STUDIES ON THE PREPARATION AND CHARACTERISATION OF PROTEIN HYDROLYSATES FROM GROUNDNUT
AND SOYBEAN ISOLATES
A THESIS SUBMITTED TO THE UNIVERSITY OF MYSORE FOR
THE DEGREE OF DOCTOR OF PHILOSOPHY IN FOOD SCIENCE
BY
GOVINDARAJU.K
DEPARTMENT OF PROTEIN CHEMISTRY AND TECHNOLOGY CENTRAL FOOD TECHNOLOGICAL RESEARCH INSTITUTE
MYSORE – 570013, INDIA
SEPTEMBER 2003
DECLARATION
I here by declare that the thesis entitled “ Studies on the preparation and
characterization of protein hydrolysates from groundnut and soybean
isolates” which is submitted here with for the degree of Doctor of Philosophy in
Food Science of the University of Mysore is the result of the work done by me at
the Central Food Technological Research Institute, Mysore, INDIA in the
Department of Protein Chemistry and Technology under the guidance of
Dr.H.Srinivas during the period 1997 to 2003.
I further declare that the results of the work have not been previously
submitted to the degree of fellowship.
Date :
Place: MYSORE Govindaraju.K
Dr.H. SRINIVAS Retd. Scientist, Department of Date: Protein Chemistry and Technology
CERTIFICATE
I here by certify that this Ph.D thesis entitled “ Studies on the
preparation and characterization of protein hydrolysates from groundnut
and soybean isolates ” submitted by Govindaraju.K for the degree of
Doctor of Philosophy in Food Science of the University of Mysore, is the
result of the research work carried out by him in the department of Protein
Chemistry and Technology, Central Food Technological Research
Institute, Mysore, India under my guidance and supervision during the
period 1997 to 2003. This has not been submitted either partially or fully to
any degree or fellowship earlier.
H.SRINIVAS
ACKNOWLEDGEMENTS
I am extremely grateful to my guides and supervisor Dr.H.Srinivas,
Retd. Scientist, CFTRI, Mysore, India, for suggesting the research problem for
Ph.D. degree and also for his constant support and guidance.
I am grateful to Dr.V.Prakash, Director, CFTRI for providing the
necessary facilities to carry out this investigation and permitting me to submit
in the form of thesis.
I am extremely thankful to Dr.A.G.Appu Rao, Head, Protein Chemistry
and Technological Department for his valuable suggestions during the course of
this work.
My special thanks to Mr. K.A. Ranganath, for editing the thesis.
I extend my sincere thanks to all the erstwhile Heads, Staff and fellow
colleagues of the Department of Protein Chemistry and Technology, CFTRI,
Mysore, both present and past for their encouragement. I am also thankful to the
staff of central instrumentation facilities, Library and administration for their
help during the investigation.
Finally I am deeply indebted to my parents and wife who were a constant
source of support and encouragement in this endeavor.
Date :
Place: Mysore Govindaraju. K
CONTENTS ABBREVATIONS II LIST OF FIGURES IV LIST OF TABLES VII INTRODUCTION 1 SCOPE AND OBJECTIVES 43 MATERIALS AND METHODS 46 RESULTS AND DISCUSSION PART A STUDIES ON SOYBEAN PROTEINS 1 ENZYMATIC HYDROLYSIS OF SOY FLOUR 62 2 ENZYMATIC MODIFICATION OF SOY PROTEIN ISOLATE 77 3 CONTROLLED ENZYMATIC HYDROLYSIS OF GLYCININ 87 PART B STUDIES ON GROUNDNUT PROTEINS 1 ENZYMATIC MODIFICATION OF GROUNDNUT PROTEIN ISOLATE 114
2 ENZYMATIC HYDROLYSIS OF ARACHIN AND ITS EFFECT ON 124
FUNCTIONAL PROPERTIES SUMMARY AND CONCLUSIONS 140 REFERENCES 147
ABBREVIATIONS
A Absorbance Aº Angstrom BAPNA N-Benzyl-L-arginine-paranitroanilide
CAM-Glycinin Carboxymethylated glycinin
CD Circular dichroism .
DH Degree of hydrolysis.
DSF Defatted soy flour
°C Degree centigrade
EC Emulsification capacity
EDTA Ethylene diamine tetra acetic acid
E/S Enzyme – substrate ratio
FAC Fat absorption capacity
FC Foaming capacity
FS Foam stability
GPI Groundnut protein isolate
GPI-FD Freeze-dried groundnut protein isolate
GPI-SD Spray dried groundnut protein isolate
HPLC High pressure liquid chromatography
Km Michaelis-Menten constant.
kD Kilo Dalton.
M molarity
meq Milli equivalents
mg milligram
min Minutes
MRW Mean residue weight
N Normality
nm nanometer
NSI Nitrogen solubility index
PAGE Poly acrylamide gel electrophoresis
PER Protein efficiency ratio rpm Revolutions per minute
SDS Sodium dodecyl sulphate
SPI Soy protein isolate
SPI-FD Freeze dried soy protein isolate
SPI-SD Spray dried soy protein isolate
TCA Trichlororic acetic acid
T Temperature
TIA Trypsin inhibitor activity
TIU Trypsin inhibitor units
TNBS 2, 4, 6- trinitro benzene sulphonic acid.
UV Ultra violet.
v/v Volume by Volume
Ve/V0 Elution Volume by Void volume
WAC Water absorption capacity. w/v Weight by Volume
LIST OF FIGURES
FIGURE No.
TITLE PAGE No.
1.1
1.2
1.3
2.1
2.2
2.3
3.1
3.2
3.3 (A,B& C)
3.4
4.1
Flowchart for the preparation of soy protein concentrate Primary structure of the acidic (a) and basic (b) polypeptide components of glycinin subunits A2B1a.
Primary structure of Kunitz trypsin inhibitor and Bowman- Birk trypsin inhibitor
Preparation of soy protein isolate from defatted soy flour by isoelectric pH precipitation.
Simultaneous isolation of soybean 7S & 11S globulin Isolation of arachin from groundnut flour
Effect of papain and alcalase on the DH of defatted soy flour Effect of fungal protease on the DH of defatted soy flour Effect of DH on EC and FC of modified DSF prepared using papain and alcalase Effect of DH on EC and FC of modified DSF prepared using fungal protease Comparison of the solubility vs pH profile of defatted soy flour and spray dried protein hydrolysates prepared using different proteolytic enzymes Effect of enzyme concentration on the DH of freeze dried soy protein isolate (SPI-FD)
8
13
17
53
54
56
66
67
69
70
76
79
FIGURE
No.
TITLE
PAGE No.
4.2
4.3
5.1
5.2
5.3
5.4
5.5
5.6
5.7
5.8
Effect of enzyme concentration on the DH of spray dried soy protein isolate (SPI-SD) Effect of degree of hydrolysis on solubility of soy protein hydrolysate generated with the three enzymes Effect of different proteolytic enzymes on the DH of glycinin Effect of temperature on the DH of soy glycinin by papain Effect of concentration of enzymes on the DH of soy glycinin Michaelis- Menten plot (A) and Lineweaver-Burk plot (B) for hydrolysis of glycinin with different proteolytic enzymes Separation of acidic and basic subunits of purified and carboxymethylated glycinin on a Dowex strongly basic anion exchange resin A. SDS- slab gel electrophoretic pattern of glycinin in comparison with enzyme modified glycinin B. SDS- slab gel electrophoretic pattern of glycinin in comparison with isolated acidic and basic subunits of glycinin Hydrolysis curve for the isolated acidic and basic subunits of glycinin with fungal protease Michaelis- Menten plot and Lineweaver-Burk plot for hydrolysis of acidic and basic subunits of glycinin with fungal protease
80
83
90
91
92
93
95
96
97
98
FIGURE
No.
5.9
5.10
5.11
5.12
5.13
5.14
5.15
6.1
6.2
6.3
7.1
7.2
TITLE
Sepharose-6B gel filtration pattern of purified glycinin Sepharose-6B column chromatography pattern of glycinin after limited proteolysis with the three enzymes SDS-slab gel electrophoretic pattern of purified glycinin and peak positions obtained after gel filtration of enzyme modified glycinin Effect of limited proteolysis on near UV CD spectrum of glycinin Near UV CD spectrum of glycinin, isolated acidic and basic subunits of glycinin Far UV CD spectrum of glycinin, isolated acidic and basic subunits of glycinin Hydropathy plot for acidic and basic subunits of glycinin Effect of enzyme concentration on the DH of freeze dried groundnut protein isolate (GPI – FD) Effect of enzyme concentration on the DH of spray dried groundnut protein isolate Effect of DH on the solubility of modified GPI generated with papain, alcalase and fungal protease Sepharose-6B gel filtration pattern of arachin purified by ammonium sulphate precipitation Sepharose-6B column chromatography pattern of arachin after limited proteolysis with different enzymes
PAGE No.
101
102
104
107
108
109
110
117
118
121
125
127
FIGURE
No.
7.3
7.4
7.5
7.6
7.7
TITLE
Michaelis-Menten plot (A) and Lineweaver-Burk plot (B) for hydrolysis of arachin with the three proteolytic enzymes Effect of proteolytic enzymes on the DH of arachin
Effect of enzyme concentrations on the DH of arachin Effect of degree of hydrolysis of arachin on its solubility SDS-PAGE pattern of arachin and arachin hydrolysed with papain, alcalase and fungal protease
PAGE No.
128
130
131
133
136
LIST OF TABLES
TABLE No.
1.1 1.2 1.3 1.4 1.5 1.6 1.7 3.1 3.2 3.3 3.4 3.5 3.6 3.7 4.1
TITLE
Essential amino acid composition of soy protein products Comparison of the typical composition of soy flour concentrate and isolated soy protein (on moisture free basis) Comparison of glycinin subunits Physico-chemical properties of soy glycinin Amino acid composition of arachin isolated by various workers Physico-chemical properties of arachin Functional properties of food proteins in various food systems Chemical analysis of soy split beans and defatted soy flour Degree of hydrolysis achieved with different modified soy flours hydrolysed for different periods using proteolytic enzymes Comparison of the water absorption capacity and fat absorption capacity of defatted soy flour and different modified samples. Physico-chemical properties of spray dried soy protein hydrolysate prepared using different proteolytic enzymes Characterization of protein hydrolysates prepared from soy flour before and after acid wash
Comparison of protein hydrolysates prepared with wet protein isolate and soy flour Amino acid composition of soy protein hydrolysates
Comparison of the degree of hydrolysis of different modified soy protein isolates prepared using different proteolytic enzymes
PAGE No.
5
7
12
15
23
24
29 63
71 72 73 74 74
75
82
TABLE No.
4.2
4.3
5.1
5.2
5.3
5.4
5.5
5.6
5.7
6.1
6.2
6.3
TITLE
Effect of limited proteolysis on functional properties of soy protein isolate Effect of extensive hydrolysis on functional properties of soy protein isolate
Kinetic constants for different enzymes with glycinin as substrate Kinetic constants for fungal protease with isolated subunits of glycinin as substrate Effect of limited proteolysis on foaming capacity and stability of glycinin Effect of limited proteolysis on functional properties of glycinin The Ve/V0 and area of different peaks of gel filtration pattern of enzyme modified glycinin samples Maximum DH obtained with defatted soy flour, soy protein isolate and glycinin with the three proteolytic enzymes
Functional properties of defatted soy flour, soy protein isolate and glycinin modified to low DH using proteolytic enzymes
Degree of hydrolysis of modified groundnut protein isolate prepared using different proteolytic enzymes. Effect of limited proteolysis on functional properties of groundnut protein isolate Effect of extensive hydrolysis on functional properties of groundnut protein isolate
PAGE No.
86
86
94
99
100
100
103
111
113
120
123
123
TABLE
No.
7.1
7.2
7.3
7.4
7.5
TITLE
Kinetic constants for hydrolysis of arachin with proteolytic enzymes Effect of limited proteolysis on functional properties of arachin
Effect of extensive hydrolysis on functional properties of arachin
Maximum DH obtained with GPI and arachin with proteolytic enzymes Functional properties of GPI and arachin modified to low DH using proteolytic enzymes
PAGE
No.
129
135
135
139
139
1
SYNOPSIS
STUDIES ON THE PREPARATION AND CHARACTERISATION OF
PROTEIN HYDROLYSATES FROM GROUNDNUT AND SOYBEAN
ISOLATES
Proteins are important in food processing and food product development, as they are
responsible for various functional properties that influence consumer acceptability. Both
animal and plant proteins are used commercially as functional ingredients. Plant proteins are
the most abundant in the world. A number of vegetable proteins have been tried for
incorporation in various food products as functional and nutritional ingredients.
Oilseed protein products are rapidly gaining importance in protein supplementation
because of their unique functional properties. The intrinsic properties of proteins like the amino
acid composition and conformation of the proteins, methods and conditions for their isolation,
degree of purification and processing alterations are some of the important factors that
influence the functional properties of food proteins. Various approaches like chemical
modification, physical treatments and enzymatic modification have been tried to improve
functional characteristics of proteins. Chemical modifications such as succinylation,
acetylation and mild alkali hydrolysis have been reported to improve the functional properties.
One of the major drawbacks of these approaches is the deterioration of the nutritional quality
owing to the blocking or destruction of essential amino acids. One of the important ways to
enhance the functional properties of oilseed proteins is enzymatic modification. Enzymatic
modification occur under mild conditions retaining nutritional value and offer a convenient
means for improving functional properties of proteins. By controlling the extent of hydrolysis
it is possible to enhance various functional properties to develop new functional ingredients to
fabricate new food analogs simulating traditional foods.
2
Protein hydrolysates find application in special foods such as those designed for
children, old people, athletes and also in pharmaceutical preparations developed for
convalescents and those who suffer from digestive disorders. Foods based on highly
hydrolysed proteins are useful in controlling food allergies. The major ways of supplying
tailored amount of amino acids are i) enzymatic protein hydrolysates and ii) a mixture of
synthetic amino acids. They are preferred over synthetic amino acids at moderate cost because
of availability on commercial scale and high quality of enzyme hydrolysed products.
Enzymatic protein hydrolysates containing short chain peptides with defined amino acid
composition and molecular size are preferred for specific formulations. These protein
hydrolysates score over elemental diets in which the protein component consists exclusively of
a mixture of free amino acids. The short chain peptides are absorbed preferentially over free
amino acids in the gut. Protein hydrolysates offer as an alternative to intact proteins and
elemental formula in the development of special formulations designed to provide nutritional
support .
Soybean and groundnut are the most widely cultivated oilseeds all over the world.
Dehulled soybean contains 17-18% oil and 25-35% protein depending on the variety.
Similarly, dehulled groundnut depending on the variety contains 50-60% oil and 30-35%
protein. Defatted soybean flour contains 50-55% protein of good nutritional quality. Soybean
proteins are rich in lysine and deficient in methionine. The major intrinsic anti nutritional
factor is trypsin inhibitor, which affects its utility. Groundnut proteins are deficient in lysine
and methionine. The protein digestibility corrected amino acid score for soy protein and
groundnut protein is 0.92 and 0.52 respectively. The protein ingredients such as defatted flour,
protein concentrate and protein isolates from these sources have a good potential for preparing
speciality foods under native and modified conditions. The protein hydrolysates obtained from
these sources can effectively replace commercially available milk protein hydrolysates because
3
of their good nutritional quality.
In general, oilseed proteins offer resistance to enzymatic hydrolysis. Studies on
hydrolysis of pure proteins would help in understanding of the various structural features of the
protein both at surface and subunit levels. Though the resistance to hydrolysis of seed proteins
by enzymes is well documented, work on the relative effectiveness of the major proteins with
different proteolytic enzymes is less. Glycinin and conglycinin are the major fractions of
soybeans and make up to 70% of the total proteins. Arachin and conarachin are the major
fractions, which make nearly 80% of the total groundnut proteins. The individual roles of
major protein fractions are important in order to understand the overall functional profile of the
total proteins and protein ingredients. Glycinin is poorer in functional properties compared to
conglycinin. This has been attributed to the compact structure of the high molecular protein
fraction in which hydrophobic groups are buried inside. The functional properties of protein
isolates and isolated fractions can be modified either by chemical means or enzymatic
hydrolysis. Although sufficient information is available on the enzymatic modification of
soybean or groundnut flour and its effect on functional properties, the information on the
controlled hydrolysis of major globulins of oilseeds are limited.
In this work, protein hydrolysates from soybean and groundnut were prepared using
different proteolytic enzymes starting with different materials like defatted meal, protein
isolates and purified fractions. With the use of different enzymes and by varying the
experimental parameters like E/S ratio, pH, temperature and combinations of proteolytic
enzymes, it was possible to tailor the functional characteristics of various protein hydrolysates.
Attempt has also been made to correlate various functional characteristics with some of the
biophysical and biochemical parameters.
4
CHAPTER 1: INTRODUCTION
The introduction reviews the information regarding the physico-chemical
characteristics, nutritional, and functional properties of soybean and groundnut proteins. This
also includes the review of literature on enzymatic hydrolysis of food proteins, methods of
isolation and physico-chemical characteristics of the purified high molecular weight protein
fractions of soybean and groundnut (glycinin and arachin).
CHAPTER 2: MATERIALS AND METHODS
This presents details regarding the materials and experimental methods used in the
investigation.
CHAPTER 3: RESULTS AND DISCUSSION
This has the results followed by the relevant discussion, which are given in the
following Sections.
PART – A: STUDIES ON SOYBEAN PROTEINS
SECTION 1: ENZYMATIC HYDROLYSIS OF SOYBEAN FLOUR
The maximum degree of hydrolysis (DH) obtained with papain, alcalase and fungal
protease enzymes were 18.6%, 29.6% and 32.4%, respectively. A comparison of the DH values
obtained with different proteases showed that fungal protease was more effective among the
proteases used for the hydrolysis of defatted soy flour (DSF). The overall effectiveness of
different proteases in getting higher DH was in the order fungal protease > alcalase > papain.
Enzymatic modification of DSF to low DH (4-6%) resulted in remarkable increase in
emulsification capacity (EC) and marginal increase in foaming capacity (FC). The extent of
improvement in EC followed by limited proteolysis was almost same for different proteolytic
enzyme modified flours. The fat absorption capacity (FAC) and water absorption capacity
(WAC) of enzyme hydrolysed DSF was higher than that of intact DSF. Extensive hydrolysis
impaired the overall functionality of DSF. The trypsin inhibitor activities of low DH and high
5
DH enzyme modified freeze dried DSF were similar to control, suggesting that trypsin
inhibitors were resistant to enzymatic attack. The nitrogen content of spray-dried protein
hydrolysate obtained with papain, alcalase and fungal protease was almost same (9-9.2 %). The
nitrogen content of DSF increased to 11.5% after acid wash. The protein hydrolysate prepared
by hydrolysis of wet protein isolate obtained by alkali extraction followed by iso-electric
precipitation had higher nitrogen content (14.5%).Amino acid composition of protein
hydrolysates showed that the nutritional quality of protein was retained after enzymatic
hydrolysis. The bitterness of protein hydrolysates was in the order papain< alcalase=fungal
protease. The spray dried protein hydrolysate of DSF obtained by different proteolytic enzymes
showed inactivation of lipoxygenase and urease activity. The trypsin inhibitor activity of DSF
hydrolysate was in the range 20-22 TIU/mg sample. The lower trypsin inhibitor activity of
spray dried hydrolysates compared to DSF may be due to application of heat in spray drying
process. The protein hydrolysate was soluble over a wide range of pH (2.0-11.0); at iso-electric
pH, the solubility was >98%.
SECTION 2: ENZYMATIC MODIFICATION OF SOY PROTEIN ISOLATE
The DH of soy protein isolate obtained with papain, alcalase and fungal protease was
7.5%, 9.5% and 18.9% respectively. The effectiveness of proteolytic enzymes for hydrolysis of
soy protein isolate (SPI) was lower compared to DSF. The overall effectiveness of proteases
towards hydrolysis of SPI was in the order fungal protease>alcalase>papain. Comparison of
the hydrolysis curves of freeze dried and spray dried SPI showed their susceptibility towards
proteolytic enzymes was almost same. The solubility of SPI followed the typical U-shape
pattern. The minimum solubility was found at pH 4.5 (iso-electric pH). A low DH of 3-5%
obtained with papain, alcalase and fungal protease increased the solubility of modified SPI up
to 29-35% at pH 4.5 and 97-98% at pH 7.0. Extensive hydrolysis of SPI increased the
solubility at pH 4.5 up to 49-54% with different proteolytic enzymes. Limited hydrolysis of
6
SPI with different proteolytic enzymes increased the EC. Among the proteases papain modified
SPI showed more EC compared to alcalase and fungal protease modified SPI. The FC of low
DH modified SPI was higher compared to unmodified SPI. Fungal protease modified SPI
showed higher FC compared to papain and alcalase modified SPI. Extensive hydrolysis of SPI
with proteolytic enzymes brought drastic reduction in EC. Although the FC of SPI extensively
hydrolysed using papain, alcalase and fungal protease was higher than intact SPI, the FC
values were lower than the corresponding low DH modified SPI. The maximum DH obtained
with SPI with proteolytic enzymes was lower than that of DSF. SPI with limited proteolysis
showed remarkable increase in FC but the increase was marginal with DSF.
SECTION 3: CONTROLLED ENZYMATIC HYDROLYSIS OF GLYCININ
Glycinin, the major protein fraction of soybean when hydrolysed with different
proteolytic enzymes showed that papain had the least effect followed by alcalase and fungal
protease. The enzymatic hydrolysis followed typical Michaelis-Menten pattern. The affinity of
glycinin to proteolytic enzymes was in the order fungal protease > alcalase > papain as shown
by the Km values. The SDS-gel electrophoretic pattern of glycinin showed bands corresponding
to acidic (30-33kD) and basic subunits (29-22kD). The pattern observed for enzymatically-
modified glycinin suggested the preferential cleavage of acidic subunits compared to basic
subunits. The hydrolysis of isolated acidic and basic subunits of glycinin with fungal protease
showed that basic subunits were less susceptible. A maximum DH of 26% was obtained with
acidic subunits at the end of 4h hydrolysis compared to 9-10% DH with basic subunits. The Km
values for acidic and basic subunits for fungal protease correlated well with cleavage
susceptibility. Glycinin possess poor functional properties; enzymatic modification with
proteolytic enzymes improved a few of the functional characteristics. Papain with limited
proteolysis increased the FC almost three fold with good foam stability. There was no
difference in the EC of low DH modified glycinin samples. The FAC of papain, alcalase, and
7
fungal protease modified glycinin decreased compared to unmodified glycinin. Limited
proteolysis of glycinin did not bring significant differences in the WAC. The molecular sieve
chromatography on Sepharose-6B gel showed single peak for glycinin. In the case of modified
glycinin it could be resolved in to two peaks. The first peak had same Ve/Vo as that of glycinin
but the second peak had higher Ve/Vo. This indicated that even low DH degraded glycinin into
low molecular weight peptides. The electrophoresis pattern of gel filtration chromatographic
peaks suggested that peak1 and peak 2 did not correspond to native glycinin; acidic subunits
were readily hydrolysed compared to basic subunits. Peak 2 did not give bands on the 10% gel
suggesting that peak 2 was extensively hydrolysed and the molecular weight of the resulting
peptides were low. A low DH of 4-5% resulted in drastic reduction in mean residue ellipticity
of modified glycinin for all the enzymes tested. However, both glycinin and modified glycinin
exhibited characteristic near UV CD peaks at 263, 275, 283 and 291nm. This suggests that
even a low DH disrupts the tertiary structure of glycinin. The EC and FC of glycinin were
lesser than that of SPI. The poor functionality of glycinin may be due to the closely packed
conformation of glycinin in which the hydrophobic groups are buried inside. The enhancement
in functionality by limited proteolysis could be due to the exposure of hydrophobic groups.
PART- B: STUDIES ON GROUNDNUT PROTEINS
SECTION 4: ENZYMATIC MODIFICATION OF GROUNDNUT PROTEIN ISOLATE
Groundnut protein isolate (GPI) was more susceptible to hydrolysis with papain,
alcalase and fungal protease compared to SPI. The maximum DH of GPI obtained with papain,
alcalase and fungal protease was 18.6%, 17.4% and 26.6% respectively. The comparatively
high affinity of GPI for proteolytic enzymes may be because groundnut proteins are less
hydrophobic compared to soybean proteins. The effectiveness of different enzymes was in the
order fungal protease > papain > alcalase. The solubility curve of GPI followed U-shaped
pattern with the minimum at pH 4.5. The low DH modified GPI with different proteolytic
8
enzymes at isoelectric pH showed solubility of 27-28%. However, high DH increased the
solubility up to 47-55% at pH 4.5.The EC and FC of GPI increased after limited proteolysis.
The effectiveness of different proteolytic enzymes in enhancing the EC was almost same.
However, alcalase and fungal protease enzymes were more effective compared to papain in
enhancing the FC by limited proteolysis. Extensive hydrolysis of GPI resulted in a remarkable
reduction of EC. Although the FC of high DH modified GPI with different enzymes were
higher than unmodified GPI, the values were lower than that of low DH modified GPI.
SECTION 5: ENZYMATIC HYDROLYSIS OF ARACHIN AND ITS EFFECT ON FUNCTIONAL PROPERTIES
Arachin, the major protein fraction of groundnut purified by ammonium sulfate
precipitation eluted as a single peak with Ve/Vo =1.5. Arachin with a low DH of 3-5% showed
two peaks, the first peak at Ve/Vo similar to arachin and second peak with a Ve/Vo =1.8 This
suggested that even at low DH arachin degraded into low molecular weight peptides. The Km
values for arachin with different proteases were 0.83-0.931%. The Km values were lower when
compared to the values obtained with glycinin as substrate. Arachin was the preferred substrate
over glycinin for the proteolytic enzymes used. The maximum DH of arachin obtained with
papain, alcalase and fungal protease was 23.6%, 18.6% and 25.5% respectively. The
effectiveness of these enzymes was in the order fungal protease > papain > alcalase. These
results obtained with arachin were comparable to that of GPI. This suggests that the proteolytic
enzymes have got equal effectiveness for GPI and arachin. The solubility of modified arachin
to low DH (3-5%) was 14-16%. Extensive hydrolysis increased the solubility up to 55-60%.
Hydrolysis increased the solubility of arachin considerably. Limited proteolysis increased the
EC of arachin. Among the different enzymes papain was more effective in enhancing the EC.
The FC of alcalase-modified arachin was remarkably high (two fold). Papain and alcalase
modified arachin showed marginal increase in FC. Excess hydrolysis impaired the
functionality of arachin except solubility, The SDS-PAGE pattern of arachin was similar to
9
those already reported in the literature. Papain, alcalase and fungal protease degraded high
molecular weight subunits. The pattern for alcalase and fungal protease was similar. The action
of papain on arachin subunits was different. The low molecular weight subunit disappeared
only after hydrolysis for 1h with different enzymes. The EC of arachin was higher than that of
glycinin. There was considerable difference in the FC between the two fractions. The overall
effect of proteases in enhancing the functionality differed considerably. Comparison of the DH
obtained with flour, isolate and purified fractions of groundnut and soybean showed that the
different proteases acted differently in getting high DH and in changing functional
characteristics. DSF gave high DH with proteolytic enzymes compared to SPI. The effect of
proteases on GPI and arachin was similar. In general, the study has indicated that groundnut
and soybean proteins are resistant to hydrolysis by proteolytic enzymes. The bitterness of
hydrolysate was more with soy proteins than groundnut proteins. The affinity of proteases
towards groundnut proteins was more than that of soybean proteins. Thus by using appropriate
proteolytic enzymes under specified conditions the functional characteristics of seed proteins
can be tailored to meet our requirements.
CHAPTER 4: SUMMARY AND CONCLUSIONS This has the general summary of the investigation focusing on the important findings of
the investigation. The references are arranged in alphabetical order.
H. SRINIVAS GOVINDARAJU.K
(Guide) (Candidate)
Retd. Scientist
Department of Protein Chemistry and Technology
Central Food Technological Research Institute
Mysore- 570013, INDIA.
1
INTRODUCTION
Plant proteins can be divided into two groups, namely reserve proteins of the seed
and functional proteins of the vegetative part of the plant. The storage proteins of seeds serve
as major nitrogen source and are utilized during germination of the seed to provide necessary
free amino acids and nitrogen to the growing plant during initial stages of germination. The
reserve proteins of seeds are major source for the nutrition of man and his livestock.
Generally, seed proteins are complex mixture of different proteins that differ in structure,
size, charge, shape, amino acid composition, solubility and other physico-chemical,
functional and nutritional properties. The utilization of plant proteins, although cheap and
abundant, in different food products are very limited due to lack of desirable functional
performance of these proteins in foods. The major impediment to increasing the utilization of
plant proteins in formulated foods is the lack of proper understanding of the molecular basis
for protein functionality in food.
Oilseeds
In India oilseed crops are cultivated in about 31 million hectares, with a total
production of nearly 24 million tons. Oilseeds occupy an important position in Indian
economy. They claim the largest share in the country’s gross sown area after food grains. On
the oilseed map of the world, India occupies a prominent position, both with regard to area
and production. The most important oilseed crops grown in India are groundnut, rapeseed,
mustard, sesame, linseed, safflower, castor, sunflower, niger and soybean.
In oilseeds, a large amount of storage proteins are accumulated and sequestered as
protein bodies. These are similar to starch in starch granules or oils in spherosomes, and
these reserve organelles are themselves located in reserve tissues (Pernollet and Mosse,
1983). The protein bodies earlier been referred to as aleurone grains of aleurins (vacuolar
2
proteins) and the concepts of aleurin biosynthesis and sequestration seem to have a bearing
on a common theme and seed protein homology and structural similarity (Dieckert and
Dieckert, 1976 a, b; Prakash and Rao, 1988).
Seed proteins can be classified into two main types, namely vicilin (7S) and legumin
(11S). The legumins contain polypeptides linked by disulfide bridges. The secondary
structure of legumins studied by circular dichroism demonstrates that they have less amount
of α-helix, about 30% pleated sheet, and a large component of unordered regions (Dieckert
and Dieckert, 1985). The legumins isolated from groundnut and soybean (arachin and
glycinin) do not undergo crystallization. Some of the legumins such as edestin and pumpkin
seed globulin crystallize readily. The legumins are generally not glycoproteins. In legumin
type of proteins the disulfide-bridged subunits contain exactly one basic subunit
(MW~22,000) and one acidic subunit (MW~33000).
Vicilin type proteins constitute a second major group of seed globulins. α conarachin
and β conglycinin are the vicilin type globulins of groundnut and soybean respectively. They
are multimeric with subunits of different molecular weights. The largest subunits are in the
68000 to 72,000 Da range. Another set exhibits molecular weights in the range 40,000 –
55,000. Generally vicilin type of proteins is considered to be dimers or trimers of the
fundamental subunits (α, α'and β) without intersubunit disulfide bridging. They are mostly
glycoproteins (Sykes and Gayler, 1981; Yu, 1977).
Soybeans
Soybeans (Glycine max) have become an increasingly important agricultural
commodity in the world. Currently global production is estimated nearly 171 million tons.
The major soybean producing countries of the world are USA, Brazil, China, Argentina and
India.
The cultivation of soybean in Madhya pradesh has been a success story of yellow
3
revolution in India. Soybean oil processors association, the national research center on
soybean and the Technology mission on oilseeds and pulses are some of the agencies, which
have contributed to this development. The production of soybean in India has gone up from
2.63 million tons in 1989-91 to 6.5 million tons in 1999. The current area of soybean
production in India is around 6.4 million Hectares (Ministry of Agriculture).
Botanical description
Soybean is a member of the subfamily, Papilonaceae of the family Leguminosae. The
cultivated form Glycine max (L.) is a plant with height ranging from 0.75 to 1.25m
branching sparsely or densely, depending on cultivars and growing conditions. Soybean
seeds vary greately among varieties and cultivars, generally ranging in size from 7.6 to 30.3g
per 100g seeds. Although most soybeans are yellow, some variation exists in seed coat
colour, including green, dark brown, purplish black. In India all commercial beans are
yellow or yellowish brown.
Nutritive value of soy proteins
Soybeans are rich source of proteins with good nutritional quality. The limiting
amino acids in soy proteins are methionine, cyst(e)ine, threonine and tryptophan (Eggum and
Beames 1983).The essential amino acid content of soy protein products are shown in Table
1.1. Soy proteins are superior to groundnut and cottonseed proteins but inferior to milk
proteins when used as a sole source of protein. The protein digestibility corrected amino acid
score and protein efficiency ratio of soybean proteins is 0.92 and 2.3 (FAO/ WHO 1990,
Torun et al., 1981) compared to 0.52 and 1.65 for groundnut proteins. Many studies on
experimental animals and human subjects indicate that substituting soy protein for animal
protein in the diet reduces the concentration of total and low-density lipoprotein (LDL)
cholesterol in plasma or serum. Thus, consumption of soy protein helps to reduce
cardiovascular disease (Sirtori et al., 1993, Carroll et al., 1995,Anderson et al., 1995).
4
Although the incidence of soybean allergy in adult is relatively uncommon, epidemiological
studies have shown that it is the most common form of allergies among young children. Only
peanut and cow`s milk allergy occur frequently. With growing consumption of soy based
products all over the world the incidence of allergy has been increased. The presence of
certain antinutritional factors such as protease inhibitors, lectins and oligosaccharides have
been attributed to reduced efficiency of utilization of soy protein compared to milk protein.
Substantial amount of work has been carried out on soy proteins to overcome the problem of
anti nutritional factors and allergenicity in recent years in order to increase its utilization.
Oriental soy foods
Oriental soy foods fall into two broad categories viz. non-fermented and fermented.
Among the non-fermented soy foods, tofu is the most popular, followed by soymilk and soy
sprouts, whereas soy sauce miso, tempeh and natto are among the popular fermented foods.
