JPS5899497A - Novel oligosaccharide, its preparation, and agent for accelerating proliferation of bacterium belonging to bifidobacterium genus - Google Patents

Abstract

NEW MATERIAL:The oligosaccharide composed of o-beta-D-galactopyranosyl(1 4)- [o-beta-D-galactopyranosyl-(1 6)]-D-glucose of formula, having the following properties: molecular weight, 504; solubility, easily soluble in water, insoluble in acetone, alcohol, etc., hardly soluble in hydrated alcohol; color reaction, positive to aniline.phthalic acid reaction, negative to ninhydrin reaction; neutral; white. USE:Agent for accelerating the proliferation of bacteria belonging to Bifidobacterium genus. PROCESS:Lactose or a lactose-containing material is treated with beta-galactosidase to obtain a reaction mixture containing oligosaccharide, which is passed through a column packed with activated charcoal to effect the selective adsorption of more than three kinds of oligosaccharides. The adsorbed oligosaccharides are eluted and the aluate is made to react with beta-galactosidase and then post-treated in a manner same as the above to obtain the objective oligosaccharide.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel oligosaccharide having growth-promoting activity of Bifidobacterium and a method for producing the same.

Bifidobacterium (hereinafter referred to as Bifidobacterium)
is a useful bacterium that grows in the human intestine, and its beneficial physiological and nutritional effects on humans have been reported. For example, the inhibitory effect on intestinal putrefaction, (Tamine Bl
It is known that Bifidobacterium and B2 have a synthetic effect, and an effect on protein production, etc. Therefore, promoting the growth of Bifidobacterium in the human body is the key to improving the above-mentioned effects.

Conventionally, many studies have been conducted for a long time on substances that promote the growth of bifidobacteria (hereinafter referred to as bifidobacterial growth factors). 1 N-acetylglucosamine and ginseng extract have been used as growth factors.

Lactulose etc. have been reported. However, although these bifidus growth factors are effective in vitro, their in vivo effects are still unclear or only extremely insufficient results have been obtained.

In recent years, oligosaccharides have attracted attention as bifidus growth factors, and Aspergillus spp.
The general formula aat-(aat)n- is obtained by reacting β-galactosidase produced by M. oryzae.
It has been proposed to use an oligo shield represented by Gtc (in the formula, Gat is a galactose residue, etc is a glucose residue, and n is an integer from 1 to 4) as a bifidus growth factor (Kokai No. 55-104885). issue).

As a result of studying the action of oligosaccharides as bifidus growth factors, the present inventors have found that the following oligosaccharides exhibit an excellent growth-promoting effect on bifidus 1 in vivo. As a result, the present invention was completed.

Therefore, the present invention aims to provide a novel oligosaccharide as a bifidus growth factor and a method for producing the same. ) is a new substance.

Oligo shields are generated by the galactose transfer reaction (transfer of galactoside bonds) that occurs when β-galactosidase acts on lactose. To date, the following oligo shields have been isolated and established. O β-Gat-(1-)2)-Gtc, β-Gat
-(1-e3)-GtCrβ-Gat-(1
-+6) -GtQ, β-aat-(1-+3)
-Gat, β-Gat-(1-s)-〇at* β-
Gat-(1-+6)-β-Gat-(1-+4
) -Gta and β-GaA -(1-+6)-β-
Gat-(1-+6)-etc (in the formula, Gat represents galactose, etc. represents glucose), etc.0, and as mentioned above, Denkin GaA-(Gat)n-Gtc (in the formula, n is an integer from 1 to 4) An oligosaccharide represented by is reported (% 1985-104885).

The difference between these known oligosaccharides and the oligo-shield according to the present invention having the above formula (1) is that the former has glucose or galactose linked in a linear chain, whereas the latter has the above formula (1). ), two galactose molecules are branched and bonded to glucose. The structure of the oligosaccharide according to the present invention, which has a novel oligosaccharide that is similar to the above-mentioned known oligosaccharide in terms of bonding, will now be described.

(a) Molecular weight Measurement using a mass spectrometer needle showed a molecular weight of 504, and from the results it was estimated that this oligosaccharide was a 3-shield compound consisting of 3 molecules of hexose.

(b) The molar ratio of the sugar produced by hydrolyzing the constituent sugar main oligosaccharide with 0.5N-HOt at 100°C for 4 hours is glucose:
Since the ratio of galactose is 1:2, this oligo shelf consists of 3 molecules of glucose and 2 molecules of galactose.
It was introduced that it is made of sugar.

(c) Binding mode of constituent sugars This oligo S was s0-IN HOt at 100C for 2 hours.
Partial hydrolysis was carried out for 0 minutes, and the resulting decomposition mixture was analyzed by thin layer chromatography, and showed a pattern as shown in the attached Figure 1. From this result, it was found that O-β-D-galactosyl-lactosyl-(1→6)
-D-Glucose 211 dimorphs were confirmed.

From the results of (a) to (c) above, it is clear that the oligosaccharide according to the present invention has the formula (I): It can be identified as pyranosyl-(1→6):)-D-glucose-steal.

In addition, this oligomer exhibits the following physical and chemical properties ^l)
Solubility in solvents: Easily soluble in water, insoluble in acetone, alcohol, chloroform, penze, and hydrous alcohol.

2) Color reaction Nianiline/phthalate reaction is positive, ninhydrin reaction is negative.

3) Distinction between basic, hostile, and neutral; neutral 4) Color: When dried and powdered, it is white in color.

Next, the method for producing oligosaccharides according to the present invention will be explained.

In the production method of the present invention, first, β-galactosidase is allowed to act on lactose or a lactose-containing substance to perform a galactose transfer reaction, thereby producing a reaction mixture consisting of a mixture of oligosaccharides. The lactose used here as a starting material may be commercially available, and lactose-containing products such as whole milk, skim milk, and whey may also be used as starting materials.

The origin of β-galactosidase to be applied to the above starting material is not particularly limited, and it does not need to be highly purified, and can be used in the form of a crude enzyme.

In order to cause β-galactosidase to act on the above-mentioned lactose or lactose-containing substances, a starting material with a lactose concentration of 5 to 50% is used as a substrate, and this is concentrated to pH 2 to 8 and #[0,1
It is appropriate to allow the enzyme to act at a temperature of 10 to 60° C. at a rate of about 200%/d.

In addition, since the reaction time for allowing the enzyme to act has a large effect on the yield of oligosaccharides, it is necessary to confirm the optimal reaction time by experiment.

That is, while quantifying the oligosaccharide in the reaction mixture produced by the action of the enzyme using high performance liquid chromatography, the reaction time Ma! :do.

A galactose transfer reaction occurs in the starting material due to the superposition reaction, producing an oligosaccharide mixture.

In the present invention, when the above enzymatic reaction is completed, the reaction solution is heated to 90°C.
After the enzyme is inactivated by heating for 2 to 30 seconds, the solution is passed through activated carbon to adsorb only the oligosaccharides of 3 or more groups in the reaction solution.

This adsorption treatment is for removing unreacted lactose, monosaccharides produced by decomposition, and various oligosaccharides produced by the transfer reaction from the reaction mixture to increase the concentration of the target oligosaccharide. Therefore, when the liquid is passed through the activated carbon, it is necessary to control the reaction mixture so that only oligosaccharides of 3 or more groups in the reaction mixture are substantially adsorbed. For that purpose, the amount of trisaccharides or more oligosaccharides in the reaction mixture must be 50 to 1,001 parts per 1 part of activated carbon! The amount of wrinkle reaction mixture passed through the activated carbon column is controlled so that the ratio is as follows. Note that if the liquid is passed in an amount lower than the above range, the disaccharides in the reaction mixture will also be adsorbed by the activated carbon, whereas if the liquid is passed in an amount higher than this range, some of the target oligosaccharides will be eluted without being adsorbed. There is a need to.

The activated carbon used in the above-mentioned adsorption treatment may be one that is normally commercially available, or the activated carbon may be mixed with a supercharging agent such as celite. Furthermore, it is preferable to use the activated carbon in a state in which a suitable amount of water is added to form a slurry, which is filled into a column, and then a sufficient amount of water is passed through the column to equilibrate with water.

Next, the trisaccharide or more oligosaccharides adsorbed on the activated carbon as described above are eluted. Ethanol is usually used at a concentration of 15 to 50% for this elution, so that the target oligosaccharide can be efficiently eluted.

The eluate thus obtained contains the oligosaccharides targeted by the present invention, but it also contains other oligosaccharides of trisaccharides or more, so the present invention further includes the following steps. Add and refine.

That is, a solution obtained by concentrating the above eluate under reduced pressure, or a solution obtained by drying (for example, spray drying) and powdering the above solution and dissolving it in warm water is treated with β-galactosidase again to obtain oligosaccharides other than the desired oligosaccharides. Decompose the sugar, and then pass the resulting enzymatically decomposed liquid through the activated carbon column again to adsorb only the desired oligosaccharide onto the activated carbon.
This adsorbed oligosaccharide is eluted in the same manner as described above to obtain the purified oligosaccharide of the present invention.

The eluate thus obtained is concentrated under reduced pressure and, if necessary, further dried and powdered.

The resulting powder is white and contains about 90% of 0-β-D-galactopyranosyl-(1→4)-(o-β-D-galactopyranosyl-(1→6))-D- Contains glucose.

The oligosaccharide obtained according to the present invention as described above exhibits an excellent growth-promoting effect on Bifidobacteria as seen in the test results shown below.

Note that the oligosaccharide according to the present invention can be used as a bifidus growth factor either in powder form or in the form of the above-mentioned concentrate.

Growth test for Bifidobacterium: Test method Ten cynomolgus monkeys were used as test animals, and each of them was fed commercially available baby milk powder containing 5% by weight of lactose for three weeks, and then the oligosaccharides according to the present invention ( Powdered milk for infants containing 5% by weight of powdered milk was added for another 3 weeks, and the feces of the monkeys that opened the can were collected to measure the proportion of bifidobacteria in the feces. For comparison, the second stage activated carbon adsorption treatment in the present invention and the subsequent bathing treatment were not performed; 3. A mixture of oligosaccharides (powdered form I&RI) for the genus lli and above was given to other groups of monkeys in the same manner as above, and the proportion of bifidus in the feces was measured.The results are shown in the attached Figure 2. As shown in Figure 2, the amount of bifidus 1 in the faeces during the period when oligosaccharide and non-inventive oligosaccharide were added was higher than that of bifidus in feces during the period when lactose was added. The proportion of
Furthermore, the growth of bifidus was clearly improved when the oligosaccharide of the present invention was added, compared to when the oligosaccharide mixture was fed.

That is, the oligosaccharide of the present invention has an extremely excellent effect on the growth pathway of Bifidobacterium in vivo, and also has an excellent effect on the growth of Bifidobacterium such as Bifidobacterium puri, which grows in the human intestinal tract. It exhibits high growth activity against a wide range of types of bifidobacteria, including Bifidobacterium longum, Bifidobacterium bifidum, Bifidobacterium addressentis, and Bifidobacterium infantis.

Therefore, the oligosaccharide according to the present invention can be incorporated into dairy products such as powdered milk and fermented milk, or added as a component of drugs such as intestinal preparations.

Examples of the present invention are shown below.

Example 10 45,000 units of β-galactosidase was added to a solution of 10-Yen lactose dissolved in 15-Yen warm water and citric acid was added to adjust the pH to 4.5, and the mixture was reacted at 40°C for 2 hours. . The resulting reaction mixture was heated at 105°C for 2 seconds to inactivate the enzyme, and then a column of activated carbon (diameter 501 x 4) (a mixture of 25 kg of activated carbon and 12.5 kg of Celite) was slurried with urine. 2kli+/ to
The liquid was passed at a flow rate of 1 hour.

At this time, oligosaccharides of trisaccharide or higher for 1 hour of activated carbon are sa
y. Next, a sufficient amount of water was passed through to elute the monosaccharides and disaccharides in the reaction mixture, and then the oligosaccharides adsorbed on the activated carbon were eluted using 20% ethanol. The obtained eluate was concentrated under reduced pressure and then spray-dried to obtain a white powder (crude product). This powder contains the oligosaccharide of the present invention, 0-β-D-galactopyranosyl (
l→4)-(0-β-D-galactopyranosyl-(1→
6)) Contained about 20% by weight of -D-glucose.

Next, 100II of the powder obtained as described above was dissolved in skg of warm water, and β-galactosidase Z was added to this solution.
After adding oo units and reacting at 40°C for 2 hours, the resulting reaction solution was treated to inactivate the enzyme and then diluted with lO 10mX.
The oligosaccharides were adsorbed by passing the solution through 10 activated carbon columns (a mixture of activated carbon and Celite in a ratio of 2=1).

Next, this adsorbed oligosaccharide was eluted using 20% ethanol, and the resulting eluate was concentrated under reduced pressure and then lyophilized to obtain 2011 as a white powder.

This powder contains about 90% by weight of 0-β-D-galactopianocyl-(1→4)-(0-β-D-galactopyranosyl-(1→6))-D-glucose. was.

2% of the oligosaccharide powder of the present invention obtained as described above
(Weight) When powdered baby milk with added g was prepared and administered to 30 infants within 3 months of age, the increase in the ratio of bifidobacteria to the total number of bacteria in their feces was due to lactose 2
% (weight 1i)

[Brief explanation of the drawing]

Figure 1 shows the turn of thin layer chromatography of the partial hydrolyzate of oligosaccharide according to the present invention, and Figure 2 is a graph showing the in vivo growth promoting effect of the oligosaccharide according to the present invention. Katashi III rl
Difference 1 Figure 1 Figure 2 jl-1-Be

Claims (1)

[Claims] 0-β-D-galactopyranosyl-(l→4) having
-(0-β-D-galactopi-2-nosyru(1→6))]-
] Oligosaccharide consisting of D-glucose. (2) A step of causing β-galactosidase to act on lactose or a lactose-containing substance to perform a galactose transfer reaction to produce a reaction mixture containing oligosaccharides, and passing the reaction mixture through an activated carbon column to carry out the reaction. A step of adsorbing an oligosaccharide mixture of trisaccharides or more in the mixture onto activated carbon, and then eluting the adsorbed oligosaccharide mixture, a step of further allowing β-galactosigase to act on this eluted oligosaccharide mixture to perform a decomposition reaction, Claim No. 1, characterized in that the obtained reaction product is passed through an activated carbon column to adsorb the target oligo shield, and then the adsorbed oligo shield is eluted.
1) Method for producing oligosaccharide of 600mg. (3) The above oligo! l! The reaction mixture* is transferred to an activated carbon column equilibrated with water, and 50 to 100 tons of oligosaccharides or more of trisaccharides in the reaction mixture are added to the activated carbon IV4.
Claim No. (2) for liquid passage as shown in the assignment of
Production method according to claim □ (4) % A bifidobacteric acid growth promoter containing the oligo shield according to claim 1 as an active ingredient.