KR20090100667A - Novel Lactobacillus luterii achwai 25101 and feed additives inhibiting swine pandemic diarrheal virus infection - Google Patents

Abstract

The present invention is a novel microorganism isolated from domestic pig stool, having acid and bile resistance, novel Lactobacillus reuteri (HY) having inhibitory activity against porcine epidemic diarrhea virus infection and growth inhibition of harmful microorganisms. 25101, a feed additive including the same, and a method for preventing and treating diarrheal diseases caused by swine epidemic diarrheal diseases and harmful microorganisms using the same, and when feeding to livestock such as piglets, death of piglets caused by swine epidemic diarrheal virus is reduced, There is an effect that can be usefully used as a probiotic.

Description

A new Lactobacillus reuteri HY 25101 with inhibitory activity against porcine epidemic diarrhea virus and A Feed additive containing according to a new Lactobacillus reuteri HY 25101 with inhibitory activity against porcine epidemic diarrheal virus infection

The present invention relates to a novel Lactobacillus ruteri HY 25101 having inhibitory activity against swine epidemic diarrheal virus infection and growth inhibitory activity of harmful microorganisms, and more particularly as a novel microorganism isolated from domestic pig stool, having acid resistance and bile resistance. The present invention relates to a novel Lactobacillus ruteri HY 25101 having an inhibitory effect on swine epidemic diarrheal virus infection and growth of harmful microorganisms, a feed additive including the same, and a method for preventing and treating diarrheal diseases caused by swine epidemic diarrhea and harmful microorganisms using the same.

The Porcine Epidemic Diarrhea Virus (hereinafter referred to as 'PEDV') was first reported in Belgium in 1978 and has since been reported in Europe, mainly in the United Kingdom and Spain (Chasey D. et al ., Virus-like). particles associated with porcine epidemic diarrhoea, Res Vet Sci., 1978, 25: 255-256, Pensaert MB., et al ., A new coronavirus-like particle associated with diarrhea in swine, Arch Virol., 1978, 58: 243- 247). PEDV is a highly infectious viral disease belonging to the Coronaviridae family. It invades the digestive system of the gastrointestinal tract, causes dehydration and high fever due to vomiting and diarrhea, and has a high mortality rate, causing severe economic losses to livestock farms. (Durate M., et al ., Sequence of the spike protein of the porcine epidemic diarrhoea virus, J Gen Virol., 1994, 75: 1195-1200). In Korea, we have been suffering serious damage every year since the early 1990s. In 2003-2007, veterinary epidemic disease was reported by the National Veterinary Research and Quarantine Service. There were 226 cases of swine epidemic diarrhea and 77,206 heads. In particular, it is a gastrointestinal diarrheal disease that occurs frequently in piglets that are weak in winter and at seasons. Recently, epidemic diarrheal disease, which occurred mainly in winter, continues to occur until June and July, indicating seasonality. The main symptom of swine pandemic diarrheal disease is water-borne diarrhea with vomiting, which has a morbidity ranging from 30% to 100%, and is particularly explosive in non-immunized weaning pigs. The mortality rate is 90% in piglets and 2-6% in piglets, and the piglets are severely dehydrated and the stomach is empty or filled with bile. The small intestine and the large intestine are vigorous and thin, often filled with aquatic contents, and the villi of the small intestine mucosa are severe. Because swine pandemic diarrheal disease is a viral disease, it cannot be fundamentally treated with antibiotics or antimicrobial agents. Currently, it is strictly administered hygiene management of pigs to prevent diarrhea in mammalian pigs. PEDV infection is prevented by carrying out maternally transferred antibodies to piglets, but there is a problem that side effects of vaccine use may occur. PEDV attenuated strains have been developed for swine pandemic diarrheal diseases, but they are recurring every year because they cannot be easily used in livestock farms due to lack of awareness of pig farming and poor breeding facilities.

On the other hand, probiotics are commonly used as probiotics or direct-fed microbials (DFMs), which contain living microorganisms and settle in the intestines of animals to inhibit the growth of harmful microorganisms through competitive exclusion with other pathogenic microorganisms, It refers to a microbial agent that promotes animal growth and improves feed efficiency by helping digestion and absorption of ingested feed. Representative probiotic microorganisms known to date include Lactobacillus acidophilus, L. casei, L. plantarum, L. rhamnosus, L. sporogenes , Bifidobacterium lactis, B. longum , Bacillus subtilis, B. polyfermenticus ), Clostridium (C lostridium butyricum, C. faecium ), yeast ( Saccharomyces cerevisiae ) are used, but lactic acid bacteria are used as the main strain. Many in vivo and in vitro experiments have shown the effects of using probiotics to reduce the growth of harmful bacteria in the intestine, reduce odors and harmful gases, prevent viral infection by activating immune function, reduce the frequency of diarrhea, and improve digestibility. Probiotics actively inhibit the pathogen-membrane process of pathogens and preempt nutrients in the intestine to competitively exclude harmful microorganisms, thereby inhibiting the growth of harmful bacteria. In addition, lactic acid and organic acids produced during metabolism lower the pH in the intestinal tract to prevent pathogen proliferation and produce antibacterial substances such as bacteriocin and have a bactericidal effect.

Accordingly, the present inventors conducted a search for a novel lactic acid bacteria that inhibited the infection of PEDV targeting the lactic acid bacteria isolated from the feces of domestic piglets. The discovery of novel lactic acid bacteria that effectively inhibits the present invention was completed.

An object of the present invention is to provide a novel acid-resistant, bile-resistant Lactobacillus reuteri HY 25101 having anti-pig diarrheal virus infection inhibitory activity and growth inhibitory activity of harmful microorganisms.

Another object of the present invention is to provide a probiotic for feed by administering the lactic acid bacteria having excellent PEDV infection inhibitory activity to livestock or by using it as a feed additive, an animal drug, and the like.

The present invention also provides a method for preventing or treating diarrhea caused by the lactic acid bacteria by inhibiting the infection of the swine epidemic diarrheal virus by using the lactic acid bacteria to inhibit the growth of the swine epidemic diarrheal disease or intestinal harmful pathogenic microorganisms of livestock. For other purposes.

In order to achieve the above object, Lactobacillus ruteri HY 25101 having anti-pig diarrheal virus infection inhibitory activity and growth inhibitory activity of harmful microorganisms, feed additives including the same and methods for preventing and treating diarrheal diseases by swine epidemic diarrheal disease and harmful microorganisms using the same It characterized in that to provide.

Hereinafter, the present invention will be described in detail.

We collected pig feces using anaerobic bags (Gas pack pouch, BBL) from piglets between 7 and 16 days of age, diluted them in anaerobic dilutions, and incubated them in MRS solid medium, respectively. In each medium, single colonies were isolated based on colony shape and size, and colony shape and characteristics were examined.

As a result of inhibition of PEDV infection in each isolated single strain, no cytopathic effect (CPE) was observed when the Lactobacillus luterii HY 25101 and PEDV of the present invention were treated together. Therefore, the lactic acid bacteria were antiviral to PEDV. It was confirmed.

In addition, the present invention performed the physiological and biochemical properties, sugar availability, and 16S rRNA gene sequencing to identify Lactobacillus luterie HY 25101. As a result of physiological and biochemical characterization, the lactic acid bacteria are gram-positive bacteria, do not form spores, can grow under both aerobic and anaerobic conditions, have no motility, and form cells. The 16S rRNA gene sequence is shown in SEQ ID NO: 1, and the molecular taxonomic analysis showed that the lactic acid bacteria had 99.1% of 16S rRNA homology with Lactobacillus reuteri . Thus on the basis of morphological, physiological and growth characteristics of bacteria, such as strains of the present invention Lactobacillus bacteria ruteri (Lactobacillus reuteri) Identification, and this Lactobacillus ruteri (Lactobacillus in reuteri ) HY 25101, which was deposited on February 27, 2008 with the Korea Biotechnology Research Institute Gene Bank (Accession Number: KCTC 11291BP).

In order to stably preserve Lactobacillus reuteri HY 25101 of the present invention for long-term stability, it is preferable to store it at -80 ° C. including glycerol or suspend in sterile 10% skim milk powder to be lyophilized.

In addition, the novel Lactobacillus luteri HY 25101 of the present invention has harmful microbial growth inhibitory ability, acid resistance, and bile resistance. Specifically, growth inhibition experiments of harmful microorganisms were performed on Escherichia coli KCTC 2441, Staphylococcus aureus KCTC 1621, Salmonella typhimurium KCTC 12401, Salmonella enteritidis KCTC 12400, Citrobacter freundii KCTC 2006, and Klebsiella pneumoniae KCTC 2208. It was confirmed that all effectively inhibited growth. In the acid resistance test, the cells were incubated for 16 hours in a 37 ° C. cell incubator in MRS liquid medium, diluted in PBS with 10-fold serial dilution, and then plated on MRS solid medium for each dilution step to measure viable cell numbers. . After inoculating the Lactobacillus ruteri HY25101 of the present invention in 0.05M sterile phosphate buffer solution adjusted to pH 4.0, pH 3.0, pH 2.0 with 10% hydrochloric acid, and then incubated at 37 ° C., the bacteria solution was diluted at regular intervals to MRS solid. The viability of the experimental strain was measured using the medium. In addition, the test for biliary resistance was added to the final concentration of 0.1%, 0.3% and 0.5% of oxgal to MRS liquid medium, and then inoculated 1% of the Lactobacillus ruteri HY25101 of the present invention and incubated at 37 ° C for 24 hours, followed by PBS. After dilution with 10-fold serial dilution method, each dilution step was plated on MRS solid medium to determine the number of viable cells. As a result of the experiment as described above, it was confirmed that the novel Lactobacillus ruteri HY25101 strain of the present invention was excellent in both acid resistance and bile resistance.

The Lactobacillus luteri HY 25101 of the present invention can be used alone or in the form of a composition suitable for livestock consumption, and can be used in addition to other useful probiotic microorganisms and prebiotics. For example, specific examples of other useful probiotic microorganisms include Bacillus subtilis, Saccharomyces cerevisiae, Lactobacillus sporogenes, Lactobacillus acidophilus, Lactobacillus brevis, Lactobacillus plantarum, Lactococcus lactis, Lactobacillus gasseti, Lactobacillus helvifius baciius bidoxin, , Enterococcus faecalis, Pediococcus acidophilus, Aspergillus niger, Pichia pastoris, Clostridium butyricum, Aspergillus oryzae, Lactobacillus bulgaricus, Bacillus coagulans . Such compositions may include stabilizers and excipients, encapsulating agents and the like, and may be in the form of lyophilized powder or encapsulated form or of culture or dry powder. Such feed probiotics can be added to feed and ingested in pigs, thereby preventing or treating diarrheal diseases by inhibiting infection of PEDV and growth of harmful microorganisms in swine epidemic diarrheal diseases.

As described above, the novel microorganism Lactobacillus luteri HY 25101 of the present invention is a lactic acid bacterium that inhibits the growth of PEDV and harmful pathogenic microorganisms, and has the characteristics of acid resistance and bile resistance and can effectively work in the intestine because it is a pig-derived microorganism. It can be used as an animal medicine or feed additive to prevent swine epidemic diarrheal disease.

In addition, when feeding to livestock such as piglets, death of piglets due to PEDV is reduced, there is an effect that can be usefully used as a probiotic for piglets.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are not intended to limit the scope of the present invention, it is possible for the ordinary change by those skilled in the art within the scope of the technical idea of the present invention.

<Example 1>

Isolation of Lactic Acid Bacteria from Pig Feces

Pig feces were collected from the piglets between 7 and 16 days of age using an anaerobic bag (Gas pack pouch, BBL), and anaerobic diluent {0.78% K ₂ HPO ₄ , salt mixture (0.47% K ₂ HPO _4, 1.18). % NaCl, 1.2% (NH ₄ ) ₂ SO ₄ , 0.12% CaCl ₂ , 0.24% MgSO ₄ ) 37.5 ml, L-Cystein 0.5 g, 25% L-ascorbic acid 2 ml, 8% Na ₂ CO ₃ 50 ml, 1 ml of 0.1% resazurin was dissolved in 860 ml of distilled water, 0.5 g of Agar was added thereto, and the mixture was diluted in a ratio of 1:10 to 90 ml. The mixture was shaken for 120 minutes using a shaker. 1 ml of the shake solution was plated in MRS solid medium (Difco, USA) and incubated for 48 hours at 37 ℃. 290 colonies were selected and stored at -80 ° C.

<Example 2>

Identification of PEDV Inhibitory Lactic Acid Bacteria

The strain isolated in Example 1 was incubated at 37 ° C. for 16 hours in a MRS liquid medium (manufactured by Difco). Biochemical properties and sugar availability were identified using API 50 CHL (Biomerius) to identify the isolated strains, and 16S rRNA gene sequencing was performed by Yoon et al., Int. Syst. Bacteriol., 47, 993, 1997). The biochemical properties of the strain isolated in Example 1 are as follows and the form is as shown in FIG.

1) Mobility:-

2) Spore Formation:-

3) aerotolerance: +

4) Catalase:-

5) Gas Generation: +

6) Gram dyeing: +

7) Form: Bacillus

The sugar availability of the strain of Example 1 is shown in Table 1 below, and the nucleotide sequence of 16S rRNA is shown in SEQ ID NO: 1.

