JP3184572B2 - Medium for the isolation and culture of a lactic acid bacterium belonging to Lactobacillus, a method for training the lactic acid bacterium into a milk medium, and a method for producing fermented milk or a lactic acid bacterium beverage using the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a medium for separating a strain belonging to Lactobacillus, a method for adapting the strain to a milk medium, and a method for producing a fermented milk or a lactic acid bacteria beverage using the strain.

[0002]

2. Description of the Related Art Various methods are known for producing fermented milk and lactic acid bacteria beverages. In fermented milk, generally, a stabilizer such as pectin, a thickener such as gelatin, and sugar are added to a fermentation substrate mainly composed of milk or the like as a raw material. A flavor material is added, and this is sterilized and homogenized according to a conventional method, and inoculated with 2 to 3% of a lactic acid bacterium and / or yeast starter, filled in a container and sealed, and fermented at 40 to 45 ° C. After the acidity is reached, the fermentation is cooled to 10 ° C. or lower to stop the fermentation, thereby obtaining a product. In the case of liquid fermented milk and lactic acid bacteria drinks, a lactic acid bacterium and / or yeast starter is similarly inoculated into a fermentation substrate to which a stabilizer such as pectin and a thickener such as gelatin are added.
After fermentation in a tank at 5 ° C. to a predetermined lactic acidity and cooling to 10 ° C. or less, the curd is crushed into a liquid state by a homogenizer or the like, and the product is filled into a container and sealed to form a product. At this time, the lactic acid bacteria beverage should be manufactured by reducing the milk solid content of the fermentation substrate or diluting it with a sterilized solution prepared by dissolving saccharides and stabilizers prepared separately to reduce the milk solid content. Is done.

[0003] At this time, the above-mentioned stabilizer such as pectin and thickener such as gelatin are used in order to suppress the occurrence of whey separation phenomenon and water separation phenomenon which are likely to occur in fermented milk and lactic acid bacteria beverages during the production process and the subsequent distribution process. Since these phenomena almost certainly occur without the use of such additives, the additives are indispensable for their production.

[0004] Plain type yogurt is free of food additives, and thus does not contain these additives. However, the yogurt generally exhibits a whey separation phenomenon and a water separation phenomenon.

[0005] However, although these pectins and gelatins are natural food additives, their addition may impair the image of foods that maintain health, such as fermented milk and lactic acid bacteria drinks. Therefore, there is a demand for fermented milk and lactic acid bacteria beverages that do not use these additives in a growing health-oriented environment.

[0006] The present invention has been made in view of the above circumstances, and a problem to be solved is firstly a lactic acid bacterium strain belonging to Lactobacillus that can be used as a lactic acid bacterium that prevents the occurrence of whey separation phenomenon and water separation phenomenon. The second object is to provide a method for adapting a lactic acid bacterium strain to a milk medium with the use of the separation culture medium. Using lactic acid bacteria strain, without adding food additives,
It is an object of the present invention to provide a method for producing a fermented milk or a lactic acid bacterium beverage capable of preventing the occurrence of whey separation and water separation.

[0007]

[Means for Solving the Problems] As a result of intensive studies along the above first problem, the present inventor has found that kefir granules formed by lactic acid bacteria and yeast called kefir grains (available from Christian Denmark, Denmark) Hansen Co., Ltd.) and obtained by separating and culturing the kefir granules from the kefir granules in a specific medium can easily and reliably obtain lactobacilli (lactobacillus strains) that produce large amounts of polysaccharide viscous substances. The invention according to claim 1 has been made based on this finding, that is, the invention according to claim 1 has the following composition and a pH of 5.5. No. belonging to Bacillus. A culture medium for separation and cultivation of 14 strains (Deposit No. 10196 of Microtechnical Laboratory). 100ml cheese whey, trypticase peptone 0.
5 g, tryptone 0.5 g, yeast extract 0.5 g,
Tween 80 0.1 ml, monopotassium phosphate 0.5
g, sodium acetate trihydrate 0.5 g, triammonium citrate 0.2 g, magnesium sulfate heptahydrate
0.058 g, manganese sulfate tetra-hexahydrate 0.028
g.

Further, in accordance with the second problem, No. 1 belonging to the above Lactobacillus isolated and cultured in the medium.
At the time of adaptation of the 14 strains to the milk culture medium, it was found that it is preferable to enrich the milk culture medium with yeast extract by 0.1% and to perform the stationary culture at a temperature of about 30 ° C. The invention according to claim 2 has been made based on the knowledge, that is, the invention according to claim 2 is a No. 1 belonging to Lactobacillus isolated and cultured with the culture medium for isolation and culture according to claim 1. 14 strains (Deposit No. 10196
No. 1), and using a medium in which yeast extract is fortified with 0.1% milk medium, and statically culturing at a temperature of about 30 ° C., the method for adapting a strain belonging to Lactobacillus to a milk medium. It was done.

[0009] Further, in accordance with the third problem, No. 3 belonging to the Lactobacillus isolated and cultured in the medium.
Since the 14 strains have the property of producing a polysaccharide viscous substance as described above, the use of the 14 strains makes it possible to claim
The invention according to claim 3, wherein the invention according to claim 1 is a fermented milk or lactic acid bacteria beverage in which the whey separation phenomenon and the water separation phenomenon are prevented from occurring, without adding any food additive. No. 1 belonging to Lactobacillus separated and cultured in a culture medium for separation. 14 strains (Deposit No. 10196
Fermented milk or lactic acid bacterium drink characterized by preventing the occurrence of whey separation phenomenon and water separation phenomenon in the state without added stabilizer by producing polysaccharide viscous substance in fermentation substrate fermented by using This is a method of manufacturing.

The present invention uses the inventions described in these claims as the gist of the invention and means for solving the above problems.

Hereinafter, the present invention will be described more specifically.
The specific medium (hereinafter sometimes referred to as GMR medium) for separating and culturing the lactobacilli (lactic acid bacteria strain) has the composition shown in Table 1, and according to the knowledge of the present inventors, this GMR medium Only by using a medium, it is possible to isolate and culture Lactobacillus (Lactobacillus strain) from kefir granules.

[0012] Separation culture from kefir granules is performed by grinding the kefir granules in a sterilized mortar, appropriately diluting with sterile physiological saline, and using 0.1 ml of the plate culture medium as a GMR.
30 ± 5 ℃ by carbon dioxide displacement anaerobic culture method
By culturing for 3-5 days at, it can be separated and cultured as the main flora in kefir granules

[0013]

Table 1 GMR medium composition Cheese whey 100 ml Trypticase peptone (BBL) 0.5 g Tryptone (Difco) 0.5 g Yeast extract (Difco) 0.5 g Tween 80 0.1 ml Monopotassium phosphate 0.5 g Sodium acetate Hydrate 0.5 g Triammonium citrate 0.2 g Magnesium sulfate heptahydrate 0.058 g Manganese sulfate tetra-hexahydrate 0.028 g pH 5.5

The inventor succeeded in separating and cultivating 52 strains of polysaccharide viscous substance-producing strains by the above method. Among them, a strain having the highest growth of the polysaccharide viscous substance was selected by observation and judgment by the ink-staining method, while the growth degree of the GMR in the liquid medium was good. 1
Four strains were designated. This strain is a lactic acid bacterium belonging to Lactobacillus and is described in Japanese Patent No. 10196 (FERM.P-1).
No. 0196), and its bacteriological properties are as shown in Table 2.

[0015]

[Table 2] Mycological properties of 14 strains Gram stain Positive sporulation None Motility None Growth under aerobic conditions + Growth under anaerobic conditions + Catalase formation-Lactic acid DL (D) Growth at 15 ° C ± 20 ° C Growth + growth at 40 ° C. + growth at 45 ° C. ± production of ammonia from arginine-gas production from glucose-gas production from gluconate-assimilation of sugars Assimilating sugars: amygdalin, fructose, galactose,
Glucose, lactose, maltose, mannose, melibiose, raffinose, saccharose, non-assimilating sugars:
Arabinose, cellobiose, esculin, gluconate, mannite, tretitol, rhamnose, ribose, salicin sorbitol, trehalose, xylose.

The above No. At the beginning of the isolation and cultivation of the 14 strains, no growth was observed in the milk medium (reduced skim milk medium having a non-fat milk solid content of 10%), and similar phenomena were observed in the other isolates. When the 14 strains were statically cultured at a temperature of 30 ° C. using a milk medium enriched with yeast extract, they grew to the extent that solidification of the milk medium was observed on the seventh day. When generation change was continued using a milk medium enriched with yeast extract, The growth of 14 strains on the same medium tended to be gradually accelerated, and the subculture was continued.
After standing for a long time, lactic acidity is 1.30-1.50%,
Viable bacteria count 5 × 10 ⁸ / ml, total bacteria count 1.5 × 10 ⁹ / ml
Succeeded to acclimate to grow up to.

Using the culture obtained here as a mother starter, reduced skim milk powder having a non-fat milk solid content of 10% is heated to 95 ° C.
5% (v / v) was inoculated into a milk medium containing no yeast extract and kept sterilized at 30 ° C. for 30 minutes, and cultured at 30 ° C. for 48 hours. 10 ⁸ /
Thus, a bulk starter having a total bacterial count of 6.5 × 10 ⁸ / ml was obtained.

The bulk starter thus prepared is used alone or in combination with a mixed bulk starter of Lactobacillus bulgaricus and Streptococcus thermophilus, etc., and inoculated to a fermentation substrate such as milk.
When fermented at 0-40 ° C, preferably 25-35 ° C,
No. The 14 strains grow during fermentation and produce a large amount of polysaccharide viscous substance in the fermentation substrate to obtain a fermented milk or lactic acid bacterium beverage that prevents the occurrence of whey separation and syneresis in the absence of a stabilizer. That was confirmed.

[0019]

[Examples] Table 1 shows the 14 strains.
Agar medium, MRS medium (Difc
o), BCP medium (Eiken), BL blood agar medium (Eiken), Brix River agar medium, tomato juice agar medium, and milk agar medium were used as test plate mediums, smeared and inoculated with carbon dioxide gas. After culturing at 30 ° C. for 5 days by the culturing method, the growth was observed on the GMR agar medium, but other MRS medium, BCP medium, BL
No growth was observed on the blood agar medium, the Brix River agar medium, the tomato juice agar medium, or the milk agar medium (similar results were obtained for 51 strains other than No. 14 out of the 52 strains).

No. 14 strains, non-fat milk solids 10%
A milk culture medium containing 0.1% yeast extract in a reduced skim milk medium and a milk medium containing no yeast extract were cultivated at 30 ° C. Showed growth to such an extent that its solidification was observed, but no growth was observed in a milk medium containing no yeast extract. Furthermore, this No. When 14 strains were used as a starter, 5% (v / v) was inoculated into a milk medium supplemented with 0.1% yeast extract and cultured at 30 ° C., and the milk medium solidified on the 7th day. When generation change was continued under the same conditions, solidification of the milk medium began to be observed 3 days after the 10th generation, and 30 days thereafter.
When the cells are cultured at 30 ° C. for 48 hours, the lactic acid content is 1.30 to 1.50%, and the number of viable cells is 5 ×.
The adaptation could be performed to the extent that 10 ⁸ / ml and the total number of bacteria were 1.5 × 10 ⁹ / ml.

Further, according to a conventional method, milk which has been sterilized and homogenized is used as a fermentation substrate, and a medium prepared with sterilized milk is used as a culture medium. 5.0% of bulk starter of 14 strains
(W / w) Inoculation was carried out, and the mixture was stirred and homogenized. At this time, the lactic acid acidity was 0.50%, the number of viable bacteria was 1.4 × 10 ⁷ / ml, and the total number of bacteria was 2.7 × 10 ⁸ / ml. Thereafter, the fermentation substrate was stirred, and a Streptococcus thermophilus bulk starter (starter made with the same sterilized milk as the fermentation substrate) was 2.0% (w
/ W) After inoculation and homogenization with stirring, the mixture was fermented at 30 ° C. for 17 hours, and the lactic acid acidity was 0.75%. 1
The viable cell count of the four strains is 1.8 × 10 ⁷ / ml, and the total cell count is 4.
5 × 10 ⁸ / ml of fermented milk was obtained. The resulting fermented milk had a thread-drawing viscosity. The milk curd was crushed with a homogenizer to obtain a liquid fermented milk. This liquid fermented milk was dispensed into a small container with a mm scale and stored at 10 ° C. for 2 weeks, and the state of water separation was observed. As a comparative example, a fermentation substrate having the same composition was fermented using Lactobacillus bulgaricus and Streptococcus thermophilus, and then the milk curd was crushed by a homogenizer to obtain a liquid, which was dispensed into small containers. Stored at 2 ° C for 2 weeks, and observed the state of water separation. Table 3 shows the results.

[0022]

[Table 3] [Note] No. Liquid fermented milk using 14 strains and fermented milk of Comparative Example were 8
The solution was dispensed into a transparent container with a mm scale so as to have a height of 0 mm, and stored at 10 ° C. The numerical values in the table are obtained by measuring the distance of separation.

As is apparent from Table 3, Although almost no water separation was observed in the liquid fermented milk using the 14 strains, about 30% of water was separated in the liquid fermented milk of the comparative example.

[0024] Further, in the same manner, the medium No.
14 strains bulk starter 5.0% (w / w), Streptococcus thermophilus bulk starter 2.0
%, And fermentation was carried out by static fermentation at 30 ° C. for 72 hours to obtain fermented milk having a lactic acidity of 1.50%. 1.5 kg of beet granulated sugar was added to 2.0 kg of liquid fermented milk homogenized with a homogenizer and heated and melted at 85 ° C.
And then homogenized again with a homogenizer, then 10 ℃
And cooled. To this, 0.02% of lemon flavor was added, stirred, filled in a glass container and sealed to produce a sterilized lactic acid bacteria beverage. The resulting sterilized lactic acid bacterium beverage had no additives such as stabilizers and thickeners, but no water separation was observed after storage at room temperature.

[0025]

The present invention is configured as described above. Therefore, the invention of claim 1 is a lactic acid bacterium strain belonging to Lactobacillus which can be used as a lactic acid bacterium capable of preventing the occurrence of whey separation phenomenon and water separation phenomenon. Koken Deposit No. 10196
No. It is possible to provide a culture medium for separation and cultivation capable of separating and culturing 14 strains. It is possible to provide a method for acclimating 14 strains to a milk culture medium. Using the 14 strains, it is possible to provide a method for producing fermented milk or a lactic acid bacteria beverage which can prevent the occurrence of whey separation and water separation without adding a food additive.

Claims (3)

(57) [Claims] 1. No. 1 belonging to Lactobacillus comprising the following composition and having a pH of 5.5: 14
A culture medium for separating and culturing a strain (Deposited by No. 10196).
100ml cheese whey, trypticase peptone
0.5 g, tryptone 0.5 g, yeast extract 0.5
g, Tween 80 0.1 ml, monopotassium phosphate
0.5 g, sodium acetate trihydrate 0.5 g, triammonium citrate 0.2 g, magnesium sulfate heptahydrate 0.058 g, manganese sulfate tetrahexahydrate 0.02
8g. 2. No. belonging to Lactobacillus isolated and cultured with the culture medium for isolation according to claim 1. A strain belonging to Lactobacillus, which is characterized by culturing 14 strains (Deposited No. 10196, Microtechnical Laboratory No. 10196) at a temperature of about 30 ° C. in a milk medium supplemented with 0.1% yeast extract. A method of acclimating a strain to a milk medium. 3. No. belonging to Lactobacillus isolated and cultured with the culture medium for isolation and culture according to claim 1. Prevention of whey separation and syneresis in the absence of a stabilizer by producing a polysaccharide viscous substance in a fermentation substrate fermented using 14 strains (Deposit No. 10196) A method for producing a fermented milk or a lactic acid bacterium beverage, comprising: