JPH08298982A - Complex microbial pharmaceutical preparation - Google Patents

Abstract

(57) [Summary] [Purpose] Blood cholesterol lowering action, intestinal action,
In addition to various physiological actions such as immunostimulatory action, carcinostatic action, blood pressure lowering action, improvement of high blood sugar level, suppression of intestinal development of substances causing mental disorders, in addition to improving livestock productivity and deodorizing fecal odor And a novel complex microbial preparation having a composting promoting action and a methane generation inhibiting action in the rumen (ruminal) of a ruminant. [Structure] At least 13 kinds of specific soil bacteria belonging to the genus Bacillus, Lactobacillus, Streptococcus, Saccharomyces and Candida, or in addition to them, nitrifying bacteria and sulfur bacteria, which are used as calcium-containing base materials. Adsorption-fermented formulation.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a blood cholesterol lowering action, an intestinal regulating action, an immunostimulating action, an anticancer action, a blood pressure lowering action, a lowering and normalization of a high blood glucose level, and a suppression of intestinal generation of a substance causing a mental disorder. In addition to various physiological actions such as the above, the present invention relates to a novel combined microbial preparation having a deodorizing action of feces and a deodorizing action of the feces, a composting promoting action thereof, a methane generation inhibiting action in the rumen (ruminal) of ruminants, and the like. More specifically, it relates to a complex microbial preparation in which at least certain 13 kinds of soil bacteria are adsorbed and fermented on a calcium-containing substrate.

[0002]

2. Description of the Related Art Livestock dung is composted with the residues of crop harvests along the flow of biological circulation and is returned to the soil as organic fertilizer. However, methane and malodorous substances are generated during composting, and they are becoming a problem as a cause of global warming and malodorous pollution. Methane is also generated abundantly in the rumen of ruminant livestock animals, and these are attracting attention as a cause of global warming of the earth's atmosphere, and have become a problem as a source of livestock pollution along with the bad smell of manure. In addition, runoff of unripe compost into rivers has been attracting attention as a water pollution problem. Under such circumstances, various microorganism / enzyme-containing materials have been developed for the purpose of promoting composting of livestock excrement and suppressing the generation of offensive odors. For example, JP-A-55-48386 discloses novel microorganisms and microorganisms belonging to the genus Thiobacillus or the genus Pseudomonas as a fecal deodorant.
Further, JP-A-59-179063 discloses a deodorant obtained by mixing and fermenting mixed soil bacteria consisting of nitrite bacteria, green sulfur bacteria, cellulolytic bacteria, filamentous fungi, actinomycetes with rice bran, sawdust, etc., and a method for producing the same. Is disclosed. It is disclosed that this mixed soil bacterium is directly applied to a source of malodor such as feces and garbage, and that it is mixed with feed to be fed to livestock to prevent fecal malodor.

On the other hand, microorganisms are fed to livestock as a feed containing live bacteria for the purpose of promoting the growth of livestock and preventing / treating diarrhea. For example, JP-A-60-224451 discloses a feed composition containing microbial cells grown in a medium containing inorganic selenium. Also, JP-A-63-636
No. 20 discloses an intestinal stabilizer containing lactic acid bacteria, saccharifying bacteria, and butyric acid bacteria as active ingredients. In addition, JP-A 1-28858
The publication discloses an animal feed comprising vegetative spores of Bacillus cereus having a growth improving effect.

In addition to the above-mentioned microbial materials, many products are on the market, but the effects and safety of these products have not been evaluated, and some of them have unclear scientific evidence. is there.

[0005]

The present inventor has also developed feed additives containing microorganisms and enzymes, especially fermented feeds, in order to promote the conversion of agricultural and livestock waste into organic fertilizer and prevent livestock pollution and pest damage. As a result of intensive studies to achieve this, among certain microorganisms existing in soil, certain cellulolytic bacteria, filamentous fungi, actinomycetes, yeasts, nitrifying bacteria, sulfur bacteria, etc. were purely cultured and mixed with an organic substrate. When the fermented and fixed product is added to the feed and fed, not only the intestinal bacteria of the livestock are stabilized and the productivity is improved, but also the malodor of livestock manure is reduced,
It was confirmed that the composting of manure was promoted and that the reduction of the compost into the soil suppressed the growth of phytopathogenic filamentous fungi in the soil and prevented the pests from being damaged. It was also found that when fed to ruminants, methane generation in the rumen (ruminal) was suppressed. Furthermore, as a result of investigating to clarify the physiological action, it was unexpectedly discovered that the various physiological actions described above are exhibited. In addition, when further research was conducted to clarify the relationship between these effects and the constituent microorganisms, it was discovered that the above-mentioned effects could not be obtained remarkably by the constituent microorganisms alone, but a synergistic effect was obtained for the first time with a composite microorganism. did.

[0006] Furthermore, the present inventor has conducted extensive studies to establish a method for producing a large amount of fermented feed while maximizing the effect of the fermented feed, and as a result, mixed cultures of the respective constituent microorganisms with a substrate. When fermenting, it was discovered that not only this object can be achieved by fermenting with a certain calcium-containing base material, but also the above-mentioned effect is enhanced. The present invention has been completed based on these findings.

[0007]

According to the present invention, at least Bacillus subtilis,
Bacillus natto (bacillus natto), Bacillus megaterium (bacillus megaterium), Lactobacillus acidophilus (Lactobacillus acidophilus), Lactobacillus plantaru (Lactobacillus plantaru)
m), Lactobacillus brev
is), Lactobacillus case
i), Streptococcus faecalis
s faecalis), Streptococcus lactis
ococcus lactis), Streptococcus thermophilus, Clostridium butyricum, Saccharomyces cerevisiae and Candida utilis, and their adsorbed microorganism groups and their microorganisms. Consists of
The substrate comprises at least a calcium-containing substrate,
There is provided a complex microbial preparation characterized in that at least a part of the calcium component of the calcium-containing base material constitutes a biocompatible organic calcium compound by the action of the microorganism group.

The microbial group used in the present invention is one that is present in soil and is not a special one, and is commonly used in test research in general, and may be of any of the above types. The strain is not particularly limited. Therefore, commercially available strains or those that can be easily obtained by those skilled in the art from a microorganism depository institution or a microorganism preservation institution can be used. For example, Bacillus subtilis
tilis) has been deposited with the American Type Culture Collection (ATCC).
The 6051 strain, ATCC6633 strain and the like can be used. AT for Bacillus natto
CC15245 strain, Institute of Fermentation, Osaka (Institute for Fermentati
on, Osaka) (hereinafter, IFO) IFO3336
You can use stocks. Bacillus megaterium (bacill
For us megaterium), ATCC14581 strain and ATCC14945 strain can be used. Regarding Lactobacillus acidophilus, ATCC4356 strain, ATCC11506
Shares, IFO3205 shares and Deutsche Samulung.
Von Microorganismen (Deutsche Sammulung vo
n Mikroorganismen) (DSM)
M20077 strain etc. can be used. For Lactobacillus plantarum,
ATCC8014 strain, ATCC14917 strain, DSM2
For example, strain 0314 can be used. For Lactobacillus brevis, see ATCC14
869 strains, IFO12520 strains, AHU1508 strains (A
HU is the Faculty of Agriculture, Hokkaido University: Faculty of Agriculture, H
abbreviation of okkaido University) etc. can be used. For Lactobacillus casei,
ATCC393 strain, DSM20021 strain and the like can be used. Streptococcus faecalis
s faecalis), ATCC 14429 strain, AT
CC19433 strain etc. can be used. For Streptococcus lactis, A
TCC19435 strain, IFO12546 strain, etc. can be used. Streptococcus thermophilus
occus thermophilus), ATCC 19258
Strains, IFO3863 strains, etc. can be used. Regarding Clostridium butyricum, ATCC 19398 strain, IFO3852 strain and the like can be used. Saccharomyce
cerevisiae), ATCC 7752 strain, IF
O0216 strain etc. can be used. For Candida utilis, see ATCC 9256.
Strain, IFO0396 strain, etc. can be used.

In addition to these microorganisms, nitrifying bacteria and sulfur bacteria can be added for the purpose of reducing the generation of malodorous substances. The nitrifying bacterium is a bacterium capable of oxidizing ammonia or nitrite to nitrite or nitric acid, respectively, and examples of the nitrifying bacterium that can be used in the present invention include, for example, Nitrosomonas europaea and Nitrobacter wisteria. Nogrady Ski (Nitorobacter wino
gradskyi) can be used. On the other hand, a sulfur bacterium is a bacterium that oxidizes sulfur or an inorganic sulfur compound to produce energy, and examples of the sulfur bacterium that can be used in the present invention include Sulfolobus acidocaldarius (Sulfolobus).
acidocaldarius) can be used. The strains of these bacteria are not particularly limited, and those commonly available to those skilled in the art can be used. For example, Nitrosomonas
AT for Europe (Nitrosomonas europaea)
CC25978 strain etc. can be used. Nitrobacter
As for Winographis ski (Nitorobacter winogradskyi), ATCC25391 strain and the like can be used. As for sulfur bacteria, ATCC33909 strain and the like can be used.

