JPS5820266B2 - Bifidobacterium growth-promoting composition and method for producing the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a composition having the effect of promoting the growth of Bifidobacterium and a method for producing the same.

Various substances have been reported to have the effect of promoting the growth of Bifidobacterium (hereinafter referred to as bifidobacterium growth factors), and there are many methods for producing them or for producing milk powder containing them. Proposed (Special Publication No. 41-32908, No. 45-6.510,
No. 45-6865.

No. 45-21606, No. 49-40956.

No. 49-40957, etc.).

However, the effects of most of the bifidus growth factors that have been reported so far have been confirmed through in vitro culture experiments, and their in vivo activity is unknown or extremely insufficient.

Bifidobacterium is a useful bacterium that grows in the large intestine, and its physiological significance and efforts are being made to increase the proportion of this bacterium in the intestinal flora of artificially fed infants. There is no need to comment at this point.

Therefore, as a bifidus growth factor that is artificially included in foods, drinks, culture media, etc., it can be expected to have a reliable and sufficient effect in vivo, even if it is used only for culturing Bifidobacterium in vitro. This is desirable, and from this point of view, there has been a strong desire to develop a better bifidus growth factor.

An object of the present invention is to provide a bifidus growth factor that can meet the above-mentioned needs and exhibits high activity in vivo.

The present invention consists of two inventions, the first of which is a compound of the general formula 0% (wherein Gal represents a galactose residue, Glc represents a glucose residue, and n represents an integer from 1 to 4, respectively.

) The second invention is a composition for promoting the growth of Bifidobacterium containing the oligosaccharide represented by () as an active ingredient. This invention provides a method for advantageously producing the composition according to the first aspect of the present invention.

Most of the above-mentioned oligosaccharides, which are the active ingredients in the Bifidobacterium growth-promoting composition according to the present invention, that is, bifidobacterial growth factors, undergo a hydrolysis reaction when the present inventors act on lactose with β-galactosidase. This was the first discovery I made during my research on the transfer reaction that occurs along with this.

Regarding the above rearrangement reaction, in 1951, Wallenfe
Although there have been many reports since the first report by ls, the only transfer oligosaccharides that have been isolated and confirmed to date are Ga1-(
β-1゜2)-Glc, Ga1-(β-1,3)-
Gl c.

Ga1-(β-1,6)-Gl clGa l-(β-
1,3) Gal s Gal (β-1,6
)-Gal disaccharide and Gal-(β-1,6)-G
al-(βL4) GlclGal-(β-1>
6) It is a trisaccharide of Ga1-(β-1,6)-Glc (the figure in parentheses indicates the form of galactoside bond)
.

The existence of tetrasaccharides has been confirmed by Huber et al., but their structures have not been clarified, and there is no example in which the production of polysaccharides of pentasaccharides or higher has been confirmed.

The content of these reports is limited to the elucidation of the transfer mechanism and the structural study of the produced oligosaccharides; none of them report the relationship between transferred oligosaccharides and the growth of Bifidobacterium.

The oligosaccharide having the above general formula (hereinafter simply referred to as oligosaccharide refers to this specific oligosaccharide), which is a bifidus growth factor, is produced when lactose is treated with β-galactosidase produced by Aspergillus oryzae.

Since unreacted lactose and galactose and glucose generated by hydrolysis are present in the reaction mixture after this treatment, in order to obtain highly active bifidus growth factors, appropriate purification is required after the enzyme treatment. how to,
A high oligosaccharide content may be achieved by selecting enzyme treatment conditions that yield a high oligosaccharide production rate, or by combining these methods.

Next, a method for producing a Bifidobacterium growth promoting substance according to the second aspect of the present invention will be described in detail.

It is not necessary to use especially highly purified lactose to be treated with β-galactosidase, and commercially available lactose can be used as is.

Further, substances containing lactose as one component, such as whole milk and skim milk, can also be used as raw materials.

When performing enzyme treatment, the substrate concentration is approximately 10-50%, p
H is 3-8, enzyme concentration is 1-100 units/ml
A suitable temperature is 20 to 50°C.

Reaction time has a great influence on the yield of oligosaccharides.

As is clear from Figure 1, which shows the relationship between the reaction time and the amount of sugars produced in an example of enzyme treatment, glucose, galactose, and oligosaccharides increase almost linearly at the beginning of the reaction, but after that, they all increase slightly. It draws a complex curve, showing a tendency for oligosaccharides to gradually decrease after a certain point.

Since the time at which the yield of oligosaccharide is maximized varies depending on other reaction conditions, it is desirable to confirm the optimal reaction time by experiment.

Note that the oligosaccharides in the reaction mixture are separated from other components by, for example, thin layer chromatography, and then
It can be quantified by the e method.

The enzyme reaction can be stopped by heating the treatment solution to about 90° C. or higher for 5 to 10 minutes.

The reaction mixture after the enzyme treatment can be used as it is, or it can be appropriately concentrated or dried to form a powder and used as a bifidus growth factor or a composition containing it (e.g., dairy products). Purification may be performed to increase the concentration of the oligosaccharide component.

Purification can be carried out in various ways, for example, after preliminary purification by treating the reaction mixture with an ion exchange resin,
There is a method in which oligosaccharides are adsorbed by passing through an activated carbon column and then eluted with an aqueous alcohol solution.

There is also a method in which the isolation of oligosaccharides is facilitated by inoculating the reaction mixture with microorganisms that assimilate monosaccharides and disaccharides and culturing them to consume monosaccharides and disaccharides.

Next, the oligosaccharide in the composition according to the present invention will be explained by showing an analysis example of the oligosaccharide according to Example 1, which will be described later, conducted under standard conditions.

■ Molecular weight distribution The results of gel p-filtration using Bio-Gel P-2 are shown in the second
Shown in the figure.

Peaks A and B in the chromatogram. C is a trisaccharide,
Corresponding to tetrasaccharides and pentasaccharides, the composition ratio calculated from the area ratio of these peaks is approximately 55% for trisaccharides and approximately 32% for tetrasaccharides.
, pentasaccharides or more accounted for approximately 13%.

■Constituent sugar lactose, oligosaccharide (unfractionated), trisaccharide class to pentasaccharide class in oligosaccharide 10 in 0.5N-HCI
Table 1 shows the molar ratio of sugar produced when hydrolyzed at 0°C for 4 hours and when hydrolyzed with β-galactosidase at 50°C for 4 hours.

From this result, the molar ratio of glucose and galactose in trisaccharides, tetrasaccharides, and pentasaccharides in oligosaccharides is 1:
It can be seen that the ratio is 2.1:3 and 1:5.

■ Binding mode of constituent sugars From the product obtained by partial acid hydrolysis of the main components in three sugars (separated by activated carbon column chromatography), in addition to glucose and galactose, lactose and Gal- (β-1°6)-Gal was confirmed.

Based on this result and the 1:2 ratio of glucose to galactose in the completely hydrolyzed product, the structure of this trisaccharide is Ga1-(β-L6)-Gal (β-1
It was estimated to be ゜4)-Glc.

Analyzing a large number of oligosaccharide components in the same manner revealed that the oligosaccharide is represented by the general formula above and contains galactose-
The bond between galactose is β-1,3 bond, β-1,4 bond or β-1,6 bond, and β-1,6 bond is the main bond, and the bond between galactose-glucose is β-I,3 bond. ,
β-1,4 bond or β-1,6 bond, and β-1,4
It was confirmed that bonding is the main factor.

As far as the isolated oligosaccharides in the composition of the present invention as detailed above have been investigated, individual oligosaccharides alone can act as bifidus growth factors, and of course, even a mixture of various oligosaccharides can act as a bifidus growth factor. Shows excellent Bifidobacterium growth promoting effect.

The oligosaccharide in the composition of the present invention is particularly excellent as a bifidus growth factor in that its action is extremely remarkable not only in vitro but also in vivo.

The Bifidobacterium proliferation-promoting effect of the composition according to the present invention is almost unrelated to the type of Bifidobacterium, and includes Bifidobacterium breve, Bifidum infantis, Addres centiis, etc. It is effective against all human intestinal colonizing Bifidobacterium bacteria.

The composition according to the present invention includes not only highly purified oligosaccharides themselves but also any mixture containing oligosaccharides as one component, such as a reaction in which lactose or a lactose-containing substance is treated with β-galactosidase to produce oligosaccharides. The product may be a food or drink made of the product itself or a processed product thereof such as powdered milk or fermented milk, or a drug, or any food or drink or drug to which purified oligosaccharide is added.

The present invention will be explained below with reference to Examples.

In the examples, "Bifidobacterium" is abbreviated as "1B".

Example 1 3.6 kg of lactose is about 61! dissolved in warm water,
IM = 50 ml of acetic acid buffer solution (pH 4,6), add 100,000 units of β-galactosidase and water to make IOA,
The reaction was carried out at 37°C for 5 hours.

Next, the reaction solution was heated to denature the enzyme, and after separating the denatured protein, it was passed through a column of cation exchange resin and anion exchange resin.

The effluent was brought into contact with a 30 x 30 CrrL column packed with activated carbon overnight, and then the activated carbon was washed with deionized water 601 to elute monosaccharides, followed by 5% ethanol 6041 and then 5% ethanol 6041.
It was eluted with 0% ethanol 601.

This 50% ethanol eluted fraction was concentrated to about 74%, filtered aseptically through a membrane filter with a pore size of 0.2μ, and then ion-exchanged again to obtain a transparent sugar solution.

The viscous liquid was further concentrated under reduced pressure to about 2.54 ml, and the viscous liquid was filtered again through a membrane filter, and the filtrate was freeze-dried to produce 500 g of white oligosaccharide powder (hereinafter referred to as TO8).

Example 2 After administering 10 types of bacteria (including B. breve) isolated from human feces to 6 sterile Fischer adult female rats and allowing these bacteria to colonize the intestines, ■ lactulose trivalent aqueous solution was administered. ■ A 3% aqueous solution of TO8 produced in Example 1 ■ A 3% aqueous solution of lactose was administered daily in the order of ■→■ for one week each.

The dosage was 0.697 days as a solid substance, and there was a blank period of one week when switching the administered substance.

During this period, rat feces were collected and the number of viable bacteria of B. breve in the feces was measured.

The results are shown in FIG.

As is clear from the figure, by administering TO8, the number of B. breve bacteria in feces increases approximately 10 times.

Although an increase in the number of bacteria is observed when lactose is administered, it is extremely small, and almost no change is observed when lactulose is administered.

Example 3 The following experiment was conducted using six normal F i 5cher adult male rats.

The B bacteria administered was B. breve or B. infantis, which were cultured in VL-G medium for 48 hours and then centrifuged, and the resulting bacterial cells were suspended in milk medium and administered.

The dosage is 2 x 103/day for bacterial count and 20 ml/day for 5 TO8 aqueous solution.

(a) First, let them drink only the bacterial solution for one day.

(b) For the second week, administer only the bacterial solution every day.

(c) For the third week, administer the bacterial solution and TO8 every day.

(d) For the 4th week, administer TO8 only daily.

During this period, B bacteria (from the administered strain) was found in rat feces.
Figure 4 shows the results of measuring the number of viable bacteria.

The figure shows that the number of B bacteria in the intestine increases significantly by the simultaneous administration of the bacterial solution and TO8, and that the high bacterial count is maintained even after the administration of the bacterial solution is discontinued.

Example 4 The following experiment was conducted on five healthy adults.

The bacterial solution administered was that of B. breve obtained in the same manner as in Example 3.

Experiment schedule: 1st week: Administer only bacterial solution 2nd week: Bacterial solution and TO8 (3g/day)
3rd week of administration...Bacterial liquid and TO8 (10
Administer B. breve bacteria count: 109/day TO8 administration method: Dissolve in lukewarm water and drink after lunch Measurement: The number of B. breve bacteria in each person's stool on the 3rd, 5th, and 7th day of each week was measured, and the average number of bacteria for that week was determined.

The results of the experiment are shown in FIG. 5, and it can be seen that administration of TO8 significantly increases the number of B bacteria in the intestine.

Example 5 The following experiment was conducted on five healthy adults.

Experiment schedule 1st week...2nd week without TO8 administration...TO83,! 3rd week of administration of li'/day...Administration of 1081097 days T
O8 administration method: Same as Example 4 Measurement: The total number of B bacteria in stool is measured in the same manner as Example 4.

The experimental results are shown in Figure 6.

From the same figure, B
It can be seen that the number of bacteria increases.

Example 6 Aspergillus oryzae β- in reduced skim milk 10001
Add 10 million units of galactosidase and incubate for 2 hours 4
After keeping at 0°C, it was heated to inactivate the enzyme and sterilize it.

Next, the fermented milk containing oligosaccharides and B bacteria is transferred to a culture tank, inoculated with B bacteria starter, cultured at 37°C for 24 hours, and homogenized by adding a sweetener etc. after the culture is completed. I got it.

[Brief explanation of the drawing]

Figure 1: Graph showing the relationship between reaction time and saccharide yield when lactose is treated with β-galactosidase. Figure 2: Graph showing the results of gel filtration of oligosaccharides. FIG. 3: Graph showing experimental results of Example 2. FIG. 4: Graph showing the experimental results of Example 3. FIG. 5: Graph showing the experimental results of Example 4. FIG. 6: Graph showing the experimental results of Example 5.

Claims (1)

[Claims] 1 General formula Ga 1-(Gal) n-G1 c
(In the formula, Gal represents a galactose residue, Glc represents a glucose residue, and n represents an integer from 1 to 4, respectively) as an active ingredient. . 2 General formula Ga1-(Gal)n-Gl, characterized in that lactose or lactose-containing substances are treated with β-galactosidase produced by Aspergillus oryzae
c (However, in the formula, Gal is a galactose residue, Gl
A method for producing a Bifidobacterium growth-promoting composition containing an oligosaccharide represented by (c represents a glucose residue and n represents an integer from 1 to 4) as an active ingredient.