JPH04173800A - Glycooligopeptide mixture having high sialic acid content and its production - Google Patents

Abstract

PURPOSE:To obtain the subject mixture useful as a nutrient preparation for hepatic diseases, etc., by hydrolyzing kappa-casein with rennet, collecting a fraction containing a glycopeptide having a specific molecular weight from the hydrolyzed product, hydrolyzing the fraction with protease and collecting a fraction having a specific molecular weight. CONSTITUTION:The objective glycopeptide mixture having a sialic acid content of >=20g/10g and a sialic acid/nitrogen molecular ratio of >=0.08, composed of 5-20 amino acid residues, containing glutamic acid, threonine, serine, proline, valine, aspartic acid, isoleucine and glycine as main constituent amino acids and having a residual antigen activity of <=10<-5> measured by ELISA suppression test can be produced by hydrolyzing kappa-casein with rennet, collecting a fraction containing a glycopeptide having a molecular weight of about 7,000 from the hydrolyzed product, hydrolyzing the glycopeptide in the fraction with protease and collecting a fraction having a molecular weight of 500-2,000 from the obtained hydrolyzate.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to a glyco-oligopeptide mixture with a high content of sialic acid and a method for producing the same.

Background of the Invention - Casein accounts for approximately 13% of the milk casein width (weight, hereinafter the same unless otherwise specified), has a sugar chain in its molecule, is contained in high concentration in human milk casein, and has various It is known to have physiological activity. It is already known that the phenylalanine-methionine bond in casein is hydrolyzed by rennet, and it is decomposed into casein and glycomacropeptide [Journal.
Journal of Biochemistry
io-chemistry), Volume 111, No. 333
Page, 1980].

Milk-derived sialic acid-binding proteins (specifically, casein) and sialic acid-binding peptides obtained by treating sialic acid-binding proteins with protease (specifically, glycomacropeptides obtained by hydrolyzing casein with rennet) ), for example, JP-A-63-284133 (infection prevention agent), JP-A-1-163110 (
skin anti-aging agent), JP-A-1-163112 (anti-pigmentation agent), JP-A-1-163114 (hair tonic), JP-A-1-163125 (anti-inflammatory agent), and JP-A-2-207089 Inventions such as No. 1 (bacterial toxin neutralizer) have been disclosed.

However, since casein or glycomacropeptide has a large molecular weight of 7,000 to 20,000 and has residual antigenicity, there have been various problems in utilizing them.

Furthermore, JP-A-63-284133 and JP-A-2-2
Publication No. 07089 describes that a sialic acid-binding peptide can be obtained by allowing a protease to act on a sialic acid-binding protein and using the resulting product by fractionation means such as gel filtration, ion exchange chromatography, and affinity chromatography. be. However, regarding the sialic acid content in the obtained sialic acid binding protein, although the former states that it is 5 g/100 g or more, there is no description that suggests a value as high as 20 g/100 g as in the present invention, and the latter states that the sialic acid content is 5 g/100 g or more as in the present invention. is not mentioned at all. Also, hydrolysis methods and conditions, separation and purification methods and conditions, especially separation of peptides with high sialic acid content.

There are no specific explanations regarding the purification method and conditions or the properties of the obtained peptides, but the effects of infection prevention and bacterial toxin neutralization have been confirmed by adding rennet to casein. Only the glycomacropeptides (and one casein) obtained by the action of

The present inventors hydrolyzed the hydrophilic fraction (glycopolypeptide) obtained by hydrolyzing casein in animal milk with rennet using protease, and determined the Finnyer value (the molar content of aromatic amino acids). He invented a peptide mixture with a molar content of branched amino acids (divided by the molar content of branched amino acids) in the range of 30 to 60, and a method for producing the same, and has already filed a patent application (1999).
Patent Application No. 119194 (hereinafter, the invention of this application will be referred to as the earlier invention). The peptide mixture of the previous invention focuses on the Fisher value, and can be used as a nutritional supplement for liver diseases.
Specifically intended for use as an oral nutritional supplement,
The product has benefits such as good digestion and absorption, and excellent flavor.

