WO2002069734A1 - Procede de preparation d'hydrolysat proteique a partir des proteines du lait - Google Patents

Procede de preparation d'hydrolysat proteique a partir des proteines du lait Download PDF

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Publication number
WO2002069734A1
WO2002069734A1 PCT/IN2001/000030 IN0100030W WO02069734A1 WO 2002069734 A1 WO2002069734 A1 WO 2002069734A1 IN 0100030 W IN0100030 W IN 0100030W WO 02069734 A1 WO02069734 A1 WO 02069734A1
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WO
WIPO (PCT)
Prior art keywords
protein hydrolysate
protein
hydrolysis
hydrolysate
casein
Prior art date
Application number
PCT/IN2001/000030
Other languages
English (en)
Inventor
Bhagya Swamylingappa
Johny Joseph
Kowsalya Shankara Murthy
Vishweshwariah Prakash
Mysore Cheluvaraya Shamanthaka Sastry
Tirumakudalu Chikkaraja Sindhu Kanya
Original Assignee
Council Of Scientific And Industrial Research
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Council Of Scientific And Industrial Research filed Critical Council Of Scientific And Industrial Research
Priority to PCT/IN2001/000030 priority Critical patent/WO2002069734A1/fr
Priority to CN01823108.XA priority patent/CN1225185C/zh
Publication of WO2002069734A1 publication Critical patent/WO2002069734A1/fr

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Classifications

    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J3/00—Working-up of proteins for foodstuffs
    • A23J3/30—Working-up of proteins for foodstuffs by hydrolysis
    • A23J3/32—Working-up of proteins for foodstuffs by hydrolysis using chemical agents
    • A23J3/34—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes
    • A23J3/341—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes of animal proteins
    • A23J3/343—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes of animal proteins of dairy proteins
    • A23J3/344—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes of animal proteins of dairy proteins of casein
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J3/00—Working-up of proteins for foodstuffs
    • A23J3/04—Animal proteins
    • A23J3/08—Dairy proteins
    • A23J3/10—Casein

Definitions

  • the present invention relates to a process for the preparation of protein hydrolysate from milk protein using fungal protease.
  • the present invention provides a process for the preparation of protein hydrolysate from casein using fungal protease obtained from Aspergillus sp.
  • 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 ⁇ , ⁇ -casein, -casein and few other minor proteins. Casein is used in 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, p.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 hydrolyzed 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.
  • casein is widely used protein for the preparation of hydrolysate owing to its superior nutritional quality.
  • casein hydrolysates are used for dietetic feeding.
  • Casein hydrolysate is used in infant and specialized nutritional formulae.
  • Rhozyme P-ll 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.
  • 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 draw back of the method is that the process involves number of steps, 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.
  • hypoallergenic whey protein hydrolysates are prepared by a process in which the initial proteolysate is heat treated at 80-100°C for 3-10 min at pH 6-8, cooled to 40-60 °C, and subjected to a second enzymic hydrolysis (with trypsin, chymotrypsin, or a trypsin/chymotrypsin/pancreatin mixture). The proteolytic enzyme is then inactivated by heating to 75-85°C.
  • the hypoallergenic proteolysate may be incorporated in dietetic preparations, foods for infants, etc.
  • the draw back of the method is use of very high temperature and proteolytic enzymes of animal origin.
  • the hydrolysate is suitable for use in infant, dietetic and convalescent foods.
  • the draw back of the method is use of very high temperature and proteolytic enzymes of animal origin.
  • the retained whey proteins are added to the pre-concentrated yeast before hydrolysis.
  • the light coloured hydrolysate product has no yeasty taste.
  • the draw back of the process is the organism and type of enzyme. Fermentation, is different.
  • an imitation milk product is obtained by mixing 9 parts of lactose - reduced whey concentrate
  • the product contains 33.6% protein, 42.0% lactose, 19.1% minerals and 4.2% moisture and has an amino acid profile between that of soy bean meal and casein.
  • the main object of the present invention is to provide a process for the preparation of protein hydrolysate from milk protein using fungal protease.
  • Another object of the present invention is to provide a process for the preparation of protein hydrolysate from whey/casein using fungal protease obtained from Aspergillus sp.
  • the present invention provides a process for the preparation of protein hydrolysate from milk, which comprises treating milk protein with fungal protease at a pH of 7.5-8.5, temperature of 40 + 5°C for a time period of 30 min to 2 hours; heating at 65 - 70°C for at least 3 min, separating the clarified supernatant by a known manner and drying the clarified liquor thus obtained to get the protein hydrolysate.
  • the present invention provides a process for the preparation of protein hydrolysate from milk protein, said process comprising: treating milk protein with fungal protease at a pH of 7.5-8.5, temperature of 40 ⁇ 5°C for a time period of 30 minutes to 2 hours; heating at 65 -70°C for at least 3 minutes, separating the clarified supernatant by a known manner and drying the clarified liquor thus obtained to get the protein hydrolysate.
  • the fungal protease obtained is from Aspergillus sp.
  • the Aspergillus fungus is selected from the group comprising of A. flavus, A. japonicus, A. niger and A. awamori
  • the milk protem is selected from the group comprising of whey and casein.
  • the pH value is maintained between 7.6 to 8.0 for maximum fungal protease activity.
  • the working temperature range during hydrolyzation of proteins is 40-45°C.
  • the hydrolysis is effected for 1.5 to 2 hours.
  • the drying is effected by freeze drying, spray drying and drum drying.
  • the protein hydrolysate has 0.4 to 0.5% perceptible threshold bitterness.
  • high yield of protein hydrolysate with 50% degree of hydrolysis is obtained from the raw material taken.
  • the protein hydrolysate obtained is creamish white in color.
  • the protein hydrolysate has 11.5 to 12.5%) nitrogen and 4.5 to 5 % ash.
  • the protein hydrolysate is soluble in water in all pH ranges.
  • the amino acid composition of the protein hydrolysate was similar to the amino acid makeup of the starting material.
  • the protein hydrolysate retained all the nutritive values of the starting material.
  • the hydrolysis process is terminated by adjusting the pH and temperature simultaneously to a point at which the protease enzyme system is most sensitive to heat damage and then to bring the slurry to elevated temperatures of 65-70°C for 3 - 5 minutes thereby destroying enzyme activity and terminating hydrolysis.
  • the present invention also provides a protein hydrolysate cremish white in color. In an embodiment of the present invention, 95% protein hydrolysate is obtained.
  • the protein hydrolysate comprises 11.5 to 12.5 % nitrogen and 4.5 to 5% ash.
  • the protein hydrolysate has 0.4 to 0.5% perceptible threshold bitterness. In yet another embodiment of the present invention, the protein hydrolysate is soluble in water in all pH ranges.
  • the amino acid composition of the protein hydrolysate is similar to the amino acid makeup of the starting material.
  • the protein hydrolysate retained all the nutritive values of the starting material.
  • the protein hydrolysate having 50% degree of hydrolysis is obtained.
  • Casein is a phosphoprotein that contains 0.7 to 0.9% of phosphorus covalently bound to the polyphenols chain by serine ester linkages. Calcium and citrate are also included in this structure.
  • Acid casein is obtained by precipitating milk with an acid such as HC1, Sulphuric acid or lactic acid. Sweet casein results from the action of chymosin.
  • Low viscosity casein is produced by treating milk with proteolytic enzymes/and an acid.
  • the basic operations in the production of casein are the same irrespective of the type of casein produced. Such operations consist of the precipitation of the curd and its washing, pressing and drying. In most countries the largest amount of casein is produced by the acid process.
  • the casein to be used for protein hydrolysate must be of edible quality and bacteriologically pure.
  • the enzyme used is fungal protease, the activity being 30,000 units/g.
  • Tri-nitrobenzenesulphonic 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 amino equivalents/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 0.100 N HC1, and the absorbance is read at 340 nm.
  • a 1.5 mM 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. (Ref: Jens Adler - Nissen, J. Agr. Food Chem. Vol. 27, No. 6, 1979).
  • Casein was dispersed in water with a suitable solvent to solute ratio and the pH of the dispersion was adjusted to 7.6 - 8.0 using 15N sodium hydroxide. This was kept stirring for a few minutes with mechanical stirrer and then temperature raised to 40 - 45°C. At this stage 1% of fungal protease on the basis of protein content of the raw material was added and stirring continued for VA - 2 h. At the end of the above time interval the temperature of the slurry was raised to 65-70°C for 3 -5 min. The slurry was cooled to room temperature and the insoluble material in the dispersion was removed by centrifugation. The clarified protein hydrolysate was spray dried to obtain protein hydrolysate.
  • casein 200 g was dispersed in 2 L of water and the pH of the dispersion was adjusted to 7.8 using 15% sodium hydroxide (NaOH) the slurry was maintained at 40°C. Fungal protease dissolved in water was added to the slurry. After l ⁇ h. the temperature of the slurry was raised to 65°C for 3 min was cooled and centrifuged. The clear supernatant was spray dried. The product had a degree of hydrolysis of 45% with an yield of 90%.
  • NaOH sodium hydroxide
  • casein 5 kg was s dispersed in 50 L of water and the pH was adjusted to 7.8-8.0 using
  • Example 4 5 kg of casein was dispersed in 50 L of water and the pH of the dispersion was adjusted to 8.0 using 15% NaOH the slurry was maintained at 45°C. Fungal protease dissolved in water was added to the slurry. After 2 h, the temperature of the slurry was raised to 70°C for 5 min., cooled and centrifuged. The clear supernatant was spray dried.
  • the product had a degree of hydrolysis of 50% with an yield of 95% casein protein hydrolysate obtained has creamish white colour with the milky flavour with an yield of 90 % (on protein basis).
  • the product has 11.5 - 12.5% nitrogen, 4.5 - 5% ash content with degree of hydrolysis of 45- 50%.
  • the product is highly soluble in water in all the pHs range.
  • the protein hydrolysate had a perceptible threshold bitterness at 0.4 to 0.5%. It retained all the nutritive value of the starting material.
  • the ratio of water to protein is comparably less and one step pH adjustment, the salt content is minimal.
  • the fungal protease enzyme from fungal origin used is commercially available and very short duration of proteolysis.
  • the yield is very high 90- 95% with higher nitrogen content Of 11.5- 12.5%.

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  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Food Science & Technology (AREA)
  • Health & Medical Sciences (AREA)
  • Nutrition Science (AREA)
  • Biochemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Polymers & Plastics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Coloring Foods And Improving Nutritive Qualities (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

L'invention porte sur un procédé de préparation d'hydrolysat protéique à partir des protéines du lait. Le procédé consiste à traiter les protéines du lait avec de la protéase fongique à un pH de 7,5-8,5, à une température de 40 ? 5° sur une durée comprise entre 30 minutes et 2 heures ; à chauffer ce produit à une température de 65 70° pendant au moins 3 minutes, à séparer le surnageant clarifié par un procédé connu et à sécher la liqueur clarifiée ainsi obtenue afin de fabriquer l'hydrolysat protéique.
PCT/IN2001/000030 2001-03-05 2001-03-05 Procede de preparation d'hydrolysat proteique a partir des proteines du lait WO2002069734A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
PCT/IN2001/000030 WO2002069734A1 (fr) 2001-03-05 2001-03-05 Procede de preparation d'hydrolysat proteique a partir des proteines du lait
CN01823108.XA CN1225185C (zh) 2001-03-05 2001-03-05 由乳蛋白制备蛋白水解物的方法

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Application Number Priority Date Filing Date Title
PCT/IN2001/000030 WO2002069734A1 (fr) 2001-03-05 2001-03-05 Procede de preparation d'hydrolysat proteique a partir des proteines du lait

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008046704A1 (fr) * 2006-10-20 2008-04-24 Nestec S.A. Peptides de structuration de glace d'origine lactique
EP3046967A4 (fr) * 2013-09-16 2017-03-22 Glanbia Nutritionals (Ireland) Plc Procédé permettant d'inhiber la formation d'agrégats pendant l'hydrolyse de protéines

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102283310A (zh) * 2010-06-17 2011-12-21 靳群牛 以酪蛋白为原料酶解生产聚蛋白胨的方法
TWI785719B (zh) * 2021-08-04 2022-12-01 東海大學 豬腎水解產物、其製備方法及其用於治療或預防腎病變之用途
CN116268174B (zh) * 2023-03-10 2024-04-12 华南理工大学 一种低苦味的酪蛋白酶解物及其制备方法与应用

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0065663A1 (fr) * 1981-05-11 1982-12-01 Miles Laboratories, Inc. Méthode de préparation d'un hydrolysat de protéines à partir du petit-lait
FR2541308A1 (en) * 1983-02-22 1984-08-24 Roehm Gmbh Protein hydrolysate free from bitter taste
EP0325986A2 (fr) * 1988-01-28 1989-08-02 Miles Inc. Hydrolyse enzymatique des protéines
WO1994025580A1 (fr) * 1993-04-26 1994-11-10 Novo Nordisk A/S Procede d'hydrolyse de proteines
US5486461A (en) * 1991-11-08 1996-01-23 Novo Nordisk A/S Casein hydrolyzate and method for production of such casein hydrolyzate
WO1996013174A1 (fr) * 1994-10-26 1996-05-09 Novo Nordisk A/S Procede de fabrication d'un hydrolysat de lacto-proteine, hydrolysat de lacto-proteine et son mode d'utilisation
WO1999065326A1 (fr) * 1998-06-17 1999-12-23 New Zealand Dairy Board Hydrolysat de proteine bioactive du petit-lait

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0065663A1 (fr) * 1981-05-11 1982-12-01 Miles Laboratories, Inc. Méthode de préparation d'un hydrolysat de protéines à partir du petit-lait
FR2541308A1 (en) * 1983-02-22 1984-08-24 Roehm Gmbh Protein hydrolysate free from bitter taste
EP0325986A2 (fr) * 1988-01-28 1989-08-02 Miles Inc. Hydrolyse enzymatique des protéines
US5486461A (en) * 1991-11-08 1996-01-23 Novo Nordisk A/S Casein hydrolyzate and method for production of such casein hydrolyzate
WO1994025580A1 (fr) * 1993-04-26 1994-11-10 Novo Nordisk A/S Procede d'hydrolyse de proteines
WO1996013174A1 (fr) * 1994-10-26 1996-05-09 Novo Nordisk A/S Procede de fabrication d'un hydrolysat de lacto-proteine, hydrolysat de lacto-proteine et son mode d'utilisation
WO1999065326A1 (fr) * 1998-06-17 1999-12-23 New Zealand Dairy Board Hydrolysat de proteine bioactive du petit-lait

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
SMYTH M. , FITZGERALD R.: "Relationship between some characteristics of WPC hydrolysates and the enzyme complement in commercially available proteinase preparations", INTERNATIONAL DAIRY JOURNAL, vol. 8, no. 9, 1998, pages 819-827, XP001039997 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008046704A1 (fr) * 2006-10-20 2008-04-24 Nestec S.A. Peptides de structuration de glace d'origine lactique
EP1917865A1 (fr) * 2006-10-20 2008-05-07 Nestec S.A. Peptides structurant la glace d'origine laitière
EP3046967A4 (fr) * 2013-09-16 2017-03-22 Glanbia Nutritionals (Ireland) Plc Procédé permettant d'inhiber la formation d'agrégats pendant l'hydrolyse de protéines

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CN1225185C (zh) 2005-11-02
CN1494384A (zh) 2004-05-05

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