JP2003246800A - Method for producing lactone of sialic acid compound - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for easily producing a lactone in a high yield from a sialic acid compound contained in a processed milk product, etc. <P>SOLUTION: This method for producing a lactone of a sialic acid compound comprises separating a fraction having a mol.wt. of 1,000 or higher from a raw material containing the sialic acid compound, bringing the fraction into contact with an anion exchanger, and converting the adsorbed fraction into a lactone. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing a lactone form easily and in high yield from a sialic acid compound contained in a processed milk product useful as a food material. In the present invention, the sialic acid compound means a substance containing sialic acid as a constituent in the molecule. Further, in the present specification, percentages are expressed by weight unless otherwise specified.

[0002]

2. Description of the Related Art Conventionally, it has been known that sialic acid is located at the terminal of a cell surface sugar chain, a glycoprotein sugar chain, or a glycolipid sugar chain, and is involved in various physiological functions such as cell-cell discrimination.
Sialic acid does not exist in the free state in nature, but exists in milk etc. as a constituent molecule of oligosaccharides, gangliosides, glycoproteins, etc., and has an infection protective activity, and acts as a bifidus factor (Kunio Yamauchi et al. Volume, "Milk Encyclopedia," Asakura Shoten, page 519, 1992),
And controlling the early immunity of the infant (a dairy science study,
Volume 17, page A-55, 1968) and other physiological functions are known.

Breast milk contains about 3 to 5 times as much sialic acid compound as cow's milk, and attempts have been made to utilize the sialic acid compound as a milk substitute, a functional food, and a drug material. .

On the other hand, it has been reported that a lactone form of a sialic acid compound having a lactone structure in the molecule exists in the brain and milk [[The Journal of Biological Chemis
try), vol. 261, page 8514, 1986.
Year], [Biochimika Biophysica Actor (Biochimi
caet Biophysica Acta), 1381, 286, 1998]].

The lactone form of the sialic acid compound is a substance similar to the sialic acid compound, but does not have a free carboxyl group, and the carboxyl group forms a part of the lactone ring. It is a substance that has a completely different chemical structure from the compound. In addition, since the lactone form of the sialic acid compound has a lactone structure in the molecule, it is more resistant to the decomposition of sialic acid by sialidase (sialic acid cleaving enzyme), as compared with the non-lactonized sialic acid compound. [Chemistry Letters, page 669, 1997] having the useful property of

Conventionally, as a method for separating a lactone form of a sialic acid compound having such properties from a natural raw material,
A method in which oligosaccharides such as sialyllactose, which is a sialic acid compound contained in bovine colostrum in a relatively large amount, are separated, lactonized, and then contacted with an anion exchanger to obtain a non-adsorbed fraction [ Methods in Enzymology, Volume 242, page 102, 1994. Hereinafter, it is described as prior art 1. ] Was reported.

[0007] Further, the present inventors have focused on sialic acid bound to glycoproteins contained in a large amount in normal milk in addition to sialyllactose contained in colostrum, as natural raw materials for lactones. The raw material containing is subjected to ring opening treatment in advance,
The raw material is brought into contact with an anion exchanger to separate the sialic acid compound contained in the adsorbed fraction, which is then lactonized, and again brought into contact with the anion exchanger to obtain a non-adsorbed fraction to obtain a lactone. Method for obtaining body (Japanese Patent Laid-Open No. 2001-2138)
94 publication. Hereinafter, it is referred to as Prior Art 2. ) Has already been disclosed.

[0008]

However, the above-mentioned prior art has the following problems. Prior art 1 reports a method for separating 3'-sialyllactose and 6'-sialyllactose, which are typical sialic acid compounds, from bovine colostrum as a raw material, but only bovine colostrum is used as a raw material. Therefore, the yield is low, and it is unsuitable for industrially producing a large amount of lactone, and since the chemicals such as chloroform, methanol, and pyridine are used, the produced lactone is a food product. However, there is a problem that it is difficult to use it as a material for feed, pharmaceuticals, cosmetics and the like. Further, sialic acid is present in milk even in a state of being bound to a sugar chain of a glycoprotein such as κ-casein, but in the conventional technique 1, a lactone form is efficiently produced from a sialic acid compound such as glycoprotein. It was impossible to do.

In the prior art 2, not only colostrum but also various raw materials such as normal milk and whey which are available in large quantities are used to produce lactones from sialic acid compounds such as sialyllactose and glycoproteins. Was the way it could be. However, the lactone ring contained in the raw material is once subjected to a ring-opening treatment, an ion exchange treatment, a lactonization treatment again, and an ion exchange treatment to collect the non-adsorbed fraction. Requires a large number of processes,
It was also observed that the yield did not rise as expected due to the lactonization treatment in the intermediate stage of production. Therefore, there has been a demand for a manufacturing method that is simpler and enables high yield.

An object of the present invention is to provide a method for easily producing a lactone form of a sialic acid compound from a raw material containing the sialic acid compound in a high yield.