Problems to be Solved by the Invention The present inventors conducted further research on this peptide mixture after filing the application for the earlier invention, and found that various fractions with a molecular weight distribution of 100 to 6000 [in these samples] The molecular weight distribution was obtained by changing the reaction time (decomposition rate), and was not obtained by fractionating by molecular weight fractionation. In the specification of the earlier invention, Test Example 1, and Table 1, the molecular weight distribution is 500.
We found that peptides with high sialic acid content were present in the ~2000 fractions.

Based on this discovery, the present inventors performed hydrolysis of glycopolypeptides with protease, followed by ultrafiltration, gel filtration, or centrifugation, and found that the molecular weight distribution was 500 to 20.
By collecting the 00 fraction, we succeeded in separating the target glyco-oligopeptide mixture with high sialic acid content, and completed the present invention.

An object of the present invention is to provide a glyco-oligopeptide mixture that has a lower molecular weight, higher sialic acid content, substantially no antigenicity, and higher physiological activity than conventionally known sialic acid-binding peptides; The object of the present invention is to provide a method for producing the above-mentioned glyco-oligopeptide mixture using casein as a raw material.

According to the present invention, a glyco-oligopeptide mixture having a high sialic acid content and having the following physicochemical properties (a) to (e) is provided. That is, (a) the molecular weight distribution is 500-2
000, (b) sialic acid content is 20g/1
(c) The molecular number ratio of sialic acid/nitrogen is 0.08 or more; (d) The number of amino acid residues is 5 to 20, including glutamic acid, threonine, serine, and proline.

The main constituent amino acids are alanine, valine, aspartic acid, isoleucine, and glycine; (e) ELISA: Enzym
e Linked Immuno 5orbent As
5ey) Antigen residual activity measured by inhibition test method is 10
-5 or less.

According to the method of the present invention, casein is hydrolyzed with rennet, and the resulting hydrolyzate has a molecular weight of about 7,000.
The glycopolypeptide-containing fractions before and after are separated, and the glycopolypeptide contained in the separated fractions is hydrolyzed with protease.
Collect ~2000 fractions. A glyco-oligopeptide mixture with a high sialic acid content is thereby obtained.

Means for Solving the Problems The starting material used in the production of the sialic acid-rich glyco-oligopeptide mixture of the present invention is casein. Ni-casein can be obtained by any known method, including the method described in Japanese Patent Application Laid-Open No. 59-914848, the method described in Japanese Patent Publication No. 59-48964, the method of Girdhal et al.・Of Food Science (Journal
of Food 5science) Volume 43, No. 397
Page, 1978], and can be prepared mainly from animal milk.

The obtained casein was hydrolyzed with rennet,
The reaction solution is heated to inactivate the enzyme, and the hydrophobic casein that coagulates and precipitates is separated and removed by filtration or centrifugation.

The step of separating a glycopolypeptide-containing fraction with a molecular weight of about 7,000 to which sialic acid is bound, and hydrolyzing the glycopolypeptide of this fraction with a protease to obtain a peptide mixture is described in the earlier invention. methods can be applied.

In the present invention, the protease used for hydrolyzing the glycopolypeptide is a protease other than rennet, and preferably an endopeptidase such as vancreatin. Although there are no particular restrictions on the hydrolysis conditions (eg, amount of enzyme, temperature, pH, time, etc.) in this step, it is desirable to avoid excessive decomposition. Hydrolysis is stopped by heating.

Molecular weight fractionation is preferable as a method for separating a fraction with a molecular weight of 500 to 2000, which is the object of the present invention, from a hydrolyzate of a glycopolypeptide. Any known method such as ultrafiltration, gel filtration, or centrifugation can be used for molecular weight fractionation, but a method using ultrafiltration is particularly recommended. Incidentally, the molecular weight fractionation is performed repeatedly as necessary.

Whether or not a fraction separated by molecular weight fractionation contains a peptide with a target molecular weight can be confirmed by high performance liquid chromatography.

When manufacturing from the same raw material under the same conditions repeatedly, other characteristic values (e.g., electrical conductivity, elution volume, etc.) or conditions (e.g., fractionation The number of repetitions) can be determined in advance, and the desired fraction can be collected from the next time using the characteristic value or condition as an index.

The fraction (aqueous solution) obtained as described above is concentrated and dried by a known method to obtain a concentrate or powder of a glyco-oligopeptide mixture with a high sialic acid content.

Next, the physicochemical properties of the glyco-oligopeptide mixture with high sialic acid content of the present invention will be described.

The glyco-oligopeptide mixture with a high sialic acid content obtained above has 5 to 20 amino acid residues, and the main constituent amino acid is glutamic acid.

Threonine, serine, proline, alanine, valine, aspartic acid, isoleucine, glycine, molecular weight distribution 500-2000, sialic acid content 20g/10
It is a sialic acid-binding oligopeptide mixture with a weight of 0g or more and a sialic acid/nitrogen molecular ratio of 0.08 or more (see Test Example 1).

The glyco-oligopeptide mixture of the present invention has a lower molecular weight than conventionally known sialic acid-binding peptides, so it has substantially no antigenicity, and the sialic acid content is 6.2g/100g (sialic acid/ The molecular number ratio of nitrogen is 3 times or more compared to 0.02), and it is characterized by high physiological activity.

The features of the present invention will be illustrated below through test examples.

(Test Example 1) This test was conducted to examine conditions for collecting fractions having a molecular weight of 500 to 2000.

(1) Preparation of sample A hydrolyzate of glycopolypeptide was prepared by the same method as in the example described below. However, repeating the fractionation 8 times,
The internal and external fluids from each session were used as samples.

(2) Test method The molecular weight distribution of each sample was determined by high performance liquid chromatography (
The peptide fraction with a molecular weight of less than 500 was measured under the following conditions according to Nobuo Ui et al., [High Performance Liquid Chromatography J of Proteins and Peptides, Kagaku Special Issue No. 102, p. 241, Kagaku Dojin Co., Ltd., 1984]. The proportion of peptides in total peptides was calculated.

Pump: Shimazu LC7A (manufactured by Shimadzu Corporation) Column: ^5ahipak G5-320 (manufactured by Asahi Kasei Industries Ltd.) Exclusion limit molecular weight 4000 Detector: 5hodex RI 5E-61 (manufactured by Showa Denko Corporation) Eluent: 6N-guanidine hydrochloric acid aqueous solution temperature : Room temperature speed: 0.65 m1/min Analyzer: Chromatopack C-R4^X (manufactured by Shimadzu Corporation) For the first sample in which the peptide fraction with a molecular weight of less than 500 accounts for 0% of the total peptides, the following The amounts of nitrogen and sialic acid, as well as the amino acid composition were measured using the method described above.

(2-1) Nitrogen analysis autoanalyzer (TECHNICON, TRAA
The sample was subjected to Kjeldahl decomposition, and ammonium ions in the decomposition solution were colorimetrically determined using a Kjeldahl decomposition machine (C, 800, manufactured by Technicon).

(2-2) Sialic acid analysis Decomposed with 0.1N sulfuric acid at 80°C for 1 hour. Thiobarbic acid was added to the decomposition solution, and colorimetric determination was performed at 549 nm.

(2-3) Amino acid composition: Decompose with 6N hydrochloric acid at 110°C for 24 hours, and analyze the decomposition solution with an amino acid analyzer (formerly HITAC, 835 Am1n).
.

Ac1d Analyzer (manufactured by Hitachi, Ltd.) was used to develop ninhydrin color and perform colorimetric determination.

(3) Test results The results of this test were as follows.

(3-1) Content of substances with a molecular weight of less than 500 in each sample Liquid before fractionation 27.5 (%) 1st external fluid 1st internal fluid - 2nd external liquid - 2nd internal fluid - 3rd external fluid - 3rd internal fluid - 4th external fluid - 4th internal fluid 13.7 5th external fluid - 5th internal fluid 3.4 6th external liquid - 6th internal fluid 0 7th external fluid 0 7th internal fluid 0 8th external fluid 0 8th internal fluid 0 (Note) - Symbol indicates not measured.

(3-2) Sixth internal solution analysis result Molecular weight distribution 500-2000 nitrogen (g/10
0g) 1.0, 0 sialic acid (g/100g)
25, 90 SA/N ratio * 0.12 (* symbol indicates the molecular number ratio, SA represents sialic acid and N represents nitrogen) (3-3) Amino acid composition of the 6th internal solution (mol%) Aspartic acid 5.5 Threonine 21.9 Serine 12.4 Glutamic acid 22.3 Glycine 4.5 Alanine 7.5 Valine 7.4 Cystine 0.1 Methionine 0.4 Isoleucine 5.1 0 Isine 1.0 Tyrosine 0.7 Phenylalanine 0 .4 Tryptophan O Lysine 1.4 Histidine 0.2 Arginine 0 Proline 9.2 From these results, it is clear that if the fractionation operation is repeated six times, there will be no peptide mixture with a molecular weight of less than 500, and this sample The molecular weight distribution of is 500-2000
The SA/N ratio is 0.14, and the main constituent amino acid is glutamic acid.

They were found to be threonine, serine, proline, alanine, valine, aspartic acid, isoleucine and glycine.

(Test example 2) This test was performed to examine antigenicity.

(1) Preparation of sample (1-1) Glyco-oligopeptide mixture sample of the present invention Glyco-oligopeptide mixture (G
(written as OP).

(1-2) Molecular weight of a hydrophilic and sialic acid-bound control sample obtained by hydrolyzing casein with rennet and separating the precipitate from the resulting hydrolyzate. Approximately 70
Glycopolypeptide (written as GPP) around 00.

(2) Test method for antigen residual activity Measured by ELIZA inhibition test method. 9
A 6-well plate (manufactured by Nunc) was coated with whey protein, washed, and a mixture of rabbit anti-whey protein serum and sample was supplied to the wells of the plate for reaction. After washing, goat anti-whey protein labeled with alkaline phosphatase was coated with whey protein. TGC antibody (manufactured by Zeymed Laboratories) was reacted, then washed, sodium p-nitrophenyl phosphate was added, and 30 minutes later, 5
The reaction was stopped by adding N sodium hydroxide, and the reaction product was measured using a microplate reader (MTP-32, manufactured by Corona Electric Co., Ltd.).

(3) Test results As a result of this test, GOP is 10-5, GPP is IQ
It was 1.11. That is, while GOP showed 1/100,000 times less antigenicity than casein, GPP showed about 1/32nd the antigenicity of casein.

It was found that the antigenicity of P was extremely reduced compared to that of GPP.

(Test Example 3) This test was conducted to examine the inhibitory effect on tyrosinase activity.

(1) Preparation of samples GOP and GPP (control) were prepared by the same method as Test Example 2.

(2) Test method (2-1) Preparation of various solutions (2-1-a) Substrate solution (tyrosine solution) Reagent Special grade Noshiichi tyrosine (manufactured by Wako Pure Chemical Industries, Ltd.) was added to 0.1M phosphate buffer ( pH 7,0)i: Dissolved at a concentration of 0.05% (W/V).

(2-1-b) Sample solution The sample was dissolved in 0.1MTP phosphate buffer wave buffer 7.0) at a concentration of 0.5 to 2.0% (W/V).

(2-1-c) Copper ion solution Reagent special grade copper sulfate aqua regia (manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved in purified water at a concentration of 1% (W/V).

(2-1-d) Enzyme solution Tyrosinase derived from pine mushroom (manufactured by Sigma, 30
00 units/mg) in purified water at 0.1% (W/V)
It was dissolved at a concentration of

(2-1-e) Reaction stop solution 30% acetic acid aqueous solution.

(2-2) Enzyme reaction and absorbance measurement Substrate solution Q preheated to 37°C, 9 ml, sample solution 1
．． Qm1, 0.02 m of the copper ion solution was collected in a test tube, and 0.08 m1 of the enzyme solution heated to the same temperature was added, making the total volume 2. OmA') and reacted at 37°C for 3 minutes. Next, 2 ml of reaction stop solution was added to stop the reaction, and the absorbance at a wavelength of 640 nm was measured using a spectrophotometer (this absorbance is designated as B). 0 instead of the sample solution as a control.
1M phosphate buffer (DH7,0) 1.0 mA' was added and treated in the same manner, and the absorbance was measured (this absorbance is designated as A). If the test solution is cloudy, use 0.08ml of 0.1M phosphate buffer instead of the enzyme solution. (Add C) and measure the absorbance in the same way, and let this absorbance be C)
, the absorbance originating from the turbid part of the reaction solution was removed.

(2-3) Tyrosinase activity inhibition rate (%) from the calculated and measured absorbance values
) was calculated using the following formula.

Inhibition rate (%) = 100 [1-(B-C)/A] (3)
Test Results The results of this test were as shown in Table 1.

As is clear from Table 1, the tyrosinase activity inhibition rate of GOP exceeded that of GPP at each concentration, and a significant tyrosinase activity inhibition rate was observed for GOP.

Table 1 Tyrosinase is an enzyme that catalyzes the acid value of tyrosine, other monohydric phenols, and corresponding ortho-dihydric phenols due to molecular state oxygen, and is used in mushrooms, potatoes,
It is present in the tissues of many plants and animals, such as apples, and is known to be involved in the formation of melanin pigment in tissues, especially skin epidermal cells in animals (Complete edition of the editorial committee of the Encyclopedia of Chemistry, Encyclopedia of Chemistry, Volume 5, page 976, Kyoritsu Publishing,
(Showa 35). It is also known that the increased skin color in Ajiran's disease, in which melanin adheres to the skin or mucous membranes, resulting in a black color, is due to a decrease in the secretion of adrenal cortical hormones, which compete with melanotropes, which promote tyrosinase activity. (Complete edition of Chemistry Dictionary Editorial Committee, Chemistry Dictionary, Volume 1,
Page 65, Kyoritsu Shuppan, 1960). Furthermore, tyrosinase is also said to be involved in reducing the freshness of foods.

Therefore, the glyco-oligopeptide mixture with high sialic acid content of the present invention can be expected to have various physiological activities.

The present invention will be explained in further detail below through Reference Examples and Examples. Incidentally, these reference examples and examples are illustrative for explanation, and the present invention is not limited to these reference examples and examples.

Reference Example 2000 g of commercially available lactic acid casein produced in New Zealand was dissolved in a 0.5% aqueous sodium hydroxide solution at a concentration of 12% while heating, and the pH of the resulting solution was adjusted to 8.0. Add 248 g of a 40% calcium chloride aqueous solution to this solution (final concentration 0°3M) and stir at 60°C for 15 to 30 minutes to fully react with calcium. After the solution becomes homogeneous, add it to a continuous centrifuge. The supernatant liquid was collected using

This supernatant was desalted to a calcium concentration of 1% or less of the initial concentration using an Ultrafiltration module (SIP 1013, manufactured by Asahi Kasei Industries, Ltd.) of a Fractionation Molecule Sekikoma 6000, and then freeze-dried to a purity of approximately 70%. Approximately 120 g of 1% casein powder was obtained.

Example: 4000 g of casein powder obtained by repeating the same method as the reference example was dissolved in water at a concentration of 5%, and the pH was adjusted to 6.
3, add 2500 mg of commercially available microbial-derived rennet (manufactured by Disaccharide Sangyo Co., Ltd.), react at 35°C for 30 minutes, heat at 70°C for 10 minutes to inactivate the enzyme, and centrifuge to remove the supernatant. The liquid was separated. This supernatant liquid was fractionated with a fraction of about 30 molecules.
00 Ultrafilt Rayon Module (SEP)
1013, manufactured by Asahi Kasei Industries, Ltd.), the protein concentration was concentrated to 3.2 times, and 0.5 g of commercially available protease (Protease Amano A1 manufactured by Amano Pharmaceutical Co., Ltd.) and lactic acid bacteria cell suspension (solid content concentration 8%) were added. Add 6.25g and heat at 51°C for 20
The reaction solution was heated at 80°C for 10 minutes to inactivate the enzyme, cooled at 4°C for 2 hours, and diatomaceous earth 2
0 g was added and filtered, and 1000 g of the obtained filtrate was fractionated using an Ultrafiltration module (SEO1013, manufactured by Asahi Kasei Industries, Ltd.) with a fractionation molecular weight of approximately 3000.

The first fractionation was carried out by circulating 1000 g of the above aqueous solution through the module, continuing until 500 mn of the external solution was obtained, and then the internal solution (500 mn).
00rrl) was added with 500mf of distilled water, and the second fractionation was continued by circulating the mixed liquid through the module until the external liquid became 500ml.After that, the same operation as the second fractionation was repeated four more times, and the obtained 350 g of the liquid was freeze-dried to obtain 34 g of a glyco-oligopeptide mixture with a high sialic acid content. The molecular weight distribution, ratio of nitrogen to sialic acid, residual antigenic activity, and amino acid composition of this glyco-oligopeptide mixture with a high sialic acid content were determined by the same method as in the above test example, and were as follows.

Molecular weight distribution 500-2000SA/N ratio*
014 Antigenic residual activity 1O-5 (* indicates the molecular number ratio, SA indicates sialic acid, N indicates nitrogen) Amino acid composition (mol%) Aspartic acid 5.4 Threonine 21.7 Serine 12.6 Glutamic acid 22. 0 Glycine 4.4 Alanine 7.6 Valine 74 Cystine 0.3 Methionine 0.2 Isoleucine 4.7 0 Isine 0.9 Tyrosine 0.8 Phenylalanine 0.5 Tryptophan 0.1 Lysine 1.5 Histidine 0.3 Arginine 0 .2 Proline 9.4 Effects of the Invention The effects achieved by the present invention are as follows.

1) The glyco-oligopeptide mixture with high sialic acid content of the present invention is substantially non-antigenic.

2) The glyco-oligopeptide mixture with high sialic acid content of the present invention has high physiological activity.

Claims (1)

[Scope of Claims] [1] A glyco-oligopeptide mixture with a high content of sialic acid characterized by having the following physicochemical properties (a) to (e): (a) a molecular weight distribution of 500 to 2000; (b) The sialic acid content is 20 g/100 g or more; (c) the sialic acid/nitrogen molecular number ratio is 0.08 or more; (d) the number of amino acid residues is 5 to 20; The main constituent amino acids are glutamic acid, threonine, serine, proline, alanine, valine, aspartic acid, isoleucine, and glycine.
Antigen residual activity measured by dImmunoSorbentAssay) inhibition test method is 10^-^5 or less. [2] Hydrolyze κ-casein with rennet, separate a glycopolypeptide-containing fraction with a molecular weight of around 7000 from the resulting hydrolyzate, and hydrolyze the glycopolypeptide contained in the fraction with protease. A method for producing a glyco-oligopeptide mixture with a high sialic acid content, which comprises decomposing the glyco-oligopeptide mixture and collecting a fraction with a molecular weight of 500 to 2,000 from the obtained hydrolyzate.