Soymilk is a water extract of soybeans closely resembling dairy milk in appearance and
composition. In the last several decades due to pioneering work by several researchers in
reducing beany flavour, soymilk has become popular in the world. As an alternative to dairy
milk, soymilk provides proteins and other nutrients to people in regions where the supply of
animal milk is inadequate. Soymilk is also useful for infants and children allergic to dairy
milk. Tofu is a curd made from soybeans and resembles a soft white cheese Tofu is water
extracted and acid coagulated soy protein gel with water, lipids and other constituents
trapped in the network. It is an inexpensive, nutritious and versatile soy food. Most
fermented soy foods usually contain salt or by-products (such as lactic acid) from a desirable
fermentation. Both inhibit the spoilage of these products in order to have relatively longer
shelf life.
Soy protein ingredients
Grinding produces soy flours and grits and screening soybean flakes either before or
5
after the oil is removed. Full fat soy flour is made from steam cooked dehulled beans to
inactivate various inhibitors. Extrusion cooking can produce full-fat soy flour with various
properties.
Table 1.1: Essential amino acid composition of soy protein (mg/g protein) products
Essential amino acid
infant soy flour soy
concentrate soy isolate FNB
pattern for infants
Histidine
Isoleucine
Leucine
Lysine
Cystine+Methionine
Phenylalanine+Tyrosine
Threonine
Tryptophan
Valine
26
51
77
69
32
89
43
13
54
26
48
79
64
28
89
45
14
50
28
49
82
64
26
92
38
16
50
17
42
70
51
26
73
35
11
48
Source: Kolar et al. 1985
Defatted soy flour
Enzyme active soy flours are prepared by grinding defatted flakes with minimum
heat denaturation to maintain high nitrogen solubility index (NSI). They are used in baking
applications to increase mixing tolerance and bleaching in bread. Defatted soy flours, which
contain 52-54% protein, are prepared by finely grinding defatted flakes to pass through 60-
mesh sieve. Defatted soy flour prepared from toasted soy grits are generally has less NSI
(<30%).
6
Soy protein Isolate
Soy protein isolates are the most refined soy protein ingredients and must contain
90% protein (N × 6.25). Isolation of soy protein minimizes the flavour, colour contribution
and anti nutritional components, and allows for the widest variety of functional properties of
soy ingredients. Preparation of isolate generally involves extraction of soluble proteins and
carbohydrates from defatted flour at alkaline pH, precipitation of the protein at isoelectric
pH, separation and removal of the soluble carbohydrates, proteins and salts by centrifugation
and drying of the proteins.
Isolated soy proteins are commercially available in several forms, such as fibers, heat
structured fibers and granules. These products are generally used to simulate the texture of
meat. The spun protein fibers are prepared by forcing the slurry of isolated soy protein
through spinnerets into an acid coagulating bath to form fibers of about 0.1mm diameter. The
fibers are washed and stretched to alter the texture. Also, fat, colour, flavour and other
ingredients may be added to simulate different types of meat products. Heat structured
fibrous proteins are produced by passing high solids slurry of isolated soy protein through an
indirect heat exchanger and forcing through a small orifice at high temperature and pressure.
Applications of soy protein isolates
Soy protein isolates can be used to reformulate the traditional meat while maintaining
the traditional quality. Soy protein isolate can be used to upgrade the mechanically deboned
poultry which has dark colour and paste like consistency so that it performs like light
coloured poultry meat. Isolated soy protein fiber is used to improve the texture of
mechanically deboned poultry meat. The traditional Japanese comminuted gel like products
prepared based on a minced fish-flesh ingredient called Surimi can be replaced with soy
protein without changing traditional quality. Soy protein isolates are also used in a wide
variety of meat and dairy products, infant formulas, beverages, and as an amino acid source
7
to substitute for casein, egg white and meat. This is because of the ability of soy protein to
impart various functions in different types of food products including emulsification, fat and
water binding ability, gelation, adhesion and cohesion. Infant formulas based on soy proteins
are produced for infants who are allergic to cow’s milk. Some of the dairy products based on
soy protein are whipped toppings, ice creams, coffee whiteners, imitation milk, cheese,
frozen desserts and yoghurts. Isolates are used in these products due to high protein contents
and excellent ability to impart different functions.
Soy protein concentrate
Soy protein concentrates are prepared by removing soluble carbohydrate fraction as
well as flavour components from defatted meal. In this process the undesirable
oligosaccharides, which cause flatulence, are eliminated and the protein content of the
product will increase. Acid leaching (pH 4.5), aqueous ethanol extraction (60-80%) and
moist heat-water leaching are the three common processes for the preparation of concentrate
(Figure 1.1). In all these treatments the proteins become insoluble and a part of carbohydrate
Table 1.2:Comparison of the typical composition of defatted soy flour concentrate and isolated soy protein (on moisture free basis)
Component (%) Defatted soy flour
Concentrate
Isolated soy
protein
Protein (N × 6.25)
Fat
Ash
Total Carbohydrate
54.0
1.0
4.5
38.0
71.0
0.6
4.8
17.6
92.0
0.5
4.5
0.3
components remain soluble so that separation by centrifugation becomes possible.
Commercially the concentrates are prepared by aqueous alcohol extraction or acid leaching
process. In general, soy protein concentrate is a product based on soy containing at least 65%
8
protein but less that 90% protein. Alcohol process yields concentrate with bland flavour, and
low nitrogen solubility because alcohol causes denaturation of proteins. The acid wash
process yields soy protein concentrate with relatively high nitrogen solubility. The typical
composition of defatted soy flour, soy protein isolate and soy protein concentrate is shown in
Table 1.2.
Defatted Soy meal
Dilute Acid Aqueous leach (pH 4.5) Alcohol leach (60 – 80%)
Neutralise Insoluble fraction Spray dry Soy protein Concentrate Figure 1.1: Flow sheet for the preparation of soy protein concentrate
Soybean proteins-Physico chemical properties
Soybean seeds contain nearly 40% protein and 20% lipids. The defatted soybean
meal contains nearly 50-55% protein of good nutritional quality and functional
characteristics. The major proteins of soybeans are storage globulins i.e. glycinin and
conglycinin and make up nearly 90% of the total. The proteins can be classified into 2S, 7S,
11S and 15S based on their sedimentation coefficients (Naismith 1955, Wolf and Briggs
1956). The 11S fraction is glycinin and accounts for at least 30% of the extractable protein
whereas 15S fraction is a polymer of glycinin and accounts for about 10% of extractable
9
protein. The 2S fraction accounts for about 20% of extractable protein and contains Kunitz,
Bowman-Birk trypsin inhibitor and cytochrome C. The 7S fraction also accounts for around
30% of the extractable protein and contains conglycinin, α-amylase and haemaglutinin.
The separation of water extractable proteins from soybean by ultracentrifugation was
reported by Naismith (1955). Wolf and colleagues made an extensive study to extract,
fractionate and characterize the soy proteins (Wolf and Briggs 1959, Wolf et al., 1961) and
classified the proteins into 2S, 7S, 11S and 15S fractions according to ultra centrifugal
differences. Tanh and Shibasaki (1976a) repoted the method for the fractionation of 7S and
11S globulins from defatted soybean meal by fractional isoelectric precipitation. The method
involves precipitation of 11S globulin at pH 6.4 from a dilute tris buffer (0.05M, pH 8.0)
extract of soy flour. The 7S protein was precipitated from the pH 6.4 supernatant at pH 4.8
Conglycinin
The 7S fraction of soybean protein contains lipoxygenase, hemagglutinin and
predominantly 7S globulins. Catsimpoolas and Ekenstam (1969) showed four components of
which β and γ conglycinin were predominant. Tanh et al., (1975) reported five fractions in
7S all of which appeared to be glycoproteins and different in terminal amino acid
composition. The different fractions of 7S globulins can undergo reversible dimerization
(association – dissociation ) in low ionic strength solutions. In low ionic strength solutions,
85% of 7S fraction dimerizes. The nondimerizing components are hemagglutinins. The non-
dimerising γ conglycinins (MW 10.4) represent 3% of total soy globulins, β conglycinin
(181 kD) comprise 28% of the globulins (Koshiyama et al., 1972b). The β and γ conglycinin
contain over 5% carbohydrate and account for over 90% of the 7S fraction. β-conglycinin is
considered as a trimer with a molecular weight of about 180kDa. The three prevalent
subunits of β-conglycinin has been designated as α α` and β. The molecular weights of α, α`
and β subunits were estimated to be 57, 57 and 42 kDa respectively (Tanh and Shibasaki
10
1976a, b). All the three subunits of conglycinin have been shown to be rich in aspartate,
glutamate, leucine and arginine. The β subunit contain no methionine and the α α` subunits
contain low levels of methionine (Nielsen 1985a).
Glycinin
Several methods are available for preparing 11S protein rich fraction from soybean.
Cooling a concentrated aqueous extract of ground soybeans results in the precipitation of a
cold insoluble fraction containing 2S, 7S, 11S and 15S proteins in which 11S predominates
(Briggs and Wolf 1957). Several attempts have been made to further purification of the
protein. Wolf et al., (1962) reported a method for purification of 11S protein by ammonium
sulphate precipitation of cold insoluble fraction, which yields 11S protein with 91-93%
purity. Chromatographic purification using gel filtration, hydroxyapatite or ionexchange
chromatography has been reported for purification of 11S globulin (Hasegawa et al., 1963,
Mitsuda et al., 1965, Wolf and Sly 1965, Catsimpoolas et al., 1967). Combinations of
affinity chromatography on ConA-Sephrose 4B and gel filtration chromatography on
sephrose 6B column were also used for purification of 11S globulin (Kitamura et al., 1974).
Glycinin can also be isolated by magnesium chloride precipitation (10-2M) of the water
extract of defatted soybean flour (Appu Rao and Narasinga Rao, 1977). Tanh and Shibasaki
(1976a) reported a method for simultaneous preparation of both 7S and 11S globulins by
fractional isoelectric precipitation. The method affords large scale isolation of the two major
proteins without significant contamination. Recently, Nagano et al. (1992) reported a method
for large-scale preparation of glycinin and conglycinin by fractional isoelectric precipitation
by using sodium bisulfite instead of mercaptoethanol as reducing agent. The method has
been shown to give a good purity of 11S globulin and high yield of 7S globulin. The method
has been successfully used to fractionate the two globulins on pilot plant scale (Wu et al.,
1999).
11
Molecular Structure of Glycinin
The Glycinin has a molecular weight of about 360kD. The amino acid composition
of glycinin shows that it is low in methionine but high in lysine. The proportion of
hydrophobic amino acids (Ala, Val, Ile, Leu and Phe) and hydrophilic amino acids (Lys, His,
Arg, Asp and Glu) are 23.5 and 46.7% respectively (Takagi et al., 1979). The isoelectric
point of glycinin is around 4.64. Kitamura and Shibasaki (1975) and Kitamura et al., (1976)
reported that four kinds of basic subunits (A1, A2, A3, A4 and B1, B2, B3, B4) exists in
glycinin molecule. The acidic and basic subunits are present in the approximate molecular
ratio of 1:1:2:2. In general, 11S globulin consists of two similar but not identical monomers
and each monomer contain three intermediary subunits of the composition A1 or A2 with
B3, A3 with B1, A4 with B4 and A3 with B2.
The dissociation of the 11S globulin can be proposed as
(A-SS-B)6 6 (A – SS - B) A1B3 + A2B3 + A3B1 + A3B2 + 2A4B4
Glycinin Intermediary subunits
(A1 + A2 + 2A3+ 2A4) + (B1 + B2+ 2B3+ 2B4)
Dieckert and Dieckert(1978) showed that in glycinin the acidic and basic subunits
are linked by disulfide bridges. Staswick et al., (1981) isolated five complexes from glycinin
consisting of basic and acidic subunits joined by disulfide bridges. The subunit pairings were
A1a/B2, A1b/B1b, A1b/B1b, A3/B4 and F(2)/B3. The data revealed that each acidic
polypeptide is nonrandomly linked to a specific basic polypeptide and in no case basic
polypeptide has been associated with more than one acidic polypeptide as predicted by
Kitamura et al., (1976). That one acidic polypeptide could be associated with several
different basic polypeptides and vice versa. Table 1.3 summarizes features of six acidic and
five basic polypeptide components purified from soybean. Acidic and basic polypeptides
except A4 are linked through polypeptide bonds. Glycinin subunits can be separated into two
12
components; Group I subunits are uniform in size (MW 58 kD) relatively rich in methionine.
Group II subunits contain less methionine and are larger (MW 69kD)
Table 1.3: Comparison of glycinin subunits
Group
Subunits MW No of met residues
I I I
II II
A1aB2 A1bB1b A2B1a A3B4 A5A4B3
58000 58000 58000 62000 69000
5-6 5-6 7-8 3 3
Source: Nielson, N.C. 1985a
Since, in glycinin acidic and basic polypeptides are linked by S-S linkage, the
structure of individual monomeric subunits have been considered to be ASSB where A and
B represents the acidic and basic polypeptides. In general, glycinin is a hexamer with a
molecular weight of about 360kDa with each monomer made up of acidic and basic
polypeptides. The polypeptides can be disassembled by reduction of disulfide bridges using
mercaptoethanol followed by denaturation with urea. The individual acidic components can
be separated by DEAE-Sephadex chromatography and the basic components with CM
Sephadex (Moreira et al. 1979). The primary structure of acidic subunit A2 and basic subunit
B2a is shown in Figure 1.2
On the basis of Fourier transform infrared spectra (FTIR) spectra, Abbott et al.
(1996) predicted the secondary structure of glycinin to be 24% α-helices, 30% β-sheet, 31%
turns and 12% unordered forms. Glycinin has the same secondary structure in solution and in
hydrated solids. Nothing is known about the tertiary structure of glycinin. The quaternary
structure of glycinin has been suggested as two layers of trimers in which each trimer
consisting of three acidic and three basic polypeptides paired and held together by disulfide
and hydrogen bonds with acidic and basic peptides alternatively (Badly et al., 1975).
13
LREQAQQNEC QIQKLNALKP GNRIESEGGF IETWNPNNKP
FQCAGVALSR CTLNRNALRR PSYTNGPQEI YIQQGNGIFG
MIFPGCPSTY QEPQESQQRG RSQRPQDRHQ KVHRFREGDL
IAVPTGVAWW MYNNEDTPVV AVSIIDTNSL ENQLDQMPRR
FYLAGNQEQE FLKYQQQQQG GSQSQKGKQQ EEENEGSNIL
SGFAPEFLKE AFGVNMQIVR NLQGENEEED SGAIVTVKGG
LRVTAPAMRK PQQEEDDDDE EEQPQCVETN KGCQRQSK.
(a)
GIDETICTMR LRQNIGQNSS PDIYNPQAGS ITTATSLDFP
ALWLLKLSAQ YGSLRKNAMF VPHYTLNANS IIYALNGRAL
VQVVNCNGER VFDGELQEGG VLIVPQNFAV AAKSQSDNFE
YVSFKTNDRP SIGNLAGANS LLNALPEEVI QHTFNLKSQQ
ARQVKNNNPF SFLVPPQESQ.
(b)
Figure 1.2: Primary structure of the acidic (a) and basic (b) polypeptide components of Glycinin Subunits A2B1a. Staswick (1982).
14
Association-dissociation behaviour of Glycinin
Several factors such as pH, ionic strength of the solution, temperature, presence of
reducing agents, urea or guanidine hydrochloride, EDTA, heat and metal ion binding
determine the association-dissociation behaviour in solutions.
Behaviour of glycinin in acid solution was studied by Wolf et al., (1958). Low pH
and low ionic strength convert glycinin (11S component) into a low sedimenting component
(2S) and 7S as a result of dissociation. This is due to electrostatic repulsion between
subunits. Acid denaturation of the protein and subsequent dissociation into subunits begins at
pH 3.75 and reaches a maximum at pH 2.0 (Catsimpoolas et al., 1971a; Koshiyama 1972c)
whereas, alkali-induced denaturation and conformational changes begin at pH10.0 and
proceed more rapidly at pH values of more than 11.4. At low-ionic strength and slightly
alkaline pH values, glycinin dissociates into 2S and 7S components (Eldridge and Wolf
1967). The physico-chemical properties of soy glycinin are summarized in Table 1.4.
The complete dissociation of glycinin in 6M-urea or guanidine hydrochloride has
been shown to be reversible (Kitamura et al., 1977). The dissociation of glycinin by heat into
subunits generally depends on the ionic strength. Glycinin appears to be stable upto 70ºC
and above this temperature, the protein becomes increasingly turbid and precipitates at 80ºC.
The heat dissociation of glycinin into subunits depends on the ionic strength. At low ionic
strength, dissociation begins at 70ºC and at high ionic strength it starts at 900C (Hashizume
et al., 1975). Mercaptoethanol prevents aggregation of glycinin by cleaving intermolecular
disulfide bonds. Increasing the sodium chloride concentration from 0-1M has been shown to
increase the denaturation temperature of glycinin by 20°C (Brooks and Morr 1985). Heating
disrupts the original quaternary structure of glycinin with the formation of two independent
fractions, one containing stable soluble 3-4S polypeptides and another having a tendency to
form a soluble aggregate which upon continued heating transforms into insoluble aggregate
15
(Hashizume et al. 1979). The gel filtration of heat-induced products of glycinin showed that
the precipitate contains high molecular weight fractions polymerized through disulfide
bonds, where as, the supernatant contains low molecular weight fraction subunit monomer.
Glycinin did not precipitate even after heating up to 30min. when the free SH groups were
blocked using N-ethyl maleimide(NEM). This shows that heat denaturation of glycinin may
be related to sulfydryl-disulfide exchange (Wolf and Tamura 1969). Heating β-conglycinin
alone did not cause precipitation, but the addition of glycinin to the system caused
precipitation. The reason has been attributed to the interaction through disulfide bonding
between β-conglycinin and glycinin subunits(Damodaran et al. 1982; Yamagishi et al. 1983).
Table 1.4: Physico chemical properties of soy glycinin
Property Values
Nitrogen content 16.3% (w/w)
Sedimentation constant S 020,W 12.3 ± 0.1S
Partial specific volume, V at 20ºC 0.730 ± 0.001 ml/g
Diffusion constant, Dº20,W 3.44 ± 0.1 cm 2/sec
Frictional ratio 1.40 Stokes radius,γ 58.5 Aº Radius of gyration, Rg 44Aº
Hydration,δ 0.34g/g
Number of subunits 12
Intrinsic viscosity,η 0.0485 dl/g
E 0.1280nm
8.04
Isoelectric point, pH 4.64 Molecular weight 350000 Da
Source: Peng et al. 1984
16
Heat induced dissociation and aggregation of glycinin may be due to the sulphydryl-disulfide
interaction between the subunits.
Trypsin inhibitors
Soybean protein extract has been shown to possess the ability to inhibit trypsin (Read
and Haas 1938). The proteins responsible for inhibition are classified into two types viz.
Kunitz trypsin inhibitor (TI) and Bowman-Birk (BB) inhibitor. Kunitz inhibitor can be
isolated by alcohol precipitation of water extract of soybean (Kunitz 1945). It has a M.W.
between 20 to 25kD and specific toward trypsin. The aminoacid sequence of the inhibitor
consists of 181 amino acid residues Arg 63 and Ile 64 (Figure 1.3a). The Bowman-Birk
inhibitor can be isolated by extraction of beans with 60% alcohol solution followed by
precipitation with acetone (Bowman 1944). It is a single polypeptide chain of 71 amino
acids containing seven disulfide bonds. It has a M.W. of about 8 kDa (Figure 1.3b). The BB
inhibitor has the capacity of inhibiting both trypsin and chymotrypsin at independent binding
sites. BB inhibitor has 61% β-turn and 0% α-helical form (Wu and Sessa 1994). The
mechanism of inhibition with trypsin involves the formation of an enzyme substrate
complex, which does not dissociate because of the tight binding of the inhibitor to the
enzyme compared to the usual enzyme-substrate complex, which easily dissociate into
products and enzyme.
The growth performance caused by the raw soybean extract from which trypsin
inhibitors have been removed was improved compared to control diet containing raw
soybean in which trypsin inhibitor has not been removed. This indicates that trypsin
inhibitors cause growth inhibition in animals. Also inhibitors have been shown to cause
hypertrophy of the pancreas of chicks. The actual physiological role of protease inhibitors
has been a subject of much controversy since medical research demonstrates that they have
17
Figure 1.3a: Primary structure of Kunitz trypsin inhibitor: Active site contains Arginine 63 and Isoleucine 64 (Source: Ikenaka et al. 1974).
Figure 1.3b: Primary structure of Bowman -Birk trypsin inhibitor: Lysine 16 and
Serine17 is the trypsin inhibitory site, where Leucine 43 and Serine 44 correspond to the Chymotrypsin inhibitory site. (Source:Odani and Ikenaka 1973).
18
anti carcinogenic activity (Kennedy 1994). However, it is generally accepted that protease
inhibitors cause growth inhibition, pancreatic hypertrophy, hyperplacia and adenoma in
animals (Liener 1994).
Extensive work has been made to inactivate inhibitors from soybeans. Various
approaches have been based on heat treatment including steam, boiling in water, dry
roasting, and microwave radiation and extrusion cooking. In addition to heating temperature
and time, moisture condition prior to and during heat treatment has significant effect on the
effectiveness of TI destruction by heat. Based on the activity loss of purified inhibitors
Kunitz inhibitor is thought to be more heat labile than the BB inhibitor. However, when
applying heat to soy products it is essential to use optimum condition (temperature, time,
moisture and pressure) to maximize destruction of TI and at the same time to minimize
reduction of protein solubility as well as loss of essential amino acids. Use of reducing
agents (cysteine, N-acetyl-cysteine and sodium sulphite) has been shown to facilitate
inactivation of inhibitors at lower temperature (Liener 1994, Friedman and Gumbman
1986).
Groundnut
Groundnut (Arachis hypogaea L.) is one of the widely cultivated oilseeds in the
world. Groundnut is also known as peanut, earthnut, monkeynut, manillanut, groundbean
(Arthur, 1953). Today groundnuts are grown in many of the tropical and subtropical areas
throughout the world. India and China produce about half of the world crop, with West
African countries, the USA, Mexico, Brazil, Argentina, Indonesia, Burma, and Australia as
other major producers of groundnut. India occupies a prominent position both in area and
production of groundnut in the entire world. In India, nearly 8 million hectares is cultivated
annually and the production is about 7.3 million tons (FAO 1999).
19
Food uses of groundnut
Groundnut is a good source of oil and protein. Freshly roasted groundnuts have great
acceptance because of its typical flavour, colour and aroma developed during the roasting
process. Freshly dug, unshelled, immature groundnuts boiled in brine solution are eaten as a
delicacy in many parts of India. (Woodroof, 1973). A variety of salted and unsalted whole,
split kernel and chopped nuts are used in confectionary products, as snacks, garnishes and
toppings for baked goods. Groundnut protein can be incorporated into a variety of products
without serious problems in terms of colour, texture, flavour and taste. Some of the potential
uses of groundnut proteins are in bakery products, breakfast cereals, meat patties, snack
foods, beverages, frozen desserts, soups, spreads and textured vegetable protein ingredients.
In India, groundnut flour is used in developing a variety of inexpensive food formulations
such as multipurpose food, fortified flour, paustic atta and weaning foods such as ‘Bal-
Ahar’, malted food, chewy candies and high protein biscuits. Groundnut butter is the most
popular product based on groundnut in the United states. It is prepared by cleaning of shelled
groundnuts, roasting, blanching, blending of ingredients, grinding, cooling and packaging. A
high typical formula includes at least 90% peanuts and ~2% salt.
Groundnut protein isolates are used in food products where highly concentrated
proteins are desired. It is used in the manufacture of cheese analogues and frozen desserts
(Lawhon et al., 1980). In India, groundnut protein isolate has been used in the preparation of
Miltone, milk like nutritive based beverage to extend the availability of milk to a larger
segment of population. Miltone resembles cow’s milk, powder-toned milk and whole milk
(Chandrashekhara et al., 1971). Miltone is prepared by mixing milk with peanut protein
isolate, glucose syrup, minerals and vitamins followed by dilution, homogenization and
pasteurization. Groundnut protein isolate is also used in the preparation of vegetable curd
and cheese (Chandrashekhara et al., 1971).
20
Groundnut is a rich source of proteins whose nutritional quality is not high because
of deficiency in the content of certain essential amino acids. The limiting amino acids are
methionine, tryptophan, lysine and threonine. Groundnut proteins are superior to wheat
proteins but inferior to cottonseed and soy proteins when used as a sole source of protein.
The Biological value, protein efficiency ratio and net protein utilization of groundnut
proteins are 55%, 1.65% and 4.3% respectively (FAO, 1990).
A typical defatted groundnut flour contains 3-5% moisture, 55-56% protein,3-4%
fiber, 5-6% ash and 30-35% nitrogen free extract. The principal carbohydrate is sucrose with
only traces of raffinose, stachyose, glucose and fructose. The potassium and sodium content
is 100ppm and 1ppm respectively. Although groundnuts are good source of fat-soluble and
water-soluble vitamins, groundnut flour contains only thiamin, riboflavin, niacin and choline
(Conckerton and Ory, 1987).
Groundnut proteins – Physico chemical properties
Deshelled groundnut contains 50-60% oil and nearly 30% protein. The major storage
globulins i.e. arachin and conarachins together constitute nearly 75-80% of the total proteins.
Typical defatted groundnut meals contain nearly 55-58% total protein of edible quality.
Groundnut proteins does not contain significant amounts of intrinsic anti-nutritional factors
such as trypsin inhibitors. Aflatoxin is commonly found in groundnut because of improper
storage of seed and is due to fungal infection especially Aspergillus parasiticus and
Aspergillus oryzae.
Early investigations on groundnut proteins were carried out by Irving et al., (1945).
They observed two main components (A and B), constituting 87% of the proteins, and two
minor components by gel electrophoresis of total extractable proteins of groundnut total
proteins. Naismith and Mc David(1958) separated groundnut total proteins into three
fractions based on sedimentation coefficients as 2S, 8S and 13S and designated as
21
conarachin I, II and arachin respectively. Tombs (1965) separated the total groundnut
proteins by polyacrylamide gel electrophoresis and designated the bands as alpha, beta,
gamma and delta regions depending upon their mobility. He reported that the α region
constitutes nearly 3% of total proteins and contains low molecular weight components. The β
region constitutes nearly 16% of the total and contains distinct group of proteins. The γ
region shows two hands, labeled as arachin monomer and dimmer along with a minor
component termed α-conarachin. The effects of SDS and heat on the physicochemical
properties of groundnut proteins have been studied in detail (Shetty and Rao 1973; Thomas
and Neucere, 1973). In general groundnut proteins can be designate as 2S, 8S, 13S and 16S
proteins. The three major fractions in groundnut are arachin (14S), conarachin II (8S) and
conarachin I (2S).
Arachin
Several methods are available in the literature for isolation of arachin from
groundnut. Johnson and Naismith (1953) obtained arachin by precipitation of the total
protein at 0.32 ammonium sulphate saturation. Naismith and McDavid (1958) isolated
homogenous arachin by precipitation of the total protein by the addition of flavine
hemisulfate solution (0.2g/100ml). In the analytical ultracentrifuge, it showed a major peak
of S20,W of 13.3, along with a minor peak in phosphate buffer of pH 8.0 containing 0.5M
NaCl. Tombs (1965) separated arachin A and B by fractional precipitation. Water dialysis of
arachin preferentially precipitated arachin B, while A remained in solution. Neucere (1969)
has reported a method for isolation of arachin by cryoprecipitation. Dawson (1971) prepared
arachin by cryoprecipitation. Dawson (1971) prepared arachin by precipitating it between 0.1
and 0.2 (NH4)2SO4 saturation from 10% NaCl extract of groundnut flour. Shetty and Rao
(1974) have shown that arachin can be precipitated quantitatively by 23%(NH4)2SO4 from
1M NaBr extract of groundnut flour. They compared the homogeneity of arachin with the
22
arachin prepared by the methods of Tombs (1965) and Dawson (1971) by
ultracentrifugation,DEAE-cellulose chromatography and polyacrylamide gel electrophoresis.
Physico chemical properties of Arachin
Arachin undergoes reversible dissociation as follows:
An ! nA.
Where An is the 14S protein and nA is the 9S protein. Naismith and Williams (1956) studied
the effect of alkaline pH on the dissociation of arachin. They proposed the following scheme
for dissociation.
14 S " 9S " 2S (Arachin) Johnson and Naismith (1956) studied the effect of GuHCl on the dissociation of arachin and
proposed the following scheme for dissociation.
14S " 12S " 7.6S " 2S
(Arachin) (Swelling) (Subunits)
Tombs (1965) elucidated the complete structure of arachin after deploymerizing the parent
molecule in 10M formamide in the presence of β - mercaptoethanol.
The nitrogen content of arachin prepared by different methods varies between 14.3 to
14.9 % (Tombs, 1965; Dawson, 1971; Neucere, 1969). The amino acid composition of
arachin prepared by different methods differs and the comparative data is shown in Table
1.5. Some of the physico- chemical data on arachin are shown in Table 1.6.
Arachin has been shown to exhibit polymorphism. The native gel electrophoretic
pattern showed two bands that have been termed monomer and dimer bands (Tombs, 1965;
Tombs and Lowe, 1967). The monomer with high solubility was termed arachin A and the
dimer with lower mobility was termed arachin B. They have reported a total of 11 disulfide
bonds and molecular weight of 330 kDa for arachin.
Yotsuhashi and Shibasaki (1973) studied the effect of denaturants like urea and
GuHCl on the dissociation of arachin into subunits. Jacks et al., (1975) studied the effect of
23
Table 1.5:Amino acid composition of arachin isolated by various workers Amino acid
Residues per 1x 105 g of protein
1 2 3 4
Aspartic acid Threonine Serine Glutamic acid Proline Glycinin Alanine Half cystine Valine Methionine Isolucine Lucine Tyrosine Phenylalanine Lysine Histidine Arginine Tryptophan
84
21
44
117
40
51
45
06
37
04
26
46
23
35
17
16
66
03
95
19
45
133
44
57
47
05
42
04
29
53
25
36
17
15
71
03
91
21
39
133
21
47
43
00
37 1
25
48
18
32
15
13
54
NR
75
14
35
124
24
48
35
NR
61
NR
14
39
17
25
12
12
99
04
Source: 1. Tombs (1965); 2. Dawson (1971); 3. Neucere (1969); 4. Shetty and Rao (1974).
24
Table 1.6: Physico chemical properties of Arachin
Property Values
Sedimentation constant S 020,W 14.6
Molecular weight 330000
Partial specific volume, V at 20ºC 0.720±0.005 ml/g
Diffusion constant, Dº20,W 3.86 ±0.1 x 10 -7 cm 2/sec
Frictional ratio 1.216 Intrinsic viscosity,η
1M NaBr 0.0485 ml/g 1M NaCl 6.2
E 0.1280nm
7.98
Isoelectric point, pH 5.1± 0.1
Carbohydrate (%) 0.3
Nitrogen content (%) 14.3 – 14.9
Secondary structure (%) a. α - helix 5 – 10 b. β - helix 25 – 30 c. γ - helix 65 – 70
Source: Prakash and Rao 1986
25
heat on antigenecity and conformational stability of arachin. Shetty and Rao (1973) observed
that SDS caused dissociation of arachin. The molecular dimension and subunit structure of
arachin has been determined.
Minor components of soybean and groundnut
Minerals, Vitamins, phytin and phenolics are the important minor components
present in soybean. The minor minerals present in soy bean and soy product include silicon,
iron, zinc, manganese, copper, molybdenum, fluorine, chromium, selinium, cobalt, cadmium,
lead, arsenic, mercury and iodine. The contents of these minor minerals range from 0.01 to
140ppm. The minor components classified, as anti nutritional factors present in soybean are
oligosaccharides, saponins, phytates, lectins, protease inhibitors and isoflavones.
Soybeans contain both water-soluble and fat-soluble vitamins. The water- soluble
vitamins present in soybean mainly include thiamine, riboflavin, niacin, pantothenic acid and
folic acid. Thiamine and riboflavin content in whole soy flour has been reported to be in the
range 6.26 to 6.85 µg/kg and 0.92 to 1.19µg/kg (Fernando and Murphy 1993). The ascorbic
acid content is essentially negligible in mature soybeans, although it is present in measurable
amounts in both immature and germinated seeds (Bates and Matthews 1975). The fat-soluble
vitamins present in soybean s are vitamin A and E with essentially no vitamins D and K. The
content is negligible in mature seeds but measurable in immature and germinated seeds
(Bates and Matthews 1975). The amounts of α, γ and δ tocopherols in soybean range from
10.9 to 28.6, 150 to 191 and 24.6 to 72.5µg/kg (Guzman and Murphy 1986) on dry basis.
The amount of β-tocopherol in soybeans is insignificant.
Phytate is the principal source of phosphorous in soybeans. The phytate content in
soybean varieties generally varies between 1.00 to 1.47% on dry matter basis. The increase
in the requirement for certain metals in experimental animals is attributed to the ability of
phytic acid to chelate with di and trivalent metal ions such as Ca2+, Mg2+, Zn2+ and Fe2+ to
26
form insoluble compounds that are not readily absorbed from the intestine. Phytate is also
capable of forming complexes with negatively charged protein molecules at alkaline pH
through calcium and magnesium binding mechanisms and with positively charged molecules
at pH values below their isoelectric point by charge neutralization. As a consequence of this
nonselective binding to proteins, phytate has been shown to inhibit the action of a number of
enzymes important for digestion (Vaintraub and Bulmaga 1991). This also affects the
isoelectric point, solubility and functionality of soy proteins. Thus phytate is an
antinutritional component present in soybeans. Because of its nutritional implications
extensive research has been centered on its reduction or removal in soy products. Isoflavones
are minor components in soybean. The isoflavone content in soybean is around 3mg/g on dry
weight basis. The presence of isoflavones has been attributed to be the factor responsible for
the potential role of soy foods in preventing and treating chronic diseases.
