Process for preparation of protein-hydrolysate from milk...

Details Process for preparation of protein-hydrolysate from milk... Process for preparation of protein-hydrolysate from milk...

2001-03-19

2003-07-08

Navarro, Mark (Department: 1645)

Food or edible material: processes, compositions, and products

Fermentation processes

Of milk or milk product

C426S037000, C426S042000, C426S055000, C426S056000, C435S068100

Reexamination Certificate

active

06589574

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to a process for the preparation of protein hydrolysate from milk protein using fungal protease. Particularly, the present invention provides a process for the preparation of protein hydrolysate from casein using fungal protease obtained from Aspergillus sp.
BACKGROUND OF THE INVENTION
Casein is a good protein with well-balanced amino acid make-up. Casein as such has limited functional properties. Casein constitutes about 80% of the milk proteins. It is a phospho protein where phosphorus is covalently bound to polypeptide chain by serine ester linkages/the casein consists of heterogeneous &agr;, &bgr;-casein, &kgr;-casein and few other minor proteins. Casein is used in the food industry in the preparation of simulated meat, wine and beer clarifier and protein enriched dairy products (Evans, E. W. Uses of milk proteins in formulated foods in Developments in Food Proteins—1 Ed. B. J. F. Hudson, Applied Science Publishers, London, pp. 131-169). They are also used in crackers, snack foods and other food formulations to improve functional and nutritional characteristics. Casein hydrolysates find their application in food products.
The reduction in the molecular size of the protein can be accomplished by breaking peptide bonds using acid or alkali or enzymatic methods. Acid and alkali hydrolysis of protein leads to decreased nutritive value because of recemisation and destruction of essential amino acids, production of toxic constituents like lysino-alanine.
Enzymatic methods accomplish protein hydrolysis selectively without causing structural changes in the amino acids that make up the proteins. The peptide profile generated by enzymatic methods are well defined. The protein retains its nutritive value in enzymatic hydrolysis better than any traditional acid/alkali hydrolysis. The strong tendency of casein and whey protein to form bitter tasting hydrolysates is well known; and nearly all the development work, in particular on casein hydrolysis processes has centered around the bitterness problem. The recovery and modification of proteins by enzymatic hydrolysis is a versatile tool. The hydrolysis is precise and unique for a given proteinase-protein system. The degree of hydrolysis determines the properties such as solubility, functionality and taste of hydrolysed protein. Protein hydrolysates can be good additives to improve the functional characteristics and nutritional value of the end products. Most of the protein hydrolysates commercially available in India are acid protein hydrolysates. The humin formation, high temperature involvement, brown colour formation, high salt content, destruction of some of the essential amino acids and low yield are some of the drawbacks in the acid hydrolysis. Chlorohydrines are produced during acid hydrolysis of vegetable protein. Chlorohydrines are eliminated from liquid hydrolysates by subjecting them to steam distillation at reduced pressure.
The substrate casein is widely used protein for the preparation of hydrolysate owing to its superior nutritional quality. Traditionally casein hydrolysates are used for dietetic feeding. Casein hydrolysate is used in infant and specialized nutritional formulae.
Reference may be made to a published paper entitled Enhancing the Functionality of Food Proteins by Enzymatic Modification, by Panyan, D. and Kilara, A., Trends in Food Sci. Tech., 7(4), 120-125, wherein the enzyme hydrolysis influence emulsifying capacity, hydrophobicity of proteins. The drawback of the method is the degree of hydrolysis is not defined.
Reference may be made to a published paper entitled dietary enzymic hydrolysates of protein with reduced bitterness Clegg, K. M. and McMillan, A. D. (1974) J. Food Tech. 9(1), 21-29, wherein, egg white and casein have been investigated as protein substrates and treated with papain, optionally with chloroform followed by endopeptidases and pig's kidney tissue as a source of exopeptidases. Hydrolysates are relatively free of bitterness and continuing small peptides and over 50% free amino acids have been obtained. The draw back of the method is that the yields are low (60.6-85.5%). Further the process involves multiple steps, double enzyme system and the protease used is obtained from animal sources.
Reference may be made to a published paper entitled debittering and nutritional upgrading of enzymic casein hydrolysates Cogan, V., Moshe, M., Mokady, S. (1981) J. Sci. Food. Agric. 32(5), 459-466, wherein, casein hydrolysates obtained by digestion using papain and pepsin was performed essentially according to the procedure of Clegg and McMillan (1974). A casein solution (pH 7.2) was first treated with papain at 40° C. The enzyme concentration was 4.0%, and 0.2% toluene was added to prevent microbial growth. At the end of an 18 h incubation the pH was adjusted to 3.0 using hydrochloric acid and the treatment continued for an additional 22 h with 0.5% with 0.5% pepsin at 37° C.
The treatment with Rhozyme enzymes was performed with specified enzyme concentrations under the following conditions of pH (adjusted using 1M NaOH) and temperature: Rhozyme P-11 and Rhozyme-41 concentrate at pH 8.5, 50° C.; Rhozyme P-53 concentrate at pH 7.5, 60° C.; Rhozyme-62 concentrate at pH 8.3, 60° C.
The extent of proteolytic digestion was examined as described previously except that the digested samples were diluted 21 fold with a 10% trichloracetic acid solution. Exhaustive enzymic digestion was achieved by incubating (at 37° C.) a 100 ml solution containing 298 mg casein and 6 mg protinase, for 15 h. At the end of the incubation period the solution was clear and its absorbance estimated at 280 nm using appropriate protein and enzyme blanks.
The bitter taste could be further reduced by treatment with 0.5 g of activated carbon/g hydrolysate. The drawback of the method is that the process involves a number of steps, a number of enzymes in sequence and as well as continuously and solvents are used to get the hydrolysate resulting in a cost ineffective process. Additional use of activated carbon is also involved for the production of casein hydrolysates to reduce bitterness.
Reference may be made to a published paper entitled Casein Hydrolysate Produced Using a Formed-in-Place Membrane Reactor, by Chland, W. D., Cordle, C. T., Thomas, R. L. J. Food. Sci. 60, 1349-1352, 1995. The degree of hydrolysis is reported as being between 4 to 51%. The time of hydrolysis varies from 18-66 h. The draw back of the method is that it involves the long duration of the hydrolysis.
Reference may be made to a published paper entitled production of an enzymic hydrolysate of casein on a kilogram scale, Clegg, K. M., Smith, G. and Walker, A. L., 1974 J. Food Tech., 9(4), 425-431, 1974, wherein, the laboratory method for the hydrolysis of casein has been described. 12 kg of commercial casein was suspended in 220 l water at pH 6.2 and digested with papain at 40° C. for 8 h. and then with a pig kidney homogenate at pH 7.8-8.0 for 24 h. Chloroform was used as a preservative instead of toluene. The hydrolysate was passed through a Russell separator to remove insoluble material and then pasteurized at 83-88° C. for 3-5 min. The product was spray dried. The process was completed in 60 h. The bacterial counts were satisfactory. The draw back of the method is that the duration of the hydrolysis is too long. Further, the enzymes used are different. Hydrolysis is multi-step hence it is cost ineffective and time consuming.
Reference may be made to British Patent No. 1595499, (patent authority VEB Berlin—Chemie, 1981), wherein, casein is hydrolysed by boiling with dilute sulphuric acid and spray dried. The powder (100 g) is heated under reflux for 10 min with 200 ml. Methanol and the undissolved material is filtered off, washed twice with 50 ml methanol and dried resulting in 80 g hydrolysate having all amino acids as a colorless, odorless and tasteless powder. The draw back of the method is it involves the usage of mineral acid likely to lead to racemisation of the amino acids and generation of toxic p

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