JP2014183751A - Bacteria that inhibit avian salmonella and viable bacterial agent utilizing them - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel viable bacterial agent showing excellent growth inhibition effect to pathogenic bacteria and promoting animal growth.SOLUTION: From the appendix of healthy chicken, novel strains of lactic acid bacteria, Enterococcus faecalis APU-14 (Accession number: NITE P-1528) and Enterococcus mundtii APU-27 (Accession Number: NITE P-1529), have been isolated. When fed to domestic animals and poultry, the viable cells of these lactic acid bacteria can inhibit growth of pathogenic bacteria such as salmonella in the fed animals, and can enhance weight gain of the animals.

Description

  The present invention relates to a novel lactic acid strain useful for suppressing the growth of pathogenic bacteria in an animal body and promoting weight gain of animals, and a viable agent containing the same as an active ingredient.

  Salmonella spp. Are pathogenic bacteria that cause infectious diseases in domestic poultry, while causing food poisoning that infects humans through foods and processed products made from the poultry. By suppressing the growth of these bacteria in the poultry body, it is possible to maintain the health of the poultry and further reduce the risk of food poisoning via the livestock processed products of the poultry. .

  As a method generally used in an animal breeding place, there is a method of controlling bacterial growth by administering an antibacterial substance such as an antibiotic in a feed. However, in the method using an antibacterial substance, problems such as the residual of the antibacterial substance in livestock products and the development of resistant bacteria due to overdose have been pointed out, and an alternative method for suppressing the growth of pathogenic bacteria is required. ing.

  As a method of inhibiting the growth of pathogenic bacteria as an alternative to antibacterial substances, a method of reducing the number of pathogenic bacteria in poultry by ingesting multiple lactic acid bacteria, etc. by oral administration, etc. A method for suppressing the propagation of pathogenic bacteria present in the body of poultry is known (Patent Document 1, etc.).

  However, there are still various problems caused by pathogenic bacteria in the poultry body, and a viable bacterial agent having an excellent ability to suppress growth is required. At the same time, there is a demand for the development of a viable bacterial agent that not only suppresses the growth of pathogenic bacteria in the animal body but also improves the health of the animal and promotes growth.

JP 2011-4636 Gazette

  In view of such a situation, an object of the present invention is to provide a novel viable agent that exhibits an excellent antiproliferative effect on pathogenic bacteria and at the same time promotes the growth of animals.

  As a result of earnest research, the inventors of the present application show that the APU-14 strain belonging to Enterococcus faecalis and the APU-27 strain belonging to Enterococcus munditi exhibit an excellent growth inhibitory effect against harmful bacteria such as Salmonella spp. Experimentally confirming that the number of pathogenic bacteria present in the poultry body is reduced by feeding the live cells of the strain to the poultry, and that the weight gain of the poultry is promoted. Completed.

  That is, the present invention relates to at least one lactic acid strain selected from Enterococcus faecalis APU-14 strain (Accession number: NITE P-1528) and Enterococcus munditi APU-27 strain (Accession number: NITE P-1529). A viable bacterial agent containing the viable bacterial cell as an active ingredient is provided. Moreover, this invention provides the feed containing the said living microbe agent of this invention. Furthermore, the present invention provides at least one lactic acid strain selected from Enterococcus faecalis APU-14 strain (Accession number: NITE P-1528) and Enterococcus munditi APU-27 strain (Accession number: NITE P-1529). A method for suppressing the growth of pathogenic bacteria in the animal body, which comprises feeding the animal with live cell bodies of Furthermore, the present invention provides at least one lactic acid strain selected from Enterococcus faecalis APU-14 strain (Accession number: NITE P-1528) and Enterococcus munditi APU-27 strain (Accession number: NITE P-1529). A method of promoting weight gain of an animal is provided, comprising feeding the animal with a live cell of

  By feeding the live bacteria agent of the present invention to animals such as livestock and poultry, the growth of harmful bacteria such as Salmonella in the animal body is suppressed, and the occurrence of infectious diseases caused by these harmful bacteria is reduced. Therefore, it is possible to reduce the risk of food poisoning by suppressing bacterial infection of livestock products. Furthermore, it is possible to improve the poultry gain and feed requirement by promoting the growth of animals.

  Enterococcus faecalis strain APU-14 and Enterococcus munditi strain APU-27 are Gram-positive cocci isolated from the cecal contents of healthy chickens and undergo homolactic fermentation under anaerobic conditions. In addition, the APU-14 strain and the APU-27 strain have 16S ribosomal RNA as a partial sequence of the nucleotide sequences shown in SEQ ID NOs: 1 and 2, respectively, as genetic properties. From these mycological properties and the partial base sequence of 16S ribosomal RNA shown in SEQ ID NOs: 1 and 2, it was identified that these strains belong to Enterococcus faecalis and Enterococcus munditi, respectively. The APU-14 and APU-27 strains have 16S ribosomal RNA as a partial sequence of the nucleotide sequence shown in SEQ ID NOs: 1 and 2, but the nucleotide sequence has substantially no ability to inhibit the growth of pathogenic bacteria. Within a range that does not change, some (preferably 1 to several) bases may have mutations due to substitution, deletion, or addition.

  The APU-14 and APU-27 strains have been deposited with the NITE P-1528 and NITE P-1529 accession numbers, respectively, at the National Institute of Technology and Technology Patent Microorganism Depositary.

  The viable agent of the present invention contains at least one viable cell of lactic acid bacteria selected from Enterococcus faecalis APU-14 strain and Enterococcus munditi APU-27 strain as an active ingredient. The living cells may be in a form that can survive and act in the fed animal body. For example, the culture solution of the strain can be used as it is or recovered and the number of bacteria appropriately adjusted can be used as a viable agent, or the living cells can be dried to be in a dormant state. May be.

  The details of the culture of the APU-14 strain and APU-27 strain are not particularly limited, and the culture can be performed by a normal lactic acid bacteria culture method. In general, the culture may be performed for about 24 to 72 hours under a facultative anaerobic condition at a temperature of 30 to 37 ° C. and a pH of 6 to 8 using a medium containing peptone or yeast extract as a component. The cells can be collected by a known method, and can be collected by a general method such as centrifugation or filtration of the culture solution. When provided as a liquid viable agent, the culture solution may be used as it is, or the collected cells may be suspended in a fresh culture solution or an appropriate buffer solution. A drying method for drying the bacterial cells is also known, and generally, a method of drying the collected bacterial cells by freeze drying, vacuum drying, fluidized bed drying or the like is widely used. The method for adjusting the number of cells of the dried cells is not particularly limited, and can be performed by a technique known in the technical field to which the present invention belongs, and is generally performed using lactose, starch, defatted nuka, etc. A method of adjusting the number is widely used.

  The viable agent of the present invention may consist only of the above-mentioned strain, or may further contain other components useful for maintaining the activity of the above-described strain, if desired.

Although the viable agent of the present invention can be directly orally administered for use, it can be more conveniently added to feed or drinking water and fed to animals. The feed may be a common feed that is commercially available. The amount used when the live bacteria agent is mixed with feed or drinking water can obtain the effect of inhibiting the growth of pathogenic bacteria (such as Salmonella, Escherichia coli, Clostridium, etc.) and promoting weight gain. There is no particular limitation as long as it is the number of cells. The amount of the viable agent used is 10 ⁴ to 10 ⁹ cells / g, for example, 10 ⁵ to 10 ⁷ cells / g as the number of cells in the feed if added to feed, and drinking water if added to drinking water. The number of microbial cells may be 10 ⁴ to 10 ⁹ cells / mL, for example, 10 ⁵ to 10 ⁷ cells / mL. In the case of direct oral administration to animals, the amount used may be 10 ⁶ to ¹⁰ 10 per bird per chicken, for example 10 ⁷ to 10 ⁹ birds per day. For animals other than chickens, What is necessary is just to administer orally according to this usage-amount. By using it in the above-mentioned range, it is possible to obtain the effect of suppressing the growth of pathogenic bacteria and the effect of promoting weight gain.

  A method of suppressing the growth of pathogenic bacteria in the animal body by feeding live animals of at least one of APU-14 strain and APU-27 strain to the animal, and a method of promoting weight gain of the animal It can be carried out by feeding an animal with the viable agent of the present invention as described above. As shown in the following Examples, both APU-14 strain and APU-27 strain have both the effect of suppressing the growth of pathogenic bacteria in the animal body and the effect of promoting the weight gain of the animal. These two effects can be achieved at the same time if live bacteria are fed to animals.

  The animal to which the viable agent of the present invention is fed is not particularly limited, and domestic animals such as cattle, pigs, horses, sheep, goats, rabbits, and poultry such as chickens, ducks, turkeys, quail, duck, and ostriches. Animals such as elephants, giraffes, and lions bred for ornamental purposes in zoos and safari parks, etc. Can be added to the feed.

  Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited to the following examples.

1. Enterococcus faecalis, creation of Enterococcus munditi strains Enterococcus faecalis APU-14 and Enterococcus munditi APU-27 strains were isolated from the cecum of healthy chickens.

Each isolated strain was treated with SCD bouillon medium (composition: tryptone 1.7%, soy peptone 0.3%, glucose 0.25%, sodium chloride 0.5%, dipotassium phosphate 0.25%, pH 7.3). ) At 37 ° C. for 24 hours. The culture solution thus obtained was used as a viable agent (the number of bacteria was 5 × 10 ⁸ cells / mL to 2 × 10 ⁹ cells / mL).

2. Infection test in chickens Live bacteria containing APU-14 strain or APU-27 strain as the active ingredient were orally administered to primary chicks (2 × 10 ⁸ cells / bird), and the next day, Salmonella tea Fimlium culture was suspended in physiological saline and orally administered using a sonde. At 7 days of age, cecal contents of chicks were collected, and the number of Salmonella was measured using DHL agar medium. The test was repeated 10 times with 10 birds in each group. Tables 1 and 2 show the results obtained by expressing the measured number of Salmonella in common logarithm.

  From the results shown in Tables 1 and 2, by feeding a chicken with a viable agent comprising the APU-14 strain or APU-27 strain according to the present invention as an active ingredient, the growth of Salmonella in the chicken body is suppressed. It was confirmed that it was possible.

3. Analysis of genetic properties of APU-14 and APU-27 strains
16S / 23S rRNA Sequencing In Nucleic Acid Techniques in Bacterial Systematics, pp115- Ed. By E. Stackebrandt and M. Goodfellow (1991) According to the method described in John Wiley & Sons Ltd., APU-14 strain and APU-27 strain The partial base sequence of 16S ribosomal RNA was analyzed.

  Amplified product obtained by PCR using chromosomal DNA obtained by extracting DNA from the culture solution of APU strain, or amplified product obtained by colony PCR method, purified from gel after electrophoresis on agarose gel Samples for analysis were used. For PCR amplification, a 27f / 1492r primer set (Table 2) targeting the 16S rRNA gene was used. PCR conditions were 94 ° C for 3 minutes, 94 ° C for 30 seconds-55 ° C for 30 seconds-72 ° C for 30 seconds. Cycle, 72 ° C. for 7 minutes, 4 ° C. ∞.

  For the sequence analysis, the partial sequence of 16S ribosomal RNA (SEQ ID NOs: 1 and 2, respectively) of APU-14 and APU-27 strains was determined by combining and correcting the obtained sequence results using the primers shown in Table 2. did. Using these partial nucleotide sequences, a primary search was performed with NCBI BLAST. Next, on the Ribosomal Database Project site (release 10, http://rdp.cme.msu.edu), a taxonomic search and a homology search with respect to the standard strain were performed, and the base sequence of the standard strain was obtained. From this and each APU strain gene, homology search was performed with gene analysis software Genetyx ver.15 (Genetics Co., Ltd.), and the obtained homology (identity,%) was shown in the table of each isolate (Table) 4 and Table 5).

  Based on the mycological properties such as Gram staining and the partial nucleotide sequence of the identified 16S ribosomal RNA, it became clear that the APU-14 strain is classified as Enterococcus faecalis and the APU-27 strain is classified as Enterococcus munditi. It was.

Claims (10)

  Effective viable cells of at least one lactic acid strain selected from Enterococcus munditi APU-27 strain (Accession number: NITE P-1529) and Enterococcus faecalis APU-14 strain (Accession number: NITE P-1528) Viable agent contained as an ingredient.   The viable agent according to claim 1, which is a growth inhibitor of pathogenic bacteria present in the animal body.   The viable agent according to claim 2, wherein the pathogenic bacterium is Salmonella.   The viable agent according to claim 1, which is a promoter of animal weight gain.   The live fungus according to any one of claims 2 to 4, wherein the animal is a poultry.   A feed comprising the viable bacterium of any one of claims 1 to 5.   At least one lactic acid bacterial strain selected from Enterococcus munditi APU-27 (Accession Number: NITE P-1529) and Enterococcus faecalis APU-14 (Accession Number: NITE P-1528) A method for suppressing the growth of pathogenic bacteria in the animal body.   At least one lactic acid bacterial strain selected from Enterococcus munditi APU-27 (Accession Number: NITE P-1529) and Enterococcus faecalis APU-14 (Accession Number: NITE P-1528) A method of promoting weight gain of the animal, comprising:   Enterococcus munditi APU-27 strain (Accession number NITE P-1529).   Enterococcus faecalis APU-14 strain (Accession number NITE P-1528).