It is also possible to add a thermostable Bacillus bacterium for the purpose of promoting fermentation even at high temperatures in compost fermentation and the like. For example, Bacillus thermophilus (Bacillus thermophilus)
s thermophilus) can be used. Bacillus
For the Bacillus thermophilus,
Sharp et al. (R.Sharp, M.Munster, T.Atkinson (PHLS Cent
re for Applied Microbiology & Research, Salisbury);
A.Vivian (Bristol Polytechnic, Bristol); S.Ahmad (Tren
Polytechnic, Nottingham), UK)
It's Potential Biotechnology
l.Extreme Environ.Its Potential Biotechnol.), pp. 62-81, 1989 (Title: Taxonomic and genomic studies of Bacillus thermophilus.
ngetic studies of Bacillus thermophilus), European Microbial Society Symposium Federation, Troia).

The fact that the above-mentioned microorganism is contained as a constituent in the complex microorganism preparation of the present invention can be identified by the following method. That is, Bacillus subtilis
llussubtilis), Bacillus nattou (bacillus natt)
o), Bacillus megaterium
), PEES agar medium (manufactured by Eiken Chemical Co., Ltd .:
Yeast extract 2.5g, peptone 10g, gelatin 30
g, lactose 2g, mannitol 10g, sodium chloride 75
g, dipotassium phosphate 5g, agar 15g, water 1000m
Isolate using l). In the case of Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus casei, LBS selective agar medium
(Becton Dickinson Co. Ltd., (BBL) (USA): Lab-lem
co powder (Oxoid) 8g, tryptic case peptone 10
g, yeast extract 5g, KH ₂ PO ₄ 6g, ammonium citrate 2g, glucose 20g, sorbitan mono-oleate 1g, sodium acetate 25g, MgSO ₄ · 7H _{2
O0.6g, MnSO 4 · 7H 2 O0.1g} , FeSO
₄ · 7H ₂ O34mg, agar 15 g, water 1000ml)
To isolate. Streptococcus faecalis
ptococcus faecalis), Streptococcus lactis, Streptococcus thermophilus,
KF Streptococcal agar (BB
L: Polypeptone 10 g, yeast extract 10 g, sodium chloride 5 g, sodium glycerophosphate 10 g, maltose 20 g, lactose 1 g, sodium nitride 0.4 g, agar 2
0 g, 1000 ml of water). N for Clostridium butyricum
N Clostridium selective agar medium (Peptone 40 g, N
a ₂ HPO ₄ 5 g, KH ₂ PO ₄ 1 g, sodium chloride 2 g, MgSO ₄ 0.1 g, glucose 2 g, agar 25
g, 100 ml of 50% egg yolk solution, 10 ml of 2% neomycin sulfate, 1000 ml of water). In the case of Saccharomyces cerevisiae and Candida utilis, isolate with potato dextrose (PD) agar medium (potato infusion, 200 g, glucose 20 g, agar 15 g, water 1000 ml). The culture conditions are
Depending on each microorganism, Streptococcus, Bacillus, Saccharomyces and Candida are aerobic, and Lactobacillus and Clostridium are anaerobic at 37 ° C under 1
Incubate for 2 to 24 hours. Minitech bacteria identification system (Be
Made by cton Dickinson Co. Ltd., (BBL), USA, Bergey's Manual of Determinative Bactrio
logy) 8th Edition, edited by Kazuo Komagata: Microbial Classification Experimental Methods, published by the Society Publishing Center (1982) and Masuo Nakano: Chemistry and Biology, Vol. 18, pp. 15-24, published by the Society Publishing Center (1980). It can be identified by the chemical classification method based on the described cell membrane chemical structure and microscopic observation.

The separation of nitrifying bacteria can be carried out as follows. In the case of Nitrosomonas bacterium, the liquid A [[NH ₄ ) ₂ SO ₄ is used as a nitrifying bacteria selective separation medium.
11.0g, MgSO ₄ · 7H ₂ O1.4g and Fe
SO ₄ .7H ₂ O 0.3 g dissolved in distilled water 100 ml] and solution B (KH ₂ PO ₄ 1.36 g dissolved in distilled water 100 ml) at a ratio of 9: 1 (volume ratio).
And mixed with 0.1N NaOH solution to pH 8.0-8.2.
After culturing using the medium prepared as described above, colonies can be separated on a silica gel plate and identified according to the above-mentioned Berjay's Manual. In the case of a bacterium of the genus Nitrobacter, as solution A, 2.5 g of KNO ₂ and MgSO ₄
· 7H ₂ O1.4g and FeSO ₄ · 7H ₂ O0.3
except that g dissolved in 100 ml of distilled water is used
It can be identified by the same method as for Nitrosomonas bacteria. on the other hand,
For the isolation of sulfur bacteria, DSM120 modified medium (K
H ₂ PO ₄ 0.227 g, K ₂ HPO ₄ 0.348 g,
_{NaCl 2.25g, MgSO 4 · 7H 2} O0.5
g, CaCl · 2H ₂ O 250 mg, MnSO ₄ · 2H
_{2 O0.09mg, FeSO 4 · 7H 2} O2mg, Co
SO ₄ 0.6 mg, ZnSO ₄ 0.3 mg, CuSO ₄
・ 7H ₂ O 0.03 mg, H ₃ BO ₄ 0.9 mg, Na
₂ MoO ₄ .2H ₂ O 0.091 mg, NiCl ₂ 0.
06mg, NaHCO ₃ 0.85g, cysteine hydrochloride _{0.3g, Na 2 S · 9H 2} O0.3g, yeast extract 2
g, vitamin solution (biotin 2 mg, folic acid 2 mg, pyridoxine hydrochloride 10 mg, thiamine hydrochloride 5 mg, riboflavin 5 mg, niacin 5 mg, DL-calcium pantothenic acid 5 mg, vitamin B ₁₂ 0.1 mg, p-aminobenzoic acid 5 mg, lipo Acid 5 mg to distilled water 100
(0 ml) 10 ml, Resazurin (1 mg dissolved in 1000 ml of distilled water) was used as a separation medium at 40 to 45 ° C. for 4 to 6 weeks, and the cells can be identified according to the above Barjay's Manual. .

In the preparation of the present invention, the above-mentioned microorganism group is adsorbed on a base material containing at least a calcium-containing base material.
The calcium-containing base material only needs to contain calcium as a main component, and may be an inorganic material or an organic material. The calcium content is preferably 25% by weight or more, and preferably 25 to 80% by weight. The calcium-containing base material is used so that the calcium content is 25% by weight or more based on the whole base material. If it is less than 25% by weight, the composition ratio of the biocompatible organic calcium compound described later becomes small, which is not preferable. Specific examples of the calcium-containing base material include calcined bone powder such as animal bones and fish bones, fossil shells, coral, synthetic apatite, calcium phosphate, calcium carbonate, etc., but are not limited thereto, and other calcium The containing base material can be used without limitation as long as the calcium content is 25% by weight or more. These calcium-containing substrates are 1
One type may be used, or a plurality of types may be mixed and used. The calcium-containing base material used in the present invention may be a solid such as powder, or a liquid dissolved in a solvent. In the present invention, the calcium-containing base material can be used in combination with other base materials. For example, chitin or its derivative,
It can be used as a mixture with alginic acid, cellulose, rice bran, bran, soybean meal, okara, etc. used as a feed base material.

In the present invention, at least a part of the calcium component of the calcium-containing substrate constitutes a biocompatible organic calcium compound which is easily absorbed by the living body by the action of the microorganism group. Examples of the biocompatible organic calcium compound include organic acid calcium such as calcium lactate, calcium fumarate, calcium malate and calcium citrate, and peptide-calcium compounds such as casein phosphopeptide of calcium-binding protein. These are hardly present in the above-mentioned calcium-containing substrate itself, but are present by the action of the microorganisms used in the present invention converting at least a part of the calcium into such a biocompatible organic calcium compound. To do. The fact that the preparation of the present invention contains this biocompatible organic calcium compound can be confirmed by analyzing these organic compounds. For example, Buren et al. (Rolf HOBuhlen and Jeremias HRKagi: FEBS
Letters, 39 (2), 229-233 (1974)) can be used for analysis and identification by the method referred to. In detail, 1 sample
Suspension in 0 mM Tris-HCl buffer (pH 8.6) for extraction, centrifugation to obtain a supernatant, which is a Sephadex G-25 or G-75 column, and DEAE-Sephadex A- Using 25 columns, fractionate by gel filtration with 10 mM Tris-HCl buffer. Minerals such as Ca and Mg in each fraction can be analyzed by atomic absorption spectrometry, and organic acids can be analyzed by gas chromatography to identify the presence of organic calcium. However, the effect of enhancing the action of the microbial group is not sufficient simply by mixing the above-mentioned organic acid calcium with the microbial group, and the complex microorganism of the present invention is brought about by fermenting the calcium-containing substrate with the microbial group. Since the effect is more remarkably improved and various physiological activities are exhibited, the biocompatible organic calcium compound as used herein is considered to be a complex of calcium and a group of microorganisms.

In the preparation of the present invention, the number of each strain of the above-mentioned microorganism group is 10 ⁴ cfu (colony forming unit) or more, preferably 10 ⁶ per 1 g of the total weight of the above-mentioned microorganism group and its adsorption base material. ^-8 cfu is included. 10 ⁴ cfu / g
If it is less than the above range, the desired effect is not noticeable.

The present invention also provides a method for producing the above-mentioned complex microbial preparation. As a production method, two kinds of methods are conceivable: a method of purely culturing each of the constituent microorganisms, then mixing them with an adsorption base material and fermenting them, and a method of mixing the constituent microorganisms with the adsorption base material from the beginning and culturing. .

That is, according to the present invention, at least Bacillus subtilis, Bacillus subtilis
Natto (bacillus natto), Bacillus megaterium (Lactobacillus acidophilus), Lactobacillus plantarum (Lactobacillus plantarum), Lactobacillus brevis, Lactobacillus Lactobacillus lactis Streptococcus faecali
s), Streptococcus l (Streptococcus l)
actis), Streptococcus thermophilus (Strep)
tococcus thermophilus), Clostridium butyricum (Clostridium butyricum), Saccharomyces cerevisiae (Cacida) and Candida utilis (Candida utilis), respectively. There is provided a method for producing the complex microbial formulation, which comprises fermenting the resulting mixture.

Furthermore, according to the present invention, at least:
Bacillus subtilis, Bacillus natto, Bacillus megaterium, Lactobacillus acidophilus, Lactobacillus plantarum,
Lactobacillus brevis,
Lactobacillus casei, Streptococcus faecal
is), Streptococcus (Streptococcus)
lactis), Streptococcus thermophilus (Stre
ptococcus thermophilus), Clostridium butyricum, Saccharomyces cerevisiae, and Candida utilis mixed microbial groups with an adsorbent substrate containing at least a calcium-containing substrate and culturing. A method for producing the above-mentioned complex microbial preparation is provided.

The former method will be described in detail below.
As a first step, liquid pure culture is performed on each of the constituent microorganisms of the complex microorganism of the present invention. The liquid medium for culture of each microorganism is not particularly limited, but usually, Streptococcus bacteria, Bacillus bacteria, Lactobacillus bacteria, Saccharomyces bacteria and Candida bacteria are under aerobic conditions, and Clostridium bacteria are Under anaerobic conditions,
Nutrient broth medium (BBL:
Gerisate peptone 5g, beef extract 3g, water 1000
ml) and DSM 1 broth medium (K ₂ HP
O ₄ 1.4g, KH ₂ PO ₄ 4g, MgSO ₄ · 7H ₂
O160mg, CaCl ₂ .6H ₂ O 80mg, MnS
_{O 4 · 7H 2 O8mg, FeSO} 4 · 7H 2 O8mg,
Diammonium hydrogen citrate 4g, sodium acetate 4
g, meat peptone 20g, yeast extract 5g, glucose 30
g, 1000 ml of distilled water) at 37 ° C. until the logarithmic growth phase. Usually, the logarithmic growth phase is reached in 7 to 14 hours.

When nitrifying bacteria and sulfur bacteria are added to the microbial group, the liquid pure culture is carried out as follows. That is, in the case of Nitrosomonas bacteria, (NH ₄ ) ₂
SO ₄ 0.5 g, KH ₂ PO ₄ 0.07 g, MgSO ₄
.7H ₂ O 0.05 g and CaCl ₂ .2H ₂ O0.
Dissolve 05 g in 1000 ml of distilled water to give 5% Na
It is possible to culture by using a medium adjusted to pH 8.0 with a ₂ CO ₃ solution as a nitrifying bacteria culture medium. In the case of Nitrobacter, (NH ₄ ) ₂ SO ₄ 0.5 g
It is possible to culture under the same culture conditions using the same medium as that used for Nitrosomonas bacteria except that 0.2 g of KNO ₂ is used instead of. All the culture conditions can be anaerobic culture at 37 ° C. Further, in the case of sulfur bacteria, 40 to 4 using the DSM120 modified medium
A large amount of pure culture can be performed at 5 ° C.

As the medium for culturing nitrifying bacteria and sulfur bacteria, when the ATCC deposited bacteria are used, the media listed in the ATCC deposited list can be used. Specifically, in the case of Nitrosomonas europae, ATC
C1573 medium, ATCC480 medium in the case of Nitrobacter winogrady ski, sulpholobus
In the case of Acidocaldarius, ATCC1723 medium can be used.

From the obtained pure culture of each microorganism, cells are collected by continuous centrifugation, washed with a phosphate buffer and freeze-dried.

In the second step, 1 kg of sterilized 100-mesh rice bran powder was added to 2 to 2 of each lyophilized microbial cell.
Inoculate 5 mg of each suspension in 1 ml of mineral water described below, add 2% by weight of the enzyme solution described below, 5% by weight of molasses, and 30% by weight of mineral water to it, and add at 4 to 4 at 38 ° C.
Fermentation is carried out under aerobic conditions for 8 hours. Mineral water includes K ₂ HPO ₄ 1.4g, KH ₂ PO ₄ 4g, MgS
_{O 4 · 7H 2 O160mg, CaCl} 2 · 6H 2 O80
_{mg, MnSO 4 · 7H 2 O8mg} , FeSO 4 · 7H
_A solution obtained by dissolving 8 mg of ₂ O in 1000 ml of water can be used. As the enzyme solution, the one prepared as follows can be used. That is, raw soybeans that have been naturally dried are roughly crushed. Weight ratio of this raw soybean crushed powder 3
To 0%, rice bran and the mixture stirred added 70% relative to the mixture 1 kg, 20 wt% Na ₂ CO ₃ solution 1000m
1 and well stirred, and fermented for 5 days under conditions of a humidity of 80% and a temperature of 40 to 50 ° C. After that, transfer this to a boiling kettle and add 1 kg of this raw material to 2000 m of mineral water.
l, crude glucose (eg molasses, etc.) 100g
(10% concentration) is added and gradually heated with stirring, and boiled and boiled. During this time, all suspended matter on the liquid surface is removed. After boiling, the mixture is boiled for about 1 hour, then the heating is stopped and the mixture is naturally cooled to room temperature and then centrifuged, and the supernatant is filtered to obtain a yellowish brown transparent liquid as an enzyme solution.

After the fermentation temperature is raised to 60 to 70 ° C. by static fermentation, the culture is stirred, and then stirred every 24 hours and cultured for 5 days to obtain the original bacteria. 60 kg of a base material containing a calcium-containing base material is mixed with 1 kg of this original bacterium, 2% by weight of the above-mentioned enzyme solution, 5% by weight of molasses and 30% by weight of mineral water are added, and the mixture is allowed to stand at 38 ° C. for 24 hours for fermentation. . After the fermentation is completed, the mixture is stirred and further fermented for 6 to 12 hours, and the fermentation temperature is 5
After the temperature is raised to 5 to 65 ° C., it is dried with low temperature cold air to a water content of 12% or less to obtain the complex microorganism preparation of the present invention. In the above method, the final bacterial count of each microorganism is usually 10 ⁴ viable bacterial count (cfu).
/ G or more, preferably 10 ^6-8 cfu / g. If necessary, by mixing each of the constituent microorganisms that have been purely cultivated by the method for preparing the original bacteria with the fermentation-completed microorganisms, the number of all microorganisms may be adjusted to fall within the above range. .

The latter method is a method of culturing the above-mentioned 13 kinds or a mixture of the above-mentioned 13 kinds of microorganisms including the above-mentioned nitrifying bacteria and sulfur bacteria from the beginning with an adsorbent substrate. Examples of the microbial group include, for example, a mixed microorganism (indicator for identification: EG) that the inventor separates from the humus soil and natural lake bottom material and retains it (due to a refusal of deposit from the Institute of Biotechnology, Institute of Industrial Science and Technology). Self-deposit) can be used.

The culture can be carried out usually using the media shown in Table 1, but it is preferable to use the above-mentioned enzyme solution for more efficient culture.

[0027]

[Table 1]

The culture conditions are not limited, but are usually 20 to 3
After culturing at 0 ° C. for 24 hours, it can be grown by scaling up in the same medium and further culturing at 20 to 30 ° C. Culturing is usually performed under facultative anaerobic conditions.
Bacteria were collected from the obtained culture by continuous centrifugation, suspended in 1 ml of mineral water in the same manner as described above, and inoculated into 1 kg of rice bran powder, 2% by weight of the enzyme solution and 5 molasses molasses were added thereto. Add 30% by weight of mineral water and 24% at 38 ° C
~ 48 hours static fermentation under aerobic conditions. Fermentation temperature is 60
The mixture is heated to ˜70 ° C., stirred, and then stirred every 24 hours and cultured for 5 days. 60 kg of a substrate containing a calcium-containing substrate is mixed with 1 kg of this culture, 2% by weight of the above-mentioned enzyme solution, 5% by weight of molasses and 30% by weight of mineral water are added, and the mixture is allowed to stand at 38 ° C. for 24 hours for fermentation. . After completion of fermentation, the mixture is stirred and further fermented for 6 to 12 hours, and the fermentation temperature is 55 to 6
After the temperature is raised to 5 ° C. and the water content is 12% or less, low temperature cold air drying is performed to obtain the complex microorganism preparation of the present invention.

