JPH0856650A - New lactic acid bacterium - Google Patents

Abstract

PURPOSE: To obtain new lactic bacteria having high anti-mutagenicity. CONSTITUTION: The new lactic acid bacteria, Streptococcus faecium MC 482 (FERM P-14267), has the following mycological characteristics: (1) form: coccui; (2) Gram stain: positive; (3) mobility: none; (4) spore: none; (5) catalase: none; (6) anaerobic; (7) viable temperature range:10-50 deg.C; (8) viable pH range: 5.0-9.5; (9) lactic fermentability: homo type; (10) lactic acid's optical rotation: L-type; (11) viable with 40% bile acid; and (12) saccharide assimilability: positive for glucose, galactose, lactose, maltose, sucrose, and raffinose.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a novel lactic acid bacterium having excellent antimutagenicity against mutagenicity, a food containing the same, and a feed for livestock.

[0002]

BACKGROUND OF THE INVENTION and Prior Art Generally, there are eight mutagens in the environment.
It is considered epidemiologically that 0% is due to foods containing tobacco and the like, suggesting that many of the mutagens may be contained in the foods we ingest daily. And since such a mutagen is closely related to cancer production,
In recent years, there has been great interest.

On the other hand, it has become clear in recent years that some foods such as vegetables have an antimutagenic property to suppress a mutagen. Fermented dairy products such as yogurt have also been known to have antimutagenic properties, and attention is focused on their application to medicine and their use in the field of carcinogenesis prevention.

Lactobacillus casei, Lactobacillus helveticus, Lactobacillus lactis, Lactobacillus delbrueckii, Streptococcus faecalis, Streptococcus lactis, Streptococcus salivarius and the like are strains of lactobacillus having antimutagenic properties that suppress such mutagens. Reported (Koshi Nishioka et al .: "Journal of the Japanese Society of Animal Science" vol.60, No.5, p4
91-494, (1989)).

[0005]

However, most of these reports are about lactic acid fermented products and sour milk mainly composed of milk, and antimutagenicity of lactic acid bacteria per se against mutagenicity has not been recognized. There are doubts about the reliability of the report itself, including bacterial cells and bacterial cells with weak antimutagenicity, and there is no definitive report that satisfactory results were obtained practically.

Further, antimutagenicity cannot be said to be an activity shared by all the bacterial cells specified by the scientific name indicating the genus or species used in taxonomy of the bacteria, and even the bacterial cells belonging to the same species It has also been reported that the presence or absence of antimutagenicity varies from strain to strain and that it is considered to be specific to individual strains (Koshi Nishioka et al .: "Journal of the Animal Husbandry Society of Japan" vol.60, No.5, p491). ~ 494, (198
9)).

[0007] In the technical field where the above-mentioned current situation is widely known, the present inventors have found that individual lactic acid bacteria having antimutagenicity against mutagens, and further belonging to the same genus in terms of mycology. We have conducted extensive studies on strains that belong to the mutagen but differ in antimutagenicity.

Then, 4-nitroquinoline 1-oxide (4NQ0), 3-amino-methyl-5H-pyrido [4,4-b] -indole (Trp-P-2), which has been attracting attention recently,
Isolate and identify a novel lactic acid bacterium that has a high mutagen inhibitory effect (antimutagenicity) against artificial substances and mutagens derived from natural substances, and that also has excellent antimutagenicity against carcinogenicity. It came to.

[0009]

The present invention has been completed as the invention described in each claim of the above-mentioned claims by the above-mentioned research.

That is, the first feature of the present application is that Streptococcus faecium MC482 ( St
reptococcus Faecium MC-482) (FERM P-142
67) which is a novel lactic acid bacterium.

The present application also provides Streptococcus faecalis MC009 ( Streptococcus faecal
It is also characterized in that it provides a novel lactic acid bacterium which is is MC-009) (FERM P-14265).

Furthermore, the present application provides the Lactobacills plantarum ML319 (claim 3).
It is also characterized in that it provides a novel lactic acid bacterium which is ML-319) (FERM P-14266).

These novel lactic acid bacteria can be isolated and collected from natural or fermented foods or fermented food starters by the following screening method.

That is, an appropriate amount of a test sample was sampled, diluted in suspension in physiological saline or a medium, diluted by culture in GYP chalk agar medium, the resulting colonies were picked, and further suspended in physiological saline. Then, pure water culture of lactic acid bacteria is carried out by repeating pour culture on GYP chalk agar medium.
The lactic acid bacterium obtained was in a GYP liquid medium at 30 ° C., initial pH
Good growth is shown at 6.8.

The culture broth cultured under the above conditions for 48 hours was added to 3
The cells were obtained by centrifugation at 000 rpm for 10 minutes and washing with 1/15 M phosphate buffer (pH 6.8). The cells were 0.5 ml of 1/15 M phosphate buffer (pH 6.
8). Add 4NQO or Trp-P-2 to this suspension.
(Supra) was added. After addition Salmonella typhirium
TA98, histidine-biotin solution, and top agar were added, poured onto a minimal glucose medium, and spread evenly. After inoculation, the cells were cultured at 36 ° C. for 48 hours, and the number of reversion colonies generated was counted.

As a result, the above Streptococcus faecium MC482, Streptococcus faecalis M
Three strains of C009 and Lactobacillus plantarum ML319 were obtained as lactic acid bacteria whose mutagens have high antimutagenicity against both base substitution type mutagens and frameshift type mutagens.

The novel lactic acid bacterium of the present invention can be used not only as the bacterium itself, but also as a preparation obtained by various treatments such as isolates, extracts and purified products from the bacterium obtained as a result of various treatments. It can also be used as a thing. For example, when using the bacterium itself, it can be used by culturing the bacterium in a suitable medium and ingesting the obtained cultivated bacterium directly or by taking a means such as formulation, and the like. These (the bacterium itself or the above-mentioned preparation thereof) can be used by adding it to food or by adding it to the feed of livestock that humans eat.

The medium and culture conditions for culturing each of the above strains can be in accordance with the medium and culture conditions used for culturing ordinary lactic acid bacteria, as described below.
In particular, it shows good growth in GYP liquid medium, pH 6.8, 30 ° C., 24 hours of culture. This is due to the fact that lactic acid bacteria require various nutrients as nutrient sources, that they grow well in the neutral range, and that they grow well at 25 to 37 ° C.

The GYP liquid medium and GYP chalk agar medium are as follows.

GYP liquid medium 10 g of glucose, yeast extract 5 in 1 liter of medium
g, peptone 5g, meat extract 2g, sodium acetate 7
g, magnesium sulfate 0.2 g, manganese sulfate 0.01
g, iron sulfate 0.01 g, sodium chloride 0.01 g, Tween 80 0.025 g, adjusted to pH 6.8 with sodium hydroxide, and after adjustment 121 in an autoclave
It was sterilized by heating at ℃ for 15 minutes.

GYP chalk agar medium 1 liter of medium, glucose 10g, yeast extract 5
g, peptone 5g, meat extract 2g, sodium acetate 2
g, magnesium sulfate 0.2 g, manganese sulfate 0.01
g, iron sulfate 0.01 g, sodium chloride 0.01 g, Tween 80 0.025 g, pH was adjusted to 6.8 with sodium hydroxide, calcium carbonate 5 g and agar 12 g were added after adjustment, 121 ° C. in an autoclave, Heat sterilized for 15 minutes.

When the preparation is obtained from the bacterial cells by various treatments such as separation, extraction and purification, for example, the separation method includes mechanical disruption by ultrasonic waves or glass beads, or disruption of the bacterial cells by enzyme treatment. , After filtration by common methods,
Alternatively, it is concentrated before that to obtain the activity. Also, it is separated and purified by microfiltration, chromatography, separator, etc.

The most optimum use form of these novel lactic acid bacteria is realized by incorporating the novel lactic acid bacteria into food. The aspect contained in the food is not particularly limited as long as these lactic acid bacteria are used in a form that does not interfere with the mutagen suppressing effect on the above-mentioned mutagens.

The lactic acid bacterium, Streptococcus faecium MC482 strain of the present invention has the properties shown in Tables 1 and 2 below.

[0025]

[Table 1]

[0026]

[Table 2]

From the results of the above table, it is confirmed that the Streptococcus faecium MC482 strain is a faecium species of the genus Streptococcus. However, it is judged to be a novel strain because it has a property not generally observed in Fesium species, that is, high antimutagenicity.

The above-mentioned lactic acid bacterium, Streptococcus faecalis MC009 strain of the present invention are shown in Tables 3 and 4 below.
It has the properties shown in.

[0029]

[Table 3]

[0030]

[Table 4]

From the results in the above table, it is confirmed that Streptococcus faecalis MC009 strain is a species of Streptococcus faecalis. However, it is judged to be a novel strain because it generally has a property not found in Faecalis species, that is, high antimutagenicity.

Further, the lactic acid bacterium Lactobacillus of the present invention
Plantarum ML319 strain has the properties shown in Tables 5 and 6 below.

[0033]

[Table 5]

[0034]

[Table 6]

From the results in the above table, it is confirmed that the Lactobacillus plantarum ML319 strain is a Lactobacillus plantarum species. However, since it has a property not generally found in plantarum species, that is, high antimutagenicity, it is judged to be a novel strain.

[0036]

The above-mentioned novel lactic acid bacteria provided by the present invention exhibit high antimutagenicity as shown in the following examples, and when added to, for example, foods, exhibit excellent carcinogenic action.

[0037]

The present invention will be further described below.

Antimutagenicity test 1 1 platinum loop of the above-mentioned stock strain of Streptococcus faecium MC482 was ingested in 3 ml of GYP medium, and 30
The cells were cultured at ℃ for 2 days. After culturing, the bacterial cells were obtained by washing with 1/15 M phosphate buffer (pH 6.8) and centrifugation at 3000 rpm for 10 minutes.
Suspension I was prepared by suspending in 15 M phosphate buffer (pH 6.8).

To this suspension I was added 4-nitroquinoline 1-oxide (4NQO) (1.2 mg / m 2) as a mutagen.
The suspension II was prepared by adding l).

Suspension II contains Salmonella typhimurium TA98, a histidine-biotin solution (His-Bio solution), and 0.6% sodium chloride used in a Salmonella mutagenicity test (Ames test). 0.7% agar was added and poured onto a minimal glucose medium (MM medium) and spread evenly.

After inoculation, the cells were cultured at 36 ° C. for 48 hours, and the number of colonies generated on the plate medium by back mutation was counted.

At the same time, an example of addition of only the mutagen (4NQO), which was subjected to the same Ames test as above except that the cells were not added, suspension I (suspension without addition of mutagen (4NQO)) Example of addition of only the bacterial cells that have been subjected to the Ames test using), the same as above for the additive-free example in which the above-described Ames test has been performed using a liquid without addition of bacterial cells and mutagen (4NQO) The number of colonies formed on the plate medium was counted.

From the above measurement results, the antimutation activity was determined based on the following calculation formula, and the calculation results are shown in Table 7 below.

Antimutation activity (%) = [(B-A) / (B-
C)] × 100 where A: the number of revertant colonies when the mutagen and the bacterial cells were added B: the number of revertant colonies when the mutagen alone was added C: the revertant when only the bacterial cells were added Number of Mutant Colonies Comparative Test Example 1 For comparison, Streptococcus faecium JC was used as a standard strain of Streptococcus faecium.
M5804 (obtained from RIKEN Microorganism Strain Preservation Facility) was selected, and the same Ames test as the above antimutagenicity test 1 was performed to determine the antimutagenic activity.

The results of these calculations are shown in Table 7 below.

[0046]

[Table 7]

Antimutagenicity Tests 2, 3 The novel lactic acid bacteria used were Streptococcus faecalis MC009 and Lactobacillus plantarum ML319.
The same Ames test as the antimutagenicity test 1 was carried out except that the above was replaced with, and the antimutagenic activity was determined, and the calculation results are shown in Table 7 above.

Comparative Test Examples 2 and 3 Streptococcus faecalis JCM5803 (obtained from the microbial strain preservation facility of RIKEN) was selected as a standard strain of Streptococcus faecalis, and Lactobacillus plantarum JCM1149 was selected as a standard strain of Lactobacillus plantarum. The same Ames test as the above antimutagenicity test 1 was carried out to obtain the antimutagenic activity except that (obtained from the microbial strain storage facility of RIKEN) was selected, and the calculation results are shown in Table 7 above.

Antimutagenicity Tests 4 to 6 For each stock strain of the above Streptococcus faecium MC482, Streptococcus faecalis MC009, Lactobacillus plantarum ML319, 1 platinum loop was ingested in a separate GYP medium and cultured at 30 ° C. for 2 days. did. After culturing, bacterial cells were obtained by centrifugation at 3000 rpm for 10 minutes and washing with 1/15 M phosphate buffer (pH 6.8), and the bacterial cells were collected in 0.5 ml of S9 Mix.
To obtain suspension III.

Suspension III for each of these strains
A suspension IV was prepared by adding Trp-P-2 (20 μg / ml) as a mutagen.

Salmonella typhimurium TA98 (described above) used in the Salmonella mutagenicity test (Ames test) was added to each of the above suspensions III, and preincubated at 36 ° C. for 20 minutes. After this incubation
His-Bio solution (supra), 0.7% agar containing 0.6% sodium chloride was added to inoculate MM medium.

After inoculation, the cells were cultured at 36 ° C. for 48 hours, and the number of colonies formed on the plate medium was counted in the same manner as above.

At the same time, addition of only the mutagen (Trp-P-2), which had been subjected to the same Ames test as described above except that each bacterial cell was not added, each suspension III (mutagen (Trp-P- (2) Suspension without addition)) Example of addition of each bacterial cell that was subjected to the above-mentioned Ames test, using a non-added liquid containing neither bacterial cell nor mutagen (Trp-P-2) The number of colonies formed on the plate medium was counted in the same manner as above for each of the non-addition examples in which the Ames test was performed.

From the above measurement results, the antimutation activity for each bacterial cell was determined based on the following formula, and the calculation results are shown in Table 8 below.

Antimutation activity (%) = [(B-A) / (B-
C)] × 100 where A: the number of revertant colonies when the mutagen and the bacterial cells were added B: the number of revertant colonies when the mutagen alone was added C: the revertant when only the bacterial cells were added Number of mutant colonies

[0056]

[Table 8]

Comparative Test Examples 4 to 6 Streptococcus was prepared in the same manner as in Comparative Examples 1 to 3 above.
Streptococcus faecalis JCM5803 was selected as a standard strain of Faecalis, and Streptococcus
Streptococcus faecalis JCM5803 was selected as the standard strain of Fecalis, and Lactobacillus plantarum JCM1149 was selected as the standard strain of Lactobacillus plantarum. The antimutation activity was determined for each, and the calculation results are shown in Table 8 above.

From Tables 7 and 8 showing the results of the above-mentioned antimutagenicity tests 1 to 6 and comparative test examples 1 to 6, each of the above novel compounds provided by the present invention as different from known strains It was confirmed that the lactic acid bacterium exhibits high antimutagenicity with respect to both the base substitution type mutagen and the frameshift type mutagen.

[0059]

INDUSTRIAL APPLICABILITY Streptococcus faecium MC482, Streptococcus faecalis MC009, Lactobacillus plantarum M provided by the present invention
L319 belongs to a known bacterial species, but is a novel lactic acid bacterium strain having high antimutagenicity not found in these conventional bacterial species, and since it has high antimutagenicity, it is added to, for example, foods. As a result, it has the effect that it can be used for carcinogenesis prevention directly or indirectly by adding it to livestock feed, and has an extremely high industrial utility value.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display area // (C12N 1/20 C12R 1:46) (C12N 1/20 C12R 1:25)

Claims (5)

[Claims] 1. A novel lactic acid bacterium, Streptococcus faecium MC4, which is highly antimutagenic and has the following mycological properties.
82 (FERM P-14267). Bacterial morphology when cultured in GYP liquid medium at pH 6.8, 30 ° C for 24 hours (1) Bacterial morphology Spherical (2) Gram stain positive (3) No motility (4) No spores (5) No catalase ( 6) Facultative anaerobic (7) Growth temperature range 10 to 40 ° C (8) Growth pH range 5.0 to 9.5 (9) Lactic acid fermentation homo type (10) Optical rotation of lactic acid L type (11) 40% Can be grown with bile acids (12) Fermentability of sugars: glucose, galactose, lactose, maltose, sucrose, and raffinose are positive 2. A novel lactic acid bacterium Streptococcus faecalis MC0 having the following mycological properties and high antimutagenicity.
09 (FERM P-14265). Bacterial morphology when cultured in GYP liquid medium at pH 6.8, 30 ° C for 24 hours (1) Bacterial morphology Spherical (2) Gram stain positive (3) No motility (4) No spores (5) No catalase ( 6) Facultative anaerobic (7) Growth temperature range 15 to 45 ° C (8) Growth pH range 6.0 to 9.5 (9) Lactic acid fermentation homo type (10) Optical rotation of lactic acid L type (11) 40% Can be grown with bile acids (12) Fermentability of sugars: glucose, galactose, lactose, maltose, sucrose, and raffinose are positive 3. A novel anti-mutagenic lactic acid bacterium Lactobacillus plantarum ML31 having the following mycological properties.
9 (FERM P-14266). Bacterial morphology when cultured in GYP liquid medium at pH 6.8 at 30 ° C for 24 hours (1) Gram stain positive (2) Bacterial morphology Rod-like (3) No motility (4) No spores (5) No catalase ( 6) Facultative anaerobic (7) Growth temperature range 10 to 40 ° C (8) Growth pH range 4.0 to 8.5 (9) Lactic acid fermentation homo type (10) Optical rotation of lactic acid DL type (11) 40% Can be grown with bile acids (12) Fermentability of sugars: glucose, galactose, lactose, maltose, sucrose, and raffinose are positive 4. A food containing the novel lactic acid bacterium according to any one of claims 1 to 3. 5. A livestock feed containing the novel lactic acid bacterium according to any one of claims 1 to 3.