Raw groundnut contains antinutritional substances such as protease inhibitors,
hemagglutinins, goitrogens, saponins and phytic acid. Protease inhibitors are present in raw
groundnut and solvent extracted groundnut meals (Astrup et al., 1962; Tur-Sinai et al.,
1972). Protease inhibitors are not a serious problem in groundnut proteins, since they are
largely inactivated by heat (Neucere, 1972). Dechary et al. (1970) partially purified a non-
specific hemagglutinin from groundnuts. Hemagglutinins can be inactivated by heat.
Groundnuts are reported to produce goitrogenic effects in animals. The goitrogenic principle
has been identified as a phenolic glycoside (Sreenivasan and Mougal, 1957). A bitter
principle present in groundnut has been shown to possess general properties of saponins
(Dieckert et al., 1959). There is no evidence to show that they possess any antinutritional
effects. Phytic acid is present in phytin and also calcium, magnesium and potassium salt of
phytic acid in groundnut flour at a concentration of 3.2% (Fontaine et al., 1946).
27
Functional properties of food proteins
Functional properties denote those physico chemical properties of food proteins that
determine their behavior of foods during processing, preparation, storage and consumption.
Functional properties affect the sensory character and physical behavior of foods or food
ingredients. The type of functional properties required in a protein varies with the particular
food system. Water binding, solubility, swelling, viscosity, gelation and surface activity are
important functional properties determining the quality of product in different food systems
(Table 1.7). Defatted soy flour is used as a source of lipoxygenase for bleaching and
conditioning of wheat flour proteins. Hence enzyme activity also can be classified as a
functional property.
Functional properties of food proteins are influenced by several factors such as, the
intrinsic structure of protein, the methods and conditions of isolations, the degree of
purification, processing alterations, protein concentrations, pH, temperature, ion
concentration and amino acid composition (Pour - El, 1981; Kinsella, 1984a). Proteins
extracted from soybean, peanut, cottonseed, sunflower, sesame and safflower have been
shown to possess different functional properties (Kinsella, 1979; Kinsella, 1984; Natarajan,
1980; Lucas, 1979; Sosulski, 1979; Betschart, 1979). Less expensive oil seed proteins can be
used to develop new functional foods to simulate traditional foods such as milk, meats and
whipped toppings. This shows that functionality has critical role of determining the
application of oilseed proteins in many foods. Most of the research on functional properties
has been made with soy proteins (Smith and Circle, 1978; Kinsella 1976; Pour-El, 1981).
Soy proteins perform several useful functions in different food products. In bakery
products soy flour is used to improve water absorption, moisture retention and dough
handling. Soy flour is also used as a source of lipoxygenase for bleaching and conditioning
of wheat flour. Water holding, emulsion formation, gelling and adhesion are the important
28
functions of soy protein in meat and meat products. Soy proteins find application in dairy
foods such as coffee whitener, whipped toppings and ice cream as functional ingredient
(Kolar et al. 1979).
Solubility
The extent of protein solublisation, which is reflected by the protein solubility index
(PSI) or the protein dispersibility index (PDI) is one of the most basic requirements for any
functional application. Solubility of a protein under given conditions like pH, ionic strength
and temperature is controlled by factors that influence the equilibrium between protein-
protein and protein water interactions. Conditions that shift the equilibrium in favour of
protein-protein interactions decrease the solubility and conditions that favour protein-solvent
interactions would increase the solubility.
The interactions between either protein molecules or protein and solvents are
governed by mainly electrostatic, hydrophobic and hydrogen bonding forces. The extent of
solubility of a protein in a given aqueous system depends on the overall result of the
different forces of interaction between the protein and solvent molecules. Changing the pH
and ionic strength of the medium can alter these interactions. The nature of salts used along
with the solvent also has been shown to greatly influence the balance of hydrophobic and
electrostatic interactions. (von Hippel and Scheich, 1969)
There is a vast literature on the solubility of soy proteins isolated by different
methods under various experimental condition (Mattil, 1974; Kinsella, 1976,1979,
Hermansson, 1967; Johnson, 1970b). Isolation of soy proteins by acid precipitation causes
insolubilization to a significant amount (Shen, 1976a). Addition of disulfide reducing agents
such as sodium sulfite during isolation improves the solubility of acid precipitated soy
protein (Nash and Wolf, 1967; Shen, 1976a). Shen (1976a) studied the effect of pH and
ionic strength on the solubility of soy protein isolates. At pH 6.8 the solubility slightly
29
decreases up to about 0.2M ionic strength and remains the same above this concentration. At
pH 4.5 the isoelectric pH of soy protein, the solubility increases with ionic strength. Shen
(1981) studied the effect of various neutral salts on the solubility of soy protein and showed
that, as the salt concentration increased, the solubility of soy protein decreased up to 0.2M
for all salts and then increased at higher salt concentrations. The initial decrease in solubility
is due to decreased electrostatic repulsion and enhanced hydrophobic interaction between
Table 1.7: Functional properties of food proteins in various food systems*
Functional property
Mode of action
Food system
Solubility Water absorption and binding Viscosity Gelation Cohesion –adhesion Elasticity Emulsification Fat absorption Flavour binding Foaming
Protein solvation Hydrogen bonding of water, entrapment of water. Thickening, water binding Protein matrix formation and setting Protein action as adhesive material Hydrophobic bonding in gluten, disulfide links in gels Formation and stabilization of emulsion Binding of free fat Adsorption, entrapment, release Formation of stable films to entrap gas
Beverages Meats, Sausages, breads, cakes Soups, gravies Meats, curds, cheese Meats, Sausages, baked goods, cheeses, pasta products Meats, bakery products Sausages, bologna, Soup, cakes Meats, sausages Simulated meats, bakery goods Whipped toppings, chiffon deserts, angel food cakes
Source: Kinsella (1979)
30
protein molecules as a result of electrostatic shielding of charged groups in proteins by ions
soy proteins are least soluble at their isoelectric region (pH 4.2 to 4.6), and solubility sharply
increases above and below this pH range.
Solubility of groundnut proteins is affected substantially by variations in pH and
ionic strengths of the extracting medium (Rhee et al., 1972; Ayres et al., 1974; Basha and
Chery, 1977; McWatters and Holmes 1979b). Groundnut proteins have minimum solubility
in the pH 1.0 and above pH 7.0. Various ions (mono and divalent) at concentrations ranging
from 0 to 1M showed suppressing effects on the extractability of groundnut proteins at pH
7.0 and higher. However at low pH (3.5 to 5.5) both mono and divalent ions enhanced
extractability.
Nitrogen solubility profiles are also affected by varietal differences (Conkerton and
Ory, 1976), degree of heat treatment (Cherry et al., 1975; Schmidt et al., 1984), growing
locations and storage conditions (Cherry et al., 1975), conditions of processing and storage
of products also have profound effects on solubility (Ahmed and Schmidt, 1979).
The extent and method of protein modification also affect the solubility of groundnut
proteins. Enzymatic hydrolysis of groundnut flour proteins with pepsin, trypsin and
bromelain substantially increased the nitrogen solubility in water at pH (4.0 to 5.0) and 4.0 to
11.0 in the presence of 0.03M calcium ions (Beuchat et al., 1975). Nitrogen solubility of
groundnut proteins partially hydrolysed with papain was also improved at all pH levels
without formation of bitter peptides (Sekul et al., 1978). The solubility of succinylated
proteins increased slightly in the isoelectric pH range (4 to 5.0), decreased at pH levels
below the isoelectric region, and increased substantially at pH 6.0 to 7.0 (Beuchat, 1977b).
Fermentation of groundnut flour with various microorganisms also substantially altered the
solubility profile (Quinn and Beuchat, 1975).
31
Emulsification Capacity
An emulsion is a dispersion of oil droplets in a continuous liquid phase. In an
emulsion dispersed phase is referred to as discontinuous phase and the dispersion medium as
continuous phase. Emulsifying agents are amphiphilic molecules in which one portion of the
molecule is soluble in water while the other portion is soluble in nonpolar solvents. Being
amphiphilic, proteins will tend to orient at the polar-nonpolar interface and possess good
emulsifying properties. Solubility and hydrophobicity of proteins play major role in
determining the emulsifying properties.
In general, emulsions are of the oil in water type, although some foods such as butter
and margarine are of the water in oil type. The ability of soy protein to aid in the formation
and stabilization of emulsions is critical for many applications in comminuted meats, cake
batters, coffee whiteners, milks, mayonnaise, salad dressings and frozen desserts.
Emulsification capacities of oilseed proteins have been reported to be affected by
protein solubility, pH and protein concentration. Crenwelge et al., (1974) reported a general
positive correlation between emulsification capacity and solubility of soy protein
concentrate. Soy protein isolates show greater emulsifying capacity compared to soy protein
concentrate (Hutton and Campbell 1977). Many workers have shown a close relationship
between emulsifying properties and solubility of soy preparations (Inklaar et al., 1969,
Yasumatsu et al., 1972a,b). The pH, ionic strength, and temperature of a food system also
affect emulsifying properties of soy proteins (Kinsella 1976, Hutton and Campbell 1977).
The pH and salt enhances the emulsifying properties of several soy isolate preparations
(Stone and Campbell, 1980). The emulsification capacity follows the typical pH solubility
profile (Frazen and Kinsella, 1976a,b). Aoki et al., (1980) showed that the emulsification
properties of soy isolate and soy 11S and 7S globulins showed maximum at around pH 10.
32
Emulsions prepared between pH 3 and 6 from native or denaturated soy proteins have been
shown to be unstable as measured by lipid release (Kamat et al., 1978). Succinylation of soy
proteins improves emulsifying capacity and enhances emulsion stability (Frazen and Kinsella
1976a). Limited pepsin hydrolysis of soy protein has been shown to improve the emulsifying
properties. (Rham et al., 1978; Zakaria and Mc Feeters, 1978).
McWatters and Cherry (1975) reported that wet heating improved the emulsification
capacity of groundnut flour. Effects of pH, salt concentration and flour concentration on
emulsifying properties of defatted groundnut flour have been reported (Mcwatters et al.,
1976; McWatters and Holmes, 1979a). Water dispersions produced more viscous emulsion
than salt dispersion, and increase in protein concentration reduced the emulsion capacity and
increase the emulsion viscosity (Ramanatham et al., 1978; McWatters and Holmes, 1979a).
Groundnut protein isolate had higher emulsion capacity than groundnut flour and emulsion
capacity and viscosity were increase by increasing the finess of groundnut flour
(Ramanatham et al., 1978)
Mild succinylation significantly increased the emulsion capacity of groundnut flour,
but extensive succinylation decreased it (Beuchat, 1977b; Shyamasunder & Rao, 1978).
Extensive enzymatic digestion of groundnut flour with pepsin, trypsin and bromelain
completely destroyed the emulsifying capacity of groundnut flour (Beuchat et al., 1975,
Sekul et al., 1978).
Foaming properties
Foams are composed of gas bubbles encapsulated by a thin film of hydrated surface-
active agent dissolved in a liquid. Proteins form a flexible, cohesive film around air bubbles
and facilitate the formation of bubbles. Although proteins contain both hydrophobic and
hydrophilic environment, the balance is generally during the whipping process, the proteins
33
partially unfold and undergo surface denaturation followed by exposure of hydrophobic
regions. This induces the reduction in surface tension at the interfaces and facilitates the
stabilization of the films around the bubbles. Also proteins provide high surface area, surface
energy and low density and allow air bubbles to encapsulate by forming a thin layer. After
formation foams move closer to each other until touching. Gradually, the capillary pressure
pulls the bubbles close to each other and cause depression in the bubbles, which expands the
surface and causes coalescence and collapse of the foam. Thus foams are generally less
stable (Kinsella 1985).
Proteins are used as foam stabilizing agents in a variety of processed foods such as
cakes, sugar, confectionary and whipped desserts. Foaming capacity of a protein depends on
the capacity of the protein to adsorb, reorient and undergo surface denaturation. These
factors generally depend on the molecular weight, surface hydrophobicity, amino acid
composition, internal bonding and molecular flexibility. Foam stability is the manifestation
of the capacity of protein to rupture and its permeability to gas. Most proteins spontaneously
diffuse to air water interface and adsorb, reorient and unfold because of the gain in hydration
and conformational energy. In a foam the charged groups are placed together at the interface
must have enough kinetic energy to overcome the charge-charge repulsion induced by the
molecules already adsorbed (Phillips 1977; Graham and Phillips 1976, 1980 a, b, c). At
isoelectric pH proteins self-associate because the net charge and electrostatic repulsion is
minimum. Also the energy barrier that opposes the adsorption at the interface is lowest at
this point. When the pH moves away from the isoelectric pH, the net charge increases and
the film strength and foam stability will decrease. In general, a protein film formed at the
interface by adsorption may contain around 8mg protein per square meter depending on the
surface property (Mac Ritchie 1978; Graham and Phillips, 1976). An ideal whipping protein
34
should have high surface hydrophobicity, good solubility and low net charge at the pH of the
food in which it has to be incorporated.
Several factors affect the foaming properties of proteins, namely protein
concentration, pH, temperature, salt, sugars and lipids. At the isoelectric pH the electrostatic
attractions are maximum, proteins self associate and assume a compact state resulting in
maximum reduction of solubility (Mita et al., 1977, 1988; Kim and Kinsella, 1985).
Several workers have described the foaming properties of oilseed proteins. (Lawhon
et al 1972; Lin et al. 1974; Kinsella, 1976). Numerous aerating or whipping agents have been
prepared from soy proteins (Wolf, 1970). Betz and Stepaniuk (1972) showed that extraction
of soy flour with an aqueous solution (pH 4.0-6.0) yielded a whipping agent that could be a
replacement or extender for egg whites. Many preparations that show excellent foam
expansion are of little practical value because of instability.
The foaming properties of soy proteins isolates are usually superior to flours and
concentrates (Fleming et al., 1974; Lin et al., 1974). Lah et al., (1980) reported that full fat
soy flour had good foaming properties after heating up to 800C. Foaming properties are
closely correlated with protein solubility (Yasumatsu et al., 1972). Presence of lipid
materials in soy preparations reduces the foaming because they destabilize protein films.
Hexane and aqueous alcohol treatments of soy proteins remove neutral and bound lipids and
enhance foaming properties (Eldridge et al, 1963; Yasumatsu et al., 1972b). The foaming
capacity of soy protein showed pH dependence that resembled the pH solubility profile, it
was lowest in the isoelectric range (pH 4-5). The foam formed showed maximum stability in
this pH range (Tornberg, 1979). However, this is concentration dependent because high
levels of salt depress foaming (Eldridge et al., 1963).
35
Proteins can be modified to a limited extent to enhance foaming properties (Frazen
and Kinsella, 1976a, b). Partial hydrolysis of oilseed proteins by reducing molecular weight
and possibly by allowing some unfolding, facilitates the formation of an interfacial
membrane, which in turn enhances surfactant properties. However, extensive hydrolysis and
reduction of molecular size beyond a critical stage impair the capacity of the hydrolysates to
form cohesive interfacial membranes with the rigidity necessary for the formation of stable
foam. Thus, retention a high degree of tertiary structure in the protein is needed for foam
formation and maximum stability. Although partially hydrolysed soy proteins form better
foams, their stability was lower than that of the unhydrolysed proteins (Halling 1981). A
decline in foam stability has been observed with increasing acid, alkaline or enzymatic
hydrolysis of soy protein (Adler-Nissen and Olsen, 1979; Puski, 1975). Lawhon et al.,
(1972) reported that foams prepared from groundnut flour had substantially higher viscosity
than those from other oilseed flours. Foaming and foam stability of groundnut meal
dispersions were influenced by pH adjustment (McWatters et al., 1976; Cherry et al., 1979).
Sekul et al., (1978) reported that foaming capacity and foam volume of groundnut proteins
were increased significantly by partial hydrolysis with papain. However drying methods
(freeze drying, spray drying and drum drying) did not seem to have any significant effect on
the foaming capacity of groundnut proteins (Ahmed and Schmidt, 1979). Khalil et al., (1985)
found that defatted groundnut flour had higher foaming capacity than either whole
groundnuts or groundnut protein isolates. The isolates however, had the best foam stability.
Foaming capacity of all products was adversely affected by dry heat at 100°C.
Water absorption capacity
The water binding capacity of proteins includes all types of hydration water plus
some water remaining loosely associated with the protein following centrifugation (Kuntz
and Kauzmann, 1974; Chou and Morr, 1979). Several factors such as amino acid
36
composition, protein conformation (shape and size), surface topography, surface charge and
polarity, ionic concentration, pH and temperature affect water binding by proteins (Kinsella
et al., 1985).
Heating to unravel tightly folded tertiary and quaternary structures enhance water
binding by forming newly structured networks. The extent of hydration correlates strongly
with the content of polar residues and charged residues. Interactions between water
molecules and hydrophilic groups (hydroxyl, amino, carboxyl, amide groups etc.) occur via
hydrogen bonding (Chou and Morr, 1979). Water binding is affected by pH and salts (Kuntz
and Kauzmann, 1974; Bull and Breese, 1976). This general behaviour may not consistently
hold good for all proteins. Thus, in certain oligomeric proteins (e.g. soybean) low salt
concentrations facilitate hydrophobic association and reduce hydration by masking
electrostatic repulsive charges (Damodaran and Kinsella, 1981, 1982). Succinylation of the
∈ amino groups of lysine causes electrostatic repulsion between the polypeptides. In
addition, the increased charge density considerably enhances the water binding capacity of
oilseed proteins (Kinsella and Shetty, 1979).
The capacity of protein to absorb water is important in meat systems where it affects
juiciness, tenderness and taste. In meat analogs, the capacity to imbibe and hold moisture and
simulate the juiciness and texture of meat is critical. Soy protein by virtue of thermal
gelation, binds and holds water very effectively. However, the capacity to retain water
following processing (e.g. cooking) is essential in meat systems.
Soy protein preparations hold various amounts of water (Hutton and Campbell,
1981). Soy flours with protein dispersibility indices of 85, 70, 55 and 15 adsorbed 2.1, 3.7,
3.8 and 2.7g water per gram solids respectively. Fleming et al. (1974) reported that soy flour,
soy concentrate and soy isolate had water-holding capacities of 2.6, 2.75, and 6.25g per g
37
solids. Lin et al. (1974) showed that flour concentrates and isolates bound 1.3, 2.2 and 4.4g
water/g solids. This indicates variability in literature data but reveal increased water holding
with increased protein content. Fleming et al. (1974) reported that salt (5%) enhanced the
water-holding capacity of soy flour but reduced that of soy isolate. Water holding capacity is
minimum at isoelectric pH, maximum at pH 7.0. Soy protein concentrate held 241 and 340g
water per 100g proteins at pH 5.0 and 7.0 respectively (Hutton and Campbell, 1977, 1981).
Heating soy isolate (Dry) at 150°C for 30min in the presence of 0.5M urea increased water-
holding capacity to 50g water per 100g protein (Ochiai-Yanagi et al., 1978). There is no
reported systematic study of the effects of heat treatments and of the interaction of heat and
salts on the water-holding capacity of soy proteins.
Fat absorption capacity
Many important properties of foods involve the interactions of proteins and lipids.
Natural or chemically formed lipoprotein complexes are functional components of egg yolk,
milk, meats, coffee whiteners, and dough and cake batters. The ability of a protein to bind fat
is very important for its application in food systems. The key role of fat in food flavouring
lies in its capacity to improve flavour carry over in simulated foods during processing. The
mechanism of fat absorption is attributable mostly to physical entrapment of oil by the
protein (Kinsella, 1976). The surface properties of the protein play an important role in fat
absorption capacity.
Processing methods and conditions, type and degree of modification and pH affect oil
absorption properties of groundnut proteins. Beuchat et al., (1975) reported that heating
groundnut flour dispersions at pH 4.5 and 7.6 enhanced oil absorption capacity. Protein
isolates produced by ultra filtration had higher oil absorption capacities than isolates
produced by conventional alkaline extraction and acid precipitation from groundnuts,
38
soybeans and cottonseed (Manak et al., 1980). Enzyme hydrolysis and succinylation of
groundnut proteins has also improved oil absorption properties (Beuchat et al., 1975).
The possibility of changing the functional properties by blending plant proteins has
been examined on a limited scale. Groundnut proteins combined at low concentrations with a
whey protein gel matrix formed stronger gels than if used alone (Schmidt et al., 1984).
Cockerton and Ory (1983) have reported that by blending groundnut and rice bran flours, the
water absorption and fat absorption could be increased as the amount of rice bran increased.
Emulsifying capacity and emulsion stability was higher in all blends.
Enzymatic hydrolysis of food protein
The various reasons for carrying out enzymatic hydrolysis of food proteins are to
improve the nutritional characteristics, improving foaming and emulsifying capacity,
removing off flavours and toxic ingredients. Generally the effect of enzymatic hydrolysis of
protein involves reduction in molecular size, increase in the number of ionizable groups,
exposure of buried regions of the molecule to the aqueous environment. This is attributed to
the overall change in functionality.
Mechanism of hydrolysis
Hydrolysis of a protein involves the degradation of peptide chain in the presence of
water to form peptides with various size and free amino acids. The catalytic mechanism of
hydrolysis by proteases generally involves three consecutive steps. 1. Formation of
Michaelis complex between the substrate and enzyme 2. Cleavage of the peptide bond to
liberate one of the two resulting peptides 3. Nucleophilic attack on the remains of the
complex to split of the other peptide and to reconstitute the enzyme.
39
C COO-
R1
H
Protease +C CO N
R1
H
H
R2
C
H
H3N C
H
R2
+
Hydrolysis of food proteins can also be carried out by using either acid or alkali.
Acid hydrolysis is difficult to control and results in hydrolysates with reduced nutritional
quality as a result of destruction of essential amino acids, alkali hydrolysis cause
racemization of amino acids and can form toxic substances like lysinoalanine. Hydrolysis
using proteolytic enzymes allows control over the process and the properties of the resulting
products. The ability of an enzyme to hydrolyse a protein varies with the specificity of the
particular enzyme employed in the process. Therefore choice of a suitable enzyme is an
important criterion for the preparation of protein hydrolysates. Other parameters such as pH
optimum, thermo stability, price and availability of enzyme also have bearing on the choice
of an appropriate enzyme for hydrolysates.
The extent of hydrolysis of a protein can be quantified by measuring the degree of
hydrolysis, which refers to the percentage of peptide bonds cleaved. When the hydrolysis
proceeds the number of amino groups increase as a result of peptide bond cleavage. Adler
Nissen et al. (1976) developed accurate method for the determination of degree of hydrolysis
of a food protein by TNBS method. Based on the DH protein hydrolysates can be classified
as partially or extensively hydrolysed protein. Partially hydrolysed proteins are useful as
functional ingredients in various food products. Extensively hydrolysed proteins generally
find applications in nutritional formulae for infants with allergy to intact proteins, patients
with impaired gastrointestinal function and nutritional supplements to provide nitrogen in an
easily assimilated form. They are also used in beverages to improve nutritional quality.
40
The resistance of a protein for enzymatic hydrolysis generally depends on the overall
structure and conformation of the protein and the specificity of the enzyme used. In a native
protein the compact super secondary coiling generally shields the peptide linkage for
proteolytic attack by enzymes. Denaturation of a protein involves the collapse of the original
three-dimensional structure of the proteins and makes the peptide bonds easily accessible for
proteolytic attack and hence increases the rate of hydrolysis.The proteolytic enzymes can be
classified as endoproteases and exoproteases depending on the mechanism of action.
Endoproteases act randomly within the protein molecules and produce peptides with larger
size. Exoproteases systematically cleave the protein either at N or C terminal to produce
peptides with relatively smaller size. Therefore sequential action of both endoprotease and
exoprotease can be used to produce protein hydrolysates with smaller molecular size.
Casein and whey protein are the two important sources of food proteins used in the
preparation of protein hydrolysates for use in various nutritional formulations because of
moderate cost, commercial availability and good nutritional value. Other sources such as
fish, meat, collagen and gelatin are not preferred because of economic reasons. Plant proteins
can be preferred over animal proteins because they are available in plenty as byproducts of
oilseed utilization and cheaper in cost compared to proteins from animal sources.
Soybean and groundnut are two important oilseeds utilized all over the world as main
sources of oil and protein. The high protein products such as protein concentrate, isolate and
defatted flours are rich in protein content and can be used as a starting material for the
preparation of protein hydrolysates. Sekul and Ory (1976, 1977) have reported reaction
conditions for partial hydrolysis of groundnut flour by three commercial proteases and
showed that the insolubility at isoelectric pH region can be eliminated by partial hydrolysis.
The use of proteolytic enzymes to modify and extract protein from groundnut flour and press
cake has been shown promise with respect to enhancing certain functional properties
41
(Beuchat et al 1975; Beuchat, 1977b). Enzymatic modification of groundnut flour by papain
and fungal protease has been shown to improve the foaming capacity, stability and
emulsification capacity near the isoelectric pH range (Subba Rau and Srinivasan 1988).
Among oilseed proteins, substantial amount of work on the preparation of protein
hydrolysate has been carried out using soybean. Soybean protein can be used for the
preparation of soluble and functional protein hydrolysates. The early example of the
preparation of soluble hydrolysate was given by Sugimoto et al. (1971). They hydrolysed soy
isolate with acid protease and used in a lemon-flavoured beverage. Puski (1975) carried out
first systematic investigation on the preparation of functional soy protein hydrolysate and
reported that limited hydrolysis of soy protein isolate with Aspergillus oryzae protease
increased the solubility, emulsification capacity, foaming capacity and water absorption
capacity. Adler Nissen and Olsen (1979) hydrolysed soy protein isolate with alcalase at pH
8.0 and Neutrase at pH 7.0. They found that by limited hydrolysis the isoelectric solubility,
emulsifying capacity and foaming capacity increased, if the reaction continued higher DH (>
5%) the emulsifying and foaming properties decreased. Bobalic and Toranto (1980)
evaluated defatted soy flour partially modified with papain for foaming, water absorption
and foaming capacity. Zakaria and Mcfeeters (1978) reported increase in surface-active
properties of soy protein treated for short incubation periods with pepsin. Adler Nissen et al.
(1983) obtained a functional protein hydrolysate with high foam expansion and foam
stability by hydrolysis of soy protein concentrate to a DH between 3-5% with alcalase. The
use of ultrafiltration after enzymatic hydrolysis has been investigated on the pilot plant scale
by using a number of process parameters (Olsen and Adler Nissen, 1981; Olsen, 1984). The
higher molecular weight hydrolysates has been found to have excellent foaming properties
and able to heat coagulate so that they could substitute egg white. Several publications report
limited proteolysis with different enzymes improved the foaming and emulsifying properties
42
of protein ingredients (Adler Nissen 1982, Adler Nissen et al. 1983). By controlling the
extent of hydrolysis, the problem of bitterness also could be avoided (Adler Nissen 1979).
43
SCOPE AND OBJECTIVES
Proteins are important in food processing and food product development, as they are
responsible for various functional properties that influence consumer acceptability. Both
animal and plant proteins are used commercially as functional ingredients. Plant proteins are
the most abundant in the world. A number of vegetable proteins have been tried for
incorporation in various food products as functional and nutritional ingredients.
Oilseed protein products are rapidly gaining importance in protein supplementation
because of their unique functional properties. The intrinsic properties of proteins like the
amino acid composition and conformation of the proteins, methods and conditions for their
isolation, degree of purification and processing alterations are some of the important factors
that influence the functional properties of food proteins (Kinsella 1979). Various approaches
like chemical modification, physical treatments and enzymatic modification have been tried
to improve functional characteristics of proteins. Chemical modifications such as
succinylation, acetylation and mild alkali hydrolysis have been reported to improve the
functional properties. One of the major drawbacks of these approaches is the deterioration
of the nutritional quality owing to the blocking or destruction of essential amino acids. One
of the important ways to enhance the functional properties of oilseed proteins is enzymatic
modification (Panyan and Kilara 1996). Enzymatic modification occur under mild conditions
retaining nutritional value and offer a convenient means for improving functional properties
of proteins. By controlling the extent of hydrolysis it is possible to enhance various
functional properties to develop new functional ingredients to fabricate new food analogs
simulating traditional foods.
Protein hydrolysates find application in special foods such as those designed for
children, old people, athletes and also in pharmaceutical preparations developed for
convalescents and those who suffer from digestive disorders (Mahmoud 1994, Frφkjaer
44
1994). Foods based on highly hydrolysed proteins are useful in controlling food allergies
(Schmidl et al., 1994). The major ways of supplying tailored amount of amino acids are i)
enzymatic protein hydrolysates and ii) a mixture of synthetic amino acids. They are preferred
over synthetic amino acids at moderate cost because of availability on commercial scale and
high quality of enzyme hydrolysed products. Enzymatic protein hydrolysates containing
short chain peptides with defined amino acid composition and molecular size are preferred
for specific formulations (Siemmensma et al.,1993). These protein hydrolysates score over
elemental diets in which the protein component consists exclusively of a mixture of free
amino acids. The short chain peptides are absorbed preferentially over free amino acids in
the gut. Protein hydrolysates offer as an alternative to intact proteins and elemental formula
in the development of special formulations designed to provide nutritional support (Clemente
2000).
Soybean and groundnut are the most widely cultivated oilseeds all over the world.
Dehulled soybean contains 17-18% oil and 25-35% protein depending on the variety.
Similarly, dehulled groundnut depending on the variety contains 50-60% oil and 30-35%
protein. Defatted soybean flour contains 50-55% protein of good nutritional quality. Soybean
proteins are rich in lysine and deficient in methionine. The major intrinsic anti nutritional
factor is trypsin inhibitor, which affects its utility. Groundnut proteins are deficient in lysine
and methionine. The protein digestibility corrected amino acid score for soy protein and
groundnut protein is 0.92 and 0.52 respectively (FAO/WHO 1990). The protein ingredients
such as defatted flour, protein concentrate and isolated proteins from these sources have a good
potential for preparing speciality foods under native and modified conditions. The protein
hydrolysates obtained from these sources can effectively replace commercially available milk
protein hydrolysates because of their good nutritional quality.
45
In general, oilseed proteins offer resistance to enzymatic hydrolysis. Studies on
hydrolysis of pure proteins would help in understanding of the various structural features of
the protein both at surface and subunit levels. Though the resistance to hydrolysis of seed
proteins by enzymes is well documented, work on the relative effectiveness of the major
proteins with different proteolytic enzymes is less. Glycinin and conglycinin are the major
fractions of soybeans and make up to 70% of the total proteins. Arachin and conarachin are
the major fractions, which make nearly 80% of the total groundnut proteins (Prakash and
Rao 1986). The individual roles of major protein fractions are important in order to
understand the overall functional profile of the total proteins and protein ingredients.
Glycinin is poorer in functional properties compared to conglycinin. This has been attributed
to the compact structure of the high molecular protein fraction in which hydrophobic groups
are buried inside (Kim et.al., 1987a, Wagner and Gueguen 1995). The functional properties
of protein isolates and isolated fractions can be modified either by chemical means or
enzymatic hydrolysis. Although sufficient information is available on the enzymatic
modification of soybean or groundnut flour and its effect on functional properties, the
information on the controlled hydrolysis of major globulins of oilseeds are limited.
With this available information the present investigation entitled “Studies on the
preparation and characterization of protein hydrolysates from groundnut and soybean
isolates” was undertaken. In this work, protein hydrolysates from soybean and groundnut
were prepared using different enzymes starting with different materials like defatted meal,
protein isolates and purified fractions. With the use of different enzymes and by varying the
experimental parameters like E/S ratio, pH, temperature and combinations of proteolytic
enzymes it was possible to tailor the functional characteristics of various protein
hydrolysates. Attempt has also been made to correlate various functional characteristics with
some of the biophysical and biochemical parameters.
46
MATERIALS AND METHODS
MATERIALS
The chemicals and reagents used and their sources in parentheses are as follows:
Sepharose-6B, blue dextran-2000, trypsin, casein, tris salts, sodium dodecyl sulphate (SDS),
urea, acrylamide, bis-acrylamide, β-mercaptoethanol, N, N, N', N'- tetra methyl ethylene
diamine(TEMED), coomassie brilliant blue R-250, 2,4,6-trinitrobenzene sulphonic acid
(TNBS), ammonium persulfate, benzoyl-DL-arginine-p-nitroanilide (BAPNA), iodoacetam-
ide (Sigma Chemicals USA); calcium chloride, copper sulfate, potassium sulfate, potassium
hydrogen phthalate, sodium chloride, sodium hydrogen phosphate, disodium hydrogen
phosphate, boric acid(SD fine chemicals India). hydrochloric acid, sulphuric acid,
trichloroacetic acid(TCA), sodium hydroxide, ethanol, hexane, petroleum ether, methanol
(Qualigens Chemicals India). Methyl red, methylene blue and bromophenol blue (Himedia
Laboratories Pvt. Ltd. India); Dialysis tubing (Thomas Scientific Co., Philadelphia, USA).
Postman brand refined groundnut oil (Ahmed Mills, Mumbai, India). All the other chemicals
and reagents were of analytical grade.
Authentic variety, TG-3 of yellow soybean and TMV-2 of groundnut seeds were procured
from agricultural university of Dharwad.
Commercial papain (from papaya latex) from Enzochem. Laboratories Pvt. Ltd, Yeola
(India), fungal protease (Aspergillus oryzae) from Amano Pharmaceutical Co. Ltd., Nagoya,
Japan, Alcalase 2.4L (Bacillus licheniformis) from Novo Laboratories, Denmark, were used
in the studies.
METHODS
Preparation of defatted groundnut flour
Groundnuts were decorticated and the seeds were decuticled by mild roasting at
55±2ºC for 10 min, cooled and later subjected to mechanical abrasion. The testa was
separated from the kernel by using an air blower. The moisture content of the decuticled
kernel was adjusted to 12% by adding water and flaked in a flaking machine, maintaining the
drum clearance of 2.0mm. The flakes were dried at 27ºC in a cross flow drier and the dried
flakes were defatted by repeated extractions with food grade hexane. The defatted meal was
47
air dried and vacuum dried to remove residual solvent and ground, passed through 60-mesh
sieve. The fat content of the final extracted meal was less than 1%. This defatted flour was
used for all the studies.
Preparation of defatted soybean flour
Soybean seeds were cleaned, graded in a grading machine to remove stones and
impurities. Water was sprayed on the seeds to raise the moisture level by 2%. The
conditioned seeds were dried in an electrically heated roaster at 50-60ºC. The dehulling was
done by passing through a plate type mill with an attached air blower. The dehulled seeds
were equilibriated to 20% moisture and passed through a flaking machine maintaining a
drum clearance of 0.3 to 0.5 mm to obtain flakes of 0.3mm thickness and dried to 5%
moisture level. The dried flakes were defatted by repeated extractions with food grade
hexane, vacuum dried to remove solvent and ground and passed through 60 mesh sieve. The
fat content of the final extracted meal was <1.0%.
Estimation of total protein
The nitrogen content was determined by the Micro-Kjeldahl procedure (AOAC,
1990). A known weight pf (~300 to 500mg) of the sample was digested with conc. H2SO4 in
a Buchi digestion flask using a mixture of copper sulphate, potassium sulphate and selenium
dioxide as catalyst till the sample becomes colourless. After cooling the contents were
quantitatively transferred to a 100 ml standard volumetric flask and volume made up with
distilled water. An aliquot (5 ml) was distilled with 40% NaOH. The liberated nitrogen in the
form of ammonia was trapped in about 25 ml of 2% boric acid solution to which 0.5 ml of
mixed indicator was added (methyl red + methylene blue). After distillation, the boric acid
solution containing absorbed ammonia was titrated against N/70 HCl. From the titre value
the amount of nitrogen present in the sample was calculated through calibration of the titre
48
value using standard (NH4)2SO4 solution. A conversion factor of 6.25 was used to convert
the nitrogen content (%) into protein content in the sample.
Estimation of fat
The fat content was estimated by the procedure outlined in AOAC (1990). Known
quantity (5 to 10g) of sample was taken in extraction thimble and the fat was extracted for 4h
with petroleum ether in a soxhlet extractor. The solvent was removed from the extract; the
fat was dried by heating at 100ºC for 2h and weighed. The moisture content and ash content
was also estimated by AOAC (1990) method.
Determination of trypsin inhibitor activity
Trypsin inhibitor activity (TIA) was determined according to the procedure of
Kakade et al., (1974) using benzoyl-DL-arginine-p-nitroanilide hydrochloride as substrate
and bovine trypsin. One gram of finely ground sample was extracted with 50 ml of 0.01M
NaOH for 3h and centrifuged. The sample extract was diluted so that 2 ml of the sample
extract inhibit 40-60% of the trypsin used as standard inhibit 40-60% of the trypsin used as
standard for analysis. Aliquots of 0.5, 1.0, 1.5 and 2.0 ml of this extract was taken in
different test tubes and diluted to 2.5 ml with distilled water. 1.5 ml of trypsin solution was
added to each test tube and mixed thoroughly. A blank was prepared by the same procedure
except adding the trypsin after adding of 1ml 30% acetic acid. Exactly after 10 min, 1 ml of
30% acetic acid was added and mixed immediately. The OD at 410nm was recorded. One
trypsin unit (TIU) is arbitrarily defined as differential absorbance of 0.01 units at 410nm.
The absorbance was converted to TIU and plotted against sample volume and extrapolated to
zero concentration.
Y intercept (TIU/ml) Χ dilution factor
Sample wt (g) TIU/g sample =
49
Lipoxygenase activity
The lipoxygenase activity was determined according to the procedure of Axelrod et.
al, (1981). Linoleic acid (70 mg) and 70 mg of Tween 20 were dissolved in 4.0 ml of
oxygen-free water. The resulting dispersion was clarified by adding sufficient amount of
0.5M NaOH. Final volume was made up to 25 ml with water. This stock solution was
divided into l-2 ml portions in small vials and flushed with nitrogen as before closing. The
enzyme was assayed by monitoring the increase in absorbance due to the formation of
conjugated diene at 234nm. The assay mixture consisted of 0.2M borate buffer pH 9.0,
0.1mM sodium linoleate and enzyme solution in a quartz cuvette of 1.0cm path length. One
unit of enzyme activity is defined as the amount of enzyme required to form one µmole of
product per minute under assay conditions. The extinction coefficient used for the diene
product was 25,000 M-1cm-1. The protein content was estimated by absorbance
measurements at 280nm with quartz cuvette of 1cm path length and using a value of E1%=14.
The lipoxygenase was extracted from soybean sample with ice-cold 0.2M sodium acetate
buffer (pH 4.5) for 1h. The suspension was centrifuged at 6000rpm and the aliquot from the
extract was used for the assay.
Determination of urease activity
The urease activity was measured according to the procedure outlined in AOCS
(1997) Buffered urea solution was prepared by dissolving 15g of urea in 500 ml of 0.5M
phosphate buffer solution (pH 7.0). 5 ml of toluene was added as a preservative to prevent
mould formation. The pH of buffered urea solution was adjusted to 7.0 using either 0.1M
acid or alkali. 0.2g of finely ground soybean sample was weighed into a test tube. 10 ml of
buffered urea solution was added and mixed thoroughly. A blank was prepared similarly
after inactivation of urease by heating at 135ºC for 30min. Both the sample and blank was
incubated for exactly 30 min in a water bath at 30ºC. The test tubes containing sample and
50
blank was transferred into a 5 ml beaker and the pH was determined. The difference between
the pH of the test sample (higher) and that of the blank was expressed as the urease activity
in the sample. The urease activity of defatted soy flour was 1 unit.
Enzyme activity measurements
Enzyme activities of different proteases were measured by its ability to hydrolyse
peptide bonds using casein as substrate. The tyrosine released during hydrolysis of casein
can be followed by spectrophotometric method after colour reaction with Folin reagent.
Preparation of substrate: To 1.5g of casein, 30 ml of 0.1N sodium hydroxide solution was
added, and heated in a boiling water bath for 10 min. After cooling, the pH was adjusted to
8.0 with 0.1N phosphoric acid solution. 20 ml of phosphate buffer (pH 8.0) was added in the
solution and the solution was made up to 100 ml with distilled water.
Tyrosine calibration curve: Various concentration of tyrosine solution (10 to 50µg/ml) was
made with 0.1NHCl. 1 ml of the tyrosine solution was added to 5 ml of 0.4M sodium
carbonate solution and 1ml of two fold diluted Folin reagent and mixed. The solution was
kept at 37ºC for 20 min. blank was prepared by omitting tyrosine solution. The OD (660nm)
was measured against reagent Blank and the standard calibration curve was made.
Assay Method: 1ml of 1.5% casein solution was placed in a test tube and preheated in an
incubator at 43ºC for 5 min. 1 ml of the enzyme solution was added and shaken thoroughly.
The solution was incubated at 43ºC for 60 min. At the end of incubation, 2ml of 0.4M TCA
solution was added and the precipitate was removed by filtration after standing for 25 min at
43ºC. Each 1 ml of the filtrate was added to 5 ml of 0.4M sodium carbonate solution. 1ml of
twofold diluted Folin′s reagent was added to the mixture solution. After mixing thoroughly
the solution was kept at 37ºC for 20 min. The developed blue colour was measured as optical
density at 660nm. The mixture containing 1 ml of water instead of the enzyme solution was
prepared in the same manner and taken as blank. The tyrosine released was quantified by
51
comparison with tyrosine calibration curve. Tyrosine released (mg) per mg of protein was
expressed as 1 tyrosine unit (TU) of activity. The activity of papain, alcalase and fungal
protease was measured under optimum conditions with respect to temperature and pH.
Papain T = 50ºC, pH = 6.2, Alcalase T = 55ºC; pH 8.0, Fungal protease T = 43ºC; pH = 8.0.
The activity of papain, alcalase and fungal protease was 11966, 25588 and 28600 TU/mg
protein, respectively (Arnon 1970).
Trichloroacetic acid (TCA) solubility index
The TCA solubility index was determined in 0.8M (13.6%) TCA. 10ml of the sample
(protein/hydrolysate) was mixed with 5ml of 2.4M TCA in a test tube and shaken well. The
content of the flask was centrifuged at 6000 rpm for 10min and the nitrogen content in the
supernatant was determined by Kjeldahl method. The TCA solubility index was expressed as
the percentage of the nitrogen content in the TCA soluble supernatant to the total protein
content (Adler-Nissen, 1982).
Degree of hydrolysis (DH)
DH is defined as the ratio of the number of peptide bonds cleaved (number of free
amino groups formed during proteolysis) expressed as hydrolysis equivalents (h), in relation
to the total number of peptide bonds before hydrolysis (htotal). The htotal is equivalent to the
amino acid composition of the protein expressed as m mol/g protein. The hydrolysis
equivalent or free amino group formed was determined by the TNBS reaction as leucine
amino equivalent (Adler Nissen 1979). The protein hydrolysate was dissolved/dispersed in
hot 1%SDS to a concentration of 0.25-2.5mM amino groups. 0.25ml sample was mixed with
2ml of 0.2125M sodium phosphate buffer (pH 8.2) and 2 ml 0.1% trinitrobenzene sulphonic
acid followed by incubation in the dark for 60 min at 500C. The reaction was quenched by
adding 4 ml 0.1N HCl, and the absorbance was recorded at 340nm. 1.5mM L-leucine
solution was used as standard. DH = h / htotal × 100
52
Preparation of enzyme modified defatted soy flour
Batches of defatted soy flour samples (10g) were dispersed in 100 ml-distilled water.
The pH of the suspension was maintained at 7.0 for enzymatic modification with papain, 7.8
for alcalase and fungal protease. The dispersion was subjected to enzymatic hydrolysis with
papain (50ºC) alcalase (55ºC) and fungal protease(43ºC) at 1% concentration. To obtain
modified samples with different DH incubation period of 10, 30 and 240 min was
maintained. The enzyme was inactivated by heating at 80ºC for 10 min. The resulting
modified DSF samples with different DH were cooled to room temperature, freeze dried and
stored at 4ºC for subsequent analysis.
Preparation of protein hydrolysate from defatted soy flour
Batches of defatted soy flour samples (10g) were dispersed in 100 ml-distilled water.
The pH of the suspension was maintained at 7.0, for enzymatic modification with papain, 7.8
for alcalase and fungal protease. The dispersion was subjected to enzymatic hydrolysis for 4h
with papain (50ºC) alcalase (55ºC) and fungal protease (43ºC) at 1% concentration. The
enzyme was inactivated by heating at 80ºC for 10 min. The resulting protein hydrolysate
samples were cooled to room temperature and centrifuged at 10,000 rpm. The hydrolysate
liquor was spray dried in a laboratory model spray drier to obtain isoelectric pH soluble
protein hydrolysates.
Evaluation of bitterness
The bitterness of spray dried protein hydrolysates prepared using papain, alcalase and
fungal protease were evaluated by sensory analysis using quinine hydrochloride as standard.
The different hydrolysate samples were dissolved in distilled water and used in the dilution
technique for identification of bitterness recognition threshold (Indian standards 1983).
53
Amino acid composition
Amino acid composition of defatted soy flour and spray dried protein hydrolysates
were determined by reverse phase HPLC on Water Picotag TM amino acids analysis system,
after hydrolysis of protein with 6N HCl to yield free amino acid. (Bidlingmeyer et al., 1984)
Preparation of protein isolate
The defatted flour (soybean/groundnut) was dispersed in water in the ratio of 1:10
(w/v), the pH of the dispersion was adjusted to 9.0 with 2N NaOH and the mixture was
stirred for 1h at room temperature. The insoluble residue was removed by centrifugation.
From the supernatant the protein was precipitated at pH 4.5 using 2N HCl. The isoelectric
precipitated protein was centrifuged and washed with water. The wet protein isolate was
dispersed in water and neutralized with sodium hydroxide. The slurry was homogenized in a
laboratory homogenizer and adjusted to 15% solids with water. The slurry was freeze-dried
or spray dried to obtain isolates. The protein content of (N x 6.25) SPI and GPI was 92.5%
and 94.2% respectively.
Defatted soy flour
Aqueous extraction pH 9.0 ,centrifugation
Residue Extract
Protein curd Whey
Washing & drying SPI-isoelectric form
Washing, neutralizing and drying soy protein isolate
Figure 2.1: Preparation of soy protein isolate from defatted soy flour
54
Preparation of Glycinin Glycinin was prepared from defatted soy flour by the procedure of Tanh and Sibasaki
(1976). Defatted soy flour was extracted with 63 mM tris-HCl buffer containing 10mM β-
mercaptoethanol, pH 7.8 (1:15 w/v) for 1h and centrifuged at 10,000 rpm. The supernatant
was adjusted to pH 6.6 using 2N HCl and dialysed against 63 mM tris-HCl buffer (pH 6.6)
and dissolved in standard phosphate buffer 0.05 M pH 7.6 containing 0.4M NaCl, 0.02%
sodium azide, 0.01M β-mercaptoethanol, and 0.001 M EDTA and dialysed against distilled
water in the cold and freeze dried to obtain crude glycinin. The purification was carried out
on a Sepharose-6B column.
Defatted soy flour
Extract with 0.03M Tris-HCl (pH 8.0) containing 0.01M mercaptoethanol centrifuge
Supernatant
Adjust pH to 6.4 with 2N HCl
Precipitate (crude 11S fraction)
Supernatant (crude 7S fraction + Whey protein)
Adjust pH to 4.8
Precipitate Supernatant
(Whey protein)
Precipitate (polymerized form)
Dissolve in 0.03M Tris –HCl Buffer, adjust pH to 6.2
Supernatant (7S fraction)
Figure 2.2: Simultaneous isolation of the soybean 7S & 11S globulin Source: Tanh & Sibasaki (1976 a)
55
Isolation of acidic and basic subunits of glycinin
Glycinin was modified by reduction of disulfide bonds and carboxymethylation of
SH group by iodoacetamide according to the method of Kitamura and Shibasaki 1975. The
purified protein (250mg) was dissolved in 25 ml of 1M tris buffer, pH 8.6 containing 6M
urea and 1mM EDTA. The solution was flushed with nitrogen for 15min. and β-
mercaptoethanol (0.25mM) was added. The solution was incubated at 37°C for 5h. After the
addition of iodoacetamide (1g). The reaction was allowed to proceed in the dark for 1h. The
protein was dialysed against distilled water and freeze dried. The modified glycinin (CAM-
glycinin) was used for the separation of acidic and basic subunits on a Dowex column. The
resin was washed exhaustively with 2M NaOH, water and 2M acetic acid and 0.05M tris-
acetate buffer, pH 8.0 and equilibrated on a column (2.5×20cm) with 0.05 M Tris-acetate
(pH 8.0) containing 6M urea. CAM- glycinin (250 mg) was loaded on the column. The basic
subunits were eluted with the same buffer and the acidic subunits with 0.05M acetic acid
containing 6M urea at pH 4.5. 3ml fractions were collected at a flow rate of 30 ml/h and the
absorbance monitored at 280nm. The acidic and basic peaks were pooled, dialysed against
distilled water and freeze-dried.
Hydropathy plot for glycinin subunits
The hydropathy profile of acidic and basic subunits of glycinin was evaluated using
the software WINPEP from the amino acid sequence according to the method of Kyte and
Doolittle (1982). In this method, each amino acid was assigned a value reflecting its relative
hydrophilicity and hydrophobicity. The values ranged from + 4.5 for isoleucine to - 4.5 for
arginine.
Isolation of arachin
Arachin was prepared from defatted groundnut flour by the procedure of Monteiro
and Prakash (1996). Defatted groundnut flour was extracted with 0.01M phosphate buffer
56
(pH 7.9) containing 0.5M NaCl (1:10) for 1h and centrifuged at 10000 rpm. To the
supernatant, (NH4)2SO4 was added to 18% saturation and the mixture was kept at 4ºC for 3h
and centrifuged. The precipitate was redissolved in the extraction buffer and again subjected
to (NH4)2SO4 precipitation to 18% saturation and centrifuged. The precipitate was dissolved
in minimum volume of extraction buffer and dialysed extensively against distilled water in
the cold and freeze dried to obtain arachin.
Defatted groundnut flour
Extract with 0.01M phosphate buffer (pH 7.9) containing 0.5M NaCl (1h ) at 270 C Centrifuge
Clear supernatant (Total proteins)
(NH4)2SO4 added to 18% saturation kept at 40 C for 3h, centrifuge
Precipitate supernatant (conarachin I & II)
Dissolve in Extraction buffer (NH4)2SO4 18% saturation
Precipitate dissolve in extraction buffer
Centrifuge
Dialysis against Distilled water
Lyophilise (Arachin)
Figure 2.3: Isolation of arachin from groundnut flour Source: Monteiro & Prakash 1996.
57
Time course enzymatic hydrolysis The time course enzymatic hydrolysis of soy protein isolate, glycinin, groundnut
protein isolate was carried out using papain, alcalase and fungal protease according to the
following procedure:
Papain hydrolysis- Different protein samples (20 mg/ml) were prepared in distilled water and
the pH was adjusted to 7.0 with 0.01N NaOH and incubated at 50, 60, and 70°C in a water
bath. Hydrolysis was carried out with 1% papain for varying time intervals (5 to 240 min).
At the end of each time interval, aliquots were withdrawn; enzyme was inactivated by
heating at 80˚C. After dilution with 2 ml of 2% SDS the DH was measured by reaction with
TNBS. The DH was also measured after hydrolysis at different enzyme concentration (0.5 to
5%) at 50˚C as a function of time.
Alcalase hydrolysis- Different protein samples (20 mg/ml) were prepared in distilled water
and the pH was adjusted to 7.8. Alcalase was added at different concentration (0.5 to 5%)
and incubated at 55˚C in a water bath. At the end of regular time intervals (5 to 240 min)
aliquot was withdrawn, enzyme was inactivated by heating (80°C) and the DH was
determined.
Fungal protease hydrolysis- This was carried out in a similar way as alcalase hydrolysis
except that the incubation temperature was 43°C. The DH was measured at regular time
intervals.
Preparation of Enzyme modified protein with low DH
Enzyme modified proteins with low DH were prepared from soy protein isolate,
glycinin, groundnut protein isolate and arachin. The samples (5g) were dispersed in 50 ml of
distilled water. The pH of the suspension was maintained at 7.0 for enzymatic modification
with papain and 7.8 for alcalase/fungal protease. The dispersions were subjected to
enzymatic hydrolysis with papain (50°C), alcalase (55°C), and fungal protease (43°C) for a
58
period of 10 min. The enzyme was inactivated by heating at 80°C for 10 min. The resulting
modified samples were cooled to room temperature and freeze dried. The degree of
hydrolysis (DH) of different modified samples were (3-5%). The samples were stored at 4°C
for subsequent analysis.
Preparation of enzyme modified protein with high DH
Enzyme modified proteins with high DH were prepared from soy protein isolate,
glycinin, groundnut protein isolate and arachin. The samples (5g) were dispersed in 50 ml of
distilled water. The pH of the suspension was maintained at 7.0 for enzymatic modification
with papain and 7.8 for alcalase/fungal protease. The dispersions were subjected to
enzymatic hydrolysis with papain (50°C), alcalase (55°C), and fungal protease (43°C) for
4h. The enzyme was inactivated by heating at 80°C for 10 min. The resulting modified
samples were cooled to room temperature and freeze dried. The samples were stored at 4°C
for subsequent analysis.
Calculation of Kinetic parameters (Km and Vmax)
The Michaelis constants for different enzymes (papain, alcalase and fungal protease)
used for hydrolysis of protein fractions (glycinin, glycinin subunits and arachin) were
determined at optimum temperature for their activity. Substrate concentration [ S ] was
varied between 0.25% to 3.0% and enzyme concentration was kept constant. The samples
were incubated for 10min and the enzyme was inactivated by heating. The reaction rate V
was measured as milliequivalents of amino group released by reaction with TNBS. The [ S ]
was plotted against [ V ]. The Km and Vmax was calculated from X and Y intercept of the
Lineweaver-Burk plot.
Gel filtration
Gel filtration was performed in Sepharose 6B (1.5 × 98 cm) column equilibrated with
0.05 M phosphate buffer (pH 7.8) containing 0.35M NaCl and 0.01M β-mercaptoethanol.
59
Sample (~80mg) dissolved in 3ml of elution buffer and dialysed extensively with the same
buffer and loaded on the column. The elution was carried out at a flow rate of 20 ± 2ml.
Fractions of 4ml were collected in automatic fraction collector, and absorbance of each
fraction was measured at 280nm. The peak fractions were dialyzed against distilled water
and freeze dried.
Sodium dodecyl sulfate polyacrylamide gel electrophoresis
SDS-PAGE was carried out using discontinuous buffer system as described by
Laemmli(1970) using 10% gels. Protein sample (1µg/ml) were prepared in a tris-HCl buffer,
(pH 6.8) containing 2% SDS, 10% glycerol, 5% 2-mercaptoethanol, and 0.002%
bromophenol blue and heated in a boiling waterbath for 5min. Each sample was applied on
the gel slots and electrophoresis was performed at constant current of 8mA/slot using
0.025M tris-0.3M glycine buffer, pH8.3 containing 0.1% SDS as electrode buffer. After
electrophoresis, the gel slab was removed and fixed in a solution of methanol, acetic acid
and water. (5:10:85v/v) stained with 0.1% coomassie blue R-50 and destained with the same
solvent.
Circular dichroism measurements
The near UV and far UV CD measurements were made with Jasco model J810
automatic spectropolarimeter. The instrument was calibrated with (+)-10-camphor sulfonic
acid. Quartz cells of path length 10mm were used for near UV (330-250nm) and 1mm for far
UV CD (190-260nm) measurement. The protein concentration in the samples were in the
range 0.3 to 2.0 mg/ml. A value of 114 was taken as the mean residue weight (MRW) for the
calculation of mean residue ellipticity. All measurements were made in 0.05M phosphate
buffer of pH 8.0.
60
Nitrogen Solubility vs pH
The nitrogen solubility was estimated by the method of Frazen and Kinsella (1986).
0.2g of protein samples were dispersed in 20ml of distilled water. The pH values of the
suspension was adjusted to desired value (2-10) with either 0.1N HCl or 0.1N NaOH. The
suspension was then shaken for 1h using a rotatory shaker at room temperature and
centrifuged at 6000rpm for 15min. The solubilized nitrogen was estimated by Kjeldahl
method. The nitrogen solubility was expressed as the percent of nitrogen content in the
supernatant to the total nitrogen content of the original sample.
Water absorption capacity (WAC)
The water absorption capacity (WAC) was determined according to the method of
Rahma and Rao (1983a). 6 ml of distilled water was added to 0.3g of lyophilized sample in a
previously weighed glass centrifuge tube. The tube was agitated in a Vortex mixer for 2 min
and centrifuged for 20 min at 3000 rpm. The supernatant was decanted and discarded. The
tube was weighed after the removal of the adhering drops of water. WAC was expressed as
weight of water bound per 100g protein.
Fat absorption capacity (FAC)
The FAC was determined according to the method of Sosulski et al (1976). 1g of the
sample and 6 ml of refined groundnut oil were added to a centrifuge tube. The tube was
agitated on a Vortex mixer for 5 min, left for 30min and centrifuged for 20 min at 3000 rpm.
The weight of free oil was recorded. The FAC was expressed as the amount of oil (g)
absorbed by 100g of sample.
Foam capacity (FC) and Foam stability (FS)
The FC and FS were determined according to the method of Lawhon et al (1972).
0.5g of the sample was dispersed in 100 ml of distilled water and whipped in a blender for
5min and the contents were poured into a 100ml-measuring cylinder along with the foam.
62
STUDIES ON SOYBEAN PROTEINS
ENZYMATIC HYDROLYSIS OF SOY FLOUR
Defatted soy flour prepared from soybean seeds as described under materials and
methods were evaluated for proximate composition. The protein content of the flour was
51% and the fat content was <1%. The comparison of the proximate composition of the soy
split beans and the defatted flour is made in Table 3.1.
The absorption of protein in the human body system can occur in the form of
peptides and amino acids. The transport mechanism of peptides in the intestinal mucus
membrane is different from that of free amino acids. The absorption of protein as short chain
peptides is considered to be more efficient way of amino acid absorption when compared to
an equivalent amount of free amino acids. This may be because peptides are less hypertonic
than free amino acids there by enabling good absorption, eliminating osmotic pressure.
Proteins are also important in food product development, because they are responsible for
various functional properties that influence consumer acceptance of food products. Soy
proteins have been widely used in various food formulations, as they possess distinctive
physicochemical and functional properties as well as have good nutritional value. One of the
approaches to enhance the functional properties of soy protein ingredients is by limited
proteolysis using different enzymes. In this respect the type of enzyme used and the methods
of processing conditions employed during the preparation are also important. Extensive
hydrolysis is generally resorted to recover more protein from the flour in the form of
hydrolysates.
The therapeutic uses of hydrolysates are limited largely to casein hydrolysates. But,
due to its high nutritional value and cheaper cost can also be used for the preparation of
protein hydrolysates. Enzyme hydrolysis is more efficient alternative for modification
63
Table 3.1: Chemical analysis of soy split beans and defatted soy flour
Constituents* Soy split beans DSF
Moisture (%)
Protein (% Nx6.25)
Fat (%)
Lipoxygenase activity(U/mg sample)
Trypsin inhibitor activity (TIU/mg sample)
9.1
38.3
19.1
61.0
41.0
6.8
50.3
0.7
66.0
45.2
* The values are averages of two independent determinations.
because of the mild conditions and the relative specificity of various enzymes, which may be
used to control various functional properties. In a typical hydrolysis process, protein and
enzyme are mixed together in a suspension and held several hours. When the desired DH is
reached the enzyme is inactivated by increasing the temperature or changing the pH. Various
proteolytic enzymes such as papain, alcalase and neutrase have been used to carry out the
hydrolysis of soy protein. (Adler Nissen 1976, Puski 1974).
In the present investigation papain, alcalase and fungal protease enzymes were used
for the modification of soy flour. Different modified soy flours were prepared by controlling
the degree of hydrolysis and the functional properties were assessed.
The DH of soy flour achieved by using different proteases for different periods of
hydrolysis are presented in the Figure 3.1 and 3.2. The DH of soy flour by different enzymes
increased with increase in hydrolysis time. The DH increased during the initial 30 min,
followed by a decrease in the rate. The DH for papain, was 4% at the end of 5 min and the
maximum DH achieved was 18-19% (Figure 3.1). But with alcalase and fungal protease
enzymes at the end of 5 min the DH were 5.8% and 6.8% respectively. The hydrolysis curve
plateaued at 24-25% DH for alcalase and 33-35% for fungal protease (Figure 3.2). From the
hydrolysis curve, it is evident that the rate of hydrolysis decreased to become very slow after
64
the initial rapid hydrolysis. The decrease in the specific peptide bonds available for the
enzyme action, product inhibition and enzyme inactivation could be the reasons for the
decrease in the rate of hydrolysis. The competition between the original substrate and the
peptides constantly formed during hydrolysis has been attributed to show this behaviour
(Adler Nissen 1976).
Functional properties of modified soy flour
It is clear from the hydrolysis curves for papain, alcalase and fungal protease (Figure
3.1 and 3.2) that it is possible to control the DH by varying the duration of hydrolysis. Soy
flour slurry (10%) was modified to different degrees by hydrolysing for 10, 30 and 240min
followed by freeze drying of the hydrolysed slurry. The papain modified samples showed a
DH of 4-5% and alcalase and fungal protease modified samples had 5-6% DH after the
termination of hydrolysis at the end of 10 min. The data pertaining to DH of modified flour
is shown in the Table 3.2.
The emulsification capacity of soy flour was 194.7ml/g. At a DH of 4-6% the
emulsification capacity was 223.5, 218.5 and 210 ml/g for alcalase, papain and fungal
protease respectively. The EC was almost equal to control when the DH was in the range of
8 to 15%. However, the emulsification capacity drastically decreased when the DH was more
than 20%. This shows that the emulsification properties of soy flour can be increased by
using different proteases and by controlling the extent of hydrolysis. The foaming capacity
of different enzyme modified flours with low DH was slightly higher than that of intact
flour. Soy flour showed a foaming capacity of 41.2% compared to 40-52% observed with
different low DH flours. No difference in the foaming capacities was observed between
enzyme modified low DH and high DH soy flour samples (Figure 3.3 A, B and C). Adler
Nissen and Olsen (1979) hydrolysed soy protein isolate with alcalase at pH 8.0 and neutrase
at pH 7.0 and found that by limited hydrolysis iso electric solubility, emulsifying capacity
65
and foaming capacity were all increased. When the DH was higher than 5% the emulsifying
and foaming capacities again decreased. The improvement in emulsifying properties of
casein by tryptic hydrolysis has been reported by Chobert et al., (1988b). They found the
optimum improvement when DH was 8%. The emulsification properties decreased when the
DH reached 9.9%. Kuehler and Stine (1974) reported that limited DH of pepsin improved
emulsifying and foaming properties of whey proteins. The increased emulsification capacity
after limited hydrolysis may be attributed to the exposure of hydrophobic groups, which
enhance the interactions between proteins and lipids (Li-Chan and Nakai 1991). The results
obtained in the present study are in agreement with those reported in the literature.
The trypsin inhibitor activities of different modified flours were similar to that of
control (40-45 TIU/mg sample). Trypsin inhibitors were resistant to enzymatic hydrolysis
when tried with different proteolytic enzymes.
Oil and water absorption capacity
The data on the amount of fat globules absorbed on the surface of different modified
flours is presented in Table 3.3. The oil absorption capacities of modified flour samples
were higher (2.3- 2.4g/g) than that control (1.9g/g). The increase may be due to the strong
association of hydrolysates to the fat globules in treated flour compared to the untreated
flour. The hydrophobic groups in proteins remain exposed at the molecular crevices and can
participate in hydrophobic interaction with other molecules. Adsorption on protein occurs by
means of hydrophobic interactions between the protein and the surface of fat globules. Thus
effective hydrophobicity may be an important factor in the oil adsorption process. Enzymatic
modification using different proteases could effectively increase the relative hydrophobicity
of constituent high molecular protein components of soy flour (glycinin and conglycinin).
Generally soybean proteins are rich in hydrophobic amino acids. During the formation of
three-dimensional structure they are mostly buried inside the matrix with some hydrophobic
66
Figure 3.1: Effect of papain and alcalase on the DH of defatted soy flour
DSF was dispersed in distilled water (1:10 w/v) and the pH increased to 7.0 for papain hydrolysis and 7.8 for alcalase hydrolysis. The enzyme concentration was 1% based on protein content. Aliquots were withdrawn at regular intervals (5- 240 min) and enzyme was inactivated by heating at 80°C and the DH was measured by TNBS method. The DH was plotted against time. The values are means of two independent determinations.
0 100 200 3000
10
20
30
PapainAlcalase
Time(min)
Deg
ree
of
hyd
roly
sis(
%)
67
Figure 3.2: Effect of fungal protease on the DH of defatted soy flour DSF was dispersed in distilled water (1:10 w/v) hydrolysis was carried out at pH 7.8 using fungal protease at 0.5 and 1% concentrations based on protein content. Aliquots were withdrawn at regular time intervals (5-240min). The enzyme was inactivated by heating at 80°C and the DH was measured by TNBS method. The DH was plotted against time. The values are means of two independent determinations.
0 100 200 3000
10
20
30
40
0.5%1%
Time(min)
Deg
ree
of
hyd
roly
sis(
%)
68
patches on the surface. Enzymatic attack can effectively expose the hydrophobic groups and
there by increase the fat absorption. The water absorption capacities of different modified
flour samples were marginally increased compared to raw flour.
Protein hydrolysates from soy flour
Defatted soy flour was dispersed in distilled water (1:10 ratio) and hydrolysed with
papain, alcalase and fungal protease at 1% concentration for 4 h. The enzyme was
inactivated by heating at 80ºC for 10 min. The supernatant obtained after centrifugation was
spray dried to get protein hydrolysates. The preparation details are described under materials
and methods. The spray-dried hydrolysates obtained using different enzymes were compared
for their physico chemical properties. The nitrogen content of spray-dried hydrolysates
prepared after hydrolysis with different proteolytic enzymes ranged from 9 to 9.2%. The
hydrolysates were yellowish in colour and bitter in taste. The protein hydrolysate prepared
using papain was less bitter compared to that prepared using alcalase and fungal protease.
The trypsin inhibitor activities of different hydrolysates were in the range 23-27 TIU/mg
sample. The reduction in the trypsin inhibitor activity of hydrolysate compared to DSF (40-
45 TIU/mg sample) was due to the inactivation of the heat labile Kunitz trypsin inhibitor
during spray drying (Liener 1994). Urease and lipoxygenase activity were not detected in the
different hydrolysates (Table 3.4).
Preparation of hydrolysate from acid washed soy flour and wet protein isolate
To increase the nitrogen content of the hydrolysates, soy flour obtained after acid
wash (pH 4.5) and the wet protein isolate obtained by isoelectric precipitation of the water
extract of soy flour at pH 9.0 was used for the preparation of protein hydrolysates. The
experimental conditions were same as those used for the preparation of protein hydrolysate
from defatted soy flour. Papain was the enzyme (1% concentration) used for hydrolysis. The
nitrogen contents of the protein hydrolysates obtained from acid washed soy flour and wet
69
Figure:3.3 Effect of DH on EC and FC of modified DSF prepared using papain and alcalase The papain (A) alcalase (B) modified DSF samples with different DH were analysed for EC and FC. The results are means from three independent determinations (error bar represents standard deviation).
DSF DH 4.0% DH 11.8% DH 18.6%0
100
200
300
FCEC
A
DSF DH 4.1% DH 8.0% DH 17%0
100
200
300
ECFC
B
70
Figure: 3.3C Effect of DH on EC and FC of modified DSF prepared using fungal
protease The fungal protease modified DSF samples with different DH were analysed for EC and FC. The results are means from three independent determinations (error bar represents standard deviation).
DSF DH(6.8%) DH(15.1%) DH(32.4%)0
100
200
300
ECFC
71
Table: 3.2 Degree of hydrolysis* achieved with different modified soy flours hydrolysed
for different periods using proteolytic enzymes.
Protease Hydrolysis (min) DH (%)
Papain
Alcalase
Fungal protease
10
30
240
10
30
240
10
30
240
4.0
11.8
18.6
5.8
13.1
29.6
6.8
15.11
32.4
* means of two replicates.
protein isolate were higher (11.5% and 14.5%) compared to hydrolysate obtained by direct
hydrolysis of soy flour using various proteolytic enzymes (9.0%). The extent of hydrolysis
was similar to the hydrolysate obtained from soy flour as indicated by the similar TCA
solubility index (46-48%). The ash content of hydrolysates obtained from acid washed soy
flour and wet protein isolate were higher (7.8%) compared to the hydrolysate obtained from
defatted soy flour (6.4%). The increase in ash content was due to the accumulation of salt
during neutralisation of the hydrolysate before spray drying. The results are summarised in
Table 3.5 and 3.6.
The amino acid composition of DSF and protein hydrolysates obtained by papain and
alcalase hydrolysis is given in Table 3.7. The content of total essential amino acid was 51.2
and 48.2% in papain and alcalase hydrolysates respectively compared to 43.7% in DSF.
72
Table: 3.3 The water absorption capacity and fat absorption capacity of defatted soy flour and different enzyme modified samples.
Sample DH (%) WAC (g/g) * FAC g/g *
DSF
DSF- modified
Papain
Alcalase
Fungal protease
−
4.0
18.6
5.8
29.6
6.8
32.4
1.8
2.1
2.4
2.0
2.32
1.95
2.2
1.9
2.31
2.38
2.18
2.31
2.1
2.41
*means of three replicates.
There was considerable increase in amino acids isolucine, leucine, tyrosine and valine in
hydrolysates. This shows that the nutritional value of hydrolysates was higher than that of
defatted soy flour. Similar type of reports have also been made by Hrckova et al., (2002).
They have showed increase in histidine, leucine and tyrosine content in the hydrolysates of
soy flour obtained by treatment with alcalase and novozyme.
Solubility of protein hydrolysates
The nitrogen solubility vs pH profile of soy flour and the protein hydrolysates
prepared using different proteolytic enzymes is shown in the Figure 3.4.The curve exhibited
minimum solubility at around pH 4.5 (4.1%) and higher solubility at acidic and alkaline pH.
At pH 7.0 the nitrogen solubility of soy flour was 76 to 77%. Different hydrolysates showed
73
nitrogen solubility values of 95-96% at isoelectric pH. The solubility at pH 7.0 was 97-98%.
The remarkable increase in the solubility of hydrolysates at isoelectric pH may be due to the
increase in the ionizable groups followed by hydrolysis, which enhance the protein-water
interactions.
Table 3.4: Physico-chemical properties of spray dried soy protein hydrolysate prepared using different proteolytic enzymes Parameters* Papain Alcalase Fungal protease
Trypsin inhibitor
activity (TIU/mg)
Lipoxygenase activity
(units/mg)
Urease activity
Nitrogen solubility
index (%)
Bitterness recognition
(Recognition threshold
(gN/100ml)
23
ND
ND
98
0.2
26
ND
ND
98.2
0.14
26
ND
ND
98.4
0.14
ND: Not detectable; * means of two replicates
At low DH, modified soy flour had improved functional properties over the
unhydrolysed soy flour. The change in functionality depends on the DH and the specificity
of the proteolytic enzyme used. The modified soy flour can be used as whipping agent in
high pH confectionary products or in foods wherever high foamability is required. The
protein hydrolysate recovered from soy flour by extraction and centrifugation had
exceptionally good solubility over a wide range of pH and the bitterness of papain-modified
sample was lower than alcalase and fungal protease modified hydrolysates. These
hydrolysates may find applications in various types of health drinks in which solubility at
acidic pH is required. The study showed that modification by enzymatic approach is one of
74
the effective means of value addition to soy flour which is a byproduct of oilseed processing
industries available plentily as a byproduct of oil extraction.
Table 3.5: Characteristics of protein hydrolysates prepared from soy flour before and after acid wash.
Hydrolysate from
Constituents (%) * Soy flour Soy flour(acid washed)
Moisture
Nitrogen
TCA solubility index
Ash
7.4
9.0
46.8
6.4
7.8
11.5
47.8
7.8
* means of two replicates.
Table 3.6: Comparison of the protein hydrolysates prepared with wet protein isolate and soy flour.
Hydrolysate from Constituents (%) *
Soy flour Wet protein isolate
Moisture
Nitrogen
TCA solubility index
Ash
6.8
9.0
46.8
6.4
ND
14.5
47.0
7.8
* means of two replicates ND : Not determined.
75
Amino acids
Soy flour
Papain hdrolysed
Mean ± S.D
Alcalase hydrolysed
Mean ± S.D
Essential
Histidine
2.8
2.4±0.2
2.2±0.25
Isoleucine 5.1 6.9±1.1 7.3±1.2
Leucine 6.8 12.6±0.7 10.5±0.8
Lysine 6.1 6.6±0.9 6.6±1
Methionine 1.4 2.1±0.3 1.2±0.4
Cysteine 1.5 0.2±0.5 0.1±0.05
Phenylalanine 5.2 4.1±0.2 4.6±0.5
Tyrosine 3.6 4.8±0.6 4.3±0.4
Threonine 4.5 3.8±0.2 4.1±0.2
Tryptophan 1.9 1.0±0.1 1.0±0.3
Valine 4.8 6.7±0.8 6.3±0.7
Non-essential
Alanine
4.3
2.6±0.1
2.4±0.2
Arginine 7.4 4.1±0.6 2.7±0.4
Aspartic acid +
aspargine
11.8 5.9±0.4 6.1±0.5
Glutamic acid +
glutamine 18.2 18.6±0.5 21.8±3.0
Glycine 4.3 2.2±0.1 2.1±0.20
Proline 5 10.4±1.4 12.3±1.2
Serine 5.3 5.1±0.2 4.9±0.3
Table 3.7: Amino acid composition of soy protein hydrolysates (g/16gN)
76
Figure 3.4: Comparison of the solubility vs pH profile of defatted soy flour and spray
dried protein hydrolysates prepared using different proteolytic enzymes. The spray dried protein hydrolysates were prepared from DSF using different enzymes. The details of preparation are given in materials and methods. The DSF and different protein hydrolysate samples were dispersed in distilled water (1%w/v). The solubility was determined as a function of pH . The soluble nitrogen content in the supernatant was determined by Kjeldahl method. The solubility was expressed as percent of nitrogen content in the supernatant to the total nitrogen content of the original sample.
0.0 2.5 5.0 7.5 10.0 12.5 15.00
25
50
75
100
DSF
Papain
Alcalase
Fungal protease
pH
So
lub
ility
(%)
61
The volume of foam was recorded after 30 sec and FC expressed as the percentage increase
in volume. After the elapse of different time intervals the volume of foam was measured and
recorded as FS.
Emulsification capacity (EC)
The EC was determined at room temperature according to the method of Beuchat et
al (1975). 1g of sample and 25 ml of water (pH 7.8) were blended for 30s in a blender. After
complete dispersion, refined groundnut oil was added from a burette at a rate of 0.4 ml/s and
blending continued until phase separation obtained. EC was expressed as ml of oil
emulsified per gram of protein.
77
ENZYMATIC MODIFICATION OF SOY PROTEIN ISOLATE
Soy protein isolates are composed of 7S and 11S globulins and show high solubility
at alkaline conditions, and low solubility in acidic media. Isolated soy proteins have
widespread applications in different formulated foods and are also used directly as
nutritional supplements. Soy protein isolates are processed into several forms including
spray-dried powdered, dry granules and fibrous materials. They are also available in textured
or spray-dried form and marketed widely all over the world. The functional properties such
as solubility, foaming and emulsifying properties of the isolate can be enhanced by
enzymatic modification (Adler-Nissen, 1976). Most of the enzymes employed in the
production of hydrolysates for food use are from plant or microbial origin such as bromelain,
papain, ficin and alcalase (Lahl and Braun 1994).
In the present investigation soy protein isolate was prepared by drying methods i.e.
freeze-drying and spray drying. The three proteases of different origin were selected for
hydrolysis studies (papain, alcalase and fungal protease). The functional properties like
solubility; emulsifying and foaming properties are generally influenced by the molecular
weight, hydrophobicity and surface charge of the protein. Thus changing the molecular
weight and hydrophobicity by enzymatic modification can alter the functional properties of
protein. The hydrolysates prepared by extensive hydrolysis of protein isolates will have
higher nitrogen content and free from objectionable flavour and have low trypsin inhibitor
content because the isolates are generally prepared by isoelectric precipitation of the water
extract.
The extent of hydrolysis of a protein by a particular enzyme is reflected by the degree
of hydrolysis (DH) or trichloroacetic acid solubility index, which is an important index that
will be useful to define any functional property. Generally low degree of hydrolysis could
78
increase the surface properties by exposing the buried hydrophobic residues to the aqueous
environment. To get high solubility extensive hydrolysis of the protein is needed. In view of
this experiments were conducted to study the effect of time on the degree of hydrolysis of
SPI using papain, alcalase and fungal protease.
The hydrolysis profiles of freeze-dried protein isolates with different proteases are
shown in Figure 4.1. The rate of hydrolysis of SPI with different proteolytic enzymes
proceeded at a rapid rate during initial 15 min and thereafter slowed down. When papain was
used at 1% concentration the DH reached to a value of 2.5% at the end of 5min, the DH
varied from 2.4 to 4% at the end of 30 min and the curve plateaued at about 6% DH. As
against this at 5% concentration of papain the DH attained a value of 5.9% at the end of
5min and reached to a maximum of 12% at the end of 240 min (Figure 4.1A). With alcalase
the hydrolysis curve showed a DH of 1.4% at the end of 5 min and attained 3.5% after 30
min. The curve showed a maximum value of 9.6 to 9.9% (Figure 4.1B). At 5% concentration
of alcalase the DH reached 12-13% after 240 min. In the case of fungal protease the DH was
2.8% after 5 min. The DH initially raised faster and reached 8.2% at the end of 30 min. The
DH reached a maximum value of 15-16% after hydrolysis for 240 min. However at 5% of
fungal enzyme the hydrolysis was rapid and the DH attained to a value of 10% at the end of
30 min and reached to a maximum of 21-22% after incubation for 240 min. (Figure 4.1C).
It is evident that from the plots that it is difficult to get high DH using papain,
alcalase or fungal protease enzyme even after increasing the enzyme concentration at
optimum pH and temperature for maximum activity. The affinity of the enzymes as indicated
by the maximum DH were in the order Fungal protease > Alcalase > Papain.
The DH vs time plot for spray dried SPI is shown in Figure 4.2. From the plot it is
evident that the pattern was similar to the hydrolysis curve obtained with freeze-dried SPI
using papain, alcalase and fungal protease. The maximum DH obtained with 1 % alcalase
79
Figure 4.1: Effect of enzyme concentration on the DH of freeze-dried soy protein isolate
(SPI-FD). SPI-FD (20mg/ml) samples were hydrolysed with papain (pH 7.0), alcalase (pH 7.8) and fungal protease (pH 7.8) at different concentrations (0.5 to 5%) to different time intervals (5min to 240min). The enzyme was inactivated by heating at 80°C.The DH was plotted against time.
SPI-FD Papain
0 50 100 150 200 250 3000.0
2.5
5.0
7.5
10.0
12.5
! 5%
" 1%
Time(min)
Deg
ree
of h
ydro
lysi
s %
A
SPI-FD Alcalase
0 50 100 150 200 250 3000
5
10
15
1%5%
Time(min)
Deg
ree
of
hyd
roly
sis
(%)
B
SPI-FD Fungal Protease
0 50 100 150 200 250 3000
5
10
15
20
25
1%
5%
Time(min)
Deg
ree
of
hyd
roly
sis(
%)
C
80
Figure 4.2: Effect of enzyme concentration on the DH of spray dried soy protein isolate
(SPI-SD). SPI-SD (20mg/ml) samples were hydrolysed with alcalase (pH 7.8) and fungal protease (pH 7.8) at different concentrations (0.5 to 5%) to different time intervals (5min to 240min). The enzyme was inactivated by heating at 80°C.The DH was plotted against time. A. alcalase B. fungal protease.
0 50 100 150 200 250 3000.0
2.5
5.0
7.5
10.0
12.5
15.0
0.5%
1%
5%
Time(min)
De
gre
e o
f h
yd
roly
sis
(%) A
0 5 0 1 0 0 1 5 0 2 0 0 2 5 0 3 0 00
5
1 0
1 5
2 0
2 5
1%5%
T im e(m in )
De
gre
e o
f h
yd
roly
sis
(%) B
81
and fungal protease was 9.4 and 16.1% respectively. At 5% concentration, the DH reached to
a maximum of 12.8 and 23.4 % for alcalase and fungal protease respectively at the end of
240 min. A comparison of the DH of freeze dried and spray dried SPI showed that spray
drying did not change the affinity of isolate towards proteolytic enzymes for hydrolytic
degradation.
The study shows that a low DH can be obtained if the hydrolysis is restricted to 10
min at 1% enzyme concentration at optimum temperature for hydrolysis. For extensive
hydrolysis the incubation period needed to be prolonged up to 240 min. By selecting these
parameters with low and high DH were prepared and evaluated for functional properties. The
DH of different modified isolates used for the study of functionality as shown in Table 4.1.
Nitrogen solubility
The solubility of a protein in water at different pH is in general the net result of the
balance between the protein-protein and protein water interaction. At isoelectric pH soy
proteins have net zero charge and protein-protein interaction predominates because of the
association between the hydrophobic patches on the surface of the protein molecules. The
net charge on the protein will increase upon hydrolysis due to increase in the ionisable
carboxyl and amino groups. This will generally increase the protein-water interactions and
increase the solubility. The solubility of soy protein followed the typical U-shape pattern
(Figure 4.3). SPI showed higher solubility with increasing and decreasing pH values;
minimum solubility being at isoelectric point, and resolubilization was observed at pH values
lower to the isoelectric point.
Solubility characteristics of proteins are the most important functional properties
since many other functional performances depend on their capacity to go in to solution. The
difference in the solubilities between intact SPI and the different modified isolates to
different DH, as a function of pH is shown in Figure 4.3 .The solubility of SPI increased
82
Table 4.1: Comparison of the degree of hydrolysis* of different modified soy protein isolates prepared using proteolytic enzymes.
Protease Period of hydrolysis (min) DH (%)
Papain
Alcalase
Fungal protease
10
240
10
240
10
240
3.3
7.5
3.1
9.5
3.9
18.9
* means of two replicates.
with increase in DH. Intact SPI had a solubility of 3.0% at pH 4.5 compared to 77.8% at pH
7.0. At low DH (3.3%) papain increased the solubility of SPI to 35.3% at pH 4.5. At pH 7.0,
the solubility was 98.2%. In the case of alcalase the solubility at 3.1% DH was 29.2% at pH
4.5 compared to a 97.7% at pH 7.0. At high DH (9.5%) the solubility was 49.5 and 88.4% at
pH 4.5 and 7.0 respectively. In the case of fungal protease, a low DH of 3.9% changed the
solubility from 31% at pH 4.5 to 99.2% at pH 7.0. However, at high DH (18.9%) the
solubility increased to 53.4 and 93.1% at pH 4.5 and pH 7.0, respectively.
The FC of SPI and SPI modified with different enzymes were studied at pH 7.0. The
FC of intact SPI was 36.0%. At low DH (3.3%) papain increased the FC to 63.3%. At higher
DH (7.5%) the FC was 43%. For alcalase, the FC at 3.1% DH was 77% compared to 56% at
9.5%DH. Similarly, the fungal protease modified SPI had a FC value of 70.7% at 3.9%DH
compared to 57.0% at 18.9%DH. From the results it is clear that the FC of SPI samples
modified to low DH were much higher than control. The FC values of high DH SPI were
higher than the control, but the values were lower than those modified to low DH (3-5%)
83
0
20
40
60
80
100
0 2 4 6 8 10 12
pH
Pro
tein
so
lub
ility
(%
)
SPI
DH(3.9%)
DH(18.9%)
C
Figure 4.3: Effect of degree of hydrolysis (% DH) on solubility (%) of soy protein (SPI)
hydrolysates The sample solution in water was adjusted to pH (2-11) with 0.1 M HCl or NaOH .The soluble N content was measured by Kjeldahl method and solubility was expressed as the percent N content in the supernatant after centrifugation to the original protein content of the sample A. Papain B. Alcalase C. Fungal protease.
0
20
40
60
80
100
0 2 4 6 8 10 12pH
Pro
tein
so
lub
ility
(%)
SPI
DH(3.3% )DH(7.5% )
A
0
20
40
60
80
100
0 2 4 6 8 10 12
pH
pro
tein
so
lub
ility
(%)
SPI
DH(3.1%)
DH(9.5%)
B
84
with various proteolytic enzymes. Limited proteolysis did not affect any change in the FS of
SPI; extensive hydrolysis brought down the FS drastically. The emulsification capacity of
SPI was 137.7 ml/g. The emulsification capacities of low DH (3-5%) SPI were 181.7, 168
and 167 ml/g for papain, alcalase and fungal protease, respectively. The emulsification
capacities of extensively hydrolysed SPI prepared using papain, alcalase and fungal protease
were 66.0, 67.4 and 62.3 ml/g, respectively. Limited proteolysis with all the three enzymes
increased the EC of SPI. The results are shown in Table 4.3 and 4.4.
Kim et. al., (1990) have reported the effects of enzymatic modification of commercial
SPI on its molecular weight and functional properties. The enzymes used were adrex F,
alcalase, α-chymotrypsin, trypsin, liquozyme and rennet. They observed that at pH 4.5 there
was threefold increase in the solubility of SPI after 5 min incubation. On the other hand,
trypsin and chymotrypsin increased the solubility and reached to the same value after 30 min
hydrolysis. Trypsin and alcalase increased the emulsification capacity after 30 min
incubation. Chymotrypsin and liquozyme were less effective in enhancing the emulsification
capacity. The improved emulsifying capacity of SPI through limited hydrolysis with alcalase
and neutrase was also reported by Adler Nissen and Olsen (1979). Puski (1975) reported
increase in emulsification and foaming capacity of SPI after hydrolysis with microbial
enzyme when the DH was 5%. Qi et al., (1997) worked on the effects of limited pancreatin
hydrolysis on functional properties of SPI. They showed that the surface hydrophobicity
indices of pancreatin hydrolysed SPI were higher than unhydrolysed SPI and low DH
increased the emulsifying activity index. Molina Ortiz et al. (2002) have reported the effects
of bromelain hydrolysis of native and denatured soy protein isolates on the physicochemical
and functional properties. They observed considerable improvement in the surface
hydrophobicity, foaming properties and solubility of SPI subjected to proteolysis with
bromelain. The results of the present study on limited proteolysis of SPI using papain,
85
alcalase and fungal protease enzymes on the functional properties of SPI are in close
agreement with the above reports.
Since hydrolysis of soy protein isolate gives hydrolysate with higher nitrogen content,
it is better suited for nutritional enrichment of various types of functional foods. Proteolytic
modification is an effective way to improve the functional properties of SPI. In this respect
papain was found to be more effective in improving the emulsification capacity compared to
alcalase and fungal protease. The foaming capacity of fungal protease modified SPI was
higher compared to papain and alcalase modified samples. Extensive hydrolysis of SPI with
various proteolytic enzymes brought down the emulsification capacity. Modification of SPI
to low DH (3-5%) with enzymes is ideal for improving emulsification capacity. Since the
solubility of extensive hydrolysate was remarkably high they can be used in nutritional
enrichment of beverages and in the preparation of geriatric foods. The study also suggests
that the selection of raw material (source of protein) has also bearing on the overall
functionality of enzyme-modified protein. Among the three enzymes fungal protease was
more effective in getting hydrolysate with high DH.
86
Table 4.2: Effect of limited proteolysis (DH 3-5%) on functional properties* of soy protein isolate.
Enzyme Emulsification
capacity (ml/g) Foaming capacity (% volume increase)
Foam stability (ml)
10 min 20 min 30 min 40 min 60 min Control Papain Alcalase Fungal protease
137.7 ± 4.0 181.7 ± 2.1 168 ± 3.7 167 ± 3.0
36 ± 1.6 63.3 ± 1.3 67 ± 3 70.7 ± 7.0
20 19 15 14 09 22 16 15 13 09 24 22 16 12 10 27 24 23 18 10
* means of three replicates ± standard deviation.
Table 4.3: Effect of extensive hydrolysis on functional properties* of soy protein isolate.
* means of three replicates ± standard deviation.
Enzyme Emulsification capacity (ml/g)
Foaming capacity (% volume increase)
Foam stability (ml) 10 min 20 min 30 min 40 min 60 min
Control Papain Alcalase Fungal protease
137.7 ± 4 66 ± 2.8 67.6 ± 3 62.3 ± 7.0
36 ± 1.6 43 ± 3.1 56 ± 3.0 57 ± 2.6
20 19 15 14 09 15 13 08 03 01 16 12 07 02 01 18 15 06 03 01
87
CONTROLLED ENZYMATIC HYDROLYSIS OF GLYCININ
Soybean has become an important protein source all over the world. The major
proteins of soybean are storage globulins i.e. glycinin and conglycinin which make nearly
90% of the total proteins. Soy proteins consist of four major fractions, 2S, 7S, 11S and 15S
classified based on their sedimentation coefficients (Naismith 1995, Wolf and Briggs 1956).
The 11S fraction of soybean known as glycinin constitutes 25-35% of total proteins (Liu
1997). The 7S fraction is heterogeneous in composition and consists of conglycinin, α-
amylase, lipoxygenase and haemaglutinin. Glycinin, the major protein fraction is a hexamer
with a molecular weight of 360kDa consisting of both acidic and basic subunits linked by a
single disulfide bond (Catsimpoolas et al 1969, Kitamura and Shibasaki 1975). The relative
molecular masses of acidic and basic subunits are approximately 34kDa and 20kDa,
respectively (Kitamura et al 1976). Glycinin and conglycinin exhibit different nutritional and
functional properties due to variations in composition and structure.
Glycinin has been shown to be rich in methionine and cysteine per unit protein
compared to 7S protein (Kitamura 1995). Glycinin can be a valuable source of protein both
from nutritional and functional point of view. Glycinin has lower emulsification capacity and
stability compared to conglycinin. The poorer functional attributes such as foaming and
emulsifying characteristics of glycinin are due to its closed globular conformation and low
molecular flexibility (Wagner and Gueguen 1995). Environmental factors such as ionic
strength, DH, temperature and chemical treatment such as reduction of component disulfide
bonds, succinylation and acetylation (Kim and Kinsella 1987 a, b) could induce structural
modification in glycinin. It is also reported that the foaming and emulsifying properties of
glycinin could be greatly improved by mild acid treatments, deamidation and reducing
agents (Wagner and Gueguen 1995, 1999). Glycinin can be isolated on larger scale by
magnesium chloride precipitation of water extract of defatted soy flour. (Appu Rao and
88
Narasinga Rao 1976). Wu et al. (1999) successfully modified Nagano’s (1992) laboratory
process for the preparation of glycinin and conglycinin to pilot plant scale. One of the
limitations of the usage of glycinin is the lack of desired functional attribute necessary for
incorporation into various food formulations although glycinin fraction is nutritionally better
than total protein isolate and conglycinin. Among the various approaches for improving the
functionality of proteins, modification by enzymes ranks very high.
Most of the published information on the enzymatic hydrolysis of seed proteins such
as groundnut and soybean and the effect of hydrolysis on functional properties of these
proteins are mainly concerned with flour or total proteins. In order to relate the change in
functionality after enzymatic modification to the physico-chemical properties a
homogeneous fraction would be ideally suited compared to total protein.
In the present study modification of glycinin was carried out using different
proteolytic enzymes to understand the mechanism of hydrolysis, and their effect on physico-
chemical and functional properties. The structural changes in glycinin due to enzymatic
modification were studied by molecular sieve chromatography, gel electrophoresis and
circular dichroism spectroscopy. The functional properties like water absorption, fat
absorption, emulsification capacities were determined using the modified glycinin prepared
by limited proteolysis. Prior to effecting hydrolysis using papain, alcalase and fungal
protease the activity of the enzymes were determined. The specific activity values were
11966, 25588 and 28600 units/mg protein for papain, alcalase and fungal protease
respectively.
The results of enzymatic hydrolysis of glycinin by different proteolytic enzymes are
shown in Figure 5.1. Of the three enzymes, papain was least effective followed by alcalase
and fungal protease. The DH increased in all the three enzymes with increasing
concentrations of enzymes (Figure 5.3). In the case of papain, with 4% enzyme
89
concentration, the hydrolysis completed in 150 min, with a DH of 16% (Figure 5.3A). DH
values reached 24% (Figure 5.3B) and 22% (Figure 5.3C) with 4% alcalase and 4% fungal
protease, respectively. Even at the end of 4 h and with 4% enzyme the DH plateaued around
22-24%. Thus glycinin was resistant to hydrolysis by all the three non specific proteases.
The effect of changing the hydrolysis temperature on the DH of glycinin as a function of
time is shown in Figure 5.2. Increasing the temperature of reaction in the case of papain from
50 to 70°C did not significantly enhanced the hydrolysis of glycinin. The DH values for
glycinin with increasing concentration of substrate for three proteolytic enzymes are given in
Figure 5.4. They followed typical Michaelis-Menten pattern. The derived Km and Vmax of
glycinin for the three enzymes are presented in Table 5.1. From the Km values it can be
inferred that the affinity of glycinin was in the order papain <alcalase < fungal protease.
Gel electrophoretic analysis of enzymatically modified glycinin
Glycinin is a multimeric protein with six acidic and six basic subunits linked by a
disulfide bond. The SDS gel electrophoretic pattern of glycinin showed two thick bands
corresponding to acidic subunits (30-33 kD) and basic subunits (20-22 kD) as shown in Lane
1 of Figure 5.6. The pattern of the enzymatically modified glycinin showed that the acidic
subunits were cleaved in preference to the basic subunits (Figure 5.6; Lane 2). To ascertain
whether, both the subunits were resistant to hydrolysis, the acidic and basic subunits of
glycinin were separated and purified according to the method of Draper and Catsimpoolas
(1977). The SDS gel electrophoretic pattern of the separated subunits is shown in Figure 5.5.
The hydrolysis of acidic and basic subunits with fungal protease is given in Figure 5.7 from
which it is clear that acidic subunits were hydrolysed readily by fungal protease and the DH
reached a maximum value of 26%.
90
Figure 5.1. Effect of different proteolytic enzymes on the DH of glycinin
Glycinin samples (20mg/ml) were hydrolysed with papain (pH 7.0), alcalase (pH 7.8) and fungal protease (pH 7.8) at 1% concentration for different time intervals (5 to 240 min) and the enzyme was inactivated by heating at 80˚C. The DH was measured by reaction with TNBS. The data were plotted as a function of time.
0 50 100 150 200 250 3000
5
10
15
20
25
Papain
Alcalase
Fungal protease
Time(min)
Deg
ree
of
hyd
roly
sis(
%)
91
Figure 5.2: Effect of temperature on the DH of soy glycinin by papain. Glycinin samples (20mg/ml) were hydrolysed with 1% papain (pH 7.0) to different time intervals (15 to 240 min) at 50, 60 and 70 0C.
0 100 200 3000
5
10
15
500C
600C
700C
Time(min)
Deg
ree
of h
ydro
lysi
s (%
)
92
Figure 5.3: Effect of concentration of enzymes on the DH of soy glycinin.
Glycinin samples (20mg/ml) were hydrolysed with papain (pH 7.0), alcalase (pH 7.8) and fungal protease (pH 7.8) at different concentrations (0.5 to 5%) for different time intervals (5 to 240 min). The enzyme was inactivated by heating at 80°C. The DH was plotted against time.
Alcalase
0 50 100 150 200 250 3000
10
20
30
0.5%1%3%5%
Time(min)
Deg
ree
of h
ydro
lysi
s(%
)Fungal protease
0 50 100 150 200 250 3000
5
10
15
20
25
0.5%1%5%
Time(min)
Deg
ree
of h
ydro
lysi
s(%
)
Papain
0 50 100 150 200 250 3000
5
10
15
20
0.5%1.0%3.0%5.0%
Time(min)
Deg
ree
of h
ydro
lysi
s(%
)
93
Figure 5.4: Michaelis-Menten Plot (A) and Lineweaver-Burk Plot (B) for hydrolysis of
glycinin with different proteolytic enzymes
Glycinin (substrate) concentration [S] was varied between 0.25 and 3.0% and incubated for 10 min with papain (500C) alcalase (550C) and fungal protease (430C) and the enzymes were inactivated by heating. The reaction rate [V] was measured as milli equivalents of amino group released. [S] was plotted against [V] .
[Substrate ] v s. Ve locity
0 1 2 3 40.0
0.5
1.0
1.5
2.0
Fungal proteaseAlca lase
! Papain
[S] (%)
V (
meq
/min
)A
Lineweaver-Burk Plot
-2 -1 0 1 2 3 4 5
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
Fungal proteaseAlcalase
! Papain
1/[S]
1/v
B
94
Table 5.1: Kinetic constants for different enzymes with glycinin as substrate
Enzyme Km (%w/v) Vmax(meq/min)
Papain Alcalase Fungal protease
3.33
1.66
1.43
5.0
2.22
3.33
Functional properties of modified glycinin
Limited proteolysis increases functional properties of oilseed proteins. Extensive
hydrolysis and reduction of molecular size beyond the critical range impairs the capacity of
proteins to form interfacial layers and the rigidity for the formation of foams. Thus, generally
a high degree of tertiary structure is necessary to get better functionality. To improve the
functional properties of glycinin it was subjected to very limited DH (4-5%) using the
conditions mentioned under materials and methods with the three proteolytic enzymes. The
functional properties of modified glycinin samples are given Tables 5.3 and 5.4. Although
the DH of glycinin modified by using different proteolytic enzymes were constant around 4-
5% their functional properties differed considerably. One of the interesting features was the
threefold increase in the foaming capacity in case of papain modified glycinin and the foam
stability was also very good (Table 5.3). Alcalase and fungal protease modified glycinin had
better foam capacity but the foam stability was low. The fat absorption capacity of the three
enzyme modified proteins was lower compared to the control. However, the water
absorption capacity and emulsification capacity were not significantly different.
95
Figure 5.5: Separation of basic (1) and acidic (2) subunits of purified and carboxy
methylated glycinin (CAM-glycinin) on a Dowex strongly basic anion exchange resin.
Sample: 250mg of CAM-glycinin in 10ml of 50mM tris-acetate buffer (pH 8) containing 6M urea. Elution: first with tris acetate containing 6M urea (pH 8.0) followed by 50mM acetic acid containing 6M urea adjusted to (pH 4.5) with NaOH. Flow rate 20 ml/h.
0
0.5
1
1.5
2
2.5
0 50 100 150 200 250 300 350
Volume(ml)
abso
rban
ce (
280n
m)
1 - Basic 2 - Acidic
1 2
96
Figure 5.6: SDS-Slab gel electrophoretic pattern of enzyme modified glycinin and
isolated basic and acidic subunits in comparison with glycinin.
A. Glycinin (Lane 1), Enzyme modified glycinin (4-5%DH; Lane 2)) B. Purified glycinin (Lane 1); Acidic subunits (Lane 2); Basic subunits (Lane 3).
Lanes 1 2 3
1 2
A
B
97
Figure 5.7: Hydrolysis curve for the isolated acidic and basic subunits of glycinin with fungal protease.
The isolated acidic and basic subunits of glycinin (20mg/ml), (pH 7.8) was hydrolysed (5 to 240 min) with 1% fungal protease. The DH was measured at various time intervals. The DH was plotted against time.
0 50 100 150 200 250 3000
10
20
30
BasicAc idic
Time(min)
De
gre
e o
f h
yd
roly
sis
(%)
98
Figure 5.8: Michaelis-Menten Plot (A) and Lineweaver-Burk Plot (B) for hydrolysis of
acidic and basic subunits glycinin with fungal protease.
Isolated glycinin acidic and basic subunit. (Substrate) concentration [S] was varied between 0.25 and 3.0% and incubated for 10 min with fungal protease (430C) and the enzyme was inactivated by heating. The reaction rate [V] was measured as milli equivalents of amino group released. [S] was plotted against [V] .(details as under materials and methods).
Lineweaver-Burk Plot
-3 -2 -1 0 1 2 3 4 5
2
4
6
8
Basic
! Acidic
1/[S]
1/v
B
[Substrate] vs. Velocity
0.0 0.5 1.0 1.5 2.0 2.5 3.00.0
0.2
0.4
0.6
0.8
1.0
Basic! Acidic
Substrate concentration[S] (%)
Rea
ctio
n v
eloc
ity([
V]m
eq/m
in)
A
99
Table 5.2: Kinetic constants for fungal protease with isolated subunits of glycinin as substrate
Subunit Km (%w/v) Vmax(meq/min)
Acidic
Basic
0.57
0.91
0.5
1.25
Effect of enzymatic modification on structure of Glycinin
In order to understand the differences in the functional properties of modified
glycinins they were subjected to molecular sieve chromatography on Sepharose-6B column
to study the molecular weight distribution pattern. The glycinin was eluted as a single
symmetrical peak in phosphate buffer of 0.5 ionic strength with a Ve /Vo of 1.4 (Figure 5.9).
With DH of 4-5%, glycinin got degraded into low molecular weight components. The
molecular sieve pattern indicated that the degradation of glycinin into smaller molecular
weight peptides was more in case of papain compared to alcalase and fungal protease (Figure
5.10). The peak areas observed are shown in Table 5.5. To understand the mechanism of
cleavage and to characterize the cleaved products the peak positions were subjected to SDS
electrophoresis along with glycinin (Figure 5.11). The electrophoretic pattern for glycinin
indicated the separation of acidic subunits with a molecular weight of 30-33 kD and basic
subunits with a molecular weight of 20- 22 kD. The gel electrophoretic pattern of the peak 1
and 2 from alcalase treated protein are given in Lane 2 and 3. The pattern for peak 1 and 2
from fungal protease treated protein is given in Lane 5 and 6. It is clear from the gel
electrophoretic pattern that peak 1 did not correspond to native glycinin and even at very
100
Table 5.3: Effect of limited proteolysis (4-5%) on foaming capacity* and stability of glycinin
Enzyme Foaming capacity (% volume increase)
Foam stability(ml) 10 min 20 min 30 min 60 min
Control Papain Alcalase Fungal protease
25±3
88±6
44±4
47±3
14 11 8 6 51 45 43 34 18 9 4 2 14 8 4 -
* means of three replicates ± standard deviation .
Table 5.4: Effect of limited proteolysis (4-5%) on functional properties*of glycinin
Enzyme Fat absorption capacity (g/ 100g)
Water absorption capacity (g/ 100g)
Emulsification capacity (ml oil/g)
Control Papain Alcalase Fungal protease
196±4.0
196±3.1
186±4.0
184±3.4
210±8
230±7
228±9
233±5
60±4
70±7
68±5
67±5
* means of three replicates ± standard deviation .
101
Figure 5.9: Sepharose-6B gel filtration pattern of glycinin purified by Tanh and Shibasaki method (1976) Purified glycinin (60 mg) was placed on a Sepharose column (1.8 × 98 cm) previously equilibrated with 0.05 M phosphate buffer (pH 7.8) containing 0.35M sodium chloride. Fractions were eluted from the column (4ml; flow rate 20 ml/h); absorbance of different fractions was recorded at 280 nm.
0
0.5
1
1.5
2
2.5
0 50 100 150 200
Glycinin
102
Figure 5.10: Sepharose-6B column chromatography pattern of glycinin after limited
proteolysis with the three enzymes (DH 4-5%). Column size (1.8 x 98 cm); volume of fraction 4ml; 0.05 molar phosphate buffer (pH7.8) containing 0.35 M sodium cloride was the elution buffer. The column and elution condition were same as those of Figure 5.9.
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
0 30 60 90 120 150 180 210 240 270
Elution volume (ml)
Ab
sorb
ence
at
280n
m
Alcalase
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
0 30 60 90 120 150 180 210 240 270
Elution volume (ml)
Ab
sorb
ence
at
280n
m
Papain
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
0 30 60 90 120 150 180 210 240 270Elution Volume (ml)
Ab
sorb
ence
at
280
nm Fungal
Protease
103
Table 5.5: The Ve/Vo and area of different peaks of gel filtration pattern of enzyme modified glycinin (DH 4-5%) samples
Enzyme Ve/Vo
Peak 1 Peak 2
%Area
Peak 1 Peak 2
Papain
Alcalase
Fungal protease
1.28 1.93
1.3 1.98
1.3 1.91
39.6 60.4
61.0 39.0
43.2 56.8
limited DH it degraded considerably. Of the two subunits, acidic subunits were readily
hydrolysed compared to basic subunits. The failure to detect electrophoretic bands in Lane 3
and 6 suggested that the peak 2 in gel filtration pattern was extensively degraded and the
molecular weight of the degraded peptides must be very low.
Effect of enzymatic modification on conformation of glycinin
The near UV CD spectrum of glycinin exhibited peaks at 263, 275, 283 and 291nm
with a small trough at 312nm (Figure 5.12). With the DH of 4-5%, glycinin showed drastic
reduction in mean residue ellipticity values for the three enzymes. However, the modified
glycinin also exhibited the characteristic peaks at 263, 275, 283 and 291 nm of glycinin. This
indicated the collapse of the tertiary structure of glycinin even at very low DH of 4-5%. For
acidic subunits of glycinin the near UV CD pattern showed at wavelengths similar to intact
glycinin. However, the mean residue ellipticity was lower than intact glycinin (Figure 5.13).
The far UV CD of glycinin in the region 250-200 nm exhibited shoulders at 230 and
215 nm and a trough at 208nm (Figure 5.14A). The results are in agreement with those
reported by German et al. (1982) and Suresh Chandra et al. (1984). Analysis of the
secondary structure of glycinin as per the method of Yang et al (1985) suggested that
glycinin had 10% α helix, 68% β structure and the rest are aperiodic. These results are in
104
Figure 5.11: SDS-slab gel electrophoretic pattern of purified glycinin and the peak
obtained after gel filtration of enzyme modified glycinin (DH 4-5%) Glycinin was modified with the three proteolytic enzymes to obtain low DH of 4-5%. The various modified glycinin samples were subjected to gel filtration on a Sepharose-6B column (The column and elution condition were same as those of Figure 5.9). Lane 1 and 4 purified glycinin, Lane 2 and 3 peak positions 1 and 2 from alcalase modified glycinin. Lane 5 and 6 were peak positions 1 and 2 from fungal protease modified glycinin.
Lane
2 3 4 5 6 1
105
agreement with the secondary structure analysis of glycinin by FT-IR methods (Abbot et al
1996). The far UV CD of acidic and basic subunits are given in Figure 5.14 B and C. The
acidic subunits of glycinin exhibited a trough at 203 nm and the basic subunits showed a
trough at 200 nm. The secondary structure analysis of acidic subunits suggested that they are
very rich in β structure (73%) and α helix was absent and rest are aperiodic. The basic
subunits have 51% β structure and rest are aperiodic structure.
The differences in observed susceptibility of hydrolysis of glycinin and its subunits
could not be attributed to the secondary structure of the proteins. Although basic subunits
had higher content of aperiodic structure as revealed by CD measurements but it was least
susceptible to hydrolysis. Similar observations have been made by Lynch et al., (1977). They
observed that rate and extent of hydrolysis of glycinin by trypsin was improved by
denaturation of the protein and reduction of the disulfide bonds. The conformational changes
in the structure of glycinin affect the rate and extent of hydrolysis by trypsin. There was a
differential rate of hydrolysis with respect to unfolded, S-S reduced and SH blocked acidic
and basic subunits of glycinin. The CAM acidic subunits exhibited fastest rate of hydrolysis
where as the CAM basic subunits the slowest. The basic subunits are more hydrophobic than
the acidic subunits and hydrolyse in such a way as to expose only a limited area to the
enzyme. No considerable differences were observed in the lysine and arginine contents of
these two types of subunits.
The hydrophobicity plots for acidic and basic subunits of glycinin is shown in Figure
5.15. The pattern shows more hydrophobic domains in basic subunits compared to acidic
subunits. In order to confirm this, the hydrophobicity index of acidic and basic subunits were
calculated using the method of Nozaki and Tanford (1971). The hydrophobicity index was
134.8 cal/mole for acidic subunits and –283.33 cal/mole for basic subunits. This confirms
106
that the basic subunits are rich in hydrophobic amino acids, which could have a resistant
hydrophobic core to be degraded by proteolytic enzymes.
Enzymatic modification is an effective way to improve the functionality of glycinin.
Treatment with proteolytic enzymes increased the solubility and foaming properties of
glycinin. The change in functionality was however dependent on DH; low DH was effective
in increasing the foaming properties. Extensive hydrolysis has improved only the solubility.
Among the enzymes studied, fungal protease was more effective in getting high DH
compared to alcalase and papain. Papain was more effective in improving the functionality at
low DH compared to alcalase and fungal protease. The specificity of enzymes towards the
cleavage of glycinin subunits was same. All the three enzymes degraded the acidic subunits
in preference to the basic subunits. The hydrolysate produced using glycinin possess good
functionality and could be an excellent source for nutrition enrichment as it contains more
methoinine and free from conglycinin, a major allergen protein in soybean protein isolate.
The study also showed that the overall effect of DH on functionality of SPI and glycinin was
different. Low DH improved both emulsification and foaming capacity of SPI but in the case
of glycinin only foaming properties is improved. Intact glycinin showed lower emulsion and
foaming properties compared to SPI. By use of appropriate proteolytic enzymes and
controlling the DH the overall functional properties of glycinin can be improved.
107
Figure 5.12: Effect of limited proteolysis on near UV CD spectrum of (A) glycinin
(B) glycinin with a DH of 4-5 %. Spectra were recorded in 0.05 M phosphate buffer of pH 7.8. The concentration of glycinin was 1.12 mg/ml and that of modified glycinin was 1.2 mg/ml.
A
B
108
Figure 5.13: Near UV CD spectrum of (A) glycinin (B) isolated acidic subunits of
glycinin. Spectra was recorded in 0.05 M phosphate buffer pH 7.8; the protein concentration was 1.2mg/ml.
A
B
109
Figure 5.14: Far UV CD spectrum of (A) glycinin (B) isolated acidic and (C) basic subunit of glycinin
A
B
C
110
Figure 5.15: A. Hydropathy plot for acidic subunits (1-278) A2 of glycinin B. Hydropathy plot for basic subunits B1a (1-180) of glycinin.
0.0
1.0
2.0
-1.0
-2.0
-3.019 38 57 76 95 114 133 152 171
Residue Number
Mean Hydropathy (GRAVY) = -0.258
Scale: Kyte and Doolittle (1982)
W = 11Hydropathy threshold for helices = 1.6
B
0.0
1.5
3.0
-1.5
-3.0
23 46 69 92 115 138 161 184 207 230 253 2
Residue Number
Mean Hydropathy (GRAVY) = -1.04
Scale: Kyte and Doolittle (1982)
W = 11Hydropathy threshold for helices = 1.6
278
A
111
Discussion
The study revealed that the degree of hydrolysis achieved with the three proteases on
defatted soy flour, soy protein isolate and glycinin was different. When defatted soy flour
was used as substrate, papain gave a maximum DH of 18.6% compared to 7.5% for SPI and
9.5% for glycinin. But in the case of soy flour, alcalase gave a maximum DH of 29.6%
compared to 9.5 and 24% with SPI and glycinin respectively. In the case of fungal protease
defatted soy flour gave a maximum DH of 32.6% compared to 18.9 and 22% for isolated soy
protein and glycinin respectively. The DH obtained with defatted soy flour, soy protein
isolate and glycinin with papain, alcalase and fungal protease showed that all the enzymes
gave higher DH with defatted soy flour followed by glycinin and soy protein isolate. The DH
of spray-dried isolate was similar to freeze dried isolate with all the three enzymes used in
the study. The comparative data are shown in Table 5.6.
Table 5.6: Maximum DH achieved with defatted soy flour (DSF), soy protein isolate (SPI) and glycinin using the three proteases
Degree of hydrolysis (%)
Substrate
Papain
Alcalase
Fungal protease
DSF
18.6
29.6
32.4
SPI
7.5
9.5
18.9
Glycinin
9.5
24.0
22.0
112
The storage proteins of soybean consist of albumins and globulins; more than 90%
are globulins and the remaining proteins are soluble at isoelectric pH. The proteins
precipitated at pH 4.5 are globulins and contain mainly glycinin and conglycinin. The higher
DH obtained with DSF may be due to the non storage proteins other than the low molecular
weight components. The DSF has both storage as well as metabolic proteins (Liu 1997). The
non-storage proteins may be more susceptible to hydrolysis by enzymes compared to storage
proteins. The protein isolate is made up of glycinin and conglycinin. The lower DH obtained
with SPI compared to glycinin suggests that the β-conglycinin component must be more
resistant to proteolytic attack compared to glycinin. β-conglycinin is trimeric in structure
and composed of α α′ and β subunits. The extension core region common to all the subunits
must be more hydrophobic, there by offering more resistance to hydrolysis by proteases.
The modified proteins obtained with the three proteases showed that the functional
properties improved when the degree of hydrolysis was low. The functional properties
especially EC and FC were dependent on the type of proteolytic enzyme used and the
substrate. In the case of DSF and SPI limited proteolysis enhanced EC considerably, but the
increase in FC was marginal. In the case of glycinin a considerable increase in the FC was
observed, but the enhancement in EC was marginal. Extensive hydrolysis impaired the EC in
DSF, SPI and glycinin. The solubility showed marked improvement even at low DH. The
variation observed in the functional property by the application of papain, alcalase and
fungal protease suggests that the point of cleavage of the protein in the three substrate could
be different resulting in peptides of different characteristics. The increased in the EC and FC
observed at low DH may be due to increase in solubility and exposure of hydrophobic
groups as a result of proteolysis. High degree of enzymatic hydrolysis impaired the
functional properties in DSF except solubility. This may be due to the smaller size of the
peptides, which are unable to impart any functional properties.
113
Table 5.7: Functional properties of defatted soy flour (DSF), soy protein isolate (SPI) and glycinin modified to low DH using various proteolytic enzymes
Functional property Substrate
Enzyme Emulsification
capacity (ml/g) Foaming capacity (% volume increase)
DSF
Papain
Alcalase
Fungal protease
212.0 223.0 218.5
41.0
49.4
51.8
SPI
Papain
Alcalase
Fungal protease
181.7 168.0 167.0
63.3
67.0
70.7
Glycinin
Papain
Alcalase
Fungal protease
70.0 68.0
67.0
88.0 44.0 47.0
114
STUDIES ON GROUNDNUT PROTEINS
ENZYMATIC MODIFICATION OF GROUNDNUT PROTEIN ISOLATE
Groundnut is the second largest oilseed produced in the world. Major portion of
groundnut produced in the world is converted into oil and cake. A number of processes have
been developed for the production of defatted flakes, meals, grits and flour from groundnut
(Rhee et al. 1972b, 1973a, Aguilera et al.1980, Ayres et al. 1974, Ayres and Davenport 1977,
Bhatia 1966, Natarajan 1980). Protein isolates are the most refined form of ingredients of
groundnut and contain usually more than 90% protein and can be utilized in the preparation
of high protein foods. One of the major limitations in the utilization of groundnut protein
products is the contamination with aflatoxins. Aflatoxin is a coumarin containing a fused
dihydrofuran moiety. Aspergillus flavus and Aspergillus parasiticus are the two fungal
species, which produce this toxic component during metabolic process. One of the
advantages of utilization of groundnut in the form of isolate is that it may overcome the
aflatoxin and to some extent the characteristic nutty flavour. Protein isolates can be produced
from groundnut by isoelectric precipitation followed by neutralization and drying (Natarajan
1980)
One of the major limiting factors in the utilization of protein hydrolysates in various
food formulations is the formation of bitter peptides as a result of hydrolysis. The
hydrolysates produced from gelatin and egg albumin had a bland taste, whereas lactalbumin,
casein and soy protein developed a bitter taste when hydrolysed (Clegg and Mc Millan
1974). The severity of bitterness generally depends on the extent of hydrolysis and the type
of enzyme used and the nature of substrate. From the survey of the amino acid composition
of bitter peptides Ney (1971) postulated a correlation between the hydrophobicity of the
115
peptides and their bitterness. He found that bitter peptides so far found had Q values above
+1400 cal/mole, whereas all the non-bitter peptides had a Q value below +1300 cal/mole.
Thus protein from casein (1605cal/mol), soy protein (1540cal/mol) give rise to bitter
peptides as indeed observed. The hydrophobicity index of groundnut protein is about 780
cal/mole, which shows that groundnut protein isolate could be a promising candidate for the
preparation of functional peptides in the form of protein hydrolysates possessing less
bitterness.
The solubility and extractability of groundnut proteins can be altered by changing the
pH and ionic strength of the extraction medium. Several reports describe the preparation of
groundnut protein products and methods for the preparation of acylated groundnut flour and
fungal fermented flours from groundnut for use as functional ingredients. The extent and
method of protein modification also affect the solubility of groundnut proteins. Enzymatic
hydrolysis of groundnut flour proteins with pepsin, trypsin and bromelain substantially
increased nitrogen solubility in water at pH 4.0-5.0 and 4.0-11.0 in the presence of calcium
ions (Beuchat et al. 1975). Nitrogen solubility of groundnut proteins partially hydrolysed
with papain was also shown to improve at all pH levels without formation of bitter peptides
(Sekul et al. 1978). Limited protein hydrolysis of groundnut flour by either papain or fungal
protease under optimal conditions has been showed to decrease the emulsification capacity
and increase the foam capacity and stability (Subba Rau and Srinivasan 1988). The
emulsification capacity of groundnut protein isolate has been reported to be higher than
groundnut flour (Ramanatham et al. 1978).
The amino acid composition of GPI is different from that of SPI. Generally, SPI is
rich in lysine and deficient in sulphur containing amino acids, whereas GPI is deficient in
both the amino acids. The structure of soy proteins involves numerous disulfide cross-links.
In groundnut proteins, the final three-dimensional structure will contain no disulfide cross-
116
links. Hence, groundnut proteins may be more flexible and better substrate for enzymatic
attack. Partial hydrolysis of groundnut protein by enzymes would offer an attractive, rapid
and economical way to enhance functional properties. In the present investigation hydrolysis
of GPI was carried out using papain, alcalase and fungal protease.
The hydrolysis curves for freeze dried GPI as a function of time with different
concentrations of papain, alcalase and fungal protease at is shown in Figure 6.1. From the
curves it is evident that the hydrolysis proceeded faster during initial 15 min and slowed
down thereafter. For papain at 1% concentration the DH attained a value of 3.3% at the end
of 5 min and the curve plateaued at around 11.2% DH after 240 min. Increase in the enzyme
concentration up to 5% gave a maximum DH of 18.6% at the end of 240 min (Figure 6.1A).
For alcalase the hydrolysis plot showed a DH of 3.4% after 5 min and a maximum DH of
13.0% was achieved at the end of 240 min at 1% concentration. At 5% concentration the
curve showed a maximum DH of 17.4% after hydrolysis for 240 min (Figure 6.1B). In the
case of fungal protease at 1% concentration the curve showed a maximum DH of 18.5%
after 240 min hydrolysis. A maximum DH of 26.7% was attained when the enzyme
concentration increased to 5% (Figure 6.1C). From the comparison of the hydrolysis curves
for different proteases it is evident that the extent of hydrolysis was in the order Fungal
protease> Papain >Alcalase.
For comparison of functional properties, the enzyme-modified samples were
prepared by hydrolysis for 10 min at 1% concentrations under optimum conditions using
papain, alcalase and fungal protease. The extensively hydrolysed samples were prepared by
hydrolysis for 240 min. The DH of papain, alcalase and fungal protease modified GPI
samples are shown in Table 6.1.
117
Figure 6.1: Effect of enzyme concentration on the DH of freeze-dried groundnut protein isolate (GPI-FD) .
GPI-FD (20mg/ml) samples were hydrolysed with papain (pH 7.0), alcalase (pH 7.8) and fungal protease (pH 7.8) at different concentrations (0.5 to 5%) to different time intervals (5 to 240 min). Enzyme was inactivated by heating to 80°C.The DH was plotted against time.
GPI-FD papain
0 50 100 150 200 250
5
10
15
20
25
0.5%1%5%
Time(min)
Deg
ree
of h
ydro
lysi
s (%
)
A
GPI -FD Fungal protease
0 50 100 150 200 250 3000
5
10
15
20
25
30
0.5%1%5%
Time(min)
Deg
ree
of
hyd
roly
sis
(%)
C
GPI-FD Alcalase
0 50 100 150 200 250 3000.0
2.5
5.0
7.5
10.0
12.5
15.0
17.5
0.5%1%5%
Time(min)
Deg
ree
of h
ydro
lysi
s (%
)
B
118
.
Figure 6.2: Effect of enzyme concentration on the DH of spray dried groundnut protein
isolate (GPI-SD).
GPI-SD (20mg/ml) samples were hydrolysed with papain (pH 7.0) and alcalase (pH 7.8) at different concentrations (0.5 to 5%) to different time intervals (5min to 240min). The enzyme was inactivated by heating to 800C.The DH was plotted against time.
GPI-SD Papain
0 50 100 150 200 250 3000
5
10
15
20
25
0.5%1%
5%
Time(min)
Deg
ree
of
hyd
roly
sis
GPI-SD alcalase
0 50 100 150 200 250 3000.0
2.5
5.0
7.5
10.0
12.5
0.5%1%
Time(min)
Deg
reeo
f hyd
roly
sis(
%)
119
Denaturation is a process in which a protein looses its native conformation without
change in primary structure. Generally, denaturation of a protein is considered in a negative
sense because it implies loss of some important functions. But the beneficial effects of
denaturation include thermal destruction of antinutritive agents, deteriorative enzymes and
enhancement of the digestibility. Denaturation decreases solubility, and α-helical content,
but increases UV absorption, exposure of thiol groups and susceptibility to proteolytic
degradation. Various processing parameters such as extraction, refining and drying methods
may cause denaturation of component proteins. Protein isolates are usually prepared by spray
drying followed by precipitation and neutralization. This may cause structural variations in
the protein components. In order to compare the effect of spray drying on the affinity
towards proteolytic enzymes, the spray dried isolate was hydrolysed under conditions similar
to those maintained for the hydrolysis of freeze dried isolate. The hydrolysis curves for spray
dried GPI is shown in Figure 6.2. Papain at 1% concentration, gave a maximum DH of
11.3% after 240 min. The maximum DH observed at 5% concentration was 18.6%. In the
case of alcalase, at 1% concentrations maximum DH was 13%. The comparison of these data
with those obtained from freeze dried GPI showed that there were no differences in the DH
among the three enzymes. The result also shows that the structural variation such as
denaturation caused by spray drying of GPI did not show influence in improving the degree
of hydrolysis with various proteolytic enzymes.
The solubility of GPI as a function of pH is shown in Figure 6.3 .The curve shows U-
shaped pattern. The solubility increased with increase and decrease in pH. At isoelectric pH
(4.5) the solubility was 37.1% compared to 77.8% at pH 7.0. At low DH (3.2%) at pH 4.5
papain increased the solubility of GPI to 25.2%; at pH 7.0, the solubility was 93.4%. At high
DH (11.2%) the solubility at pH 4.5 was 47.8% compared to 94% at pH 7.0. The solubility
of alcalase at low DH (3.9%) was 28.8% at pH 4.5 compared to a value of 93.4% at pH 7.0.
120
Table 6.1: Degree of hydrolysis* of modified groundnut protein isolate prepared using different proteolytic enzymes.
Protease Period of hydrolysis
(min) DH (%)
Papain
Alcalase
Fungal protease
10
240
10
240
10
240
3.2
11.2
3.9
9.8
3.4
18.6
* means of two replicates.
At high DH (9.8%), however the solubility was 27.8 and 92% at pH 4.5 and pH 7.0,
respectively. The fungal protease at pH 4.5 and under low DH (3.4%) showed a solubility of
28.8%, which increased to 92.3% at pH 7.0. However, at high DH (18.6%) the solubility
increased to 52.2% and 93.0% at pH 4.5 and pH 7.0, respectively. It is clear from the data
that even a low DH of 3-5% with different enzymes the solubility of GPI increased by 25-
26% at isoelectric pH.
The functional properties of GPI modified to low DH (3-5%) using papain, alcalase
and fungal protease are shown in Table 6.2. The EC of GPI was 116.6 ml/g compared to
134.3, 136.7 and 134.7 ml/g for those modified using papain, alcalase and fungal protease
respectively. The FC of GPI modified by using papain, alcalase and fungal protease were
32.0, 40.0 and 43.0%, respectively compared to 24% for control. A low DH increased the
emulsification capacity and foam capacity of GPI. Alcalase and fungal protease were more
effective in enhancing the foaming properties. Similar observation has been reported by
Subba Rau and Srinivasan (1988). They have shown that the solubility, foam capacity and
121
Figure 6.3: Effect of degree of hydrolysis on solubility of enzyme modified GPI
A. Papain B. Alcalase C. Fungal protease.
The sample solution in water was adjusted to pH (2-11) with 0.1 M HCl or NaOH .The soluble N content was measured by Kjeldahl method and solubility was expressed as the percent of N content in the supernatant after centrifugation to the original protein content of the sample.
0
20
40
60
80
100
0 2 4 6 8 10 12pH
Pro
tein
so
lub
ility
(%
)GPI
DH(3.2%)
DH(11.2%)
A
0
20
40
60
80
100
0 2 4 6 8 10 12pH
Pro
tein
so
lub
ility
(%
)
GPI
DH(3.9%)
DH(9.8%)
B
0
20
40
60
80
100
0 2 4 6 8 10 12
pH
pro
tein
so
lub
ility
(%)
GPI
DH(3.4%)
DH(18.6%)
C
122
stability increased and the emulsification capacity decreased in the case of modified
groundnut flour obtained by hydrolysis for 30 min with papain and fungal protease. The
decrease in emulsification capacity may be due to the change in DH beyond 8-10% after
hydrolysis. However, their study did not report the DH of the modified flour.
The functional properties of extensively hydrolyzed GPI in comparison with GPI are
shown in Table 6.3. The emulsification capacities of all the three enzyme-hydrolyzed GPI
were in the range of 46-61 ml/g compared to 116.6 ml/g for GPI. The foaming capacity of
papain, alcalase and fungal protease hydrolysed samples were 28.0, 32 and 39%
respectively. Excess hydrolysis decreased the EC to a great extent. The foam capacities of
different enzyme hydrolysed GPI were higher compared to GPI. Extensive hydrolysis
considerably reduced the foam stability.
Groundnut protein isolate was resistant to enzymatic hydrolysis by proteolytic
enzymes. Limited proteolysis with papain, alcalase and fungal protease increased the
foaming capacity and emulsification capacity. Extensive hydrolysis however increased the
solubility of groundnut protein isolate but decreased the emulsification capacity. The
hydrolysates of GPI prepared using different proteolytic enzymes were less bitter compared
to the hydrolysates prepared using SPI. Since groundnut is the second largest oilseed crop
grown in the world, the large amount of meal obtained as a result of oil extraction, if suitably
modified, could find applications in various processed foods such as beverages, whipped
topping, ice cream etc. Since the bitterness produced with GPI is less the hydrolysates can be
incorporated in various types of therapeutic foods and beverages without necessitating post
hydrolysis process for the removal of bitterness.
123
Table 6.2: Effect of limited proteolysis (DH 3-5%) on functional properties* of groundnut protein isolate.
Enzyme Emulsification
capacity (ml/g) Foaming capacity (% volume increase)
Foam stability (ml) 10 min 20 min 30 min 40 min 60 min
Control
Papain
Alcalase
Fungal protease
116.6 ± 4.0 134.3 ± 4.9 136.7 ± 7.0 134.7 ± 5.0
24 ± 3.2 32.0 ± 4.0 40.0 ± 2.0 43.0 ± 3.0
22 21 18 16 13 10 07 07 06 04 12 11 09 08 03 11 08 07 06 03
* means of three replicates ± standard deviation. Table 6.3: Effect of extensive hydrolysis on functional properties* of groundnut protein
isolate.
Enzyme Emulsification
capacity (ml/g) Foaming capacity (% volume increase)
Foam stability (ml) 10 min 20 min 30 min 40 min 60 min
Control
Papain
Alcalase
Fungal protease
116.6 ± 4.0 46.0 ± 3.0 52.0 ± 3.0 61.0 ± 2.0
24.0 ± 3.2 28.0 ± 3.0 32.0 ± 4.0 39.0 ± 3.0
11 08 07 05 03 10 07 07 06 04 12 11 09 08 03 09 08 07 06 03
* means of three replicates ± standard deviation.
124
ENZYMATIC HYDROLYSIS OF ARACHIN AND ITS EFFECT ON THE FUNCTIONAL PROPERTIES
Enzymatic modification has been shown to alter the functional and physico-chemical
properties of oilseed proteins. Most of the enzymatic modifications reported so far have been
carried out on the defatted flour or protein isolate. Among oilseeds, groundnut has excellent
potential as a source of protein. Arachin, conarachin II and conarachin I are the major protein
fractions of groundnut and they make up nearly 75% of the total protein (Prakash and Rao
1986). The different fractions of peanut protein undergo hydrolysis to different extents and at
different rates with pepsin, trypsin and α-chymotrypsin (Monteiro and Prakash 1994).
Papain is a well-known plant protease, though it is a non-specific protease its action has been
mainly directed towards the arginine-peptide link. Arginine is the third most prevalent amino
acid in arachin, one of the major protein components of groundnut. Papain was shown to be
the most efficient proteolytic enzyme for the degradation of arachin compared to pronase and
trypsin (Neucere 1969; Van Huystee 1973). Alcalase and fungal protease are two important
serine proteases having bacterial and fungal origin, respectively. In the present investigation,
the hydrolysis of arachin was carried out using papain, alcalase and fungal protease to
compare their effectiveness in hydrolysis and change in functional characteristics.
Arachin was isolated by ammonium sulphate precipitation from defatted groundnut
flour. In order to confirm the purity of arachin isolated by ammonium sulphate precipitation,
it was subjected to gel filtration on a Sepharose-6B column equilibrated with 0.01M
phosphate buffer (pH 7.8) containing 0.5M NaCl. The gel filtration pattern of arachin is
shown in Figure 7.1. The pattern shows that arachin eluted as a single symmetric peak in
phosphate buffer of 0.5 ionic strength with a Ve/Vo of 1.5. When the DH was 4-5% (low
DH), arachin underwent degradation into low molecular weight peptides with papain,
alcalase and fungal protease.
125
Figure 7.1: Gel filtration chromatography pattern of arachin purified by ammonium
sulphate precipitation on a Sepharose- 6B column.
A column of Sepharose-6B (1.8 × 98 cm) was equilibrated with 0.01M phosphate buffer (pH 7.8) containing 0.5M sodium chloride.60 mg of arachin was loaded on the column. The sample was eluted with the same buffer and 4 ml fractions were collected. The absorbance was recorded at 280 nm.
0
0.4
0.8
1.2
1.6
2
0 50 100 150 200 250
Elution volume (ml)
Ab
sorb
ance
(28
0 n
m)
126
The gel filtration pattern of arachin modified by papain, alcalase and fungal protease at low
DH is shown in Figure 7.2. The pattern showed two peaks: Peak 1 with the same Ve/Vo as
intact arachin and peak with Ve/Vo of 1.8. This proves that even at low DH of 4-5% a drastic
change in the overall molecular structure of arachin by degradation takes place.
Since the three proteases used for hydrolysis are having different origin it is expected
that the relative affinity of arachin could be different. In order to compare the relative
affinity kinetic studies were carried out under optimum conditions of hydrolysis. For
arachin-papain at pH 7.0 and at a temperature of 50°C, the Km value was 0.931. Similarly for
alcalase at pH 7.8 and at 55°C the Km value was 0.905. In the case of fungal protease at pH
7.8 and at 43°C the Km value was 0.831. The reaction velocity vs substrate concentration plot
showed that the kinetics followed typical Michaelis-Menten pattern (Figure 7.3). The results
are shown in Table 7.1.
Generally, enzymatic hydrolysis of proteins improve the solubility near their
isoelectric points and alters the emulsifying properties and other functional properties
compared to intact proteins. The extent of hydrolysis of arachin by different proteolytic
enzymes as a function of time is shown in Figure 7.4. The curve is a typical hydrolysis plot
with an initial linear region followed by a plateau region. It is evident from the curve that the
plateau region was reached in about 30 min. The DH obtained at the end of 5 min with
papain, alcalase and fugal protease were respectively 3.6, 3.3 and 4.8% compared to 14.8,
12.2 and 19.1% obtained after hydrolysis for 240 min. The degree of hydrolysis was
minimum with alcalase and the order was fungal protease > papain > alcalase. From the time
course of enzymatic digestion it is evident that the susceptibility of different enzymes for
proteolytic cleavage was not alike.
127
Figure 7.2: Gel filtration chromatographic pattern of arachin after limited proteolysis with different enzymes (DH 3-5%) A. Papain, B. Alcalase, C. Fungal protease.
Arachin modified with different proteolytic enzymes (DH 3-5%) was placed on a Sepharose-6B column (1.8 x 98 cm) previously equilibrated with 0.01M phosphate buffer (pH 7.8) containing 0.5 M sodium chloride was the elution buffer. Four ml fractions were eluted from the column (flow rate 20 ml/min). The absorbance of different fractions was recorded at 280 nm.
0
0.4
0.8
1.2
1.6
2
0 50 100 150 200 250
Volume(ml)
Ab
sorb
ance
(28
0nm
) A
0
0.4
0.8
1.2
1.6
2
0 50 100 150 200 250
Volume (ml)
Ab
sorb
ance
(28
0 n
m) B
0
0.4
0.8
1.2
1.6
2
0 50 100 150 200 250
Volume (ml)
abso
rban
ce(2
80n
m) C
128
Lineweaver-Burk plot
Figure 7.3: Michaelis- Menten plot (A) and Lineweaver – Burk plot (B) for hydrolysis of arachin with different proteolytic enzymes.