[0011]

Means for Solving the Problems The inventors of the present invention have made extensive studies in view of the above problems, and as a result, fractionated from a raw material containing a sialic acid compound, a fraction having a molecular weight of 1000 or more, and fractionated the fraction. It was found that a lactone compound obtained from a sialic acid compound can be produced by a simple method and in a high yield by contacting an ion exchanger and lactonizing the adsorption fraction thereof. Furthermore, it has been found that it is particularly preferable to produce a lactone according to this method when a processed milk product containing a large amount of a sialic acid compound such as glycoprotein is used as a raw material.

That is, the present invention is characterized in that a fraction having a molecular weight of 1000 or more is fractionated from a raw material containing a sialic acid compound, the fraction is brought into contact with an anion exchanger, and the adsorption fraction is lactonized. And a method for producing a lactone obtained from the sialic acid compound.

In the method for producing the lactone, it is preferable that the adsorption fraction is hydrolyzed and then lactonized. In the method for producing the lactone form, it is preferable to use a processed milk product as the raw material containing the sialic acid compound. The processed milk product is preferably one or a mixture of two or more selected from casein, a casein degradation product, cheese whey, and rennet whey.

In the method for producing a lactone, the lactone is preferably a glycopeptide lactone or a glycoprotein lactone. Further, it is preferable that the lactone has microbial sialidase resistance activity.

[0015]

BEST MODE FOR CARRYING OUT THE INVENTION Preferred embodiments of the present invention will be described in detail below. However, the present invention is not limited to the following preferred embodiments and can be freely modified within the scope of the present invention.

The sialic acid compound used in the present invention is a substance containing sialic acid in the molecule as a constituent, and specifically, sialic acid or an oligosaccharide containing sialic acid as a constituent, a peptide or a protein. Examples thereof include glycopeptides and glycoproteins. These glycopeptides,
Glycoprotein and the like have a relatively high molecular weight, but by using the method of the present invention, a lactone form of a sialic acid compound can be obtained from these in high yield. Examples of proteins include casein and whey protein. Examples of peptides include degradation products of casein, whey protein and the like. The raw material containing a sialic acid compound used in the present invention may be any as long as it contains a sialic acid compound, but it is preferably a natural raw material acceptable as a food, A milk processed product is particularly preferable because it is an available raw material.

Examples of the processed milk products include casein obtained from milk, human milk, goat milk and the like, casein degradation products, cheese whey produced as a by-product during cheese production, rennet whey, etc. Origin casein (manufactured by Morinaga Milk Industry Co., Ltd.), bovine κ-casein (manufactured by Sigma), cheese whey (manufactured by Mirai) and the like can be used. The milk processed product may be used alone or in combination of two or more.

The solution raw material can be used as it is as a raw material solution. The solid or powdered raw material can be dispersed or dissolved in water or warm water and used as a raw material solution. The concentration of the raw material solution is not particularly limited, but it is usually adjusted to a concentration range of about 5 to 30% (weight, hereinafter, the same unless otherwise specified) in terms of solid content in the subsequent steps. It is desirable in terms of efficiency and operability.

Next, a method for producing a lactone form of a sialic acid compound according to the present invention will be described as a first embodiment (hereinafter referred to as production method 1) and a second embodiment (hereinafter referred to as production method 2). It will be described separately. A flowchart showing the steps of manufacturing method 1 and manufacturing method 2 is shown in FIG.

[1] Production Method 1 (a) The raw material containing the pretreated sialic acid compound may be used as it is, but in order to improve the efficiency and operability of the production process, it may be changed depending on the type of each raw material. Therefore, it is desirable to perform the following pretreatment.

When whey or casein degradation product is used as a raw material, unnecessary proteins and peptides are removed by a trichloroacetic acid precipitation method, an alcohol precipitation method, a heating method or the like. Specifically, in the trichloroacetic acid precipitation method, trichloroacetic acid having a final concentration of 5 to 30% is added to the raw material solution, and in the alcohol precipitation method, alcohol having a final concentration of 20 to 80% is added, while in the heating method, the raw material solution is added. 80-100 ° C
After that, each method is left to stand for a certain period of time and then centrifuged to precipitate and remove unwanted proteins and peptides. When the whey or casein decomposition product used as the raw material is solid or powdery, a trichloroacetic acid solution or an alcohol solution is prepared in advance so that the final concentration after addition of the raw material reaches a target final concentration. Disperse the raw material directly, leave it for a certain time, centrifuge,
It is also possible to precipitate and remove unnecessary proteins and peptides.

When casein containing κ-casein or the like is used as a raw material solution, the raw material solution is treated with a protein hydrolase such as calf chymosin or porcine pepsin to hydrolyze the protein contained in the raw material solution, Unwanted proteins and peptides are separated and removed by the above-mentioned trichloroacetic acid precipitation method, alcohol precipitation method, heating method and the like.

The above trichloroacetic acid precipitation method, alcohol precipitation method, heating method and the like can be used in combination of two or more. For example, the supernatant separated by the alcohol precipitation method may be treated by a heating method to remove unnecessary proteins and peptides. If fine precipitates are found in the raw material solution, it is desirable to remove them by centrifugation or a clarifier as appropriate.

(B) Fractionation Treatment by Molecular Weight In the present invention, only a fraction having a molecular weight of 1000 or more is fractionated from a raw material solution containing a sialic acid compound which has been appropriately pretreated and used in the next step. By collecting fractions with a molecular weight of 1000 or more, unnecessary low-molecular weight peptides, free amino acids, lactose (lactose), salts (minerals), etc. can be removed from the raw materials, thus producing a more pure lactone form. can do.

As a method for fractionating the above-mentioned fraction having a molecular weight of 1000 or more, all fractionation methods capable of molecular weight fractionation can be used. As a preferred example of the preparative method,
When the amount of the raw material solution is small, a dialysis method, a gel filtration method and the like are included. When the amount of the raw material solution is large, an ultrafiltration method and the like can be used, in which the concentration can be performed simultaneously with the molecular weight fractionation.

As the dialysis membrane used in the dialysis method, seamless cellulose tubing 24/32 is used.
(Manufactured by Sanko Junyaku Co., Ltd.), Spectropore 1 [MWCO: 600-800
0], Spectropore 6 [MWCO: 1000-50000] (above,
Made by Spectrum Co., Celsep T3 [MWCO: 12000-1
4000] (manufactured by Membrane Filtration Products Inc.) and the like.

The gel filtration carrier used in the gel filtration method is Sephadex G10, G15, G2.
5, G50, and G75 (above, manufactured by Amersham Biosciences), biogels P2, P6, P30, and P
60 (above, manufactured by Bio-Rad) and the like are exemplified.

The ultrafiltration membrane used in the ultrafiltration method is a UF membrane module SEP0013, SI.
Examples include P0013 and SLP0053 (all manufactured by Asahi Kasei Corp.).

(C) Treatment with ion exchanger The fractionated solution obtained by fractionating the above-mentioned fraction having a molecular weight of 1000 or more is then contacted with an anion exchanger to collect the adsorbed fraction. By collecting the fraction having a molecular weight of 1000 or more and then subjecting it to an ion exchanger treatment, the efficiency of collecting the adsorbed fraction can be further enhanced.

The anion exchanger is not particularly limited, and specific examples of the anion exchanger used include AG1X4.
(Manufactured by Bio-Rad), Microprep Q support (manufactured by Bio-Rad), DEAE-cellulose, DEA
E-Sephadex, Q-Sepharose (above, manufactured by Amersham Biosciences), Amberlite IRA4
Examples thereof include weakly basic anion exchangers such as 00 (manufactured by Organo) and strong basic ion exchangers.

The method of passing the preparative solution into contact with the anion exchanger is not particularly limited, and examples thereof include a column method and a batch method. During the passage, the concentration of the fractionated solution is adjusted so that lactose does not precipitate, and is preferably 0.01 to 30% in terms of solid content, more preferably 0.
It is adjusted to 1 to 25%. Further, the temperature during the passage is set in a range in which lactose does not precipitate, preferably 4 to 50 ° C.
More preferably, it is set to 10 to 50 ° C. Furthermore, the ionic strength during the passage of the liquid preferably has an electrical conductivity of 30 mS.
/ Cm or less, more preferably 0.001 to 20 mS / c
m is set.

The elution method of the sialic acid compound adsorbed on the anion exchanger is carried out by passing the preparative solution through the anion exchanger, washing the anion exchanger with water or the like, and then adsorbing with a salt solution. It is carried out by eluting the fractions.
Examples of the salt solution include sodium chloride solution and sodium acetate solution. The salt concentration of the salt solution is not particularly limited as long as it is in the range of 0.01 to 1.5 M, and it is possible to elute at any salt concentration in this range. In addition, for elution of the adsorption fraction from the anion exchanger,
Forming a continuous salt concentration gradient using a method in which multiple concentrations of salt solution are used and then a high concentration salt solution is passed in sequence to elute stepwise, or a concentration gradient creation device is used. It is also possible to appropriately select and use the method of elution and the like.

The eluate thus recovered by anion exchange chromatography is desalted by a gel filtration method, an electrodialysis method or the like, and then used in the next step. Further, when the volume of the eluate is large, it may be concentrated by appropriately performing an evaporator, freeze-drying or the like.

(D) Lactonization treatment The eluate subjected to the desalting treatment, or the concentrate or freeze-dried product thereof, is then subjected to a lactonization treatment to lactonize the sialic acid compound contained in the adsorbed fraction. . As the lactonization method, any method capable of lactonizing a sialic acid compound can be used, but in consideration of applying the final product, the lactone form, to foods such as glacial acetic acid. The method of lactonizing a sialic acid compound by carrying out a dehydration / condensation reaction using an acceptable acid is particularly preferable.

Specifically, the desalted eluate, or the concentrate or freeze-dried product thereof is dissolved in glacial acetic acid, preferably at 25 ° C. or higher for 10 hours or longer, more preferably 30 ° C. or higher. The lactonization is carried out by reacting for 72 hours or more. During the lactonization treatment, if the concentration of the sialic acid compound is high, a reaction such as intermolecular condensation may occur, and if the concentration of the sialic acid compound is low, the reaction efficiency of the lactonization may decrease. is there. Therefore, the desalted ion-exchange eluate, or the concentrated solution or lyophilized product thereof is added to glacial acetic acid so as to be less than 10%, more preferably 0.01 to 8% in terms of solid matter. It is desirable that the lactonization is carried out by dissolving the lacton in the solution.

By repeating the above-mentioned lactonization treatment, the lactone form of the sialic acid compound can be obtained in higher purity. Specifically, the solution obtained by the lactonization treatment is freeze-dried, and the freeze-dried powder is dissolved again in glacial acetic acid, preferably at 25 ° C. or higher for 10 hours or more, more preferably at 30 ° C. or higher for 72 hours. Lactonization is carried out by the above reaction.

The lactone form of the sialic acid compound thus obtained can be used as it is or in the form of a solution as a raw material for foods and the like. However, since the lactone form in a solution state may be decomposed with time, it is desirable to prepare the powder form by freeze-drying or the like and store it when it is stored for a long period of time.

[2] Manufacturing Method 2 In Manufacturing Method 2 which is the second embodiment of the present invention, steps (a) to (c) described in Manufacturing Method 1 are performed as common steps. That is, a fraction having a molecular weight of 1000 or more is collected from a raw material containing a sialic acid compound that has been appropriately pretreated, and the fraction is contacted with an anion exchanger to collect the adsorbed fraction.
Thereafter, the collected adsorption fraction is subjected to a hydrolysis treatment (c ') using a protein hydrolase, and then the same step as the (d) lactonization treatment described in Production method 1 is performed to obtain a sialic acid compound. A lactone form is produced from Hereinafter, the hydrolysis treatment (c ') that is different from the step of the production method 1 will be described.

(C ') Hydrolysis treatment After treating a raw material containing a sialic acid compound by the same method as the steps (a) to (c) described in the production method 1, the obtained desalted ion exchange elution The solution, or a concentrated solution or a lyophilized product thereof, is used as a 10-20 mM Tris-HCl buffer (p
H7.5) and add a protein hydrolase to a solid content of about 0.1 to 1%.
Hydrolysis is carried out at ℃ for 3 to 24 hours.

The protein hydrolase used in the hydrolysis treatment (c ') is not particularly limited as long as it hydrolyzes a protein, but Proteinase K (enzyme number: EC3.4.21.64: manufactured by Sigma) , Pronase (enzyme number: EC3.4.24.31: manufactured by Kaken Chemical Co., Ltd.) and the like. The solution after the hydrolysis treatment (c ') is 50 to 10
After heating at 0 ° C. for about 1 to 30 minutes to inactivate the proteolytic enzyme used, or as it is without deactivating the proteolytic enzyme, the gel filtration method, the electrodialysis method or the like described in Production Method 1 To desalt.

After the hydrolysis treatment (c '),
A lactone form can be produced by performing a lactonization treatment by the same method as the step of the lactonization treatment (d) described in Production Method 1. In addition, the hydrolysis treatment (c
It is also possible to perform the ion exchanger treatment (c) step described in the production method 1 again between the step ′) and the lactonization treatment step, whereby unnecessary peptides and the like are separated and removed from the hydrolysis treatment solution. Therefore, it is possible to produce a higher-purity lactone form.

As described above, the manufacturing method 2 is the manufacturing method 1.
A method similar to, but capable of producing a high-purity glycopeptide lactone or glycoprotein lactone having a higher lactone content compared to Production Method 1 by performing a hydrolysis treatment after the ion exchanger treatment. Is. Further, the lactone body obtained from the sialic acid compound produced according to the production method 2 is used as a material for foods, feeds, pharmaceuticals, cosmetics, etc., similarly to the lactone body obtained from the sialic acid compound produced according to the production method 1. In addition to being possible,
Its low molecular weight makes it suitable for analyzing its structure. In the NMR analysis described below, the structure was analyzed using the lactone form of the sialic acid compound produced according to Production Method 2.

As described above, in the above Production Method 1 or Production Method 2, the production of a lactonized glycopeptide and a lactonized glycoprotein in which a peptide or protein is bound to a lactonized sugar chain containing sialic acid or sialic acid as a constituent component. Especially suitable for. The intramolecular lactonization of the sialic acid compound can be appropriately monitored by a nuclear magnetic resonance spectrum method as shown in the test method described later. In addition, the lactone form of the sialic acid compound obtained by the method of the present invention has a resistance activity against sialidase derived from a microorganism. Examples of such microorganisms include Salmonella typhimurium and Arthrobacter ureafasciens.
reafaciens), or Streptococcus sp. The lactone form of the sialic acid compound of the present invention is obtained by such a method.

[0044]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to the following examples. Example 1 200 g of desalted cheese whey powder was dispersed in 1 liter of a 12% trichloroacetic acid solution, and the supernatant after centrifugation was dialyzed against water using Seamless Cellulose Tube 24/32 (manufactured by Sanko Junyaku Co., Ltd.). After freeze-drying, about 7 g of freeze-dried powder was obtained. Of this, 100 mg of 3 ml of 1
Dissolve in 0 mM Tris-HCl buffer (pH 7.5)
After passing through a column packed with pharose High Performance (2.6 × 15 cm, manufactured by Amersham Biosciences) and thoroughly washing with demineralized water, sodium chloride of 0 to 1.0 M concentration was prepared using a concentration gradient preparation device. Was eluted with a linear gradient of. The fraction eluted at a salt concentration of 0.2 to 0.5 M was applied to Bio-gel P2 (2.6 × 90 cm, manufactured by Bio-Rad), and the void volume (V ₀ = 1) was applied.
30 ml eluted around 60 ml) was collected and freeze-dried to obtain about 40 mg of dry powder. This powder was dissolved using glacial acetic acid (manufactured by Nacalai Tesque, Inc.) so as to have a concentration of 2% in terms of solid content, and reacted at 30 ° C. for 3 days to lactonize. The solution containing the obtained lactone form was diluted 10 times with distilled water, acetic acid was removed by using Micro Acylizer G1, and then freeze-drying was performed to freeze the lactone form obtained from the sialic acid compound. About 45 mg of dry powder was produced.

Example 2 200 g of desalted cheese whey powder was dispersed in 1 liter of a 12% trichloroacetic acid solution, and the supernatant after centrifugation was mixed with water using Seamless Cellulose Tubing 24/32 (manufactured by Sanko Junyaku Co., Ltd.). It was dialyzed and freeze-dried to obtain about 7 g of freeze-dried powder. Of this, 100 mg is 3 m
It was dissolved in 10 ml of 10 mM Tris-HCl buffer (pH 7.5), and Q Sepharose High Performance (2.6 × 15c) was used.
m, manufactured by Amersham Bioscience Co., Ltd.) and was thoroughly washed with demineralized water, and then eluted with a linear gradient of 0 to 1.0 M sodium chloride using a concentration gradient preparation device. Fractions eluted at a salt concentration of 0.2-0.5M were collected on Bio-gel P2 (2.6 x 90 cm,
The product was applied to Bio-Rad) and 30 ml eluted near the void volume (V ₀ = 160 ml) was collected and freeze-dried. This is dissolved in 40 ml of 20 mM Tris-HCl buffer (pH 7.5) and proteinase K is added.
(Enzyme number: EC3.4.21.64, manufactured by Sigma) was added in an amount of 2 mg and reacted at 37 ° C. for 24 hours. Reaction mixture at 98 ℃ 1
Heat for 0 minutes to inactivate the enzyme, and use Q Sepharose High Per
The solution was passed through a column packed with formance (2.6 × 15 cm), thoroughly washed with demineralized water, and then eluted with a 1.0 M sodium chloride solution. Eluate the Bio-gel P2 (2.6 x
90 cm) and the fraction positive for the resorcinol-hydrochloric acid reaction was collected and freeze-dried. This powder was dissolved using glacial acetic acid (manufactured by Nacalai Tesque, Inc.) to a concentration of 2% in terms of solid content, and reacted at 30 ° C. for 3 days,
It was lactonized. The solution containing the obtained lactone form was diluted 10 times with distilled water, and after removing acetic acid using a microacylizer G1, it was freeze-dried to freeze-dry the solution containing the lactone form obtained from the sialic acid compound. About 10 mg of powder was produced.

The amino acid sequence of the protein portion (caseinoglycopeptide: κ-Cn106-109) of glycoprotein lactone (CGP-lactone) obtained in Example 1 is shown in FIG. The amino acid sequence in FIG. 4 is the parent molecule κ.
-Shows residues 106 to 169 resulting from chymosin digestion of casein. Two types of genetic variants, A and B, exist in κ-casein, and as a characteristic of the amino acid sequence, the genetic variant A is 13 in the figure.
Genetic variant B containing threonine (Thr) at the 6th residue and aspartic acid (Asp) at the 148th residue
Is isoleucine (Ile) and 1 at residue 136 in the figure.
The 48th residue contains the sequence of alanine (Ala). Also,
In both the genetic variants A and B sequences, the serine (Ser) at residue 149 is phosphorylated. Further, the sugar chain binding site is CGP derived from κ-casein genetic variant A.
In the CGP derived from the κ-casein genetic variant B, the sugar chains are bound to threonines at residues 131, 133, 135 and 136, and 131, 133 and 1
A sugar chain is bound to threonine at the 35th residue. There are five types of sugar chains, and the constituent sugars are galactose (Ga
l), N-acetylneuraminic acid (NeuAc), N-
Acetylgalactosamine (GalNAc). Among them, one sugar chain structure is shown in Chemical formula 1. In Chemical formula 1, the threonine residue on the right side of GalNAc is a threonine residue in CGP. The glycopeptide lactone in which the sugar chain is bound to CGP is dehydrated, condensed and cyclized between the carboxyl group of NeuAc and the hydroxyl group bonded to the 2-position and 4-position of galactose or the 7-position carbon of NeuAc. To generate. Of these, glycopeptide lactones produced by dehydration, condensation, and cyclization between the carboxyl group of NeuAc and the hydroxyl group bonded to the 2-position carbon of galactose are shown in Chemical formula 2. The glycopeptide lactone obtained in Example 2 has a number derived from the sequence of FIG. 4 at the N-terminal side and the C-terminal side of each threonine residue at the 131, 133, 135 or 136th residue. Is a glycopeptide lactone having tens of amino acids.

[0047]

[Chemical 1]

[0048]

[Chemical 2]

Example 3 3 kg of a casein decomposition product (Morinaga Milk Industry Co., Ltd.) was dissolved in 50 liters of water, fine precipitates were removed by a clarifier (A.P.V.), and 50 liters of ethanol was added. After standing for 30 minutes, the supernatant was centrifuged. Subsequently, the supernatant was ultrafiltered using an ultrafiltration membrane SEP0013 (manufactured by Asahi Kasei) to adjust the pH of the residual liquid side to 7.0, and a column packed with Amberlite IRA410 (manufactured by Organo). Was passed through. Pass water through the column, then 15
After passing a 0 mM sodium chloride solution, a 1.0 M sodium chloride solution was passed and the eluted fractions were collected.
The collected fractions were desalted using an electrodialysis device (Asahi Kasei) and then freeze-dried. The freeze-dried powder thus obtained was dissolved using glacial acetic acid (manufactured by Nacalai Tesque, Inc.) so as to have a concentration of 2% in terms of solid content, and reacted at 30 ° C. for 3 days to be lactonized. Dilute the solution after reaction 10 times with distilled water,
Then, using an electrodialyzer (Asahi Kasei) to desalt
After freezing in a -40 ° C freezing bath and freezing,
Lyophilization was performed to produce 370 g of lyophilized powder containing a lactone form obtained from a sialic acid compound.

Example 4 To 1 kg of acid casein was added 9 liters of water, the pH was adjusted to 6 with sodium hydroxide, 10 g of calf chymosin (manufactured by Sigma) was added, and the mixture was stirred at 37 ° C. for 10 hours. It was made to react. The supernatant of the reaction solution was centrifuged and ultrafiltered using an ultrafiltration membrane SIP0013 (manufactured by Asahi Kasei) to adjust the pH of the residual liquid side to 7.0, and the anion exchange resin AGIX4
The solution was passed through a column filled with (BioRad). After thoroughly washing the column with water, a 0.8 M sodium chloride solution was passed through to separate the eluted fraction. The separated elution fraction was desalted using an electrodialysis device (manufactured by Asahi Kasei) and then freeze-dried. The freeze-dried powder thus obtained was dissolved using glacial acetic acid (manufactured by Nacalai Tesque, Inc.) so as to have a concentration of 2% in terms of solid content, and reacted at 30 ° C. for 3 days to be lactonized. The solution after the reaction was diluted 10-fold with distilled water, and then desalted using an electrodialysis device (Asahi Kasei Co., Ltd.), -40
It was rapidly cooled in a freezing bath at ℃, frozen, and then freeze-dried to produce 70 g of a freeze-dried powder containing a lactone form obtained from a sialic acid compound.

Test Example 1 [Analysis of intramolecular lactonization of sialic acid compound] The method of Nakahara et al. [Tetrahedron Lette
r), Vol. 35, p. 3321, 1994], the state of lactonization in the molecule of the sialic acid compound contained in the sample is analyzed by ¹ H-nuclear magnetic resonance spectroscopy, and the lactone of the sialic acid compound is analyzed. The state of generation of the body was monitored. ¹ H-nuclear magnetic resonance (hereinafter sometimes referred to as NMR) was recorded at 300 K in heavy water (D ₂ O) using a spectrometer INOVA600 (manufactured by Varian), and chemical shift values (ppm) were recorded. ) Was calculated as the difference from the signal positions of 3- (trimethylsilyl) -1-propanesulfate sodium salt (TPS) which is a reference substance and acetone.

The results obtained by analyzing the sample manufactured in Example 2 are shown in FIG. 2C. As control samples, 3'-sialyllactose which is a typical sialic acid compound (Fig. 2 [A]) and 3'-sialyllactose lactone which is a lactone of a typical sialic acid compound (Fig. 2 [B] ]) Was used to perform the same analysis as that performed on the sample manufactured in Example 2, and the obtained spectra are shown in the drawings. The arrow in the figure indicates the position of the signal characteristic of the lactone form.

As is clear from FIG. 2C as a result of the analysis, in the spectrum obtained by analyzing the sample prepared in Example 2, the equatorial position located on the 3-position carbon of the sialic acid molecule is shown. A characteristic signal derived from hydrogen (hereinafter sometimes referred to as H-3eq), that is,
Signals of 2.552 ppm and 2.581 ppm were respectively confirmed, and the structure of the lactone form obtained from the sialic acid compound was confirmed.

Test Example 2 This test was carried out in order to investigate the hydrolysis resistance of a lactone obtained from a sialic acid compound to sialidase. (1) Preparation of Sample The lactone form obtained from the sialic acid compound produced in Example 1 was divided into three microtubes of 150 μg each to obtain test samples (1 to 3). In addition, sialic acid compounds prepared by the same method as in Example 1 except that the lactonization treatment was not performed
Control sample (1
~ 3).

The test sample (1-3) and the control sample (1
~ 3) 500 μl of 10 mM Tris-HCl buffer (pH
In the solution obtained by dissolving in 7.5), Salmonella
An enzyme solution of sialidase derived from S. typhimurium, Arthrobacter ureafaciens or Streptococcus spices was added at a concentration of 20 mU each, and the mixture was incubated at 37 ° C, and the solution was sampled after 2, 4 and 8 hours. Hereinafter, the Salmonella typhimurium-derived sialidase enzyme solution was added to the solution obtained by dissolving each of the test sample 1 and the control sample 1, and the sampled solutions are referred to as the test solution 1 and the comparative solution 1, and the test sample 2 and the control sample A solution obtained by dissolving Arthrobacter ureafaciens-derived sialidase enzyme solution in a solution obtained by dissolving 2 is referred to as Test Solution 2 and Comparative Solution 2, and Test Sample 3 and Control Sample 3 are respectively referred to as A solution obtained by adding a sialidase enzyme solution derived from Streptococcus spices to a solution obtained by dissolution and sampling the solution is referred to as a test solution 3 and a comparative solution 3.

(2) Test Method The free sialic acid present in each test solution (1-3) and comparative solution (1-3) was analyzed by the periodate-thiobarbituric acid method (Metabolism, Vol. 16, Vol. (Pp. 761, 1979)
Was quantified by This method is a method for producing β-formylpyruvate from sialic acid by oxidation with periodate, and coloring this with thiobarbituric acid for quantification. The method was carried out using the following four reagents. Periodic acid solution: Periodic acid (manufactured by Wako Pure Chemical Industries, Ltd.) 25
Solution dissolved in 0.125 N sulfuric acid at a final concentration of mM. Sodium arsenite solution: A solution of sodium arsenite (manufactured by Wako Pure Chemical Industries, Ltd.) in 0.5N hydrochloric acid at a final concentration of 2%. Thiobarbituric acid solution: A solution prepared by dissolving 2-thiobarbituric acid solution (manufactured by Nacalai Tesque, Inc.) in water at a final concentration of 0.1 M and adjusting the pH to 9.0 with sodium hydroxide. Acidic butanol: A solution obtained by mixing 95 ml of n-butanol (manufactured by Nacalai Tesque, Inc.) and 5 ml of 12N hydrochloric acid.

Each test solution (1-3) and comparative solution (1
~ 3) was passed through a cation exchange column AG1X2 (manufactured by Bio-Rad), and the permeated liquid was collected. The collected permeate was appropriately diluted, and 0.25 ml of the periodate solution was added to 0.5 ml of the diluted solution and the mixture was kept at 37 ° C. for 30 minutes. Then, 0.2 ml of the sodium arsenite solution is added to decompose periodate, and 2 m of the thiobarbituric acid is added.
1 was added, and the mixture was capped and kept in a boiling water bath for 7.5 minutes for color development. After coloring, soak in ice water to cool,
Add 5.0 ml of the acidic butanol and mix well,
The dye was redissolved in the butanol layer and the absorbance at 549 nm was measured. In the same manner as above, a solution containing a known concentration of sialic acid (manufactured by Sigma) was colored to give 549 nm.
The absorbance at was measured and a standard curve was prepared. Based on the absorbances of the measured test solutions (1 to 3) and the comparative solutions (1 to 3) based on the prepared standard curve, the test solutions (1 to 1)
3) and the amount of free sialic acid contained in the comparative solutions (1 to 3) were calculated.

(3) Test Results The test results obtained are shown in FIG. From FIG. 3, after the hydrolysis treatment using the microorganism-derived sialidase, the test solution (1 to
It was shown that the amount of free sialic acid contained in 3) (shown by ● in the figure) was smaller than the amount of free sialic acid contained in the comparative solutions (1 to 3) (shown by ○ in the figure). From this, the lactone form of the sialic acid compound contained in the test samples (1 to 3) was higher than that of the non-lactonized sialic acid compound contained in the control samples (1 to 3). It was revealed that it has an excellent resistance to the decomposition of sialic acid by the. Moreover, since the lactone of the present invention has excellent resistance to the degradation of sialic acid by sialidase, it is considered that it has an effect of inhibiting infection with viruses, particularly human influenza A virus. That is, human influenza A virus recognizes and binds to a sugar chain receptor (receptor) containing sialic acid, which is present in target cells such as the respiratory tract, by the hemagglutinin present on the cell surface, invades inside and self-proliferates. Then, the sialic acid is cleaved by the receptor-destroying enzyme (neuraminidase) and released from the host cell.
Cause infection. CGP lactone showed resistance to hydrolysis of microbial-derived sialidase, so that hemagglutinin was strongly recognized and bound, resulting in competitive inhibition,
Since the frequency of infection decreases and it becomes difficult for it to be cleaved by neuraminidase, it is considered that the withdrawal step is strongly inhibited to comprehensively control viral infection.

[0059]

As described in detail above, the present invention is
The present invention relates to a method for easily producing a lactone form obtained from a sialic acid compound with high yield and high purity from a natural raw material, and the effects of the present invention are as follows. (1) By the method of the present invention, a milk processed product or the like that can be obtained in a large amount can be used as a raw material, and a lactone form obtained from a sialic acid compound can be easily produced in high yield. (2) By the method of the present invention, it becomes possible to mass-produce a lactone form from a processed milk product as a raw material without using a substance that is not allowed in foods, pharmaceuticals, cosmetics, feeds, etc.
It can be applied to a wide range of applications as a material for food, pharmaceuticals, cosmetics, feed, etc.

[Brief description of drawings]

FIG. 1 is a manufacturing process diagram showing a method for manufacturing a lactone obtained from a sialic acid compound.

FIG. 2 shows 3′-sialyllactose [A],
^{1 is} a ¹ H-NMR spectrum of 3′-sialyllactose lactone [B] and a lactone derivative [C] obtained from the sialic acid compound produced in Example 2.

FIG. 3 is a graph showing the resistance of lactones obtained from the sialic acid compound produced in Example 1 to sialidase derived from microorganisms.

FIG. 4 shows the amino acid sequence of the peptide portion of the glycopeptide lactone obtained in Example 2.

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Totoshi Ito             2-20-7 Izumi-ku, Sendai City, Miyagi Prefecture (72) Inventor Hironori Hayazawa             Morinaga 5-83 Higashihara 5-chome, Zama City, Kanagawa Prefecture             Dairy Industry Co., Ltd. (72) Inventor Yoshitaka Tamura             Morinaga 5-83 Higashihara 5-chome, Zama City, Kanagawa Prefecture             Dairy Co., Ltd. Nutritional Science Research Institute (72) Inventor Susumu Teraguchi             Morinaga 5-83 Higashihara 5-chome, Zama City, Kanagawa Prefecture             Dairy Co., Ltd. Nutritional Science Research Institute (72) Inventor Hidefumi Kuwata             Morinaga 5-83 Higashihara 5-chome, Zama City, Kanagawa Prefecture             Dairy Co., Ltd. Nutritional Science Research Institute F-term (reference) 4B064 AG01 BA10 CA21 CB06 DA01                       DA13                 4C057 AA05 AA18 BB04 CC03 EE03                       JJ09 JJ10                 4H045 AA10 AA20 AA30 BA53 CA40                       EA01 EA20 FA16 GA23 HA03

Claims (7)

[Claims] 1. A sialic acid characterized in that a fraction having a molecular weight of 1000 or more is collected from a raw material containing a sialic acid compound, the fraction is brought into contact with an anion exchanger, and the adsorbed fraction is lactonized. A method for producing a lactone form of a compound. 2. The method for producing a lactone form of a sialic acid compound according to claim 1, wherein the adsorbed fraction is hydrolyzed and then lactonized. 3. The method for producing a lactone derivative of a sialic acid compound according to claim 1, wherein the raw material containing the sialic acid compound is a processed milk product. 4. The processed milk is casein, a casein decomposition product,
The method for producing a lactone form of a sialic acid compound according to claim 3, which is one kind or a mixture of two or more kinds selected from cheese whey and rennet whey. 5. The method for producing a lactone form of a sialic acid compound according to any one of claims 1 to 4, wherein the lactone form is a glycopeptide lactone or a glycoprotein lactone. 6. The method for producing a lactone form of a sialic acid compound according to any one of claims 1 to 5, wherein the lactone form has a microorganism-derived sialidase resistance activity. 7. A lactone form of a sialic acid compound obtained by the method for producing a lactone form of a sialic acid compound according to any one of claims 1 to 6.