WO2009150888A1 - Produit de dégradation de la protéine du lait, procédé de fabrication du produit de dégradation de la protéine du lait et agent promoteur de la croissance bifidobactérienne - Google Patents

Produit de dégradation de la protéine du lait, procédé de fabrication du produit de dégradation de la protéine du lait et agent promoteur de la croissance bifidobactérienne Download PDF

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WO2009150888A1
WO2009150888A1 PCT/JP2009/056803 JP2009056803W WO2009150888A1 WO 2009150888 A1 WO2009150888 A1 WO 2009150888A1 JP 2009056803 W JP2009056803 W JP 2009056803W WO 2009150888 A1 WO2009150888 A1 WO 2009150888A1
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milk protein
degradation product
protein degradation
lactic acid
bifidobacteria
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PCT/JP2009/056803
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English (en)
Japanese (ja)
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金忠 清水
寿美子 米澤
浩 越智
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森永乳業株式会社
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Priority to JP2010516787A priority Critical patent/JPWO2009150888A1/ja
Publication of WO2009150888A1 publication Critical patent/WO2009150888A1/fr

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    • 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
    • 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
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/20—Animal feeding-stuffs from material of animal origin
    • A23K10/26—Animal feeding-stuffs from material of animal origin from waste material, e.g. feathers, bones or skin
    • A23K10/28—Animal feeding-stuffs from material of animal origin from waste material, e.g. feathers, bones or skin from waste dairy products
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/142—Amino acids; Derivatives thereof
    • A23K20/147—Polymeric derivatives, e.g. peptides or 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
    • 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
    • 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
    • A23L33/19—Dairy proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/20—Milk; Whey; Colostrum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/14—Prodigestives, e.g. acids, enzymes, appetite stimulants, antidyspeptics, tonics, antiflatulents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • 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)
    • C12Y304/21096—Lactocepin (3.4.21.96)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
    • Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
    • Y02P60/87—Re-use of by-products of food processing for fodder production

Definitions

  • the present invention uses a living lactic acid bacterium having a cell wall-enhanced proteinase (cell wall-enveloped proteinase, PrtP), a disrupted microbial cell of the lactic acid bacterium, an enzyme fraction fractionated from the lactic acid bacterium, and the like.
  • the present invention relates to a protein degradation product to be obtained, a method for producing the protein degradation product, and a bifidobacteria growth promoter comprising the protein degradation product as an active ingredient.
  • Bifidobacterium spp. That is, bifidobacteria, is one of the dominant fungal species of the intestinal flora formed in the human intestinal tract, the intestinal regulating action to restore the balance of intestinal bacteria, It is known to have an immunopotentiating action, a carcinogenesis suppressing action, and the like. For this reason, in recent years, the demand for foods containing living bifidobacteria such as fermented bifidobacteria has increased along with an increase in the health orientation of consumers.
  • Bifidobacteria have poor growth in milky media. For this reason, in order to contain a certain amount of, for example, 1 ⁇ 10 7 CFU / mL bifidobacteria in the fermented milk, usually, various growth promoting substances such as yeast extract are added. However, the growth promoting substance is generally expensive and the flavor may be impaired. For this reason, development of a growth promoter for bifidobacteria that is less expensive and has less influence on the flavor has been demanded.
  • a peptide obtained by pepsin treatment of human milk has a bifidobacteria growth promoting action, and in particular, a pepsin degradation product of human protein lactoferrin has a bifidobacterial growth promoting action.
  • a bifidobacterial growth promoter characterized by comprising (2) actomyosin or a peptide-containing fraction obtained by treating a meat protein suspension containing actomyosin with a protease.
  • actomyosin or a peptide-containing fraction obtained by treating a meat protein suspension containing actomyosin with a protease.
  • the peptide of the above (1) is a protein contained in human milk or human milk, it is difficult to secure the raw material and is not very suitable for industrial use such as addition to food and drink.
  • the bifidobacteria growth promoter of (2) above has no particular problem in securing the raw material because the raw material is meat protein, but since the peptide can be an allergen, it is originally derived from meat such as bifidobacteria fermented milk. It is not preferable to add this bifidobacterial growth promoter to foods and drinks not using protein as a raw material from the viewpoint of food allergy.
  • the present invention is a milk protein degradation product which is excellent in mass productivity and can promote the growth of bifidobacteria without requiring a protein source other than a milk-derived material which is a main culture raw material of bifidobacteria, and the milk protein It aims at providing the manufacturing method of a degradation product, and the bifidobacteria growth promoter which uses this milk protein degradation product as an active ingredient.
  • the present inventors have found that a living lactic acid bacterium having a cell wall-localized proteolytic enzyme PrtP represented by Lactococcus lactis or a microbial cell of the lactic acid bacterium.
  • the present inventors have found that a crushed product and a milk protein degradation product from a cell wall-localized proteolytic enzyme PrtP fraction fractionated from the lactic acid bacteria have a bifidobacteria growth-promoting action, thereby completing the present invention.
  • the present invention provides a living cell of lactic acid bacteria having cell wall-enveloped proteinase (cell wall-enveloped proteinase, PrtP), a crushed cell of the lactic acid bacteria, and the enzyme fraction fractionated from the lactic acid bacteria.
  • the present invention provides a milk protein hydrolyzate obtained by hydrolyzing milk protein using one or more selected from the group consisting of:
  • the present invention also provides the milk protein degradation product as described above, wherein the lactic acid bacterium is Lactococcus lactis.
  • the present invention also provides the milk protein degradation product as described above, wherein the Lactococcus lactis does not have xylose utilization and does not produce diacetyl and acetoin.
  • the present invention provides the milk protein degradation product according to any one of the above, wherein the milk protein is casein or total milk protein.
  • the hydrolysis is performed under the conditions of 20 to 37 ° C. and a milk protein concentration of 0.5% by weight or more using a milk protein adjusted to pH 6.0 to 8.0 as a substrate.
  • the milk protein degradation product according to any one of the above-mentioned features is provided.
  • the present invention also provides a living cell of lactic acid bacteria having cell wall-enhanced proteinase (cell wall-enveloped proteinase, PrtP), a crushed cell of the lactic acid bacteria, and the enzyme fraction fractionated from the lactic acid bacteria.
  • the present invention provides a method for producing a milk protein hydrolyzate, which comprises hydrolyzing milk protein using one or more selected from the group consisting of:
  • the present invention also provides the method for producing a milk protein degradation product as described above, wherein the lactic acid bacterium is Lactococcus lactis. Further, the present invention provides the method for producing a milk protein degradation product as described above, wherein the Lactococcus lactis has no xylose utilization property and does not produce diacetyl and acetoin. It is.
  • the present invention also provides the method for producing a milk protein degradation product according to any one of the above, wherein the milk protein is casein or total milk protein. In the present invention, the hydrolysis is performed under the conditions of 20 to 37 ° C.
  • the present invention provides a method for producing a milk protein degradation product according to any one of the above. Moreover, this invention provides the bifidobacteria growth promoter which uses the milk protein degradation product in any one of the said description as an active ingredient. The present invention also provides the aforementioned bifidobacteria growth promoter, wherein the bifidobacteria is Bifidobacterium longum.
  • the Bifidobacterium longum strain is the Bifidobacterium longum ATCC BAA-999 strain and / or the Bifidobacterium longum type strain ATCC15700 strain. An agent is provided.
  • the milk protein degradation product of the present invention is obtained by hydrolyzing milk protein with a cell wall-localized proteolytic enzyme PrtP derived from lactic acid bacteria, so that it is excellent in mass productivity and manufactured at a relatively low cost. Is also possible. Further, the bifidobacteria growth promoter of the present invention is unprecedented and can grow bifidobacteria, particularly Bifidobacterium longum. Moreover, since the milk protein degradation product which is an active ingredient is a milk-derived substance, the bifidobacteria growth promoter of the present invention is also applied to foods and drinks produced by culturing bifidobacteria with milk-derived raw materials such as fermented milk. It can be used without having to consider protein allergies other than milk protein, and is useful in designing dairy products such as fermented milk.
  • the milk protein degradation product of the present invention includes a living lactic acid bacterium having a cell wall-localized proteolytic enzyme PrtP (hereinafter sometimes simply referred to as “PrtP enzyme”), a crushed cell product of the lactic acid bacterium, and It is obtained by hydrolyzing milk protein using one or more selected from the group consisting of the enzyme fractions fractionated from the lactic acid bacteria. Since the milk protein degradation product of the present invention has a bifidobacteria growth promoting activity, an excellent bifidobacteria growth promoter having a high bifidobacteria growth promoting effect can be obtained by using the milk protein degradation product of the present invention as an active ingredient. Can be obtained. The reason why the milk protein degradation product of the present invention exerts the bifidobacteria growth-promoting effect is not clear. It is guessed that there is not.
  • the lactic acid bacterium used in the present invention has a PrtP enzyme.
  • the PrtP enzyme is an enzyme that exists in the cell membrane and has an active site exposed on the cell surface.
  • Examples of lactic acid bacteria having a PrtP enzyme include Lactococcus lactis subspices cremoris and Lactococcus lactis subspice lactos. In the genus, several strains having PrtP have been reported.
  • PrtP enzymes derived from Lactococcus lactis include P type I (which does not degrade ⁇ -casein very much and ⁇ -casein is degraded well from the vicinity of the C-terminal), type P III ( ⁇ -casein and ⁇ -casein is well resolved from both C-terminus and N-terminus) and its intermediate form (P I / P III type) (eg, Reid, JR. et al., Applied and Environmental Microbiology, 1994).
  • PrtP enzymes derived from Lactococcus lactis include PrtP enzymes whose gene sequences are registered in NCBI (National Center for Biotechnology Information) as accession numbers AY542690, AY5422691, and the like.
  • a certain lactic acid bacterium is a PrtP enzyme-containing lactic acid bacterium (lactic acid bacterium having a PrtP enzyme) has, for example, a PrtP gene encoding a PrtP enzyme using a gene analysis technique such as PCR (Polymerase Chain Reaction). It can be confirmed by examining whether or not.
  • the PrtP enzyme Since the PrtP enzyme has an enzyme active site outside the cell, it can degrade proteins in the medium. For example, when a lactic acid bacterium grows in a milky medium, the PrtP enzyme degrades milk protein in the milky medium, and oligopeptides and amino acids necessary for the growth of the lactic acid bacterium are provided. Therefore, Lactococcus lactis with PrtP enzyme grows so that the medium can be solidified when cultured in a 10% (W / W) reduced skim milk medium at a temperature range of 25-30 ° C. for 16 hours. It is fast and has strong fermentability. PrtP enzyme-containing lactic acid bacteria can also be detected by utilizing such characteristics of high growth and fermentability. In addition, PrtP enzyme-containing lactic acid bacteria can also be detected by detecting PrtP enzyme activity.
  • the lactic acid bacterium used in the present invention is not particularly limited as long as it has a PrtP enzyme, but is preferably a bacterium belonging to the genus Lactococcus, more preferably Lactococcus lactis, and Lactococcus lactis. -Subspecies Cremolis and Lactococcus lactis subspecies lactis are more preferable. This is because dairy products made from bifidobacteria such as fermented milk have been used as raw materials and are considered to be highly safe. Examples of Lactococcus lactis having PrtP enzyme include Lactococcus lactis subspices cremolith NBRC100676 T strain, Lactococcus lactis subspices lactis JCM20101 strain and the like.
  • the medium used for the preculture of the PrtP enzyme-containing lactic acid bacteria is not particularly limited as long as it is a medium that is usually used for culturing Lactococcus lactis, but Difco (registered trademark) M17 Broth (Becton And a commercially available semi-synthetic medium such as Dickinson) or a dairy medium such as a reduced skim milk medium.
  • growth promoting substances such as glucose and yeast extract, reducing agents such as L-cysteine, and the like can be added to the medium used for preculture.
  • the culture medium used for preculture uses what sterilized.
  • the sterilization treatment can be performed by a commonly used method, for example, by heat treatment at 80 to 122 ° C. for 5 to 40 minutes, preferably 85 to 95 ° C. for 5 to 35 minutes.
  • the one used for the hydrolysis of milk protein is not particularly limited as long as the enzyme activity of the PrtP enzyme is maintained, but the live cell of the PrtP enzyme-containing lactic acid bacterium, It is preferable that it is the crushed cell body of lactic acid bacteria, and the PrtP enzyme fraction fractionated from the said lactic acid bacteria.
  • the live cell of the PrtP enzyme-containing lactic acid bacterium used in the present invention may be a lactic acid bacterium cultured by a conventional method as it is, or may be a dry bacterium powder prepared by a conventional method such as lyophilization.
  • the cultured lactic acid bacteria may be used together with the medium used for the culture, or may be one obtained by collecting only the cells from the medium by centrifugation or the like and then suspending them in an appropriate buffer or the like. Since it is possible to remove excess medium and the concentration can be adjusted by concentration or dilution, it is preferable to use a suspension of cells recovered from the medium in a buffer.
  • the buffer for suspending the cells recovered from the medium is not particularly limited as long as it is a solvent that can suspend lactic acid bacteria in a live state, and is usually used for suspending lactic acid bacteria. Although a buffer etc. can be used, it is preferable that it is a buffer containing calcium ion.
  • the pH of the buffer used for the suspension is preferably 4.5 to 8.0. Examples of the buffer include MES buffer, HEPES buffer, and phosphate buffer. In addition, physiological saline may be used.
  • the cell disruption product of the PrtP enzyme-containing lactic acid bacterium used in the present invention is not particularly limited as long as it is obtained by crushing the lactic acid bacterium without impairing the enzyme activity of the PrtP enzyme.
  • a crushing method include a crushing process using ultrasonic waves, a glass bead crushing process, and a crushing process using osmotic shock. These crushing treatments are preferably performed at a low temperature of 50 ° C. or lower, preferably room temperature or lower, more preferably 10 ° C. or lower. This is because the PrtP enzyme is deactivated by heating.
  • the PrtP enzyme fraction fractionated from the PrtP enzyme-containing lactic acid bacterium used in the present invention is obtained by fractionating the PrtP enzyme from the lactic acid bacterium without impairing the enzyme activity of the PrtP enzyme, It is not particularly limited. For example, after culturing the lactic acid bacteria collected from the culture medium after suspending in a buffer, removing the bacterial cells by centrifugation or the like, the supernatant containing the PrtP enzyme released from the cell surface of the lactic acid bacteria, It can be obtained as a PrtP enzyme fraction.
  • a chelating agent such as EDTA
  • a buffer to which a chelating agent such as EDTA is added As a buffer for obtaining such a supernatant, for example, a buffer obtained by adding a chelating agent such as EDTA to the above-mentioned buffers that can be used for suspending viable cells can be used.
  • the temperature at which lactic acid bacteria are retained in the buffer is not particularly limited as long as it is less than 50 ° C., but is preferably about 4 to 37 ° C., more preferably about 30 ° C.
  • the holding time can be appropriately determined in consideration of the holding temperature, the type of buffer, etc., but is preferably within 1 hour, more preferably 5 to 30 minutes, and 5 to 15 minutes. More preferably it is.
  • the chelating agent can be removed from the PrtP enzyme fraction by dialysis of the supernatant thus obtained.
  • the fractionated PrtP enzyme fraction can be concentrated by a known technique such as freeze concentration without impairing the PrtP enzyme activity.
  • it may be a PrtP enzyme fraction obtained by fractionating a crushed bacterial body of lactic acid bacteria using a known fractionation means such as ammonium sulfate precipitation method or chromatography method.
  • the milk protein used in the present invention is not particularly limited as long as it is a milk-derived protein collected from animals such as cows, goats, horses and sheep (excluding humans), and is usually used for food. Milk-derived protein can be appropriately selected and used. In this invention, it is preferable that it is the milk protein contained in the milk with high industrial utility value.
  • the milk protein may be casein, whey protein (protein contained in whey), or total milk protein.
  • the milk protein used in the present invention is preferably casein derived from milk or total milk protein, and more preferably casein derived from milk. This is because a milk protein degradation product having a higher bifidobacteria growth promoting effect can be obtained.
  • milk proteins such as casein, total milk protein, and whey protein, those prepared by a conventional method can be used. Moreover, you may use what is marketed.
  • the milk protein degradation product of the present invention is one or more selected from the group consisting of a milk protein, a living cell of a PrtP enzyme-containing lactic acid bacterium, a crushed cell of the lactic acid bacterium, and a PrtP enzyme fraction fractionated from the lactic acid bacterium. It is obtained by adding and hydrolyzing.
  • the added PrtP enzyme-containing lactic acid bacteria live cells, crushed cell bodies, and PrtP enzyme fractions are preferably added as prepared by appropriately freeze-concentrating or lyophilizing those prepared as described above.
  • the hydrolysis reaction conditions are not particularly limited as long as the enzymatic reaction can be performed by the PrtP enzyme, and may be appropriately determined in consideration of the type of PrtP enzyme, the type of milk protein, and the like. it can.
  • the PrtP enzyme can be expected to have enzyme activity under conditions that are substantially equivalent to the environment in which the derived lactic acid bacteria can grow (proliferate).
  • the reaction temperature in the hydrolysis is preferably less than 50 ° C., more preferably 10 to 42 ° C., further preferably 20 to 37 ° C., and about 30 ° C. Particularly preferred.
  • the milk protein to be subjected to hydrolysis is preferably adjusted to pH 6.0 to 8.0, more preferably adjusted to 6.0 to 7.5. More preferably, it is adjusted to around 5.
  • the pH of the milk protein that is the substrate is 6.0 or more, protein aggregation hardly occurs when the obtained milk protein degradation product is heat-sterilized.
  • the pH of milk protein is 8.0 or less, protein denaturation hardly occurs when the obtained milk protein degradation product is heat sterilized.
  • the milk protein concentration in the reaction system in the hydrolysis can be appropriately determined in consideration of the desired milk protein degradation product concentration, the amount of PrtP enzyme to be added, the reaction time, and the like.
  • the milk protein concentration in the hydrolysis is preferably 0.5% by weight or more, and more preferably 1% by weight or more. This is because a milk protein degradation product with more excellent bifidobacteria growth promoting ability can be obtained.
  • the amount of the PrtP enzyme-containing lactic acid bacterium live cells, crushed cells, and PrtP enzyme fraction added to the reaction system in the hydrolysis is determined in consideration of the amount of milk protein to be added and the like. Although it can determine suitably, it is preferable to add so that it may become 1 Unit / ml or more as a unit of the PrtP enzyme activity mentioned later.
  • the hydrolysis reaction time can be appropriately determined in consideration of the reaction temperature, milk protein concentration, the amount of PrtP enzyme to be added, and the like. For example, the addition amount of live cells of PrtP enzyme-containing lactic acid bacteria will be described later.
  • the unit of PrtP enzyme activity is 3.5 Unit / ml and the reaction temperature is 30 ° C., it is preferably 5 hours or longer.
  • the milk protein degradation product obtained by hydrolysis may use the reaction solution after the reaction as it is, but after the reaction, the PrtP enzyme in the reaction solution is inactivated by heat treatment or the like. It is preferable.
  • the bifidobacteria growth promoter of the present invention contains the milk protein degradation product produced as described above as an active ingredient.
  • milk-derived ingredients produced as active ingredients
  • food and drink produced by culturing bifidobacteria with milk-derived raw materials such as fermented milk should be used without considering protein allergies other than milk proteins. Can do.
  • the amount of the milk protein degradation product of the present invention contained in the bifidobacteria growth promoter of the present invention is not particularly limited as long as the bifidobacteria growth promoting effect is obtained, but it may be 10% by weight or more. It is preferably 15% by weight or more, more preferably 20% by weight or more, and particularly preferably 40% by weight or more.
  • the dosage form of the Bifidobacterium growth promoter of the present invention is not particularly limited, and may be a powder such as a lyophilized product, a tablet, a liquid, or a paste. There may be.
  • the bifidobacteria growth promoter of this invention may contain the other component, unless the bifidobacteria growth promotion effect by the milk protein degradation product of this invention is inhibited. Examples of other components include commonly added adjuvants and stabilizers such as starch and lactose.
  • a known substance having a bifidobacteria growth promoting action may be added.
  • the bifidobacteria growth promoter of the present invention can exert a bifidobacteria growth promoting effect by adding it to a culture medium of bifidobacteria.
  • the amount to be added to the culture medium is not particularly limited as long as the effect of promoting the growth of bifidobacteria is obtained.
  • the amount of milk protein degradation product in the bifidobacteria growth promoter, the composition of the culture medium of bifidobacteria, and the starter It can be determined as appropriate in consideration of the inoculation amount of a certain bifidobacteria, the type of bifidobacteria and the like.
  • the bifidobacteria growth promoter of the present invention is preferably added so as to be 0.01 to 10 (V / V)%, for example, 0.1 to 5 (V / V)% is particularly preferable.
  • the culture medium for bifidobacteria to which the agent for promoting growth of bifidobacteria of the present invention is added is not particularly limited as long as it is a commonly used medium. You can choose.
  • the fermenting base used for producing bifidobacteria fermented milk is not particularly limited as long as it is a base usually used for producing fermented milk.
  • the base is, for example, milk, skim milk, fresh cream, butter, whole milk powder, skim milk powder, sweeteners such as sucrose, pectin, fruit, fruit juice, agar, gelatin, oil, fat, flavor, coloring It can be prepared by blending a material, a stabilizer, a reducing agent, etc., and sterilizing, homogenizing, cooling and the like according to a conventional method.
  • the bifidobacteria to which the bifidobacteria growth promoter of the present invention is added can be those pre-cultured by a conventional method.
  • the culture medium used for pre-culture of bifido is not particularly limited as long as it is a commonly used medium, but is preferably a milky medium.
  • a reduced skim milk medium is particularly preferred because of easy handling.
  • the concentration of the reduced skim milk medium is preferably 3% (W / W) or more, particularly preferably 8% (W / W) or more.
  • growth-promoting substances such as yeast extract, reducing agents such as L-cysteine, and the like can be added to the medium used for preculture.
  • a medium to which a growth promoting substance is added For example, a medium containing 0.1 to 1% (W / W) yeast extract can be used.
  • the culture medium used for preculture uses what sterilized.
  • the sterilization treatment can be performed by a commonly used method, for example, by heat treatment at 80 to 122 ° C. for 5 to 40 minutes, preferably 85 to 95 ° C. for 5 to 35 minutes.
  • the growth target bacterium of the Bifidobacterium growth promoter of the present invention is not particularly limited as long as it is a Bifidobacterium, but is preferably Bifidobacterium longum. This is because the growth promoting effect of the milk protein degradation product of the present invention can be obtained more significantly.
  • Bifidobacterium longum ATCC BAA-999 strain and Bifidobacterium longum type strain ATCC15700 strain are preferable.
  • the bifidobacteria growth promoter of the present invention is capable of growing bifidobacteria, particularly Bifidobacterium longum, which has never existed before, the use of the bifidobacteria growth promoter of the present invention makes it more effective in regulating the intestine. It is possible to produce foods and beverages containing bifidobacteria such as bifidobacteria fermented milk, which are high in health and useful for health care.
  • the primer used was a primer set of forward primer GBf (GCAAATACGGTGACGGCTGGCGA) and reverse primer GB2r (TGAGCATTATAATAGGTCTTTCTTCC), or forward primer GHf (CAAATACGGTGACGCGCTGCTAAA) and reverse primer GH2r (TAGCATTATATAGTGTG).
  • FIG. 1 is a diagram showing band patterns obtained by separating PCR products by electrophoresis and detecting them by staining.
  • “1” is a lane in which a molecular weight marker is passed
  • “2” is a PCR product of Lactococcus lactis subspecies Cremolis NBRC100676 T strain
  • “3” is Lactococcus lactis subspices lactis.
  • PCR product of JCM20101 strain “4” is a PCR product of Lactococcus lactis subspecies lactis NBRC 12007 strain, “5” is a PCR product of Lactococcus lactis subspecies lactis JCM20128 strain, “6” is a lactococcus -Lanes in which PCR products of Lactis subspecies Cremolis ATCC-9625 strain were respectively flowed.
  • Lactococcus lactis sub-species Cremolis NBRC100676 T strain and Lactococcus lactis sub-species lactis JCM20101 strain possessed the PrtP gene.
  • Lactococcus lactis subspecies Cremolis ATCC-9625 strain, Lactococcus lactis subspices lactis NBRC12007 strain and Lactococcus lactis subspices lactis JCM20128 strain each possesses Not found out.
  • PrtP Enzyme Activity 0.4 ml of 30 mM MES-NaOH buffer (pH 6.5) containing 0.02% FTC-casein was added to 0.1 ml of a commercial proteolytic enzyme trypsin solution (manufactured by Sigma, enzyme activity). 11,100 Unit / mg) was added and reacted at 30 ° C. for 1 hour. 1.2 ml of 5% trichloroacetic acid was added, mixed by vortexing, and allowed to stand at room temperature for 15 minutes.
  • the fluorescence intensity value obtained by decomposing one unit of trypsin was converted into a PrtP enzyme in the case of degrading with a live cell of Lactococcus lactis having a PrtP enzyme gene, a crushed cell or a PrtP enzyme fraction.
  • One enzyme unit of activity was defined.
  • the cells were washed twice with 30 mM MES-NaOH buffer (pH 6.5) containing 10 mM calcium chloride, suspended in the same buffer, and washed cells (live cells) concentrated 10 times. Moreover, the heat processing for 10 minutes were performed at 90 degreeC with respect to a part of microbial cell. Next, 1% of viable cells or heat-treated cells was added to a 50 mM Tris-HCl buffer (pH 6.5) solution containing 1% casein, and the mixture was maintained at 30 ° C. for 24 hours with gentle stirring. After the reaction, it was sterilized by heating at 90 ° C. for 10 minutes, adjusted to pH 4.6 with hydrochloric acid, and the supernatant was collected by centrifugation. The collected solution was adjusted to pH 6.5 with an aqueous sodium hydroxide solution and concentrated to 1/50 volume by freeze concentration.
  • Measurement of bifidobacteria growth-promoting activity 4-1 Measurement of the number of bifidobacteria The medium containing total milk protein 5% (W / W) and lactose 5% (W / W) was sterilized at 90 ° C. for 10 minutes, as described above. A fixed amount of casein degradation product of each prepared strain of Lactococcus lactis was added, inoculated with 0.1% culture of Bifidobacterium longum ATCC BAA-999 strain, and cultured at 37 ° C. for 16 hours. . After cultivation, the number of bifidobacteria was measured by a serial dilution method using a TOS propionic acid agar medium (manufactured by Yakult Pharmaceutical Co., Ltd.).
  • a culture of Bifidobacterium longum ATCC BAA-999 strain was prepared as described in Example 1 below.
  • a medium containing 5% (W / W) total milk protein and 5% (W / W) lactose was sterilized at 90 ° C. for 10 minutes, and the bacterial cells of each strain of Lactococcus lactis prepared as described above.
  • 1% of the casein degradation product and 0.1% culture of Bifidobacterium longum ATCC BAA-999 strain were inoculated and cultured at 37 ° C. for 16 hours. The culture was rapidly cooled, and the number of bifidobacteria and pH were measured. The measurement results are shown in Table 1.
  • the casein degradation product of the Lactococcus lactis subspices cremolith NBRC100676 strain and the Lactococcus lactis subspices lactis JCM20101 strain having the PrtP enzyme gene has a pH of 5.
  • the number decreased to 0 or less, and the number of bifidobacteria reached around 3 ⁇ 10 8 CFU / g.
  • each of Lactococcus lactis subspecies Cremolis ATCC-9625 strain, Lactococcus lactis subspices lactis NBRC12007 strain, and Lactococcus lactis subspices lactis strain JCM20128 not having a PrtP enzyme gene When the casein degradation product was added by viable cells, the pH was 5.5 or more and the number of bifidobacteria was 5 ⁇ 10 7 CFU / g or less, which was not significantly different from the non-treated control. On the other hand, the casein degradation product by heat-treated cells did not show the bifidobacteria growth promoting activity.
  • the milk protein degradation product by the living strain of Lactococcus lactis having the PrtP enzyme gene has growth promoting ability against Bifidobacteria. Since the casein degradation product of Lactococcus lactis subspecies lactis JCM20101 strain showed stronger bifidobacteria growth promoting activity, the degradation conditions were examined using the JCM20101 strain.
  • washed cells (PrtP enzyme-containing living cells) of Lactococcus lactis subspecies lactis JCM20101 strain were obtained.
  • 3.5 unit / ml of PrtP enzyme-containing living cells as a PrtP enzyme unit was added to a 50 mM Tris-HCl buffer (pH 6.5) solution containing 1% casein, and gently stirred at 30 ° C. for 24 hours. Retained.
  • the mixture was sterilized by heating at 90 ° C. for 10 minutes, adjusted to pH 4.6 with hydrochloric acid, and the supernatant was collected by centrifugation.
  • the collected supernatant was adjusted to pH 6.5 with an aqueous sodium hydroxide solution and concentrated to 1/20 volume by freeze concentration.
  • a commercial proteolytic enzyme trypsin manufactured by Sigma was added to a 50 mM Tris-HCl buffer (pH 6.5) solution containing 1% casein at a rate of 3.5 Units / ml. went. After the reaction, the mixture was sterilized by heating at 90 ° C. for 10 minutes, adjusted to pH 4.6 with hydrochloric acid, and the supernatant was collected by centrifugation. The collected supernatant was adjusted to pH 6.5 with an aqueous sodium hydroxide solution and concentrated to 1/20 volume by freeze concentration.
  • PrtP enzyme activity As described in 3 above, washed cells (PrtP enzyme-containing live cells) of Lactococcus lactis sub-species lactis JCM20101 strain were obtained. Next, 0.01 to 10 Units / ml of PrtP enzyme-containing living cells as a PrtP enzyme unit was added to a 50 mM Tris-HCl buffer (pH 6.5) solution containing 1% casein at 30 ° C. with gentle stirring. Hold for 24 hours. After the reaction, the mixture was sterilized by heating at 90 ° C. for 10 minutes, adjusted to pH 4.6 with hydrochloric acid, and the supernatant was collected by centrifugation.
  • Tris-HCl buffer pH 6.5
  • the collected supernatant was adjusted to pH 6.5 with an aqueous sodium hydroxide solution and concentrated to 1/20 volume by freeze concentration.
  • Each obtained casein degradation product was subjected to a bifidobacteria growth promotion test by the method described in 5 above, and the pH after the culture was measured. The results are shown in Table 5.
  • the bifidobacteria growth-promoting action by each degradation product became stronger depending on the PrtP enzyme activity, and a stronger bifidobacteria growth-promoting activity was observed with an enzyme activity of 1 Units / ml or more.
  • the collected supernatant was adjusted to pH 6.5 with an aqueous sodium hydroxide solution and concentrated to 1/20 volume by freeze concentration.
  • Each obtained casein degradation product was subjected to a bifidobacteria growth promotion test by the method described in 5 above, and the pH after the culture was measured. The results are shown in Table 6.
  • the bifidobacteria growth-promoting action of each degradation product was observed to be bifidobacteria growth-promoting activity at a casein protein concentration of 0.5% or more, and particularly stronger at a concentration of 1% or more.
  • PrtP enzyme-containing disrupted cells As described in 3 above, washed cells of Lactococcus lactis sub-species lactis JCM20101 strain were obtained. Subsequently, the washed cells are subjected to ultrasonic crushing treatment for 10 minutes or more using an ultrasonic crusher (BRANSON SOFIER 450) to crush the cells, and then centrifuged (5000 ⁇ g, 10 minutes). Unbroken cells were removed to obtain crushed cells. In addition, it was confirmed by microscopic observation and colony culture that there were no living cells in the crushed cells.
  • BRANSON SOFIER 450 ultrasonic crusher
  • Degradation activity by PrtP enzyme fraction As described in 3 above, washed bacterial cells of Lactococcus lactis subspecies lactis JCM20101 strain were obtained. Subsequently, the washed cells were suspended in 30 mM MES-NaOH buffer (pH 6.5) containing 10 mM EDTA ⁇ 2Na and kept at 30 ° C. for 10 minutes. Thereafter, it was rapidly cooled to obtain a supernatant containing PrtP enzyme released by centrifugation at 4 ° C. (8500 ⁇ g, 10 minutes).
