Title of Invention	"AN IMPROVED ENZYMATIC PROCESS FOR THE PREPARATION OF MODIFIED GLYCININ HAVING ENHANCED SOLUBILITY, EMULSIFYING AND WHIPPING CAPABILITIES AND LOWER DEGREE OF HYDROLYSIS."
Abstract	Glycinin, the major storage protein of soybean was enzymatically modified using papain, alcalase and fungal protease. The degree of hydrolysis was controlled by varying the ratio of enzyme to substrate, time and temperature of hydrolysis. The functional properties of glycinin could be tailored by controlling the degree of hydrolysis. With a very low degree of hydrolysis, 3 - 5%, modified glycinin had better functional characteristics compared to glycinin.

Title of Invention

"AN IMPROVED ENZYMATIC PROCESS FOR THE PREPARATION OF MODIFIED GLYCININ HAVING ENHANCED SOLUBILITY, EMULSIFYING AND WHIPPING CAPABILITIES AND LOWER DEGREE OF HYDROLYSIS."

Abstract

Glycinin, the major storage protein of soybean was enzymatically modified using papain, alcalase and fungal protease. The degree of hydrolysis was controlled by varying the ratio of enzyme to substrate, time and temperature of hydrolysis. The functional properties of glycinin could be tailored by controlling the degree of hydrolysis. With a very low degree of hydrolysis, 3 - 5%, modified glycinin had better functional characteristics compared to glycinin.

