Biological intestinal antiseptics

Cells or endospores of a butyric-acid bacterium Clostridium butyricum MII588-Sens 1 strain, or cells or endospores of a butyric-acid bacterium Clostridium butyricum MII588-Res 1 strain are effective as a biological intestinal antiseptic.

TECHNICAL FIELD 
This invention relates to a novel biological intestinal antiseptic. More 
particularly, this invention relates to a biological intestinal antiseptic 
which contains, as the active component, cells or endospores of 
Clostridium butyricum MII588-Sens 1 strain having a sensitivity to a 
butyric-acid bacteriophage KMl, or cells or endospore of another 
Clostridium butyricum MII588-Res 1 strain which is a spontaneous mutant 
derived from the Sens 1 strain and which has gained resistance to the 
phage KM1. The antiseptic of this invention is useful for the prevention 
and therapeutic treatment of bacterial food-poisonings. 
BACKGROUND ART 
Among bacteriophages which infect Clostridium bacteria as obligatory 
anaerobes, the bacteriophage which was separated from Clostridium tetani 
by Cowels in 1934 was first known. Since then, bacteriophages capable of 
lysing various Clostridium bacteria have been reported. There are however 
rather few reports on such bacteriophages which lyse butyric-acid 
bacteria. It has been reported by Tahara et al. and Hongo et al. that one 
species of butyric-acid bacteria is lysed by the enzymes induced by phages 
HM3 and HM7 which infect Clostridium saccharoperbutylacetonicum. On the 
other hand, Schlechte described in 1980 such phage 5 which lyses oncolytic 
Clostridium oncolyticum M55 and Clostridium butyricum H8 {Schlechte, H. et 
al. "Archiv fur Geschuwulstforschung", 50, 53-57 (1980)}. 
The present inventors discovered the new bacteriophage KM1 which is totally 
different from these reported phages. Starting from this discovery, the 
present inventors further have found out a special butyric-acid bacterium, 
namely Clostridium butyricum MII588-Sens 1 strain having a sensitivity to 
the bacteriophage KM1 from amongst Clostridium butyricum MII588 strains, 
as well as a mutant, Clostridium butyricum MII588-Res 1 strain which is 
derived from the Clostridium butyricum MII588-Sens 1 strain and having 
gained a phage resistance. It has also been found by the present inventors 
that these strains, i.e., the Sens 1 and Res 1 strains exhibit, both in 
vitro and in vivo, a strong and stable antagonism against the causative 
bacteria of acute gastritis and enterocolitis, such as infectious bacteria 
of food-poisonings and enteric infections, enteropathogenic Escherichia 
coli and multiple-drug-resistant Shigella strains. Based on these 
findings, we have found that cells or endospores of the MII588-Sens 1 
strain or the MII588-Res 1 strain are effective as an intestinal 
antiseptic or as a bacteriostatic agent against the causative bacteria of 
food-poisonings. 
Bacteriophage KM1 was isolated from soil. Isolation, purification and 
biological characteristics of the bacteriophage KM1 have recently been 
reported in detail by Maeds, Ishii, Tanaka, Mikami and Arai {"J. General 
Microbiology", 132, 2271-2275 (1986)}. 
The characteristics of this phage are as follows. 
Shape and host range: 
A regular plaque (lysed lesion) of bacteriophage KM1 is transparent and 
reaches 1-2 mm in diameter 24 hours after incubation. Under electron 
microscope using the negative staining method, bacteriophage KM1 is in the 
form of a tadpole and has a hexagonal head whose diameter is 60 nm. A tail 
having a contractile sheath has a length of 90 nm. Phage 5 of Schlechte et 
al., which was used for the sake of comparison, has an elliptical head 
whose diameter is 50.times.80 nm. Its tail is as long as 125 nm, has no 
sheath and is flexible. The G+C contents were 30.5 mol % for phage KM1 and 
35.4 mol % for phage 5, which are both greater than the G+C contents 
(27-28 mol %) of standard C. butyricum. These two bacteriophages are 
clearly different from each other, because it is shown that when their 
DNAs are treated with restriction enzymes Bgl II, Eco RI and Hind III and 
the resultant DNA fragments are compared by an agarose gel 
electrophoresis, revealing that their electrophoresis patterns are 
entirely different. The DNA of phage 5 cannot be digested by Bgl II. 
