NZ619383B2 - Enzymatic production method for brain-function-improving peptides - Google Patents
Enzymatic production method for brain-function-improving peptides Download PDFInfo
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- NZ619383B2 NZ619383B2 NZ619383A NZ61938312A NZ619383B2 NZ 619383 B2 NZ619383 B2 NZ 619383B2 NZ 619383 A NZ619383 A NZ 619383A NZ 61938312 A NZ61938312 A NZ 61938312A NZ 619383 B2 NZ619383 B2 NZ 619383B2
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- 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
- 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
- A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
- A23L2/52—Adding ingredients
- A23L2/66—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
-
- 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
- A61K38/00—Medicinal preparations containing peptides
- A61K38/01—Hydrolysed proteins; Derivatives thereof
- A61K38/012—Hydrolysed proteins; Derivatives thereof from animals
- A61K38/018—Hydrolysed proteins; Derivatives thereof from animals from milk
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4732—Casein
-
- 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
-
- 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
Abstract
Disclosed is an isolated or purified peptide consisting of the amino acid sequence shown in SEQ ID NO: 3. Also disclosed is a composition comprising a peptide consisting of the amino acid sequence shown in SEQ ID NO: 3 and further comprising a peptide consisting of the amino acid sequence shown in SEQ ID NO: 1 and/or a peptide consisting of the amino acid sequence shown in SEQ ID NO: 2. Further disclosed is a method for preparing a peptide for improving brain function, comprising: hydrolysing milk casein with an enzymatic catalyst comprising an alkaline protease derived from Bacillus licheniformis, an alkaline protease derived from Aspergillus sp., or a neutral protease derived from Aspergillus melleus to produce a hydrolysate comprising: (i) a peptide consisting of the amino acid sequence shown in SEQ ID NO: 1,; (ii) a peptide consisting of the amino acid sequence shown in SEQ ID NO: 2,; or (iii)a mixture of the peptides of (i) and (ii), wherein the production yield of each of the peptides is 2% or more, or the total production yield of the mixture is 10% or more; wherein the sequences are as defined in the specification. n SEQ ID NO: 1 and/or a peptide consisting of the amino acid sequence shown in SEQ ID NO: 2. Further disclosed is a method for preparing a peptide for improving brain function, comprising: hydrolysing milk casein with an enzymatic catalyst comprising an alkaline protease derived from Bacillus licheniformis, an alkaline protease derived from Aspergillus sp., or a neutral protease derived from Aspergillus melleus to produce a hydrolysate comprising: (i) a peptide consisting of the amino acid sequence shown in SEQ ID NO: 1,; (ii) a peptide consisting of the amino acid sequence shown in SEQ ID NO: 2,; or (iii)a mixture of the peptides of (i) and (ii), wherein the production yield of each of the peptides is 2% or more, or the total production yield of the mixture is 10% or more; wherein the sequences are as defined in the specification.
Description
DESCRIPTION
TITLE OF INVENTION: ENZYMATIC PRODUCTION METHOD FOR BRAIN-
FUNCTION-IMPROVING PEPTIDES
TECHNICAL FIELD
The present invention relates to a method for enzymatically preparing a peptide for
improving brain function, especially a peptide for improving or preventing amnesia.
Specifically, it relates to a method comprising hydrolyzing milk casein with an enzymatic catalyst
comprising a protease to prepare a hydrolysate rich in a peptide having the above action.
The present invention also relates to a food or drink or a pharmaceutical composition
comprising the above hydrolysate.
BACKGROUND ART
Symptoms and diseases caused by decreased brain function include depression,
schizophrenia, deliria, and dementias (such as cerebrovascular dementia and Alzheimer disease).
Particularly, an increase in dementia patients has become a major social problem with the aging
of modem society. The symptoms of dementia depend on individuals. Examples of symptoms
observed in common include memory disorder, disorientation, and decreased ability to
judge/think. Among dementias, those from which many patients suffer are cerebrovascular
dementia and Alzheimer disease. For example, for cerebrovascular dementia,
cognition/memory disorder appears because decreased cerebral blood flow damages neuronal
cells in the cerebral cortex and the hippocampus. Thus, a drug improving cerebral blood flow
and a drug protecting cerebral neurons are applied in addition to treating underlying diseases
including hypertension, diabetes, and hypercholesterolemia which potentially cause
cerebrovascular disorder. For Alzheimer disease, whose cause remains to be clearly elucidated,
the decreased function of cholinergic nerve is considered to be one of the causes thereof because a
decrease in the level of acetylcholine as an intracerebral neurotransmitter is observed in patients
with the disease (for example, Bartus, R.T. et al., Science, 217: 408-414 (1982)). Therefore, for
Alzheimer disease, a therapeutic method is predominant which is intended to prevent the decrease
of function of cholinergic nerve by increasing the level of acetylcholine.
Currently, as therapeutic drugs for Alzheimer disease, for example,
acetylcholinesterase inhibitors such as donepezil hydrochloride are commercially available.
However, acetylcholinesterase inhibitors such as donepezil hydrochloride have a disadvantage
that they cannot be administered for a long period of time because of their liver toxicity and
strong side effects and are also expensive.
As a report on a peptide having the effect of improving amnesia, it is reported, for
example, that lateral ventricular injection or oral administration of 300 mg/kg of XPLPR (where
X is L, 1, M, F, or W) has the effect of improving scopolamine-induced amnesia, suggesting the
release of acetylcholine through brain C3a receptor as one of mechanisms therefor (JP Patent No.
3898389). Scopolamine is considered to cause a decrease of the function of cholinergic nerve as
a muscarinic receptor antagonist, and act as an agent inducing brain dysfunction; for an
improving action in a model animal for the development of therapeutic drugs for Alzheimer
disease, the effect of improving and/or enhancing brain function can be demonstrated, for
example, by a behavioral test such as a Y-shaped maze test, an eight-arm maze test, or a passive
avoidance test. However, each of the peptides is required to be orally administered at a large
dose or intraperitoneally administered, intraventricularly injected, or by other delivery route to
exhibit the action, and does not have a sufficient effect as an orally ingestable substance.
Further, there is no report in which the peptide and analogs thereof according to the present
invention have been evaluated, and the action thereof involved in improving brain function has
been unknown.
Thus, with the progress of the aging of the society, there is a strong need for the
development of such a pharmaceutical product as to have the effect of preventing and also
improving symptoms or diseases caused by decreased brain function and further a safer
compound excellent in application to food.
Meanwhile, it is reported that the hydrolysate obtained by hydrolyzing casein with an
enzyme has pharmacological actions such as a blood-pressure-lowering action, a smooth muscle-
contracting action, and an anemia-improving action, and can be used in a nutrient preparation and
a mother's milk substitute, and the like (JP Patent Publication (Kohyo) No. 10-507641 (1998), JP
Patent Publication (Kokai) No. 06-128287 (1994), JP Patent Nos. 1893866, 1572761 and
4633876, JP Patent Publication (Kokoku) No. 07-032676 B (1995), and JP Patent No. 1907911);
however, no report on the brain function-improving action thereof is found.
PRIOR ART DOCUMENT
PATENT DOCUMENT
Patent Document 1: JP Patent No. 3898389
Patent Document 2: JP Patent Publication (Kohyo) No. 10-507641 (1998)
Patent Document 3: JP Patent Publication (Kokai) No. 06-128287 (1994)
Patent Document 4: JP Patent No. 1893866
Patent Document 5: JP Patent No. 1572761
Patent Document 6: JP Patent No. 4633876
Patent Document 7: JP Patent Publication (Kokoku) No. 07-032676 B (1995)
Patent Document 8: JP Patent No. 1907911
NON-PATENT DOCUMENT
Non-Patent Document 1: Bartus, R.T. et al., Science, 217: 408-414 (1982)
SUMMARY OF THE INVENTION
PROBLEM TO BE SOLVED BY THE INVENTION
The present invention provides a method for enzymatically preparing a new
peptide for improving brain function.
The present invention also provides a reaction product prepared by the method,
and a food or drink and a pharmaceutical composition comprising the same.
MEANS FOR SOLVING PROBLEM
In summary, the present invention has the following features.
(1) A method for preparing a peptide for improving brain function,
comprising:
hydrolyzing milk casein with an enzymatic catalyst comprising a protease to produce a
hydrolysate comprising:
(i) a peptide consisting of the amino acid sequence shown in SEQ ID NO: 1, or
peptide consisting of an amino acid sequence comprising deletion, substitution, or addition of one
or two amino acid residues in the amino acid sequence of SEQ ID NO: 1;
(ii) a peptide consisting of the amino acid sequence shown in SEQ ID NO: 2, or a
peptide consisting of an amino acid sequence comprising deletion, substitution, or addition of one
or two amino acid residues in the amino acid sequence of SEQ ID NO: 2; or
(iii) a mixture of the peptides of (i) and (ii),
wherein the production yield of each of the peptides is 2% or more, or the total production
yield of the mixture is 10% or more.
(2) The method according to (1) above, wherein the protease is a neutral protease or
an alkaline protease.
(3) The method according to (1) or (2) above, wherein the protease is derived from a
microorganism.
(4) The method according to (3) above, wherein the microorganism is a
microorganism belonging to the genus Bacillus or genus Aspergillus, or a lactic acid bacterium.
(5) The method according to (4) above, wherein the microorganism is selected from
the group consisting of Bacillus licheniformis, Aspergillus sp., Aspergillus oryzae, Aspergillus
melleus, Lactobacillus helveticus, Lactobacillus bulgaricus, and Streptococcus thermophilus.
(6) The method according to any of (1) to (5) above, wherein the milk casein is cow
milk casein.
(7) The method according to any of (1) to (6) above, wherein the production yield of
the peptide consisting of the amino acid sequence shown in SEQ ID NO: 1 or the peptide
consisting of an amino acid sequence comprising deletion, substitution, or addition of one or two
amino acid residues in the amino acid sequence of SEQ ID NO: 1 is 5 to 10%, or more.
(8) The method according to any of (1) to (7) above, wherein the production yield of
the peptide consisting of the amino acid sequence shown in SEQ ID NO: 2 or the peptide
consisting of an amino acid sequence comprising deletion, substitution, or addition of one or two
amino acid residues in the amino acid sequence of SEQ ID NO: 2 is 10 to 50%, or more.
(9) The method according to any of (1) to (8) above, wherein the total production
yield of the peptide consisting of the amino acid sequence shown in SEQ ID NO: 1 or the peptide
consisting of an amino acid sequence comprising deletion, substitution, or addition of one or two
amino acid residues in the amino acid sequence of SEQ ID NO: 1 and the peptide consisting of
the amino acid sequence shown in SEQ ID NO: 2 or the peptide consisting of an amino acid
sequence comprising deletion, substitution, or addition of one or two amino acid residues in the
amino acid sequence of SEQ ID NO: 2 is 15 to 60%, or more.
(10) The method according to any of (1) to (9) above, wherein the hydrolysate further
comprises a peptide consisting of the amino acid sequence shown in SEQ ID NO: 3 or a peptide
consisting of an amino acid sequence comprising deletion, substitution, or addition of one or two
amino acid residues in the amino acid sequence of SEQ ID NO: 3.
(11) The method according to any of (1) to (10) above, wherein the enzymatic
catalyst further comprises an enzyme having a peptidase activity.
(12) The method according to any of (1) to (11) above, wherein the enzymatic
catalyst is immobilized on a support.
(13) The method according to any of (1) to (12) above, wherein the hydrolysis of the
milk casein is carried out using milk.
(14) The method according to any of (1) to (13) above, wherein weight ratio of the
enzyme/milk casein is 1/100 to 1/1,000.
