JP3423972B2 - Growth-promoting substances for Escherichia coli and bifidobacteria - Google Patents

Description

Detailed Description of the Invention

[0001]

The present invention relates to Escherichia coli (Escherichia coli) and Bifidoba which are useful bacteria among intestinal indigenous bacteria.
The present invention relates to a substance having an action of strongly promoting the growth of cterium bacteria (Bifidobacteria).

[0002]

BACKGROUND OF THE INVENTION Certain strains of Bacillus subtilis are used in the production of natto. So-called natto bacteria. In animal experiments, during oral administration of Bacillus natto spores, Bacillus natto was detected in the feces at about 10 ^4-6 cfu / g, but it is said that if the administration is stopped, it is rapidly reduced or eliminated. See Reference 1 and Reference 2). [Reference 1] Ozawa, K. et al .: Effects of administrat
ion of Bacillus subtilis strain BN on intestinal f
lora of weanling piglets. Jpn. J. Vet. Sci. 43: 77
1-775 (1981) [Reference 2] Hideo Murata, et al .: Bacillus natto preparation in weaned pigs ( Ba
cillus natto BN strain)
30: 645-649 (1977)

Further, some of the administered Bacillus natto germinated in the intestinal tract, but most of them died (see Reference 3). [Reference 3] Shinji Kuninaga , et al .: Bacillu in the intestinal tract of dogs
s natto About germination of spores, Journal 33: 689-696 (197
8)

Further, according to Reference 1, Bacillus subtili
Oral administration of s spores increased the number of lactic acid bacteria in the upper small intestine and reduced the variation in the number of bacteria among individuals, but the mechanism of this increase in bacterial numbers has not been clarified.

[0005] The following Document 4 reports the effect of administration of Bacillus subtilis BN strain on the intestinal flora of weaned pigs. [Reference 4] Kimura, et al .: Effect of Bacillus subtilis BN strain administration on intestinal flora of weaned pigs, Veterinary and Animal Husbandry Bulletin, No. 733: 434-440 (1982)

[0006]

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention
According to ~ 3, oral administration of Bacillus subtilis spores increased the number of lactic acid bacteria in the upper part of the small intestine and reduced the variation in the number of bacteria among individuals, but the degree of growth of lactic acid bacteria was not so large. . References 1 to 3 show that Ba
cillus subtilis is Escherichia coli and Bifi
There is no description of the effect on the growth of dobacterium (Bifidobacteria).

[0007] In the above-mentioned document 4, two experiments were conducted, and in one experiment, it was said that the lactic acid bacteria and bifidobacteria were proliferated by the intake of Bacillus natto, but in another experiment, the test group and the control group. There is no difference between and,
The reproducibility was not always sufficient.

[0008] Under the above circumstances, the present invention is useful for enteric bacteria such as Escherichia coli and Bifidobacteriu.
An object of the present invention is to provide a substance having a function of strongly promoting the growth of genus m bacteria (all of which are resident intestinal bacteria that play a leading role in antagonism against various enteritis-causing bacteria under anaerobic conditions).

[0009]

The growth-promoting substance for Escherichia coli and Bifidobacteria of the present invention contains Bacillus subtilis and amylase as active ingredients.

The present invention will be described in detail below.

In the present invention, Baci is used as the first component.
llus subtilis is used. Examples of strains of Bacillus subtilis include those used in the Examples below, such as Ba
cillus subtilis ATCC 6633 , Bacillus subtilis NIHJ
-PCI 219, Bacillus subtilis IFO 13169, Bacillus s
ubtilis IFO 3009, Bacillus subtilis BN (Meguro Research Co., Ltd.), etc. Of these strains IFO 13169
, IFO 3009, BN belong to Bacillus natto (see Document 5 below), and such Bacillus natto is particularly preferable for the purpose of the present invention. [Reference 5] Yu Ushijima, Yoshikatsu Ozaki: Bacillus subtilis and B
Studies on the biological and antibacterial properties of acillus natto ,
Journal 35 (4): 625-637 (1980).

The first component, Bacillus subtilis, is
Usually, it is used in the form of spores (spores) having heat resistance and acid resistance, but in some cases, it can also be used in the form of vegetative type germinated and grown from the spores.

The second component is amylase. Here, as amylase, α-amylase, β-amylase,
Examples thereof include saccharified amylase, and α-amylase is particularly important.

As the third component, at least one saccharide selected from the group consisting of monosaccharides, oligosaccharides and polysaccharides can be used in combination with the above-mentioned Bacillus subtilis and amylase. Here, examples of monosaccharides are glucose (D-glucose), fructose (D-fructose), and the like. Examples of oligosaccharides are sucrose (lactose), lactose, trehalose, maltose (maltose), cellobiose, isomaltose, xylobiose and the like. Examples of polysaccharides are starch (various raw starches, physically modified starches, enzyme modified starches, chemically degraded starches, chemically modified starch derivatives, etc.), glycogen and the like.

The growth promoting substance of the present invention is the above-mentioned Bacillus.
subtilis and amylase (possibly further sugars)
Consists of. It is particularly desirable that the ratio of Bacillus subtilis in 1 g of the preparation when the growth-promoting substance is prepared is 10 ⁴ cfu (colony forming unit) or more, particularly 10 ⁵ cfu or more. Bacillus subtilis only or Bacillus
Even if subtilis is used in an excessively small amount, it is not as effective as the present invention in terms of promoting the growth of Escherichia coli and Bifidobacteria. In addition, the proportion of amylase in 1 g of the preparation when the growth-promoting substance is prepared is
It is desirable that it is 0 unit or more, especially 30 unit or more.
If the enzymatic activity of amylase is too low, the excellent effects of the present invention cannot be obtained in terms of promoting the growth of Escherichia coli and Bifidobacteria. Since sugar is an optional ingredient,
The ratio in the formulation can be set appropriately.

The growth promoting substance of the present invention comprising Bacillus subtilis and amylase described above is preferably used in the form of a mixture of both. If the water content is kept below the allowable limit, it will be stable for a long period of time even when both components are mixed.

The growth-promoting substance of the present invention may be used in a conventional excipient such as a binder, excipient, wetting agent, disintegrant, or an aqueous or oily medium, if necessary, as a powder, granules, It may be any orally administrable dosage form including tablets, capsules, suspensions, solutions and syrups. Further, it is used in the form of being added to foods (including beverages), pharmaceuticals, feeds and the like.

[0018]

[Operation] In the growth promoting substance of the present invention comprising Bacillus subtilis and amylase, the first component, Ba
Because cillus subtilis has a high resistance to heat and acid (has a heat resistance up to at least 80 ° C and is not affected by gastric acid), when it is administered orally, it passes through the stomach in a dormant state without being destroyed. Escherichia coli and Bifidobacterium , which are useful bacteria among indigenous bacteria in the intestine, grow in the intestinal tract for the first time and, in combination with the action of the second component amylase (further, the third component, saccharide).
It has the effect of strongly promoting the growth of genus bacteria.

Therefore, the intestinal bacterial flora becomes rich in Escherichia coli and Bifidobacterium which are useful enteric bacteria.

[0020]

EXAMPLES The present invention will be further described with reference to examples.

<Preliminary Experiment> First,Bacillus subtilis
 , Factors such as amylase, sugar, and pHEscherichia
 coliandBifidobacterium What to the growth of genus bacteria
In order to obtain various basic knowledge about whether it affects,
In vitro experiments were performed as described above.

<First Component> The first component, Bacillus
The following strains were prepared as subtilis . These subjects
Among Bacillus subtilis , IFO 13169, IFO 3009, and BN are strains that are conserved as Bacillus natto, although their properties are slightly different.・Bacillus subtilis ATCC 6633 ・Bacillus subtilis NIHJ-PCI 219 ・Bacillus subtilis IFO 13169 ・Bacillus subtilis IFO 3009 ・Bacillus subtilis BN (Meguro Research Institute, Ltd.)

<Useful bacteria in the intestine> As useful bacteria among intestinal indigenous bacteria, the following strains Escherichia coli and Bifidobacter
An ium genus and a Lactobacillus genus (lactic acid bacterium) were prepared.・Escherichia coli ATCC 25922 ・Bifidobacterium adolescentis ATCC 15703 ・Bifidobacterium longum GA-34 ・Bifidobacterium breve BT-4 ・Bifidobacterium infantis La-11 ・Bifidobacterium bifidum XA-30 ・Lactobacillus acidophilus ATCC 19992

<Second component> The following amylase was prepared as the second component. -Α -amylase ( Amyloliquefase X: Bacill
derived from us amyloliquifaciens , manufactured by Hankyu Bio Industry Co., Ltd.-α-amylase (Amylolysin-5: Bacillus sub)
derived from tilis , manufactured by Hankyu Bio Industry Co., Ltd.)

<Third component> As the saccharide of the third component,
Glucose, maltose, lactose, sucrose, and starch were prepared.

<Medium> As the medium, the pH of the following composition
A 6.5 casein-starch medium was prepared.・ Casein 0.5 g / dl ・ Starch 0.5 g / dl ・ NaCl 0.4 g / dl ・ KH ₂ PO ₄ (anhydrous) 0.4 g / dl ・ Na ₂ HPO ₄ (anhydrous) 0.3 g / dl ・ Yeast extract powder 0.02 g / dl cysteine HCl-H ₂ O 0.02g / dl in addition, heart infusion (HI) broth (Nissui Pharmaceutical Co., Ltd.) was also used as a basal medium.

<Preparation of Bacillus subtilis spores>
The I agar slope was inoculated and cultured in air at 37 ° C. for 2 to 3 days. This bacterium was suspended in sterile physiological saline and stored at room temperature.

<Preparation of trophozoites from spores> Spores orally ingested by humans are presumed to germinate in the small intestine. After that, since it is exposed to anaerobic conditions, it is considered that strong growth cannot occur. Therefore, a trophozoite that had germinated but was not yet almost divided was prepared. That is,
Approximately 10 ⁸ colony forming units (cfu) in 50 ml of HI broth
Spores were inoculated and cultured in air at 37 ° C. for 2 hours. As a result, it was confirmed that 90% or more of the spores germinated and extended to 3 to 10 μm, but almost no division occurred.

<Proliferation promoting action of Bacillus subtilis trophozoite> The trophozoite prepared as described above was centrifuged and the supernatant was discarded. 5 ml of casein-starch medium was added to the bacterial cells in the precipitate to suspend the cells. As a control, a culture medium containing no bacteria was used. Bifi
About 1 bacterium of the genus dobacterium or Lactobacillus
0 ^3-4 cfu / ml and incubate at 37 ° C under N ₂ gas environment for 2
Anaerobic culture was performed for 0 hours. The number of bacteria was counted in a selective medium for each bacterium. The effect of removing casein and starch from this culture,
The effects on growth were examined by adding sugar, acid, etc. to the medium containing no Bacillus subtilis vegetative strain.

[Results] <Promotion of growth by Bacillus subtilis trophozoite> To the casein-starch medium, a 10-fold serially diluted trophozoite of BN strain was added, and 10 ^3-4 cfu / ml of Escherichia coli or
Anaerobic culture inoculated with Bifidobacterium adolescentis 20
After the lapse of time, the number of bacteria was counted (see FIGS. 1 and 2).

As shown in FIG. 1, Escherichia coli was apparently stimulated to grow in the presence of a considerably high level of trophozoites of 5 × 10 ⁵ cfu / ml or more. Although not shown in the figure, among other tested Bacillus subtilis , PCI-
219 showed a weak growth promoting action, and ATCC6633 showed a strong growth promoting action. It was presumed that these two strains and the BN strain had stronger starch decomposition than the other two strains.

On the other hand, Bifidobacterium adolescentis is
As shown in Figure 2, the vegetative type was only 5 x 10 ³ cfu / ml.
The growth was promoted even in the coexistence of a low bacterial count.

The BN strain is Lactobacillus acidophilus ,
Bifidobacterium long other than Bifidobacterium bifidum
um , Bifidobacterium breve , Bifidobacterium infant
It also showed a strong growth-promoting action against is and the like. Also, Bifi
Other test Bacillus against dobacterium adolescentis
All four subtilis strains showed a strong growth promoting action.

<Nutrient sources involved in growth promotion> BN strain about 1
Escherichia coli and Bifidobacterium were added to a medium containing casein or starch alone in the presence of 0 ⁶ cfu / ml.
Adolescentis was inoculated and cultured. Both of them received strong growth promotion only in the presence of starch (see FIGS. 3 and 4). Although not shown in the figure, Bifidobacterium adoles
In centis , the same strong growth promotion was observed even when maltose, glucose, lactose and sucrose were used instead of starch as the sugar added to the medium.

From the above results, the growth promotion can be achieved by Bacillus s
Starch was decomposed by amylase produced by ubtilis , and the resulting low-molecular sugar was converted into Escherichia coli and Bifidobacterium ad
It is presumed that it was used by olescentis .

Then, α-amylase (Amyloliquefase X 200 μg / ml or amilolysin-5 40 μg / ml) was added to the starch-containing medium, and Escheric was added to this.
hiacoli (Figure 5) or Bifidobacterium adolescenti
s (FIG. 6) was inoculated and cultured. However, contrary to expectations, only Escherichia coli was growth-promoted.

Glucose as a degradation product of starch and proteose peptone (Difco) as a degradation product of protein were added to the casein-starch medium to examine the effect on growth. Escherichia coli was strongly stimulated to grow by the addition of glucose (Fig. 7). However, Bifidobacterium
Adolescentis did not undergo growth promotion by these, but rather the number of bacteria decreased with the addition of glucose (Fig. 8). Although not shown in the figure, maltose, lactose, sucrose, etc. also did not promote growth.

< Location of growth promoting factor for Bifidobacterium adolescentis > Glucose 0.5 g / dl, cysteine HCl-H ₂ O 0.02 g added HI broth (pH 6.7) to Ba
A culture solution inoculated with cillus subtilis spores and cultured in air at 37 ° C. for 3 hours, and a supernatant obtained by centrifuging the culture solution was further filtered through a membrane filter (0.2 μm). About the added one, Bifido
The growth promoting activity against bacterium adolescentis was examined. The promoter was not present in the centrifugal supernatant-filtrate but was localized in the precipitate, that is, in the bacterial cells (Fig. 9).

<Extraction of growth promoting factor> Raw cells were disrupted with an ultrasonic disruptor and centrifuged at 15,000 x G for 20 minutes. Despite destroying the bacterium, the supernatant has no promoting activity,
It was localized only in the precipitate. Therefore, the Bacillus subtilis culture solution was subjected to high-pressure steam sterilization at 120 ° C. for 20 minutes. This was centrifuged at 5 ° C., 4000 rpm for 15 minutes, and a small number of remaining bacterial cells was filtered off with a membrane filter. By this sterilization treatment, the promoting factor left the cells and was transferred to the supernatant (Fig. 10). The supernatant was adjusted to pH 2 and extracted with 2 volumes of diethyl ether: hexane (1: 1). In order to completely remove the organic solvent from the supernatant, blow it with air 7
Heat in a 0 ° C. hot bath for 5 minutes. This treatment almost completely eliminated the growth promoting activity of the supernatant. The extracted organic solvent is
When almost completely removed and dissolved in the previously extracted supernatant, the activity was weaker than that of the original supernatant, but the growth promoting activity was recovered. From these results, it was presumed that the growth promoting factor would be a thermostable acid.

<Influence of pH> pH of casein-starch medium
Of Bacillus subtilis BN strain trophotype 8.4
Add 10 ⁴ cfu / ml and add 10 ⁴ cfu / ml of Bifidoba
After 20 hours of anaerobic culture after inoculation with cterium adolescentis , the number of bacteria was counted and compared with the case without BN strain addition,
As shown in Fig. 11, Bifidobacter at pH 6.0, 6.5 and 7.0
It was found that ium adolescentis grew strongly and was considerably stronger even at pH 7.5. However, it became extremely weaker when the pH was more acidic than 5.5 or pH 8 was alkaline.

[Consideration] Among the 5 strains of Bacillus subtilis to be tested, 3 strains, BN, IFO 13169, and IFO 3009 are strains stored as Bacillus natto. Germination than spores of these bacteria, grown nutrient types, in anaerobic conditions, Esch
It has been clarified that it strongly promotes the growth of erichia coli and many Bifidobacterium species, and that it has the function of expanding the strong growth range to pH 7.5 even though the optimum pH of the latter is around 6. It was

Escherichia coli is a facultative anaerobic bacterium. Energy is obtained by respiration in the air, but under anaerobic conditions, energy is obtained by mixed acid fermentation. In this case, the presence of available sugar is required for efficient fermentation. Therefore, Escherichia coli
In the case of, it suffices if the starch is decomposed simply by the amylase of Bacillus subtilis and the resulting low-molecular-weight sugar is present. However, Bifidobacterium
The presence of both saccharides and Bacillus subtilis cells stimulated strong growth for the first time.

The spores of the BN strain germinated under anaerobic conditions even in the presence of 0.5% bile powder, and became trophozoites. In addition, under anaerobic conditions, Escherichia coli and Bifidobacteriu
When mixed with m for 20 hours, many of these trophozoites, sometimes 90% or more, died. In animal experiments, it is estimated that some of the orally administered Bacillus natto spores germinated in the small intestine and grew weakly in some animals. It is said that some of the trophozoites die until they pass the large intestine and are defecated (see References 1, 2 and 3). Thus Bacillus
It is highly possible that subtilis spores germinate in the upper small intestine of humans and that they promote the growth of Bifidobacterium spp. The bacterial cells of Bacillus subtilis orally ingested are, so to speak, natural microcapsules containing a growth promoting factor.

Natto is a favorite product of many people. By eating this, Escherichia coli and Bifidobacteriu
If the growth of m in the intestine was promoted, this would lead to normalization of the indigenous bacteria and maintenance of their stability. Therefore, it retains a stable and strong antagonistic ability against various enteritis-causing bacteria that sometimes enter orally, and eliminates this, which will effectively work to keep the intestinal tract in a normal state (Reference 6 below). ，
7, 8, 9). Therefore, natto is likely to be a food that not only has an excellent nutritional source but also has a normalizing effect on intestinal indigenous bacteria.

[Reference 6] Ushijima, T. & Seto, A .: Sel
ected faecal bacteria and nutrients essential for
antagonism of Salmonella typhimurium in anaerobic
continuous flow cultures. J. Med. Microbiol. 35: 1
11-117 (1991) [Reference 7] Ushijima Satoshi: Salmonella typhimu of 7 indigenous human colons in anaerobic continuous mixed culture at around pH 5
Bifidobacteriu in strong antagonism against rium
Role of m adolescentis , Medicine and Biology, 122 (1): 13-18
(1991) [Reference 8] Ushijima Tsuyoshi: Strong antagonism against Clostridium difficile of four anaerobic bacteria resident in the human colon in anaerobic continuous culture, Medicine and Biology, 124 (4): 163-168 (1992) [Reference] 9] Yu Ushijima: Strong antagonism of Escherichia coli resident in human colon against enteropathogenic Escherichia coli and enterohemorrhagic Escherichia coli,
Medicine and Biology, 127 (1): 5-9 (1993)

Example 1 Example 1 The effect of feeding a mixture of the Bacillus natto preparation and the amylase preparation mixed with the feed to pigs having the antibiotic-induced diarrhea was examined. From the results of Table 1 conducted in advance, it was found that sultamicillin tosylate is more suitable for causing diarrhea, so sultamicillin tosylate was used as the antibiotic.

<Materials and Methods> Experiments were conducted under the following materials and conditions. The antibiotic setting test is shown in Table 1, and the sample composition is shown in Table 2. -Animal: 16 weeks old, body weight about 60 kg, LW species, 13 (4 males, 9 females) -Feed: Commercially available fully formulated feed (no antibiotics added), ingredients (DCP (digestible crude protein mass)) 13.5% or more, TDN (total digestible nutrients) 79% or more) ・ Breeding: Single cage, unlimited feed, free drinking water ・ Drug: Antibiotics: Sultamicillin tosylate 4 g / day / head Sample: Bacillus natto- Add amylase mixture, 3% in feed

[0048]

[Table 1] Diarrhea symptoms caused by renampicillin or sultamicillin (antibiotic setting test) Antibiotic swine feces Shape (score) Name No. Dosage No. Gender Day 0 of administration Day 1 of administration Day 2 of administration 2.1 1 Male 0 0 0 Hydrochloric acid Renampi 2.1 2 Male 0 0 0 Sirin 2.1 3 Female 0 0 0 2.1 4 Female 0 0 0 2.1 5 Female 0 0 0 2.1 6 Female 0 1-Sulfur tosylate 4.0 7 Female 0 0 0 Tamicillin 4.0 8 Female 0 3 2 4.0 9 Female 0 22 (Note) The dose is g / day / head. The score is 0: Normal, 1:
Soft stool, 2: Mud stool, 3: Watery stool.

[0049]

[Table 2] Sample composition (in 1 g) Content Content Bacterial name or number or (mg) Enzyme activity Enzyme activity Bionatmin ^{* 1} 60 Natto spore 1 × 10 ⁷ or more Amilolysin-5 ^{* 2} 24 Starch saccharification ability 65- 125 units (Note) ^{* 1} : Bacillus subtilis BN formulation, manufactured by Meguro Laboratory Ltd. ^{* 2} : α-amylase preparation, derived from Bacillus subtilis , manufactured by Hankyu Bio Industry Co., Ltd.

Escherichia coli and bifidobact in faecal swine administered with sultamicillin tosylate when a mixture of Bacillus subtilis BN strain and amylase was mixed in the feed.
The proliferative effect of eria is shown in Table 3 and Table 4 (Table 4 is a continuation of Table 3). The experimental group and the control group each have 5 animals.
In Tables 3 and 4, administered feed A is an antibiotic-containing feed, administered feed B is a natto-amylase mixture-containing feed (experimental group) or normal feed (control group), and administered feed C is a normal feed. The administration period of feed A is 0th day (November 29th).
From the morning of (Sun) to the morning of 3 days (December 2), the administration period of feed B is from the afternoon of 3 days (December 2) to 1
Until the afternoon of 0 day (December 9), the administration period of feed C is thereafter (December 10).

[0051]

[Table 3] Proliferation effect of Escherichia coli and bifidobacteria Log ₁₀ Viable count ± standard deviation / (lower limit-upper limit) Bacillus Esherichia bifido- administered Fecal subtilis coli bacteria feed Collection date Specimen BN strain A 92/11/30 Experimental group < 2.3 <2.3 7.49 ± 0.35 (1 day elapsed) (6.9-7.82) Control group <2.3 <2.3 7.04 ± 0.25 (6.78-7.34) A 92/12/1 Experimental group <2.3 <2.3 <4.3 (2 days elapsed) Control Group <2.3 <2.3 <4.3 B 92/12/3 Experimental group 5.91 ± 0.19 7.95 ± 0.70 7.75 ± 0.60 ^* (4 days elapsed) (5.69-6.10) (7.0-8.8) (7.0-8.44) Control group <2.3 <2.3 <4.3 B 92/12/5 Experimental group 6.19 ± 0.07 6.41 ± 0.54 7.28 ± 0.24 ^* (6 days elapsed) (6.11-6.3) (5.91-7.25) (7.0-7.64) Control group <2.3 7.67 ± 0.88 <4.3 (6.9- 8.66) B 92/12/8 Experimental group 6.18 ± 0.17 6.66 ± 0.18 ^# 6.45 ± 0.64 (9 days elapsed) (6.0-6.38) (6.38-6.84) (<4.3-6.9) Control group <2.3 7.25 ± 0.38 7.47 ± 0.58 (7.0-7.82) (<4.3-7.88)

[0052]

[Table 4] Proliferative effects of Escherichia coli and bifidobacteria (continued) Log ₁₀ Viable count ± standard deviation / (lower limit-upper limit) Bacillus Esherichia bifido- Administration Fecal subtilis coli bacteria Feed Collection date Specimen BN strain C 92/12/11 Experimental group 5.25 ± 0.52 6.09 ± 0.24 7.98 ± 0.42 (12 days elapsed) (<2.3-5.78) (5.88-6.5) (7.38-8.54) Control group <2.3 5.76 ± 0.42 7.86 ± 0.35 (5.14-6.0) (7.41- 8.27) C 92/12/13 Experimental group 3.83 ± 0.50 5.61 ± 0.54 7.87 ± 0.37 (14 days elapsed) (3.3-4.6) (4.78-6.14) (7.41-8.29) Control group <2.3 5.70 ± 0.70 7.49 ± 0.31 ( 4.9-6.2) (7.14-7.74) C 92/12/15 Experimental group 3.30 ± 0 ^* 5.30 ± 0.32 7.85 ± 0.22 (16 days elapsed) (<2.3-3.3) (4.9-5.58) (7.55-8.14) Control group <2.3 4.9 ± 0.86 7.04 ± 0.51 (4.0-5.99) (6.3-7.47) ( ^{Note) * P <0.05, # P} <0.02 ( trophozoites pair spores or experimental group versus control group t test)

In the above, the fecal sample, medium and culture method are as follows. Fecal sample: 2 g of collected fecal material is transported medium (Lab-
Lemco) was placed in a test tube with 18 ml of a screw cap, sealed and stored at -80 ° C. Medium: DHL (Nissui) was used for detection of Escherichia coli . Bacillus subtilis BN strain is CL15
Bifidobacteria was detected using U / ml-supplemented PRM medium and 5% horse blood-supplemented BS (Nissui), respectively. Culturing method: The feces were left at 4 ° C. for thawing, and then a 10-fold serial dilution series was prepared using the diluent. Appropriate dilution was selected for each target bacterial group, 0.05 ml of which was spread on each selective medium, and cultured at 37 ° C. according to a conventional method. Anaerobic culture is Ga
It was carried out for 72 hours using sPack System (BBL).

From the results shown in Tables 3 and 4, when the growth-promoting substance of the present invention comprising a mixture of Bacillus natto-amylase was added to the feed in swine treated with antibiotics, the day after the administration (December 3) It can be seen that as early as Escherichia coli in the feces proliferated remarkably and reached the normal value, and after December 5 there was no significant difference between the experimental group and the control group.

Similarly, from the results shown in Tables 3 and 4, when the growth-promoting substance of the present invention comprising a mixture of Bacillus natto-amylase was added to the feed in the pigs to which the antibiotics were administered, the day after the administration (December 3). It can be seen that Bifidobacterium in feces proliferated markedly to reach a normal value as early as possible, and that there was no significant difference between the experimental group and the control group after December 8th.

From these facts, it is understood that the administration of the growth promoting substance of the present invention is extremely excellent in the normalizing action of the intestinal bacterial flora.

Example 2 Target livestock animal: 30-day-old sow formula: Artificial milk (containing starch, sugar, amylase)
1% by weight of a natto preparation (“Bionnatmin” manufactured by Meguro Research Co., Ltd.) was added. Implementation method: The above feed was freely taken for 14 days. Results: Bifidobacteria in the small intestine were enhanced and stabilized.

Example 3 Target livestock animal: 5-week-old pig formulation: 0.005% by weight of a Bacillus natto preparation (“Bionatmin” above) was added to a milk-phase feed (containing 5% by weight dextrin, glucose and amylase). Implementation method: The above-mentioned feed was freely taken until 11 weeks old. Results: Improvement of intestinal flora, weight gain and improvement of feed conversion rate were observed (p <0.05).

Example 4 Target livestock animal: Broiler primiforma Bina prescription: 0.05% by weight of a natto bacterium preparation (“bionatmin” above) was added to broiler larvae feed (containing starches and amylase). Method of implementation: The above feed was fed for 8 weeks. Results: Improvement of intestinal flora (enhancement of bifidobacteria and lactic acid bacteria), weight gain, and improvement of feed conversion rate (p <0.
01).

<Prescription example> Prescription example 1 Target: Natural food using bran Recipe: A rice cracker was produced by the following recipe. Flour 4,000g Bran 6,000g Bionatmin 100g Egg 1,500g 800g brown sugar Honey 1,000g Others 2,250g α-amylase Suitable amount

[0061]

INDUSTRIAL APPLICABILITY The growth-promoting substance of the present invention containing Bacillus subtilis and amylase as active ingredients is Escherichia coli and Bifidobacteriu which are useful enteric bacteria.
It strongly promotes the growth of m bacteria and keeps the intestinal microflora in a normal state.

[Brief description of drawings]

Figure 1: Bacillus subtilis BN vegetative count and Escher
It is a graph which showed the relation with the growth promotion of ichia coli .

Figure 2: Bacillus subtilis BN vegetative count and Bifido
It is a graph which showed the relation with the growth promotion of bacterium .

Fig. 3: Escherichia co by Bacillus subtilis BN
It is a graph which showed the influence of the involvement of the nutrient source on the growth promotion of li .

Fig. 4 Bifidobacteriu by Bacillus subtilis BN
It is a graph which showed the influence of the involvement of the nutritional source on m growth promotion.

FIG. 5 is a graph showing the relationship between amylase and Escherichia coli growth promotion.

FIG. 6 is a graph showing the relationship between amylase and Bifidobacterium growth promotion.

FIG. 7 is a graph showing the influence of glucose and peptone on the growth promotion of Escherichia coli .

FIG. 8 is a graph showing the influence of glucose and peptone on the growth promotion of Bifidobacterium .

FIG. 9: Bacillus against Bifidobacterium adolescentis
It is the graph which showed the localization site of the growth promoting factor of us subtilis .

FIG. 10: Baci against Bifidobacterium adolescentis
3 is a graph showing the localization site of the growth promoting factor of llus subtilis .

FIG. 11: Bifidobacterium ad in various pH media
It is a graph which showed the influence of Bacillus subtilis BN coexistence on the growth of olescentis .

─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Kyosuke Ozawa 5-25, Kiyowadai-higashi, Kawanishi-shi, Hyogo Prefecture (72) Inventor Shinzuke Hiroishi 26-9, Nango, Otsu-shi, Shiga (56) References Biomedical Letter s, 1993, Vol. 48, p. 73-78 Starch, No. 37, p. 36-42 (58) Fields surveyed (Int.Cl. ⁷ , DB name) C12N 1/38 C12N 1/20 BIOSIS / WPI (DIALOG) PubMed

Claims (4)

(57) [Claims] 1. A growth-promoting substance for Escherichia coli and bifidobacteria, which comprises Bacillus subtilis and amylase as active ingredients. 2. The growth promoting substance according to claim 1, wherein Bacillus subtilis is Bacillus natto. 3. The method further comprises at least one saccharide selected from the group consisting of monosaccharides, oligosaccharides and polysaccharides.
Alternatively, the growth promoting substance described in 2. 4. A preparation containing 1 g of a growth-promoting substance.
The ratio of Bacillus subtilis is 10 ⁴ cfu (community forming unit) or more and the ratio of amylase is 20un in the enzyme activity.
The growth promoting substance according to claim 1, 2 or 3, which has an it or more.