Full Text	The present invention relates to an improved enzymatic process for the preparation of modified glycinin having enhanced solubility, emulsifying and whipping capabilities and lower degree of hydrolysis. Soybean is a source of high quality proteins all over the world. The production of soybean in India has gone up from 2.63 million tones in 1989-91 to 6.5 million tones. Soy proteins in particular soy protein isolate is useful as a food ingredient in various formulations by virtue of its high protein content and functional properties. In the food industry materials such as egg white, hydrolyzed milk proteins and soy proteins have been used as aerating agents and heat setting binders when properly whipped along with other jaqueous foaming or whipping ingredients, the aerating agents permit the entrapment of gases thereby to provide a foamed or aerating agents permit the entrapment of gases thereby to provide a foamed or aerated prodeuct. Thus aerating agents must necessarily ingest and entrap gas volume within a reasonably low whipping time. j Defatted soybean meal contains nearly 50-55% protein. The major proteins of soybeans are storage globulin i.e. glycinin and conglycinin make 90% of the total. The proteins can be classified into 2S, 7S, 11S and 15S protein based on sedimentation coefficients. The 11S fraction also known as cjlycinin constitutes 25-35% of the total potein (Liu 1997, In: 'Soybeans: chemistry, technology and Utilization, pp. 25-113, Chapman & Hall, New York). Reference may be made to a published paper titled "Major proteins of soybean seeds. A straight forward fractionation and characterization", by Tanh and Shibasaki, 1976, Journal of Agriculture and Food Chemistry, 24,1117-1121 wherein it is reported a method for the fractionation of 7S and 11S globulins from defatted soybean meal by fractional isoelectric precipitation. Reference may be made to the published paper titled "A method for isolation of 2S, 7S and 11S proteins of soybeans" by Appu Rao, A.G. and Narasinga Rao, M.S., 1979, wherein glycinin can be obtained by magnesium chloride precipitation of the water extract. Reference may be made to the published paper by Wu et al., 1999, titled "A pilot- plant fractionation iof soybean glycinin and conglycinin", Journal of American Oil Chemists Society, 76, 285-293, wherein glycinin can be prepared on a pilot plant scale. Reference may be made to a published paper by Kitamura et al., 1995, titled " Genetic improvement of nutritional and food processing quality in soybean, Japanese Agricultural Research Quarterly, 29, 1-8 wherein glycinin is shown to be rich in methionine and contains 3-4 times more methionine and cysteine per unit protein than ;7S protein. Reference may be made to Wagner and Gueguen, 1995, titled "Surface functional properties of native, acid treated and reduced soy glycinin", wherein it is shown that glycinin is poorer in functional attributes such as foaming and emulsifying characteristics due to its closed globular conformation and low molecular flexibility. Reference may be made to Kim and Kinsella, 1987, in the published papers titled "Surface active properties of food proteins: effects of reduction of disulfide bonds on film formation and foam stability of glycinin, Journal of Food Science, 52, 128-131, and "Surface active properties of food proteins: effect of progressive succinylation of film properties and foam stability of glycinin, Journal of Food Science, 52, 1341-1352, wherein the structural modification of glycinin could induced by varying environmental factors such as ionic strength, DH, temperature and chemical treatments such as reduction of component disulfide :l bonds, succinylation and acetylation. Reference could be made to the published papers of Wagner and Gueguen 1995, and Wagner, and Gueguen 1999, titled "Effects of dissociation, deamidation and reducing treatment on structural and surface active properties of soy glycinin, Journal of Agriculture and Food Chemistry, 43, 1993 - 1994, and "Surface functional properties of native acid treated and reduced soy glycinin. 1. Foaming properties", Journal of Agriculture and Food Chemistry, 47, 2173 -2180, wherein the foaming and emulsifying properties of glycinin could be greatly improved by mild acid treatments, deamidation and reducing agents. Reference may be made to RC Gunther et al., 1974, US patent No. 3,814 816, wherein a successful vegetable protein aerating agent is disclosed wherein he has prepared vegetable jprotein aerating agent by hydrolyzing an oil free vegetable protein isolate (Soy Isolate) with acid \ alkali followed by an enzymatic modification using pepsin to \| produce the desired aerating agent. The present invention does not involve enzymatic modification with pepsin. The present invention also uses a protein fraction, glycinin, from soybean and not soy protein isolate. Reference may be made to Boyce et al., 1986, US patent No 46 32 903, wherein a process for the production of egg white substitute from soy protein by enzymatic hydrolysis with microbial rennet to a DH of 0.25-2.5% is described. The present invention uses $ different proteolytic enzyme and a product with a DH of 3 - 5%. Reference may be made to Pour-El et al, 1980, U.S. patent No. 39 32 672 ;l wherein preparation of an egg white substitute material through treatment of