JPH11285378A - Novel bacillus subtilis having antimicrobial function - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a novel Bacillus subtilis species, composed of Bacillus subtilis species having ability of utilizing lactose and amygdalin but no ability of utilizing inulin, and useful for, e.g. infection prophylactic therapeutic agents, immunopotentiator, antidiarrheal agent, antiflatulent and growth promotor, etc. SOLUTION: This novel Bacillus subtilis species, e.g. Bacillus subtilis AS2 stock (FERMOBP-6139), has ability of utilizing lactose and amygdalin but no ability of utilizing inulin, showing antimicrobial functions, and useful for, e.g. infection prophylactic/therapeutic agents, immunopotentiator, antidiarrheal agent, antiflatulent, dejection deodorant, growth promotor, side-effect reducing agents for administration of anti-cancer agents. Bacillus subtilis AS2 stock is found, after microorganisms of strong antimicrobial activity against a variety of pathogenic microorganisms have been searched for, to have antimicrobial functions against various bacteria, e.g. Salmonella, Escherichia coil staphylococcus, Vibrio parahaemolyticus, campylobacter, botulinus bacillus and Welch bacillus.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an antibacterial agent comprising a novel Bacillus subtilis strain as an active ingredient. More specifically, Bacillus subtilis (Bacillus subtilis)
The present invention relates to an antibacterial agent comprising an AS2 strain, which is one of the strains described above.

[0002]

2. Description of the Related Art It has long been known that antibiotics such as antibacterial agents and antifungal agents are effective for prevention and treatment of various infectious diseases, and they have been widely used in clinical settings until now. These antibiotics can be obtained by extracting those produced by bacteria or fungi, or by chemically synthesizing them.
However, repeated use of antibiotics of the same strain for a long period of time causes the bacteria to acquire resistance, and the effects of administration of the drug become ineffective, and the emergence of so-called resistant bacteria has become a serious social problem. ing. Attempts have been made to find antibacterial agents that exist in nature, do not adversely affect humans and animals, and show antibacterial activity only against harmful bacteria, instead of antibiotics consisting of a single chemical substance. . Some microorganisms secrete substances that kill different types of bacteria around you. Penicillium notatum (Penicillium notatum) and Penicillin chrysogenum (P. chrysogenum), which produce penicillin, are good examples. However, penicillin exhibits bacteriostatic and bactericidal effects against Gram-positive bacteria, and thus has a problem in selectivity. Another problem is that a wide range of bacteria acquire resistance to penicillin. Bacteria such as Bacillus subtilis and Bacillus licheniformis are known as microorganisms that produce substances having a selective spectrum. Ducluseau and colleagues have shown that Bacillus licheniformis has been working on Clostridium perfringens.
m. perfringens (Ducluzeau, R., et al., Ant)
imicrobial agents and Chemotherapy, 9 , 20-25,197
6). In addition, Japanese Patent Publication No. 8-18996 discloses that Bacillus subtilis IFO-3009, IFO-3013, IFO-3335, IFO-3336,
It is disclosed that administration of a probiotic agent including the IFO-3936 strain and the IFO-13169 strain to humans has an effect of reducing the number of Clostridium perfringens bacteria in feces. Further, Japanese Patent Application Laid-Open No. 1-502340 discloses that Bacillus subtilis TSMPM No. V-2335 is responsive to Bacillus licheniformis against Pseudomonas aeruginosa, Shigella sonei, Salmonella, Klebsiella pneumonia, Proteus vulgaris.・ TSMPM
No. V-2336 is described as having an antagonistic effect on Staphylococcus aureus, Candida albican, and Aspergillus flavus, and is effective for the prophylactic treatment of gastrointestinal disease in domestic animals.

[0003]

However, the bacteria that are pathogenic to humans and animals are not limited to these bacteria. Serotype O15, which can be lethal in humans
7: Pathogenic Escherichia coli including H7, Vibrio spp., Shigella spp., Enterococcus spp., Campylobacter spp. Which cause food poisoning. Salmonella spp., Cattle and sheep, which frequently infect chickens and pigs in livestock. , A large number of bacteria, such as Campylobacter, which causes abortion in pigs, Staphylococcus, which causes bovine mastitis,
Livestock products such as eggs and meat, feed, barn, soil, etc.
It is the cause of infection. Therefore, the development of a novel bacterial strain that exhibits stronger antibacterial activity against various pathogenic bacteria than conventionally known bacterial strains of the genus Bacillus, and that does not act on useful bacteria such as lactic acid bacteria and natto bacteria. I have been waiting.

