KR960001472B1 - Additive for feeding-stuff - Google Patents

Abstract

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Description

Enteric Salmonella Inhibitor for Algae Feed

1 is a graph showing the results of Example 2 showing the intestinal Salmonella genus bacteria inhibitory effect of the present invention on E. coli.

2 is a graph showing the results of Example 3 showing the effect of the entero Salmonella genus bacterial inhibitor of the present invention on Campylobacter jejuni N 222 (Asabu Daigaku).

3 is a graph showing the results of Example 4 showing the effect of the intestinal Salmonella genus bacterial inhibitor of the present invention on Salmonella typhimirium S4-27 (Climatic Daigaku).

[Industrial use]

The present invention relates to an intestinal Salmonella genus bacteriostatic agent, and more particularly to an enteric Salmonella genus bacterium for algae feed added to the bird feed such as chicken for the purpose of reducing the intestinal Salmonella bacteria.

[Prior art]

Conventionally, in animal feed such as chickens, antibiotics and the like have been added to the feed for the purpose of weight gain, improvement of breeding efficiency, and prevention of bacterial diarrhea. In order to circumvent, the use of antibiotics etc. is restrict | limited by the law about feed stability (law about securing of feed stability and quality improvement).

Therefore, a substance which achieves the above object is required in place of antibiotics and the like, and probiotics are attracting attention as such substances.

Such probiotics include lactobacillus (Lactobacillus spp.), Lactobacillus (such as Streptococcus Faecalis), Bifidobacterium] or lactic acid bacterium (such as Clostridium butyriccomb). butyricum)] is a commercially available probiotic with active ingredients [Kamura Makoto: Livestock Research Vol. 33, No. 1, 31-36 (1979)].

B. natto BN strain [B.natto is classified taxonomically as B.subtilis as a probiotic of Bacillus genus; Baadis manual, Absistic bacterium. Theology Vol. Ⅱ (1986)] of spores (trade name "Glogen", manufactured by Ezai Co., Ltd.) [Makoto Kimura: published in front, Ohta Teruo; Science and Technology of Miso No. 293 (1978. 8) 7.) Printing, 1-9 et al.] And B.toyoi (B.toyoi is classified taxonomically as B. cereus) (trade name "Toyoserine"), Asahi Kasei Kogyo Co., Ltd.] is known, and together with the above-described target effect.

On the other hand, administration of a probiotic containing Bacillus subtilis C-3102 (Microtechnologies No. 1096) to mammals, birds, fish and other animals results in an increase in body weight and improved feed efficiency. It is known that it is obtained (Japanese Patent Laid-Open No. 63-209580), and a compound feed for piglets containing Bacillus and Subtilis C-3102 as an active ingredient is proposed by the present applicant (Japanese Patent Laid-Open No. 62-232343). ).

[Problems that the invention tries to solve]

However, it is not known that preparations containing C-3102 strains of live cells have the effect of reducing the intestinal Salmonella bacteria of birds such as chickens when ingested in the feed.

An object of the present invention is to reduce the enteric Salmonella genus bacteria of birds such as chickens to promote the maintenance of algae health, and further to provide an enteric Salmonella genus bacteria inhibitor for bird feed for avoiding food poisoning by these bacteria.

[Means for solving the problem]

According to the present invention, there is provided an entero Salmonella genus bactericidal agent for the addition of algae feed which contains the live cells of Bacillus subtilis-3102 (uncoated air conditioning apparatus No. 1096) as an active ingredient.

Hereinafter, the present invention will be described in more detail.

In the present invention, the probiotic to be contained as an essential active ingredient is Bacillus subtilis C-3102 (unpublished air conditioning apparatus No. 1096), which is a known bacterium, and its bacteriological properties are already disclosed in Japan. 63-209580 and Japanese Patent Laid-Open No. 62-232343.

To cultivate the Bacillus subtilis C-3102 (U.S. air conditioning apparatus 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 culture of microorganisms, can be used. .

The carbon source may be an assimilable carbon source. For example, glucose, sucrose, starch, molasses, and the like, and nitrogen sources include peptone, hexaextract, caseinic acid-hydrolysate, ammonium sulfate, and the like.

Furthermore, salts, such as phosphate, magnesium, sodium, potassium, calcium, iron, or manganese, a vitamin, an amino acid, an antifoamer, surfactant, etc. can also be added as needed.

The culture conditions are preferably carried out under aerobic conditions.

The initial pH of the medium is 5 to 9, especially 6 to 8, the culture temperature is 20 to 50, 35 to 40 ℃, the culture time is most suitable 12 hours to 7 days.

The culture obtained in this way can be used as an entero Salmonella genus bactericidal agent for addition of algae feed of the present invention by adding itself or its concentrate, or cells separated therefrom, or by adding an additive such as an excipient.

In addition, the culture, concentrate or cells can be used as a probiotic after being dried or formulated as it is or by adding an additive such as an excipient.

The excipient is not particularly limited, and calcium carbonate, skim sugar, corn glitz, cornflower, bran, skim milk powder and the like can be used.

The probiotic of the present invention preferably contains 10 ⁶ to 10 ¹¹ cells / g of live cells of C-3102 strain (unpublished stage No. 1096), that is, apoptosis and / or feeder cells.

The algae which ingest the intestinal Salmonella bacteriostatic agent of the present invention are not particularly limited, but may be added to feeds such as chickens, geese, ducks, turkeys, quails, etc .; pet birds; birds bred for appreciation in a facility such as a zoo. Can be.

The dosage form is not particularly limited, but may be appropriately used by being added to a known feed as a powder, particularly a fine powder.

The amount of the intestinal salmonella genus inhibitor of the present invention added and mixed with a known feed varies depending on the type of algae, the age, and the like, but Bacillus subtilis C-3102 (Natural Performance Aid No. 1096) is the number of live cells, is added to the mixture to ^3-10 10 ⁹ / g, in particular ^4-10 10 ⁶ / g.

The effective intake of the entero Salmonella genus bactericidal agent of the present invention is to ingest a large number of live cells of Bacillus subtilis C-3102 (U.S. performance No. 1096) according to the number of bacteria per gram of intestinal Salmonella genus bacteria. Preferably, at least 1/20 of the bacterium of Bacillus subtilis C-3102 (uncoated air conditioning apparatus No. 1096), more than the number of bacteria per fecal gram of entero Salmonella bacteria to be suppressed, Preferably it is desirable to ingest more than the same number of live cells.

In addition, the administration period varies depending on the type and age of the birds, but in the case of livestock, it is preferable to continuously administer from at least 1 day before the slaughter, preferably 3 days before, more preferably 7 days before. Since it is different from the general antimicrobial agent on it, it is most preferable to prevent the increase of the number of bacteria by administering when the number of bacteria of the genus Salmonella genus is small.

Intestinal Salmonella genus bactericidal agent for bird feed of the present invention is, for example, livestock, especially chicken, such as Escherichia coli (Escherichia coli), Campylobacter genus bacterium, Salmonella genus bacteria that may cause food poisoning , The number of bacteria such as Clostridium perfringenes can be significantly reduced.

[Effects of the Invention]

Since the inhibitor of the present invention contains the live cells of Bacillus subtilis C-3102 (unpublished air conditioner No. 1096) as an active ingredient, it is possible to remarkably reduce the number of bacteria of the genus Salmonella genus of algae, At the same time, it can significantly reduce food poisoning that can occur when these birds are eaten.

In addition, the live cells of Bacillus subtilis C-3102 (Natural Performance No. 1096) are known bacteria known as feed additives, and are excellent in their stability.

EXAMPLE

The following Examples of the invention are illustrated.

Example 1 (Preparation of Probiotic)

Dissolve 200 g of soybean peptone, 10 g of potassium diphosphate, and 200 g of molasses in 10 liters of tap water, and insert the medium whose pH was adjusted to 7.5 with IN sodium hydroxide solution into a self-proventer, sterilize for 15 minutes at 121 ° C., and pre-incubate. The inoculated culture solution of Bacillus subtilis C-3102 (uncoated air conditioning apparatus No. 1096) was inoculated, and the mixture was ventilated at 37 ° C for 40 hours to carry out stirring culture.

The culture solution thus obtained was centrifuged to collect the cells, dried and mixed with calcium carbonate to obtain 200 g of entero Salmonella bacteria inhibitor (hereinafter referred to as inhibitor (1)). The number of new bacteria contained in the inhibitor 1 is 1 × 10 ¹⁰ cells / g.

Example 2 (E. coli infection test)

1 x 10 total of 3 strains of Escherichia coli (derived from two weeks) in 30 layers (brand name `` Select High '' Gunma Co., Ltd.) ^{A 9} / ml sterile physiological saline suspension was administered to the bag using Sonde per ml / ml.

3 x 10 ⁵ inhibitors (1) obtained in Example 1 were fed to the commercially mixed feeds of chickens (20 animals) among chickens (590 days old) after the settling period (10 days) after E. coli administration. Feed for 30 days by replacing with feed added to / g, and then add feed of no additive group (10) to inhibitor (1) feed, and then add feed of inhibitor (1) without additive and feed for 15 days Breeding.

Coliform bacteria were counted by extracting the powder immediately before and after excretion and immediately after excretion.

The result is shown in FIG.

Example 3 (Camphylobacterium Bacterial Infection Test)

35-day-old SPF layer (Line-M, Campylobacter-free), bred using standard feed for layer testing (Central feed for layer testing, Nippon Haigo Food Co., Ltd., SDL No. 2). In 10 groups, a 3 × 10 ⁷ / ml sterile physiological saline suspension of Campylobacter jejuni N 222 (Asabu Daigaku) was administered into the sac using 0.5 ml / margin, sonde.

Infected chickens (cecum test) after this treatment were infected as described above at 42 days of age.

To a group of chicken feed, the inhibitor (1) obtained in Example 1 was added to the standard feed so as to be 3 × 10 ⁵ pieces / g of live bacteria.

The feed was reared at 50 days of age.

At 50 days of age, cecum powder immediately after excretion was collected and the number of the Campylobacter bacteria was measured. Further, when the number of bacteria was 2 × 10 ² / g (detection limit) or less, the enrichment culture was performed, but neither of the Campylobacter bacteria was detected, and the number of bacteria was determined to be 0.

The result is shown in FIG.

Example 4 Salmonella Genus Bacterial Infection Test

Four-day-old male broiler (cove) was bred from the time of harvest using commercially mixed feed which was added or suppressed to 3 × 55 / g as the viable cell count obtained in Example 1 Salmonella typhimirium SU-27 (Gifu Daigaku) 1.6 × 10 ⁷ / ml sterile physiological saline suspension was administered into the bag using 1 ml / head, sonde.

Commercially available blended feeds include 1 to 7 days of age, 8 to 21 days of age and 22 to 28 days of age, under the trade names "Shia Hi-Ace HS-1", "Si Eye Hi-Ace HS-2" and trade name "Si Eye Broace G2" (which Idozu Sample O Co., Ltd.) was used respectively. At the time of gambling, each group was slaughtered five times, and the contents of cecum were collected, and the number of Salmonella was measured.

The result is shown in FIG.

Example 5 Test in Broiler Completion Step

Breeding 6,300 broilers (cove) (1 house) with commercial late feed (brand name `` Sea Ibro ace G2 '' Ito @ Shiyoo Co., Ltd.) and at 51 days of age, marketed finished feed (brand name `` Sea ibro ace F2 '') Sample O Co., Ltd.) was rearranged by replacing the inhibitor (1) of Example 1 with feed added to 5 × 10 ⁵ / g of viable bacteria.

At 47 days of age (before addition of inhibitor (1)), 54 days of age (after 3 days of addition) and 58 days of age (after 7 days of addition), two to three minutes immediately after discharge from the central part of the cage were collected as one specimen and immediately refrigerated. Enterobacteriaceae, Clostridium perfringenes, and Campylobacter bacteria in each sample were measured.

The results are shown in the first table.

TABLE 1

[Addition of Finished Feeders]

* p <0.05, ** p <0.01, *** p <0.001

Logarithm display, () indicates the number of detection samples / inspection samples

Detection limit 1 × 10 ² (campylobacter 1 × 10 ³ ) / g

Example 6 Test in Broiler Late Stage and Finisher

Broiler (cove) of three chickens (6300 chickens) raised in commercial electric feed (brand name `` Sea i-Hybace HS-2 '' Itoken Sioyo Co., Ltd.) was 17-day-old. Ace G2 ') was reared at 49 days of age with finished feed (trade name `` Sea Ibro Ace F2' ').

On the other hand, the broiler is classified into three groups for each cage, and the additive-free group is commercial feed only, test group-1 is 34 days old and test group-2 is 17 days old, respectively. The feed was added to 3 × 10 ⁵ / g.

At 56 days of age, two to three animals immediately after excretion were taken from the center of the cage and 20 colonies were collected from the center and 15 colonies from each end and middle section. Enterobacteriacease, Clostridium perfringenes and Campylobacter bacteria in each specimen were measured.

The results are shown in the second table.

TABLE 2

[Addition of late, finished feed feeder]

** p <0.01, *** p <0.001

Logarithm display, () indicates the number of detection samples / inspection samples

Detection limit 1 × 10 ² (campylobacter 1 × 10 ³ ) / g

Example 7 Test in Broiler Finisher

Commercial feed (brand name "broiler electric clamble" Shikoku Haigoo Food Co., Ltd.) and broiler of 1 chicken (6000 chickens) which were reared feed (brand name "broiler S") from 21 days old at 33 days old The broiler was replaced with the brand name "broiler completed." In the finished feed, the inhibitor (1) of Example 1 was added so as to be 5 x 10 ⁵ pieces / g of viable bacteria.

One sample was taken from 26 days of age (without inhibitor (1)) and 43 days of age (after addition of inhibitor (1) for 10 days) with 4 to 5 minutes immediately after excretion. It was. The number of bacteria in each sample was measured in the same manner as in Example 5.

The number of harmful bacteria (CFU / g) in manure at 26 days of age was 1 to 2 orders higher than that of feed inhibitors (CFU / g). Investigate.

The results are shown in the third table.

TABLE 3

[Addition of late and finished feeders]

Logarithm display, () indicates the number of detection samples / inspection samples

Detection limit 1 × 10 ² (campylobacter 1 × 10 ³ ) / g

As is apparent from Examples 9 and 10 described later, the effect of the inhibitor is remarkable when the number of bacteria and the order of bacteria are the same.

However, the results in Table 3 indicate that the number of harmful bacteria targeted is ineffective as the order difference.

Example 8 (Preparation of Probiotic)

30 g / L of medium 20 of Trypticase Soy Broth (manufactured by BBL) was collected in a test tube, and after sterilization, Bacillus subtilis C-3102 (Natural Aid No. 1096) was used. Cultured colonies were inoculated with 100 bacteria (白 均 耳) and shake cultured at 37 ° C. for 20 hours.

The culture solution thus obtained (hereinafter referred to as inhibitor (2)) was used as triplicate case soy broth (manufactured by BBL) for the Salmonella bacteria, and 15 g of proteus peptone (manufactured by OXIOD) for Campylobacter bacteria. / g, the number of live bacteria in a solution of medium composition (pH about 7.4) of reversible digest (product of OXIOD) 2.5g / l, yeast extract (product of OXIOD) 5g / l, and salt (defense) of 5g / l, respectively. The inhibitor (2) containing the number shown in 11 was adjusted.

Example 9 (Salmonella genus bacterium concentration test)

After Salmonella genus bacteria isolated from 53-day-old broiler were subjected to stationary culture for 37 and 20 hours under the trade name "Trip Case Soy Broth" (BBL), and then trade name "Trip Case Soy Broth" (BBL). The mixture was diluted to prepare a Salmonella solution containing the bacterium in Tables 4 to 7.

Inhibitor (2) of Example 8 was added to each Salmonella solution, followed by incubation for 37, 20 hours and 44 hours. The results are shown in Tables 4-7.

In addition, Table 4 and Table 5 show the result after 20-hour incubation, and Table 6 and 7 show the result after 44-hour incubation.

The symbols of the bridge are as follows.

Sterilization concentration;

± inhibitory concentration (development concentration)

+ Thinning concentration

TABLE 4

TABLE 5

TABLE 6

TABLE 7

In particular, as apparent from Table 7, after 44 hours, the bacteriostatic effect was expressed even in 3/50 of the bacterium and Salmonella bacterium of the inhibitor (2).

Example 10 (inhibiting Campylobacter spp.)

Campylobacter SP. Isolated from 43-day-old broiler as a medium composition, proteus peptone (manufactured by OXIOD) 15g / L, river digest (manufactured by OXIOD) 2.5g / L, yeast extract (manufactured by OXIOD) 5 g / L and 5 g / L of salt (defense) were dissolved in water, adjusted to pH 7.4, and cultured in a medium sterilized at 121 ° C. for 15 minutes in the presence of 37%, 20 hours, and 5% oxygen, and then diluted with the medium. Campylobacter liquid containing the bacterium of Tables 8-11 was prepared.

Inhibitor 2 of Example 8 was added to each campylobacter solution, and the mixture was cultured statically in the presence of 5% oxygen at 37 ° C for 20 hours.

The results are shown in 8 to 11.

In addition, the symbol in a table | surface is as follows.

－: Sterilization concentration

±: inhibitory concentration (development concentration)

+: Growth concentration

TABLE 8

TABLE 9

TABLE 10

TABLE 11

In particular, as is apparent from Table 11, it can be seen that the growth inhibitory effect is expressed even when the number of bacteria of Inhibitor 2 and that of Campylobacter is 5/60.

Claims (3)

An entero Salmonella bacteria inhibitor for the addition of algae feed comprising a living cell of Bacillus subtilis C-3102 (uncoated air conditioning apparatus No. 1096) as an active ingredient. The intestinal Salmonella genus inhibitor according to claim 1, wherein the intestinal Salmonella genus inhibitor is contained in an amount of 1/20 or more bacteria per gram of feed per microgram of the intestinal Salmonella genus bacteria to be inhibited. The method according to claim 1, wherein the number of bacteria per gram of feed of the living cells contained in the feed at least one day before the slaughter of the bird contains at least 1/20 of the number of bacteria per gram of bowel Salmonella bacteria to be suppressed. Intestinal Salmonella bacteria inhibitor.