the II soy protein isolate with ficinjbromelain, pepsin, trypsin and papain is described. The present invention uses alprotein fraction, glycinin, from soybean. Reference may be made to a research paper entitled "Industrial production and application bf soluble enzymatic hydrolysate of soy protein" (Olsen, H.S. and Adler Nissen J, Process Biochemistry, 14(7), 10-11, 1979) wherein a method for preparation of soy protein hydrolysate from soy flakes washed at pH 4.5 followed by hydrolysis using alcalase is described. The solubility of the hydrolysate is very low at acidic pH. The hydrolysate had higher DH, which impairs the foaming and emulsification properties. Reference may be made to the published paper by Adler Nissen et al (1983), titled "Improvement of the functionality of vegetable proteins by controlled enzymatic hydrolysis", Qual. Plant: Plant Foods for Human Nutrition, 32, 411 -423, wherein is shown that soy concentrate when hydrolyzed to a DH of 3-5% with alcalase, a functional pVotein hydrolysate with high foam expansion and remarkably good foam stability obtained. On the other hand hydrolysis to DH of 8% lowered the functionality]considerably. The present invention relates only to the modification of glycinin. Reference may be made to Olsen (1981) U.S. patent No 43 24 805, wherein a method is described for producing soy protein hydrolysate and oil from partially defatted soy material by hydrolysis with proteolytic enzyme. The DH was in the range of 8-12%. Alcalase was the enzyme used. The process involves high DH and the hydrolysate will Have low functionality except solubility. Reference may be made to Tsumura et al (1997) European patent No. 0797928 A1, wherein a process for the manufacture of soy protein hydrolysate with a protease used selectively to decompose glycinin at a pH of 1.5 to 2.5. The aim of the process was to achieve a low glycinin content. The present invention relates to the modification of glycinin. Reference may be made to Tsumura et al., 2001, US patent No. 6 303 178, wherein disclosed the' preparation of functional protein hydrolysate by independently hydrolyzing p conglycinin and glycinin. The hydrolysate contained both 7S and 11S components which has shown to be good emulsifying and whipping capabilities and was shown to be useful for incorporation in food products including ices, meringues, spread pastes, beverages. The present invention includes only the modified 11S component of soybean. Reference may be made to Tsumura et al., 2000, US patent (No. 6 126 973) wherein the preparation] of soy protein hydrolysate with a low content of p conglycinin has been disclosed. The present invention relates only to the modification of glycinin. The major object of the present invention is to provide an improved enzymatic process for the preparation of modified giycinin. Another object of the present invention is to use a plant protein fraction, which is richer in cysteine/methionine compared to soy protein. Yet another object of the present invention is to have modified giycinin, which as 300% more foaming capacity and foam stability. Accordingly, the present invention provides an improved enzymatic process for the preparation of modified giycinin having enhanced solubility, emulsifying and whipping capabilities and lower degree of hydrolysis which comprises: a) preparing an aqueous slurry of at least 10% giycinin (w/v) using 80 to 100ml of water to 10g of protein, [j b) adjusting the pH of the dispersion to 7.0 to 7.5 by using 0.5N to 1N sodium hydroxide, c) heating the above said slurry at a temperature ranging between 50°C and 55°C in a water bath under the above controlled condition, d) adding proteolytic enzyme selected from papain to the above slurry at the rate of 0.2 to 0.5% by weight, e) encubating the above said slurry for a period of 15 to 25 min, f) inactivating the: enzyme by rasing the slurry temperature in the range of 75 to 80°C, followed by cooling to a temperature ranging between 25°C and 30°C and g) freeze drying the above said slurry at temperature in the range of --40 to -47°C for a period of 3h to 4h, to obtain the desired product. In another embodiment of the present invention, the glycinin obtained may be selected as 11S protein of soybean using known method of extraction. Yet in another embodiment of the present invention, the proteolytic enzyme used may be selected from a plant such as papain. The invention involves the following steps: Preparation of glycinin from defatted soy flour Soybean flour is obtained from soybean seeds. The cleaned beans are dehulled flaked and desolventised. The defatted flakes are ground and passed through 60 mesh. From the defatted flour glycinin was obtained by Tanh and Shibasaki method. Defatted soy flour ws ijextracted with 63 m M Tris-HCl buffer containing 10mM (3 mercaptoethanol pH 7.8 (1:15) for 1h and centrrfuged at 10000 rpm. The supernatant was adjusted to pH 6.6 using 2N HCI and dialyzed against 63 mM tris-HCI buffer (pH 6.6) and dissolved in standard phosphate buffer 0.05 MpH7.6 containing 0.4 M ' NaCI, 0.02% sodium azide, 0.01 M p-mercaptoethanol, and 0.001 M EDTA and dialyzed against distilled water in the cold and freeze dried to yield crude glycinin. Papain Commercially available food grade enzyme papain having activity not less than 6000 Units/ mg protein. Alcalase Commercially