JPS5953834B2 - Bifidobacterium growth promoter - Google Patents

Description

[Detailed description of the invention] The present invention relates to a substance that promotes the growth of Bifidobacterium.

Bifidobacteria are well known as bacteria that make up the majority of the intestinal bacteria of breast-fed infants.

There are many reports on the physiological significance of this bacterium, and it is widely known that it inhibits putrefaction caused by enteric putrefactive bacteria, inhibits the production of solenoid amines, and inhibits the growth of pathogenic bacteria by producing organic acids such as lactic acid and acetic acid. It is being

Since Bifidobacterium has such an effect,
Various bifidobacteria preparations and fermented dairy products have been proposed to increase the number of bifidobacteria in the intestines for the purpose of maintaining and improving the health of people, including adults.

However, even if bifidobacteria are ingested into the body in this way, they generally do not colonize the body, and if ingestion is discontinued, bifidobacteria are often no longer detected in feces.

Sugars are thought to be the most important factors necessary for the growth of bifidobacteria in the intestines, and lactulose was long thought to be effective.

Lactulose is an indigestible sugar, and when ingested, it is hardly absorbed into the body and reaches the large intestine, where it is assimilated by intestinal bacteria.

However, in addition to bifidobacteria in the intestines, Escherichia coli,
A wide variety of intestinal bacteria such as Clostridium exist, and lactulose is non-selectively utilized by these intestinal bacteria, so it has been difficult to selectively grow only Bifidobacteria.

As a result of extensive research into sugar sources that selectively proliferate bifidobacteria in the intestines, the present inventors discovered that fructooligosaccharides, in which 1 to 4 molecules of fructose are bound to sucrose, are not digested and absorbed in vivo. (Japanese Patent Application No. 56-136130 (Japanese Unexamined Patent Publication No. 58-40065)).

Furthermore, we investigated the assimilation of these saccharides by intestinal bacteria and found that these fructooligosaccharides are selectively assimilated by Bifidobacteria, and therefore can serve as a selective growth factor for Bifidobacteria in the intestine. , completed the present invention.

That is, in the present invention, sucrose contains 1 fructose.
This invention relates to a bifidobacteria growth-promoting substance whose main component is a fructooligosaccharide bound to four molecules.

The fructooligosaccharide used in the present invention is an oligosaccharide obtained by allowing fructosyltransferase to act on sucrose, that is, a substance in which one molecule of fructose is bound to sucrose (hereinafter referred to as GF2).

), a substance in which two molecules of fructose are bound to sucrose (hereinafter referred to as GF3).

), a substance in which three furan-1-hose molecules are bound to sucrose (hereinafter referred to as GF4).

) and a substance in which four furan-1 hese molecules are bound to sucrose (hereinafter referred to as GF5).

). These oligosaccharides can be obtained from a transferred sugar composition obtained by reacting sucrose with furano-1-zyltransferase, for example by Bonn chromatography,
It can be isolated and purified by means such as ion exchange chromatography.

Fructosyl I transferase used here mainly acts on sucrose to cleave the β-1,2 bond between fructose and glucose, then transfers the fructose to sucrose to produce GF2, and further transfers fructose to GF2. It has actions such as transferring and producing GF3.

This enzyme is from Aspergillus
Genus (Aspergillus niger)
niger) ACE-2-1, FERM-P588
6 etc.), Penicillium genus (
Penicillium nigricans
nigricans etc.), Fusarium (Fusari
um) genus (Fusarium lini)
i IAM5008) etc.), Daleosporium (Gl
oeosporium) genus (Gloeosporium kaki IAM5Ql
l) etc.), Aureobasidium (Aureobasid
ium) genus (Aureohacideum pullulans pal)
Melanigenum (Aureobasidium pull)
ulans var melanigenum A-8,
Saccharomyces genus (ATCC20612), etc.), Saccharomyces spp.
Saccharomyces cerev
issiae), Rhodotoru
lla) genus (Rhodotorula glutis)
orulla glusinis), Pihia (Pi
genus (Pichiamiso)
), Hansenula genus (Hansenula m1so etc.), Candida genus (Candida tropicalis),
Enzymes originating from microorganisms such as yeast, such as s), etc., and enzymes originating from plants, such as asparagus and Jerusalem artichoke, are used.

Fructosyltransferase of microbial origin is prepared using a suitable medium, for example, 5.0% sucrose, 1 peptone.
．． The optimum temperature for each microorganism, i.e. 25~
After culturing at 30°C for 24 to 96 hours, after the completion of the culture, cells obtained by separating the cells from the culture solution by means such as filtration or centrifugation, or a culture filtrate from which the cells have been removed, and furthermore, ultraviolet cells are obtained from this culture filtrate. Enzymes that have been purified and purified by conventional enzyme purification methods such as filtration, ammonium sulfate salting out, solvent filtration, gel filtration, and ion exchange chromatography can be used.

Furthermore, enzymes of plant origin can be used by extracting the enzyme after destroying the plant tissue by physical means such as grinding, and then using an extract thereof or an enzyme purified from the extract by a conventional method.

As a result of various studies on industrial transfer reaction conditions when these fructosyltransferases are allowed to act on sucrose, it is preferable to carry out the reaction under the following conditions.

That is, the sucrose concentration during the transfer reaction was set at 5 to 70
%, preferably 30 to 60%.

In addition, the reaction period and reaction temperature vary depending on the origin of the enzyme, but 1
f (4,0-7.0, temperature 25-65°C, preferably 5
The temperature shall be 0 to 60°C.

The amount of enzyme used is 5 to 20 per gram of sucrose.
0 units, preferably 2.0 to 80 units.

Here, the enzyme unit is 5% sucrose solution 1. Om
L IH5.0 buffer 1. 0.5m of enzyme solution in Oml
1 is added and reacted at 40°C for 60 minutes, the amount of enzyme that produces 1 μmole of glucose in 60 minutes in 2.5 ml of the reaction solution is expressed as 1 unit.

After the rearrangement reaction is completed, the heated enzyme is inactivated, decolorized with activated carbon, desalted with an ion exchange resin, and then concentrated to obtain the desired product.

The transition composition can be analyzed using, for example, a Microphone Tapak CH column (manufactured by Waters Limited).
This can be carried out by high performance liquid chromatography using a solvent system of acetonitrile:water (80:20 (V/V)).

The composition thus obtained has, for example, 30% glucose, 11% sucrose, 228% GF,
GF325%, GF45%, GF51%, but the composition of each constituent sugar can take various values depending on the reaction conditions.

GF2 used in the present invention. GF3. GF4. GF5 or a mixture thereof can be obtained, for example, by adding the above composition to a column packed with activated carbon and sequentially eluting with water and a mixed solution of water and ethane.

For example, GF2 can be eluted with water and then 5% (V/V) ethanol aqueous solution, and GF3 can be eluted with 10% (V/V) ethanol aqueous solution, and GF4. GF5 was obtained by elution with a 20% (■/V) ethanol aqueous solution, and GF2. GF3. GF
4. The GF5 mixture was prepared by adding the above composition containing glucose, sucrose, and fructooligosaccharides to an activated carbon column, removing glucose and sucrose by passing water through the column, and eluting with a 20% (V/V) ethanol aqueous solution. obtained by doing.

GF2 of fructooligosaccharide is 0-β-D-fructofuranosyl-(2→1)-〇-β-fructofuranosyl-(2→1)-α-D glucopyranoside, O-β-D
-Fructofuranosyl-(2→6)-〇-βglucopyranosyl-(1→2)-β-D-fructofuranoside, 0
-β-D-fructofuranosyl-(2→6) -〇-β
-Fructofuranosyl-(2→1)-α-D-glucopyranoside, etc., and GF3 includes 〇-β-D-fructofuranosyl(2→[1-O-β-D-fructofuranosyl-2]2) →1)-α-D-glucopyranoside, O
-β-D-fructofuranosyl-(2→6)-〇-β-
D-Fructofuranosyl (2→2)) -〇-α-D
-glucopyranosyl(1→2)-β-D-fructofuranoside, etc., and GF4 includes 〇-β-D-frac I furanosyl-(2→[1-0-β-D-fructofuranoside). GF5 includes 0-β-D-fructofuranosyl-(2→[1-0-β-D-fructofuranosyl-2).
] 4→1)-α-D-glucopyranoside, etc.

In addition, 〇-β-D-fructofuranosyl-(2→1) -0-β-fructofuranosyl-(2→
1)-α-D-glucopyranoside (hereinafter referred to as 1-guesdose).

), 〇-β-D-fructofuranosyl of GF3 (2 → [1-0-β ~ D-fructofuranosyl 2] 2
→1) -α-D-glucopyranoside (hereinafter referred to as varnish 1 to -
It is called ``Su''.

), 0-β-D-fructofuranosyl-(2→
[1-〇-β-D-fructofuranosyl 2] 3→1)
-α-D-glucopyranoside (hereinafter referred to as IF-fructofuranosyl-nisdose).

) and 〇-β-D-fructofuranosyl of GF5 (
2→[1-0-β-D-frac)furanosyl-2]4
→1)-α-D-glucopyranoside (hereinafter referred to as IF-(fructofuranosyl)2-nisdose).

) is particularly preferable because it is selectively assimilated by bifidobacteria as a result of examining its assimilation ability by intestinal bacteria as shown in Example 1 below.

) Fructooligosaccharides obtained by allowing fructosyltransferase to act on sucrose are
It is effective as a substance that promotes the growth of bifidobacteria in the intestines, and this substance can be made into the desired form, such as liquid or powder, depending on the purpose, and these forms can be used as is.
It can be used for the purpose of the present invention, or can be added to general food and drink products in a desired amount.

Furthermore, it can be added to tablets, fermented milk, powders, tablets, etc. containing bifidobacteria.

1 Next, the present invention will be explained in detail with reference to Examples.

Example 1 Aureobasidium pullulans pearl melanogenum A-8 strain (FERM-P5885) was grown in medium 3 containing 2% broth and 5% sucrose in an Erlenmeyer flask.
00ml and cultured at 28°C for 24 hours, which was used as a seed culture solution.

301 jar fermenter with 1.0 sucrose, 1.0% peptone, 0.7% meat extract, 10.3% NaC
%, CoC1□・6H200, medium containing 1% (IH6
, 5) 201 was placed in a square and sterilized at 120°C for 30 minutes, 300ml of the above sugar culture solution was aseptically inoculated and cultured at 28°C for 24 hours.

Note that the aeration stirring conditions were 240 rpm and 5QVVm.

The culture solution was centrifuged to obtain 400 g of crude enzyme cells containing fructosyltransferase.

This crude enzyme had an activity of 12,000 units/g.

After adding 100 kg of water to 150 kg of sucrose to dissolve the sucrose, the concentration was adjusted to 6.0, and 375 g of the crude enzyme cells of fructosyl 1 to transferase were added and reacted at 60°C for 48 hours.

Next, the enzyme reaction was stopped by heating at 100° C. for 10 minutes, and the bacterial cells were removed by filtration.

Thereafter, the furnace liquid was decolorized using activated carbon in a conventional manner and further desalted using an ion exchange resin.

The sugar composition per solid content of the composition thus obtained was as follows.

Furano I・-su 1%, glucose 36%, sucrose 10%, ■-kesdose 23%, nisdose 24%,
IF-fructofuranosylunist-4%, 1F-(hook 1-furanosyl)2-varnish 1-2%.

1.2 kg of the above composition was packed into a 401 activated carbon column, and after passing 8001 water at 5V=1, it was eluted with 5% ethanol of 2801, then 10% ethanol of 2801, and then 20% ethanol of 201. , GF2B.

GF3. Both portions containing only GF4 were obtained, concentrated, and then frozen.

After drying, GF2.50g, GF340g, GF
410 g of GFslg was obtained.

As a result of examining the chemical structures of these substances using means such as the composition ratio of glucose and fructose and gas chromad mass spectrometry, it was found that GF2 is 1-quesdose and GF3 is nisdose.

GF4 is IF-fructofuranosyl-nisdose, GF
It was revealed that 5 was a base of IF-(fructofuranosyl) 2-varnish 1.

Next, we investigated the assimilation of these substances by intestinal bacteria.

In addition, when considering each flannel. Tooligosaccharide 0
．． Enterobacteriaceae were inoculated into a medium containing 5% and cultured at 37°C for 72 hours, and the increase in the number of bacteria was determined by measuring the absorbance at 600 nm, and the absorbance when glucose was used as the sugar source was set as 100. The comparison value at the time indicated the degree of bacterial growth.

In addition, Escherichia coli (E, co
In the case of ll), the following medium A was used, and in the case of other intestinal bacteria, medium B was used.

The results are shown in Table 1. As is clear from the table, GF2.
GF3. GF4°GF5 has higher assimilation selectivity for bifidobacteria than lactulose.

Claims (1)

[Scope of Claims] 1. A bifidobacteria growth-promoting substance whose main component is fructooligosaccharide in which 1 to 4 molecules of fructose are bound to sucrose. 2. The substance according to claim 1, wherein the fructooligosaccharide is obtained by allowing fructosyltransferase to act on sucrose. 3 Fructooligosaccharide is 1-quesdose, nisdose,
1F-furano I furanosyl-nisdose and IF-
The substance according to claim 1, which is any one of (Fructofuranosyl)2-nisdose.