JP2927845B2 - Oligosaccharide composition and method for producing the same - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a method for efficiently producing an oligosaccharide which can be used as a food material that can be used for promoting the growth of bifidobacteria.

(Prior art) In recent years, the usefulness of Bifidobacterium has been clarified, fermented milk and intestinal medicine using Bifidobacterium has been marketed, and infant formula powder added with a special sugar source for the purpose of growing Bifidobacterium, Drinks, confectionery, and the like have come to be marketed.
For example, fructooligosaccharides, isomaltooligosaccharides, soybean oligosaccharides, and the like are used as special sugar sources for the purpose of growing Bifidobacteria. Recently, oligosaccharides have been attracting attention as growth promoting substances for Bifidobacteria. It is becoming popular. In addition, some oligosaccharides contained in human milk are known to be growth factors for bifidobacteria, and galactooligosaccharides, N-acetyllactosamine, and the like have been developed. This N-acetyllactosamine [Gal β- (1 → 4) GlcNAc] is contained as a partial structure in many human milk oligosaccharides, and is present in the glycans of ribopolysaccharides, various glycoproteins and glycolipids. In addition to bifidobacterial growth factor, it is a very important oligosaccharide not only biochemically, but also a promising material as a functional raw food material.

Conventionally, it has been known that N-acetyllactosamine is produced by chemical synthesis or by lactose synthase using high energy compounds UDP-galactose and N-acetylglucosamine as substrates. However, it is a complicated and expensive manufacturing method that still has many industrial difficulties. On the other hand, as a simple method, using lactose and NP acetylglucosamine as substrates, Lactobacillus bifidus and Sporobolomyces sin
gularis) [J. Biol. Chem., 271 , 79
(1955), Can. J. Chem., 42 , 2307 (1964)] and a report of the action of β-galactosidase produced by Lactobacillus bifidus [J. Biol. Chem., 208 , 299 (1954)]. is there. Recently, Escherichia coli (Escherichichi) has been developed using galactose and N-acetylglucosamine as substrates.
a production of N-acetyllactosamine by a dehydration-condensation reaction with a β-galactosidase produced by A. coli (Starch Science, 36 , 113 (1989)).

However, the methods using microbial cells and β-galactosidase described above all have a low production amount of N-acetyllactosamine and a β-1,6 bond Isomeric N-
There are drawbacks such as a very high production ratio of acetyl allolactosamine.

Therefore, the present inventor studied a method for industrially producing N-acetyllactosamine easily and at low cost in order to solve the above-mentioned disadvantages, and found that Bacillus circulans (Bacillus c.
It has been found that β-galactosidase produced by L. irculance efficiently produces N-acetyllactosamine using lactose and N-acetylglucosamine as substrates (see Japanese Patent Application No. 1-184918).

(Problems to be Solved by the Invention) In view of such circumstances, the present inventor has further developed the above-mentioned patent application in the course of studying the production of oligosaccharides and contains an N-acetyllactosamine unit. The oligosaccharide efficiency represented by the general formula Gal β- (1 → 4)-[GlcNAc] n (Gal represents a galactose residue, GlcNAc represents an N-acetylglucosamine residue; n = 2 to 6) We found a good production method and completed the present invention.

(Means for Solving the Problem) The present invention relates to a method for preparing lactose or a lactose derivative,
An object of the present invention is to provide a method for producing an oligosaccharide containing an N-acetyllactosamine unit by reacting β-galactosidase, which has glycosyltransferring activity, on -acetylchitooligosaccharide or an N-acetylchitooligosaccharide mixture.

As the lactose derivative of the present invention, lactitol,
Lactulose can be used. In addition, N-acetylchitooligosaccharides include disaccharides to hexasaccharides obtained by hydrolyzing chitin with an acid or an enzyme, and separated and purified by means such as activated carbon chromatography, and N-acetylglucosamine as a monosaccharide. Mixtures can be used.

Β-galactosidase used in the present invention is an enzyme produced by Bacillus circulans having glycosyl transfer activity. As the enzyme, a crude enzyme or a commercially available enzyme prepared by culturing a microorganism and preparing the culture solution by ammonium sulfate precipitation or the like can be used.

The concentration of lactose used in the reaction is 1-40%, N-
The concentration of the acetyl-chitooligosaccharide or the N-acetyl-chitooligosaccharide mixture is 1 to 50%, and the total substrate concentration is 10%.
It is preferably set to ~ 80%. In addition, the enzyme reaction is pH4 ~
9. It is preferable to carry out at a temperature of 5 ° C to 50 ° C for 2 to 100 hours.

The reaction solution obtained as described above is heated to stop the reaction, and if necessary, powdered using a freeze dryer, a vacuum dryer or a spray dryer. In the obtained powder,
Gal β- (1 → 4)-[GlcNAc] n of the present invention (where Gal
Represents a galactose residue, and GlcNAc represents an N-acetylglucosamine residue. 1 to 30% of an oligosaccharide represented by n = 2 to 6), and glucose, galactose, lactose, N-acetylchitooligosaccharide, and galacto-oligosaccharide.

Furthermore, when it is desired to increase the content of the oligosaccharide of the present invention, separation and purification means such as activated carbon column chromatography, gel filtration, and high performance liquid chromatography (HPLC) can be used in combination.

(Example) Hereinafter, an example of the present invention will be described more specifically, but it goes without saying that the present invention is not limited by this description.

(1) Lactose 0.9g and di-N-acetylchitobiose
2.1 g was dissolved in water to make a 5 ml solution.

This solution contains commercially available β from Bacillus circulans.
-2U of galactosidase (Daiwa Kasei: Violacta) was added, the reaction was allowed to proceed at 30 ° C for 30 hours, and the reaction was stopped by heating at 80 ° C for 10 minutes. After the completion of the reaction, the reaction mixture was subjected to activated carbon column chromatography to elute 0 to 50% alcohol to collect a trisaccharide sugar transfer product fraction, which was further subjected to gel filtration of Biogel P-2 (manufactured by Bio-Rad). Purification was performed by chromatography. This substance was identified as Gal β- (1 → 4) GlcNAc _{2 as} a result of ¹³ C-NMR structural analysis.

The substance was found to be present in an amount of about 12% based on the solid content of the reaction solution from the results of HPLC. The figure shows the results of HPLC analysis.

 The analysis conditions were as follows.

Column: YMC-Pack PA-03 (4.6 x 250 mm) Mobile phase: acetonitrile: water = 75:25 Flow rate: 0.8 ml / min Temperature: 25 ° C Detection: UV 215 mm (2) 0.9 g lactose and tri-N-acetyl chitotri 0.8 g of aose was dissolved in water to make 10 ml.

This solution contains commercially available β- from Bacillus circulans.
4 U of galactosidase (Daiwa Kasei: Violacta) was added, the reaction was allowed to proceed at 25 ° C. for 50 hours, and the reaction was stopped by heating at 80 ° C. for 10 minutes. After completion of the reaction, the reaction solution was subjected to activated carbon column chromatography, and a glycosyltransferred product fraction of tetrasaccharide was collected by elution with 0 → 50% alcohol, and further subjected to gel filtration of Biogel P-2 (manufactured by Bio-Rad). Purification was performed by chromatography. This substance was identified as Gal β- (1 → 4) GlcNAc ₃ by ¹³ C-NMR structural analysis.

From the result of HPLC measurement, it was found that the substance was contained in an amount of about 7% based on the solid content of the reaction solution.

(3) Lactose 250g and N-acetylchitooligosaccharide mixture (composition N-acetylglucosamine 45%, di-N-acetylchitobiose 22%, tri-N-acetylchitotriose 14%, tetra-N-acetylchito Tetraose 10
%, Penta-N-acetylchitopentaose 6%, hexa-N-acetylchitohexaose 3%) was dissolved in water to prepare a 1260 ml solution.

This solution contains commercially available β- from Bacillus circulans.
500 U of galactosidase (Daiwa Kasei: Violacta) was added, the reaction was allowed to proceed at 30 ° C. for 24 hours, and the reaction was stopped by heating at 80 ° C. for 10 minutes. After completion of the reaction, the reaction solution was freeze-dried to obtain 495 g of a white powder. This white powder was determined by HPLC
9% N-acetyllactosamine, Gal β- (1 → 4)
[GlcNAc] n (n = 2-6) contained 7%.

(Effect of the Invention) According to the present invention, an oligosaccharide that can be a food material that can be used for promoting the growth of bifidobacteria can be efficiently produced.

[Brief description of the drawings]

The figure shows an example of analysis of the oligosaccharide obtained by the present invention by HPLC.

Continuation of the front page (56) References ANALYTICAL BIOCHE MISTRY 145, 322-330 (1985) Supervised by Fumuji Maruya "Enzyme Handbook", December 1, 1982, Asakura Shoten, p. 501 (58) Cl. ⁶ , DB name) C12P 19/26 C07H 5/06 CA (STN) REGISTRY (STN)

Claims (1)

(57) [Claims] (1) lactose or a lactose derivative and N-
A substrate containing acetylchitooligosaccharide is added to β-produced by Bacillus circulance.
Galactosidase is acted on to cause a galactose transfer reaction.
→ 4) A method for producing an oligosaccharide represented by-[GlcNAc] n (Gal represents a galactose residue, GlcNAc represents an N-acetylglucosamine residue, n = 2 to 6).