  • This PrtP enzyme fraction was dialyzed overnight against 30 mM MES-NaOH buffer (pH 6.5) containing 10 mM calcium chloride, and then concentrated to 1/50 volume by freeze concentration, and this was used as the PrtP enzyme fraction.
  • 1 unit / ml of this PrtP enzyme fraction was added as a PrtP enzyme unit to a 50 mM Tris-HCl buffer (pH 6.5) solution containing 1% casein, and the mixture was kept at 30 ° C. for 16 hours with gentle stirring.
  • heat treatment was performed at 90 ° C. for 10 minutes, the pH was adjusted to 4.6 with hydrochloric acid, and the supernatant was collected by centrifugation.
  • the living cells of the Lactococcus lactis subspices cremolith NBRC100676 strain and Lactococcus lactis subspices lactis JCM20101 strain having the PrtP enzyme gene, and milk from the PrtP enzyme fraction It was revealed that the protein degradation product has a growth promoting activity against bifidobacteria. Moreover, it turned out that the activity is stronger than the decomposition product by a general commercial proteolytic enzyme.
  • Lactococcus lactis having these PrtP enzyme genes does not produce diacetyl and acetoin, it can be expected that a savory fermented product can be produced by using these lactic acid bacteria.
  • a Bifidobacterium starter is added to a living cell of Lactococcus lactis having these PrtP enzyme genes, a crushed cell of a cell, or a milk protein degradation product from a PrtP enzyme fraction. And may be fermented.
  • Example 1 Preparation 1 of casein degradation product using live cells containing PrtP enzyme
  • 100 L of Difco (registered trademark) M17 Broth (Becton, Dickinson) added with 0.5% lactose and glucose was sterilized for 15 minutes at 121 ° C. 3 L of culture was inoculated and cultured at 30 ° C. for 16 hours.
  • live cells containing PrtP enzyme were obtained by centrifugation (8500 ⁇ g, 10 minutes, 4 ° C.).
  • the obtained PrtP enzyme-containing viable cells were added to 100 L of a 10% casein (fontera) solution, and kept at 30 ° C. for 24 hours with gentle stirring.
  • heat sterilization treatment was performed at 90 ° C. for 10 minutes, and 10.5 kg of casein decomposition product was obtained by freeze-drying.
  • Example 2 Preparation 2 of casein degradation product using live cells containing PrtP enzyme 11 kg of casein degradation product was used in the same manner as in Example 1 except that the seed culture of Lactococcus lactis sub-species Lactis JCM20101 strain was used instead of the seed culture of Lactococcus lactis sub-species Cremolis NBRC100676 strain. Obtained.
  • Example 3 Preparation 1 of casein degradation product using PrtP enzyme-containing crushed cells First, 100 L of Difco (registered trademark) M17 Broth (Becton, Dickinson) added with 0.5% lactose and glucose was sterilized for 15 minutes at 121 ° C. 3 L of culture was inoculated and cultured at 30 ° C. for 16 hours. Subsequently, the cells were obtained by centrifugation (8500 ⁇ g, 10 minutes, 4 ° C.). Next, the cells were crushed by ultrasonic treatment (BRANSON SOFIER 450, 10 minutes crushing) for 30 minutes, and then centrifuged (8500 ⁇ g, 10 minutes, 4 ° C.) to remove unbroken cells.
  • Difco registered trademark
  • M17 Broth Becton, Dickinson
  • PrtP enzyme-containing microbial cell disruption product was obtained.
  • the obtained PrtP enzyme-containing microbial cell disruption was added to 100 L of 10% casein (Fontera) solution, and kept at 30 ° C. for 24 hours with gentle stirring.
  • heat sterilization treatment was performed at 90 ° C. for 10 minutes, and 10 kg of casein decomposition product was obtained by freeze-drying.
  • Example 4 Preparation 2 of casein degradation product using crushed cell containing PrtP enzyme 9. Casein degradation product in the same manner as in Example 3 except that the seed culture of Lactococcus lactis subspices lactis JCM20101 strain was used instead of the seed culture of Lactococcus lactis subspecies Cremolis NBRC100676 strain. 1 kg was obtained.
  • Example 5 Preparation 1 of casein degradation product by PrtP enzyme fraction 1 First, 100 L of Difco (registered trademark) M17 Broth (Becton, Dickinson) added with 0.5% lactose and glucose was sterilized for 15 minutes at 121 ° C. 3 L of culture was inoculated and cultured at 30 ° C. for 16 hours. Subsequently, the cells were obtained by centrifugation (8500 ⁇ g, 10 minutes, 4 ° C.). Next, this microbial cell was suspended in 30 mM MES-NaOH buffer (pH 6.5) containing 5 L of 10 mM EDTA ⁇ 2Na and kept at 30 ° C. for 10 minutes.
  • Difco registered trademark
  • M17 Broth Becton, Dickinson
  • PrtP enzyme fraction was added to 100 L of 10% casein (Fontera) solution, and the mixture was kept at 30 ° C. for 24 hours with gentle stirring. After the reaction, sterilization was performed at 90 ° C. for 10 minutes, and freeze-dried to obtain 10.1 kg of casein degradation product.