(15) The method according to any of (1) to (14) above, wherein reaction time of the
hydrolysis reaction at 45 to 55°C is 2 to 10 hours.
(16) The method according to any of (1) to (15) above, further comprising
inactivating the enzymatic catalyst.
(17) The method according to any of (1) to (16) above, further comprising isolating
and/or concentrating the peptide consisting of the amino acid sequence shown in SEQ ID NO: 1
or the peptide consisting of an amino acid sequence comprising deletion, substitution, or addition
of one or two amino acid residues in the amino acid sequence of SEQ ID NO: 1, the peptide
consisting of the amino acid sequence shown in SEQ ID NO: 2 or the peptide consisting of an
amino acid sequence comprising deletion, substitution, or addition of one or two amino acid
residues in the amino acid sequence of SEQ ID NO: 2, the peptide consisting of the amino acid
sequence shown in SEQ ID NO: 3 or the peptide consisting of an amino acid sequence
comprising deletion, substitution, or addition of one or two amino acid residues in the amino acid
sequence of SEQ ID NO: 3, or a mixture comprising at least two of the peptides.
(18) A hydrolysate produced by the method according to any of (1) to (17),
comprising at least one of the peptides consisting of the amino acid sequences shown in SEQ ID
NOS: 1 to 3 or the peptides consisting of amino acid sequences comprising deletion,
substitution, or addition of one or two amino acid residues in the amino acid sequences of SEQ
ID NOS: I to 3, in an amount of about 0.5 mg or more per gram of a dry solid of the hydrolysate.
(19) The hydrolysate according to (18) above, wherein the hydrolysate is dried.
(20) A food or drink comprising the hydrolysate according to (18) or (19) above.
(21) A supplement comprising the hydrolysate according to (18) or (19) above.
(22) The food or drink according to (20) above or the supplement according to (21)
above, wherein the food or drink or the supplement is a lactic acid bacterium-fermented food or
drink.
(23) The food or drink or the supplement according to any of (20) to (22) above,
wherein the food or drink or the supplement is a functional food.
(24) A pharmaceutical composition comprising the hydrolysate according to (18) or
(19) above and a pharmaceutically acceptable carrier.
(25) The pharmaceutical composition according to (24) above, wherein the
composition is for improving brain function.
(26) The pharmaceutical composition according to (25) above, wherein the
improvement of brain function is the improvement or prevention of amnesia.
[0038A] (27) According to the invention there is also provided a method for preparing a peptide
for improving brain function, comprising:
hydrolyzing milk casein with an enzymatic catalyst comprising an alkaline protease derived
from Bacillus licheniformis, an alkaline protease derived from Aspergillus sp., or a neutral protease
derived from Aspergillus melleus to produce a hydrolysate comprising:
(i) a peptide consisting of the amino acid sequence shown in SEQ ID NO: 1.;
(ii) a peptide consisting of the amino acid sequence shown in SEQ ID NO: 2.; or
(iii) a mixture of the peptides of (i) and (ii),
wherein the production yield of each of the peptides is 2% or more, or the total production
yield of the mixture is 10% or more.
[0038B] (27) According to the invention there is also provided a hydrolysate produced by the
method according to the invention, comprising at least one of the peptides consisting of the amino
acid sequences shown in SEQ ID NOS: 1 to 2, in an amount of about 0.5 mg or more per gram of
a dry solid of the hydrolysate.
[0038C] (28) According to the invention there is also provided a hydrolysate produced by the
method according to the invention, comprising at least one of the peptides consisting of the amino
acid sequences shown in SEQ ID NOS: 1 to 3 in an amount of about 0.5mg or more per gram of a
dry solid of the hydrolysate.
[0038D] (29) According to the invention there is also provided an isolated or purified peptide
consisting of the amino acid sequence shown in SEQ ID NO: 3.
[0038E] (30) According to the invention there is also provided a composition comprising a
peptide consisting of the amino acid sequence shown in SEQ ID NO: 3 and further comprising a
peptide consisting of the amino acid sequence shown in SEQ ID NO: 1 and/or a peptide
consisting of the amino acid sequence shown in SEQ ID NO: 2.
[0038F] (31) According to the invention there is also provided use of the hydrolysate
according to the invention in the manufacture of a medicament for improving brain function.
[0038G] (32) According to the invention there is also provided use of a peptide consisting of
the amino acid shown in SEQ ID NO: 3 in the manufacture of a medicament for improving brain
function.
[0038H] (33) Throughout this specification the word "comprise", or variations such as
"comprises" or "comprising", will be understood to imply the inclusion of a stated element,
integer or step, or group of elements, integers or steps, but not the exclusion of any other
element, integer or step, or group of elements, integers or steps.
EFFECT OF INVENTION
The method of the present invention enables the efficient enzymatic production of a
peptide having a brain function-improving action such as improving or preventing amnesia.
BRIEF DESCRIPTION OF THE DRAWINGS
is a graph showing the time course of the production yield of Asn-Ile-Pro-Pro-
Leu-Thr-Gln-Thr-Pro-Val-Val-Val-Pro-Pro-Phe-Leu-Gln-Pro-Glu (NIPPLTQTPVVVPPFLQPE;
SEQ ID NO: 1), Asn-Ile-Pro-Pro-Leu-Thr-Gln-Thr-Pro-Val-Val-Val-Pro-Pro-Phe-Leu-Gln-Pro-
Glu-Val-Met (NIPPLTQTPVVVPPFLQPEVM; SEQ ID NO: 2) and Ser-Trp-Met-His-Gln-Pro-
His-Gln-Pro-Leu-Pro-Pro-Thr-Val-Met-Phe-Pro-Pro-Gln-Ser-Val-Leu (SWMHQPHQPLPPTV
MFPPQSVL; SEQ ID NO: 3) when a Bacillus licheniformis-derived enzyme, Protin SD-AY10
(from Amano Enzyme Inc.), was added to a phosphate buffer solution (pH: 7.0 to 7.3) containing
cow milk casein sodium to a weight ratio of enzyme/casein of 1/200 for hydrolysis reaction.
is a graph showing the time course of the production yield of Asn-Ile-Pro-Pro-Leu-
Thr-Gln-Thr-Pro-Val-Val-Val-Pro-Pro-Phe-Leu-Gln-Pro-Glu (NIPPLTQTPVVVPPFLQPE;
SEQ ID NO: 1), Asn-Ile-Pro-Pro-Leu-Thr-Gln-Thr-Pro-Val-Val-Val-Pro-Pro-Phe-Leu-Gln-Pro-
Glu-Val-Met (NIPPLTQTPVVVPPFLQPEVM; SEQ ID NO: 2) and Ser-Trp-Met-His-Gln-Pro-
His-Gln-Pro-Leu-Pro-Pro-Thr-Val-Met-Phe-Pro-Pro-Gln-Ser-Val-Leu (SWMHQPHQPLPPT
VMFPPQSVL; SEQ ID NO: 3) when a Bacillus licheniformis-derived enzyme, Protin SD-AY10
(from Amano Enzyme Inc.), was added to a phosphate buffer solution (pH: 7.0 to 7.3) containing
cow milk casein sodium to a weight ratio of enzyme/casein of 1/1,000 for hydrolysis reaction.
is a graph showing the time course of the production yield of Asn-Ile-Pro-Pro-Leu-
Thr-Gln-Thr-Pro-Val-Val-Val-Pro-Pro-Phe-Leu-Gln-Pro-Glu (NIPPLTQTPVVVPPFLQPE;
SEQ ID NO: 1), Asn-Ile-Pro-Pro-Leu-Thr-Gln-Thr-Pro-Val-Val-Val-Pro-Pro-Phe-Leu-Gln-Pro-
Glu-Val-Met (NIPPLTQTPVVVPPFLQPEVM; SEQ ID NO: 2) and Ser-Trp-Met-His-Gln-Pro-
His-Gln-Pro-Leu-Pro-Pro-Thr-Val-Met-Phe-Pro-Pro-Gln-Ser-Val-Leu (SWMHQPHQPLPPTV
MFPPQSVL; SEQ ID NO: 3) when an Aspergillus sp.-derived enzyme, Sumizyme MP (from
Shinnihon Chemicals Corporation), was added to a phosphate buffer solution (pH: 7.0)
containing cow milk casein sodium to a weight ratio of enzyme/casein of 1/200 for hydrolysis
reaction.
is a graph showing the time course of the production yield of Asn-Ile-Pro-Pro-Leu-
Thr-Gln-Thr-Pro-Val-Val-Val-Pro-Pro-Phe-Leu-Gln-Pro-Glu (NIPPLTQTPVVVPPFLQPE;
SEQ ID NO: 1), Asn-Ile-Pro-Pro-Leu-Thr-Gln-Thr-Pro-Val-Val-Val-Pro-Pro-Phe-Leu-Gln-Pro-
Glu-Val-Met (NIPPLTQTPVVVPPFLQPEVM; SEQ ID NO: 2) and Ser-Trp-Met-His-Gln-Pro-
His-Gln-Pro-Leu-Pro-Pro-Thr-Val-Met-Phe-Pro-Pro-Gln-Ser-Val-Leu (SWMHQPHQPLPPTV
MFPPQSVL; SEQ ID NO: 3) when an Aspergillus sp.-derived enzyme, Sumizyme MP (from
Shinnihon Chemicals Corporation), was added to a phosphate buffer solution (pH: 8.0)
containing cow milk casein sodium to a weight ratio of enzyme/casein of 1/200 for hydrolysis
reaction.
is a graph showing an effect of preventing scopolamine-induced amnesia of the
peptide, Asn-Ile-Pro-Pro-Leu-Thr-Gln-Thr-Pro-Val-Val-Val-Pro-Pro-Phe-Leu-Gh
-i-Pro-Glu
(NIPPLTQTPVVVPPFLQPE). Water (control), scopolamine alone, or 0.05 nmol/kg body
weight, 0.5 nmol/kg body weight, 1.5 nmol/kg body weight, 5 nmol/kg body weight, 50 nmol/kg
body weight or 500 nmol/kg body weight of NIPPLTQTPVVVPPFLQPE with scopolamine
was administered to mice, and the amnesia-preventing effects thereof were evaluated by the
method as described in Example 4. The vertical axis of represents change in spontaneous
alternation behavior (%). In the graph, the spontaneous alternation behavior (%) for a control
group, a scopolamine control group, and 0.05 nmol/kg body weight, 0.5 nmol/kg body weight,
1.5 nmol/kg body weight, 5 nmol/kg body weight, 50 nmol/kg body weight, and 500 nmol/kg
body weight of NIPPLTQTPVVVPPFLQPE-administered groups are shown in order from the
left. To confirm whether amnesia is induced, the significance of difference between the water-
administered control group and the scopolamine alone-administered scopolamine control group
was tested by Student's t-test. ** indicates p<0.01 relative to the water-administered control
group. The significance of difference between the NIPPLTQTPVVVPPFLQPE-administered
group and the scopolamine control groups was tested by Dunnett's multiple comparison test. #
indicates p<0.05 relative to the scopolamine control group. ## indicates p<0.01 relative to the
scopolamine control group.
is a graph showing an effect of preventing scopolamine-induced amnesia of the
peptide, Asn-Ile-Pro-Pro-Leu-Thr-Gln-Thr-Pro-Val-Val-Val-Pro-Pro-Phe-Leu-Gln-Pro-Glu
(NIPPLTQTPVVVPPFLQPE), or Asn-Ile-Pro-Pro-Leu-Thr-Gln-Thr-Pro-Val-Val-Val-Pro-Pro-
Phe-Leu-Gln-Pro-Glu-Val-Met (NIPPLTQTPVVVPPFLQPEVM). Water (control),
scopolamine alone, or 50 nmol/kg body weight of NIPPLTQTPVVVPPFLQPE, or 50 nmol/kg
body weight of NIPPLTQTPVVVPPFLQPEVM with scopolamine was administered to mice,
and the amnesia-preventing effects thereof were evaluated by the method as described in
Example 5. The vertical axis of represents change in spontaneous alternation behavior
(%). To confirm whether amnesia is induced, the significance of difference between the water-
administered control group and the scopolamine alone-administered scopolamine control group
was tested by Student's t-test. ** indicates p<0.01 relative to the water-administered control
group. The significance of difference between each of the peptide-administered groups and the
scopolamine control group was tested by Dunnett's multiple comparison test. ## indicates
p<0.01 relative to the scopolamine control group.
is a graph showing a memory-enhancing effect of the peptide Asn-Ile-Pro-Pro-Leu-
Thr-Gln-Thr-Pro-Val-Val-Val-Pro-Pro-Phe-Leu-Gln-Pro-Glu Water (control) or 500 nmol/kg
of NIPPLTQTPVVVPPFLQPE was administered to mice, and the memory-enhancing effects
thereof were evaluated by the method as described in Example 6. The vertical axis of
represents the percentage of exploration time. The significance of difference between the
control group and the peptide group was tested for the percentage of exploration time by Student's
t-test. * indicates p<0.05 relative to the water-administered control group.
is a graph showing an effect of preventing scopolamine-induced amnesia of the
peptide Ser-Trp-Met-His-Gln-Pro-His-Gln-Pro-Leu-Pro-Pro-Thr-Val-Met-Phe-Pro-Pro-Gln-Ser-
Val-Leu. Water (control), scopolamine alone, or 150 nmol/kg body weight or 500 nmol/kg
body weight of SWMHQPHQPLPPTVMFPPQSVL with scopolamine was administered to mice,
and the amnesia-preventing effects thereof were evaluated by the method as described in
Example 7. To confirm whether amnesia is induced, the significance of difference between the
water-administered control group and the scopolamine alone-administered scopolamine control
group was tested by Student's t-test. ** indicates p<0.01 relative to the water-administered
control group. The significance of difference between each of the peptide-administered groups
and the scopolamine control group was tested by Student's t-test. ## indicates p<0.01 relative to
the scopolamine control group, and t indicates p<0.1.
MODE FOR CARRYING OUT THE INVENTION
The present invention will be described in further detail.
1. Peptide Having Brain Function-Improving Action
The peptides enzymatically produced from milk casein or a milk casein-containing raw
material by the method of the present invention have the following amino acid sequences.
The amino acid sequence of Asn Ile Pro Pro Leu Thr Gln Thr Pro Val Val Val Pro Pro
Phe Leu Gln Pro Glu (or NIPPLTQTPVVVPPFLQPE) (SEQ ID NO: 1; hereinafter also referred
to as "N-E peptide") or an amino acid sequence comprising deletion, substitution, or addition of
one or two amino acid residues in the amino acid sequence.
The amino acid sequence of Asn Ile Pro Pro Leu Thr Gln Thr Pro Val Val Val Pro Pro
Phe Leu Gln Pro Glu Val Met (or NIPPLTQTPVVVPPFLQPEVM) (SEQ ID NO: 2; hereinafter
also referred to as "N-EVM peptide") or an amino acid sequence comprising deletion, substitution,
or addition of one or two amino acid residues in the amino acid sequence.
The amino acid sequence of Ser Trp Met His Gln Pro His Gln Pro Leu Pro Pro Thr Val
Met Phe Pro Pro Gin Ser Val Leu (or SWMHQPHQPLPPTVMFPPQSVL) (SEQ 1D NO: 3;
hereinafter also referred to as "S-SVL peptide") or an amino acid sequence comprising deletion,
substitution, or addition of one or two amino acid residues in the amino acid sequence.
The N-E peptide is a peptide in which the ValMet sequence is removed from C-
terminus of the N-EVM peptide. The S-SVL peptide has a sequence different from the above
two peptides; however, all of these peptides are peptides cut out from milk casein and have the
common characteristics of having a brain function-improving action, especially an amnesia-
improving or -preventing action. Their amnesia-improving or -preventing action is
demonstrated in Examples 4 to 7 hereinbelow.
As used herein, "brain function-improving action" refers to the effect of improving
disorders associated with the decrease of brain function relating to memory and recognition.
As used herein, "amnesia" refers to memory decline and is one of memory disorders.
Thus, as used herein, "improving or preventing amnesia" means improving or preventing the
memory decline.
The "peptide consisting of an amino acid sequence comprising deletion, substitution, or
addition of one or two amino acid residues" as used herein for the amino acid sequence of a
peptide is a variant of the peptide (also referred to as "variant peptide") consisting of the amino
acid sequence shown in SEQ ID NO: 1, 2, or 3, and has a brain function-improving action at the
same or similar level (for example, 50% or more, preferably 80% or more, more preferably 100%
the brain function-improving action of the peptide consisting of the amino acid
or more) to
sequence shown in SEQ ID NO: 1, 2, or 3. Such a variant peptide may also be a peptide
consisting of an amino acid sequence having 89% or more, preferably 90% or more, more
preferably 94% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1,
2, or 3. As used herein, "sequence identity" refers to the percentage (%) of the number of
identical amino acid residues to the total number of amino acids containing the number of gaps
when two amino acid sequences are aligned so as to maximize the match of amino acids
therebetween by introducing gaps or by introducing no gaps. The percentage of sequence
identity can be determined, for example, by using a known algorithm such as BLAST or FASTA
publicly available through NCBI in USA.
Preferred examples of the variant peptide include a peptide consisting of an amino acid
sequence in which the conservative amino acid substitution of one or two amino acid residues is
contained in the amino acid sequence shown in SEQ ID NO: 1, 2, or 3.
As used herein, "conservative amino acid substitution" refers to substitution between
amino acids similar in properties such as polarity, electric properties, and structural properties,
including hydrophobic amino acids, polar amino acids, acidic amino acids, basic amino acids,
amino acids having branched side chains, and aromatic amino acids. Examples of the
hydrophobic (non-polar) amino acid include glycine, alanine, valine, leucine, isoleucine, and
proline; examples of the polar amino acid include serine, threonine, cysteine, methionine,
asparagine, and glutamine; examples of the acidic amino acid include aspartic acid and glutamic
acid; examples of the basic amino acid include lysine, arginine, and histidine; examples of the
amino acid having a branched side chain include valine, isoleucine, and leucine; and examples of
the aromatic amino acid include phenylalanine, tyrosine, tryptophan, and histidine.
The variant peptide can encompass a variant obtained by artificially altering the amino
acid sequence of the peptide of SEQ ID NO: 1, 2, or 3 in addition to a natural variant based on the
difference in the source of casein as a raw material substrate. Such a variant can be synthesized
by a known peptide synthesis technique, and its brain function-improving effect, particularly its
amnesia-improving effect can be confirmed by an effectiveness testing method as stated in
Examples hereinbelow.
The peptide of the present invention may encompass the form of a salt as well as the
free form thereof. This is because when the peptide is produced, the peptide can form a salt with
calcium (Ca) ion contained in casein as a raw material, for example, sodium (Na) ion derived
from an alkaline solution used for dissolving casein, a metal ion derived from a buffer solution
used as needed, and the like. Making the peptide in salt form is expected to facilitate the
dissolution of the peptide in water.
2. Method for Enzymatically Preparing Peptide
2.1 General Procedure for Enzymatic Preparation
The peptide for improving brain function according to the present invention may be
produced by hydrolyzing milk casein with an enzymatic catalyst containing a protease.
Specifically, the present invention provides a method for preparing a peptide for
improving brain function, comprising hydrolyzing milk casein with an enzymatic catalyst
containing a protease to produce a hydrolysate containing the N-E peptide (SEQ ID NO: 1) or a
variant peptide thereof, the N-EVM peptide (SEQ ID NO: 2) or a variant peptide thereof, or a
mixture thereof, wherein the production yield of each of the peptides is 2% or more, preferably 5
to 10%, or more, or the total production yield of the mixture is 10% or more, preferably 15 to
60%, or more.
The milk casein may be casein of an animal milk such as cow milk, goat milk, or horse
milk, and is preferably cow milk.
When milk casein is used in the enzymatic reaction, the casein may be added little by
little to an alkaline aqueous solution such as aqueous sodium hydroxide for dissolution under
stirring, if necessary, while heating at 50°C or less, followed by adjusting the pH at around
neutral pH with an acid such as hydrochloric acid.
Alternatively, the milk casein may be a casein-containing raw material such as milk.
In this case, the alkali treatment as described above may be unnecessary.
As described in Background Art, many examples are known of the use of a hydrolysate
obtained by enzymatically hydrolyzing milk protein in a pharmaceutical, a food, or the like;
however, the production of the N-E peptide and/or the N-EVM peptide has not previously been
reported or known. The method of the present invention is a method capable of enhancing the
production yield of these new peptides and has novelty and inventive step.
According to the present invention, when the enzyme amount is reduced relative to the
casein amount, the S-SVL peptide (SEQ ID NO: 3) or a variant peptide thereof having the same
brain function-improving action is also produced together with the N-E peptide or a variant
peptide thereof and the N-EVM peptide or a variant peptide thereof.
The enzymatic catalyst and reaction conditions will be described below in further detail.
2.2 Enzymatic Catalyst
The enzymatic catalyst used for hydrolyzing milk casein in the method of the present
invention may be an enzymatic catalyst containing a protease. Here, the expression "comprise"
or "contain" mean that the catalyst may comprise/contain, if necessary, a hydrolytic enzyme such
as an enzyme having a peptidase activity, in addition to a protease.
The protease may be of any origin or type provided that it enables the production of the
N-E peptide or a variant peptide thereof and/or the N-EVM peptide or a variant peptide thereof
from milk casein. That is, the protease may be an enzyme capable of hydrolyzing proteins and
polypeptides and, according to the present invention, encompass all proteases from
microorganisms such as bacteria, yeasts, and fungi, algae, plants, animals, and others.
Among such proteases, preferred is a neutral protease or an alkaline protease. The
neutral protease is a protease having the optimal pH in the neutral region, and the alkaline
protease is a protease having the optimal pH in the alkaline region. The reaction pH in the
method of the present invention is pH 6.5 to 8.5, preferably pH 7.0 to 8.0; thus, an enzyme is
preferable which has hydrolytic activity in such a pH range.
Among the above proteases, preferred is, for example, a serine protease. This enzyme
is a protease typically having catalytic triplet residues, i.e., serine, histidine, and aspartic acid, at
the active center.
The protease is preferably a microorganism-derived protease enabling the production
of. the N-E peptide or a variant peptide thereof and/or the N-EVM peptide or a variant peptide
thereof from milk casein with the above yield. For example, a protease derived from a
microorganism belonging to the genus Bacillus or the genus
Aspergillus, a lactic acid bacterium-
derived protease, or the like may be used as the enzyme in the method of the present invention.
Specifically, the preferable protease may include a protease derived from a microorganism
selected from the group consisting of
Bacillus licheniformis, Aspergillus sp., Aspergillus otyzae,
Aspergillus melleus, Lactobacillus helveticus, Lactobacillus bulgaricus, and Streptococcus
thermophilus. The enzyme may be in any forms such as a purified enzyme, a partially-purified
enzyme, a crude enzyme, and disrupted microbial cells (preferably, a freeze-dried product),
provided that it has the activity of cutting out the peptides from milk casein.
Because an enzyme has a substrate specificity, there are enzymes which may be
unsuitable for the method of the present invention, including, for example, animal enzymes such
as trypsin and pancreatin and proteases derived from Bacillus amyloliquefaciens, Bacillus
stearothermophilus, and others as stated in Examples hereinbelow.
A suitable protease for the method of the present invention can be selected, for example,
by, as stated in Example 1, dissolving about 10 mg of cow milk casein sodium in a phosphate
buffer solution (pH: 7.0 to 7.3), to which a test protease is then added so as to provide a weight
ratio of enzyme/casein of about 1/200,treating the buffer solution at a temperature of about 35°C
to about 50°C for about 1 to 4 hours, followed by terminating the reaction with trichloroacetic
acid, and calculating the content of desired peptides by separating the components by gradient
analysis using a reverse phase ODS column as a separation column and a 0.1% formic acid
aqueous solution and 0.1% formic acid-containing acetonitrile as eluents, detecting each peptide
with the mass spectrometer with the use of a high-performance liquid chromatograph triple
quadrupole mass spectrometer (LC/MS/MS, Waters TQD), and calculating the content thereof
using a calibration curve prepared using a synthetic peptide as a standard to determine a desired
peptide production.
Examples of proteases which may be used in the method of the present invention
Bacillus licheniform
include, but not limited to, the is-derived alkaline protease "Protin SD-AY10"
(trade name, from Amano Enzyme Inc.) and the Aspergillus
sp.-derived alkaline protease
"Sumizyme MP" (trade name, from Shinnihon Chemicals Corporation).
The enzymatic catalyst used in the method of the present invention may further contain
an enzyme having a peptidase activity. The enzyme having a peptidase activity has an activity
to produce the N-E peptide or a variant peptide thereof and/or the N-EVM peptide or a variant
peptide thereof from casein or an activity to produce the N-E peptide from the N-EVM peptide.
In fact, as shown from the time course of peptide production in Tables 1 and 4 stated in Examples
hereinbelow, the N-EVM peptide is first produced, followed by the production of the N-E
peptide; thus, the N-E peptide may probably be produced from the precursor N-EVM peptide
using enzymes having a peptidase activity contained in the commercial enzyme preparations
actually used.
The enzyme having a peptidase activity which may be used in combination with each
above protease is exemplified below.
Carboxypeptidases (EC3.4.16.-, EC3.4.17.-, EC3.4.18.-), which release one amino acid
from C-terminus of a peptide, such as, for example, carboxypeptidase A (EC3.4.17.1),
carboxypeptidase B (EC3.4.17.2), carboxypeptidase C (EC3.4.16.5), and carboxypeptidase Y
(EC3.4.16.5). Alternatively, peptidyl dipeptidases (EC3.4.15.-), which release two amino acids
from C-terminus of a peptide, such as, for example, peptidyl dipeptidase A (EC3.4.15.1), peptidyl
dipeptidase B (EC3.4.15.4), and peptidyl dipeptidase Dcp (EC3.4.15.5).
Carboxypeptidase A (EC3 .4.17.1) (http ://www. chem. qmul ac.uldiubmb/enzyme/
EC3/4/17/1.html) is considered to less easily degrade C-terminal -PE (-Pro-Glu) of the N-E
peptide (NIPPLTQTPVVVPPFLQ-PE) because although the enzyme releases a C-terminal
amino acid, it little or completely not acts on terminal -Asp, -Glu, -Arg, -Lys or -Pro, while for
the N-EVM peptide (NIPPLTQTPVVVPPFLQ-PE-VM), the C-terminal -VM (-Val-Met) can be
hydrolyzed to produce the N-E peptide.
Carboxypeptidase B (EC3 .4 .17.2) (http://www. chem. qmul ac.uIc/iubmb/enzyme/
EC3/4/17/2.html) is a typical carboxypeptidase enzyme catalyzing the hydrolysis of basic amino
acids; although the enzyme is not likely to directly act on the conversion of the N-EVM peptide to
the N-E peptide, it can contribute to the production of these peptides.
Carboxypeptidase Y (EC3.4.16.5) (http://www.chem.qmul.ac.uk/iubmb/enzyme/
EC3/4/15/1.html) can contribute to the production of peptides including the N-E peptide because
it has a wide substrate specificity.
Peptidyl dipeptidase A (EC3.4.15.1) (http://www.chem.qmul.ac.uldiubmb/enzyme/
EC3/4/15/1.html) is not considered to degrade C-terminal -PE (-Pro-Glu-) of the N-E peptide
(NIPPLTQTPVVVPPFLQ-PE) because it releases C-terminal dipeptide Xaa-Yaa of an
oligopeptide + Xaa-Yaa (where Xaa is an amino acid residue which is not Pro, and Yaa is an
amino acid residue which is not Asp or Glu), while for the N-EVM peptide
(NIPPLTQTPVVVPPFLQ-PE-VM), C-terminal -VM (-Val-Met) can be hydrolyzed to produce
the N-E peptide.
Peptidyl dipeptidase B (EC3.4.15.4) (http://www.chem.qmul.ac.uldiubmb/enzyme/
EC3/4/15/4.html) can contribute to the production of peptides including the N-E peptide because
it has a wide substrate specificity.
Peptidyl dipeptidase Dcp (EC3.4.15.5) (http://www.chem.qmul.ac.uldiubmb/enzyme/
EC3/4/15/5.html) can contribute to the production of peptides including the N-E peptide because
it has a wide substrate specificity.
Examples of the enzyme having a peptidase activity can include endoproteinase Glu-C
(EC3.4.21.19) and enzymes having the same activity in addition to the above exemplified
enzymes. The enzyme is an enzyme selectively hydrolyzing the C-terminus of glutamic acid
(Glu) in a protein or a peptide (http://www.chem.qmul.ac.uldiubmb/enzyme/
EC3/4/21/19. html).
The enzyme having a peptidase activity used in the method of the present invention
may be in any forms such as a purified enzyme, a partially-purified enzyme, a crude enzyme, and
disrupted microbial cells (preferably, a freeze-dried product) provided that it has the above
enzyme activity.
The enzymatic catalyst used in the method of the present invention may be preferably a
combination of at least one above-described protease and at least one above-described enzyme
having a peptidase activity. In this case, the way in which these enzymes are added is not
particularly limited; for example, they may be simultaneously present in the reaction system in
the hydrolysis reaction, or the way is used in which the protease is first added for reaction,
followed by adding the enzyme having a peptidase activity to the reaction system at the time
point that the production of the N-EVM peptide or a variant peptide thereof is almost maximal.
The enzymatic catalyst may also be immobilized on a support. The support is not
particularly limited provided that it can be typically used for the immobilization of enzymes.
The method of binding the enzyme to the support may be based on covalent bonding or non-
covalent bonding. In the case of covalent bonding, a reactive group such as cyanogen bromide,
N-hydroxymaleimide, phthalaldehyde, diisocyanate, or imide ester can be bound to a support,
followed by binding the enzyme to such a group. In the case of non-covalent binding, it can be
bound to a support in a way such as physical adsorption or van der Waals binding, ion binding,
cross-linking, inclusion, or microcapsules. Non-limited examples of the support include
minerals, metals, polymers, and polysaccharides. Minerals include, for example, pumice stone
and porosity glass. Metals include, for example, magnetic substance and ceramic. Polymers
include, for example, polyacrylamide gel, ion-exchange resin, photocrosslinkable resin
prepolymer, and urethane prepolymer. Polysaccharides include, for example, ic-carrageenan and
alginate.
The protease and the enzyme having a peptidase activity may be together or separately
immobilized to the support.
2.3 Reaction Condition
The enzymatic hydrolysis reaction may be carried out by a continuous method or a
batch method. The continuous method typically uses an immobilized enzymatic catalyst, while
the batch method uses a non-immobilized enzymatic catalyst.
When the immobilized enzymatic catalyst is used, the catalyst may be packed in a
column whose temperature can be controlled, and a casein solution is continuously applied
thereinto at a fixed flow rate. The reaction solution is sampled over time to monitor the
production of a desired peptide, and the reaction solution is recovered once the highest production
content is reached.
In the case of the non-immobilized enzymatic catalyst, a casein solution and an
enzymatic catalyst are added to a reaction vessel equipped with a stirrer whose temperature can
be controlled, and reacted by a batch method to similarly monitor the production of a desired
peptide, and the reaction solution is recovered once the highest production content is reached.
The milk casein as a substrate raw material may be an animal milk casein as described
above, preferably cow casein, and is preferably in the form of a milk casein salt easily soluble in
water, for example, milk casein sodium. Typically, the milk casein sodium may be prepared, for
example, by suspending casein in water and adding aqueous sodium hydroxide thereto little by
little under stirring, or adding casein to aqueous sodium hydroxide little by little, and, if necessary,
the pH is adjusted after the addition of casein. Alternatively, in place of milk casein salt, milk
containing casein may be used directly or after concentration.
Because an enzyme has an optimal pH, a buffer solution is preferably contained so that
pH is not changed during reaction; however, if the control of pH is possible, the buffer solution is
not always required. The buffer solution may be a buffer solution capable of maintaining a pH
ranging from the neutral region to the weak alkaline region, for example, a pH of 6.5 to 8.5,
preferably a pH of 7.0 to 8.0. Such buffer solutions include, for example, a phosphate buffer
solution, a Tris-hydrochloric acid buffer solution, and an ammonium chloride buffer solution.
The buffer solution may be added in an amount capable of showing a buffer action to a substrate
raw material solution.
The weight ratio of each enzyme/milk casein may be 1/50 to 1/2,000, preferably 1/100
to 1/1,000, more preferably 1/150 to 1/250, and is, for example, 1/200. Alternatively, the ratio
may be 1 to 100,000 units (U), preferably 10 to 50,000 units (U), more preferably 50 to 10,000
units (U) (where 1 U refers to the enzyme activity of degrading 1 innol of a substrate for 1
minute) as protease per gram of milk casein. It may be adjusted depending on the type and
properties of the enzyme.
The reaction temperature may be a temperature not inactivating the enzyme, and may
be typically 30 to 60°C, preferably 45 to 55°C. If the enzyme is a heat-resistant enzyme, a
temperature exceeding the range can also be used. Iit is preferably a temperature at which
casein as a substrate is not coagulated.
The reaction time varies depending on the type of the enzyme, the temperature, and
other factors. It may be typically 1 to 12 hours, preferably 2 to 10 hours, more preferably 3 to 7
hours.
The reaction is monitored, and the reaction may be performed until the production
amount of a desired peptide reaches a predetermined value.
According to the method of the present invention, the production yield of the N-E
peptide (SEQ ID NO: 1) or a variant peptide thereof is 2 to 10%, or more, and/or the production
yield of the N-EVM peptide (SEQ ID NO: 2) or a variant peptide thereof is 10 to 50%, or more,
and the total production yield of the N-E peptide or a variant peptide thereof and the N-EVM
peptide or a variant peptide thereof is 15 to 60%, or more. Particularly, when the conversion of
the N-EVM peptide to the N-E peptide is achieved by the action of the enzyme having a
peptidase activity, the production yield of the N-E peptide may be predicted to be further
increased. According to the method of the present invention, a mixture of the peptides is
typically produced. It is preferable to obtain a mixture rich in the N-E peptide.
As used herein, "production yield" is the percentage (%) of the molar concentration of
each produced peptide to the molar concentration calculated from the average molecular weight
of (3-casein. Specifically, for example, the production yield has been determined to be about
% by taking the average molecular weight of p-casein in cow milk casein containing the
sequences of the N-E peptide (SEQ ID NO: 1), the N-EVM peptide (SEQ ID NO: 2), and the S-
SVL peptide (SEQ ID NO: 3) to be 25,100, taking the peptide molecular weight to be 2,086.4 for
the N-E peptide (SEQ ID NO: 1), 2,316.8 for the N-EVM peptide (SEQ ID NO: 2) and 2,550.0
for the S-SVL peptide (SEQ ID NO: 3), and further taking the content ratio of f3-casein in the cow
milk casein.
