Title of Invention

"A PROCESS FOR PREPARATION OF PROTEIN HYDROLYSATE FROM SOY FLOUR"

Abstract The present invention provides a process for preparation of protein hydrolysate from soy flour, said process comprising hydrolyzing aqueous slurry of defatted soy flour containing 6-30% solid content w/v using proteolytic enzyme of plant origin at pH 5-9 and temperature of 53± 5°C under stirring for 30 minutes to 6 hours; inactivating the enzyme by a known manner; neutralizing the pH value of the slurry; separating the solids by a known manner and drying the clarified liquor so obtained to get the said hydrolysate
Full Text TECHNICAL FIELD
The present invention relates to a process for the preparation of protein hydrolysate from soy flour using proteolytic enzyme of plant origin. Particularly, the present invention relates to a process for the preparation of protein hydrolysate from defatted soy flour using papain.
BACKGROUND ART
Presently about 6.8 M tons of soybean is produced in India and extracted for oil and the solvent extracted flour is exported to foreign countries for feed purposes. By providing additional facilities for the hygienic processing of soybean in the solvent extraction units, it is possible to obtain edible grade defatted flour having the desired functional characteristics. After the recovery of oil, 4.9 M tons of soy flour is available in India for utilization. A small portion of the total soybean produced also finds its use for different edible grade flours, protein isolate and texturized products and the popularity of these products are greatly picking up globally. Soybean is an excellent source of protein, which contains about 40% protein. New manufacturing techniques for high quality soybean foods have been developed by lowering or destroying of the anti-nutritional factors such as trypsin inhibitors.
United States Patent No. 5180597 claimed a process for hydrolyzed vegetable protein with enhanced flavor, which contains no detectable level of monochlorodihydroxypropanol is described. In the above reference, wheat gluten is hydrolyzed using Prozyme 6 (a fungal protease) at a temperature of 40-50°C, pH 6.5-7.0, enzyme concentration of 0.1-2.0% of substrate for a time period of 4h. The hydrolyzed protein is treated with gaseous HCl for deamidation before the addition of acid for inactivating the enzyme. The drawback in such hydrolysis is that it is likely to lead to racemisation of amino acids and the addition of acid increases the salt content in the product.
United States Patent No. 5077062 claimed a low sodium, low mono sodium glutamate soy
hydrolysate that is prepared from soy material such as soy flour, soy meal or soy grits using
fungal protease in water. The hydrolysis is conducted in the absence of acid or base at 90°C
for 2 hours. After deactivating the enzyme and

