Classifications

• • 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
  • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
  • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
  • A23L29/06—Enzymes
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
  • A23L31/00—Edible extracts or preparations of fungi; Preparation or treatment thereof
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
  • 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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
  • A61K36/06—Fungi, e.g. yeasts
  • A61K36/062—Ascomycota
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
  • A61K38/46—Hydrolases (3)
  • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
• • 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
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • 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
  • C12P13/00—Preparation of nitrogen-containing organic compounds
  • C12P13/04—Alpha- or beta- amino acids
• • 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
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the present invention relates to a digestive enzyme agent capable of promoting the release of branched-chain amino acids, and specifically to a digestive enzyme agent containing a protease derived from Jiuqu.
• BCAA branched chain amino acids
• Patent Document 1 describes a branched chain in which a proteolytic product containing a branched chain amino acid is placed in an aqueous environment having a polar organic solvent concentration of 70 v / v% or more, and then the precipitate is removed to recover a soluble fraction.
• a method for producing a high-amino acid fraction is disclosed, and specific methods for preparing a proteolytic product containing branched-chain amino acids include a reaction with a soybean glycinin composition solution with a samoase, a reaction with a bioprase, and a reaction with a bioprase. It is disclosed that glycinin decomposition products were obtained by carrying out the reactions with Sumiteam FP at 58 ° C. for 60 minutes each.
• the proteolytic product decomposition itself is performed in vitro (for example, in a factory) in advance. It is necessary to heat it to a temperature exceeding the body temperature using papain or samoase. Therefore, the enzymes used in these methods cannot support the digestion of ingested foods into BCAAs in the body.
• an object of the present invention is to provide a digestive enzyme agent capable of promoting the release of protein into BCAA in the internal environment.
• a protease derived from Jiuqu is an effective component as a digestive enzyme agent capable of promoting the release of protein into BCAA in the internal environment.
• the present invention has been completed based on these findings.
• Item 1 A digestive enzyme preparation containing a protease derived from Jiuqu.
• Item 2. The digestive enzyme preparation according to Item 1, wherein the aspergillus is Aspergillus oryzae and / or Aspergillus niga.
• Item 3. Item 2.
• Item 4. Item 3.
• Item 5. Item 2.
• Item 6. Item 2.
• a digestive enzyme agent capable of promoting the release of a protein into BCAA in the internal environment.
• the digestive enzyme preparation of the present invention is characterized by containing a specific protease.
• the digestive enzyme preparation of the present invention will be described in detail.
• the digestive enzyme preparation of the present invention contains a protease derived from Jiuqu as an active ingredient.
• the aspergillus that is the source of the protease is not particularly limited, and examples thereof include Aspergillus spp. And Rhizopus spp. Specific examples of Aspergillus spp.
• Aspergillus oryzae and Aspergillus are preferable. niger), Aspergillus melleus, and / or Rizopus oryzae, more preferably Aspergillus oryzae, Alpergillus niga, and / or Rizopus oryzae, and even more preferably. , Aspergillus oryzae.
• the combination of the type of the source Jiuqu and the type of the protease of the protease derived from Jiuqu is arbitrary.
• the acidic protease derived from Aspergillus oryzae, the acidic protease derived from Aspergillus oryzae, and / or the acidic protease derived from Aspergillus oryzae, and / or the acid protease derived from Aspergillus oryzae are preferable.
• Acid proteases more preferably acidic proteases derived from Aspergillus oryzae.
• acidic protease derived from Aspergillus oryzae include the polypeptides shown in any of the following (1) to (3).
• the polypeptide shown in (1) above is an acidic protease derived from wild-type Aspergillus oryzae
• the polypeptides shown in (2) and (3) above are acidic proteases derived from mutant Aspergillus oryzae. Since all of these polypeptides have excellent substrate specificity for recognizing the amino acid residue portion corresponding to BCAA of the protein, they exhibit an excellent BCSAA release promoting effect.
• the modification of the introduced amino acid may include only one modification (for example, substitution only) from substitutions, additions, insertions, and deletions.
• the above modifications eg, substitution and insertion
• the number of amino acids to be introduced, substituted, added, inserted or deleted may be one or several, for example, 1 to 81, preferably 1 to 48, or 1 to 32 pieces, more preferably 1 to 16 pieces, 1 to 10 pieces, or 1 to 8 pieces, particularly preferably 1 to 3 pieces, 1 or 2 pieces or 1 piece.
• sequence identity with respect to the amino acid sequence shown in SEQ ID NO: 1 may be 80% or more, preferably 85% or more, preferably 90% or more, still more preferably 95%. Above, 99% or more is particularly preferable.
• sequence identity is defined as BLAST PACKAGE [sgi32 bit edition, Version 2.0.12; available from National Center for Biotechnology Information (NCBI)] bl2sex The value of the identity of the amino acid sequence obtained by Microbiol. Lett., Vol. 174, p247-250, 1999) is shown. The parameters may be set to Gap insertion Cost value: 11 and Gap extension Cost value: 1.
• amino acid substitution when an amino acid substitution is introduced into the amino acid sequence shown in SEQ ID NO: 1 in the polypeptides (2) and (3), conservative substitution is mentioned as a preferable embodiment of the introduced amino acid substitution. Be done. That is, as the substitution in the polypeptides (2) and (3), for example, if the amino acid before substitution is a non-polar amino acid, it may be replaced with another non-polar amino acid, or the amino acid before substitution may be an uncharged amino acid. For example, substitution with another uncharged amino acid, substitution with another acidic amino acid if the amino acid before substitution is an acidic amino acid, and substitution with another basic amino acid if the amino acid before substitution is a basic amino acid. Can be mentioned.
• BCAA free ability evaluation value obtained by the following method is the above. Equivalent to the BCAA free capacity evaluation value of the polypeptide of (1) (that is, when the BCAA free capacity evaluation value of the polypeptide of (1) is 100%, BCAA of the polypeptide of (2) or (3) It means that the free capacity evaluation value is about 30 to 170%, 50 to 150%, or 80 to 120%).
• the ratio (%) of the amount of free BCAA (mg / L) to the total amount of free amino acids (mg / L) is obtained as the BCAA free ability evaluation value.
• the BCAA free capacity evaluation value reflects the degree of substrate specificity that recognizes the amino acid residue portion corresponding to BCAA of the protein.
• the acidic protease is used as the ratio of the acidic protease activity at pH 3 measured by the following method to the neutral protease activity at pH 6 measured by the following method.
• it can be included so as to be 0.027 or more.
• the acidity of the acidic protease at pH 3 with respect to the neutral protease activity at pH 6 The larger the ratio of protease activity, the more preferable, but preferably 0.09 or more or 0.5 or more, more preferably 0.7 or more, still more preferably 1 or more, still more preferably 1.3 or more, 1.5 or more, or 2.0 or more, particularly preferably 2.2 or more.
• a tyrosine calibration curve is prepared by separately using a tyrosine solution of 10 to 40 ⁇ g / mL and performing the same operation as that for the filtrate described above. Under this condition, the amount of enzyme that causes an increase in the folin test solution color-forming substance corresponding to 1 ⁇ g of tyrosine at 37 ° C. for 1 minute is defined as 1 U. The following formula is used for the calculation.
• the digestive enzyme preparation containing the protease derived from the above-mentioned Jiuqu may be produced using the Jiuqu that produces the protease, may be produced by a known genetic engineering method, or may be a commercially available product. good.
• examples of the digestive enzyme agent containing a relatively large amount of acidic protease derived from Aspergillus oryzae at pH 3 with respect to neutral protease activity at pH 6 include ASPSDU-pine, protease M Amano SD, and peptidase.
• Acid Protease UF Amano SD (all manufactured by Amano Enzyme), Orientase AY (manufactured by HBI), Protease YP-SS (manufactured by Yakult Pharmaceutical Co., Ltd.), etc.
• Neutral protease derived from Aspergillus oryzae As digestive enzyme agents containing a relatively large amount of acidic protease activity at pH 6 with respect to neutral protease activity at pH 3, Proteax, Protease P Amano 3SD (all manufactured by Amano Enzyme), Sumiteam (manufactured by Shin Nihon Kagaku Kogyo Co., Ltd.), etc. Can be mentioned.
• the content of the protease derived from Jiuqu in the enzyme preparation of the present invention is appropriately set within a range in which the effect of promoting BCAA release by the protease derived from Jiuqu is exhibited.
• the digestive enzyme preparation of the present invention contains, if necessary, the bacterial cell component of the aspergillus that produced the active ingredient, other nutritional components, pharmacological components, and / or enzyme components. It may be contained.
• the nutritional component, pharmacological component, and enzyme component are not particularly limited as long as they can be used in foods and drinks and / or pharmaceuticals, and for example, vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin C, and vitamin A.
• Vitamin D Vitamin E
• Vitamin K Niacin
• Pantothenic acid Folic acid
• Calcium Sulfur, Magnesium, Zinc, Serene, Iron and other minerals
• Proteases BCAA (Leucine, Isoleucine) , Valin), glycine, alanine, arginine, aspartic acid, cystine, phenylalanine, taurine, tryptophan and other amino acids
• Extracts Other functional materials such as dietary fiber, royal jelly, propolis, honey, chondroitin sulfate, glucosamine, ceramide, hyaluronic acid
• stomachic agents such as betaine hydrochloride, carnitine chloride, betanecol chloride
• intestinal regulators amylase, glucosidase, galactosidase, glucoamylase, Carbohydrate digestive enzymes such as maltase and cellulase; lipid-degrading enzyme
• These nutritional components, pharmacological components, and / or enzyme components may be used alone or in combination of two or more.
• the content of these components is appropriately set according to the type of component used, the form and / or use of the digestive enzyme preparation of the present invention, and the like.
• the digestive enzyme preparation of the present invention may contain a base and / or an additive or the like, if necessary, in order to prepare it into a desired pharmaceutical form.
• bases and additives are not particularly limited as long as they can be used in foods and drinks and / or pharmaceuticals, and for example, excipients (starch, dextrin, maltose, trehalose, lactose, D-glucose, sorbitol).
• bases and / or additives may be used alone or in combination of two or more.
• the content of these bases and / or additives is appropriately set according to the type of the agent to be used, the form and / or use of the digestive enzyme agent of the present invention, and the like.
• the digestive enzyme preparation of the present invention is orally ingested or orally administered.
• the timing of ingestion or administration of the digestive enzyme preparation of the present invention is not particularly limited as long as the ingested substrate protein and the ingested or administered digestive enzyme preparation of the present invention coexist in the body. Or after meals.
• the intake or dose of the digestive enzyme preparation of the present invention per meal containing a protein varies depending on the intake of the substrate protein, and is, for example, 1 to 2,000 mg, 2 to 1,000 mg, 3 to 500 mg. Alternatively, 5 to 400 mg can be mentioned.
• the intake or dose of the digestive enzyme preparation of the present invention per meal containing a protein varies depending on the intake of the substrate protein, and the amount of acidic protease is, for example, 100 U or more. Further, from the viewpoint of obtaining a higher BCAA release promoting effect from the protein, the intake or dose of the digestive enzyme agent of the present invention per meal containing the protein is preferably 200 U as the amount of acidic protease. As mentioned above, the amount of 500 U or more, 1,000 U or more, 2,000 U or more, 5,000 U or more, 10,000 U or more, 20,000 U or more, or 30,000 U or more can be mentioned.
• the upper limit of the range of the amount of the acidic protease is not particularly limited, and examples thereof include 400,000 U or less, 200,000 U or less, 100,000 U or less, or 80,000 U or less.
• the digestive enzyme preparation of the present invention has an amount of acidic protease per 1 g of the substrate protein of 1,800 or more, 2,000 U or more, and 2,500 U or more. , 3,000 U or more, or 5,000 U or more may be used.
• the upper limit of the amount of acidic protease per 1 g of the substrate protein is not particularly limited, and examples thereof include 20,000 U or less, 10,000 U or less, or 7,000 U or less. Be done. Further, from the viewpoint of efficiently obtaining the BCAA release promoting effect with respect to the amount of the enzyme agent used, the upper limit of the amount of acidic protease per 1 g of the substrate protein is, for example, 6,000 U or less, 5,000 U or less, 3,000 U. Hereinafter, it may be 2,000 U or less, 1,500 U or less, or 1,000 U or less.
• the digestive enzyme preparation of the present invention is used for the purpose of promoting the release of branched chain amino acid (BCAA) from a substrate protein by the action of the above-mentioned protease which is an active ingredient.
• promoting the release of BCAA means releasing more BCAA by digestion than the amount of BCAA released by a protease other than the protease derived from Aspergillus, and in a preferred embodiment, the release based on the total amount of free amino acids. It means that BCAA is released so that the ratio of the amount of BCAA is larger than the ratio of the amount of BCAA residue to the total amount of amino acid residues in the substrate. That is, the digestive enzyme preparation of the present invention can be used as a release promoter from protein to BCAA.
• the digestive enzyme agent of the present invention can promote the release of BCAA in the internal environment. Therefore, the digestive enzyme preparation of the present invention is digested in an environment of, for example, 35 to 40 ° C, preferably 35.5 to 38 ° C, more preferably 36 to 37.5 ° C, still more preferably 36.5 to 37.5 ° C. Can be used for the purpose of doing. Particularly preferably, the digestive enzyme preparation of the present invention can be used for the purpose of supporting digestion in the digestive tract.
• the pH applied at the time of digestion of the digestive enzyme preparation of the present invention varies depending on the type and content ratio of the protease contained, but when the digestive enzyme preparation of the present invention is contained in a predetermined ratio, which is a preferable embodiment, the digestive enzyme preparation of the present invention is used.
• the digestive enzyme preparation of the present invention can be used for the purpose of digesting any protein. Therefore, the digestive enzyme preparation of the present invention can be used for the purpose of digesting animal proteins such as meat, seafood and dairy products; and plant proteins such as wheat, beans and nuts.
• the digestive enzyme agent of the present invention promotes the release of BCAA, it can be used for the purpose of digesting a protein having a high BCAA content. Further, since the digestive enzyme preparation of the present invention has an excellent digestive ability capable of releasing not only BCAA but also a large amount of total amino acids, it is used for the purpose of digesting protein foods that are difficult to digest by itself. Can be done. From these viewpoints, a preferable example of a protein food to which the digestive enzyme agent of the present invention is applied is meat (livestock meat). Specific examples of meat include mammals such as cows, pigs, horses, sheep, boars, deer, and whales; meat of animals such as birds such as chickens, duck, pigeons, and sardines, and preferably mammals.
• Meat is mentioned, more preferably beef meat.
• the part of the animal is not particularly limited, and examples thereof include a neck, a back, an abdomen, a thigh, a shin, and a buttocks, and a thigh is preferable.
• the digestive enzyme preparation of the present invention has an excellent effect of promoting the release of BCAA, a large amount of BCAA can be released even from a plant protein food having a relatively low protein content.
• plant protein foods include wheat, beans and nuts, more preferably beans, more preferably peas and soybeans, even more preferably soybeans, and particularly preferably edamame. Be done.
• the release of BCAA can be promoted, so that it can be used for a subject requiring active intake of BCAA.
• targets include those that require suppression of muscle proteolysis and / or promotion of muscle protein synthesis, and specifically, those that seek suppression of muscle fatigue, improvement of muscle damage, muscle strengthening, and the like. Be done.
• the digestive enzyme preparation of the present invention since the digestive enzyme preparation of the present invention has an excellent digestive ability capable of releasing not only BCAA but also a large amount of total amino acids, it not only requires active intake of BCAA but also supports digestion. Can also be used for objects that require.
• Such subjects include subjects during and after illness, elderly subjects (in the case of humans, for example, 60 years or older) and the like.
• Examples of the application target of the digestive enzyme preparation of the present invention include humans and non-human mammals.
• Non-human mammals include experimental animals such as mice, rats, rabbits, guinea pigs and non-human primates; pets such as dogs and cats; domestic animals such as cows, pigs, goats, sheep and horses; humans; Can be mentioned.
• humans, pet animals, and livestock are preferable, and humans are more preferable.
• the formulation mode of the digestive enzyme preparation of the present invention is not particularly limited, and a person skilled in the art can appropriately determine it according to the mode of use.
• the specific embodiment is not particularly limited as long as it can be orally ingested or orally administered, but specifically, foods and drinks and foods. Examples include additives and oral medicines.
• the active ingredient is prepared as it is or in combination with the above other contained ingredients, other food materials and / or seasonings into a desired form. do it.
• foods and drinks include foods for specified health use, foods with functional claims, dietary supplements, foods for the sick, foods for the elderly, and the like, in addition to general foods and drinks.
• foods and drinks include not only foods and drinks for humans, but also feeds for laboratory animals or livestock, and pet foods for pet animals.
• the form of these foods and drinks is not particularly limited, but specifically, supplements such as capsules (soft capsules, hard capsules), tablets, granules, powders, jellies; nutritional drinks, fruit juice drinks, carbonated drinks, lactic acid.