available food grade enzyme alcalase having activity not less than 25,588 Units/ mg protein. Fungal protease Commercially available food grade enzyme fungal protease (Amano Pro) having activity not less than 28600 Units/ mg protein. Measurement of Degree of Hydrolysis (DH) Trinitrobenzenesulfonic acid, (TNBS) procedure is an accurate reproducible i method for determining the degree of hydrolysis of food protein hydrolysates. The protein hydrolysate is dissolved/dispersed in hot 1% sodium dodecyl sulphate to a concentration of 0.25- 2.5 x 10"3 amino equivalents/L. A sample solution (0.25ml) is mixed wjth 2ml of 0.2125M sodium phosphate buffer ( pH 8.2) and 2ml of 0.1% TNBS; followed by incubation in the dark for 60 min at 50°C. The reaction is quenched by adding 4ml of 0.1N HCI, and the absorbance is read at 340nm. 1.5 mM L-Leucine solution is used as the, standard. Tranformation of the measured leucine amino equivalents to a degree of hydrolysis is carried out by ineans of a standard curve. ( Adler Nissen, 1979, "Determination of degree of hydrolysis of food protein hydrolysates by trinitrobenzene sulfonic acid, Journal of Agriculture and Food Chemistry, 27, 1256-1262.) Glycinin was dispersed in water 1:10 ratio. The pH of the dispersion was adjusted to 6.5 - 7.5 using ;1 N sodium hydroxide or hydrochloric acid. The temperature of the slurry was brought to 50 - 55 °C in a water bath. At this stage 0.4 to 0.5% papain was added and incubated for 15 to 25 min. At the end of incubation the enzyme was, inactivated by heating to 80°C, cooled to room temperature and the contents were freeze-dried. The novelty and inventive steps of an improved enzymatic process for the preparation of modified glycirlin lies in the modification of glycinin from soybean with the following: i) Degree of hydrolysis is only\|3 - 5% ii) The modified glycinin has an emulsifying capacity of 60 - 70 ml/ g iii) The whipping capability of 88% volume increase iv) Modified glycinin, which has 300% more foaming capacity and foam stability The process is further illustrated by the examples given below, which should not however be construed to limit the scope of the invention. Example 1 Three grams of glycinin was dispersed in 30 ml of water and the pH of the dispersion was adjusted to 7.0 using 1N sodium hydroxide solution. The temperature of the slurry was: brought to 50 °C in a water bath. At this stage 0.5% papain was added and incubated for 15 min. At the end of incubation the enzyme was inactivated by heating to80°C, cooled to room temperature and the contents were freeze-dried. The degree of hydrolysis (DH) of the sample was 3.75%. Example 2 Five grams of glycinin was! dispersed in 50ml of water and the pH of the dispersion was adjusted to 7.0 using 1N sodium hydroxide solution. The temperature of the slurry was brought to 50°C in a water bath. At this stage 0.5% papain was added and incubated for 15 min. At the end of incubation the enzyme was inactivated by heating to 80°C, cooled to room temperature and the contents were freeze-dried. the degree of hydrolysis (DH) of the sample was 4.2%. Example 3 Six grams of glycinin was dispersed in 50ml of water and the pH of the dispersion was adjusted to 7.0 using 1N sodium hydroxide solution. The temperature of the slurry was brought to 50°C in a water bath. At this stage 0.5% papain was added and incubated for 15 min. At the end of incubation the enzyme was inactivated by heating to 80°C, cooled to room temperature and the contents were freeze-dried. The degree of hydrolysis (DH\| of the sample was 3.3%. The main advantages of the! process: 1. The process provides modified glycinin with specified DH with high foam capacity and stability. 2. The raw material for the above process is defatted soy flour with high nitrogen solubility, which is available in abundance. 3. The time of hydrolysis is low which is having advantage both in input cost and energy. 4. The modified glycinin which contains 1.5 to 2 times higher methionine+ cystine compared to soy protein isolate. 5. The modified sample is very low in trypsin inhibitor activity. We claim: 1. An improved enzymatic process for the preparation of modified glycinin having enhanced solubility, emulsifying and whipping capabilities and lower degree of hydrolysis i which comprises: a) preparing an aqueous slurry of at least 10% glycinin (w/v) using 80 to 100ml of water to 10g of protein, b) adjusting the pH of the dispersion to 7.0 to 7.5 by using 0.5N to 1N sodium hydroxide, c) heating the above said slurry at a temperature ranging between 50°C and 55°C in a water bath under the above controlled condition, d) adding proteolytic enzyme selected from papain to the above slurry at the rate of 0.2 to 0.5% by weight. e) incubating the above said slurry for a period of 15 to 25 min, f) inactivating the enzyme by rasing the slurry temperature in the range of 75 to 80°C, followed by cooling to a temperature ranging between 25°C and 30°C and g) freeze drying the above said slurry at temperature in the range of -40 to -47°C for a period of 3h to 4h, to obtain the desired product. 2. An improved enzymatic process for the preparation of modrfied glycinin having enhanced solubility, emulsifying and whipping capabilities and lower degree of hydrolysis substantially as herein described with reference to the examples.

Full Text

The present invention relates to an improved enzymatic process for the preparation of modified glycinin having enhanced solubility, emulsifying and whipping capabilities and lower degree of hydrolysis.
Soybean is a source of high quality proteins all over the world. The production of soybean in India has gone up from 2.63 million tones in 1989-91 to 6.5 million tones. Soy proteins in particular soy protein isolate is useful as a food ingredient in various formulations by virtue of its high protein content and functional properties.
In the food industry materials such as egg white, hydrolyzed milk proteins and soy proteins have been used as aerating agents and heat setting binders when properly whipped along with other jaqueous foaming or whipping ingredients, the aerating agents permit the entrapment of gases thereby to provide a foamed or aerating agents permit the entrapment of gases thereby to provide a foamed or aerated prodeuct. Thus aerating agents
must necessarily ingest and entrap gas volume within a reasonably low whipping time.
j Defatted soybean meal contains nearly 50-55% protein. The major proteins of soybeans
are storage globulin i.e. glycinin and conglycinin make 90% of the total. The proteins can be
classified into 2S, 7S, 11S and 15S protein based on sedimentation coefficients. The 11S
fraction also known as cjlycinin constitutes 25-35% of the total potein (Liu 1997, In:
'Soybeans: chemistry, technology and Utilization, pp. 25-113, Chapman & Hall, New York).
Reference may be made to a published paper titled "Major proteins of soybean seeds. A
straight forward fractionation and characterization", by Tanh and Shibasaki,
1976, Journal of Agriculture and Food Chemistry, 24,1117-1121
wherein it is reported a method for the fractionation of 7S and 11S globulins from defatted soybean meal by fractional isoelectric precipitation.
Reference may be made to the published paper titled "A method for isolation of 2S, 7S and 11S proteins of soybeans" by Appu Rao, A.G. and Narasinga Rao, M.S., 1979, wherein glycinin can be obtained by magnesium chloride precipitation of the water extract.
Reference may be made to the published paper by Wu et al., 1999, titled "A pilot- plant fractionation iof soybean glycinin and conglycinin", Journal of American Oil Chemists Society, 76, 285-293, wherein glycinin can be prepared on a pilot plant scale.
Reference may be made to a published paper by Kitamura et al., 1995, titled " Genetic improvement of nutritional and food processing quality in soybean, Japanese Agricultural Research Quarterly, 29, 1-8 wherein glycinin is shown to be rich in methionine and contains 3-4 times more methionine and cysteine per unit protein than ;7S protein.
Reference may be made to Wagner and Gueguen, 1995, titled "Surface functional properties of native, acid treated and reduced soy glycinin", wherein it is shown that glycinin is poorer in functional attributes such as foaming and emulsifying characteristics due to its closed globular conformation and low molecular flexibility.
Reference may be made to Kim and Kinsella, 1987, in the published papers titled "Surface active properties of food proteins: effects of reduction of disulfide bonds on film formation and foam stability of glycinin, Journal of Food
Science, 52, 128-131, and "Surface active properties of food proteins: effect of progressive succinylation of film properties and foam stability of glycinin, Journal of Food Science, 52, 1341-1352, wherein the structural modification of glycinin could induced by varying environmental factors such as ionic strength, DH, temperature and chemical treatments such as reduction of component disulfide
:l
bonds, succinylation and acetylation.
Reference could be made to the published papers of Wagner and Gueguen 1995, and Wagner, and Gueguen 1999, titled "Effects of dissociation, deamidation and reducing treatment on structural and surface active properties of soy glycinin, Journal of Agriculture and Food Chemistry, 43, 1993 - 1994, and "Surface functional properties of native acid treated and reduced soy glycinin. 1. Foaming properties", Journal of Agriculture and Food Chemistry, 47, 2173 -2180, wherein the foaming and emulsifying properties of glycinin could be greatly improved by mild acid treatments, deamidation and reducing agents.
Reference may be made to RC Gunther et al., 1974, US patent No. 3,814 816, wherein a successful vegetable protein aerating agent is disclosed wherein he has prepared vegetable jprotein aerating agent by hydrolyzing an oil free vegetable protein isolate (Soy Isolate) with acid \ alkali followed by an enzymatic modification using pepsin to | produce the desired aerating agent. The present invention does not involve enzymatic modification with pepsin. The present invention also uses a protein fraction, glycinin, from soybean and not soy protein isolate.
Reference may be made to Boyce et al., 1986, US patent No 46 32 903, wherein a process for the production of egg white substitute from soy protein by enzymatic hydrolysis with microbial rennet to a DH of 0.25-2.5% is described. The present invention uses $ different proteolytic enzyme and a product with a DH of 3 - 5%.
Reference may be made to Pour-El et al, 1980, U.S. patent No. 39 32 672
;l
wherein preparation of an egg white substitute material through treatment of the
II
soy protein isolate with ficinjbromelain, pepsin, trypsin and papain is described. The present invention uses alprotein fraction, glycinin, from soybean.
Reference may be made to a research paper entitled "Industrial production and application bf soluble enzymatic hydrolysate of soy protein" (Olsen, H.S. and Adler Nissen J, Process Biochemistry, 14(7), 10-11, 1979) wherein a method for preparation of soy protein hydrolysate from soy flakes washed at pH 4.5 followed by hydrolysis using alcalase is described. The solubility of the hydrolysate is very low at acidic pH. The hydrolysate had higher DH, which impairs the foaming and emulsification properties.
Reference may be made to the published paper by Adler Nissen et al (1983), titled "Improvement of the functionality of vegetable proteins by controlled enzymatic hydrolysis", Qual. Plant: Plant Foods for Human Nutrition, 32, 411 -423, wherein is shown that soy concentrate when hydrolyzed to a DH of 3-5% with alcalase, a functional pVotein hydrolysate with high foam expansion and remarkably good foam stability obtained. On the other hand hydrolysis to DH of
8% lowered the functionality]considerably. The present invention relates only to
the modification of glycinin.
Reference may be made to Olsen (1981) U.S. patent No 43 24 805, wherein a method is described for producing soy protein hydrolysate and oil from partially defatted soy material by hydrolysis with proteolytic enzyme. The DH was in the range of 8-12%. Alcalase was the enzyme used. The process involves high DH and the hydrolysate will Have low functionality except solubility.
Reference may be made to Tsumura et al (1997) European patent No. 0797928 A1, wherein a process for the manufacture of soy protein hydrolysate with a protease used selectively to decompose glycinin at a pH of 1.5 to 2.5. The aim of the process was to achieve a low glycinin content. The present invention
relates to the modification of glycinin.
Reference may be made to Tsumura et al., 2001, US patent No. 6 303 178, wherein disclosed the' preparation of functional protein hydrolysate by independently hydrolyzing p conglycinin and glycinin. The hydrolysate contained both 7S and 11S components which has shown to be good emulsifying and whipping capabilities and was shown to be useful for incorporation in food products including ices, meringues, spread pastes, beverages. The present invention includes only the modified 11S component of soybean.
Reference may be made to Tsumura et al., 2000, US patent (No. 6 126 973) wherein the preparation] of soy protein hydrolysate with a low content of p conglycinin has been disclosed. The present invention relates only to the modification of glycinin.
The major object of the present invention is to provide an improved enzymatic process for the preparation of modified giycinin.
Another object of the present invention is to use a plant protein fraction, which is richer in cysteine/methionine compared to soy protein.
Yet another object of the present invention is to have modified giycinin, which as 300% more foaming capacity and foam stability.
Accordingly, the present invention provides an improved enzymatic process for the
preparation of modified giycinin having enhanced solubility, emulsifying and whipping
capabilities and lower degree of hydrolysis which comprises:
a) preparing an aqueous slurry of at least 10% giycinin (w/v) using 80 to 100ml of
water to 10g of protein,
[j
b) adjusting the pH of the dispersion to 7.0 to 7.5 by using 0.5N to 1N sodium hydroxide,
c) heating the above said slurry at a temperature ranging between 50°C and 55°C in a water bath under the above controlled condition,
d) adding proteolytic enzyme selected from papain to the above slurry at the rate of 0.2 to 0.5% by weight,
e) encubating the above said slurry for a period of 15 to 25 min,

