JPH05146260A - Agent for suppressing harmful enteric bacterium to be added to poultry feed - Google Patents

Abstract

PURPOSE:To obtain the subject suppressing agent effective for decreasing harmful enteric bacteria, keeping the health and avoiding food poisoning, having excellent safety and useful for the treatment of domestic animals such as chicken by using live bacterial cell of Bacillus subtilis C-3102 as an active component. CONSTITUTION:The objective suppressing agent contains live cells of Bacillus subtilis C-3102 (FERM BP-1096) as an active component. The number of the live cells in 1g of feed is >=1/20 of the number of the object harmful enteric bacteria in 1g of feces. The culture of Bacillus subtilis C-3102 is preferably carried out e.g. in a conventional liquid medium at pH6-8 and 35-40 deg.C for 12hr to 7 days under aerobic condition.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an intestinal harmful bacterium inhibitor, and more specifically, it is contained in the feed of birds such as chickens for the purpose of reducing intestinal harmful bacteria, and is added to avian feed to inhibit enteric harmful bacteria. Regarding agents.

[0002]

2. Description of the Related Art Conventionally, in feeds for livestock such as chickens, antibiotics and the like have been added to feeds for the purpose of weight gain, improvement of breeding efficiency, prevention of bacterial diarrhea, etc.
In order to avoid the residue on meat and eggs, the law concerning the safety of feed (the law concerning ensuring the safety of feed and improving the quality) limits the use of antibiotics.

Therefore, instead of antibiotics and the like, substances that achieve the above-mentioned objects are required, and probiotic agents are drawing attention as such substances. Such probiotic agents include lactobacilli [genus Lactobacillus ], lactococcus [eg Streptococcus faecal
is)], bifidobacteria (Bifidobacterium) or butyric acid bacteria (e.g. cross-tri Jiu scan butyricum (Clostridiu
[ M butyricum )] as an active ingredient is commercially available [Kimura Makoto; Livestock Research Vol. 33, No. 1, 31-36
(1979)]. In addition, as a viable agent of Bacillus, B. natto BN strain [B. natto is taxonomically B. subtil
It is classified as is; Bajisu Manual of Systematic Bacteriology Biology Vol. II (19
86)] spore preparation [trade name “Growen”, manufactured by Eisai Co., Ltd.] [Makoto Kimura; supra, Teruo Ota; Miso Science and Technology No. 293 (July 1978) Print, 1-9
Other], and B. toyoi [(B. toyoi
Is a taxonomically classified B. cereus ) spore preparation (trade name "Toyoselin"), manufactured by Asahi Kasei Kogyo Co., Ltd., both of which have the same purpose as above.
Have an effect.

On the other hand, Bacillus subtilis C-3102
It is known that administration of a probiotic agent containing microbial cells of (Ministry of Industrial Science and Technology No. 1096) to mammals, birds, fish and other animals produces effects such as weight gain and improvement of feed efficiency. The present applicant has proposed a mixed feed for piglets containing Bacillus subtilis C-3102 as an active ingredient (JP-A-63-209580) (JP-A-62-232343).

[0005]

[Problems to be Solved by the Invention] However, C-3
It is not known that a formulation containing 102 strains of viable cells has an effect of reducing harmful intestinal bacteria in birds such as chickens when mixed with feed and ingested.

The object of the present invention is to reduce the harmful bacteria in the intestines of birds such as chickens and to maintain the health of the birds, and consequently to suppress the harmful bacteria of the intestinal bacteria to be added to avian feed in order to avoid food poisoning by these bacteria. To provide.

[0007]

According to the present invention, Bacillus subtilis C-3102 (Microtechnical Research Institute No. 1096).
No. 6) is provided as an active ingredient, and an inhibitor of harmful enteric bacteria for addition to avian feed is provided.

The present invention will be described in more detail below.

In the present invention, the probiotic agent to be contained as an essential active ingredient is Bacillus subtilis C-3102 (Kikusoji No. 1
No. 096), and the mycological properties thereof have been already disclosed in JP-A-63-209580 and JP-A-62.
No. 232343.

The Bacillus subtilis C-3102
For culturing (Mikori Kenjiro No. 1096), for example, a liquid medium or a solid medium containing a carbon source, a nitrogen source, an inorganic substance or the like, which is usually used for culturing microorganisms, can be used as the medium. .. The carbon source may be any assimilable carbon source, for example glucose, sucrose, starch, molasses, etc., and the nitrogen source, for example, peptone, meat extract, hydrolyzed caseinate, ammonium sulfate and the like. You can Further, if necessary, phosphates, salts of magnesium, sodium, potassium, calcium, iron, manganese, etc., vitamins, amino acids, antifoaming agents, surfactants and the like can be added. The culture conditions are preferably aerobic conditions, and the initial pH of the medium is 5 to 9, particularly 6 to 8,
The culturing temperature is preferably 20 to 50 ° C., particularly 35 to 40 ° C., and the culturing time is preferably 12 hours to 7 days.

The culture thus obtained may be used as such, or a concentrate thereof, or cells isolated therefrom,
As it is or by adding additives such as excipients, it can be used as the intestinal harmful bacteria inhibitor for bird feed addition of the present invention. In addition, the above-mentioned culture, concentrate, or microbial cell may be used as it is or after adding an additive such as an excipient, or may be formulated into a post-biological agent. The excipient is not particularly limited, and calcium carbonate, defatted rice sugar, corn grits, corn flour, bran, skim milk powder and the like can be used. The viable cell agent of the present invention preferably contains 10 ⁶ to 10 ¹¹ cells / g of viable cells of the C-3102 strain (Ministry of Industrial Science and Technology No. 1096), that is, spores and / or vegetative cells.

Birds to be ingested with the intestinal harmful bacteria inhibitor of the present invention are not particularly limited, but for example, livestock such as chicken, eagle bird, duck, turkey, quail, etc .; pet bird; raised for viewing at facilities such as zoos. It can be added to the feed of birds and the like. The dosage form is not particularly limited, but it can be preferably used by adding and mixing it to a feed known as a powder, particularly a fine powder. The amount of the intestinal harmful bacterium inhibitor of the present invention to be added to and mixed with a known feed varies depending on the type of bird, the age, etc., but is not limited to that of Bacillus subtilis C-3102 (Microtechnical Research Institute No. 1096). Number of viable bacteria is 10 ³ to 10 ⁹ /
It is preferable to add and mix so as to be g, particularly 10 ⁴ to 10 ⁶ pieces / g.

The effective dose of the intestinal harmful bacteria inhibitor of the present invention depends on the number of bacteria of the intestinal harmful bacteria per gram of feces, Bacillus subtilis C-3102 (Microtechnical Research Institute No. 1096).
No.), it is desirable to ingest a large amount of viable bacterial cells, and preferably at least 1/20 of the number of Bacillus bacilli that is to be suppressed against the number of intestinal harmful bacteria per gram of feces.
It is desirable to ingest live cells of Subtilis C-3102 (Microtechnical Laboratory Article No. 1096), more preferably the same number or more. Although the administration period varies depending on the type of bird, the age, etc., in the case of livestock, it is desirable to administer the administration continuously from at least one day before slaughter, preferably 3 days before, and more preferably 7 days before slaughter. Further, since it is different from general antibacterial agents, it is most preferable to administer it while the number of intestinal harmful bacteria is small so as to prevent the increase of the number of bacteria.

The inhibitor of enteric toxic bacteria for addition to avian feed of the present invention is, for example, in the case of livestock, especially chickens, which may cause food poisoning, for example, pathogenic Escherichia coli and Campylobacter bacteria. , The number of bacteria such as Salmonella , Clostridium perfringenes and the like can be remarkably reduced.

[0015]

INDUSTRIAL APPLICABILITY The inhibitor of the present invention contains Bacillus subtilis C-3102 as an active ingredient (Ministry of Industrial Science and Technology, Article 1096).
No.) viable cells, the number of harmful intestinal harmful bacteria in birds can be significantly reduced, the health of birds can be maintained, and food poisoning that may cause when the birds are eaten Can be greatly reduced. In addition, Bacillus subtilis C-3102 (Ministry of Industrial Science and Technology Article 1096)
Since the live bacterium is a known bacterium known as a feed additive, it is also excellent in safety.

[0016]

EXAMPLES Examples of the present invention will be described below.

Example 1 (Production of live cells) Soybean peptone 200 g and phosphoric acid 2 in 10 liters of tap water
A medium in which 10 g of potassium and 200 g of molasses were dissolved and the pH was adjusted to 7.5 with IN sodium hydroxide solution was placed in a jar fermenter, sterilized at 121 ° C for 15 minutes, and pre-cultured with Bacillus subtilis. C-3
Inoculated with the culture solution of No. 102 (Microtechnology Research Institute No. 1096),
The culture was performed at 37 ° C. for 40 hours with aeration and stirring.

The thus obtained culture broth was centrifuged to collect bacterial cells, which were dried and mixed with calcium carbonate to obtain 200 g of an intestinal harmful bacterium inhibitor (hereinafter referred to as inhibitor 1). The viable cell count contained in the inhibitor 1 was 1 × 10 ¹⁰ cells / g.

[0019] Example 2 (E. coli infection test) commercially available formula feed (trade name "select high" Gunma union Feed Co., Ltd.) layer, which were bred using the (trade name "DeKalb TX") to 30 birds, E. coli (Escherichiacoli) A total of 1 × 10 ⁹ suspensions / ml of 3 strains (2 strains were derived from layers) / ml in sterile physiological saline were applied to the sacrococcus using a sonde at 1 ml / wing. Fixation period after administration of E. coli (10 days)
Of the later chickens (590 days old), the feed of one group of chickens (20 birds)
Inhibitor 1 obtained in Example 1 was added to the above-mentioned commercially available mixed feed so that the number of viable bacteria was 3 × 10 ⁵ cells / g, and the feed was changed to 3
It was bred for 0 days, and then the feed of the non-addition group (10 birds) was replaced with the feed of the inhibitor 1 added, and the feed of the addition group was replaced with the feed without the inhibitor 1 for another 15 days. Immediately before the addition of the probiotic agent and immediately after the excretion, feces immediately after excretion were collected and the number of Escherichia coli was measured.

The results are shown in FIG.

Example 3 (Campylobacter bacterium infection
Test) 35-day-old SPF layer (line-M, Campylobacter free) bred using a standard feed for layer test (standard feed for medium chicks for layer test "SDL No. 2" Japan Formulated Feed Co., Ltd.) Campylobacter jejuni N222 (Azabu University) with 10 birds per group
0.5 x 3 × 10 ⁷ cells / ml of sterile saline suspension
It was administered intraperitoneally using a sonde. In addition,
After this treatment, uninfected chickens (cecal fecal test) were infected at 42 days of age in the same manner as above. For a group of chicken feed,
Inhibitor 1 obtained in Example 1 was added to the above standard feed in a viable cell count of 3 × 1.
It was added at a rate of 0 ⁵ pieces / g. This feed was further bred for 50 days. From the 50th day, cecal feces immediately after excretion were collected daily and the number of Campylobacter bacteria was measured. When the number of bacteria was 2 × 10 ² / g (detection limit) or less, the enrichment culture was carried out, but the Campylobacter bacteria were not detected in any of them, and the number of bacteria was determined to be 0.

The results are shown in FIG.

Example 4 (Salmonella bacterium infection test) A commercially available compounded feed to which the inhibitor 1 obtained in Example 1 was added at a viable cell count of 3 × 10 ⁵ cells / g or not was added. Four-day-old male broilers (cobbs) that were raised from the time of chick insertion were used to produce Salmonella typhimirium ( Salmonel).
la typhimirium SU-27 (Gifu University) 1.6 × 10
⁷ pieces / ml of sterilized physiological saline suspension, 1 ml / wing,
It was administered intrathecally using a sonde. As a commercially available compound feed, in the 1 to 7-day age, the 8 to 21-day age and the 22 to 28-day age, the trade name "CIA HiBloace HS-1", the trade name "CIA HiBloace HS-2" and the trade name ""Cibro Ace G2" (both manufactured by ITOCHU Feed Co., Ltd.) was used. Five birds were killed in each group from the time of infection, the contents of the cecum were collected, and the number of Salmonella bacteria was measured.

The results are shown in FIG.

Example 5 (Trial in broiler finishing stage
Test) 6300 broiler (cobb) chickens (1 poultry house) were bred with a commercial late feed (trade name "CAI Bloace G2" ITOCHU FEED CO., LTD.) Ace F2 "ITOCHU FOOD CO., LTD. Was added with the inhibitor 1 of Example 1 at a viable cell count of 5 × 10 ⁵ cells / g, and the above-mentioned feed was replaced with the above-mentioned feed.
47 days old (before addition of inhibitor 1), 54 days old (3 days after addition)
At the age of 58 days (7 days after the addition), 2-3 feces immediately after being discharged were collected as one sample from the central part of the poultry house and immediately stored in a refrigerator. Enterobacteriaceae ( Enterobact) in each specimen
eriaceae ), Clostridium perfringens and Campylobacter counts were measured.

The results are shown in Table 1.

[0027]

[Table 1]

Example 6 (in the latter stage of broiler and finishing stage)
Kake test ) Commercial pre-term feed (brand name "CHI HYBLOACE HS-
2 "3 poultry houses (1 poultry house 63) bred by ITOCHU Feed Co., Ltd.
00 broilers (cobbs) were changed to a late feed (trade name "CIAIBROACE G2") at the age of 17 days and a finish feed (trade name "CIAIBROACE F2") at the age of 49 days to be bred. On the other hand, the above broilers were divided into 3 groups for each poultry house, the non-addition group was commercial feed only, and the test group-1 was 3 groups.
From the 4th day of age, the test group-2 was from the 17th day of age, respectively.
The inhibitor 1 was replaced with a feed added so that the viable cell count would be 3 × 10 ⁵ / g. 2 to 56 days after excretion
With three birds as one sample, 20 samples were collected from the central part of the poultry house, and 15 samples were collected from each of the end and middle parts, for a total of 50 samples in each group, and immediately stored under refrigeration. The number of Enterobacteriaceae bacteria, the number of Clostridium perfringens bacteria, and the number of Campylobacter bacteria in each sample were measured.

The results are shown in Table 2.

[0030]

[Table 2]

Example 7 (Test in broiler finishing stage)
Test) Commercial early feed (brand name "Broiler Early Crumble" Shikoku Blended Feed Co., Ltd.) and late feed (brand name "Broiler S") 1 chicken house (6000 birds) from 21 days old
The broiler of No. 3 was replaced with a finishing feed (brand name "broiler finishing") at the age of 33 days and bred. Example 1 for finishing feed
Inhibitor 1 was added at a viable cell count of 5 × 10 ⁵ cells / g.

At 26 days of age (without addition of inhibitor 1) and at 43 days of age (after addition of inhibitor 1 for 10 days), feces immediately after excretion 4 to
Twenty specimens were collected from the central part of the poultry house, with five specimens as one specimen, and immediately refrigerated. The number of bacteria of each sample was measured in the same manner as in Example 5.

26-day-old fecal harmful bacteria count (CFU /
g) is 1-2 from the number of bacteria (CFU / g) of the inhibitor in the feed
There were many orders, and the effect of the inhibitor in the situation where a remarkable increase in harmful bacteria was expected was investigated.

The results are shown in Table 3.

[0035]

[Table 3]

As will be apparent from Examples 9 and 10 described below, the effect of the inhibitor is remarkably exhibited if the target number of bacteria and the order of the number of bacteria are the same. However, the results in Table 3 show that the target harmful bacteria count is not effective when the difference is two orders of magnitude.

Example 8 (Production of live cells) "Trypticase Soy broth (Trypticase S ) "
oy Broth) "(manufactured by BBL) 20g medium of 30g / l
In a test tube and after sterilization, Bacillus subtilis C-3
1 of 102 (Microtechnical Lab. No. 1096) cultured colonies
White ear was inoculated and cultured with shaking at 37 ° C. for 20 hours.

The culture broth thus obtained (hereinafter referred to as inhibitor 2) is used for Salmonella bacteria, Trypticase soy broth (manufactured by BBL) medium, or Campylobacter bacteria. For Proteose Peptone (OXI
OD) 15g / l, River digest (OXI
Medium composition (pH: 2.5 g / l, OD), yeast extract (OXIOD), 5 g / l
The solution of about 7.4) was used to prepare the inhibitor 2 containing the numbers of viable bacteria shown in Tables 4 to 11, respectively.

Example 9 (Salmonella bacterium inhibitory concentration test)
Test) Salmonella bacteria isolated from 53-day-old broiler feces are trade name "Trypticase soy broth" (BBL)
After statically culturing at 37 ° C. for 20 hours at 37 ° C., it was diluted with the trade name “Trypticase soy broth” (BBL) to prepare Salmonella solutions containing the numbers of bacteria shown in Tables 4 to 7.

Inhibitor 2 of Example 8 was added to each Salmonella solution, and the mixture was subjected to static mixed culture at 37 ° C. for 20 hours and 44 hours.

The results are shown in Tables 4 to 7.

Tables 4 and 5 show the results after culturing for 20 hours, and Tables 6 and 7 show the results after culturing for 44 hours. The symbols in the table are as follows.

-: Bactericidal concentration ±: Inhibitory concentration (growth inhibitory concentration) +: Growth concentration

[0044]

[Table 4]

[0045]

[Table 5]

[0046]

[Table 6]

[0047]

[Table 7]

In particular, as is clear from Table 7, it can be seen that after 44 hours, the growth inhibitory effect is exhibited even when the number of inhibitors 2 against the number of Salmonella is 3/50.

Example 10 (Inhibition of Campylobacter spp.
Campylobacter bacteria isolated from control) 43-day-old broilers (Camplylobacter SP.) As the medium composition, proteose peptone (manufactured by OXIOD Co.) 15g / l, River digest (made by OXIOD Inc.) 2.5g / l, Yeast extract (manufactured by OXIOD) 5 g / l, salt (Pharmacopoeia) 5
g / l was dissolved in water, adjusted to pH 7.4, sterilized at 121 ° C. for 15 minutes, and statically cultivated in a medium at 37 ° C. for 20 hours in the presence of 5% oxygen. 8-Table 1
A Campylobacter solution containing 1 of the bacteria was prepared.

Inhibitor 2 of Example 8 was added to each Campylobacter solution, and static mixing culture was carried out at 37 ° C. for 20 hours in the presence of 5% oxygen.

The results are shown in Tables 8-11.

The symbols in the table are as follows.

-: Bactericidal concentration ±: inhibitory concentration (growth inhibitory concentration) +: developmental concentration

[0054]

[Table 8]

[0055]

[Table 9]

[0056]

[Table 10]

[0057]

[Table 11]

In particular, as is clear from Table 11, the inhibitor 2
It can be seen that the growth-inhibiting effect is exhibited even when the number of bacteria against the number of Campylobacter is 5/60.

[Brief description of drawings]

FIG. 1 is a graph showing the results of Example 2 showing the effect of the enteric bacterium inhibitor of the present invention on Escherichia coli.

FIG. 2 is a graph showing the results of Example 3 showing the effect of the intestinal harmful bacterium inhibitor of the present invention on Campylobacter jejuni N222 (Azabu University).

FIG. 3 is a graph showing the results of Example 4 showing the effect of the enteric bacterium inhibitor of the present invention on Salmonella typhimurium SU-27 (Gifu University).

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[Procedure amendment]

[Submission date] July 17, 1992

[Procedure Amendment 1]

[Document name to be corrected] Drawing

[Name of item to be corrected] Figure 2

[Correction method] Change

[Correction content]

[Fig. 2]

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Office reference number FI technical display location C12R 1: 125)

Claims (3)

[Claims] 1. An inhibitor of harmful enteric bacteria for avian feeds, which contains live cells of Bacillus subtilis C-3102 (Microtechnical Laboratory No. 1096) as an active ingredient. 2. The intestinal harmful bacterium according to claim 1, which contains at least 1/20 of the viable cells per gram of feed relative to the number of intestinal harmful bacteria to be suppressed per gram of feces. Inhibitor. 3. The number of bacteria of the live bacteria contained in the feed at least one day before the slaughter of the birds is controlled with respect to the number of harmful enteric bacteria to be suppressed per gram of the feces. The intestinal harmful bacterium inhibitor according to claim 2, containing at least 1/20.