• Beverages and the like examples include luxury items such as dumplings, ice cream, sherbet, gummy, and candy.
• Examples include luxury items such as dumplings, ice cream, sherbet, gummy, and candy.
• supplements are preferable, and capsules, tablets, granules, and powders are more preferable.
• These foods and drinks are suitably used as foods and drinks for promoting the release of proteins into branched-chain amino acids.
• the active ingredient may be prepared in a desired form as it is or in combination with the other contained ingredients and / or seasonings. ..
• such food additives include not only those added to foods and drinks for humans, but also those added to feeds for laboratory animals or livestock, and those added to pet foods for pet animals. .. Examples of such food additives include granules, powders, and liquids that are easily mixed with foods, and from the viewpoint of stability, granules and powders are preferable. These food additives are suitably used as food additives for promoting the release of proteins into branched chain amino acids.
• the active ingredient may be prepared as it is or in combination with the other contained ingredients in a desired form.
• an internal medicine include capsules (soft capsules, hard capsules), tablets, granules, powders, jellies, syrups and the like.
• capsules, tablets, granules, and powders are preferable.
• These internal medicines are preferably used as internal medicines for promoting the release of proteins into branched-chain amino acids.
• the internal medicine can be taken before, at the same time as, or after a meal of a food containing protein, and is preferably taken after a meal. It can be taken within 20-40 minutes after meals, more preferably.
• the enzyme reagent When the digestive enzyme preparation of the present invention is configured as an enzyme reagent, the enzyme reagent contains an artificial digestive system constructed to imitate the internal environment, preferably the gastric environment, specifically, artificial gastric fluid and is equivalent to body temperature. It can be used to test the promotion of release of BCAA from a protein in a temperature-conditioned artificial digestion system, eg, before, at the same time as the protein is provided, or when the protein is provided to the artificial digestion system. It can be added to the artificial digestive system within 20-40 minutes, more preferably after serving the protein.
• an artificial digestive system constructed to imitate the internal environment, preferably the gastric environment, specifically, artificial gastric fluid and is equivalent to body temperature. It can be used to test the promotion of release of BCAA from a protein in a temperature-conditioned artificial digestion system, eg, before, at the same time as the protein is provided, or when the protein is provided to the artificial digestion system. It can be added to the artificial digestive system within 20-40 minutes, more preferably after serving the protein.
• the content of the digestive enzyme preparation in these oral enzyme preparations or enzyme reagents is appropriately as long as the protease contained in the digestive enzyme preparation is effective in promoting BCAA release by the protease derived from the aspergillus. Set.
• Test Example 1 Beef thigh meat (lean meat) was prepared as a test substrate.
• the BCAA weight ratio to the total amino acid weight of beef thigh (lean) is 22.9%.
• the amount of beef thigh meat used was 13 g for each protease, and the shape was finely ground (3 mm ground).
• the digestive enzyme preparations shown in Table 1 were prepared.
• the amount of the digestive enzyme preparation used was such that the protease activity was 3000 U as measured by the enzyme activity measuring method (measured pH: 6.0) based on the following folin method.
• the activity measured by the enzyme activity measuring method (measurement pH: 3.0) based on the following Folin method was obtained as the acidic protease activity (unit: U).
• the ratio of the acidic protease activity (unit: U) to the protease 3000U measured at pH 6.0 was defined as the acidic protease ratio. The results of these measurements are shown in Table 1.
• the acidic protease contained in the digestive enzyme preparations shown in Examples 1 to 3 is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1; one or several in the amino acid sequence shown in SEQ ID NO: 1.
• a polypeptide in which an amino acid has been substituted, added, inserted or deleted and has protease activity equivalent to that of the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1; or sequence identity to the amino acid sequence shown in SEQ ID NO: 1. Is 80% or more, and has a protease activity equivalent to that of the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1.
• ⁇ Enzyme activity measurement method (casein formin method)> 5 mL of a substrate solution for measurement prepared to a predetermined measurement pH (when the measurement pH is 3.0, an aqueous solution of pH 3.0 containing 6.0 g / L dairy casein and 0.08 mol / L lactic acid; measurement pH In the case of 6.0, an aqueous solution of pH 6.0 containing 6.0 g / L dairy casein and 0.04 mol / L disodium phosphate) was placed in a test tube and heated at 37 ° C. for 10 minutes.
• a tyrosine calibration curve was prepared by performing the same operation as the above-mentioned operation on the filtrate using a tyrosine solution of 10 to 40 ⁇ g / mL. Under the conditions of the above enzyme activity measurement method, the amount of enzyme that causes an increase in the folin test solution color-forming substance corresponding to 1 ⁇ g of tyrosine in 1 minute at 37 ° C. was set to 1 U. The following formula was used for the calculation.
• a digestive enzyme preparation corresponding to a protease activity (pH 6.0) of 3,000 U was added, a stirrer bar (3.5 cm) was added, and the mixture was stirred at 250 rpm for 90 minutes. From 5 minutes to 65 minutes after the start of the reaction, 0.54 mL of 1 mol / L hydrochloric acid was added every 10 minutes. After 90 minutes, it was left in a boiling water bath for 10 minutes. After the boiling water bath, the reaction solution was mesh-filtered (2 mm square), and the obtained filtrate was centrifuged at 10,000 rpm for 10 minutes to obtain a supernatant containing free amino acids. Further, as a control (Comparative Example 1), the same operation was carried out except that a digestive enzyme agent was not added, and a supernatant was obtained.
• the obtained supernatant is diluted 25-fold with water, filtered through a filter (0.45 ⁇ m), and then the amount of free amino acids is analyzed according to the protocol with an amino acid analyzer (amino acid analysis using Aginent 1260 Infinity II LC system). bottom.
• the ratio (%) of the total amount of free amino acids (mg / L) obtained and the amount of free BCAAs (mg / L) in the total amount of free amino acids (mg / L) is shown in Table 1.
• Test Example 2 (Test substrate) As the test substrate, 13 g of the same finely ground (3 mm ground) beef thigh meat (lean meat) as in Test Example 1 was prepared.
• Example 7 A digestive enzyme preparation having the composition shown in Table 2 (Example 7) was used.
• the digestive enzyme preparations of Example 7 include ASPSDU-pine (a digestive enzyme preparation containing a large amount of acidic protease derived from Aspergillus oryzae) used in Example 1 and a protease derived from Aspergillus oryzae contained in Biodiastase 2000 (acidic). Includes proteases and neutral proteases).
• the amount of the digestive enzyme preparation used in Example 7 was such that the protease activity was 3000 U as measured by the enzyme activity measuring method (measured pH: 6.0) based on the Folin method shown in Example 1.
• Example 7 As is clear from Table 2, it was found that the digestive enzyme preparation containing the protease derived from Jiuqu (Example 7) was able to release a large amount of BCAA even if it contained other digestive enzymes. In addition, the digestive enzyme agent of Example 7 has obtained a ratio of free BCAA exceeding the BCAA weight ratio (22.9%) contained in the substrate beef thigh protein, which is a particularly remarkable promotion of BCAA release. It was found to be effective.
• Test Example 3 (Test substrate) As a test substrate, 13 g of edamame was prepared. The protein weight ratio of common edamame is about 9%, and the BCAA weight ratio to the total amino acid weight is about 17%.
• the obtained supernatant is diluted 25-fold with water, filtered through a filter (0.45 ⁇ m), and then the amount of free amino acids is analyzed according to the protocol with an amino acid analyzer (amino acid analysis using Aginent 1260 Infinity II LC system). bottom.
• the ratio (%) of the total amount of free amino acids (mg / L) obtained and the amount of free BCAAs (mg / L) in the total amount of free amino acids (mg / L) is shown in Table 3.
• Test Example 4 (Test substrate) As a test substrate, 5 g of Soya Flower FT-N (soybean powder manufactured by Nisshin Oillio Group Co., Ltd.) or 5 g of LYSAMINE GPS (pea protein powder manufactured by Rocket Co., Ltd.) was prepared.
• the BCAA weight ratio to the total amino acid weight of general soybean is about 17%
• the BCAA weight ratio to the total amino acid weight of general peas is about 17%.
• the obtained supernatant is diluted 25-fold with water, filtered through a filter (0.45 ⁇ m), and then the amount of free amino acids is analyzed according to the protocol with an amino acid analyzer (amino acid analysis using Aginent 1260 Infinity II LC system). bottom.
• Tables 4 and 5 show the total amount of free amino acids (mg / L) obtained and the ratio (%) of the amount of free BCAAs (mg / L) to the total amount of free amino acids (mg / L).

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