JPH05276932A - New lactobacillus strain - Google Patents

Abstract

PURPOSE:To provide a new lactobacillus strain enabling the industrial production of fermented milk kefir at a low cost without using kefir grains by combining a known lactobacillus with yeast. CONSTITUTION:A homo-fermenting lactobacillus accounting for the largest portion in lactobacillus constituting kefir grains, e.g. Lactobacillus sp. glico 1701 (FERM P-12894). The lactobacillus is preferably separated from the cultured product obtained by grinding and homogenizing kefir grains, diluting the product and culturing the suspension in a newly invented GMR medium consisting of cheese whey, trypticase peptone, trypton, yeast extract, Tween 80, monopotassium phosphate, sodium acetate trihydrate, triammonium citrate, magnesium sulfate heptahydrate and manganese sulfate tetra or hexahydrate.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to the production of fermented milk kefir using two kinds of novel lactobacilli belonging to the genus Lactobacillus isolated from kefir granules. More specifically, the present invention aims to industrially advantageously produce fermented milk kefir using a single starter of the present strain and a strain isolated from other kefir granules without using kefir granules.

[0002]

2. Description of the Related Art Unlike other fermented milk / lactic acid bacteria beverages, the method for producing fermented milk kefir is characterized by the use of cauliflower-like solid starters called kefir grains. Kefir granules are aggregates of several kinds of lactic acid bacteria and yeasts, and by their actions, the unique flavor of fermented milk kefir and the refreshing feeling due to carbon dioxide are produced. Fermented milk kefir is generally produced by the following method. That is, if necessary, a pectin, a stabilizer such as gelatin, a sugar, and, if necessary, a flavoring material such as a flavor and a fruit juice are added to a fermentation substrate containing milk or the like as a raw material as a raw material. Sterilized and homogenized to 2 to 10% of kefir granules and 1 to 3% of lactococcus such as Streptococcus lactis as needed, and then fermented overnight at 15 to 30 ° C to sterilize kefir granules. Filter off with a crushed mesh. The filtered kefir granules are stored as a starter for the next fermentation. The filtrate is homogenized as necessary, filled into a container, sealed, and then aged and fermented at 10 to 20 ° C for 1 to 3 days to contain a trace amount of alcohol and carbon dioxide gas by the alcohol fermentation of yeast. It is a product. In addition, a method for producing fermented milk kefir by inoculating the milk medium with kefir granules for fermentation for the purpose of mass production and fermenting it and using the filtrate as a starter and adding a lactic acid coccus starter such as Streptococcus lactis as needed is also used. ing. Fermented milk kefir is characterized by using kefir granules composed of several kinds of lactic acid bacteria and yeast as a starter, but as described above, kefir granules are reused as a starter, and therefore, it is necessary to separate them from the fermentation substrate. However, when fermented milk is produced, the fermentation substrate before fermentation is not viscous and is dry, but it is usually viscous after fermentation.
This tendency is particularly noticeable when the fermentation substrate contains fat. The viscosity of the fermentation substrate often makes it difficult to separate the kefir granules from the fermentation substrate. Kefir granules are usually 5 to 5 in one fermentation culture.
Although it grows by 10%, the kefir granules are crushed during the filtration due to the thickening of the fermentation substrate and cannot be recovered, and it is often recognized that the amount smaller than the amount inoculated as the starter can be recovered. In such a case, the next fermentable amount decreases, which is not suitable for industrial production. Also,
Since the kefir granules are reused as a starter, equipment for sanitarily separating the kefir granules, equipment for storing the kefir granules, and other equipment that are more sophisticated and larger than other fermented milks are required.

[0003]

[Means for Solving the Problems] The present inventors have completed the present invention as a result of intensive studies to solve the above problems. That is, the bacterial flora of kefir granules and the bacterial flora of fermented milk fermented using the kefir granules were clarified using a newly devised GMR medium and yeast extract-enhanced PDA medium, and the kefir granules were used according to the combination of isolated strains. It has succeeded in producing fermented milk kefir without performing. Conventionally, many researchers from Kefir granules are lactococcus represented by Streptococcus lactis, hetero-type lactobacilli represented by Lactobacillus kefir, homo-lactobacilli represented by Lactobacillus acidophilus, and candy. Yeast typified by da kefir has been isolated and reported. However, there are still no published reports on the bacterial flora of Kefir granules, including Lactobacillus kefiranofaciens, which produces the polysaccharide viscous substance forming Kefir granules. The inventors first conducted extensive studies to clarify the bacterial flora of the kefir granules, and concluded that the yeast extract-enriched PDA medium is the best for yeast and the newly devised GMR medium is the best for the separation of lactic acid bacteria. It was The composition of the GMR medium is shown in Table 1. P
The DA medium used was a commercial product of Nissui Pharmaceutical.

[0004]

[Table 1] GMR medium composition Cheese whey 100 ml Trypticase peptone (BBL) 0.5 g Trypton (Difco) 0.5 g Yeast extract (Difco) 0.5 g Tween 80 0.1 ml Monopotassium phosphate 0.5 g Sodium triacetate 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

Kefir granules (obtained from Christian Hansen, Denmark) were lightly rinsed with sterile saline, drained with sterilized filter paper and ground in a sterilized mortar. 1 g of this is measured and added with 9 g of sterilized physiological saline, homogenized with a Teflon homogenizer, homogenized with a Teflon homogenizer several times with 10 times sterilized physiological saline, and then appropriately diluted. It was smeared on a flat plate. The culturing conditions were 30 ° C. for both PDA medium and GMR medium, the PDA medium was cultivated for 3 to 5 days, and the GMR medium was cultivated under anaerobic conditions of carbon dioxide gas substitution for 5 days. After counting the colonies, all the grown colonies were picked and purified, and then the yeast was identified according to The Yeast 8th Edition, and the lactic acid bacteria were identified according to the Burjay's Manual of Systematic Bacteriology Volume 2. The results are shown in Table 2.

[0006]

[Table 2] Bacterial flora of kefir granules ─────────────────────────────────── Viable cell count (/ g )% ─────────────────────────────────── Lactic acid bacteria 60 × 10 ⁸ Lactobacillus species Type I 29. 5 49 Lactobacillus kefiranofaciens 26 43 Lactobacillus kefir 3.5 6 6 Lactobacillus species Type II 1 2 ──────────────────────── ──────────── Yeast 25 × 10 ⁷ Candida holmi 19.5 78 Candida kefir 5.5 22 ────────────────── ──────────────────

As is clear from Table 2, four types of lactobacilli and two types of yeast were detected in the kefir granules. Of these, Lactobacillus species Type I and Lactobacillus species Type II are novel lactobacilli that have not been known so far, and they are named Lactobacillus species Glyco 1701 and Lactobacillus species Glyco 1731, respectively. , Institute of Microbial Science and Technology, AIST
(FERM P-12894) and Microtechnical Research Institute of Microbiology 128
95 (FERM P-12895). The mycological properties of 1701 and 1731 are shown in Table 3 and Table 4.
It was shown to.

[0008]

[Table 3] Mycological properties of strain 1701 Gram stain Positive Spore formation None Motility None Growth under aerobic conditions + Growth under anaerobic conditions + Growth of catalase-Lactic acid produced D (L) lactate at 15 ° C Growth W Growth at 45 ° C.-Ammonia production from arginine-Gas production from glucose-Assimilation of sugar; Amygdalin + cellobiose + fructose + esculin-galactose + gluconate-glucose + mannitol-lactose + melethose + maltose + Rhamnose-mannose + ribose-melibiose + salicin + raffinose + sorbitol-saccharose + trehalose + arabinose-xylose-

[0009]

[Table 4] Mycological properties of strain 1731 Gram stain Positive Spore formation None Motility None Growth under aerobic conditions + Growth under anaerobic conditions + Growth of catalase-Lactic acid L-lactic acid Growth at 15 ° C +45 Growth at ° C-Ammonia production from arginine + Gas production from glucose + Sugar assimilation; Amygdalin-cellobiose-fructose-escrine-galactose + gluconate-glucose + mannitol-lactose + melezitose + maltose + rhamnose-mannose -Ribose + melibiose + salicin-raffinose-sorbitol-saccharose-trehalose-arabinose + xylose-

As is clear from the bacterial flora of kefir granules shown in Table 2, the large number of Lactobacillus kefiranofaciens can be separated by the newly devised GMR.
There are no reports other than medium, and Lactobacillus species type I represented by strain 1701 is GMR.
Many strains other than the culture medium did not grow. That is, the lactic acid bacterial flora of kefir granules revealed in GMR medium is the most true as far as is known so far. 10% (w / w) of kefir granules in sterilized milk
After inoculating and culturing at 25 ° C. overnight with periodic stirring, the bacterial flora of fermented milk kefir from which kefir granules were filtered out was similarly clarified in yeast extract-enriched PDA medium and GMR medium. The results are shown in Table 5.

[0011]

[Table 5] Bacterial flora of fermented milk kefir ─────────────────────────────────── viable cell count (/ g)% ─────────────────────────────────── Lactic acid bacteria 47 × 10 ⁷ Lactobacillus species Type I 19 40 Lactobacillus kefiranofaciens 0 0 Lactobacillus kefir 24 51 51 Lactobacillus species Type II 4 9 ─────────────────────────── ───────── Yeast 150 × 10 ³ Candida holmi 30 20 Candida kefir 120 80 ───────────────────────── ───────────

As is apparent from Table 5, 3 types of lactobacillus and 2 types of yeast were detected in fermented milk kefir, but Lactobacillus kefiranofaciens was not detected at 10 ⁷ dilution. That is, 3 types of lactobacillus and 2 yeast
It is possible to produce fermented milk kefir without using kefir granules by controlling the types so that the bacterial count ratio shown in Table 5 is obtained.

[0013]

[Action and effect] Lactobacillus species type I 1701 strain, Lactobacillus species
Type II 1731 strain, three kinds of lactic acid bacteria of Lactobacillus kefir and two kinds of yeasts of Candida holmi and Candida kefir were added to the fermentation substrate so that the ratio of the numbers of bacteria was as shown in Table 5 when fermented. If fermented, fermented milk kefir can be produced without using kefir granules. Unlike Kefir granules, these bacteria can be managed in the same manner as conventional lactic acid bacterium starters, and therefore no special equipment or the like is required to separate the Kefir granules by filtration. Hereinafter, the present invention will be described in more detail with reference to examples.

[0014]

EXAMPLES Lactobacillus species Type I 170
1 strain, 1 of Lactobacillus species type II
The 731 strain and three types of Lactobacillus kefir strains of lactic acid bacteria were used as a starter in a milk medium enriched with 0.5% of yeast extract. Two types of yeast, Candida holmi and Candida kefir, were grown in YM medium, cells were collected by centrifugation, and suspended in a milk medium to be used as a starter. These starters were treated with yeast extract at 0.
Lactic acid bacteria in the fermentation substrate reinforced by 5% are 5 in total in the ratio shown in Table 5.
%, Yeast is Candida holmi 10 × 10 ³ / m
l, Candida kefir was inoculated in the order of 40 × 10 ³ / ml and fermented at 25 ° C. for 17 hours,
It was possible to produce fermented milk kefir having the bacterial flora shown in Table 5 above.

The kefir produced above was used as a kefir starter at 1.0% (w / w), and Streptococcus lactis starter produced at a milk medium was 2.0%.
(W / w) Sterilized milk was inoculated, uniformly stirred, dispensed into a container, sealed, fermented at 25 ° C for 17 hours, and further aged and fermented at 15 ° C for 24 hours. At the same time, as a control, the kefir granules were inoculated into a 10% (w / w) milk medium, and the kefir granules were filtered off after 17 hours of culturing at 25 ° C. with regular stirring, and used as the kefir starter. Fermented under the conditions. Both of the two types of fermented milks thus produced had a yeast-fermenting odor characteristic of kefir, and no difference was observed in the sensory test.

Claims (3)

[Claims] 1. A homofermentative lactic acid bacterium belonging to the genus Lactobacillus, which accounts for the largest number of lactic acid bacteria constituting kefir granules. 2. A heterofermentative lactic acid bacterium belonging to the genus Lactobacillus that produces L (+) lactic acid that can be separated from kefir granules. 3. A method for producing fermented milk kefir without using kefir granules, using the lactic acid bacterium strain of claim 1 or 2.