[0004]

Means for Solving the Problems The inventors of the present invention have made intensive studies based on these backgrounds for many years, and as a result, the AS2 strain, a kind of Bacillus subtilis, has been transformed into Salmonella, Escherichia coli, It shows stronger antibacterial activity against various harmful bacteria such as staphylococci, Vibrio parahaemolyticus, Campylobacter, Clostridium botulinum, and C. perfringens than conventionally known bacterial strains, and useful bacteria such as lactic acid bacteria and natto bacteria. Did not act on the present invention, and completed the present invention. That is, the present invention relates to 1) having lactose and amygdalin utilization ability, not having inulin utilization ability, having a negative orthonitrophenol-β-D-galactopyranoside test, or growing at 50 ° C. Bacillus subtilis AS2 strain, characterized by the absence of a deposit, FERM BP
-6139, 2) 16S ribosome R shown in SEQ ID NO: 1
Bacillus subtilis AS2 having NA base sequence
Strain (Bacillus subtilis AS2), accession number FERM B
P-6139, 3) an antibacterial agent comprising a living bacterium, a dead bacterium or a cell component of the AS2 strain as an active ingredient; 4) a food containing the antibacterial agent; 5)
Feed for livestock or farmed fish containing the antibacterial agent, 6) a preventive / therapeutic agent for infectious diseases containing AS2 strain, 7) an antidiarrheal and intestinal medicine containing AS2 strain, 8) a fecal deodorant containing AS2 strain, 9) AS2 And 10) a side effect reducing agent when an anticancer drug containing AS2 strain is administered. The Bacillus subtilis AS2 strain was transferred to the Biotechnology and Industrial Technology Research Institute (FERM) of the Agency for Industrial Science and Technology.
Deposited on October 9, 1997 and its deposit number is FERM
BP-6139. In the present specification, Bacillus subtilis AS2 strain may be abbreviated as Bacillus subtilis AS2 strain or simply AS2 strain.

[0005] Bacillus subtilis AS according to the present invention
The two strains have the following properties. (1) Gram-positive, aerobic bacilli, (2) oval spore formation, (3) motility, (4) catalase-positive, (5) VP reaction: +, (6) Growth at 50 ° C .: -, (7) Growth in medium supplemented with 7% sodium chloride: +, (8) Starch hydrolysis: +, (9) Use of sabouraud dextrose: +, (10) E
gg yolk: +, (11) casein hydrolysis: +, (12) citrate utilization: +, (13) nitrate reduction: +, (14) hippurate reduction: +, (15) tyrosine Uses: +, (16) gas production from glucose, (17) o-nitrophenol-β-D-
Galactopyranoside:-, (18) ADH:-, (19) LD
C:-, (20) ODC:-, (21) Utilization of citrate of Simon:-, (22) Utilization of citrate of Christiansen:
+, (23) hydrogen sulfide production:-, (24) urease production:-,
(25) TDA:-, (26) Indole production:-, (27) Gelatin hydrolysis: +, (28) Film formation in liquid medium: +,
(29) Coagulation of milk:-, (30) Peptone conversion of milk: +, (31)
Growth at pH 5.7: +, acid production from (32) sugars: erythritol, L-arabinose, ribose, D-xylose, D-glucose, D-fructose, D-mannose, cellobiose, maltose, lactose, melibiose, saccharose Trehalose, D-raffinose, glycogen, β-gentiobiose, inositol, mannitol, sorbitol, α-methyl-D-glucoside, amygdalin, arbutin, eesculin,
Salicin, D-Turanose: +, glycerol, D-
Arabinose, L-xylose, adonitol, β-methylxyloside, galactose, inulin, melezitose, xylitol, L-sorbose, rhamnose, dulcitol, α-methyl-D-mannoside, N-acetylglucosamine, D-lyxose, D-tagatose , D
-Fucose, L-fucose, D-arabitol, L-arabitol, gluconate, 2-ketogluconate, 5
-Ketogluconate:-. Among these properties, o-nitrophenol-β-D-galactopyranoside which does not grow at 50 ° C .: has the ability to utilize lactose and amygdalin, but does not have the ability to utilize inulin.
It is a new strain with different properties from common Bacillus subtilis. The AS2 strain has the nucleotide sequence shown in SEQ ID NO: 1 as 16S ribosomal RNA. This nucleic acid sequence may be partially substituted, deleted or added within a range that does not substantially change its properties.

[0006] Bacillus subtilis AS2 strains exhibit the antibacterial activity as a kind of bacterium including Salmonella typhimurium.
la typhimurium, Salmonella typhi (S. typh)
i), Salmonella paratyphi, S. cholerasuis, S. enteritidis, S. gallinarum, S. abortusequi , Salmonella abortusovis (S. abortusovis), Salmonella Dublin (S.
dublin), Salmonella, toxinogenicity, tissue invasiveness,
Escherichia coli including pathogenic Escherichia coli such as pathogenic serotype, enterohemorrhagic, and intestinal adherence,
Shigella sonnei, S. dysenteriae, Shigella flexneri
flexneri), Shigella genus such as S. boydii, Serratia marcescen
s), Serratia genus such as S. rubidaea, Vibriocholerae, Vibrio genus such as V. parahaemolyticus, Staphylococcus aureus (Staphylo)
staphylococci such as C. coccus aureus, S. epidermidis, and Clostridium perfingen.
s), Clostridium shobei (C. chauvoei), Clostridium botulinum (C. botulinum), Clostridium tetani (C. tetani), Clostridium genus such as C. septicum, Campylobacter fetus (Campylobacter)
fetus), Campylobacter jejuni (C. jejun)
i), Campylobacter sputrum (C. sputoru)
m), Campylobacter mucosalis (C. mucosali)
s), Campylobacter faecalis (C. faecali)
s), Yersinia pestis, Yersinia pestis, Y. pseudotuberculosis, Y. enterocolitica, Y. enterocolitica, Enterobactor c
loacae), Citrobacter freundii etc., Proteus, Bacteroides (B
acteroides), Fusobacterium
And the like. In particular, methicillin-resistant staphylococci (Me
ticilin resistant Staphylococcus aureus, MRS
A), macrolide-resistant Enterococcus seriolicida, E. coli serotype O2
6, O111, O157, etc. have the feature of exhibiting antibacterial activity.

On the other hand, this strain is a genus of Bifidobacterium such as Bifidobacterium longum, Bifidobacterium bifidum, B. breve, or the like; Lactobacillus bulgarix (Lactobacillus bu
lgaricus), Lactobacillus acidophilus (L. aci)
dphilus), Streptococcus thermophilus, etc.
It has no antibacterial activity against useful bacteria such as a group of so-called lactic acid bacteria and a genus Bacillus such as Bacillus subtilis.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION The method of administering the antibacterial agent according to the present invention includes oral administration, or a method of ingesting it in a food, feed, drinking water, etc., depending on the purpose of administration and conditions such as symptoms. In humans and animals, it is in the form of a prophylactic agent when administered when bacterial infection is not established or after healing, and as a therapeutic agent when administered after infection and during onset. The dose varies depending on the purpose, the target animal, the type of the disease, and the state of the disease. In the case of oral administration, the number of bacteria per dose is 1 × 10 ² to 10 ¹³ ,
Preferably 1 × 10 ⁵ to 10 ¹¹ , more preferably 1 × 10 ⁷
~ 10 ⁹ pieces.

The antibacterial agent according to the present invention may be administered as live bacteria or in a dead state. Further, even if the cells are decomposed by freezing, heating, drying, exposure to a drug, or the like, the components may be retained as long as the components are retained. In addition, powders, tablets, granules, capsules, and liquid preparations can be prepared by known methods, and can be mixed with foods, feeds, drinking water, and the like, depending on commonly used carriers for preparations.
When preparing an oral preparation, after adding excipients, binders, binders, disintegrants, lubricants, coloring agents, flavoring agents, antioxidants, solubilizing agents, etc. to the main drug, Tablets in the usual way,
Coated tablets, granules, powders, capsules, etc. As the excipients, starch, corn starch, dextrin, yeast extract powder, flour, wheat middling, bran, rice bran, rice bran oil cake, soybean meal, soybean powder, soybean oil cake, kinako, glucose, lactose, sucrose, maltose, Vegetable oil, animal oil, hydrogenated oil, higher saturated fatty acid, fatty acid, mannitol, crystalline cellulose, silicon dioxide, anhydrous silicon, calcium silicate, silicic acid, calcium monohydrogen phosphate, dibasic calcium phosphate, tricalcium phosphate, calcium phosphate, dibasic phosphate Calcium hydrogen or the like is used. As a binder, polyvinylpyrrolidone, ethyl cellulose,
Methylcellulose, gum arabic, tragacanth, hydroxypropylcellulose, hydroxypropylmethylcellulose, sodium alginate, sodium caseinate, sodium carboxymethylcellulose, propylene glycol, sodium polyacrylate and the like are used. As the lubricant, magnesium stearate, talc, stearic acid and the like are used. The coloring agent and flavoring agent are not particularly limited as long as they are permitted to be added to pharmaceuticals and feeds. As antioxidants, ascorbic acid, α-tocopherol, ethoxyquin,
Any substance such as dibutylhydroxytoluene and butylhydroxyanisole that is permitted to be added to pharmaceuticals and feeds may be used. Tablets and granules may be coated as needed. In the case of preparing a liquid preparation, a pH adjuster, a buffer, a suspending agent, a solubilizer, a stabilizer, a tonicity agent, an antioxidant, a preservative, etc. are added to the main ingredient as necessary, It can be manufactured by an ordinary method. At this time, if necessary, a freeze-dried agent can be used.

Bacillus subtilis AS according to the present invention
Culture of the two strains may be performed by a commonly used method. For example, at a culture temperature of 10 to 45 ° C and a pH of 5 to 8 under aerobic conditions,
Agar medium, blood agar medium, heart infusion medium,
It can be easily cultured in a medium such as a tryptosome medium, a Sabouraud agar medium, or a brain heart infusion liquid medium.

[0011]

Next, the present invention will be described with reference to examples, but the present invention is not limited to these examples. Example 1 Antibacterial activity of AS2 strain on general bacteria The presence or absence of the antibacterial activity of Bacillus subtilis AS2 strain on general bacteria derived from livestock was examined by cross streaking inoculation. First, the general bacteria shown in Table 1 were linearly inoculated on a Muller Hinton agar medium, and the AS2 strain was linearly inoculated so as to cross each other, and cultured at 37 ° C. for 24 hours. Thereafter, the growth state of general bacteria at the intersection was observed. The results are shown in Table 1.

[0012]

[Table 1]

[0013] From the above results, it is clear that the Bacillus
The S. subtilis AS2 strain has an antibacterial activity against bacteria such as Salmonella, Escherichia, Enterobacter, Serratia, Proteus, Staphylococcus, Enterococcus, Clostridium, and the like, such as Bifidobacterium and Bacillus natto. It has been found that it does not act on useful bacteria.

Example 2 Antibacterial activity of AS2 strain against bacteria associated with food poisoning Enterohemorrhagic Escherichia coli of Bacillus subtilis AS2 strain,
The presence or absence of antibacterial activity against Staphylococcus aureus, Bacillus cereus, Vibrio parahaemolyticus, and Yersinia enterocolitica was examined by cross streaking. First, the bacteria shown in Table 2 were linearly inoculated on a Muller Hinton agar medium, and the AS2 strain was linearly inoculated so as to cross each other, and cultured at 37 ° C for 24 hours. Thereafter, the growth state of general bacteria at the intersection was observed. The results are shown in Table 2.

[0015]

[Table 2]

From the above results, it is clear that the Bacillus strain of the present invention
It was revealed that the subtilis AS2 strain has an antibacterial activity against various food poisoning-related bacteria such as enterohemorrhagic Escherichia coli and Vibrio parahaemolyticus.

Example 3 Bactericidal action of AS2 strain on Escherichia coli The bactericidal action of Bacillus subtilis AS2 strain on Escherichia coli E01292 strain was examined. As a control, Bacillus subtilis natto strain which is a commercially available natto fungus was used. Escherichia coli was pre-cultured at 37 ° C. for 18 hours in a Brain Heart Infusion (hereinafter referred to as “BHI”) broth medium (manufactured by BBL), and inoculated individually or together with the AS2 strain into 30 ml of BHI medium in a 100 ml flask. The cells were shake-cultured in a water bath. Before the start of the mixed culture, samples were taken after 1, 2 and 3 days, and a 10-fold serial dilution series was prepared with physiological saline, 0.1 ml of which was spread on an agar medium and cultured at 37 ° C for 24 hours to determine the number of viable cells. Measured. For separation of Escherichia coli, MacConkey agar medium (manufactured by BBL) was used, and for isolation of Bacillus subtilis, Sabouraud agar medium (manufactured by Nissui Pharmaceutical) was used. The results are shown in FIGS. The number of Escherichia coli mixed and cultured with the AS2 strain decreased from one day after the start of the culture, whereas the number of Escherichia coli mixed and cultured with Bacillus natto did not change.

Example 4 Bactericidal Action of AS2 Strain on Escherichia coli and Salmonella The bactericidal action of Bacillus subtilis AS2 strain on Escherichia coli NIHJ and Salmonella enteritidis ATCC14028 strain was examined. As controls, Bacillus subtilis IFO-3009, a common natto, and PCI219, a common Bacillus subtilis, were used. Escherichia coli and Salmonella were grown at 37 ° C. for 18 hours using a BHI broth medium (BBL).
The cells were pre-cultured for an hour, inoculated individually or together with the AS2 strain into 30 ml of BHI medium in a 100 ml flask, and cultured with shaking in a 37 ° C. water bath. Before the start of culture, one day and three days after the start of sampling, serial 10-fold dilution series were prepared with physiological saline, 0.1 ml of which was spread on an agar medium, cultured at 37 ° C. for 24 hours, and the number of viable bacteria was counted. . MacConkey agar medium (manufactured by BBL) was used for separation of Escherichia coli and Salmonella, and Sabouraud agar medium (manufactured by Nissui Pharmaceutical) was used for separation of Bacillus subtilis. The results are shown in FIG. 3 and FIG.
E. coli or Salmonella is IFO-3009 strain or PCI219
In contrast to the increase in the case of mixed culture with
When the strain was mixed with the strain, the number was clearly decreased. From the above results, it was considered that the bactericidal action against harmful bacteria such as Escherichia coli and Salmonella is not common to Bacillus subtilis in general but is specific to the AS2 strain.

Example 5 Intestinal Adherence upon Ingestion of the AS2 Strain Artificially Inoculated into B. subtilis at 10 ⁸ CFU / ml (suspended in 0.05 ml of physiological saline) in 25 mice (SLC: ICR).・ 5 AS2 strains
Oral administration for one day. On the first, second, third, fifth and seventh days, the mice were sacrificed by over-anaesthesia with pentobarbital sodium on days 1, 2, 3, 5 and 7, and the intestinal contents were collected by necropsy. When the contents were cultured on a plate agar medium for 24 hours, the AS2 strain was detected on all collection days. That is, it was revealed that the AS2 strain had intestinal colonization.

Example 6 Comparison of antibacterial activity between AS2 strain and other Bacillus subtilis strains Bacillus subtilis AS2 strain, IFO-3009 strain, IFO-30
13, IFO-3336, IFO-3936, IFO-13169 and PCI
The antimicrobial activity of 219 strains (ATCC6633) against Clostridium perfringens NCTC3182 Type C was compared.
The above Clostridium was streaked on a Mueller Hinton agar medium (manufactured by BBL) and cross-inoculated with each strain of Bacillus subtilis. 37 with gas pack (BBL)
After culturing at 24 ° C. for 24 hours, a zone of inhibition at the intersection was observed. As a result, the AS2 strain strongly inhibited the growth of Clostridium perfringens, whereas the other strains did not show any growth effect (Table 3).

[0021]

[Table 3]

Example 7 Protective Effect of AS2 Strain on Intravenous Escherichia coli Escherichia coli AS2 strain 10 ⁷ , 10 ⁸ or 10 ⁹ suspended in physiological saline in 10 male mice (SLC: ICR, 5 weeks old) in each group 3 CFU / 0.5ml
Oral administration for consecutive days, human-derived E. coli E01292 strain suspended in physiological saline 24 hours after the final administration 4.0 × 10 ⁷ CFU / 0.2 ml
Was administered intravenously. The survival rate of the mice 4 days after the inoculation was calculated to determine the protective effect. As a result, by prophylactically administering the AS2 strain, the infection resistance of the mice to E. coli infection was enhanced, and a significant increase in the survival rate was observed as compared with the control group. That is, it was suggested that the AS2 strain exhibited an immunopotentiating effect (Table 4).

[0023]

[Table 4]

Example 8 Protective Effect of AS2 Strain on Intravenous Staphylococcus aureus In a group of 10 male mice (SLC: ICR, 5 weeks old), each of AS2 strains 10 ⁷ , 10 ⁸ or 10 suspended in a physiological saline solution 10 ⁹ CFU / 0.5ml 6
Bovine mastitis-derived Staphylococcus aureus OB-72 strain 6.5 × 10 ⁷ CF suspended orally in physiological saline on day 4 of oral administration for 4 consecutive days
U / 0.2 ml was administered intravenously. The survival rate of the mice 7 days after the final administration of the AS2 strain was calculated, and the protective effect was determined (Table 5).

[0025]

[Table 5]

Example 9 Protective effect of AS2 strain against subcutaneous infection of Aujeszky virus AS2 strains 10 ⁷ , 10 ⁸ or 10 were suspended in physiological saline in 10 male mice (SLC: ICR, 5 weeks old) in each group. ⁹ CFU / 0.5ml 6
Oral administration was performed continuously for one day, and on day 4 of administration, subcutaneous administration of 133 PFU / 0.2 ml of swine-derived Auessky virus suspended in physiological saline was performed. The survival rate of the mice 7 days after inoculation with the virus solution was calculated to determine the protective effect (Table 6).

[0027]

[Table 6]

[0028] Example 10 AS2 strain lipopolysaccharide (LPS) Protective Effect group each 10 examples of male mice to intravenous inoculation (SLC: ICR, 5 weeks old) in AS2 strain ¹⁰⁷ suspended in physiological saline, Oral administration of 10 ⁸ or 10 ⁹ CFU / 0.5 ml was performed, 24 hours later, 50 mg / kg of LPS derived from E. coli was administered into the tail vein, and 6 hours later, the same dose of the AS2 strain was orally administered. The survival rate of the mice 4 days after inoculation of the LPS solution was calculated to determine the protective effect (Table 7).

[0029]

[Table 7]

Example 11 Antidiarrheal activity of AS2 strain The effect of Bacillus subtilis AS2 strain on a mouse LPS diarrhea generation model was examined. 1.0 × 10 ⁹ CFU / 0.5 ml of AS2 strain was added to 20 6-week-old male mice (SLC: ICR).
Oral administration was continued for one day, and one day later, LPS 10 mg / kg was intravenously administered. The onset time of the onset of diarrhea
Fecal excretion up to 3 hours after S administration was measured for each cage. The control group received 0.5 ml of sterilized water instead of the AS2 strain. Table 8 shows the results.

[0031]

[Table 8]

Example 12 Deodorant effect of AS2 strain on chicken manure 10 g of chicken manure was supplemented with 2 × 10 ¹¹ CFU / g of AS2 strain at 0.05 and 0.1%, respectively.
Add 5, 5, 50 mg (10 ⁶ , 10 ⁷ , 10 ⁸ , 10 ⁹ CFU /
(equivalent to g chicken dung)) was stored at room temperature, and the deodorizing effect was evaluated after 2, 3, 4 and 7 days. According to the sensory test, ◎: marked effect (well deodorized or no discomfort), ○: effective (deodorizing effect), △: somewhat effective (odor remains but discomfort is improved), X: Ineffective (no deodorizing effect or uncomfortable even with deodorizing effect) was evaluated on a four-point scale. A no-addition group was provided as a negative control group. As a result, the AS2 strain showed a deodorizing effect on chicken dung (Table 9).

[0033]

[Table 9]

Example 13 Growth-Promoting Activity of AS2 Strain Five male mice (SLC: ICR, 3 weeks old, body weight 10-12) per group
g), 3.125 × 10 ⁴ , 1.25 × 10 ⁵ , 5 × 10 ⁵ or 2 × 10 ⁶ C
The mice were bred for 28 days with a mouse feed (trade name: MF, Oriental Yeast Co., Ltd.) to which the AS2 strain was added at a concentration of FU / g, and the weight gain and weight gain were calculated. As a result, in the group to which the AS2 strain was added, the body weight gain effect was observed in a dose-dependent manner as compared with the control group (Table 10).

[0035]

[Table 10]

Example 14 Protective Effect of AS2 Strain on Fluorouracil (5-FU) Immunosuppression A group of 5 male mice (SLC: ICR, 5 weeks old) was suspended in physiological saline at a concentration of 10 ⁹ CFU / 0.5 ml was orally administered for 9 consecutive days. On the fourth day of administration, 5-FU 500 mg / kg (fluorouracil injection) was also orally administered. The survival rate of mice 18 days after 5-FU administration was calculated (Table 11).

[0037]

[Table 11]

Example 15 Analysis of Genetic Properties of AS2 Strain The nucleotide sequence of Ribosomal-RNA 16S was changed to Microsec 16Sr R
Analysis was performed using NA Gene kit (trade name, manufactured by Applied Biosystems). In other words, after collecting and lysing the colonies on the plate of each strain, a template is prepared and amplified by PCR,
ABI plasma 310 Genetic analyzer (product name, Perkin
(Made by Elmer). As a result, the AS2 strain (SEQ ID NO: 1) and the NC.
The nucleotide sequence of the DO1769 strain (SEQ ID NO: 2) was found to differ at four locations.

[0039]

[Sequence list] SEQ ID NO: 1 Sequence length: 1455 Sequence type: Nucleic acid Number of strands: Single-stranded Topology: Linear Sequence type: Genomic RNA Sequence: GACGAACGCT GGCGGCGCGTGC CTAATACATG CAAGTCGAGC GGACAGATGG GAGCTTGCTC 60 CCTGATGTTA GCGGCGGAAC GGTGAGTAACGT CCTGCCTGTA AGACTGGGAT 120 AACTCCGGGA AACCGGGGCT AATACCGGAT GGTTGTCTGA ACCGCATGGT TCAGACATAA 180 AAGGTGGCTT CGGCTACCAC TTACAGATGG ACCCGCGGCG CATTAGCTAG TTGGTGAGGT 240 AACGGCTCAC CAAGGCGACG ATGCGTAGCC GACCTGAGAG GGTGATCGGC CACACTGGGA 300 CTGAGACACG GCCCAGACTC CTACGGGAGG CAGCAGTAGG GAATCTTCCG CAATGGACGA 360 AAGTCTGACG GAGCAACGCC GCGTGAGTGA TGAAGGTTTT CGGATCGTAA AGCTCTGTTG 420 TTAGGGAAGA ACAAGTGCCG TTCAAATAGG GCGGCACCTT GACGGTACCT AACCAGAAAG 480 CCACGGCTAA CTACGTGCCA GCAGCCGCGG TAATACGTAG GTGGCAAGCG TTGTCCGGAA 540 TTATTGGGCG TAAAGGGCTC GCAGGCGGTT TCTTAAGTCT GATGTGAAAG CCCCCGGCTC 600 AACCGGGGAG GGTCATTGGA AACTGGGGAA CTTGAGTGCA GAAGAGGAGA GTGGAATTCC 660 ACGTGTAGCG GTGAAATGCG TAGAGATGTG GAGGAACACC AGTGGCGA G GCGACTCTCT 720 GGTCTGTAAC TGACGCTGAG GAGCGAAAGC GTGGGGAGCG AACAGGATTA GATACCCTGG 780 TAGTCCACGC CGTAAACGAT GAGTGCTAAG TGTTAGGGGG TTTCCGCCCC TTAGTGCTGC 840 AGCTAACGCA TTAAGCACTC CGCCTGGGGA GTACGGTCGC AAGACTGAAA CTCAAAGGAA 900 TTGACGGGGG CCCGCACAAG CGGTGGAGCA TGTGGTTTAA TTCGAAGCAA CGCGAAGAAC 960 CTTACCAGGT CTTGACATCC TCTGACAATC CTAGAGATAG GACGTCCCCT TCGGGGGCAG 1020 AGTGACAGGT GGTGCATGGT TGTCGTCAGC TCGTGTCGTG AGATGTTGGG TTAAGTCCCG 1080 CAACGAGCGC AACCCTTGAT CTTAGTTGCC AGCATTCAGT TGGGCACTCT AAGGTGACTG 1140 CCGGTGACAA ACCGGAGGAA GGTGGGGATG ACGTCAAATC ATCATGCCCC TTATGACCTG 1200 GGCTACACAC GTGCTACAAT GGACAGAACA AAGGGCAGCG AAACCGCGAG GTTAAGCCAA 1260 TCCCACAAAT CTGTTCTCAG TTCGGATCGC AGTCTGCAAC TCGACTGCGT GAAGCTGGAA 1320 TCGCTAGTAA TCGCGGATCA GCATGCCGCG GTGAATACGT TCCCGGGCCT TGTACACACC 1380 GCCCGTCACA CCACGAGAGT TTGTAACACC CGAAGTCGGT GAGGTAACCT TTATGGAGCC 1440 AGCCGCCGAA GGTGG 1455

SEQ ID NO: 2 Sequence length: 1553 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: Genomic RNA Sequence: TTTATCGGAG AGTTTGATCC TGGCTCAGGA CGAACGCTGG CGGCGTGCCT AATACATGCA 60 AGTCGAGCGG ACAGATGGGA GCTTGCTCCC GG TGAGTAACAC 120 GTGGGTAACC TGCCTGTAAG ACTGGGATAA CTCCGGGAAA CCGGGGCTAA TACCGGATGG 180 TTGTTTGAAC CGCATGGTTC AAACATAAAA GGTGGCTTCG GCTACCACTT ACAGATGGAC 240 CCGCGGCGCA TTAGCTAGTT GGTGAGGTAA CGGCTCACCA AGGCAACGAT GCGTAGCCGA 300 CCTGAGAGGG TGATCGGCCA CACTGGGACT GAGACACGGC CCAGACTCCT ACGGGAGGCA 360 GCAGTAGGGA ATCTTCCGCA ATGGACGAAA GTCTGACGGA GCAACGCCGC GTGAGTGATG 420 AAGGTTTTCG GATCGTAAAG CTCTGTTGTT AGGGAAGAAC AAGTACCGTT CAAATAGGGC 480 GGCACCTTGA CGGTACCTAA CCAGAAAGCC ACGGCTAACT ACGTGCCAGC AGCCGCGGTA 540 ATACGTAGGT GGCAAGCGTT GTCCGGAATT ATTGGGCGTA AAGGGCTCGC AGGCGGTTTC 600 TTAAGTCTGA TGTGAAAGCC CCCGGCTCAA CCGGGGAGGG TCATTGGAAA CTGGGGAACT 660 TGAGTGCAGA AGAGGAGAGT GGAATTCCAC GTGTAGCGGT GAAATG CGTA GAGATGTGGA 720 GGAACACCAG TGGCGAAGGC GACTCTCTGG TCTGTAACTG ACGCTGAGGA GCGAAAGCGT 780 GGGGAGCGAA CAGGATTAGA TACCCTGGTA GTCCACGCCG TAAACGATGA GTGCTAAGTG 840 TTAGGGGGTT TCCGCCCCTT AGTGCTGCAG CTAACGCATT AAGCACTCCG CCTGGGGAGT 900 ACGGTCGCAA GACTGAAACT CAAAGGAATT GACGGGGGCC CGCACAAGCG GTGGAGCATG 960 TGGTTTAATT CGAAGCAACG CGAAGAACCT TACCAGGTCT TGACATCCTC TGACAATCCT 1020 AGAGATAGGA CGTCCCCTTC GGGGGCAGAG TGACAGGTGG TGCATGGTTG TCGTCAGCTC 1080 GTGTCGTGAG ATGTTGGGTT AAGTCCCGCA ACGAGCGCAA CCCTGGATCT TAGTTGCCAG 1140 CATTCAGTTG GGCACTCTAA GGTGACTGCC GGTGACAAAC CGGAGGAAGG TGGGGATGAC 1200 GTCAAATCAT CATGCCCCTT ATGACCTGGG CTACACACGT GCTACAATGG ACAGAACAAA 1260 GGGCAGCGAA ACCGCGAGGT TAAGCCAATC CCACAAATCT GTTCTCAGTT CGGATCGCAG 1320 TCTGCAACTC GACTGCGTGA AGCTGGAATC GCTAGTAATC GCGGATCAGC ATGCCGCGGT 1380 GAATACGTTC CCGGGCCTTG TACACACCGC CCGTCACACC ACGAGAGTTT GTAACACCCG 1440 AAGTCGGTGA GGTAACCTTT TAGGAGCCAG CCGCCGAAGG TGGGACAGAT GATTGGGGTG 1500 AAGTCGTAAC AAGGTAGCCG TATCGGAAGG TGCGGCTGGA TCACCTCCTT TCT Fifteen 53

