The Production of Protein Isolates from the Aqueous

Extraction of de-hulled Yellow Mustard Flour and

Determination of their Functional Properties

by

Benjamin Hijar Soltero

A thesis submitted in conformity with the requirements for the degree of Master of Applied Science (M. A. Sc.)

Graduate Department of Chemical Engineering and Applied Chemistry University of Toronto

© Copyright by Benjamin Hijar Soltero (2013)

ii

The Production of Protein Isolates from the Aqueous Extraction of de-hulled Yellow

Mustard Flour and Determination of their Functional Properties

Benjamin Hijar Soltero

Master of Applied Science

Graduate Department of Chemical Engineering and Applied Chemistry

University of Toronto

2013

ABSTRACT

Two types of protein isolates were prepared from de-hulled yellow mustard flour by aqueous

extraction, membrane processing and acid precipitation of proteins at the isoelectric point (IEP

5.5). Their electrophoretic, main functional properties and protein composition were determined.

The precipitated and acid soluble protein isolates had 83.0 and 96.0% protein content on a

moisture and oil free basis, respectively. The acid soluble protein isolate had comparable

functional properties to those of commercially available soybean and other protein isolates. The

precipitated protein isolate exhibited less desirable functionality than the soluble isolate, due to

its high lipid content (~25%); however, it was still comparable to soybean isolates. Storage

temperature had limited effect on lipid oxidation, and hence the stability of the precipitated

protein isolate at 25-45ºC. Taste and texture of wieners and bologna prepared with 1-2% of this

isolate as binder were comparable to those prepared with soy protein isolates.

iii

ACKNOWLEDGMENTS

First and foremost I would like to thank Professor L. L. Diosady for giving me the opportunity of

being part of the Food Engineering group and the opportunity of pursuing this degree. His

guidance, advice and support throughout this project made possible its successful completion. I

would also like to thank both Professor O. Trass and Professor C. Q. Jia for their valuable

feedback. I would also like to thank the Mexican Council of Science and Technology

(CONACYT) for making this project possible through its financial support. My sincere thanks

also go to all my colleagues in the Food Engineering group, especially Solmaz, Sayeh, Veronica

and Bih King for their assistance. Finally, I would like to thank my family and friends for their

support, especially my wife Elizabeth.

iv

TABLE OF CONTENTS

ABSTRACT .............................................................................................................................................. ii

ACKNOWLEDGMENTS .................................................................................................................... iii

TABLE OF CONTENTS ...................................................................................................................... iv

LIST OF FIGURES .............................................................................................................................. vii

LIST OF TABLES .............................................................................................................................. viii

1. INTRODUCTION............................................................................................................................... 1

2. LITERATURE REVIEW ................................................................................................................. 2

2.1 Mustard seed ............................................................................................................................................... 2 2.1.1 Types of mustard ............................................................................................................................................. 2 2.1.2 Mustard uses .................................................................................................................................................... 5

2.2 Mustard seed components ........................................................................................................................... 6 2.2.1 Oil .................................................................................................................................................................... 8 2.2.2 Protein .............................................................................................................................................................. 8 2.2.3 Protein-oil interactions in mustard seeds ....................................................................................................... 13 2.2.4 Glucosinolates ................................................................................................................................................ 15 2.2.5 Phytates .......................................................................................................................................................... 17 2.2.6 Phenolic compounds ...................................................................................................................................... 18

2.3 Protein extraction .......................................................................................................................................20 2.3.1 Solvent extraction process ............................................................................................................................. 21 2.3.2 Aqueous extraction process ........................................................................................................................... 23

2.4 Protein purification and isolation ...............................................................................................................26 2.4.1 Ultrafiltration ................................................................................................................................................. 28 2.4.2 Diafiltration .................................................................................................................................................... 30

2.5 Functional properties in protein isolates ....................................................................................................32 2.5.1 Hydration properties ...................................................................................................................................... 33 2.5.1.1 Water and oil absorption ............................................................................................................................. 34 2.5.1.2 Protein Solubility ........................................................................................................................................ 35 2.5.2 Properties related to protein surface ............................................................................................................... 35 2.5.2.1 Foaming properties ..................................................................................................................................... 37 2.5.2.2 Emulsifying properties ................................................................................................................................ 37

v

2.5.3 Properties related to protein structure: Gelation ............................................................................................ 38

2.6 Lipid oxidation in lipid-protein systems .....................................................................................................39 2.6.1 Lipid and protein oxidation mechanisms ....................................................................................................... 40 2.6.2 Accelerated lipid oxidation evaluation ........................................................................................................... 42

3. PROJECT OBJECTIVES .............................................................................................................. 44

4. EXPERIMENTAL METHODS .................................................................................................... 45

4.1 Starting materials .......................................................................................................................................45

4.2 Solvents ......................................................................................................................................................45

4.3 Reagents .....................................................................................................................................................45

4.4 Equipment and materials ...........................................................................................................................47

4.5 Experimental Methods ...............................................................................................................................48 4.5.1 Aqueous extraction process ........................................................................................................................... 48 4.5.2 Membrane processing of protein solution and isoelectric precipitation ......................................................... 51 4.5.3 Protein isolates recovery ................................................................................................................................ 54 4.5.4 Temperature effect on lipid oxidation in the precipitated protein isolate ....................................................... 56 4.5.5 Functional properties ..................................................................................................................................... 56 4.5.6 Other analytical methods ............................................................................................................................... 58

5. RESULTS AND DISCUSSION ..................................................................................................... 60

5.1 Starting material analysis ...........................................................................................................................60

5.2 Aqueous extraction process ........................................................................................................................60

5.3 Membrane processing of protein solution and isoelectric precipitation .....................................................63

5.4 Protein isolates recovery ............................................................................................................................65

5.5 Functional properties .................................................................................................................................71 5.5.1 Colour and Flavour ........................................................................................................................................ 71 5.5.2 Gel electrophoresis ........................................................................................................................................ 71 5.5.3 Nitrogen Solubility Index (NSI) .................................................................................................................... 74 5.5.4 Water absorption capacity (WAC) and oil absorption capacity (OAC) ......................................................... 75 5.5.5 Emulsifying Properties ................................................................................................................................... 78 5.5.6 Foaming Properties ........................................................................................................................................ 80 5.5.7 Gelation .......................................................................................................................................................... 83

5.6 Temperature effect on lipid oxidation in the precipitated protein isolate ...................................................84

vi

6. MEAT PRODUCT TESTING ....................................................................................................... 88

7. CONCLUSIONS ............................................................................................................................... 91

8. RECOMMENDATIONS ................................................................................................................. 95

8. REFERENCES .................................................................................................................................. 97

9. APPENDICES................................................................................................................................. 107

APPENDIX A ................................................................................................................................................ 108

Analytical Methods ........................................................................................................................................ 108

APPENDIX B ................................................................................................................................................ 128

Results ........................................................................................................................................................... 128 B1. Yellow mustard flour analyses ....................................................................................................................... 129 B2. Aqueous extraction ......................................................................................................................................... 131 B3. Membrane processing and isoelectric precipitation ....................................................................................... 134 B4. Protein isolates analyses ................................................................................................................................. 138 B5. Functional properties ...................................................................................................................................... 140 B6. TBA values for the precipitated protein isolate stored at different temperatures ........................................... 145 B7. Meat testing forms and results ....................................................................................................................... 149

vii

LIST OF FIGURES

Figure 1: Types of mustard seeds .................................................................................................................. 4

Figure 2: Average fix oil content of mustard seeds in Canada ..................................................................... 7

Figure 3: Average crude protein content for mustard seeds in Canada ......................................................... 7

Figure 4: Amino acid structure ..................................................................................................................... 9

Figure 5. Structure of oil bodies (Huang 1992) .......................................................................................... 14

Figure 6: Isothyocianate release reaction for sinapis alba .......................................................................... 16

Figure 7: Isothyocianate release reaction for brassica juncea .................................................................... 17

Figure 8: Chemical structure of phytic acid ................................................................................................ 18

Figure 9: Chemical structure of phenolic compounds ................................................................................. 19

Figure 10: Main operations in a solvent extraction system (Becker 1970) ................................................. 22

Figure 11: Main operations in aqueous extraction systems (Cater, et al. 1974) ......................................... 24

Figure 12: Ultrafiltration principle of operation ......................................................................................... 28

Figure 13: Fatty acid radical chain oxidation mechanism ........................................................................... 41

Figure 14: Flow diagram of the aqueous extraction process ....................................................................... 50

Figure 15: Ultrafiltration/Diafiltration process schematics ......................................................................... 51

Figure 16: Protein extract membrane processing ........................................................................................ 55

Figure 17: Aqueous extraction. Mass balance of key components (*Estimate) .......................................... 63

Figure 18: Protein fractions A and B after membrane processing .............................................................. 66

Figure 19: Yields for the membrane process and isoelectric precipitation of the protein solution ............. 68

Figure 20: Protein isolates comparison ....................................................................................................... 71

Figure 21: Non-reducing conditions SDS-PAGE patterns of the precipitated protein isolate (lanes a and

b), acid soluble protein isolate (lanes c and d) and protein standards (lane e) ............................................ 72

Figure 22: Reducing conditions SDS-PAGE patterns of the precipitated protein isolate (lanes a and b),

acid soluble protein isolate (lanes c and d) and protein standards (lane e) ................................................. 73

Figure 23: Foam stability expressed as the foam volume (%) remaining against time ............................... 83

Figure 24: Malondialdehyde formation in the precipitated protein isolate at three temperatures ............... 87

viii

LIST OF TABLES

Table 1: Seeded area and production of Canadian mustard .......................................................................... 5

Table 2: Fatty acid composition of Yellow and Brown mustard .................................................................. 8

Table 3: Values of oriental and yellow mustard amino acid composition compared to FAO indispensable

amino acid requirements ............................................................................................................................. 11

Table 4: Reagents used for experiments ..................................................................................................... 45

Table 5: Equipment and materials used for the experiments ...................................................................... 47

Table 6: Yellow mustard flour characterization .......................................................................................... 60

Table 7: Protein and oil composition of the resulting fractions after aqueous extraction ........................... 62

Table 8: Protein and oil composition of the resulting fractions before and after membrane processing .... 67

Table 9: Final product characterization ....................................................................................................... 70

Table 10: Nitrogen solubility index value of different protein isolates....................................................... 75

Table 11: Water absorption capacity for different protein isolates ............................................................. 76

Table 12: Oil absorption capacity for different protein isolates .................................................................. 77

Table 13: Emulsifying properties of protein isolates .................................................................................. 79

Table 14: Foam expansion values for protein isolates ............................................................................... 80

Table 15: Foam stability data ...................................................................................................................... 81

Table 16: Foam volume stability values for selected protein isolates ......................................................... 82

Table 17: Least gelation concentration values for selected protein isolates ............................................... 83

Table 18: TBA values for the starting materials ......................................................................................... 85

Table 19: TBA values for samples stored at different temperatures ........................................................... 86

Table 20: Ratings for wieners produced with precipitated protein isolate and meal residue derived from

the aqueous extraction process, membrane processing and isoelectric precipitation .................................. 89

Table 21: Ratings for bologna produced with precipitated protein isolate and meal residue derived from

the aqueous extraction process, membrane processing and isoelectric precipitation .................................. 89

Table 22: Pairs of mean ratings with significant differences at the 95% confidence level ......................... 90

1

1. INTRODUCTION

Canada is the largest exporter of mustard seeds in the world and stands in second place in world

production with an average of 176,600 metric tonnes, with yellow mustard representing over

40% of the total production.

Yellow mustard (sinapis alba) seed is mostly used to prepare food condiments, such as the hot-

dog mustard. Its uses are limited in food products because of its anti-nutritional components,

spicy flavour precursors and astringent phenolic compounds. Furthermore, the retained oil

readily oxidizes, resulting in rancidity over time.

The seed is high in oil and protein. Since the oil is high in erucic acid, a monounsaturated fatty

acid associated with certain heart conditions its use has been prohibited in North America and

Europe from human consumption but there is a great potential for its use in biofuel production.

Mustard protein, on the other hand, has a well-balanced amino acid profile and a high nutritional

value. However protein use in food systems relies more in the desirable functional properties and

sensory attributes they are able to provide. Animal proteins have been traditionally the main

source of functional ingredients, however it is estimated that about 8 kg of protein from a

vegetable source are needed to produce 1 kg of animal protein. Considering this low ratio, the

increasing protein demand due to population growth and land use competition between food

crops and biofuel, oilseeds such as mustard offer an interesting alternative as a renewable source

of oil for biofuel production and protein to provide sensory characteristics and nutritional value

to food products as a replacement of animal protein.

2

Oil extraction from oilseeds is usually performed using hexane, achieving high extraction yields

but causing significant damage to the protein during desolventizing, affecting its functionality. In

addition, there are cost, environmental and safety implications limiting the use of hexane as an

extraction solvent. Aqueous extraction processes allow simultaneous recovery of oil and protein

with improved functionality without the use of hazardous solvents. In our Food Engineering

group, several research projects have focused on the aqueous extraction of Canadian oil seeds

(Canola, rapeseed and mustard) in order to obtain high extraction yields for both oil and protein.

Yields of more than 90% protein and up to 80% oil have been achieved in the case of yellow

mustard. Due to the presence of anti-nutritional components in the seed, such as glucosinolates,

phytates and phenolic compounds, further processing is necessary to obtain high quality protein

isolates. Techniques developed in our Food Engineering laboratory to treat the protein extract

include membrane processing by ultrafiltration and isoelectric precipitation for protein recovery.

On the other hand, as a result of the aqueous extraction and the presence of oleosin and other

proteins, the protein isolates will inherently contain some oil that could impact their

functionality.

This project aims to find an optimized process for the production of high quality protein isolates

with low levels of anti-nutritional factors and low oil content starting with the aqueous extraction

of de-hulled yellow mustard flour, and the determination of the food functionality and thermal

stability of the protein products produced. Ultimately, the project is expected to result in protein

isolates with high purity, low oil content and with the required food functionality for their

application in the food industry.

3

2. LITERATURE REVIEW

2.1 Mustard seed

Mustard is a series of plants of the genera Brassica and botanical family Cruciferae. Evidence of

human use of mustard seeds has been traced back to 4000 B.C. in China and Pakistan (Fenwick,

Heaney and Mullin 1982), carbonized seeds dated to 3000 BC have also been found in Iraq,

evidencing the use of mustard by the Mesopotamian civilization (Zohary and Hopf 2000). The

cultivation of mustard is believed to have been introduced to Europe by the moors. Its spread

around Europe during the middle ages can be explained by factors such as the Crusades and the

development of commerce around the Mediterranean (Fenwick, Heaney and Mullin 1982). The

English word mustard has its origin in the French term “moustarde”, from Latin “mustum”.

2.1.1 Types of mustard

There are three different kinds of mustard seeds: black mustard (brassica nigra), popular in the

Middle East and parts of Asia; brown and oriental mustard (brassica juncea), whose origin is

uncertain, with proposed sources between Eastern Europe, the Middle East or China (Labana and

Gupta 1993); and yellow mustard (sinapis alba), which originated in the Mediterranean region

and is broadly consumed around the world.

Black mustard seeds are roughly globular with a diameter of 1 to 1.5 mm and a dark brown

colour; the seed coat is pitted and when soaked in water the seeds produce a strong pungent

odour. Brown mustard seeds are similar to black mustard seeds, their diameter is less than 2 mm

and have a reddish brown to dark brown colour, it is primarily grown for the European market

and has also become popular in North America as a replacement of yellow mustard. Oriental

4

mustard seeds vary in colour from yellow to dark yellow and brown. It is mostly used in the

Asian and Japanese markets as a condiment. These varieties have a pungent taste and contain

about 28% of oil and 30% of protein (Heath 1981). Yellow mustard seeds, on the other hand,

vary in colour from light creamy yellow to yellow and in some cases yellowish brown, have a

roughly globular shape and have a diameter of 2 to 3 mm; the seed coat is minutely pitted, and

seeds turn mucilaginous when soaked in water. Yellow mustard has a pungent taste and is low in

starch, contains about 30% of oil and 25% of protein (Heath 1981) (Figure 1).

Figure 1: Types of mustard seeds

Mustard is a broad-leaved, yellow-flower plant that requires a short growing season, between 85

to 95 days for yellow mustard seeds to reach maturity and between 95 to 105 days for the

oriental and brown varieties to reach maturity (McKenzie 2010). Crops require an annual

precipitation of between 350 and 450 mm and give higher yields in temperate zones with a cool

and dry weather. Mustard is capable of growing in a variety of soils from sandy loam to clay

loam (Agroecommerce Network Private Ltd. 2002). Mustard seeds are considered more tolerant

to frost, drought and heat than other crops like canola or flax, which makes the dry brown and

dark soils, warm dry summers and cold dry winters in the southern Canadian prairies an ideal

place for mustard growth.

Yellow Mustard Oriental Mustard Brown Mustard Black Mustard

5

The sowing of mustard in Canada began in the 1930s with a modest 40 hectares, but in the next

30 years, it quickly grew to 60,000 hectares (Agriculture and Agri-Food Canada 2011). As of

2007, mustard crop occupied 176,000 hectares of harvested area with an annual production of

114,000 tonnes, representing a farm gate value of around 100 million dollars (Agriculture and

Agri-Food Canada 2007). Canada is considered the world largest exporter of mustard seed and

the second largest producer (Canadian Special Crops Association 2007) surpassed only by India.

Mustard seed production for the year 2011 is presented in Table 1.

Table 1: Seeded area and production of Canadian mustard

Region Seeded Area (2011)1

Production (2011)2

Mean Production

(2001-2010)2

Manitoba - - 2.6

Saskatchewan 107.3 103.2 140.3

Alberta 20.2 21.6 33.7

Total 127.5 124.8 176.6

1 Thousand hectares. November Estimates of Production of Principal Field Crops, Catalogue no. 22-002-X, vol. 90 no. 8 Released December 6, 2011; Statistics Canada

2 Thousand tonnes. Small Area Data 1976-2010 Statistics Canada, Agriculture Division, Crop Section

2.1.2 Mustard uses

Pythagoras mentioned the use of mustard seeds for scorpion stings and Hippocrates used it for

the preparation of medicines. The medicinal properties of mustard were known to the Greeks and

Romans, and ancient documents written by Cato, Columella and Pliny (Fenwick, Heaney and

Mullin 1982) suggest that mustard seeds were cultivated and used as a condiment, mixing the

6

ground seeds with wine must to make a paste, hence the name “mustard”. The use of mustard

seeds to prepare food condiments is still their main use, and has a wide range of applications in

the food industry. Dried seeds are milled for flour production and wet milling is used to

manufacture mustard paste. Whole ground seeds are also used for spice mix preparations and

meat processing. Traditional or hot-dog mustard is prepared using the whole ground seed.

Mustard is also used as a protein source, flavour enhancer and as a binder in the manufacturing

of processed meats. The different mucilage contents in the three varieties of mustard allow the

manufacturing of products with different viscosities. Seed hulls are also used as a thickening

agent and stabilizers in prepared foods. Heat inactivated whole ground seed is used in a variety

of food products to enhance their flavour, colour, texture and viscosity and it can also be used as

an emulsifier agent. The presence of sinigrin in the brown and oriental varieties makes them

suitable for the manufacture of hot mustard for the European market and the production of

mayonnaise. High oil content oriental mustard is used to cover the oilseed demand in the Indian

subcontinent where one of its main uses is cooking oil production (Jimmerson 2005).

2.2 Mustard seed components

Mustard seeds contain a hull that represents between 15 and 20% of the seed weight and is

composed of a hygroscopic integument containing lignin, cellulose, hemicellulose and mucilage,

while the kernel makes up between 80 and 85% and contains most of the oil, proteins and soluble

sugars. They have a thin endosperm membrane and occur in seed pods varying in quantity from

10 to 40 seeds (Appelqvist 1971). Mustard seeds contain 28-32% protein by weight and 30-35%

of oil, although these values can vary slightly between varieties, growing regions and crop years

as shown by Figures 1 and 2 (Canadian Grain Commission 2012).

7

Figure 2: Average fix oil content of mustard seeds in Canada

Survey data from the Grain Research Laboratory shows that cool and moist weather tends to

increase the fixed oil content in the seed as well as the iodine values, on the other hand protein

content tends to be lower (Siemens 2011).

Figure 3: Average crude protein content for mustard seeds in Canada

0.0%

10.0%

20.0%

30.0%

40.0%

50.0%

2007 2008 2009 2010 2011

Fixed oil content for Canadian mustard crops

Yellow Mustard Oriental Mustard Brown Mustard

0.0%5.0%

10.0%15.0%20.0%25.0%30.0%35.0%40.0%

2007 2008 2009 2010 2011

Crude protein content for Canadian mustard crops

Yellow Mustard Oriental Mustard Brown Mustard

8

2.2.1 Oil

Between 95 and 98% of the oil in brassica seeds is composed of triglycerides, and only a small

amount, in the range of 0.3 to 0.5% are free fatty acids (Appelqvist 1971), although the quantity

may increase due to incorrect seed handling after harvest. The content of mono and diglycerides

is usually low. The typical brassica juncea and sinapis alba varieties have a high erucic acid

content (Table 2).

Nonsaponifiable material in mustard seeds is low and in the order of 0.5% of the oil. Mustard

seeds also contain polar lipids apart from nonpolar triglycerides, mainly phospholipids and

galactolipids which are comparable to soybean phospholipids.

Table 2: Fatty acid composition of Yellow and Brown mustard

Seed type Palmitic (%) Oleic (%) Linoleic (%) Linolenic (%) Eicosenoic (%) Erucic acid (%)

Brassica juncea 2-4 7-22 12-24 10-15 6-14 18-49

Sinapis Alba 2-3 16-28 7-10 9-12 6-11 33-51

2.2.2 Protein

Around 28-32% of the mustard seed total weight is composed of proteins. Proteins are polymers

of amino acids. Proteins form the structural elements of cells and tissue in the human body and

are considered as the basis of life, but they are also essential components in different food

systems. Proteins are complex bio-molecules formed by amino acid aggregates and are

essentially composed of carbon (50-55%), hydrogen (6-7%), oxygen (20-23%), nitrogen (12-

29%) and sulfur (0.2-3%) but also may contain phosphorous, iron, magnesium and copper

among other elements.

9

The building blocks of proteins are L-α-amino acids, organic compounds containing a central

carbon atom connected to a basic amino group (-NH2), an acid carboxyl group (-COOH) and one

of the 20 possible organic substituents (R) as shown in Figure 4. These substituents differ in their

physical and chemical properties and hence are the basis of the physicochemical differences in

proteins such as polarity, acidity, basicity, conformational flexibility, reactivity and functionality.

Amino acids can be classified according to the chemical characteristics of the substituent chain

in: nonpolar, polar uncharged, polar positively charged and polar negatively charged (Ludescher

1996).

Regardless of the side chain, amino acids are zwitterions at neutral pH, which means that are

molecules with both a positive and a negative electrical charge (Figure 4).

Amino acids can polymerize through the formation of a peptide bond into polypeptides, which

are the basic constituents of proteins.

The peptide bond is a kind of covalent bonding between the amine group of an amino acid and

the carboxyl group of another, producing an amide and a water molecule. The electronic

structure of the peptide bond gives proteins and peptides their conformational properties

(Ludescher 1996).