  • Example 6 Preparation 2 of casein degradation product by PrtP enzyme fraction 2 9. Casein degradation product in the same manner as in Example 5 except that the seed culture of Lactococcus lactis subspices lactis JCM20101 strain was used instead of the seed culture of Lactococcus lactis subspecies Cremolis NBRC100676 strain. 1 kg was obtained.
  • Example 7 Preparation of bifidobacteria starter by casein degradation product from PrtP enzyme-containing living cells
  • the casein degradation product prepared by the method described in Example 1 is 0.1% (W / W).
  • 1000 mL of the medium containing the added 10% (W / W) nonfat dry milk was sterilized at 90 ° C. for 30 minutes.
  • 100 mL of a seed culture of Bifidobacterium longum ATCC BAA-999 strain was inoculated and cultured at 37 ° C. for 5 hours to obtain a culture.
  • the pH of the obtained culture was 4.7, and the number of bifidobacteria was 1.2 ⁇ 10 9 CFU / g.
  • Example 8 Preparation of an intestinal preparation by casein degradation product using PrtP enzyme-containing living cells
  • 1 kg of casein degradation product prepared by the method described in Example 2 above was added to 59 kg of dry sterilized starch and 40 kg of sugar, and mixed uniformly. As a result, about 100 kg of an intestinal preparation was obtained.
  • Example 9 Preparation of beverage by casein degradation product using PrtP enzyme-containing living cells 1 kg of casein degradation product prepared by the method described in Example 1 above was added to 100 kg of apple juice, and sterilized at 140 ° C for 2 seconds. Filled into a PET bottle, the milk protein decomposed product-containing beverage of the present invention was obtained.
  • This beverage is a beverage that can be expected to have a bifidobacteria growth action.
  • Example 10 Preparation of feed by casein degradation product with live PrtP enzyme-containing cells 1 kg of casein degradation product prepared by the method described in Example 2 above is added to 100 kg of skim milk powder and mixed uniformly. About 100 kg was obtained as a feed containing a milk protein degradation product.
  • Example 11 Preparation of Total Milk Protein Degradation Product Using PrtP Enzyme-Containing Live Bacteria
  • Lactococcus lactis Prtis PrtP enzyme-containing viable cells were obtained using the seed culture of Subspecies lactis JCM20101 strain.
  • the obtained PrtP enzyme-containing viable cells were added to 100 L of a 10% total milk protein (Mirai) solution, and kept at 30 ° C. for 24 hours with gentle stirring. After the reaction, heat sterilization treatment was performed at 90 ° C. for 10 minutes, and 10.2 kg of total milk protein degradation product was obtained by lyophilization.
  • the milk protein degradation product of the present invention Since the milk protein degradation product of the present invention has bifidobacteria growth-promoting activity, it can be used in the field of manufacturing foods and beverages containing bifidobacteria such as fermented milk containing bifidobacteria.
  • the milk protein degradation product of the present invention uses milk protein such as cow milk as a raw material, the use of the milk protein degradation product of the present invention makes it possible to design a bifidobacteria-containing product using only a milk product. Therefore, it can be used particularly in the field of dairy products.

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Abstract

L’invention concerne un produit de dégradation de la protéine du lait qui présente une excellente aptitude à la production de masse et qui ne nécessite aucune source de protéine source autre qu’un matériau de départ dérivé du lait, qui est le principal matériau de départ pour la culture du bifidobacterium et qui peut promouvoir la croissance du bifidobacterium, un procédé de fabrication dudit produit de dégradation de la protéine du lait, et un agent promoteur de la croissance bifidobactérienne qui renferme ledit produit de dégradation de la protéine du lait en tant qu’ingrédient actif. L’invention concerne un produit de dégradation de la protéine du lait qui est obtenu en hydrolysant la protéine de lait à l’aide d’un ou plusieurs éléments choisis parmi un groupe constitué de cellules vivantes de lactobacille incluant une protéase localisée sur la paroi cellulaire (protéinase enveloppée par la paroi cellulaire, PrtP), des cellules fractionnées du lactobacille ci-dessus, et une fraction de l’enzyme ci-dessus fractionnée du lactobacille ci-dessus, un procédé pour la fabrication dudit produit de dégradation de la protéine du lait et un agent promoteur de la croissance bifidobactérienne renfermant ledit produit de dégradation de la protéine du lait en tant qu’ingrédient actif.
PCT/JP2009/056803 2008-06-11 2009-04-01 Produit de dégradation de la protéine du lait, procédé de fabrication du produit de dégradation de la protéine du lait et agent promoteur de la croissance bifidobactérienne WO2009150888A1 (fr)

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CN112205471A (zh) * 2020-10-16 2021-01-12 光明乳业股份有限公司 一种两歧双歧杆菌发酵乳及其制备方法
CN113999884A (zh) * 2021-11-05 2022-02-01 南京康瑞生物科技有限公司 一种甲鱼生物活性肽的制备方法

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JP2009296910A (ja) * 2008-06-11 2009-12-24 Morinaga Milk Ind Co Ltd ビフィドバクテリウム属菌含有組成物及びビフィドバクテリウム属菌含有組成物の製造方法
CN112205471A (zh) * 2020-10-16 2021-01-12 光明乳业股份有限公司 一种两歧双歧杆菌发酵乳及其制备方法
CN113999884A (zh) * 2021-11-05 2022-02-01 南京康瑞生物科技有限公司 一种甲鱼生物活性肽的制备方法
CN113999884B (zh) * 2021-11-05 2023-09-12 南京康瑞生物科技有限公司 一种甲鱼生物活性肽的制备方法

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