[0041]

[Brief description of the drawings]

FIG. 1 is a view showing the action of Bacillus subtilis AS2 strain on Escherichia coli.

FIG. 2 shows the action of Bacillus subtilis natto strain on Escherichia coli.

Fig. 3 Bacillus subtilis AS2 strain, IFO-3009
FIG. 7 is a view showing the bactericidal action of Escherichia coli strain PCI219 on Escherichia coli.

Fig. 4 Bacillus subtilis AS2 strain, IFO-3009
FIG. 4 is a view showing the bactericidal action of the strain PCI219 against Salmonella.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code FI A61K 31/00 631 A61K 31/00 631 631A 637 637C 643 643D 35/74 35/74 A C12N 15/09 ZNA C12N 15/00 ZNAA // (C12N 1/20 C12R 1: 125) (C12N 15/09 ZNA C12R 1: 125)

Claims (16)

[Claims] 1. A Bacillus subtilis having lactose and amygdalin utilization and not having inulin utilization. 2. A Bacillus subtilis characterized by having a negative orthonitrophenol-β-D-galactopyranoside test. 3. A Bacillus subtilis that does not grow at 50 ° C. 4. The Bacillus subtilis according to any one of claims 1 to 3, wherein the Bacillus subtilis is Bacillus subtilis AS2 strain, accession number FERM BP-6139. 5. The 16S ribosomal RN represented by SEQ ID NO: 1.
Bacillus subtilis AS2 strain having the nucleotide sequence of A, accession number FERM BP
-6139. 6. An antibacterial agent comprising a live bacterium, a dead bacterium or a cell component of Bacillus subtilis according to any one of claims 1 to 5 as an active ingredient. 7. The antibacterial agent according to claim 6, comprising an excipient that enhances the action. 8. The antibacterial agent according to claim 6, comprising yeast as an excipient for enhancing the action. 9. A food containing the antibacterial agent according to claim 6. 10. A feed for livestock or cultured fish containing the antibacterial agent according to claim 6. (11) An agent for preventing or treating infectious diseases comprising a live bacterium, a dead bacterium or a cell component of Bacillus subtilis according to any one of (1) to (5) as an active ingredient 12. An immunopotentiator comprising a live bacterium, a dead bacterium or a cell component of Bacillus subtilis according to any one of claims 1 to 5 as an active ingredient. 13. An antidiarrheal and intestinal preparation comprising a live bacterium, a killed bacterium or a cell component of Bacillus subtilis according to any one of claims 1 to 5 as an active ingredient. (14) A fecal deodorant comprising a live bacterium, a killed bacterium or a cell component of Bacillus subtilis according to any one of (1) to (5) as an active ingredient. 15. A growth promoting agent comprising a live bacterium, a killed bacterium or a cell component of Bacillus subtilis according to any one of claims 1 to 5 as an active ingredient. 16. An agent for reducing side effects upon administration of an anticancer agent comprising a live bacterium, a killed bacterium or a cell component of Bacillus subtilis according to any one of claims 1 to 5 as an active ingredient.