WO1994025580A1 - Procede d'hydrolyse de proteines - Google Patents

Procede d'hydrolyse de proteines Download PDF

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Publication number
WO1994025580A1
WO1994025580A1 PCT/DK1994/000165 DK9400165W WO9425580A1 WO 1994025580 A1 WO1994025580 A1 WO 1994025580A1 DK 9400165 W DK9400165 W DK 9400165W WO 9425580 A1 WO9425580 A1 WO 9425580A1
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WIPO (PCT)
Prior art keywords
protein
proteolytic
preparation
hydrolysis
food product
Prior art date
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PCT/DK1994/000165
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English (en)
Inventor
Per Munk Nielsen
Peter Hvass
Kim Hansen
Gitte Budolfsen
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Novo Nordisk A/S
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Filing date
Publication date
Application filed by Novo Nordisk A/S filed Critical Novo Nordisk A/S
Priority to AU65640/94A priority Critical patent/AU681653B2/en
Priority to KR1019950704686A priority patent/KR960701993A/ko
Priority to EP94913509A priority patent/EP0700433A1/fr
Priority to JP6523763A priority patent/JPH08509366A/ja
Publication of WO1994025580A1 publication Critical patent/WO1994025580A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
    • C12Y301/01003—Triacylglycerol lipase (3.1.1.3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/58—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from fungi
    • C12N9/62—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from fungi from Aspergillus
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
    • A23C9/13—Fermented milk preparations; Treatment using microorganisms or enzymes using additives
    • A23C9/1322—Inorganic compounds; Minerals, including organic salts thereof, oligo-elements; Amino-acids, peptides, protein-hydrolysates or derivatives; Nucleic acids or derivatives; Yeast extract or autolysate; Vitamins; Antibiotics; Bacteriocins
    • 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
    • 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
    • A23J3/342—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes of animal proteins of collagen; of gelatin
    • 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
    • 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
    • A23J3/30—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
    • A23J3/345—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes of animal proteins of blood proteins
    • 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
    • A23J3/346—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes of vegetable proteins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L15/00—Egg products; Preparation or treatment thereof
    • A23L15/25—Addition or treatment with microorganisms or enzymes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
    • A23L27/20—Synthetic spices, flavouring agents or condiments
    • A23L27/21—Synthetic spices, flavouring agents or condiments containing amino acids
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
    • A23L27/20—Synthetic spices, flavouring agents or condiments
    • A23L27/24—Synthetic spices, flavouring agents or condiments prepared by fermentation
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/18—Peptides; Protein hydrolysates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A61K8/645—Proteins of vegetable origin; Derivatives or degradation products thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A61K8/65—Collagen; Gelatin; Keratin; Derivatives or degradation products thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A61K8/66—Enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
    • C12N9/54—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea bacteria being Bacillus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21004—Trypsin (3.4.21.4)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21014—Microbial serine proteases (3.4.21.14)
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q5/00—Preparations for care of the hair
    • A61Q5/02—Preparations for cleaning the hair

Definitions

  • the present invention relates to a method of hydrolysing proteins, a protein hydrolysate obtained by the method, and food and non-food products containing the protein hydrolysate.
  • the present invention provides a method for hydrolysis of proteins by incubation with a proteolytic preparation derived from Aspergrillus oryzae and supplied by Novo Nordisk A/S, Denmark under the tradename FlavourzymeTM.
  • the invention provides protein hydrolysates obtained by the method of the invention.
  • the invention provides food and non-food products comprising a protein hydrolysate of the invention.
