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US 2002O132028A1

(19) United States


(12) Patent Application Publication (10) Pub. No.: US 2002/0132028A1
Appu Rao et al. (43) Pub. Date: Sep. 19, 2002
(54) PROCESS FOR PREPARATION OF (21) Appl. No.: 09/811,780
PROTEIN-HYDROLYSATE FROM SOY
FLOUR (22) Filed: Mar 19, 2001
(76) Inventors: Appu Rao Gopala Rao Appu Rao, Publication Classification
Mysore (IN); Hole Narasipura
Nanjundaiah Chandrasekhara, (51) Int. Cl." ....................................................... A23L 1/20
Mysore (IN); Karadka Govindaraju, (52) U.S. Cl. ................................................................ 426/46
Mysore (IN); Johny Joseph, Mysore
(IN); Kolara Subramanyam Krishna (57) ABSTRACT
Murthy, Mysore (IN); Vishweshwariah
Prakash, Mysore (IN); Mysore A process for preparation of protein hydrolysate from Soy
Cheeluvaraya Shamanthaka Sastry, flour Said process comprising hydrolyzing aqueous Slurry of
Mysore (IN) defatted soy flour containing 6-30% solid content w/v using
proteolytic enzyme of plant origin at pH5-9 and temperature
Correspondence Address: of 53(5(C under stirring for 30 minutes to 6 hours; inacti
POWELL GOLDSTEIN, FRAZER & Vating the enzyme by a known manner; neutralizing the pH
MURPHY LLP value of the Slurry; Separating the Solids by a known manner
P.O. BOX 97223 and drying the clarified liquor So obtained to get the Said
WASHINGTON, DC 20090-7223 (US) hydrolysate.
US 2002/0132028A1 Sep. 19, 2002

PROCESS FOR PREPARATION OF jet cooking or dynamic heating at 104 C. for a few seconds
PROTEIN-HYDROLYSATE FROM SOY FLOUR and later cooled in a vacuum chamber before hydrolysis
using bromelin. The protein was precipitated at its isoelec
FIELD OF THE INVENTION tric point from an aqueous extract of the material before the
0001. The present invention relates to a process for the hydrolysis. The drawback of the proceSS is the Starting
preparation of protein hydrolysate from Soy flour using material protein isolate, which is more expensive. The
proteolytic enzyme of plant origin. Particularly, the present process is a multiStep process, energy intensive. The proceSS
invention relates to a proceSS for the preparation of protein further needs machines like the jet cooker and a vacuum
hydrolysate from defatted Soy flour using papain. chamber.

BACKGROUND OF THE INVENTION 0007 European Patent No. 0223560 A2 claimed a


method describing the Separation of protein hydrolysates
0002 Presently about 6.8 M tons of soybean is produced with meat and cheese flavor, from proteinaceous feed StockS
in India and extracted for oil and the solvent extracted flour (e.g. containing Soybean, gluten, whey, casein, hemoglobin,
is exported to foreign countries for feed purposes. By yeast, cereal or microbial proteins) by Stepwise hydrolysis
providing additional facilities for the hygienic processing of using an endopeptidase followed by amino peptidase from
Soybean in the Solvent extraction units, it is possible to Streptococcus lactus. The drawback of the process is that it
obtain edible grade defatted flour having the desired func is a multi-step process.
tional characteristics. After the recovery of oil, 4.9M tons of
soy flour is available in India for utilization. A small portion 0008 European Patent No. 0087246 B1 claimed a pro
of the total Soybean produced also finds its use for different ceSS for the hydrolysis of Soybeans, wheat gluten and cotton
edible grade flours, protein isolate and texturized products Seeds using fungal protease from Aspergillus and pancreatin
and the popularity of these products are greatly picking up (trypsin, chymotrypsin A, B and C, elastase and carbox
globally. Soybean is an excellent Source of protein, which ypeptidase A and B) is described. Activated charcoal is used
contains about 40% protein. New manufacturing techniques to treat the hydrolysate, which is used for nutritional
for high quality Soybean foods have been developed by improvement. The draw back of the process is it involves
lowering or destroying of the anti-nutritional factorS Such as more Steps.
trypsin inhibitors. 0009 European Patent No. 0187048 A2 described the
0003 U.S. Pat. No. 5,180,597 claimed a process for preparation of soy protein hydrolysate with 0.25 to 2.5%
hydrolyzed vegetable protein with enhanced flavor, which degree of hydrolysis (DH) using microbial rennet (Mucor
contains no detectable level of monochlorodihydroxypro miehei) and to be used as an egg white Substitute. The
panol is described. In the above reference, wheat gluten is enzyme used in the proceSS is different and involves very
hydrolysed using Prozyme 6 (a fungal protease) at a tem low DH of soy protein.
perature of 40-50 C., pH 6.5-7.0, enzyme concentration of 0010 United Kingdom Patent No. 2053228A described a
0.1-2% of Substrate for a time period of 4 h. The hydrolysed process for the production of Soy protein hydrolysate from
protein is treated with gaseous HCl for deamidation before partially defatted soy material by hydrolysis with proteolytic
the addition of acid for inactivating the enzyme. The draw enzyme. The drawback of the process is that due to partial
back in such hydrolysis is that ió is likely to lead to defatting Soy flour, left over oil comes in contact with
racemisation of amino acids and the addition of acid
increases the Salt content in the product. protein phase, which could lead to off-flavors.
0004 U.S. Pat. No. 5,077,062 claimed a low sodium, low 0011 U.S. Pat. No. 4,324.805 described a method for
mono Sodium glutamate Soy hydrolysate that is prepared producing Soy protein hydrolysate and oil from partially
from Soy material Such as Soy flour, Soy mean or Soy grits defatted Soy material by hydrolysis with proteolytic enzyme.
using fungal protease in water. The hydrolysis is conducted The Soyflour is partially defatted by water washing at pH
in the absence of acid or base at 90° C. for 2 h. After 3.5-4.5 and later hydrolysed using water and a base to
deactivating the enzyme and dewatering the mixture the increase the pH. The DH is in the range of 8-12%. Oil is
resulting hydrolysate contains between 45 and 55 wt.% recovered from the wash water. Alcalase is the enzyme used.
enzymatically hydrolysed Soy based protein with an average The drawback of the proceSS is that it is a multi Step process
molecular weight of 670,000+50,000. The fungal protease and due to partial defatting of Soy flour, left over oil comes
used is different from the enzyme used in the present in contact with protein phase which could lead to off-flavors.
invention. The proceSS is energy intensive due to the high Enzyme inactivation is done by addition of acid, which is
temperature (90° C) used. likely to lead to increase Salt content in the product.
0005 U.S. Pat. No. 4,757,007 claimed a preparation of 0012 U.S. Pat. No. 3640725 described an enzymatic
two hydrolyzed products using a protease from Soy protein. hydrolysis proceSS for production of Soy protein hydroly
The Soy protein is hydrolysed with papain or pepsin after sates. The soy seeds are comminuted and heated at 90-140
precipitating with alcohol. The drawback of the process is it C. Protease (fungal and bacterial) is added at 25-75 C. The
involves the separation of the mixture of hydrolysed prod fiber is separated and slurry has two phases-oil and aqueous
ucts. Hydrolysis is carried out using papain or pepsin. phase. Aqueous phase is brought to pH 4.5 to precipitate the
Acidification is carried out to bring down the pH to 2.5-5.0 protein, which is then concentrated. The Starting material is
to Separate the two kinds of hydrolysates, which could lead not defatted and hence the residual oil could come in contact
to increase in Salt content. with the aqueous phase, which could lead to off-flavors.
0006 European Patent No. 0,148,600 B 1 relates to the 0013 Canadian Patent No. 905742 described a soy pro
preparation of hydrolyzed protein from protein isolate after tein hydrolysate modified with pepsin to yield a product
US 2002/0132028A1 Sep. 19, 2002

which in presence of water and Sugar whips at a rapid rate 0023. In another embodiment of the present invention,
to produce aerated products of low density. the plant origin proteolytic enzyme is Selected from the
group comprising of papain and bromelin.
0014 European Patent No. 0797928 A1 described a
proceSS for the manufacture of a Soy protein hydrolysate 0024. In still another embodiment of the present inven
with a protease used Selectively to decompose glycinin at a tion, 0.4–0.6 w/w of the proteolytic enzyme is added to the
pH of 1.5-2.5. The pH used in the process is very low and Soy flour.
therefore differs from the pH used in the present invention. 0025. In yet another embodiment of the present inven
Further, the aim of the process is to achieve a low glycinin tion, the hydrolysis is effected for a period of 3-4 hours.
content which is not the case in the present invention.
0.015 Reference may be made to a published paper 0026. In one more embodiment of the present invention,
entitled “Industrial production and application of Soluble the drying is effected by freeze drying, spray drying and
enzymatic hydrolysate of Soy protein, (Olsen, H. S., Adler drum drying.
Nissen, J., (1979), Process Biochemistry, 14(7), 6.8, 10-11) 0027. In one another embodiment of the present inven
wherein a method for preparation of Soy protein hydrolysate tion, the protein hydrolysate produced has decreased bitter
from soy flakes washed at pH 4.5 followed by hydrolysis CSS.
using alcalase is described. The Solubility of the Substrate is
low at the acidic pH which is likely to result in low yields. 0028. In an embodiment of the present invention, the
The enzyme used is different from the enzyme used in the protein hydrolysate produced is leSS hygroscopic in nature.
present invention. 0029. In another embodiment of the present invention,
OBJECTS OF THE INVENTION
the protein hydrolysate has 2-2.2 g/100 ml bitterness rec
ognition threshold.
0016. The main object of the present invention is to 0030. In still another embodiment of the present inven
provide a process for the preparation for the preparation of tion, the protein hydrolysate produced has low mineral
protein hydrolysate from Soy four from plant based protease. COntent.
0.017. Another object of the present invention is to pro 0031. In one more embodiment of the present invention,
vide a process for the preparation of protein hydrolysate high yield of protein hydrolysate with 30 to 35% degree of
with Specified degree of hydrolysis. hydrolysis is obtained from the raw material taken.
0.018 Still another object of the present invention is to 0032. In one another embodiment of the present inven
provide a proceSS for the preparation of protein hydrolysate tion, a protein hydrolysate having creamy color and a yield
Soluble in water over a wide range of pH value. of 20 to 25% (on flour basis) is obtained.
0.019 Yet another object of the present invention is to 0033. In an embodiment of the present invention, the
provide a proceSS for the preparation of protein hydrolysate protein hydrolysate has 3.0 to 5.0% moisture, 8.0 to 8.5%
which can be used for nutritional enrichment. nitrogen and 30.0-35.0% degree of hydrolysis.
SUMMARY OF The PRESENT INVENTION 0034. In another embodiment of the present invention,
the protein hydrolysate obtained has 25-30 trypsin inhibitor
0020. The invention provides a process for the prepara units/mg activity, 95 to 98% Nitrogen Solubility Index and
tion of protein hydrolysate from Soy flour, Said proceSS 1.0 to 1.4% of Salt content.
comprising: hydrolyzing aqueous Slurry of defatted Soy flour
containing 6-30%. Solid content w/v using proteolytic 0035) In still another embodiment of the present inven
enzyme of plant origin at pH 5-9 and temperature of 53+5 tion, lipoxygenase and urease activities of the protein
C. under Stirring for 30 minto 6 h; inactivating the enzyme hydrolysate are not detectable.
by a know manner; neutralizing the pH value of the slurry;
Separating the Solids by a know manner and drying the 0036). In yet another embodiment of the present inven
clarified liquor So obtained to get the Said hydrolysate. tion, the amino acid composition of the protein hydrolysate
is similar to the amino acid makeup as that of the Starting
DESCRIPTION OF THE INVENTION material.

0021 Accordingly, the present invention provides a pro 0037. In one more embodiment of the present invention,
ceSS for the preparation of protein hydrolysate from Soy the protein hydrolysate retained the nutrition value as in the
flour, Said process comprising: hydrolyzing aqueous Slurry Starting material.
of defatted Soy flour containing 6-30% solid content w/v 0038. In one another embodiment of the present inven
using proteolytic enzyme of plant origin at pH 5-9 and tion, the protein hydrolysate does not impart any undesirable
temperature of 53+5 C. under stirring for 30 min to 6 h; flavor for the finished product.
inactivating the enzyme by a known manner; neutralizing
the pH value of the slurry; Separating the Solids by a known 0039. In an embodiment of the present invention, the
manner and drying the clarified liquor So obtained to get the solubility of the protein hydrolysate is independent of the pH
Said hydrolysate. value.
0022. In an embodiment of the present invention, the 0040. The present invention also provides a protein
solid content of the slurry is 20% w/v. hydrolysate creamy in color.
US 2002/0132028A1 Sep. 19, 2002

0041. In still another embodiment of the present inven reaction is quenched by adding 4 ml. Of 0.10 N hydrdoch
tion, the protein hydrolysate has 3 to 5% moisture, 8 to 8.5% loric acid (HCl) and the absorbance is read at 340 nm. A 1.5
nitrogen and 30 to 35% degree of hydrolysis. mM L-leucine Solution is used as the Standard. Transforma
0042. In yet another embodiment of the present inven tion of the measured leucine amino equivalents to a degree
tion, the protein hydrolysate has 20 to 30 trypsin inhibitor of hydrolysis is carried out by means of a Standard curve for
units/mg activity, 95 to 98% Nitrogen Solubility Index and each particular protein substrate (Adler Nissen, J. (1979) J.
1 to 1.4% of Salt content. Agri. Food Chem. 27, 6, 1256-1262).
0043. In one more embodiment of the present invention, 0056 Defatted soybean flour was dispersed in water with
the protein hydrolysate has 2 to 2.2 g/100 ml bitterness a suitable solvent to solute ratio and the pH of the dispersion
recognition threshold. was adjusted using 6N sodium hydroxide or 6N hydrochlo
ric acid. This was kept Stirring for a few minutes with
0044) In one another embodiment of the present inven mechanical stirrer and the temperature raised to 50-55 C. At
tion, the lipoxygenase and urease activities of the protein this stage 0.4–0.6 (w %) of papain on the basis of soy flour
hydrolysate are not detectable. was added and Stirring continued for 3-4 hours. At the end
of the above time interval the temperature of the slurry was
0.045. In an embodiment of the present invention, the raised to 90-95 C. for 5-10 minutes. The slurry was cooled
amino acid composition of the protein hydrolysate is similar to room temperature and the insoluble carbohydrate rich
to the amino acid makeup of the Starting material. fraction in the dispersion was removed by centrifugation.
0046. In another embodiment of the present invention, The clarified protein hydrolysate was spray dried to obtain
the protein hydrolysate retained the nutrition value present protein hydrolysate.
in the Starting material. 0057 The following examples are given by way of
0047. In still another embodiment of the present inven illustrations of the present invention and should not be
tion, the protein hydrolysate does not impart any undesirable construed to limit the Scope of the present invention.
flavor for the finished product. EXAMPLE 1.
0.048. In yet another embodiment of the present inven 0.058 100g of defatted soy flour is dispersed in 500 ml
tion, the Solubility of the protein hydrolysate is independent of water and the pH of the dispersion was adjusted to 5.5
of the pH value. using IN HCl. The solution stirred with mechanical stirrer
0049. The process involves following process steps: and then the temperature raised to 50 C. by heating the
Solution. 500 mg of papain was added and Stirring continued
0050. Defatted Soy Flour for 3 hrs. The enzyme was inactivated by boiling for 5 min.
0051 Soybean flour is derived from clean round beans. The pH of the hydrolysate was adjusted to 6.8 using 6N
The cleaned beans pass through a cracking process, the bean NaOH. The slurry was cooled and centrifuged. The clear
fragments are graded on Sieves and aspirate System. The solution was spray dried. The yield was 24% (on flour basis)
cleaned cracked meat passed through a conditioner cooker and degree of hydrolysis was 30%.
and flaked. This is Subjected to Solvent extraction process. EXAMPLE 2
The extracted flakes were desolventized and ground to 100
mesh. The specification for soy flour consists of (a) Mois 0059) 300 g of defatted soy flour is dispersed in 1500 ml
ture=9% by mass (max); (b) Protein on dry basis=48% by of water and the pH of the dispersion was adjusted to 5.5.
mass (min); (c) The total ash on dry basis=7.2% by mass using 1N HC1. The solution was stirred with mechanical
(max); (d) Acid insoluble ash on dry basis=0.4% by mass stirrer and the temperature raised to 55° C. 1.5 g of papain
(max); (e) Fat on dry basis=1.5% by mass; (f) Crude fibre on was added and Stirring continued for 3 h. The enzyme was
dry basis=4.2% by mass (max); (g) Aflatoxin=30 ppb (max); inactivated by boiling for 5 min. The pH of the hydrolysate
(h) Residual solvent=170 ppm, (I) Total bacterial count per was adjusted to 6.8 using 6N NaOH. The slurry was cooled
gram=50,000; (ii) Coliform bacteria/g=10; and (k) Salmo and centrifuged. The clear Solution was spray dried. The
nella bacterial - Nil. yield was 21% (on flour basis) and degree of hydrolysis was
30%.
0.052 Papain
EXAMPLE 3
0053. The specification of the plant thiol protease papain 0060) 1 kgg of defatted soy flour was dispersed in 5000
is to obtain commercially available food grade enzyme
having proteolytic activity not leSS than 2,000 tyrosine units ml of water and the pH of the dispersion was adjusted to 5.0.
(TU)/mg proteolytic activity. using 1N HCl. The solution stirred with mechanical stirrer
and then the temperature raised to 50 C. 5g of papain was
0.054 Measurement of Degree of Hydrolysis added and Stirring continued for 4 hrs. The enzyme was
0055 Trinitrobenzenesulphonic acid (TNBS) procedure, inactivated by boiling for 5 min. The pH of the hydrolysate
is an accurate, reproducible and generally applicable proce as adjusted to 6.5 using 6N NaOH. The slurry was cooled
dure for determining the degree of hydrolysis of food protein and centrifuged. The clear Solution was spray dried. The
hydrolysates. The protein hydrolysate is dissolved/dispersed yield was 20% (on flour basis) and degree of Hydrolysis was
30%.
in hot 1% sodium dodecyl sulphate to a concentration of
0.25-2.5x10 aminoequivalents/L. A sample solution (0.25 0061 The particle size of the Soy flour, ratio of enzyme
ml) is mixed with 2 ml of 0.2125M sodium phosphate buffer to Substrate, temperature, pH and time interval controls the
(pH 8.2) and 2 ml of 0.1% Trinitrobenzenesulphonic acid, end of enzymatic hydrolysis resulting into minimizing bit
followed by incubation in the dark for 60 min at 50 C. The terness of the hydrolysate.
US 2002/0132028A1 Sep. 19, 2002

0062) The Soya protein hydrolysate obtained has creamy 10. A process as claimed in claim 1, wherein the protein
colour and an yield of 20-25% (on flour basis). The product hydrolysate produced has low mineral content.
has 3.0-5.0% moisture, 8.0-8.5% nitrogen and 30.0-35.0% 11. A proceSS as claimed in claim 1, wherein high yield of
degree of hydrolysis (TNBS procedure) protein hydrolysate with 30 to 35% degree of hydrolysis is
0.063. The Soy protein hydrolysate obtained has 25-30 obtained from the raw material taken.
trypsin inhibitor Unit/mg (TIU/mg) activity, 95-98% nitro 12. A proceSS as claimed in claim 1, wherein protein
gen Solubility index, 1.0-1.4% of Salt content (measured as hydrolysate obtained has creamy color and a yield of
Cl ions) and 2-2.2 g/100 ml bitterness recognition thresh 20-25% (on flour basis).
old. The lipoxygenase and urease activities were not detect 13. A proceSS as claimed in 1, wherein protein hydrolysate
able. The amino acid composition of the Soy protein has 3.0 to 5.0% moisture, 8.0 to 8.5% nitrogen and 30.0-
hydrolysate obtain was similar to the amino acid make up of 35.0% degree of hydrolysis.
Starting raw material thereby retaining the nutritional value. 14. A process as claimed in claim 1, wherein the protein
The protein hydrolysate is leSS bitter compared to protein hydrolysate obtained has 25-30 trypsin inhibitor units/mg
hydrolysate obtained from casein and is less hygroscopic in activity, 95 to 98% Nitrogen Solubility Index and 1.0 to
nature. 1.4% of Salt content.
0064. The main advantages of this invention are: 15. A proceSS as claimed in claim 1, wherein lipoxygenase
0065 1. The enzyme employed is a food grade and urease activities of the protein hydrolysate are not
detectable.
commercially available acceptable plant enzyme
with broad specificity resulting in 30-35% degree of 16. A process as claimed in claim 1, wherein the amino
hydrolysis. acid composition of the protein hydrolysate was similar to
the amino acid makeup as that of the Starting material.
0066 2. The product can be a good additive without 17. A process as claimed in claim 1, wherein the protein
imparting any undesirable flavour for the finished hydrolysate retained the nutrition value as in the Starting
product. material.
0067 3. Hydrolysate has a solubility which is inde 18. A process as claimed in claim 1, wherein the protein
pendent of pH making it a Suitable additive either in hydrolysate does not impart any undesirable flavor for the
acid pH or alkaline pH. finished product.
0068 4. The final product is in the dry form suitable 19. A process as claimed in claim 1, wherein the solubility
for different food formulations and easy to handle. of the protein hydrolysate is independent of the pH value.
1. A process for the preparation of protein hydrolysate 20. A protein hydrolysate obtained from soy flour, com
from Soy flour, Said process comprising: hydrolyzing aque prising lour 20 to 30 trypsin inhibitor units/mg activity, 95
ous slurry of defatted soy flour containing 6-30% solid to 98% Nitrogen Solubility Index, 1 to 1.4% of salt content
content w/v using proteolytic enzyme of plant origin at pH 3 to 5% moisture, 8 to 8.5% nitrogen and 30 to 35% degree
of hydrolysis.
5-9 and temperature of 53+5 C. under stirring for 30 21. A protein hydrolysate as claimed in claim 20, wherein
minutes to 6 hours; inactivating the enzyme by a known
matter; neutralizing the pH value of the slurry; Separating the lipoxygenase and urease activities are not detectable.
the Solids by a known manner and drying the clarified liquor 22. A protein hydrolysate as claimed in claim 20, further
So obtained to get the Said hydrolysate. comprising 2 to 2.2 g/100 ml bitterneSS recognition thresh
2. A proceSS as claimed in claim 1, wherein the Solid old.
content of the slurry is 20% w/v. 23. A protein hydrolysate as claimed in claim 20, wherein
3. A proceSS as claimed in claim 1, wherein the plant the amino acid composition of the protein hydrolysate is
origin proteolytic enzyme is Selected from the group com Similar to the amino acid makeup of the Starting material.
prising of papain and bromelin. 24. A protein hydrolysate as claimed in claim 20, wherein
4. A process as claimed in claim 1, wherein 0.4–0.6% w/w/ the protein hydrolysate retained the nutrition value present
of the proteolytic enzyme is added to the Soy flour. in the Starting material.
5. A process as claimed in claim 1, wherein the hydrolysis 25. A protein hydrolysate as claimed in claim 20, wherein
is effect for a period of 3-4 hours. the protein hydrolysate does not impart any undesirable
6. A proceSS as claimed in claim 1, the drying is effected flavor for the finished product.
by freeze drying, spray drying and drum drying.
7. A process as claimed in claim 1, wherein the protein 26. A protein hydrolysate as claimed in claim 20, wherein
hydrolysate produced has decreased bitterness. the solubility of the protein hydrolysate is independent of the
8. A process as claimed in claim 1, wherein the protein pH value.
hydrolysate produced is less hygroscopic in nature. 27. A protein hydrolysate as claimed in claim 20, has
9. A process as claimed in claim 1, wherein the protein cream color.
hydrolysate has 2-2.2 g/100 ml bitterness recognition thresh
old.