JPS61185182A - Mutant strain of bifidus and cultivation composition thereof - Google Patents

Abstract

PURPOSE:To provide the titled mutant belonging to Bifidobacterium longum, having high hydrogen peroxide resistance, and capable of keeping the number of bifidus cells even in the storage of a food under aerobic condition or acidic condition. CONSTITUTION:Bifidobacterium longum ATCC-15708 is applied to a blood liver agar (BL agar) plate or Brigg's liver agar plate, irradiated with ultraviolet radiation to an extent to give a survival rate of <=0.1%, and cultured at 37 deg.C for 48hr under anaerobic condition. The strain is streaked to a BL agar plate, cultured under anaerobic condition, subjected to pure culture, and sub-cultured for several generations. A strain resistant to H2O2 having high concentration as far as possible is selected by the H2O2 resistant test. A number of H2O2 resistant strains were sub-cultured for 10 generations, and a strain [Bifidobacterium longum No.1022 (FERM-P No.8033)] having H2O2 resistance and high stability was separated. The strain has improved oxygen resistance and acid resistance.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a Bifidobacterium mutant strain that has a high survival rate even when stored in foods for a long period of time, and a culture composition thereof.

More specifically, it belongs to Vihydrobacterium longum,
The present invention relates to a mutant strain having high hydrogen peroxide resistance and a culture composition thereof.

The present invention is applicable to the production of various milk drinks, fermented milk, lactic acid bacteria drinks, and various powdered foods to which Bifidus 1113@nutrients are added, when these foods are stored under aerobic conditions or under acidic conditions. The purpose of the present invention is to provide a Bifidobacterium mutant strain and a culture composition thereof that exhibit excellent survivability without decreasing the bifidobacterium function during storage even after being extracted.

In general, bifidobacteria are highly valued for their role in IIm, not only in infants but also in adults and the elderly, and it is considered that keeping the intestinal flora dominated by bifidobacteria is the secret to good health. For this reason, continuous intake of foods containing large amounts of Bifidobacteria is considered to be favorable for human health.

General K, Bifidobacterium used for food
dobacterium longum, Bifid
obacteriumbrev@, Bifidoba
ctarium bifidum, etc., but these are obligate anaerobic S and gradually die under aerobic conditions. Therefore, in many foods that are kept under aerobic conditions, Bifidobacterium dies out during storage, so foods should contain large amounts of Bifidobacteria, which are thought to have positive effects on human health. That was a big deal.

The inventors have conducted intensive research on the oxygen resistance of Bifidobacteria, and found that the oxygen resistance of Bifidobacteria is significantly affected by hydrogen peroxide (hereinafter referred to as 1 (hereinafter referred to as O)) produced by Bifidobacterium itself, and that H2O We learned that resistant bifidobacteria tend to have high oxygen resistance, and based on this knowledge, we conducted extensive research to find strains with high resistance to H, O, and bifidobacteria.
We found that among the mutant strains of Vihydrobacterium longum, there is a strain that has a significantly high resistance to 1(,O).

Next, a method for creating a mutant strain with high hydrogen peroxide resistance according to the present invention will be described.

Namely, Blood Liver Agar (Blood Liver Ag)
ar.

BL agar) plate, or Br1gg・8 liver agar (Bri
ggIs Liver Agar) Create a flat plate and B
ifidobacterium longurn A
TCC-15708 was applied to the surface and the survival rate was 0.1.
irradiate with ultraviolet light so that the temperature is less than After irradiation, 67℃
A large number of bacterial strains are obtained by culturing anaerobically for 48 hours. A large number of these strains are streaked onto a BL agar plate, cultured anaerobically, isolated, and subcultured over several generations.

The bacterial strains obtained in this way are then subjected to H, O, and resistance tests to select strains that can withstand as high a concentration of H2O as possible. In other words, BL liquid medium containing H7 prisoners with a stepwise increase in IIk degree, Brtgg/Galliver liquid medium,
Alternatively, prepare a BL maten plate, Br1gg's Liver agar plate, etc. containing Hlo in a stepwise increasing concentration, inoculate or coat the bed with the mountain stocks that survived irradiation with ultraviolet rays, and leave it anaerobically for 57'048 hours. Cultivate.

The concentration of H and O is low in liquid media, up to about 50 ppm, and slightly higher in agar plates, up to about 500 ppm.
It is preferable to incorporate each into the medium in stages. Parent stock (
Bifidobacterlum longum AT
CC1570B) is clearly more resistant to H, O, and grown at the highest possible temperature of 114 degrees H2O, and a strain that is resistant to H2O can be obtained.

In the present invention, a large number of H, O! The resistant strain was subcultured for 10 generations, and a stable strain without loss of resistance was isolated. This strain was designated Bihydrobacterium longum No. 1022, Fine Engineering Research [F
'ERM P-8035.

Bihydrobacterium longum No. 1022°FgR
M P-8033 is an excellent Hz OJ strain and has the following mycological properties.

It is an ant-like anaerobic box that exhibits rod-shaped or branched polymorphism in Durham-positive orchards harboring ants.

When this strain is applied to a BLIfi Ohbaji (Eiken) plate and cultured for 48 hours at 57°C using the steel cool method, white, opaque, round, hemispherical, glossy colonies are formed. Also, condensate milk land and do not withhold gas Vi.

Catalase, gelatin liquefaction, nitrate reduction, and indole inhibition are all i-type, and the decomposition properties of our class are as follows.

arabinose, xylose, sobose, gluco-x,
Fuc)-su, caractose, sucrose x, -ql)-x, ruff)-su, melichiose, raffinose, and meletitose are positive.

Rhamnose, mannose, cellobiose, trehalose, clicogen, mannitol, sorbitol, inositol, esculin, salicin, and amygdalin were all negative.

These properties are described in Bergey's Manual o
fDeterminative Bacteriolo
gy (8th edition) 1974, and Bifid by Hikama (clinical examination vo118, 1163 (1974)).
According to the classification of obacterium, Bifidoba
The properties match those of cterium longum.

As for other properties, this strain has H10! It is characterized by high resistance and improved oxygen resistance and acid resistance.

In addition, as a feature of this strain, to obtain the culture, we conduct anaerobic culture at a certain - temperature and centrifuge the concentrated bacterial solution obtained by non-circulating centrifugation, and then add it to lactic acid buffer under aerobic conditions. When you save,
Oxygen resistance varied depending on the rice field during anaerobic culture, and it was observed that the oxygen resistance in acidic solutions was particularly affected.

The beak of Vihydrobacterium longum in the present invention,
Obtaining a culture composition of a mutant strain with high H, O, tolerance
It is preferable to carry out constant anaerobic culture at a pl''l of 57 to 5.7. Oxygen resistance is improved by this weakly acidic constant anaerobic culture.

That is, Bihydrobacterium longum no.

1022 was cultured at a constant pH in a liquid medium of pH 4.7, and the pH at which the number of cells reached at the time of neutralization culture was the highest.
A concentrated bacterial solution obtained by centrifuging the culture cultured anaerobically in a liquid medium with pH 5.0 IC [
Add to 0.1 M lactic acid-lactate buffer and add 15'
The results of measuring the number of viable bacteria during CK storage are shown in the table below.

In other words, those cultured at -6.0 may reach a higher number of bacteria during culture than those cultured at -4.7, or their storage stability in acidic solutions may be inferior to those cultured at -4.7. Recognize.

Next, Bihydrobacterium longum No. 1022° F'ERM P-8033 and its parent strain Bihydrobacterium longum aTcc 1570B were adjusted to constant-anaerobic temperature at 4.7 and centrifuged. Pour the Lactic Acid Bacteria into yogurt and lactic acid bacteria drinks, and add 1
Changes in stroke number when stored at 5°C are shown in Figures 1 and 2.

Figure 1 shows the change in the number of viable bacteria in yogurt, and Figure 2 shows the change in the number of viable bacteria in milk #1 ceramic drink.

Next, an embodiment of the present invention will be described.

Example 1 Bifidobaeterium longum I
AT CC15708 was cultivated anaerobically in liquid medium;
Using a sterile diluent, prepare KMMl so that the number of bacteria per 1 d is about 10. 0.1 ml of this diluted solution was evenly applied onto a BL agar plate using a Conrage rod, and 257 n
UV light (Toshiba germicidal lamp GL-15) of 50CI
Irradiation was performed for 100 seconds from a distance of IL. This flat plate was heated to 67℃
After anaerobic culture for 48 hours, approximately 100 colonies were obtained per 1,000 plates. This is 1/10' of the case without UV irradiation.
It is commercially available. 50-500 pp for about 500 colonies grown on BL agar plate after UV + ljl irradiation
It was coated on a BL agar plate containing m H, O, and heated at 57°C.
Cultivate anaerobically for 48 hours. An H, O, and resistant strain was obtained that clearly grew on plates with a higher HzOt concentration than the parent strain. Even if this strain was subcultured for 10 generations in skim milk medium, H, O
1 resistance was stable without loss. This strain was named Bihydrobacterium longum No. 1022.

When we conducted biochemical tests on this Itot-resistant strain, focusing on the ability to assimilate it, we found that Bifidobacter
The properties were consistent with those of iumlongum.

Example 2 Enzymatically decomposed casein powder 3? , lactose 2t, beer yeast extract 0.0! 100d of liquid medium containing IM
Sterilize at 1°C for 15 minutes.

Vihydrobacterium longum No. was added to this medium.

1022, FIRM P-8035 was attached, the constant was set to 4.7, and neutralization culture was carried out at 37°C for 18 hours. 2N NaOH is used as a neutralizing agent, and during cultivation, the culture is performed by replacing the gas phase with CO and gas. Centrifuge the culture solution to obtain a concentrated bacterial solution.

When added to commercially available yogurt and stored at 10°C for 10 days, Hlo, non-resistant parent strain (8ifidobacterium)
rium longum ATCC15708), no survival was observed, whereas the survival rate of this strain was 2.
1 inch was good.

Example 3 A liquid medium containing 10 t of proteolyzed whey powder and 0.1 f of brewer's yeast extract was sterilized at 120° C. for 10 minutes.

Vihydrobacterium longum No. was added to this medium.

1022, inoculated with F'ERM P-8035 and established.
5.0 and 2 N NaICO3 as a neutralizing agent.
Neutralize and culture at 7°C for 17 hours. During cultivation, the culture is performed by replacing the gas phase with CO and gas. Add the culture solution to the lactic acid bacteria drink,
When stored at 0°C for 7 days, HlO, non-resistant parent strain (B
ifidobactarium longum A T
CC15708) showed no survival, whereas
The survival rate of this one plant was 7, which was good.

[Brief explanation of the drawing]

Figure 1 is yogurt KFERM P-803KzAT
CC1570B was fixed at 4.7. The anaerobic culture composition was cooled and the number of bacteria changed during storage. Figure 2 is a diagram showing the change in the number of strokes in the lactic acid 1 drink.

Claims (3)

[Claims] (1) A mutant strain that belongs to Vihydrobacterium longum and has high hydrogen peroxide resistance. (2) A mutant strain belonging to Vihydrobacterium longum with high hydrogen peroxide resistance is cultured anaerobically at a constant pH in a slightly acidic region, and the resulting culture is centrifuged to form a concentrated bacterial solution. Composition. (3) The mutant strain is Vihydrobacterium longum no. 1
022, FERM P-8033, and a culture composition thereof according to claims 1 and 2.