f) inactivating the: enzyme by rasing the slurry temperature in the range of 75 to 80°C, followed by cooling to a temperature ranging between 25°C and 30°C and
g) freeze drying the above said slurry at temperature in the range of --40 to -47°C for a period of 3h to 4h, to obtain the desired product.
In another embodiment of the present invention, the glycinin obtained may be selected as 11S protein of soybean using known method of extraction.
Yet in another embodiment of the present invention, the proteolytic enzyme used may be selected from a plant such as papain.
The invention involves the following steps:
Preparation of glycinin from defatted soy flour
Soybean flour is obtained from soybean seeds. The cleaned beans are dehulled flaked and desolventised. The defatted flakes are ground and passed through 60 mesh. From the
defatted flour glycinin was obtained by Tanh and Shibasaki method. Defatted soy flour ws
ijextracted with 63 m M Tris-HCl buffer containing 10mM (3 mercaptoethanol pH 7.8 (1:15) for 1h
and centrrfuged at 10000 rpm. The supernatant was adjusted to pH 6.6 using 2N HCI and
dialyzed against 63 mM tris-HCI buffer (pH 6.6) and dissolved in standard phosphate buffer 0.05
MpH7.6 containing 0.4 M ' NaCI, 0.02% sodium azide, 0.01 M p-mercaptoethanol, and
0.001 M EDTA and dialyzed against distilled water in the cold and freeze dried to yield crude glycinin.
Papain
Commercially available food grade enzyme papain having activity not less than
6000 Units/ mg protein.
Alcalase
Commercially available food grade enzyme alcalase having activity not less than
25,588 Units/ mg protein.
Fungal protease
Commercially available food grade enzyme fungal protease (Amano Pro) having
activity not less than 28600 Units/ mg protein.
Measurement of Degree of Hydrolysis (DH)
Trinitrobenzenesulfonic acid, (TNBS) procedure is an accurate reproducible
i
method for determining the degree of hydrolysis of food protein hydrolysates. The protein hydrolysate is dissolved/dispersed in hot 1% sodium dodecyl sulphate to a concentration of 0.25- 2.5 x 10"3 amino equivalents/L. A sample solution (0.25ml) is mixed wjth 2ml of 0.2125M sodium phosphate buffer ( pH 8.2) and 2ml of 0.1% TNBS; followed by incubation in the dark for 60 min at 50°C. The reaction is quenched by adding 4ml of 0.1N HCI, and the absorbance is read at 340nm. 1.5 mM L-Leucine solution is used as the, standard.
Tranformation of the measured leucine amino equivalents to a degree of hydrolysis is carried out by ineans of a standard curve. ( Adler Nissen, 1979, "Determination of degree of hydrolysis of food protein hydrolysates by trinitrobenzene sulfonic acid, Journal of Agriculture and Food Chemistry, 27, 1256-1262.)
Glycinin was dispersed in water 1:10 ratio. The pH of the dispersion was adjusted to 6.5 - 7.5 using ;1 N sodium hydroxide or hydrochloric acid. The temperature of the slurry was brought to 50 - 55 °C in a water bath. At this stage 0.4 to 0.5% papain was added and incubated for 15 to 25 min. At the end of
incubation the enzyme was, inactivated by heating to 80°C, cooled to room
temperature and the contents were freeze-dried.
The novelty and inventive steps of an improved enzymatic process for the preparation of modified glycirlin lies in the modification of glycinin from soybean with the following: i) Degree of hydrolysis is only|3 - 5%
ii) The modified glycinin has an emulsifying capacity of 60 - 70 ml/ g
iii) The whipping capability of 88% volume increase
iv) Modified glycinin, which has 300% more foaming capacity and foam stability
The process is further illustrated by the examples given below, which should not however be construed to limit the scope of the invention.
Example 1
Three grams of glycinin was dispersed in 30 ml of water and the pH of the dispersion was adjusted to 7.0 using 1N sodium hydroxide solution. The temperature of the slurry was: brought to 50 °C in a water bath. At this stage 0.5% papain was added and incubated for 15 min. At the end of incubation the enzyme was inactivated by heating to80°C, cooled to room temperature and the contents were freeze-dried. The degree of hydrolysis (DH) of the sample was 3.75%.
Example 2
Five grams of glycinin was! dispersed in 50ml of water and the pH of the dispersion was adjusted to 7.0 using 1N sodium hydroxide solution. The temperature of the slurry was brought to 50°C in a water bath. At this stage 0.5% papain was added and incubated for 15 min. At the end of incubation the enzyme was inactivated by heating to 80°C, cooled to room temperature and the contents were freeze-dried. the degree of hydrolysis (DH) of the sample was 4.2%.
Example 3
Six grams of glycinin was dispersed in 50ml of water and the pH of the dispersion was adjusted to 7.0 using 1N sodium hydroxide solution. The temperature of the slurry was brought to 50°C in a water bath. At this stage 0.5% papain was added
and incubated for 15 min. At the end of incubation the enzyme was inactivated by heating to 80°C, cooled to room temperature and the contents were freeze-dried. The degree of hydrolysis (DH| of the sample was 3.3%.
The main advantages of the! process:
1. The process provides modified glycinin with specified DH with high foam capacity and stability.
2. The raw material for the above process is defatted soy flour with high nitrogen solubility, which is available in abundance.
3. The time of hydrolysis is low which is having advantage both in input cost and energy.
4. The modified glycinin which contains 1.5 to 2 times higher methionine+ cystine compared to soy protein isolate.
5. The modified sample is very low in trypsin inhibitor activity.

We claim:
1. An improved enzymatic process for the preparation of modified glycinin having
enhanced solubility, emulsifying and whipping capabilities and lower degree of hydrolysis
i which comprises:
a) preparing an aqueous slurry of at least 10% glycinin (w/v) using 80 to 100ml of water to 10g of protein,
b) adjusting the pH of the dispersion to 7.0 to 7.5 by using 0.5N to 1N sodium hydroxide,
c) heating the above said slurry at a temperature ranging between 50°C and 55°C in a water bath under the above controlled condition,
d) adding proteolytic enzyme selected from papain to the above slurry at the rate of 0.2 to 0.5% by weight.
e) incubating the above said slurry for a period of 15 to 25 min,
f) inactivating the enzyme by rasing the slurry temperature in the range of 75 to 80°C, followed by cooling to a temperature ranging between 25°C and 30°C and
g) freeze drying the above said slurry at temperature in the range of -40 to -47°C for a period of 3h to 4h, to obtain the desired product.
2. An improved enzymatic process for the preparation of modrfied glycinin having
enhanced solubility, emulsifying and whipping capabilities and lower degree of hydrolysis
substantially as herein described with reference to the examples.

Documents:

231-del-2002-abstract.pdf

231-del-2002-claims.pdf

231-del-2002-complete specification(granted).pdf

231-del-2002-correspondence-others.pdf

231-del-2002-correspondence-po.pdf

231-del-2002-description (complete).pdf

231-del-2002-form-1.pdf

231-del-2002-form-2.pdf

231-del-2002-form-3.pdf

« Previous Patent

Next Patent »

Patent Number

242192

Indian Patent Application Number

231/DEL/2002

PG Journal Number

34/2010

Publication Date

20-Aug-2010

Grant Date

18-Aug-2010

Date of Filing

14-Mar-2002

Name of Patentee

COUNCIL OF SCIENTIFIC AND INDUSTRIAL RESARCH

Applicant Address

RAFI MARG, NEW DELHI-110001, INDIA

Inventors:

#	Inventor's Name	Inventor's Address
1	HEMMIGE SRINIVAS	CENTRAL FOOD TECHNOLOGICAL RESEARCH INSTITUTE, MYSORE,INDIA
2	VISHWESHWARIAH PRAKASH	CENTRAL FOOD TECHNOLOGICAL RESEARCH INSTITUTE, MYSORE,INDIA
3	KARADKA GOVINDARAJU	CENTRAL FOOD TECHNOLOGICAL RESEARCH INSTITUTE, MYSORE,INDIA
4	APPU RAO GOPALA RAO APPU RAO	CENTRAL FOOD TECHNOLOGICAL RESEARCH INSTITUTE, MYSORE,INDIA

PCT International Classification Number

C07K 3/00

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA