Leseprobe
ii
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
iv
LIST OF CONTENTS
Contents
Page no.
Acknowledgements
ii
Abstract
iii
List of contents
iv
List of tables
v
List of figures
vi
Abbreviations
vii
Chapter I
Introduction and Objectives
1-4
1.1 Introduction
1
1.2 Objectives
4
Chapter II
Literature review
5-15
Chapter III
Materials and methods
16-17
3.1 Materials
16
3.1.1 Chemicals and instruments
16
3.2 Methods
16
3.2.1
Location of the study
3.2.2
Methods of data collection
3.2.3
Study variables
16
16
16
3.3 Methodology
16
3.2.1
Samples
3.2.2
Sample processing
16
16
3.4 Statistical analysis
17
Chapter IV
Results and discussion
18-24
Chapter V
Conclusion and recommendation 25
5.1 Conclusion
25
5.2 Recommendation
25
5.3 Acknowledgement
25
References
26-37
Appendices
viii-xi
v
LIST OF TABLES
Page no.
Table 1 Protein and other nutrients contain in some common legumes
6
Table 2 Urease activity index values for determining the degree of legumes
processing
13
Table 3 PSKOH values for determining the degree of legumes processing
14
Table 4 PDI values for determining the degree of legumes processing
14
Table 5 Effect of heat treatment at 120°C on urease index (pH) in various
legumes
19
Table 6 Effect of heat treatment at 120°C on protein solubility index
(PSKOH) in various legumes
20
Table 7 Effect of heat treatment at 120°C on protein dispersibility index
(PDI) in various legumes
20
Table 8 Correlation between UAI, PSI and PDI in various legume samples
21
Table 9 Adequate processing time (minutes) of various legume samples at
120°C
24
vi
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
23
Figure 2
Effect of heat treatment at 120°C on protein dispersibility index (PDI) in
various legumes
23
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
UI
Urease Index
1
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
2
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.
3
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).
4
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
5
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)
6
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
1
: 0.202 mg
Vitamin B
2
: 0.055 mg
Niacin: 0.495 mg
Pantothenic acid: 0.411 mg
Vitamin B
6
: 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
1
: 0.116 mg
Vitamin B
2
: 0.063 mg
Niacin: 0.526 mg
Pantothenic acid: 0.286 mg
Vitamin B
6
: 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
1
: 0.146 mg
Vitamin B
2
: 0.059 mg
Niacin: 0.781 mg
Pantothenic acid: 0.319 mg
Vitamin B
6
: 0.05 mg
7
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
1
: 0.1 mg
Vitamin B
2
: 0.145 mg
Niacin: 1.41 mg
Pantothenic acid: 0.453 mg
Vitamin B
6
: 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
1
: 0.216 mg
Vitamin B
2
: 0.058 mg
Niacin: 0.578 mg
Pantothenic acid: 0.22 mg
Vitamin B
6
: 0.12 mg
Folate: 130 mcg
Vitamin E: 0.03 mg
Vitamin K: 8.4 mcg
Contains some other vitamins in
small amounts.
8
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
9
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 &
10
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
11
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
12
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
13
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,
14
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
15
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.
16
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).
17
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.
18
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
19
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)
5
10
15
20
25
30
Black eye bean 0.3
a
0.1
b
0.1
b
0.1
b
0.1
b
0.1
b
Chickpea
0.2
b
0.1
b
0.1
b
0.1
b
0.1
b
0
c
Pea
0.2
b
0.1
b
0.1
b
0.1
b
0
c
0
c
Soybeans
0.3
a
0.3
a
0.2
b
0.1
b
0
c
0
c
Kidney beans
0.1
b
0.1
b
0
c
0
c
0
c
0
c
Note;
a
: Under processing (>0.20),
b
: Adequately processed (0.05-0.20) and
c
: 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.
20
Table 6, Effect of heat treatment at 120°C on protein solubility index (PSKOH) in various
legumes
Samples
Heating time (minutes)
5
10
15
20
25
30
Black eye bean 107.88
a
98.63
a
87.84
a
82.79
b
60.62
c
55.48
c
Chickpea
80.49
b
80.03
b
76.37
b
64.02
c
60.82
c
60.82
c
Pea
85.21
a
84.42
b
68.72
c
68.72
c
54.97
c
51.44
c
Soybeans
98.19
a
87.60
a
82.73
b
75.29
b
74.14
b
70.71
c
Kidney beans
75.87
b
68.37
c
67.79
c
64.90
c
63.17
c
60.87
c
Note;
a
: Under processing (>85),
b
: Adequately processed (71-85) and
c
: 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)
5
10
15
20
25
30
Black eye bean 52.4
a
49.7
a
47.6
a
37.95
b
30.02
b
28.51
b
Chickpea
40.1
a
38.8
b
37.1
b
34.1
b
30.9
b
20.8
b
Pea
25.7
b
23.7
b
23.5
b
21.8
b
20.5
b
16.1
b
Soybeans
51.47
a
38.13
b
36.80
b
36.27
b
34.67
b
26.67
b
Kidney beans
39.66
b
37.97
b
35.16
b
33.47
b
32.91
b
30.10
b
Note;
a
: Under processing (>40),
b
: Adequately processed (15-40) and
c
: 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
21
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
22
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
0.1
0.2
0.3
0.4
0.5
0.6
5
10
15
20
25
30
UAI (pH)
Heating time (minutes) at 120°C
Black eye bean
Chick pea
Pea
Soybeans
kidney beans
23
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
5
10
15
20
25
30
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
5
10
15
20
25
30
PDI (%
)
Heating time (minutes) at 120°C
Black eye bean
Chick pea
Pea
Soybeans
kidney beans
24
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
10
5-30
Soybeans
15-20 15-25
10-30
Kidney beans
5-10
5
15-30
25
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.
26
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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
ix
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).
x
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.
xi
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.
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