An intestinal microflora-improving agent containing, as an effective component, bacterial cells and/or the water-soluble extracts therefrom, obtained from microorganisms belonging to the genus Streptococcus.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention relates to a new intestinal microflora-improving 
agent. 
2. Description of the Related Art 
It is said that there are about 100 trillion intestinal bacteria in the 
human intestines. These are classified into over 300 kinds. The 
significance of the intestinal microflora in humans has been revealed. For 
example, a fundamental investigation on relationships between intestinal 
bacteria and aging of the host has revealed that intestinal bacteria 
influence the activities of enzymes in various organs and the metabolism 
of important substances and that intestinal bacteria depress the 
accumulation of lipids and the inactivation of the detoxification function 
of the liver with aging (See Yazawa, K. et al. Mech. Ageing Devel. 17, 173 
(1981), Kawai, Y. et al. Mech. Ageing Devel. 16, 149 (1981), Kawai, Y et 
al. Infect. Immun. 19, 771 (1978), and Kawai, Y am. J. of Clin. Nut 32, 
187 (1979)). Many other studies on the importance of the intestinal 
microflora in hosts have been reported. (See Freter, R. Am. J. Clin. Nutr. 
27, 1049 (1974), Gorbach, S. L. Gastroenterology, 60, 1110 (1971), Savage, 
D. C. Am. J. Clin. Nutr. 25, 1372 (1972), de Dombal, F. T., et al. Gut, 
10, 270 (1969), Donaldson, R. M., Jr. New Engl. J. Med., 270, 938 (1964), 
Gordon, H. A., et al. Bacteriol. Rev., 35, 390 (1971), Taniguchi, T., et 
al. Microbiol. Immunol., 22, 793 (1978), Elyssen, H., Proc. Nutr. Soc., 
32, 59 (1973), Wostmann, B. S. et al. J. Germfree Life Gnotobiol., 5, 4 
(1975), Phear, E. A., et al. Br. J. Exp. Pathol., 37, 253 (1965), and 
Wolpert, E. et al. Lancet, ii, 1387 (1971)). 
As evidenced by these studies, in many cases, the host's health is 
deteriorated by the abnormal over-growth of harmful bacteria in the 
intestines. On the contrary, it is kept in a normal condition or is 
improved by the ordinary growth of useful intestinal bacteria such as 
Streptococcus, Lactobacillus, and Bifidobacterium. These facts strongly 
suggest that the selective growth of such useful intestinal bacteria in 
the intestines is quite important for the prevention and treatment of 
various kinds of so-called middle-aged or geriatric diseases. 
SUMMARY OF THE INVENTION 
Accordingly, the objects of the present invention are to suppress the 
abnormal over-growth of harmful bacteria in the intestines and to provide 
a novel intestinal microflora-improving agent capable of selectively 
stimulating the growth of useful microorganisms in the intestines. 
Other objects and advantages of the present invention will be apparent from 
the following description. 
In accordance with the present invention, there is provided an intestinal 
microflora-improving agent containing, as an effective component, 
bacterial cells and the water-soluble extracts therefrom, obtained from 
microorganisms belonging to the genus Streptococcus.

DESCRIPTION OF THE PREFERRED EMBODIMENTS 
The inventors of the present invention have found in an investigation on 
the growth of Streptococcus, Lactobacillus, Bifidobacterium, etc. in the 
intestines that the bacterial cells or their water-soluble extracts 
obtained from bacteria belonging to the genus Streptococcus effectively 
stimulate the growth of those useful microorganisms. 
The types and microbiological characteristics, the preparation procedures, 
the physiological activity, etc. of the intestinal microflora-improving 
agents according to the present invention will be explained in detail 
hereinbelow. 
Microorganisms suitable for use in the preparation of the bacterial cell 
products according to the present invention are those belonging to the 
genus Streptococcus, especially Streptococcus faecalis, S. faecium, S. 
avium, S. salivarius, S. durans, S. mitis, S. bovis, and S. equinus. 
Typical examples of such microorganisms have been deposited since July 15, 
1982 in the Fermentation Research Institute (FRI) in Japan (all the 
numbers quoted as "FERM-P" in Table 1 refer to the deposition numbers of 
the Institute) and internally transferred in the FRI as an international 
depository authority under the Budapest Treaty (Budapest Treaty on the 
International Recognition of the Deposit of Microorganisms for the Purpose 
of Patent Procedure) under the FERM-BP deposition numbers in Table 1. 
TABLE 1 
______________________________________ 
Strains Deposition number 
______________________________________ 
Streptococcus faecium 
ADV1009 FERM P-6624 FERM BP- 
296 
Streptococcus faecalis 
ADV9001 FERM P-6625 FERM BP- 
297 
Streptococcus avium 
AD2003 FERM P-6626 FERM BP- 
298 
Streptococcus salivarius 
ADV10001 FERM P-6627 FERM BP- 
299 
Streptococcus durans 
ADV3001 FERM P-6628 FERM BP- 
300 
Streptococcus mitis 
ADV7001 FERM P-6629 FERM BP- 
301 
Streptococcus equinus 
ADV8001 FERM P-6630 FERM BP- 
302 
______________________________________ 
The general microbiological characteristics of the microorganisms in the 
present invention are the same as those of known microorganisms belonging 
to the same class. That is, the general microbiological characteristics, 
cultivation method, and other properties correspond to those described in 
the following articles: 
(1) Bergey's Manual of Determinative Bacteriology, 8th ed., 490-509 (1974) 
(2) Int. J. Syst. Bacteriol. 16: 114 (1966). 
(3) Microbiol. Immunol. 25: 257-269 (1981). 
(4) J. Clin. Pathol. 33: 53-57 (1980). 
(5) J. Gen. Microbiol. 128: 713-720 (1982). 
(6)Appl. Microbiol. 23: 1131-1139 (1972). 
The typical microbiological characteristics of the above-exemplified 
strains according to the present invention are summarized in Table 2. 
TABLE 2 
__________________________________________________________________________ 
Strains 
ADV ADV AD ADV ADV ADV ADV 
Characteristics 
1009 
9001 
2003 
10001 
3001 
7001 
8001 
__________________________________________________________________________ 
Shape of cell spheroid 
Gram stain + + + + + + + 
Hemolysis .alpha. 
.alpha. 
.alpha. 
.alpha. 
.alpha. 
.alpha. 
.alpha. 
Growth at 
10.degree. C. + + .+-. 
- + - - 
45.degree. C. + + + .+-. 
+ .+-. 
+ 
50.degree. C. + - - - + - - 
Thermal resistance at 
+ + + - + - - 
60.degree. C. for 30 min 
Growth in culture medium 
+ + + - + - - 
at pH 9.6 
Methylene blue reduction 
+ + - - + - - 
Liquefaction of gelatin 
- - - - - - - 
Growth in culture medium 
+ + - - + - - 
containing NaCl (6.5%) 
Growth in culture medium 
+ + + - + - + 
containing bile (40%) 
Productivity of ammonia 
+ + ND - + .+-. 
- 
Hydrolysis of hippuric acid 
- .+-. 
- - + - - 
Growth in culture medium 
- + - ND - ND - 
containing tellurite 
Growth in culture medium 
- + - ND*2 
- ND - 
containing TTC*1 
Acid production from 
Glucose + + + + + + + 
Esculin .+-. 
+ + + .+-. 
ND + 
Inulin - - - + - - .+-. 
Lactose + + + .+-. 
+ .+-. 
- 
Glycerol - + .+-. 
- - - - 
Arabinose + - + - - - - 
Melezitose - + .+-. 
ND - ND - 
Sorbitol - + + - - - - 
Antigenic group 
D D Q(D) 
K D - D 
__________________________________________________________________________ 
*1: 2,3,5Triphenyltetrazolium chloride 
*2: Not done 
Each strain of the above-mentioned microorganisms is inoculated into 5 L of 
Rogosa broth medium consisting of 
______________________________________ 
Trypticase (BBL) 10 g 
Yeast extract 5 g 
Tryptose 3 g 
KH.sub.2 PO.sub.4 3 g 
K.sub.2 HPO.sub.4 3 g 
Triammonium citrate 2 g 
Tween 80 1 g 
Glucose 20 g 
Cysteine hydrochloride 0.2 g 
Salt solution *2 5 ml 
Distilled water to 1 liter 
(pH 7, autoclave at 121.degree. C for 15 min) 
______________________________________ 
*2 MgSO.sub.4 --7H.sub.2 O 
11.5 g 
FeSO.sub.4 --7H.sub.2 O 
0.68 g 
MnSO.sub.4 --2H.sub.2 O 
2.4 g 
Distilled water 
100 ml 
Each strain is then stationarily cultivated under an aerobic condition at 
37.degree. C. for 10 hrs to yield the subsequent culture broth containing 
10.sup.9 cells/ml of the viable cells. The microorganisms are harvested by 
continuous centrifugation at 12,000 rpm. The bacterial cells are washed 
with physiological saline and, then, are suspended in physiological saline 
(0.85% NaCl solution) to obtain 50 ml of the cell suspension containing 
10.sup.11 cells/ml. 
The viable cells obtained are further washed twice with physiological 
saline and, then, are suspended in the same solution. Fifty ml of the cell 
suspension is thus obtained and is heated at 115.degree. C. for 10 min to 
form the desired cell suspension containing the dead cells. This cell 
suspension is, then, lyophilized and dried in vacuo to obtain the dead 
cell powder. 
The suspension of the above-mentioned bacterial cells, in distilled water 
or physiological saline (0.85% NaCl solution) containing 2.times.10.sup.11 
cells/ml, is autoclaved at 115.degree. C. for 10 min to destroy the 
bacterial cells and to extract the hot-water soluble substances from the 
bacterial cells. The treated bacterial cell suspension is centrifuged at 
2,000 G for 20 min to obtain the effective components in the supernatant 
of the present invention. 
The above bacterial cell suspension is treated by sonication at 15 kc for 1 
hr to destroy the bacterial cells and, then, the destroyed bacterial cell 
suspension is centrifuged at 20,000 to 25,000 G for 30 min to obtain the 
effective components in the supernatant of the present invention. 
Distilled water, physiological saline, and various kinds of pH-adjusted 
buffers, etc. can be also used for the extraction. 
The above-mentioned bacterial cells are heat-treated at 0.degree. C. to 
130.degree. C., preferably at 80.degree. C. to 120.degree. C., for 10 min 
to several hours and, then, are centrifuged to obtain the effective 
components in the supernatant of the present invention. 
The above-mentioned bacterial cells are treated singly with water and 
alcohol, such as methanol and ethanol, or with the mixture of these 
solvents and, then, are centrifuged to obtain the effective components in 
the supernatant of the present invention. The mixing ratios are usually 
water/alcohol=0/10 (v/v). 
Methanol is removed from the supernatant when it is used as a solvent. 
The effective components obtained by the above-mentioned procedures or by 
the combined procedures according to the present invention are used in the 
forms of liquid, lyophilized powder, powder dried in vacuo, etc. 
The agents of the present invention selectively stimulate the growth of the 
useful intestinal bacteria. Such a physiological activity results in the 
effective improvement of the intestinal microflora. That is, the oral 
administration of the agents of the present invention into humans with 
abnormal intestinal microflora normalizes the population levels of useful 
intestinal bacteria such as Bifidobacterium, Lactobacillus, and 
Streptococcus, whereby the growth of these bacteria is stimulated to their 
normal levels. For example, as mentioned in the examples hereinbelow, the 
oral administration of the agents of the present invention can enhance the 
intestinal bacterial population levels to their normal levels 
(Bifidobacterium, ca. 10.sup.8 to 10.sup.11 /g feces; Lactobacillus, about 
10.sup.5 to 10.sup.8 / g feces; and Streptococcus, ca. 10.sup.6 to 
10.sup.8 /g feces) even in people whose population levels of 
Bifidobacterium, Lactobacillus, and Streptococcus are extremely lower than 
those of healthy people who have normal intestinal microflora. 
As shown in the examples hereinbelow, the LD.sub.50 values of the 
preparations composing the bacterial cells and the water-soluble extracts 
of the present invention were over 6.times.10.sup.9 bacterial cells/mouse 
(intraperitoneal administration) and the amount equivalent to over 
2.6.times.10.sup.10 bacterial cells/mouse (intraperitoneal 
administration), respectively. Both the preparations were substantially 
nontoxic on oral administration. 
The agents according to the present invention can be generally applied in a 
dose of 10.sup.7 to 10.sup.15 cells/kg body weight, more preferably 
10.sup.9 to 10.sup.12 cells/kg body weight by, for example, oral 
administration. The agents according to the present invention can be made 
in the form of, for example, suspensions in physiological saline 
solutions, powder, granules, tablets, and capsules. The agents according 
to the present invention can be optionally prepared by using conventional 
appropriate carriers, bulk fillers, diluents, etc. 
The present invention will now be further shown by, but is by no means 
limited to, the following examples. 
EXAMPLE 1 
Preparation of Bacterial Cells 
Streptococcus faecalis ADV9001 was inoculated into the above-mentioned 
Rogosa broth medium (5 l) at a final concentration of 10.sup.6 viable 
cells/ml and was stationarily incubated at 37.degree. C. for 10 hrs to 
yield 10.sup.9 cells/ml of the culture fluid. Then, the bacterial cells 
were collected by continuous centrifugation (12,000 rpm) of the culture 
fluid. The separated cells were washed twice with physiological saline 
(0.85% NaCl solution) by centrifugation. The centrifuged cells were then 
suspended in distilled water or physiological saline to obtain 50 ml of 
the cell suspension (10.sup.11 cells/ml) and were autoclaved at 
115.degree. C. for 10 min. Finally, the heat-treated bacterial cell 
suspension was lyophilized to obtain the dead bacterial cell powder. 
EXAMPLE 2 
Growth-Stimulating Effect 1 
The growth-stimulating effect of the agents obtained from the 
microorganisms exemplified below according to the present invention was 
examined in vitro. In this example, the lyophilized powder of the 
heat-treated cells of Streptococcus faecalis ADV9001 was used. 
______________________________________ 
Bifidobacterium adolescentis 
RIMD 0232001 
Lactobacillus salivarius 
An isolate from human 
intestine 
Lactobacillus casei 
IID892 
Lactobacillus acidophilus 
IID893 
Streptococcus faecalis 
ADV9001 
Streptococcus faecalis 
ADV9002 
Streptococcus durans 
ADV3001 
Streptococcus bovis 
ADV4002 
Streptococcus faecium 
ADV1003 
Streptococcus avium 
ADV2002 
______________________________________ 
The dead bacterial cell powder of S. faecalis ADV9001 was added into media 
shown in Table 3. The media were, then, autoclaved at 115.degree. C. for 
15 min. Each of the strains shown in Table 3 was then inoculated into the 
respective medium. The viable cell counts in the culture fluid were 
examined periodically. For a comparison with the dead bacterial cell 
powder of S. faecalis ADV9001, the heat-treated (115.degree. C., 10 min) 
and lyophilized powder of Bacteroides fragilis ss. fragilis RIMD 0230001 
and E. coli DEFINE (IAM) 1239, and commercial yeast extract were also 
examined as to their growth-stimulating effect on each strain shown in 
Table 3. 
TABLE 3 
______________________________________ 
Microorganisms 
Media 
______________________________________ 
Bifidobacterium 
VLG medium diluted 10-fold 
with phosphate 
buffered saline (PBS) 
Lactobacillus 
PBS supplemented with glucose (1 mg/ml) 
and Trypticase (5 mg/ml, BBL) 
Streptococcus 
PBS or PBS supplemented with glucose 
(1 mg/ml) 
______________________________________ 
The results were shown in FIGS. 1-15. 
The growth-stimulating effect of the lyophilized powder of S. faecium ADV 
1009, prepared according to the methods described in Example 1, was also 
examined. As illustrated in FIGS. 16 to 18, the results were almost the 
same as those of S. faecalis ADV9001. 
In FIGS. 1 to 18, the ordinate indicates viable cell counts (log/ml), and 
the abscissa indicates the incubation time (hr). The bacterial strains 
used and signs (A, B, C, and D) were those shown in Table 4. 
As clearly shown in each Figure, the microbial strains listed above were 
greatly stimulated in their growth by the addition of the lyophilized 
powder prepared as shown above of S. faecium ADV1009 and S. faecalis 
ADV1009. 
TABLE 4 
__________________________________________________________________________ 
Growth curves 
FIG. 
Strains A B C D 
__________________________________________________________________________ 
1 S. faecalis ADV9001 
"Powder"* of 
"Powder" of 
"Powder" of 
No "powder" 
2 S. faecalis ADV9002 
S. faecalis 
S. faecalis 
S. faecalis 
was added 
3 S. faecalis ADV1003 
ADV9001 
ADV9001 
ADV9001 
4 S. avium ADV2002 
was added 
was added 
was added 
5 S. durans ADV3001 
(1 mg/ml) 
(5 mg/ml) 
(10 mg/ml) 
6 S. bovis ADV4002 
7 L. salivarius 
8 L. casei 
9 L. acidophilus 
10 B. adolescentis 
11 L. salivarius "Powder" of 
"Powder" of 
Yeast 
12 L. casei Bacteroides 
E. coli 
extract 
13 S. faecalis ADV9001 
was added 
was added 
was added 
(1 mg/ml) 
(1 mg/ml) 
(1 mg/ml) 
14 B. adolescentis 
Same powder 
Same powder 
Same powder 
Same powder 
15 L. acidophilus 
above above above above 
was added was added 
was added 
was added 
was added 
(5 mg/ml) (5 mg/ml) 
(5 mg/ml) 
(5 mg/ml) 
(5 mg/ml) 
16 S. faecalis ADV9001 
"Powder" of 
"Powder" of 
"Powder" of 
No "Powder" 
17 S. avium ADV2001 
S. faecium 
S. faecium 
S. faecium 
was added. 
18 S. facium ADV1003 
ADV1009 
ADV1009 
ADV1009 
was added 
was added 
was added 
(1 mg/ml) 
(5 mg/ml) 
(10 mg/ml) 
__________________________________________________________________________ 
*Powder: heattreated and lyophilized powder according to methods describe 
in Example 1. 
EXAMPLE 3 
Growth-Stimulating Effect 2 
The growth-stimulating effect of the hot-water soluble extracts obtained 
from S. faecium ADV1009 on S. faecalis ADV9001 was examined. The bacterial 
powder prepared according to the above-mentioned methods of S. faecium 
ADV1009 was suspended (5 mg powder/ml) in PBS. The suspension was heated 
at 115.degree. C. for 10 min to destroy the cells and to extract hot-water 
soluble components. The heat-treated suspension was centrifuged at 3,000 
rpm for 15 min, and the precipitation was suspended again in PBS. These 
two preparations were used for media in which S. faecalis ADV9001 was 
inoculated. The viable cell number was counted periodically (FIG. 19). In 
the Figure, A and B show the growth in media into which the supernatant 
and the precipitation were added, respectively, and C shows the growth in 
PBS as a medium. The ordinate and the abscissa show viable cell counts 
(log/ml) and incubation time (hr). The supernatant tended to stimulate the 
growth of those bacterial strains as well as the heat-treated and 
lyophilized bacterial powder mentioned above in the examples, and the 
precipitate comparatively weakly stimulated the growth. 
EXAMPLE 4 
Clinical Tests 
The bacterial powder of Streptococcus faecalis ADV9001 prepared in Example 
1 was orally adminstered (60 mg/day) into a familial hyperlipidemic 
volunteer (male, 29 year-old) and normal volunteers (male, 23-42 
year-old). The fecal microflora (total bacteria, Streptococcus, 
Lactobacillus, Bifidobacterium, Bacteroides, Enterobacteriaceae, 
Staphylococcus, Clostridium (lecithinase-positive), and fungi) in all the 
volunteers was examined. The results were shown in FIG. 20 (values of a 
hyperlipidemic volunteer) and FIG. 21 (average values of normal 
volunteers). In the Figures, A, B, C, D, and E show the viable counts of 
total bacteria, Streptococcus, Lactobacillus, Bifidobacterium, 
Bacteroides, and Enterobacteriaceae, respectively, the ordinate and the 
abscissa indicate viable counts (log/g) and administration period (weeks), 
respectively. An arrow on the abscissa indicates the start of 
administration. The viable cell number of Staphylococcus, Clostridium, and 
fungi was not shown in these Figures, because there was no significant 
difference between before and after the powder-administration in every 
volunteer. 
It is clear in FIG. 20 that the viable cell number of the lactic acid 
bacteria such as Bifidobacterium, Lactobacillus, Streptococcus, etc. which 
was in extremely low number in the hyperlipidemic volunteer's feces, 
compared with that in normal controls, increased up to that of normal 
controls, and the total viable cell number also increased, 8 weeks after 
starting of the administration. 
Moreover, clinical tests similar to these mentioned above were done with 
the heat-treated and lyophilized powder of S. faecium ADV1009 and S. avium 
AD2003, and almost the same results were obtained. 
EXAMPLE 5 
Acute Toxicity 
The heat-treated bacterial cells and hot-water soluble extracts obtained 
from the 7 strains of the genus Streptococcus, prepared according to the 
above-mentioned preparation methods were intraperitoneally administered 
into ICR mice (6 week-old, average body weight of 30.0.+-.0.6 g). Thus, 
the thanatobiological observation of the mice was carried out for 14 days. 
The LD.sub.50 values calculated according to the Behrens-Karber method are 
shown in Table 5 (heat-treated cells) and Table 6 (hot-water soluble 
extracts). All the strains tested of the present invention were nontoxic, 
substantially, in the case of daily oral administration. 
TABLE 5 
______________________________________ 
Strains LD.sub.50 (bacterial cells/mouse) 
______________________________________ 
S. faecium ADV1009 
6.3.times. 10.sup.9 
S. faecalis ADV9001 
3.8 .times. 10.sup.9 
S. avium AD2003 4.2 .times. 10.sup.9 
S. salivarius ADV10001 
3.6 .times. 10.sup.9 
S. durans ADV3001 
8.9 .times. 10.sup.9 
S. mitis ADV7001 6.7 .times. 10.sup.9 
S. equinus ADV8001 
6.5 .times. 10.sup.9 
______________________________________ 
Table 
______________________________________ 
Strains LD.sub.50 (mg/mouse) 
______________________________________ 
S. faecium ADV1009 7.1 
S. faecalis ADV9001 6.8 
S. avium AD2003 7.2 
S. salivarius ADV10001 
6.3 
S. durans ADV3001 10.1 
S. mitis ADV7001 8.6 
S. equinus ADV8001 8.2 
______________________________________ 
Regarding pharmaceutical preparations, a 60 mg amount (equivalent to 
6.times.10.sup.10 cells) of the heat-treated and lyophilized powder of S. 
faecium ADV1009 cells prepared according to the above-mentioned 
preparation methods were uniformly mixed with 940 mg of purified starch 
powder and, then, the tablets were formed for oral administration. This 
tablet corresponds to a dosage of 10.sup.9 cells/kg body weight for a 
human adult having a body weight of 60 kg. 
A tablet obtained from 600 mg of the above-mentioned lyophilized powder by 
mixing with 400 mg of purified starch powder corresponds to a dosage of 
10.sup.10 cells/kg body weight. 
Thus, the cell products of the present invention can be converted into the 
desired dosage form having a predetermined activity by mixing with 
pharmaceutically acceptable carriers based on the above-mentioned standard 
dosage.