Arachin (substrate) concentration [S] was varied between 0.25 and 3.0% and incubated for 10 min with papain (500C) alcalase (550C) and fungal protease (430C) and the enzymes were inactivated by heating. The reaction rate [V] was measured as milli equivalents of amino group released. [S] was plotted against [V] .( Details see under Materials and Methods )
[Substrate] vs. Velocity
0 1 2 3 40.0
0.5
1.0
1.5
2.0
Fungal proteaseAlcalase
! Papain
[S] (%)
Rea
ctio
n ve
loci
ty [
V]
meq
/min A
-3 -2 -1 0 1 2 3 4 5
1
2
3
4
5
6
7
8
" Fungal protease# Alcalase! Papain
1/[S]
1/v
B
129
Table 7.1: Kinetic constants for hydrolysis of arachin with proteolytic enzymes.
Parameter Papain Alcalase Fungal protease
Km (%) 0.931 0.905 0.853
Vmax(meq/min) 1.705 0.579 0.794
The hydrolysis curves of arachin as a function of time with different concentrations
of papain, alcalase and fungal protease at is shown in Figure 7.5. It is evident from the curve
that the reaction rate was faster during the initial 15 min and the rate slowed down gradually
with the progress of time. At 1% concentration of papain the DH attained a value of 3.4% at
the end of 5 min and it attained a maximum of 14.7% at the end of 240 min. For alcalase the
plot showed a DH of 2.4% after 5 min and a maximum DH of 12.2% at the end of 240 min at
1% concentration. At 5% concentration the curve plateaued at around 18.6%DH. In the case
of fungal protease the DH reached up to 13.3% at the end of 10min and attained 25.3% after
240 min. At 5% concentration the hydrolysis curve showed a maximum DH of 25.5%. The
study showed that even after increasing the enzyme concentration to 5% the DH did not rise
beyond 27%. Among the different enzymes used for hydrolysis fungal protease was more
efficient for degradation of arachin as shown by the DH values.
The solubility profile of arachin and various proteolytic enzyme modified arachin to
low and high DH is shown in Figure 7.6. Arachin showed a broad range of solubility
between pH 2.0 and 10.0 and followed the typical U-shaped pattern. At pH 4.5 the solubility
was about 3.5% and it increased to 96.8% at pH 7.0 and to more than 97% at above pH 7.0.
At low DH (3.6%) papain increased the solubility of arachin by 9.0% at pH 4.5. At pH 7.0
the solubility was 92.7%. Extensive hydrolysis with papain (DH 14.8%) increased the
solubility up to 54.1% at pH 4.5 and 89.7 % at pH 7.0. In case of alcalase the solubility at
low DH (3.3%) was 14.3% compared to 92.1% at pH 7.0. In the case of fungal protease at
130
Figure 7.4. Effect of proteolytic enzymes on the DH of arachin
Arachin samples (20mg/ml) were hydrolysed with papain (pH 7.0), alcalase (pH 7.8) and fungal protease (pH 7.8) at 1% concentration for different time intervals (5 to 240 min) and the enzyme were inactivated by heating at 80˚C. The DH was measured by reaction with TNBS. The data were plotted as a function of time.
0 50 100 150 200 250 3000
10
20
30
PapainAlcalaseFungal protease
Time(min)
Deg
ree
of H
ydro
lysi
s(%
)
131
Figure 7.5: Effect of enzyme concentrations on the DH of arachin A. Papain B. Alcalase
C. Fungal protease. Arachin (20mg/ml) samples were hydrolysed with papain (pH 7.0), alcalase (pH 7.8) and fungal protease (pH 7.8) at different concentrations (0.5 to 5%) for different time intervals (5min to 240min). The enzymes were inactivated by heating at 80°C.The DH was plotted against time.
0 50 100 150 200 250 3000
5
10
15
20
0.5%
1%
4%
5%
B
Time (min)
Deg
ree
of
Hyd
roly
sis
%
0 50 100 150 200 250 3000
10
20
30
40
0.5%1%
2%5%
C
Time(min)
Deg
ree
of h
ydro
lysi
s (%
)
0 50 100 150 200 250 3000
5
10
15
20
25
0.5%1.0% 5.0%
A
Time (min)
Deg
ree
of h
ydro
lysi
s (%
)
132
low DH of 4.8% the solubility was 15.8 % at pH 4.5 and it increased to 90.6% at pH 7.0.
Extensive hydrolysis with fungal protease (DH 19.1%) increased the solubility to 59.4% at
pH 4.5 and to 94.5% at pH 7.0. The study revealed that enzymatic hydrolysis resulted in
substantial increase in the solubility of arachin over a wide range of pH (2.0 to 10.0). The
solubility at isoelectric point ranged from 14-16% at low DH to 55-60% at high DH.
Functional properties
The functional properties of arachin and papain, alcalase and fungal protease
modified arachins (low DH) are shown in Table 7.2. The emulsifying capacity of arachin
was 80.7 ml/g as against 102.7, 92.0 and 94.0 ml/g obtained for those modified with papain,
alcalase and fungal protease, respectively. The foaming capacity of arachin was 24.0%
compared to 28.0, 46.0 and 33.0% for limited proteolysed arachin with papain, alcalase and
fungal protease, respectively. All the proteolytic enzymes improved the emulsification
capacity of arachin upon limited proteolysis; papain was more effective compared to alcalase
and fungal protease. The foam stability of alcalase-modified arachin was remarkably high
(two fold). Papain and fungal protease modified arachin samples showed marginal increase
in foam capacity compared to arachin (control). There was no significant improvement in
foam stability among various enzyme modified arachin samples after limited hydrolysis.
The functional properties of extensively hydrolysed arachin in comparison with intact
arachin are shown in Table 7.3. The emulsification capacities of various enzyme-hydrolysed
arachin samples were 55-58 ml/g compared to 80.7 ml/g for arachin. The foam capacity of
papain, alcalase & fungal protease hydrolysed samples were 20.0, 23.0 and 18.0%
respectively. Excess hydrolysis of arachin caused reduction in emulsification and foaming
capacity as well as stability.
133
Figure 7.6: Effect of degree of hydrolysis of arachin on its solubility A. Papain
B. Alcalase C. Fungal protease
The sample solution in water was adjusted to pH (2-11) with 0.1 M HCl or NaOH .The soluble N content was measured by Kjeldahl method and solubility was expressed as % of N content in the supernatant after centrifugation to the original protein content of the sample.
0
20
40
60
80
100
0 2 4 6 8 10 12
pH
Pro
tein
so
lub
ility
(%)
Arachin
DH(3.3%)
DH(12.2%)
B
0
20
40
60
80
100
0 2 4 6 8 10 12pH
Pro
tein
so
lub
ility
(%)
Arachin
DH(3.6%)
DH(14.8%)
A
0
20
40
60
80
100
0 2 4 6 8 10 12
pH
Pro
tein
so
lub
ility
(%)
Arachin
DH(4.8%)
DH(19.1%)
C
134
Gel electrophoresis of enzyme hydrolysed arachin
The subunit composition of arachin analysed by SDS-PAGE in the presence of
disulfide reducing agents indicated 7 bands (Lane1; Figure 7.7). Tombs and Lowe (1967)
observed the subunit pattern of arachin and reported that it contained six major subunits and
classified them in to two groups of hydrophilic and hydrophobic groups. Monteiro and
Prakash (1994) reported seven bands having molecular weights ranging from 158000 to
72400 and also showed that six prominent bands had molecular weights of 72400, 603000,
39800, 33100, 26900 and 21900. The SDS PAGE pattern of arachin observed in the present
study is in agreement with the reported pattern (Monteiro and Prakash 1994). The SDS-
PAGE profiles for hydrolysates at different DH values did not show high molecular weight
subunits. Lane 1 (Figure 7.7) indicates the SDS pattern of arachin hydrolysed to a DH of
3.4% with papain. The pattern shows simultaneous action by papain on both high and low
molecular weights subunits. The low molecular weight subunit was resistant to attack
compared to high molecular weight subunits. Lane 3-6 represents the SDS-PAGE pattern at
5.2, 7.1, 9.4 and 13.7% DH. The low molecular weight component disappeared only when
the DH reached above 13.7%. Figure 7.7B represents the SDS-PAGE pattern of arachin
hydrolysed with alcalase. The pattern shows the degradation of high molecular weight
components first. The SDS PAGE pattern for fungal protease hydrolysis of arachin was
similar to that of alcalase (Figure 7.7C). The action of papain on arachin subunits was
different from that of alcalase and fungal protease. However, the low molecular weight
components disappeared only after hydrolysis for 1h with papain, alcalase and fungal
protease. The study indicates that the low molecular weight component is resistant to
hydrolysis by proteases.
Limited proteolysis increased the emulsifying and foaming properties of arachin.
Among the three enzymes tested, papain was more effective in enhancing the emulsification
135
Table 7.2: Effect of limited proteolysis (DH 3-5%) on functional properties* of arachin.
Enzyme Emulsification
capacity (ml/g) Foaming capacity (% volume increase)
Foam stability (ml) 10 min 20 min 30 min 40 min 60 min
Control Papain Alcalase Fungal protease
80.7 ± 1.4 102.7 ± 2.0 92.0 ± 2.6 94.0 ± 2.0
24.0 ± 1.6 28.0 ± 1.6 46.0 ± 3.3 33.0 ± 1.5
11 09 08 07 06 14 09 07 07 06 23 21 18 17 15 16 15 14 11 09
* Means of three replicates ± standard deviation. Table 7.3 : Effect of extensive hydrolysis on functional properties* of arachin.
* Means of three replicates ± standard deviation.
Enzyme Emulsification capacity (ml/g)
Foaming capacity (% volume increase)
Foam stability (ml) 10 min 20 min 30 min 40 min 60 min
Control Papain Alcalase Fungal protease
80.7 ± 1.4 58.0 ± 2.0 56.0 ± 1.3 55.0 ± 2.8
24.0 ± 1.6 20.0 ± 1.6 23.0 ± 3.3 18.0 ± 1.5
11 09 08 07 06 08 06 04 01 - 11 10 08 04 02 10 06 04 02 -
136
Figure 7.7: SDS- PAGE pattern of arachin and arachin hydrolysate with different DH
obtained with proteolytic enzymes.
Arachin was hydrolysed with papin, alcalase and fungal protease at 1% concentration for 5 to 240 min to get hydroysates with different DH. These hydrolysates with different DH were subjected to electrophoresis on a 10% polyacrylamide gel.
A. Papain: Lane 1; arachin: Lane 2; 3.4%DH: Lane 3; 5.2% DH: Lane 4; 7.1% DH: Lane 5; 9.4%DH: Lane 6; 13.7%DH.
B. Alcalase: Lane 1; arachin : Lane 2; 2.4% DH: Lane 3; 3.8% DH: Lane 4; 4.8% DH: Lane 5; 6.4%DH: Lane 6; 10.4%DH. C. Fungal protease: Lane1; arachin: Lane2; 4.1%DH: Lane 3; 13.1% DH: Lane 4;
14.2%DH:Lane5;17.8%DH:Lane6;22.1%DH.
Lane 1 2 3 4 5 6
A
Lane 1 2 3 4 5 6
B
Lane 1 2 3 4 5 6
C
137
capacity and alcalase was more effective in enhancing the foaming capacity of arachin.
Limited proteolysis of glycinin enhanced only the foaming properties and in this respect
papain was more effective. Emulsification capacity of glycinin could not be enhance by
limited hydrolysis. This shows that the effect of proteolytic enzymes on arachin is different
from that of glycinin. Arachin has more affinity towards enzymatic hydrolysis compared to
glycinin which is evident by the Km values and degree of hydrolysis. The overall
effectiveness of proteolytic enzymes in enhancing the functionality was more towards
arachin compared to the groundnut protein isolate. All the three proteolytic enzymes used for
modification enhance the functionality to different extent. By maintaining appropriate
hydrolysis conditions, such as enzyme-substrate ratio, right type of enzyme, and correct DH
it is possible to enhance the functionality of arachin including foaming, emulsification, and
solubility. The peptides generated after extensive hydrolysis of arachin can be used for the
identification of bioactive peptides.
138
Discussion
From the study it can be concluded that GPI and the homogenous fraction arachin
show different affinity to proteolytic enzymes. The functional properties of groundnut
proteins could be enhanced by proteolytic modification. The maximum DH observed with
GPI was18.6, 17.4 and 26.7% with papain, alcalase and fungal protease, respectively. In the
case of arachin the corresponding maximum DH noted was 23.6, 18.6 and 25.3%. These
results are shown in Table 7.4. The degree of hydrolysis of GPI and arachin was almost
equal with the three proteolytic enzymes. Among these enzymes fungal protease gave
maximum DH with both GPI and arachin which was followed by papain and alcalase.
Although papain has broad specificity its activity is mainly directed towards arginine peptide
link. The high DH obtained in the case of papain may be due to the high arginine content.
Alcalase although has broad specificity but it has preference to terminal hydrophobic amino
acids. The lower DH obtained with alcalase may be because groundnut proteins have low
content of hydrophobic amino acids. Fungal protease was more effective protease for GPI
and arachin for getting maximum DH.
The functional properties of modified GPI and arachin improved with lower degree
of hydrolysis; the extent of improvement was dependent on the enzyme and the substrate. In
the case GPI the EC considerably improved after limited proteolysis with papain alcalase and
fungal protease. However, the extent of improvement was almost equal with respect to all
the enzymes used. Limited proteolysis did not contribute towards change in FC of GPI.
Limited proteolysis of arachin increased both EC and FC. The extent of improvement was
dependent on the protease used. Papain was more effective in enhancing the EC. On the
other hand, alcalase was more effective in enhancing the FC. The results are shown in Table
7.5 Both GPI and arachin after proteolytic modification behaved differently with respect to
change in functional characteristics. The difference observed in the functionality with
139
papain, alcalase and fungal protease may be due to the difference in the point of cleavage of
the protein leading to the generation of peptides with different characteristics. Extremely
high degree of hydrolysis impaired the functionality of GPI. This may be due to the smaller
size of peptides which are less hydrophobic and provide no surface activity and hence not
capable of improving functional characteristics.
Table 7.4: Maximum DH obtained with groundnut protein isolate (GPI) and arachin with proteolytic enzymes
Degree of hydrolysis (%) Substrate
Papain Alcalase Fungal protease
GPI
18.6
17.4
26.7
Arachin
23.6
18.6
25.3
Table 7.5 : Functional properties of groundnut protein isolate (GPI) and arachin
modified to low DH using proteolytic enzymes
Functional property
Substrate
Enzyme
Emulsification capacity (ml/g)
Foaming capacity (% volume increase)
GPI
Papain
Alcalase Fungal protease
134.3 136.7 134.7
20.0
23.0
18.0
Arachin
Papain
Alcalase
Fungal protease
102.7 92.0 94.0
28.0 46.0
33.0
140
SUMMARY AND CONCLUSIONS
This investigation reports the results of enzymatic hydrolysis of soybean proteins
and groundnut proteins. These studies were carried out starting with defatted soy flour,
protein isolate, and purified fractions. The degree of hydrolysis (DH) was accurately
monitored by trinitro benzene sulphonic acid reaction of α amino groups released after
enzymatic hydrolysis. The changes in functional and physico chemical properties have been
correlated with the precise change in degree of hydrolysis. The study has led to the following
conclusions.
Studies on soy proteins
1. The maximum DH obtained with papain, alcalase and fungal protease enzymes were
18.6%, 29.6% and 32.4% respectively. A comparison of the DH value obtained with
different proteases showed that fungal protease was more effective among the proteases
used for the hydrolysis of defatted soy flour (DSF). The overall effectiveness of different
proteases in getting higher DH was in the order fungal protease > alcalase > papain.
2. Enzymatic modification of DSF to low DH (4-6%) resulted in remarkable increase in
emulsification capacity and marginal increase in foaming capacity. The extent of
improvement in EC followed by limited proteolysis was almost same for different
proteolytic enzyme modified flours.
3. The fat absorption capacity and water absorption capacity of enzyme hydrolysed DSF
was higher than that of intact DSF. Extensive hydrolysis impaired the overall
functionality of DSF.
4. The trypsin inhibitor activities of low DH and high DH enzyme modified freeze dried
DSF were similar to control, suggesting that trypsin inhibitors were resistant to enzymatic
attack.
141
5. The nitrogen content of spray-dried protein hydrolysate obtained with papain, alcalase
and fungal protease was almost same (9-9.2 %). The nitrogen content of DSF increased
to 11.5% after acid wash. The protein hydrolysate prepared by hydrolysis of wet protein
isolate obtained by alkali extraction followed by iso-electric precipitation had higher
nitrogen content (14.5%).
6. Amino acid composition of protein hydrolysates showed that the nutritional quality of
protein was retained after enzymatic hydrolysis. The bitterness of protein hydrolysates
was in the order papain< alcalase=fungal protease.
7. The spray dried protein hydrolysate of DSF obtained by different proteolytic enzymes
showed inactivation of lipoxygenase and urease activity. The trypsin inhibitor activity of
DSF hydrolysate was in the range 20-22 TIU/mg sample. The lower trypsin inhibitor
activity of spray dried hydrolysates compared to DSF may be due to application of heat
in spray drying process.
8. The protein hydrolysate was soluble over a wide range of pH (2.0-11.0); at iso-electric
pH, the solubility was >98%.
9. The DH of soy protein isolate obtained with papain, alcalase and fungal protease was
7.5%, 9.5% and 18.9% respectively. The effectiveness of proteolytic enzymes for
hydrolysis of SPI was lower compared to DSF. The overall effectiveness of proteases
towards hydrolysis of SPI was in the order fungal protease>alcalase>papain. Comparison
of the hydrolysis curves of freeze dried and spray dried SPI showed their susceptibility
towards proteolytic enzymes was almost same.
10. The solubility of SPI followed the typical U-shape pattern. The minimum solubility was
found at pH 4.5 (iso-electric pH). A low DH of 3-5% obtained with papain, alcalase and
fungal protease increased the solubility of modified SPI up to 29-35% at pH 4.5 and
142
97-98% at pH 7.0. Extensive hydrolysis of SPI increased the solubility at pH 4.5 up to
49-54% with different proteolytic enzymes.
11. Limited hydrolysis of SPI with different proteolytic enzymes increased the EC. Among
the proteases papain modified SPI showed more EC compared to alcalase and fungal
protease modified SPI. The FC of low DH modified SPI was higher compared to
unmodified SPI. Fungal protease modified SPI showed higher FC compared to papain
and alcalase modified SPI.
12. Extensive hydrolysis of SPI with proteolytic enzymes brought drastic reduction in EC.
Although the FC of SPI extensively hydrolysed using papain, alcalase and fungal
protease was higher than intact SPI, the FC values were lower than the corresponding
low DH modified SPI.
13. The maximum DH obtained with SPI with proteolytic enzymes was lower than that of
DSF. SPI with limited proteolysis showed remarkable increase in FC but the increase
was marginal with DSF.
14. Glycinin, the major protein fraction of soybean when hydrolysed with different
proteolytic enzymes showed that papain had the least effect followed by alcalase and
fungal protease. The enzymatic hydrolysis followed typical Michaelis-Menten pattern.
The affinity of glycinin to proteolytic enzymes was in the order fungal protease >
alcalase > papain as shown by the Km values.
15. The SDS-gel electrophoretic pattern of glycinin showed bands corresponding to acidic
(30-33kD) and basic subunits (29-22kD). The pattern observed for enzymatically-
modified glycinin suggested the preferential cleavage of acidic subunits compared to
basic subunits.
16. The hydrolysis of isolated acidic and basic subunits of glycinin with fungal protease
showed that basic subunits were less susceptible. A maximum DH of 26% was obtained
143
with acidic subunits at the end of 4h hydrolysis compared to 9-10% DH with basic
subunits. The Km values for acidic and basic subunits for fungal protease correlated well
with cleavage susceptibility.
17. Glycinin possess poor functional properties; enzymatic modification with proteolytic
enzymes improved a few of the functional characteristics. Papain with limited proteolysis
increased the FC almost three fold with good foam stability.
18. There was no difference in the EC of low DH modified glycinin samples. The FAC of
papain, alcalase, and fungal protease modified glycinin decreased compared to
unmodified glycinin. Limited proteolysis of glycinin did not bring significant differences
in the WAC.
19. The molecular sieve chromatography on Sepharose-6B gel showed single peak for
glycinin. In the case of modified glycinin it could be resolved in to two peaks. The first
peak had same Ve/Vo as that of glycinin but the second peak had higher Ve/Vo. This
indicated that even low DH degraded glycinin into low molecular weight peptides.
20. The electrophoresis pattern of gel filtration chromatographic peaks suggested that peak1
and peak 2 did not correspond to native glycinin; acidic subunits were readily hydrolysed
compared to basic subunits. Peak 2 did not give bands on the 10% gel suggesting that
peak 2 was extensively hydrolysed and the molecular weight of the resulting peptides
were low.
21. A low DH of 4-5% resulted in drastic reduction in mean residue ellipticity of modified
glycinin for all the enzymes tested. However, both glycinin and modified glycinin
exhibited characteristic near UV CD peaks at 263, 275, 283 and 291nm. This suggests
that even a low DH collapses the tertiary structure of glycinin.
22. The EC and FC of glycinin were lesser than that of SPI. The poor functionality of
glycinin may be due to the closely packed conformation of glycinin in which the
144
hydrophobic groups are buried inside. The enhancement in functionality by limited
proteolysis could be due to the exposure of hydrophobic groups.
Studies on groundnut proteins
23. GPI was more susceptible to hydrolysis with papain, alcalase and fungal protease
compared to SPI. The maximum DH of GPI obtained with papain, alcalase and fungal
protease was 18.6%, 17.4% and 26.6% respectively. The comparatively high affinity of
GPI for proteolytic enzymes may be because groundnut proteins are less hydrophobic
compared to soybean proteins. The effectiveness of different enzymes was in the order
fungal protease > papain > alcalase.
24. The solubility curve of GPI followed U-shaped pattern with the minimum at pH 4.5. The
low DH modified GPI with different proteolytic enzymes at isoelectric pH showed
solubility of 27-28%. However, high DH increased the solubility up to 47-55% at pH
4.5.
25. The EC and FC of GPI increased after limited proteolysis. The effectiveness of different
proteolytic enzymes in enhancing the EC was almost same. However, alcalase and fungal
protease enzymes were more effective compared to papain in enhancing the FC by
limited proteolysis.
26. Extensive hydrolysis of GPI resulted in a remarkable reduction of EC. Although the FC
of high DH modified GPI with different enzymes were higher than unmodified GPI, the
values were lower than that of low DH modified GPI.
27. Arachin, the major protein fraction of groundnut purified by ammonium sulfate
precipitation eluted as a single peak with Ve/Vo =1.5. Arachin with a low DH of 3-5%
showed two peaks, the first peak at Ve/Vo similar to arachin and second peak with a
Ve/Vo =1.8 This suggested that even at low DH arachin degraded into low molecular
weight peptides.
145
28. The Km values for arachin with different proteases were 0.83-0.931%. The Km values
were lower when compared to the values obtained with glycinin as substrate. Arachin
was the preferred substrate over glycinin for the proteolytic enzymes used.
29. The maximum DH of arachin obtained with papain, alcalase and fungal protease was
23.6%, 18.6% and 25.5% respectively. The effectiveness of these enzymes was in the
order fungal protease > papain > alcalase. These results obtained with arachin were
comparable to that of GPI. This suggests that the proteolytic enzymes have got equal
effectiveness for GPI and arachin.
30. The solubility of modified arachin to low DH (3-5%) was 14-16%. Extensive hydrolysis
increased the solubility up to 55-60%. Hydrolysis increased the solubility of arachin
considerably.
31. Limited proteolysis increased the EC of arachin. Among the different enzymes papain
was more effective in enhancing the EC. The FC of alcalase-modified arachin was
remarkably high (two fold). Papain and alcalase modified arachin showed marginal
increase in FC. Excess hydrolysis impaired the functionality of arachin except solubility,
32. The SDS-PAGE pattern of arachin was similar to those already reported in the literature.
Papain, alcalase and fungal protease degraded high molecular weight subunits. The
pattern for alcalase and fungal protease was similar. The action of papain on arachin
subunits was different. The low molecular weight subunit disappeared only after
hydrolysis for 1h with different enzymes.
33. The EC of arachin was higher than that of glycinin. There was considerable difference in
the FC between the two fractions. The overall effect of proteases in enhancing the
functionality differed considerably.
34. Comparison of the DH obtained with flour, isolate and purified fractions of groundnut
and soybean showed that the different proteases acted differently in getting high DH and
146
in changing functional characteristics. DSF gave high DH with proteolytic enzymes
compared to SPI. The effect of proteases on GPI and arachin was similar. In general, the
study has indicated that groundnut and soybean proteins are resistant to hydrolysis by
proteolytic enzymes. The bitterness of hydrolysate was more with soy proteins than
groundnut proteins. The affinity of proteases towards groundnut proteins was more than
that of soybean proteins. Thus by using appropriate proteolytic enzymes under specified
conditions the functional characteristics of seed proteins can be tailored to meet our
requirements.
147
REFERENCES Abbott, T.P., Nabetani, H., Sessa, D.J., Wolf, W.J., Liebman, M.N., and Dukor,R.K. 1996.
Effects of bound water on FTIR spectra of glycinin. J. Agric. Food Chem. 44, 2220-2224.
Adler-Nissen,J.(1976). Enzymatic hydrolysis of proteins for increased solubility. J Agric.
Food chem., 24,1090-1093.
Adler- Nissen, J. and Olsen, H.S. (1979). The influence of peptide chain on taste and
functional properties of enzymatically modified soy protein. ACS Symp. Ser., 92, 125-146.
Adler-Nissen,J.(1979).Determination of degree of hydrolysis of food protein hydrolysates by
trinitrobenzene sulphonic acid. J. Agric. Food Chem., 27, 1256-1262.
Adler-Nissen, J (1982). Limited enzymic degradation of proteins. A new approach in the
industrial application of hyrolysates. J.Chem.Technol.Biotechnol. 32, 138-156.
Adler-Nissen, J., Eriksen, S. and Olsen, H.S. (1983). Improvement of the functionality of
vegetable proteins by controlled enzymatic hydrolysis. Qual. Plant: Plant foods Hum. Nutr.
32, 411-423.
Aguilera, J.M., Rossi, F., and Hiche, E. (1980). Development and evaluation of an extrusion-
textured peanut protein. J. Food Sci., 45, 246-250.
Ahmed, E.M. and Schmidt, R.H. (1979). Functional properties of peanut and soybean
proteins as influenced by processing method. Peanut Sci., 6, 1-6.
Anderson, J.W., Johnstone, B.M., and Cook-Newell, M.L. (1995). Meta-analysis of the
effects of soy protein intake on serum lipids. N. Engl. J. Med., 333, 276.
AOAC. (1990). Official Methods of Analysis. 15th ed. Association of Official Analytical
Chemists, Washington, D.C.
148
AOCS. (1997). Official Methods and Recommended practices of the American Oil Chemist’s
Society, 5th ed. Champaign, IL.
Aoki, H., Taneyama, O., and Inami, M. (1980). Emulsifying properties of soy protein:
Characteristics of 7S and 11S proteins. J. Food Sci. 45, 534-538.
Appu Rao, A.G. and Narasinga Rao, M.S (1977) A method for isolation of 2S, 7S and 11S
proteins from soybean. Preparative Biochem. 7(2), 90-101
Arnon.R. (1970).Assay of proteolytic activity. Methods Enzymol. 19, 226-230.
Arthur, J.C. Jr. (1953). Peanut protein: Isolation, composition and properties. In: Advances in
protein chemistry, 8, 393-414.
Astrup, T., Brakman, P. and Sjlφn, K.E. (1962). Haemophilia and the protease inhibitors in
peanuts. Nature (London), 194(1), 980-981.
Axelrod, B., Cheesbrough, T.M. and Laakso, S. (1981). Lipoxygenase from soybeans.
Methods Enzymol., 71, 441-451.
Ayres, J.L., Branscomb, P.L., and Rogers, G.M. (1974). Processing of edible peanut flour
and grits. J. Am. Oil. Chem. Soc., 51, 133-136.
Ayres, J.L. and Devenport, B.L. (1977). Peanut protein: A versatile food ingredient. J. Am.
Oil Chem. Soc. 54, 109A-111A
Basha, S.M.and Cherry, J.P. (1976). Composition, solubility and gel electrophoretic
properties of proteins isolated from flornner (Arachis hypogaea L.) peanut seeds. J. Agric.
Food Chem., 24, 359-365.
Badley, R.A., Atkinson, D., Hauser H., Oldani, D. Green, J.P., and Stubbs, J.M. (1975). The
structure, physical and chemical properties of the soybean protein glycinin. Biochem.
Biophys. Acta 412, 214-228.
149
Bates, R. P., and Matthews, R.F. (1975). Ascorbic acid and β-carotene in soyabean as
influenced by maturity, sprouting, processing and storage. Proc. Fla. State Hort Soc 88,266-
271.
Beuchat, L.R. (1977b). Functional property, Modification of defatted peanut flour as a result
of proteolysis, Lebenm. Wiss. Technol., 10, 78-83.
Beuchat, L.R., Cherry, J.P. and Quinn, M.R. (1975). Physicochemical properties of peanut
flour as affected by proteolysis. J.Agric. Food Chem, 23, 616-620.
Betschart, A.A. (1979). Development of safflower protein. J. Am. Oil Chem. Soc. 56, 454-
459.
Betz and Stepaniuk, P. (1972). U.S. Patent, 3, 674,501.
Bhatia, D.S., Parpia, H.A.B. and Baliga, B.P. (1966). Peanut protein isolate-production and
properties. J. Food Sci. Technol. 3, 2-12.
Bidlingmeyer, B.A., Cohen S.A. and Tarvin T.L. (1984). Rapid analysis of amino acids
using precolumn derivatisation. Journal of Chromatography.336, 93-104.
Briggs, D.R., and Wolf, W.J. (1957). Studies on the cold-insoluble fraction of the water-
extractable soybean proteins. i. Polymerisation of the 11S component through reactions of
sulfhydryl groups to form disulfide bonds. Arch. Biochem. Biophys. 72, 127-144.
Bobalik, J.M. and Toranto, M.V. (1980). The effect of enzyme modification on the foaming,
water absorption and baking quality of defatted soya flour. J. Food Technol. 15, 637-646.
Bowman, D.E. (1944), Fractions derived from soybeans and navy beans which retard the
tryptic digestion of casein, Proc. Soc. Exp. Medicine, 57,139-144
Brooks, J.R. and Morr, C.V. (1985). Current aspects of soy protein fractionation and
nomenclature. J. Am. Oil Chem. Soc., 62, 1347-1354.
150
Bull, H.B. and Breese, K. (1976). Binding of water and electrolytes to proteins. An
equilibrium dialysis study. Biopolymers, 15, 1573-1583.
Carrol, K.K., and Kurowska, E.M. (1995). Soy consumption and cholesterol reduction:
review of animal and human studies. J. Nutr. (Suppl.) 125(35) 594S-597S.
Catsimpoolas, N., and Ekenstam, C. (1969). Isolation of alpha, beta, and gamma
conglycinins. Arch. Biochem. Biophys. 129, 490-497.
Catsimpoolas, N., Rogers, D.A., Circle, S.J., and Meyer, F.W. (1967). Purification and
structural studies of the 11S component of soybean proteins. Cereal Chem. 44, 631-637.
Catsimpoolas, N., Berg, T, and Meyer, E.W. (1971a). Hydrogen ion titration of ionizable
side chains in native and denatured glycinin. Int. J. Protein Res. 3, 63-71.
Cherry, J.P., Mc Watters, K.H.and Beuchat, L.R. (1979). Oilseed protein properties related
to functionality in emulsions and foams. In: Functionality and protein structure. (Ed: Pour-
El), ACS Symposium series 92. American Chemical Society, Washington, D.C., pp 1-26.
Chobert, J.M., Sitohy, M.Z., and Whitaker, J.H. 1988a. Solubility and emulsifying properties
of casein modified by Staphyloccus aureus V8 protease. J.Agric. Food Chem. 36, 220-224.
Chou, D. and Morr, C (1979). Protein-water interactions and functional properties. J. Am.
Oil Chem. Soc., 56, 53A-62A.
Cherry, J.R., Mc Watters, K.H. and Holmes, M.R. (1975). Effect of moist heat on solubility
and structure components of peanut proteins. J. Food Sci., 40(II), 1199-1204.
Chandrasekhara, M.R., Ramanna, B.R., Jaganath, K.S., and Ramanathan P.K.,
(1971).Miltone vegetable toned milk.. Food Technol., 25, 596-598.
Clegg, K.M., and McMillan, A.D. (1974). Dietary enzymatic hydrolysates of protein with
reduced bitterness, J.Food Technol. 9, 21-29.
151
Clemente A. (2000). Enzymatic protein hydrolysates in human nutrition. Trends Food Sci.
Technol. , 11, 254- 262.
Conckerton, E.J. and Ory, R.L. (1983). Chemical and functional properties of peanut and
ricebran flour blends. Peanut Sci., 10, 56-58.
Conckerton, E.J., and Ory, R.L., (1987). Peanuts as food proteins. In: Developments in food
proteins-5. (Ed) Hudson, B.J.F., Elsevier Applied Science, London and New York, pp. 1-29.
Conckerton, E.J. and Ory, R.L. (1976). Peanut proteins as food supplements: A
Compositional study of selected Virginia and Spanish peanuts. J. Am. Oil Chem. Soc., 53,
754-756.
Crenwelge, D., Dill,C., Tybor, P., and Landmann, W. A (1974). A comparison of the
emulsification capacities of some protein concentrates. J. Food Sci. 39, 175-177.
Damodaran, S. and Kinsella, J.E. (1981). Interaction of carbonyls with soy protein:
Thermodynamic effects. J.Agric. Food Chem., 29(II), 1249-1253.