Bacteriophage KM1 cannot lyse entirely any of the butyric-acid bacteria 
which have been obtained from American Type Culture Collection (ATCC) of 
U.S.A., Institute of Fermentation, Osaka, and Institute of Applied 
Microbiology, University of Tokyo, etc. as well as C. butyricum H8 and 
oncolytic C. oncolyticum M55 (C. butyricum ATCC 13732) which have been 
supplied by Dr. Schlechte. On the other hand, phage 5 lysed C. butyricum 
IAM 19004, C. butyricum H8 and oncolytic C. oncolyticum M55. 
C. butyricum MII588-Sens 1 and MII588-Res 1 strains are novel 
microorganisms isolated by the present inventors, and principal 
morphological and biochemical properties of these two strains may be 
summarized as shown in the following Tables 1 and 2, in comparison with 
those of a standard strain, namely, C. butyricum IAM 19001 strain which is 
stored in Institute of Applied Microbiology, University of Tokyo. 
TABLE 1 
__________________________________________________________________________ 
Comparison in morphological properties between Clostridium butyricum 
MII5588-Sens 1 and Res 1 strains, and Clostridium butyricum IAM 19001 
MII588-Sens 1 MII588-Res 1 
IAM 19001 
__________________________________________________________________________ 
Shape Gram positive. 
Gram positive. 
Gram positive. 
Straight or slightly 
Straight or slightly 
Straight or slightly 
curved rod, and 
curved rod, and 
curved rod, and 
round at both ends. 
round at both ends. 
round at both ends. 
Size: 0.7-1.4 .times. 
Size: 0.6-1.4 .times. 
Size: 0.9-1.6 .times. 
3.0-6.5 .mu.m. 
2.8-7.0 .mu.m. 
5.0-12.5 .mu.m. 
Normally occurs as 
Occurs as discrete 
Occurs as discrete 
discrete cells. 
cells, in pair 
cells, in pairs 
and/or short chains. 
and/or short chains. 
Motility 
++ ++ ++ 
Flangella 
Peritrichal 
Peritrichal 
Peritrichal 
Endospores 
Egg-shaped. 
Egg-shaped. 
Egg-shaped. 
0.8-1.1 .times. 1.9-2.4 .mu.m. 
1.0-1.1 .times. 1.9-2.3 .mu.m. 
1.0-1.2 .times. 2.0-2.9 .mu.m. 
Subterminal. 
Subterminal. 
Subterminal. 
__________________________________________________________________________ 
TABLE 2 
______________________________________ 
Comparison in biochemical properties between Clostridium 
butyricum MII588-Sens 1 and Res 1 strains, and 
Clostridium butyricum IAM 19001 
Biochemical MII588- MII588- 
Properties Sens 1 Res 1 IAM 19001 
______________________________________ 
Sugar fermentations 
Sucrose + + + 
Raffinose + + + 
Lactose + + + 
Xylose + + - 
Glucose + + + 
Maltose + + + 
Rhamnose - - - 
Mannose + + + 
Arabinose + + + 
Ribose + + - 
Glycerol + + - 
Sorbitol - - - 
Dulcitol - - - 
Starch + + + 
Salicin + + + 
Diaminopimelic acid 
DL DL DL 
of Cell wall 
Litmus milk Acidic, gas 
Acidic, gas 
Acidic, gas 
and and and 
coagulated coagulated 
coagulated 
Sensitivity to 
++ - - 
bacteriophage KM1 
Reduction of nitrates 
- - + 
Optimum growth 
37.degree. C. 
37.degree. C. 
37.degree. C. 
temperature 
______________________________________ 
Clostridium butyricum MII588-Sens 1 and Res 1 strains are spore-forming 
Gram-positive rods and are obligatory anaerobes. According to the Bergy's 
classification book "Bergy's Manual of Determinative Bacteriology", 8th 
edition, Williams & Wilkins Co., (1974), therefore, these two strains are 
thus classified to be comprised by either the genus Clostridium or the 
genus Desulfotomaculum. Since the present strains, namely, the Sens 1 and 
Res 1 strains do not reduce a sulfate in a lactate-sulfate medium, it is 
without question that they belong to the genus Clostridium. Moreover, 
their various morphological and biochemical properties are in substantial 
conformity with those of the butyric-acid bacterium Clostridium butyricum 
standard strain IAM 19001, as shown in the above Tables. IAM 19001 strain 
is, however, different from the description of "Bergy's Manual of 
Determinative Bacteriology" in that IAM 19001 strain lacks the ability for 
fermentation of xylose and ribose, and is also different from the 
MII588-Sens 1 and Res 1 strains in this respect. Although the IAM 19001 
strain is also different from the MII588-Sens 1 and Res 1 strains with 
respect to their ability for the fermentations of glycerol, this is 
immaterial because some of the sugar fermentations is described to vary 
depending on each specific strain. The MII588-Sens 1 strain can be 
distinguished from the already known butyric-acid bacterium strains in 
that it is sensitive to bacteriophage KM1 and can be lysed by the latter 
phage. 
DISCLOSURE OF THE INVENTION 
In one aspect of this invention, there is provided a biological intestinal 
antiseptic which comprises, as the active component, cells or endospores 
of a butyricacid bacterium, Clostridium butyricum MII588-Sens 1 strain 
having such sensitivity that this strain is infected and lysed by 
bacteriophage KM1, or cells or endospores of a spontaneous mutant 
MII588-Res 1 strain which has been screened from the cultured colonies of 
the MII-588-Sens 1 strain and exhibits resistance to bacteriophage KM1. 
Clostridium butyricum MII588-Sens 1 strain useful in th practice of this 
invention has been selected as a strain having the sensitivity to 
bacteriophage KM1, by cloning the parent strain, Clostridium butyricum 
MII588 strain (FERM BP-7765) (see the specification of Japanese Patent 
Application No. 174066/84 or Japanese Patent Application first publication 
"Kokai" No. 53219/86). The MII588-Res 1 strain has been obtained by mixing 
a large number of cells of the MII588-Sens 1 strain with bacteriophage 
KM1, coating the resultant mixture to an agar plate, incubating the cells 
there and then screening from the colonies subsequently grown. 
The Clostridium butyricum MII5887-Sens 1 strain has been deposited with 
"Fermentation Research Institute" under FRI Deposit Number FERM P-9070 and 
also under FERM BP-1612 in terms of the Budapest Treaty, while Clostridium 
butyricum MII588-Res 1 strain has been deposited there under FRI Deposit 
Number FERM P-9069 and also under FERM BP-1611 in terms of the Budapest 
Treaty.

BEST MODE FOR WORKING THE INVENTION 
When carrying out the production of an antiseptic according to this 
invention, Clostridium butyricum MII588Sens 1 or Res 1 strain which serves 
as the active component of the antiseptic can be cultured by any method 
which is employed routinely for the cultivation of obligatory anaerobes. A 
culture medium where extremely little formation of spores is permitted, 
for example, PYG medium is generally recommendable to provide the 
vegetative cells of the Clostridium butyricum strain. The composition of 
PYG medium may be described as illustrated below. 
______________________________________ 
Glucose 20 g 
Peptone 20 g 
Yeast extract 10 g 
Distilled water 1000 ml 
pH 7.0 
______________________________________ 
When cultured at 37.degree. C. for 24 hours in this medium, most cells of 
the incubated strain are obtained in the form of vegetative cells. 
On the other hand, CS medium may be used to provide the endospores. The 
composition of CS medium may be described as shown below. 
______________________________________ 
Corn starch 20 g 
Aqueous amino acid mixture 
20 g 
CaCO.sub.3 7.5 g 
Distilled water 1000 ml 
pH 7.0 
______________________________________ 
The aqueous amino acid mixture is a slurry of strained soy sauce less 
residue ("AJIEKI", a product of Ajinomoto Co., Inc.). When the C. 
butyricum strain is cultured at 37.degree. C. for 72 hours in this CS 
medium, at least 80% of the cells turn to the endospores. 
When effecting the production of an antiseptic 
according to this invention, the above-described C. butyricum strain is 
cultured at a prescribed temperature for a predetermined period of time in 
the above-mentioned culture medium and the resulting culture broth is 
thereafter subjected to a centrifugal separator so as to harvest the 
cells. The thus-harvested cells are suspended in distilled water. After 
stirring the resultant suspension, it is subjected to a centrifugal 
separater to wash the cells. This procedure is repeated twice or thee 
times, and the cells ar then lyophilized into powder. 
The cells or endospores in the powder form may usually be used as an 
antiseptic of this invention as they are, but they may further be mixed 
with a pharmaceutically acceptable ordinary excipient such as lactose or 
starch. They may also be admixed with a stabilizer and another medicament 
as needed, thereby formulating it into a dosage form. So long as the 
antiseptic of this invention is kept in such a state isolated from the 
surrounding air, by enclosing the antiseptic in ampoules, capsules or the 
like, no special conditions are required to store the antiseptic of this 
invention after its formulation into a dosage form. In use of the 
antiseptic of this invention, the cells or endospores may be administered 
orally at a dose of 0.5-2.5 g per day for adult. 
A description will next be made of the activities of C. butyricum 
MII588-Sens 1 and MII588-Res 1 strains, which are employed in this 
invention, against food-poisoning bacteria and Shiqella strains. 
Bacterial food-poisonings may be classified into infectious food-poisonings 
and toxin-induced food-poisonings. In the infectious food-poisonings, the 
causative bacteria present in the food orally enter the digestive tract 
with the food, and they grows there, whereby the symptoms of acute 
gastritis and/or enterocolitis occur. Effectiveness of the C. butyricum 
strain can be tested by tracing in vitro the antagonism of the C. 
butyricum strain against the causative bacteria. In contrast, in the case 
of such causative bacteria of the toxin-induced food-poisonings, the 
causative bacteria have already grown in the food and elaborated such 
toxins which can cause acute gastritis and/or enterocolitis. By the 
ingestion of the toxins present in the food, a man develops the disease. 
Accordingly, the medical efficacy on such toxin-induced food-poisonings 
must be determined by an absolutely different testing method. 
Herein, descriptions are made with particular reference to Vibrio 
parahaemolyticus, Salmonella enteritidis and Bacillus cereus etc., as 
exemplary causative bacteria of the infectious food-poisonings. 
1. Test of the antagonism of C. butyricum MII588-Sens 1 and Res 1 strains 
against the causative bacteria of food-poisonings: 
Erlenmeyer flasks (100 ml capacity) were each filled with 100 ml of a 
glucose-added nutrient broth {comprising 0.5% glucose, 1.0% peptone, 0.5% 
NaCl (but 1.5% NaCl for V. parahaemolyticus), 0.5% meat extract}, followed 
by inoculation of the C. butyricum MII588-Sens 1 strain or Res 1 strain in 
an amount of 10.sup.6 cells/ml and a tested causative bacterium of the 
food-poisonings in an amount of 10.sup.3 cells/ml. They were stationarily 
cultured at 37.degree. C. Time-dependent variations in viable cell count 
of each causative bacterium of the food-poisonings during the above 
culturing process were traced by counting the colonies of said bacterium 
on nutrient agar medium. Since it was extremely difficult to perform 
accurately the counting of the colonies in the case of counting the cells 
of C. butyricum, time-dependent variations in the viable cell count 
thereof were traced by means of a blood cell counting chamber. 
In accordance with the above-described stationary culturing process, Vibrio 
parahaemolyticus was cultured singly and also in combination or mixture 
with C. butyricum MII588-Res 1 strain. Viable cells of the microorganisms 
on the respective incubated media were counted separately with the lapse 
of incubation time. Logarithmic values of the counts of viable cells (in 
term of CFU: colony-forming units) are plotted along the axis of 
ordinates, and the culture time is plotted along the axis of abscissas, 
whereby test results are exhibited by the diagrammatic curves shown in 
FIG. 1 of the accompanying drawings. Namely, FIG. 1 shows the 
time-dependent changes in pH value of the culture media and in the viable 
cell counts upon the single culture of Vibrio parahaemolyticus and upon 
the mixed culture of C. butyricum MII588-Res 1 strain and Vibrio 
parahaemolyticus in mixture. In FIG. 1, the curve connecting the closed 
circles indicates the viable cell counts obtained in the single culture of 
Vibrio parahaemolyticus as a tested bacterium, the curve connecting the 
open circles indicates the viable cell count of Vibrio parahaemolyticus 
obtained in its mixed culture, and the curve connecting the closed 
triangles indicates the viable cell counts of the MII588-Res 1 strain 
obtained in its mixed culture. Further, variations in pH values of the 
medium with the incubation time are also shown in the top of FIG. 1. The 
viable cell counts of the C. butyricum increased by about 100 times at an 
elapsed time of 7 hours after the beginning of the culture. In the case of 
culturing Vibrio parahaemolyticus singly, its rapid growth continued to 
reach a peak around 6 hours after the inoculation. When cultured in 
mixture with C. butyricum MII588-Res 1 strain, the viable cell counts of 
Vibrio parahaemolyticus reached the maximum in about 4 hours and Vibrio 
parahaemolyticus was then killed rapidly. Then, the pH value dropped to 
4.6. This causative bacterium of the food-poisonings was also be killed in 
the same way as above when the MII588-Sens 1 strain and Vibrio 
parahaemolyticus were cultured in mixture. Substantially same results are 
also observed in the case of culturing Bacillus cereus singly, or in 
mixture. 
A similar test was also conducted using Salmonella enteritidis as a tested 
bacterium. Results of the test are also shown diagrammatically in FIG. 2. 
Namely, FIG. 2 illustrates time-dependent variations in the pH value and 
in the viable cell counts obtained in the single culture of Salmonella 
enteritidis and also in the mixed culture of C. butyricum MII588-Sens 1 
strain and Salmonella enteritidis in mixture. When cultured in association 
with C. butyricum MII588-Sens 1 strain, the causative bacterium of the 
food-poisonings reached the maximum growth at a time of 6 hours after the 
inoculation and was killed thereafter. The killing of S. enteritidis was 
not so rapid as the above-described Vibrio parahaemolyticus or Bacillus 
cereus. 
With respect to single culture of enteropathogenic E. coli and its mixed 
culture in mixture with C. butyricum MII588-Res 1 strain, the experiments 
were carried out in the same manner as above. The test results are also 
shown diagrammatically in FIG. 3. Namely, FIG. 3 illustrates 
time-dependent variations in the pH value and in the viable cell counts 
obtained in the single culture of the E. coli and also in the mixed 
culture of C. butyricum MII588-Res 1 strain and E. coil in mixture. In 
this case, the effect of the mixed culture was bacteriostatic, and the 
growth of the causative bacterium of the food-poisonings was suppressed 
strongly but the antibacterial activity against E. coli was not so 
remarkable as the aforementioned three species of the causative bacteria 
of the food-poisonings. Substantially same results were also obtained 15 
with a single culture of a parent strain of Shigella flexneri and with the 
mixed culture of the S. flexneri parent strain in mixture with C. 
butyricum MII588-Res 1 strain (the test results obtained therefor are 
shown in FIG. 4), as well as with a single culture of a 
multiple-drug-resistant strain of Shigella flexneri and its mixed culture 
with C. butyricum MII588-Res 1 strain (the test results obtained therefor 
, are shown in FIG. 5). Namely, FIG. 4 shown time-dependent variations in 
the pH value and in the viable cell counts obtained in the single culture 
of the Shigella flexneri, as well as in the mixed culture of C. butyricum 
MII588-Res 1 strain with the Shigella flexneri. On the other hand, FIG. 5 
depicts time-dependent variations in the pH value ad in the viable cell 
counts obtained in the single culture of the multiple-drug-resistant 
strain of Shigella flexneri as well as in the mixed culture of C. 
butyricum MII588-Res 1 strain with the multiple-drug-resistant strain of 
Shigella flexneri. 
2. Test of intestinal localization of C. butyricum MII588-Sens 1 strain: 
In order to allow the C. butyricum strain to exhibit the antagonism against 
such causative bacteria of the food-poisonings even in vivo in a living 
body, the C. butyricum must settle and live for a certain period of time 
in the digestive tract. The discovery of the novel bacteriophage KM1 and 
the C. butyricum MII588-Sens 1 strain has provided an extremely important 
means for the analyses of "in vivo" behaviors of the C. butyricum strain. 
Namely, the MII588-Sens 1 strain can be readily and accurately 
distinguished, identified and counted by testing its sensitivity to 
bacteriophage KM1 even when the MII588-Sens 1 strain is present along with 
many colonies of many other bacteria, including other strains of C. 
butyricum, within the contents of the digestive tract. 
As a result of extensive investigations, the colistin-CS medium of the 
under-mentioned composition has been found to give very good results when 
it is used as a culture medium for incubating and screening the C. 
butyricum strain from the contents of digestive tract and culturing the 
same strain. 
The colistin-CS medium is the medium of such composition as prepared by 
adding 800 .mu.g/ml of colistin sodium methanesulfonate and 1.5% of agar 
to the CS medium given hereinbefore. A quantity of the digestive tract 
contents or feces is mixed with and incubated in the colistin-CS medium. 
This incubation may be conducted at 37.degree. C. in a glove box under 
such an environment atmosphere comprising 80% N.sub.2, 10% CO.sub.2 and 
10% H.sub.2. Colonies which appear on the screening culture medium in 
48-hours of incubation are mostly of C. butyricum. The colonies of the C. 
butyricum can be easily distinguished from the colonies of the other 
bacteria, because the former colonies can form the surrounding transparent 
zone wherein insoluble calcium carbonate precipitates have been dissolved 
by the produced organic acids mainly comprising butyric acid. Furthermore, 
when the colonies of C. butyricum are tested for their sensitivity to 
bacteriophage KM1, it is possible to distinguish and identify whether or 
not the tested colonies comprise the MII588-Sens 1 strain. By mean of the 
above procedures, the counting was periobically made of viable cells of 
the MII588-Sens 1 strain present in the contents of the stomachs, small 
intestines, cecums and large intestines of male golden hamsters of about 
100 g body weight which had received oral administration of 
8.times.10.sup.8 endospores of C. butyricum MII588-Sens 1 strain, whereby 
the cell distribution of C. butyricum MII588-Sens 1 strain present in each 
part of the digestive tract was traced. Results of this test are 
diagrammatically depicted in FIG. 6. Thus, time-dependent variations in 
the viable cell counts of C. butyricum MII588-Sens 1 strain present in the 
digestive tract parts of the hamsters are represented in FIG. 6. 
Until two hours after the administration of C. butyricum MII588-Sens 1 
strain, this strain remained mostly within the stomach contents. 
Thereafter, the C. butyricum MII588-Sens 1 strain moved gradually 
therefrom to the small intestine and further to the cecum and large 
intestine. Six hours later, this C. butyricum strain was distributed 
almost evenly throughout the entire digestive tract with its population 
level of 10.sup.5 -10.sup.6 cells/g. This population level was kept 
constant approximately for 20 hours. In addition, C. butyricum MII588-Sens 
1 strain present in feces was also assayed. Twenty four hours after a 
similar oral administration of the MII588-Sens 1 strain, 5.times.10.sup.7 
cells of C. butyricum MII588-Sens 1 strain were found per gram of feces. 
3. Toxicity test: 
An acute toxicity test for mice was conducted in the following manner. 
Endospores of C. butyricum MII588-Sens 1 and Res 1 strains were separately 
administered to male DDY mice of 4-weeks age. Each group consisted of 10 
mice and was tested by administration of the endospores at doses not 
greater than 500 mg/kg. None of the tested animals was killed at an of the 
doses of administration. During the observation for the period of 1 week, 
no visual abnormality was observed with the tested mice, and the body 
weight increases involved were not different from those for a control 
group of mice. 
As has been demonstrated above, the C. butyricum strains used as the active 
components for biological intestinal antiseptics according to this 
invention can always exhibit stable and strong antiseptic and 
bacteriostatic activities against the food-poisoning bacteria. Moreover, 
the use of C. butyricum MII588-Res 1 strain can surprisingly avoid such 
accident that all the cells of the cultured MII588-Res 1 strain could 
suddenly be lysed and killed due to the contamination of bacteriophages 
from soil. 
In another aspect of this invention, there is hence provided a method for 
treating Vibrio parahaemolyticus, Salmonella enteritidis, enteropathogenic 
Escherichia coli or Shigella flexneri, which comprises orally 
administering the cells or endospores of Clostridium butyricum MII588-Sens 
1 strain or Clostridiunm butyricum MII588-Res 1 strain or both of them to 
an animal including a man, and thereby inhibiting the growth of Vibrio 
parahaemolyticus, Salmonella enteritidis, enteropathogenic Escherichia 
coli or Shigella flexneri that has entered accidentally in the intestinal 
tract of the animal, and also prohibiting the production of toxins in the 
intestinal tract by the pathogenic bacterium. 
The present invention will hereinafter be illustrated specifically by the 
following Examples. 
EXAMPLE 1 
Clostridium butyricum MII588-Sens 1 strain (FRI Deposit Number FERM P-9070) 
was inoculated to 5 l of the CS medium and cultured at 37.degree. C. for 
72 hours. The endospores were collected by a cooled continuous centrifugal 
separator and then washed with distilled water, to recover 13.2 g of the 
wet cells. The cells were dried at 80.degree. C. with hot air to obtain 
2.7 g of the endospores as a powder. The dry endospores thus-obtained were 
sealed in 100 mg-portions into bials and then stored at 5.degree. C. for 2 
months. One of two groups of such male golden hamsters (10 hamsters per 
group) which had a body weight of about 100 g and had been inoculated 
orally with 10.sup.5 cells of Salmonella enteritictis was used as a 
control group, while the hamsters of the other group were orally 
administered with the dry endospores of the MII588-Sens 1 strain at a dose 
of 8.times.10.sup.8 endospores at the end of 1 hour after the oral 
inoculation of the Salmonella species. 
Three hours later, the animals were sacrificed and the abdominal cavity was 
opened, and Salmonella enteritictis present in the contents of the small 
intestine were incubated in the DHL-agar medium, followed by counting the 
number of the cells of the Salmonella species. 
10.sup.3 Cells/g of Salmonella enteritictis were found with the control 
group, whereas Salmonella enteritictis detected was in the level of 10 
cells/g or less in the treated group of the hamsters which were 
administered with C. butyricum MII588-Sens 1 strain. 
EXAMPLE 2 
C. butyricum MII588-Res 1 strain (FRI Deposit Number FERM P-9069) was 
inoculated to 5 l of the CS medium and cultured at 37.degree. C. for 72 
hours. The endospores were collected by a cooled continuous centrifugal 
separator and then washed with distilled water, to recover 12.5 g of the 
wet cells. The cells were lyophilized to obtain 2.4 g of the endospores as 
a powder. The dry endospores thus-obtained were sealed in 100 mg-portions 
into bials and then stored at 5.degree. C. for 2 months. Male golden 
hamsters (10 hamsters per group) having a body weight of about 100 g were 
orally inoculated with 105 cells of Vibrio parahemolyticus. One group of 
hamsters was used as a control group, while the hamsters of the other 
group were orally administered with the dry endospores of the MII588-Res 1 
strain at a dose of 8.times.10.sup.8 endospores at the end of 1 hour after 
the inoculation of the Vibrio species. 
Four hours later, the hamsters were sacrificed and subjected to the 
abdominal operation. Vibrio parahaemolyticus present in the contents of 
the large intestine of each hamster was incubated in BTB Teepole medium, 
and the cell number was counted. Vibrio parahaemolyticus was observed to 
grow to 3.times.10.sup.2 cells/g with the control group, but Vibrio 
parahaemolyticus was not detected at all in the treated group which was 
administered with C. butyricum MII588-Res 1 strain. 
INDUSTRIAL UTILITY OF THE INVENTION 
As has been described above, the biological intestinal antiseptics of this 
invention are useful for the prevention and therapeutic treatment of 
bacterial food-poisonings in animals, including men. 
Reference to Microorganisms Deposited under Rule 13.2 
(a) Name and address of depository authority with which the microorganisms 
have been deposited: 
Name: Fermentation Research Institute, Agency of Industrial Science and 
Technology, Ministry of International Trade and Industry 
Address: 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, Japan (Mail Mo. 
305). 
(b) Date of the deposit with the depository authority (a): Dec. 5, 1986 
(c) Accessible numbers allotted for the deposit by the depository authority 
(a): 
BIKOKEN-JOKI No. 1611 (FERM BP-1611) 
BIKOKEN-JOKI No. 1612 (FERM BP-1612)