JP4671384B2 - New lactic acid strain - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a Lactobacillus gasseri strain having bile acid resistance and osmotic pressure resistance.
[0002]
[Prior art]
In recent years, lactic acid bacteria having physiological effects have attracted attention. In particular, lactic acid bacteria that reach the intestinal tract in a living state and show effective health effects on the host are defined as probiotic lactic acid bacteria, and lactic acid bacteria such as Bifidobacterium and Lactobacillus are attracting attention. Yes. And about these lactic acid bacteria, many health effects, such as an intestinal regulation effect and improvement of digestion absorption ability, reduction of serum cholesterol, an antitumor effect, are reported.
[0003]
By the way, in order for these probiotic lactic acid bacteria to work effectively in the digestive tract and exert health effects, it is necessary to reach the digestive tract while remaining alive. However, before reaching the digestive tract, there are various growth-inhibiting environments and growth-inhibiting substances for probiotic lactic acid bacteria such as temperature, pH, oxygen, osmotic pressure, stomach acid and bile acid. Therefore, when using a probiotic lactic acid bacterium, it is important whether the probiotic lactic acid bacterium has resistance to a growth-inhibiting environment or a growth-inhibiting substance.
[0004]
In particular, probiotic lactic acid bacteria, due to a rapid change in water activity in the process of freeze-drying probiotic lactic acid bacteria, or addition of sugars, salts, etc. in the process of producing products using probiotic lactic acid bacteria, Receive stress from osmotic pressure. Therefore, it can be said that having osmotic pressure resistance is one of the advantageous and useful properties for probiotic lactic acid bacteria. In addition, when ingesting probiotic lactic acid bacteria, bile acids that have a strong surface-active effect and a bactericidal effect that acts directly on the cell membrane are present in the intestinal tract and are thought to have a significant impact on the bile acid resistance. It can be said that it is one of advantageous and useful properties for probiotic lactic acid bacteria.
[0005]
Bile acid is a substance produced in the livers of many animals including humans. Bile acids such as cholic acid and chenodeoxycholic acid generated from cholesterol in the liver are called primary bile acids, which are combined with taurine and glycine to form conjugated bile acids and are secreted into the bile. And most bile acids secreted in the bile are absorbed from the intestinal tract, return to the liver, and repeat the enterohepatic circulation where it is secreted again in the bile. Some primary bile acids are dehydrated and oxidized in the intestine by the action of intestinal bacteria, and converted into bile acids called secondary bile acids such as deoxycholic acid and lithocholic acid.
[0006]
[Problems to be solved by the invention]
The present inventors have been diligently researched to obtain a strain that can be used as a probiotic lactic acid bacterium, and as a result, among the strains belonging to Lactobacillus gasseri , bile acid resistance and osmotic pressure. The present inventors have succeeded in obtaining a Lactobacillus gasseri mutant strain having durability, and have completed the present invention. Accordingly, an object of the present invention is to provide a strain of Lactobacillus gasseri that can be expected to have a health effect as a probiotic lactic acid bacterium and has bile acid resistance and osmotic pressure resistance.
[0007]
[Means for Solving the Problems]
The present invention is a novel strain of Lactobacillus gasseri having bile acid resistance and osmotic pressure resistance, for example, glycocholic acid 24%, glycochenodeoxycholic acid 24%, glycodeoxycholic acid 18% It has a bile acid resistance capable of growing in a medium containing 10 mM or more of a conjugated bile acid mixture composed of 13% taurocholic acid, 13% taurochenodeoxycholic acid and 8% taurodeoxycholic acid, and has an osmotic pressure of 2.2. It has osmotic pressure resistance and can grow in a medium of Osmol / kg or more. This novel strain is a mutant strain of Lactobacillus gasseri , for example, Lactobacillus gasseri SBT10801 (FERM P-18137).
[0008]
The Lactobacillus gasseri strain having bile acid resistance and osmotic pressure resistance according to the present invention can be obtained as follows.
That is, human feces are smeared on Mitsuoka's LBS agar medium and colonies obtained by culturing at 37 ° C. for 3 days under anaerobic conditions are isolated. As a result of identifying this strain according to Bergy's Manual of systematic bacteriology, vol. 2 (1986), it was consistent with the mycological properties of Lactobacillus gasseri . Moreover, it had properties equivalent Lactobacillus gasseri (Lactobacillus gasseri) JCM1131 and a reference strain of Lactobacillus gasseri (Lactobacillus gasseri). This is named Lactobacillus gasseri SBT0269, which is the parent strain, and is glycocholic acid 24%, glycochenodeoxycholic acid 24%, glycodeoxycholic acid 18%, taurocholic acid 13%, taurochenodeoxycholic acid 13% and A medium containing 5 mM of a conjugated bile acid mixture consisting of 8% taurodeoxycholic acid is inoculated to isolate the grown strain. Then, isolate the strain that grew by gradually increasing the concentration of the conjugated bile acid mixture added to the medium, and finally isolate the strain that grew on the medium containing 10 mM of the conjugated bile acid mixture. A strain of Lactobacillus gasseri having bile acid resistance is obtained.
[0009]
Next, the isolated strain of Lactobacillus gasseri having resistance to bile acid is used as a parent strain and inoculated into a medium having an osmotic pressure of 1.5 Osmol / kg to isolate the grown strain. Then, isolate the strain that grew by gradually increasing the osmotic pressure of the medium, and finally isolate the strain that grew on the medium of osmotic pressure 2.2 Osmol / kg. A strain of Lactobacillus gasseri having performance is obtained.
[0010]
The strain thus obtained is a mutant strain of Lactobacillus gasseri having bile acid resistance and osmotic pressure resistance, and is a novel strain of Lactobacillus gasseri .
The Lactobacillus gasseri of the present invention can be grown in a conjugated bile acid mixture designed with reference to the human bile acid composition as described above, and thus can grow in human bile. .
Furthermore, it can grow on a medium having an osmotic pressure of 2.2 Osmol / kg or more. When the osmotic pressure 2.2 Osmol / kg is converted into the sodium chloride concentration, it is about 1 M, which corresponds to a sucrose concentration of 43%. Therefore, it can grow sufficiently even in high osmotic pressure foods such as sweetened fermented milk and pickles having such osmotic pressure.
[0011]
This strain has the following bacteriological properties, and since it was confirmed that it belongs to Lactobacillus gasseri for the reasons described below, Lactobacillus gasseri SBT10801 and Named and deposited as FERM P-18137 at the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology. Its mycological properties are as follows.
[0012]
A Morphological properties Cell shape: Neisseria gonorrhoeae 2. Presence or absence of spores: None Gram staining: positive B Growth state on medium Colony morphology on plate count agar medium: white sphere with smooth surface
C Physiological properties
Viability at 15 ° C: negative
Growth ability at 45 ° C: Degradability of positive arginine: Negative Type of lactic acid produced: DL
Degradability of various carbohydrates (positive +, negative-)
Amygdalin: +
Arabinose:-
Cellobiose:-
Fructose:-
Galactose: +
Glucose: +
Lactose: +
Gluconate:-
Maltose: +
Mannitol:-
Mannose: +
Rhamnose:-
Ribose:-
Sorbitol: +
Sucrose: +
Xylose:-
[0014]
From mycological properties of the above, Bergy's Manual of systematic bacteriology, vol.2 of (1986) and is identified by, Lactobacillus gasseri (Lactobacillus gasseri) SBT10801 (FERM P -18137) , the Lactobacillus gasseri (Lactobacillus gasseri) Consistent with mycological properties.
This Lactobacillus gasseri SBT10801 (FERM P-18137) is composed of glycocholic acid 24%, glycochenodeoxycholic acid 24%, glycodeoxycholic acid 18%, taurocholic acid 13%, taurochenodeoxycholic acid 13% and taurodeoxy It can grow on a medium containing 15 mM of a conjugated bile acid mixture consisting of 8% cholic acid, and can grow on a medium having an osmotic pressure of 2.2 Osmol / kg or higher. Unlike Lactobacillus gasseri, it is a mutant strain of Lactobacillus gasseri having bile acid resistance and osmotic pressure resistance.
[0015]
Next, the present invention will be described in more detail with reference to examples and test examples.
[Example 1]
Against bile acids and osmotic pressure, Lactobacillus gasseri (Lactobacillus gasseri) SBT0269 with Lactobacillus gasseri (Lactobacillus gasseri) JCM1131 equivalent resistance is the reference strain of Lactobacillus gasseri (Lactobacillus gasseri) with the parent strain, MRS medium containing 5 mM conjugated bile acid mixture consisting of 24% glycocholic acid, 24% glycochenodeoxycholic acid, 18% glycodeoxycholic acid, 13% taurocholic acid, 13% taurochenodeoxycholic acid and 8% taurodeoxycholic acid And cultured at 37 ° C. for about 120 hours until growth was observed. Then, 5% of this culture solution was inoculated into a medium containing 5.5 mM bile acid, and similarly cultured at 37 ° C. for about 120 hours.
In this way, the strain grown by gradually increasing the concentration of the conjugated bile acid mixture added to the MRS medium by 0.5 mM stepwise is isolated, and finally the MRS medium containing the 10 mM conjugated bile acid mixture is added. The strains that grew on were isolated. The culture time was up to 240 hours depending on the growth of the strain.
MRS medium has the following composition per liter of medium;
Protospepton No. 3 10 g, Beef Extract 10 g, Yeast Extract 5 g, Dextrose 20 g, Tween 80 1 g, Ammonium Citrate 2 g, Sodium Acetate 5 g, Magnesium Sulfate 0.1 g, Manganese Sulfate 0.05 g and Dibasic Potassium Phosphate 2 g .
[0016]
Next, the isolated strain was inoculated into an MRS medium having an osmotic pressure of 1.5 Osmol / kg to isolate the grown strain. The osmotic pressure of the medium was adjusted by adding sodium chloride to the MRS medium. Then, 5% of this culture solution was inoculated into an MRS medium having an osmotic pressure of 1.6 Osmol / kg, and cultured at 37 ° C. for about 120 hours.
In this way, the strain that grew by gradually increasing the osmotic pressure of the MRS medium by 0.1 Osmol / kg was isolated, and finally the strain that grew on the MRS medium having the medium osmotic pressure of 2.2 Osmol / kg was isolated. . The culture time was up to 240 hours depending on the growth of the strain.
The obtained mutant strain was subcultured for 3 to 7 passages in a normal MRS medium, and then inoculated and grown in a medium containing 10 mM bile acid or a medium containing 1 M sodium chloride.
The mutant strain of Lactobacillus gasseri thus obtained is a novel strain of Lactobacillus gasseri having bile acid resistance and osmotic pressure resistance, and Lactobacillus gasseri. (Lactobacillus gasseri) SBT10801 (FERM P-18137) was deposited at the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology.
[0017]
The parent strain is inhibited from growing in a medium containing 6 mM of a conjugated bile acid mixture, whereas the mutant strain can grow in a medium containing 15 mM of a conjugated bile acid mixture. The growth is inhibited by a medium having an osmotic pressure of 1.6 Osmol / kg, whereas the mutant strain can grow on a medium having an osmotic pressure of 2.2 Osmol / kg.
[0018]
[Test Example 1]
The concentration of the conjugated bile acid mixture obtained by adding Lactobacillus gasseri SBT10801 (FERM P-18137) having bile acid resistance and osmotic pressure resistance obtained in Example 1 to the medium is determined. The inoculated MRS medium prepared stepwise in the range of 0 to 20 mM was cultured at 37 ° C. for 8 hours, and the growth states were compared. In addition, the growth state of Lactobacillus gasseri SBT0269 and the reference strain Lactobacillus gasseri JCM1131 were also compared in the same manner. The growth state was evaluated by measuring the turbidity of the medium. That is, the OD value at 660 nm of the culture solution when cultured in the MRS medium not added with the conjugated bile acid mixture is set to 100, and the 660 nm of the culture solution when cultured in each MRS medium added with the conjugated bile acid mixture solution. The OD value was expressed as a percentage. The result is shown in FIG.
[0019]
In the parent strain Lactobacillus gasseri SBT0269 and the reference strain Lactobacillus gasseri JCM1131, growth was inhibited in a medium containing 5-6 mM conjugated bile acid mixture, Lactobacillus gasseri SBT10801 (FERM P-18137) of the present invention grows in a medium containing 15 mM of a conjugated bile acid mixture, although the growth state worsens as the concentration of conjugated bile acid increases. I found that I can do it.
[0020]
[Test Example 2]
Lactobacillus gasseri SBT10801 (FERM P-18137) having bile acid resistance and osmotic pressure resistance obtained in Example 1 was cultured in MRS medium, and then centrifuged to recover the cells. . Then, the recovered cells were suspended in MRS medium adjusted to an osmotic pressure of 2.2 Osmol / kg with salt and stored at 10 ° C., and the number of viable cells was measured over time by the plate count method, and the growth state was compared. . Similarly, the growth conditions of the parent strain Lactobacillus gasseri SBT0269 and the reference strain Lactobacillus gasseri JCM1131 were also compared. The result is shown in FIG.
In the parent strain Lactobacillus gasseri SBT0269 and the reference strain Lactobacillus gasseri JCM1131, the number of viable bacteria decreased to 10 ⁶ cfu / ml or less 21 days after the start of storage. In the Lactobacillus gasseri SBT10801 (FERM P-18137) of the invention, the viable cell count was 10 ⁸ cfu / ml 21 days after the start of storage, and the viable cell count was hardly reduced.
[0021]
【The invention's effect】
Lactobacillus gasseri SBT10801 (FERM P-18137) of the present invention has a high bile acid resistance and osmotic pressure resistance, so the bile acid concentration is high in the intestinal tract and high osmotic pressure environment High survival can be expected below, and it is useful as a probiotic lactic acid bacterium.
[Brief description of the drawings]
1 shows the growth state of each strain of Lactobacillus gasseri in Test Example 1. FIG.
[Explanation of symbols]
Black circle: SBT10801
Black triangle: SBT0269
Black square: JCM1131
2 shows the number of viable bacteria of each strain of Lactobacillus gasseri in Test Example 2. FIG.
[Explanation of symbols]
Black circle: SBT10801
Black triangle: SBT0269
Black square: JCM1131

Claims (2)

Lactobacillus gasseri strain having bile acid resistance and osmotic pressure resistance ,
Conjugated bile acid mixture consisting of 24% glycocholic acid, 24% glycochenodeoxycholic acid, 18% glycodeoxycholic acid, 13% taurocholic acid, 13% taurochenodeoxycholic acid and 8% taurodeoxycholic acid A strain that can grow on a medium containing 10 mM or more of the osmotic pressure, and that can grow on a medium having an osmotic pressure resistance of osmotic pressure of 2.2 Osmol / kg or more. Lactobacillus gasseri (Lactobacillus gasseri) SBT10801 strain according to claim 1, wherein the (FERM P-18137).

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