteurized soymilk which presented highest values during

storage. Pasteurized soymilk showed an accelerated increase in the first 7 days of storage

and remained fairly constant during the rest of the period of analysis, whereas UHPH

treatments presented a slow rate of hydroperoxides formation. Although significant

differences were obtained between UHPH treatments, its evolution was quite similar

during storage time, reaching equivalent values on the last day of analysis (P ≥ 0.05).

Similar results were obtained by Pereda et al. (2008) who did not observe relevant

differences between day 1 and day 18 of cold storage in milk pasteurized and treated at 200

MPa, 40ºC.


149

Secondary compounds formed by the oxidative process are commonly involved in

unpleasant quality changes due to the generation of aldehydes, ketones, alcohols and furans

compounds. Some of these chemical families are primarily derived from lipoxygenase

action on unsaturated fatty acids as described in chapter 7 and most of them are part of the

volatile profile of foods.

A

B

Figure 8-6. Evolution of (A) Total volatiles compounds and (B) percent ratio of hexanal to total volatiles of

soymilk ( ) BP, ( ) pasteurized, ( ) 200 MPa at 55ºC and ( ) 200 MPa at 75ºC over a period of 28 days

at 4ºC.

0

10

20

30

40

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60

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1 28

Tota

l vol

atile

s pr

ofile

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are

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106

Days

aba

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Chapter 8

150

Special interest has hexanal, since it is a characteristic compound related to secondary

oxidation. Pasteurization and UHPH effect on total volatiles profile of soymilk during 28

days of storage at 4ºC is shown in Figure 8-6A. Significant changes were observed after

pasteurization treatment compared to BP, and no changes were observed during the rest of

storage. No significant differences were observed in the volatile profile between UHPH

soymilk samples just after treatments. However, 200 MPa, 55ºC UHPH-treated soymilk

showed a significant increase on the last day of storage while 200 MPa, 75ºC treated

sample remained invariable.

Achouri et al. (2007) treated 3 different soymilk formulations at 142ºC for 4 s and found

results of volatile profile fairly stable in two of them between 1 and 4 weeks of storage at

4ºC. Although they applied drastic thermal treatment, similar results were found in the

present study in the same period of evaluation.

In base to found results, 28 days of storage at 4ºC did not produce important changes in

hydroperoxide index and total volatiles compounds which could cause perceptible sensory

modifications, primarily for 200 MPa, 75ºC by the total volatile results (Figure 8.6A). The

high levels of hydropexides and secondary products of pasteurized soymilk is an indication

of high oxidation rate compared to UHPH soymilks. Probably these oxidation levels could

be linked to the holding time and temperature applied in pasteurization process.

As described in chapter 7, hexanal was the main compound formed as a consequence of

lipid oxidation among all chemical families identified. Due to the large impact on the

flavor of soymilk by its low threshold and by its high detection, hexanal is commonly used

as an indicator of secondary products formed during the oxidation process (Plutowska &

Wardencki, 2007; Yuan & Chang, 2007). Figure 8-6B shows the ratio of hexanal to total

volatiles of untreated and treated soymilk. UHPH treatment caused significant increase of

hexanal compared to pasteurization treatment (P < 0.05) in the first day of analysis.

However, on the last day of storage an important decrease was observed in UHPH

soymilks, whereas in pasteurized soymilk increased considerably (P < 0.05). Hexanal

compound can be degraded in presence of oxygen to render carboxylic acid (Wilkens &

Lin, 1970) and it can be reduced to n-hexanol (Kakumyan et al., 2009).

The dramatic reduction in fat globules size and the increase observed in surface

hydrophobicity in UHPH-treated soymilks is indicative, as mentined above of a well

protected surface of oil droplets. The new surface of oil droplets formed as a consequence

of the UHPH treatment could be covered by protein through hydrophobic zones exposed

(Dunkley et al., 1962; Huppertz & Kelly, 2006). Due to protein rearrangement,


151

phospholipids were partially transferred to the aqueous phase, but some of them were

retained in the particles for binding among protein and lipids molecules (Ono et al., 1996).

The new interface formed could have caused a protective effect of the proteins against lipid

oxidation. Therefore, a low oxidation rate of UHPH soymilks took place during storage

compared to pasteurized soymilk. For UHPH-treated milk samples, Hayes et al. (2005)

suggested that at higher pressures there would be more exposed fat interface allowing a

great proportion of casein to be adsorbed on the fat globule. The protective caseins at the

surface of fat globule in milk treated at 200 MPa was the reason for the low

hydroperoxides and hexanal values obtained in the study reported by Pereda et al. (2008).

8.2.5 Sensory analysis

Sensory analysis applied in this study included the main attributes relevant to a global

perception of the product characteristics. It is a subjective analysis that can be related to

instrumental color evaluation and volatile compounds detection. L* (luminosity) and b*

(yellow-blue) of color evaluation are the main parameters that indicate darkness/whiteness

and yellowness, respectively, which are visually perceptible for a judges panel. On the

other hand, volatile compounds can have an impact on sensory quality of soymilk due to

the off-flavors generated by the compounds. Some of them are: hexanal (beany and

grassy), 1-hexanol (beany, grassy), 1-pentanol (grassy), hexanoic acid (sweaty), 1-octen-3-

ol (mushroom), 2-penthyl furan (beany and grassy), pentanal (buttery), 2,3-pentanedione

(buttery), benzaldehyde (almond) and 1-octen-3 one (beany and grassy) (Wang et al.,

1997; Boatright & Lei, 1999; Lozano et al., 2007).

Sensory analysis of treated soymilk was carried out after 15 days of storage at 4ºC using

two different methods of evaluation: triangular and descriptive testing. The first one, a

triangular test was used to identify possible differences between treated soymilks.

According to results, 82% of the judges were not able to detect any difference between

UHPH soymilks and pasteurized soymilk. The second part, descriptive test was performed

to determine the most relevant attributes in soymilk by panelist, such as beany flavor,

grassy aroma, oxidized aroma, astringent mouthfeel, thickness, yellowness and darkness.

Results of panelist evaluation are shown in Figure 8-7. Pasteurized soymilk were identified

by judges as containing higher notes of grassy and oxidized aroma while soymilk treated at

200 MPa, 55ºC showed higher notes of beany and similar notes of grassy. On the other

hand, 200 MPa, 75ºC soymilk obtained lower notes of these attributes.

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152

Figure 8-7. Effect on sensory attributes of soymilk pasteurized (-♦-), UHPH-treated soymilk at 200 MPa,

55ºC (--) and 200 MPa, 75ºC (--).

Beany flavor, grassy and oxidized aroma can be considered the main off-flavors in soymilk

(N'Kouka et al., 2004). These off-flavors perceived by the judges were the consequence of

chemical changes that occurred due to the oxidative process, generating secondary

compounds during elaboration, treatment and storage of soymilk (see chapter 7). Table 8-3

lists the main volatile compounds detected at day 1 and day 28 of BP and treated soymilks.

All compounds listed are considered as the main volatile compounds identified in soymilk

by the sensory impact and on the other hand by the total levels detected (see chapter 7).

Among of compounds detected, hexanal and ethanol were the most abundant, followed by

1-hexanol and 1-pentanol. UHPH-treated soymilk showed a significant decrease in hexanal

detection during storage while pasteurized soymilk remained stable. 1-Octen-3-one,

benzaldehyde and 1-pentanol did not experience modification due to treatment applied and

period of storage. 2-Penthyl furan increased significantly after pasteurization treatment,

however decreased during storage (P < 0.05), reaching similar levels to UHPH soymilks at

day 28 of storage. 1-Hexanol and ethanol increased in UHPH soymilks during storage,

whereas a slight decrease was observed in pasteurized soymilk. On the other hand,

hexanoic acid increased in pasteurized soymilk while it decreased in UHPH soymilks. In

general, inlet temperature of UHPH soymilks did not exhibit significant differences with

similar tendency during storage and levels for all volatile compounds.

0.0

1.0

2.0

3.0

4.0Beany

Grassy

Oxidized

AstringencyThickness

Yellowness

Darkness


153

Table 8-3. Main volatile compounds1 detected in untreated and treated soymilks

Compound ID2 Base Pasteurization 200 MPa 55ºC 200 MPa 75ºC

product 1 28 1 28 1 28Hexanal ABC 113.93 x 146.53 abx 165.30 a 123.35 bx 89.96 c 141.37 abx 83.31 cEthanol AB 131.20 x 108.40 ax 90.09 a 65.93 ay 121.25 a 63.84 by 114.14 a1-Hexanol ABC 39.52 x 71.00 ax 47.35 a 39.28 ax 78.44 b 41.70 ax 63.45 a 1-Pentanol ABC 35.25 x 47.73 ax 41.61 a 31.20 ax 41.84 a 32.90 ax 35.18 aHexanoic acid AB 27.29 x 37.22 aby 44.09 a 30.65 bcx 21.59 c 36.72 aby 23.39 c1-Octen-3-ol ABC 15.40 x 18.45 ax 16.38 a 11.82 by 16.80 a 12.36 bx 11.92 b2-Penthyl furan ABC 10.06 x 16.96 ay 13.79 a 10.97 bx 15.65 a 11.96 bx 13.38 aPentanal ABC 10.19 x 16.05 ay 18.81 b 12.34 cx 9.20 d 14.54 bcy 9.80 dPropanal A 3.34 x 7.06 ay 5.46 a 4.05 bx 4.49 b 4.00 bx 5.39 a2,3-Pentanedione ABC 4.13 x 5.64 abx 5.44 ab 6.40 cbx 4.78 cd 7.24 cy 4.10 dBenzaldehyde AB 2.27 x 5.47 ay 5.30 a 3.66 ax 7.05 a 4.28 ax 4.34 a2,3-Octanedione AB 4.23 x 4.70 abx 6.03 ac 7.34 dcy 4.06 b 8.00 dy 3.47 b2-Butanone AB 2.30 x 3.08 ax 2.24 a 3.57 bx 2.69 a 3.05 ax 2.14 b1-Octen-3-one ABC 1.97 x 2.32 ax 2.15 ax 1.94 ax 1.97 ax 1.90 ax 1.89 ax a-d Different letters in the same row of treated samples are significantly different (P < 0.05). x-y Different letters in the same row of treated sample at day 1 are significantly different from BP (P < 0.05). 1 Integrated area counts. Mean values x 105. 2 Identification: A = Mass spectra, B = Retention index compared to literature database, C = Positively

identified by comparison with authentic standard.

The compounds of interest mentioned above, beany and grassy are, in general, the main

sensory attributes associated to unpleasant flavors, although benzaldehyde confers a

desirable almond flavor. On the other hand, sensory threshold plays an important role in

the judges’ perception. Hexanal, 1-octen-3-one and 2-penthyl furan were reported to

possess low sensory threshold, with hexanal being the most perceptible. Content of around

25 µg/kg allows its detection (Hashim & Chaveron, 1995). Considering results of volatile

compounds and specifically compounds of interest, pasteurized soymilk should have had

the highest notes of beany, grassy and oxidized attributes, mainly by the thermal effect of

the treatment on the oxidation (hydroperoxides and volatile compounds). However, 200

MPa, 55ºC soymilk reached the highest note of beany and similar values of grassy for

pasteurized soymilk. Results of astringent mouthfeel may be associated to non-volatile

compounds such as phenolic acids and isoflavones (Min et al., 2005) which are minor

Chapter 8

154

components well known as being present in soybeans (Murkies et al., 1998; Rochfort &

Panozzo, 2007). Thickness may be associated to the homogenization intensity in the

processing. Because of that, it was expected that soymilks treated at 200 MPa (55 and

75ºC) had similar notes of thickness, instead of pasteurized soymilk. However, sensory

results showed that UHPH treatment at 55ºC of inlet temperature obtained similar notes of

pasteurized soymilk. Standardization of measurements and subjectivity of panelists were

possible problems affecting results of sensory analysis, although 3 training sections were

applied to the judges. On the other hand, soymilk is a complex of proteins, lipids and

carbohydrates combined with several micro components, which make soymilk a difficult

matrix for an objective evaluation of the relation between sensorial analysis and volatile

profile. Similar observations were reported by Torres-Penaranda and Reitmeier (2001) who

carried out a sensory descriptive analysis of soymilk-heat treated samples and commercial

soymilk samples.

A study carried out by Lozano et al. (2007) with heat-treated soymilk by different

conditions of time and temperature (143ºC 14 s, 143ºC 59 s, 154ºC 24 s) and obtained high

notes of astringent mouthfeel in treated samples and high notes of beany flavor as

temperature and holding time increased (P ≥ 0.05). In the present study, the trend of beany

formation due to an increase of processing temperature was not detected. UHPH treatment

at 200 MPa and 75ºC of inlet temperature achieved approximately 116ºC in the

homogenization valve while 55ºC of inlet temperature achieved 105ºC, both temperatures

for 0.7 s. Thus, inlet temperature used in the UHPH treatment did not affect sensory

results, but chemical changes occurring during storage, sample manipulation and panelist

subjectivity were probably the reasons for the slight differences observed in beany, grassy

and oxidized results among UHPH treatments.

Color notes suggested that UHPH soymilks were darker than pasteurized soymilk, and

yellowness achieved equivalent notes among all treated soymilks. Table 8-4 shows results

of color parameters, L*, a*, b* and ΔE of BP and treated soymilks. The first attribute of

color, lightness value (L*), is associated with luminous intensity which described light-

reflecting or transmitting capacity of an object (Kwok et al., 1999). UHPH treatments

caused a significant decrease in the L* parameter after treatment indicating a slight

darkening compared to pasteurized and BP soymilks. Similar results were observed by

sensory analysis. During storage, L* values of UHPH treatments remained stable, except

200 MPa, 55ºC which showed an increasing beyond 21 days. On the contrary, pasteurized


155

soymilk showed similar L* values to BP and remained fairly stable during the period of

storage.

Table 8-4. Color parameters1 of untreated and treated soymilks

Treatment Day L* a* b* ΔEBP

BP 1 85.03 ± 0.26ax -0.39 ± 0.43x 14.43 ± 0.54x - Pasteurized 1 85.23 ± 0.07ax 0.01 ± 0.43ax 14.34 ± 0.27ax 0.64 ± 0.26a

7 85.24 ± 0.08a 0.01 ± 0.42a 14.22 ± 0.14a 0.60 ± 0.25a

14 85.39 ± 0.25a -0.87 ± 0.27b 13.39 ± 0.24b 1.25 ± 0.23b

21 85.42 ± 0.14a -0.94 ± 0.62b 13.39 ± 0.24b 1.45 ± 0.15b

28 85.38 ± 0.30a -0.77 ± 0.28b 13.18 ± 0.63b 1.44 ± 0.48b

200MPa 55ºC 1 81.00 ± 0.27ay -2.88 ± 0.05ay 9.78 ± 0.29ay 6.66 ± 0.37ab

7 80.50 ± 0.38a -2.87 ± 0.06a 9.26 ± 0.18b 7.31 ± 0.34a

14 80.77 ± 0.24a -2.70 ± 0.32a 9.21 ± 0.18b 7.14 ± 0.20a

21 82.41 ± 0.72b -2.57 ± 0.12a 9.74 ± 0.44ab 5.81 ± 0.72bc

28 82.45 ± 0.55b -1.85 ± 0.57b 10.15 ± 0.74a 5.24 ± 0.87c

200MPa 75ºC 1 80.36 ± 0.38ay -2.68 ± 0.19ay 9.65 ± 0.26ay 7.27 ± 0.17a

7 80.64 ± 0.56a -3.11 ± 0.50a 9.65 ± 0.26a 7.15 ± 0.24a

14 80.16 ± 0.10a -2.80 ± 0.25a 9.31 ± 0.29a 7.47 ± 0.20a

21 80.77 ± 0.24a -2.88 ± 0.21a 9.17 ± 0.11a 7.22 ± 0.13a

28 81.33 ± 0.43a -2.80 ± 0.47a 9.28 ± 0.23a 6.94 ± 0.27a

1 Mean values ± SD of color parameters. ΔE was calculated taking into account BP as reference sample. a-c Different superscript in the same column of treated samples are significantly different (P < 0.05). x-y Different superscript in the same column between treatments at day 1 are significantly different (P < 0.05).

Particle distribution and concentration (droplet characteristics) of UHPH and pasteurized

soymilks were different due to the new interface of the soymilk dispersion caused by the

treatment conditions. Thereby, pasteurized soymilk showed similar lightness to BP instead

of UHPH samples. For a* (red-green) parameter, a significant decrease was obtained after

both UHPH treatments compared to BP, indicating a small trend towards green color.

However, a* values remained stable during the period of storage, except for 200 MPa,

55ºC in which a significant increase was observed on the last day of analysis. No important

modifications were experimented in pasteurized soymilk during 28 days compared to BP.

UHPH treatment caused a significant decrease in the b* (yellow-blue) values on the first

day of analysis. As observed for L* and a* parameters, b* values did not exhibit changes

due to storage conditions. Although b* values indicated loss of yellow color in UHPH

soymilks, the judges’ evaluation did not appreciate relevant changes.

Chapter 8

156

The ΔE parameter is a single value which takes into account differences between L*, a*,

and b* of the sample and standard. The ΔE values were calculated taking into account BP

as reference sample. As shown in Table 8-4, color difference (ΔE values) was more

important for UHPH treatments than for pasteurized soymilk. These differences may be

attributed primarily to the L* parameter contribution due to treatment applied. Similar

results of L* and ΔE were obtained by Cruz et al. (2007) for soymilk treated by UHPH at

200 MPa and 40ºC of inlet temperature. Achouri et al. (2007) reported a significant

increase in the ΔE values after 21 days of storage at 4ºC of different blends of soymilk

treated at 142ºC for 4 s (UHT).

In the last part of sensory analysis, preference test was applied using hedonic scale of 9

categories. Results indicated that soymilk treated at 200 MPa, 55ºC was the most accepted

sample for about 67% of the judges (P < 0.05). Unexpectedly, soymilk treated at 200 MPa

and 55ºC of inlet temperature was selected by the judges as the best soymilk, even though

it achieved high notes of beany off-flavor. These results demonstrate the subjectivity of the

judges in the sensory analysis, indicating that new training sections and sensory analyses

should be performed to achieve reliable results. Nevertheless, these results gave a global

idea of sensory evaluation and show a tendency towards UHPH processing being able to

produce soymilk with improved sensory qualities.

8.3. Conclusions

The study carried out in this chapter has shown that UHPH-treated soymilks achieved high

microbial inactivation compared to pasteurization treatment during 28 days of storage at

4ºC, being 200 MPa, 75ºC the most stable microbiologically. UHPH soymilk was

characterized by great exposure of hydrophobic zones of the protein, which helped to

improve the protein dispersion in the interface o/w observed by the oil droplets distribution

in the micrographs. As a result, high colloidal stability was detected for UHPH samples

showing low solids sedimentation during storage. Physical and chemical aspects evaluated

of UHPH-treated samples during storage showed, on the other hand, high color stability

and high stability against initial stages of oxidation. Considering hexanal compound as

indicator of secondary oxidation product, an important decrease was detected in the same

period of storage compared to pasteurized sample. In base to panel opinion, UHPH

treatment did not affect overall soymilk characteristics and in addition, soymilk achieved

better sensory acceptance than pasteurized soymilk, especially at 200 MPa, 55ºC.


157

Therefore, UHPH technology was able to produce soymilk as fresh product stored under

refrigeration conditions microbiologically and physically stable and with better sensorial

acceptance than pasteurization treatment.

8.4 References



Boatright, W.L., & Lei, Q. (1999). Compounds contributing to the "beany" odor of

aqueous solutions of soy protein isolates. Journal of Food Science. 64, 667-670.



Journal. 16, 275-284.




Donsì, F., Ferrari, G., & Maresca, P. (2009). High-pressure homogenization for food


Barbosa-Cánovas, Alan Mortimer, David Lineback, Walter Spiess, Ken Buckle, &

Paul Colonna), Academic Press, USA. pp. 309-352.

Dunkley, W.L., Franklin, J.D. & Pangborn, R.M. (1962). Influence of homogenization,

copper, and ascorbic acid on light-activated flavor in milk. Journal of Dairy Science.

45, 1040-1044.

Durand, A., Franks, G.V. & Hosken, R.W. (2003). Particle sizes and stability of UHT

bovine, cereal and grain milks. Food Hydrocolloids. 17, 671-678.




Chapter 8

158



Gray, J.I., & Monahan, F.J. (1992). Measurement of lipid oxidation in meat and meat

products. Trends in Food Science & Technology. 3, 315-319.

Hashim,L., & Chaveron, H. (1995). Isolation and identification of off-flavor components


France. pp. 1007-1019.

Hayes, M.G., Fox, P.F., & Kelly, A. L. (2005). Potential applications of high pressure





1155.

Huppertz, T. , & Kelly, A. L. 2006. Physical chemistry of milk fat globules. In Advanced

Dairy Chemistry, Volume 2: Lipids (Edited by, P. F. Fox, & P. L. H. McSweeney),

Springer US, USA. pp. 173-212.




Kwok, K.C., MacDougall, D.B. & Niranjan, K. (1999). Reaction kinetics of heat-induced

colour changes in soymilk. Journal of Food Engineering. 40, 15-20.







159

Malaki-Nik, A., Tosh, S., Poysa, V., Woodrow, L. & Corredig, M. (2008).



Middelberg, A.J. (1995). Process-scale disruption of microorganisms. Biotechnology

Advances. 13, 491-551.

Miriani, M., Keerati-u-rai, M., Corredig, M., Iametti, S. & Bonomi, F. (2011).

Denaturation of soy proteins in solution and at the oil–water interface: A fluorescence

study. Food Hydrocolloids. 25, 620-626.

Murkies, A.L., Wilcox, G. & Davis, S.R. (1998). Phytoestrogens. Journal of Clinical

Endocrinology Metabolism. 83, 297-303.

N'Kouka, K.D., Klein, B.P. & Lee, S.-. (2004). Developing a lexicon for descriptive

analysis of soymilks. Journal of Food Science. 69, 259-263.

Ono, T., Takeda, M. & Shuntang, G. (1996). Interaction of protein particles with lipids in

soybean milk. Bioscience, Biotechnology, and Biochemistry. 60, 1165-1169.




7125-7130.







Comparison of ultra high pressure homogenization and conventional thermal

treatments on the microbiological, physical and chemical quality of soymilk. LWT -

Food Science and Technology. 46, 42-48.

Chapter 8

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Rochfort, S., & Panozzo, J. (2007). Phytochemicals for health, the role of pulses. Journal

of Agricultural and Food Chemistry. 55, 7981-7994.






The Soyfoods Association of America (1996). Voluntary standards for the composition

and labeling of soymilk in the Unitated States. Acessed September/2012. Available

from: http://www.soyfoods.org/wp-content/uploads/2006/11/smstandards.pdf



Wang, Z.H., Dou, J., Macura, D., Durance, T.D. & Nakai, S. (1997). Solid phase extraction

for GC analysis of beany flavours in soymilk. Food Research International. 30, 503-

511.



Yuan, S.H., & Chang, S.K.C. (2007). Selected odor compounds in cooked soymilk as

affected by soybean materials and direct steam injection. Journal of Food Science. 72,

S481-S486.