According to the method of the present invention, the S-SVL peptide (SEQ ID NO: 3)
or a variant peptide thereof can be produced in a production yield of about 3 to 70% by using
different types or amounts of the enzyme (see FIGs. 1 to 4). For example, this peptide can be
produced in a yield of about 65% by about 4-hours reaction when the enzyme preparation "Protin
SD-AY10" (trade name, 90,000 PU/g or more, from Amano Enzyme Inc.) is used for hydrolysis
reaction at enzyme/casein = 1/1,000 (weight ratio) {see Table 5). On the other hand, it may be
produced in a yield of 11% by4-hours reaction when the same enzyme is used at enzyme/casein =
1/200 (weight ratio) for the same hydrolysis reaction (see Table 4). The yield of the S-SVL
peptide decreases from about 10% to 0% with the lapse of reaction time when "Sumizyme MP"
(trade name, from Shinnihon Chemicals Corporation) is used as an enzyme preparation (see
Tables 6 and 7).
After the completion of reaction, the enzymatic catalyst is typically inactivated. The
inactivation can be performed, for example, by heating at a temperature of about 60°C to about
75°C.
3. Hydrolysate
The present invention further provides a hydrolysate produced by the above method,
containing at least one of the peptides consisting of the amino acid sequences shown in SEQ ID
NOS: 1 to 3 or peptides consisting of amino acid sequences containing deletion, substitution, or
addition of one or two amino acid residues in the amino acid sequence of SEQ ID NO: 1 to 3, in
an amount of about 0.5 mg or more per gram of a dry solid of the hydrolysate.
Here, "hydrolysate" generally refers to a reaction solution (preferably the reaction
solution after enzyme inactivation) obtained by the hydrolysis reaction of milk casein by the
above method or a concentrate thereof.
This hydrolysate is characterized by containing about 0.5 mg or more, preferably about
1.0 mg or more, more preferably about 10 to 15 mg or more of the N-E peptide or a variant
peptide thereof per gram of the dry solid of the hydrolysate.
The hydrolysate may further contain about 1.0 to 20 mg, or more of the N-EVM
peptide or a variant peptide thereof per gram of a dry solid of the hydrolysate and/or about 1.0 to
mg, or more of the S-SVL peptide or a variant peptide thereof per gram of a dry solid of the
hydrolysate.
Preferably, the total content of the N-E peptide or a variant peptide thereof and the N-
EVM peptide or a variant peptide thereof in the hydrolysate may be about 1.5 to 4.0 mg, or more,
preferably about 10 to 30 mg, or more per gram of a dry solid of the hydrolysate.
The hydrolysate of the present invention may be directly used, or may be concentrated
by ultrafiltration or the like, or may be dried. The drying is preferably freeze drying, and may be
freeze drying after concentration.
As needed, each of the N-E peptide or a variant peptide thereof, the N-EVM peptide or
a variant peptide thereof, and the S-SVL peptide or a variant peptide thereof may be isolated from
the hydrolysate, or the N-E peptide or a variant peptide thereof, the N-EVM peptide or a variant
peptide thereof, and the S-SVL peptide or a variant peptide thereof may be separated from each
other. In the latter case, the N-E peptide or a variant peptide thereof and the N-EVM peptide or
a variant peptide thereof can be separated as a mixture, and the mixture may be concentrated so
that it is rich in these components, preferably rich in the N-E peptide or a variant peptide thereof.
The isolation and concentration of each peptide may be performed by using a common
technique, for example, techniques including gel filtration, chromatography such as ion-exchange
chromatography, affinity chromatography, silica gel chromatography, or I-IPLC, crystallization,
salting-out, organic solvent precipitation, and ultrafiltration alone or in combination. The buffer
component and the like may also be removed by this operation.
4. Application to Pharmaceutical and Food
The hydrolysate or the above peptides of the present invention each have a brain
function-improving action, for example, have the action to improve or prevent amnesia as well as
enhancing memory. As described above, amnesia refers to memory decline and is one of
memory disorders. Such an effect is demonstrated in Examples hereinbelow. Thus, the
hydrolysate or the above peptides of the present invention can be used for treating or preventing
symptoms and diseases caused by decreased brain function, for example, diseases or symptoms
such as depression, schizophrenia, deliria, and dementias (cerebrovascular dementia, Alzheimer
disease, and the like), and can also be used when amnesia symptoms are observed although such
diseases are not developed.
4.1 Application to Pharmaceutical
The present invention further provides a pharmaceutical composition comprising the
above hydrolysate and a pharmaceutically acceptable carrier.
The hydrolysate at least comprises the N-E peptide among the above peptides. The
hydrolysate may be contained in the form of a liquid or a solid in the carrier.
According to an embodiment of the present invention, the pharmaceutical composition
is for improving brain function. More specifically, the pharmaceutical composition is for
improving or preventing amnesia.
Possible indications include symptoms and diseases caused by decreased brain function,
for example, diseases or symptoms such as depression, schizophrenia, deliria, and dementias
(cerebrovascular dementia, Alzheimer disease, and the like), as described above.
The amount of the hydrolysate as an active ingredient in the pharmaceutical
composition of the present invention is not particularly limited. It may be typically preferably
0.1 pg/kg body weight to 1 mg/kg body weight in terms of the weight of each of the peptides
although not being limited to the range. The dose to be administered may be determined
according to age, sex, the degree of symptoms, and the like.
The route of administration may be, for example, oral administration, intravenous
administration, transmucosal administration, intranasal administration, or intrarectal
administration, and it is preferably oral administration. The administration may be performed to
subjects in once daily or a plurality of times daily in divided doses.
The subject may be typically a human, and also encompasses a mammal other than a
human, for example, a pet animal such as a dog.
The dosage form (or preparation) may be any form of a solid preparation and a liquid
preparation; examples thereof include tablet, pill, capsule, powder, granules, solution, injection,
epipastic, and aerosol preparation.
The pharmaceutically acceptable carrier includes an excipient or a diluent; examples
thereof include dextrans, saccharose, lactose, maltose, xylose, trehalose, mannitol, sorbitol,
gelatin, carboxymethylcellulose, caxboxyethylcellulose, hydroxypropylmethylcellulose, gum
arabic, guar gum, tragacanth, acrylate copolymer, ethanol, saline, and Ringer's solution.
In addition to the above carrier, additives may be added such as preservative, stabilizer,
binder, pH regulator, buffer, thickening agent, agelling agent, and antioxidant, if necessary.
These additives may be preferably those used in preparing pharmaceutical products.
The pharmaceutical composition of the present invention may be used in combination
with another pharmaceutical product having a brain function-improving effect. Examples of
Hypnotics.
4.2 Application to Food or Feed
such pharmaceutical product include the following, and it is preferably a commercially available
pharmaceutical product.
Therapeutic drugs for dementia such as, for example, an acetylcholinesterase inhibitor
(donepezil, galanthamine, rivastigmine, tacrine, or the like) and an NMDA receptor antagonist
(memantine or the like).
Antianxiety drugs such as, for example, a benzodiazepine antianxiety drug.
Antidepressant drugs such as, for example, a selective serotonin reuptake inhibitor
(SSRI), serotonin/norepineplrine (noradrenaline) reuptake inhibitor (SNRI), a tricyclic
antidepressant (TCA), a tetracyclic antidepressant, a triazolopyridine antidepressant (SARI), a
monoamine oxidase inhibitor (MAO inhibitor), a noradrenergic/specific serotonergic
antidepressant (NaSSA), and a norepinephrine/dopamine reuptake inhibitor (NDRI).
Antipsychotic drugs.
These pharmaceutical products can each be administered at any time point of with,
before, and after the administration of the pharmaceutical composition of the present invention.
The dose thereof is preferably a dose as instructed by a pharmaceutical supplier when it is a
commercially available pharmaceutical product.
The present invention further provides a food or drink or a supplement containing the
above hydrolysate.
Such a food or drink or a supplement can be a functional food or drink or a health food
because the hydrolysate has a brain function-improving action, especially has an amnesia-
improving or -preventing action. Examples thereof include a food or drink or a supplement
obtained by adding the hydrolysate to lactic acid bacterium-fermented solution. The supplement
may be one of the food classifications, consisting of a dietary supplement, and herein refers to a
function auxiliary substance capable of providing a brain function-improving action.
The hydrolysate can also be used as a material for feed for a non-human animal. For
example, for a dog, for example, the hydrolysate may be blended upon the production of a dog
food.
Examples of the food or drink include, but not limited to, various drinks, various dairy
products such as yogurt, cheese, butter, and lactic acid bacterium-fermented products, liquid food,
jelly, candy, retort pouched food, tablet confectionary, cookies, sponge cake, bread, biscuits, and
chocolate.
The functional food or drink, the health food, or the supplement may be in the form of
a solid, a gelatinous material, or a liquid, for example, may be in the form of any of various
processed foods or drinks, powders, tablets, pills, capsules, jellies, granules, or the like.
The food or drink may be properly blended with food additives such as carbohydrate,
protein, lipid, vitamins, minerals, a saccharide (glucose or the like), a natural or artificial
sweetener, citric acid, carbonated water, fruit juice, a stabilizer, a preservative, a binder, a
thickener, and an emulsifier.
The blending amount of the hydrolysate is not particularly limited. It may be 0.1
pig/kg body weight to 1 mg/kg body weight in terms of the weight of each of the peptides.
Alternatively, the blending amount may be 10 i..tg to 500 mg, more preferably 100 1.cg to 100 mg
per 100 g of the food or drink although being not limited thereto. The intake amount for each
ingestion of a food, for example, a functional food, can also be further reduced compared to the
above amount, depending on the number of ingestions per day. The suitable intake amount can
be further adjusted considering various factors.
The food or drink or the supplement according to the present invention may be further
blended with a combination of other materials and compounds described below, which are
considered to have brain function-improving actions.
Food ingredients such as, for example, ginkgo leaf extract, arachidonic acid (ARA),
GABA, theanine, ceramide, caffeine, carnitine, oc-glycerylphosphorylcholine (a-GPC), Bacopa
inonniera, DHA-bound phospholipids, phosphatidylserine (PS), phosphatidylcholine, St. John's
wort, astaxanthin, niacin, pyrroloquinoline quinone (PQQ), and coenzyme Q10 (CoQ10).
Unsaturated fatty acids such as, for example, docosahexaenoic acid (DHA) and
eicosapentaenoic acid (EPA); Polyphenols such as, for example, resveratrol.
Chlorogenic acid and the like.
Catechins and the like.
The present invention will be described below in further detail with reference to
Examples. However, the scope of the invention is not limited to these Examples.
Method for Enzymatically Preparing Peptides Asn-Ile-Pro-Pro-Leu-Thr-Gln-Thr-Pro-Val-Val-
Val-Pro-Pro-Phe-Leu-Gln-Pro-Glu (NIPPLTQTPVVVPPFLQPE; SEQ ID NO: 1), Asn-Ile-Pro-
mg of cow milk-derived casein sodium was dispersed and dissolved in 1 ml of
final concentration of 1% to the reaction solution to terminate the reaction. Subsequently, each
peptide contained in the resultant solution was quantitated by, with the use of a high-performance
liquid chromatograph triple quadrupole mass spectrometer (LC/MS/MS, Waters TQD),
separation column and a 0.1% formic acid aqueous solution and 0.1% formic acid-containing
The blending amount of these materials and compounds is within the known range in
which efficacy is confirmed.
EXAMPLES
(EXAMPLE 1)
Pro-Leu-Thr-Gln-Thr-Pro-Val-Val-Val-Pro-Pro-Phe-Leu-Gln-Pro-Glu-Val-Met (NIPPLT
QTPVVVPPFLQPEVM; SEQ ID NO: 2), and Ser-Trp-Met-His-Gln-Pro-His-Gln-Pro-Leu-Pro-
Pro-Thr-Val-Met-Phe-Pro-Pro-Gin-Ser-Val-Leu (SWMHQPHQPLPPTVMFPPQSVL; SEQ ID
NO: 3) by Hydrolysis of Casein Using Microorganism-Derived Enzyme
phosphate buffer solution, pH 7.0 to 7.3, and adjusted to a temperature of 50°C to prepare a
substrate solution. Each of the commercially available enzymes shown in Table 1 was added to
the resultant substrate solution at a weight ratio of each enzyme/casein of 1/100 to 1/400, and then
reacted at 50°C for 4 hours, followed by adding a 10% aqueous trichloroacetic acid solution to a
separating the components by gradient analysis using a reverse phase ODS column as a
acetonitrile as eluents, detecting each peptide with the mass spectrometer, and calculating the
content thereof using a calibration curve prepared using a synthetic peptide as a standard.
The yield of the peptides of SEQ ID NOs: 1, 2, and 3 was approximately determined
by taking the I3-casein concentration in the 10 mg/ml casein sodium solution used for the analysis
to be 3 mg/ml and taking the molecular weight of (3-casein to be 25,100, the molecular weight of
molecular weight of SEQ ID NO: 3 to be 2550.0.
Val-Val-Val-Pro-Pro-Phe-Leu-Gln-Pro-Glu (NIPPLTQTPVVVPPFLQPE; SEQ ID NO: 1), Asn-
Ile-Pro-Pro-Leu-Thr-Gln-Thr-Pro-Val-Val-Val-Pro-Pro-Phe-Leu-Gln-Pro-Glu-Val-Met (NIPPLT
QTPVVVPPFLQPEVM; SEQ ID NO: 2), and Ser-Trp-Met-His-Gln-Pro-His-Gln-Pro-Leu-Pro-
Pro-Thr-Val-Met-Phe-Pro-Pro-Gln-Ser-Val-Leu (SWMHQPHQPLPPTVMFPPQSVL; SEQ ID
NO: 3) were obtained in high yields by the hydrolysis of casein using each of the enzymes having
Bacillus and the genus
Aspergillus.
SEQ ID NO: 1 to be 2086.4, the molecular weight of SEQ ID NO: 2 to be 2316.8, and the
The results shown in Table 1 demonstrated that Asn-Ile-Pro-Pro-Leu-Thr-Gln-Thr-Pro-
neutral protease or alkaline protease activity, derived from the genus
[Table 1]
(pH of neutral region)
Enzyme name
SEQ ID NO: 1 SEQ ID NO: 2 SEQ ID NOS: 1 + 2
SEQ ID NO: 3
Origin
(product name) yield [%]
yield [%] yield [%]
yield [%]
Bacillus licheniformis Protin SD-AY10 (1) 8.4 53.0
61.3 10.9
Aspergillus sp. Sumizyme MP(2)
12.0 20.8 32.8.
Aspergillus oryzae Sumizyme LP50 (2) 3.3
24.5 27.8 2.7
Bacillus licheniformis Protease P5459 (3) 4.5 22.8 27.3 3.9
Aspergillus melleus Protease P "amano" 3SD 2.9 24.3 27.2 1.2
Aspergillus oryzae Sumizyme FP (2) 2.3 20.8 23.1
Aspergillus oryzae Proteax (1) 5.5 15.3 20.8 0.1
Aspergillus oryzae Umamizyme G (1) 3.9 16.1
.0 0.4
Aspergillus oryzae Protease A "amano" SD (l) 0.5 10.8 11.3
Aspergillus oryzae Protease M "amano" SD (1) 0.1 2.8
2.9 1.0
Bacillus amyloliquefaciens Protin SD-NY10 (1) 0.1 0.1 0.1
Bacillus stearothermophilus Thermoase PC10F (1) 0.0 0.0
0.0 0.0
(1) From Amano Enzyme Inc., (2) From SHINNIHON CHEMICALS Corporation, (3) From Sigma-Aldrich Corporation
(COMPARATIVE EXAMPLE 1)
Method for Enzymatically Preparing Peptides Asn-Ile-Pro-Pro-Leu-Thr-Gln-Thr-Pro-Val-Val-
Val-Pro-Pro-Phe-Leu-Gln-Pro-Glu (NIPPLTQTPVVVPPFLQPE; SEQ ID NO: 1), Asn-Ile-Pro-
Pro-Leu-Thr-Gln-Thr-Pro-Val-Val-Val-Pro-Pro-Phe-Leu-Gln-Pro-Glu-Val-Met
(NIPPLTQTPVV'VPPFLQPEVM; SEQ ID NO: 2), and Ser-Trp-Met-His-Gln-Pro-His-Gln-Pro-
Leu-Pro-Pro-Thr-Val-Met-Phe-Pro-Pro-Gln-Ser-Val-Leu (SWIVIHQPHQPLPPTVMFFPQS VL;
SEQ ID NO: 3) by Hydrolysis of Casein Using Acidic Protease
mg of cow milk-derived casein sodium was dispersed and dissolved in 1 ml of
acetate buffer solution, pH 4.0, and adjusted to a temperature of 40°C to prepare a substrate
solution. Each of the commercially available enzymes shown in Table 2 was added to the
resultant substrate solution at a weight ratio of each enzyme/casein of 1/200, and then reacted at
40°C for 3 hours, followed by adding a 10% aqueous trichloroacetic acid solution to a final
concentration of 1% to the reaction solution to terminate the reaction. Subsequently, each
peptide contained in the resultant solution was quantitated by, with the use of a high-performance
liquid chromatograph triple quadrupole mass spectrometer (LC/MS/MS, Waters TQD), separating
the components by gradient analysis using a reverse phase ODS column as a separation column
and a 0.1% formic acid aqueous solution and 0.1% formic acid-containing acetonitrile as eluents,
detecting each peptide with the mass spectrometer, and calculating the content thereof using a
calibration curve prepared using a synthetic peptide as a standard. The yield of each peptide was
approximately determined as described in Example 1.
The results shown in Table 2 demonstrated that Asn-Ile-Pro-Pro-Leu-Thr-Gln-Thr-Pro-
Val-Val-Val-Pro-Pro-Phe-Leu-Gln-Pro-Glu (NIPPLTQTPVVVPPFLQPE; SEQ ID NO: 1), Asn-
Ile-Pro-Pro-Leu-Thr-Gln-Thr-Pro-Val-Val-Val-Pro-Pro-Phe-Leu-Gln-Pro-Glu-Val-Met (SEQ ID
NO: 2), and Ser-Trp-Met-His-Gln-Pro-His-Gln-Pro-Leu-Pro-Pro-Thr-Val-Met-Phe-Pro-Pro-Gln-
Ser-Val-Leu (SWMHQPHQPLPPTVMFPPQSVL; SEQ ID NO: 3) were little produced by the
hydrolysis of casein using each of the enzymes having an acidic protease activity.
[Table 2]
(pH of acidic region)
Enzyme name SEQ ID NO: 1 SEQ ID NO: 2
SEQ ID NOS: 1 + 2
SEQ ID NO: 3
Origin
(product name) yield [%] yield [%]
yield [%] yield [%]
Aspergilius saitoi
Protease (P2143) (1) 0.0 0.0
0.0 0.1
Rhizopus sp. Protease (P0107) (1) 0.0
0.0 0.0 0.0
porcine stomach mucosa Pepsin (P7000) (1) 0.0 0.0
0.0 0.4
(1) From Sigma-Aldrich Corporation
(COMPARATIVE EXAMPLE 2)
Method for Enzymatically Preparing Peptides Asn-Ile-Pro-Pro-Leu-Thr-Gln-Thr-Pro-Val-Val-
Pro-Leu-Thr-Gln-Tlir-Pro-Val-Val-Val-Pro-Pro-Phe-Leu-Gln-Pro-Glu-Val-Met
Leu-Pro-Pro-Thr-Val-Met-Phe-Pro-Pro-Gln-Ser-Val-Leu (SWMHQPHQPLPPTVMFPPQS VL;
SEQ ID NO: 3) by Hydrolysis of Casein Using Each of Mammal-Derived Pancreatin and Trypsin
solution. Each of the commercially available enzymes pancreatin and trypsin shown in Table 3
each peptide contained in the resultant solution was quantitated by, with the use of a high-
separation column and a 0.1% formic acid aqueous solution and 0.1% formic acid-containing
acetonitrile as eluents, detecting each peptide with the mass spectrometer, and calculating the
content thereof using a calibration curve prepared using a synthetic peptide as a standard. The
yield of each peptide was approximately determined as described in Example 1.
Ile-Pro-Pro-Leu-Thr-Gln-Thr-Pro-Val-Val-Val-Pro-Pro-Phe-Leu-Gln-Pro-Glu-Val-Met (NIPPLT
QTPVVVPPFLQPEVM; SEQ ID NO: 2), and Ser-Trp-Met-His-Gln-Pro-His-Gln-Pro-Leu-Pro-
Val-Pro-Pro-Phe-Leu-Gln-Pro-Glu (NIPPLTQTPVVVPPFLQPE; SEQ ID NO: 1), Asn-Ile-Pro-
(NIPPLTQTPVVVPPFLQPEVM; SEQ ID NO: 2), and Ser-Trp-Met-His-Gln-Pro-His-Gln-Pro-
mg of cow milk-derived casein sodium was dispersed and dissolved in 1 ml of
phosphate buffer solution, pH 7.3, and adjusted to a temperature of 50°C to prepare a substrate
was added to the resultant substrate solution at a weight ratio of each enzyme/casein of 1/200, and
then reacted at 50°C for 4 hours, followed by adding a 10% aqueous trichloroacetic acid solution
to a final concentration of 1% to the reaction solution to terminate the reaction. Subsequently,
performance liquid chromatograph triple quadrupole mass spectrometer (LC/MS/MS, Waters
TQD), separating the components by gradient analysis using a reverse phase ODS column as a
The results shown in Table 3 demonstrated that Asn-Ile-Pro-Pro-Leu-Thr-Gln-Thr-Pro-
Val-Val-Val-Pro-Pro-Phe-Leu-Gln-Pro-Glu (NIPPLTQTPVVVPPFLQPE; SEQ ID NO: 1), Asn-
Pro-Thr-Val-Met-Phe-Pro-Pro-Gln-Ser-Val-Leu (SWMHQPHQPLPPTVMFPPQSVL; SEQ ID
NO: 3) were little produced by the hydrolysis of casein using each of pancreatin and trypsin,
mammal-derived enzymes.
[Table 3]
Enzyme name
SEQ ID NO: 1 SEQ ID NO: 2 SEQ ID NOS: 1 + 2
SEQ ID NO: 3
Origin
(product name) yield [%] yield [%] yield [%]
yield [%]
porcine pancreas Pancreatin (P1750) (1) 0.3 6.5
6.8 0.0
bovine pancreas Trypsin (208-13954) (2) 0.0 0.0 0.0
(1) From Sigma-Aldrich Corporation, (2) From Wako Pure Chemical Industries Ltd.
(EXAMPLE 2)
Study of Optimal Reaction Time for Production of Asn-Ile-Pro-Pro-Leu-Thr-Gln-Thr-Pro-Val-
Val-Val-Pro-Pro-Phe-Leu-Gln-Pro-Glu (NIPPLTQTPVVVPPFLQPE; SEQ ID NO: 1), Asn-Ile-
Pro-Pro-Leu-Thr-Gln-Thr-Pro-Val-Val-Val-Pro-Pro-Phe-Leu-Gln-Pro-Glu-Val-Met
(NIPPLTQTPVVVPPFLQPEVM; SEQ ID NO: 2), and Ser-Trp-Met-His-Gln-Pro-His-Gln-Pro-
Leu-Pro-Pro-Thr-Val-Met-Phe-Pro-Pro-Gln-Ser-Val-Leu (SWMHQP HQPLPPTVMFPPQSVL;
SEQ ID NO: 3)
mg of cow milk-derived casein sodium was dispersed and dissolved in 1 ml of
phosphate buffer solution, pH 7.3, and adjusted to a temperature of 50°C to prepare a substrate
solution. A Bacillus licheniformis-derived enzyme, Protin SD-AY10 (Amano Enzyme Inc.),
was added to the resultant substrate solution at a weight ratio of the enzyme/casein of each of
1/200 and 1/1,000, and then reacted at 50°C for 7 hours. During the reaction, the reaction
solution was recovered over time every one hour. Upon the recovery, a 10% aqueous
trichloroacetic acid solution was added to a final concentration of 1% to the reaction solution to
terminate the reaction. Subsequently, each peptide contained in the resultant solution was
quantitated by, with the use of a high-performance liquid chromatograph triple quadrupole mass
spectrometer (LC/MS/MS, Waters TQD), separating the components by gradient analysis using a
reverse phase ODS column as a separation column and a 0.1% formic acid aqueous solution and
0.1% formic acid-containing acetonitrile as eluents, detecting each peptide with the mass
spectrometer, and calculating the content thereof using a calibration curve prepared using a
synthetic peptide as a standard. The yield of each peptide was approximately determined as
described in Example 1.
From the results shown in Table 4, Table 5 and the total of the yields of
Asn-Ile-Pro-Pro-Leu-Thr-Gln-Thr-Pro-Val-Val-Val-Pro-Pro-Phe-Leu-Gln-Pro-Glu (NIPPLTQT
PVVVPPFLQPE; SEQ ID NO: 1) and Asn-Ile-Pro-Pro-Leu-Thr-Gln-Thr-Pro-Val-Val-Val-Pro-
Pro-Phe-Leu-Gln-Pro-Glu-Val-Met (NIPPLTQTPVVVPPFLQPEVM; SEQ ID NO: 2) had the
maximum values 4 hours after the start of reaction when the enzyme was added at the weight ratio
of enzyme/casein of 1/200 (. Similarly, the yield of Ser-Trp-Met-His-Gln-Pro-His-Gln-
Pro-Leu-Pro-Pro-Thr-Val-Met-Phe-Pro-Pro-Gln-Ser-Val-Leu (SWMHQPHQPLPPTVMFPPQS
VL; SEQ ID NO: 3) had the maximum value 4 hours after the start of reaction when the enzyme
was added at the weight ratio of enzyme/casein of 1/1,000 (. The above demonstrated
that these peptides were obtained with high yields by terminating the reaction in a short time.
[Table 4]
(Amount of enzyme added: 1/200 of total)
Enzyme name Reaction time SEQ ID NO: 1 SEQ ID NO: 2
SEQ ID NOS: 1 + 2 SEQ ID NO: 3
Origin
(product name) [hr] yield [%]
yield [%] yield [%] yield [%]
1 0.9 12.0 12.9
17.9
1.7 17.7 19.5 9.9
3 2.8 19.9
22.7 6.3
Bacillus licheniformis Protin SD-AY10 4 8.4 53.0 61.3
.9
10.5 47.8
58.3 8.1
6 11.4 42.0 53.4
7 12.7 38.2 50.9
[Table 5]
(Amount of enzyme added: 1/1,000 of total)
Enzyme name Reaction time SEQ ID NO: 1 SEQ ID NO: 2 SEQ ID NOS: 1 + 2 SEQ ID NO: 3
Origin
(product name) [hr] yield [%] yield PM yield [%] yield [%]
1 4.7 5.3 30.4
2 0.7 14.5 15.2 52.4
3 0.8 22.7 23.5 61.5
Bacillus licheniformis Protin SD-AY10 4 1.2 30.2 31.4 63.9
1.6 35.7 37.3 62.4
6 2.0 40.4 42.4 60.8
7 2.4 42.2
44.6 56.0
(EXAMPLE 3)
Val-Val-Pro-Pro-Phe-Leu-Gln-Pro-Glu (SEQ ID NO: 1), Asn-Ile-Pro-Pro-Leu-Thr-Gln-Thr-Pro-
phosphate buffer solution, pH 7.0 or 8.0, and adjusted to a temperature of 50°C to prepare a
sp.-derived enzyme, Sumizyme MP (from Shinnihon
Chemicals Corporation), was added to the resultant substrate solution at a weight ratio of the
terminate the reaction. Subsequently, each peptide contained in the resultant solution was
0.1% formic acid-containing acetonitrile as eluents, detecting each peptide with the mass
described in Example 1.
From the results shown in Table 6, Table 7 and the total of the yields of
1) and Asn-Ile-Pro-Pro-Leu-Thr-Gln-Thr-Pro-Val-Val-Val-Pro-Pro-Phe-Leu-Gln-Pro-Glu-Val-
Met (SEQ ID NO: 2) had the maximum values 2 to 3 hours after the start of reaction in both the
one hour after the start of reaction; however, it decreased with the lapse of time. The above
short time.
Study of Optimal Reaction Time for Production of Asn-Ile-Pro-Pro-Leu-Thr-Gln-Thr-Pro-Val-
Val-Val-Val-Pro-Pro-Phe-Leu-Gln-Pro-Glu-Val-Met (SEQ ID NO: 2), and Ser-Trp-Met-His-Gln-
Pro-His-GM-Pro-Leu-Pro-Pro-Thr-Val-Met-Phe-Pro-Pro-Gln-Ser-Val-Leu (SEQ ID NO: 3)
mg of cow milk-derived casein sodium was dispersed and dissolved in 1 ml of
substrate solution. An Aspergillus
enzyme/casein of 1/200, and then reacted at 50°C for 7 hours. During the reaction, the reaction
solution was recovered over time every one hour. Upon the recovery, a 10% aqueous
trichloroacetic acid solution was added to a fmal concentration of 1% to the reaction solution to
quantitated by, with the use of a high-performance liquid chromatograph triple quadrupole mass
spectrometer (LC/MS/MS, Waters TQD), separating the components by gradient analysis using a
reverse phase ODS column as a separation column and a 0.1% formic acid aqueous solution and
spectrometer, and calculating the content thereof using a calibration curve prepared using a
synthetic peptide as a standard. The yield of each peptide was approximately determined as
Asn-Ile-Pro-Pro-Leu-Thr-Gln-Thr-Pro-Val-Val-Val-Pro-Pro-Phe-Leu-Gln-Pro-Glu (SEQ ID NO:
cases of pH 7.0 and pH 8.0. Similarly, the yield of Ser-Trp-Met-His-Gln-Pro-His-Gln-Pro-Leu-
Pro-Pro-Thr-Val-Met-Phe-Pro-Pro-Gln-Ser-Val-Leu (SEQ ID NO: 3) had the maximum value
demonstrated that these peptides were obtained with high yields by terminating the reaction in a
[Table 6]
(Reaction pH: 7.0)
Enzyme name Reaction time SEQ ID NO: 1 SEQ ID NO: 2 SEQ ID NOS: 1 + 2
SEQ ID NO: 3
Origin
(product name) [hr] yield [%] yield [%] yield [%]
yield [%]
1 4.9 26.5 31.4
2 8.9 29.4 38.3 1.5
3 11.1 24.1
.2 0.5
Aspergillus sp. Sumizyme MP 4 10.5 19.8 30.3
10.4 13.9
24.3 0.1
6 9.0 12.0 21.0 0.1
7 7.5 7.9 15.4
[Table 7]
(Reaction pH: 8.0)
Enzyme name Reaction time SEQ ID NO: 1 SEQ ID NO: 2 SEQ ID NOS: 1 + 2
SEQ ID NO: 3
Origin
(product name) [hr] yield [%] yield [%] yield [%] yield [%]
1 8.4 12.5 20.9 3.3
2 14.2 11.4 25.6 2.1
3 16.4 8.7 25.2
Aspergillus sp. Sumizyme MP 4 15.7 6.9 22.6 0.9
14.8 5.5
.3 0.5
6 12.6 4.1 16.6 0.1
7 10.0 3.2 13.2
(EXAMPLE 4)
Amnesia-Preventing Action of Asn-Ile-Pro-Pro-Leu-Thr-Gln-Thr-Pro-Val-Val-Val-Pro-Pro-Phe-
Leu-Gln-Pro-Glu (NIPPLTQTPVVVPPFLQPE; SEQ ID NO: 1)
Male ddY mice (about 7 weeks old) were used (n=15 to 75), and provided with food and
water ad libitum. Asn-Ile-Pro-Pro-Leu-Thr-Gln-Thr-Pro-Val-Val-Val-Pro-Pro-Phe-Leu-Gln-
Pro-Glu was used as a test substance in amounts of 0.05 nmol/kg body weight (0.1 lig/kg body
weight), 0.5 nmol/kg body weight (1 pig/kg body weight), 1.5 nmol/kg body weight (3 jig/kg body
weight), 5 nmoVkg body weight (10 µg/kg body weight), 50 nmollkg body weight (100 µg/kg
body weight), and 500 nmol/kg body weight (1,000 pg/kg body weight). The test substance was
administered at a single dose orally to mice 60 minutes before performing a Y-shaped maze test
for evaluating spontaneous alternation behavior. Thirty minutes before performing the Y-shaped
maze test, scopolamine was subcutaneously administered at a dose of I mg/kg body weight into
the back to induce brain dysfunction (memory disorder and/or cognition disorder) in mice. In
the Y-shaped maze test, as an experiment device, a Y-shaped maze was used in which the length
for each arm was 40 cm; the wall height was 12 cm; the floor width was 3 cm; the upper part
width was 10 cm; and three arms were connected to each other at an angle of 120°. Each mouse
was placed in the end of any of the arms of the Y-shaped maze and allowed to explore freely in
the maze over 8 minutes, and the sequence of the arms to which the mouse moved was recorded.
The number of movements of the mouse to the arms within the measurement time was counted
and used as the total number of entries; in the sequence, the combination in which three different
arms were selected in succession (for example, with the three arms respectively called A, B, and C,
if the sequence of the arms entered is ABCBACACB, the count is 4 inclusive of overlapping) was
investigated, and the count number was used as the number of spontaneous alternation behaviors.
The change in spontaneous alternation behavior (%) was calculated by dividing the number of
spontaneous alternation behaviors by a number obtained by subtracting 2 from the total number of
entries, and multiplying the resultant number by 100, and the percentage was used as an indicator
of the spontaneous alternation behavior. A higher value of the indicator suggests better
maintenance of short-term memory. The measured values were expressed as mean ± standard
error for each group. The significance of difference between the control group and the
scopolamine control group was tested by Student's t-test. The significance of difference between
the scopolamine control group and the NIPPLTQTPVVVPPFLQPE-administered group was
tested by Dunnett's multiple comparison test after one-way analysis of variance. The results are
shown in NIPPLTQTPVVVPPFLQPE was shown to have an amnesia-preventing
action in the range of 0.05 nmol/kg body weight to 500 nmol/kg body weight (0.1 pg/kg body
weight to 1,000 pg/kg body weight).
(EXAMPLE 5)
Amnesia-Preventing Action of Asn-Ile-Pro-Pro-Leu-Thr-Gln-Thr-Pro-Val-Val-Val-Pro-Pro-Phe-
Leu-Gln-Pro-Glu (NIPPLTQTPVVVPPFLQPE; SEQ ID NO: 1)-Related Peptide
Male ddY mice (about 7 weeks old) were used (n=15 to 45), and provided with food and
water ad libitum. As a test substance, 50 nmol/kg body weight (100 lig/kg body weight) of Asn-
Ile-Pro-Pro-Leu-Thr-Gln-Thr-Pro-Val-Val-Val-Pro-Pro-Phe-Leu-Gln-Pro-Glu or 50 nmol/kg
body weight (120 lig/kg body weight) of Asn-Ile-Pro-Pro-Leu-Thr-Gln-Thr-Pro-Val-Val-Val-
Pro-Pro-Phe-Leu-Ghi-Pro-Glu-Val-Met (NIPPLT QTPVVVPPFLQPEVM) was used. The test
substance was administered at a single dose orally to mice 60 minutes before performing a Y-
shaped maze test for evaluating spontaneous alternation behavior. Thirty minutes before
performing the Y-shaped maze test, scopolamine was subcutaneously administered in an amount
of 1 mg/kg body weight into the back to induce brain dysfunction (memory disorder and/or
cognition disorder) in mice. In the Y-shaped maze test, as an experiment device, a Y-shaped
maze was used in which the length for each arm was 40 cm; the wall height was 12 cm; the floor
width was 3 cm; the upper part width was 10 cm; and three arms were connected to each other at
an angle of 120°. Each mouse was placed in the end of any of the arms of the Y-shaped maze
and allowed to explore freely in the maze over 8 minutes, and the sequence of the arms to which
the mouse moved was recorded. The number of movements of the mouse to the arms within the
measurement time was counted and used as the total number of entries; in the sequence, the
combination in which three different arms were selected in succession (for example, with the three
arms respectively called A, B, and C, if the sequence of the arms entered is ABCBACACB, the
count is 4 inclusive of overlapping) was investigated, and the count number was used as the
number of spontaneous alternation behaviors. The change in spontaneous alternation behavior
(%) was calculated by dividing the number of spontaneous alternation behaviors by a number
obtained by subtracting 2 from the total number of entries, and multiplying the resultant number
by 100, and the percentage was used as an indicator of the spontaneous alternation behavior. A
higher value of the indicator suggests better maintenance of short-term memory. The measured
values were expressed as mean ± standard error for each group. The significance of difference
between the control group and the scopolamine control group was tested by Student's t-test. The
significance of difference between the scopolamine control group and each peptide-administered
group was tested by Dunnett's multiple comparison test after one-way analysis of variance. The
results are shown in NIPPLTQTPVVVPPFLQPE and NIPPLTQTPVVVPPFLQPEVM
were shown to have an amnesia-preventing action at 50 nmol/kg body weight (100 pg/kg body
weight) and 50 nmol/kg body weight (120 mg/kg body weight), respectively.
(EXAMPLE 6)
Memory-Enhancing Action of Asn-Ile-Pro-Pro-Leu-T1u-Gln-Thr-Pro-Val-Val-Val-Pro-Pro-Phe-
Leu-Gln-Pro-Glu (NIPPLTQTPVVVPPFLQPE; SEQ ID NO: 1)
Male ddY mice (about 7 weeks old) were used (n=14 to 15), and provided with food
and water ad libitum. As a test substance, 500 nmol/kg body weight (1,000 jig/kg body weight)
of Asn-Ile-Pro-Pro-Leu-Thr-Gln-Thr-Pro-Val-Val-Val-Pro-Pro-Phe-Leu-Gln-Pro-Glu was used.
The test substance was administered at a single dose orally to mice 60 minutes before performing
a novel object recognition test for evaluating memory retention. In the novel object recognition
test, a 30x30x30 cm box was used as an experiment device. As a conditioning operation, a
mouse was placed in an experiment device in which a floorcloth was laid for 5 minutes, and
allowed to explore freely in the device. A training trial was performed the day following the
conditioning operation. In the training trial, 2 out of 3 objects were selected and placed in the
experiment device (the objects were placed at positions 8 cm from the walls on the both sides
along the central line of the floor, and the positions were called X1 and X2.). For the selection of
the objects to be placed, the objects were randomly selected in advance to prevent bias among the
animals and among the groups. Sixty minutes after orally administering the test substance or
water, a mouse was placed in the experiment device for 5 minutes, and the time (second) was
measured during which the mouse explored by approaching each object to be within 1 cm
therefrom. A retention trial was performed 48 hours after the training trial. In the retention trial,
2 objects were placed in the experiment device as in the training trial; however, 1 of the objects
was substituted for a different object (a novel object) from that used in the training trial, and the
position thereof was called Y. (For example, when an object A was placed in X1 and an object
B in X2 in the training trial, an object C was placed in place of the object A in the retention trial,
and the position thereof was called Y.) In the training trial and the retention trial, the time
(second) was measured during which each mouse explored by approaching each object to be
within 1 cm therefrom. (However, the state in which a mouse rides on an object is excluded.)
The percentages of the times were determined during which two objects were explored in each of
the training trial and the retention trial. The percentage (%) of the exploration time for each
object was expressed as mean ± standard error for each of the groups. The significance of
difference between the control group and the peptide group was tested by Student's t-test for the
percentage of the exploration time for the novel object (the object placed at Y) in the retention trial
and the percentage of the exploration time for the object (the object placed at X1 or X2) placed at
the position at which the novel object is placed in the training trial. The results are shown in NIPPLTQTPVVVPPFLQPE (SEQ II) NO: 1) was shown to have a memory-enhancing
action at 500 nmol/kg body weight (1,0001_tg/kg body weight).
(EXAMPLE 7)
Amnesia-Preventing Action of Ser-Trp-Met-His-Gln-Pro-His-Gln-Pro-Leu-Pro-Pro-Thr-Val-
Met-Phe-Pro-Pro-Gln-Ser-Val-Leu (SWMHQPHQPLPPTVMFPPQSVL; SEQ ID NO: 3)
Male ddY mice (about 7 weeks old) were used (n=15 to 30), and provided with food and
water ad libitum. As a test substance, Ser-Trp-Met-His-Gln-Pro-His-Gln-Pro-Leu-Pro-Pro-Thr-
Val-Met-Phe-Pro-Pro-Gln-Ser-Val-Leu was used at 150 nmol/kg body weight (380 µg/kg body
weight) and 500 nmol/kg body weight (1,280 pg/kg body weight). The test substance was
administered at a single dose orally to mice 60 minutes before performing a Y-shaped maze test
for evaluating spontaneous alternation behavior. Thirty minutes before performing the Y-shaped
maze test, scopolamine was subcutaneously administered in an amount of 1 mg/kg body weight
into the back to induce brain dysfunction (memory disorder and/or cognition disorder) in mice.
In the Y-shaped maze test, as an experiment device, a Y-shaped maze was used in which the
length for each arm was 40 cm; the wall height was 12 cm; the floor width was 3 cm; the upper
part width was 10 cm; and three arms were connected to each other at an angle of 120°. Each
mouse was placed in the end of any of the arms of the Y-shaped maze and allowed to explore
freely in the maze over 8 minutes, and the sequence of the arms to which the mouse moved was
recorded. The number of movements of the mouse to the arms within the measurement time
was counted and used as the total number of entries; in the sequence, the combination in which
three different arms were selected in succession (for example, with the three arms respectively
called A, B, and C, if the sequence of the arms entered is ABCBACACB, the count is 4 inclusive
of overlapping) was investigated, and the count number was used as the number of spontaneous
alternation behaviors. The change in spontaneous alternation behavior (%) was calculated by
dividing the number of spontaneous alternation behaviors by a number obtained by subtracting 2
from the total number of entries, and multiplying the resultant number by 100, and the percentage
mean ± standard error for each group. The significance of difference between the control group
administered group was tested by Student's t-test. The results are shown in
SWMHQPHQPLPPTVMFPPQSVL was shown to have an amnesia-preventing action at 150
nmol/kg body weight to 500 nmol/kg body weight (380 i_tg/kg body weight to 1,280 jig/kg).
especially because it has the effect of improving or preventing amnesia.
was used as an indicator of the spontaneous alternation behavior. A higher value of the indicator
suggests better maintenance of short-term memory. The measured values were expressed as
and the scopolamine control group was tested by Student's t-test. The significance of difference
between the scopolamine control group and the SWMHQPHQPLPPTVMFPPQSVL -
INDUSTRIAL APPLICABILITY
According to the present invention, a new peptide for improving brain function is
provided, which is obtained by the enzymatic hydrolysis using milk casein safe as a food as a raw
material. The peptide is useful for ameliorating or improving symptoms of hypomnesia
SEQUENCE LISTING FREE IEXT
SEQ ID NOs: 1 to 3: cow casein-derived peptides
Claims (11)
1. A method for preparing a peptide for improving brain function, comprising: hydrolyzing milk casein with an enzymatic catalyst comprising an alkaline protease derived from Bacillus licheniformis, an alkaline protease derived from Aspergillus sp., or a neutral protease derived from Aspergillus melleus to produce a hydrolysate comprising: (i) a peptide consisting of the amino acid sequence shown in SEQ ID NO: 1,; (ii) a peptide consisting of the amino acid sequence shown in SEQ ID NO: 2,; or (iii) a mixture of the peptides of (i) and (ii), wherein the production yield of each of the peptides is 2% or more, or the total production yield of the mixture is 10% or more.
2. The method according to claim 1, wherein the milk casein is cow milk casein.
3. The method according to claim 1 or 2, wherein the milk casein is milk casein sodium.
4. The method according to any one of claims 1 to 3, wherein the production yield of the peptide consisting of the amino acid sequence shown in SEQ ID NO: 1 is 5 to 10%, or more.
5. The method according to any one of claims 1 to 4, wherein the production yield of the peptide consisting of the amino acid sequence shown in SEQ ID NO: 2 is 10 to 50%, or more.
6. The method according to any one of claims 1 to 5, wherein the total production yield of the peptide consisting of the amino acid sequence shown in SEQ ID NO: 1 and the peptide consisting of the amino acid sequence shown in SEQ ID NO: 2 is 15 to 60%, or more.
7. The method according to any one of claims 1 to 6, wherein the hydrolysate further comprises a peptide consisting of the amino acid sequence shown in SEQ ID NO: 3.
8. The method according to any one of claims 1 to 7, wherein the enzymatic catalyst further comprises an enzyme having a peptidase activity.
9. The method according to any one of claims 1 to 8, wherein the enzymatic catalyst is immobilized on a support.
10. The method according to any one of claims 1 to 9, wherein the hydrolysis of the milk casein is carried out using milk.
11. The method according to any one of claims 1 to 10, wherein the weight ratio of the enzyme/milk casein is
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2011141101A JP5718741B2 (en) | 2011-06-24 | 2011-06-24 | Enzymatic production of peptide for improving brain function |
JP2011-141101 | 2011-06-24 | ||
PCT/JP2012/064997 WO2012176658A1 (en) | 2011-06-24 | 2012-06-12 | Enzymatic production method for brain-function-improving peptides |
Publications (2)
Publication Number | Publication Date |
---|---|
NZ619383A NZ619383A (en) | 2015-11-27 |
NZ619383B2 true NZ619383B2 (en) | 2016-03-01 |
Family
ID=
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