  • Fig. 1 shows the degree of hydrolysis (%DH) vs. time s of hydrolysis (hours) achieved by the method of the invention applied to sodium caseinate in the pH range of from pH 5 to pH 9 ( ⁇ : pH 5, A pH 6, O pH 7, D pH 8, • pH 9) ;
  • Fig. 2 shows the degree of hydrolysis (%DH) of soy protein isolate after 22 hours of hydrolysis according to the ⁇ o method of invention ( ⁇ : FlavourzymeTM, A : CorolaseTM 7092, O: CorolaseTM 7093, ⁇ : AlcalaseTM, D: NeutraseTM) ; and
  • Fig. 3 shows the degree of hydrolysis (%DH) of Na- caseinate after 22 hours of hydrolysis according to the method of invention ( ⁇ : FlavourzymeTM, A : CorolaseTM 7092, O: CorolaseTM is 7093, ⁇ : AlcalaseTM, D: NeutraseTM).
  • proteolytic enzyme components each of which may have any of the following approximate molecular weights: 23 kD, 27 kD, 31 kD, 32 kD, 35 kD, 38 kD, 42 kD, 47 kD, 53 kD, and 100 kD.
  • the present invention provides a method
  • proteolytic enzyme preparation which is derived from Aspergillus oryzae and comprises at least five proteolytic components having an approximate molecular weight, respectively, selected from 23 kD, 27 kD, 31 kD, 32 kD, 35 kD, 38 kD, 42 kD, 47 kD, 53 kD, and
  • the protein is incubated with a proteolytic preparation derived from Aspergillus oryzae and comprising at least five proteolytic components having the approximate molecular weights 23 kD, 31 kD, 35 kD, 38. kD, and
  • the molecular weight of the protease components in the proteolytic preparation was determined by using SDS polyacrylamide gel electrophoresis (SDS-PAGE) in a manner known to persons skilled in the art. In this way, the molecular weight of each protease component was determined.
  • the method of the invention is able to perform extensive protein hydrolysis of proteins, and the method leads to non-bitter hydrolysates and hydrolysates having pronounced soup-flavour/meat-flavour.
  • the extent of the protein hydrolysis may be determined by the degree of hydrolysis achieved.
  • the degree of hydrolysis (DH) is defined by the following formula:
  • the DH may be calculated according to Adler-Nissen ;
  • a DH which is higher than 35%, preferably higher than 60%, more preferably higher than 70%, most preferably higher than 80%.
  • the degree of protein solubility may be described by way of a
  • PSI Protein Solubility Index
  • the protein or proteinaceous material which may advantageously be hydrolysed by the present method may be any vegetable protein such as soy protein, grain proteins, e.g. wheat gluten or zein, rape seed protein, alfalfa protein, pea protein, fabaceous bean protein, cotton seed protein or sesame seed protein, or any animal protein or proteinaceous material such as milk protein, whey protein, casein, meat protein, fish protein, blood protein, egg white or gelatin.
  • vegetable protein such as soy protein, grain proteins, e.g. wheat gluten or zein, rape seed protein, alfalfa protein, pea protein, fabaceous bean protein, cotton seed protein or sesame seed protein
  • animal protein or proteinaceous material such as milk protein, whey protein, casein, meat protein, fish protein, blood protein, egg white or gelatin.
  • the proteolytic enzyme may suitably be added to the protein or proteinaceous material in an amount of 0.05-15 AU/100 g of protein, in particular 0.1-8 AU/100 g of protein.
  • the incubation may be performed at a pH from between about 4 and about 10, preferably between about 5 and about 9.
  • the method of the invention performs excellent even at extreme pH conditions, i.e. at pH values in all of the range of 5 to 9.
  • the incubation may be performed at any convenient temperature at which the enzyme preparation does not become inactivated, i.e. in the range of from about 20°C to about 70°C.
  • the proteo ⁇ lytic enzyme preparation may suitably be inactivated by increasing the temperature of the incubation mixture to above about 70°C, or by decreasing the pH of the incubation mixture to below about 4.0.
  • incubation of a protein or proteinaceous substrate may be performed with a combination of FlavourzymeTM and one or more other protease preparations.
  • Preferred protease preparations comprises neutral or alkaline proteases.