Chapter 9

Shelf-life evaluation of aseptically packaged UHPH-treated soymilk

163

9

Shelf-life evaluation of aseptically packaged UHPH-treated soymilk

9.1 Introduction

In order to extend the shelf-life and facilitate distribution, soymilks are usually subjected to

intense heat treatment for sterilization (Kwok & Niranjan, 1995). Although UHT treatment

can produce a soymilk microbiologically stable for at least a year, it may change quality

aspects that could compromise shelf-life (Wang et al., 2001).

Several aspects define the quality characteristics of soymilk such as physical stability of

the product, including color, chemical changes and microbial growth, and all of them are

crucial factors to take into account in the shelf-life determination of products packaged

under aseptic conditions. Additionally to shelf-life determination, volatile profile could be

considered as an important factor related to the consumer acceptation, due to the typical

beany flavor generated by the compounds. Sensory analysis allows evaluating the impact

of volatile compounds by analyzing general sensory attributes during storage of the treated

soymilk. In general, acceptance of the product, appropriate terminologies as well as

appropriate standard to evaluate objectionable flavors are essentials to achieve reliable

results (Torres-Penaranda and Reitmeier, 2001). Moreover, it is important for emerging

technologies, such as UHPH, to gain the acceptance and understanding of consumers apart

from its opinion about the final product.

Thus, the goal of this study was to evaluate quality aspects of soymilk treated at 300 MPa,

80ºC of inlet temperature in comparison with UHT-treated soymilk. The UHPH condition

was selected based in the microbiological and chemical stability results given in chapter 5

and 6. Samples were stored at room temperature for 180 days (see 3.4 condition B).

Microbial analysis applied included mesophilic aerobic bacteria and mesophilic aerobic

spores, enterobacteria and Bacillus cereus counts (see 3.6.1). The determinations applied

were: particle size (see 3.9), particle sedimentation (see 3.10 centrifugation and particle

migration methods), hydroperoxide index (see 3.7.2), color (see 3.13), pH (see 3.5),

surface hydrophobicity (see 3.12), volatile profile evolution (see 3.14) and sensory analysis

(see 3.15). Particle size, volatile profile and sensory analysis were performed on 1, 90 and

180 days of storage.

Chapter 9

164


9.2.1 Microbiological quality and pH measurements

Microbiological quality of soymilk is an important factor that contributes to the chemical

changes during storage and determines the shelf-life, especially for product aseptically

packaged.

Results of unprocessed soymilk were: mesophilic aerobic bacteria of 3.18 ± 0.73,

mesophilic aerobic spores of 2.18 ± 0.53, enterobacteria of 0.49 ± 0.19 and Bacillus cereus

of 2.29 ± 0.78 log cfu/mL and pH 6.80 ± 0.02. After both treatments, microbial growth was

below the detection limit for all samples (< 0.5 cfu/mL). Because injured cells may not

grow on media immediately after treatment (Smith et al., 2009), around of 10 bricks of

each treatment at day 1 was incubated at 30ºC for 20 days and 55ºC for 10 days to evaluate

mesophilic and thermophilic growth respectively. As a result of incubation time, there was

no microbial growth. These results are in accordance with the study described in chapter 5,

where soymilk UHT treated and UHPH treated at 300 MPa, 75ºC of inlet temperature did

not show microbial growth neither after treatment nor after 20 days of incubation at 30ºC.

Likewise to the day 1, UHPH and UHT soymilk remained sterile during for all storage

days of analysis (20, 40, 60, 90, 120, 150 and 180 days). Supporting these microbiological

results, pH measurements did not change significantly and remained constant around the

neutral value during storage at room temperature (Figure 9-1). The good microbiological

quality of the soymilk BP have played important role in the UHPH efficienct of microbial

inactivation. In this way Donsì, et al (2006) and Tahiri et al. (2006) reported that UHPH-

treatment effectiveness increased at low initial bacterial concentration. Similarly, high inlet

temperature combined with high pressure causes better microbial inactivation (Thiebaud et

al., 2003).

In the literature there is not enough information about soymilk treated by UHPH and

aseptically packaged to compare with this study. However, different studies have been

published about UHT treatments in soymilk. For instance, Achouri et al. (2007) subjected

soymilk samples to UHT treatment (142ºC, 4 s) followed by aseptic packaged in coated

paperboard and stored for 3 months at 3 controlled temperatures of 4, 22 and 38ºC. They

reported counts below the limit of detection (< 10 cfu/mL). Nevertheless, a significant

decrease in the pH measurements after 1 month of storage was observed. Beyond this

point, the pH increased (values around 7) and remained stable during the rest of storage

Shelf-life evaluation of UHPH soymilk

165

period. They attributed the pH decreasing was attributed to chemical interactions caused by

lipolysis and proteolysis reactions.

Examples of different studies based on the same UHPH technology, but with different

foodstuffs such as, apple juice, milk and orange juice are shown below. Pereda et al.

(2007) studied milk treated by different UHPH conditions and stored at 4ºC for 21 days.

On day 21, milk treated at 300 MPa and 40ºC of inlet temperature showed mesophilic

aerobic bacteria of 7.1 log cfu/mL and mesophilic aerobic spores of 2.6 log cfu/mL.

Suárez-Jacobo (2011) applied UHPH treatment at 300 MPa and 4ºC of inlet temperature in

apple juice followed by aseptic packaging and storage for 60 days at 4, 10, 20 and 30ºC. In

the end of storage period, mesophilic aerobic bacteria were below of 2 log cfu/mL.

Velazquez-Estrada (2011) studied UHPH-treated orange juice at 300 MPa and 20ºC of

inlet temperature and aseptically packaged. Samples stored at 20ºC achieved more than 75

days of shelf-life compared to pasteurized orange juice. Both apple and orange juice

samples have low pH compared to milk and soymilk. This factor did not favor

microbiological growth compared tothose latter products with neutral pH. Therefore, the

different levels of microbial inactivation achieved by UHPH treatment, mainly at 300 MPa

is strongly dependent of inlet temperature and microbial initial load of unprocessed

sample.

Figure 9-1. pH measurements of UHPH soymilk (-♦-) and UHT soymilk (--).

6.6

6.7

6.8

6.9

0 30 60 90 120 150 180

pH

Days

Chapter 9

166

9.2.2 Colloidal stability

Depending on the desired final concentration, soymilk can appears as a diluted emulsion of

soybeans constituted by a dispersion of proteins in the interface o/w in the aqueous phase.

Destabilization phenomena are the main problems associated to the vegetable beverages

manufacturing, commonly as a consequence of the heat treatment and homogenization

process. In the case of soymilk, creaming and solids sedimentation may take place after

treatment or during long periods of storage, causing phase separation which adversely

affects the quality of final product.

To evaluate the extent of treatment and to understand possible changes in physical stability

during storage, surface hydrophobicity characterization, particle size and sedimentation by

centrifugation and particle migration can provide reliable results to identify possible

changes in particle stability due to treatment and along storage. Firstly, protein structural

characterization of untreated and treated soymilk was measured by using surface

hydrophobicity determination (Figure 9-2). The resultant fluorescence released by the

complex between protein and ANS reactive, provide information regarding to the

exposition degree of inner hydrophobic zones of the protein due to treatment applied (see

chapter 8). Unmasking of inner regions makes the protein more active to hydrophobic

interactions and to disulfide binding (S-S) between fat globules and small particles finely

distributed in the aqueous phase, creating a new interface o/w which may modify the

emulsion stability (Floury et al., 2002; Bouaouina et al., 2006; Shimoyamada et al., 2008).

In colloidal BP distribution, most of the inner hydrophobic zones of the protein are masked

for binding to ANS reactive, leading then to low fluorescence values. This was occurred by

large fat globules, protein bodies (high density particles from seed grinding during soymilk

elaboration) and large aggregates of protein-fat globules observed in Figure 8-3A (chapter

8). This result was confirmed by particle size parameters of BP samples (0.64 ± 0.09 µm

for D50, 0.44 ± 0.03 µm for d3,2 and 17.11 ± 2.61 µm for d4,3) where particles of large

volume were detected. UHT soymilk, on the other hand, obtained higher fluorescence

values than BP. The double effect homogenization (18 and 4 MPa) and the high

temperature (142ºC) of treatment caused higher particle disruption allowing better

dispersion of the interface o/w than BP soymilk. The high temperature applied in the

treatment also favored protein denaturation, changing from globular structure to unfolded

state. This new protein structural organization increased the exposition of buried

hydrophobic zones of the protein, leading to the formation of large aggregates (fat-protein

and protein-protein) and leaving part of them free for binding to the ANS. As a result UHT


167

soymilk was characterized by a dispersion of large particles which experienced slight

modifications during storage (Table 9-1).

Figure 9-2. Protein surface hydrophobicity of BP (―), UHT-treated soymilk (―) and UHPH-treated

soymilk at 300 MPa 80ºC (―).

Soymilk treated at 300 MPa, 80ºC achieved the highest fluorescence values compared to

UHT treatment. UHPH treatment caused such protein rearrangement which led to a high

exposition of hydrophobic zones of the soy protein to the aqueous phase and hence to the

ANS, forming micro-soluble aggregates well dispersed in the continuous phase. Although

the high surface hydrophobicity is related to protein denaturation with consequent loss of

solubility, the presence of soy lipids, particularly phospholipids that have strong

emulsifying capacity, improved the stability of soymilk emulsion due to increased

interaction of proteins with lipids (Liu, 1999). In this way, particle size parameters (Table

9-1) evidenced that aggregates formed by UHPH treatment (d4,3 parameter) were

significantly lower than those by UHT treatment, remaining stable during the period of

storage. The high intensity of homogenization in UHPH treatment produced strong particle

size reduction which favored a better stabilization of the interactions between micro

0

50

100

150

200

250

300

350

400

400 450 500 550

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Chapter 9

168

particles and protein and fat globule during storage. On the other hand, the double-stage

homogenization applied in UHT soymilk was not enough to produce an additional

dispersion of aggregates formed into continuous phase in the first step such as single-stage

step of UHPH treatment.

Table 9-1. Changes in particle size distribution of UHT and UHPH soymilks

Parameters Day UHT UHPH

D50A 1 0.45 ± 0.03xa 0.17 ± 0.01ya

90 0.47 ± 0.06xa 0.18 ± 0.01yb 180 0.32 ± 0.06xb 0.17 ± 0.01ya

d3,2B 1 0.36 ± 0.02xa 0.23 ± 0.19xa

90 0.35 ± 0.02xa 0.17 ± 0.01ya 180 0.26 ± 0.05xb 0.16 ± 0.01ya

d4,3C 1 14.03 ± 0.72xa 6.14 ± 1.64ya

90 15.46 ± 2.03xab 5.07 ± 1.15ya 180 16.57 ± 1.94xb 6.38 ± 0.83ya

a-b Different superscripts in the same column are significantly different (P < 0.05). x-y Different superscripts in the same row are significantly different (P < 0.05). A Mean values ± SD (µm) of diameter below which 50% of the volume of particles are found. B Mean values ± SD (µm) of average diameter (surface weighted mean diameter). C Mean values ± SD (µm) of average diameter (volume weighted mean diameter).

During storage no changes in d4,3 parameter was observed for UHPH soymilk, whereas an

increase tendency in this parameter was observed for UHT treatment. It means that large

aggregates in UHT-treated soymilks were formed and their evolution during storage was

slightly increased. In traditional UHT treatment, homogenization step commonly occur

prior to heat treatment. Thus, aggregates formed in the heating of soymilk were not re-

dispersed in the continuous phase. Large aggregates of soy globulin (glycinin and β-

conglycinin) were also observed during heating of soymilk at high temperature (Tay et al.,

2005). Similar results of particle size parameters (d3,2 and d4,3) for UHT and 300 MPa

soymilks were described in chapter 5 and by Cruz et al. (2007).


169

As reported in chapter 8, solids sedimentation measured by low-speed centrifugation is a

good tool to quantify, under same conditions, sedimentation potential during long-term

storage. Table 9-2 show results of solids settled during 180 days.

Table 9-2. Solids sedimentation1 of treated soymilks during storage.

Day UHT UHPH 1 4.67 ± 0.38ax 2.34 ± 0.48ay 20 5.06 ± 0.79abx 2.42 ± 0.23ay 40 5.23 ± 0.98abx 2.11 ± 0.14aby 60 4.99 ± 0.61abx 2.37 ± 0.17ay 90 5.97 ± 0.61bx 2.09 ± 0.09aby 120 5.96 ± 0.47bx 2.19 ± 0.08aby 150 6.17 ± 0.75bx 2.13 ± 0.08aby 180 6.03 ± 0.84bx 2.00 ± 0.17by

a-b Different superscript in the same column are significantly different (P < 0.05). x-y Different superscript in the same row are significantly different (P < 0.05). 1 Mean values ± SD (g/100g w/w) of solids sedimentation after low-speed centrifugation. BP value was 5.77

± 0.50 g/100g.

Solids sedimentation in UHT soymilks was higher than UHPH soymilk, as expected.

Sedimentation values of both treatments did not show relevant changes during storage,

although a slight increase was observed in UHT soymilk. According to the previous results

of particle size and surface hydrophobicity, UHT-treated soymilk was characterized by

large aggregates, whose behaviour was like a large particle. Large particles of great

volume can become insoluble, allowing easily their deposition when samples were

submitted to centrifugation or storage during a long period. Using equivalent method, Cruz

et al. (2007) observed similar values of solids sedimentation for UHT and 300 MPa

soymilks. In their study, samples were analyzed at days 1, 30 and 60 of cold storage (4ºC).

Results indicated a significant increase of sediments for 300 MPa samples after 30 days of

storage, remaining unchanged in the last day of analysis. This increase was attributed to the

presence of large particles and aggregates which could act as nuclei of aggregation of small

particles suspended in the soymilk samples.

Chapter 9

170

An appropriate instrumental (TurbiscanR) method to evaluate colloidal stability is that

based on migration and/or interaction of particles in samples at resting conditions during

storage. Measurement of light backscattered through time along the sample bottle, from the

bottom to the top is recorded. Changes of backscattering (ΔB) are associated to changes in

the homogeneity, particle size and concentration, and thus the stability of the sample

(Durand et al., 2003). ∆B indicates variation of backscattering from punctual

measurements at different times during storage. The increase in ∆B at the bottom or at the

top of the tube indicates an increase in particle or oil droplets concentration in this part of

the tube, and hence sedimentation or creaming phenomena respectively can be detected. In

all samples analyzed, no creaming phenomenon was observed. Therefore, only the

occurrence of sedimentation was considered to express results. Using a tool calculation

provided by the Turbiscan manufacturer, solids sedimentation was expressed as the height

of solid layer in the bottom of the tube during the storage period of 180 days at 4ºC (Figure

9-3).

Figure 9-3. Height of solids settled of ( ) BP, ( ) UHT and ( ) UHPH soymilks.

a-e Different letters above each bars for each treatment indicate significant differences (P < 0.05).

The intense homogenization applied in UHPH treatment was enough to achieve high

colloidal stability of soymilk. UHPH-treated soymilk showed spontaneous solids layer

from 60th day of storage, reaching a maximum height of just 0.87 mm in the last day of

0

2

4

6

8

10

12

14

20 40 60 90 120 150 180

Days

Hei

ght o

f sol

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settl

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a

e e

b bc bcd cd cd d

a b c d d


171

storage. On the other hand, UHT soymilk which was subjected to much lower

homogenization intensity compared to UHPH soymilk, reached 6.87 mm of solids settled

after 180 days of storage. These results are in accordance to those obtained by

centrifugation and by particle size parameters, where UHT reached high values indicating

low colloidal stability. Taking into account the velocity of sedimentation, UHT soymilk

showed values of 0.094 (mm/day) in the lineal stage (the first 60 days of storage), reaching

5.35 mm of solids layer height. UHPH soymilks obtained a maximum rate of 0.0056

(mm/day) between day 60 and day 90, with 0.53 mm of height. On the other hand, in BP

samples subjected to colloidal mill in the soymilk elaboration, the sedimentation rate was

0.16 (mm/day) in the lineal stage (first 45 days), with 7.2 mm of solids settled. The large

aggregates formed during storage of UHT soymilks, was evidenced by the spontaneous

particle migration to the bottom of the tube. In the case of UHPH soymilks, the thin layer

of sediments detected in the bottom of the analysis tube was dramatically smaller than

UHT, what prove a much better stability of the product than those treated by conventional

processes.

Therefore, homogenizing conditions had a fundamental role to predict the kinetic of

particle migration. Durand et al. (2003) observed creaming phenomenon at slow rate in

commercial soymilk treated by UHT evaluated during 12 h in Turbiscan equipment. This

phenomenon was attributed to the tendency of oil droplets and fat globules to coalesce

producing a clarification zone in the bottom of the analysis tube. In the present study, no

coalescence phenomenon was observed.

9.2.4 Chemical stability

The most important problem that limits the extensive use of soy products is related to

strong off-flavors caused by oxidative reactions. Oxidation of the lipids is one of the main

causes of deterioration that negatively affect quality and storage life as well as consumer

acceptance (Hornero-Méndez et al., 2001). Moreover, studies have reported that secondary

products cause undesirable implications in human health and contribute to decrease the

nutritional value of the product (Angulo et al., 1998). Determination of hydroperoxide

concentration gives information about initial state of oxidation reactions, being the first

stage of the further formation of volatile compounds as secondary products.

Chapter 9

172

Table 9-3. Hydroperoxide index values (meq/L) of UHT and UHPH soymilks during

storage at room temperature (Mean values ± SD).

Day UHT UHPH 1 0.18 ± 0.05ax 0.32 ± 0.01ay 20 0.16 ± 0.02ax 0.21 ± 0.05by 40 0.20 ± 0.04ax 0.14 ± 0.01cy 60 0.30 ± 0.05ax 0.10 ± 0.01dy 90 0.47 ± 0.02bx 0.08 ± 0.02dy 120 0.55 ± 0.21bcx 0.07 ± 0.02dy 150 0.49 ± 0.13bcx 0.05 ± 0.03dy 180 0.67 ± 0.24cx 0.08 ± 0.06dy

a-d Different superscript in the same column are significantly different (P < 0.05). x-z Different superscript in the same row are significantly different (P < 0.05).

The evolution of hydroperoxide index during storage of soymilks exhibited an inverse

tendency in UHT and UHPH samples (Table 9-3). In UHT soymilks, the hydroperoxide

index remained quite stable during the first 40 days and then experienced an important

increase between 40 and 90 days of storage. In UHPH samples a decrease of the

hydroperoxide index values was found during storage. Fat globules breakdown, as well as

molecules conformation, especially protein and lipids, may have an important role in the

oxidation reactions. Due to the great disruption of fat globules in UHPH soymilk, the

exposition of oil droplets surface increased considerably compared to UHT soymilk. This

new state of fat globules could have initially contributed to peroxidation reactions,

resulting for UHPH soymilk higher hydroperoxides index values than UHT soymilk at day

1. However, the protein rearrangement caused by UHPH treatment and by the natural

presence of phospholipids, allowed an efficient covering of the oil droplets surface by

proteins. These interactions between proteins and oil droplets that occurred during the first

days of storage resulted in a protective effect against the formation of new hydroperoxide

radicals. In this way, Pereda et al. (2008) reported that homogenized milk was less

susceptible to oxidation due to the covering action of casein on the oil droplets surface. In

UHT soymilk, the large aggregates formed during treatment and possibly during the period

of storage, probably reduced this protective action of the proteins on the oil droplets

surface, allowing a gradual increase of hydroperoxide index values during storage.


173

To evaluate the impact of oxidation products on soymilk, volatile profile determination is

an indicator of secondary products with potential off-flavor characteristics (Plutowska &

Wardencki, 2007). Volatile compounds can be formed by different routes. Oxidation of the

polyunsaturated fatty acids is the main way of formation, commonly initiated in the

soaking and grinding steps of soymilk elaboration. In this case, lipoxygenase is the main

enzyme responsible for catalyzing oxidation reactions by means of molecular oxygen (see

chapter 7). Once hydroperoxides lipids are formed, hydroperoxylyase is known as the

enzyme that catalyzes the formation of secondary products, such as aldehydes, alcohols,

ketones, furans, esters and acids (Mizutani & Hashimoto, 2004; Min et al., 2005ab). On the

other hand, saccharides and amino acids conversions through Maillard reaction and

Strecker degradation respectively, may also contribute to the formation of secondary

products (Plutowska & Wardencki, 2007). Table 9-4 lists the main volatile compounds

detected at day 1, 90 and 180 days of BP and treated soymilks selected according to the

level of detection, as well as the impact on the sensory characteristics. Among of

compounds detected in soymilk, the literature often points to hexanal as indicator of the

degree of oxidation (Plutowska & Wardencki, 2007; Pereda et al., 2008). Additionally to

hexanal, compounds such as pentanal (Brunton et al., 2000, Min et al., 2005b), 1-hexanol

(Kobayashi et al., 1995), 1-octen-3-ol (Wilkens & lin, 1970), 2-heptanone and 2-penthyl

furan (Mtebe & Gordon, 1987) are formed from fatty acids oxidation, therefore could also

indicate the oxidation degree. According to the P values of each compound presented in

Table 9-4, hexanal was affected only by the time storage factor, while pentanal was not

affected by any parameter. In the alcohols group, 1-octen-3-ol did not change by any

parameter evaluated and 1-hexanol changed significantly by all parameters studied,

showing the latter an increasing during storage in UHPH soymilk. Levels of 2-heptanone

and 2-penthyl furan were affected by the treatment, time of storage and by the interaction

among parameters. 2-Heptanone presented an increase in UHPH soymilk while a decrease

was observed in UHT soymilk during the days analyzed. On the other hand, 2-penthyl

furan increased in both treatments, being high levels in UHT soymilk.

Table 9-4. Main volatile compounds1 detected in soymilk treated by UHT and UHPH.