Damodaran, S. and Kinsella, J.E. (1982). Effects of ions on protein conformation and
functionality. In: Food Protein Deterioration: mechanisms and Functionality. (Ed: Cherry,
J.P.), ACS Symposium Series No. 201, American Chemical Society, Washington, D.C., pp.
327.
Dawson, R. (1971). Comparison of fractionation of groundnut proteins by two different
methods. Anal. Biochem., 41, 305-313.
Dechary, J.M., Leonard, G.H. and Corkern, S. (1970). Purification and properties of a non-
specific hemagglutinin from the peanut. Lloydia, 33, 270-274.
Dieckert, J.W., Morris, N.J. and Manson, A.F. (1959). Saponins of the peanut: Isolation of
some peanut sapogenins and their comparison with the soya sapogenins by Glass-paper
chromatography. Arch. Biochem. Biophys., 82, 220-228.
152
Dieckert, J.W. and Dieckert, M.C., (1985). The chemistry and biology of the seed storage
proteins. In: New protein foods (Eds: Altschul: Altschul, A.M. and Wilcke, H.L.), vol. 5,
Academic Press Inc., Orlando, Florida, pp. 1-25.
Dieckert, J.W. and Dieckert, M.C., (1976a). In “Genetic Improvement of seed Proteins,
proceedings of a workshop” pp. 18-56. Natal Acad. Sci., 41, 475- 482.
Dieckert, J. W., and Dieckert, M.C. (1976b). The chemistry and biology of the vacuolar
proteins of seeds. J. Food Sci, 41, 475-482.
Draper, M., and Catsimpoolas, N. (1977). Isolation of the acidic and basic subunits of
glycinin. Phytochem. 16, 25-27.
Eggum, B.O., and Beames R.M. (1983). The nutritive value of seed proteins, In seed
Proteins, Biochemistry, Genetics, Nutritive value. E. Gottschalk and H.P. Muller (Ed.), pp.
499-531. Martinus Nijhof/Junk, Hague
Eldrige, A.C., and Wolf, W.J., (1967). Purification of the 11S component of soybean
protein. Cereal Chem. 44, 645-652.
Eldridge, A.C., Hall, P.K., and Wolf, W.J. (1963). Stable foams from unhydrolysed soybean
protein. Food Technol. 17(II), 1592-1595.
FAO Production year Book, Vol.44, (1999). Food and agriculture organization of the United
Nations, Rome,Italy, pp. 107-119.
FAO/WHO, (1990). Protein quality evaluation. FAO/WHO Nutrition Meetings, Report
Series. 51. Food and Agriculture Organisation/World Health Organization, Rome.
Fernando, S.M. and Murphy, P.A.(1990). HPLC determination of thiamin and riboflavin in
soybeans and tofu. J.Agric. Food Chem. 38, 163-167.
153
Fleming, S.E. Sosulski, F., Kilara, A. and Humbert, E.S. (1974). Viscosity and water
absorption characteristics of slurries of sunflower and soybean flours, concentrates and
isolates, J. Food Sci., 39, 188-191.
Fontaine, T.D., Pons, W.A.Jr. and Irwing, G.W. (1946). Protein-Phytic acid relationship in
peanuts and cottonseed. J. Biol. Chem., 164, 487-507.
Frazen, K.L., and Kinsella, J.E., (1976a). Functional properties of succinylated soy protein.
J. Agric. Food Chem. 24, 788-795.
Frazen, K.L., and Kinsella, J.E. (1976b). Functional properties of succinylated and acetylated
leaf protein. J. Agric. Food Chem. 24, 914-919.
Friedman, M. and Gumbman, M.R. (1986). Nutritional Improvement of soy flour through
inactivation of trypsin inhibitors by sodium sulfite. J. Food Sci. 51(5), 1239-1241.
Frφkjaer, S., (1994). Use of hydrolysates for protein supplementation. Food Technol. 48, 86-
88.
German, B., Damodaran, S. and Kinsella, J.E. (1982). Thermal dissociation behaviour of
soya Proteins. J. Agric. Food Chem., 30, 807-811.
Graham, D.E. and Phillips, M.C. (1976). In: Theory and practice of Emission Technology,
(Ed: Smith, A.L.), Acacemic Press, New York, and pp.75.
Graham, D.E. and Phillips, M.C. (1980a). Proteins at liquid interface.I. Kinetics of
adsorption and surface denaturation. J. Colloid Interface Sci., 70, 403-414.
Graham, D.E. and Phillips, M.C. (1980b). Proteins at liquid interface. II. Adsorption
isotherms. J. Colloid Interface Sci., 70, 414-426.
Graham, D.E. and Phillips, M.C. (1980c). Proteins at liquid interface. III. Molecular
structures of adsorbed films. J. Colloid Interface Sci., 70, 427-439.
154
Guzman, G.J and Murphy, P.A. (1986). Tocopherols of soyabean seeds and soyabean curd
(tofu) J. Agric Food Chem., 34, 791-795.
Halling, P.J. (1981). Protein stabilized foams and emulsions. CRC Crit. Rev. Food Sci. Nutr.,
15, 155-203.
Hashizume, K., Nakamura, N., and Watanabe, T. (1975). Influence of ionic strength on
conformation changes of soybean proteins caused by heating and relationship of its
conformation changes to gel formation. Agric. Biol. Chem. 39(II), 1339-1347.
Hashizume, K., and Watanabe, T. (1979). Influence of heating temperature on
conformational changes of soybean proteins. Agric. Boil. Chem. 43, 683-690.
Hasegawa, K., Kusano,T., and Mitsuda, H. (1963). Fractionation of soybean proteins by gel
filtration. Agric. Biol. Chem. 27, 878-880.
Hermansson, A.M., (1967). In “Problems in Human Nutrition” (J. Rolls and B. Porter, eds.),
P. 407, Academic Press, New York.
Hrckova, M., Rusnakova. M. and Zemanovic, J. (2002). Enzymatic hydrolysis of defatted
soy flour by three different proteases and their effect on the functional properties of resulting
protein hydrolysates. Czech J. Food Sci. 20(1), 7-14.
Hutton, C. and Campbell, A.M. (1977). Functional properties of a soy concentrate and soy
isolate in simple systems. J. Food Sci., 42(I), 454-456.
Hutton, C. and Campbell, A.M. (1977). Functional properties of soy concentrate and soy
isolate in simple systems and in a food system. Emulsion properties, thickening function and
fat absorption. J.Food Sci. 42(I), 457-460.
Indian Standards. Indian standard Recommended Methods for Determination of Aroma and
Taste Thresholds. Indian Standard Institution: New Delhi, 1983; No. 10641.
155
Inklaar, P., and Fortuin, J. (1969). Determining the emulsifying and Emulsion stabilizing
capacity of protein meat additives. Food Technol, 23, 103-197.
Irwing, G.W.Jr., and Fontaine, T.D. and Warner, R.C. (1945). Electrophoretic investigation
of peanut proteins. I. Peanut meal extract, arachin and conarachin. Arch. Biochem.,7, 475-
489.
Jacks, T.J., Neucere, N.J. and Mc Call, E.R. (1975). Thermally induced permutations of
antigenicity and conformation of arachin (Peanut globular protin). Int. J. Pept. Protein Res.,
7, 153-157.
Johnson, D.W., J. (1970b). Functional properties of oilseed proteins.J. Am. Oil Chem. Soc.
47, 402- 407.
Johnson, P. and Naismith, W.E.F., (1953). High weight system. The physicohemical
examination of the groundnut globulins (Arachis hypogaea). Discuss. Faraday Soc., 13, 98.
Kakade, M.L, Rackis, J.J., Mc Ghee, J.E., and Puski, G. (1974). Determination of trypsin
inhibitor activity of soya products: A collaborative analysis of improved procedure. Cereal
Chem. 51, 376-382.
Kamat, V., Graham, G., and Davis, M. (1978). Vegetable protein: Lipid interactions. Cereal
Chem. 55, 295-307.
Kamata, Y., Okubo, K., and Shibasaki, K. (1979b). Decrease of the soybean glycinin
digestibility in excess denaturation, Effect of refolding. Agric. Biol. Chem. 43, 1219-1223.
Kennedy, A.R. (1994). Prevention of carcinogenesis by protease inhibitors. Cancer Res.
(Suppl.) 54, 1999s-2005s.
Khalil, M. Ragab, M.and Hassanien, F.R. (1983). Some functional properties of oilseed
proteins. Nahrung- Food., 29, 275-282.
156
Kim, S.H.and Kinsella, J.E. (1985). Surface activity of food proteins: Relationships between
surface pressure development, viscoelasticity of interfacial films and foam stability of bovine
serum albumin. J.Food Sci., 50, 1526-1530.
Kim, S.H., and Kinsella, J.E. (1987a). Surface active properties of food proteins: Effects of
reduction of disulfide bonds on film properties and foam stability of glycinin. J. Food Sci.
52, 128-131.
Kim, S.Y., Park, P.S.W. and Rhee, K.C. (1990). Functional properties of proteolytic enzyme
modified soy protein isolate. J. Agric. Food Chem. 38, 651-656.
Kitamura, K., Takagi, T., and Shibasaki, K. (1976). Subunit structure of soybean 11S
globulin. Agric. Biol. Chem. 40(II), 1837-1844.
Kitamura, K., Okubo, K., and Shibasaki, K. (1974). The purification of soybean 11 S
globulin with ConA-Sepharose 4B and Sepharose 6B. Agric. Biol. Chem. 38, 1083-1085.
Kitamura, K., and Shibasaki, K. (1977). Homology among the acidic subunits of soybean
11S globulin. Agric. Biol. Chem. 41, 351-357.
Kinsella, J.E. and Shetty, R.J. (1979). Chemical modification for improving functional
properties of plant and yeast proteins. In: Functionality and protein structure, (Ed: Pour-
El.A.), ACS, Washington, pp. 37-63.
Kinsella, J.E. (1979). Functional properties of soy proteins. J. Am. Oil Chem. Soc 56,242-
258.
Kinsella, J.E., (1984). Milk proteins: Physicochemical and functional properties. CRC Crit.
Rev. Food Sci. Nutr. 21, 197-287.
Kinsella J.E., (1976). Functional properties of proteins in foods - A survey. CRC Crit. Rev.
Food Sci. Nutr. 7, 219-280.
157
Kinsella, J.E., Damodaran, S. and German, B. (1985). Physicochemical and functional
properties of oilseed proteins with emphasis on soy products. In: New Protein Foods, Vol. 5,
Seed Storage Proteins, (ED. Altschul, M. and Wilcke, H.L.), Academic Press, New York,
PP. 108-118.
Kitamura, K., and Shibasaki, K. (1975). Isolation and some physicochemical properties of
the acidic subunits of soybean 11S globulin. Agric. Biol. Chem. 39, 945-951.
Koshiyama, I. (1972b). Purification and physico-chemical properties of 11S globulin in
soybean seeds. Int. J. Peptide Protein Res. 4, 167-176.
Koshiyama, I. (1972c). Comparison of acid induced conformation changes between 7S and
11S globulins in soybean seeds. J. Sci. Food. Agric. 23, 853-859.
Koide, T. and Ikenaka, T. (1973). Studies on soybean trypsin inhibitors. III. Aminoacid
sequence of the carboxyl region and the complete amino acid sequence of soybean trypsin
inhibitor (Kunitz), Eur. J. Biochem., 417- 431.
Kolar,C., Cho, I.C., and Watrous, W.L. (1979). Vegetable protein application in yoghurt,
coffee creamers and whip toppings. J.Am.Oil Chem. Soc. 56, 389-391.
Kolar, C.W., Richert, S.H., Decker, C.D., Steinke, F.H., and Vander Zanden, R.J.(1985).
Isolated protein. Ch. VIII. In New Protein Foods, Vol. 5, A.M. Altschul and H.L. Wilcke,
(Ed.), pp. 259-299. Academic Press, New York.
Kuehler, C.A. and Stine, C.M. (1974). Effect of enzymatic hydrolysis on some functional
properties of whey. J. Food Sci. 39, 379-382.
Kunitz, M. (1945). Crystallization of trypsin inhibitor from soybeans. Science 101, 668-669.
Kuntz, I.D. and Kauzman, W. (1974). Hydration of proteins and polypeptides. Adv. Protein
Chem., 28, 239-345.
158
Kyte, J. and Doolittle, R.J. (1982). A simple method for displaying the hydropathic character
of protein. J. Mol. Biol., 157, 105-132.
Laemmli, U.K., (1970) Cleavage of structural proteins during the assembly of the head of
bacteriophage T4. Nature (London), 227, 680–985.
Lah, C., Cheryan, M., and deVor, R. (1980). A response surface methodology approach to
the optimization of whipping properties of an ultrafiltered soy product. J.Food Sci. 45, 1729-
1726.
Lahl, W.J., and Braun, S.D. (1994). Enzymatic production of protein hydrolysates for food
use. Food Technology, 48(10), 68-71.
Lawhon, J.T., Cater, C.M., Mattil, K.F. (1972). A Comparative study of the whipping
potential of an extract from several oilseed flours. Cereal Sci. Today, 17, 240-246.
Lawhon, J.T., Golightly, N.H, and Lusas, E.W. (1980). Utilization of membrane- produced
oilseed isolates in soft-serve frozen desserts. J.Am.Oil Chem. Soc. 57(II), 302-306.
Li-chan, E and Nakai, S (1991). Importance of hydrophobicity of proteins in food emulsion.
Microemulsions Emulsions Food. 448, 193-212.
Liener, I.E. (1994). Implications of antinutritional components in soybean foods. CRC Crit.
Rev. Food Sci. Nutr. 34(1), 31-67.
Lin, M.J., Humbert, E.S., and Sosulski, F.W. (1974). Certain functional properties of
sunflower meal products. J.Food Sci., 39, 368-370.
Lucas, E.W. (1979) Food uses of peanut protein.. J.Am.. Oil Chem. Soc. 56 (I), 425-430.
Lynch, C. J., Rha, C.K. and Catsimpoolas, N. (1977). Tryptic hydrolysis of glycinin and its
subunits. J. Sci. Food Agric. 28, 971- 979.
159
Mattil, K.F. (1971). The functional requirements of proteins for foods. J. Am. Oil Chem.
Soc. 48, 477–480.
Mattil, K.F. (1974). Composition, Nutritional, and Functional properties, and Quality criteria
of soy protein isolates. J. Am.Oil Chem.Soc. 51, 81A-84A.
Mac Ritchie, F. (1978). Proteins at interfaces. Adv. Protein Chem., 32, 283-326.
Mahmoud, M.I.(1994). Physicochemical and functional properties of protein hydrolysates in
nutritional products. Food Technol. 48, 89-95.
Manak, L.J., Lawhon, J.T. and Luscas, E.W. (1980). Functional potential of soy, cottonseed,
and peanut protein isolates produced by industrial membrane systems. J. Food Sci., 45, 236-
238.
McWatters, K.H. and Holmes, M.R. (1979b). Influence of moist heat on solubility and
emulsifying properties of soy and peanut flours. J. Food Sci., 44 (I), 774-776.
McWatters, K.H., Cherry, J.P. and Holmes, M.R. (1976). Influence of suspension medium
on functional and protein properties of defatted peanut meal. J. Agric. Food Chem., 24, 517-
523.
McWatters, K.H. and Holmes, M.R. (1979a) Influence of pH and salt concentration on
nitrogen solubility and emulsification properties of soy flour. J. Food Sci., 44, 770-773.
Mitsuda, H., Kusano, T, and Hasegawa, K. (1965). Purification of the 11S component of
soybean proteins. Agric. Biol. Chem. 29, 7-12.
Mita, T., Nikai, K, Hiraoka, T., Matsuo, S. and Matsumoto, H. (1977). Physicochemical
studies on wheat protein foams. J.Colloid Interface Sci., 59, 172-178.
Mita, T., Ishido, F. and Matsumoto, H.(1978). Physico-Chemical studies on wheat protein
foams. II Relationship between bubble size and stability of foams prepared with gluten and
gluten components. J.Colloid Interface Sci., 64, 143-153.
160
Molina Ortiz, S.E. and Wagner, J.R. (2002). Hydrolysates of native and modified soy protein
isolates: Structural characteristics, solubility and foaming properties. Food Research
International 35, 511-518.
Monteiro, P.V., and Prakash V. (1994). Effect of proteases on arachin, conarachin I and
conarachin II from peanut (Arachis hypogaea L.) J.Agric.Food Chem., 42, 268-273.
Monteiro, P.V., and Prakash V. (1994) Functional properties of homogeneous protein
fractions from peanut (Arachis hypogaea L.) J.Agric.Food Chem., 42, 274-278.
Moreira, M.A., Hermondson, M.A., Larkins, B.A., and Nielson, N.C. (1979). Partial
characterization of the acidic and basic polypeptides of glycinin. J. Biol. Chem. 254, 9921-
9926.
Natarajan, K.R. (1980). Adv.Food Res. Peanut protein Ingredients: Preparation, properties,
and food uses. 27, 216-273.
Naismith, W.E.F., and Mc David, H.M.R. (1958). Fractionation studies on the proteins of the
groundnut (Arachis hypogaea). J.Appl. Chem, 8, 605-610.
Naismith, W.E.F. and Kelly, K.O. (1958). The cross-linking action of formaldehyde on the
proteins of the groundnut (Arachis hypogaea). J.Appl.Chem., 8, 286-290.
Naismith, W.E.F. and Williams, R.K. (1956). The alkaline denaturation of arachin. J. Appl
Chem., 6, 412-427.
Naismith, W.E.F. (1955). Ultra centrifuge studies on soybean protein Biochem. Biophys.
Acta 16, 203-210.
Nagano, T., Hirotsuka, M., Mori, H., Kohyama, K.Nishinari, K.(1992). Dynamic
Viscoelastic study on the gelation of 7S globulin from soybean. J. Agric. Food. Chem. 40,
941-944.
161
Nash, S.M., and Wolf, W.J. (1967). Solubility and ultracentrifugal studies on soybean
globulins. Cereal Chem. 44, 183-192.
Neucere, N.J. (1972). Effect of heat on peanut proteins. I. Solubility properties and
immunochemical-electrophoretic modifications. J. Agric. Food Chem., 20, 252-255.
Neucere, N.J. (1969). Isolation of alpha arachin, the major peanut globulin. Anal. Biochem.,
27, 15-24.
Nielson, N.C. (1985a). Structure of soy proteins. Ch. 2. In New Protein Foods, Vol 5. Seed
storage Proteins, A.M. Altschul and H.L. Wilcke (Ed.), pp. 27-64. Academic Press, Orlando,
FL.
Nozaki, Y. and Tanford, C. (1971). The solubility of amino acids and two glycine peptides in
aqueous ethanol and dioxane solutions. Establishment of a hydrophobicity scale. J. Biol.
Chem. 246, 2211-2217.
Ochiai-Yanagi, S., Miyauchi, H., Saio, K., and Watanabe, T. (1978). Modified soybean
protein with high water-holding capacity. Cereal Chem. 55, 157-167.
Odani,S. and Ikenaka, T. (1973). Studies on the soybean trypsin inhibitors. VII. Disulfide
bridges in soybean Bowman-Birk proteinase inhibitor, J. Biochem. (Tokyo), 74, 697-701.
Olsen, H.S. and Adler-Nissen, J. (1981). Application of ultra and hyper filtration during
production of enzymatically modified proteins. ACS Symp. Ser. 154, 133-169.
Olsen, H.S. (1984). Method of producing an egg white substitute material. US Patent
4,431,629 (to Novo industri A/A, Denmark).
Panyan, D. and Kilara, A. (1996). Enhancing the functionality of food proteins by enzymatic
modification. Trends Food Sci. Technol. 71, 120-125.
162
Pernollet, J.C. and Mosse, J. (1983). Structure and location of legume and cereal storage
proteins. In: Seed Proteins (Edn: Daussant, J., Mosse, J. and Vaughan, J.) Academic Press
Inc. (London) Ltd., London, pp. 155-191.
Peng. I.C., Quass, D.W., Dayton, W.R., Allen, C.E. (1984). The physico- chemical and
functional properties of soyabean 11S globulin- A review. Cereal Chem. 61, 480-490.
Phillips, M.C. (1981). Protein conformation at liquid interfaces and its role in stabilizing
emulsions and foams. Food Technol., 35, 50-57.
Phillips, M.C. (1977). The conformational properties of proteins at liquid interfaces. Chem.
Ind. (London), 5, 170-176.
Pour-El, A. (1981). Protein functionality, classification,definition and methodology. In:
Protein functionality in foods (Ed. Cherry, J.P) ACS. Symp Ser, 147. Washington, DC,pp. 1-
19.
Prakash, V., and Rao, M.S.N., (1986). Physico-chemical properties of oilseed proteins, CRC.
Crit. Rev. Biochem., , 20, 265-364.
Prakash, V. and Rao, M.S.N. (1988). Structural similarities among the high molecular weight
protein fractions of oilseeds. J. Bio Sci., 13, 171-180.
Puski, G. (1975). Modification of functional properties of soy proteins by proteolytic enzyme
treatment. Cereal Chem. 52, 655-644.
Qi, M., Hettiarachchy N.S and Kalapathy.U. (1997). Solubility and emulsifying properties of
soy protein isolates modified by pancreatin. J. Food Sci. 62(6), 1110-1115.
Quinn, M.R. and Beuchat, L.R. (1975). Functional property changes resulting from fungal
fermentation of peanut flour. J. Food Sci., 40, 475-478.
Rahma, E.H., and Rao, M.S.N. (1983a). Effects of acetylation and succinylation of
cottonseed flour on its functional properties. J. Agric. Food Chem., 31, 352-355.
163
Ramanatham, G., Rao, L.H. and Urs, L.N. (1978). Emulsification properties of groundnut
protein. J. Food Sci., 43, 1270-1273.
Read, J.W. and Haas, W. (1938). Studies on the baking quality of flour as affected by certain
enzyme actions. V. Further studies concerning potassium bromate and enzyme activity.
Cereal Chem., 15, 59-68.
Rham,O., Kruseman, J., and Hidalgo,J. (1978). Nongelling soy protein hydrolysate for use in
concentrated fluid complex foods. J. Food Sci. 43, 642-643.
Rhee, K.C., Cater, C.M. and Mattil, K.F. (1972a). Simultaneous recovery of protein and oil
from raw peanuts in aqueous system. J. Food Sci., 37, 90-93.
Rhee, K.C. Cater, C.M. and Mattil K.F. (1973a). Effect of processing pH on the properties of
peanut protein isolates and oil. Cereal Chem. 50, 395-404
Schmidl M.K., Taylor, S.L., and Nordlee J.A. (1994). Use of hydrolysate based products in
special medical diets. Food Technol. 48, 77-85.
Schmidt, R., Packard, V. and Morris, H. (1984). Effect of processing on whey protein
functionality. J. Dairy Sci., 67, 2723-2733.
Sekul, A.A and Ory, R.L. (1976). Rapid method for partial hydrolysis of oilseed protein for
food uses. J. Am. Oil Chem. Soc. 53, 151A.
Sekul, A.A. and Ory, R.L. (1977). Rapid enzymatic method for partial hydrolysis of oilseed proteins for food uses. J. Am. Oil Chem.Soc. 54(1), 32-35.
Sekul, A.A., Vinnett, C.H., and Ory, R.L. (1978). Some functional properties of peanut
proteins partially hydrolysed with papain. J. Agric. Food Chem., 26, 855-858.
Shen, J.L. (1976). Soy protein solubility: The effect of experimental conditions on the
solubility of soy protein isolates. Cereal Chem. 53 (II), 902-909.
164
Shen, J.L., (1981). Solubility and viscosity Ch.5. In protein functionality in foods. J.P.
Cherry (Ed.), ACS Symp. Ser. 147, 89-109. American Chemical Society, Washington, DC.
Shetty, K.J. and Rao, M.S.N. (1973). Studies on groundnut proteins. Part.I. Effect of Sodium
dodecyl Sulphate on the physicochemical properties. Indian J. Biochem. Biophys., 10, 149-
154.
Shetty, K.J. and Rao, M.S.N. (1974). Studies on groundnut proteins. III. Physicochemical
properties of arachin prepared by different methods. Anal. Biochem., 62, 108-120.
Shyamasunder, R. and Rajgopal Rao, D. (1982). Acylated arachins- functional properties.
Lebensm-Wiss. Technol., 15, 102-104.
Siemmensma, A.D., Weijer, W.J., Bak, H.J., (1993). The importance of peptide lengths in
hypoallergenic infant formulae. Trends Food Sci. Technol. 4, 16-21.
Sipose, E.F., Endres, J., Tybor, P.T., and Nakojima, Y. (1979). Use of vegetable protein in
processed seafood products. J.Am.Oil Chem. Soc. 56, 320-327.
Sirtori, C.R., Evem, R., and Lovati. M.R. (1993). Soybean protein diet and plasma
cholesterol: from therapy to molecular mechanisms. Ann. N.Y. Acad. Sci. 676, 188.
Smith, A.K., and Circle, S.J., Eds. (1978). Soybean: Chemistry and technology, Vol. 1 AVI
Publ. Co., Westport, Connecticut.
Sosulski,F., (1979). Food uses of sunflower proteins.. J. Am. Oil Chem. Soc. 56, 438- 446.
Sreenivasan, V and Moudgal, N.R., and Sharma, P.S. (1957). Studies on goitrogenic agents
in food I. Goitrogenic action of groundnut. J. Nutr., 61, 87-89.
Subba Rau, B.H., and Srinivasan.K.S. (1988). Enzymatic modification of groundnut flour(by
papain/protease) and its effect on functional properties. Lebensm.-Wiss.u.-Technol.,21, 126-
130
165
Sugimoto, H. Van Buren, J.P., and Robinson, W.B. (1971). An enzymatic process for a
protein-containing beverage based on soybean protein and lemon juice. J. Food Sci. 36, 729-
731.
Staswick, P.E., Hermondson, M.A., and Nielson, N.C. (1981). Identification of the acidic
and basic subunit complexs of glycinin. J. Biol. Chem. 256, 8752-8755.
Staswick, P.E., (1982). Characterization of the subunits of glycinin and the complete primary
structure of the A2B1a subunit. Ph.D. thesis, Purdue University, West Lafayette, Indiana.
Stone, M., and Campbell, A.M. (1980). Emulsification in systems containing soy protein
isolates, salt and starch. J. Food Sci. 45(II), 1713-1716.
Suresh Chandra, B.R., Appu Rao, A.G. and Rao M.S.N. (1984). Effect of temperature on the
conformation of soyabean glycinin in 8 M urea or 6 M guanidine hydrochloride solution. J.
Agric. Food Chem., 32, 1402-1405.
Sykes, G.E., and Gayler, K. R. (1981). Detection and characterization of a new β-
conglycinin from soybean seeds. Arch. Biochem. Biophys 210(2), 525-530.
Tanh, V.H., and Shibasaki, K. (1976a). Major proteins of soybean seeds. A straight forward
fractionation and their characterization. J. Agric. Food Chem. 24(6), 1117-1121.
Tanh, V.H. and Shibasaki, K. (1976b). Heterogeneity of β-conglycinin. Biochem. Biophys.
Acta. 439, 326-348.
Takagi, S., Okamoto, N, Akashi, M., and Yasumatsu, K. (1979). Hydrophobic bonding and
SS bonding in heat denaturation of 11S of soybean protein, Nippon Shokuhin Kogyo Gakkai-
Shi 26, 139.
Thomas, D.L., and Neucere, N.J. (1973). Effect of heat in situ on electrophoretic patterns of
reserve proteins and enzymes in dormant peanut (Arachis hypogaea). J. Agric. Food Chem.,
21, 479- 482.
166
Tanh, V.H., Okubo, K and Shibasaki, K. (1975a). Isolation and characterization of the
multiple 7S globulins of soybean, Plant Physiol. 56, 19-22.
Tombs, M.P., (1963). Variant forms of arachin. Nature( London), 2000(I),1321- 1322.
Tombs, M.P. (1965). An electrophoretic investigation of groundnut proteins. The structure of
arachinA and B. Biochem. J., 96, 119-133.
Tombs, M.P. and Lowe, M. (1967). A determination of the subunits of arachin by
osmometry. Biochem. J., 105, 181-187.
Tornberg, E. (1979). The adsorption behaviour of proteins at an interface as related to their
emulsifying properties. ACS Symp. Ser., 92, Washington, DC, pp. 105-123.
Torun, B., Vitery, F.E., and Young, V.R. (1981). Nutritional role of soya protein for humans.
J. Am. Oil Chem. Soc. 58, 400-406.
Tur-Sinai, A., Birk, Y. Gertler, A. and Righi, M. (1972). A basic trypsin and chymotrypsin
inhibitor from groundnuts(Arachis hypogaea). Biochim. Biophys. Acta., 263, 666-672.
Vaintraub, I.A. and Bulmaga, V.P. (1991). Effect of phytate on the in vitro activity of
digestive proteinases. J.Agric. Food Chem. 39, 859-861.
Van Huystee, R.B. (1973). A Study on the degradation of arachin by proteolytic enzyme.
Can. J. Bot., 51, 2217-2222.
Von Hippel, P.H. and Schleich, T. (1969) In: Structural and stability of biological
Macromolecules. (Eds: Timasheff, S.N. and Fasman), G.D. Marcel Dekker, New York. pp.
416-574.
Wagner, J.R. and Gueguen, J. (1995). Effects of dissociation, deamidation and reducing
treatment on structural and surface active properties of soya glycinin. J. Agric. Food Chem.,
43, 1993-2000.
167
Wagner, J.R.and Gueguen, J. (1999). Surface functional properties of native, acid treated and
reduced soya glycinin. 1. Foaming properties. J. Agric. Food Chem., 47, 2173-2180.
Waggle, D.H., Decker, C., and Kolar, C.W. (1981). Soya products in meat, poultry and sea
food J.Am.Oil Chem. Soc. 58,341-343.
Wolf, W.J. (1970). Soybean proteins-Their functional, chemical and physical properties. J.
Agric. Food Chem. 18(6), 969-976.
Wolf.W.J., and Briggs, D.R. (1956). Ultracentrifugal investigation of the effect of neutral
salts on the extraction of soybean proteins. Arch. Biochem. Biophys. 63, 40-49.
Wolf, W.J., and Biggs, D,R. (1958). Studies on the cold-insoluble fraction of water-
extractable soybean proteins. II. Factors influencing conformation changes in the 11S
component. Arch. Biochem. Biophys. 76, 377-393.
Woodroof , J.G. (1973). Composition and nutritious value of peanuts. In: production,
processing and products, (Ed: Woodroof, J.G), AVI publishing Co. Inc., Westport, CT, pp.
165-180.
Wolf, W.J. and Sly, D.A. (1965). Chromatography of soybean proteins on hydroxylapatite.
Arch. Biochem. Biophys. 110, 47-56.
Wolf, W.J., and Sly, D.A. (1967). Cryoprecipitation of soybean 11S protein. Cereal Chem.
44, 653-668.
Wolf, W.J., Rackis, J.J., Smith, A.K., Sasame, H.A., and Babock, G.E. (1958). Behaviour of
the 11S protein of soybean in acid solutions. 1. Effects of pH, ionic strength, and time on
ultracentrifugal and optical rotatory properties. J. Am. Chem. Soc. 80(5), 5730-5735.
Wolf, W.J., Babcock, G.E., and Smith, A.K. (1961). Ultra centrifugal differences in soybean
protein composition. Nature 191(II), 1395- 1396.
168
Wolf, W.J., Babcock, G.E., and Smith, A.K. (1962). Purification and stability studies of the
11S component of soybean proteins. Arch. Biochem. Biophys. 99, 265-274.
Wolf, W.J., and Briggs, D.R. (1956). Ultracentrifugal investigation of the effect of neutral
salts on the extraction of soybean proteins. Arch. Biochem. Biophys.63, 40-44
Wolf, W.J., and Briggs, D.R. (1959). Purification and characterization of the 11S component
of soybean proteins. Arch. Biochem. Biophys. 85, 186-199.
Wolf, W.J. and Tamura, T. (1969). Heat denaturation of soybean protein. Cereal Chem. 46,
331.
Wu, S., Murphy, P.A., Johnson, L.A., Fratzke, S.R., Reuber, M.A. (1999). Pilot plant
fractionation of soybean glycinin and β-conglycinin. J. Am. Oil Chem. Soc. 76, 285-293.
Wu. Y.V. and Sessa, D.J. (1994). Conformation of Bowman-Birk inhibitor. J. Agric. Food
Chem. 42, 2136- 2138.
Yang, J.T. and Kubota, S. (1985). In. Microdomains in polymer solution, (Dublin, P.L.,Ed.)
Plenum press, New York.
Yamagishi, T. Miyakawa, A, Noda, N and Yamagishi, F. (1983). Thermal dissociation and
association behaviour of soy proteins. Agric. Boil. Chem., 47, 1229-1237.
Yatsumatsu, K., Toda, J., Wada, T., Misaki, M., and Ishii, K. (1972b). Studies on the
functional properties of food grade soybean products. Part III. Properties of heat coagulated
gels from soybean products. Agric. Biol. Chem. 36, 537-550.
Yatsumatsu, K., Sawada, S., Moritaka, S., Misaki, M., Toda, J., Wada, T., and Ishii, K.
(1972a). Whipping and emulsifying properties of soybean products. Agric. Biol. Chem., 36,
719-727.
Yotsuhashi, K. and Shibasaki, K. (1973). Peanut proteins II. Dissociation of conarachin into
subunits. Chem. Abstr., 84, 70551.
169
Yu, P.H. (1977). Isolation and partial characterization of arachin and α-conarachin from the
seeds of Arachis hypogea. L. Ph.D. Dissertation, Texas A&M University college Station.
Zakaria, F., and Mc Feeters, R.F. (1978). Improvement of the emulsification properties of
soy protein by limited pepsin hydrolysis. Lebens. Wiss. Technol. 11, 42-44.