In the above method, the final number of bacteria of each microorganism is usually 10 ⁴ cfu / g or more, preferably 10 ^6-8 cfu / g.
However, if the bacterial population of any of the microorganisms does not reach the desired level, a pure culture of the microorganism may be prepared by the method described above and supplemented with additional mixing.

In any method, it is necessary that the adsorbing substrate for microorganisms contains at least a calcium-containing substrate. Calcium-containing substrates and other substrates that can be used in the method of the present invention include those mentioned above. In this method, at least a part of calcium contained in the calcium-containing base material is converted into the above-mentioned biocompatible organic calcium compound in the process of culturing and fermenting the microorganism group. It is presumed that this biocompatible organic calcium compound enhances various physiological activities of the complex microorganism group used in the present invention, which will be described later.

[0031]

The complex microbial preparation of the present invention exhibits various beneficial physiological actions as will be described later, and thus can be used for medicinal purposes. In that case, the complex microorganism preparation of the present invention can be administered as it is, but can also be administered in various dosage forms for oral administration. For example, granules, tablets,
It can also be in the form of a pharmaceutical composition such as a pill or capsule. In that case, the composite microorganism preparation of the present invention can be formulated by mixing the constituent components used in ordinary medicines. Examples of the pharmaceutical component include, in the case of granules, excipients such as lactose, sodium hydrogen carbonate, sodium chloride, sucrose, mannitol, starch, crystalline cellulose, calcium sulfate, precipitated calcium carbonate, calcium phosphate, starch, crystalline Disintegrator such as cellulose, 10 to 20% aqueous solution of gum arabic, 5
Examples of the binder include a 10% starch paste solution, a 5-10% aqueous solution of PVP or an ethanol solution, a 1-2% aqueous solution of a cellulose derivative, or an ethanol solution. In the case of tablets, excipients such as lactose, sucrose, glucose, starch, crystalline cellulose and the like, 5 to 10% starch paste solution, hydroxypropylcellulose solution, carboxymethylcellulose solution, gum arabic solution, gelatin solution, glucose solution, sucrose Liquid, tragacanth liquid, binder such as sodium alginate, disintegrating agent such as starch and carboxymethyl cellulose calcium, lubricant such as magnesium stearate, purified talc, stearic acid and calcium stearate, and the like. In addition, in the case of pills and capsules, a coating agent may be mentioned as a constituent component in addition to the above-mentioned excipients and binders. Examples of the coating agent include sucrose, starch, talc and the like in pills, and gelatin, glycerin, sorbitol and the like in capsules.
These compositions can be produced by a conventional method for producing veterinary drugs. Moreover, you may mix | blend usual stabilizers, such as antioxidant, as needed.

Further, since the complex microbial preparation of the present invention has a odor reducing effect on feces, a composting promoting effect on stool and a methane generation suppressing effect on rumen of ruminant animals, it can be used as a feed additive for livestock in addition to the above medicinal use. It can also be used. When used for feed, the composite microorganism preparation of the present invention can be directly fed to livestock, or it can be mixed with a feed base material and fed. As the feed base material, corn or barley which is usually used as feed,
Examples thereof include, but are not limited to, wheat, such as wheat, rye, and oats, bran, such as bran and bran, oil meal such as soybean meal, rousan meal, flour, and the like. The shape in that case is not limited, and it may be in the shape of an ordinary feed such as powder or pellet. Molding can be performed by a method similar to the method used for the production of ordinary mixed feed and mixed feed. The manufacturing method is described, for example, in "Combined feed course (second volume)", pages 15 to 27, published by Chikusan Publishing Co., Ltd.

The complex microorganism preparation of the present invention can be normally orally administered as described above. In the case of humans and domestic animals such as cows and pigs, the dose is usually 3 to 8 g / day. In the case of small animals, the amount of food intake is 0.0
5 to 0.2% by weight can be supplied.

[0034]

【Example】

Example 1 (Production Example of Formulation of the Present Invention) The complex microbial preparation of the present invention was prepared as follows.
The constituent microorganisms were isolated from brown forest soils in western Japan and northern Japan, respectively. The isolation method was carried out by the following method using the above-mentioned separation medium. That is, PEES medium (manufactured by Eiken Chemical Co., Ltd.) was used for Bacillus and LBS selective agar (Becton Dickinson) for Lactobacillus.
Co. Ltd., (BBL) and TOS agar medium (previously mentioned), Streptococcus agar medium KF Streptococcus agar medium (previously mentioned), Clostridium spp.
Yeasts (Saccharomyces and Candida) were isolated using N. clostridium selective agar using potato dextrose (PD) agar (described above).
Culture conditions are aerobic conditions for Streptococcus bacteria, Bacillus bacteria, Lactobacillus bacteria and Saccharomyces bacteria (yeast), Candida bacteria, and Clostridium bacteria under anaerobic conditions at 37 ° C. went. In addition, Nitrosomonas and Nitrosobacter were isolated by culturing at 37 ° C under aerobic conditions using the above-mentioned nitrifying bacteria selective separation medium. In addition, sulfur bacteria are the above-mentioned D
It was isolated by culturing at 40 to 45 ° C under anaerobic conditions using SM120 modified medium. Minitech Bacterial Identification System (Becton Dickinson Co. Ltd., (BBL))
Manufactured in the United States), Bergey's Manual of Determinative Bacteriology
Determinative Bactriology) 8th edition, edited by Kazuo Komagata: Experimental method for classification of microorganisms, published by Society Publishing Center (1982) and Masuo Nakano: Chemistry and Biology, Volume 18, 15-24.
Page, published by the Society Publishing Center (1980), and identified by chemical classification by cell membrane chemical structure and microscopic observation. As a result, the followings were recently identified. That is,
Bacillus subtilis, Bacillus natto, Bacillus megaterium, Lactobacillus acidophilus, Lactobacillus plantarum,
Lactobacillus brevis,
Lactobacillus casei, Streptococcus faecal
is), Streptococcus (Streptococcus)
lactis), Streptococcus thermophilus (Stre
ptococcus thermophilus), Clostridium butyricum, Saccharomyces cerevisiae, Candida
Candida utilis, Nitrosomonas europaea, Nitorobacter winogradskyi, Sulfolobus acidocal
darius) was identified.

The colonies of the identified constituent microorganisms were separated and subjected to liquid pure culture. Streptococcus bacteria,
Bacillus bacterium, Lactobacillus bacterium, Saccharomyces bacterium and Candida bacterium under aerobic conditions, Clostridium bacterium under anaerobic conditions, the above-mentioned Nutrient broth medium and DSM1 broth (DSM1 broth) Using the medium, the cells were cultured at 37 ° C until the logarithmic growth phase. In addition, nitrifying bacteria (Nitrosomonas bacteria,
Nitrobacter) was cultured for 12 to 24 hours at 37 ° C. under aerobic conditions using the above-mentioned nitrifying bacteria culture medium. Sulfur bacteria were analyzed under the anaerobic condition using the above modified DSM120 medium.
The cells were cultured at 0 to 45 ° C for 4 to 6 weeks. The thus obtained pure culture of each microorganism was collected by continuous centrifugation, washed with a phosphate buffer and freeze-dried.

Next, 5 mg of each freeze-dried cell was suspended in 1 ml of the above-mentioned mineral water and sterilized.
1 kg of rice bran powder of 00 mesh was inoculated, 2% by weight of the enzyme solution, 5% by weight of molasses and 30% by weight of mineral water were added to the powder, and the mixture was fermented at 38 ° C. for 48 hours under aerobic conditions. . After the fermentation temperature rises to 65 ° C, the mixture is stirred, and then 24
After stirring for 5 hours and culturing for 5 days, the original bacterium (identification number: EG) of the complex microorganism consisting of the above 17 kinds of bacteria including nitrifying bacteria and sulfur bacteria was obtained. Self-deposited by refusal). 70% by weight of fossil shellfish and 3 rice bran as a calcium-containing base material for 1 kg of this original bacterium
60 kg of a base material containing 0% by weight is mixed, and the above enzyme solution 2
% By weight, 5% by weight molasses and 30% by weight of mineral water are added, and the mixture is allowed to stand at 38 ° C. for 24 hours for fermentation. After the completion of fermentation, the mixture was stirred and further fermented for 12 hours. After the fermentation temperature was increased to 60 ° C, it was dried with low temperature cold air to a water content of 12% or less to obtain a complex microorganism preparation of the present invention (Preparation 1).

On the other hand, a composite microorganism of the present invention containing neither nitrifying bacteria nor sulfur bacteria was prepared. That is, the following 13 kinds of bacteria were separated from the original bacteria of the complex microorganism prepared by the above method. Separation of Bacillus subtili
s), Bacillus natto, and Bacillus megaterium are PEES
Using a medium (Eiken Chemical Co., Ltd.), Lactobacillus
Lactobacillus acidophilus, Lactobacillus plantarum
), Lactobacillus brevi
s), Lactobacillus casei
Is an LBS selective agar medium (Becton Dickinson Co. Ltd., (B
BL)), Streptococcus faecalis, Streptococcus faecalis
KF Streptococcus agar medium was used for lactis (Streptococcus lactis) and Streptococcus thermophilus, and NN for Clostridium butyricum.
Saccharomyces cerevisiae and Candida utilis were isolated on Clostridium selective agar using potato dextrose agar. Culture conditions include Streptococcus bacteria, Bacillus bacteria and Lactobacillus bacteria, Saccharomyces bacteria (yeast),
Candida was cultivated at 37 ° C. under aerobic conditions and Clostridium bacteria under anaerobic conditions. After culturing, the identification was confirmed by the above-mentioned identification method. Colonies of the identified constituent microorganisms were separated and subjected to liquid pure culture. Streptococcus spp, Bacillus spp, Lactobacillus spp, Saccharomyces spp, and Candida spp under aerobic conditions, and Clostridium spp under anaerobic conditions.
Broth) medium and DSM1 broth medium were used to culture at 37 ° C. until the logarithmic growth phase. The thus obtained pure culture of each microorganism was collected by continuous centrifugation, washed with a phosphate buffer and freeze-dried.

The lyophilized cells of each bacterium thus obtained were cultured in the medium shown in Table 1 at 25 ° C. for 24 hours under aeration and anaerobic conditions, and then scaled up in the same medium to further 25 ° C.
Proliferated by culturing in. Bacteria were collected from the obtained culture by continuous centrifugation, suspended in 1 ml of mineral water in the same manner as described above, and inoculated into 1 kg of rice bran powder, 2% by weight of the enzyme solution and 5 molasses molasses were added thereto. % By weight and 30% by weight of mineral water were added and static fermentation was carried out at 38 ° C. for 48 hours under aerobic conditions. After the fermentation temperature has risen to 65 ° C., the mixture is stirred, and then stirred every 24 hours and cultured for 5 days. 70 kg of fossil shellfish as a calcium-containing substrate for 1 kg of this culture
60 kg of a base material containing 30% by weight of rice bran and 30% by weight of rice bran is mixed, 2% by weight of the enzyme solution, 5% by weight of molasses and 30% by weight of mineral water are added, and the mixture is allowed to stand at 38 ° C. for 24 hours for fermentation. After completion of the fermentation, the mixture was stirred and further fermented for 12 hours, and after the fermentation temperature was increased to 60 ° C., low-temperature cold air drying was performed to a water content of 12% or less to obtain a complex microorganism preparation of the present invention (Preparation 2).

Examples 2 and 3 and Comparative Examples 1 and 2 (Blood cholesterol lowering action) F344 male rats purchased from CLEA Japan, Inc. (Tokyo) were fed a high fat diet containing 1% cholesterol (Table 2) for 4 weeks. High cholesterol rats were obtained. The test rats were allowed free food and water during the test.

Rats (8 weeks old) thus obtained were 8
Heads were divided into two groups, one of which was fed a high cholesterol-containing feed containing 15% of the complex microbial preparation of the present invention (formulation 2) prepared in Example 1 (Example 2), and the other 15% of rice bran. A high-cholesterol-containing feed containing was added (Comparative Example 1). Also, 10 animals were selected from another same test rat group and divided into 2 groups of 5 animals, one of which was a basic feed containing 15% of the complex microbial preparation of the present invention (formulation 2) prepared in Example 1 (Table 2) is paid (Example 3), and the other 15%
The basic feed containing the rice bran of Example 2 was fed (Comparative Example 2).
The feeding was performed for 8 weeks, and the excreted feces were collected every other week for 24 hours. Further, once a week, the rat was not fixed and blood was collected from the jugular vein, placed in a centrifuge tube containing an anticoagulant, and centrifuged to obtain a serum sample. After 8 weeks, the test rat was killed by ether anesthesia, the liver was removed, washed with 0.9% physiological saline, weighed and stored frozen.

The total cholesterol content in the serum sample was measured using a commercially available measurement kit (TDX system analysis kit manufactured by Abbott, USA). In addition, neutral sterols in the liver were extracted from the extracted liver sample with total lipids using a chloroform-methanol mixed solvent (volume ratio 2: 1), and then Matsubara et al. (Agricultural and Biological Chemistry 54,
1143 to 1148, 1990) acetylated by gas chromatography (Shimadzu, Shimadzu)
14A GLC).

The results are shown in FIGS. 1, 2 and 3. In FIG. 3, the hatched line shows the data of the formulation administration group of the present invention, and the black mark shows the data of the control group. The measurement data of the administration group and the non-administration group were tested for a significant difference by Student's t-test. As is clear from the results, the complex microorganism preparation of the present invention has an effect of significantly lowering blood cholesterol level.

[0043]

[Table 2]

Example 4, Comparative Example 3 (Intestinal regulation action) As an index showing the intestinal regulation action, the ratio of useful bacteria to Escherichia coli in feces was examined. That is, the number of bacteria in rat feces collected in Example 3 and Comparative Example 2 was measured and compared. The number of bacteria in feces was measured as follows. Escheric
hia coli) and streptococcus are bacteria that are resident in the intestine, and these are stool samples cultured at 37 ° C. for 2 days using desoxycholate agar medium and KF Streptococcal agar medium (Becton Dickinson, USA), respectively. The number of colonies was counted. Bifidobacteria (Bifidobacteriu) are useful as enterobacteria.
m) genus bacterium, Eubacterium genus bacterium,
And lactic acid bacteria such as Lactobacillus genus bacteria, a fecal sample was cultured at 37 ° C. for 5 days by a gas pack method using BS agar medium, ES agar medium and LBS agar medium (manufactured by Becton Dickinson, USA), respectively, The number of colonies was counted. The ratio of other bacteria to Escherichia coli was calculated from the obtained number of colonies and shown over time (FIG. 4). As is clear from FIG. 4, the group administered with the complex microbial preparation of the present invention (Example 4) promotes the growth of lactic acid bacteria and bifidobacteria, which are useful for intestinal regulation, as compared with the control group (Comparative Example 3). It is thought to have an effect.

Example 5 and Comparative Example 4 (Carcinogenicity) The carcinogenicity of the composite microorganism preparation of the present invention was evaluated using nude mice. Incidentally, the antimicrobial effect of the present invention is presumed that the cell membrane substance of the microorganism of the present invention is acting, and the reason for eliminating the effect of administering a live bacterial agent to nude mice, the complex microbial preparation of the present invention It was performed by administering the bacterial cell component. The bacterial cell component was prepared by the following method.

Complex microorganism prepared in Example 1 (Formulation 2)
The cultured bacterial cells of (1) were collected, and chloroform-methanol (1:
2) The mixture was extracted three times and degreased. After removing the solvent from the defatted bacterial cells with an aspirator, 1 is added to 60 mg of the defatted bacterial cells.
Suspend in ultrapure water at a ratio of ml, add the same amount of 80% phenol, heat in a water bath at 75 ° C, stir for 5 minutes after reaching 60 to 70 ° C, and quickly in an ice bath at 10 ° C. Cooled down. This was centrifuged at 2000 rpm for 60 minutes to separate an aqueous layer and a phenol layer. For the phenol layer, the same amount of ultrapure water was further added and the above operation was repeated. The obtained aqueous layer was dialyzed against ultrapure water for 48 hours and then concentrated under reduced pressure to an appropriate amount. 0.1 M Tris-HCl buffer solution (p
H7.8) was added in equal amounts. Then RNase 2mg and DNase
3 mg was added, and enzyme treatment was performed at 37 ° C. for 24 hours to separate the bound nucleic acid. The enzyme-treated solution was concentrated to an appropriate amount, an equal amount of 80% phenol was added, the mixture was stirred for 40 minutes at 4 ° C., and then centrifuged at 2000 rpm for 60 minutes to obtain an aqueous layer, which was then subjected to ultrapure water at 4 ° C. for 48 hours. After dialysis for an hour, it was freeze-dried to obtain a crude bacterial cell component. The crude bacterial cell component was dissolved in a 0.2 M ammonium acetate buffer containing 0.01% sodium azide, and the mixture was loaded on a Sepharose 6B column (Pharmacia) (fractionable range: molecular weight 10 ⁴ to 10 ⁶ ) and eluted. Fractions of 6 ml each were purified at a rate of 9 ml / hour for purification.

Nude mice are inbred BALB / c (nu / n
u) 4-5 week old ones were used. Human nude liver transplantable tumor human primary liver cancer PLC-303 Hepatoma cells were transplanted to the nude mice. The transplantation was performed aseptically, and tumor pieces finely cut into 2 to 3 mm squares were subcutaneously transplanted into the left and right axilla of the nude mouse to perform an administration experiment.

For administration, the above-mentioned bacterial cell components were treated with physiological saline for 1 m.
What was dissolved in a concentration of g / ml was put in a vial and autoclaved under high pressure sterilization (Example 5). The control group (Comparative Example 4) was administered with physiological saline sterilized in the same manner. The administration method was intraperitoneal administration once a day until the 36th day with the tumor transplantation day as the 0th day. Mouse body weight and tumor size were measured at 4-day intervals from day 0 of transplantation.
For the size of the tumor, the major axis and the minor axis were measured using a caliper, and the tumor volume was calculated by the following formula: tumor volume = (major axis × minor axis) ² × 1/2. The tumor volume on day 0 of transplantation was set to 1.0, and the ratio of the tumor volume to that was calculated. FIG. 5 shows the tumor volume ratios of the bacterial cell component administration group (Example 5) and the control group (Comparative Example 4) on the 36th day after transplantation. As is clear from FIG. 5, the tumor volume of the control group did not change from the time of transplantation, but the tumor volume was reduced to less than half in the group containing the bacterial cell component of the microbial preparation of the present invention (Example 5). This difference was found to be significant by Student's t-test. No change was observed in the body weight of the mice during the test.

Example 6, Comparative Example 5 (Immunostimulatory Action) Cancer cell pieces were transplanted to nude mice in the same manner as in Example 5 and Comparative Example 4, and bacterial cell components of the microorganism group of the present invention (Examples). 6) and physiological saline (Comparative Example 5) were administered. Transplant 36
On the day, the activity of mouse peritoneal macrophages was measured by the following method. As an index of macrophage activation, mouse peritoneal macrophages were reacted with peroxidase-antiperoxidase-soluble complex (PAP) as a labeled immune complex, and the uptake amount was microquantified by the principle of enzyme immunoassay.

That is, the mouse was anesthetized with ether and 70
% Disinfect the abdominal wall with ether cotton. Cold HBSS ^over (Hank's balanced salt solution: Hank's Balanced Salt Solution) 5-6
Take in a ml syringe and rapidly inject into the abdominal cavity from the abdominal muscle portion in the center of the lower abdomen. After massaging for 30 seconds abdominal wall with 70% ethanol cotton, recovering HBSS ^over containing peritoneal exudate cells (PEC) by syringe. This was transferred to a polypropylene centrifuge tube, washed with HBSS ^- twice by centrifugation at 1000 rpm for 1 minute at 4 ° C., and then 10% fetal bovine serum (FBS) -10 mM N-2-hydroxyethylpiperi.
Suspend in RPMI1640 medium containing zine-N'-2-ethanesulfonic acid (hereinafter abbreviated as HEPES), dispense into a 6-well flat-bottomed plastic petri dish, and culture at 37 ° C. for 60 minutes in a 5% carbon dioxide gas incubator. After culturing, the dish is shaken well to suspend the non-adherent cells, and suctioned off. After washing three times with similarly warm HBSS ^over, 2.5 mM E
After adding 2 ml of DTA / saline solution and ice-cooling for 20 minutes, the adherent cells were physically peeled off with a rubber policeman, washed again with a centrifuge tube at 1000 rpm for 5 minutes at 4 ° C., and washed 5 × 10 ⁴ with a hemocytometer. RPMI16 containing 10% FBS-10mM HEPES so that it becomes / ml
It was suspended in 40 medium.

The macrophage suspension thus prepared was placed in a 96-well flat-bottomed microplate in an amount of 200 μl / well.
Dispense repeatedly and culture overnight at 37 ° C. in a 5% carbon dioxide gas incubator. After culturing, the culture solution is removed by suction, and 100 μl of a PAP solution diluted with HBSS containing bovine serum albumin (BSA) is dispensed into each well. Also prepare a blank hole in which the cells are seeded but the PAP is not dispensed. This plate is incubated at 37 ° C. for 60 minutes in a 5% carbon dioxide gas incubator to react with the immune complex. After the reaction, the PAP solution in each hole was removed by suction, and immediately, each hole was ice-cooled. HBS containing 10 mM HEPES
Wash with S. HBSS was removed by suction from the hole after washing, and 1% NP-40 (emulsifier Nonid
100 μl of a phosphate buffered saline containing et-P-40 (hereinafter abbreviated as NP40-D'PBS) is dispensed. From the dispensed plate, transfer 50 μl per well to the plate for color development, and add 150 μl of the color development substrate (2,2'-azino-di- [3-ethyl-ben
zthiazoline sulfate] was dissolved in citric acid-phosphate buffer solution at a concentration of 0.2 mg / ml, and 0.03% hydrogen peroxide was mixed in equal amounts immediately before use) was added to each hole, and light was shielded with aluminum foil. It was left at room temperature for 50 minutes. To remove air bubbles in each hole, the plate was centrifuged at 1000 rpm for 5 minutes, and the absorbance was measured with a microplate recorder at a wavelength of 405 nm. The macrophage activity of the control group was 1.
The macrophage activity of the microbial preparation administration group of the present invention was determined as 0. FIG. 6 shows the results. As is clear from the results, the combined microbial preparation of the present invention had significantly higher activity by Student's t-test than the non-administered group.

Example 7, Comparative Example 6 (Hypertensive action) SHR / Ncri spontaneously hypertensive rat (Charles River Japan) was used to evaluate the hypotensive action of the complex microorganism preparation of the present invention. Normal blood pressure Wistar rats WKY / Ncri (Charles River Japan)
It was used. Animals are at room temperature 22 ± 1 in SPF environment
Preliminary breeding was carried out for 1 week at a temperature of 50 ° C., humidity of 50 to 60%, and a long-day cycle of 14 to 10 hours of light and dark cycle. The animals were randomly divided into 3 groups and 7 animals for use in the experiment. One group was a microbial preparation of the present invention (Preparation 2) administered group (Example 7), one group was a non-administered group (Comparative Example 6), and the remaining one group was a control group that was not administered to normotensive rats. Animals in the same administration group were placed in the same cage so that excrement would not affect the animals. Before and during the experiment, the feed (CE2 manufactured by CLEA Japan, autoclave sterilized) and water were freely taken. The administration of the complex microorganism preparation was carried out for 6 weeks from 6 weeks of age. An oral probe was used as the administration method, and the dose was 3% by weight of an average feed intake of 20 g per 250 g of rat body weight diluted with 1.0 ml of 0.9% physiological saline / 100 g of body weight. In the comparative example and the control group, physiological saline was administered in the same volume as above. Administration is 1 am daily
The measurement was performed in the same order from 0 o'clock, but on the day of blood pressure measurement, it was administered after the measurement in order to eliminate the effect of stress caused by administration of the sonde. Blood pressure was measured once a week after the start of administration. Non-invasive blood pressure measurement from rat tail vein NARCO, PE300
(Manufactured by Tokai Sakuri) was used. The measurement data of the administration group and the non-administration group were tested for a significant difference by Student's t-test. FIG. 7 shows the results. As is clear from FIG. 7, the complex microorganism preparation of the present invention significantly lowered blood pressure as compared with the non-administered group.

Example 8 (Blood glucose level lowering action) The combined microbial preparation of the present invention prepared in Example 1 (Preparation 1)
5 diabetic patients (2 female, 3 male, age composition 4
5 to 53 years old). The administration was started in April 1990, and 5 g was orally administered daily. Blood glucose levels were in July 1988 before administration, July 1989 and 19 after administration.
Blood was sampled and measured 3 times in October 1990, before meals at breakfast, 1 hour after meals, and 2 hours after meals. Glucose was converted to glucose-6-phosphate (G-6-P) by hexokinase, and NADP was converted to NADP by G-6-P dehydrogenase.
After reduction to DPH ₂ , the enzymatic method of measuring the absorbance in the ultraviolet at a wavelength of 334 nm (Bergmeyer, H.
U.) et al. (Methodender enzymatischen Anal
yse), 2nd edition, Volume II, pp. 1163-1165,
Published by Verlag Chemie (Weinheim / Bergstraße)), and the average value of 5 persons was calculated. The results are shown in Fig. 8. As is clear from the results, administration of the complex microorganism preparation of the present invention markedly lowered the blood glucose level and entered the normal range.

Example 9 (Intestinal Suppression of Substances Causing Mental Illness) When the balance of intestinal microorganisms collapses due to stress or the like, putrefactive bacteria become dominant, and amines, ammonia, phenol, hydrogen sulfide, mercaptan, etc. are activated by the activity of putrefactive bacteria. It is considered that so-called malodorous substances such as sulfur compounds are generated and enter the brain through the liver, causing various brain disorders. That is, the generation of malodorous substances in the intestine is considered as one of the causes of mental disorders due to stress and the like. Reducing this malodorous substance restores mental stress rapidly. The complex microorganism preparation of the present invention exhibits an action of suppressing the generation of malodorous substances in the intestine.
This was confirmed by suppressing the generation of intestinal malodorous substances in 5 persons.
That is, arbitrarily select 5 volunteers with a history of neurosis,
The complex microbial preparation of the present invention (Preparation 1) was orally taken in an amount of 5 g per person per day, and immediately before and 8 days after the administration, 16
On the day and the 24th day, 5 g was collected in a stool tube at a fixed time in the morning, transferred to a vial and incubated at 37 ° C. for 24 hours. The ammonia concentration in the vial was measured by the method described in Japanese Industrial Standard JIS K01042 and the Kjeldahl microquantification method. The sulfur compounds of hydrogen sulfide, methyl mercaptan, methyl sulfide and methyl disulfide were measured by gas chromatography, and the average value of the data of 5 persons was calculated and shown in the graph (FIG. 9). As is clear from the results, the administration of the complex microorganism preparation of the present invention suppressed the generation of the malodorous substance. In addition, the symptoms were reduced along with the reduction of the malodorous substances.

Example 10, Comparative Example 7 (Improvement of milk quality) 5 g / cow / day of the complex microbial preparation of the present invention (formulation 2) prepared in Example 1 was prepared by adding normal feed to 5 cows / group. Then, the effect on milk quality was investigated. As a control, another group of cows was given the same amount of the microbial base material used in Example 1 for comparison. 1 week before salary, at salary, 1, 5, 7, 9, after salary,
100 g of milk expressed after 11, 13, 15, 17 weeks
Was weighed and freeze-dried, and about 150 ml of a chloroform-methanol (2: 1) mixed solution was added to the powder sample, followed by ultrasonic treatment for 30 minutes. This is subjected to suction filtration, the obtained filtrate is transferred to a separating funnel, 50 ml of chloroform-methanol (2: 1) mixed solution is added to the residue, followed by ultrasonic treatment for 30 minutes, and suction filtration to separate the filtrate from the previous separating funnel. Moved to. This operation was repeated. Next, add 1 / to the filtrate in the separating funnel.
After adding 5% of 1% BaCl ₂ and shaking and stirring, the mixture was left overnight in a dark place and partitioned into a chloroform layer and an aqueous layer. The resulting chloroform layer was concentrated under reduced pressure in a water bath at 30 ° C to obtain total lipid. A chloroform-methanol (2: 1) mixed solution was added to this, dissolved at a concentration of 200 mg / ml, and stored at -20 ° C. The obtained total lipid sample was converted to fatty acid methyl ester and gas chromatography (Shimadzu LC-6A) and gas chromatography mass spectrometer (Shimadzu QP-1)
000) to determine the total lipid content, unsaturated fatty acid, saturated fatty acid, polyunsaturated fatty acid and cholesterol content. For unsaturated fatty acids and polyunsaturated fatty acids, the ratio of saturated fatty acids was calculated. The results are shown in FIGS. As shown in FIG. 10, the total lipid content was not different from the control group,
The amount of unsaturated fatty acid and the amount of polyunsaturated fatty acid with respect to saturated fatty acid increased in the group administered with the complex microorganism preparation of the present invention as compared with the control group (FIGS. 11 and 12). On the other hand, the cholesterol content was decreased conversely (Fig. 13). Unsaturated fatty acids and polyunsaturated fatty acids are said to be hard to harden, and are said to have an effect of preventing arteriosclerosis, and it was confirmed that administration of the complex microorganism preparation of the present invention increases the fatty acid composition.

Example 11, Comparative Example 8 (Improvement of meat quality) In addition to feeding normal feed to 249 brown bulls, Example 1
5 g / head / day of the complex microorganism preparation of the present invention (Preparation 1) prepared in (3) was fed, and the effect on meat quality was examined. As a control,
The same amount of the base material used in Example 1 was given to another group of 290 cows for comparison. After the fattening, 100 g of red meat and 100 g of white meat were sampled from the carcass obtained by normal slaughter, and the lipid was analyzed and quantified in the same manner as in Example 10. The results are shown in FIGS. As shown in FIG. 14, the administration of the complex microorganism preparation of the present invention markedly reduced cholesterol in both red meat and white meat. On the other hand, the content of unsaturated fatty acids, especially palmitooleic acid, which is effective in preventing cerebral thrombosis, increased.

Examples 12, 13 and Comparative Example 9 (improvement of egg quality) Three groups of 250 hens were prepared for each group, and two groups were prepared with the complex microbial preparation of the present invention (Formulation 1) prepared in Example 1. The feed was mixed with 0.3% and 1.0%, respectively, and the effect on the sterols content was investigated. As a control, the remaining one group was bred without adding the complex microorganism preparation of the present invention to the feed, and the egg quality was compared. The measurement data of the administration group and the non-administration group were tested for a significant difference by Student's t-test. Results are shown in FIG. As shown in FIG. 17, by supplying the complex microorganism preparation of the present invention, the content of sterols was significantly reduced as compared with the case of no addition.

Example 14 (Improvement of productivity) The effect on milk production was investigated by comparing the annual milk yields of cow groups before and after administration of the complex microorganism preparation of the present invention to a group of 50 cows each. The test was started in 1989, the annual milk yield was recorded without administration for 2 years in 1989 and 1990, and continued from February 1991, and the combined microbial preparation of the present invention prepared in Example 1 (formulation 1 ) Was administered at 5 g / head / day in addition to the usual diet, and the annual milk yield was recorded. The results are shown in Fig. 18. As is clear from FIG. 18, the annual milk yield increased by about 66% after the administration of the complex microorganism preparation of the present invention (19
Comparison between 1990 and 1993).

Example 15 and Comparative Example 10 (Improvement of fish lipid composition and reduction of cholesterol) The complex microorganism preparation of the present invention (Preparation 1) was administered to cultured fish hamachi and the action on the lipid was examined. A group of 3000 sea bream was fed with a 0.2% amount of the feed for aquaculture, the complex microorganism preparation of the present invention prepared in Example 1 and fed, and the feeding start date, 90 days after the start, 180 days after the start Twelve fish were caught in each of the above, and the ventral and dorsal flesh and liver were collected, and lipids were analyzed by the same method as in Example 10. As a comparative example, the same amount of the base material used in Example 1 was fed and the same test was conducted. The measurement data of the administration group and the non-administration group were tested for a significant difference by Student's t-test. The results are shown in FIG.
20 and 21. As is clear from FIG. 19, the ratio of unsaturated fatty acid and polyunsaturated fatty acid was significantly increased by the administration of the preparation of the present invention, and the cholesterol content was significantly decreased as shown in FIG.

Example 16 and Comparative Example 11 (Reduction of offensive odor of faeces and promotion of composting) In addition to the usual feed feeding to 50 cows per group, the complex microbial preparation of the present invention prepared in Example 1 (formulation 1) 5g / head /
Daily feeding was performed to examine the effects of reducing the odor of feces and promoting composting.
As a control, another 50 groups of cattle were given the same amount of the base material for preparing the microbial preparation used in Example 1 for comparison. The cowshed was covered with sawdust for bedding. Three weeks after the administration, the feces in the barn were collected by a loader and piled up for composting. Immediately after pile-up, the temperature, humidity and pH of compost were measured every day. 22 days after compost,
After 36 days and 73 days, cutback was performed three times. Also, 1
The compost was sampled once every day, and the C / N ratio, which is an index of the maturity of the compost, was determined using a high-sensitivity NC-analyzer, Sumigraph model NC-80 type (sold by Sumitomo Chemical Co., Ltd.). The measurement results of compost temperature, humidity and pH are shown in FIG.
The C / N ratio is shown in FIG. As is clear from FIG. 22, the present invention has higher fermentation temperature and higher microbial activity. The composting promoting effect of the present invention is also apparent from the sharp decrease in the C / N ratio (FIG. 23).

Separately from the above, 8 days after the pile of compost, 1
The odorous substances were measured after 6 days and 24 days. For ammonia, Japanese Industrial Standard JIS K10
242 and the method in accordance with the Environmental Agency Notification No. 47 (1989). For hydrogen sulfide, methyl mercaptan and methyl sulfide, a flame photometric detector (F
It was measured using gas chromatography with PD). The results are shown in Fig. 24. As is clear from FIG. 24, hydrogen sulfide, methyl mercaptan and methyl sulfide are significantly reduced,
Ammonia was also reduced to about 1/3.

Example 17 and Comparative Example 12 (Suppression of methane generation in compost process) The same method as in Example 16 was repeated to pile up compost. Immediately after that, the amount of methane generated over time was measured. The measurement was carried out by putting 50 ml of the compost sample in a 100 ml container (vial bottle), sealing the container, shaking and heating at 37 ° C. for 24 hours, and then analyzing the gas portion by gas chromatography analysis with a thermal conductivity detector (TCD). The results are shown in Fig. 25.
As is apparent from the figure, the compost of the group of cattle to which the complex microorganism preparation of the present invention was administered had significantly less methane generation than the group of non-administered groups.

Example 18, Comparative Example 13 (inhibition of methane generation in rumen) Among the cows administered with the complex microbial preparation of the present invention in Example 16, 6 rumen (ruminal) gastric juice (rumen juice) was extracted from 6 cows. The sample was collected, incubated at 37 ° C., and the concentration of methane generated over time was measured by the same method as in Example 17. Results are shown in FIG. As is clear from the figure, administration of the complex microorganism preparation of the present invention suppressed methane generation in the lumen.

Example 19, Comparative Examples 14 and 15 (Comparison with a Single Microorganism) The combined microbial preparation of the present invention prepared in Example 1 (Preparation 2)
The cholesterol-reducing effect of lactobacillus was compared with that of Lactobacillus and Streptococcus. Each of these bacteria has been reported to have a cholesterol-lowering effect (Grillland et al., Appl.
ied and Environmental Microbiology49, 377-381 (198
5); Ishihara et al., Intestinal bacteria on cholestero
l metabolism, 121-144 (1989) Japan Scientific Pres
s; Suzuki et al., Animal Science and Technology 62, 565.
-571 (1991)). Lactobacillus acidophilus ATCC11506 strain (Comparative Example 14) was used as Lactobacillus bacterium, and Streptococcus faecalis ATCC14429 strain was used as Streptococcus bacterium (Comparative Example 15). Each was cultured in the medium described in Example 1 and used for the test. The test method was the same as in Example 2 except that each of these test samples was used. The results are shown in Fig. 27. As the results show,
The combined microbial preparation of the present invention was more effective than the bacterium having a cholesterol-reducing effect alone, and the combined effect of the combined microorganism was obtained.