Amino acid Zwitterion

Figure 4: Amino acid structure

10

The shape and functionality of proteins are determined by their secondary, tertiary and

quaternary structures, while their composition or sequence of amino acids along their backbone

determines their primary structure. The secondary structure of a protein is the spatial

configuration of the amino acid sequence. Secondary structures can be periodic, where there is a

repetition in the values of the dihedral angles generating a helix, such as α-helices and β-sheets

structure types; and aperiodic, where there is no repetition of the dihedral angles, like in β-turns

structures (Ludescher 1996). In proteins, the secondary structure is defined by non-covalent

interactions and patterns of hydrogen bonds between the backbone amide and carboxyl groups.

The tertiary structure describes the atomic coordinates of each atom in a protein molecule. It is

the folded and complete tri-dimensional structure of the polypeptide chain and is consequence of

all non-covalent interactions between the amino acids in the molecule and between the molecule

and the solution. Quaternary structure is the result of the association through weak non-covalent

bonds of several polypeptide chains with a tertiary structure to form a larger protein complex.

Each of the polypeptide chains is a subunit, and the quaternary structure is their assembling

arrangement; it is the consequence of the non-covalent interactions between the subunits in the

molecule and between the molecule and the solution (Ludescher 1996).

Most of the 20 amino acids can be synthetized by the human body, but there are nine essential

amino acids that cannot be made by the organism and must be supplied in the diet. Amino acid

requirement values for essential amino acids, as well as the amino acid composition of yellow

mustard protein are shown in Table 3. The amino acid composition is well-balanced and

comparable to other vegetal protein sources such as soybeans. It has been proposed that mustard

proteins, along with other brassica proteins have a lower digestibility value than casein, due their

11

structural rigidity and lower nitrogen release in early digestion phases (Wanasundara 2011),

particularly napin proteins show more resistance to degradation.

Table 3: Values of oriental and yellow mustard amino acid composition compared to FAO indispensable amino acid

requirements

Indispensable

amino acid

requirements

(WHO/FAO 2007)

Soybeans

(Rackis, et al.

1961)

Yellow mustard

(VanEtten, et

al. 1967)

(Cserhalmi,

et al. 2001)

(Sarwar, et

al. 1981)

Amino acid Composition (mg/g protein)

Alanine - 45 45 42 38

Arginine - 84 61 62 33

Aspartic acid - 120 79 78 101

Glutamic acid - 210 180 191 133

Glycine - 45 61 55 66

Histidine - 26 29 37 23

Isoleucine 30 51 41 40 32

Leucine 59 77 73 74 56

Lysine 45 69 59 61 87

Methionine 16 16 17 - 10

Phenylalanine 38* 50 41 45 34

Proline - 63 64 111 95

Serine - 56 43 51 69

Threonine 23 43 46 47 71

Tryptophan 6 13 - - 5

Tyrosine 38* 39 33 29 55

Valine 39 54 56 31 60

Hydroxyproline - - 11 - -

* Value for phenylalanine + tyrosine

12

Of the proteins in mustard seed, around 70% is composed of storage proteins, cruciferin and

napin, which are found inside the protein bodies and have no catalytic functions. Up to 10% is

considered to be oleosin, a main structural component of the membrane surrounding the oil

bodies (Bell, Rakow and Downey 1999), the rest of the protein in the seed is part of other

cellular organelles while some of have catalytic functions, such as myrosinase (Appelqvist

1971). There are two main types of storage proteins present in mustard seeds: legumin type

globulins (11S, cruciferins), and napin-type proteins (2S, napins), which are water soluble and

have an isoelectric point around a pH value of 7 (Wanasundara 2011). Proteins are found in

special organelles called protein bodies, which are generated by the storage protein vacuoles

inside the seed. The relative content of cruciferin and napin proteins in mustard is variable and

depends on the seed variety.

Although allergic reactions to 2S napins in mustard seed have been reported, including celiac

disease and asthma (Monsalve, Villalba and Rodriguez 2001), the incidence of mustard allergies

in animals seems to be low, since canola and mustard meals have long been used as a

proteinaceous feed. Because of the occurrence of 2S napins in mustard seed and others from the

brassica family, the European Union has listed mustard as an allergenic food ingredient (EU

Directive 2003/89/EC).

Mustard, as well as other brassica oilseeds can be considered an important source of protein, but

is most currently used for livestock feeding due to its content of anti-nutritional components and

due to protein denaturation during industrial oil extraction, limiting its uses in the food industry.

The utilization of friendly processing conditions and proper separation processes such as the

ones used in this study would open the opportunity for value increase of mustard seeds. These

techniques will be discussed in the following sections.

13

2.2.3 Protein-oil interactions in mustard seeds

Different binding forces are present in lipid protein interactions, such as covalent binding,

electrostatic binding, polarization interaction, dispersion interaction and hydrophobic binding.

Evidence has shown that electrostatic and hydrophobic binding and metal ion participation are

particularly important in lipid protein structures (Chapman 1969).

The main lipid-protein interaction within mustard seeds occurs in cellular organelles. Mustard

seeds, like most oil bearing seeds, store oil reserves in oil bodies. Oil bodies have a spherical

shape, with a diameter that ranges from 0.2 to 2.5 µm (Huang 1992) depending on the seed

species and consist in a triacylglycerol core surrounded by a phospholipid monolayer and an

outer surface layer composed of proteins (Figure 5). The average size is also affected by

nutritional and environmental factors. The main component, triacylglycerols comprise about 92 –

98% of the total organelle weight. Phospholipids represent 0.6 – 4% and proteins around 0.6 –

3% (Gitte, Mundy and Jason 2001). The phospholipid monolayer in oil bodies is composed of

phosphatidylcholine, and lesser quantities of phosphatidylserine, phosphatidylethanolamine, and

phosphatidylinositol are also present (Huang 1992). The outer layer of oil bodies is formed by a

special type of proteins called oleosins.

Oleosins are alkaline proteins with a molecular weight varying from 15 to 30 kDa. Recent

studies have found that these proteins are not only present in the oil bodies, and as much as 5%

can be found on endoplasmatic reticulum segments inside the cells (Gitte, Mundy and Jason

2001). Oleosin structure consists of three different regions according to its amino acid sequence:

A hydrophilic N-terminal portion which contains between 50 – 70 amino acid residues, a central

portion which is a hydrophobic chain made of around 70 amino acid residues and a C-terminal

14

amphipatic portion of variable length that interacts with the phospholipid layer, with the

positively charged residues facing the phospholipid monolayer and the negatively charged

residues facing the oil body surface (Hsieh and Huang 2004). It has been proposed that the center

of the hydrophobic portion is formed by two antiparallel β-strands connected by three proline

and one serine residues, interacting to form a “proline knot” that is inserted into the

triacylglycerol matrix (Hsieh and Huang 2004).

Due to the presence of the oleosin and phospholipid monolayer, oil bodies present a negative

electrical charge at neutral pH and a hydrophilic surface, preventing coalescence with one

another and are able to retain their shape even through seed desiccation. The main function of

these discrete and small organelles is to provide a large surface area per triacylglycerol unit in

order to enable lipase binding during seed germination (Hsieh and Huang 2004). Oil body size in

oilseeds is related to the particular seed species and is also determined by the relationship

between oil and oleosin contents. As the triacylglycerol content in the oil bodies increase, the

phospholipid and protein content decreases and the diameter of the oil bodies grows larger. It has

oil bodies

protein body

Figure 5. Structure of oil bodies (Huang 1992)

15

been found that oil bodies in mustard seeds have an average diameter of 0.73 µm and a

composition of around 95% lipids, 3% protein and 1.5% phospholipids (Tzen, et al. 1993).

The presence of oil bodies in mustard seeds may play an important role in the efficiency of the

extraction process, particularly in an aqueous extraction process. The extent of the disruption of

the cell oil bodies prior to extraction has a direct impact in oil yields as they may remain intact

after flaking or grinding, although coalescence can be induced by the use of enzymes (Campbell,

Glatz and Johnson, et al. 2011).

2.2.4 Glucosinolates

Glucosinolates are considered anti-nutritional compounds and their presence is important for the

food applications of brassica seed meals and derived products. In vivo models in rats show that

high levels of glucosinolates and their breakdown products have an adverse thyrotoxic effect, but

are not seen when protein isolates with low glucosinolate levels are used (Wanasundara 2011).

They are responsible for the bitter taste of mustard, and their breakdown products,

isothyocianates, for the pungency and hot flavour. Glucosinolates in brassica seeds are digested

by the endogenous enzyme myrosinase to isothiocyanates, glucose and sulfates. The

glucosinolate content in brown/oriental mustard is about 5-7% (Mustakas, et al. 1965) and in

yellow mustard around 9% (Josefsson 1970). Glucosinolates are thioglucosides with a cyano

and a sulfate group (Zrybko, Fuduka and Rosen 1997). There is a considerable variation in the

glucosinolate content of mustard seeds due to factors such as genetic origin, age, and

environmental conditions in which the plant is grown (Fenwick, Heaney and Mullin 1982). The

predominant thioglucoside in yellow mustard (sinapis alba) is sinalbin and its reaction with

myrosinase is shown in Figure 6.

16

Similarly, in the brown/oriental mustard (brassica juncea), the main thioglucoside sinigrin reacts

in the presence of myrosinase to produce allyl isothyocianate (Figure 7), which is a volatile

pungent liquid and gives brown/black mustard its pungent flavour and odour. The main function

of these substances in the plant is self defense mechanisms against pests and other diseases

(Zrybko, Fuduka and Rosen 1997). Several studies have found that isothyocianates can inhibit

the neoplastic effects of different carcinogens in different organs (Stoewsand 1995 and Spitz, et

al. 2000). On the other hand, isothyocianates have also been shown to have goitrogenic

Sinalbin

Sinapine acid sulfate p-Hydroxybenzyl isothiocyanate

Myrosinase H2O

+ +

Glucose

Figure 6: Isothyocianate release reaction for sinapis alba

17

properties, interfering with iodine uptake and affecting the function of the thyroid glands,

inhibiting hormone production (Zukalová and Vasák 2002). Heat treatment for the inactivation

of myrosinase has been shown to be an effective method to avoid the breakdown of

glucosinolates from brassica seeds (Fenwick and Heaney 1983) but has an adverse effect due to

protein denaturation during the thermal process and glucosinolates may undergo an enzyme

mediated reaction to produce isothyocianates after ingestion. Alternatively membrane processing

has also been shown effective for the reduction of glucosinolates from mustard protein isolates

(Lui 1998).

2.2.5 Phytates

Phytates, salts of calcium, magnesium and potassium from phytic acid (Figure 8) are other of the

components in mustard seeds. About 3% of the yellow mustard seed is composed by phytates, on

an oil free basis (Luo 1998). These compounds accumulate in the protein storage vacuoles as

crystals and show strong electrostatic interactions with proteins, particularly at pH values lower

than their isoelectric point, above which both dissociate. Phytic acid is capable of forming

insoluble protein complexes and attention should be kept in the pH extraction values of the

Figure 7: Isothyocianate release reaction for brassica juncea

Myrosinase

H2O

Sinigrin

+ +

Glucose Allyl isothiocyanate Potassium bisulfite

18

protein (Okubo, Myers and Iacobucci 1976). Because of the nature of phytic acid, there have

been a series of studies that show contrasting consequences of phytate ingestion. While

beneficial effects related to its natural antioxidant activity have been reported, suppressing iron-

mediated oxidation reaction in the colon (Graf and Empson 1987), phytic acid is a strong

chelating agent and can decrease the bioavailability of minerals such as calcium, zinc and iron

and lead to mineral deficiencies in mammals. Studies have shown that rats fed with yellow

mustard protein concentrate show symptoms of zinc deficiency (Wanasundara 2011).

Alkaline extraction of grounded yellow mustard seed, followed by ultrafiltration and diafiltration

of the protein extract has been considered an effective method in the reduction of phytic acid

levels in protein isolates, where the excess of basic cations prevents the formation of protein-

phytate complexes and free phytates are effectively removed by membrane processing (Luo

1998).

Figure 8: Chemical structure of phytic acid

2.2.6 Phenolic compounds

There is a wide variety of phenolic compounds in mustard seeds which includes esterified and

free forms of phenolic acids. These compounds are usually found as methoxylated derivatives of

19

benzoic and cinnamic acids. The most abundant phenolic compounds present in yellow mustard

are p-hydroxybenzoic acid and sinapic acid (Figure 9), present also as sinapine, its choline ester

form (Kozlowska, Zadernowski and Sosulski 1983). Phenolic compounds are known to have a

strong antioxidant effect, but are also responsible for a bitter and astringent taste in the mustard

seed meal as well as a dark colour (Shahidi and Naczk 1989), both of them un-wanted

characteristics in a food additive or a food ingredient. Four types of interactions exist between

these compounds and proteins: hydrogen bonding, covalent bonding, ionic bonding and

hydrophobic interactions (Xu and Diosady 2002). It has been shown that alkaline extraction,

followed by treatment with 0.05 M sodium chloride and membrane processing can reduce the

unbound phenolic fraction and the ionic protein-bonded fraction, while treatment with sodium

lauryl sulphate is able to reduce the hydrophobic protein-bonded fraction (Xu and Diosady

2002).

Figure 9: Chemical structure of phenolic compounds

Sinapic acid p-hydroxybenzoic acid

20

2.3 Protein extraction

Two main problems arise when considering mustard seed and other oilseeds for the production

of food grade protein isolates; current oil extraction methods increase protein denaturation by the

use of organic solvents and high temperatures (Pedroche, et al. 2004), and the presence of anti-

nutritional components such as phytates, glucosinolates and phenolic compounds (Naczk, et al.

1998).

Protein denaturation is a physical-chemical process in which the configuration, conformation and

state of folding of the polypeptide chains within the molecule is changed to a different

arrangement by an energy input that can consist in heat, light, pressure, etc. Depending on the

type of protein, denaturation can hinder or induce desirable functional properties. Proteins can be

denatured by different types of processes such as thermal effects, presence and concentration of a

denaturant like urea, guanidine hydrochloride and various salts that induce conformational

changes of proteins (Kilara and Harwalkar 1996), high pressures related to extrusion processes

and changes in pH that can lead to an unstable protein molecule. The effects of any of these

factors depend on the nature of the protein; not all will suffer denaturation at the same conditions

of temperature, pH, pressure or salt ion concentration.

Currently, most of the oilseed processing plants are focused in the production of edible oil and

little attention has been given to the production of food grade protein from the meal fraction. But

the need for additional sources of high quality protein for human nutrition has pushed forward

the development of alternative processes, such as aqueous extraction systems.

21

2.3.1 Solvent extraction process

In the traditional solvent extraction process, the time-temperature-moisture relationship is

essential (Becker 1970). As the value of each of these variable increases, the protein denaturation

will also increase, affecting the quality and functional properties of the final product. The use of

organic solvents such as hexane, derived from a non-renewable source, has inherent safety risks

to both the manufacturing facilities and personnel due to flammability and explosion hazards. In

addition hexane vapors can react with nitrogen oxides in the atmosphere and increase ground

level ozone (Campbell et al. 2011). The Environment Protection Agency (EPA) in the United

States has classified hexane as a hazardous air pollutant so its emission to the atmosphere has to

be monitored and reported (Environmental Protection Agency 2001) and is subject to costly fines

if the limits are exceeded.

In the typical solvent extraction process, seeds are first cleaned by aeration and sieving (Becker

1970). After cleaning seeds are submitted to hull decortication followed by the separation of the

kernels, although in the case of Canola seeds de-hulling is not performed. Size reduction is

usually the next step, the seeds are cracked using a rolling mill which helps disrupt the cellular

structure and increases the surface area to improve oil extraction yield. After de-hulling and size

reduction, oilseeds are tempered or cooked. Usual cooking temperatures vary from 120°C for

rapeseed, 100°C for canola to 65°C for soybeans (Dunford 2012). Cooking inactivates the

myrosinase enzyme which prevents the hydrolysis of glucosinolates into isothyocianates and

nitriles in brassica seeds. Tempering also improves pressing and solvent extraction efficiencies

(Dunford 2012); it is also useful to decrease the oil viscosity prior pressing and to complete the

cell disruption and facilitate the oil extraction (Ward 1984). A prepress-solvent extraction

process is usually the next step (Figure 10). Lower temperatures and pressures applied in the

22

prepressing operation compared to hard pressing reduce protein denaturation and the resulting oil

concentration in the meal is about 17% to 20% (Ward 1984), which can be subsequently

removed by hexane extraction. A continuous percolation type extractor is commonly used for

this task, which experiences a hexane loss in the order of 1.9 to 5.7 liters per tonne of seed

processed. The final meal typically contains between 0.5% and 1.0% residual oil (Lusas 1983).

Figure 10: Main operations in a solvent extraction system (Becker 1970)

After hexane extraction the resulting meal has a hexane concentration around 30% (Becker

1970) and a desolventizing process is required to reduce the residual solvent to acceptable levels.

In a desolventizer toaster system, the defatted meal moves through a series of trays where it is

Cleaning / De-hulling

Conditioning/ Cooking

Pressing

Flaking

Extraction

Desolventizing / Toasting

Evaporation / Distillation Degumming

Oilseed

Press oil

Miscella Solvent

Oil Solvent

Solvent extracted meal

23

heated in order to remove most of the solvent, live steam is then injected to strip the remaining

hexane, and finally the meal is toasted in the lower trays at a temperature of 107°C (Becker

1970) to reduce the moisture content of the product. It has been shown that under the same

desolventizing conditions, factors such as the moisture content of the seeds prior crushing, de-

hulling and solvent extraction times affect the residual hexane content (Wolff 1983).

2.3.2 Aqueous extraction process

The development of aqueous extraction processes from oilseeds to obtain both oil and protein

date back to the 1950’s. Chayen (1953) and Subrahmanyan (1959) considered the extraction of

oil and protein with water as the main solvent in an analogous way to traditional extraction,

where all or a part of the oil is first removed. Just a limited number of these methods have been

fully developed to a commercial level. Further aqueous processes for the recovery of oil and

protein were developed for a wide variety of oilseeds, like coconuts (Hagenmaier, Cater and

Mattil 1972), sunflower seeds (R. D. Hagenmaier 1974), peanuts (Rhee, Cater and Mattil 1972),

soybeans (Campbell and Glatz 2009) and rapeseed (Caviedes 1996) have also been studied.

Protein and oil can be simultaneously recovered in an aqueous system, where protein in the

resulting aqueous and solid phases can be further processed and purified. The efficiency of the

process greatly depends on the main operations involved: cell disruption, oil and protein

extraction, centrifugation, de-emulsification (Campbell K. A., 2011; Cater, et. al 1974 and

Rosenthal, 1996) and protein purification and isolation. A general process diagram is shown in

Figure 11.

The conditions, methods and degree of cell disruption are fundamental in an aqueous extraction

processes. Cells in the seeds to be extracted must be efficiently destroyed to increase the

24

extraction yields of both oil and protein. Insufficient disruption may leave large quantities of oil

and protein in the solid residue (Cater, et al. 1974), while excessive comminution might result in

a highly stable oil and water emulsion due to the smaller oil droplets (Rosenthal, Pyle and

Niranjan 1996) and an increase in the oil content of the aqueous phase. Moisture content,

physical structure and chemical composition of the seed are important in deciding the disruption

method, and wet or dry operations. Commonly used methods include flaking, extrusion, dry

grinding and wet grinding.

Figure 11: Main operations in aqueous extraction systems (Cater, et al. 1974)

The extraction operation consists in the agitation of a dispersion composed of the disrupted seed

material and water; factors that influence the effectiveness and extent of the extraction are solid

to water ratio, pH, temperature (Cater, et al. 1974), particle size, agitation degree, extraction time

(Rosenthal, Pyle and Niranjan 1996), extraction stages and ionic strength. After the extraction,

the dispersion is separated, usually by centrifugation, into a water in oil emulsion, a solid phase

Cell disruption

Extraction

Centrifugation

Protein purification and isolation

De-emulsification and oil recovery Meal drying

Oilseed

Aqueous phase

Emulsion Solids

25

containing insoluble components such as fibers, protein and oil, and an aqueous phase with the

soluble components of the seed. Studies in our food laboratory have found that for full fat yellow

mustard flour, an optimum water to solid ratio of 4 to 1, pH of 12, ambient temperature, 30

minutes of extraction time and 3 stages yield the highest amount of protein and oil extraction

(Prapakornwiriya 2002 and Balke 2006).

Filtration of the aqueous phase rich in soluble protein is an essential step for the recovery of

protein concentrates and isolates with low levels of anti-nutritional components. Several methods

have been developed in our food engineering laboratory group for the recovery of high quality

products that include microfiltration, ultrafiltration and diafiltration.

The removal of water from the protein solution is the final step. The use of a freeze drying or

spray drying systems may be considered depending on the scale of the production process.

The aqueous extraction processing of oilseeds has important advantages. High quality protein

can be obtained, since heating and toasting steps that can irreversibly cause denaturation are

omitted. Safety risks regarding the use of highly volatile solvents are eliminated which have an

important impact on equipment and training costs. There is also a considerable decrease in the

environmental footprint of the process and costs related to volatile organic compounds emission

and control. An aqueous extraction process has a smaller number of operations than the solvent

extraction, making it a simpler, more energy efficient process and having the possibility of being

designed for continuous or batch operation. Even though there are important advantages, there

are also some disadvantages due to the nature of the process. There is a lower oil extraction yield

compared to solvent treatment and there is the need of a de-emulsification step when oil is

recovered in the form of an emulsion, additionally there is an increased potential for microbial

26

contamination because the material is wet during most of the operations (Cater, et al. 1974 and

Rosenthal, 1996).

The use of enzymes in aqueous extraction systems can increase both oil and protein yields.

Depending on the seed and its components different kinds of enzymes or combination of

enzymes can be used. Carbohydrases, such as cellulases, pectinases and hemicellulases help

degrade the cell wall materials and can increase the oil recovery, while proteolitic enzymes

hydrolyze proteins including oleosins, which may increase the release of oil (Rosenthal, Pyle and

Niranjan 1996).

2.4 Protein purification and isolation

In order to obtain food quality products by either a solvent or an aqueous extraction, protein must

be purified. Several protein purification and separation processes rely on the differences in

solubility between them, or between proteins and non-protein materials in a solution.

Precipitation is one of the techniques used for the recovery of proteins, and is usually

accompanied by a concentration step in order to reduce the volume of the initial solution and the

level of undesired, micro-molecular components. The principles of protein precipitation are

related to forces acting between the polypeptide chains in the proteins and also their interaction

with the solvent molecules. Changes in the solvent-protein and protein-protein interactions which

lead to precipitation can be induced by modifying the temperature, the composition of the

solvating medium or the pH (Li-Chan 1996). At the isoelectric point, where there are an equal

number of positive and negative charged groups, the surface of the protein will be least solvated

facilitating hydrophobic interactions and aggregation.

27

One of the most common processes for protein precipitation is known as “salting out”, where a

high salt concentration leads to a decrease in the effective concentration of water. The

concentration and nature of the salt used is important to determine the effect on protein-protein

and protein-water interactions. In general terms, salts with high molal surface tension values are

effective in protein precipitation, while salts with low values have the opposite effect, called

“salting in” (Li-Chan 1996). An alternate process for protein precipitation proposed by Murray,

et al. (1979) called micellization consists of the extraction of proteins from seed meals using a

“salting in” technique followed by precipitation by the dilution of the concentrated extract with

water and a temperature adjustment, favoring hydrophobic interactions and protein aggregation.

In some cases the solubility of proteins at their isoelectric point is low enough to allow their

recovery by a pH adjustment, this process is known as isoelectric precipitation. Previous studies

in our food engineering laboratory have shown that isoelectric precipitation is a suitable process

to recover most of the mustard seed proteins after an alkaline extraction. The isoelectric point for

the alkaline extracted proteins from defatted mustard is around a pH value of 4.75 (Lui 1998 and

Xu, Lui, et al. 2003) while a value of 5.5 has been found and used for alkaline extracted full fat

mustard (Prapakornwiriya 2002). It must be considered that for the mustard protein extraction,

isoelectric precipitation of the protein extract would result in a product with high levels of anti-

nutritional components that would limit its use for human consumption. Since the molecular

weight of mustard proteins is considerably larger than most anti-nutritional components or

contaminants, membrane processing via ultrafiltration and diafiltration is used as a purification

step.

28

2.4.1 Ultrafiltration

Ultrafiltration is a cross-flow membrane separation process. In a solution containing low

molecular weight and high molecular weight solutes, the latter will be retained by the membrane,

while the smaller low molecular weight particles will permeate through. The driving force in

order to achieve the separation is a pressure difference applied to a solution on the feed side of a

membrane. Ultrafiltration membrane pore sizes are usually classified according to the molecular

weight of the species that will be retained by assigning to them a molecular weight cut off

(MWCO). A schematic of this process is shown in Figure 12. The solvent and low molecular

weight species passes through the membrane and constitute the permeate, while solutes with a

larger weight than the MWCO are retained and form the retentate.

Figure 12: Ultrafiltration principle of operation

Since micro molecular components have significantly lower molecular weights, it is possible to

separate them from other macromolecular compounds in aqueous solution by using

Pressure

Retentate Ultrafiltration membrane

Permeate

29

ultrafiltration. Membrane molecular weight cut offs in this case are typically between 5 and 500

kDa and are able to retain proteins, polymers, and chelates of heavy metals (Cheryan 1998).

Since low-molecular-weight solutes flow through the membrane, osmotic pressure is not an

issue. However, since retained large molecules and colloidal particles have low diffusivities in

the liquid medium, ultrafiltration membranes are more susceptible to fouling and concentration

polarization than reverse osmosis or microfiltration membranes (Cheryan 1998).

Usually, not all the particles larger than the molecular weight cut off of the membrane are

rejected, and some particles smaller than this parameter may be partially rejected. In order to

estimate the separation degree attained by the process, a mathematical model has been developed

for the rejection of the solutes (Cheryan 1998):

𝑅 = 1 −𝐶𝑃𝐶𝑅

Equation 1

where R is the rejection coefficient, CP is the concentration in the permeate and CR is the

concentration in the retentate. During this process, the total volume of a solution will be reduced

as the solvent and low molecular weight components are being removed resulting in the

concentration of the macromolecular species, whose quantity remains unchanged. The

concentration and volume relationship in ultrafiltration systems are characterized by the

following equation (Cheryan 1998):

𝐶𝑓𝐶0

= �𝑉0𝑉𝑓�𝑅

= 𝐶𝐹𝑅

Equation 2

30

Where Cf is the final concentration of the feed, C0 is the initial concentration of the feed, V0 is the

initial feed volume, Vf is the final feed volume, CF is the concentration factor and R is the

rejection coefficient.

2.4.2 Diafiltration

Diafiltration is a method where permeable solutes are eliminated from a solution and consists in

an initial volume reduction, usually performed by ultrafiltration and a subsequent addition of a

suitable buffer solution or water. This process can be made in a continuous or discontinuous

manner. In discontinuous diafiltration the adequate buffer solution or water quantity is added to

the concentrated solution to reach the initial volume, and the ultrafiltration operation is repeated

until the unwanted micro-molecular components are removed. In continuous diafiltration, buffer

solution or water is added at the same rate as the permeate flux, keeping the concentrated

solution volume constant during the process.

The amount of micro-molecular components that is removed is related to the volume of permeate

resulting from the operation and the initial volume of retentate. This relationship is referred to as

diafiltration volume (DV) (Cheryan 1998):

𝐻𝑉 =𝑉𝑓𝑉0

Equation 3

Where Vf is the permeate volume and V0 is the initial retentate volume. For continuous

diafiltration the relationship between the initial and final concentration of the micro-molecular

components is given by following equation (Cheryan 1998):

31

𝐶𝑅 = 𝐶0𝑒−𝐷𝑉(1−𝑅)

Equation 4

Where CR is the final concentration of the micro-molecular component, C0 is the initial

concentration, and R is the rejection coefficient. As a result of continuous diafiltration, the final

volume and concentration of the macro-molecular components retained by the membrane does

not change. As shown by the equation, a diafiltration volume of 6 is enough to remove more than

99.5% of a micro-molecular component with a rejection coefficient of 0. The given formula also

shows that when the solute is partially retained by the membrane (the rejection coefficient is

greater than 0), the diafiltration volume needed to reach the same removal will increase.

The main limitations for membrane separation processes are concentration polarization and

membrane fouling. Concentration polarization controls the performance of ultrafiltration. It is an

effect where particles rejected by the membrane tend to form a layer near the surface causing

further resistance to the flow of the permeate. The flux decrease is usually explained by two

mechanisms: The first one is an increase in the osmotic pressure due to the increased solute

concentration near the surface of the membrane in comparison to the bulk concentration in the

feed, and the second one is the hydrodynamic resistance of the boundary layer (Cheryan 1998).

To reduce the effect of concentration polarization several factors such as pressure, feed

concentration, temperature and turbulence in the feed channel must be optimized.

Membrane fouling on the other hand is characterized by an irreversible decline in the flux that

cannot be counteracted with fluid management techniques. It is due to the accumulation of feed

components on the membrane surface or within the pores of the membrane and is influenced by

the chemical natures of both the membrane and the solutes and membrane-solute and solute-

32

solute interactions (Cheryan 1998). Usually the only way of restoring the flux of a fouled

membrane is through cleaning. Fouled membranes and auxiliary equipment are generally cleaned

by clean-in-place procedures (Lindau and Jönson 1994) which are usually based on various

chemical or enzymatic treatments to restore the membrane to its original state.

2.5 Functional properties in protein isolates

The importance of protein isolates when used in food systems does not rely only in their

nutritional value, but in the desirable properties and sensory attributes that the additives are able

to provide. Emulsification capacity, water and lipid holding capacity, gelation capacity, foaming

capacity and foaming stability are functional properties that enhance food sensory and

organoleptic characteristics including colour, flavour, odour, texture or mouth feel. For centuries

animal proteins have been traditionally the main source of functional ingredients; milk, egg and

animal meat proteins have unique properties and functionality applications, however it is

estimated that about 8 kg of protein from a vegetable source are needed to produce 1 kg of

animal protein (Damodaran 1996). Considering this low ratio, the increasing protein demand due

to population growth and land use competition between food crops, non-food crops for biofuel,

and cattle, oilseeds such as mustard offer an interesting alternative as a renewable source of oil

for biofuel production and protein to provide sensory characteristics and nutritional value to food

products as a replacement of animal protein.

Kinsella and Melachouris (1976) defined the functional properties of proteins as those physical

and chemical properties which have an influence on their behavior in diverse food systems,

whether it is in their preparation, storage, cooking or consumption. The size, shape, amino acid

composition and sequence, net charge, charge distribution, hydrophobicity, hydrophilicity,

33

structural arrangements and molecular flexibility of proteins are intrinsic characteristics that

define their functionality and interactions with other food ingredients.

Functional properties can be classified in three groups according to their action mechanism in

food systems: properties due to hydration such as solubility and wettability, properties related to

protein structure such as viscosity and gelation, and properties related to protein surface such as

emulsifying and foaming capacities (Moure, et al. 2006 and Siong, et al. 2011).

2.5.1 Hydration properties

Important functional properties such as solubility, wettability, dispersibility, foaming,

emulsification and gelling properties are affected by the solvation and dissolution characteristics

of the protein and depend on the interaction between the molecules and the solvent. The

hydration mechanism of a protein describes different states of water in hydrated proteins

(Kinsella, Fox and Rockland 1986): structural water is formed by water molecules that are part

of the protein structure, bound by hydrogen bonds; this water is not available for chemical

reactions, is un-freezable and not relevant for the functional properties of the protein. Monolayer

water is composed by water molecules bound via dipole-induced dipole, ion-dipole and dipole-

dipole interactions with polar groups in the protein and hydrophobic hydration of nonpolar

groups. The monolayer forms when the water activity is in the range from 0.05 to 0.3 and is

unavailable for most chemical reactions. On the other hand, water states related to protein

functionality include: multilayer water at water activities between 0.3 and 0.7, un-freezable water

consisting of multilayer ordered water molecules up to a water activity of 0.9, capillary water

bound due to capillary forces in crevices and cavities, which appears when the water activity is

34

between 0.5 and 0.95 and finally, hydrodynamic hydration water that exists at a water activity

over 0.99 and affects viscosity and diffusion properties of the protein.

2.5.1.1 Water and oil absorption

In food systems, the water absorption capacity of a protein is the ability to hold water against

gravity and form network structures with other proteins via non-covalent interactions. The

capacity of retaining moisture influences the texture and mouth-feel of foodstuffs (Kinsella and

Melachouris 1976 and Johnson 1970) and is function of the fraction of charged residues, polar

amino acid side chains and nonpolar residues of the protein (Moure, et al. 2006). Amino acid

residues with charged side chains will experience strong ion-dipole interaction and bind more

water. External factors like pH, ionic strength, protein concentration, temperature and particle

size of protein powders have a considerable effect in water absorption (Damodaran 1996 and

Johnson 1970). Most proteins have the lowest water binding capacity at their isoelectric pH.

Water absorption is usually described by the water absorption capacity (WAC), the amount of

water retained per unit mass of protein after mixing and centrifugation (F. Sosulski 1962) and the

water hydration capacity (WHC) (Naczk, Diosady and Rubin 1985).

Similarly, oil absorption can be defined as the amount of oil retained per unit mass of protein

after thorough mixing and centrifugation (Lin and Humbert 1974 and Sosulski, Humbert and Bui

1976). The importance of fat absorption by protein in food systems lies in the in the ability of

lipid molecules to modify and in some cases provide odours and flavours as well as a pleasant

mouth feel (Forss 1972), and an improvement in flavour transport during food processing

(Kinsella and Melachouris 1976). The oil-protein binding mechanism is related to capillary

forces in crevices and cavities of the protein molecule surface which are able to entrap oil

35

molecules as well as hydrophobic interactions between non-polar side chains and lipid

molecules.

2.5.1.2 Protein Solubility

Protein solubility can be described as a thermodynamic equilibrium between protein-solvent and

protein-protein interactions. As protein solubility increases, it can be more easily incorporated

into foodstuffs, increasing its functionality and applications. Solubility of a protein is influenced

by the balance of hydrophobic and hydrophilic residues on the protein surface, given by the

amino acid composition. A low number of hydrophobic residues, as well as a high number of

electrostatic repulsions and ionic hydration lead to high solubility (Moure, et al. 2006). Solubility

is also affected by the environmental conditions of the solution such as pH, ionic strength, ion

types, temperature, solvent polarity and processing conditions, all of which interfere with the

hydrophilic and hydrophobic interactions at the protein surface (Damodaran 1996). In the case of

pH and ionic strength, their effect on solubility can be explained by the changes in the protein

electrostatic forces (Kinsella and Melachouris 1976). At pH values around the isoelectric point,

the solubility of a protein will be at its minimum value. Processing conditions that promote

protein denaturation lead to conformational changes that even at a low extent can alter the

hydrophobic and hydrophilic balance at the protein surface affecting solubility. The nitrogen

solubility index (NSI) given as the percentage of water-soluble nitrogen from a given sample

under slow stirring is the usually adopted method to determine protein solubility (AOCS, 1999).

2.5.2 Properties related to protein surface

Many processed foods are in foam or emulsion type systems. Emulsions and foams are two-

phase systems consisting of a dispersed and a continuous phase. Foam can be defined as a

36

substance formed by the dispersion gas cells in a continuous liquid phase that contains a

surfactant, while an emulsion is a mixture of two or more liquids that are normally immiscible

where one of the liquids is dispersed in the other. Because of the amphipathic nature of proteins,

they act as macromolecular surfactants in emulsions and foam-type products. Proteins act by

lowering the interfacial tensions and also are able to produce a continuous film at the interface

via intermolecular interactions. The high viscosity and high dilatational modulus of protein films

makes them able to withstand external forces, producing more stable foams and emulsions than

low molecular weight surfactants. These characteristics are the result of protein surface activity,

which is affected by molecular properties such as conformational stability, flexibility, the

symmetry in the distribution of hydrophilic and hydrophobic side chains and external factors

such as pH, ionic strength and temperature (Moure, et al. 2006). The dynamics of protein

adsorption proceed through the sequential attachment of polypeptide segments. The first step is

the transport of the protein from the bulk to the interface where the global free energy of the

protein is lower. The kinetics of adsorption of proteins has been proposed to follow a diffusion

controlled model that depends not only in the concentration and diffusion coefficient, but on an

activation energy for adsorption at the interface that arise from physiochemical constraints of the

protein related to hydrophobic, hydrophilic and conformational flexibility of the molecule

(Damodaran 1996). The surfactant properties of proteins are improved when they possess a high

rate of diffusion and adsorption, are able to unfold rapidly and are able to form a cohesive and

viscous film at the interface. Protein adsorbs to interfaces in multiple contact points according to

the degree of flexibility of the polypeptide chain and may change their conformation upon

interface adsorption (Damodaran 1996).

37

2.5.2.1 Foaming properties

Foaming properties in proteins describe their ability to form a large and stable interfacial film

between the solution and the surrounding air that will withstand internal and external forces. The

foaming ability of a protein depends on its rate of adsorption at the interface, on its molecular

flexibility, that is, the rates at which it can unfold and undergo molecular rearrangements to

reduce the surface tension, and its capacity to form a cohesive film (Moure, et al. 2006). On the

other hand, the stability of the foam is affected by the molecular rigidity of the protein and the

rheological properties of protein films such as film viscosity, shear resistance, elasticity and the

disjoining pressure between protein layers. Ultimately, an adequate balance of flexibility and

rigidity must be present to produce stable foams (Damodaran 1996). External factors such as pH

(Sathe, Deshpande and Salunkhe 1982), temperature (Richert, Morr and Cooney 1974) and the

presence of other components such as sugars or lipids (Yasumatsu, et al. 1972) also affect the

foaming properties and foam stability. The foam capacity can be determined by the measure of

the foam volume produced after whipping of a protein dispersion with a specific concentration

(Lin and Humbert 1974), while foam stability can be expressed as the volume of foam remaining

after a certain amount of time has passed.

2.5.2.2 Emulsifying properties

An emulsion can be defined as a two phase system in which one liquid is dispersed as droplets in

another. Thermodynamically speaking, an emulsion is an unstable system and given enough time

the phases will separate, but it can be stabilized by the addition of surface active molecules or

surfactants. The emulsifying potential of a protein can be described by the emulsifying activity

index, the emulsion stability index and the emulsifying capacity (Kinsella and Melachouris

38

1976). Factors that affect the emulsifying properties of proteins include the rate of adsorption at

the interface, the amount of protein adsorbed, the conformational rearrangement at the interface,

molar mass, and external factors such as pH, ionic strength and temperature (Moure, et al. 2006).

Disjoining forces generated by electrostatic, steric and solvation interactions in the aqueous

phase also have a major role in the stability of emulsions (Damodaran 1996). Most emulsions are

more stable at pH values that are far away from the isoelectric point of the protein, where

electrostatic repulsion and hydration repulsion forces are maximized. Heat denaturation, as well

as some chemical and enzymatic treatments, like succinylation, phosphorylation and

glycosylation can improve the emulsifying properties (Damodaran 1996).

2.5.3 Properties related to protein structure: Gelation

Gels are considered as an intermediate phase between a solid and a liquid. In food systems the

liquid phase is usually water and the solid phase is formed by proteins or carbohydrates. Protein

gels are formed by polymeric molecules covalently or non-covalently cross-linked in a three-

dimensional network. Gels provide a structural matrix able to hold water, flavours, sugars and

other food ingredients. The ability to form a gel by a protein solution is affected by its molecular

weight and its ability to denature (Moure, et al. 2006). The mechanism for protein gelation is a

stepwise process (Damodaran 1996) in which the protein solution is first irreversibly converted

to a pro-gel by heating above the denaturation temperature to expose the functional groups which

interact to form the network, then the protein forms one of two types of gel networks depending

on the type of protein, its amino acid composition and external factors such as pH and ionic

strength: a coagulant gel is formed by proteins with high levels of nonpolar residues, and a

transparent type gel is formed by proteins that contain hydrophilic amino acid residues. The

minimum protein concentration necessary to form a self-supporting gel network is known as the

39

least gelation concentration (LGC). Below this critical concentration, proteins unfolded by heat

treatment undergo random aggregation which may lead to precipitation. Globular proteins

usually have higher LGC values than fibrous proteins.

2.6 Lipid oxidation in lipid-protein systems

One of the disadvantages of an aqueous extraction process, as mentioned in section 2.3.2, is that

proteins are recovered in a solution containing oil. Oil concentration depends on factors such as

the cell disruption methods used prior extraction, the extraction conditions, the use of enzymes,

and oil and protein contents of the starting material. Proteins form large molecular aggregates

along with remaining oil bodies in the solution (Dendukuri and Diosady 2003) preventing oil

from being permeated during membrane processing, and being unavoidably recovered in the

protein isolates after isoelectric precipitation. As an oil containing ingredient, the isolates are

prone to lipid oxidation and the subsequent interactions between the oxidation products and

proteins.

Lipid oxidation is one of the most important processes for food deterioration as it causes the

development of unpleasant odours, flavours and rancidity in both oils and oil rich foodstuffs.

Lipid oxidation reactions may also decrease the nutritional value of the food (Pokorný,

Kolakowska and Bienkiewicz 2005). Toxic substances can also be generated, which can be

associated with health risks to consumers (Tazi, et al. 2009 and St. Angelo and Ory 1975).

Lipid oxidation in food systems is a complex process, since oxidation products may react with

other components in the food system such as proteins, carbohydrates, water and vitamins. The

result of lipid oxidation under these conditions has different effects in the functional properties,

texture, mouth feel, aroma, nutritional value, colour and safety of food and food ingredients

40

(Hidalgo, Zamora and Alaiz 1991). The degree of unsaturation of fatty acids, the presence of

antioxidant substances, traces metals, light, temperature and oxygen availability are the main

factors that affect lipid oxidation.

2.6.1 Lipid and protein oxidation mechanisms

There are four different pathways for the oxidation and formation of hydroperoxides in lipids:

photo-oxidation, enzymatic oxidation, irradiation and autoxidation, the latter being the most

important (Matthäus 2010). The autoxidation mechanism in unsaturated lipids begins with the

reaction of a fatty acid radical with oxygen (Figure 13). The fatty acid radical is formed by

hydrogen abstraction from an allylic carbon, which has a low dissociation energy. It is believed

that heat, metal catalysis or ultraviolet irradiation provide the driving force for this de-

protonation (Matthäus 2010). The fatty acid radical is unstable and reacts with atmospheric

oxygen to produce a peroxy radical, which forms a new fatty acid radical and starts an

exponential chain reaction. Bond strength vary between fatty acids, highly unsaturated fatty acids

are subjected to a faster autoxidation due to the weakness of the allylic carbon-hydrogen bonds.

Hydroperoxides themselves are odourless and tasteless compounds, but are unstable and react

into secondary products that can be easily detected by their aroma and taste, some of them even

at very low concentrations (Reindl and Stan 1982). The types of these compounds produced

depend on the fatty acid composition and other components in a food system. The main pathway

of hydroperoxide decomposition to volatile compounds is the β-scission of a carbon-carbon bond

to produce oxo-compounds and an alkyl or alkenyl radical (Hidalgo, Zamora and Alaiz 1991).

Secondary oxidation products include ketones, aldehydes, alcohols, hydrocarbons, acids and

epoxides.

41

Deterioration of proteins in dry food products is similar to lipid oxidation, and is promoted by

the increase in water activity. Proteins are oxidized by an initial electron abstraction, by the

transfer of a hydrogen atom by plant phenols or by free radical scavenging, trapping radicals by

hydrogen donation during the first steps of lipid oxidation (Elias and Decker 2010) acting as

lipid antioxidants. The de-protonation of the α-carbon in a protein molecule leads to a radical

formation that can combine to form cross-links with other protein molecules or lipid oxidation

products, changing the texture and functional properties of foods. They could also undergo an

oxidation reaction to produce a protein hydroperoxide (Pokorný, Kolakowska and Bienkiewicz

2005).

Reactive functional groups in both proteins and lipid hydroperoxides explain the complexity of

the interaction between them. Reactions between both moieties follow two types of mechanisms

(Hidalgo, Zamora and Alaiz 1991); the first one is the formation of non-covalent complexes with

the hydroperoxides or secondary oxidation products, and takes place through hydrophobic

bonding. The second mechanism is covalent bonding by radical and non-radical reactions

RH

R· + O2

R· + H·

2R·

2RO2

RO2·

RO2· + R· + O2

RO2H + R·

R· + RO2·

RO2·

Initial phase

Propagation

Termination

Stable products

Figure 13: Fatty acid radical chain oxidation mechanism

42

between amino acids and lipid hydroperoxides or secondary oxidation products. Amino groups

from proteins react to produce an imine (Pokorný, Kolakowska and Bienkiewicz 2005), which

increases the hydrophobicity of the protein molecule due to the covalently bound lipid residue.

The solubility of the protein also decreases with these interactions and the resulting insoluble

proteins are cleaved with proteases slowly or incompletely, decreasing their digestibility. Protein

reaction with lipid hydroperoxides decreases their availability during the propagation step of

lipid oxidation, and can be considered as a protein antioxidant mechanism (Elias and Decker

2010).

2.6.2 Accelerated lipid oxidation evaluation

The main reason to assess lipid oxidation in food and food ingredients is to follow the oxidation

state by comparison with an initial sample at different stages after being submitted to oxidation

conditions, until the oxidation products render the food unacceptable or a previously set limit is

reached. Different titrimetric and spectroscopic techniques have been developed for the

determination of hydroperoxides or secondary products of lipid oxidation, while

chromatographic techniques are used when a more detailed description of the reaction products

is needed. Under ideal conditions, a product should be submitted for an oxidation evaluation

under the intended storage conditions, but for products with long shelf life an accelerated

stability test is usually performed, which consists of the oxidation evaluation of a product stored

at higher temperatures than the intended storage temperature. The kinetics of lipid oxidation over

time, as well as for other food quality indexes can be represented by:

−𝑑𝐴𝑑𝑡

= 𝑘𝐴𝑛

Equation 5

43

Where A is the lipid oxidation value, t is the temperature, k is the rate constant and n is the

reaction order. Although most chemical reactions responsible for food deterioration are complex

enough not to follow a zero or first order reaction model, they can be integrated and simplified to

a pseudo-zero (Equation 6) or pseudo-one (Equation 7) order reaction kinetics.

𝐴𝑒 = 𝐴0 − 𝑘𝑡

Equation 6

ln �𝐴𝑒𝐴0� = −𝑘𝑡

Equation 7

Where k is the rate constant, Ae is the final oxidation value or oxidation limit parameter, A0 is the

initial oxidation value and t is the time. On the other hand, the oxidation rate, in the same manner

as other chemical reactions, is temperature dependent and follows the Arrhenius relationship

(Equation 8).

𝑘 = 𝐴𝑒−𝐸𝐴𝑅𝑇

Equation 8

Where k is the rate constant, A is the pre-exponential factor, EA is the activation energy, R is the

universal gas constant ant T the absolute temperature. By measuring the lipid oxidation rate at

different temperatures, the activation energy can be calculated and applied to a shelf life model

which can be used for shelf life estimation at different temperatures.

44

3. PROJECT OBJECTIVES

The main objectives of this project are to characterize the yellow mustard protein isolates

obtained from the aqueous extraction of full fat, de-hulled yellow mustard flour and the

membrane processing of the resulting protein solution, to evaluate their potential applications,

not only in terms of their nutritional benefits already discussed in section 2.2.2, but more

importantly in terms of the functionality they may be able to provide as food ingredients, such as

their solubility, water and oil absorption, and their emulsifying, foaming and gelling properties.

Finally, to evaluate lipid oxidation in the precipitated protein isolate, which contains significant

amounts of oil.

In order to develop an integrated process, the following detailed objectives were pursued:

• Recovery and characterization of a protein solution from mustard flour using an aqueous

process, based on a method previously developed in the food engineering laboratory.

• Recovery of protein isolates by the concentration and isoelectric precipitation of a protein

solution, based on a method previously developed in the food engineering laboratory that

includes the use of an ultrafiltration and diafiltration process with a 5 kDa membrane.

• Characterization of the protein isolates, including their main composition, electrophoretic

properties and functional properties, comparing the obtained values with those reported in

the literature for mustard protein isolates and commercially available soybean protein

isolates.

• Evaluation of the storage temperature effect on lipid oxidation in the oil containing

protein fraction.

45

4. EXPERIMENTAL METHODS

4.1 Starting materials

Pure yellow mustard flour: product code 106, containing traces of volatile oil and with mild

strength, as described by the supplier, was used for all extraction experiments. This material was

provided by G. S. Dunn dry mustard millers, Hamilton Ontario. Product from lot number

1480811 was used.

4.2 Solvents

The main solvent used throughout the experiments was reverse osmosis water, obtained from the

Walberg building facility services, at the University of Toronto.

4.3 Reagents

All reagents used are described in Table 4.

Table 4: Reagents used for experiments

Reagent Grade/Description Supplier

1-butanol Reagent A.C.S. BDH Chemicals, Poole, England

2-thiobarbituric acid Minimum 98% Sigma Aldrich, St. Louis, MO, USA

2-mercaptoethanol Electrophoresis purity Bio-Rad Laboratories, Hercules,

CA, USA

Ammonium hydroxide Reagent 28.0-30.0% EMD, Gibbstown, NJ, USA

Anhydrous ethyl alcohol - Commercial Alcohols, Brampton,

ON, Canada

Ascorbic acid USP Fisher Scientific, Fair Lawn, NJ,

USA

Boric acid Reagent A.C.S. Fisher Scientific, Fair Lawn, NJ, USA

46

Diethyl ether Reagent A.C.S. >99.0% Sigma Aldrich, St. Louis, MO, USA

Glass wool Low in lead BDH Chemicals, Poole, England

Hydrochloric acid Reagent 36.5-38.0% Caledon, Georgetown, ON, Canada

Kjeldahl Tablets 3.5g K2SO4/0.175g HgO Fisher Scientific, Fair Lawn, NJ,

USA

Laemmli sample buffer - Bio-Rad Laboratories, Hercules,

CA, USA

Liquid nitrogen - Linde Canada Limited, Mississauga,

ON, Canada

N-point indicator - EMD, Gibbstown, NJ, USA

Petroleum ether (B.P. 35-

60°C) Reagent A.C.S. BDH Chemicals, Poole, England

Phosphoric acid Reagent >85.0% Caledon, Georgetown, ON, Canada

Sodium chloride Reagent >99.0% Bioshop, Burlington, ON, Canada

Sodium hydroxide (50%

Solution) Analytical

VWR Scientific, West Chester, PA,

USA

Sodium thiosulfate Reagent A.C.S. Fisher Scientific, Fair Lawn, NJ,

USA

Sulfuric acid Reagent, 95-98% Caledon, Georgetown, ON, Canada

Sulfuric acid 0.1000 N Analytical VWR Scientific, West Chester, PA,

USA

Enzymatic detergent Terg-A-Zyme Alconox Inc., New York. U.S.A.

47

4.4 Equipment and materials

The list of equipment and materials used for all experiments is described in Table 5.

Table 5: Equipment and materials used for the experiments

Equipment Model/Part no. Supplier

Analytical balance 2001 MP2 Sartorius, Germany

Analytical balance Mettler AC100 Mettler Toledo, Zurich, Switzerland

Balance Mettler PC 4400 Mettler Toledo, Zurich, Switzerland

Balance Mettler PE 3600 Mettler Toledo, Zurich, Switzerland

Centrifuge J-20 XP Beckman Instruments, Fullerton, CA,

USA

Clinical centrifuge 1968H International Equipment Company,

Boston, MA, USA

Convection oven Blue M-0V-490-A2 Electric Company, Blue Island, IL, USA

Electric 1 HP motor CSM3546-2 Baldor Motors and Drives, Fort Smith,

AR, USA

Electrophoresis station PowerPac HC Bio-Rad Laboratories, Hercules, CA,

USA

Freeze dryer Freezone 12-plus Labconco, Kansas City, MO, USA

Positive displacement

diaphragm pump Hydracell M-03E

Wanner Engineering Inc., Mineapolis,

MN, USA

Kjeldahl digestor 425 Büchi, Switzerland

Kjeldahl distillation unit K-350 Büchi, Switzerland

Laboratory mixer/emulsifier L2R Silverson Machines Ltd., Waterside,

England

Low temperature incubator 307 Fisher Scientific, Fair Lawn, NJ, USA

Low temperature incubator 315 Precision Scientific, Chicago, IL, USA

Mechanical stirrer RZR 50 Caframo, Warton, ON, Canada

Membrane cell system SEPA CF II General Electric Osmonics,

Minnetonka, MN, USA

Pellicon 2 mini P2C0 05V 01 EMD Millipore, Billerica, MA, USA

48

ultrafiltration membrane

Pellicon filter holder XX42 PMI NI EMD Millipore, Billerica, MA, USA

Peristaltic pump 7518-12 Cole-Parmer Instrument Co., Bernon

Hills, IL, USA

pH meter 8000 VWR Scientific, West Chester, PA,

USA

Polyacrylamide gel, 4-20%,

10 well 161-1105

Bio-Rad Laboratories, Hercules, CA,

USA

Prestained protein ladder, 10

to 170 kDa 26616

Thermo Scientific Pierce, Rockford, IL,

USA

Refrigerator/Freezer RB1855SW Samsung, Daehu, South Korea

Rotary evaporator Rotavapor-R Büchi, Switzerland

Ultrafiltration membrane YMPTSP1905 Sterlitech, Kent, WA, USA

Plastic storage bags Ziploc

165 x 149 mm

SC Johnson Canada, Brantford, ON,

Canada

4.5 Experimental Methods

4.5.1 Aqueous extraction process

The aqueous extraction process used for the current study is based on the experimental

procedures followed by Balke (2006) and Ataya (2010). The materials and conditions for this

process were:

• 400 grams of mustard flour

• 3 minutes homogenizing time

• 4:1 water to flour ratio

• 30 minutes extraction time

• Extraction pH of 11.00 ± 0.05

49

• Ambient temperature (~25°C)

• 3 stages

Figure 14 shows the flow diagram for the aqueous extraction. Yellow mustard flour was mixed

with 4 times its weight with water using a spatula in a 2L beaker until a uniform paste was

obtained, free of lumps. The mixture was homogenized for three minutes using a L2R Silverson

mixer at top speed followed by the addition of ascorbic acid (1% w/w) as antioxidant. The native

pH of the mixture was around 4.75 and it was adjusted and maintained at a value of 11.00 ± 0.05

by the addition of sodium hydroxide solution (50% w/w). The extraction was performed for a

period of 30 minutes, after which the mixture was poured evenly into 3 1L bottles and

centrifuged for 20 minutes at ~10500 x g (6500 rpm). Three fractions were separated: a solid

residue at the bottom of the bottle, a liquid protein solution in the middle and an oil rich

emulsion at the top. The emulsion and protein extract were recovered and weighed in separate

containers, while the solid residue was transferred back to the extraction beaker, enough water

was added to reach the weight of the initial mixture, and it was re-homogenized for three minutes

and re-extracted under the same conditions. After centrifugation, the protein solution and

emulsion were recovered and weighed. The solid residue was re-suspended in water up to 4:1

water to solids ratio, homogenized for three minutes and centrifuged again. The final solid

residue was neutralized with 6 M phosphoric acid to a pH value around 7.50. The three protein

solutions were combined.

50

NaOH, 50%

Homogenizer – 3 min.

Mixing at pH 11 for 30 min. Room temp.

Emulsion

Meal Residue

Add water (4:1) Homogenizer – 3 min.

Centrifugation 20 min @ 6500 rpm

25ºC Protein Extract / Emulsion

Water addition (4:1)

Centrifugation 20 min @ 6500 rpm

25ºC

Protein Extract

Mixing at pH 11 for 30 min. Room temp.

Centrifugation 20 min @ 6500 rpm

25ºC

Meal Residue

Add water (4:1) Homogenizer - 3 min.

Centrifugation 20 min @ 6500 rpm

25ºC

Washing

Neutralize meal residue

PO4H3, 6M

Protein Extract / Emulsion

Figure 14: Flow diagram of the aqueous extraction process

Yellow mustard flour

NaOH, 50%

51

4.5.2 Membrane processing of protein solution and isoelectric precipitation

For the purification and isolation of the proteins extracted by the aqueous process, membrane

processing and isoelectric precipitation operations were followed. The protein solution obtained

from the aqueous extraction process was first filtered using Whatman filter paper No. 1. After

filtration, enough reverse osmosis water was added to the solution to obtain a concentration

factor between 5.5 and 6.0 during the ultrafiltration process. Water addition was necessary in

order to obtain a concentrated protein solution with a final concentration below 10%, which is

near the solubility limit of mustard proteins. The water quantity was calculated by multiplying

the protein solution quantity after filtration by a factor of 0.5672. An additional 2.92 g/L,

considering the overall quantity of water and protein solution, of sodium chloride was added and

dissolved to obtain a 0.05 M sodium chloride concentration. The solution was heated and

maintained between 55 and 60ºC for a 30 minute period, treatment that could break the phenolic-

protein complexes bonded ionically (Diosady, Xu and Chen 2005) and later be removed during

membrane processing.

PI

Feed

Permeate Concentrate

Membrane Buffer solution Diafiltration

Figure 15: Ultrafiltration/Diafiltration process schematics

52

After the heating treatment the solution was cooled down to 40ºC, a sample was taken for protein

determination, and the rest was submitted to ultrafiltration and diafiltration. Two different

equipments were used; the first was a Pellicon filtration system consisting of a Cole-Parmer

peristaltic pump, a filter holder and a regenerated celulose acetate membrane with a MWCO of 5

kDa and a membrane area of 0.1 m2. The second equipment consisted in a SEPA CF II filtration

system equiped with a Hydracell diaphragm pump, a Baldor electric motor with a frequency

variator capable of achieving different motor speeds and pump flows, and a 5 kDa MWCO

polyethersulfone membrane with an effective area of 0.015 m2. This equipment had been used in

our food laboratory for high pressure membrane processing, such as reverse osmosis and

nanofiltration applications, and needed to be addapted for ultrafiltration operations. The

adaptation consisted in the replacement of the pressure gauge for one with a lower range capable

of measuring pressures between 0 and 100 psi with more accuracy, and the addition of a pressure

dampener for the gauge, in order to decrease the pointer pulsation and be able to obtain reliable

measurements. Unfortunately, due to the high volumes used during the experiments, the use of

this equipment became unpractical because of its design, which offers a smaller effective

membrane area and does not allow filter stacking in the membrane cell system.

Molecular weight profiles of alkaline mustard extracts show that most of the mustard proteins

have a molecular weight above 5 kDa (Dendukuri and Diosady 2003, Ranjana, Bhattacherjee and

Ghosh 2009), hence a membrane with a MWCO of 5 kDa was selected for the ultrafiltration and

diafiltration processes in order to obtain maximum protein recovery. During the operation of

both systems in ultrafiltration mode, solvent and low molecular weight components that were

able to pass through the membrane were collected as permeate. The concentrated solution was

recycled to the feed container and recovered as the retentate (Figure 15). After ultrafiltration, the

53

retentate was submitted to a continuous diafiltration step by the addition of enough 0.05 M

sodium chloride solution at pH 11.0 to obtain a diafiltration volume of 5.5, at the end of which a

sample was withdrawn for protein determination. The ultrafiltration and diafiltration permeate

fractions were mixed, a sample was taken for protein determination and the rest was discarded.

Upon completion of each experiment, the unit was immediately drained and flushed with

distilled water. A 7.5 g/L enzymatic detergent solution was then recycled through the unit for

about one hour. After draining, the unit was again flushed with distilled water until the initial

water flux was restored. A volume of 15-20 liters of distilled water was usually used. The

cleaned membrane cartridge was stored in a 0.5% (w/v) formaldehyde solution. One day prior to

use, the cartridge was rinsed and soaked in distilled water.

For the isoelectric precipitation of the concentrated protein extract, 6 M phosphoric acid was

added in a drop-wise manner under continuous agitation in order to decrease the pH to a value of

around 5.50. Moderate agitation was maintained for an additional 20 minutes.

The Pellicon system was used under the following operating conditions:

• Peristaltic pump speed setting of 300 rpm

• Typical flow rate of 0.5 L/min

• Backpressure of 30 psi (~200 kPa)

• System temperature of 40ºC

On the other hand, the operating conditions for the SEPA CF II filtration system were the

following:

• 20 Hz motor frequency

• Typical flow rate of 2.22 L/min

• Backpressure of 50 psi (~350 kPa)

54

• System temperature of 40ºC

In this work, it was assumed that any problems with phytate, glucosinolate and phenolics content

could be resolved by application of membrane technology already developed for use with

extracts from defatted flour (Luo 1998, Lui 1998, Xu 1998, Xu et al. 2003).

4.5.3 Protein isolates recovery

After 20 minutes of mixing at a pH around 5.50, the suspended solids dispersion was centrifuged

at ~10500 x g (6500 rpm) for 20 minutes. The supernatant consisting of protein solution A was

separated, while the solid fraction was re-suspended and washed with 5 times its weight of

reverse osmosis water and was likewise centrifuged. The supernatant consisting of protein

solution B was again separated and the solid fraction, consisting of precipitated protein isolate,

was frozen with liquid nitrogen and freeze-dried. Both protein solution fractions were then

submitted separately to an ultrafiltration and diafiltration process using the Pellicon system. For

both of the protein solutions A and B, the operating conditions during membrane processing

were:

• Peristaltic pump speed setting of 300 rpm

• Typical flow rate of 0.5 L/min

• Backpressure of 30 psi (~200 kPa)

• System temperature of 40ºC

After ultrafiltration and diafiltration, both fractions were mixed, shell-frozen with liquid nitrogen

and freeze-dried. Figure 16 shows a complete flow diagram for the protein recovery process.

55

Figure 16: Protein extract membrane processing

Ultrafiltration 40°C, CF >= 5.0

Diafiltration, pH=11, 0.05 M NaCl. DV >= 5.5

Isoelectric Precipitation. Mixing 20 min.

Filtration. Whitman Filter paper #1

Protein Solution Heat treatment 55°C 30 min.

Centrifugation 20 min @ 6500 rpm

25C

Ultrafiltration. 40°C, CF = 2.30

Diafiltration. DV >= 5.50 PPI washing

(water 5:1)

Protein solution A

Centrifugation 20 min @ 6500 rpm

25C

Ultrafiltration. 40°C, CF = 7.70

Diafiltration. DV >= 5.50

Protein solution B

PIAB Freeze drying

PPI Freeze drying

NaCl 0.05M

PO4H3 6M

56

4.5.4 Temperature effect on lipid oxidation in the precipitated protein isolate

To determine the storage temperature effect on the oxidation of the oil contained in the

precipitated protein isolate, 3 sets of 7 ten-gram samples of the powder were stored in plastic

storage bags and stored at 25ºC, 35ºC and 45ºC. Thiobarbituric acid reactive substances

(TBARS) associated with the isolate oil were monitored at 1 week intervals.

TBARS of the extracted oil were measured using AOCS method Cd 19-90 (Appendix A10). For

the oil extraction, a 10-gram sample was mixed with 100 mL of a mixture of petroleum ether and

diethyl ether (50:50 v/v) and stirred for a 5 hour period at room temperature. The mixture was

then centrifuged at 2000 rpm for ten minutes and the supernatant was recovered in a round

bottom flask. The solvent was evaporated using a rotary evaporator system at 30ºC. The oil

recovered was frozen at -20ºC until analyzed.

4.5.5 Functional properties

4.5.5.1 Nitrogen solubility index (NSI)

The NSI was determined by AOCS method Ba 11-65. A 2.5% dispersion in reverse osmosis

water was stirred at 30ºC in a water bath. After centrifugation the supernatant was filtered

through a glass fiber plug and protein content was determined by AOCS method Ba 4d-90. A

detailed description of the procedure can be consulted in Appendix A4.

4.5.5.2 Water absorption capacity (WAC)

Water absorption for the protein isolates was determined by the method developed by Naczk et

al. (1985). A two-gram sample was dispersed in 16 ml of reverse osmosis water in a 50 mL

centrifugation tube and mixed 30 seconds every 10 minutes 7 times. The mixture was then

57

centrifuged at 2000 x g. The supernatant was carefully decanted and the tube was tilted and

drained at an angle between 15º and 20 º mouth down for 15 minutes and weighed inmediatly. In

the case of soluble protein isolates, the product remaining in the tube was determined by

completely washing the contents into a 50 mL beaker and drying the wet sample in an oven at

105 ºC for 24 hours. The WAC was reported as the percentage increase of sample weight. A

detailed description of the procedure can be consulted in Appendix A6.

4.5.5.3 Oil absorption capacity (OAC)

Oil absorption was measured using the method developed by Sosulski et al. (1976) by dispersing

a two-gram sample in 12 mL of canola oil in a 50 mL centrifuge tube. The contents were stirred

for 30 seconds every 5 minutes, and after 30 minutes the tubes were centrifuged at 1600 x g for

25 minutes and the supernantant was carefuly decanted. Oil absorbed by the sample was

measured as weight gain. A detailed description of the procedure can be consulted in Appendix

A5.

4.5.5.4 Emulsifying activity and emulsifying stability

Emulsifying activity was measured using the method developed by Yasumatsu et al. (1972),

while emulsion stability was assayed by the method used by Naczk et al. (1985). A 3.5 g sample

was dispersed in 50 mL of reverse osmosis water. 25 mL of canola oil were added and the

mixture was emulsified for 30 seconds. A further 25 mL of canola oil were added and the

mixture was again emulsified for 90 seconds. The emulsion was divided evenly in 50 mL

centrifugation tubes and centrifuged at 2000 rpm. The emulsifying activity was then calculated

as the relationship between the emulsifyied layer in the tube and the tube total contents. For the

emulsion stability, emulsions prepared as described were heated to 85ºC for 15 minutes prior

58

centrifugation, and the emulsion stability was expressed as a percentage of the emulsifying

activity remaining after heating. A detailed description of the procedures can be consulted in

Appendices A7 and A8.

4.5.5.5 Foam expansion and foam volume stability

The foam expansion value was determined by the method used by Naczk et al. (1985). 3%

dispersions were homogenized for 6 minutes. The resulting mixture was transferred to a

graduated cylinder and the foam volume noted. The foam stability after 20, 40, 60 and 120

minutes was reported as the foam volume stability (FVS) calculated according to the method of

Patel et al. (1988). A detailed description of the procedure can be consulted in Appendix A9.

4.5.5.6 Least gelation concentration

Least gelation concentration (LGC) was assayed by the method of Moure, et al. (2002). 2, 4, 6, 8,

10, 12 and 14% dispersions (w/v) were prepared using reverse osmosis water. The dispersions

were adjusted to pH 7.0 by the addition of 1 N NaOH and mixed. 5 mL of each dispersion were

added to a test tube and heated in boiling water for 1 hour, followed by rapid cooling under cold

tap water and additional cooling at 4ºC for 2 hours. The LGC was calculated as the concentration

above which the sample remained in the bottom of the inverted tube. A detailed description of

the procedure can be consulted in Appendix A11.

4.5.6 Other analytical methods

• Oil content was determined by the Mojonnier method. AOAC Method 922.06 was used

for lipids in flour or solid samples and AOAC Method 995.19 was used for lipids in

emulsions. Results were reported as oil percentage by acid hydrolysis.

59

• Protein content was determined by Kjeldahl method AOCS Ba4d-90 and reported as

Nx6.25%.

• Moisture content was measured using AACC method 44-155A.

• SDS-PAGE analyses for reduced and non-reduced conditions were run on 4-20%

gradient gels according to the manufacturer’s instructions (Bio-Rad Laboratories).

Samples were mixed with sample buffer and heated to 95ºC for ten minutes, cooled to

room temperature and centrifuged at 16000 x g for 10 seconds. A 14 µl aliquot was

loaded onto the gel. Reduced samples were prepared by adding 5% 2-mercaptoethanol to

the sample buffer prior heat treatment.

For details, refer to Appendices A1, A3, A2 and A12 respectively.

60

5. RESULTS AND DISCUSSION

5.1 Starting material analysis

The same batch of de-hulled yellow mustard flour was used for all the experiments presented in

the following sections. Protein, moisture and oil determination were performed and the TBA

value of an oil sample extracted from the flour was also determined. The characterization of the

yellow mustard flour is presented in Table 6.

Table 6: Yellow mustard flour characterization

Content

(wt %, as is) Crude proteina 31.24 ± 0.14 Oila 36.78 ± 0.11 Moisturea 5.01 ± 0.29 TBA Value* 0.1037 ± 0.0021

*TBA value reported as mg of malondialdehyde per kilogram of sample (Mean value ± standard deviation) aData reported as mean values ± standard deviation

The oil and protein values are consistent with protein and oil contents for yellow mustard seeds

harvested in 2011. The measured TBA value was similar to the value of 0.99 reported in AOCS

method Cd 19-90 for rapeseed oil.

5.2 Aqueous extraction process

Aqueous extraction of yellow mustard flour was performed following the procedure described in

Section 4.5.1. In a previous project carried out in our food engineering group many variables

where considered to have an influence in the aqueous extraction of yellow mustard seeds. It was

concluded that pH had a very significant effect. Maximum oil and protein extraction was

observed at high pH values. Water to flour ratio, extraction time, temperature and

61

blending time were also studied and it was concluded that pH 11, 4:1 water to flour ratio, 3 min

blending time, room temperature and 30 min of extraction, with two extraction and one washing

stages of the solid residue under the same conditions, represented the best set of parameters for

oil and protein recovery (Balke, 2006).

For all extractions, approximately 400 g of yellow mustard flour were mixed with 4500 g of

water, 4 grams of ascorbic acid, and around 35 g of 50% NaOH solution in order to maintain the

pH at 11.0 ± 0.5. After centrifugation, a solid residue phase, a liquid protein solution and an

emulsion phase were separated in each stage. Every batch produced around 180 g of emulsion,

4000 g of protein solution and 580 g of meal residue. The protein solution represented 84% of

the total weight of the three phases, the solid residue 12% and the emulsion phase 4%.

The composition of each of the fractions resulting from the aqueous extraction is presented in

Table 7, and the key component balance is presented in Figure 17 and further expanded in

Appendix B2. The protein solution is rich in protein, with 81.5% of the total crude protein

present in the original flour, while the emulsion and residue contain 4.9% and 7.8% of the

original crude protein respectively, giving a crude protein extraction yield of at least 86.3%.

Mustard protein is highly soluble at high pH and protein extraction yields of over 90% have been

previously reported (Balke 2006) when extracted at a pH value of 12. Part of the protein present

in the emulsion phase is likely to be oleosin, which is not soluble at alkaline pH, while most of

the non-oleosin protein is dissolved in the protein solution.

The crude protein concentration in the protein solution was low, around 2.6%. Further processing

was necessary to recover protein isolates with high purity. A process involving ultrafiltration and

diafiltration followed by isoelectric precipitation before drying of the final products was used to

62

recover isolates with high protein purity. Given the well-balanced amino acid profile of yellow

mustard proteins and its functional properties, the production of protein isolates could yield

products comparable to those of soy for use in different applications such as protein bars, protein

enhanced beverages, soups, bakery products, among others. Application of the meal residue as a

food ingredient is also possible even though the protein concentration is low. Its emulsifying and

binding functional properties may be useful in the production of processed meats.

Table 7: Protein and oil composition of the resulting fractions after aqueous extraction

Component Protein (%) Oil (%) Emulsion* 3.01 ± 0.01 45.12 ± 0.47

Meal residue** 11.74 ± 0.04 21.57 ± 1.1 Protein solution** 50.95 ± 0.09 13.92 ± 0.08

*Reported as wt%, as is (Mean values ± standard deviation) **Reported as wt%, dry basis (Mean values ± standard deviation)

Recovering mustard protein via aqueous extraction is a feasible process; however, because the

oil is extracted in the form of an oil-in-water emulsion, separation of free oil is difficult. This

represents a major disadvantage in the aqueous process because de-emulsification steps are

necessary to recover the oil. The presence of oleosin and other proteins surrounding the oil

bodies is the main reason for the stability of the emulsion, as it was described in section 2.2.3.

The surfactant nature of proteins stabilizes the oil drops and prevents their coalescence. As

proposed by Balke (2006), a high pH extraction leads to an electrostatically stabilized dispersion

that resists flocculation and coagulation, where the surfactant nature of proteins helps keep some

oil in the protein solution, which ends up in the protein isolates and might affect the functionality

and physicochemical characteristics of the final products.

63

While 62.1% of the oil present in the original flour is extracted in the emulsion, 12.2% remains

in the solid residue after extraction and 18.9% in the protein solution. In a previous study

performed in the food engineering laboratory, oil recovery in the emulsion was as high as 75% at

alkaline pH (Balke 2006).

5.3 Membrane processing of protein solution and isoelectric precipitation

Membrane processing of the protein solution resulting from the aqueous extraction was

performed following the procedure described in Section 4.5.2. For all the experiments,

Figure 17: Aqueous extraction. Mass balance of key components (*Estimate)

Aqueous Extraction

64

approximately 4000 g of protein solution resulting from the aqueous extraction process were

filtered using a Whatman No. 1 filter paper. After filtration, around 2330 g of reverse osmosis

water were added to the filtrate in order to obtain a concentration factor between 5.0 and 6.0 in

the following ultrafiltration operation. Subsequently, sodium chloride was added to the diluted

protein solution prior to the heat treatment, after which the solution was submitted to

ultrafiltration with an average concentration factor of 5.5. The average flux for this operation was

17.2 liters per square meter of membrane per hour (Lmh) when using the Pellicon equipment,

and around 25.0 Lmh when using the SEPA CF II system. In every batch an average of 5441.1 g

of permeate and 957.2 g of retentate were produced. After ultrafiltration, the retentate was

diafiltered in a continuous manner by the addition of approximately 5330 g of 0.05 M sodium

chloride solution at pH 11.0 in order to get a diafiltration volume of 5.5. The average flux for the

diafiltration process was 13.6 Lmh when using the Pellicon equipment, and around 20.0 Lmh

when using the SEPA CF II equipment. The diafiltration permeate accounted for the 5330 g of

brine continuously added during the process. The differences in the ultrafiltration and

diafiltration flux for the two systems can be attributed to the different pressures and recirculation

flows used. Additionally, the polyethersulfone membrane used in the SEPA CF II equipment was

brand new, while the regenerated cellulose membrane used in the Pellicon equipment had been

used for several years and although it was meticulously washed after each use, its use resulted in

irreversible fouling and a lower flux. After the membrane process, the concentrated protein

solution, which had a dark brown colour and high viscosity, was acidified by the addition of

approximately 14.5 g of 6 M phosphoric acid, and was treated as described in section 4.5.2. After

isoelectric precipitation, the colour of the protein suspension changed to a lighter brown and

changes in viscosity were also noted, increasing with acid addition to a maximum near pH 7.0,

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and decreasing again at pH 5.5. Similarly to other brassica oilseeds like canola or rapeseed,

mustard has a wide variety of proteins and a broad spectrum of isoelectric points. There is not a

clear maxima for protein precipitation along pH range from 3.0 to 9.0 (Balke 2006), however a

pH value around 5.5 is considered to be in the region of highest protein precipitation.

5.4 Protein isolates recovery

Following the procedure described in section 4.5.3, after centrifugation of the 957.2 g of

acidified protein suspension, the supernatant, consisting of approximately 632 g of protein

solution A was kept aside while the precipitate was washed with around 2217 g of reverse

osmosis water and centrifuged again. The second supernatant, consisting of 2185.1 g of protein

solution B on average, was poured in a separate bottle and the 318.8 grams of precipitate

obtained on average were freeze-dried. Protein solutions A and B were not mixed prior to

membrane processing. The mixture of both protein solutions resulted in protein precipitation,

which can be explained by a “micellization” effect similar to the reported by Murray et al.

(1979), where the dilution of the protein solution A, with a higher ionic strength and

concentration, with protein solution B induced a “salting out” effect. Altough this method could

be applied for the recovery of some of the protein, after centrifugation and freeze-drying of the

precipitate, the resulting product had a salty flavour and a protein concentration of 81.96% on a

moisture free basis, and it was concluded that a diafiltration step was still necessary to improve

its quality. Since the precipitate resulting from the mixture of both protein solutions would likely

decrease the flux during membrane processing, it was decided to treat them separetly.

The 632 g of protein solution A obtained from each batch were submitted to ultrafiltration using

the Pellicon system with a concentration factor of 2.33, after which around 360 g of permeate

were obtained and discarded, and the remaining retentate was diafiltered up to a diafiltration

66

volume of 5.5 using reverse osmosis water. For the protein solution B, the 2185.1 g obtained for

each batch were submitted to ultrafiltration with a concentration factor of 7.7, and the resulting

retentate to a continuous diafiltration process with a diafiltration volume of 5.5 using reverse

osmosis water. The average ultrafiltration flux measured after the treatment of both protein

solutions was 18.0 Lmh, while the average diafiltration flux was lower at 5.6 Lmh mainly due to

the high amount of protein precipitation during this step. Both retentate fractions were mixed

after membrane processing shell-frozen using liquid nitrogen and freeze-dried. As shown in

Figure 18, the concentrated protein suspension had a milky appearance.

Figure 18: Protein fractions A and B after membrane processing

The composition of each of the fractions is presented in Table 8, while the distribution and yields

of oil and protein for the membrane processing and the recovery of protein isolates are presented

in Figure 19 and further expanded in Appendix B3. After ultrafiltration of the initial protein

solution, the retentate contained approximately 66.2% of the original crude protein present in the

67

starting material, while the total permeate contained 11.2%, giving a crude protein recovery of

around 83.0% by the 5 kDa membrane.

Table 8: Protein and oil composition of the resulting fractions before and after membrane processing

Protein (%) Oil (%)

Concentrated protein solution* 8.7 ± 0.35 3.0a Total permeate* 0.14 ± 0.00 - Precipitated protein isolate* 21.5a 7.7a Protein solutions (A+B)* 0.5a -

*Reported as wt%, as is (Mean values ± standard deviation) a Estimate

These results indicate that around 11.2% of the extracted mustard crude protein consists in

peptides with a molecular weight lower than 5 kDa or most likely non-protein nitrogen. The

crude protein concentration in the final retentate was around 9.0%, which was found to be its

solubility limit, as shown by precipitation in the concentrated solution that along with the high

oil content contributed to an increase in the viscosity and a sharp decrease of the flux during the

last part of the diafiltration process. After isoelectric precipitation, the solid fraction obtained

represented 33.3% by weight of the concentrated protein solution and contained 83.1% of its

total protein content, while the sum of both protein solutions A and B contained roughly 17.0%.

In terms of yields, the precipitated fraction accounted for 55.0% of the total crude protein in the

starting material, while 11.2% of the total protein was recovered from the protein solutions A

and B, a quantity very similar to the crude protein yield of the permeate from the ultrafiltration

and diafiltration of the initial protein solution.

68

While 18.9% of the oil present in the original flour is extracted into the protein solution, only

16.8% could be accounted for in the protein isolates. Around 2% of the oil was lost during

membrane processing and in the permeates. During the oil analysis of the protein, difficulties

were encountered in closing the oil mass balance. Similar difficulties have been reported by

Balke (2006) in the analysis of precipitated protein isolates from alkaline full fat yellow mustard

flour aqueous extraction. Balke proposed that a material either proteinaceous or a saccharide

extracted at alkaline pH associated with the oil bodies and was responsible for hexane extraction

resistance. Flash freezing using liquid nitrogen prior freeze-drying improved the effectiveness of

Filtration Heat treatment Ultrafiltration Diafiltration

Isoel. Precipitation Washing

Centrifugation

Figure 19: Yields for the membrane process and isoelectric precipitation of the protein solution

69

hexane for defatting, probably because the smaller particle size obtained by this technique as

opposed to a tougher glassy structure obtained by slow freezing, but the improvement was not

enough to give good closure to the mass balance. Lipids remaining in the protein solution after

alkaline extraction are likely bound to proteins or polysaccharides by hydrophobic or hydrogen

bonding. Non-polar solvents like hexane are typically able to extract hydrophobically bound

lipids, although in some cases hydrophobic regions may be surrounded by polar regions,

preventing extraction. In such cases, proteins may be able to provide an ionic barrier to non-polar

solvents, or water molecules strongly hydrogen-bonded to protein and polar lipids may form

stabilized aqueous regions so that the non-polar lipids are not exposed to the extracting solvent

(Nelson 1975). To overcome this problem, the method of lipid extraction by acid hydrolysis was

used as described in section 4.5.6 throughout the experiments.

After freeze-drying an average of 97.46 grams of precipitated protein isolate, and 17.08 grams of

protein isolate from solutions A and B (called acid soluble protein isolate hereafter) were

recovered per batch, these quantities represented 28.6% of the initial mass of yellow mustard

flour and around 66.2% of its initial crude protein content. As mentioned before, 4.9% of the

initial protein was recovered in the emulsion fraction, 7.8% remained in the meal residue, 11.2%

was recovered in the permeate fractions and around 10.0% was lost during the different steps of

the process. On the other hand, 62.1% of the oil was recovered in the emulsion, 12.2% remained

un-extracted in the meal residue, 9.0% was lost in the process, and 16.7% ended up in the protein

isolates. The quality of the protein products obtained is described in Table 9. It is interesting to

note the hydrophobic nature of both protein isolates, hence the low moisture content, which was

enhanced by the high oil concentration in the case of the precipitated protein isolate.

70

While the protein concentration of the precipitated protein isolate was only 70.43% in “as is”

basis, it had a purity of 96.0% on a moisture and oil free basis. For the acid soluble protein

isolate, on the other hand, a purity of 83.5% was reached on a moisture and oil free basis. Oil

determination shows that more than 98% of the oil recovered in the isolates was precipitated

along with the proteins during the pH adjustment, and less than 2% remained bound with the

proteins in solutions A and B; the precipitated protein isolate had 0.35 grams of oil per gram of

protein while the acid soluble protein isolate had 0.06 grams of oil per gram of protein.

Table 9: Final product characterization

Precipitated protein isolate Acid soluble protein isolate Protein* (%) 70.43 ± 0.97 81.88 ± 0.79 Oil* (%) 25.11 ± 0.36 0.48 ± 0.03 Moisture* (%) 1.56 ± 0.01 1.47 ± 0.20 Others* (%) 2.90a 16.17a Mass yield (%) 24.30b 4.26b TBA value** 0.1165 ± 0.0036 N.D.

*Reported as wt%, as is (Mean values ± standard deviation) a Estimate b Reported as wt% of the initial mustard flour quantity **Reported as mg of malondialdehyde per kg of sample (Mean values ± standard deviation)

The comparison between the TBA value of the initial mustard flour and that of the precipitated

protein isolate indicates there is some lipid oxidation during the process, which was expected due

to the frequent transferring, mixing and heating to which the product was submitted. The TBA

value for the acid soluble protein isolate was not determined due to its low oil content. The high

oil amount and the different protein concentration for both isolates were expected to have an

impact in their functional properties, especially for the precipitated protein isolate. These factors

were studied and the results presented in the following sections.

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5.5 Functional properties

5.5.1 Colour and Flavour

The precipitated protein isolate obtained during the experiments had a slightly darker colour than

the acid soluble protein isolate. Both products were compared to soybean protein isolate Supro

500E (Figure 20) which is a standard commercial isolate. While the soybean protein isolate was

slightly yellow, the precipitated protein isolate was light tan in colour and the acid soluble

protein isolate was off-white, lighter than the soybean product. All three products had a similar

consistency, mostly composed of fine particles. The precipitated protein isolate had a very bland

flavour, not salty and with a slightly bitter aftertaste, conversely the soybean protein isolate had a

more creamy mouth feel, a characteristic soybean flavour and was a little bit salty, while the acid

soluble protein isolate had an astringent taste, a slightly bitter aftertaste and was slightly salty.

5.5.2 Gel electrophoresis

The molecular weight distribution of polypeptides in the protein isolates can provide important

information about their functionality in food systems. Differences between the polypeptide

compositions may relate to differences in some of the functional properties observed in this

Precipitated protein isolate

Acid soluble protein isolate

Soybean protein isolate (Supro 500 E)

Figure 20: Protein isolates comparison

72

study. Figures 21 and 22 show the molecular weight profiles of both products, obtained by gel

electrophoresis for non-reducing and reducing conditions, respectively.

In Figure 21, two of the mustard characteristic storage proteins can be identified in both isolates;

the 14 kDa band shows the presence of the 1.7s storage protein, which has an estimated

molecular weight of 15 kDa, and the 25 kDa band, also present in both products, belongs to the

2S storage protein of mustard seeds (Aluko and McIntosh 2004). The Figure also shows that

there is a marked difference in the 55 and 50 kDa bands between the products. For the acid

soluble protein isolate, both bands have a higher intensity, which indicates that the isolate is

more abundant in polypeptides with such molecular weights and that these are more resistant to

acid precipitation.

a b c d

~130

~120

~50

~14

~55

~25

e

Figure 21: Non-reducing conditions SDS-PAGE patterns of the precipitated protein isolate (lanes a and b), acid soluble protein isolate (lanes c and d) and protein

standards (lane e)

73

Also, the precipitated protein isolate gel pattern shows intense bands around 36, 25 and 20 kDa,

which are missing or are less intense in the acid soluble protein isolate, suggesting that

polypeptides of those molecular weights form proteins susceptible to acid precipitation. These

proteins might have a higher concentration of hydrophobic groups at their surface which favor

lipid-protein and protein-protein interactions. Acid soluble protein isolate presents high intensity

bands in the 26 to 35 kDa range, which are less intense or missing in the precipitated protein

isolate.

Figure 22 shows the gel patterns for both isolates under reducing conditions. The addition of 2-

mercaptoethanol as a reducing agent during the SDS-PAGE procedure helped identify the

presence of disulfide bonds in the polypeptides. In both cases, bands at 130, 120, 55, 50, 25 and

14 kDa that can be identified in Figure 21, disappeared almost completely in Figure 22,

indicating that each of these proteins contain polypeptides held together by disulfide bonds,

a b c d

~70

~12 ~8

~35 ~30

e

Figure 22: Reducing conditions SDS-PAGE patterns of the precipitated protein isolate (lanes a and b), acid soluble protein isolate (lanes c and d) and protein

standards (lane e)

74

while bands at 70, 30, 35, 12 and 8 kDa appeared in both cases. The presence of disulfide bonds

in proteins is usually related to limited levels of molecular flexibility, unfolding and

reorientation, all fundamental aspects for protein adsorption at the interface during the formation

of foams and emulsions. On the other hand, it has been proposed that in globular proteins

disulfide bonds may increase the average molecular weight or increase the polymer chain length

during gelation (Damodaran 1996).

5.5.3 Nitrogen Solubility Index (NSI)

As shown in Table 10, there was a large difference between the NSI values of the precipitated

protein isolate and the acid soluble protein isolate. The first was as low as 1.36% and the second

reached 39.53%, which is comparable to the value obtained by Lin (2007) for soybean protein

isolate Supro 500E (44.5%). It is also close to the solubility value of 35.0% reported by

Pedroche, et al. (2004) for abyssinian mustard precipitated protein isolate and the value of 39.8%

reported by Liadakis et al. (1998) for soybean protein isolate Supro 670, but it is low compared

to the NSI values of defatted whole ground yellow mustard soluble protein isolate, double-zero

oriental mustard soluble protein isolate and the value reported for rapeseed soluble protein

isolate (Yumiko et al. 2008).

The precipitated protein isolate, on the other hand, exhibited lower solubility than the reported

for defatted whole ground yellow mustard precipitated protein isolate (2.64%), the reported value

of 2.8% for Chinese rapeseed precipitated protein isolate (Xu et al. 1994), and the values of

12.1% and 26.4% for double-zero oriental mustard precipitated protein isolate (Lin 2007) and

rapeseed precipitated protein isolate (Yumiko et al. 2008), respectively. Since the most important

factor that affects the solubility of a protein is the balance of hydrophobic and hydrophilic

residues on its surface, these results suggest that the protein isolates obtained in this study have a

75

structure or conformation where hydrophilic groups are most likely blocked, and their interaction

with the aqueous environment is hindered.

Table 10: Nitrogen solubility index value of different protein isolates

*DWGYM = Defatted whole ground yellow mustard PPI = precipitated protein isolate. SPI = soluble protein isolate

a Mean values ± standard deviation

In the precipitated protein isolate this effect might be amplified by its high oil content. In

addition, the pH used for the test was very near to their isoelectric points.

5.5.4 Water absorption capacity (WAC) and oil absorption capacity (OAC)

The water absorption capacity (WAC) can be used as an index of the water-binding properties of

a protein product and suggests the degree of protein interaction with water. In this study it was

found that the WAC value for the precipitated protein isolate was low (Table 11), which could be

explained by the presence of oil, since water binding by proteins is influenced by their physical-

chemical environment, and it might also suggest low availability of polar amino acids, which are

the primary sites for water interaction of proteins (Sathe et al. 1982). This value is comparable to

those reported by Lin (2007) for the precipitated protein isolate produced from double-zero

Product NSI (%) Reference DWGYM* SPI 95.71 Lin (2007) Rapeseed SPI 93.3 Yumiko et al. (2008) 0-0 Oriental mustard SPI 75.9 Lin (2007) Soybean Supro 500 E 44.5 Soybean Supro 670 39.8 Liadakis et al. (1998) Acid soluble protein isolatea 39.53 ± 0.45 Current study Abyssinian mustard PPI 35 Pedroche et al. (2004) Rapeseed PPI 26.4 Yumiko et al. (2008) 0-0 Oriental mustard PPI 12.1 Lin (2007) Chinese rapeseed PPI 2.8 Xu et al. (1994) DWGYM* PPI 2.64 Lin (2007) Precipitated protein isolatea 1.36 ± 0.12 Current study

76

oriental mustard, which contained a low amount of oil (3.2%), but it is near 2.5 times lower than

the values obtained for the precipitated protein isolate produced from defatted whole ground

yellow mustard and 1.7 times lower than the value reported for Chinese rapeseed precipitated

protein isolate by Xu et al. (1994).

Table 11: Water absorption capacity for different protein isolates

* DWGYM = Defatted whole ground yellow mustard PPI = precipitated protein isolate. SPI = soluble protein isolate a Mean values ± standard deviation

On the other hand, acid soluble protein isolate presented a WAC value 2 times higher than the

precipitated protein isolate (Table 11), but it was still 2.2 times lower than the water absorption

capacity of the soybean protein isolate Supro 500 E (Lin 2007) and very similar to the value

obtained for the soybean protein isolate Supro 670 by Liadakis et al. (1998). In comparison to

other mustard products, it was 3 times lower and 14.6 times higher than double-zero oriental

mustard soluble protein isolate and defatted whole ground yellow mustard soluble protein

isolate, respectively (Lin 2007). It did not moisten well on first contact with water, but could be

dispersed after vigorous mixing.

Product WAC (%) Reference 0-0 Oriental mustard SPI 787.3 Lin (2007) Soybean Supro 500 E 580.6

DWGYM PPI* 316.5 Acid soluble protein isolatea 263.1 ± 6.2 Current study

Soybean Supro 670 241.2 Liadakis et al. (1998) Chinese rapeseed PPI 219.3 Xu et al. (1994) 0-0 Oriental mustard PPI 151.4 Lin (2007) Precipitated protein isolatea 131.1 ± 3.7 Current study Abyssinian mustard PPI 99.0 Pedroche et al. (2004) DWGYM SPI* 18.0 Lin (2007)

77

The acid soluble protein isolate had a higher oil absorption capacity than most of the isolates that

were compared (Table 12). The value of 239.3% was very similar to that of soybean protein

isolate Supro 670 reported by Liadakis et al. (1998), and an order of magnitude higher than the

reported for the commercial protein isolate Supro 500 E (Lin 2007). It was also close to the oil

absorption value for rapeseed precipitated protein isolate in its Chinese variety, 20% higher than

the defatted whole ground yellow mustard precipitated protein isolate and higher than double-

zero oriental mustard protein isolates. On the other hand, it was 50% lower than the oil

absorption capacity value for the defatted whole ground yellow mustard soluble protein isolate

reported by Lin (2007).

Table 12: Oil absorption capacity for different protein isolates

Product OAC (%) Reference DWGYM SPI* 478.0 Lin (2007) Chinese rapeseed PPI 256 Xu et al. (1994) Acid soluble protein isolatea 239.3 ± 3.8 Current study Abyssinian mustard PPI 217.0 Pedroche et al. (2004) Soybean Supro 670 210.1 Liadakis et al. (1998) DWGYM PPI* 192.0 Lin (2007) 0-0 Oriental mustard SPI 157

Precipitated protein isolatea 143.5 ± 2.9 Current study Soybean Supro 500 E 123 Lin (2007) 0-0 Oriental mustard PPI 82

* DWGYM = Defatted whole ground yellow mustard PPI = precipitated protein isolate. SPI = soluble protein isolate a Mean values ± standard deviation

The precipitated protein isolate obtained in these experiments, which initially contained around

25% oil, had a lower OAC than most of the protein isolates compared, although it was 20%

higher than the commercial soybean protein isolate Supro 500 E and 70% higher than the

double-zero oriental mustard precipitated protein isolate. These results suggest that most of the

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mustard proteins are lipophilic in nature. Kinsella et al. (1976) proposed that the mechanism for

oil absorption relies mostly on the physical entrapment of oil by capillary attraction, but the high

oil absorption of mustard proteins may also be explained by the abundance of hydrophobic

groups on the protein surface which bind the hydrocarbon chains of lipids.

5.5.5 Emulsifying Properties

The emulsifying properties of the precipitated protein isolate and the acid soluble protein isolate

were investigated by measuring both the emulsifying activity index (EAI) and emulsion stability

(ES) (Table 13). The emulsions of both protein isolates appeared in the form of thick mixtures

and were apparently affected by the oil content. The precipitated protein isolate obtained from

the aqueous extraction in this study showed lower emulsifying activity than both the reported

value for soybean protein isolate Supro 500E (Lin 2007) and the acid soluble protein isolate,

while the latter was actually superior to the other two and to all other compared protein isolates.

The emulsifying activity of the acid soluble protein isolate was 15% higher than the 84.9% value

reported for Supro 500E and up to 55% higher than any of the other values reported for yellow

mustard and Abyssinian mustard protein isolates (Lin 2007 and Pedroche et al. 2004). On the

other hand, the emulsifying activity for the precipitated protein isolate was almost 20% lower

than the reported for Supro 500 E, it was comparable to the value of 68.5% reported by Lin

(2007) for the double-zero oriental mustard soluble protein isolate, and at least 25% higher than

the values reported for Abyssinian mustard precipitated protein isolate (Pedroche et al. 2004),

Chinese rapeseed precipitated protein isolate (Xu et al. 1994) and defatted whole ground yellow

mustard isolates.

Both the precipitated protein isolate and the acid soluble protein isolate showed good emulsion

stability. Heat denaturation usually increases the protein surface hydrophobicity, improving the

79

emulsifying properties. This effect was confirmed, as the emulsion volumes tended to increase

during heating. In the case of the precipitated protein isolate, there was a 3% increase in the

emulsifying activity. In the case of the acid soluble protein isolate the increment was around 2%,

although it was noted that after heat treatment and centrifugation the thick emulsion had gelled,

probably because the heat treatment of the globular proteins caused polymerization via

Table 13: Emulsifying properties of protein isolates

Product EAI (%) ES (%) Reference Acid soluble protein isolatea 98.8 ± 0.1 100.4 ± 0.2 Current study Soybean Supro 500 E 84.9 100 Lin (2007) Precipitated protein isolatea 70.4 ± 1.7 103.5 ± 4.8 Current study 0-0 Oriental mustard SPI 68.5 96 Lin (2007) Chinese rapeseed PPI 56.1 99.4 Xu et al. (1994) Abyssinian mustard PPI 54.0 5.0 Pedroche et al. (2004) DWGYM PPI* 53.5 59.7 Lin (2007) DWGYM SPI* 49.0 71.5

0-0 Oriental mustard PPI 45.7 120 * DWGYM = Defatted whole ground yellow mustard PPI = precipitated protein isolate. SPI = soluble protein isolate a Mean values ± standard deviation

sulfhydryl-disulfide interchange reactions (Damodaran 1996). For the other mustard protein

isolates, the emulsion stability values reported show that heat treatment has different effects on

the emulsifying activity, most likely due to different oil contents, carbohydrate contents and

protein physicochemical characteristics. Emulsifying properties show strong correlation with

surface hydrophobicity and the ability of the protein to change its conformation at the interface.

The oil content, low solubility, low water absorption and high values for oil absorption of both

protein isolates produced in this study show their hydrophobic nature, which contributes to their

emulsifying properties.

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5.5.6 Foaming Properties

The foaming properties of a protein depend on an adequate balance between its flexibility and

rigidity. While the foaming expansion is generally related to protein flexibility and rate of

adsorption at the interface, foam stability is affected by their molecular rigidity (Moure et al.

2006 and Damodaran 1996).

The acid soluble protein isolate obtained from the aqueous extraction of yellow mustard flour

had higher foam expansion values than most of the values for protein isolates reported in the

literature (Table 14), surpassed only by the value reported for the defatted whole ground yellow

mustard soluble protein isolate (Lin 2007) which was 40% higher, and the value reported by Xu

et al. (1994) for the Chinese rapeseed precipitated protein isolate.

Table 14: Foam expansion values for protein isolates

* DWGYM = Defatted whole ground yellow mustard PPI = precipitated protein isolate. SPI = soluble protein isolate

a Mean values ± standard deviation

The value of 213.3% was 2.8 times higher than the 75% reported for Supro 500E (Lin 2007) and

3.1 times higher than the value reported for Supro 670 (Liadakis et al. 1998). Soybean protein

Product FE (%) Reference DWGYM SPI* 306.0 Lin (2007) Chinese rapeseed PPI 275 Xu et al. (1994) Acid soluble protein isolatea 213.3 ± 1.5 Current study Abyssinian mustard PPI 163 Pedroche, et al. (2004) DWGYM PPI* 147.5 Lin (2007) 0-0 Oriental mustard SPI 125

Precipitated protein isolatea 95.0 ± 5.0 Current study 0-0 Oriental mustard PPI 82 Lin (2007) Soybean Supro 500 E 75

Soybean Supro 670 66.8 Liadakis, et al. (1998)

81

isolates had the lowest reported foaming activity. On the other hand, the precipitated protein

isolate had a foam expansion value almost 50% lower than the acid soluble protein isolate and it

was also lower than the values reported for Abyssinian mustard precipitated protein isolate

(Pedroche et al. 2004), defatted whole ground yellow mustard precipitated protein isolate and the

double-zero oriental mustard soluble protein isolate (Lin 2007). The low foam expansion value

obtained in this study for the precipitated protein isolate can be explained by its high oil content,

since the presence of lipids in protein isolates hinder their foaming capacity (Yasumatsu et al.

1972).

Although the foam expansion of the precipitated protein isolate was relatively low, it had high

foam stability during the first part of the evaluation test, but after 60 minutes it showed a steep

collapsing rate, without leveling off during the two hour test period. The foam stability data and

the foam volume stability percentage values (FVS) are tabulated in Tables 15 and 16, and the

data plotted in Figure 23.

Table 15: Foam stability data

Foam stability (ml) Product 0.5 min 20 min 40 min 60 min 120 min Acid soluble protein isolate 300.0 ± 7.0 237.3 ± 2.5 224.7 ± 1.2 213.3 ± 2.9 202.0 ± 7.2

Precipitated protein isolate 96.3 ± 5.5 92.3 ± 3.8 82.7 ± 2.3 79.7 ± 4.5 58.0 ± 2.6

Mean values ± standard deviation

This can be attributed to the de-foaming effects of the lipids contained in this isolate.

Conversely, the acid soluble protein isolate and the data for all other protein isolates compared

showed a different and characteristic behavior, where the collapsing rate was high during the

first ~25 minutes but then abruptly decreased and leveled off. The acid soluble protein isolate

82

had the highest foam stability, maintaining 76% of its initial foam volume after 20 minutes and

65% after two hours. The precipitated protein isolate, on the other hand, maintained up to 95% of

its initial foam volume after 20 minutes, but then dropped off and retained only 60% after two

hours. The foam stability values reported by Lin (2007) for the soybean protein isolate Supro 500

E show that only 55% of the initial foam volume remains after a period of 20 minutes and 44%

after two hours, while for the Abyssinian mustard precipitated protein isolate the values reported

by Pedroche et al. (2004) show 48% foam retention after 20 minutes and 34% after two hours.

Table 16: Foam volume stability values for selected protein isolates

FVS (%) Product 20 min 30 min 40 min 120 min Reference Acid soluble protein isolate 75.7 72.9 71.7 64.5 Current study Precipitated protein isolate 47.3 44.7 42.4 29.7 Chinese rapeseed PPI 60.0 - 58.0 50.7 Xu et al. (1994) 0-0 Oriental mustard SPI 52.0 - 45.1 30.0 Lin (2007) 0-0 Oriental mustard PPI 44.0 - 38.5 27.5 Soybean Supro 500 E 52.6 - 47.7 42.0 Abyssinian mustard PPI 47.5 - 39.8 33.9 Pedroche, et al. (2004) Soybean Supro 670 - 40.6 - - Liadakis, et al. (1998) SPI = soluble protein isolate. PPI = precipitated protein isolate

According to the values reported by these authors, the soybean protein isolate Supro 500 E had

lower foam stability than the reported for other mustard protein isolates including the ones from

this study. This suggests that mustard proteins are able to form more cohesive viscoelastic films

through intra-molecular interactions than soybean proteins and could be more suited for foaming

applications.

83

5.5.7 Gelation

The effect of protein isolate concentration on the gel formation is shown in Table 17. The

minimal protein isolate concentration required for inverting a tube without producing sliding of

the gel in the walls is known as the least gelation concentration (LGC). LGC was measured only

in the acid soluble protein isolate, it was not measured for the precipitated protein isolate because

of its high oil contents and limited solubility.

Table 17: Least gelation concentration values for selected protein isolates

Product LGC (%) Reference Rosa Mosqueta protein isolate 6 Moure et al. (2001) Acid soluble protein isolate 8 Current study Lupin protein isolate 12 Lqari et al (2002) Soybean Promine D 14 Okezie and Bello (1988) Soybean protein isolate 16 Moure et al. (2005)

30.00%

40.00%

50.00%

60.00%

70.00%

80.00%

90.00%

100.00%

0 20 40 60 80 100 120 140

Foam

per

cent

age

(%)

Time (min)

Acid soluble protein isolate Precipitated protein isolateSupro 500E

Figure 23: Foam stability expressed as the foam volume (%) remaining against time

84

A value of 8% was found to be the least gelation concentration for acid soluble protein isolate,

which formed a hard, white, coagulant-type gel. According to Shimada et al. (1980) proteins

with non-polar residues tend to form this type of gel. The LGC value was found to be similar to

the reported by Moure et al. (2001) for rosa mosqueta protein isolate, 33% lower than the value

reported for lupin protein isolate (Lqari, et al. 2002) and at least 1.75 times lower than the values

for soybean protein isolates reported in the literature. It has been suggested that for globular

proteins, in addition to non-covalent interactions, the ability to form intra-molecular disulfide

bonds during heat treatment is important for gelation and it is likely that disulfide cross-linking

increases polymer chain length and the molecular weight-average (Monahan, et al. 1995 and

Damodaran 1996). In addition, acid soluble protein isolate composition (Table 9) shows that

approximately 20% of its mass is not formed by proteins, lipids or moisture, but it is most likely

formed by carbohydrates, which may improve the gelling properties (Sathe, Deshpande and

Salunkhe 1982). The type of gel formed also suggests the hydrophobic, non-polar nature of the

proteins in the isolate.

5.6 Temperature effect on lipid oxidation in the precipitated protein isolate

The lipid oxidation study on the precipitated protein isolate was performed following the

procedure described in Section 4.5.4. 10-gram samples were stored at three different

temperatures in closed plastic bags. Each week the oil from a sample for each temperature was

extracted using a solvent mixture of petroleum and diethyl ether at room temperature. The

solvent in the recovered miscella was then evaporated. The same extraction procedure was

applied to the starting material (de-hulled yellow mustard flour) and a sample of the precipitated

protein isolate from the final batch, which was kept frozen until the analysis. Although the

85

extraction method used was not quantitative, the extraction results showed consistency

throughout the experiments. For the samples of precipitated protein stored at 25°C the extracted

oil represented an average of 10.80% of their initial mass, for the samples stored at 35°C it

represented an average of 10.02% and for the samples stored at 45°C the average value was

10.18%. In the case of the initial precipitated protein isolate sample and for the yellow mustard

flour sample, the values obtained were 10.62% and 29.52% of their initial mass, respectively.

The TBA value was determined for each of the recovered oil samples. The results are shown in

Table 18 for the starting materials and in Table 19 for the heated samples.

Table 18: TBA values for the starting materials

Sample TBA value* Mustard flour 0.1037 ± 0.0021 Initial PPI 0.1165 ± 0.0036

*TBA value reported as mg of malondialdehyde per kg of sample (Mean values ± standard deviation)

The TBA values of the precipitated protein isolate obtained after the aqueous extraction,

membrane processing, isoelectric precipitation and freeze drying show an increment of 12.3% in

comparison to the value of the starting material. This increment was expected due to material

handling and to the heating step prior the protein solution membrane processing, where it was

heated up to a temperature of 60°C for at least 30 minutes. These factors might have promoted

the formation of lipid free radicals, hydroperoxides and secondary oxidation products. In

comparison with the initial TBA value for the precipitated protein isolate, samples stored at 25°C

presented an increment of almost 52% after 35 days. Samples stored at 35°C presented an

increment of 56%, and for the samples stored at 45°C the TBA value increase was 68.5%.

86

Table 19: TBA values for samples stored at different temperatures

TBA values* Time (days) 25ºC Storage 35°C Storage 45°C Storage

7 0.1265 ± 0.0025 0.1341 ± 0.0047 0.1368 ± 0.0089 14 0.1502 ± 0.0056 0.1556 ± 0.0087 0.1583 ± 0.0066 21 0.1359 ± 0.0031 0.1424 ± 0.0063 0.1500 ± 0.0062 27 - 0.1683 ± 0.0010 0.1708 ± 0.0074 28 0.1678 ± 0.0051 - - 35 0.1765 ± 0.0033 0.1821 ± 0.0075 0.1963 ± 0.0047

*TBA value reported as mg of malondialdehyde per kg of sample (Mean values ± standard deviation)

Samples stored at 45°C presented a slightly faster oxidation rate when compared to those

samples stored at lower temperatures. The maximum TBA value for the samples stored at 45°C

after 35 days was 7.8% higher than the maximum value of samples stored at 35°C and 11.2%

higher than the value of the samples stored at 25°C after the same amount of time (Figure 30).

However, the analysis of variance for the TBA values obtained every 7 days for the three

temperature treatments showed that the differences between them were not significant at the 95%

confidence level (p>0.05), suggesting that under these circumstances the lipid oxidation was only

slightly dependent on the storage temperature. Similar observations were made by Kristensen, et

al. (2001) in oxidative stability studies of processed cheese at 5°C, 20°C and 37°C, and by

Thomsen, et al. (2005) in the lipid oxidation evaluation of whole milk powder at 37°C and 45°C.

On the other hand, several authors have reported a maximum TBA value in lipid oxidation

studies; Danowska-Oziewics et al. (2005) reported a maximum in the TBA values when

assessing quality changes of soybean and rapeseed oils during heating. Similar observations were

made by Tazi, et al. (2009) in the oxidation of almond paste and by Liu, et al. (1991) in the

stability study of lean ground beef patties.

87

Figure 24: Malondialdehyde formation in the precipitated protein isolate at three temperatures

Since the TBA values in the current study did not show a maximum, it is suggested that they

may still increase with storage time.

The low oxidation rates and small differences between the TBA values obtained may be due to

the high protein content in the isolate and its antioxidant properties. Proteins inhibit lipid

oxidation reactions in food lipid-protein systems by the mechanisms outlined in section 2.6.1,

and its effects have been shown by several authors; Tong et al. (2000) showed that whey proteins

decrease the oxidation rate in salmon oil emulsions, while Cheng et al. (2010) found lower lipid

oxidation rates in soybean oil-in-water emulsions treated with potato protein hydrolysates as

antioxidants. Taylor et al. (1980) also showed the antioxidant activity of skim milk proteins in a

linoleate emulsion.

0.1000

0.1200

0.1400

0.1600

0.1800

0.2000

0.2200

0 10 20 30 40

TB

A V

alue

(mg

of m

alon

dial

dehy

de/k

g)

Time (days)

25°C

35°C

45°C

88

6. MEAT PRODUCT TESTING

One of the potential uses of mustard protein is as binders in meat products such as sausages and

hams, as a replacement for the soy protein currently used by the industry. While not all binders

are protein based, the addition of soy protein is a common industrial practice to aid in stabilizing

the fat and water of the meat emulsion, producing a firmer and moister product.

Tests were performed using the precipitated protein isolate produced by the aqueous extraction

of de-hulled yellow mustard flour, followed by membrane processing of the protein solution with

a 5kDa membrane, isoelectric precipitation and freeze drying. The mustard flour meal residue, a

byproduct of the aqueous extraction process, was also used. Unfortunately, not enough acid

soluble protein isolate was produced for a pilot-scale meat test. Both products were tested as a

binder in a typical emulsion product. The formulation (standard Hermann Laue base formulation

plus 1% or 2% added protein) was stuffed into fibrous casings, which allow penetration of

smoke, and cooked in a smoke house for wiener production, or in waterproof casings, and

cooked in an industrial oven to produce bologna. The meat products were evaluated and

compared against a control of pure meat by an untrained panel. Participants were asked to assess

the taste of the products and their impressions and comments were recorded using the form given

in Appendix B7, following a hedonic or affective test method. In the case of the wieners, as

shown in Table 20, the ratings were similar indicating that all products had pleasant flavours and

mouth feels, but all agreed that the wieners manufactured with either the meal residue or

precipitated protein isolate had a more firm consistency than the control. Wieners manufactured

with 2% meal residue or 2% precipitated protein isolate were described in some cases as dry.

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Table 20: Ratings for wieners produced with precipitated protein isolate and meal residue derived from the aqueous extraction process, membrane processing and isoelectric precipitation

Overall preference (9 is the highest rating) Control MR 1% MR 2% PPI 1% PPI 2%

Total responses 6 6 6 6 6 Mean rating 6.0 6.6 5.7 5.7 6.9 Standard deviation 2.5 1.5 1.8 2.4 1.8

Although the samples manufactured using 2% of precipitated protein isolate had a higher mean

rating than the rest, statistical analysis of the data showed that the differences between ratings

were not significant at the 95% confidence level (p>0.05). As expected, the standard deviations

of this test were high, reflecting the wide differences among people in their preferences and

feelings about food. These results show that the mustard meal residue and precipitated protein

isolate do not have a negative impact in the acceptance of the wieners and actually conferred

some characteristics that made them more preferable for some of the participants.

In the case of the bologna type products, the samples containing yellow mustard protein products

were also described as having a firm texture. The ratings are shown in Table 21.

Table 21: Ratings for bologna produced with precipitated protein isolate and meal residue derived from the aqueous extraction process, membrane processing and isoelectric precipitation

Overall preference (9 is the highest rating) Control MR 1% MR 2% PPI 1% PPI 2%

Total responses 7 7 7 7 7 Mean rating 5.7 4.9 5.1 6.2 8.2 Standard deviation 1.6 0.9 1.6 1.0 1.4

Statistical analysis of the data showed that the differences between mean ratings were significant

at the 95% confidence level (p<0.05). In order to determine which of the means had significant

90

differences, the Tukey HSD (honestly significant difference) test was performed on them.

Significant differences were found between the pairs of mean ratings showed in Table 22.

Table 22: Pairs of mean ratings with significant differences at the 95% confidence level

Sample Mean Sample Mean Control 5.7 PPI 2% 8.2 MR 1% 4.9 PPI 2% 8.2 MR 2% 5.1 PPI 2% 8.2

The results indicate that the samples prepared with 2% precipitated protein isolate had a higher

rating and were preferred over the control samples and over the samples containing meal residue.

The sensory characteristics provided by this isolate, probably due to its oil content, had

noticeable differences in its sensory perception. As in the case of the wieners, samples

manufactured with 2% meal residue were also described in some cases as dry. The differences in

the preparation method for both wieners and bologna, where the first are cooked in a smoke

house, might affect the final product characteristics and their sensory perception, leading to the

noted differences. As in the case of wieners, the addition of yellow mustard derived protein

products did not show a negative impact in the acceptance of the bologna.

91

7. CONCLUSIONS

• Aqueous extraction of full fat mustard flower is a good approach to the simultaneous

recovery of oil and protein. The crude protein extraction yield for the process was 86.4%.

As much as 81.5% was recovered in a protein solution, 4.9% was recovered in an oil-in-

water emulsion and 7.8% remained un-extracted in the meal residue. The protein solution

was mainly composed of water (94.9%), had a crude protein content of 2.57%, and an oil

content of 0.7%.

• Membrane processing via ultrafiltration and diafiltration with a 5kDa membrane resulted

in a crude protein recovery of 83.0% of the total protein in the initial protein solution,

while 17.0% was lost in the permeate fraction. The crude protein recovery yield as

permeate was around 11.2%, a lower value than the 20% reported by Balke (2006) when

using a 10 kDa membrane. The protein loss was most likely non-protein nitrogen and

some low MW polypeptides.

• Isoelectric precipitation resulted in two protein isolates. A precipitated protein isolate that

represented 55% of the total crude protein from the starting material, had a protein

content of around 96% on a dry, oil free basis. However contained a considerable amount

of lipids (~25%). The acid soluble isolate represented 11.2% of the crude protein in the

starting material, had a low lipid and moisture content, and a protein concentration of

83.5% on moisture and oil free basis. An average of 5.7 times more precipitated protein

isolate was obtained per gram of acid soluble protein isolate.

92

• The precipitated protein isolate was light tan in colour and with a bland taste and a

slightly bitter aftertaste. The acid soluble protein isolate was off-white with an astringent

and salty taste. Their texture and colour was similar to soybean protein isolate Supro 500

E, which also had a salty flavour.

• SDS-PAGE analysis of the two mustard isolates show that they are mostly composed of

polypeptides with MW below 60 kDa. MW distribution varied among isolates, and that at

least 6 protein groups contain disulfide bonds which may affect their molecular flexibility

and rigidity, most likely increasing the polymer chain length and average molecular

weight during gelation.

• Low NSI values suggest that the protein isolates obtained in this study, at their dispersion

pH, have a structure or conformation where hydrophilic groups are most likely blocked,

and their interaction with the aqueous environment is hindered. This effect in the

precipitated protein isolate is amplified by its high oil content.

• The WAC and OAC values suggest that most of the mustard proteins from these isolates

are lipophilic in nature at their native pH, with abundance of hydrophobic groups on the

protein surface which bind the hydrocarbon chains of lipids. The WAC value for the acid

soluble protein isolate was comparable to the value reported for the soybean protein

isolate Supro 670. In the case of the OAC, both isolates had similar or higher values than

those reported for the soybean protein isolates.

• The precipitated protein isolate had good emulsifying properties, but low foaming

expansion and foaming stability values, attributed to its high oil content. Conversely, the

acid soluble protein isolate presented high emulsifying and foaming properties compared

93

to other mustard and soybean protein isolates, which confirms its hydrophobic nature, its

ability to undergo conformational changes at the interface, and a better balance between

molecular flexibility and rigidity.

• The low least gelation concentration obtained for the acid soluble protein isolate is

attributed to the formation of intra-molecular disulfide bonds and also to its carbohydrate

content, which may play an important role. The LGC value was lower than those reported

in the literature for soybean protein isolates.

• The analysis of the temperature effect on lipid oxidation of the precipitated protein isolate

showed that there were no significant differences between the TBA values at 25°C, 35°C

and 45°C during a 35 days period, which suggests that temperature has a moderate effect

on lipid oxidation under the tested conditions. The low oxidation rates can be explained

by the antioxidant effects of proteins as outlined in section 2.6.1. While rancidification is

impaired by proteins and might not be a concern during normal storage conditions, the

reaction of amino acids with lipid free radicals and hydroperoxides, may hinder the

functionality and nutritional properties of the protein isolate.

• The meat testing confirmed the good binding and emulsifying properties of the

precipitated protein isolate and of the yellow mustard meal residue of the aqueous

extraction, without affecting in a negative way the flavour or mouth feel of the products.

Both isolates obtained in this study exhibited good functionality, indicating their usefulness in

many food applications. In comparison to the soybean protein isolates Supro 500 E and Supro

670, they presented a superior performance in the oil absorption, emulsifying, foaming and

gelation properties. The precipitated protein isolate presented a lower NSI and WAC than the

94

reported values for soybean isolates, while the acid soluble protein isolate had a NSI and WAC

similar to the values reported for the Supro 670 isolate, but lower than the values reported for the

Supro 500 E isolate.