dewatering the mixture the resulting hydrolysate contains between 45 and 55 wt. %
enzymatically hydrolyzed soy based protein with an average molecular weight of
670.000 ± 50,000. The fungal protease used is different from the enzyme used in the
present invention. The process is energy intensive due to the high temperature (90°C)
used.
United States Patent No.4757007 claimed a preparation of two hydrolyzed products
using a protease from soy protein. The soy protein is hydrolyzed with papain or
pepsin after precipitating with alcohol. The drawback of the process is it involves the
separation of the mixture of hydrolyzed products. Hydrolysis is carried out using
papain or pepsin. Acidification is carried out to bring down the pH to 2.5-5.0 to
separate the two kinds of hydrolysates, which could, lead to increase in salt content.
European Patent No.0148600 B 1 relates to the preparation of hydrolyzed protein
from protein isolate after jet cooking or dynamic heating at 104°C for a few seconds
and later cooled in a vacuum chamber before hydrolysis using bromelin. The protein
was precipitated at its isoelectric point from an aqueous extract of the material before
the hydrolysis. The drawback of the process is the starting material protein isolate,
which is more expensive. The process is a multi step process, energy intensive. The
process further needs machines like the jet cooker and a vacuum chamber.
European Patent No. 0223560 A2 claimed a method describing the separation of
protein hydrolysates with meat and cheese flavor, from proteinaceous feed stocks
(e.g. containing soybean, gluten, whey, casein, hemoglobin, yeast, cereal or microbial
proteins) by stepwise hydrolysis using an endopeptidase followed by amino peptidase
from Streptococcus lactus. The drawback of the process is that it is a multi-step
process.
European Patent No. 0087246 Bl claimed a process for the hydrolysis of soybeans,
wheat gluten and cotton seeds using fungal protease from Aspergillus and pancreatin
(trypsin, chymotrypsin A, B and C, elastase and carboxypeptidase A and B) is
described. Activated charcoal is used to treat the hydrolysate, which is used for
nutritional improvement. The draw back of the process is it involves more steps.
European Patent No. 0187048 A2 described the preparation of soy protein
hydrolysate with 0.25 to 2.5% degree of hydrolysis (DH) using microbial rennet
(Mucor miehei) and to be used as an egg white substitute. The enzyme used in the
process is different and involves very low DH of soy protein.
United Kingdom Patent No. 2053228A described a process for the production of soy
protein hydrolysate from partially defatted soy material by hydrolysis with proteolytic
enzyme. The drawback of the process is that due to partial defatting soy flour, left
over oil comes in contact with protein phase, which could lead to off-flavors.
United States Patent No. 4,324,805 described a method for producing soy protein
hydrolysate and oil from partially defatted soy material by hydrolysis with proteolytic
enzyme. The soy flour is partially defatted by water washing at pH 3.5-4.5 and later
hydrolyzed using water and a base to increase the pH. The DH is in the range of 8-
12%. Oil is recovered from the wash water. Alcalase is the enzyme used. The
drawback of the process is that it is a multi step process and due to partial defatting of
soy flour, left over oil comes in contact with protein phase which could lead to offflavors.
Enzyme inactivation is done by addition of acid, which is likely to lead to
increased salt content in the product.
United States Patent No. 3640725 described an enzymatic hydrolysis process for
production of soy protein hydrolysates. The soy seeds are comminuted and heated at
90-140°C. Protease (fungal and bacterial) is added at 25-75°C. The fiber is separated
and slurry has two phases- oil and aqueous phase. Aqueous phase is brought to pH
4.5 to precipitate the protein, which is then concentrated. The starting material is not
defatted and hence the residual oil could come in contact with the aqueous phase,
which could lead to off-flavors.
Canadian Patent No.905742 described a soy protein hydrolysate modified with pepsin
to yield a product which in presence of water and sugar whips at a rapid rate to
produce aerated products of low density.
European Patent No.0797928 Al described a process for the manufacture of a soy
protein hydrolysate with a protease used selectively to decompose glycinin at a pH of
1.5-2.5. The pH used in the process is very low and therefore differs from the pH
used in the present invention. Further, the aim of the process is to achieve a low
glycinin content which is not the case in the present invention.
Reference may be made to a published paper entitled "Industrial production and
application of soluble enzymatic hydrolysate of soy protein", (Olsen, H.S., Adler
Nissen, J., (1979), Process Biochemistry, 14(7), 6,8, 10-11) 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 substrate is low at the
acidic pH which is likely to result in low yields. The enzyme used is different from
the enzyme used in the present invention.
OBJECTS OF THE INVENTION
The main object of the present invention is to provide a process for the preparation
for the preparation of protein hydrolysate from soy four from plant based protease."
Another object of the present invention is to provide a process for the preparation of
protein hydrolysate with specified degree of hydrolysis.
Still another object of the present invention is to provide a process for the preparation
of protein hydrolysate soluble in water over a wide range of pH value.
Yet another object of the present invention is to provide a process for the preparation
of protein hydrolysate which can be used for nutritional enrichment.
SUMMARY OF THE PRESENT INVENTION
The invention provides a process for the preparation of protein hydrolysate from soy
flour, said process comprising: hydrolyzing aqueous slurry of defatted soy flour
containing 6-30% solid content w/v using proteolytic enzyme of plant origin at pH 5-
9 and temperature of 53± 5°C under stirring for 30 min to 6 h; inactivating the
enzyme by a known manner; neutralizing the pH value of the slurry; separating the
solids by a known manner and drying the C; ,ined liquor so obtained to get the said
hydrolysate.
DESCRIPTION OF THE INVENTION
Accordingly, the present invention provides a process for the preparation of protein
hydrolysate from soy flour, said process comprising: hydrolyzing aqueous slurry of
defatted soy flour containing 6-30% solid content w/v using proteolytic enzyme of
plant origin at pH 5-9 and temperature of 53± 5°C under stirring for 30 min to 6 h;
inactivating the enzyme by a known manner; neutralizing the pH value of the slurry;
separating the solids by a known manner and drying the clarified liquor so obtained to
get the said hydrolysate.
In an embodiment of the present invention, the solid content of the slurry is 20% w/v.
In another embodiment of the present invention, the plant origin proteolytic enzyme is
selected from the group comprising of papain and bromelin.
In still another embodiment of the present invention, 0.4-0.6% w/w of the proteolytic
enzyme is added to the soy flour.
In yet another embodiment of the present invention, the hydrolysis is effected for a
period of 3-4 hours.
In one more embodiment of the present invention, the drying is effected by freeze
drying, spray drying and drum drying.
In one another embodiment of the present invention, the protein hydrolysate produced
has decreased bitterness.
In an embodiment of the present invention, the protein hydrolysate produced is less
hygroscopic in nature.
In another embodiment of the present invention, the protein hydrolysate has 2-
2.2g/100ml bitterness recognition threshold.
In still another embodiment of the present invention, the protein hydrolysate produced
has low mineral content.
In one more embodiment of the present invention, high yield of protein hydrolysate
with 30 to 35% degree of hydrolysis is obtained from the raw material taken.
In one another embodiment of the present invention, a protein hydrolysate having
creamy color,and a yield of 20 to 25% (on flour basis) is obtained.
In an embodiment of the present invention, the protein hydrolysate has 3.0 to 5.0%
moisture, 8.0 to 8.5% nitrogen and 30.0-35.0% degree of hydrolysis.
In another embodiment of the present invention, the protein hydrolysate obtained has
25-30 trypsin inhibitor units/mg activity, 95 to 98% Nitrogen Solubility Index and 1.0
to 1.4% of salt content.
In still another embodiment of the present invention, lipoxygenase and urease
activities of the protein hydrolysate are not detectable.
In yet another embodiment of the present invention, the amino acid composition of
the protein hydrolysate is similar to the amino acid makeup as that of the starting
material.
In one more embodiment of the present invention, the protein hydrolysate retained the
nutrition value as in the starting material.
In one another embodiment of the present invention, the protein hydrolysate does not
impart any undesirable flavor for the finished product.
In an embodiment of the present invention, the solubility of the protein hydrolysate is
independent of the pH value.
The present invention also provides a protein hydrolysate creamy in color.
In still another embodiment of the present invention, the protein hydrolysate has 3 to
5% moisture, 8 to 8.5% nitrogen and 30 to 35% degree of hydrolysis.
In yet another embodiment of the present invention, the protein hydrolysate has 20 to
30 trypsin inhibitor units/mg activity, 95 to 98% Nitrogen Solubility Index and 1 to
1.4% of salt content.
In one more embodiment of the present invention, the protein hydrolysate has 2 to
2.2g/100ml bitterness recognition threshold.
In one another embodiment of the present invention, the lipoxygenase and urease
activities of the protein hydrolysate are not detectable.
In an embodiment of the present invention, the amino acid composition of the protein
hydrolysate is similar to the amino acid makeup of the starting material.
In another embodiment of the present invention, the protein hydrolysate retained the
nutrition value present in the starting material.
In still another embodiment of the present invention, the protein hydrolysate does not
impart any undesirable flavor for the finished product.
In yet another embodiment of the present invention, the solubility of the protein
hydrolysate is independent of the pH value.
The process involves following process steps:
Defatted soy flour
Soybean flour is derived from clean round beans. The cleaned beans pass through a
cracking process; the bean fragments are graded on sieves and aspirate system. The
cleaned cracked meat passed through a conditioner cooker and flaked. This is
subjected to solvent extraction process. The extracted flakes were desolventized and
ground to 100 mesh. The specification for soy flour consists of (a) Moisture = 9%
by mass (max); (b) Protein on dry basis = 48% by mass (min); (c) The total ash on
dry basis = 7.2% by mass (max); (d) Acid insoluble ash on dry basis = 0.4% by mass
(max); (e) Fat on dry basis = 1.5% by mass; (f) Crude fibre on dry basis = 4.2% by
mass (max); (g) Aflatoxin = 30 ppb (max); (h) Residual solvent = 170 ppm; (I) Total
bacterial count per gram = 50,000; (j) Coliform bacteria/g =10; and (k) Salmonella
bacterial - Nil.
Papain
The specification of the plant thiol protease papain is to obtain commercially available
food grade enzyme having proteolytic activity not less than 2,000 tyrosine units
(TU)/mg proteolytic activity.
Measurement of Degree of Hydrolysis
Trinitrobenzenesulphonic acid (TNBS) procedure, is an accurate, reproducible and
generally applicable procedure 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 aminoequivalents/L.
A sample solution 0.25 ml) is mixed with 2 ml of 0.2125 M sodium phosphate buffer
(pH 8.2) and 2 ml of 0.1% Trinitrobenzenesulphonic acid, followed by incubation in
the dark for 60 min at 50 C. The reaction is quenched by adding 4 ml. Of 0.10 N
hydrochloric acid (HC1) and the absorbance is read at 340nm. A 1.5mM L-leucine
solution is used as the standard. Transformation of the measured leucine amino
equivalents to a degree of hydrolysis is carried out by means of a standard curve for
each particular protein substrate (Adler Nissen, J. (1979) J. Agri. Food Chem. 27, 6,
1256-1262).
Defatted soybean flour was dispersed in water with a suitable solvent to solute ratio
and the pH of the dispersion was adjusted using 6N sodium hydroxide or 6N
hydrochloric acid. This was kept stirring for a few minutes with mechanical stirrer
and the temperature raised to 50 - 55° C. At this stage 0.4-0.6 (w%) of papain on the
basis of soy flour was added and stirring continued for 3-4 hours. At the end of the
above time interval the temperature of the slurry was raised to 90-95°C for 5-10
minutes. The slurry was cooled to room temperature and the insoluble carbohydrate
rich fraction in the dispersion was removed by centrifugation. The clarified protein
hydrolysate was spray dried to obtain protein hydrolysate.
The following examples are given by way of illustrations of the present invention and
should not be construed to limit the scope of the present invention.
Example 1
100 g of defatted soy flour is dispersed in 500 ml of water and the pH of the
dispersion was adjusted to 5.5 using IN HC1. The solution stirred with mechanical
stirrer and then the temperature raised to 50°C by heating the solution. 500 mg of
papain was added and stirring continued fc , :rs. The enzyme was inactivated by
boiling for 5 min. The pH of the hydrolysate was adjusted to 6.8 using 6N NaOH. The
slurry was cooled and centrifuged. The clear solution was spray dried. The yield was
24% (on flour basis) and degree of hydrolysis was 30%.
Example 2
300 g of defatted soy flour is dispersed in 1500 ml of water and the pH of the
dispersion was adjusted to 5.5 using IN HC1. The solution stirred with mechanical
stirrer and then the temperature raised to 55°C. 1.5g of papain was added and stirring
continued for 3 h. The enzyme was inactivated by boiling for 5 min. The pH of the
hydrolysate was adjusted to 6.8 using 6N NaOH. The slurry was cooled and
centrifuged. The clear solution was spray dried. The yield was 21% (on flour basis)
and degree of hydrolysis was 30%.
Example 3
1 kg g of defatted soy flour was dispersed in 5000 ml of water and the pH of the
dispersion was adjusted to 5.0.using IN HC1. The solution stirred with mechanical
stirrer and then the temperature raised to 50°C. 5 g of papain was added and stirring
continued for 4 hrs. The enzyme was inactivated by boiling for 5 min. The pH of
the hydrolysate was adjusted to 6.5 using 6N NaOH. The slurry was cooled and
centrifuged. The clear solution was spray dried. The yield was 20% (on flour basis)
and degree of Hydrolysis was 30%.
The particle size of the soy flour, ratio of enzyme to substrate, temperature, pH and
time interval controls the end of enzymatic hydrolysis resulting into minimizing
bitterness of the hydrolysate.
The soya protein hydrolysate obtained has creamy colour and an yield of 20-25% (on
flour basis). The product has 3.0-5.0% moisture, 8.0-8.5% nitrogen and 30.0-35.0%
degree of hydrolysis (TNBS procedure).
The soy protein hydrolysate obtained has 25-30 trypsin inhibitor Unit/mg (TlU/mg)
activity, 95-98% nitrogen solubility index, 1.0-1.4% of salt content (measured as Cl~
ions) and 2 - 2.2 g /100 ml bitterness recognition threshold. The lipoxygenase and
urease activities were not detectable. The amino acid composition of the soy protein
hydrolysate obtained was similar to the amino acid make up of starting raw material
thereby retaining the nutritional value. The 'protein hydrolysate is less bitter compared
to protein hydrolysate obtained from casein and is less hygroscopic in nature.
The main advantages of this invention are:
1. The enzyme employed is a food grade commercially available acceptable plant
enzyme with broad specificity resulting in 30- 35% degree of hydrolysis.
2. The product can be a good additive without imparting any undesirable flavour
for the finished product.
3. Hydrolysate has a solubility which is independent of pH making it a suitable
additive either in acid pH or alkaline pH.
4. The final product is in the dry form suitable for different food formulations
and easy to handle.






