JP2980362B2 - Glyco-oligopeptide mixture having high sialic acid content and method for producing the same - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a glycooligopeptide mixture having a high sialic acid content and a method for producing the same.

Prior art κ-casein accounts for about 13% (weight, hereinafter the same unless otherwise specified) of milk casein, has a sugar chain in the molecule, is contained in human milk casein at a high concentration, and has various It is known to have physiological activity. It is already known that κ-casein is hydrolyzed by rennet at a phenylalanine-methionine bond to be degraded to para-κ-casein and glycomacropeptide [Journal.
Journal of Biochemistr
y), Vol. 111, p. 333, 1980].

Sialic acid-binding protein derived from milk (specifically, κ-casein) and sialic acid-binding peptide obtained by treating sialic acid-binding protein with protease (specifically, glycomacropeptide obtained by hydrolyzing κ-casein with rennet) )
For example, JP-A-63-284133 (anti-infective agent), JP-A-1-163110 (skin aging inhibitor), JP-A-1-163112 (anti-pigmenting agent), Inventions such as Japanese Unexamined Patent Publication No. Hei 1-163114 (hair nourishing agent), Japanese Unexamined Patent Publication No. 1-163125 (anti-inflammatory agent), and Japanese Unexamined Patent Publication No. Hei 2-20789 (bacterial toxin neutralizing agent) are disclosed.

However, since κ-casein or glycomacropeptide has a large molecular weight of 7000 to 20,000 and remains antigenic, there have been various problems in using these.

JP-A-63-284133 and JP-A-2-207089 disclose a protease acting on a sialic acid-binding protein and subjecting the resulting product to gel filtration, ion exchange chromatography, affinity chromatography, etc. There is a description that a sialic acid-binding peptide can be obtained by fractionation means. However, as for the sialic acid content in the obtained sialic acid binding protein, there is a description of 5 g / 100 g or more in the former, but there is no description suggesting a value of 20 g / 100 g as in the present invention, and in the latter, there is no description. There is no description. In addition, hydrolysis methods and conditions, separation and purification methods and conditions, especially separation of peptides having a high sialic acid content,
No specific explanation is given on the purification method and conditions, the characteristics of the obtained peptide, etc., and the test for the effects of protection against infection and neutralization of bacterial toxins confirmed that rennet was added to κ-casein. Only glycomacropeptide (and para-κ-casein) obtained by the action.

The present inventors have proposed a hydrophilic fraction (glycopolypeptide) obtained by hydrolyzing animal milk κ-casein with rennet.
Was hydrolyzed using a protease, and a peptide mixture having a Fischer value (value obtained by dividing the molar content of branched amino acids by the molar content of aromatic amino acids) in the range of 30 to 60 and a method for producing the same were invented. Application (Heisei 1
Patent Application No. 119194, hereinafter the invention of this application is referred to as prior invention). The peptide mixture of the present invention focuses on the Fisher value and is intended to be used as a nutritional agent for liver disease, particularly as an oral nutritional agent, and the product has good digestion and absorption, and excellent flavor. It has the effect of being.

Problems to be Solved by the Invention The present inventors conducted further research on this peptide mixture after filing the application of the prior invention, and as a result, obtained various fractions having a molecular weight distribution of 100 to 6000 of the peptide mixture in the prior invention [ The molecular weight distribution was obtained by changing the reaction time (decomposition rate), and was not fractionated by molecular weight fractionation. Refer to the above specification of the invention, Test Example 1 and Table 1].
Was found to contain a peptide having a high sialic acid content.

Based on this finding, the present inventors carried out hydrolysis of glycopolypeptide with protease, followed by ultrafiltration, gel filtration, centrifugation, etc., to obtain a molecular weight distribution of 500 to 2,000.
By collecting the above fractions, the objective glycooligopeptide mixture having a high sialic acid content was successfully separated, thereby completing the present invention.

An object of the present invention is to provide a glyco-oligopeptide mixture having a lower molecular weight, a higher sialic acid content, substantially no antigenicity, and a higher physiological activity than conventionally known sialic acid-binding peptides, and κ- An object of the present invention is to provide a method for producing the glycooligopeptide 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. (A) the molecular weight distribution is 500 to 2,000, (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, and glutamic acid, threonine, serine, proline, alanine, valine, aspartic acid, isoleucine, and glycine are mainly used as constituent amino acids. (E) ELISA (Enzyme Linked Immunoassay ELISA) Sorbent
Assey) The residual antigen activity measured by the inhibition test method is 10 ^-5
Must be:

According to the method of the present invention, κ-casein is hydrolyzed by rennet, and a glycopolypeptide-containing fraction having a molecular weight of about 7000 is fractionated from the obtained hydrolyzate, which is contained in the fractionated fraction. Glycopolypeptide is hydrolyzed by a protease, and the resulting hydrolyzate is used to obtain a molecular weight of 500 to 200.
Collect fraction 0. This gives a glycooligopeptide mixture with a high sialic acid content.

The starting material used for the production of the sialic acid-rich glyco-oligopeptide mixture of the present invention is kappa-casein. K-casein can be obtained by any known method, for example, the method described in JP-A-59-914848,
The method described in JP-B-59-48964, Girdal et al.
dhal et al.] [Journal of Food Science, Vol. 43, p. 397, 1978], etc., and can be prepared mainly from animal milk.

The obtained κ-casein is hydrolyzed by rennet, the reaction solution is heated to inactivate the enzyme, and the hydrophobic para-κ-casein that aggregates and precipitates is separated and removed by filtration or centrifugation. Isolating a glycopolypeptide-containing fraction having a molecular weight of about 7,000 to which sialic acid is bound, hydrolyzing the glycopolypeptide of this fraction with a protease to obtain a peptide mixture, The method described in the invention can be applied.

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

As a method for separating a fraction having a molecular weight of 500 to 2000, which is the object of the present invention, from a hydrolyzate of a glycopolypeptide, a molecular weight fraction is desirable. Ultrafiltration for molecular weight fractionation
Any of known methods such as gel filtration and centrifugation can be employed, but a method by ultrafiltration is particularly recommended. In addition, molecular weight fractionation is performed repeatedly as needed.

Whether or not the fraction fractionated by the molecular weight fractionation contains a peptide having a target molecular weight can be confirmed by high performance liquid chromatography. When repeatedly produced from the same raw material under the same conditions, the analysis results of the molecular weight distribution by high performance liquid chromatography and other characteristic values (eg, electric conductivity, elution volume, etc.) or conditions (eg, fractionation) Is determined in advance, and a target 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 by a known method and dried to obtain a concentrate or powder of a glyco-oligopeptide mixture having a high sialic acid content.

Next, the physicochemical properties of the glyco-oligopeptide mixture having a high sialic acid content of the present invention will be described. The glyco-oligopeptide mixture having a high sialic acid content obtained as described above has 5 to 20 amino acid residues, and the main constituent amino acids are glutamic acid, threonine, serine, proline,
Alanine, valine, aspartic acid, isoleucine, glycine, sialic acid-binding oligopeptide mixture with a molecular weight distribution of 500-2000, a sialic acid content of 20 g / 100 g or more, and a sialic acid / nitrogen number ratio of 0.08 or more ( Test example 1
reference).

The glyco-oligopeptide mixture of the present invention has substantially no antigenicity because it has a lower molecular weight than conventionally known sialic acid-binding peptides, and the sialic acid content is 6.2 g / 100 g (sialic acid / nitrogen) of the conventional sialic acid-binding peptide. Is 0.02)
It is three times or more as compared with, and has a feature of high physiological activity.

 Hereinafter, the features of the present invention will be illustrated through test examples.

(Test Example 1) This test was performed to examine conditions for collecting a fraction having a molecular weight of 500 to 2,000.

(1) Preparation of sample A hydrolyzate of glycopolypeptide was prepared by the same method as in the examples described later. However, the fractionation was repeated eight times, and each of the inner and outer liquids at each time was used as a sample.

(2) Test method The molecular weight distribution of each sample was determined by high performance liquid chromatography (Edited by Nobuo Ui et al., “High Performance Liquid Chromatography for Proteins and Peptides”, Chemical Special Issue No. 102, page 241,
Chemical Co., Ltd., 1984) under the following conditions,
The ratio of the peptide fraction having a molecular weight of less than 500 to the total peptide was calculated.

Pump: Shimazu LC7A (manufactured by Shimadzu Corporation) Column: Asahipak GS-320 (manufactured by Asahi Kasei Kogyo) exclusion limit molecular weight 4000 Detector: Shodex RI SE-61 (manufactured by Showa Denko) Eluent: 6N-guanidine hydrochloride aqueous solution Temperature: Room temperature Rate: 0.65 ml / min Analyzer: Chromatopack C-R4AX (manufactured by Shimadzu Corporation) For the first sample in which the ratio of the peptide fraction having a molecular weight of less than 500 to the total peptide was 0%, nitrogen was measured by the following method. And the amount of sialic acid and the amino acid composition were measured.

(2-1) Nitrogen analysis The sample was subjected to Kjeldahl decomposition using an autoanalyzer (TECHNICON, TRA _CS 800 ^™ , manufactured by Techicon), and the ammonium ion in the decomposition solution was colorimetrically quantified.

(2-2) Sialic acid analysis Decomposed with 0.1 N sulfuric acid at 80 ° C for 1 hour. Thiobarbituric acid was added to the digested solution and colorimetrically determined at 549 nm.

(2-3) Amino acid composition The mixture was decomposed with 6N hydrochloric acid at 110 ° C. for 24 hours, and the decomposed liquid was analyzed with an amino acid analyzer (HITACHI, 835 Amino Acid Analyzer,
Ninhydrin was developed by Hitachi Ltd.) and colorimetrically determined.

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

(3-1) The content of substances having a molecular weight of less than 500 in each sample 27.5 (%) of the liquid before fractionation 1st outer liquid-1st inner liquid-2nd outer liquid-2nd inner liquid-3rd Outward solution-3rd inner solution-4th outer solution-4th inner solution 13.7 5th outer solution-5th inner solution 3.4 6th outer solution-6th inner solution 0 7th outer Liquid 0 7th inner liquid 0 8th outer liquid 0 8th inner liquid 0 (Note)-The symbol indicates that measurement was not performed.

(3-2) Results of the sixth analysis of the inner solution Molecular weight distribution 500 to 2000 Nitrogen (g / 100 g) 10.0 Sialic acid (g / 100 g) 25.90 SA / N ratio * 0.12 (* indicates the ratio of the number of molecules, SA Represents sialic acid and N represents nitrogen. (3-3) Amino acid composition (mol%) of the sixth inner solution Aspartic acid 5.5 Threonine 21.9 Serine 12.4 Glutamate 22.3 Glycine 4.5 Alanine 7.5 Valine 7.4 Cystine 0.1 Methionine 0.4 Isoleucine 5.1 Leucine 1.0 Tyrosine 0.7 Phenylalanine 0.4 Tryptophan 0 Lysine 1.4 Histidine 0.2 Arginine 0 Proline 9.2 From these results, it is clear that if the fractionation operation is repeated 6 times, the peptide mixture having a molecular weight of less than 500 does not exist. 500-2000, the SA / N ratio is 0.14, and the main constituent amino acids are glutamic acid, threonine, serine, proline, alanine, Phosphate, aspartic acid, it is isoleucine and glycine were found.

(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 (described as GOP) prepared in Examples.

(1-2) Control sample In the examples, κ-casein was hydrolyzed with rennet, and the precipitate was separated from the obtained hydrolyzate. The hydrophilic and sialic acid-bound molecular weight was obtained. About 70
Glycopolypeptides around 00 (described as GPP).

(2) Test method of residual antigen activity It was measured by an ELISA inhibition test method. A 96-well plate (manufactured by Nunc) is coated with whey protein, washed, and a mixture of rabbit anti-whey protein serum and a sample is supplied to the plate to react, and after washing, alkaline phosphatase-labeled goat anti-rabbit IgG is washed. The antibody (Zeimed Laboratories) was reacted, washed, washed with sodium p-nitrophenyl phosphate, added with 5N sodium hydroxide 30 minutes later to stop the reaction, and the reaction product was transferred to a microplate reader. (MTP-32, manufactured by Corona Electric)
Was measured.

(3) Test result As a result of this test, GOP was 10 ⁻⁵ and GPP was 10 ^−1.5 . That is, GOP showed 1 / 100,000 antigenicity of casein, while GPP showed about 1/32 of antigenicity of casein, and the antigenicity of GOP was much lower than that of GPP. Turned out to be.

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

(1) Preparation of sample GOP and GPP (control) were prepared in the same manner as in Test Example 2.
Prepared.

(2) Test method (2-1) Preparation of various solutions (2-1-a) Substrate solution (tyrosine solution) 0.1 M of L-tyrosine (Wako Pure Chemical Industries, Ltd.)
It was dissolved in a phosphate buffer (pH 7.0) at a concentration of 0.05% (W / V).

(2-1-b) Sample solution A sample was added to a 0.1MTP phosphate buffer (pH 7.0) at 0.5 to 2.0% (W
/ V).

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

(2-1-d) Enzyme solution Mushroom-derived tyrosinase (manufactured by Sigma, 30
(00 units / mg) was dissolved in purified water at a concentration of 0.1% (W / V).

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

(2-2) Enzyme reaction and absorbance measurement: 0.9 ml of substrate solution, 1.0 ml of sample solution, previously heated to 37 ° C,
A copper ion solution (0.02 ml) was collected in a test tube, and an enzyme solution (0.08 ml) heated to the same temperature was added (total amount: 2.0 ml), and reacted at 37 ° C. for 3 minutes. Next, 2 ml of a reaction stop solution was added to stop the reaction, and the absorbance at a wavelength of 640 nm was measured with a spectrophotometer (this absorbance is referred to as B). As a control, 1.0 ml of a 0.1 M phosphate buffer (pH 7.0) was added instead of the sample solution, and the same treatment was carried out, and the absorbance was measured (this absorbance is referred to as A). If the test solution is cloudy, add 0.08 ml of 0.1 M phosphate buffer instead of the enzyme solution and measure the absorbance in the same manner (this absorbance is referred to as C). Was removed.

(2-3) Calculation From the measured absorbance values, the tyrosinase activity inhibition rate (%) was calculated by the following equation.

Inhibition rate (%) = 100 [1- (BC) / A] (3) Test results The results of this test were as shown in Table 1. Table 1
As is clear from the above, the tyrosinase activity inhibition rate of GOP was higher than that of GPP at each concentration, and GOP exhibited a significant tyrosinase activity inhibitory action.

Tyrosinase is an enzyme that catalyzes the acid value of tyrosine and other monohydric phenols and corresponding ortho-dihydric phenols by molecular oxygen, and is present in many plant and animal tissues such as mushrooms, potatoes and apples, and Is known to be involved in the formation of melanin pigments in tissues, especially skin epidermal cells (edited by the Editorial Board of the Chemical Dictionary, Chemical Dictionary, Vol. 5, p. 976, Kyoritsu Shuppan, 1960) ). It is also known that the increase in skin color in Addison's disease, in which melanin is deposited on skin or mucous membranes and turns black, is due to a decrease in secretion of adrenocortical hormone which antagonizes melanotropin which promotes tyrosinase activity ( Compilation of Chemical Dictionary, Chemical Dictionary, Volume 1, Volume 65
Page, Kyoritsu Publishing, 1960). In addition, tyrosinase
It is also said to be involved in reducing the freshness of food.

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

Hereinafter, the present invention will be described in more detail through Reference Examples and Examples. It should be noted that these reference examples and examples are exemplifications for explanation, and the present invention is not limited to these reference examples and examples.

Reference Example 2000 g of commercially available casein lactate (from New Zealand)
It 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. To this solution was added 248 g of a 40% aqueous calcium chloride solution (final concentration
0.3M), the mixture was stirred at 60 ° C for 15 to 30 minutes to sufficiently react with calcium, and after the solution became homogeneous, the supernatant was separated using a continuous centrifuge. The supernatant is treated with an ultrafiltration module (SIP
1013, manufactured by Asahi Kasei Kogyo Co., Ltd.), desalted to a calcium concentration of 1% or less of the initial concentration, freeze-dried, and kappa with a purity of about 70%.
About 120 g of casein powder were obtained.

Example 4000 g of κ-casein powder obtained by repeating the same method as in the Reference Example was dissolved in water at a concentration of 5%, the pH was adjusted to 6.3, and commercially available microorganism-derived rennet (each sugar industry company) 25)
After addition of 00 mg, the mixture was reacted at 35 ° C. for 30 minutes, heated at 70 ° C. for 10 minutes to inactivate the enzyme, and centrifuged to separate the supernatant. The supernatant was concentrated to a protein concentration of 3.2 times using an ultrafiltration module (SPE 1013, manufactured by Asahi Kasei Kogyo) having a molecular weight cutoff of about 3000, and a commercially available protease (protease Amano A, manufactured by Amano Pharmaceutical Co., Ltd.) was used. 0.5 g and 6.25 g of a lactic acid cell suspension (solid content: 8%) were added, and reacted at 51 ° C. for 20 hours. Then, the reaction solution was heated at 80 ° C. for 10 minutes to inactivate the enzyme. After cooling for 2 hours, 20 g of kieselguhr was added and the mixture was filtered. 1000 g of the obtained filtrate was fractionated using an ultrafiltration module (SEO1013, manufactured by Asahi Kasei Kogyo Co., Ltd.) having a molecular weight cutoff of about 3,000. The first fractionation circulates 1000 g of the above aqueous solution through the module and continues until 500 ml of the outer solution is obtained. Then, 500 ml of distilled water is added to the inner solution (500 ml).
Batch fractionation was continued by circulating the mixture through the module until the volume of the external solution reached 500 ml. Thereafter, the same operation as in the second fractionation was repeated four more times, and 350 g of the obtained internal solution was freeze-dried to reduce the sialic acid content. 3.4 g of a high glyco-oligopeptide mixture was obtained. The molecular weight distribution, the ratio of nitrogen to sialic acid, the residual antigenic activity, and the amino acid composition of the glycooligopeptide mixture having a high sialic acid content measured by the same method as in the above Test Example were as follows.

Molecular weight distribution 500-2000 SA / N ratio * 0.14 Antigenic residual activity 10 ^-5 (* indicates the number of molecules, 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 7.4 Cystine 0.3 Methionine 0.2 Isoleucine 4.7 Leucine 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 present invention The effects of the present invention are as follows.

1) The sialic acid-rich glyco-oligopeptide mixture of the present invention has substantially no antigenicity.

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

──────────────────────────────────────────────────続 き Continuation of the front page (51) Int.Cl. ⁶ identification code FI A61K 38/55 A61K 37/64 (58) Investigated field (Int.Cl. ⁶ , DB name) C07K 14/47 C07K 9/00 A23J 3/34 A23J 1/20

Claims (2)

(57) [Claims] 1. A hydrolyzate of κ-casein, which has the following (a) to (e), (a) a molecular weight distribution of 500 to 2,000, and (b) a sialic acid content of 20 g / 100 g or more. (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, and glutamic acid, threonine, serine, proline, alanine, valine, Aspartic acid, isoleucine, and glycine as main constituent amino acids; (e) ELISA (Enzyme Linked Immuno Sorbent)
Assay) The residual antigen activity measured by the inhibition test method is 10 ^-5
A glyco-oligopeptide mixture having a high sialic acid content, characterized by having the following physicochemical properties: 2. Kappa-casein is hydrolyzed by rennet, a glycooligopeptide-containing fraction having a molecular weight of about 7,000 is separated from the obtained hydrolyzate, and the glyco-oligopeptide contained in the fraction is converted to a protease. Hydrolyzed by
A method for producing a glyco-oligopeptide mixture having a high sialic acid content, comprising collecting a fraction having a molecular weight of 500 to 2,000 from the obtained hydrolyzate.