Heat treatment of legumes can reduce antinutritional factors, and the optimization of protein digestibility, however, over- or under heating would damage the amino acids content of legumes. Therefore, adequate time is necessary. But direct analysis of both specifications is difficult in routine operations and is thus replaced with indirect tests such as urease assay index (UAI), protein solubility in 0.2% potassium hydroxide (PSKOH) and protein dispersibility index (PDI).

The UAI is useful to determine if the legumes have been heated enough to reduce the antinutritional factors, while PSKOH is a good protein solubility index (PSI) for determining over processing of legumes. The PDI is the best method of evaluating these legume ingredients for both under heating and overheating. So, combining the PDI test with the UAI and PSI could be useful for better monitoring of legumes quality.

In this research, five various legume samples commonly used in the household are placed in an oven at 120°C for up to 30 minutes; analysis was accomplished every 5 minutes. Adequate processing of black eye bean (20 minutes), chickpea (10-15 minutes), pea (10 minutes), soybean (15-20 minutes) and kidney bean (5 minutes) are determined combining UAI, PSI and PDI.

Extracto

AKNOWLEDGEMENTS

This research project was funded by the University Grants Commission (UGC),

Sanothimi, Bhaktapur, fiscal year 071/072. It has been such an admiration to be part of

the UGC. I would like to express thanks UGC for giving me an opportunity to do this

study. Through the project I achieved chance to further my knowledge and expand a lot

of experience.

I would like to express my deep sense of gratefulness to Mr. Surya Prakash Poudel,

Scientist, Nepal Agricultural Research Council (NARC), Khumaltar, Lalitpur for

identifying the botanical names of my legume samples.

There are no words to describe how thankful I am to Prof. Dr. Anjana Singh, Head of

Central Department of Microbiology (CDM), Kirtipur, TU. I am grateful to her all

guidance, motivation and support she gave me as well working space to carry out my

research. I also received continuous, immeasurable support and help from my friends

Manita Aryal, Supriya Sharma and the CDM staff.

A lot of thanks to my husband, Mr Deepak Kr. Shrestha, who has offered his support

from the beginning of my studies. Thank you very much for all the support and your

comments.

Last but not the least I would like to express my extreme gratefulness to my dear father

Mr. Kedar Man Baidya, mother Mrs. Sharada M. Shrestha and sisters Dr. Pooja Baidya,

Poonam Baidya, Dr. Malika Baidya and Kshitiz Man Pradhan, who always motivated me

and are my constant source of inspiration and encouragement in every step of my life.

iii

Abstract

Heat treatment of legumes can reduce antinutritional factors, and the optimization of protein

digestibility, however, over or under heating would damage the amino acids content of

legumes therefore, adequate time is necessary. But direct analysis of both specifications is

difficult in routine operations, therefore, replaced with indirect tests such as urease assay

index (UAI), protein solubility in 0.2% potassium hydroxide (PSKOH) and protein

dispersibility index (PDI). The UAI is useful to determine if the legumes have been heated

enough to reduce the antinutritional factors, while PSKOH is a good protein solubility index

(PSI) for determining over processing of legumes. The PDI is the best method of evaluating

these legume ingredients for both under heating and overheating. So, combining the PDI test

with the UAI and PSI could be useful for better monitoring of legumes quality. In this

research, five various legume samples commonly used in household are placed in an oven at

120°C for up to 30 minutes; analysis was accomplished every 5 minutes. Adequate

processing of black eye bean (20 minutes), chickpea (10-15 minutes), pea (10 minutes),

soybean (15-20 minutes) and kidney bean (5 minutes) are determined combining UAI, PSI

and PDI.

Keywords: Antinutritional factors, protein dispersibility index, urease activity index,

protein solubility, legumes, overheating, adequate heating, under heating

LIST OF CONTENTS

Contents

Page no.

Acknowledgements

Abstract

iii

List of contents

List of tables

List of figures

Abbreviations

vii

Chapter I

Introduction and Objectives

1-4

1.1 Introduction

1.2 Objectives

Chapter II

Literature review

5-15

Chapter III

Materials and methods

16-17

3.1 Materials

3.1.1 Chemicals and instruments

3.2 Methods

3.2.1

Location of the study

3.2.2

Methods of data collection

3.2.3

Study variables

3.3 Methodology

3.2.1

Samples

3.2.2

Sample processing

3.4 Statistical analysis

Chapter IV

Results and discussion

18-24

Chapter V

Conclusion and recommendation 25

5.1 Conclusion

5.2 Recommendation

5.3 Acknowledgement

References

26-37

Appendices

viii-xi

LIST OF TABLES

Page no.

Table 1 Protein and other nutrients contain in some common legumes

Table 2 Urease activity index values for determining the degree of legumes

processing

Table 3 PSKOH values for determining the degree of legumes processing

Table 4 PDI values for determining the degree of legumes processing

Table 5 Effect of heat treatment at 120°C on urease index (pH) in various

legumes

Table 6 Effect of heat treatment at 120°C on protein solubility index

(PSKOH) in various legumes

Table 7 Effect of heat treatment at 120°C on protein dispersibility index

(PDI) in various legumes

Table 8 Correlation between UAI, PSI and PDI in various legume samples

Table 9 Adequate processing time (minutes) of various legume samples at

120°C

LIST OF FIGURES

Page no.

Figure 1

Effect of heat treatment at 120°C on urease index (pH) in various legumes 22

Figure 1

Effect of heat treatment at 120°C on protein solubility index (PSKOH) in

various legumes

Figure 2

Effect of heat treatment at 120°C on protein dispersibility index (PDI) in

various legumes

vii

ABBREVATIONS

ANF

Antinutritional Factors

BSA

Bovine Serum Albumin

KOH

Potassium Hydroxide

MS Excel Software

Microsoft Excel Software

NSI

Nitrogen Solubility Index

PDI

Protein Dispersibility Index

PSI

Protein Solubility Index

PSKOH

Protein Solubility in 0.2% KOH

TIA

Trypsin Inhibitor Activity

UAI

Urease Activity Index

Urease Index

CHAPTER I

INTRODUCTION AND OBJECTIVES

1.1 INTRODUCTION

A legume belongs to a plant seed in the family Fabaceae (or Leguminosae). They are

grown agriculturally, primarily for their food grain seed, e.g., beans and lentils, or

generally pulse, for livestock feed and silage, and as soil-enhancing green fertilizer

(Kedibone Y. M., 2010). Commonly used legumes as foods includes soybeans (Glycine

max), chickpea (Cicer arientinum L.), black gram (Phasedous Mungo), cow pea (Vigna

unguiculata), dry beans (Phaseolus vulgaris), winged beans (Psophocarpus

tetragonolobus), horse gram (Doliches biflorus), moth bean (Vigna aconitifolia), pigeon

pea (Cajanus Cajan), favabeans (Vicia faba L. minor), grain amaranth (Amaranthus spp.),

lentil (Lens culinaris medic), jackbean (Canavalia gliadata) and grass peas (Lathyrus

sativus) (Ahn, Robertson, Elliot, Gutterridge, & Ford, 1989).

The recommended dietary allowances (RDA), for protein are listed as total grams of dietary

protein per kilogram body weight, regardless of whether the source is animal or vegetable.

Animal and vegetable proteins, however, are different from each other in unique ways

(Craig & Mangels, 2009).

There are ten indispensable or essential amino acids, defined as those that the body is

unable to synthesize from simpler molecules and is diet dependent; they are arginine,

histidine, Isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan and

valine. Under certain extreme physiological conditions such as in prematurity or during

some catabolic illnesses, the non-essential amino acids, cysteine, glutamine, glycine,

proline and tyrosine may be required in the diet. These standard amino acids act as

precursors for many coenzymes, hormones, nucleic acids and other various biomolecules.

The nutritional value or quality of structural different proteins varies and is governed by

amino acid composition, ratios of essential amino acids, susceptibility to hydrolysis during

digestion, source, and the effects of processing. To optimize the biological utilization of

proteins, a better understanding is needed of the various interrelated parameters that

influence their nutritive value. Now a day there is an increasing attention on the metabolic

effects of particular individual dietary amino acids, and for this reason it is important to

have precise information on the amounts of digestible or preferably bioavailable amino

acids in foods and proteins. It is thus recommended that dietary amino acids be treated as

individual nutrients (UN, 2011).

In western countries, legumes play a negligible position in the diet, except the fact that they

are brilliant reservoirs of protein, dietary fiber and a variety of macro and micro nutrients

and phytochemicals. They are among the best protein sources in the plant kingdom. Since

legumes are relatively cheap compared to meat and its products, therefore, eating more

legumes may be an alternative to meat for some (Monary, 1996). They contain superior

percentages of protein than cereal and provide a relatively affordable protein source in

developing countries (Khokhar & Owusu- Apenten, 2009). So, legumes constitute an

important part of the diet of large sections of the population in the developing countries, as

a good source of protein, carbohydrate and water soluble vitamins (Monary, 1996).

The proteins from pulses are known to be inferior quality, due to the deficiency of sulphur

containing amino acids as well as due to other factors like digestibility, availability of

amino acids, antinutritional factors (ANF) etc. (Friedman, 2001). Generally, plants

including legumes synthesize a range of low and high molecular weight compounds known

as ANF. These secondary metabolites play a role in defense against herbivorous, insects,

pathogens or adverse growing conditions (Herbourne, 1989). The ANF mainly occurs in

legumes in a raw state (Kedibone, 2010), foods and feed material prepared from grain

legumes. The ANF is presented in this type of food, including their contents of enzyme

inhibitors, lectins, flatulence factors, tannins, phytic acid and saponins (Price, Johnson, &

Fenwick, 1987). They can cause detrimental effects to humans and animal growth and

performance by impairing intake, uptake or utilization of other foods and feed components

or by causing discomfort and stress in humans and animals (Friedman, 2001).

In Nepal, most people are vegetarian and/or poor, depending largely on cereals, pulses and

legumes, as their staple foods provide the main source of dietary proteins and calories.

Various physical and chemical methods are employed to reduce or remove ANF including

soaking, cooking, germination, fermentation, selective extraction, irradiation and enzyme

treatment. Application of single technique has been frequently insufficient for effective

treatment and so combinations are commonly employed. Industrial processes, including

canning, toasting, fractionation and isolation of protein concentrates have been shown to

be effective in reducing or removing ANF. However, it should be borne in mind that

processing can also introduce undesirable compound, for example, volatile aldehydes and

ketones and peroxides as a direct result of lipid oxidation or reduce levels of desirable

compound ex-proteins and essential minerals. It is reported that ANF is generally

eliminated by soaking and subsequently discarding of the liquid and/or by heat treatment

at a relatively elevation temperature (Khokhar & Owusu- Apenten, 2009).

So in this research some of very common legumes used in household are heat treated and

tried to uncover the least amount of ANF content on them by indirect tests as urease assay

index (UAI), protein solubility index (PSI) and protein dispersibility index (PDI).

1.2 OBJECTIVES

1.2.1 General objective

1.2.1.1 Analysis of chemical indices to determine legumes protein quality

1.2.2 Specific objectives

1.2.2.1 Determination of antinutritional factors in legumes by urease index

1.2.2.2 Determination of legumes solubility by KOH protein solubility index and

protein dispersibility index

1.2.2.3 Determination of legumes solubility by protein dispersibility index

1.2.2.4 Correlation with indices

CHAPTER II

LITERTURE REVIEW

Legumes form the third largest plant family (Dam, Laursen, Ornfelt, Jochimsen, &

Staeffeldt, 2009). These are also among the largest and most diverse families of flowering

plants (Brandon, Wagmaister, Kawashima, Bui, & et al, 2007). These are crops of the

family Leguminosae that is also called Fabacae (Khan, Khan, & Bhatty, 2009). Legumes

play an important role in human nutrition as they are a rich source of protein, calories,

certain minerals and vitamins (Baloch & Zubair, 2010); (Morrow, 1991); (Nielsen, 1991);

(Tharanathan & Mahadevamma, 2003) and used as curry or pulse (dhal) in Nep-Indo-Pak

region (Khan, Khan, & Bhatty, 2009). Therefore, grain legumes contribute 33% of daily

protein intake of humans (Graham & Vance, 2003). Therefore, legume studies are

important for establishing overall legume development (Dam, Laursen, Ornfelt,

Jochimsen, & Staeffeldt, 2009). Proteins are the major structural component of muscles

and other tissues in the body. They are also component of hormones, enzymes and

haemoglobin (Jay & Michael, 2004).

Proteins composed of twenty different amino acids linked together by a peptide bond and

the resulting chain are called polypeptides. They have a similar basic structure but differ in

their side chains. This difference in side chains gives the proteins their specificity and

functionality. These amino acids are classified as essential and nonessential (Clark, 2003).

Proteins are available in varieties of dietary sources, including animals, plant origin, and

from highly marketed spot supplement industry. Typically, all dietary animal proteins (e.g.

Eggs, milk, meat, fish and poultry) are considered a complete protein because they contain

all essential amino acids (Wardlaw & Insel, 1996). But the provision of adequate animal

proteins is difficult due to high cost and changing consumer's attitudes towards animal

based proteins (Iqbal, Khalil, Ateeq, & Khan, 2005); (Nunes, Raymundo, & Sousa, 2006).

Proteins from vegetable sources are incomplete proteins since they are lacking one or two

essential amino acids. It is well documented that cereal proteins are deficient in certain

essential amino acids, particularly in lysine (Anjum, Ahmad, Butt, & Sheikh, 2005)

whereas legumes contained adequate amount of tryptophan and the highest digestible

lysine (Liener, 1983); (Liu, 1997); (Sai-Ut, Ketnawa, Chaiwut, & Rawdkue, 2009).

Protein content in legume ranged from 17- 40%, contrasting with that of cereals 7-13% and

comparable to meat 18-25% (Genovese & Lajolo, 2001).

Table 1, Protein and other nutrients contain in some common legumes

Legumes

Protein/Fiber

Minerals

Vitamins

Black

eye

beans

100 grams of cooked, Black

eye beans contain 7.73

grams protein, 116 calories

and 6.5 grams dietary fiber.

Potassium: 278 mg

Phosphorus: 156 mg

Calcium: 24 mg

Magnesium: 53 mg

Iron: 2.51 mg

Sodium: 4 mg

Manganese: 0.475 mg

Zinc: 1.29 mg

Copper: 0.268 mg

Selenium: 2.5 mcg

Also contains trace

amounts of other minerals.

Vitamin B

: 0.202 mg

Vitamin B

: 0.055 mg

Niacin: 0.495 mg

Pantothenic acid: 0.411 mg

Vitamin B

: 0.1 mg

Folate: 208 mcg

Vitamin A: 15 IU

Vitamin E: 0.28 mg

Vitamin K: 1.7 mcg

Contains some other vitamins in

small amounts.

Chick

peas

100 grams of Garbanzo

Beans, boiled without salt

contain 8.86 grams protein,

164 calories and 7.6 grams

of fiber.

Potassium: 291 mg

Phosphorus: 168 mg

Calcium: 49 mg

Magnesium: 48 mg

Iron: 2.89 mg

Sodium: 7 mg

Manganese: 1.03 mg

Zinc: 1.53 mg

Copper: 0.352 mg

Selenium: 3.7 mcg

Also contains trace

amounts of other minerals.

Vitamin C: 1.3 mg

Vitamin B

: 0.116 mg

Vitamin B

: 0.063 mg

Niacin: 0.526 mg

Pantothenic acid: 0.286 mg

Vitamin B

: 0.139 mg

Folate: 172 mcg

Vitamin A: 27 IU

Vitamin E: 0.35 mg

Vitamin K: 4 mcg

Contains some other vitamins in

small amounts.

Peas

100 grams of Pigeon Peas

boiled without salt contain

6.76 grams proteins, 121

calories and 6.7 grams of

dietary fiber.

Potassium: 384 mg

Phosphorus: 119 mg

Calcium: 43 mg

Magnesium: 46 mg

Iron: 1.11 mg

Vitamin B

: 0.146 mg

Vitamin B

: 0.059 mg

Niacin: 0.781 mg

Pantothenic acid: 0.319 mg

Vitamin B

: 0.05 mg

Source: http://www.health-alternatives.com/legumes-nutrition-chart.html

Legumes are an inexpensive source of proteins with high nutritional profile and after

cereals, important food source for humans (Doyle, 1994); (Vietmeyer, 1986). Improving

protein intake of the people is to supplement the diet with plant proteins. For that reason,

Sodium: 5 mg

Manganese: 0.501 mg

Zinc: 0.9 mg

Copper: 0.269 mg

Selenium: 2.9 mcg

Also contains trace

amounts of other minerals.

Folate: 111 mcg

Vitamin A: 3 IU

Contains some other vitamins in

small amounts.

Soybeans

100 grams of Soy beans,

roasted without salt contain

35.22 grams of protein, 471

calories and 17.7 grams

dietary fiber.

Potassium: 1470 mg

Phosphorus: 363 mg

Calcium: 138 mg

Magnesium: 145 mg

Iron: 3.9 mg

Sodium: 4 mg

Manganese: 2.158 mg

Zinc: 3.14 mg

Copper: 0.828 mg

Selenium: 19.1 mcg

Also contains trace

amounts of other minerals.

Vitamin C: 2.2 mg

Vitamin B

: 0.1 mg

Vitamin B

: 0.145 mg

Niacin: 1.41 mg

Pantothenic acid: 0.453 mg

Vitamin B

: 0.208 mg

Folate: 211 mcg

Contains some other vitamins in

small amounts.

Kidney

beans

100 grams of Kidney beans,

boiled without salt, contain

8.67 grams of protein, 127

calories and 7.3 grams

dietary fiber.

Potassium: 403 mg

Phosphorus: 142 mg

Calcium: 28 mg

Magnesium: 45 mg

Iron: 2.94 mg

Sodium: 2 mg

Manganese: 0.477 mg

Zinc: 1.07 mg

Copper: 0.242 mg

Selenium: 1.2 mcg

Also contains trace

amounts of other minerals.

Vitamin C: 1.2 mg

Vitamin B

: 0.216 mg

Vitamin B

: 0.058 mg

Niacin: 0.578 mg

Pantothenic acid: 0.22 mg

Vitamin B

: 0.12 mg

Folate: 130 mcg

Vitamin E: 0.03 mg

Vitamin K: 8.4 mcg

Contains some other vitamins in

small amounts.

consumption of plant protein isolates with special reference to legumes is beneficial (Iqbal,

Khalil, Ateeq, & Khan, 2005); (Nunes, Raymundo, & Sousa, 2006). It is advisable to

enhance the protein content of the diet through easily available and accessible plant protein

sources, especially legumes to improve the nutritional status of the low-income groups of

population (Khattab & Arntfield, 2009). Assessing the quality of protein is important when

considering the nutritional benefit that it can provide (Wardlaw & Insel, 1996). Protein

quality of food is the ability of the food to meet the nutritional requirement of an individual

species. It is indicated by how well the protein is digested, absorbed, and utilized for the

growth and sustenance of the body (Morrow, 1991); (Nielsen, 1991); (Tharanathan &

Mahadevamma, 2003); (Wardlaw & Insel, 1996).

Nepal is an Agra-based economy produces different types of legumes like chickpea,

soybeans, lentil, broad and kidney beans etc. Utilization of legumes in food formulations

as a source of protein is increasing as they provide a balanced amino acid profile. The

nutritional quality depends upon specific amino acids and their physiological utilization

after digestion, absorption and minimal mandatory rates of oxidation (Longnecker, Kelly,

& Huang, 2002).

Protein malnutrition is one of the major nutritional problems in the developing world. The

specific maladies like kwashiorkor and marasmus are prevalent in the children owing to

protein deficiency, whereas in adults, results in poor health and reduced work capacity.

Bridging the gap between increased food consumption and production is amongst the most

challenging tasks, round the globe, especially in developing countries (Black, Caulfield,

Bhutta, & Cesa, 2008). The existing problems of food security and malnutrition coupled

with escalating population, uncertain crop yield and high cost of animal based food

supplies have urged to identify and incorporate unconventional protein sources to enrich

the traditional formulations (Awan, 2000). Thus the protein energy malnutrition threats in

developing nations can be minimized using protein enriched sources in the daily diet

(Black, Caulfield, Bhutta, & Cesa, 2008).

Though legume proteins are highly valued, plants including legumes commonly synthesize

a wide range of secondary metabolites as part of their protection against attack by

herbivorous, insects and pathogens or as means to survive in adverse growing

conditions (Parul, 2014). The nutritional value and protein digestibility of legumes is

generally poor due to the presence of antinutritional factors (ANF). If the farm or domestic

animals or humans consume these plants, these compounds may cause poor

physiological effects (Machlachlan, 1993).

The term antinutrients refers to defense metabolites, having specific biological effects

depending upon the structure of specific compounds which range from high molecular

weight proteins to simple amino acids and oligosaccharides. Legumes are a rich source of

antinutrients in the human diet (Parul, 2014). The antinutrients like trypsin inhibitors,

phytic acid, saponins, heamagglutinins and tannins are some of the undesirable components

in legumes that could hinder utilization of important minerals including calcium,

magnesium, iron and zinc etc. They are interfering with their absorption and utilization and

thereby contributes to mineral deficiency (Vasagam & Rajkumar, 2011) as well as

interfering with normal growth, reproduction and health, when consumed regularly in

amount existing in a normal component of the diet therefore should be considered

as harmful and toxic (Parul, 2014). Similarly, protease inhibitors in various legumes have

the ability to retard proteolytic enzyme activity, for example, lectins are polymeric proteins

present in common beans that bind to monosaccharide in glycoproteins of the cell

membrane, causing lesions in the intestinal mucosa and reduced nutrient absorption too

(Ma, Boye, Simpson, & Prasher, 2011) and may cause unfavourable physiological effects

(Buttle, Burrels, Good, Williams, Southgate, & Burrells, 2001).

The removal or inactivation of the ANF described above is considered important if they

are present at higher levels (Wiseman, 1986). In addition to inactivating the ANF, the

processing treatment improves the taste of the end product and increases the use of the

energy and proteins contained in the legumes. The improved taste results from the heat

applied to the legumes which trigger the release of additional aromas and flavours, which

encourage humans to eat more (Murray & Murray, 1987). Part of this improvement could

be due to the inactivation of the lipoxygenase in the legumes, promoting the quality and

storage life of the end product. However, the exact improvement depends on the processing

method used, the processing condition and the species of legumes is in used (Leeson &

Atteh, 1996). Moreover, dehusking, soaking, germination, cooking and roasting have been

shown to exert beneficial effects on nutritional quality of legumes. Previously, different

processing methods such as boiling, hydration and germination are used to inactivate the

antinutrients (Shimelis & Rakshit, 2007) in the plant based foods, thus enhances the

nutritional value of isolated protein (Agbede & Aletor, 2005). Different technological

processes have been developed for the treatment of legumes to inactivate the ANF of these

valuable protein sources (Senkoylu, Samli, Akyurek, Agma, & Yasar, 2005). They include:

cooking, expansion, extrusion, flaking, jet sploding, micrronization, microwave and

roasting (Barbi, 1996). Factors which vary from one process to another are time,

temperature, pressure, humidity, exposed surface, particle size and type of energy used

(Hayward, Steenbock, & Bohstedt, 1936).

In order for the nutritional value of legumes to be maximized, these ANF need to be

inactivated, minimized and/or destroyed (Hayward, Steenbock, & Bohstedt, 1936);

(Liener, 1983); (Liu, 1997), therefore, heat treatment is an important step in variation of

protein quality as well as in legumes processing. Raw legumes also have urease activity,

which is not likely to be of great nutritional significance other than as an indirect

assessment of the degree of adequacy of processing (Monary, 1996). The urease assay is

based on the release of ammonia from urea by the residual urease enzyme in legumes

(Araba & Dale, 1990a); (Parsons, Hashimoto, Wedekind, & Baker, 1991). The heat

treatment required to destroy the urease parallels the treatment required to destroy the

trypsin inhibitor. So, urease index, an indirect indicator has been used to determine

presence of ANF, such as trypsin inhibitors (Araba & Dale, 1990b); (Caskey & Knapp,

1944); (Parsons, Hashimoto, Wedekind, & Baker, 1991).

One of the major concerns is, the role of heat on legumes if they are under- or over-

processed. Second, either under processing is more detrimental than over processing or

vice versa. To define under- and over-processing is easy in theoretical terms, but not in

practical terms. Under processing will not sufficiently eliminate trypsin inhibitor activity

and will reduce protein digestibility. Overheating, however, will denature proteins and

reduce the availability of amino acids, especially the essential ones, particularly lysine and

arginine (Hayward, Steenbock, & Bohstedt, 1936). The following mechanisms involves in

under- and over-processing:

During the under processing of legumes the ANF are not destroyed and this leads to a

reduction in the use of amino acids. The residual trypsin inhibitor mediates its effects via

the digestive processes, effecting both endogenous and exogenous amino acid losses. It

also binds and inactivates the pancreatic enzyme; trypsin and chymotrypsin. This lost or

bound trypsin is also rich in sulphur amino acids, which further reduce the protein status

of the human. The result is that protein digestibility is reduced and swelling of the pancreas

occurs, caused by the production of additional trypsin and chymotrypsin (Monary, 1989).

While in over processing, the proteins are denatured and the amino acid availability is

reduced. When proteins are exposed to excessive heat treatment or over processing, the

negative effects that cause reduced analytical concentrations and reduced digestibility of

amino acids occur for lysine and cysteine. Most of the other amino acids are not affected

by excessive heat treatment or over processing. Thus the reduction in protein quality of

legumes as a result of over processing is primarily due to the combination of the destruction

of lysine or cysteine and the reduced digestibility of the lysine and cysteine that is not

destroyed and, possibly to a lesser extent, other amino acids (Del Valle, 1981); (Monary,

1989); (Skrede & Krogdahl, 1985). These effects on lysine and cysteine may be largely

explained by the Maillard reaction in which proteins are heated in the presence of certain

carbohydrates, the sugar (such as xylose and glucose) and complex with free amino groups

(especially the epsilon group of lysine). The sugar and amino acids enter into a series of

reactions and, as a consequence, the availability of amino acids is reduced (Anderson-

Haferman, Zhang, & Parsons, 1992).

The mild heating (~90°C) improves the nutritional value of legumes by protein degradation

(denaturation of tertiary and quaternary structures) and exposing new sites for enzymatic

hydrolysis as well as inactivating heat labile ANF (Holmes, 1987); (Ruiz, Belalcazar, &

Diaz, 2004); (Zarkadas & Wiseman, 2005). Therefore, it is crucial to involve the methods

to distinguish adequately processed legumes from under or over processed meals. An

important and frequently observed effect of food processing is the reduction of protein

nutritive quality. The denaturation of the protein and reduce in amino acid availability of

cross-linking, racemization, degradation and formation of complexes with sugar may result

in loss of digestibility (Caprita & Caprita, 2007); (Del Valle, 1981); (Friedman M. , 1996a).

Therefore, when attempting to estimate protein quality, one of the first factors that must be

evaluated is its digestibility (Araba & Dale, 1990b); (Caprita & Caprita, 2008).

The quality of legumes is related to heat treatment and it is an extremely important element

during legumes processing. The objective of quality assurance is to prevent and minimize

the deterioration of the quality of the products during processing. And the main objective

of heat processing of the legumes is to achieve an optimum balance between the

degradation of the ANF and the maintenance of amino acid availability. Proper heat

treatment results in a product with a low trypsin inhibitor activity and high nutritional value

of legume products. The quality of the end product can be affected by four key factors

which are the processing conditions, the amount of legume hulls and their origin and type

of legumes taken (Shini, 2002). Following methods are commonly used for determining

the protein quality: UAI, PSKOH, PDI, nitrogen solubility index (NSI), trypsin inhibitor

activity (TIA), and cresol red test and lysine availability. The objectives of these tests are

to examine whether: the reduction of anti-nutritional factors and the optimization of protein

digestibility (Bruce, et al., 2006); (Monary, 1989).

The UAI is the test most commonly used to evaluate the quality of the legumes processing

treatment. This method determines the residual urease activity of the legumes and its

products as an indirect indicator to assess whether ANF such as trypsin inhibitors that is

present in legumes have been destroyed by heat processing. This test measures the increase

in the pH consequence of the release of ammonia into the media arising from the

breakdown of urea by the urease present in legumes and their products (American Oil

Chemists Society, 1980a). This assay is useful for detecting under processing of legumes,

but is of limited use for detecting over processing (Araba & Dale, 1990b); (Parsons,

Hashimoto, Wedekind, & Baker, 1991). All over heated samples yield urease indices below

0.05 but that does not imply that all samples with urease tests below 0.05 have been over

heated (Waldroup, Ramsey, Helwig, & Smith, 1985). Moreover, the recommended

maximum level of urease is controversial, with acceptable values varying from 0.2 or less

to 0.5 units of pH change (Waldroup, Ramsey, Helwig, & Smith, 1985). The fact that the

urease index is not linear and that it rapidly falls from approximately 2.0 units of pH change

to near zero (Araba & Dale, 1990b). According to (Garlich, 1988), the following UAI

values table 1 are used to estimate the degree of processing:

Table 2, Urease activity index values for determining the degree of legumes processing

(Garlich, 1988)

Legumes

UAI (pH)

Under processing

>0.20

Adequately processed 0.05-0.20

Over processing

<0.05

When pH is used as an indicator of legumes processing, the data may be influenced by

whether or not they had been previously treated with organic acids, preservatives or

sterilizing agents. Thus the value of UAI as a reliable indicator of the adequacy of legumes

is questionable. Furthermore, a UAI value of "zero" does not necessarily indicate that the

legumes are over processed, which has been shown in trials. Consequently, the UAI

suitable only for determining under processed legumes and its use, therefore, is limited

(Araba & Dale, 1990a).

The PSKOH method is relatively inexpensive and simple to perform. Unlike the UAI

method, it can be used to determine all degrees of legumes processing from under- to over-

processed. The original (Araba & Dale, 1990a) method showed a low reproducibility

between South African laboratories. This has been modified by (Palic, 2005a). It was

evaluated PSKOH as a good indicator of in vivo legumes quality (Parsons, Hashimoto,

Wedekind, & Baker, 1991). It determines the percentage of protein that is solubilized in a

potassium hydroxide (KOH) solution (Araba & Dale, 1990a). Earlier, the potassium

hydroxide (KOH) solubility assay was also reported to be useful for detecting

under

processing

of legumes (Araba & Dale, 1990b). However, later concluded that this assay

was not very accurate for assessing under processing of legumes (Anderson-Haferman,

Zhang, & Parsons, 1992). PSKOH has been shown to be a good indicator of in vivo protein

quality for

over processed legumes (Araba & Dale, 1990b); (Parsons, Hashimoto,

Wedekind, & Baker, 1991). Following PSKOH values shown in table 2 are commonly used

(Monary, 1989) for determining the degree of legumes processing.

Table 3, PSKOH values for determining the degree of legumes processing (Monary, 1989)

Legumes

Protein solubility in KOH (%)

Under processing

>85

Adequately processed 71-85

Over processing

<70

It was observed by Shini, 2002 that a lower KOH value means lesser digestible amino acids or

lesser amino acids availability due to the Maillard reaction and that a higher KOH value means

more digestible amino acids but a lesser breakdown of trypsin inhibitors present in legumes,

leading to a lower digestion and absorption of amino acids.

Another method that is often used in ruminant and human nutrition to monitor optimum heat

processing of legumes is PDI (American Oil Chemists Society, 1980b). It distinguishes the

quality of legumes used in meals. The PDI is simply measure of protein solubility in water with

high speed mixing, since the protein solubility of legumes decrease as the heat exposure

increases. It was reported that the PDI method has proven to be especially useful in the

determination of the degree of under heating of legumes to remove ANF (Batal, Douglas,

Engram, & Parsons, 2000). Current recommendations for meals that are considered adequately

heat processed are those with PDI values between 15-28% (NOPA, 2007) as shown in table 3.

Table 4, PDI values for determining the degree of legumes processing (NOPA, 2007)

Legumes

Protein solubility in water (%)

Under processing

>40

Adequately processed 15-40

Over processing

<15

The PDI of legumes and their products are very important quality index, particularly if relative

tests are run. It is demonstrated that flaking legumes at 121°C for increasing lengths of time

brought about a reduction in the contents of protein dispersibility (Kratzer, Bersh, Vohra, &

Ernst, 1990). PDI is a useful quality indicator of legumes (Batal, Douglas, Engram, & Parsons,

2000). The PDI method uses a special blender at a speed of 8500 rpm, which makes the method

the simplest to perform of all the methods for legumes quality control. It was reported that the

PDI procedure demonstrates more consistent results than the urease or PSKOH methods. While

UAI rapidly decline to "zero" (at which point the legumes may or may not be over processed)

PDI values do not approach "zero" even sever over processing. It also observed that the PDI

displayed the most constant response to the heating of legumes and that it may better indicate

the legumes quality compared to other methods (Batal, Douglas, Engram, & Parsons, 2000).

The urease index is useful to determine if the legume has been heated enough to reduce the

ANF, but it is not very useful for determining if legume has been over processed and urease

index value of zero does not necessarily indicate over processing of legumes. PSKOH remains

high, during initial heat treatment (Caprita and Caprita 2007). PSKOH also often did not

change consistently as legumes were heated. As observed for urease assay index, the

inconsistent response for KOH was particularly evident for the shorter heating times (Batal, et

al. 2000). PSKOH variation with the heating time was not linear. PSKOH decreased very little

up to 10 minutes of heating therefore, it is not a sensitive index for monitoring under processing

of legumes and appears to be a better indicator for over processing (Caprita, Caprita and Ilia,

et al. 2010). In marked contrast the PDI index decreased incrementally for the heating times

(Caprita & Caprita, 2007). PDI decreased incrementally when heating. The experimental data

suggest that PDI is more sensitive than UAI, PSKOH. PDI was reduced by approximately half

when raw legume was heated 20 minutes (Caprita, Caprita and Ilia, et al. 2010).

Being a cheap source of protein for low income group of population, legumes are commonly

used as a substitute for meat and play a significant role in alleviating the protein-energy

malnutrition as well as demand for the relatively cheap sources of protein that can be

incorporated to value-added food products is increasing worldwide, and numerous researches

are still going on various sources of plant proteins that may help in improving the nutritional

value of food products at low cost. Meanwhile the consumers are also more conscious in their

food selection owing to growing awareness about nutritional dependent ailments. Therefore,

the goal of this research is to evaluate total protein, discovering some most commonly

consumed legumes in households of Nepal with low ANF while heat treatment will be done

and finally compare based on their ANF via some chemical and biophysical laboratory

procedures for assessing legumes quality.

CHAPTER III

MATERIALS AND METHODS

3.1 Materials

3.1.1 Chemicals and instruments

All chemicals used were of analytical reagent grade. All the experiments were

carried out in doubly distilled water. The instruments and various reagents used

in different tests of this research are listed in Appendices.

3.2 Methods

3.2.1 Location of the study:

All experiments were carried out at the Central Department of Microbiology

(CDM), T.U; Kirtipur.

3.2.2 Methods of data collection:

The research method was quantitative and primary data were collected for

further analysis.

3.2.3 Study variables:

Five varieties of leguminous seeds (red kidney beans, black eyed beans,

soybeans, chick peas and peas)

3.3 Methodology

3.3.1 Samples:

Five varieties of leguminous seeds (red kidney beans, black eyed beans,

soybeans, chick peas and peas) which are very commonly used in a household

were obtained from the local farmers near Kathmandu, Nepal. Their systematic

botanical names were identified from Nepal Agriculture Research (NARC),

Khumaltar, Latilpur. The collected seeds were cleaned, rinsed with tap water

followed by distilled water to remove any the dust particles and any other

impurities and then dried again. The samples were ground to pass the 200

sieve. Powered legume samples were heated in a forced air oven at 120°C for

varying periods of time: 5, 10, 15, 20, 25 and 30 minutes.

3.3.2 Sample processing:

i. Total protein of raw and heat treated legume seed powder was determined by

the Bardford method with bovine serum albumin as standard (Bradford 1976).

ii. Urease assay of raw and heat treated legume seed powder was determined by

UAI (American Oil Chemists Society 1980a).

iii. The PSKOH solution of raw and heat treated legume seed powder was

determined by the PSI according to the procedure of Araba and Dale 1990a.

iv. The protein dispersibility in water of raw and heat treated legume seed powder

was determined by the PDI according to the procedure of American Oil

Chemists Society 1980b.

3.4 Statistical analysis

Correlation coefficients (r) to determine correlations between particular parameters

were calculated using SPSS version 21 and MS Excel Software for plotting graphs.

CHAPTER IV

RESULTS AND DISCUSSION

To continue optimal nutritional value, the legumes must not be subjected to excessive heat,

as this will denature the protein, making it less soluble and less digestible. Excessive heat

or heating time reduces the availability of amino acids due to the Maillard reaction (Del

Valle, 1981) and tends to destroy certain amino acids (Shede & Krogdahl, 1985). Protein

quality of legume depends on two parameters, the decrease of antinutritional factors and

the optimization of protein digestibility (Parsons, Hashimoto, Wedekind, & Baker, 1991).

Direct analysis of both specifications is difficult in routine operations, therefore, replaced

with indirect tests such as UAI, PDI and PSKOH (Lee & Garlich, 1992). Additional heat

treatment decreased PSKOH, and the UAI rapidly approached zero (Del Valle, 1981).

Legumes contain urease, an enzyme that hydrolyzes urea to produce carbon dioxide and

ammonia. The production of ammonia causes the pH of a legume solution to increase. The

destruction of urease by heating is highly correlated with the destruction of trypsin

inhibitors and other antinutritional factors (Araba & Dale, 1990a). The primary purpose of

the urease assay is to determine if legume has been sufficiently heated to destroy most of

the ANF. Table 4, shows the variation of UAI when various legume samples were placed

in an oven at 120°C for up to 30 minutes (5, 10, 15, 20, 25 and 30 minutes); analysis was

accomplished every 5 minutes. UAI of black eye bean decreased from 0.3 to 0.1 after 5

minutes to till 30 minutes of heating. For chickpea, it decreased from 0.2 to 0.1 till 25

minutes, then zero after 30 minutes of heating. Similarly for pea, UAI decreased from 0.2

to 0.1 till 20 minutes of heating then zero after 25 minutes of heating, the UAI activity of

soybeans decreased from 0.3 to 0.2 till 15 minutes and 0.1 to zero after 20 minutes of

heating. Lastly, for kidney bean, UAI decreased rapidly dropped to 0.1 at 10 minutes, so

that zero value was reached in 15 minutes of heating. Additional heating could not have

any effect on UAI, showing that this test is unuseful in detecting over processing (Araba

& Dale, 1990b); (Del Valle, 1981); (Garlich, 1988). These findings were agreed with (Mc

Naughton & Reece, 1980) who reported that urease activity values were decreased quickly

by autoclaving.

Table 5, Effect of heat treatment at 120°C on urease index (pH) in various legumes

Samples

Heating time (minutes)

Black eye bean 0.3

0.1

Chickpea

0.2

0.1

Pea

0.2

0.1

Soybeans

0.3

0.2

0.1

Kidney beans

0.1

Note;

: Under processing (>0.20),

: Adequately processed (0.05-0.20) and

: Over

processing (<0.05) of the legumes however zero does not necessarily indicate over

processing of legumes. Data were compared with UAI values for determining the degree

of legumes processing (Garlich, 1988).

The protein solubility of legumes decreases significantly following increasing about the

heating time from 5 to 30 minutes. Protein solubility was reduced from 107.88 to 55.48%,

80.49 to 60.82%, 85.21 to 51.44%, 98.19 to 70.71% and 75.87 to 60.87% for black eye

bean, chickpea, pea, soybean and kidney bean respectively as shown in table 5. This

marked decrease of protein solubility in an alkaline solution supports the finding of (Araba

& Dale, 1990a), who reported a decrease in solubility of legume protein in aqueous KOH

after autoclaving. The legumes with protein solubility below 70% probably would impair

the nutritive value as reflected by decrease in protein digestibility. Araba and Dale 1990

reported that protein solubility value of less than 65%, almost certainly indicated over

processing of legumes, perhaps autoclaving denatures the protein of legumes and reduced

their solubility in KOH.

Table 6, Effect of heat treatment at 120°C on protein solubility index (PSKOH) in various

legumes

Samples

Heating time (minutes)

Black eye bean 107.88

98.63

87.84

82.79

60.62

55.48

Chickpea

80.49

80.03

76.37

64.02

60.82

Pea

85.21

84.42

68.72

54.97

51.44

Soybeans

98.19

87.60

82.73

75.29

74.14

70.71

Kidney beans

75.87

68.37

67.79

64.90

63.17

60.87

Note;

: Under processing (>85),

: Adequately processed (71-85) and

: Over processing (<70) of the

legumes. Data were compared with PSKOH values for determining the degree of legumes processing

(Monary, 1989).

The PDI in water is inversely related to the degree of heat treatment and displayed large

incremental decreases as heating times increased from 5 to 30 min. The PDI of black eye

bean, chick pea, pea, soybean and kidney bean were decreased from 52.4 to 28.51%, 40.1

to 20.8%, 25.7 to 16.1%, 51.47 to 26.67% and 39.66 to 30.10% respectively as shown in

table 6. It was observed that PDI decrease gradually as the heating time increased during

autoclaved at 120°C (Del Valle, 1981).

Table 7, Effect of heat treatment at 120°C on protein dispersibility index (PDI) in various

legumes

Samples

Heating time (minutes)

Black eye bean 52.4

49.7

47.6

37.95

30.02

28.51

Chickpea

40.1

38.8

37.1

34.1

30.9

20.8

Pea

25.7

23.7

23.5

21.8

20.5

16.1

Soybeans

51.47

38.13

36.80

36.27

34.67

26.67

Kidney beans

39.66

37.97

35.16

33.47

32.91

30.10

Note;

: Under processing (>40),

: Adequately processed (15-40) and

: Over processing (<15) of the

legumes. Data were compared with PDI values for determining the degree of legumes processing (NOPA,

2007).

The UAI is useful to determine if the legume has been heated enough to reduce the ANF,

but it is not very useful for determining if legume has been over processed, urease pH

change slowly and remained constant during the shorter legumes heating times and then

usually decreased precipitously to levels (Caprita, Caprita, Ilia, Cretescu, & Simulescu,

2010). PSKOH remains high, during initial heat treatment in most of legumes used in this

experiment except for the chickpea and kidney bean sample used (80.49 to 60.82% and

75.87 to 60.87% respectively) (Caprita & Caprita, 2007). PSKOH also often did not

change consistently as legumes were heated. PSKOH decreased very little up to 10 minutes

of heating in all legumes samples used in this experiment, therefore, it is not a sensitive

index for monitoring under the processing of legumes and appears to be a better indicator

for over processing (Caprita, Caprita, Ilia, Cretescu, & Simulescu, 2010). Same kind of

findings was observed in this research as well. In marked contrast the PDI decreased

incrementally for the heating times (Caprita & Caprita, 2007). The overall experimental

data suggest that PDI is more sensitive and demonstrated more consistent responses to

heating of legumes than did UAI or PSKOH which is in agreement with earlier studies

(Anderson-Haferman, Zhang, & Parsons, 1992); (Araba & Dale, 1990b); (Mc Naughton &

Reece, 1980). PDI was reduced by approximately half when raw legumes were heated 20

minutes (Caprita, Caprita, Ilia, Cretescu, & Simulescu, 2010). Decreases in PDI were again

quite consistent and large. These results also suggest that PDI is a better indicator of

minimum adequate heating of legumes than is UAI or PSKOH (Araba & Dale, 1990b);

(Mc Naughton & Reece, 1980).

Combing UAI and PSKOH or UAI and PDI could be useful to monitor legumes quality

(Caprita, Caprita, Ilia, Cretescu, & Simulescu, 2010). Results collected in this study

indicated that UAI and PSKOH observed a positive correlation (r= .510, p= .002), UAI and

PDI observed a positive correlation (r=.275) as well as PSI and PDI, observed a very strong

positive correlation (r= .703, p<0.01) as shown in table 7. Similar kind of positive

correlations were been observed PDI and UAI values in study carried out by (Caprita,

Caprita, & Cretescu, 2010).

Table 8, Correlation between UAI, PSI and PDI in various legume samples

PSI

PDI

UAI

Pearson Correlation

.510

.275

Sig. (1-tailed)

.002

.071

**. Correlation is significant at the 0.01 level (1-tailed), total number of sample (N): 30

By the help of a group of tests, done in vitro condition, adequate processing at 120°C for

five varieties of legumes has been set. According to UAI, the adequate time for black eye

bean (10-30 minutes), chickpea (5-25 minutes), pea (5-20 minutes), soybean (15-20

minutes) and kidney bean (5-10 minutes) at 120°C was set, as shown in figure 1. Similarly,

according to PSKOH, the adequate time for black eye bean (20 minutes), chickpea (5-15

minutes), and pea (10 minutes), soybean (15-25 minutes) and kidney bean (5 minutes) was

set, as shown in figure 2. Lastly the adequate time for black eye bean (20-30 minutes),

chick pea (10-20 minutes), pea (5-30 minutes), soybean (10-30 minutes) and kidney bean

(5-30 minutes) was set for PDI as shown in figure 3.

Figure 1. Effect of heat treatment at 120°C on urease index (pH) in various legumes

0.1

0.2

0.3

0.4

0.5

0.6

UAI (pH)

Heating time (minutes) at 120°C

Black eye bean

Chick pea

Pea

Soybeans

kidney beans

Figure 2. Effect of heat treatment at 120°C on protein solubility index (PSKOH) in various

legumes

Figure 3. Effect of heat treatment at 120°C on protein dispersibility index (PDI) in various

legumes

Perhaps combining the PSKOH and PDI tests with the UAI would be useful for better

monitoring of legumes quality. For example, a legume containing low urease (0.05 to 0.2),

PSKOH (71-85%) and PDI (15-40%) may indicate that the sample is definitely high quality

because it has been adequately heat processed to destroy ANF, but not over processed

(Batal, Douglas, Engram, & Parsons, 2000). Therefore adequate time at 120°C for black

50.00

55.00

60.00

65.00

70.00

75.00

80.00

85.00

90.00

95.00

100.00

105.00

110.00

PSKOH

)

Heating time (minutes) at 120°C

Black eye bean

Chick pea

Pea

Soybeans

kidney beans

10.00

20.00

30.00

40.00

50.00

PDI (%

)

Heating time (minutes) at 120°C

Black eye bean

Chick pea

Pea

Soybeans

kidney beans

eye bean (20 minutes), chick pea is (10-15 minutes), pea (10 minutes), soybean (15-20

minutes) and kidney bean (5 minutes) as shown in table 8.

Table 9, Adequate processing time (minutes) of various legume samples at 120°C

Sample

UAI

PSKOH PDI

Black eye bean 10-30 20

20-30

Chick pea

5-25

5-15

10-20

Pea

5-20

5-30

Soybeans

15-20 15-25

10-30

Kidney beans

5-10

15-30

CHAPTER V

CONCLUSION AND RECOMMENDATION

5.1 CONCLUSION

Protein quality of legume is linked to both the reduction of antinutritional factors, and the

optimization of protein digestibility, however, excessive heating would damage the amino

acid content of legumes therefore, adequate time is necessary. But direct analysis of both

specifications is difficult in routine operations, therefore, replaced with indirect tests such

as UAI, PDI and PSKOH. The UAI is useful to determine if the legumes have been heated

enough to reduce the antinutritional factors, while PSKOH is a good index for determining

over processing of legumes. The PDI is the best method of evaluating these legume

ingredients for both under heating and overheating. So, combining the PDI test with the

UAI and PSI could be useful for better monitoring of legumes quality.

In this research also adequate time at 120°C for black eye bean (20 minutes), chickpea is

(10-15 minutes), pea (10 minutes), soybean (15-20 minutes) and kidney bean (5 minutes)

are determined combing UAI, PSI and PDI.

5.2 RECOMMENDATION

In this research, five various legume samples commonly used in household are placed in

an oven at 120°C for up to 30 minutes; analysis was accomplished every 5 minutes. Same

study can be done in almost all legumes as they synthesis the antinutritional factors, as well

as each of the antinutritional factors can be quantified.

5.3 ACKNOWLEDGEMENTS

This work was awarded by University Grants Commission (UGC), Sano thimi Bhakatpur,

Nepal, Mini Research Grant for the fiscal year 2071/2072.

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viii

APPENDICES: I

1. Chemicals

Chemicals

Company

Bovine serum albumin

Himedia

Coomassie brilliant blue G 250

Loba chemie

Dibasic potassium phosphate

Qualigens

Ethanol (Absolute)

Himedia

Hydrochloric acid (35%)

Emplura

Monobasic potassium phosphate

Merck

Phosphoric acid (85%)

Qualigens

Potassium hydroxide

Emplura

Urea

Himedia

2. Glass wares

Glass wares

Company

Beakers (100 ml, 250 ml, 1000 ml)

Borosil

Conical flasks (100 ml, 250 ml, 1000 ml)

Borosil

Glass pipettes (2 ml, 5 ml and 10 ml)

Borosil

Test tubes (10 ml, 25 ml)

Borosil

Volumetric flasks (25 ml, 50 ml, 100 ml, 250 ml, 500 ml, 1000 ml) Borosil

3. Equipments

Equipments

Company

Air forced oven

Amco Memmert

Centrifuge machine Eppendrof Centrifuge 5417R

Grinder

Greenline

Micro pipettes

Status

pH meter

Philip Harris

Spectrophotometer Elico SL150 UV-VIS Spectrophotometer

Weighing balance

Explorer Dhasu

APPENDICES: II

1. Preparation of reagents

1.1 Phosphate buffer solution (0.05 M phosphate buffer, pH 7.0):

In approximately 100 ml of distilled water, 1.27 g of monobasic potassium

phosphate (Merck) was dissolved. Similarly, in approximately 100 ml of

distilled water, 7.07 g of dibasic potassium phosphate (Merck) was dissolved.

The two solutions were combined and diluted to 1000 ml. Solutions were mixed

and pH was maintained 7.0.

1.2 Buffered urea solution:

In 500 ml of 0.05M phosphate buffer solution (pH 7.0), 15 g of urea (Merck)

were dissolved.

1.3 Bardford reagent:

In 100 ml of 85% phosphoric acid, 50 ml of ethanol and 100 mg of Coomassie

brilliant blue G 250 was dissolved. And final volume was maintained 1 L with

distilled water.

1.4 KOH solution (0.2%):

In about 20 ml of distilled water, 0.2 g of KOH was dissolved and final volume

was maintained 100 ml with further addition of distilled water.

2. Principle of test

2.1 Bardford test:

This is the assay of choice in most cases due to its simplicity, scalability and

sensitivity. This method is based on the binding of Coomassie brilliant blue G

250 to protein. At low pH, free dye has a maximum absorbance at 470 nm and

650 nm but when bound to protein, it has absorbance maximum at 505 nm. The

method depends on quantitating the binding dye to an unknown protein and

comparing this binding to that of different amounts of standard proteins usually

BSA. Both hydrophobic and ionic interactions stabilize the anionic form of the

dye, causing a visible colour change. It is designed to quantify 1-10 g protein,

but can be carried out in the range of 10-100 g by increasing the volume of the

dye solution 5-fold and using larger tubes (Bradford, 1976).

2.2 Urease assay index:

The urease assay is based on the pH increase from ammonia released from urea

by a residual urease enzyme in legumes. In the presence of significant urease

activity the pH of the legume solution increases due to the release of ammonia

from urea. (American Oil Chemists Society, 1980a)

2.3 Protein solubility index:

The KOH protein solubility test is based on the solubility of soybean proteins

in a dilute solution of potassium hydroxide (Araba and Dale 1990a).

2.4 Protein dispersibility index:

The protein dispersibility test is based on the solubility of legume proteins in

distilled water. This method uses ten minutes of high speed mixing in distilled

water at 8,500 rpm (Batal, Douglas, Engram, & Parsons, 2000).

3. Protocols

3.1 Bardford test:

i. Standard BSA solutions (20-100 µg/ml), diluted original samples or the

diluted supernatant (0.5 ml) was combined with 2.5 ml of Bardford reagent.

ii. The solutions were mixed properly and the absorbance of the solution was

measured at 595 nm against the blank. All samples were analyzed in

duplicate.

3.2 Urease assay index:

i. Buffered urea solution (pH=7. 5) 10 ml was added to 200 mg finely ground

legumes (test sample), and 10 ml phosphate buffered solution was added to

200 mg of the same sample (blank sample).

ii. The two solutions were incubated at 30°C for 30 minutes under stirring.

iii. After incubation, the pH of the solutions was determined rapidly. All

samples were analyzed in duplicate.

iv. And the difference between a pH of the test and pH of the blank was

calculated as an index of urease activity.

3.3 Protein solubility index:

i. The procedure involves incubation of the 20 mg sample with 1ml 0.2%

KOH (wt/Vol; 0.036 N) solution for 20 min at room temperature.

ii. Following this incubation, the sample is centrifuged for 5 minutes at 6,000

rpm and the supernatant is analyzed for the protein concentration by the

Bradford method. All samples were analyzed in duplicate.

iii. The solubility of the protein, expressed as a percentage, was calculated by

dividing the protein content of the KOH-extracted solution by the protein

content of the original sample.

3.4 Protein dispersibility index:

i. The procedure involves high speed mixing of the 20 mg sample with 1ml

of distilled water at 8,500 rpm.

ii. Following this mixing, the sample is centrifuged for 5 minutes at 6,000 rpm

and the supernatant is analyzed for the protein concentration by the

Bradford method. All samples were analyzed in duplicate.

iii. The dispersibility of the protein, expressed as a percentage, was calculated

by dividing the protein content of the water-extracted solution by the protein

content of the original sample.

Frequently asked questions

What is the 'AKNOWLEDGEMENTS' section about?

This section expresses gratitude to the University Grants Commission (UGC) for funding the research project during fiscal year 071/072. It also thanks Mr. Surya Prakash Poudel for identifying legume botanical names, Prof. Dr. Anjana Singh for guidance and lab space, friends Manita Aryal and Supriya Sharma, husband Deepak Kr. Shrestha for support, and family members Mr. Kedar Man Baidya, Mrs. Sharada M. Shrestha, Dr. Pooja Baidya, Poonam Baidya, Dr. Malika Baidya and Kshitiz Man Pradhan for their continuous motivation and support.

What is the study about?

The study focuses on determining the optimal heat treatment time for various legumes (black eye bean, chickpea, pea, soybean, and kidney bean) by analyzing chemical indices like urease activity index (UAI), protein solubility in potassium hydroxide (PSKOH), and protein dispersibility index (PDI).

What are the keywords associated with this study?

The keywords are: Antinutritional factors, protein dispersibility index, urease activity index, protein solubility, legumes, overheating, adequate heating, under heating.

What chapters are included in the 'LIST OF CONTENTS' section?

The list includes Acknowledgements, Abstract, List of Contents, List of Tables, List of Figures, Abbreviations, Chapter I (Introduction and Objectives), Chapter II (Literature Review), Chapter III (Materials and Methods), Chapter IV (Results and Discussion), and Chapter V (Conclusion and Recommendation), and Appendices

What is Chapter I, "Introduction and Objectives," about?

Chapter I, "Introduction and Objectives," provides an overview of legumes, their importance in human nutrition, and the challenges associated with antinutritional factors (ANF). It also presents the objectives of the study related to chemical indices, protein quality, antinutritional factors, and correlation with indices.

What are some physical and chemical methods are employed to reduce or remove ANF from legumes.

The methods are soaking, cooking, germination, fermentation, selective extraction, irradiation and enzyme treatment.

What are the objectives mentioned in Chapter I section 1.2?

The objectives are: analysis of chemical indices to determine legumes protein quality, determination of antinutritional factors in legumes by urease index, determination of legumes solubility by KOH protein solubility index and protein dispersibility index, determination of legumes solubility by protein dispersibility index, and correlation with indices

What is Chapter II, "LITERTURE REVIEW" about?

Chapter II, "LITERTURE REVIEW" discusses the role of legumes in human nutrition, their protein content, essential amino acids, and the presence of antinutritional factors (ANF). It also explores methods to inactivate or remove ANF and the importance of optimizing heat treatment during legume processing.

What did the LITERTURE REVIEW say about PROTEIN

Proteins are available in varieties of dietary sources, including animals, plant origin, and from highly marketed spot supplement industry. Typically, all dietary animal proteins (e.g. Eggs, milk, meat, fish and poultry) are considered a complete protein because they contain all essential amino acids.

What were some of the types of legumes mentioned in the preview?

Black eye beans, chickpea, soy beans, and kidney beans were mentioned. A table also shows a comparison between Black eye beans, Chick peas, Peas, Soybeans, and Kidney beans, and thier protein, mineral, and vitamin contents.

What does Table 2 represent?

Table 2, lists Urease activity index values for determining the degree of legumes processing. It also shows the conditions as Under processing, Adequately processed, and Over processing, and thier assosiated ranges.

What does Table 3 represent?

Table 3, shows PSKOH values for determining the degree of legumes processing. It also shows the conditions as Under processing, Adequately processed, and Over processing, and thier assosiated ranges.

What does Table 4 represent?

Table 4, shows PDI values for determining the degree of legumes processing. It also shows the conditions as Under processing, Adequately processed, and Over processing, and thier assosiated ranges.

What is Chapter III, "MATERIALS AND METHODS" about?

Chapter III, "MATERIALS AND METHODS" discusses the location of the study at the Central Department of Microbiology (CDM), T.U; Kirtipur, data collection methods, study variables including the various leguminous seeds, and methodology including sample preparation, chemical analysis, and statistical analysis. It lists the chemicals and instruments to be used in the experiment.

What is Chapter IV, "RESULTS AND DISCUSSION" about?

Chapter IV, "RESULTS AND DISCUSSION" the results of the experiments carried out, the role of heat to ensure maximum protein retainment, and provides a discussion of the results. The relationship between UAI, PSI, PSKOH, Urease, and overall food quality in relation to heating legumes was explored, as was the UAI for different types of legumes under various heat treatment methods.

What does Table 5 represent?

Table 5, represents Effect of heat treatment at 120°C on urease index (pH) in various legumes.

What does Table 6 represent?

Table 6, represents Effect of heat treatment at 120°C on protein solubility index (PSKOH) in various legumes.

What does Table 7 represent?

Table 7, represents Effect of heat treatment at 120°C on protein dispersibility index (PDI) in various legumes.

What does Table 8 represent?

Table 8, represents Correlation between UAI, PSI and PDI in various legume samples.

What does Table 9 represent?

Table 9, represents Adequate processing time (minutes) of various legume samples at 120°C. It shows a comparison between UAI, PSI, and PDI.

What does Chapter V, "CONCLUSION AND RECOMMENDATION" contain?

Chapter V, "CONCLUSION AND RECOMMENDATION," summarizes the study's findings, highlighting the importance of balancing ANF reduction with protein digestibility optimization. It recommends further research on various legumes and quantification of specific ANFs. It also acknolwedges this work was awarded by University Grants Commission (UGC), Sano thimi Bhakatpur, Nepal.

Final del extracto de 48 páginas - subir

Detalles

Título: Assessment on Quality of Legumes Protein
Universidad: Tribhuvan University (Central Department of Microbiology)
Autor: Purnima Baidya (Autor)
Año de publicación: 2016
Páginas: 48
No. de catálogo: V337614
ISBN (Ebook): 9783668270824
ISBN (Libro): 9783668270831
Idioma: Inglés
Etiqueta: Antinutritional factors protein dispersibility index urease activity index protein solubility legumes
Seguridad del producto: GRIN Publishing Ltd.

Citar trabajo: Purnima Baidya (Autor), 2016, Assessment on Quality of Legumes Protein, Múnich, GRIN Verlag, https://www.grin.com/document/337614

Assessment on Quality of Legumes Protein

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Frequently asked questions

What is the 'AKNOWLEDGEMENTS' section about?

What is the study about?

What are the keywords associated with this study?

What chapters are included in the 'LIST OF CONTENTS' section?

What is Chapter I, "Introduction and Objectives," about?

What are some physical and chemical methods are employed to reduce or remove ANF from legumes.

What are the objectives mentioned in Chapter I section 1.2?

What is Chapter II, "LITERTURE REVIEW" about?

What did the LITERTURE REVIEW say about PROTEIN

What were some of the types of legumes mentioned in the preview?

What does Table 2 represent?

What does Table 3 represent?

What does Table 4 represent?

What is Chapter III, "MATERIALS AND METHODS" about?

What is Chapter IV, "RESULTS AND DISCUSSION" about?

What does Table 5 represent?

What does Table 6 represent?

What does Table 7 represent?

What does Table 8 represent?

What does Table 9 represent?

What does Chapter V, "CONCLUSION AND RECOMMENDATION" contain?

Detalles