Name ID2 KI3 BP UHT UHPH P value 1 90 180 1 90 180 Treatment Time Interaction Aldehydes Hexanal MS, RI, P 1092 122.2 x 200.03 ay 120.68 b 109.16 bc 192.48 ay 132.36 b 82.07 c 0.239 0.001 0.066 Pentanal MS, RI, P 990 9.88 x 22.22 ay 16.89 a 16.49 a 20.23 ay 14.84 a 16.28 a 0.474 0.070 0.908 Acetaldeyde MS 4.29 x 4.77 ax 4.25 a 4.59 a 4.66 ax 7.59 b 6.15 b 0.002 0.115 0.019 Benzaldehyde MS, RI 1563 1.86 x 3.10 ay 5.33 b 6.12 b 4.66 bcz 3.53 ac 3.62 ac 0.004 0.031 0.001 2-Heptenal MS, RI 1345 1.66 x 2.67 ax 0.46 bc 0.33 c 2.77 ax 1.50 b 3.66 a 0.001 0.001 0.001 Heptanal MS, RI 1192 2.01 x 4.37 ay 2.23 b 0.24 c 3.80 ay 2.86 b 0.26 c 0.873 0.001 0.026 2-Hexenal MS, RI 1219 1.48 x 3.87 ay 1.30 b 1.40 b 2.73 cz 2.97 ac 1.52 b 0.240 0.001 0.001 Ketones Ketone MS, RI 826 9.61 x 25.10 ay 28.65 ab 33.25 b 15.91 cx 13.30 c 12.91 c 0.001 0.116 0.001 2-Heptanone MS, RI 1190 2.07 x 5.63 ay 6.93 a 1.17 b 3.36 cx 7.41 b 10.19 b 0.015 0.001 0.038 2-Butanone MS, RI 910 3.58 xy 2.68 ay 3.84 ac 5.81 b 6.14 bx 4.22 c 5.11 bc 0.011 0.015 0.001 2,3-Pentanedione MS, RI, P 1072 3.89 x 3.30 x ND ND 6.14 y ND ND 0.001 - - 2,3-Octanedione MS, RI 1333 3.72 x 4.36 ax 0.28 b 0.24 b 8.47 cy 0.41 b 0.45 b 0.001 0.001 0.001 1-Octen-3-one MS, RI, P 1314 1.49 x 1.64 ax 1.79 a 1.94 a 1.94 ax 1.86 a 2.18 a 0.124 0.211 0.749 Acetophenone MS, RI 1693 0.16 x 0.32 ax 0.49 ab 0.68 b 0.63 by 0.51 ab 0.34 a 0.938 0.788 0.001 Alcohols Ethanol MS, RI 944 143.76 x 103.54 ay 87.60 a 91.41 a 133.41 bx 151.25 b 152.29 b 0.001 0.977 0.522 1-Hexanol MS, RI, P 1359 32.60 x 34.15 ax 32.65 a 33.10 a 37.47 ax 90.55 b 136.57 c 0.001 0.005 0.004 1-Pentanol MS, RI, P 1260 34.02 xy 27.53 ay 29.13 a 36.05 ac 43.14 bcx 50.92 b 50.56 b 0.001 0.085 0.525 1-Penten-3-ol MS, RI 1175 A 13.63 x 13.10 ax 11.59 a 13.55 a 14.14 ax 14.01 a 13.15 a 0.187 0.666 0.325 1-Octen-3-ol MS, RI, P 1454 15.88 x 17.54 ax 15.59 a 13.47 a 15.63 ax 16.72 a 15.57 a 0.780 0.532 0.559 Furans 2-Penthyl furan MS, RI, P 1235 A 11.71 x 40.89 aey 57.40 b 81.20 c 16.36 dx 49.06 be 59.66 b 0.001 0.001 0.010 2-Ethyl furan MS, RI 965 A 7.55 x 35.00 ay 39.72 a 61.16 b 11.03 cx 24.10 d 39.55 a 0.001 0.001 0.102

Name ID2 KI3 BP UHT UHPH P value 1 90 180 1 90 180 Treatment Time Interaction Furans 2-Propyl furan MS, RI 1044 1.66 x 15.11 ay 15.65 a 26.35 b 3.17 cx 5.94 c 13.13 a 0.001 0.001 0.030 Esters Ethyl acetate MS, RI 897 4.80 x 3.70 ax 4.11 a 8.81 b 4.84 ax 9.33 b 16.22 c 0.054 0.001 0.001 Methyl acetate MS, RI 839 3.22 x 7.33 aby 6.43 b 8.43 a 3.85 cx 7.58 ab 8.67 a 0.001 0.001 0.044 Acids Hexanoic acid MS, RI 1946 25.67 x 39.38 ay 27.21 b 21.93 b 44.46 ay 26.58 b 18.19 b 0.924 0.001 0.349 a-c Different letters in the same row are significantly different (P < 0.05). x-y Different letters in the same row of treated sample at day 1 are significantly different from BP (P < 0.05). 1 Integrated area counts. Mean values x 105. 2 Identification: MS = Mass spectra, RI = Retention index compared to Pherobase database and (A) (Vichi et al., 2003), P = Positively identified by comparison with authentic

standard. 3 Kovats retention index calculated.

Chapter 9

176

Aldehyde compounds are commonly associated to off-flavor in soymilk. Beany and grassy

flavors as well as oxidized aroma generated by the compounds have unpleasant sensory

perception which noticeably affects the soymilk acceptance (Vara-Ubol et al., 2004; Yuan

& Chang, 2007). Results showed that hexanal, pentanal and acetaldehyde were the most

abundant compounds in aldehydes group. Benzaldehyde, 2-heptanal, heptanal and 2-

hexenal experienced significant changes in the levels detected due to treatment and period

of storage (P < 0.05). For most of them, their levels decreased during storage (Table 9-4).

Among the aldehydes identified, hexanal is the most studied and is commonly considered

as the main contributor to off-flavor in soymilk (Wilkens & Lin, 1970; Achouri et al.,

2006; Yuan & Chang, 2007). Considering the total volatiles at day 1 listed in Table 9-4,

hexanal was the dominant compound with 32% for UHT and UHPH treatments and 26%

for BP (Figure 9-4). However at day 90, the total of hexanal in relation to the total of

volatiles reduced significantly to 23% and 20% for UHT and UHPH soymilks respectively.

The same tendency was observed at day 180 of storage, reaching UHPH soymilks the

lowest value (12%). Similarly, Achouri et al. (2007) detected hexanal values from 14% to

52% in 3 types of heat-treated soymilk and aseptically packaged, stored at 4, 22 and 38ºC

for 12 weeks. They concluded that storage conditions was the main factor affecting

hexanal profile with a significant decrease observed after 6 weeks of storage, mainly at 4

and 22ºC. In the present study, the results obtained of hydroperoxide index and hexanal

levels of UHPH soymilk indicated that the chemical changes occurred during storage

reached a level of stabilization that could lead to better sensory response.

Ketone was the most abundant compound detected in ketones group followed by 2-

heptanone and 2-butanone. Latter two compounds were affected by the time of storage and

all three were affected by the type of treatment (P < 0.05), while acetophenone and 1-

octen-3-one were not affected significantly by treatment and time parameters. 2-

Heptanone, acetophenone, 1-octen-3-one were also identified by Boatright and Lei (1999)

in soy protein isolate and they attributed them, respectively, flowery odor, penetrating

green and mushroom odor properties. 1-Octen-3-one had small peak in the chromatogram

compared to ketone compounds and hexanal in the aldehydes group. However, 1-octen-3-

one as hexanal, has a very low sensory threshold (0.005 mg/L), making its contribution

very important to the overall soymilk sensory quality (Yuan & Chang, 2007). Additionally

to 1-octen-3-one, 2,3-pentanedione was detected only in the first day of analysis and

possess buttery unpleasant odor (Boatright & Lei, 1999).


177

Figure 9-4. Percentage ratio of hexanal to total volatiles of ( ) BP, ( ) UHT and ( ) UHPH soymilks.

a-c Different letters above bars indicate that samples are significantly different (P < 0.05).

In the alcohols group, ethanol, 1-hexanol, 1-pentanol, 1-octen-3-ol and 1-penten-3-ol were

the main compounds detected, with ethanol being the most abundant. They are commonly

associated to grassy and beany flavor (1-hexanol) and mushroom flavor (1-octen-3-ol). 1-

Hexanol is the main compound of alcohol chemical family related to off-flavor in soymilk.

It can be formed through oxidative evolution process of hexanal (Yuan & Chang, 2007;

Kakumyan et al., 2009) that could partially justify the decrease of hexanal during storage,

especially in samples treated by UHPH. In general, alcohols are produced by the reduction

of their corresponding aldehydes by chemical reactions (Molimard & Spinnler, 1996).

Ethanol and 1-pentanol values showed a significant difference between treatments, but did

not present significant difference over time for a same treatment (Table 9-4). On the

contrary, 1-hexanol values showed significant increase during storage for UHPH soymilks,

whereas remained stable for UHT soymilk. Observing all alcohol compounds, UHPH

soymilks obtained higher values than UHT soymilks, mainly for ethanol and 1-hexanol

which the highest values were reached after 180 days of storage. Furan compounds and

specially 2-pentyl furan are associated to beany odor and taste in soymilk. As reported by

Achouri et al. (2007), 2-pentyl furan was the main compound detected followed by 2-ethyl

furan in heat-treated soymilk. UHT treatment produced a significant increase of all furan

compounds detected at day 1 as reported in chapter 7. However, furan compounds tended

0

5

10

15

20

25

30

35

1 90 180

%Hexan

al/Totalvolatile

Days

a

b b

c cd

de

Chapter 9

178

to increase significantly during storage time (P < 0.05). On the other hand, the increase in

the levels of furan compounds for UHPH soymilks was observed after 90 and 180 days of

storage, showing significant lower values than UHT soymilks. Taking into account the

total furan composition after 6 months of storage (Table 9-4), 64% of the levels detected

was represented for UHT soymilks and 36% for UHPH soymilk. The formation of this

chemical family is related to thermal damage derived of high temperature used during

processing. In UHT treatment the process conditions (142ºC for 6 s) were the main factor

in the formation of furans. At these conditions, take place Maillard reactions that favor

furan compounds formation (Achouri et al., 2007).

Ester and acid compounds were detected at low levels for both treatments. Ethyl acetate

obtained a significant increase (P < 0.05) during storage primary for UHPH soymilk,

reaching the highest value in the last day of analysis. Methyl acetate increased significantly

after UHT treatment and remained stable during storage, whereas it was not affected by

UHPH treatment at day 1, but increased significantly until day 90 maintaining this level at

day 180. As reported in chapter 7, there is no information about sensory effect of ester

compounds on soymilk, but they could be associated to floral and fruit flavors. Hexanoic

acid was the most abundant acid compound detected. It could be originated by hexanal

oxidation in presence of oxygen, and produce a harsh and fetid sensory odor (Wilkens &

Lin, 1970). Significant increases were observed for UHPH and UHT treatment at day 1.

However an important decrease was detected (P < 0.05) for both samples in the period of

storage, reaching values close to untreated soymilk.

Not all compounds detected in this study possess a disagreeable odor. For example,

benzaldehyde has a cherry or almond aroma and 2-heptanone has flowery odor (Aparicio

et al., 1997; Wilkens & Lin, 1970; Boatright & Lei, 1999). However, these compounds

have small peak in the chromatogram compared to undesirable compounds detected in the

total volatiles of soymilk.

9.2.5 Sensory analysis and color quality

Thermal treatment and long periods of storage may produce color changes affecting food

quality and sensory response of consumers. In addition to the heat effects, homogenization

process of soymilk may also produce changes in color. When a light interact on the surface

of an emulsion, part of it is reflected, while the rest is transmitted into the emulsion. As the

transmitted light propagates through the emulsion it may be absorbed by any


179

chromophores or scattered by any droplets and/or particles. The light returning from the

emulsion is therefore the result of reflected, transmitted, scattered, and absorbed light

(Chantrapornchai et al., 1998). Table 9-5 shows evolution of color parameters (L*, a* and

b*) during storage of UHT and UHPH soymilks over 180 days.

Luminosity (L*) values, is associated with darkness or lightness of the product. A

significant difference was found in L* value between UHT and UHPH treatments. UHT

soymilk showed higher L* values than UHPH soymilk and BP, remaining this tendency

during storage (P < 0.05). In emulsions, L* parameter is mainly related with particle

concentration and size distribution, and probably with protein denaturation degree (Rhim et

al. 1988ab; Chantrapornchai et al., 1998). In general, L* value of the emulsions increased

with decreasing droplet size diameter (Chantrapornchai et al., 1998), so it was expected in

UHPH soymilk higher L* values than UHT soymilk. However, state of particle

aggregation could have influenced the light reflection and scattering through the UHPH

sample, affecting L* parameter result. On the other hand, it is important to highlight that

the L* value was fairly stable during storage for both treatments. Regarding a* and b*

parameters, slight differences, although significant, were found between UHT and UHPH

soymilks during storage. Treated soymilks changed slightly their color from the initial

yellow of the BP (b* value), remaining this tendency during the period of storage. For a*

(red-green) value, UHPH soymilks showed a small trend towards the green color, while

UHT showed a small trend towards the red color. Among treatments applied, significant

differences were found for all days analyzed for a* values, except in the last day of

analysis. The yellow color and the slight tendency to red color of UHT samples would

indicate a trend towards orange color, whereas UHPH soymilk the yellow color and the

slight tendency to blue color would indicate a trend to green-beige. In UHPH samples, this

result could contribute to visual dark color perception. Cruz (2008) observed similar results

of color parameters. In that study, UHT-treated soymilk showed higher L* values than

UHPH-treated soymilk immediately after treatment.

Chapter 9

180

Table 9-5. Changes in color1 parameters during storage in Tetra Brik containers Parameters Day UHT UHPH

L* 1 85.55 ± 0.41ax 82.20 ± 0.53ay 90 86.71 ± 0.28bx 82.69 ± 0.14by 180 86.23 ± 0.35cx 82.16 ± 0.09ay

a* 1 0.17 ± 0.24ax -0.83 ± 0.13ay 90 0.32 ± 0.22ax -1.01 ± 0.24ay 180 0.34 ± 0.12ax -0.18 ± 0.90bx

b* 1 12.63 ± 0.37ax 12.16 ± 0.75ax 90 12.13 ± 0.14ax 12.34 ± 0.06ay 180 12.58 ± 0.35ax 12.50 ± 0.39ax

ΔEBP 1 3.64 ± 0.27ax 2.12 ± 0.52ay 90 4.86 ± 0.23bx 2.09 ± 0.24ay 180 4.27 ± 0.43cx 1.79 ± 0.41ay

ΔEUHT 1 - 2.90 ± 0.90a 90 - 4.25 ± 0.06b 180 - 4.26 ± 0.10b x-y Different superscripts in the same row indicate significant differences (P < 0.05). a-c Different superscripts in the same column indicate significant differences (P < 0.05). 1 Mean values ± SD of color parameters. BP results were: L* = 82.13 ± 0.15; a* = 0.20 ± 0.40; b* = 13.36 ±

0.76 and ΔEUHT = 2.65 ± 0.16.

The ΔE (color difference) parameter is a single value which takes into account differences

between L*, a*, and b* of the sample and standard. Considering BP as standard, the ΔE

gives an idea of the influence of treatment contributing to the overall color. UHT soymilk

at day 1 and during storage showed higher color difference than UHPH samples. On the

other hand, considering UHT soymilk as standard, the resultant ΔE can be the value of

UHPH processing for comparing to the most conventional commercial product. Making a

general observation of the results, the color variation for both treatments remained stable

during storage. On the contrary, Achouri et al. (2007) reported a drastic change in the color

difference (ΔE) of heat-treated soymilk during 12 weeks of storage. At the first 6 weeks, a

significant increase in the ΔE values was observed. Beyond this point, the values decreased

and then slight modifications were detected during the rest of the storage period.

Sensory analysis gives information about consumer acceptation of soymilks and the

detection of possible changes occurred during long periods of storage. Sensory analysis

was carried out at day 1, 90 and 180. In the first part of the analysis, judges were instructed

to identify possible differences between UHT and UHPH soymilks by means of triangular


181

test. To evaluate results, chi-square test was applied considering as null hypothesis that no

differences between treatments were perceived by the judges. Triangular test showed the

following P values: day 1 (P = 0.2733), day 90 (P = 0.4652) and day 180 (P = 0.1441).

This result indicated that judges did not identify differences between UHT and UHPH

soymilks.

The second test performed to assess sensory analysis was descriptive test. In this test some

characteristic attributes of soy products such as beany flavor, grassy aroma, oxidized

aroma, astringent mouthfeel, thickness and color (darkness) were evaluated. In this part of

sensory evaluation, judges were instructed to identify and describe no recognize taste that

could affect the overall organoleptic quality. Results for each day of analysis are shown in

Figure 9-5. For astringency mouthfeel, panel evaluation at day 1 classified UHT soymilks

with higher mouthfeel astringency than UHPH soymilks (P < 0.05). Similar tendency was

obtained at day 90 of storage, whereas no significant differences were observed at day 180.

Astringent mouthfeel may be originated by phenolic compounds such as isoflavones and

saponins which were reported being the main non-volatile off-flavor in soy products

(Matsuura et al., 1989). Regarding dark color at day 1, judges classified UHPH soymilk

slightly darker than UHT soymilk in all days of analyzis, showing only significant

differences at day 90. This result is in accordance to that observed in L* values of

instrumental color evaluation. Considering thickness attribute, no significant differences

were observed between days analyzed (P ≥ 0.05). Likewise, beany flavor and grassy and

oxidized aromas did not show significant differences between treatments during the period

of storage (Figure 9-5ABC). However, these attributes in UHT soymilk obtained notes

slightly higher than UHPH soymilk at 90 and 180 days of storage, with oxidized aroma

being the most relevant difference. These attributes are related to hexanal detection. As

previously mentioned, hexanal was the most abundant compound detected in the whole

volatile profile (Figure 9-4) and moreover, it has very low sensory threshold (25 µg/kg)

(Hashim & Chaveron, 1995) so its detection plays an important role in the sensory

perception.

Chapter 9

182

A

B

C

Figure 9-5. Sensory attributes evaluation of (--) UHT and (-♦-) UHPH soymilks at (A) day 1, (B) day 90

and (C) day 180.

0.0

1.0

2.0

3.0

4.0Beany

Grassy

Oxidized

Astringency

Thickness

Darkness

0.0

1.0

2.0

3.0

4.0Beany

Grassy

Oxidized

Astringency

Thickness

Darkness

0.0

1.0

2.0

3.0

4.0Beany

Grassy

Oxidized

Astringency

Thickness

Darkness


183

Adittionaly to hexanal, total furan compounds in UHT soymilks showed higher values than

UHPH soymilks in the same days of analysis (Table 9-4), emphasizing the possible

relation of the sensory perception with volatile profile. Oxidized aroma could be originated

by a mix of aldehydes such as, nonanal, heptanal, pentanal and 2,4-heptadienal which were

related to aged or rancid flavor ( Boatright & Lei, 1999; Vara-Ubol et al., 2004). Grassy

aroma and green odors in general are primarily associated to furan compounds, mainly to

2-pentyl furan, although hexanal and 2-hexenal were also reported to have these

characteristics (Wilkens & Lin, 1970; Boatright & Lei, 1999; Suratman et al., 2004;

Lozano et al., 2007). Therefore, hexanal and furan results could explain the panel sensation

of beany, grassy and oxidized flavor for UHT-treated soymilk.

Of all days analyzed, bitter taste was the main no recognized taste indicated by the judges

for UHT soymilk. Few papers have studied the impact of soymilk treatment on volatile

compounds and their sensory characteristics. Lozano et al. (2007) studied soymilk treated

by three UHT conditions by combining time and temperature. They considered a total of

10 attributes evaluated after treatment, resulting astringency and beany flavor the most

perceived attributes by panelist. That result is in accordance with the present study for

UHT samples at day 1 of analysis as well as at day 90.

Storage time of treated samples did not affect levels of each attribute evaluated by the

panel (P ≥ 0.05). This result indicates that chemical changes occurred during storage did

not impact the organoleptic characteristics of soymilk, as also observed in the results of

triangular test, where judges did not identify differences between treatments. Preference

test was the last part of sensory analysis. This test gives information about the acceptance

of UHT and UHPH treatments. To perform the interpretation of the results, it was

considered that the null hypothesis indicated that samples were equal in the panelist

preference. According to the results, no significant differences were detected (P ≥ 0.05), so

the type of treatment was not relevant in the panel preference. However panel evaluation at

each day of analysis (bold values) demonstrated a trend of UHPH soymilk to be the

preferred sample (Table 9-6). Hence overall sensory results suggested that high

temperature is more detrimental to soymilk flavor than high pressure treatment. These

results are very positive for UHPH technology, because it would be possible to produce

soymilk with improved sensory characteristics and good acceptation.

Chapter 9

184

Table 9-6. Preference results of panel evaluation1

Day UHT UHPH Like 1 3 7

90 3 7 180 5 4

Dislike 1 7 2 90 5 2

180 4 3 1 Number of judges that selected its preference according to the category gave.

9.3 Conclusions

The present study has shown that UHPH-treated and aseptically packaged soymilk was

able to achieve 6 months of storage at room temperature, without any microbial growth as

commonly reached for conventional UHT treatments. Moreover, it showed higher colloidal

stability with smaller spontaneous solids sedimentation than UHT treatment. Although

hydrophobicity results indicated that UHPH treatment produced high degree of protein

denaturation, the new state of particle distribution contributed to improve the physical

stability of soymilk. In this sense, UHPH treatment achieved high color stability with small

color difference compared to UHT soymilk. On the other hand, UHPH-treated soymilk

obtained stable levels of primary oxidation degree and an important decrease of hexanal

values used as indicator of advanced levels of oxidation over storage. Almost all

compounds associated to soymilk off-flavors were detected in the volatile profile of UHT

and UHPH soymilks. Nevertheless, UHPH treatment did not produce changes in the

soymilk which could affect the panel perception for differing UHT and UHPH soymilks

and for selecting the preference. Moreover, overall attributes as well as preference test

analyzed for each treatment in each day, allow concluding that UHPH-treated soymilk has

a slight tendency to be the sample with better characteristics in the panelist opinion. This

result is very promising for UHPH technology, turning it into a clear alternative to heat

treatments producing soymilk with long shelf-life and better global qualities than

conventional technologies. On the other hand, it is important to note that soybeans variety

and its chemical composition of lipids and protein, so as the method of soymilk elaboration

and treatment, are the main factors which indicate what phenomena (physical and

chemical) can undergo soymilk during its shelf-life.


185

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chemical compounds potentially associated with beany aroma in foods. Journal of

Sensory Studies. 19, 15-26.

Velazquez-Estrada, R. M. (2011). Evaluation of the efficacy og ultra-high pressure

homogenization technology to improve the safety and quality of liquid foods and

especially of orange juice [dissertation]. Universitat Autònoma de Barcelona, Spain.

Vichi, S., Pizzale, L., Conte, L.S., Buxaderas, S. & López-Tamames, E. (2003). Solid-

phase microextraction in the analysis of virgin olive oil volatile fraction:

Modifications induced by oxidation and suitable markers of oxidative status. Journal

of Agricultural and Food Chemistry. 51, 6564-6571.





Yuan, S.H. & Chang, S.K.C. (2007). Selected odor compounds in cooked soymilk as

affected by soybean materials and direct steam injection. Journal of Food Science.

72, S481-S486.

Chapter 10

Conclusions

193

10

Conclusions

The conclusions obtained in this thesis are numerated below:

1. The elaboration method developed at the UAB pilot plant produced a good quality soymilk without lipoxygenase activity and with reduced trypsin inhibitor activity. The extraction performed at 80ºC was the selected temperature as it produced a better yield than 60ºC and a good standard of composition similar to commercial soymilk. The method developed allowed the large-scale production of soymilk for UHT and UHPH treatments.

2. The combination of pressure and inlet temperature in UHPH treatment reduced

significantly microbial populations and particle size compared to conventional thermal treatments. In this sense, UHPH soymilks achieved high colloidal stability and low oxidation index compared to heat treatments. Nevertheless, UHPH treatments did not achieve the same level of trypsin inhibitor inactivation as obtained in UHT treatment.

3. The combined conditions of pressure and temperature showed that 300 MPa and 75ºC of inlet temperature was able to produce commercial sterile soymilks. On the other hand, the main potential spores identified and purified from soymilk treated at 300 MPa, 65ºC of inlet temperature after incubation time belonged to Bacillus genus, in particular Paenibacillus taichunengis, P. glucanolyticus and Bacillus cereus.

4. UHPH treatments at 300 MPa and 55, 65, 75 and 85ºC of inlet temperature were able to reduce noticeably of P. taichungensis and B. cereus spores inoculated in sterilized soymilk. Inactivation of P. taichungensis was more effective than B. cereus at any inlet temperature, reaching complete inactivation at 85ºC.

5. Fifty seven volatile compounds were identified in soymilk. The aroma profile was characterized primarily by aldehyde and alcohol compounds. Ketone, furan, ester

Chapter 10

194

and acid compounds were detected in low levels, although not less relevant. Pasteurization and 200 MPa treatments were less favorable to the formation of volatile compounds, reaching results close to untreated soymilk. UHT and 300 MPa were the treatments which favored volatile formation. UHT-treated soymilk produced high levels of furan compounds in contrast to 300 MPa.

6. UHPH treatments at 200 MPa (55 and 75ºC) showed high colloidal stability during

28 days of storage at 4ºC compared to pasteurized soymilk. In terms of lipid oxidation, UHPH-treated soymilk showed lower values of hydroperoxide index than pasteurized soymilk and a significant reduction of hexanal levels during storage under refrigeration conditions. On the other hand, the UHPH condition of 75ºC remained microbiologically stable during storage, although 200 MP, 55ºC soymilk reached acceptable results in the period of storage studied.

7. UHPH-treated soymilk at 300 MPa, 80ºC aseptically packaged was able to achieve

6 month of storage at room temperature, without microbial growth. Although UHPH soymilk showed high hydrophobicity values after treatment, the colloidal stability was highly stable compared to UHT soymilk. Moreover, it showed high color stability and stable levels of primary and secondary compounds of lipid oxidation over the period of storage.

8. In UHPH treatments, where fresh soymilk and soymilk with extended shelf-life was obtained, panelists were not able to identify differences between UHPH-treated and heat-treated soymilks. Almost all compounds which are associated to soymilk off-flavors (beany, grassy and oxidized attributes) were detected in the volatile profile of the UHPH and heat-treated soymilks. However, overall sensory results allow the conclusion that UHPH-treated soymilk had a tendency to be the sample with better sensory characteristics.

9. Finally, UHPH treatment (200 MPa, 75ºC) was able to produce soymilk as a fresh

product stored under refrigeration conditions for 28 days with better qualities than pasteurization treatment (95ºC, 30 s). On the other hand, soymilk treated at 300 MPa, 80ºC aseptically packaged and stored at room temperature proved to be a clear alternative to conventional thermal treatments, such as UHT.

Chapter 11

Appendix

Appendix 1


treatments on the microbiological, physical and chemical quality of soymilk

Poliseli-Scopel, F.H., Hernádez-Herrero, M., Guamis, B. & Ferragut, V.

LWT – Food Science and Technology

at SciVerse ScienceDirect

LWT - Food Science and Technology 46 (2012) 42e48

Contents lists available

LWT - Food Science and Technology

journal homepage: www.elsevier .com/locate/ lwt

Comparison of ultra high pressure homogenization and conventional thermaltreatments on the microbiological, physical and chemical quality of soymilk

Fábio H. Poliseli-Scopel, Manuela Hernández-Herrero, Buenaventura Guamis, Victoria Ferragut*

Centre Especial de Recerca Planta de Tecnologia dels Aliments (CERPTA), XaRTA, TECNIO, MALTA-Consolider group, Departament de Ciència Animal i dels Aliments,Facultat de Veterinària, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain

a r t i c l e i n f o

Article history:Received 28 June 2011Received in revised form18 October 2011Accepted 3 November 2011

Keywords:Ultra high pressure homogenization (UHPH)SoymilkMicrobial qualityPhysical stabilityChemical stability

* Corresponding author. Tel.: þ34 935813292; fax:E-mail address: [email protected] (V. Ferra

0023-6438/$ e see front matter 2011 Elsevier Ltd.doi:10.1016/j.lwt.2011.11.004

a b s t r a c t

The effect of ultra high pressure homogenization (UHPH) at 200 and 300 MPa in combination withdifferent inlet temperatures (55, 65 and 75 C) on soymilk was studied. UHPH-treated soymilk wascompared with the base product (untreated), with pasteurized (95 C for 30 s) and with ultra hightemperature (UHT; 142 C for 6 s) treated soymilks. Microbiological (total aerobic meshophilic bacteria,aerobic spores, and Bacillus cereus), physical (dispersion stability and particle size distribution) andchemical (lipoxygenase activity, hydroperoxide index and trypsin inhibitor activity) parameters of specialrelevance in soymilk were studied. Microbiological results showed that pressure and inlet temperaturecombination had a significant impact on the lethal effect of UHPH treatment. While most of UHPHtreatments applied produced high quality of soymilks better than that pasteurized, the combination of300 MPa and 75 C produced a commercially sterile soymilk. UHPH treatments caused a significantdecrease in particle size resulting in a high physical stability of samples compared with conventional heattreatments. UHPH treatment produced lower values of hydroperoxide index than heat treated soymilksalthough trypsin inhibitor activity was lower in UHT-treated products.

2011 Elsevier Ltd. All rights reserved.

1. Introduction

Consumption of soy derived products has increased recently inwestern countries. Popularity of such a products is related with thegenerally recognized nutritive properties of soybeans, and espe-cially since the Food and Drug Administration approved the soyprotein health claim in 1999 (FDA, 1999).

Soymilk is a liquid extract, obtained by mixing and groundingsoybeans with water, resulting in a product with a similarappearance to cow milk, although with different composition.

Commercial soymilk is produced mainly by conventional tech-nologies, such as UHT (ultra high temperature); high temperatures,however, may cause undesirable chemical changes which includedestruction of amino acids and vitamins, browning reactions, anddevelopment of cooked flavors.

Ultra high pressure homogenization (UHPH) is an emergingtechnology whose ultimate aim is to obtain food products withbetter quality than conventional heat treatments. UHPH is based onthe same principle as conventional homogenization, but it worksat significantly higher pressures (up to 400 MPa). Cavitation,

þ34 935812006.gut).

All rights reserved.

turbulence, impact and shear forces are physical phenomena takingplace during UHPH treatment (Floury, Legrand, & Desrumaux,2004). These mechanical forces acting on the liquid food product,cause fine and stable emulsion (Thiebaud, Dumay, Picart, Guiraud, &Cheftel, 2003;) and microbial and enzymatic inactivation (Hayes,Fox, & Kelly, 2005) since all colloidal particles, from macromole-cules to microorganisms, may be mechanically destroyed whenpassing through the high pressure valve. Another important aspectof this technology is that it is a continuous process, highly efficientfor industrial application purposes.

Consumers demand high quality food products, which meanthat they have to guaranty good nutritional quality, long shelf lifeand high colloidal stability. All these characteristics could be ach-ieved by using UHPH to obtain soymilk as pointed out in a previousstudy (Cruz et al., 2007). In this work, soymilk resulted in microbialreduction similar to pasteurization and an excellent physicalstability was obtained. However, even using different retentiontimes at high temperatures, it was not possible to obtain thesterility of products. This is why the purpose of the present workwas to extend UHPH conditions, by combining inlet temperatureand pressure applied to soymilk for obtaining high quality soy-milks, paying special attention to find the right conditions forcommercial sterility.

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F.H. Poliseli-Scopel et al. / LWT - Food Science and Technology 46 (2012) 42e48 43

2. Materials and methods

2.1. Soymilk elaboration

Soybeans (Majesta variety) were hydrated (1:3 water: soybean)for 15 h at room temperature and then ground in a crushingmachine with heating control (adapted from Frigomat, Milan, Italy)for 20 minutes at 80 C with recirculation in a colloidal mill (E.Bachiller B. S.A, Barcelona, Spain). The pulp obtained was separatedby filtration (model: CE98, Mejisa e Mectufry, Jijona, Spain). Thiswas considered to be the soymilk base product (2.68 0.17 g/100protein; 1.92 0.17 g/100 fat; 5.53 0.39 g/100 dry matter;1.35 0.05 g/100 carbohydrate; 0.18 0.05 g/100 ash. Mean pHvalue was 6.65 0.05 for further processing by UHPH, pasteuriza-tion or UHT.

2.2. Soymilk treatments: UHPH, pasteurization and UHT

To obtain pasteurized soymilk, base product was homogenizedat 18 MPa (LAB type: 22.51, Rannie, Copenhagen, Denmark) andsubsequently pasteurized using a tubular heat exchanger (ATI,Granollers, Barcelona, Spain) at 95 C for 30 s. To obtain UHT soy-milk, two step homogenization (18 MPa and 4 MPa) was performedin a homogenizer (X68IEþX68P, Niro Soavi, Parma, Italy) previousto the heat treatment (142 C for 6 s) with an indirect systemequipment (6500/010, GEA Finnah GmbH (Ahaus, Germany).

UHPH treatments were conducted with a high pressurehomogenizer (model: FPG11300, Stansted Fluid Power Ltd., Essex,UK). This device (flow rate of 120 L/h) is provided with a highpressure ceramic valve able to support 400 MPa. Inlet and outlettemperatures of soymilk were controlled by two heat exchangers(Garvía, Barcelona, Spain) located before the machine entranceand after the high pressure valve, respectively. During treatments,the inlet temperature, the temperature after the high pressurevalve, and outlet temperature were monitored. Six UHPH treat-ments were performed by combining, 200 and 300 MPa withdifferent inlet temperatures (55 C, 65 C and 75 C). Samples werecollected in sterile bottles in a laminar flow cabin (adapted fromMini-V cabin, Telstar Technologies, S.L. Terrassa, Spain) for thispurpose.

2.3. Microbiological analysis

Microbiological quality of soymilk samples was assessed byenumerating the following microorganisms: mesophilic aerobicbacteria were counted on PCA medium (Oxoid Ltd, Basingstoke,Hamphire, UK) incubated for 48 h at 30 C;mesophilic aerobic sporecounts were assessed by heat shock at 80 C for 10 min, quicklycooled in ice and plated on PCA medium (Oxoid) and incubated for48 h at 30 C; enterobacteria counts were determined in violet redbile glucose agar (VRBG, Oxoid) incubated at 37 C for 24 h; yeastsand molds were detected in rose-bengal chlorampenicol medium(Oxoid), incubated at 25 C for 5 days. Staphylococcus aureus wasdetermined in Baird-Parker RPF (bioMérieux, S. A., Marcy l’Etoile,France) and incubated at 37 C for 48 h; Bacillus cereus was deter-mined in brilliance Bacillus cereus medium (Oxoid), supplementedwith B. cereus selective supplement (Oxoid) and incubated at 30 Cfor 48 h; Salmonella sp. was investigated through sample pre-enrichment at 37 C for 24 h and then enriched in Muller Kauff-man broth (bioMérieux) and Rappaport Vassiliadis broth (bio-Mérieux) at 37 C and42 C for 24 h, respectively. Enrichmentswerestreaked onto XLD (Oxoid) and SM2 agar media (bioMérieux) andincubated at 37 C for 24 h.

Sterility test. Soymilk samples were incubated at 30 C for 20days. During this period, coagulation and phase separation were

checked visually. Total aerobic mesophilic bacteria was determinedon PCA medium (Oxoid) in all incubated bottles.

2.4. Particle size determination

Particle size distributionwas performed by laser light-scattering,in the same way as described by Cruz et al. (2007). Particle sizedistribution was characterized by the Sauter mean diameter, d3.2(particle diameter that has the same specific surface as that of thefull distribution) and by the d4.3 (diameter of the sphere of equiva-lent volume to measured particles).

2.5. Particle sedimentation

Physical stability was performed using the method described byCruz et al. (2007) which consists of inducing particle sedimentationby centrifugation. Values were obtained on days 1 and 15 aftertreatment while stored at 4 C.

2.6. Lipid oxidation

2.6.1. Lipoxygenase (LOX) activityThe LOX extraction was performed following the method

described by Van der Ven, Matser, and Van den Berg (2005) withsome modifications. Soymilk (30 mL) was placed in polypropylenetubs and centrifuged for 60 min at 12,000g at 4 C. Supernatantswere used for enzyme activity test.

The assay to determine LOX activity was performed followingmethod described by Axelrod, Cheesbrough, and Laasko (1981). Thereaction was carried out at 25 C in quartz cuvettes in a spectro-photometer (Cecil 9000, Cambridge, England) at 234 nm. The assaymixture contained (2.975e x) mL of borato buffer, pH 9.0, 0.025 mLof sodium linoleate substrate, and xmL of LOX extract (enzyme).After each addition the mixture was stirred. The blank cuvettecontained no enzyme. The activity was expressed as absorbance perminute (abs/min) read in the spectrophotometer and transformedto units of lipoxygenase activity (ULA). One unit of enzyme wasdefined as absorbance increase of 0.1/min.

2.6.2. Hydroperoxide indexLipid hydroperoxides in soymilk were determined by using the

method described by Ostdal, Andersen, and Nielsen (2000) withsome modifications. The reaction consists of the oxidation offerrous to ferric ion by hydroperoxides in the presence of ammo-nium thiocyanate to produce ferric thiocyanate whose absorbancecan be measured at 500 nm. Soymilk (0.4 mL) was mixed withwater (1.6 mL). Then 2 mL of methanol were added and stirred.Chloroform (4 mL) was added and vortexed for 30 s approximately.After centrifugation for 10 min at 12,000g, 1 mL of the chloroformphase was transferred to a test tube and mixed with 1 mL of Fe (II)/thiocyanate in methanol/chloroform. The mixture was allowed toreact for 5 min at room temperature before the absorbance at500 nmwas read. Soymilk samples were analyzed in triplicate, anddata were expressed as meq peroxide/L of sample as described byHornero-Méndez, Pérez-Gálvez, and Mínguez-Mosquera (2001).

The calibration curve was made according with methodologydescribed by FIL- IDF (1991). Measure is based on spectrophoto-metric reading at 500 nm of a series of dilutions that contain Fe3þ inchloroform/methanol (70:30). Analysis was carried out 10 h aftertreatment and after 15 days of storage at 4 C.

2.7. Trypsin inhibitors (TI) activity

The trypsin inhibitor extraction was prepared following themethod described by Van der Ven et al. (2005) with some

Table 2Microbial populations (log cfu/mL SD) of base product and treated soymilks.

Treatment Total bacteria Total spores B. cereus

Base product 5.02 2.24a 3.46 1.21a 3.55 2.37aPasteurized 3.46 1.23b 2.85 0.95a 2.56 1.91bUHT NDa ND ND200 MPa 55 C 3.39 1.55b 1.75 0.69b 2.31 1.38b200 MPa 65 C 2.44 1.59c 0.85 0.57c NDb

200 MPa 75 C ND ND ND300 MPa 55 C ND ND ND300 MPa 65 C ND ND ND300 MPa 75 C ND ND ND

aec Values in the same column with different alphabets are significantly differentp< 0.05).

a ND¼ no detected.b Bacillus cereus growth was detected only in one production (3.40 0.12 log cfu/

mL).

F.H. Poliseli-Scopel et al. / LWT - Food Science and Technology 46 (2012) 42e4844

modifications. Soymilk samples were diluted twice with 0.02 mol/LNaOH (the pH adjusted, when required, to 8.4e10.0) and stirred for2 h. The extract was then centrifuged at 12,000g for 35 min andfiltered through Whatman paper No. 42. The TI extract was dilutedso that 2 mL of the extract dilution inhibited 40e60% of the trypsinused as standard in the analysis. This extract was used for further TIactivity analysis.

TI activity on soymilk was assessed following the methoddescribed by Hamerstrand, Black, and Glover (1981) with littlemodifications as follows: blank sample consisted of distilled water,without sample extract. To define TI activity, one trypsin unit (TU)was arbitrarily described as the increase of 0.01 absorbance unitsper 10 mL reaction mixture at 410 nm (Kakade, Simons, & Liener,1969). Values obtained from soymilk sample extracts were sub-tracted from the trypsin standard and the TI activity was expressedas percentage of the residual value of each treated soymilk taking asreference the base product (not treated soymilk).

2.8. Statistical analysis

Three individual productions of soymilk were performed. Allanalyses were made in triplicate. Results were analyzed by ANOVAusing GLM procedure of SAS (SAS Institute, 2004) to determine thedifferences. The SNK (Student Newnan Keuls) was used forcomparing sample data. Evaluations were based on a significantlevel of p< 0.05.

3. Results and discussion

3.1. Temperature changes in UHPH processing

Temperature of soymilk reached in the high pressure valve incombinationwith very short time (approximately 0.7 s in this UHPHmachine) at this temperature determined the quality characteristicsof UHPH-treated samples. Temperature increase during UHPHtreatment (Table 1) was about 20 C between 200 and 300 MPa atthe different inlet temperatures used in this study. This temperatureincrease was close to those observed by other authors. Pereda,Ferragut, Quevedo, Guamis, and Trujillo (2007), who pressurizedmilk in the same UHPH equipment at inlet temperatures of 30 Cand 40 C and pressures from 100 to 300 MPa, found the sametemperature change. Thiebaud et al. (2003) reported a temperatureincrease of 18.5 C per 100 MPa, in the pressure range 100e300 MPafor milk samples. The increase of temperature during UHPH treat-ment is the consequence of the adiabatic heating generated in themachine in addition to the turbulence, shear, and cavitation forcesthat the food experiments when passing through the ultra highpressure valve (Hayes & Kelly, 2003; Thiebaud et al., 2003).

3.2. Microbiological quality

Table 2 shows the microbiological counts (log cfu/mL) soymilksamples. In general, UHPH treatments at 200 and 300 MPa were

Table 1Temperature changea of UHPH-treated soymilks during processing.

Ti (C) Treatment (MPa) T1 (C) Tf (C)

55 200 105.7 0.58 27.1 1.0300 128.3 1.53 27.3 1.1

65 200 111.7 1.15 27.0 1.0300 130.7 1.15 26.2 0.8

75 200 117.0 2.00 25.6 2.7300 135.7 1.53 26.2 2.2

a Ti¼ inlet temperature; T1¼ temperature reached in the homogenization valve;Tf¼ outlet temperature.

more effective than pasteurization against almost all the microor-ganisms. However, significant differences (p< 0.05) were detectedbetween UHPH samples at 200 MPa (55 and 65 C inlet tempera-ture) and pressurized samples at 300 MPa. According with someauthors, the reduction in microbiological counts was pressuredependent, showing better inactivation as homogenization pres-sure increased (Hayes et al., 2005; Thiebaud et al., 2003). In contrast,Pereda et al. (2007) reported that no significant differences weredetected betweenUHPHmilk samples at 200 and 300 MPaat 30 and40 C inlet temperature, immediately after treatment. However,they concluded that milk treated at 300 MPa, 30 C inlet tempera-ture had similar or better microbial shelf life than pasteurized milk.

Additionally to the pressure applied in the UHPH treatment,inlet temperature could also be considered as a factor affectingmicrobial lethality, especially when samples were treated at200 MPa. In this study, the highest temperature reached was 135 Cat 300 MPa, 75 C inlet temperature. All treatments at 200 MPawere more effective than pasteurization in reducing aerobic sporecounts. UHPH samples treated at 200 MPa, 55 C inlet temperatureshowed no significant differences (p> 0.05) in total counts ofmesophilic aerobic bacteria and B. cereus counts with raw andpasteurized samples, while UHPH treatment at 200 MPa, 65 Csignificantly reduced B. cereus and total mesophilic bacteria andspore counts compared with raw, pasteurized and 200 MPa, 55 Csamples. Cruz et al. (2007) who worked with soymilk at 200 MPa,40 C inlet temperature, obtained a reduction in the total meso-philic bacteria counts of 2.42 log units, which was slightly higherthan the reduction obtained in this study at inlet temperature of55 C and similar to those obtained at 65 C inlet temperature,where reduction reached was of 1.6 and 2.58 log units, respectively.

Counts below the detection level for B. cereus and total meso-philic bacteria and spore counts were obtained for 200 MPa, 75 Cand 300 MPa at 55, 65 and 75 C inlet temperatures. Although Cruzet al. (2007) obtained in soymilk samples a significant reduction oftotal mesophilic bacteria and spore counts (4 and 2 log units,respectively) after UHPH treatment at 300 MPa, 40 C, it was notachieved a complete inactivation. Picart et al. (2006) reporteda reduction on the total bacteria in milk samples of 2.9 log units at300 MPa, 24 C inlet temperature, while Pereda et al. (2007) re-ported in milk samples reductions about 3.3 log units at 300 MPa,30 or 40 C inlet temperatures. However, Smiddy, Martin,Huppertz, and Kelly (2007) found a reduction of about 5 log unitsfor UHPH-treated milk at 250 MPa, 55 and 70 C. As it was abovementioned, differences observed in microbial counts between thisstudy and others could be explained by the effect caused of themaximum temperature reached in the UHPH treatment.

Despite the fact that in some UHPH treatments, apparently wereachieved a complete microbial inactivation, after incubation at


30 C for 20 days to test the sterility of treated samples, it wasobserved that only the UHT samples and those treated at 300 MPa,75 C did not show bacterial growth. Samples treated at 200 MPa,65 C and 75 C inlet temperature coagulated after 2 days of incu-bation and those treated at 300 MPa, 65 C showed microbialgrowth of 3.97 0.08 cfu/mL after one week of incubation andcoagulated on day 20. Spores showed a high resistance. Accordingwith Suárez-Jacobo, Gervilla, Guamis, Roig-Sagués, and Saldo(2010), who worked with fruit juice, all vegetative cells werealready destroyed at 200 MPa, and the differences between 200and 300 MPa were only accounting for the destruction of spores.Cruz et al. (2007) and Pereda et al. (2007) also detected thatbacterial spores were not completely eliminated by UHPH treat-ments with inlet temperatures of 30 or 40 C at 300 MPa.

Coliform counts (data not shown) were only detected in oneproduction of base product (2.57 0.02 log cfu/mL). However,coliforms counts were below the detection level after pasteuri-zation, UHT and UHPH treatments. Cruz et al. (2007) foundenterobacteria counts below detection level (initial count of2.3 log cfu/mL) in soymilk samples when homogenized at 200 and300 MPa, and Hayes et al. (2005) and Pereda et al. (2007), did notdetect coliforms in milk samples, after treatment at 200 MPa andabove.

Yeast and molds, Salmonella spp. and S. aureus (data notshown) were not detected in both raw and treated soymilks Tahiri,Makhlouf, Paquin, and Fliss (2006) obtained inactivation ofS. cerevisiae and Penicillum spp. in orange juice by 2.5 and 4.0 logunits, respectively, working at 200 MPa, 25 C. Suárez-Jacobo et al.(2010) obtained reductions of yeast and mold counts around 5 logunits in apple juice treated at 200 and 300 MPa and Donsì, Ferrari,and Maresca (2009) achieved a reduction of 5 log units for S. cer-evisae inoculated in sterile water at 250 MPa, showing the highsusceptibility of some yeasts to UHPH treatment.

3.3. Colloidal stability: particle size and sedimentation

In soymilk, particles dispersed in the aqueous phase are ofdifferent characteristics such as oil droplets, native protein aggre-gates (protein bodies), and other aggregates formed from oildroplets and proteins and/or polysaccharides (Cruz et al., 2007;Malaki Nik, Tosh, Poysa, Woodrow, & Corredig, 2008). The mostcommon stability problems of vegetable beverages are thecreaming of oil droplets and the sedimentation of solid particles;both phenomena depending to a great extent on particle sizedistribution. Colloidal stability was assessed by measuring solidssedimentation after centrifugation of soymilk and by means ofparticle size distribution (Table 3). It was observed that UHPH

Table 3Solids sedimentation and particle size parameters of base product and treatedsoymilks.

Treatment Solids sedimentationa d3.2b d4.3

c

Base product 7.03 0.55a 0.51 0.05a 12.98 3.82aPasteurized 3.32 0.30b 0.68 0.04b 48.00 10.2bUHT 3.08 0.23b 0.39 0.03c 15.65 1.83a200 MPa 55 C 1.32 0.11c 0.13 0.02d 4.82 2.27c200 MPa 65 C 1.34 0.10c 0.14 0.02d 0.14 0.01d200 MPa 75 C 1.38 0.12c 0.14 0.01d 3.09 0.37e300 MPa 55 C 1.55 0.40c 0.12 0.01d 4.39 1.16ce300 MPa 65 C 1.61 0.36c 0.11 0.01d 1.32 0.55d300 MPa 75 C 1.38 0.20c 0.11 0.01d 3.39 1.83ce

aee Different alphabets in the same column are significantly different at p< 0.05.a Mean values SD (g/100 w/w) of solids sedimentation after centrifugation at

3000 rpm for 45 min at 20 C.b Mean values SD (mm) of average diameter (surface weighted mean diameter).c Mean values SD (mm) of average diameter (volume weighted mean diameter).

treatments reduced solids sedimentation induced by centrifuga-tion compared with conventional thermal treatments applied inthis study; thus UHPH dramatically increased colloidal stability ofsoymilk. Samples treated at 200 and 300 MPa, at any inlettemperature did not show significant differences (p> 0.05) insolids sedimentation values. In the same way, samples did notexhibit additional sedimentation increase after 15 days of storage(data not shown). Another test to qualitatively evaluate the phys-ical stability of samples consisted of detecting any destabilizingphenomena in glass bottles of treated soymilks for one week whilemaintained at rest. UHPH-treated soymilks remained in perfectdispersion state without any visible phase separation, while UHTand pasteurized soymilk exhibited a layer on the bottom of thebottles easily observable. All these results indicated that applyingpressure in such a magnitude compared with those conventionalapplied at the food industry (in this case 18 MPa in a single ordouble step) is very effective for dispersing solid particles, evenduring storage periods. However this latter aspect should furtherbe study.

Analyzing particle size distribution in soymilk is useful not onlyto determine the individual particle size but also to detect aggre-gates, which behave as big particles. Analysis of particle size wasperformed by examining the distribution curves and through theparameters d3.2 and d4.3 which are the average diameter of particlesbased on surface area and that based on volume of particles,respectively. The latter is especially sensitive to the presence ofaggregates. All samples exhibited a bimodal curve of particle sizedistribution, which was characterized by the presence of two peaks(Fig. 1). UHPH-treated soymilks were characterized by a big peak inthe range of 0e0.6 mm which represented 80e95% of the totalvolume of particles, and a second small peak (>3 mm) which rep-resented 5e20% of the total volume of particles, depending onpressure applied. UHT and pasteurization treatments were char-acterized by a peak in the range of 0.1e1.6 mm which representedapproximately 65 and 20% respectively of the total volume ofparticles, and a second large peak (>3 mm) representing about 35%of total volume of particles for UHT and 80% for pasteurizedsoymilk.

Particle size parameters (Table 3) exhibited a significant(p< 0.05) decrease in UHPH-treated soymilks compared to thebase product and to those soymilks processed by heat treat-ment. However, pasteurized soymilk showed unexpected highvalues of particle size parameters compared to the base product.This might be due to the aggregation of particles caused byusing a single effect homogenizer as this used in the presentstudy as part of the pasteurization line. This phenomenon, wellknown in the dairy industry (Walstra, Geurts, Noomen, Jellema,& van Boekel, 1999), makes using double effect homogenizers tobe preferred. The second valve in double effect homogenizers,such as this used in UHT-treated soymilk in this study, have theobjective of dispersing aggregates formed in the first valve, intosmaller particles. Another data that supports this explanation isthe fact that particle sedimentation was similar in both heattreated soymilks, pasteurized and UHT, since oil dropletsaggregates, experienced creaming in the centrifugation ofsamples.

UHPH technology produced a considerably reduction of themean particle size and also caused the reduction of aggregatesvolume fraction. On the other hand, in UHPH treatments thecombination of pressure and inlet temperature did not producesignificant variations in the mean diameter of particles, while itaffected the production of aggregates. However, no specific relationwas observed between UHPH treatment applied and aggregatesformation. Despite the presence of small quantity of aggregates inthe UHPH-treated soymilks, physical stability of these samples was

Fig. 1. Particle size distribution as determined by light-scattering of raw (d), pasteurized (- -), UHT (.), 200 MPa, 75 C Ti (-C-) and 300 MPa, 75 C Ti (-D-).


not affected as demonstrated by the observed values of particlessedimentation. The presence of aggregates in UHPH-treated soy-based food products has also been described by Floury, Desrumaux,and Legrand (2002) who worked with emulsions stabilized withsoy proteins, and Cruz et al. (2007), who worked with soymilk. Theformation of large particles was observed at 300 MPa for milksamples (Thiebaud et al., 2003) and for soymilk (Cruz et al., 2007).They attributed the aggregates presence to unfolding and dena-turation of whey proteins, which in this condition may interactwith other proteins and/or fat globules. In the UHPH treatment,phenomena of cavitation, shear and turbulence are involvedsimultaneously, affecting the macromolecular conformation ofproteins. In the case of soybean, globulins are strongly affected bythe decrease in solubility, due to the denaturation produced byUHPH treatment above 200 MPa, with the consequent aggregation(Floury et al., 2002). Malaki Nik et al. (2008) have study thecolloidal stability of soymilk in terms of particle size distribution asinfluenced by heat treatment and homogenization. They concludedthat heating of soymilk disrupts large aggregates of soy proteinsand causes a decrease in the particle size. Their conclusions may bepartially in agreement to those observed in this study. However,results have to be interpreted in their specific context. They applieda mild thermal treatment (60 C) to obtain the base product whichwas further heat treated (95e100 C for 7 min) or heated andsubsequently homogenized (69 MPa). However, in the presentstudy, the base product was obtained by a method which includedthe heating at 80 C, causing partial denaturation of soy proteins.Subsequent heat treatments and homogenization of soymilks weremade in different conditions and sequence as those performed byMalaki Nik et al. (2008). The same authors (Malaki Nik, Tosh,Woodrow, Poysa, & Corredig, 2009) have further investigated theinfluence soy protein subunits by studying different soy varietiesand confirmed the influence of protein subunit composition in thecolloidal stability of soymilks. However, it is important to note thatchanging the elaboration process of soymilk might producedifferent results.

3.4. Chemical stability: oxidation and trypsin inhibitor activity

Lipid oxidation involves the formation of hydroperoxides asprimary products in reactions which are developed through freeradicals. There are many factors which favor lipid oxidation: pres-ence of oxygen, light and enzymes such as lipoxygenase, and hightemperatures. In this study, the oxidation process was evaluated bymeasuring the lipoxygenase activity and the hydroperoxideconcentration at days 1 and 15 after processing of soymilks. Asprimary reaction, lipoxygenase catalyzes the hydroperoxidation bymolecular oxygen of linoleic acid and other polyunsaturated lipids(Axelrod et al., 1981). Lipoxygenase activity was determinedbecause it is an initiator of hydroperoxides formation and it maygive some information about rawmaterial manipulation previouslyor during processing. In this study, lipoxygenase was completelyinactivated during the elaboration of soymilk base product (nottreated) and consequently, all subsequent treatments applied tosoymilk did not present any lipoxygenase activity. During theelaboration of the base product, soybeans were ground at 80 C for20 minwhich explains total inactivation of lypoxigenase. Kwok andNiranjan (1995) reported different ways of lipoxygenase inactiva-tion: grinding with water between 80 C and 100 C for 10 min,blanching soaked soybeans in boiling water for 10 min, and boilingdry whole beans inwater for 20 min. Hydroperoxide determinationwas carried out on the first day and 15 days after soymilk storage at4 C (Fig. 2). On the first day after treatment, soymilk samplespresented significantly (p< 0.05) lower hydroperoxide values thanat day 15 at cold storage as expected. The initial hydroperoxidevalue of samples did not exhibit a tendency when comparing UHPHand heat treatments, although the index ranged in a narrowinterval between 0.2 and 0.4 meq/L for all samples. After 15 days ofcold storage all treated soymilks presented higher hydroperoxideindex values and the tendency was defined: heat treated soymilkshad higher values of oxidation index than UHPH treatments,300 MPa at any inlet temperature showed the lowest hydroper-oxide index. Pereda, Ferragut, Quevedo, Guamis, and Trujillo (2008)

0

0.2

0.4

0.6

0.8

1

1.2

1.4

Raw Past UHT 300MPa55ºC

200MPa55ºC

300MPa65ºC

200MPa65ºC

300MPa75ºC

200MPa75ºC

sedixorepordyh

dipiL

)elp

mas

L/edixorep

qe

m(

h

b

g a

i

cd c

j

d

cede

j

j

khk

a

l

ce

Fig. 2. Hydroperoxide values (mean SD) of base product, heat treated and UHPH soymilks at day 1 (,) and day 15 ( ). aee Different letters above bars of day 1 indicate thatsamples are significantly different (p< 0.05). gel Different letters above bars of day 15 indicate that samples are significantly different (p< 0.05).


who applied UHPH to milk reported that 300 MPa producedsamples with less hydroperoxides index compared to 200 MPa.However, the secondary oxidation, studied through malondialde-hyde formation, was higher at this UHPH condition, which indi-cated an evolution in the oxidation process to final products as alsoconfirmed by the higher hexanal content of these samples.

Trypsin inhibitors are substances with anti nutritional proper-ties which are present in animal and vegetal tissues, such assoybeans. They reduce the availability of trypsin, an importantprotease in the animal digestive function for splitting proteins torender dipeptides and tripeptides (Guerrero-Beltrán, Estrada-Girón, Swanson, & Barbosa-Cánovas, 2009). Consumption of rawor inadequately cooked soy products may cause a decrease inprotein digestibility and nutritive value (Yuan, Chang, Liu, & Xu,2008). For this reason, reaching the maximum possible inactiva-tion of trypsin inhibitors is an objective in the production of soyproducts. However, trypsin inhibitors are very stable due to thepresence of disulfide bonds in their molecular structure (Wolf,1977). In the present study, the initial content of trypsin inhibitor

0

0.5

1

1.5

2

2.5

3

Raw Past UHT 300MPa55ºC

205

elp

masfo

L/ITfo

g

b

c

b

a

Fig. 3. Trypsin inhibitor values (mean SD) of base product, heat treated and UHPH sosignificantly differences (p< 0.05).

was of 9.25 g/L in a soymilk obtained with raw soybeans. Fig. 3shows results of residual TI activity after treatment of soymilks.UHPH treatments caused similar inactivation compared to thepasteurized sample (37% of initial activity). UHPH treatmentsshowed a positive correlation between inlet temperature ofsamples and percentage of inactivation, although no significantdifferences (p> 0.05) were observed. However, to reach the sameinactivation effect on soymilk, it was necessary that pasteurizationacted during 30 s at 90 C, whereas in UHPH treatment the holdingtime at high temperature (from 105 to 135 C depending on inlettemperature and pressure applied) was about 0.7 s. So, to reacha significantly higher reduction of TI, it was necessary to combinehigher temperatures and longer holding time such as in the UHTtreatment applied in this work (142 C for 6 s). Under theseconditions, the TI inactivation achieved was 60.8% of the initial TIactivity in the base product. This result is in accordance with valuesobserved in commercial UHT soymilk (data not shown).

Kwok, Qin, and Tsang (1993) reached TI inactivation in soymilkabout 90% at 93 C with holding time of 60 min. Miyagi et al. (1997)

0MPa5ºC

300MPa65ºC

200MPa65ºC

300MPa75ºC

200MPa75ºC

bb

bb b

ymilks. aec Different letters above bars of each treatment indicate that samples are


boiled soymilk by traditional cooking method at 100 C for 30 minand reduced TI to about 10% or less of the initial activity. All theabovementioned studies applied higher temperature during longerholding times comparedwith UHPH treatment applied in this work.However, applying overheating to remove TI activity completelymay destroy important amino acids such as cystine, arginine andlysine (Friedman & Brandon, 2001; Kwok & Niranjan, 1995).

4. Conclusions

The present study demonstrated that UHPH was effective in thereduction of microbial population. Inlet temperature was decisiveto achieve complete reduction in combinationwith pressure. UHPHat 300 MPa and 75 C inlet temperature was able to producecommercially sterile soymilks. In terms of physical stability, UHPHconditions applied produced high stability products, with a consi-derable reduction of particle size and no deposition layer of parti-cles observed during the first week compared to that produced inheat treated samples. The hydroperoxides index also showed lowervalues in UHPH-treated samples compared to heat treated samples,although in order to assess better the complete oxidation caused bytreatments, further analysis must be completed by means ofdetermination of the secondary oxidation products, as well asprofile of volatile compounds and finally sensorial analysis. UHPHtreatments did not achieve the same level of TI inactivation as UHTtreatment. However, a further study of digestibility should beperformed to assess the real importance of the residual activity of TIafter UHPH selected treatment.

Acknowledgments

The authors acknowledge theMinisterio de Ciencia e Innovación(AGL2008-05430) for the financial support to the investigation. Theauthor Poliseli-Scopel gratefully acknowledges the financialsupport for his doctoral studies proportioned by Centre Especial deRecerca Planta de Tecnologia dels Aliments (CERPTA). We thankJoan Miquel Quevedo for his valuable technical support in theCERPTA pilot plant.

References

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Cruz, N., Capellas, M., Hernández, M., Trujillo, A. J., Guamis, B., & Ferragut, V. (2007).Ultra high-pressure homogenization of soymilk: microbiological, physico-chemical and microstructural characteristics. Food Research International, 40(6),725e732.

Donsì, F., Ferrari, G., & Maresca, P. (2009). High-pressure homogenization for foodsanitization. In G. Barbosa-Cánovas, A. Mortimer, D. Lineback, W. Spiess,K. Buckle, & P. Colonna (Eds.), Global issues in food science and technology (pp.309e351). San Diego: Academic Press.

FDA. (1999). Food Labeling: health claim; soy protein and coronary heart disease 21CFR Pt. 101. Final Rule. Fed. Regist, 64, 57700e57733.

Floury, J., Desrumaux, A., & Legrand, J. (2002). Effect of ultra high-pressurehomogenization on structure and on rheological properties of soy protein-stabilized emulsions. Journal of Food Science, 67(9), 3388e3395.

Floury, J., Legrand, J., & Desrumaux, A. (2004). Analysis of a new type of highpressure homogeniser. Part B. Study of droplet break-up and recoalescencephenomena. Chemical Engineering Science, 59(6), 1285e1294.

Friedman, M., & Brandon, D. L. (2001). Nutritional and health benefits of soyproteins. Journal of Agricultural and Food Chemistry, 49(3), 1069e1086.

Guerrero-Beltrán, J. A., Estrada-Girón, Y., Swanson, B. G., & Barbosa-Cánovas, G. V.(2009). Pressure and temperature combination for inactivation of soymilktrypsin inhibitors. Food Chemistry, 116(3), 676e679.

Hamerstrand, G. E., Black, L. T., & Glover, J. D. (1981). Trypsin-inhibitors in soyproducts e modification of the standard analytical procedure. Cereal Chemistry,58(1), 42e45.

Hayes, M. G., Fox, P. F., & Kelly, A. L. (2005). Potential applications of high-pressurehomogenisation in processing of liquid milk. Journal of Dairy Research, 72(1),25e33.

Hayes, M. G., & Kelly, A. L. (2003). High-pressure homogenisation of raw wholebovine milk (a) Effects on fat globule size and other properties. Journal of DairyResearch, 70(3), 297e305.

Hornero-Méndez, D., Pérez-Gálvez, A., & Mínguez-Mosquera, M. I. (2001). A rapidspectrophotometric method for the determination of peroxide value in foodlipids with high carotenoid content. Journal of the American Oil Chemists’ Society,78(11), 1151e1155.

IDF. (1991). Peroxide index determination. IDF Standard 74A. Brussels: InternationalDairy Federation.

Kakade, M. L., Simons, N., & Liener, I. E. (1969). An evaluation of natural vs syntheticsubstrates for measuring antitryptic activity of soybean samples. Cereal Chem-istry, 46(5), 518e526.

Kwok, K. C., & Niranjan, K. (1995). Review: effect of thermal processing on soymilk.International Journal of Food Science & Technology, 30(3), 263e295.

Kwok, K. C., Qin, W. H., & Tsang, J. C. (1993). Heat inactivation of trypsin-inhibitorsin soymilk at ultra-high temperatures. Journal of Food Science, 58(4), 859e862.

Malaki Nik, A., Tosh, S., Poysa, V., Woodrow, L., & Corredig, M. (2008). Physico-chemical characterization of soymilk after step-wise centrifugation. FoodResearch International, 41(3), 286e294.

Malaki Nik, A., Tosh, S., Woodrow, L., Poysa, V., & Corredig, M. (2009). Effect of soyprotein subunit composition and processing conditions on stability and particlesize distribution of soymilk. LWT-Food Science and Technology, 42(7),1245e1252.

Miyagi, Y., Shinjo, S., Nishida, R., Miyagi, C., Takamatsu, K., Yamamoto, T., et al.(1997). Trypsin inhibitor activity in commercial soybean products in Japan.Journal of Nutritional Science and Vitaminology, 43(5), 575e580.

Ostdal, H., Andersen, H. J., & Nielsen, J. H. (2000). Antioxidative activity of urate inbovine milk. Journal of Agricultural and Food Chemistry, 48(11), 5588e5592.

Pereda, J., Ferragut, V., Quevedo, J. M., Guamis, B., & Trujillo, A. J. (2007). Effects ofultra high-pressure homogenization on microbial and physicochemical shelflife of milk. Journal of Dairy Science, 90(3), 1081e1093.

Pereda, J., Ferragut, V., Quevedo, J. M., Guamis, B., & Trujillo, A. J. (2008). Effects ofultra-high pressure homogenization treatment on the lipolysis and lipidoxidation of milk during refrigerated storage. Journal of Agricultural and FoodChemistry, 56, 7125e7130.

Picart, L., Thiebaud, M., René, M., Pierre Guiraud, J., Cheftel, J. C., & Dumay, E. (2006).Effects of high pressure homogenisation of raw bovine milk on alkaline phos-phatase and microbial inactivation. A comparison with continuous short-timethermal treatments. Journal of Dairy Research, 73(4), 454e463.

Smiddy, M. A., Martin, J., Huppertz, T., & Kelly, A. L. (2007). Microbial self life ofhigh-pressure-homogenised milk. International Dairy Journal, 17(1), 29e32.

Suárez-Jacobo, Á., Gervilla, R., Guamis, B., Roig-Sagués, A. X., & Saldo, J. (2010). Effectof UHPH on indigenous microbiota of apple juice: a preliminary study ofmicrobial shelf-life. International Journal of Food Microbiology, 136(3), 261e267.

Tahiri, I., Makhlouf, J., Paquin, P., & Fliss, I. (2006). Inactivation of food spoilagebacteria and Escherichia coli O157:H7 in phosphate buffer and orange juiceusing dynamic high pressure. Food Research International, 39(1), 98e105.

Thiebaud, M., Dumay, E., Picart, L., Guiraud, J. P., & Cheftel, J. C. (2003). High-pres-sure homogenisation of raw bovine milk. Effects on fat globule size distributionand microbial inactivation. International Dairy Journal, 13(6), 427e439.

Van der Ven, C., Matser, A. M., & Van den Berg, R. W. (2005). Inactivation of soybeantrypsin inhibitors and lipoxygenase by high-pressure processing. Journal ofAgricultural and Food Chemistry, 53(4), 1087e1092.

Walstra, P., Geurts, T. J., Noomen, A., Jellema, A., & van Boekel, M. A. J. S. (1999).Homogenization. In Dairy technology: Principles of milk properties and processes(pp. 245e264). New York: Marcel Dekker, Inc.

Wolf, W. J. (1977). Physical and chemical properties of soybean proteins. Journal ofthe American Oil Chemists’ Society, 54(2), A112eA117.

Yuan, S., Chang, S. C., Liu, Z., & Xu, B. (2008). Elimination of trypsin inhibitor activityand beany flavor in soy milk by consecutive blanching and ultra high temper-ature (UHT) Processing. Journal of Agricultural and Food Chemistry, 56(17),7957e7963.

Appendix 2

Characterization of volatile profile in soymilk treated by ultra high pressure

homogenization

Poliseli-Scopel, F.H., Gallardo-Chacón, J.J, Juan, B., Guamis, B., & Ferragut, V.

Journal of Food Chemistry, Submitted.

1

Characterization of volatile profile in soymilk treated by ultra high 1

pressure homogenization 2

3

Fábio H. Poliseli-Scopela, Joan-Josep Gallardo-Chacón

ab, Bibiana Juan

a, 4

Buenaventura Guamisa, Victoria Ferragut

a* 5

6

a Centre Especial de Recerca Planta de Tecnologia dels Aliments (CERPTA), XaRTA, 7

TECNIO, MALTA-Consolider, Departament de Ciència Animal i dels Aliments, 8

Facultat de Veterinària, Universitat Autònoma de Barcelona, 08193-Bellaterra 9

(Cerdanyola del Vallès), Barcelona, Spain. 10

b Felnuti sl. 08027-Barcelona. 11

12

*Corresponding author: Tel.:+34 935813292; Fax: +34 935812006. 13

e-mail adress: [email protected] 14

15

16

Abstract 17

The effect of ultra high pressure homogenization (UHPH) on the volatile profile of 18

soymilk was studied and compared with conventional treatments. Soymilk was treated 19

at 200 MPa combined with two inlet temperatures (55ºC and 75ºC) and treated at 300 20

MPa at 80ºC of inlet temperature. UHPH-treated soymilks were compared with base 21

*ManuscriptClick here to view linked References

http://ees.elsevier.com/foodchem/viewRCResults.aspx?pdf=1&docID=32831&rev=0&fileID=889982&msid=A93D1ECD-40F6-403E-A6E2-30DCBF12E1BC

2

product (untreated sample), pasteurized soymilk (90ºC, 30 s) and ultra high temperature 22

(UHT; 142ºC, 6 s) treated samples. Volatile compounds were extracted by solid-phase 23

microextraction and were identified by gas chromatography and mass spectrometry. 24

Pasteurization and UHPH treatments at 200 MPa produced few changes in the volatile 25

composition, reaching similar values to untreated soymilk. UHT treatment produced the 26

most important effects on volatile profile compared to UHPH at 300 MPa and 80ºC. 27

Hexanal was the most abundant compound detected in all treatments. The effect of 28

UHPH technology on volatile profile induced modifications depending on the 29

combinations of processing parameters. 30

31

Keywords: Ultra high-pressure homogenization (UHPH); soymilk; chemical 32

composition, solid-phase microextraction (SPME); gas chromatography-mass 33

spectrometry (GC-MS). 34

35

1. Introduction 36

The consumption of soymilk in western countries has been limited, partially because of 37

its off-flavors generated during processing, despite some investigation reporting 38

important improvements in the off-flavor elimination (Wang, Xiong, & Wang, 2001). 39

The main off-flavors that could be associated to the limited soymilk acceptation by 40

consumers have been described as green, grassy, paint, rancid, astringent, and bitter 41

(Torres-Penaranda, & Reitmeier, 2001; N'Kouka, Klein, & Lee, 2004; Lozano, Drake, 42

Benitez, & Cadwallader, 2007). Soy odors in soymilk are primarily derived from 43

enzymatic oxidation. Lipoxygenase, an enzyme naturally present in soybeans, acts as a 44

catalyst in the oxidation of polyunsaturated fatty acids, mainly in the soaking and 45

3

grinding steps of soymilk processing (Mizutani & Hashimoto, 2004). Moreover, auto-46

oxidation of linoleic and linolenic acids also plays an important role in the off-flavor 47

generated by degradation products in the last steps of oxidation reaction (Min, Yu, Yoo, 48

& Martin, 2005). Thus, the profile of volatile compounds in soymilk is strongly related 49

to its quality and acceptability. 50

Many researches tried to relate chemical compounds with sensory response. 51

Compounds such as ethyl vinyl ketone, hexanol, hexanal, heptanal, 1-penten-3-ol, 2-52

pentylfuran, 1-pentanol, 2,4-heptadienal, 2-heptanone, 1-octen-3-one, and 1-octen-3-ol 53

were described as causing unpleasant flavor, aroma and taste of soymilk (Wilkens & 54

Lin, 1970; Kobayashi, Tsuda, Hirata, Kubota, & Kitamura,1995; Vara-Ubol, Chambers, 55

& Chambers, 2004). In addition, phenolic compounds such as isoflavones and saponins 56

were reported to be the main nonvolatile off-flavor in soy products because they 57

contributed to the bitter taste and astringent mouthfeel (Matsuura, Obata, & Fukushima, 58

1989). 59

Among the soy odors identified, hexanal is the most studied and the major off-flavor 60

compound in soy products. Commonly, hexanal is related to the sensory beany and 61

grassy flavor in soymilk. Moreover, it has a low sensory detection threshold; in general, 62

content above 25 µg/kg allows its sensory detection (Hashim & Chaveron, 1995; Yuan 63

& Chang, 2007a). 64

Development in processing technologies has helped to improve soymilk quality and to 65

reduce its off-flavors. Examples of these modifications are hot water extractions of 66

soymilk and acid grinding of soybeans. In addition, controlling the soaking time and 67

temperature, the water-to-bean ratio, the method of grinding and the treatment have 68

been suggested to improve off-flavors of soymilk (Achouri, Boye, & Zamani, 2008). 69

Additionally to improvements in soymilk elaboration, conservation treatments play an 70

4

important role in the overall sensory quality of soymilk. Ultra high temperature (UHT) 71

is one example of treatment which has commonly been used in soymilk production. 72

Despite the benefits, such as microbial and enzyme inactivation, thermal treatments 73

cause undesirable chemical changes which may lead to the destruction of amino acid 74

and vitamins, browning reactions and development of cooked flavor (Kwok & Niranjan, 75

1995). 76

In order to improve chemical and physical parameters as well as sensory quality, 77

compromised by thermal technologies, ultra high pressure homogenization (UHPH) is a 78

technology which has been studying a set of parameters such as emulsion stability and 79

microbial and enzymatic activity, with the aim of obtaining products with better quality 80

than those produced by conventional technologies such as pasteurization and UHT 81

(Cruz, Capellas, Hernández, Trujillo, Guamis,& Ferragut, 2007; Pereda, Ferragut, 82

Quevedo, Guamis, & Trujillo, 2007; Poliseli-Scopel, Hernández-Herrero, Guamis, & 83

Ferragut, 2012). 84

In the literature, many reports have studied the profile of volatile compounds and their 85

changes due to treatments applied in soymilk samples (Min et al., 2005; Yuan & Chang, 86

2007a; Achouri et al., 2008). However, only one study has been reported on the effect 87

of UHPH in volatile composition, such as that by Pereda, Jaramillo, Quevedo, Ferragut, 88

Guamis, and Trujillo (2008) on milk. 89

Identification of volatile compounds in soymilk treated by UHPH and comparing them 90

with those identified by conventional thermal treatments could provide information 91

related to sensory quality and better acceptation of soymilk. The purpose of this study 92

was to characterize the volatile profile of soymilk treated at different UHPH conditions 93

and to compare the products with pasteurized and UHT treated samples. 94

95

5

2. Materials and methods 96

2.1 Soymilk elaboration 97

Soymilk elaboration details were obtained following the method described by Poliseli-98

Scopel et al. (2012). Briefly, soybeans were soaked overnight and then were ground in a 99

crushing machine for 20 min at 80ºC. The pulp was separated by filtration, leaving what 100

is considered to be the soymilk base product. 101

102

2.2. Soymilk treatments: UHPH, pasteurization and UHT 103

To obtain pasteurized soymilk, the base product was homogenized at 18 MPa (LAB 104

type: 22.51, Rannie, Copenhagen, Denmark) and subsequently pasteurized using a 105

tubular heat exchanger (ATI, Granollers, Barcelona, Spain) at 95ºC for 30 s. Two step 106

homogenization (18 MPa and 4 MPa) was performed in a homogenizer (X68IE+X68P, 107

Niro Soavi, Parma, Italy) previous to UHT treatment (142ºC for 6 s) with indirect 108

system equipment (6500/010, GEA Finnah GmbH (Ahaus, Germany). 109

Soymilk base product was UHPH-treated using an ultra high pressure homogenizer 110

(model: FPG 11300, Stansted Fuild Power Ltd., Essex, UK) at a flow rate of 120 L/h. 111

The UHPH system was described by Poliseli-Scopel et al. (2012). Three UHPH 112

treatments were performed: 200 MPa at two inlet temperatures (55ºC and 75ºC) and 300 113

MPa at 80ºC of inlet temperature. Samples were collected in sterile bottles in a laminar 114

flow cabin adapted for this purpose. 115

116

2.3 Chemical composition 117

Fat content was determined with ASE 200 (Accelerated Solvent Extraction, Sunnyvale, 118

USA). 1 g of sample was mixed with 1.1 g of sea sand and 0.9 g of celite and then 119

transferring the mix to 11 mL cell extraction. 3 Cycles of 1 min at 120ºC and 1500 psi 120

6

were applied according to the method proposed by the manufacturer. Petroleum ether 121

and 2-propanol (60:40) were used as extracting solvents which were further evaporated 122

with nitrogen and the residue weighted. Dry matter and ash content were analyzed by 123

the reference AOAC method (AOAC, 2000); total nitrogen content was analyzed by the 124

Dumas method (IDF, 2002) and carbohydrates calculated by differences. Total amount 125

was expressed as g/100 g of the sample (w/w). The pH was measured with a pH meter 126

(model GLP 21+ Crison, Alella, Spain). 127

128

2.4 Headspace analysis of volatile compounds 129

Changes in volatile compounds produced by thermal and high pressure treatments could 130

strictly modify odor and flavor of soymilk. SPME is a simple and sensitive technique 131

which allows a direct extraction of the volatile compounds present in soymilk. In 132

addition, this method is economic and ecologic because it does not consume large 133

quantities of solvent. The analyses were carried out after high pressure and thermal 134

treatments. 135

136

2.4.1 SPME – gas chromatography mass spectrometry 137

Volatile compounds analysis was performed following the method described by 138

Achouri, Boye, and Zamani (2006) with some modifications. The SPME fiber used was 139

85 µm CAR-PDMS (Supelco, Bellefonte, PA, USA). 1.5 mL of soymilk sample 140

(previously homogenized using ultrasound equipment with temperature control for 10 141

min) were placed in tubes (4 mL) and incubated for 30 min at 40ºC. The adsorbed 142

volatiles were desorbed in the injector port in splitless at 300ºC for 1 min. Headspace of 143

the volatile compounds was analyzed using an automated gas chromatography (model: 144

HP 6890 Series II, Agilent, Santa Clara, CA, USA). The column model used was 0.25 145

7

µm in a 60 m x 0.25 mm (TRB-Was, Agilent technologies). The mass spectrometry 146

(MS) detector was used in the electron impact ionization (model: HP 5972 Agilent, 147

Santa Clara, CA, USA) with a mass range of 30 – 250 m/z. Before each analysis, the 148

fiber was preconditioned for 1 h at 300ºC. The temperature was programmed in 2 149

stages. The initial temperature was kept at 35ºC for 9 min, and then increased at 5 150

ºC/min to 110ºC for 10 min. In the second stage, the temperature increased at 10 ºC/min 151

to 250ºC for 10 min. Compounds were identified by comparison based on the Saturn 152

spectra reported on Willey 1n.l and NIST 0.5 (National Institute of Standards and 153

Technology) database. Main, molecular, and qualifier ions were selected for each 154

compound indentified. Relative retention times of detected compounds were also 155

determined by injecting 1 µL of alkane standard solutions (Alkane standard solution 156

C8-C20 from Sigma-Aldrich, and Connecticut ETPH calibration mixture C9-C36 were 157

used as standards) in triplicate with a split ratio of 1:200. Signals were processed using 158

Agilent MSD Productivity ChemStation Enhanced Data Analysis software (Agilent 159

technology, Santa Clara, CA, USA). 160

Confirmation of the identification of hexanal, pentanal, 1-octen-3-one, 2,3-161

pentanodione, 1-hexanol, 1-pentanol, 1-octen-3-ol and 2-penthyl furan was performed 162

by comparing GC retention times and mass spectra of individual components with those 163

authentic reference compounds injected under the same operating conditions. 164

165

2.5 Statistical analysis 166

Chemical composition analysis was carried out in triplicate and volatile profile in 167

duplicate. ANOVA was performed on soymilk chemical composition and volatile 168

compounds by SAS (SAS Institute, 2004). The Tukey test was applied to compare the 169

mean values of the samples. Principal component analysis (PCA) was performed to 170

8

reduce the data in two dimensions and identify patterns of variation in the results. R 171

software (R software, Auckland, New Zealand) was used for this purpose. 172

173

3. Results and discussion 174

3.1 Chemical composition 175

Soymilk basic composition was not affected by the treatment applied. Data are given as 176

mean values regardless of the treatment applied to soymilk (heat treatments or UHPH): 177

2.72 ± 0.30 protein; 1.74 ± 0.14 fat; 5.96 ± 0.60 dry matter; 1.47 ± 0.22 carbohydrate; 178

0.22 ± 0.03 ash. Mean pH value was 6.80 ± 0.03. Authors such as Wang et al. (2001) 179

and Cruz et al. (2007) presented similar compositions of soymilk in their studies. 180

181

3.2 Volatile compounds 182

In addition to those natural compounds from soy seeds, responsible for the characteristic 183

volatile profile, other compounds may be generated and the amount of the original 184

compounds may be changed during processing of soymilk. An important part of those 185

compounds is consequence of lipid auto-oxidation. This important lipid degradation in 186

soy and derived products depends on soymilk processing conditions such as light 187

incidence, partial pressure of oxygen and processing temperature. Oxidation may also 188

take place by the enzymatic pathway through the lipoxygenase action. In both, 189

enzymatic and no enzymatic auto-oxidation, hydroperoxidation of polyunsaturated fatty 190

acids such as linoleic and linolenic acids by molecular oxygen (Boatright & Lei, 1999; 191

Kakumyan, Kato, Hajika, & Matsui, 2009) is the primary reaction. The hydroperoxides 192

formed are very unstable and they can easily be transformed to final products such us 193

aldehydes, ketones, furans, alcohols, polymers, etc. Maillard reaction and Strecker 194

9

degradation might also contribute to the formation of volatiles compounds through 195

saccharides and amino acids reaction in thermally processed soymilk (Lozano et al., 196

2007; Plutowska & Wardencki, 2007). 197

Fifty-seven compounds were identified in the headspace of soymilks studied in the 198

present work, which consisted of untreated, pasteurized, UHT and UHPH treated at 3 199

different combinations of temperature and pressure. Table 1 to 5 lists the abundance of 200

volatile compounds grouped by chemical families which were identified as aldehydes, 201

ketones, alcohols, furans, esters and acids. 202

Adehydes and alcohols were the most representative compounds in all chromatographic 203

profiles studied for all treatments in agreement with the study reported by Achouri et al. 204

(2006). In general and considering the total of volatiles for each family of compounds, 205

soymilk treated at 200 MPa, 55ºC and 75ºC inlet temperature respectively, caused 206

similar effects to the pasteurized and unprocessed soymilks. Only some differences 207

were found between specific treatments and families which are later discussed without 208

altering general results (tables 1 to 5). At 300 MPa, the abundance of compounds of all 209

chemical families increased with the increase of pressure and inlet temperature, 210

reaching similar results to UHT soymilk for all groups except for furans for which UHT 211

obtained the highest abundance values (p < 0.05). These results indicated that the 212

adiabatic temperature increase caused by high pressure could be the main reason for the 213

formation of volatile compounds. Temperature after homogenization valve in UHPH 214

treatments at 200 MPa reached values between 106ºC and 117ºC when inlet 215

temperatures of 55ºC and 75ºC, respectively, were applied and 144ºC were reached for 216

soymilk treated at 300 MPa, 80ºC inlet temperature. The residence time at these 217

temperatures in the UHPH treatment was only 0.7 seconds as described by Poliseli-218

Scopel et al. (2012), while pasteurization reached 95ºC for 30 seconds and UHT 142ºC 219

10

for 6 seconds. The differences of temperatures after homogenization valve between 200 220

and 300 MPa were the main cause of the changes observed in the volatile profile among 221

UHPH treatments. On the other hand, differences observed between UHT and 300 MPa, 222

80ºC indicated that high pressure additionally to the temperature could have an 223

important effect on formation of the volatile profile, especially in the oxidative process 224

during treatment application, as discussed below. 225

226

3.2.1 Aldehydes 227

UHT treatment and 300 MPa, 80ºC UHPH condition caused the most significant effect 228

in the total aldehydes compounds. The abundance of this chemical family influenced by 229

treatment is shown in Table 1. Hexanal was the most abundant compound identified 230

followed by pentanal. Other compounds were also detected in low levels such as 231

acetaldehyde, propanal, heptanal, benzaldehyde and 2-hexanal. Except acetaldehyde, all 232

other aldehyde levels changed after treatment (p < 0.05). 233

Of the aldehydes identified in soymilk, hexanal is the most studied because it is 234

indicative of the oxidation degree of product and, as reported, it plays the most 235

important role in the sensory off-flavors. Commonly it is related to beany, grassy and 236

green flavors in soymilk (Yuan & Chang, 2007a). According to Hashim and Chaveron 237

(1995), hexanal has a very low sensory threshold, so content above 25 µg/kg allows its 238

detection. Therefore, hexanal content could be decisive in the sensory quality and as a 239

result soymilk with a low hexanal content would receive a better acceptance by the 240

consumers. Table 1 shows that UHPH treatments at 200 MPa achieved similar levels of 241

hexanal compared to pasteurized and raw soymilk. However, soymilk treated at 200 242

MPa, 75ºC inlet temperature reached values slightly higher than those observed at 200 243

MPa, 55ºC, although this difference was not significant (p > 0.05). These results are in 244

11

accordance to the temperature increase during UHPH application. High temperatures 245

combined with oxygen have an important role in the oxidative processes that take place 246

during and after treatment. Alternatively, 300 MPa, 80ºC and UHT treatments reached 247

the highest levels of hexanal. Similar results were observed by Pereda et al. (2008), who 248

analyzed the effect of UHPH in the volatile profile in milk. They found that 300 MPa, 249

30ºC and 40ºC inlet temperatures showed higher content of hexanal compared to 200 250

MPa at the same inlet temperatures. Several authors (Min et al., 2005; Yuan & Chang, 251

2007a; Yuan & Chang, 2007b; Achouri et al., 2007; Achouri et al., 2008) found also 252

hexanal as the main compound in heat treated soymilk. 253

Percentage of hexanal detected related to total volatile compounds in all treated 254

soymilks varied between 25 and 30%. UHT, 200 MPa, 75ºC and 300 MPa, 80ºC were 255

those treatments which reached the highest hexanal percentage with values around 30% 256

compared to 25% of base product. Wilkens and Lin (1970) reported that hexanal 257

comprises about 25% of the total volatile profile in soymilk. On the other hand, 258

Suratman, Jeon, and Schmidt (2004) found 34%-49% of hexanal related to total 259

volatiles in soymilk added of cyclodextrins and heated at 95ºC for 15 min. Reported 260

hexanal percentage in soymilk by Achouri et al. (2008) was between 50% and 66% 261

taking into account a total of 14 volatile compounds identified in soymilk made from 262

soybeans stored for 10 months. They concluded that making soymilk from stored 263

soybeans for 3 months, resulted in a product with lower volatile compounds formed. 264

Thus, controlling process conditions, method of treatment as well as soybean quality, 265

could help to minimize the hexanal formation and improve soymilk sensory quality. 266

Pentanal was the second most detected compound in the aldehydes group (Table 1). It 267

was formed from oxidation of linoleic acid by the catalytic action of lipoxygenase, 268

mainly in the grinding step of soybeans, when lipoxygenase was still active (Min et al., 269

12

2005; Mizutani & Hashimoto, 2004). The two treatments performed at 200 MPa did not 270

produce pentanal level differences from untreated soymilk. On the other hand, UHT and 271

300 MPa soymilks showed higher values (p < 0.05) than those observed at 200 MPa 272

(Table 1). Minor detected compounds such as heptanal, nonanal and 2-hexenal were 273

also identified by Wilkens and Lin (1970), Suratman et al. (2004) and Min et al. (2005). 274

These compounds in soymilk may be related to grassy flavor and oxidized aroma. In 275

general, results indicated that temperature reached during UHPH treatment, in addition 276

to the pressure, was decisive in aldehydes formation. 277

278

3.2.2 Ketones 279

Ketone compounds are derived mainly from linoleic acid (Wilkens & Lin, 1970). Table 280

2 shows the chromatographic areas of ketones in soymilk studied samples. UHT and 281

300 MPa, 80ºC treatments significantly (p < 0.05) increased total ketones composition 282

while pasteurization and UHPH treatments at 200 MPa reached equivalent values to 283

base product. The main compound detected was ketone, followed by 2,3-octanedione, 284

2,3-pentanedione, 2-butanone, 2-heptanone and 1-octen-3-one. Some of these 285

compounds were also identified by Wilkens and Lin (1970), Boatright and Lei (1999) 286

and Lozano et al. (2007) on soymilk. As observed in Table 2, 2-pentanone, 1-octe-3-287

one, 2-octanone and 3-ocatanone, were not affected by treatment applied compared to 288

base product (p > 0.05). UHT treatment reached higher values of ketone and 2-289

heptanone and 300 MPa, 80ºC had higher levels of 2-butanone and 2,3-octanedione 290

compared to the rest of the treatments. UHPH treatments at 200 MPa did not show 291

significant differences between them and base product, except for acetophenone, 2,3-292

octanedione and 2,3-pentanedione. The most interesting compound of the ketones group 293

is 1-octen-3-one. It has been described by many authors to cause an undesirable flavor 294

13

in soymilk. It was related to green-beany odor (Wilkens & Lin, 1970) and mushroom 295

aroma (Boatright & Lei, 1999; Lozano et al., 2007). In addition, its sensory threshold 296

was described to be approximately 7 µg/mL in heat-treated soymilk (Yuan & Chang, 297

2007a). 298

Due to low sensory threshold, hexanal and 1-octen-3-one may compromise sensory 299

quality of soymilk as described by Yuan and Chang (2007a). Authors reported that a 1-300

octen-3-one content was affected by soybean material, heating method, and heating 301

time. They achieved a drastic reduction of 1-octen-3-one compared to unprocessed 302

soymilk according to the combination of temperature and time parameters during 303

treatment. 304

In addition to 1-octen-3-one, acetophenone and 2,3-pentanedione respectively possess 305

penetrating green and buttery unpleasant odors which may compromise the acceptance 306

of soymilk (Boatright & Lei, 1999; Lozano et al., 2007). However, in our working 307

conditions, the levels of this compound were not affected by any treatment applied. 308

309

3.2.3 Alcohols 310

Volatile alcohols were the second most detected group in all treated soymilks, even in 311

untreated soymilk. Table 3 shows the relative abundance area of alcohols for all 312

soymilks studied. Most of them were associated with green and beany aromas which are 313

characteristic off-flavors of soymilk. No changes were observed in the total alcohols 314

due to heat and UHPH treatment taking into account base product as reference level, 315

although a slight decrease was observed in treatment at 200 MPa (p > 0.05). Some 316

compounds were not affected significantly by the type of treatment: 2-heptanol, (Z)-2-317

penten-1-ol, (Z) 3-hexen-1ol, 2-octanol, 3-octanol, 1-octen-3-ol, 2-ethylhexanol and 1-318

octanol. In general, treatments, except pasteurization, presented only one significantly 319

14

different compound (p < 0.05) to the untreated sample. Ethanol was one of them and 320

moreover, it was the most abundant among this family of compounds. Its origin was 321

reported to be from oxidation of linolenic acid (Wilkens & Lin, 1970). As shown in 322

Table 3, ethanol levels decreased in all treatments compared to base product and 323

showed specially a stronger reduction in 200 MPa UHPH treatments. Min et al. (2005) 324

identified ethanol in soymilk samples as the second most abundant compound detected. 325

On the other hand, Wilkens and Lin (1970) found ethanol as the least abundant alcohol 326

compound in soymilk. 1-Hexanol and 1-pentanol were also identified as relevant 327

compounds in the present study in terms of high volatile contents. Both compounds may 328

be originated from linoleic acid as reported by Wilkens and Lin (1970) and Kobayashi 329

et al. (1995), although a possible alternative of 1-hexanol formation could be through 330

hexanal reduction by alcohol dehydrogenase before treatment (Kakumyan et al., 2009). 331

1-Hexanol and 1-pentanol were associated to beany and green odors (Achouri et al., 332

2008) and 1-hexanol may also be related to harsh grassy and painty odors (Wang et al., 333

2001). Therefore, these compounds in soymilk samples may play an important role in 334

the overall quality aroma of soymilk. UHPH treatment did not affect 1-hexanol levels as 335

shown in Table 3, but pasteurization treatment caused a significant effect with higher 336

values detected. For 1-pentanol, no significant effect of treatment was found. Relevant 337

levels of 1-octen-3-ol and 1-penten-3-ol were also identified in the alcohols group. 338

These compounds were associated respectively to mushroom and pungent aroma 339

(Lozano et al., 2007) and their formations were linked to the oxidation of linoleic acid 340

and linolenic acid, respectively (Wilkens & Lin, 1970). All treatments applied did not 341

affect levels of 1-octen-3-ol, but slight differences, although not significant, were found 342

between 200 MPa, 55ºC (low values) and 300 MPa, 80ºC (high levels). 343

15

1-Hexanol, 1-pentanol and 1-octen-3-ol were also identified by Suratman et al. (2004) 344

and Achouri et al. (2008) and 1-penten-3-ol by Wilkens and Lin (1970) and Lozano et 345

al. (2007). Volatile compounds identification reported by these authors was carried out 346

in heat-treated soymilk. 347

348

3.2.4 Furans 349

Generally, furans in soymilk are associated with unpleasant flavor besides being an 350

indication of color changes due to treatment applied. Furans may be formed by the 351

oxidation of unsaturated fatty acids or by Maillard reaction products (Achouri et al., 352

2007; Yuan & Chang, 2007a). UHT treatment (Table 4) caused the most significant 353

effects in furans composition (p < 0.05). Levels of all compounds increased 354

substantially comparing those of UHPH treatments, pasteurization and untreated 355

soymilk. The Maillard reaction which favors furans formation take place when 356

processing at high temperatures, for instance 121ºC, 143ºC and 154ºC (Kwok & 357

Niranjan, 1995). Processing conditions (time and temperature) of UHT treatment used 358

in the present study were probably the reason for the occurrence of browning reactions, 359

contributing to the increase of furan compounds. Treatments at 200 MPa (Table 4) and 360

that processed by pasteurization did not affect furan composition compared to base 361

product. UHT and 300 MPa, 80ºC produced a significant increase in the levels of 2-362

methyl furan compared to base product, noticeably reaching UHT soymilk levels. The 363

most abundant compound detected was 2- penthyl furan which is the main compound 364

related with grassy and beany off-flavors (Boatright & Lei, 1999; Min et al., 2005; 365

Achouri et al., 2006). 2-Penthyl furan is formed from linoleic acid by singlet oxygen 366

that could be obtained by soy riboflavin or by atmospheric air under light (Min et al., 367

2005). Moreover, it has a low perception threshold (Yuan & Chang, 2007a). Results 368

16

showed that UHPH treatment did not affect levels of 2-penthyl furan which, on the 369

contrary, occurred in UHT soymilk (p < 0.05). For the UHPH system this is a great 370

result because it signifies that it is possible to produce, commercial soymilk with better 371

sensory and color quality than soymilk UHT treated. 372

373

3.2.5 Esters and Acids 374

Ester and acid were the minority compounds in the whole volatile profile of soymilks in 375

this study. In Table 5, a total of 3 acids were identified in soymilk: butanoic acid, 376

pentanoic acid and hexanoic acid. Prevalence of all acids detected increased 377

significantly due to treatment applied, either heat or UHPH. However, no changes were 378

observed in 200 MPa, 55ºC compared to base product. The predominant compound 379

detected was hexanoic acid. In general, its levels increased in all treatments compared to 380

soymilk base product, with a more important increase in 300 MPa, 80ºC (p < 0.05). 381

Lozano et al. (2007) found butanoic acid and hexanoic acid in soymilk heat treated. 382

These compounds were related to cheese aroma and sweaty odor, respectively. 383

According to Wilkens and Lin (1970), hexanoic acid is formed by hexanal oxidation in 384

presence of oxygen which in turn causes a fetid odor. 385

In the esters group, treatments at 200 MPa caused a significant decrease in the total 386

esters composition, while esters remained stable in heat treatments and 300 MP, 80ºC 387

compared to untreated soymilk. Methyl acetate and ethyl acetate were the most 388

abundant compounds detected (table 5). For methyl acetate, only UHT treatment caused 389

a significant increase in the levels detected (p < 0.05) and, on the other hand, ethyl 390

acetate decreased significantly in all treatments applied. Achouri et al. (2006) found 391

ethyl heptanoate, ethyl octanoate and isoamyl acetate in 6 different commercial 392

soymilks, and Kato, Doi, Tsugita, Kosai, Kamiya, and Kurata (1981) found ethyl 393

17

methanoate and ethyl acetate in roasted soybeans, but in this study only ethyl acetate 394

was identified. There is no information in the literature about esters formation or about 395

sensory effect on soymilk, however, they have been related with floral and fruity flavors 396

in other products. 397

398

3.3 Principal component analysis (PCA) 399

PCA is a statistical analysis for resolving sets of data into orthogonal components, 400

whose linear combinations (principal components, PC) approximate the original data to 401

any desired degree of accuracy. In most cases, two components are sufficient to explain 402

a great proportion of the variation in the original parameters. Figure 1 shows the 403

distribution of the samples in the PC1 and PC2 according to volatile composition. 38% 404

and 19% of the variability was explained by PC1 and PC2, respectively. As observed in 405

PC1 there is a clear separation between drastic treatment conditions and mild 406

treatments. UHT and 300 MPa at 80ºC are located on the negative side of PC1 and 407

treatments at 200 MPa, pasteurized and untreated soymilks are located on the positive 408

side. Regarding PC1 model, several aldehydes, 3 ketones (ketone, 3-octen-2-one, 2-409

heptanone), two alcohols (1-butanol and 1-heptanol), 2 acids (pentanoic and hexanoic 410

acids) and all furans achieved very high loadings (Table 6). UHT shows higher values 411

of these compounds compared to 300 MPa, 80ºC. Due to processing parameters of UHT 412

treatment, thermal damage was higher in UHT soymilk than 300 MPa, 80ºC soymilk. 413

Therefore, samples are distributed along the PC1 component according to treatment 414

intensity. In addition, 300 MPa, 80ºC soymilk shows a subtle approach between 415

pasteurized and 200 MPa treatments. Concerning PC2, propanal and primarily 416

benzaldehyde achieved high values in the aldehydes group. Some ketones (2,3-417

pentanedione, acetophenone, 1-octen-3-one and 2,3-octanedione), some alcohols and all 418

18

furan compounds obtained very high values in the PC2 (Table 6). Moreover, the higher 419

values of all furan compounds are related to UHT soymilk. As mentioned above, this 420

class of compounds as well as aldehydes, some ketones and alcohols, are related to off-421

flavors of soymilk. On the contrary, benzaldehyde, which was reported as having a 422

desirable almond flavour (Boatright & Lei, 1999), shows negative loading in the PC2. 423

This compound is more representative in 300 MPa, 80ºC treatment. 424

Differences in the holding time at high temperature and pressure applied during the 425

process played an important role on the type of compound affected. To support this 426

affirmation, treatments at low UHPH pressures and temperatures had similar results to 427

pasteurized and untreated soymilk. Therefore, the real impact of pressure on volatile 428

formation was associated to the temperature generated during UHPH process as a 429

consequence of inlet temperature. High holding times at high temperature was more 430

beneficial to volatile formation than combination of pressure and middle and high 431

temperature at low holding time. 432

433

4. Conclusions 434

The soymilk aroma profile was characterized primarily by aldehyde and alcohol. 435

Compounds of these chemical families were the most detected in all treatments applied 436

as well as untreated soymilk. Furan and ketone compounds were identified in low 437

levels, but they are not less relevant. In general, the main compounds detected in 438

soymilk by other authors were also identified in the present study, primarily compounds 439

responsible for generating off-flavors. On the other hand, not all compounds identified 440

possess a disagreeable aroma. For example, it is well recognized that benzaldehyde has 441

an almond aroma, octanol has an odor slightly reminiscent of roses and 2-heptanone has 442

a flowery odor. Hence, the characteristic aroma of soymilk is not formed by an 443

19

individual compound, but by a mixture of them which can be formed in higher or lower 444

levels according to the treatment applied. Pasteurization and UHPH treatments at 200 445

MPa produced slight changes in the volatile profile compared to untreated soymilk. 446

Although UHPH treatment at 300 MPa, 80ºC inlet temperature achieved similar results 447

to UHT treatment, PCA analysis indicated higher levels of furans in UHT soymilk. 448

High temperature and high holding time were the main processing parameters 449

responsible for affecting changes in volatile profile. Therefore, UHT could be the 450

treatment that produces soymilk with lower sensory acceptance due to results of furan 451

compounds combined with high levels of aldehydes, ketones and alcohols chemicals 452

group. On the other hand, UHPH treatment at 300 MPa, 80ºC with low levels of furan 453

compounds could have good sensorial acceptation, nevertheless, complementary 454

sensory analysis should be made in the future to corroborate these results. 455

456

Acknowledgments 457

The authors acknowledge the Ministerio de Ciencia e Innovación (AGL2008-05430) 458

and European Community's Seventh Framework Programme (FP7/2007-2013-SME- 459

232603) for the financial support to the investigation. Moreover, we thank to Joan 460

Miquel Quevedo for his valuable technical support in the CERPTA pilot plant and to 461

Joan Josep Gallardo Chacón for his technical support in the PCA and chromatographic 462

analysis. 463

464

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550

Table 1. Abundance1 of aldehydes detected in the volatile fraction of soymilks

Treatments4

Name ID2

KI3

BP

Past

UHT

P1 P2 P3

Hexanal MS, RI, P 1092 122.20 a 143.14 a 200.03 b 123.35 a 141.37 a 192.48 b

Pentanal MS, RI, P 990 9.88 a 14.77 a 22.22 b 12.34 a 14.54 a 20.23 b

Acetaldeyde MS 4.29 4.63 4.77 2.74 4.23 4.66

Benzaldehyde MS, RI 1563 1.86 a 6.36 c 3.10 ab 3.66 b 4.28 b 4.66 bc

Propanal MS, RI 804 2.78 a 5.53 b 4.22 ab 4.05 ab 4.00 ab 4.41 ab

2-Butenal MS, RI 1056 1.16 ab 2.01 bc 0.62 a 3.15 c 5.65 d 2.22 bc

3-Methylbutanal MS, RI 929 0.91 a 1.52 ab 1.36 ab 0.93 a 1.23 ab 2.15 b

Heptanal MS, RI 1192 2.44 ab 2.64 a 4.37 d 1.43 c 1.59 ab 3.80 d

2-Hexenal MS, RI 1219 1.48 a 2.03 ab 3.87 c 2.14 ab 2.49 ab 2.73 bc

2-Heptanal MS, RI 1345 1.00 a 1.55 ab 2.67 bc 1.34 ab 1.63 abc 2.77 c

Nonanal MS, RI 1407 0.91 a 1.21 ab 1.69 b 0.96 ab 1.17 ab 1.54 ab

2,4-Hexadienal MS, RI 1430 0.48 a 0.65 ab 1.02 c 0.63 a 0.73 abc 0.95 bc

Octenal MS 0.22 a 0.37 abc 0.72 b 0.31 ac 0.37 abc 0.61 bc

2,4-Heptadienal MS, RI 1520 0.13 a 0.32 bc 0.96 d 0.31 b 0.37 b 0.68 c

2-Pentenal MS, RI 1145 0.90 ab 1.41 c 0.80 ab 0.84 ab 0.97 a 0.59 b

Total 150.64 a 188.14 a 252.42 b 158.19 a 184.64 a 244.47 b

a-d Different superscripts in the same row are significantly different (p < 0.05).

1 Integrated area counts. Mean values x 10

5.

2 Identification: MS = Mass spectra, RI = Retention index compared to Pherobase database,

P = Positively identified by comparison with authentic standard.

3 Kovats retention index calculated.

4 BP = Base product; Past = Pasteurization; P1 = 200 MPa, 55ºC; P2 = 200 MPa, 75ºC; P3 =

300 MPa, 80ºC.

Table(s)

Table 2. Abundance1 of ketones detected in the volatile fraction of soymilks

Treatments4

Name ID2

KI3

BP Past UHT P1 P2 P3

Ketone MS, RI 826

12.05 abc 12.99 bc 25.10 d 9.87 ab 6.95 a 15.91 c

2,3-Octanedione MS, RI 1333

3.72 a 5.46 ab 4.36 a 7.34 bc 8.00 c 8.47 c

2,3-Pentanedione MS, RI, P 1072

3.77 a 5.17 b 3.30 a 6.39 bc 7.24 c 6.14 bc

2-Butanone MS, RI 910

2.30 a 2.83 a 2.68 a 3.57 a 3.05 a 6.14 b

2-Heptanone MS, RI 1190

2.07 a 3.85 b 5.63 c 2.59 ab 2.76 ab 3.36 ab

1-Octen-3-one MS, RI, P 1314

1.49 1.79 1.64 1.94 1.90 1.94

2-Octanone MS, RI 1295

1.02 1.51 1.30 1.25 1.27 0.99

2-Pentanone MS, RI 1070

0.62 0.87 0.95 0.78 1.22 1.00

3-Octen-2-one MS, RI 1427A

0.54 a 0.64 a 1.20 b 0.64 a 0.67 a 1.30 b

3-Octanone MS 0.46 0.62 0.64 0.52 0.50 0.55

Acetophenone MS, RI 1693

0.16 a 0.47 bc 0.32 ab 0.41 bc 0.49 bc 0.63 c

Total 28.20 a 36.19 a 47.12 b 35.28 a 34.06 a 46.45 b



5.

2 Identification: MS = Mass spectra, RI = Retention index compared to Pherobase database

and (A) Kobayashi et al. (1995), P = Positively identified by comparison with authentic

standard.



300 MPa, 80ºC.

Table(s)

Table 3. Abundance1 of alcohols detected in the volatile fraction of soymilks

Treatments4

Name ID2

KI3


Ethanol MS, RI 944 143.76 a 102.99 ab 103.54 ab 65.93 b 63.84 b 133.41 a

1-Pentanol MS, RI, P 1260 32.58 a 33.36 a 27.53 a 31.20 a 32.90 a 43.14 b

1-Hexanol MS, RI, P 1359 31.76 a 62.91 b 34.15 a 39.28 a 41.70 a 37.47 a

1-Octen-3-ol MS, RI, P 1454 15.88 16.94 17.54 11.82 12.36 15.63

1-Penten-3-ol MS, RI 1175A 12.72 ab 12.07 ab 13.10 ab 10.64 a 10.86 ab 14.14 b

3-Methyl-1-butanol MS, RI 1260 2.00 ab 4.45 c 1.40 b 2.59 a 2.63 a 3.09 a

(Z)-2-penten-1-ol MS, RI 1328 2.52 2.77 1.72 1.88 1.93 2.44

2-Methyl-1-butanol MS, RI 1217 1.88 ab 2.34 b 1.32 a 2.01 ab 2.07 b 2.11 b

1-Butanol MS 1.05 ab 1.20 ab 1.56 b 0.93 a 1.10 ab 1.42 ab

1-Heptanol MS, RI 1460 0.94 a 1.13 ab 1.34 b 0.93 a 1.01 ab 1.24 ab

2-Heptanol MS, RI 1323 0.73 0.99 0.83 0.77 0.80 0.78

1-Octanol MS, RI 1562 0.73 0.71 0.69 0.74 0.63 0.71

2-Octanol MS, RI 1421 0.66 0.86 0.73 0.71 0.73 0.63

(Z)-3-hexen-1-ol MS, RI 1466 0.59 0.78 0.54 0.57 0.62 0.67

3-Octanol MS, RI 1395 0.40 0.48 0.40 0.38 0.38 0.41

2-Ethylhexanol MS, RI 1492 0.40 0.27 0.66 0.59 0.61 0.88

(E )-2-penten-1-ol MS 0.33 ab 0.37 ab 0.42 ab 0.29 a 0.28 a 0.47 b

Total 248.92 ab 244.61 ab 207.47 ab 171.26 a 174.46 a 258.62 b

a-c Different superscripts in the same row are significantly different (p < 0.05).


5.


and (A) Vichi, Pizzale, Conte, Buxaderas and López-Tamames (2003), P = Positively

identified by comparison with authentic standard.



300 MPa, 80ºC.

Table(s)

Table 4. Abundance1 of furans detected in the volatile fraction of soymilks

Treatments4

Name ID2

KI3


2-Penthyl furan MS, RI, P 1235A 10.61 a 13.96 a 40.89 b 10.97 a 11.96 a 16.36 a

2-Ethyl furan MS, RI 965A

6.56 a 9.70 a 35.00 b 6.42 a 6.65 a 11.03 a

2-Propyl furan MS, RI 1044 1.22 a 1.58 a 22.11 b 1.42 a 1.54 a 3.17 a

2-Vinyl furan MS, RI 1085 0.74 a 0.75 a 2.98 b 0.71 a 0.68 a 0.98 a

2-n-Butyl furan MS, RI 1135 0.46 a 0.53 a 2.61 b 0.59 a 0.65 a 0.57 a

2-Methyl furan MS, RI 881 0.24 a 0.35 ab 1.25 c 0.26 a 0.28 a 0.46 b

Total 19.84 a 26.87 ab 104.83 c 20.36 a 21.77 ab 35.56 b

a-c Different superscripts in the same row are significantly different (p < 0.05).


5.


and (A) Vichi et al. (2003), P = Positively identified by comparison with authentic standard.



300 MPa, 80ºC.

Table(s)

Table 5: Abundance1 of esters, acids and others compounds detected in the volatile fraction

of soymilks

Treatments4

Name ID2

KI3


Esters

Ethyl acetate MS, RI 897 6.02 a 4.45 b 3.70 bc 2.84 bc 2.89 bc 4.84 ab

Methyl acetate MS, RI 839 3.02 abc 3.69 ab 7.33 d 2.33 bc 2.18 c 3.85 a

n-Hexyl acetate MS, RI 1278 0.41 0.38 0.48 0.46 0.46 0.45

n-Amyl acetate MS, RI 1178 0.32 a 0.44 ab 0.55 ab 0.70 b 0.33 a 0.42 ab

Total 9.95 ab 8.96 a 12.22 b 6.48 c 6.01 c 9.79 ab

Acids

Hexanoic acid MS, RI 1946 24.52 a 35.44 bc 39.38 bc 30.65 ab 36.72 bc 44.46 c

Pentanoic acid MS, RI 1747 1.84 a 3.63 bc 4.64 c 2.92 ab 3.66 bc 4.39 bc

Butanoic acid MS, RI 1638 0.14 a 0.32 ab 0.28 ab 0.27 ab 0.35 b 0.33 b

Total 26.50 a 39.39 b 44.30 bc 33.83 ab 40.73 bc 49.18 c

Others

Carbon dissulphide MS 1.48 a 1.42 a 0.43 b 1.22 a 1.32 a 1.38 a



5.

2 Identification: MS = Mass spectra, RI = Retention index compared to Pherobase database,

P = Positively identified by comparison with authentic standard.



300 MPa, 80ºC.

Table(s)

Table 6. Loading and percentage variance accounted by the first two principal

components of soymilk volatile profile.

Principal Component

PC1 PC2

Aldehydes

2-Butenal 0.093 -0.149

2-Pentenal 0.062 -0.056

Benzaldehyde -0.038 -0.246

Acetaldeyde -0.072 0.086

Propanal -0.078 -0.195

3-Methylbutanal -0.093 -0.148

Nonanal -0.180 -0.038

2-Heptanal -0.187 -0.078

Heptanal -0.188 0.061

2,4-Hexadienal -0.190 -0.087

Octenal -0.193 -0.045

2-Hexenal -0.194 -0.044

Hexanal -0.197 -0.021

Pentanal -0.198 -0.056

2,4-Heptadienal -0.204 -0.010

Ketones

2,3-Pentanedione 0.065 -0.249

2,3-Octanedione 0.004 -0.255

1-Octen-3-one -0.033 -0.243

2-Octanone -0.039 -0.115

2-Butanone -0.052 -0.200

Acetophenone -0.053 -0.273

2-Pentanone -0.069 -0.178

3-Octanone -0.122 -0.029

Ketone -0.176 0.106

3-Octen-2-one -0.180 -0.045

2-Heptanone -0.198 0.009

Alcohols

2-Methyl-1-butanol 0.113 -0.164

3-Methyl-1-butanol 0.069 -0.175

(Z)-3-hexen-1-ol 0.032 -0.078

1-Hexanol 0.017 -0.196

(Z)-2-penten-1-ol -0.002 -0.076

1-Pentanol -0.025 -0.177

Ethanol -0.029 0.128

2-Octanol -0.033 -0.093

3-Octanol -0.042 -0.103

1-Octanol -0.044 -0.017

2-Heptanol -0.061 -0.122

2-Ethylhexanol -0.080 -0.121

1-Octen-3-ol -0.110 0.045

Table(s)

Principal Component

PC1 PC2

Alcohols

1-Penten-3-ol -0.127 -0.006

(E )-2-penten-1-ol -0.150 -0.053

1-Butanol -0.175 0.019

1-Heptanol -0.189 -0.037

Furans

2-n-Butyl furan -0.178 0.102

2-Ethyl furan -0.181 0.119

2-Propyl furan -0.182 0.122

2-Vinyl furan -0.185 0.116

2-Methyl furan -0.190 0.102

2-Penthyl furan -0.195 0.094

Esters

Ethyl acetat 0.027 0.129

n-Hexyl acetat -0.050 -0.044

n-Amyl acetat -0.053 0.009

Methyl acetat -0.169 0.145

Acids

Butanoic acid -0.078 -0.245

Hexanoic acid -0.156 -0.186

Pentanoic acid -0.172 -0.145

Ohters

Carbon dissulphide 0.139 -0.054

Porcentage of variance explained 38 19

Figure(s)

Figure 1. Loadings plot after principal component analysis of the individuals in the

plane defined by two first principal components (PC1 and PC2).

Figure(s)

Appendix 3

Effect of UHPH on selected Bacillus spores isolated from soymilk and almond

beverage

Poliseli-Scopel, F.H., Valencia-Flores, D., Ferragut, V., Guamis, B., & Hernández-

Herrero, M.