  • suitable neutral proteases are neutral proteases derived from Bacillus, preferably from Bacillus ⁇ ubtilis, such as the enzyme preparation supplied by Novo Nordisk, Denmark, under the tradename NeutraseTM.
  • alkaline proteases examples include alkaline proteases derived from Bacillus , preferably from Bacillus licheniformis, such as the enzyme preparation supplied by Novo Nordisk, Denmark, under the tradename AlcalaseTM and containing Subtilisin A (Subtilisin Carlsberg) as the active component.
  • AlcalaseTM alkaline proteases derived from Bacillus , preferably from Bacillus licheniformis, such as the enzyme preparation supplied by Novo Nordisk, Denmark, under the tradename AlcalaseTM and containing Subtilisin A (Subtilisin Carlsberg) as the active component.
  • the incubation carried out in the method of the invention may also be performed with a combination of FlavourzymeTM and one or more other lipase preparations.
  • Preferred lipase preparations comprises fungal lipases.
  • Suitable fungal lipases are lipases derived from Mucor, preferably from .Rhizojnucor miehei , such as the enzyme preparation supplied by Novo Nordisk, Denmark, under the tradename PalataseTMM; and lipases derived from Aspergillus , preferably from Aspergillus niger such as the enzyme preparation supplied by Novo Nordisk, Denmark, under the tradename PalataseTMA.
  • the present invention provides a protein hydrolysate obtained by the method of the invention.
  • the proteolytic activity may be determined with haemoglobin as substrate.
  • TCA trichloroacetic acid
  • a folder AF 4/5 describing the analytical method in more detail is available from Novo Nordisk A/S, DK-2880 Bagsvaerd, Denmark, upon request which folder is hereby included by reference.
  • a substrate for lipase was prepared by emulsifying glycerine tributyrat (MERCK) using gum-arabic as emulsifier. Lipase activity was assayed at pH 7 using pH stat method. One unit of lipase activity (LU/mg) is defined as the amount needed to liberate one micro ole fatty acid per minute.
  • Step 1 Centrifuge the fermentation supernatant, discard the precipitate. Adjust the pH of the supernatant to 7 and add gradually an equal volume of cold 96 % ethanol. Allow the mixture to stand for 30 minutes in an ice bath. Centrifuge and discard the precipitate.
  • Step 2 - Ion exchange chromatography. Filter the supernatant and apply on DEAE-fast flow (Pharmacia TM) column equilibrated with 50 mM tris-acetate buffer pH 7. Wash the column with the same buffer till absorption at 280 nm is lower than 0.05 OD. Elute the bound enzymatic activity with linear salt gradient in the same buffer (0 to 0.5 M NaCl ) using five column volumes, Pool the fractions containing enzymatic activity .
  • Step 3 Hydrophobic chromatography. Adjust the molarity of the pool containing enzymatic activity to 0.8 M by adding solid Ammonium acetate. Apply the enzyme on TSK gel Butyl- Toyopearl 650 C column (available from Tosoh Corporation Japan) which was pre-equilibrated with 0.8 M ammonium acetate. Wash the unbound material with 0.8 M ammonium acetate and elute the bound material with distilled water.
  • Step 4 Pool containing lipase activity is diluted with water to adjust conductance to 2 mS and pH to 7. Apply the pool on High performance Q Sepharose (Pharmacia) column pre- equilibrated with 50 mM tris -acetate buffer pH 7. Elute the bound enzyme with linear salt gradient.
  • the present invention relates to food products comprising a protein hydrolysate of the inven ⁇ tion.
  • the amount of protein hydrolysate incorporated in the food product will typically be in the range of 1-30% by weight.
  • An important food product of the present invention is an ingredient of a mother milk substitute for infants. Due to the high degree of hydrolysis obtained by the method of the invention, the protein hydrolysates of the invention may advan- tageously be incorporated in mother milk substitutes, the hydrolysate having a significantly lower allergenicity than unhydrolysed milk proteins.
  • the milk substitute may be for ⁇ mulated in substantially the same way as that indicated in the prior literature for products of this type (cf. for instance EP Patent Application 322,589) with the exception that the protein hydrolysate included in the known products is replaced by the protein hydrolysate of the present invention.
  • the food product of the invention may also include the protein hydrolysate of the invention as a protein supple- ment or to provide other properties of the food product.
  • the protein hydrolysate incorporated in the food product may for instance be based on meat or scrap meat (e.g. the so-called mechanically recovered meat, i.e. meat remaining on bones after the regular pieces of meat have been carved from animal carcases in the slaughterhouse.
  • meat or scrap meat e.g. the so-called mechanically recovered meat, i.e. meat remaining on bones after the regular pieces of meat have been carved from animal carcases in the slaughterhouse.
  • meat or scrap meat e.g. the so-called mechanically recovered meat, i.e. meat remaining on bones after the regular pieces of meat have been carved from animal carcases in the slaughterhouse.
  • mechanically recovered meat i.e. meat remaining on bones after the regular pieces of meat have been carved from animal carcases in the slaughterhouse.
  • Other proteinaceous by-products from the meat industry may
  • the resulting protein hydrolysate may then suitably be added to emulsified meat products, e.g. sausages or pates, or as flavour ingredients in soups or other food products.
  • a food product obtained from cow milk by the method of invention is preferably a cheese flavour product.
  • the extensive hydrolysis of the milk protein surprisingly leads to flavour compounds with very distinct cheese flavour.
  • Such cheese flavour products of the invention preferably find application in snack products, imitation cheese products, or as enhancers of cheese flavour in general.
  • the cheese flavour may be adjusted by use of lipases according to well established practice.
  • a traditional way of producing Hydrolysed Vegetable Protein (HVP) for use as flavour products is based on cooking in hydrochloric acid at a high temperature and for a long time. This is known to cause unwanted formation of chloric containing compounds. By the process of the invention it is now possible to obtain enzymatically produced HVP products, which are considered more healthy products.
  • the high DH obtainable by the process of the invention leads to desirable flavour characteristics and flavour enhancing properties.
  • the process of the invention may also be used for protein enrichment of dietetic products due to the non-bitter flavour obtained by the process.
  • Also advantageous is the very high protein solubility in a very broad pH range that is associated with the process of the invention. It has surprisingly been found that an excellent fermentation medium may be produced by subjecting milk to the method of the present invention. Such milk hydrolysates may be used for acceleration of the fermentation in yoghurt production or in the production of lactic acid starter bacteria cultures, by addition in relatively small amounts. The shortening of process time increases production capacity and reduces the risk of infection, e.g. bacteriophage infection.
  • the method of the invention may be used in combination with a fermentation process, preferably a fermentation process for the production of a food product.
  • the proteolytic enzyme preparation e.g. FlavourzymeTM
  • the protein hydrolysate of the invention or both may be added in a fermentation process, preferably a food fermentation process, for enhancing the productivity of the fermentation process.
  • fermentation processes are processes involving or fermenting fish (fish sauce) , cocoa beans or soy (such as soy sauce, tempeh, iso) .
  • the addition of the proteolytic enzyme preparation or the addition of protein hydrolysate may be useful for decreasing the total process time, i.e. increasing the rate of fermentation.
  • Non-food products e.g. FlavourzymeTM
  • the process of the invention may also find application in the non-food area.
  • the flavour characteristics obtained by the method of invention may advantageously be used for production 5 of pet food.
  • Preparation B 1% FlavourzymeTM, 2% NeutraseTM 0.5L, and 0.15% AlcalaseTM 2,4L.
  • FlavourzymeTM is a proteolytic preparation derived from
  • NeutraseTM 0.5L is a proteolytic preparation derived from Bacillus subtilis
  • AlcalaseTM 2.4L is a proteolytic preparation derived from Bacillus licheniformis .
  • hydrolysates were served before a tasting panel in a 4% solution.
  • the meat flavour was most pronounced in hydrolysates obtained with Preparation A.
  • flavour-samples 1, 2, and 3 were now diluted in cream cheese, and a fourth sample was prepared as a control.
  • sample No. 4.1 6.0 g of fresh milk + 20 g of cheese.
  • Sample No. 1.1 (FlavourzymeTM) appeared to have a very strong and intense flavour and a taste of well ripened cheese.
  • Sample No. 2.1 (FlavourzymeTM + PalataseTM) appeared to have a 0 well balanced cheese flavour.
  • sample No. 3.1 turned out to give the weakest taste impression.
  • sample No. 4.1 was evaluated being sour/sourish.
  • PalataseTM creates a new and much more simple way of producing cheese flavour.
  • the hydrolysis was carried out for 20 hours at 50°C. Then the enzymes were denatured by heat treatment at 80°C for 5 min.
  • Skim milk was heat treated at 90°C for 2 min and three samples of 200 ml were prepared.
  • the temperature was adjusted to 41°C, and yoghurt culture YC DVS from Chr. Hansens Lab. was added to all three samples in a level of 10 6 bacteria per g.

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Abstract

L'invention concerne un procédé d'hydrolyse d'une protéine végétale ou animale par incubation avec une préparation à base d'enzymes protéolytiques dérivée de l'Aspergillus oryzae telle que la FlavourzymeTM comprenant au moins cinq composés protéolytiques présentant une masse molaire approximative de 23 kD, 27 kD, 31 kD, 32 kD, 38 kD, 42 kD, 47 kD, 53 kD ou de 100 kD, qui permet de produire un hydrolysat protéique utile dans ou comme un produit alimentaire tel que le lait maternel de substitution, le fromage, la protéine végétale hydrolysée, un aromatisant et un adjuvant de procédé pour la fermentation de produits alimentaires ou un produit non alimentaire tel qu'un aliment pour animaux familiers, des cosmétiques. Le procédé selon l'invention permet d'obtenir un degré d'hydrolyse et de goût élevés et solubilité protéique élevée.
PCT/DK1994/000165 1993-04-26 1994-04-25 Procede d'hydrolyse de proteines WO1994025580A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
AU65640/94A AU681653B2 (en) 1993-04-26 1994-04-25 A method for hydrolysing proteins
KR1019950704686A KR960701993A (ko) 1993-04-26 1994-04-25 단백질의 가수분해 방법(a method for hydrolysing proteins)
EP94913509A EP0700433A1 (fr) 1993-04-26 1994-04-25 Procede d'hydrolyse de proteines
JP6523763A JPH08509366A (ja) 1993-04-26 1994-04-25 タンパク質の加水分解方法

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DK93467A DK46793D0 (da) 1993-04-26 1993-04-26 Enzym
DK0467/93 1993-04-26

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DK (1) DK46793D0 (fr)
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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
WO1998018343A1 (fr) * 1996-10-30 1998-05-07 Novo Nordisk A/S Procede de preparation d'un agent aromatique alimentaire
WO1998018342A1 (fr) * 1996-10-30 1998-05-07 Novo Nordisk A/S Procede de preparation d'un agent aromatisant alimentaire
WO1998027828A1 (fr) * 1996-12-23 1998-07-02 Dsm N.V. Renforçateur de gout
WO1998027827A1 (fr) * 1996-12-23 1998-07-02 Dsm N.V. Procede de production d'un hydrolysat proteique
WO1998051163A2 (fr) * 1997-05-16 1998-11-19 Novo Nordisk Biotech, Inc. Procedes de production d'hydrolysats proteiques
WO1998051803A1 (fr) 1997-05-16 1998-11-19 Novo Nordisk Biotech, Inc. Polypeptides a activite de prolyl dipeptidyl aminopeptidase et acides nucleiques codant ces derniers
US5985337A (en) * 1996-08-12 1999-11-16 Cpc International Inc. Process for preparing a protein hydrolysate from protein containing animal products
US6007851A (en) * 1996-12-23 1999-12-28 Gist-Brocades, B.V. Process for producing a flavor enhancer
EP1149902A1 (fr) * 2000-04-28 2001-10-31 Sumitomo Rubber Industries, Ltd. Agent de déprotéinisation, caoutchouc naturel déprotéiné avec celui-ci et procédé pour la production d'articles en caoutchouc
WO2002001963A2 (fr) * 2000-06-23 2002-01-10 Societe Des Produits Nestle S.A. Preparation de biohydrolysats
KR20020026640A (ko) * 2000-10-02 2002-04-12 김강권 항원성이 저감된 카제인 가수분해물의 제조방법 및 이가수분해물을 함유하는 유제품
US6372452B1 (en) * 1996-11-29 2002-04-16 Consejo Superior De Investigaciones Cientificas Process for obtaining plant peptones with a high hydrolysis degree and applications thereof
WO2002069734A1 (fr) * 2001-03-05 2002-09-12 Council Of Scientific And Industrial Research Procede de preparation d'hydrolysat proteique a partir des proteines du lait
WO2002069732A1 (fr) * 2001-03-05 2002-09-12 Council Of Scientific And Industrial Research Procede de preparation d'hydrolysat de proteine a partir de farine de soja
US6495342B2 (en) 2000-02-04 2002-12-17 Roquette Freres Nitrogenous composition resulting from the hydrolysis of maize gluten and a process for the preparation thereof
KR20030028089A (ko) * 2001-09-27 2003-04-08 주식회사 태훈바이오 홍게 가공부산물을 이용한 반응향미제 및 이의 제조방법
WO2003054186A1 (fr) * 2001-12-21 2003-07-03 Dsm Ip Assets B.V. Nouvelles presures
FR2836014A1 (fr) * 2002-02-15 2003-08-22 Gervais Danone Sa Nouveau procede de fabrication de produits laitiers fermentes mettant en oeuvre des enzymes d'origine bacterienne
US6787168B1 (en) * 2003-03-18 2004-09-07 James W. Sawhill Peptide product
WO2004098309A1 (fr) * 2003-05-05 2004-11-18 Unilever N.V. Produit a base de caseine hydrolysee comportant des tripeptides ipp et/ou vpp
WO2005010170A2 (fr) * 2003-07-25 2005-02-03 Centro Sperimentale Del Latte S.P.A. Milieu de fermentation a base d'un substrat proteique fortement hydrolyse
US6875456B2 (en) 2000-10-19 2005-04-05 Dsm Ip Assets B.V. Protein hydrolysates
EP1553913A1 (fr) * 2002-06-14 2005-07-20 Carol J. Buck Compositions et procedes pour adoucir, fluidifier et enlever le tissu hyperkeratosique
EP1669463A1 (fr) * 2003-08-01 2006-06-14 Calpis Co., Ltd. Hydrolysat de caseine et son procede de production et d'utilisation
US7070953B1 (en) 1999-10-20 2006-07-04 Nordur Ehf Protein hydrolysates produced with the use of cod proteases
WO2006128685A3 (fr) * 2005-05-31 2007-04-19 Gelita Ag Procede de fabrication d'hydrolysat de gelatine a faible poids moleculaire et compositions d'hydrolysat de gelatine
WO2007066841A1 (fr) * 2005-12-09 2007-06-14 Serombio Co., Ltd. Hydrolisat de levure renfermant du cyclo-his-pro et procede de production afferent
WO2007086762A1 (fr) * 2006-01-24 2007-08-02 Vital Food Processors Limited Récupération enzymatique d'une protéine
FR2925325A1 (fr) * 2007-12-21 2009-06-26 Vincience Sa Utilisation d'un hydrolysat de colza en tant que principe actif activateur de la synthese des aquaporines
WO2009147105A2 (fr) * 2008-06-03 2009-12-10 Novozymes A/S Procédé de fabrication d'un hydrolysat de caséine
WO2009147103A2 (fr) * 2008-06-03 2009-12-10 Novozymes A/S Procédé de fabrication d'un hydrolysat de protéine de blé
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WO2013050407A1 (fr) * 2011-10-03 2013-04-11 Specialites Pet Food Hydrolysats à base de tissu carné présentant une meilleure appétibilité utilisés dans des aliments pour animaux
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Publication number Priority date Publication date Assignee Title
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
US5985337A (en) * 1996-08-12 1999-11-16 Cpc International Inc. Process for preparing a protein hydrolysate from protein containing animal products
CN1067859C (zh) * 1996-10-30 2001-07-04 诺沃挪第克公司 生产食品调味剂的方法
WO1998018343A1 (fr) * 1996-10-30 1998-05-07 Novo Nordisk A/S Procede de preparation d'un agent aromatique alimentaire
WO1998018342A1 (fr) * 1996-10-30 1998-05-07 Novo Nordisk A/S Procede de preparation d'un agent aromatisant alimentaire
US6024990A (en) * 1996-10-30 2000-02-15 Novo Nordisk A/S Method of producing food flavoring agent
US6372452B1 (en) * 1996-11-29 2002-04-16 Consejo Superior De Investigaciones Cientificas Process for obtaining plant peptones with a high hydrolysis degree and applications thereof
US6190709B1 (en) * 1996-12-23 2001-02-20 Gist-Brocades B.V. Flavor enhancer
WO1998027827A1 (fr) * 1996-12-23 1998-07-02 Dsm N.V. Procede de production d'un hydrolysat proteique
WO1998027828A1 (fr) * 1996-12-23 1998-07-02 Dsm N.V. Renforçateur de gout
US6007851A (en) * 1996-12-23 1999-12-28 Gist-Brocades, B.V. Process for producing a flavor enhancer
WO1998051803A1 (fr) 1997-05-16 1998-11-19 Novo Nordisk Biotech, Inc. Polypeptides a activite de prolyl dipeptidyl aminopeptidase et acides nucleiques codant ces derniers
US6465209B1 (en) 1997-05-16 2002-10-15 Novozymes Biotech, Inc. Methods of producing protein hydrolysates
US6664092B1 (en) * 1997-05-16 2003-12-16 Novozymes Biotech, Inc. Polypeptides having dipeptidyl aminopeptidase activity and nucleic acids encoding same
WO1998051163A3 (fr) * 1997-05-16 1999-02-04 Novo Nordisk Biotech Inc Procedes de production d'hydrolysats proteiques
WO1998051163A2 (fr) * 1997-05-16 1998-11-19 Novo Nordisk Biotech, Inc. Procedes de production d'hydrolysats proteiques
US7070953B1 (en) 1999-10-20 2006-07-04 Nordur Ehf Protein hydrolysates produced with the use of cod proteases
US6495342B2 (en) 2000-02-04 2002-12-17 Roquette Freres Nitrogenous composition resulting from the hydrolysis of maize gluten and a process for the preparation thereof
US6544791B2 (en) 2000-02-04 2003-04-08 Roquette Freres Nitrogenous composition resulting from the hydrolysis of maize gluten and a process for the preparation thereof
EP1149902A1 (fr) * 2000-04-28 2001-10-31 Sumitomo Rubber Industries, Ltd. Agent de déprotéinisation, caoutchouc naturel déprotéiné avec celui-ci et procédé pour la production d'articles en caoutchouc
US7015264B2 (en) 2000-04-28 2006-03-21 Sumitomo Rubber Industries, Ldt. Deproteinizing agent, deproteinized natural rubber latex using the same, and method of producing rubber product
WO2002001963A3 (fr) * 2000-06-23 2002-07-25 Nestle Sa Preparation de biohydrolysats
WO2002001963A2 (fr) * 2000-06-23 2002-01-10 Societe Des Produits Nestle S.A. Preparation de biohydrolysats
KR20020026640A (ko) * 2000-10-02 2002-04-12 김강권 항원성이 저감된 카제인 가수분해물의 제조방법 및 이가수분해물을 함유하는 유제품
US6875456B2 (en) 2000-10-19 2005-04-05 Dsm Ip Assets B.V. Protein hydrolysates
WO2002069734A1 (fr) * 2001-03-05 2002-09-12 Council Of Scientific And Industrial Research Procede de preparation d'hydrolysat proteique a partir des proteines du lait
WO2002069732A1 (fr) * 2001-03-05 2002-09-12 Council Of Scientific And Industrial Research Procede de preparation d'hydrolysat de proteine a partir de farine de soja
KR20030028089A (ko) * 2001-09-27 2003-04-08 주식회사 태훈바이오 홍게 가공부산물을 이용한 반응향미제 및 이의 제조방법
WO2003054186A1 (fr) * 2001-12-21 2003-07-03 Dsm Ip Assets B.V. Nouvelles presures
US7404977B2 (en) 2001-12-21 2008-07-29 Dsm Ip Assets B.V. Rennets
FR2836014A1 (fr) * 2002-02-15 2003-08-22 Gervais Danone Sa Nouveau procede de fabrication de produits laitiers fermentes mettant en oeuvre des enzymes d'origine bacterienne
EP1553913A1 (fr) * 2002-06-14 2005-07-20 Carol J. Buck Compositions et procedes pour adoucir, fluidifier et enlever le tissu hyperkeratosique
EP1553913A4 (fr) * 2002-06-14 2007-01-10 Carol J Buck Compositions et procedes pour adoucir, fluidifier et enlever le tissu hyperkeratosique
US6787168B1 (en) * 2003-03-18 2004-09-07 James W. Sawhill Peptide product
AU2004237455B2 (en) * 2003-05-05 2007-08-02 Dsm Ip Assets B.V. Hydrolysed casein product comprising tripeptides IPP and/ or VPP
US7785824B2 (en) 2003-05-05 2010-08-31 Conopco Inc. Hydrolysed casein product comprising tripeptides IPP and/or VPP
WO2004098309A1 (fr) * 2003-05-05 2004-11-18 Unilever N.V. Produit a base de caseine hydrolysee comportant des tripeptides ipp et/ou vpp
WO2005010170A3 (fr) * 2003-07-25 2006-05-18 Ct Sperimentale Del Latte S P Milieu de fermentation a base d'un substrat proteique fortement hydrolyse
WO2005010170A2 (fr) * 2003-07-25 2005-02-03 Centro Sperimentale Del Latte S.P.A. Milieu de fermentation a base d'un substrat proteique fortement hydrolyse
EP1669463A1 (fr) * 2003-08-01 2006-06-14 Calpis Co., Ltd. Hydrolysat de caseine et son procede de production et d'utilisation
EP1669463A4 (fr) * 2003-08-01 2006-08-23 Calpis Co Ltd Hydrolysat de caseine et son procede de production et d'utilisation
US8580557B2 (en) 2003-08-01 2013-11-12 Calpis Co., Ltd. Casein hydrolyzate, process for producing the same and use thereof
WO2006128685A3 (fr) * 2005-05-31 2007-04-19 Gelita Ag Procede de fabrication d'hydrolysat de gelatine a faible poids moleculaire et compositions d'hydrolysat de gelatine
US7485323B2 (en) 2005-05-31 2009-02-03 Gelita Ag Process for making a low molecular weight gelatine hydrolysate and gelatine hydrolysate compositions
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AU681653B2 (en) 1997-09-04
NZ265247A (en) 1996-07-26
AU6564094A (en) 1994-11-21
CN1090675C (zh) 2002-09-11
JPH08509366A (ja) 1996-10-08
DK46793D0 (da) 1993-04-26
CN1121732A (zh) 1996-05-01
EP0700433A1 (fr) 1996-03-13
KR960701993A (ko) 1996-03-28

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