We claim:
1. A process for the preparation of protein hydrolysate from soy flour characterized in that the
flour is hydrolyzed using proteolytic enzymes of plant origin, wherein the process steps
comprising:
[a] preparing an aqueous slurry of defatted soy flour having 6 to 30 % w/v of solid content and hydrolyzing using proteolytic enzyme of plant origin at a pH of 5 to 9 and temperature of 53 ± 5 degree C under stirring for 30 minutes to 6 hours;
[b] inactivating the enzyme in the slurry of step [a] by known methods;
[c] neutralizing the pH of the slurry as obtained in step [b];
[d] separating the solids by known methods followed by drying the clarified liquor to get the desired protein hydrolysate.

2. A process as claimed in claim 1, wherein the solid content of the slurry is preferably 20% w/v.
3. A process as claimed in claim 1, wherein the proteolytic enzyme is selected from the group comprising of papain and bromelin.
4. A process as claimed in claim 1, wherein 0.4 to 0.6% w/w of the proteolytic enzyme is added to the soy flour.
5. A process as claimed in claim 1, wherein hydrolysis is carried out for a period of 3 to 4 hours.
6. A process as claimed in claim 1, wherein drying is preferably done by freeze drying, spray drying and drum drying.
7. A process for the preparation of protein hydrolysate from soy flour substantially as herein described with reference to the foregoing examples.

Documents:

00457-delnp-2003-abstract.pdf

00457-delnp-2003-claims.pdf

00457-delnp-2003-correspondence-others.pdf

00457-delnp-2003-description (complete).pdf

00457-delnp-2003-form-1.pdf

00457-delnp-2003-form-18.pdf

00457-delnp-2003-form-2.pdf

00457-delnp-2003-form-3.pdf

457-DELNP-2003-Abstract-(10-09-2008).pdf

457-delnp-2003-abstract-(12-03-2009).pdf

457-DELNP-2003-Claims-(10-09-2008).pdf

457-delnp-2003-claims-(12-03-2009).pdf

457-DELNP-2003-Correspondence-Others-(10-09-2008).pdf

457-delnp-2003-correspondence-others-(12-03-2009).pdf

457-DELNP-2003-Description (Complete)-(10-09-2008).pdf

457-delnp-2003-description (complete)-(12-03-2009).pdf

457-DELNP-2003-Form-1-(10-09-2008).pdf

457-delnp-2003-form-1-(12-03-2009).pdf

457-DELNP-2003-Form-3-(10-09-2008).pdf

457-DELNP-2003-Petition-137-(10-09-2008).pdf


Patent Number 233134
Indian Patent Application Number 00457/DELNP/2003
PG Journal Number 13/2009
Publication Date 27-Mar-2009
Grant Date 27-Mar-2009
Date of Filing 26-Mar-2003
Name of Patentee COUNCIL OF SCIENTIFIC AND INDUSTRIAL RESEARCH
Applicant Address RAFI MARG, NEW DELHI-110 001, INDIA.
Inventors:
# Inventor's Name Inventor's Address
1 APPU RAO GOPALA RAO APPU RAO CFTRI, MYSORE.
2 KARADKAGOVIND RAJU CFTRI, MYSORE.
3 RAMASWAMY HARENDRANATH CFTRI, MYSORE.
4 JOHNY JOSEPH CFTRI, MYSORE.
5 VISHWESWARIAH PRAKASH CFTRI, MYSORE.
6 CHERUPPANPULLIL RADHA CFTRI, MYSORE.
7 MYSORE CHELUVARAYA SHAMANTHAKA SASTRY CFTRI, MYSORE.
8 SRIDERI ANNAPURNA SINGH CFTRI, MYSORE.
PCT International Classification Number A23J 3/34
PCT International Application Number PCT/IN01/00031
PCT International Filing date 2001-03-05
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA