Author: Title:

Thapa, N ava, R Effect of Tempering and Other Processing Treatments on the Anti­nutritional Factors and a Canning Quality Attribute of Dark Red Kidney Beans

The accompanying research report is submitted to the University of Wisconsin-Stout, Graduate School in partial

completion of the requirements for the

Graduate Degree/ Major: MS Food and Nutritional Sciences

Research Adviser: Cynthia Rohrer, Ph.D.

Submission Term/Year: Summer, 2012

Number of Pages: 71

Style Manual Used: American Psychological Association, 6th edition

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I:8J I understand that this research report must be officially approved by the Graduate School and that an electronic copy of the approved version will be made available through the University Library website I:8J I attest that the research report is my original work (that any copyrightable materials have been used with the permission of the original authors), and as such, it is automatically protected by the laws, rules, and regulations of the U.S. Copyright Office. I:8J My research adviser has approved the content and quality of this paper.

STUDENT:

NAVATHAPA DATE: JULY, 2012

ADVISER:

CYNTHIA ROHRER, PhD DATE: JULY, 2012

1. CMTE MEMBER'S NAME: NA VEEN CHIKTHIMMAH, PhD DATE: JULY, 2012

2. CMTE MEMBER'S NAME: MARCIA MILLER-RODEBERG, PhD DATE: JULY, 2012

This section to be completed by the Graduate School This final research report has been approved by the Graduate School.

Director, Office of Graduate Studies: DATE:

2

Thapa, Nava R. Effect of Tempering and Other Processing Treatments on the Anti-nutritional

Factors and a Canning Quality Attribute of Dark Red Kidney Beans

Abstract

Kidney beans are an excellent source of proteins (20-30%), carbohydrates (50-60%) and

fairly good sources of minerals, fibers and vitamins. Thus, they are important components of a

healthy diet. However, their nutritional quality is indirectly impacted by the presence of heat

labile and heat-stable antinutritional factors (ANF) that could exhibit undesirable physiological

effects. The effect of tempering process alone and in combination with blanching, and soaking

processes on the level of two antinutritional factors of beans, namely phytic acid and tannin, was

studied. The phytic acid and tannins in dark red kidney beans ranged from 12.37 to 23.60 mg/g,

and 0.11 to 28.78 mg/g, respectively. A reduction in the level of these antinutrients was observed

after 12 different treatments. Out of 12 different treatment conditions used in canning process of

beans, the 12 h tempering-soaking-blanching-canning process was found to be the most effective

for the reduction of phytic acid (62%) and tannins (82%). This study also demonstrated that the

percentage split of beans obtained from the tempered canned beans (11%) was found to be lower

than the commercially soaked and processed canned beans (21%).

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Acknowledgements

I would like to recognize and express my sincere gratitude to a number of people that have

contributed and helped me throughout this project. I would first like to thank Dr. Cynthia Rohrer

for her tremendous help to my thesis completion. Without her continued guidance and support, I

would have not been able to finish my master’s degree. I would like to thank Dr. Naveen

Chikthimmah for providing me valuable inputs for my research and for editing my thesis. I am

also very thankful to Dr. Marcia Miller-Rodeberg for the valuable time and suggestions.

I would like to express my deep gratitude to Dr. Lamin Kassama for providing me direction,

encouragement and countless effort to my thesis work. His continuous guidance, valuable ideas,

and amazing mentorship brought this research into the real shape.

I would also like to thank UW-Stout Research Services for providing me student research grant

and Chippewa Valley Bean Company for helping me in my research. I am also thankful to Dr.

Carol Seaborn, and Dr. Carolyn Barnhart for their continuous support.

I would like to offer my high regards to Biswas Palikhey and Prawesh Rijal for their great help in

assisting me in data analysis and providing me valuable comments in my thesis. I am also

thankful to Connie Galep, Bikram Upadhaya, and my other friends who helped me in the

laboratory management and chemical analysis during the conduction of experiments.

Last, but not the least, I would like to show my sincere gratitude to my parents for their continued

support, love and inspiration.

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Table of Contents

.................................................................................................................................................... Page

Abstract ............................................................................................................................................2

List of Tables ...................................................................................................................................7

List of Figures ..................................................................................................................................8

Chapter I: Introduction ....................................................................................................................9

Statement of the Problem ...................................................................................................12

Purpose of the Study ..........................................................................................................13

Objectives .........................................................................................................................13

Definition of Terms............................................................................................................14

Limitations .........................................................................................................................14

Chapter II: Literature Review ........................................................................................................15

History of Kidney Beans ....................................................................................................15

Application of Kidney Beans .............................................................................................15

Importance of Dark Red Kidney Beans .............................................................................16

Nutritional Aspects of Beans .............................................................................................16

Canning of Dry Beans ........................................................................................................18

Canning Technique for Preservation of Red Kidney Beans ..............................................18

Standard Canning Procedure..............................................................................................19

Importance of Canned Beans .............................................................................................20

Sensory Quality of Canned Beans .....................................................................................20

Texture ...................................................................................................................20

Color ......................................................................................................................21

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Visual Appearance .................................................................................................22

Splitting ..................................................................................................................22

Current Studies on Tempering of Beans ............................................................................23

Antinutritional Factors of Beans ........................................................................................23

Phytic Acid.........................................................................................................................26

Tannins ...............................................................................................................................29

Chapter III: Methodology ..............................................................................................................34

Sample Preparation ............................................................................................................34

Tempering ..............................................................................................................34

Soaking ..................................................................................................................35

Blanching ...............................................................................................................35

Thermal Processing and Canning ..........................................................................36

Phytic Acid Content ...........................................................................................................36

Tannin Content...................................................................................................................37

Splitting of Beans ...............................................................................................................38

Moisture Content of Beans ................................................................................................38

Statistical Analysis .............................................................................................................39

Chapter IV: Results and Discussion ..............................................................................................40

Moisture Content of Beans after Tempering and Soaking.................................................40

Phytic Acid Content of Canned Beans...............................................................................42

Tannin Content of Canned Beans ......................................................................................48

Splitting of Canned Beans .................................................................................................51

Chapter V: Conclusion ...................................................................................................................56

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Recommendations ..............................................................................................................57

References ..........................................................................................................................59

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List of Tables

Table 1: Ranges in levels of antinutritional factors in dry beans……………...……………….…25

Table 2: Effect of various treatments on the phytic acid content of dark red kidney beans and

subsequent reduction (%) in dark red kidney beans …………………………………………...…45

Table 3: Effect of various treatments on the tannin content of dark red kidney beans and

subsequent reduction (%) in dark red kidney beans……………………………………………....50

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List of Figures

Figure 1: Structures of Phytic acid………………………………………………………….…..27

Figure 2: An example of hydrolysable tannins and related compounds…………………….….31

Figure 3: An example of a condensed tannin……………………………………………….......32

Figure 4: Effect of tempering (45°C/95% R.H.) at 6 h, 12 h and 24 h on moisture content of beans

soaked for 0, 1, 2, 3, 4 and 5 h, respectively...………………………………..……….…............41

Figure 5: Percentage split in canned beans with various treatment conditions ………................53

Figure 6: Canned beans obtained from commercial soaking and canning process (left) and canned

beans obtained from tempering and canning process …………………………………………...54

Figure 7: Example of split beans after thermal processing due to high water uptake

levels…………………………………………..………………………………………………….55

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Chapter I: Introduction

Dry beans are important source of protein throughout the world. Red kidney beans

(Phaseolus vulgaris L.) are one of the most important legumes of nutritional interest in the United

States. Per capita, bean use has been increasing in the USA, which is attributed to the increasing

consumption trend in dry bean market due to nutritional awareness and to changes in the

traditional American diet. Furthermore, increase in the Hispanic population has also significantly

contributed to the increase consumption of beans in the U.S. It is reported that approximately 61%

of the total dark red kidney beans are produced in Minnesota and Wisconsin as reported by

USDA, Agricultural Outlook (1999).

It is well recognized that legumes such as common beans, lentils, and kidney beans are the

main source of supplemental protein based diets. Kidney beans are fairly good source of nutrients

with 22.7% protein, 3.5% mineral matter, 1% fat, 5.1% crude fiber, and 57.7% total

carbohydrates ( Khalil et al., 1986). However, the biological utilization (bioavailability) of the

nutrients is interfered with various anti-nutritional factors present in legumes. These anti-

nutritional factors coupled with indigestible bean proteins impede the absorption of the nutrients

like calcium, iron, and zinc in the human gut. The removal of antinutritional factors can be a

challenging process and requires treatment methods to reduce or remove the level of antinutrients

in beans. Examples of treatment methods to reduce antinutritional factors include fermentation,

germination, thermal treatments (Cooking) and soaking procedures (Abd El-Hady& Habiba,

2003; Martin-Cabrejas et al, 2004).

Soaking before canning may be one of the processes for removal of soluble antinutritional

compounds, which can be eliminated by discarding the soaking solution. The purpose of soaking

before canning is to remove foreign material, facilitate cleaning, aid in can filling through uniform

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expansion, ensure product tenderness and to improve color (Loggerenberg, 2004). During

soaking, dry beans increase 80 % in mass and reach 53 – 57 % final moisture content. Water

uptake (caused by diffusion) takes place during soaking, causing the beans to soften and swell

(Wang et al., 1988). Soaking beans before cooking would also accelerate the cooking rate

(Loggerenberg, 2004).

Blanching is the immersion of foods into hot water (80 to 100°C) or steam for several

minutes. The main purpose of blanching is the inactivation of enzymes, which might produce off-

flavors’, but also to soften the product and remove gases to reduce strain on can seams during

retorting (Jones & Beckett, 1995). The blanching process is also responsible for the removal of

dry bean flavor and odor (Loggerenberg, 2004).

Another process during canning is thermal processing. De Lange (1999) heat sterilized

canned beans in a vertical autoclave at 121.1°C for 50 min. Bolles et al. (1990) sterilized at

121.1°C for 30 min, also using a vertical autoclave. Sterilizing beans at 115.6°C for 35 min or

121°C for 15 min in the presence of CaCl2 and EDTA (Ethylene diamine tetra acetic acid)

containing brine resulted in optimal sterilization values, reduction of trypsin inhibiting activity

and bean firmness values (Wang &Chang, 1988).

Tannins in beans are any naturally occurring phenolic compounds with molecular mass of

500 to 3,000 that contain 1 or 2 phenolic hydroxyl or other suitable groups per 100 MW, which

enables it to form cross-linkages to proteins and other macromolecules (Loggerenberg, 2004).

These heat resistant substances, which are not destroyed by cooking, interfere with the physiology

and utilization of nutrients by the animal (Sgarbieri, 1989). This is caused by the cross-linkages of

tannins to protein, which leads to low protein digestibility and availability in dry beans

(Loggerenberg, 2004). Tannins may also interfere with the biological utilization of minerals and

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certain vitamins, but the importance of these reactions has not yet been identified (Sgarbieri,

1989).

Phytic acid is a chelating agent, which might lower the bioavailability of minerals, such as

Zn, Mn, Cu and Fe. Although phytic acid is regarded as an antinutritional factor in beans, the

phytic acid phosphorous content of red kidney beans is an indicator of cookability. This is

because phytic acid favors a more rapid rate of dissolution of pectic substances in beans during

cooking (Loggerenberg, 2004).

One of the major quality attributes in canned beans is splitting. Splitting of cooked beans is

one of the factors that determine the intactness of cooked beans, and is often determined

subjectively (Hosfield, 1991). Beans that appear intact before canning might also develop large

percentages of splits during retort processing, causing the product to be unappealing and may lead

to price reductions. In addition, not only would splitting of canned beans result in the exudation of

starch into the canning medium, causing graininess of the sauce. Splitting could also lead to

clumping of individual beans (Loggerenberg, 2004). Starch material might also be deposited on

the bottom of the can and thereby reduce the quality of the canned product (Forney et al., 1990).

Splitting might even lead to a complete breakdown of beans. In small white beans a larger

percentage of split beans would be more tolerable than in larger beans, such as kidney and pinto

beans (Loggerenberg, 2004).

The objective of this research was to compare the reduction of phytic acid and tannins in

canned beans by using a tempering technique with processing treatments including soaking,

blanching and thermal processing. Measured canning quality attributes were: splitting, phytic

acid, and tannin content.

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The tempering process was conducted in an environmental chamber at a predetermined

relative humidity and temperature. Tempering prior to thermal processing was hypothesized to

contribute to reducing the level of anti-nutritional factors. The anti-nutritional factors of dark red

kidney beans were determined before and after the canning process using a spectrophotometric

method.

Statement of the Problem

A significant number of world populations rely on legumes and cereals as their staple foods.

Legumes are often advocated in Western diets because of their nutritional benefit and are relatively

inexpensive compared to other sources of protein (Borade et al., 1984). Red kidney beans (Phaseolus

vulgaris L.) are one of the most important legumes of nutritional interest in the United States.

The health-related benefits of beans include their positive effect on lowering the blood cholesterol

and glucose levels (Walker, 1982; Leeds, 1982), because of their high dietary fiber content. However,

they are under-consumed because of the presence of antinutritional compounds, such as enzyme

inhibitors (trypsin, chymotrypsin, α-amylase), phytic acid, flatulence factors, tannins and lectins (Lyimo

et al., 1992). In both human and animal nourishment, phytic acid is considered to be an antinutrient

because of its negative effect on the absorption of zinc, iron, copper and calcium (Van der Poel, 1990).

Tannins are one of the several anti-nutritional factors present in beans that lower the nutritional quality of

beans (Vincent et al., 1981).

Adopting simple processing methods like soaking, blanching and cooking may be able to

reduce the anti-nutritional concentration in beans (Yasmin et al., 2008). Soaking is a pretreatment

process that requires significant amount of time prior to cooking. Thus, soaking as a pretreatment

prolongs the process time in processing of beans. Research done by Thapa, Juliech & Kassama

(2009) observed that a tempering process reduced soaking time by 12h during the pretreatment of

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beans prior to the canning process. The current research focused on the effect of tempering to

minimize the antinutrients prior to cooking.

Purpose of the Study

The quality characteristics and anti-nutritional factors in dry beans has been an area of

research relevance. Major quality attributes such as bean splitting during thermal processing may

be reduced by a tempering process. Previous preliminary research shows the importance of a

tempering process to achieve reduction in bean splitting during canning. Tempering alters bean

structure, which may also improve physical appearance of the canned bean by minimizing

splitting and increasing firmness. There is a scarcity of research information in the area of canning

pretreatments to reduce bean splitting. No research information exists in the literature on the

reduction of anti-nutritional factors of kidney beans using a tempering process of 12h and 24h at

45°C and 95% R.H. This study is therefore done to gain research based information to develop

bean pretreatments.

Objectives

The main objective of this research was to study the effect of tempering on the antinutrient

content and quality (splitting) of canned red kidney beans. These parameters were studied before

and after the canning process. The specific objectives were as follows:

Objective-1: Determine the effect of the tempering process on reducing the splitting of thermally

processed red kidney beans.

Objective-2: Investigate the effect of the tempering process on tannins and phytic acids in canned

dark red kidney beans.

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Definition of Terms

The following terms are important in understanding this study and will be used commonly

throughout this research paper and are defined as follows.

Tempering of Beans. To bring to a desired consistency, state or other physical condition

by the application of heat and relative humidity.

Canning. Method of sterilizing food by heat in hermetically sealed (airtight) containers,

which allows ready to eat foods that are neither frozen nor dehydrated to remain safe and

wholesome during months or even years of storage at room temperature without the use of

additives or preservatives.

Anti-nutritional Factors. Natural or synthetic substances found in the human diet or

animal feed that have the potential to adversely affect health and growth by preventing the

absorption of nutrients from food.

Limitations

In this study only one condition of environmental chamber was used to study the effect of

tempering on the canning quality of red kidney beans. Further studies of quality characteristics of

canned kidney beans should be done. A comparative study of tempering and soaking process

could be carried out to judge the effectiveness of canning process. Also the antinutrients analysis

may be done with advanced analytical techniques to enhance accuracy.

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Chapter II: Literature Review

This chapter will include an in-depth discussion on history, applications and the canning

techniques for the preservation of red kidney beans, followed by a discussion of canning quality

parameters, and tempering of beans.

History of Kidney Beans

Dry beans (Phaseolus spp. L.) are the most important grain legumes for human

consumption. Dry beans have been cultivated for thousands of years, and have been played an

important role in the traditional diets of many regions throughout the world (Zamindar et al.,

2011). Beans are less significant in western diets compared to most of the developing countries.

The daily per capita consumption of all bean products is 9 g in the United States compared to

about 110 g in Asia (Boateng et al., 2008).

The kidney bean, also known as Phaseolus vulgaris L., was cultivated about 7,000 years

ago in Southwestern Mexico. Kidney beans gained wide acceptance during the history of

Americas before the appearance of significant European influences on the American continents

(Pre-Columbian period). Early chroniclers indicated that great importance was given to this

species in the Aztec and Incan empires. The people of Axocopan used dry beans to pay tributes at

the early colonial period in North America (Wu, 2002).

Application of Kidney Beans

Beans play an important role in human diet in Africa, Latin-America and Asian countries,

improving the nutritional status of many low income populations (Milan-Carrillo et al., 2007).

Beans contain two or three times more proteins than cereals and offer a more practical way of

eradicating protein malnutrition than cereal based diets (Carmona-Garcia et al., 2007).

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Kidney beans are consumed as cooked dried beans or canned beans (cooked, baked or refried).

Also they are used in the fruit and vegetable processing industry in the production of frozen or

canned food. Beans are highly nutritious food able to complete with meat. In the western

hemisphere, kidney beans are used in salads, soups and other food products (Kahlon et al., 2005).

Importance of Dark Red Kidney Beans

The dry bean is an important source of protein, dietary fiber, iron, complex carbohydrates,

minerals, and vitamins for millions of people in the world. It is one of the basic food categories in

the diet of the indigenous populations in South America, Asia and Eastern/Southern Africa. The

per capita dry bean consumption has been increasing in the United States in the past 20 years.

Factors contributing to this continuous trend in the dry bean market include the increasing

awareness and changes in the traditional American diet, an increase in immigration of Hispanic

population, and the increasing interests in ethnic foods featuring dry beans. According to the

Continuing Survey of Food Intakes of Individuals, complied by USDA’s Agriculture Research

Service, about 4% of the population consumes kidney bean on any given day, which is among the

highest of any dry bean consumption (Lucier et al., 2000; Belshe et al., 2001).

Nutritional Aspects of Beans

Beans (Phaseolus vulgaris L.), are excellent sources of proteins (20-30%) and

carbohydrates (50-60%) and fairly good sources of minerals and vitamins (Rehman & Shah 2004;

Yin et al., 2008). Dry beans are widely known for their fiber, mineral and protein contents. The

flour and protein concentrate of red kidney bean exhibited good functional properties (Tang,

2008). About 80% of the total proteins found in dry beans are storage proteins. These proteins

supply the young seedling with nitrogenous compounds and amino acids. Dry beans are deficient

in sulphur-containing amino acids such as methionine, cysteine and have small deficiencies in

17

valine, leucine, isoleucine and threonine. All dry beans are good sources of lysine, indicating that

dry beans could be added to lysine-deficient cereal products (Loggerenberg, 2004).

Resistant starch is important due to its various beneficial health properties mostly

mediated by short chain fatty acids produced during its fermentation in the large intestine.

Legumes contain higher amount of resistant starch in comparison to cereals and tubers (Yadav et

al., 2010). The merit of dry bean is its high caloric value and protein content. Ramirez-Cardenas

et al. (2008) pointed out that low concentrations of phytates and phenolic compounds (which are

present in beans) can be protective against cancer and cardiovascular diseases. Also fermentation

of oligosaccharides present in beans may result in the production of short chain fatty acids and

decrease in intestinal pH (Fernandes, et al., 2010).

Beans are the rich source of B vitamins, folate, riboflavin and valuable mineral substances

like potassium, calcium, magnesium, phosphorus and iron salts (Souci, Fachmann, & Kraut,

2000). Thus, they are important components of a healthy diet. However, their nutritional quality is

indirectly impacted by the presence of heat labile and heat-stable antinutritional factors (ANF)

that exhibit undesirable physiological effects (Pusztai, et al., 2004). The ANFs are structurally

different compounds broadly divided into two catergories: proteins (such as lectins and protease

inhibitors) and others such as phytate, tannins or proanthocyanidins, oligosaccharides, saponins

and alkaloids. In general raw beans contain far higher levels of ANFs than their processed forms

hence processing is necessary before the incorporation of these grains into food or animal diets

(Hajos and Osagie, 2004). Few studies about the industrial process of dehydration after soaking

and cooking treatments have been carried out in order to investigate the nutritional improvement

of beans (Martin-Cabrejas et al., 2006).

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Canning of Dry Beans

Canning is the heat sterilization process in which product is placed in hermetic container,

heated at sufficiently high temperature for a sufficient length of time to destroy all microbial and

enzyme activity (Loggerenberg, 2004). Properly sealed and heated canned foods should remain

stable and indefinitely unspoiled in the absence of refrigeration. The sealing step is critical and

heat is applied under pressure for a specific temperature-time combination. The latter is

determined by the type of food, pH, container size and consistency or bulkiness of the food, but

heating of food for longer than necessary is undesirable, as the nutritional and eating quality of

food are affected negatively by prolonged heating (Brock et al., 1994).

Canning Technique for Preservation of Red Kidney Beans

Canning technique was invented by Nicholas Appert, a Frenchman who successfully

developed this food preservation technique, for almost two centuries. Nowadays canned foods

have become widely accepted in daily lives. Canned foods provide a convenient food style free

from food spoilage and natural deterioration with a longer shelf life. The thermal processes

greatly enhance the palatability of the edible dry beans, inactivate toxic factors, and increase the

nutritional availability and digestibility of different nutrients. The current research also shows that

thermal treatment can reduce heat-labile antinutritional factors and increase the digestibility of

protein and amino acid in raw edible beans (Phaseolus vulgaris L.) thereby increasing the

nutritive value of beans (Wu et al., 1994). However, excessive cooking may introduce reduced

protein utilization and increased mineral loss (Wu, 2002)

Standard Canning Procedure

Canning of beans is mainly composed of two processes, namely the soaking /blanching

process and thermal processing/heat sterilization. The purpose of soaking before canning is to

19

remove foreign material, facilitate cleaning, aid in can filling through uniform expansion, ensure

product tenderness and to improve color (Loggerenberg, 2004).

Industrial canners make use of either a long/cold or short/hot soaking process. With the

former, soaking takes place for 6 to 20 h, changing water every 4 to 6 h to prevent bacterial

activity. Cold soaking is followed by blanching in continuous rotary water blanchers at

approximately 90-95oC for 5 min. The overnight soaking process has the following

disadvantages: It is a lengthy process, difficult to control swelling and microbiological stability

and germination could take place, resulting in worm-like material in the beans if broken off

during further processing. Hot soaking takes place in slowly running continuous blanchers or pipe

blanchers, where product heating takes place at 85 – 90 °C for 30 min. The main disadvantage of

this process is that the product does not become as tender as in the case of slowly hydrated beans

(Loggerenberg, 2004).

The blanching process is responsible for the increase of bean moisture content to the final

50 – 55 % and the removal of dry bean flavor and odor. Blanching is the immersion of foods into

hot water (80 to 100°C) or steam for several minutes whose purpose is the inactivation of

enzymes (Loggerenberg, 2004).

In heat sterilization canning process, soaked blanched beans are filled into a can, hot sauce

added (95oC), and the can seamed and heat sterilized immediately. Sterilization is done in static

retorts, agitating retorts or hydrostatic sterilizers. Rotation increases the rate of heat transfer,

thereby reducing processing time and the gelation tendency of the sauce. Canning time varies,

depending on the size of the cans and the operational temperature. A No.2 ½ can require 45

minutes at 116°C. High temperature not only kills the microbes, but also completely tenderizes

20

the beans. The canned beans are immediately cooled by water bath to a temperature of 35° to

41°C. They are then ready to be labeled and shipped (Loggerenberg, 2004).

Beans are further hydrated during the thermal process. Equilibration with brine or sauce

takes 2-4 weeks after canning. The final moisture content of the canned beans ranges from 65-

70% (Uebersax, Ruengsakulrach & Occena, 1991).

Importance of Canned Beans

Canned products from kidney beans such as refried beans, soups and baked beans, are

very commonly available for consumers. Many people look for beans with rapid expansion

ability, higher drained weight, ease in cooking, and uniformity after the thermal process. The

major characteristics responsible for canning quality of beans are the physical characteristics of

the seed, processing and cooking characteristics and chemical composition of beans (Wu, 2002)

Organoleptic properties within the final canned products are considered to be one of the

major quality evaluation standards. However, not all the cultivars are blessed with equally

acceptable quality. The problems affecting consumer’s preference are often related to the

occurrence of bean discoloration, hardness of the beans and breakage of the seed coat after the

canning process (Wassimi et al., 1990). When the seed coat splits, it affects more than just the

appearance, since this splitting can also result in starchiness and excessive viscosity in the final

product. The excessive viscosity is due to the graininess of the sauce and clumping of individual

beans which make the canned product unacceptable to consume. The beans must still maintain

their individual integrity in the canning medium (Loggerenberg, 2004).

Sensory Quality of Canned Beans

Texture. Texture is used as an indication of the degree of consumer acceptance of canned

beans as it affects the perceived stimulus of chewing. Texture, which is measured by a shear

21

press, is an indication of the firmness of beans and is measured as kg force required to shear 100 g

of beans. The shear press ignores other kinesthetic perceptions such as viscosity, gumminess and

adhesion. The higher maximum peak in height indicates firmer beans. Consumers usually rate

texture of beans from “too soft” or “mushy” to “too firm / tough” or “hard” (Loggerenberg, 2004).).

Texture is influenced by bean temperature. Shear values of beans decrease as the

temperature of canned beans on evaluation increases. These firmer textures at lower evaluation

temperatures could probably be the consequence of gelatinization or retrogradation of bean starch.

Bean firmness after cooking relates to the phytin, calcium, magnesium, and free pectin levels,

while the thickness of the palisade layer of the seed coat, as well as the lignin and alpha-cellulose

in the seed coat also play a role in firmness. Hence, beans should be softened to certain limit

during its processing to maintain its individual integrities that help to increase the product quality

(Loggerenberg, 2004).

Color. Color of food is caused by the absorption of more light at some wavelengths by

pigments. Color is one of the properties of beans that consumers have specific preferences about

(Hosfield, 1991). The color of dry and cooked beans is usually measured with a HunterLab color

meter (Hunter Associates Laboratory Inc., Reston, VA). The L-value indicates white to black, a-

value indicates red to green and b-value indicates yellow to blue (Chung et al., 1995).

Bean which was processed 115.6 °C for 45 min and 121.1 °C for 30 min had no

significant influence on the color of processed beans (Bolles et al., 1990). Soaking or cooking

times affected the redness (b-values), while storage under unfavorable conditions that would

cause hard-to-cook beans reduced the lightness character of beans (Paredes-López et al., 1991).

Beans canned without soaking pre-treatment are significantly darker in color than those that

received a soaking treatment (Occena et al., 1992). Generally, the more intact the seed the greater

the surface reflection would be, resulting in brighter appearing beans. The same was found by

22

Wang et al. (1988) when EDTA and CaCl2 were added to the brine. EDTA chelates free metal

ions that would cause the formation of color complexes. The tannin content is one very important

factor, which affects the darkness in color of uncooked dry beans. So it is more likely to have

higher tannin content in darker beans (Loggerenberg, 2004).

Visual Appearance (VA). Visual appearance is one of the preferences of consumers of

beans, which is determined subjectively (Hosfield, 1991). Visual appearance of canned beans is

an evaluation of the general suitability of beans for commercial processing. Beans are evaluated

for intactness, splits, free seed coats and brine consistency (Balasubramanian et al., 1999)

Splitting. Splitting of cooked beans is one of the factors that determines the intactness of

cooked beans, and is determined subjectively. Beans that appear intact before canning might also

develop large percentages of splits during retort processing, causing the product to be unappealing

and may lead to price reductions (Loggerenberg, 2004). Not only would splitting of canned beans

result in the exudation of starch into canning medium, causing graininess of the sauce, but could

also lead to clumping of individual beans (Lu & Chang, 1996).

Splitting is also reduced in canned beans by the addition of calcium to the canning

medium. Beans that contain a greater mass, due to greater water uptake levels, have a larger

tendency to split (Forney et al., 1990). Van Buren et al. (1986) found that firmer beans would

have fewer splits due to swelling that is generally caused by placing bean skins under pressure

causing them to rupture. Larger sized beans would take up less water during canning, due to a

larger volume-to-surface ratio, with consequential lower split values. The addition of CaCl2 to the

brine reduces splitting and clumping of canned navy and pinto beans (Loggerenberg, 2004).

Splits were determined as the actual percentage of split beans found in a sample, as was

recommended by industry. Only completely broken beans and loose skins were considered as

23

splits. The actual percentage of splits is determined by canning 100 seeds and calculating the

percentage broken seeds after canning (Loggerenberg, 2004).

Current Studies on Tempering of Beans

A very few studies has been conducted on beans using the tempering process. The use of a

tempering process is the art of food preparation and more particularly to an improved method for

preparing a reconstitutable, dehydrated refried bean product containing whole beans as well as

crushed beans (Thapa, Juliech & Kassama, 2009). The process of tempering is significant to

achieve the desired condition of beans before canning. The tempering is the process of treatment

in high temperature and high humidity. Tempering of beans at 95% relative humidity and at 45°C

for 6, 12 and 24 hours in environmental chamber significantly decrease the soaking time (Thapa

et al., 2009). High relative humidity tempering of red kidney beans reduces the soaking time to a

relatively short time frame and allow for a more environmentally friendly technique by

discharging less amount of waste water than the conventional method of soaking (Thapa et al.,

2009). The industrial canner soaks for 6-20 h to get the fully hydrated beans while the

conventional method includes overnight soaking at room temperature that has the disadvantages

of lengthy process and uncontrolled swelling( Loggerenberg, 2004). Hence, the process developed

by Thapa et al. (2009) reduces the soaking time by 12 to 14 h. Based on this optimized process

this paper attempts to evaluate the level of antinutrients and splitting percentage in kidney beans

before and after the tempering and canning process.

Antinutritional Factors of Beans

Legumes contain toxic substances, such as trypsin inhibitors, phytohaemagglutinins

(substances agglutinating and destroying red blood cells), factors causing lathyrism and favism,

cyanogenic factors, goitrogenic factors, saponins and alkaloids. These compounds adversely

24

affect enzyme activity, digestibility, nutrition and health, but many of them could be inactivated

or eliminated by particular processing procedures, such as dehulling, pre-soaking and diffusion,

sterilization, steaming and cooking (Loggerenberg, 2004). Some important antinutritional factors

are tannins, phytic acid, trypsin inhibitors and hemagglutinin which are also listed in the table 1.

According to this table the tannin content in different varieties of Phaseolus vulgaris varies from

0.11 to 28.78 mg catechin equivalent g-1, while the phytic acid varies from 12.37 to 27.60 mg g-1.

Higher the amount of tannins and phytic acid, lower will be the nutritional value in beans. The

table 1 also shows that the lower concentrations of trypsin inhibitor and hamegglutinin which also

reduce the nutritional quality of dry beans.

25

Table 1.

Ranges in levels of antinutritional factors in dry beans

Observations

Minimum

Content

Maximum

Tannins

Cowpea varieties (mg.g-1)

Four Phaseolus vulgaris

varieties (mg catechin

equivalent.g-1)

Trypsin-inhibiting activity

(TUI×10-3.g-1 protein)

Phytic acid

Four dry bean varieties

(mg.g-1)

Soybean, navy and

northern beans (mg.g-1)

Hemagglutinin

Four dry bean varieties

(HU×10-3.mg-1)

1.03

0.11

4.77

12.37

10.00

0.40

1.96

28.78

27.98

23.60

12.00

6.98

References: (Giami & Okwechime, 1993; Barampana & Simard, 1993; Elkowicz & Sosulski,

1982)

26

Phytic Acid

Phytic acid (PA, myo-inositol hexakisphosphate, IP6) was first identified in 1855 (Figure

1). It is a natural plant compound with a unique structure that is responsible for its characteristic

properties. Phytic acid has 12 replaceable protons, allowing it to complex with multivalent cations

and positively charged proteins and thus can be found in many forms. Phytate is the calcium salt

of PA and phytin is the calcium/magnesium salt of PA. Phytic acid can exist as free acid, phytate,

or phytin according to physiological pH and the metal ions present. All of these forms have been

used interchangeably in most of the literature linked to phytic acid systems. Complete hydrolysis

of phytic acid results in inositol and inorganic phosphates. Phytic acid phosphorus constitutes the

major portion of total phosphorus in several seeds and grain. It accounts for 50-80% of the total

phosphorus in different cereals and legumes. The phytic acid content is influenced by cultivar,

climatic conditions and year. The accumulation site of phytic acid in monocotyledonous seeds is

the aleurone layer, particularly the aleurone grain. Aleurone grain contains two types of

inclusions: (a) globoids containing high amount of phytates, and (b) protein carbohydrate bodies

(Oatway, 2001).

27

Figure 1: Structure of Phytic acid

The association of phytate with proteins begins in seeds during ripening, when phytate

accumulates in the protein-rich aleurone layer of cereals and protein bodies of legumes. Although

the fine structure of phytate-rich particles in plants has been intensively studied, the nature of the

interaction of proteins in such organelles with phytic acid is practically unknown. The formation

of globoid crystals and their size is highly dependent on the presence of inorganic cations. Higher

amounts of magnesium and calcium favor the formation of large globoid crystals (Oatway, 2001).

This fact suggests that higher concentrations of divalent cations increase phytate-phytic acid

interactions rather than protein-phytic acid interactions. The conditions of processing such as

addition of water, heat treatment (baking, autoclaving, extrusion etc), isolation and separation,

action of phytate degrading enzymes (e.g.phytates, phosphomonoesters) may cause changes in the

intensity and character of interactions.

28

The interaction of phytate with proteins, vitamins, and several minerals is considered to be

one of the factors that limit the nutritive value of dry beans. Numerous studies suggest that

phytate reduces the biological availability of dietary copper and manganese, iron, magnesium, and

zinc. Phytate is also suggested to interfere with protein metabolism and to decrease the utilization

of proteins subjected to proteolytic digestion (Loggerenberg, 2004).

Phytic acid has been shown to be bioavailable in cattle and other ruminants. At least 90%

of phytate P in concentrates was hydrolyzed in vitro after inoculation with ruminal fluid from a

lactating dairy cow. Less than 5% was recovered in excreta (Oatway, 2001). Most of this

degradation is due to 3-phytase characteristic of microorganisms and takes place almost entirely

in the rumen. Phosphorus of plant origin, mainly phytic acid, is generally considered to be poorly

available for utilization by monogastric animals, including pigs, poultry, and fish. It is usually

ignored in animal diet formulation as unavailable. Total phosphorus available from a feed

ingredient is equivalent to non-phytate-phosphorus plus digestible phytate-phosphorus.

Availability of phosphorus from phytate ranged from 20 to 60%. It is important to determine how

much P is available to minimize loss of nutrients to the environment. Excess P and other nutrients

are excreted by the animal; 65–75% of the total P in formulated diets with supplemental inorganic

P is excreted in the manure (Oatway, 2001). This creates an environmental problem when the

animal waste is left on farmland. A recent water quality assessment showed that P often exceeded

water quality guidelines in high and medium intensive agriculture areas (Anderson et al., 1998). It

is imperative that phytic acid be degraded in the animal to enhance the mineral and phosphate

utilization by the animals and to decrease the pollution of groundwater by P.

The phytate molecule is negatively charged at physiological pH and is reported to bind

with essential nutritionally important divalent cations such as Fe2+, Zn2+, Mg2+ and Ca2+….

29

etc., and forms insoluble complexes, thereby making minerals unavailable for absorption. It also

forms complexes with proteins and starch and inhibits their digestion (Oatway et al., 2001). The

dephosphorylation of phytate is a prerequisite for improving nutritional value because removal of

phosphate groups from the inositol ring decreases the mineral binding strength of phytate. These

results increased bioavailability of essential dietary minerals (Sandberg et al., 1999). Overall, a

good balance of nutrients is important for both animals and humans.

The total amount of any substance is not as important as the amount that is readily

available for utilization. Any deficiency, excess, or imbalance in minerals or other nutrients can

have a negative effect on health. PA has been shown to have many beneficial effects and it is

tempting to recommend increasing PA intake to reap some of these benefits. However, any use of

phytates as a therapeutic agent needs to be carefully considered because of the adverse effects

associated with large intakes. Precautions must be taken to avoid mineral and trace element

deficiencies (Oatway, 2001).

Tannins

Tannins are ubiquitous in nature and although a lot of attention has been given to their

study, the term “tannin” continues to be difficult to define precisely. Indeed, whereas related

phenolic compounds such as simple phenolics, neolignans and flavonoids are characterized and

classified according to their chemical structure, tannins are a diverse group of compounds that are

related primarily in their ability to complex with proteins (Fahey & Jung, 1989). Thus, tannins are

usually defined as water-soluble polyphenolic substances that have high molecular weight and

that possess the ability to precipitate proteins. Tannins have diverse effects on biological systems

because they are potential metal ion chelators, protein precipitating agents, and biological

antioxidants. Because tannins can play such varied biological roles and because of the enormous

30

structural variation among tannins, it has been difficult to develop models which allow accurate

prediction of the effects of tannins in any system (Loggerenberg, 2004).

Tannins are high molecular weight, phenol-rich polymers that exist in many foods,

including dry beans, and some examples are beverages, cereals, fruits, coffee and tea. Tannins are

divided into two major types, condensed and hydrolysable. Hydrolysable tannins are polyesters of

phenolic acids such as gallic acid, digallic acid (gallotannins) or hexahydroxydiphenic acid

(ellagitannins) and D-glucose or quinic acid, the latter serving as a polyalcohol core shown in

Figure 2. Hydrolysable tannins receive their name because they are readily cleaved by enzymes

(i.e. Penicillium tanninase) as well as by dilute acid to give a sugar such as glucose and a

phenolcarboxylic acid such as gallic acid. On the other hand, condensed tannins are composed of

flavan-3-ols linked via carbon-carbon bonds that produce anthocyanidins upon treatment with

acidic alcohol as in Figure 3 (Rolando, 1999).

Condensed tannin concentration in plant tissue has been shown to vary with many factors.

These include plant species, plant part, plant maturity, growing season and soil fertility. Anuraga

et al. (1993) observed that a combination of moisture stress and high temperature can resulted in

an increase in the concentration of condensed tannins in plants. A dramatic change in condensed

tannin concentration is due to the changes in soil fertility (Rolando, 1999).

31

Figure 2. An example of hydrolysable tannins and related compounds; G = gallic acid.

Adapted from (Mangan, 1988) and Mueller-Harvey & McAllan (1992).

COOH

COOH

OH

Hexahydroxydiphenic acid

G OG

Penta-O-galloyl-/3-D-gluco.se

I OG

Hydrolysable tannin

32

Figure 3. An example of a condensed tannin. Adapted from Mueller-Harvey & McAllan (1992).

The tannins of different plant species have different physical and chemical properties and

therefore they have very diverse biological properties. The high affinity of tannins for proteins

lies in the formers’ great number of phenolic groups. These provide many points at which

33

bonding may occur with the carbonyl groups of peptides. The formation of such complexes is

specific, both in terms of the tannin and protein involved, the degree of affinity between the

participating molecules residing in the chemical characteristics of each (Rolando, 1999). With

respect to tannins, the factors promoting the formation of complexes include their relatively high

molecular weight and their great structural flexibility (Mueller-Harvey & McAllan, 1992). The

proteins that show the most affinity for tannins are relatively large and hydrophobic, have an

open, flexible structure and are rich in proline. The complexes formed between tannins and

proteins or other compounds are generally unstable. The bonds uniting them continually break

and re-form (Mueller-Harvey & McAllan, 1992). The complexes that could come through four

types of bond: 1) hydrogen bonds (reversible and dependent on pH) between the hydroxyl

radicals of the phenolic groups and the oxygen of the amide groups in the peptide bonds of

proteins, 2) by hydrophobic interactions (reversible and dependent of pH) between the aromatic

ring of the phenolic compounds and the hydrophobic regions of the protein, 3) by ionic bonds

(reversible) between the phenolate ion and the cationic site of the protein (exclusive to HT), and

4) by covalent bonding (irreversible) through the oxidation of polyphenols to quinones and their

subsequent condensation with nucleophilic groups of the protein (Rolando, 1999). For a long time

it was believed that the formation of tannin-protein complexes was owed mainly to hydrogen

bonds. However, it is now known that hydrophobic interactions are important.

34

Chapter III: Methodology

This chapter includes a discussion on the methods of sample preparation, chemical

analysis and data analysis. It also comprises the process of raw material acquisition,

instrumentation, and data collection procedure.

The red kidney bean, because of its hard nature, is difficult to cook. Therefore, in the food

industry the canning process of red kidney beans requires a long soaking process in order to

soften the seeds prior to cooking for enhancement of the bean quality. The advantages of soaking

of bean includes increased yield of whole beans, tenderness level improvement, and uniform bean

expansion in the can during processing. In an industrial practice, the soaking process takes about

6 to 18 h with intermittent water changing every 4 to 6 h to prevent bacterial activity. This current

research utilized the environmental chamber for tempering of beans.

Sample Preparation

Red kidney beans used for the experiment were provided by the Chippewa Valley Bean

Company (Menomonie, Wisconsin). Beans were stored at room temperature (72°F) in cardboard

boxes until they were processed for tempering. Beans of the same size were used in this research.

The smallest, largest and broken seeds were further removed manually. Beans were treated with

different processes like tempering, soaking, blanching and thermal processing for canning.

Canned beans processed at specific time and treatment condition were prepared at the Department

of Food and Nutritional Sciences, University of Wisconsin-Stout.

Tempering. The complete tempering process of red kidney bean was carried out in an

Environmental chamber (EC) (ESPEC model EWPA 647-4WWL, Hudsonville, Michigan).

Kidney beans were tempered in the environmental chamber at 45°C and 95% Relative Humidity

(RH). Three sets of bean sample each 600 g were tempered in EC for 6, 12 and 24 h. At specific

35

length of time in EC, the beans were soaked in water of 24°C for 0, 1, 2, 3, 4 and 5 h. The beans

which were tempered only for 6 hours were not porous, and had moisture content of 30-40% after

soaking for 4-5h. Therefore, due to the inadequate swelling of beans and low hydration

coefficient, the tempered beans for 6 hours were not used for further canning process (Thapa et

al., 2009).

Soaking. The beans which tempered for 12 h and 24 h were soaked for 2 h to achieve

adequate moisture content (50-65%) required for the canning (Loggerenberg et al., 2004). Some

samples were further soaked and blanched to evaluate the overall effect on the quality and anti-

nutritional factors. To work on a commercial canning condition, one sample of untempered beans

was soaked overnight in distilled water at room temperature. Based on the type of sample, the

beans were soaked in small plastic buckets. The mass of the soaked beans were then obtained or

further treated for blanching. Blanching was carried out in order to shorten the hydration time of

beans.

Blanching. In the blanching process, dry beans samples were cooked in distilled water at

85°C for 8 minutes in electric oven in aluminum pan and then drained for can fillings. Broken or

split beans resulting from soaking and blanching were removed while completely hydrated and

firmer beans were selected for canning.

The canning medium was made with the following composition; CaCl2 = 0.42 g/L, NaCl =

27.24 g/L and sugar = 74.60 g /L (Van Buren et al., 1986). Calcium chloride was added to

increase the firmness of beans. An increase in calcium content in brines also helps to enhance the

lightness of canned beans. The NaCl helps to maintain the individual integrity of canned beans,

while the EDTA chelates free metal ions that caused the formation of color complexes

(Loggerenberg, 2004).

36

Thermal Processing and Canning. The soaked and blanched beans (soaked beans

equivalent to 96 g of dry sample used) were transferred to cans of size # 303, and filled with brine

canning medium. The cans were sealed in Dixie automatic can sealer (Dixie Canner Co, Athens,

GA, USA). The sealed cans were then heat sterilized in a vertical autoclave (Loveless

manufacturing Co, OK) at 240°F/10 psig for 40 minutes followed by instant cooling. After

canning, a storage period of two weeks was allowed for the beans and the moisture in the brine

medium to reach equilibrium (De Lange, 1999). Split beans were counted and massed to

determine the percentage split in canned beans. After this procedure, the canned beans were dried

in an oven (Blue-M/Lindberg MO1430A-1 Mechanical Convection Oven) at 130°C for 1 hour

(Agrawal, 2004) and ground in a standard coffee grinder to produce a flour that was used in the

analysis of antinutritional factors (phytic acid and tannins).

Phytic Acid Content

Phytic acid content of raw, treated and untreated canned beans was determined by the

method outlined by Wheeler and Ferrel (1971). Two g of flour sample was extracted with 50 mL

3% trichloroacetic acid (TCA) with mechanical shaking (VWR DS 500 orbital shaker) for 45

minutes with occasional swirling by hand. The slurry obtained was centrifuged (Beckman Coulter

Allegra 6KR centrifuge) and 10 mL aliquot of the supernatant was transferred into a 40 mL

conical centrifuge tube. Four mL FeCl3 solution was added to the aliquot and heated in a boiling

water bath for 45 minutes. One or two drops of 3% sodium sulfate in 3% TCA were added and

further heated until the supernatant was not clear. Then the solution was centrifuged and carefully

decanted as a clear supernatant.

The precipitate was washed twice by dispersing in 20 to 25 mL 3% TCA followed by

heating in boiling water bath for 5 to 10 minutes and then was centrifuged. Again the wash was

37

repeated by using distilled water. The precipitate was then dispersed in few ml of water and added

with 3 mL 1.5 N NaOH with mixing. The volume was made up to 30 mL with distilled water and

heated in boiling water bath for another 30 min. Then the precipitated solution was quantitatively

filtered hot through Whatman No. 2. The precipitate from the paper was again dissolved with the

help of 40 mL hot 3.2 N HNO3 into a100 mL volumetric flask. The paper was washed with

several portions of distilled water, collecting the washings into the same flask. The cooled flask

was diluted to volume of 100 mL with distilled water. Five mL of aliquot was transferred to

another 100 mL volumetric flask and diluted to approximately 70 mL. Another addition of 20 mL

of 1.5 M KSCN (potassium thiocyanate) was made and diluted to volume and the color was read

immediately within 1 min at 480 nm in spectrophotometer (Spectronic 20D+) available at

University of Wisconsin-Stout. Also the reagent blank was analyzed with each set of samples.

The iron content was determined from the previously prepared standard curve and the phytate

phosphorus from the iron resulted assuming a 4:6 iron: phosphorus molecular ratio (Wheeler and

Ferrel, 1971).

Tannin Content

Quantitative estimation of tannins was carried out using the Vanillin assay method (Price

et al., 1978). A 200 mg sample of bean flour was extracted using 10 mL 1% (v/v) concentrated

HCl in methanol for 20 min in capped rotating test tubes. Vanillin reagent (0.5%, 5 mL) was

added to the extracts (1mL) and the HCl solution (4%, 5 mL) was also added to the second set of

blank samples. The samples were left in the water bath for exactly 20 min, and were removed and

the absorbance was read at 500 nm. A standard curve was prepared expressing the results as

catechin equivalents, i.e. amount of catechin (mg mL-1) which gives a color intensity equivalent to

38

that given by tannins after correcting for blank. Then tannin content (%) was calculated according

to the equation (Idris et al., 2006):

Catechin equivalent (CE) %

Where C, concentration obtained from the standard curve (mg mL-1).

Splitting of Beans

The extent of splitting in the canned beans was evaluated by separating beans from

duplicate cans into two categories: (1) little or no split-beans with no longitudinal splits and with

no transverse split longer than 1/5 of the small circumference of the beans; (2) significant split-all

beans with greater splitting than for category 1. All beans were evaluated for splitting by the

investigator. The % split was obtained as the mass of significant split beans to the mass of total

bean samples (Loggerenberg, 2004).

Moisture Content of Beans

Moisture determination was based on ASABE standard procedure 2003. In this method 5

g of the soaked beans were weighted in aluminum drying discs and then placed in a mechanical

oven (Lindberg/blue, mo 14505A-1, Asheville, NC) at 103°C for 72 h. After drying, the final

weight was calculated and then moisture content was determined. The general formula for

moisture content determination is given by the following equation 1.

Where: Mc = moisture content (%) of material, Ww = wet weight of the sample and Wd =weight of

the sample after drying

100

Ww

WdWwMc

39

Statistical Analysis

All work was conducted twice in triplicates and the data presented are means ±standard

deviation on the dry weight basis. Duncan’s multiple range tests was used to determine significant

differences (p < 0.05).

40

Chapter IV: Results and Discussion

This chapter includes a discussion on the results obtained and compares the findings to

those of other researchers. This chapter concludes with the interpretation of data obtained.

The dark red kidney beans were tempered in an environmental chamber (EC) at 45°C and

95% R.H. for 12 h and 24 h, respectively. The tempered beans were then pretreated, which

included soaking and blanching followed by canning. The canning process was carried out in a

retort canner at 240°F for 40 minutes. The antinutrient content of canned beans was analyzed by

determining phytic acid content of beans using Wheeler and Ferrel (1971) method, while the

tannin content was determined by Vanillin-assay method (Price et al., 1978). The percentage split

of canned beans was determined to evaluate the canning quality of canned beans. The reduction in

antinutrients level in kidney beans was compared with the results obtained from various similar

studies. Twelve different kinds of treatment conditions were selected to assess the reduction of

antinutrients and to evaluate the quality of canned beans by the measurement of its splitting along

with moisture content (MC), another key quality attribute in the canning process, which is

discussed below.

Moisture Content of Beans after Tempering and Soaking

It has been found that the desired moisture content of beans before canning needs to be 53

to 57% (Hosfield & Uebersax, 1991). The objective of the determination of the moisture content

of kidney beans was to estimate the time of tempering and soaking and to achieve the desired

moisture content of 53-57% required for canning of beans. Three different tempering times of 6 h,

12 h and 24 h were studied (Thapa et al., 2009) and the aim was to achieve the moisture content

between 53-57% in tempered and soaked beans.

The moisture content determination was based on the standard air-oven method (ASAE

standards, 2003) and the computed results of the moisture contents are shown in Figure 4 that

41

indicates that tempering beans for 12 and 24 h reduces the time it takes to soak the red kidney

beans. Tempering beans had noteworthy implications on their hydration; for example, 12 h

tempering followed by subsequent soaking of 2 h increased the moisture content from

14.27±0.01% to 52.40±1.62%.

Figure 4. Effect of tempering (45°C/95% R.H.) at 6 h, 12 h and 24 h on moisture content of beans

soaked for 0, 1, 2, 3, 4 and 5 h, respectively.

The beans tempered for 24 h were soaked for 0, 1, 2, 3, 4 and 5 h and it was noted that

the moisture content varied after different soaking times from 1 to 5 h from 14.27±0.01% to as

much as 60.88±2.65%. However, the desirable moisture content of 54.07% in kidney beans was

found after soaking for 2 h which was determined based on the research findings of Hosfield &

Uebersax (1991).

It has been noted that too low of a MC (less than 11%) at the time of processing beans

could lead to water imbibition problems during processing (Nordstrom & Sistrunk, 1979) as well

as affecting the rate of water uptake (Hosfield & Uebersax, 1979). Another factor that can result

in affecting water uptake is that if beans become too dry (M.C < 11%) before soaking they will

42

become water-impermeable. Too low initial MC (less than 11%) of beans lead to brittle seed

coats with consequential cracking, thereby delivering a poor quality canned product (Nordstrom

& Sistrunk, 1979). Dry beans (11 – 14 % moisture content) therefore will split more during

canning than semi-dry beans (50 – 60 % moisture content) (Gonzalez et al., 1982).

The tempering process of beans in high humidity conditions of 95% R.H. with 45°C

decomposes the pectin substances, weakens the cell connections and decreases the shearing

strength. Current results showed that tempering red kidney beans in a high humidity environment

reduced the soaking times with the attainment of the desired moisture content (53-57%). This was

possible because of the vapor pressure difference between the humid environment conditions

(high vapor pressure) and dry bean seed (low vapor pressure,) which caused the formation of a

capillary pore as the water vapor migrated into the bean matrix and hence altered the

microstructure (Thapa et al., 2009). The desired M.C (53-57%) was achieved from 12 h and 24 h

tempering and 2h soaking. This tempering process also helped to reduce antinutrients such as

phytic acid and tannins which are discussed below.

Phytic Acid Content of Canned Beans

Phytic acid (PA) in legumes is one of the major concerns in bean consumption.

Dry beans are widely known for their fiber, mineral and protein contents. Thus, they are

important components of a healthy diet. On the other hand, phytic acid in the beans chelates

mineral cations and interacts with proteins forming insoluble complexes that lead to reduced bio-

availability of minerals and reduced digestibility of protein (Reyden & Selvendran, 1993).

Phytates have also been implicated in decreasing protein digestibility by forming complexes and

also by interacting with enzymes such as trypsin and pepsin (Reddy & Pierson, 1994).

43

Table 2 summarizes the phytic acid content of raw and canned dark red kidney beans with

various treatment conditions. The PA concentration in the raw dark red kidney bean was found to

be 23.94 mg/g and this value was comparable with those of a previous report (Abd El-Hady &

Habiba, 2003). A reduction in phytic acid content was observed that depended upon the treatment

conditions. For example, in the current study, the phytic acid reduction was found to be

significant (p<0.05) for both 12 h and 24 h tempering as compared to raw dry kidney beans as

shown in table 2.

Reduction in phytic acid was determined to be 84% as a result of the 24 h tempering-

soaking-blanching-canning method; however, this method offers a long processing time. A

shorter processing time, 12 h tempering-soaking-blanching-canning, allowed for about 62%

reduction in phytate. Approximately more than half of the phytic acid was lost in kidney beans by

the combined effect of the tempering, soaking, blanching and canning process. Phytic acid was

not significantly different between 24 h tempering-canning method and 12 h tempering-

blanching-canning method but in both processes there was a significant reduction of 45-46% of

phytic acid when compared to the raw kidney beans as shown in table 2. The reduction by the

blanching and canning processes was most likely due to hydrolysis of phytic acid as discussed in

other studies (Khatoon & Prakash, 2004; Rehman & Shah, 2005). Also there was no significant

difference between 24 h tempering-blanching-canning and 12 h tempering-soaking-blanching-

canning method. Higher reduction of phytic acid was noted in 12 h tempering-soaking-blanching-

canning method; therefore, it seems that the cumulative effect of tempering, soaking, blanching

and canning helped to reduce the levels of phytic acid. Different values in the phytic acid

reduction of several legumes attributed to the cooking methods such as cooking at 100°C for 10,

20 and 30 min, and autoclaving at 121°C for 15 min were previously reported (Vijayakumari et

44

al., 1998). When noting the 24 h tempering-soaking-blanching-canning process, it was found that

a significant reduction (84%) (p<0.05) of phytic acid in dark red kidney beans resulted. However,

this process requires a long treatment time which might be the greater disadvantages to the

canning company compared to a commercial soaking process. Therefore, the optimal treatment

condition would be the 12 h tempering-soaking-blanching-canning process, which significantly

reduced the level of phytic acid and processing time over the commercial 20 h soaking-canning

process.

45

Table 2

Effect of various treatments on the phytic acid content of dark red kidney beans and subsequent

reduction (%) in dark red kidney beans

Treatment1 Phytic Acid Content2

(mg/g)

Reduction (%)

Raw 23.94±0.35 A

20S+C (Commercial)

12T

13.50±0.35 E

21.22±0.12 B

44

11

12T+C

12T+B+C

12T+S+C

12T+S+B+C

24T

17.05±0.23 D

13.02±0.12 F

11.45±0.00 G

9.19±0.20 H

19.30±0.11 C

29

46

52

62

19

24T+C 13.08±0.00 F 45

24T+B+C 9.27±0.11 H 61

24T+S+C 6.94±0.00 I 71

24T+S+B+C 3.80±0.12 J 84

1 Represents treatment combinations where T= Tempering, B= Blanching, S= Soaking and C=

Canning and 12, 20 and 24 indicates the treatment time in hour.

2 Values are means of three determinations ± standard deviation; values of each column followed

by different letters are significantly different (p<0.05).

46

Table 2 showed that tempering help to reduce the level of phytic acid content in

comparison to commercial canning process. The tempering process that offers the temperature of

45°C inside the EC for 12 h and 24 h reduced the phytic acid by thermal activity. Thermal

degradation of these molecules as well as changes in their chemical reactivity or the formation of

insoluble complexes, could explain the significant reduction of antinutrients by thermal

processing (Barroga, Laurena & Mendoza, 1985; Kataria Chauhan & Punia, 1989). In the current

study, the decrease in phytic acid during tempering might be due to the moisture obtained from

high humidity environment that is postulated to weaken the cellular connection in beans and this

resulted in increased porosity, which may have aided in the loss of the phytic acid.

A significant reduction (p<0.05) of 44% for phytic acid (13.5 mg/g) was obtained on

commercial 20 h soaking-canning method compared to raw kidney beans (23.94 mg/g). It was

reported that soaking lowered the phytic acid content, and the extent of reduction increased with

an increased soaking period. This reduction may be attributed to leaching out of phytate ions into

the soaking water, and the losses of the phytate ions may be due to the increased permeability of

seed coat (Duhan et al., 1989). This decrease in phytate could be attributed to the hydrolization by

phytases during soaking, and to the formation of insoluble complexes between phytate and other

compounds of dry beans (Kataria et al., 1988; Lestienne et al., 2005). Phytic acid reduction was

reported as 28% in black gram bean after being soaked for 12 h by Kataria et al. (1988). A

significant decrease was previously reported in phytic acid for soaked-cooked beans and the

greatest reduction was noted as 47.18% compared with raw beans (Barampama & Simard, 1994).

Additionally, Lestienne et al. (2005) reported that the reduction of the phytic acid content varied

from 17 to 28% in different cereals and legumes after being soaked for 24 h at 30°C.

47

It has been reported that cooking beans, particularly after soaking them, will reduce the

phytic acid in Phaseolus vulgaris L. (Iyer et al., 1980). However, Akindahunsi (2004) concluded

that the phytic acid contents increased by the soaking and cooking processing of African oil

beans. Vidal-Valverde et al. (1998) observed that soaking fava beans in either water, acid, or base

solutions did not produce significant changes (p<0.05) in phytic acid levels. Boiling of legumes

also did not result in a significant breakdown of their phytic acid content (Kumar et al., 1978).

Ologhobo and Fetuga (1984) also could not record a significant reduction in phytic acid of

soybeans due to cooking, autoclaving, and soaking. Microwave heating of soybeans caused a 23%

phytic acid reduction after 9 min and 46% after 15 min, while gamma irradiation (1 kGy) reduced

the phytic acid content of soybean by only 4% (Hafiz et al., 1989).

In contrast, reductions in phytic acid contents of cereals and legume seeds with sprouting

have been frequently reported (Ibrahim et al., 2002). These reductions are mainly due to an

increase in the phytase activity, leading to a solubilization of phytates once the beans have

sprouted (Camacho et al., 1992). The simple and inexpensive technique of sprouting has been

therefore recommended for both in home and industrial use. In the current study, tempering for 24

hours is comparable to the effectiveness of sprouting for the reduction of phytic acid and other

antinutrients. Previous research conducted in red kidney beans by Yasmin et al. (2008) found that

43% of phytic acid was reduced by sprouting for 96 h. Likewise 19% of reduction of phytic acid

was noticed in the current study by a tempering process of 24 h. In earlier studies, sprouting has

also been reported to have a diminishing effect on the phytic acid content of various legumes like

the moth bean (Khokhar, 1984), rice bean and fava beans (Saharan, 1994) and the pigeon pea

(Duhan et al., 2002). Additionally, Duhan et al. (2002) reported that the cumulative effect of

soaking, cooking and dehulling were more pronounced than soaking alone for lowering the phytic

48

acid content in pigeon pea. Similarly, it can be seen in the current study that the combined effect

of tempering, soaking, blanching and canning was very effective in the reduction of phytic acid.

Tannin Content of Canned Beans

Generally, tannins found in beans may form insoluble complexes with proteins thus

decreasing the digestibility of proteins (Uzoechina, 2007). Tannins may decrease protein quality

by decreasing digestibility and palatability, damaging the intestinal tract, and enhancing

carcinogenesis (Makkar & Becker, 1996). Furthermore, tannins could impair iron availability

(Svanberg et al., 1993; Udayasakhara-Rao, 1995).

Table 3 shows results for tannin content of dark red kidney beans as a function of

processing and it is noted that compared to raw beans, a significant (p<0.05) reduction in tannin

levels was observed after 12 h tempering, 24 h tempering and other treatments. There were more

than 50% and 29% reduction of tannin content in 24 h and 12 h tempering, respectively. Since

tempering is a type of thermal processing method, the decrease in tannin content could be related

to the fact that these compounds are heat labile (Rakic et al., 2007) and degrade upon heat

treatment. Also the moisture obtained from high humidity environment may have increased the

porosity of beans by the vapor pressure difference in the cell connection. Therefore, the exposure

of moist beans to high temperature in the environmental chamber might have allowed for

degradation of the tannin content.

There was no significant difference between 20 h soaking-canning method and 12 h

tempering-blanching-canning method. Treatment of 12 h tempering-soaking-canning caused a

75% reduction in tannin content. The greatest reduction in tannin content by 95% compared to

raw beans was caused by 24 h tempering-soaking-blanching-canning method. There was no

significant difference between three different treatments: 24 h tempering-blanching-canning

49

method, 12 h tempering-soaking-blanching-canning method and 24 h tempering-soaking-canning

method. The reduction of tannins after tempering, soaking, blanching and canning is mainly due

to the fact that those compounds are water soluble (Kumar, Reddy & Rao, 1979) and

consequently leach into the liquid medium (Vijayakumari, Pugalenthi & Vadivel, 2007). In the

current study, the commercial 20 h soaking-canning method also significantly reduced the tannin

content by 63% when compared to the raw dark red kidney beans. These losses may have

originated from the diffusion of the tannins into the water during soaking, and canning, as well as

possible binding of tannins with proteins and other organic substances during blanching (Reddy et

al., 1985; Barampama & Simard, 1994).

50

Table 3

Effect of various treatments on the tannin content (mg/g) and subsequent reduction (%) in dark

red kidney beans

Treatment 1 Tannin Content 2 (mg/g) Reduction (%)

Raw 21.6±0.99 A

20S+C (Commercial)

12T

8.00±0.99 F

15.3±0.12 B

63

29

12T+C 13.21±0.78 C 39

12T+B+C

12T+S+C

12T+S+B+C

24T

7.28±0.45 F

5.40±0.99 G

3.96±0.33 H

10.6±0.37 D

66

75

82

51

24T+C 9.16±0.75 E 58

24T+B+C 4.23±0.67 H 80

24T+S+C 3.74±0.33 H 83

24T+S+B+C 1.08±0.43 I 95

1 Represents treatment combinations where T= Tempering, B= Blanching, S= Soaking and C=

Canning and 12, 20 and 24 indicates the treatment time in hour.

2 Values are means of three determinations ± standard deviation; values of each column followed

by different letters are significantly different (p<0.05).

It can be seen that greater reduction of 82% in shorter processing time was observed in 12

h tempering-soaking-blanching-canning method. Important reductions of 33.1-45.7% in the

51

tannin content of dry beans were also reported in an earlier study, when different cooking

methods such as ordinary cooking at 100°C for 10 min and autoclaving at 121°C for 10, 20, 40,

60 and 90 min were applied (Rehman & Shah, 2005). Similarly the thermal processing condition

of 115°C for 40 min in an autoclave used in the current study also allowed a reduction of tannins.

The tannin content was found to be significantly reduced (<0.05) in all processes that were

thermally processed in an autoclave (canning) (Table 3). These results are consistent with the

findings of other studies in which complete elimination of tannins on cooking fava beans

(Sharma & Sehgal, 1992) were observed while autoclaving at 121°C for 25 min. The reduction in

tannins in cooked seeds had been recorded by earlier investigations on plant foodstuff (Habiba,

2000; Nithya, Ramachandramurty & Krishnamoorthy, 2007). The reductions noted may be due to

the loss of compounds at high temperatures or to degradation or to interaction with other seed

components, such as proteins, to form insoluble complexes. Shimelis and Rakshit (2007) found

that both soaking and cooking/autoclaving caused a significant reduction of 75% of tannin

contents of kidney beans and the combined effect was significantly greater than cooking or

soaking alone. The combinations of soaking and cooking/autoclaving were able to

eliminate/reduce heat-stable and heat-sensitive antinutrients (Shimelis & Rakshit, 2007). These

results compare with the current study’s results in that the combination of 12 h tempering-

soaking-blanching-autoclaving significantly reduced (p<0.05) by 82% the tannin content of

beans.

Splitting of Dark Red Kidney Beans

The effect of various processing methods on the percentage splits of dark red kidney beans

is presented in Figure 5. Among the various processing methods used, the commercial soaking

process had the greatest splits of 20.57%, while those tempered for 12 and 24 h and then canned

had only between 2-6% splitting. Therefore it can be inferred that the splitting would result from

52

over-water uptake level in beans during soaking and to some extent due to the thermal processing

in retort canning and/or tempering. It may be speculated that a cause of transverse splitting was

the swelling of the bean during cooking producing stress on the skin and/or the weakening of the

skin during cooking so that this stress resulted in loosened skin. It was reported previously that

beans that have more mass, due to greater water uptake levels, have a greater tendency to split

(Forney et al., 1990). The beans, which were soaked for a longer time and thermally processed,

had greater percentages of splitting. The reason for this could be related to the excessive time

allowing for swelling of the beans that placed the skin under pressure, causing it to rupture.

Another reason for the splitting would be the amount of CaCl2 used in the canning medium. In the

current study the use of CaCl2 might have decreased the drain weight and increased the firmness

of beans. This was similar to the study of Uebersax, (1985) in which it was found that the

increased calcium in the soak water or the brine depressed final drained mass and moisture

content and increased firmness of pinto and navy beans. Similarly, Van Buren et al. (1986) found

that the firmer beans and less drain weight beans would have fewer splits. The observation

showed that lower percentage of split was associated with lower drained weight and greater

firmness that agrees with those values presented by Davis (1976) and Junek et al. (1980). Size of

beans also matters in the overall splitting of beans. Size depends on the different types of dry bean

varieties with larger sized having a value of 48.77 g/100 beans would take up less water during

canning, due to a larger volume-to-surface ratio, with consequential lower split values (Faris &

Smith, 1964).

53

Figure 5. Percentage split in canned beans with various treatment conditions

1 Represents treatment combinations where T= Tempering, B= Blanching, S= Soaking and C=

Canning and 12, 20 and 24 indicates the treatment time in hour.

2 Values are means of three determinations ± standard deviation; values of each column followed

by different letters are significantly different (p<0.05).

A 12 h tempering-soaking-canning method and 12 h tempering-soaking-blanching-

canning had the splitting of about 7.86% and 11.00%, respectively. There was no significant

difference between the percentage splitting of 24 h tempering method, 24 h tempering-soaking-

blanching-canning method, 12 h tempering-canning method and 12 tempering-blanching-canning

methods. The tempered beans are firmer and have a lower chance of splitting compared to

commercially soaked beans. The greater percentage of splitting (12.54%) was observed in 24 h

tempering-soaking-canning process. The beans processed for a longer time with various treatment

54

steps before canning could lead to a breakdown of seed coats resulting in the splits seen during

canning. Also excessive bean breakage during cooking would result in starch exudation into the

canning medium, with consequential clumping of individual beans. Softening of beans while

processing is thus important, but beans must still maintain their individual integrity (Hosfield &

Uebersax, 1980).

Figure 6 shows the color difference in commercial canned beans compared with the

tempered canned beans. The discoloration in commercial canned beans was due to the 20 h

soaking in water and thermal processing. The long soaking time and the migration of brine into

the beans caused the discoloration in commercial canned beans. Similarly the greater percentage

of spitting was noticed in commercial canned beans as apparent in Figure 6. The tempered canned

beans appeared firmer and darker in color. The long tempering process caused the beans to

become darker in color. The beans obtained from tempering and canning process have less split

but the overall appearance was altered.

Figure 6: Canned beans obtained from commercial soaking and canning process (left)and

canned beans obtained from tempering and canning process (right)

55

Figure 7: Example of split beans after thermal processing due to high water uptake levels

Figure 7 shows that the splitting of kidney beans often occurs after the commercial

canning process. This kind of splitting is due to high water uptake and excessive swelling. The

high temperature and pressure treatment during the thermal processing also caused the breakdown

of seeds. Again, over-processing could have caused splits or high water uptake levels (Forney et

al., 1990). The amount of CaCl2 used in the canning medium, type of canning medium, type of

legume seeds, type of soaking solution, and effect of treatment condition all several factors

reported to be responsible in the final quality of canned beans.

Hence, from the current study, the ideal treatment condition for optimum canning quality

of beans would be 12 h tempering-canning processes, however; the most effective method for

antinutrient reduction and desired canning quality would be based on 12 h tempering-soaking-

blanching-canning method.

56

Chapter V: Conclusion

The purpose of this study was to evaluate the effect of tempering process on antinutrients

level by analyzing the phytic acid and tannins and thereby examining the quality of canned dark

red kidney beans. Beans were obtained from Chippewa Valley Bean Company Inc. (Menomonie,

WI). The bean samples were tempered, soaked, blanched and thermally processed according to

the study design and prepared for further chemical analysis and quality evaluation. The samples

were analyzed using a spectrophotometry technique for antinutrients and quality was judged

based on percentage splits. Data obtained were compared with the standard curve of individual

components to quantify antinutrients.

The tempering process was successfully used in the optimization of canning process of

dark red kidney beans. The times of 12 and 24 h tempering with a 2 h soaking time were studied

and incorporated in the canning process to shorten the long soaking process of 20 h and to

improve the nutritional and overall quality of canned beans. Out of twelve different treatment

conditions used in canning process of beans, the 12 h tempering-soaking-blanching-canning

process was found to be very effective for the greatest reduction of phytic acid (62%). While, 24 h

tempering-soaking-blanching-canning process was found to be the most advantageous in reducing

the phytic acid content by as much as 84%. This process was found to be very efficient as

compared to a commercial soaking process used in typical canning companies.

This study showed that the tannin content in dark red kidney beans reduced significantly

with the treatment process of 12 h tempering-soaking-blanching-canning by 82%. This would be

the shortest treatment process that reduced a large percentage of antinutrients. On the other hand,

the tannin content reduction was found to be reduced by 95% in 24 h tempering-soaking-

57

blanching-canning process. However, this process has a disadvantage of having a longer canning

cycle.

This study also demonstrated that the percentage split in tempered canned beans being

lowered more than with the commercial soaking process. The percentage split in 12 h tempering-

canning process and 24 h tempering-canning process was found to be 2-3%. However, the process

of 12 h tempering-soaking-blanching-canning, which was found to be very efficient process for

the reduction of antinutrients discussed earlier, had a splitting value of 11%. Hence the overall

nutritional and quality benefits can be achieved in the 12 h tempering-soaking-blanching-canning

process.

The research of this finding showed that the application of tempering in the canning

process can drastically reduce the antinutritional load of dark red kidney beans with the

incorporation of soaking and blanching. Therefore, since kidney beans are proposed as

ingredients in the human diet, any of these conducted treatments are strongly advocated to be

applied in processing prior to their consumption to ensure their safety and quality based on phytic

acid, tannin content, and splitting.

Recommendations

1. Investigate the effect of tempering on the antinutritional levels of other legumes and dry

beans.

2. Investigate the effect of tempering on different types of antinutrients in legumes, cereals,

dry beans and other plant foods.

3. The quality and sensory evaluation of canned beans obtained from the tempering and

canning process can be done.

58

4. Investigate the exact tempering time and soaking time for the optimization of canning

process.

5. Analyze the antinutritional factors of kidney beans with the help of some other

sophisticated instruments like HPLC, GC/MS etc.

6. Investigate the physical, chemical and microbiological parameters of canned beans

prepared from the tempering process.

7. Microbiology study of the soaked water of beans before canning process.

59

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