1

Effect of UHPH on selected Bacillus spores isolated from soymilk and 1

almond beverage 2

3

Fábio H. Poliseli-Scopel, Dora Valencia-Flores, Victoria Ferragut, Buenaventura 4

Guamis, Manuela Hernández-Herrero 5

6

Centro Especial de Recerca Planta de Tenoclogia dels Aliments (CERTPTA), XaRTA, 7

TECNIO, MALTA-Consolider, Departament de Ciència Animal i dels Aliments, Facultat de 8

Veterinària, Universitat Autònoma de Barcelona, Bellaterra 08193. Barcelona, Spain. 9

10

11

*Corresponding author: Tel +34 935811460; Fax: +34 935812006. 12

E-mail address: [email protected] 13

14

15

Abstract 16

The effect of ultra high pressure homogenization (UHPH) on the bacterial spore’s inactivation 17

of soymilk and almond beverage was studied. Previously, spores of Bacillus cereus, Bacillus 18

2

subtilis, Paenibacillus taichungensis, Paenibacillus glucanolyticus and Lysinibacillus spp 19

were isolated and selected by showing resistance to the UHPH treatment of 300 MPa at 55 20

and 65ºC of inlet temperature of vegetable beverage (soy and almond). Vegetable beverages 21

were submitted to sterilization (121ºC, 15 min) and then inoculated with the different 22

bacterial strains before UHPH treatment at 300 MPa combined with four inlet temperatures 23

(55ºC, 65ºC, 75ºC and 85ºC). Soymilk were inoculated with Bacillus cereus and P. 24

taichungensis, whereas almond beverage were inoculated with B. subtilis, P. glucanolyticus 25

and Lysinibacillus spp. Results showed that as the inlet temperature increased, the bacterial 26

spores inactivation increased. Complete inactivation was achieved at 85ºC of inlet 27

temperature for B. subtilis, P. glucanolyticus, P. taichungensis and Lysinibacillus spp., being 28

the latter most sensible to UHPH treatment. Bacillus cereus presented high resistance to the 29

UHPH treatment of soymilk. 30

31

Keywords: Soymilk; almond beverage; UHPH treatment; spores inoculation; inactivation. 32

33

34

1. Introduction 35

Bacterial spores (endospores) are common contaminants in foods. Sources of contamination 36

in food chain may include soil, feces, animal feeds and food ingredients and processing. The 37

bacterial spores are among the most resistant forms of living organisms. Their resistance 38

affect espcially food processing and primary to the products stored during long period of time 39

(Carlin, 2011). Nowadays, the Bacillus specie is known to be split into 18 genus, among 40

which Alicyclobacillus, Aneurinibacillus, Brevibacillus, Geobacillus, Lysinibacillus, 41

Paenibacillus, Ureibacillus, Virgibacillus and Sporosarcina (Fritze, 2004). 42

3

Because of ubiquity and resistance and persistence, contamination of bacterial spores in food 43

and food processing facilities is almost impossible to avoid. Processed foods are increasingly 44

a combination of multiple ingredients, each bringing its own spore-forming bacteria into the 45

final products (Carlin, 2011). The spore concentrations in some ingredients such as spices, 46

dehydrated vegetables or flours can be quite high (exceeding 102–103 spores/g) (Anonymous, 47

2002; Berghofer et al., 2003; Hara-Kudo et al., 2006; Oomes et al., 2007; Sharma et al., 2009; 48

Postollec et al., 2012). Because of their implication in food poisoning and food spoilage, 49

Bacillus spp. inactivation is a major objective in food processing. Processed foods are stable 50

at room temperature with mesophilic spore formers as target organisms in moderate climates 51

(Nauta et al., 2003; Scheldeman et al., 2005; Oomes et al., 2007; De Jonghe et al., 2010). 52

Dormant bacterial endospores are extremely resistant to heat and other preservation 53

treatments, such as irradiation, chemicals or pressure, compared with vegetative cells 54

(Chaves-López et al., 2009). The heat resistance of bacterial spores can be attributed to three 55

main factors. protoplast dehydration, mineralization and thermal adaptation (Cazemier et al., 56

2001; Jagannath et al., 2005; Oomes et al., 2007). The thermal resistance of spores is to some 57

extent species-dependent, but it is also affected by sporulation conditions such as cationic 58

environment, temperature and pH. These factors influence the protoplast water content, which 59

appears to be inversely correlated to the spore heat resistance (Palop et al., 1999ab; Cazemier 60

et al. 2001). Consequently, spore deactivation in foods requires high levels of technological 61

treatments (heat, irradiation or disinfectants), which can in turn have negative effects on the 62

sensory and nutritional profile. For this reason, alternative methods have been studied in 63

recent years such as hydrostatic high pressure or ultra-high pressure homogeneization (Ju et 64

al., 2008; Sharma et al. 2009; Pinho et al., 2011). 65

UHPH is a novel technology that lately has been applied in food, cosmetic and 66

pharmaceutical areas to fragment particles for producing fine and stable emulsions, to modify 67

4

the viscous properties of fluids due to the particle size reduction, to facilitate metabolite 68

extraction as well as to achieve inactivation of microorganisms, enzymes or even some 69

viruses. The most important parameters that influence the microbial inactivation by high-70

pressure can be divided into 3 different groups: process parameters (pressure, temperature, 71

hold time, number of passes and type of equipment); microbial-physiological parameters and 72

parameters that are related to characteristics of the fluid. Despite several hypotheses, the exact 73

mechanism of how UHPH inactivates microorganisms has not yet been fully elucidated. High 74

pressure and velocity gradients, shear stresses, turbulence, shocks and cavitation phenomena 75

occurring through the high-pressure valve can induce mechanical disruption and/or at least 76

alteration of cell membranes (Diels & Michiels, 2006). Pathanibul et al., (2009) suggested 77

that when the fluid temperature in the high-pressure valve reached 80 °C, microbial 78

inactivation could mainly be explained by thermal effect. High resistance of bacterial spores 79

to UHPH-treatments has been reported at mild inlet temperatures (Feijoo et al., 1997; 80

Bevilacqua et al., 2007). However, increasing inlet temperature (>55ºC) and operating 81

pressure seem to have positive sporicidal effects in Bacillus spores. 82

The purpose of this study was to evaluate the inactivation of spores of Bacillus genus 83

previously isolated from soymilk and almond beverage treated by UHPH. Spores were 84

inoculated in sterile soymilk and almond beverage and treated by UHPH at 300 MPa and 55, 85

65, 75 and 85ºC of inlet temperature. 86

87

2. Material and Methods 88

2.1 Isolate collection and selection 89

Isolates were obtained from soymilk and almond beverage samples treated by UHPH at 300 90

MPa and 55 and 65ºC of inlet temperature. Spore counts were assessed by heat shock at 80ºC 91

5

for 10 min, quickly cooled in ice and plated on PCA medium (Oxoid Ltd, 96 Basingstoke, 92

Hamphire, UK) and incubated for 48-72 h at 30ºC. Typically, colonies representing visually 93

distinct morphology (ranging from 1 to 5 colonies per sample) were selected and streaked for 94

purity on TSA. Biochemical testing on cultures using the API 50 CHB kit (bioMérieux, 95

Marcy l'Etoile, France) was performed and thereafter, genetic identification was established 96

based on comparative 16S rRNA sequences. Briefly, isolates were cultured on TSA. Bacterial 97

genomic DNA was prepared by using DNeasy tissue kit (QIAgen, Valencia, CA, USA). 16S 98

rRNA genes were amplified by conventional PCR. Amplicons were analyzed on 1% agarose 99

gels containing ethidium bromide. The sequencing was performed at Macrogen Inc (Seoul, 100

Korea). DNA sequences of the 16S rRNA were aligned with the Clustal method from 101

MegAlign (DNAStar Inc., Madison, USA) assembling with 95 % minimum match. The 102

obtained nucleic acid sequences were analyzed with the algorithm BLASTN at the National 103

Center for Biotechnology Information (NCBI). 104

105

2.2 Sporulation conditions 106

Sporulation of all Bacillus strains was performed following the method EN-13704 standard 107

(Anonymous, 2002). A suspension of Bacillus spores was prepared from an exponential phase 108

culture of vegetative bacteria in tryptone glucose broth (0.25% yeast extract; 0.5% tryptone; 109

0.1% glucose and pH was adjusted to 7.2). Approximately 2–3 mL of this culture were 110

transferred to Roux flasks containing meat yeast extract agar (1% meat extract; 0.2% yeast 111

extract, 0.004% MnSO4·H2O; 1.5% agar and pH was adjusted to 7.2) and was incubated for 112

8–10 days at 30 °C. The culture was harvested and purified by repeated centrifugation 113

(10000g × 20 min) and washed with sterile distilled water. The suspensions were heat-114

shocked for 30 min at 75 °C in order to kill vegetative cells. All the spore preparations were 115

free (>97%) of sporulating cells, cell debris and germinated spores, as determined by phase-116

6

contrast microscopy. Finally, the spores were stored in double distilled sterile water at 4 °C 117

until use. 118

119

2.3 Preparation and UHPH processing of vegetable beverages 120

Soymilk and almond beverage were prepared according to the method developed by Poliseli-121

Scopel et al. (2012) and Valencia-Flores et al. (2012), respectively. Briefly, soybeans 122

(Majesta variety) were hydrated (3:1 water-to-soybean ratio) for 15 h at room temperature and 123

then ground in a crushing machine with heating control (adapted from Frigomat, Milan, Italy) 124

for 20 minutes at 80ºC with recirculation in a colloidal mill (E. Bachiller B. S.A, Barcelona, 125

Spain). The pulp obtained was separated by filtration (model CE98, Mejisa-Mectufry, Jijona, 126

Spain). To obtain the almond beverage, 4% (w/w) grinded almond seed in water at 60 ºC was 127

mixed in a tank for 8 min, circulated in a colloidal mill (E. Bachiller, B. S. A) for 12 min and 128

subsequently filtrated to separate the liquid phase using a 0.100 mm steel sieve. 129

A benchtop ultra-high pressure homogeniser model nG12500 from Stansted Fluid Power Ltd. 130

(Essex, UK) was used for the assays. This high pressure equipment comprises two 131

intensifiers, driven by a hydraulic pump and a high pressure valve made of resistant ceramics 132

able to support 400 MPa. All these components guarantee a constant flow rate of 15 L/h 133

during the process. Inlet and outlet temperatures of vegetable beverages were controlled by 134

two heat exchangers located before the machine entrance and after the second 135

homogenization valve, respectively. Temperature probes located before and after the pressure 136

valve allowed monitoring inlet temperature (Ti) and the temperature reached after the high-137

pressure valve (T1) respectively, the same as outlet temperature of the product (Tf) which 138

were measured after the cooling heat exchanger. The spores (105–106 spores/mL) suspended 139

in 500 mL of sterilized samples (sterilization at 121ºC for 10 min) were subjected to UHPH 140

treatment of 300 MPa and 55, 65, 75 and 85 ºC of inlet temperature. 141

7

2.4 Spores recovery 142

Between 80 and 100 ml of the processed samples were taken for analysis. Spore counts were 143

quantified in glucose yeast agar (0.1% casamino acids without vitamins; 0.1% soluble starch; 144

0.25% glucose; 0.5% yeast extract; 0.01% FeSO4; 0.00001% MnSO4·H2O; 1.5% agar and 145

pH was adjusted to 6.8), incubating at 30 °C for 72 h. Further investigation was carried out to 146

confirm if complete inactivation had been achieved. For this purpose, 50 mL of beverage 147

samples were incubated at 30ºC for 10 days. During this period, coagulation and phase 148

separation were checked visually. Moreover, a loopful of incubated samples was streaked out 149

on glucose yeast agar, which was incubated at 30 °C for 48-72 h. Lethality was calculated as 150

the difference between the logarithms of colony counts of the untreated and treated samples 151

(log No- log N). 152

153

2.5 Statistical analysis 154

All the experiments were performed three times. The results were expressed as the mean of 155

three replicates for each treatment. Statistical analyses were performed with either Statistical 156

Package for the Social Sciences 19.0.1 software (SPSS Inc, Chicago, ILL) One-way analysis 157

of variance and SNK (Student-Newnan-Keuls) was used for comparing sample data. 158

Evaluations were based on a significant level of p < 0.05. 159

160

3. Results and discussion 161

3.1 Spore-former occurrence 162

Spore-forming bacteria prevalence in vegetable beverages treated at 300 MPa at 55 and 65ºC 163

of inlet temperature was investigated. Several genus and species of Bacillus were identified: 164

Lysinibacillus and Paenibacillus (P. taichunengis and P. glucanolyticus). Of these Bacillus 165

species, B. cereus and Paenibacillus taichungensis were the most frequently encountered on 166

8

UHPH soymilk, while B. cereus, B. subtilis and Lysinibacillus spp on UHPH almond 167

beverage. Precisely, these indigenous spore-forming bacteria were selected to evaluate the 168

UHPH effect on their lethality at 300 MPa and 55, 65, 75 and 85ºC inlet temperatures. 169

The contamination of foods with bacterial spores is well documented in several foodstuffs. 170

The multiplication of the vegetative cells formed after spore germination and outgrowth can 171

occur at a wide range of temperature (spore-forming bacterial species which include 172

psychrotrophic, mesophilic or thermophilic). Water activity and pH can be the cause of 173

foodborne poisoning or food spoilage (Carlin, 2011). In the present study, the more resistant 174

aerobic mesophilic spore-forming bacteria found in vegetable beverages treated by UHPH at 175

300 MPa at relative high inlet temperatures (55 and 65ºC) coincide partially with those found 176

by Postollec et al. (2012). They investigated the occurrence of spore-forming bacteria in 90 177

foodstuffs (raw materials, dehydrated ingredients and processed foods) and reported that the 178

most frequently encountered Bacillus species were B. cereus, B. subtilis and B. licheniformis, 179

isolated from dehydrated vegetables. In food industry, hygiene management and processing 180

largely contribute to lower spore-forming bacteria contamination ensuring quality and safety 181

of final products (Postollec et al., 2012). Bacillus cereus, for instance, is widely distributed in 182

natural and commercial products due to the strong resistance of its spores to physical and 183

chemical disinfectant agents. The organism is associated with two types of gastrointestinal 184

syndromes, denominated as emetic type and diarrhoeal type (Ju et al., 2008; De Jonghe et al., 185

2010). Other Bacillus species, such as B. subtilis, have generally been considered of low 186

significance in food poisoning incidents (From et al., 2005). Food spoilage can be produced 187

by two different ways: by survival of viable spores to the treatment (pasteurization/UHT) with 188

subsequent growth, causing therefore the characteristic spoilage; or by extracellular enzymes 189

(proteases, lipases and lecithinases) that are synthesized prior to heat treatment. According to 190

9

Rodríguez-Lozan et al. (2010), conventional treatments are able to eliminate viable 191

organisms, but do not inactivate the preformed heat-stable enzymes. 192

B. subtilis is considered a common cause of food spoilage. Their heat resistant spores, often 193

pose a challenge to the thermal efficacy of heat processes in order to extend shelf-life of 194

processed foods (Jagannath et al., 2005). On the other hand, Paenibacillus has recently been 195

recognized as a separate genus from Bacillus and many new species of Paenibacillus continue 196

to be identified (Ivy et al., 2012). They have been isolated from various different pasteurized 197

foodstuffs (Guinebretiere et al., 2001) and even by ultra high temperature (UHT) treatment 198

applied in milk (Scheldeman et al., 2004). Recently, Paenibacillus spp. has described as 199

spoilage organisms. Some species appear to be predominantly psychrotolerant with an ability 200

to grow in milk and possibly other foods at temperatures as low as 6ºC (Ivy et al., 2012). 201

202

3.2 UHPH effect on spores survive 203

Spores of B. cereus and P. taichungensis were suspended in sterilized soymilk and B. cereus, 204

B. subtilis and Lysinibacillus spp. were suspended in sterilized almond beverage. The UHPH 205

effect has been evaluated by measuring the capability of spores to survive and proliferate in 206

solid medium after treatment. Among of 7-8 log cfu/mL of each bacterial strain were 207

inoculated in sterilized before treatment. In order to evaluate the total elimination of 208

inoculated strains and the possible recovery of sub-lethally injured cells, samples UHPH 209

treated at 75ºC and 85ºC of inlet temperature were incubated for 10 days at 30ºC. Table 1 210

shows UHPH effect in the reduction of B. cereus, P. taichungensis, B. subtilis and 211

Lysinibacillus spp. in sterile vegetable beverages. All treatments applied caused significant 212

reduction in the colony counts of the strains inoculated (P < 0.05). 213

In general, B. cereus achieved great log reductions for all UHPH treatments, reaching 214

complete inactivation at 75 and 85ºC of inlet temperature for almond beverage. Lysinibacillus 215

10

spp. was the most sensible to the UHPH treatment of all bacteria experimented, with complete 216

inactivation at 300 MPa and 55ºC. Similar levels of inactivation were achieved for P. 217

taichungensis in soymilk and B. subtilis in almond beverage, being more sensible to the 218

treatment than B. cereus. Regarding this latter, B. cereus inoculated in almond beverage was 219

more sensible to the UHPH treatment than soymilk. P. taichungensis obtained higher log 220

reductions compared to B. cereus, reaching complete inactivation at 300 MPa, 85ºC. These 221

results indicated that inlet temperature played an important role in the strains resistance 222

against treatment. In addition, as inlet temperature increased, high microbial inactivation was 223

reached. During the period of incubation, no coagulation and no microbial growth were 224

observed as indicated in table 1. For the UHPH condition of 300 MPa, 85ºC, B. cereus strain 225

in soymilk reached reduction about 5 log cfu/mL, while 55, 65 and 75ºC reached, 226

respectively, values around of 1, 2.5 and 3.5 log cfu/mL. Lethality strains of each UHPH 227

condition are shown in Figure 1. 228

Regarding the lethality of each bacterial spore, no differences were observed between 65 and 229

75ºC for B. cereus 1. For P. taichungensis, B. subtilis and B. cereus 2, significant lethality 230

was obtained at 75ºC of inlet temperature (about 6, 5.75 and 5.66 log units, respectively), 231

being constant at 85ºC. Lysinibacillus spp. reached a lethality of 5.22 log units in the UHPH 232

condition of lowest intensity. Among inlet temperature applied, only significant differences 233

were observed at 85ºC between B. cereus 1 and the rest of bacterial strains. 234

Feijoo et al. (1997) investigated spores inactivation of Bacillus licheniformis in ice cream 235

high-pressure homogenized. They reached reductions of 0.5 log units applying pressures from 236

50 to 200 MPa at 33ºC of inlet temperature. Chaves-López et al. (2009) evaluated the 237

influence of high-pressure homogenization treatment at 150 MPa (one, two or three stages) in 238

the inactivation of Bacillus cereus spore and Bacillus subtilis spore suspended in sterilized 239

distilled water. When single stage was applied, a negligible reduction of colony counts at 150 240

11

MPa was observed. In fact, they obtained inactivation values of 0.5 log cfu/mL for B. subtilis 241

and 0.3 log ufc/mL for B. cereus. However, applying three consecutive treatment stages, high 242

reduction of colony counts (about 5 log units) were obtained. In the present study, UHPH 243

treatment at single stage was enough to reach about 6 log units (P. taichungensis), indicating 244

that pressure of 300 MPa was more effective than number of stages at 150 MPa. 245

Treatment parameters such as inlet temperature, temperature after homogenization vale, outlet 246

temperature and pressure were monitored during UHPH treatment. Table 2 shows changes of 247

temperature and pressure during vegetable beverages processing. Treatments at 55ºC, 65ºC, 248

75ºC and 85ºC of inlet temperature achieved maximum temperature after high pressure valve 249

near to 109ºC, 120ºC, 130ºC and 138ºC respectively. These values may explain why spores 250

lethality reached high values in samples treated at 85ºC, as it was expected. In the UHPH 251

processing, the food fluid is brought to high pressure in few seconds and then forced through 252

a very small orifice, the valve gap of few micrometres in width. The resulting pressure drop 253

generates intense mechanical forces and elongational stress in laminar flow at the valve 254

entrance and in the valve gap. Turbulence, cavitation and impacts with solid surfaces occur at 255

the gap outlet. Due to these phenomena, the food fluid experiences a short-heating 256

phenomenon that increases as pressure increase (Dumay et al., 2012). According to Sharma et 257

al. (2009), spore inactivation requires a combination of shear and temperature. In this way, the 258

resultant temperature elevation due to high pressure in the UHPH treatment has a positive 259

effect in the destruction of bacterial spores. Some studies have reported germination 260

stimulation of spore accompanied by sub-lethal heating, rendering germinated spore more 261

sensitive to destructive action of temperature and mechanical forces as were vegetative cells 262

(Hyung-Yong et al., 1999; Ananta et al., 2001). Therefore, the flow of inoculated vegetable 263

beverages through the homogenizing valve may have stimulated spores germination in the 264

food fluid, rendering them sensitive to heat and mechanical forces (Diels & Michiels, 2006; 265

12

Chaves-López et al., 2009). Nevertheless, it is worth to point that the holding time at high 266

temperature was lower than 0.3 s, resulting in a minimal thermal damage respect to usual heat 267

treatments applied in food industries. Although the residence time was very low, it was 268

essential to reach high levels of spore inactivation. 269

Additionally to processing parameters, the type of foodstuff may influence the effectiveness 270

of the treatment. Available results indicated that UHPH treatments were more effective 271

against microorganisms in saline buffered solutions than complex matrices (Vachon et al., 272

2002; Diels et al., 2005). Foodstuff is a complex chemical system in which most of 273

components, such as protein, lipids and carbohydrates may affect microbial tolerance to heat 274

or pressure, making difficult to study the possible protective mechanism of the interactions 275

among those components (Roig-Sagués et al., 2009). Therefore, the possible reason of higher 276

resistance of B. cereus 1 to the UHPH treatment than rest of bacterial strains could be due to 277

soymilk composition and to the components interaction in addition to cellular structure of the 278

spore strain. Structurally, an endospore consists of a core, surrounded by a cortex of 279

peptidoglycan, a spore coat of protein and in some species a delicate thin layer called 280

exosporium. Mature core of endospore differs greatly of cytoplasm of vegetative cell from 281

which it is derived. In addition, it is rich in calcium dipicolinate, contains small water content 282

(10-30%) from the vegetative cell, becoming gel consistency the cytoplasm core (Diels & 283

Michiels, 2006). These characteristics of spore confer high resistance to the treatment in 284

addition to the matrix characteristics. On the other hand, when cell water content is about 285

10% the resistance to pressure and temperature remained unaffected. However, beyond 10% 286

moisture, the resistance of spores is gradually reduced, enabling a particular pressure-287

temperature combination to achieve an adequate level of spore reduction (Ananta et al., 288

2001). In addition, water content from the matrix, in this case soymilk, could be transferred to 289

the spore cells increasing its sensibility to pressure-temperature effect (Chaves-López et al., 290

13

2009). This approach was first elucidated from heat inactivation studies of spores in fat 291

systems (Molin & Snygg, 1967; Harnulv et al., 1977). 292

293

4. Conclusion 294

Endospores inactivation is the main objective in food sterilization. It is well known that 295

dormant spores are highly resistant to many physical and chemical agents. UHPH treatment 296

under pressure of 300 MPa was effective in the spore’s inactivation, mainly at 75 and 85ºC of 297

inlet temperature. Applying these inlet temperatures, the maximum temperature reached in the 298

high-pressure valve was 130 and 138ºC respectively. Although the holding time at high 299

temperature was lower than 0.3 s, as the inlet temperature increased the lethality of all 300

bacterial strains assayed increased. So that, a complete inactivation for P. taichungensis in 301

soymilk samples and B. subtilis, B. cereus and Lysinibacillus spp. in almond beverage were 302

achieved using 85ºC of inlet temperature. As well reported in the literature, the mechanical 303

effects that take place during the pass of vegetable beverage through the high-pressure valve, 304

is responsible for the gradual temperature rise that demonstrated play an important role to 305

achieve complete inactivation of bacterial spore. Thus, this study has shown that UHPH 306

technology could be an alternative to conventional treatment, able to produce vegetable 307

beverage with extended shelf-life. 308

309

Acknowledgments 310

The authors acknowledge the Ministerio de Ciencia e Innovación (AGL2008-05430) and 311

European community’s framework Programa (FP7/2007-2013-SME-232603) for the financial 312

support to the investigation. Moreover, we thank to Roger Escriu for his valuable technical 313

support in the CERPTA pilot plant. 314

14

315

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437

438

Table 1. Counts (log cfu/mL ± SD) of bacterial strains inoculated in vegetable beverages before and after treatment. 439

Treatments Soymilk Almond beverage

B. cereus 1 P. taichungensis B. Subtilis B. cereus 2 Lysinibacillus spp.

Before Treatment 5.28 ± 0.55a 6.01 ± 0.24a 5.69±0.04a 5.33±0.12a 5.65±0.02a

300 MPa, 55ºC 4.08 ± 0.22b 3.19 ± 0.57b 3.48±0.23b 2.03±0.32b 1.66±0.27b

300 MPa, 65ºC 2.90 ± 0.99bc 1.98 ± 0.93c 2.21±0.17c 1.33±0.38c ND1

300 MPa, 75ºC 1.78 ± 1.55c 0.20 ± 0.35d ND ND ND

300 MPa, 85ºC 0.54 ± 0.47d ND ND ND ND

440 a-d Different superscripts in the same column are significantly different (p < 0.05). 441 1 No coagulation was detected after sample incubation at 30ºC for 10 days. 442

443

444

Figure 1. Letalithy (mean ± SD) of UHPH treatment on B. cereus 1, P. taichunengis, B. 445

cereus 2 and Lysinibacillus spp. spores. 446

a-c Different letters above bar of each Bacillus strain indicate that samples are significantly 447

different (p < 0.05). 448

A-B Different letters above each inlet temperature indicated that samples are significantly 449

different (p < 0.05). 450

451

452

453

454

455

456

457

Table 2. Temperature and pressure changes1 of UHPH-treated soymilk during processing. 458

Pressure Ti (ºC) T1 (ºC) Tf (ºC)

309.67 ± 12.01 55.25 ± 1.06 112.5 ± 3.54 11.15 ± 1.92

310.00 ± 8.54 66.25 ± 0.35 120.0 ± 1.41 13.30 ± 0.42

304.00 ± 3.61 75.75 ± 1.06 129.50 ± 0.71 12.15 ± 0.07

313.33 ± 3.51 86.00 ± 0.0 138.00 ± 1.41 16.30 ± 0.28

459 1 Ti = inlet temperature; T1 = temperature after the homogenization valve; Tf = temperature 460

after final heat exchanger. Mean values ± SD from 3 individual productions. 461

462

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