These results show that the aqueous extraction of de-hulled yellow mustard flour along with

membrane processing of the protein solution are a potential source of high quality protein

products which in terms of functionality, can be viewed as direct competitors of their soybean

counterparts. Furthermore, the increasing value of plant protein isolates as replacement of animal

derived proteins, and the increasing demand for natural and organic products should encourage

the application of solvent-free technologies, that are able to provide environmentally friendly

solutions to the energy and food industries.

95

8. RECOMMENDATIONS

• Since the quantity of precipitated protein isolate produced is almost 6 times larger than

acid soluble protein isolate, the addition of enzymes could be considered after the

extraction process in order to increase the production and solubility properties of the acid

soluble protein isolate.

• Although the effects of storage temperature on lipid oxidation were found to be low, the

effects of lipid-protein interactions in the functionality of the precipitated protein isolate

should be studied since the loss of functionality will negatively affect the effectiveness of

the product.

• Being a water intensive process, nanofiltration or reverse osmosis treatment of the

permeate fraction should be studied in order to recover other valuable compounds, such

as phenolic compounds and water for its recirculation into the aqueous extraction

process.

• Considering the promising results obtained by the precipitated protein isolate and the

meal residue in the meat test, enough acid soluble protein isolate should be produced and

tested in order to evaluate its functionality in a food system and its effects on the flavour

and mouth feel of the final product. The functional properties of the meal residue should

also be assessed to evaluate additional applications.

• While the acid soluble protein isolate had a low gelation concentration, additional studies

on cold and hot gel strength should be considered, in order to determine the gellation

properties and more specific applications.

96

• A solubility profile over a wide range of pH values should be determined for both protein

isolates in order know how their functionality is affected under more acidic or basic

conditions, increasing its applications in the production of acidic beverages or other food

systems.

• A preliminary economic analysis of the protein isolates production should be performed

in order to verify its viability as a competitor for other plant protein isolates currently in

the market.

97

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9. APPENDICES

108

APPENDIX A

Analytical Methods

109

A1. Determination of oil content using the Mojonnier Method

All measurements were performed in triplicate. For all analysis, corks were soaked in water for

at least 1 hour.

AOAC Method 922.06. Fat in flour by acid hydrolysis

This method was used for solid samples.

1. Weigh 2 g of sample (to nearest 0.1mg) in 50 ml beaker.

2. Add 2 ml ethanol and stir to moisten particles to prevent lumping on addition of acid.

3. Add 10 ml HCl (25+11), mix well, set beaker in water bath held at 70-80ºC stirring in

frequent intervals during 30-40 min.

4. Add 10 ml ethanol and cool to room temperature.

5. Weigh 4 150 ml beakers to nearest 0.1 mg.

6. Transfer the mixture to a mojonnier flask. Rinse beaker into extraction flask with 25 ml

diethyl ether added in 3 portions, stopper flask (cork stopper) and shake vigorously for 1

min.

7. Add 25 ml petroleum ether and shake for 1 min.

8. Let stand until upper liquid is practically clear. Draw off as much possible of ether-lipid

solution through a filter consisting of cotton ball packed just firmly enough in the funnel

stem to let ether pass freely into a previously weighed 150 ml beaker.

9. Re-extract liquid remaining in flask twice, each time with only 15 ml of each ether.

Shake well on addition of each ether and draw off the ether solution into same beaker.

Wash tip of funnel and end of funnel with few ml of mixture of ethers in equal volumes.

110

10. Evaporate ethers inside a fume hood, and then dry the lipids in an oven at 90ºC for 90

min.

11. Remove beaker from oven, let cool down and weight.

12. Run a blank using only reagents for each set of experiments

13. Calculation

𝐿 = �𝑊1 −𝑊0 −𝑊𝐵

𝑊𝑆�100

Where: L is the lipid percentage in the sample, W1 is the weight of the beaker containing the

lipids, W0 is the weight of the empty beaker, WB is the weight of the blank and WS is the weight

of the sample.

AOAC Method 995.19. Fat in cream

This method was used for emulsion samples.

1. Place test sample in a water bath at 38.0 ± 1ºC. Mix thoroughly and weight aliquot

immediately. Do not let samples remain in water bath more than 15 min after reaching

38ºC.

2. Weight an empty mojonnier flask.

3. Pipet into flask enough cream to yield 0.3-0.6g of extracted fat (0.8g of 60% emulsion)

and weight to the nearest 0.01 g.

4. Dilute test portion with 10 ml distilled water at room temperature.

5. Weigh 4 150 ml beakers to the nearest 0.1 mg.

111

6. To test portions in the mojonnier flask add 1.5 ml NH4OH and mix thoroughly. It

neutralizes any acid present.

7. Add 3 drops of phenolphthalein indicator to sharpen visual appearance of interface.

8. Add 10ml ethanol, stopper with water-soaked cork and shake vigorously 15 s.

9. For first extraction add 25 ml diethyl ether, stopper with cork and shake for 1 min. Hold

body of flask horizontally with lower bulb and stopper up.

10. Loosen cork gently to release built-up pressure. Add 25 ml petroleum ether, shake for 1

min.

11. Let stand until ether phase and the pink aqueous phase are separated and transfer ether-

lipid solution to weighed beaker.

12. For second extraction add 5 ml ethanol, stopper with same cork used for first extraction

and shake 15 s.

13. Add 15 ml diethyl ether, replace cork and shake 1 min.

14. Add 15 ml petroleum ether, stopper with same cork and shake 1 min.

15. Let phases separate. If interface is below neck of flask, add water to bring level half way

up neck. Add water slowly to cause minimum disturbance of separation. Decant ether

solution into same beaker used for first extraction.

16. For third extraction omit addition of ethanol and repeat procedure for second extraction.

17. Evaporate solvents in the fume hood. Dry extracted lipids at 90ºC for 90min

18. Remove beaker from oven and place them in a desiccator. Cool down and weigh.

19. Run 1 blank with reagents and substitute emulsion with 10 ml distilled water. Reagent

blank should be < 0.0020g residue

20. Calculation:

112

𝐿 = �𝑊1 −𝑊0 −𝑊𝐵

𝑊𝑆�100

Where: L is the lipid percentage in the sample, W1 is the weight of the beaker and flask, W0 is the

weight of the empty beaker, WB is the weight of the blank and WS is the weight of the sample.

113

A2. Moisture Analysis (AACC Method 44-15A)

1. Record the weight of an aluminum tray.

2. Weight the sample in the tray and record the data.

3. Cover the plate with a sheet of aluminum foil and punch several small holes on the foil.

4. Put the sample in an oven at 105°C for 24 hours.

5. Remove samples from oven. Cool in a desiccator and record weight.

6. Calculate the moisture content using the following equation:

𝑚 = �𝑊2 −𝑊0

𝑊1�100

Where m is the percent moisture content, W2 is the weight of the dry sample and the tray, W0 is

the weight of the tray and W1 is the weight of the initial sample.

114

A3. Protein Analysis (Kjeldahl Method, AOCS method Ba 4d-90)

1. For solid samples weight ~0.2 g of each sample on a nitrogen free paper or for liquid

samples weight 5-30 g into each digestion tube.

2. For solid samples place a clean nitrogen free paper in the blank tube or for liquid samples

weight 5-30 g of distilled water in the blank tube.

3. Add 4 Kjeldahl Tablets (3.5g K2SO4, 0.175 HgO per Tablet) and 25 ml concentrated

H2SO4 to each tube.

4. Clamp the suction manifold onto the digestion tubes. Insert the suction tube into the end

of the manifold and a tuft of glass wool into the other to allow air passage through the

manifold. Turn on the tap water of the aspirator.

5. Place the connected tubes onto the Digestion unit. Heat the tubes at setting 4 for 20

minutes or until the foam subsides. Raise the temperature to setting 6 for 10 minutes or

until the foam subsides and the air in the tubes show mist. Then turn the setting up to 10

and digest for 35 minutes ensuring that the walls of the glass are clean and that the

solution is colourless or very pale yellow for at least 30 minutes before taking off the

heat.

6. Remove the tubes from the digester and place in a rack with the suction continuing until

the solution is cool (~ 30 minutes). Then remove the suction tube and place the rack in a

fume hood to finish cooling.

7. Remove the glass manifold. Rinse the manifold with water and leave it aside to air dry.

115

8. Add 50 ml of distilled water to each tube and swirl until the precipitate is dissolved. Then

add 25 ml of sodium thiosulfate solution (8% Na2S2O3·5H2O) to each tube and swirl.

Cover the tubes and cool before proceeding with the distillation procedure.

9. For the distillation, turn on the Büchi Distillation Unit K-350 and the cooling water line.

Wait until the equipment warms up.

10. Label 4 500 ml Erlenmeyer flasks (one per sample and one blank). Add 60 ml of 4%

(w/v) boric acid and three drops of N-point indicator to each flask.

11. When the machine is ready, rinse for 2 minutes using distilled water in a clean tube.

12. Replace the water tube with the blank tube. Place the blank labeled Erlenmeyer flask in

the distillate outlet of the unit. Make sure the outlet tube is as far below the surface of

boric acid solution as possible.

14. Add 90 ml of 32% NaOH solution by pressing the reagent button or until the total

solution volume is around 180 ml.

15. Set the distillation time to 5 minutes and start the distillation.

16. When the instrument is finished replace the current tube with the next sample tube.

Replace the current Erlenmeyer flask (rinse off any liquid from the straw into the flask

using distilled water) with the corresponding sample flask in the distillate outlet.

17. Repeat steps 14 to 16 for the remaining samples.

18. Titrate the boric acid solutions in the Erlenmeyer flasks with 0.1000N H2SO4 from green

to the same shade of pink as that in the blank.

19. Calculate the protein content of the sample using the following equations:

𝐻𝑁 =(𝑉1 − 𝑉0)(1.4007)(𝑁)

𝑊

𝑃 = (𝐻𝑁)6.25

116

Where: V1 is the volume (ml) of titrant used for the sample, V0 is the volume (ml) of titrant used

for the blank, N is the acid normality used for titration, W is the sample weight (grams), SN is the

percent nitrogen in the sample and P is the percent protein in the sample.

Note: Oilseed protein is assumed to have 16% nitrogen, thus the factor 6.25 is the reciprocal of

16%.

117

A4. Nitrogen Solubility Index (NSI) (AOCS Official Method Ba 11-65 or AACC Method

46-23 )

1. Accurately weigh 5.0 g of the sample into a 500 ml beaker. Measure 200 ml of distilled

water at 30°C. Add a small portion of the water at a time and disperse it thoroughly with

a stirring rod. Stir in the remainder of the water, using the last of it to wash off the

stirring rod.

2. Stir the mixture at 120 rpm with the mechanical stirrer for 120 min in a water bath at

30°C.

3. Transfer the mixture to a 250 ml volumetric flask by carefully washing out the contents

of the beaker into the flask. Add 1 or 2 drops of antifoam, dilute to mark with distilled

water and mix the contents of the flask thoroughly.

4. Allow to stand for a few minutes and decant off about 40 ml into a 50 ml centrifuge tube.

Centrifuge at 1500 rpm for 10 min and decant the supernatant through a funnel

containing a plug of glass fiber. Collect the clear filtrate in a 100 ml beaker.

5. Pipet 5 -30 ml of the clear liquid into a Kjeldahl tube.

6. Determine total nitrogen in the sample using standard protein analysis.

7. Calculate the water soluble nitrogen percentage (WSN):

𝑊𝐻𝑁 =(𝑉1 − 𝑉0)(𝑁)(1.4007)(6.25)

𝑊

Where V1 is the volume of acid used for titration of the sample, V0 is the volume of acid used for

titration of the blank, N is the normality of the acid used for the titration and:

118

𝑊 =(𝑀0)(𝑉)

250

Where w0 is the weight of the initial sample and V is the volume of filtered supernatant pipetted

for protein analysis.

8. Calculate the Nitrogen Solubility Index:

𝑁𝐻𝐼 =𝑊𝐻𝑁𝑃

Where NSI is the nitrogen solubility index (%), WSN is the water soluble nitrogen calculated in

step 7 and P is the protein percentage of the initial sample.

119

A5. Oil absorption capacity (Sosulski et al, 1976)

1. Accurately weigh 2.0 g of sample in a 50 ml centrifuge tube.

2. Add 12 ml of canola seed oil.

3. Stir the sample for 30 seconds using a stirring rod every 5 min.

4. Repeat the stirring procedure 6 times.

5. Centrifuge at 1600 x g for 25 min.

6. Carefully decant the supernatant oil right after centrifugation.

7. Weight the tube immediately after decanting.

8. Calculate the oil absorption capacity:

𝑂𝐴𝐶 = �𝑊𝑓

𝑊𝑖�100

Where OAC is the oil absorption capacity (%), Wf is the final weight of the sample after oil

decanting and Wi is the initial weight of the sample.

120

A6. Water absorption capacity (Naczk et al, 1985)

1. Accurately weigh 2.0 g of sample in a 50 ml centrifuge tube.

2. Add 16 ml of distilled water.

3. Stir the sample for 30 s every 10 min using a stirring rod.

4. Repeat the stirring procedure 7 times.

5. Centrifuge at 2000 x g for 15 min.

6. Carefully decant the supernatant right after centrifugation.

7. Tilt the tube 15º to 20º mouth down for 15 minutes.

8. Weight the tube immediately.

9. Calculate the water absorption capacity:

𝑊𝐴𝐶 = �𝑊𝑓

𝑊𝑖�100

Where WAC is the water absorption capacity (%), Wf is the final weight of the sample after water

decanting and Wi is the initial weight of the sample.

121

A7. Emulsifying Activity (EA) (Yasumatsu et al, 1972 and Naczk et al, 1985)

1. Suspend 3.5 g of sample with 50 ml distilled water in a 400 ml beaker using a spatula or

stirring rod.

2. Add 25 ml of canola seed oil and homogenize/emulsify for 30 sec.

3. Add 25 ml of canola seed oil and homogenize/emulsify for an additional 90 sec.

4. The emulsion obtained is divided evenly into 50ml centrifuge tubes and centrifuged at

2000 rpm for 5 minutes.

5. Calculate the emulsifying activity using the equation:

𝐸𝐴 = �𝑉1𝑉0�100

Where: EA is the emulsifying activity (%), V1 is the volume of emulsion formed, and V0 is the

volume of the total tube contents.

122

A8. Emulsion Stability (ES) (Naczk et al, 1985)

1. Suspend 3.5g of sample in 50ml distilled water in a 400 ml beaker using a spatula or

stirring rod.

2. Add 25 ml of canola seed oil and homogenize/emulsify for 30 sec.

3. Add 25 ml of canola seed oil and homogenize/emulsify for an additional 90 sec.

4. The emulsion obtained is heated in an 85°C water bath for 15 minutes, covering the

beaker with foil to avoid water loss. Slow stirring is applied during heating.

5. After heating and cooling, divide evenly into 50ml centrifuge tubes and centrifuge at

2000 rpm for 5 minutes.

6. Calculate the emulsifying activity of the heated emulsion:

𝐸𝐴 = �𝑉1𝑉0�100

Where: EA is the emulsifying activity (%), V1 is the volume of emulsion formed, and V0 is the

volume of the total tube contents.

7. Calculate the emulsion stability:

𝐸𝐻 = �𝐸𝐴1𝐸𝐴0

�

Where: ES is the emulsion stability (%), EA1 is the emulsifying activity after the heating

treatment and EA0 is the emulsifying activity before the heating treatment.

123

A9. Foam expansion and foam stability (Naczk et al. 1985 and Patel et al. 1988)

1. Disperse 3.0 g of sample in 100ml of distilled water with a spatula.

2. Whip the suspension using a homogenizer at 10,000 rpm for 6 minutes.

3. Immediately transfer the contents into a 250 ml graduated cylinder (or the best suitable

volume according to the foam volume formed) and record the volume increase.

4. Record the foam volume in the standing cylinder at 0.5, 20, 40, 60 and 120 minutes after

whipping.

5. Calculate the foam expansion (%) as follows:

𝐹𝐸 = �𝑉1 − 𝑉0𝑉0

�100

Where FE is the foam expansion (%), V1 is the volume of the foam plus the liquid just after

whipping and V0 is the initial volume of the dispersion.

6. Calculate the foam volume stability (%) at 20, 30, 40, 60 and 120 minutes using the

following formula:

𝐹𝑉𝐻 = �𝑉𝑡𝑉0�100

Where FVS is the foam volume stability (%), Vt is the foam volume at time t and V0 is the initial

volume of the dispersion.

124

A10. AOCS method Cd 19-90. 2-Thiobarbituric acid value. Direct method.

1. Accurately weigh 50-200 mg of the sample into a 25 ml volumetric flask. Dissolve the

sample in a small volume of 1-butanol and make up the volume with 1-butanol.

2. Transfer, using a pipette, 5.0 ml of the sample solution to a dry test tube; add by pipette

5.0 ml of the TBA reagent solution (200 mg 2-thiobarbituric in 100 ml 1-butanol). Close

the test tube with a ground-glass stopper and mix thoroughly.

3. Place the prepared test tube into a thermostated bath at 95°C.

4. After 120 min., remove the test tube from the bath and cool it under running tap water for

about 10 minutes until it reaches room temperature.

5. Measure the absorbance of the reaction solution in a 10 mm cuvette at 530 nm using

distilled water in the reference cuvette.

6. Prepare a reagent blank at the same time as the sample. The reading of the blank

determination should not exceed 0.1 in a 10 mm cuvette.

CALCULATION

Results are calculated as follows:

𝑇𝐵𝐴 𝑣𝑎𝑙𝑢𝑒 = 50 × (𝐴 − 𝐵)

𝑚

Where A is the absorbance of the test solution, B is the absorbance of the reagent blank and m is

the mass of the test portion in mg.

125

A11. Least gelation concentration (LGC) (Moure et al. 2002)

1. Prepare 2, 4, 6, 8, 10, 12 and 14% (w/v) dispersions of the sample in 50 ml of distilled

water.

2. Adjust the pH of the dispersion to a value of 7.0 ± 0.05 by the drop wise addition of 1 N

NaOH solution, or 1 N HCl solution.

3. After the pH value is reached, mix the dispersion for another 3 to 5 min.

4. Take a 5ml aliquot of each dispersion and pour it in clean and dry glass test tube.

5. Heat the test tubes with the samples for 1 h in a water bath at 95 – 100°C.

6. After the heating treatment, immediately cool the tubes under running tap water for 10

minutes.

7. Place the tubes in a refrigerator at 4°C for an additional 2 h.

8. Take the samples out the refrigerator and find the LGC.

9. The LGC is the concentration above which the sample remains in the bottom of the

inverted tube.

126

A12. Sodium dodecyl sulfate polyacrylamide gel electrophoresis

1. Prepare a protein solution with the initial sample in distilled water with a target

concentration between 4.8 and 5.6 mg of protein per ml (pH adjustment might be

necessary to ensure complete dissolution of the sample). Prepare the solution

immediately before the analysis.

2. Add 20 µl of the sample solution in a 0.5 ml centrifuge tube with lid.

3. Add 10 µl of sample buffer (containing 62.5 mM Tris-HCl, 25% glycerol, 2% SDS and

0.01% bromophenol blue) for non-reduced condition analysis.

4. Add 9.5 µl of sample buffer and 0.5 µl of β-mercaptoethanol for reduced condition

analysis.

5. Close the tube lid and heat the sample for 10 min at 95°C.

6. Immediately after heating, centrifuge the sample for 10 s at 14000 rpm.

7. Take a suitable already made or purchased Tris-HCl gel and insert the gel cassette into

the cassette holder assembly with the short plastic plate facing inwards.

8. Insert the assembly into the cell and fill it up with running buffer (0.1% SDS, 0.3% tris,

1.44% glycine).

9. Using a micropipette, inject 14 µl of the previously heated and centrifuged samples in

each well.

10. Inject a suitable amount of prestained protein ladder solution in another well(s).

11. Close the cell with the appropriate lid, being careful to align and connect the positive and

negative electrodes correctly.

127

12. Connect the cell to the power supply and run the samples at a constant voltage of 130 V

until they reach the bottom of the gel.

13. Turn off the power supply, disconnect and disassemble the cell lid and the cassette

holder. Take out the gel cassette and open it carefully by separating the two plates.

Gently nudge the gel off one corner of the plate and allow it to roll off into an awaiting

staining solution (40% methanol, 10% acetic acid and 0.1% Coomassie blue R-250). Let

stand for 30 min on a shaking platform.

14. After the staining period, destain the gel by immersion in a 40% methanol, 10% acetic

acid solution on a shaking platform. Change the destaining solution as often as necessary

until the background is clear, usually 2 to 3 times. Destaining takes 2 to 3 hours.

128

APPENDIX B

Results

129

B1. Yellow mustard flour analyses

Protein content AS IS

Sample Weight (g) SO4H2 0.1 N (ml) Nitrogen content

(%) 1 0.2963 10.60 31.30 2 0.3083 10.95 31.08 3 0.2933 10.50 31.32 4 0.2829 10.15 31.40 5 0.3122 11.15 31.25 6 0.3324 11.80 31.06

Average 31.24

SD 0.14

Oil content AS IS Sample Weight (g) Oil (g) Oil content (%)

1 1.5032 0.5505 36.62% 2 1.4391 0.5310 36.90% 3 1.9833 0.7297 36.79% 4 1.5565 0.5708 36.67% 5 1.6785 0.6194 36.90% 6 1.9865 0.7305 36.77%

Average 36.78%

SD 0.11

130

Moisture content

Sample Initial Weight (g) Final Weight (g) Moisture (%) 1 1.2501 1.1881 4.96% 2 1.2280 1.1665 5.00% 3 1.2785 1.2150 4.97% 4 1.3342 1.2738 4.53% 5 1.2267 1.1607 5.38% 6 1.2459 1.1807 5.23%

Average 5.01%

SD 0.29

TBA value reported as mg of malondialdehyde per kg of sample

Sample Absorbance @ 530 nm TBA value 1 0.2733 0.1040

Average 0.1036 2 0.2767 0.1055

SD 0.0020

3 0.2674 0.1014 Blank 0.0371

Sample Weight 113.5 mg

131

B2. Aqueous extraction

Mass, protein and oil balance

AQUEOUS EXTRACTION

MASS IN Protein (g) Yield Oil (g) Yield Flour 400.66 g 125.17 100% 147.04 100% Water 4498.64 g NaOH 34.64 g Total 2466.97 g 125.17 147.04

MASS OUT Emulsion 202.28 g 6.08 4.9% 91.27 62.1% Extract 3958.4 g 101.92 81.5% 27.85 18.9% Meal 586.48 g 9.75 7.8% 17.91 12.2% Total 2373.58 g 117.75 137.03

Difference -186.78 -7.42 -10.01 -3.79% -5.92% -6.81%

Oil content in fractions

Emulsion (as is) Sample Weight (g) Beaker (g) Final (g) Difference (g) W/O Blank (g) Oil (g)

1 1.01 68.7402 69.1965 0.4563 0.4563 0.451782 2 1.12 67.0347 67.545 0.5103 0.5103 0.455625 3 1.02 67.8934 68.3486 0.4552 0.4552 0.446275

blank 0 68.4056 68.4056 0 Oil % 45.12% SD 0.47

132

Meal Residue (dry basis) Sample Weight (g) Beaker (g) Final (g) Difference (g) W/O Blank (g) Oil (g)

1 1.5124 67.8931 68.2387 0.3456 0.3456 0.228511 2 1.5036 68.7417 69.0610 0.3193 0.3193 0.212357 3 1.5417 67.0362 67.3540 0.3178 0.3178 0.206136

blank 0 68.4045 68.4045 0 Oil % 21.57% SD 1.15 Protein extract (dry basis) Sample Weight (g) Beaker (g) Final (g) Difference (g) W/O Blank (g) Oil (g)

1 1.5498 66.3225 66.5376 0.2151 0.2151 0.138792 2 1.5191 67.9961 68.2090 0.2129 0.2129 0.140149 3 1.5333 66.956 67.1688 0.2128 0.2128 0.138786

blank 0 68.4045 68.4045 0 Oil % 13.92% SD 0.08

Protein content in fractions

Meal Residue (dry basis) Sample Weight (g) SO4H2 (ml) Protein (%)

1 0.610 8.20 11.76 Average 11.74% 2 0.610 8.15 11.69 SD 0.04

3 0.610 8.20 11.76

Emulsion (as is) Sample Weight (g) SO4H2 (ml) Protein (%)

1 1.52 5.20 2.99 Average 3.01% 2 1.53 5.25 3.00 SD 0.01

3 1.55 5.35 3.02

Protein EXTRACT (dry basis) Sample Weight (g) SO4H2 (ml) Protein (%)

1 0.1631 9.50 50.97 Average 50.95% 2 0.1755 10.20 50.85 SD 0.09 3 0.1800 10.50 51.04

133

Aqueous extraction. Final Batches

Run Flour

(g) Total water

(g) NaOH

Soln.(g) Asc. Acid

(g) Extract

(g) Emulsion

(g) Residue

(g) 11 400.93 4531.90 35.65 4.00 4056.82 152.05 579.70 12 400.10 4525.20 34.42 4.10 4044.19 172.86 570.80 13 400.90 4537.00 35.36 4.02 4083.84 175.69 570.00 14 401.80 4547.00 37.58 4.04 4096.15 179.37 575.60 15 400.60 4525.00 36.11 4.04 4051.66 173.85 575.60 16 401.30 4524.00 34.01 4.02 4054.76 191.64 578.00 17 401.90 4528.00 35.33 4.04 4086.80 185.31 575.90

134

B3. Membrane processing and isoelectric precipitation

Mass, protein and oil balance

MEMBRANE PROCESS MASS IN Protein (g) Yield Oil (g)

Extract 4048.7 g 101.4 80.8% 27.6 18.9% Water 7662.0 g 0 0 NaCl 34.2 g 0 0

Total 11744.9 g 101.4 27.6

MASS OUT Permeate 10770.0 g 14.1 11.2% N.D. Retentate 957.2 g 83.3 66.5% N.D.

Total 11727.2 g 97.4

Difference -17.70 -4.02 -0.2% -3.96%

ISOELECTRIC PRECIPITATION MASS IN Protein (g) Yield Oil (g) Yield

Retentate 957.2 g 83.3 N.D. PO4H3 14.5 g 0 0 Water 2216.8 g 0 0 Total 3188.5 g 83.3 MASS OUT PPI 318.8 g 68.7 54.9% 24.5 16.6% SPI 2817.0 g 14.0 11.2% 0.09 0.1% Total 3135.8 g 82.7

Difference -52.7 -0.6 -1.65% -0.73%

135

Protein content in fractions

Final Retentate (as is) Sample Weight (g) SO4H2 (ml) Protein (%)

1 1.02 10.4 8.92 Average 8.74% 2 0.84 8.6 8.96 SD 0.35 3 1.47 14.0 8.33

Total permeate (as is)

Sample Weight (g) SO4H2 (ml) Protein (%) 1 10.03 1.5 0.131 Average 0.13% 2 10.12 1.6 0.138 SD 0.00 3 10.59 1.6 0.132

Membrane processing: final batches

Ultrafiltration and diafiltration of the initial protein solution

Run Extract

(g) After Filt.

(g) Water

(g) NaCl (g)

DF Soln. (g)

Retentate (g)

UF Permeate (g)

Tot. permeate

(g) 11 4056.82 4035.00 2325.00 18.58 5403.00 951.55 5413.00 10816.00 12 4044.19 4030.40 2322.30 18.56 5345.00 969.78 5402.00 10747.00 13 4083.84 4063.50 2341.40 18.72 5350.00 969.10 5455.00 10805.00 14 4096.15 4085.20 2353.90 18.82 5350.00 948.91 5509.00 10859.00 15 4051.66 4040.00 2327.80 18.61 5350.00 955.75 5431.00 10781.00 16 4054.76 4023.70 2318.50 18.53 5250.00 949.69 5411.00 10661.00 17 4086.84 4062.80 2341.00 18.71 5250.00 955.50 5467.00 10717.00

Isoelectric precipitation

Run PO4H3 (g) Water (g) PPI wet (g) Total SPI (g) 11 15.00 2147.23 303.03 2778.3 12 13.07 2088.00 290.00 2625.0 13 12.50 2196.00 313.53 2682.4 14 15.89 2046.00 313.53 2710.9 15 14.72 2314.80 319.49 2953.8 16 14.72 2337.17 356.38 2945.2 17 15.46 2388.23 335.89 3023.3

136

Ultrafiltration and diafiltration parameters for the acid soluble protein solution A

Run SPI1 (g) Ret1 (g) CF1 Water1 (g) DV1 permeate1 (g) 11 661.8 275.8 2.4 1551.0 5.6 386.0 12 597.4 270.2 2.2 1500.0 5.6 327.2 13 553.5 269.8 2.1 1500.0 5.6 283.7 14 591.3 271.3 2.2 1500.0 5.5 320.0 15 631.5 271.5 2.3 1500.0 5.5 360.0 16 709.5 269.5 2.6 1500.0 5.6 440.0 17 678.0 270.0 2.5 1500.0 5.6 408.0

Ultrafiltration and diafiltration parameters for the acid soluble protein solution B

Run SPI2 (g) Ret2 (g) CF2 Water2 (g) DV2 permeate2 (g) 11 2116.5 350.0 6.0 1900.0 5.4 1766.5 12 2027.6 297.6 6.8 1640.0 5.5 1730.0 13 2128.9 270.9 7.9 1500.0 5.5 1858.0 14 2119.6 269.6 7.9 1500.0 5.6 1850.0 15 2322.3 272.3 8.5 1500.0 5.5 2050.0 16 2235.7 270.7 8.3 1500.0 5.5 1965.0 17 2345.3 270.3 8.7 1500.0 5.5 2075.0

Overall fluxes, concentration factors and diafiltration volumes

Run

11 12 13 14 15 16 17

CF (In. Extract) 6.7 6.3 6.4 6.6 6.4 6.5 6.5 Flux (Lmh) 12.03 18.50 17.21 17.66 20.34 16.65 18.22 DV (In. Extract) 5.7 5.5 5.5 5.6 5.6 5.5 5.5 Flux (Lmh) 9.00 15.27 13.38 13.38 15.29 13.93 14.79 CF (SPI) 4.4 4.8 4.9 5 5.5 5.45 5.6 Flux (Lmh) 20.80 21.70 20.80 14.80 13.40 18.00 19.40 DV (SPI) 5.7 5.6 5.6 5.6 5.6 5.55 5.55 Flux (Lmh) 8.90 10.20 7.50 6.80 6.10 7.30 7.10

137

Isolate recovery in grams:

Run

11 12 13 14 15 16 17

Precipitated PI 95.70 97.10 97.48 98.98 98.68 96.28 98.02 Acid soluble PI 16.10 17.40 17.90 16.59 17.10 17.64 16.88

138

B4. Protein isolates analyses

Protein PPI Sample Weight (g) SO4H2 (ml) Protein (%)

1 0.1527 12.1 69.34 Average 70.43% 2 0.1539 12.45 70.78 SD 0.97

3 0.1512 12.3 71.18

Protein acid sol. isolate

Sample Weight (g) SO4H2 (ml) Protein (%) 1 0.1529 14.45 82.69 Average 81.88%

2 0.1465 13.7 81.83 SD 0.79 3 0.1494 13.85 81.12

Oil Content in PPI

Sample Weight (g) Beaker (g) Final (g) Difference W/O Blank Oil 1 1.5394 65.4964 65.8850 0.3886 0.3886 0.2524 2 1.5792 68.7421 69.1430 0.4009 0.4009 0.2538 3 1.7846 67.9955 68.4365 0.441 0.441 0.2471

blank 0 68.4045 68.4045 0

Oil % 25.11% SD 0.36

Oil Content in acid soluble isolate

Sample Weight Beaker Final Difference W/O Blank Oil 1 2.0113 67.7747 67.7844 0.0097 0.0097 0.0048

blank 0 68.4045 68.4045 0

Oil % 0.48%

139

Moisture determination

PPI Sample Dish Initial final Moisture

1 2.0952 1.0155 0.9832 3.18% 2 2.087 1.053 1.0365 1.57% 3 2.088 1.06 1.0436 1.55%

Moisture 2.10%

SD 0.014 Acid soluble isolate Sample Dish Initial final Moisture

1 2.072 0.514 0.507 1.36% 2 2.114 0.504 0.4954 1.71% 3 2.0893 0.517 0.51 1.35%

Moisture 1.47%

SD 0.201

TBA value of precipitated protein isolate (reported as mg of malondialdehyde per kg of precipitated protein isolate)

Sample Absorbance @ 530 nm TBA value 1 0.3536 0.119886

Average 0.1165 2 0.3347 0.112727

SD 0.0036

3 0.3455 0.116818 Blank 0.0371

140

B5. Functional properties

Nitrogen solubility index

Precipitated protein isolate

Sample 1 Sample 2 Sample 3

SO4H2 0.60 0.50 0.55 ml

Blank 0 0 0 ml

Vol. pip 24.90 24.90 24.92 ml

W 0.501 0.501 0.501 g

Sample 5.03 5.03 5.03 g

Sample P 70.43 70.43 70.43 %

WSN 1.048 0.874 0.960 %

NSI 1.36% NSI 1.49 1.24 1.36 % SD 0.12

Acid soluble protein isolate

Sample 1 Sample 2 Sample 3 SO4H2 1.2 1.2 1.75 ml Blank 0 0 0 ml Vol. pip 2.01 2.01 2.99 ml W 0.032 0.032 0.048 g Sample 4.01 4.01 4.01 g Sample P 81.88 81.88 81.88 % WSN 32.584 32.584 31.944 % NSI 39.53%

NSI 39.79 39.79 39.01 % SD 0.45

141

Water absorption capacity

Precipitated protein isolate

50 ml bottle Sample bot+sam+wat Sam+wat WAC 10.2628 2.0582 17.0521 4.7311 129.87% WAC 131.12% 10.2449 2.0584 17.0010 4.6977 128.22% SD 3.69 10.2137 1.9346 16.7000 4.5517 135.28%

Acid soluble protein isolate

50 ml bottle Sample bot+sam+wat Real sample Water WAC 10.3614 1.8955 13.2503 0.6216 2.2673 264.75% WAC 263.12% 10.2607 2.0397 13.5377 0.7182 2.5588 256.28% SD 6.19 10.362 1.9337 13.5757 0.6862 2.5275 268.33%

Oil absorption capacity

Precipitated protein isolate

50 ml bottle Sample After Sample aft OAC 10.2157 1.9197 16.8045 4.6691 243.22% OAC 243.47% 10.312 1.8537 16.7355 4.5698 246.52% SD 2.93

10.3115 2.0910 17.4350 5.0325 240.67%

Acid soluble protein isolate

50 ml bottle Sample After Sample aft OAC 10.2123 1.7209 17.8306 5.8974 342.69% OAC 339.34% 10.3088 1.852 18.4606 6.2998 340.16% SD 3.83 10.308 1.903 18.5891 6.3781 335.16%

142

Emulsifying activity

Precipitated protein isolate

Sample Total Volume Emulsion volume EA 1 42.50 29.38 69.12% 2 41.88 29.38 70.15% 3 42.50 31.25 73.53% 4 42.50 30.00 70.59% 5 42.50 30.00 70.59%

Average 70.42% 6 43.75 30.00 68.57%

SD 1.73

Acid soluble protein isolate

Sample Total Volume Emulsion volume EA 1 47.50 47.0 98.95% 2 41.25 40.8 98.79% 3 41.25 40.8 98.91% 4 40.00 39.5 98.75% 5 37.50 37.0 98.67%

Average 98.79% 6 37.50 37.0 98.67%

SD 0.12

Emulsion stability

Emulsifying activity after heating at 85 °C

Precipitated protein isolate.

Sample Total Volume Emulsion volume EA 1 43.75 33.75 77.14% 2 45.00 32.50 72.22% 3 43.75 31.25 71.43% 4 45.00 33.12 73.61% 5 45.00 32.00 71.11%

Average 72.82% 6 43.75 31.25 71.43%

SD 2.30

Emulsion stability 103.41%

143

Acid soluble protein isolate

Sample Total Volume Emulsion volume EA 1 47.50 47.20 99.37% 2 50.00 49.50 99.00% 3 45.00 44.70 99.33% 4 43.75 43.40 99.20% 5 45.00 44.60 99.11%

Average 99.23% 6 47.50 47.20 99.37%

SD 0.15

Emulsion stability 100.45%

Foaming properties

Precipitated protein isolate

Foam expansion

Sample Weight (g) Initial Vol. (ml) Whip. Vol. (ml) F.E. 1 3.00 100 190 90% 2 3.00 100 195 95% 3 3.02 100 200 100%

F. E. 95.00% SD 5.00

Foam stability

Sample 1 Sample 2 Sample 3 Time (min) Foam (ml) Foam (ml) Foam (ml) Average SD FVS

0.5 90 99 100 96.33 5.51 49.40% 20 88 94 95 92.33 3.79 47.35% 40 84 84 80 82.67 2.31 42.39% 60 80 84 75 79.67 4.51 40.85%

120 60 59 55 58.00 2.65 29.74%

144

Acid soluble protein isolate

Foam expansion

Sample Weight (g) Initial Vol. (ml) Whip. Vol. (ml) F.E. 1 3.02 100 312 212% 2 3.01 100 313 213% 3 2.99 100 315 215%

F. E. 213.33% SD 1.53

Foam stability

Sample 1 Sample 2 Sample 3 Time (min) Foam (ml) Foam (ml) Foam (ml) Average SD FVS

0.5 292 303 305 300.00 7.00 95.74% 20 237 235 240 237.33 2.52 75.74% 40 224 226 224 224.67 1.15 71.70% 60 215 210 215 213.33 2.89 68.09%

120 200 196 210 202.00 7.21 64.47%

Foam percentage remaining against time:

30.00%

40.00%

50.00%

60.00%

70.00%

80.00%

90.00%

100.00%

0 20 40 60 80 100 120 140

Foam

per

cent

age

(%)

Time (min)

Acid soluble protein isolate Precipitated protein isolate

145

B6. TBA values for the precipitated protein isolate stored at different temperatures

Absorbance values for samples stored at 25°C

T = 25° Absorbance @ 530 nm Sample Week 1 Week2 Week3 Week 4 Week 5

1 0.4239 0.4964 0.4912 0.4662 0.4572 2 0.4245 0.5228 0.5108 0.4432 0.5033 3 0.4379 0.5096 0.5098 0.4436 0.491

Blank 0.0341 0.0122 0.0201 0.0182 0.0222

TBA values for samples stored at 25°C reported as mg of malondialdehyde per kg of sample

Weight (mg) 156.0 165.6 178.0 129.0 130.8 tba1 0.1249 0.1462 0.1323 0.1736 0.1663 tba2 0.1251 0.1542 0.1378 0.1647 0.1839 tba3 0.1294 0.1502 0.1376 0.1649 0.1792

TBA (mg/kg) 0.1265 0.1502 0.1359 0.1678 0.1765 SD 0.0025 0.0040 0.0031 0.0051 0.0033

Absorbance values for samples stored at 35°C

T = 35° Absorbance @ 530 nm Sample Week 1 Week2 Week3 Week 4 Week 5

1 0.4085 0.4887 0.4825 0.4662 0.4788 2 0.4228 0.5149 0.4908 0.4693 0.4536 3 0.3967 0.4616 0.5228 0.4642 0.4343

Blank 0.0341 0.0122 0.0201 0.0182 0.0222

TBA values for samples stored at 35°C reported as mg of malondialdehyde per kg of sample

Weight (mg) 139.9 153.0 168.0 133.2 119.0 tba1 0.1338 0.1557 0.1376 0.1682 0.1918 tba2 0.1389 0.1643 0.1401 0.1693 0.1813 tba3 0.1296 0.1469 0.1496 0.1674 0.1732

TBA (mg/kg) 0.1341 0.1556 0.1424 0.1683 0.1821 SD 0.0047 0.0087 0.0063 0.0010 0.0075

146

Absorbance values for samples stored at 45°C

T = 45° Absorbance @ 530 nm Sample Week 1 Week2 Week3 Week 4 Week 5

1 0.3408 0.4181 0.5260 0.4795 0.4866 2 0.3341 0.45 0.5112 0.5166 0.4868 3 0.3273 0.4479 0.5407 0.515 0.5062

Blank 0.0341 0.0122 0.0201 0.0182 0.0222

TBA values for samples stored at 45°C reported as mg of malondialdehyde per kg of sample

Weight (mg) 109.6 134.7 168.6 142.1 120.0 tba1 0.1399 0.1507 0.1500 0.1623 0.1935 tba2 0.1368 0.1625 0.1456 0.1754 0.1936 tba3 0.1338 0.1617 0.1544 0.1748 0.2017

TBA (mg/kg) 0.1368 0.1583 0.1500 0.1708 0.1963 SD 0.0031 0.0066 0.0044 0.0074 0.0047

0.1000

0.1200

0.1400

0.1600

0.1800

0.2000

0.2200

0 5 10 15 20 25 30 35 40

TB

A V

alue

Time (days)

25°C

35°C

45°C

147

Single factor ANOVA analysis for weekly TBA values at three storage temperatures.

Week 1

SUMMARY Groups Count Sum Average Variance

Week 1 25 3 0.3794 0.1264 6.43628E-06 Week1 35 3 0.4023 0.1341 2.18199E-05 Week1 45 3 0.2736 0.1368 1.89652E-05

ANOVA Source of Variation SS df MS F P-value F crit

Between Groups 0.000151008 2 7.55038E-05 5.0017 0.0641 5.7861 Within Groups 0.000075477 6 1.50955E-05

Total 0.000226485 8

Week 2

SUMMARY Groups Count Sum Average Variance

Week 2 25 3 0.3003 0.1501 3.17685E-05 Week2 35 3 0.4668 0.1556 7.58565E-05 Week2 45 3 0.4749 0.1583 4.38632E-05

ANOVA Source of Variation SS df MS F P-value F crit

Between Groups 7.97483E-05 2 3.98741E-05 0.7351 0.52495 5.7861 Within Groups 0.000271208 6 5.42416E-05

Total 0.000350956 8

Week 3

SUMMARY Groups Count Sum Average Variance

Week 3 25 3 0.4077 0.1359 9.61474E-06 Week3 35 3 0.4273 0.1424 4.01104E-05 Week3 45 3 0.3000 0.1500 3.82683E-05

148

ANOVA Source of Variation SS df MS F P-value F crit

Between Groups 0.000240279 2 0.000120139 4.3617 0.08012 5.7861 Within Groups 0.000137718 6 2.75437E-05

Total 0.000377997 8

Week 4

SUMMARY Groups Count Sum Average Variance

Week 4 25 3 0.5032 0.1677 2.60381E-05 Week4 35 3 0.5049 0.1683 9.30454E-07 Week4 45 3 0.5124 0.1708 5.44598E-05

ANOVA Source of Variation SS df MS F P-value F crit

Between Groups 1.61573E-05 2 8.07864E-06 0.2976 0.7529 5.1432 Within Groups 0.000162857 6 2.71428E-05

Total 0.000179014 8

Week 5

SUMMARY Groups Count Sum Average Variance

Week 5 25 3 0.5293 0.1764 8.32654E-05 Week5 35 3 0.5462 0.1820 8.7911E-05 Week5 45 3 0.5887 0.1962 2.20069E-05

ANOVA Source of Variation SS df MS F P-value F crit

Between Groups 0.000623629 2 0.000311815 4.8422 0.0559 5.1432 Within Groups 0.000386367 6 6.43945E-05

Total 0.001009996 8

149

B7. Meat testing forms and results

Distribution of responses on Hedonic scale for wieners testing

Frequency of responses

Scale description Assigned

value Control MR 1% MR 2% PPI 1% PPI 2% Like least 1.8 1 0 0 1 0 Like slightly 3.6 0 0 1 0 0 Like moderately 5.4 2 3 4 3 3 Like very much 7.2 2 2 0 1 1 Like extremely 9 1 1 1 1 2

Total responses

6 6 6 6 6 Mean rating

6.0 6.6 5.7 5.7 6.9

Standard deviation

2.46 1.47 1.77 2.39 1.77 Percentage "dislike" responses 16.7% 0.0% 16.7% 16.7% 0.0%

Single factor ANOVA analysis for wiener ratings

SUMMARY Groups Count Sum Average Variance

Control 6 36 6.0 6.05 MR 1% 6 39.6 6.6 2.16 MR 2% 6 34.2 5.7 3.13 PPI 1% 6 34.2 5.7 5.72 PPI 2% 6 41.4 6.9 3.13

ANOVA

Source of Variation SS df MS F P-value F crit Between Groups 7.13 4 1.78 0.44 0.78 2.76 Within Groups 100.98 25 4.04

Total 108.11 29

150

Distribution of responses on Hedonic scale for bologna testing

Frequency of responses

Scale description Assigned

value Control MR 1% MR 2% PPI 1% PPI 2% Like least 1.8 0 0 1 0 0 Like slightly 3.6 1 2 0 0 0 Like moderately 5.4 5 5 5 4 1 Like very much 7.2 0 0 1 3 1 Like extremely 9 1 0 0 0 5

Total responses

7 7 7 7 7 Mean rating

5.66 4.89 5.14 6.17 8.23

Standard deviation

1.62 0.88 1.62 0.96 1.42 Percentage "dislike" responses

14.3% 28.6% 14.3% 0.0% 0.0%

Single factor ANOVA analysis for bologna ratings

SUMMARY Groups Count Sum Mean Variance Std. dev

Control 7 39.6 5.66 2.62 1.62 MR 1% 7 34.2 4.89 0.77 0.88 MR 2% 7 36.0 5.14 2.62 1.62 PPI 1% 7 43.2 6.17 0.93 0.96 PPI 2% 7 57.6 8.23 2.01 1.42

ANOVA

Source of Variation SS df MS F P-value F crit Between Groups 49.61 4 12.40 6.93 0.0004 2.69 Within Groups 53.69 30 1.79

Total 103.30 34

151

Tukey HSD test for bologna mean ratings

q calculation k 5 df 30 q 4.102

HSD 2.074

Means compared Difference Control MR 1% 0.771 Not different

Control MR 2% 0.514 Not different Control PPI 1% 0.514 Not different Control PPI 2% 2.571 Significantly different MR 1% MR 2% 0.257 Not different MR 1% PPI 1% 1.286 Not different MR 1% PPI 2% 3.343 Significantly different MR 2% PPI 1% 1.029 Not different MR 2% PPI 2% 3.086 Significantly different PPI 1% PPI 2% 2.057 Not different

𝐻𝐻𝐻 = 𝑞�𝑀𝐻𝑀𝑛

154

Weiner and Bologna evaluation form

Wiener ratings Control 1% MR 2% MR 1% PPI 2% PPI 1% Tex 2% Tex Colour Texture Taste Like least Like extremely Bologna ratings Control 1% MR 2% MR 1% PPI 2% PPI 1% Tex 2% Tex Colour Texture Taste Like least Like extremely