[0065]

INDUSTRIAL APPLICABILITY The complex microbial preparation of the present invention has a blood cholesterol lowering action, an intestinal regulating action, an immunostimulating action, an anticancer action, a blood pressure lowering action, a high blood glucose level lowering and normalizing action, and an intestine of a substance causing mental disorders. Various physiological actions such as the inhibitory effect on internal production,
Also, it is very effective for improving productivity of livestock, deodorizing livestock feces and promoting composting, and suppressing methane generation in rumen and compost.

With respect to these effects, it is possible to roughly show which bacterium contained in the complex microbial preparation of the present invention works. For example, it is considered that Bacillus megaterium, Streptococcus faecalis, and Streptococcus lactis are involved in the blood cholesterol lowering action. In addition, it is considered that Lactobacillus is mainly involved in the intestinal regulation action. In addition, it is considered that Bacillus bacteria are involved in the immunostimulatory action. However, the respective effects obtained by using only these bacteria are smaller than the effects exhibited by the complex microbial preparation of the present invention, and synergistic action with other bacteria that are not considered to be involved in each action works. It is speculated that In addition, the action is enhanced by using a calcium-containing base material as an adsorption base material and fermenting it together with a microorganism group.

[Brief description of drawings]

FIG. 1 is a diagram comparing changes with time in serum cholesterol levels of rats fed a diet containing high cholesterol.

FIG. 2 is a diagram comparing changes with time in serum cholesterol levels of rats fed a basic diet.

FIG. 3 is a diagram comparing the cholesterol levels in the livers of rats fed a high-cholesterol-containing diet and a basic diet.

FIG. 4 is a diagram comparing changes over time in the ratio of the number of useful lactic acid bacteria to the number of Escherichia coli in rat feces.

FIG. 5 is a diagram comparing the size of cancer cells after administering the preparation of the present invention to nude mice artificially transplanted with cancer cells for 36 days.

FIG. 6 is a diagram comparing macrophage activity after administering the preparation of the present invention to nude mice artificially transplanted with cancer cells for 36 days.

FIG. 7 is a diagram comparing changes in blood pressure with time when the preparation of the present invention was administered to spontaneously hypertensive rats.

FIG. 8 is a graph showing changes in blood glucose level before and after administration of the preparation of the present invention in diabetic patients.

FIG. 9 is a graph showing changes in the content of malodorous components in feces before and after administration of the preparation of the present invention in a neurotic patient.

FIG. 10 is a diagram comparing changes with time in fat content in milk by feeding the preparation of the present invention.

FIG. 11 is a diagram comparing changes over time in the ratio of the amount of unsaturated fatty acid to the amount of saturated fatty acid in milk by feeding the preparation of the present invention.

FIG. 12 is a diagram comparing changes over time in the ratio of the amount of polyunsaturated fatty acid to the amount of saturated fatty acid in milk by feeding the preparation of the present invention.

FIG. 13 is a diagram comparing changes with time in cholesterol content in milk by feeding the preparation of the present invention.

FIG. 14 is a diagram comparing the cholesterol contents in beef according to the preparation feeding of the present invention.

FIG. 15 is a diagram comparing the palmitooleic acid contents in beef according to the feeding of the preparation of the present invention.

FIG. 16 is a view comparing the ratio of the amount of unsaturated fatty acid to the amount of saturated fatty acid in beef by feeding the preparation of the present invention.

FIG. 17 is a diagram comparing changes in cholesterol content in egg yolk with time when the preparation of the present invention is fed.

FIG. 18 is a diagram showing changes in the annual milk yield before and after feeding the formulation of the present invention.

FIG. 19 is a diagram showing changes in the ratio of unsaturated fatty acids to saturated fatty acids of cultured fish according to the feeding of the formulation of the present invention.

FIG. 20 is a graph showing changes in the ratio of unsaturated fatty acids to polyvalent saturated fatty acids in cultured fish according to the formulation of the present invention.

FIG. 21 is a diagram showing changes in the cholesterol content of cultured fish by feeding the preparation of the present invention.

FIG. 22 is a diagram showing changes over time in temperature, humidity and pH when manure of dairy cows fed with the preparation of the present invention is composted.

FIG. 23 is a diagram comparing the change with time of the C / N ratio in the case of composting the manure of dairy cows fed with the preparation of the present invention, compared with the case of non-feeding.

FIG. 24 is a diagram showing the change over time in the malodorous component content when manure of dairy cows fed with the preparation of the present invention was composted.

FIG. 25 is a diagram comparing the time-dependent change in the amount of methane generated when manure of dairy cows fed with the preparation of the present invention was composted with the case of non-feeding.

FIG. 26 is a diagram comparing changes over time in the amount of methane generated in the rumen juice of dairy cows fed with the preparation of the present invention as compared with the case of non-feeding.

[Fig. 27] Fig. 27 is a diagram comparing changes in cholesterol content in rats with time when the preparation of the present invention was administered, as compared with the case where only bacteria having a cholesterol-reducing action were administered.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display area A61K 35/56 ADN A61K 35/56 ADN 35/70 ADU 35/70 ADU 35/74 ACJ 35/74 ACJA ADQ ADQ AEV AEV AEX AEX AFC AFC // (C12N 1/20 C12R 1: 125 1:11 1:23 1:25 1:24 1: 245 1:46 1: 145 1: 865 1:72)

Claims (9)

[Claims] 1. At least Bacillus subtilis (Ba
cillus subtilis), Bacillus natto (bacillus n)
atto, Bacillus megaterium
), Lactobacillus acidophilus
acidophilus), Lactobacillus plantarum (Lac
tobacillus plantarum), Lactobacillus brevis, Lactobacillus casei, Streptococcus faecalis, Streptococcus lactis, Streptococcus lactis, Streptococcus lactis, Streptococcus lactis, Streptococcus lactis
s), Clostridium butyricum (Clostridium but
yricum), Saccharomyces cerevisiae (Saccharomyc
es cerevisiae) and Candida Utilis (Candi
da utilis) and a substrate adsorbing the microorganisms, the substrate containing at least a calcium-containing substrate, and at least a part of the calcium component of the calcium-containing substrate is the microorganism. A composite microbial preparation characterized by comprising a biocompatible organic calcium compound by the action of a group. 2. The preparation according to claim 1, wherein the microorganism group further contains nitrifying bacteria and sulfur bacteria. 3. The calcium-containing base material is bone meal, shellfish fossil,
The preparation according to claim 1 or 2, containing at least one selected from synthetic apatite, calcium phosphate, and calcium carbonate. 4. At least Bacillus subtilis (Ba
cillus subtilis), Bacillus natto (bacillus n)
atto, Bacillus megaterium
), Lactobacillus acidophilus
acidophilus), Lactobacillus plantarum (Lac
tobacillus plantarum), Lactobacillus brevis, Lactobacillus casei, Streptococcus faecalis, Streptococcus lactis, Streptococcus lactis, Streptococcus lactis, Streptococcus lactis, Streptococcus lactis
s), Clostridium butyricum (Clostridium but
yricum), Saccharomyces cerevisiae (Saccharomyc
es cerevisiae) and Candida Utilis (Candi
The method for producing a complex microbial preparation according to claim 1, which comprises culturing each of the liquid utilis) in liquid pure culture, mixing each obtained microorganism culture with a calcium-containing substrate, and fermenting the obtained mixture. 5. The production method according to claim 4, further comprising nitrifying bacteria and sulfur bacteria as the microorganisms to be purely cultured. 6. The calcium-containing base material is bone meal, shellfish fossil,
The production method according to claim 4 or 5, containing at least one selected from coral, synthetic apatite, calcium phosphate, and calcium carbonate. 7. At least Bacillus subtilis (Ba
cillus subtilis), Bacillus natto (bacillus n)
atto, Bacillus megaterium
), Lactobacillus acidophilus
acidophilus), Lactobacillus plantarum (Lac
tobacillus plantarum), Lactobacillus brevis, Lactobacillus casei, Streptococcus faecalis, Streptococcus lactis, Streptococcus lactis, Streptococcus lactis, Streptococcus lactis, Streptococcus lactis
s), Clostridium butyricum (Clostridium but
yricum), Saccharomyces cerevisiae (Saccharomyc
es cerevisiae) and Candida Utilis (Candi
The method for producing a complex microbial preparation according to claim 1, which comprises mixing a mixed microorganism group consisting of da utilis) with an adsorption substrate containing at least a calcium-containing substrate and culturing the mixture. 8. The method according to claim 7, wherein the microorganism group further contains nitrifying bacteria and sulfur bacteria. 9. The calcium-containing base material is bone meal, shell fossil,
The production method according to claim 7 or 8, containing at least one selected from coral, synthetic apatite, calcium phosphate, and calcium carbonate.