Strains of the invention from the above results, the Lactobacillus were identified as Bacillus ruteri (Lactobacillus reuteri), Lactobacillus and ruteri named (Lactobacillus reuteri) HY 25101, it was deposited on February 27, 2008 Date of the Korea Research Institute of Bioscience and Biotechnology Gene Bank (trustee Number: KCTC 11291BP).

<Example 3>

Preparation of Feed Additives Including Lactobacillus Luteri HY 25101

Lactobacillus luteri HY 25101 was inoculated in MRS broth (Difco) and incubated at 37 ° C. for 1 day. In a 6,500 liter tank, 1.5 wt% SLS (CSL, corn steep concentrate, pH 4.5 or less), 0.1 wt% yeast extract, 0.05 wt% calcium carbonate, 1.0 wt% glucose, 0.1 wt% phosphate 1 5,000 liters of a liquid medium consisting of potassium (KH ₂ PO ₄ ), 0.1 wt% potassium diphosphate (K ₂ HPO ₄ ), and 0.1 wt% sodium hydroxide was added and sterilized at 121 ° C. for 15 minutes. The obtained lactic acid bacteria were inoculated and incubated at 37 ° C for 1 day. After culturing, the lactic acid bacteria were recovered by centrifugation, dispersed in 10% skim milk, and then freeze-dried to prepare a feed additive including Lactobacillus luteri HY 25101 of the present invention. Subsequently, 2 kg of the feed additive was homogeneously mixed with 1 ton of commercial feed commercially available and then fed to pigs.

<Test Example 1>

PEDV Infection Inhibition Experiment of Lactobacillus Luteri HY 25101

(Virus acquisition)

After growing Vero cells into monolayers, wash once with PBS, adsorb PEDV for 30 minutes, and then maintain the medium (Dulbecco's Modified Eagle Medium (DMEM), 1% penicillin / streptomycin, 5 ㎍ / ml trypsin, 5% Fetal bovine serum) The cells were cultured for 3 days in a 37 ° C. cell incubator containing 5% CO ₂ to confirm the cytopathic effect [Cytopatic effect (CPE)]. When 70% of the cell denaturation effect was observed on the Vero monolayer cells, the supernatant from which cell components were removed by centrifugation after freezing and thawing the infected cells including the maintenance medium in liquid nitrogen was repeatedly stored at -80 ° C. Used.

(Virus quantification)

Prepare Vero monolayer cells in 24-well cell culture plate, dilute PEDV stored at -80 ° C by 10-fold dilution method, inoculate each well in each dilution step, and 37 ° C cells containing 5% CO ₂ for 3 days. The cell degeneration effect was observed while culturing in an incubator. Based on the observation results of cytopathic effect, the virus content in the culture was quantified by the Reed & Muench method.

(Verify PEDV Infection Inhibition Activity)

PEDV was diluted with DMEM without 5% fetal bovine serum so that the viral titers were TCID ₅₀ /0.1mL, 10 TCID ₅₀ /0.1mL and 100 TCID ₅₀ /0.1mL. After preparing Vero monolayer cells in a 24-well cell culture plate, add 100µl of TCID ₅₀ /0.1ml, 10 TCID ₅₀ /0.1ml, 100 TCID ₅₀ /0.1ml and add 10µl of the isolated microbial culture to each well. Cytopathic effect was observed after 3 days of incubation in a 37 ° C. cell incubator containing 5% CO ₂ . The results are shown in Table 2.

Cytopathic Effect of Lactobacillus Luteri HY 25101 on PEDV TCID ₅₀ 10 TCID ₅₀ 100 TCID ₅₀ TCID ₅₀ Lactobacillus luster HY25101 adding adding adding No addition Cytopathic effect voice voice voice positivity

As can be seen in Table 2 above, TCID ₅₀ , Since no cytopathic effect was observed at 10 TCID ₅₀ and 100 TCID ₅₀ (negative), it was confirmed that the Lactobacillus ruteri HY 25101 of the present invention had an inhibitory activity against PEDV.

<Test Example 2>

Acid resistance check

The new Lactobacillus luteri HY 25101 isolated in Example 1 was incubated for 16 hours in a 37 ° C. cell incubator in an MRS liquid medium (manufactured by Difco), and then diluted in PBS by 10-fold serial dilution. The number of viable cells was measured by plating on MRS solid medium at each dilution step. After inoculating the lactic acid bacteria in 0.05M sterile phosphate buffer solution pH adjusted to pH 4.0, pH 3.0, pH 2.0 with 10% hydrochloric acid, and then incubated at 37 ℃ using a MRS solid medium The viability of the experimental strain was measured. The results are shown in FIG.

As can be seen in Figure 2, the novel Lactobacillus lusteri HY 25101 of the present invention showed a high acid resistance by showing a survival rate of 86% at pH 2.0, 98% at pH 3.0 and pH 4.0 there was.

<Test Example 3>

Checking bile resistance

After adding oxgall to the final concentration of 0.1%, 0.3%, and 0.5% in MRS liquid medium, 1% of the Lactobacillus luteri HY 25101 of the present invention of Example 1 was inoculated and incubated at 37 ° C for 24 hours. After dilution in PBS by 10-fold serial dilution method (10-fold serial dilution) was measured in each MRS solid medium by dilution step to measure the number of viable cells. The results are shown in FIG.

As can be seen in Figure 3, the Lactobacillus ruteri HY 25101 of the present invention shows a result of maintaining a viable cell count of 5 x 10 ⁸ cfu / ㎖ or more even under the condition that 0.5% oxgal is added to confirm that it has bile resistance Could.

<Test Example 4>

Confirmation of harmful microbial growth inhibitory ability

In order to confirm the growth inhibitory ability of harmful microorganisms, the following six microorganisms were tested using Kuroiwa et al . (Kuroiwa et . Al ., Journal of Infectious Diseases, 64, 257, 1990). Escherichia coli KCTC 2441, Staphylococcus aureus KCTC 1621, Salmonella typhimurium KCTC 12401, Salmonella enteritidis KCTC 12400, Citrobacter freundii KCTC 2006, Klebsiella pneumoniae KCTC 2208 After smearing and drying on agar medium, a paper disc of 8 mm in diameter was placed on the medium. The novel Lactobacillus luteri HY 25101 of the present invention was incubated at 37 ° C. for 18 hours in MRS liquid medium, and then 30 μl of the culture solution was inoculated on a 8 mm diameter paper disc on the prepared nutrient agar medium, followed by further culture at 18 ° C. for 18 hours. The inhibitory activity is shown in Table 3 by measuring the clear zone formed after this.

Inhibitory Activity of Lactobacillus Luteri HY 25101 on Harmful Microorganisms Harmful microorganisms Transparent ring diameter (mm) Escherichia coli KCTC 2441 14.1 Staphylococcus aureus KCTC 1621 16.4 Salmonella typhimurium KCTC 12401 18.2 Salmonella enteritidis KCTC 12400 16.7 Citrobacter freundii KCTC 2006 20.0 Klebsiella pneumoniae KCTC 2208 15.8

As can be seen in Table 3, the diameter of the inhibitory ring inhibiting the growth of harmful microorganisms by the Lactobacillus luteri HY 25101 of the present invention is Escherichia coli 14.1 mm for KCTC 2441, 16.4 mm for Staphylococcus aureus KCTC 1621, 18.2 mm for Salmonella typhimurium KCTC 12401, 16.7 mm for Salmonella enteritidis KCTC 12400, 20.0 mm for Citrobacter freundii KCTC 2006, 10.0 mm for Klebsiella pneumoniae mm. Therefore, it was confirmed that Lactobacillus luteri HY 25101 of the present invention was a lactic acid bacterium having excellent growth inhibitory activity against six harmful microorganisms.

Figure 1 is a photograph of the Lactobacillus Lutheri HY 25101 observed with an electron microscope.

2 is a graph showing the acid resistance of Lactobacillus luterie HY 25101.

3 is a graph showing the bile resistance of the Lactobacillus luterie HY 25101.

YANG, Ki Rak; Korea Yakult Co., Ltd <120> A new Lactobacillus reuteri HY 25101 with inhibitory activity          against porcine epidemic diarrhea virus and A Feed additive          containing <130> P0803-02 <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 1571 <212> DNA <213> Lactobacillus reuteri HY 25101 <400> 1 agagtttgat cctggctcag gatgaacgcc ggcggtgtgc ctaatacatg caagtcgtac 60 gcactggccc aactgattga tggtgcttgc acctgattga cgatggatta ccagtgagtg 120 gcggacgggt gagtaacacg taggtaacct gccccggagc gggggataac atttggaaac 180 agatgctaat accgcataac aacaaaagcc acatggcttt tgtttgaaag atggctttgg 240 ctatcactct gggatggacc tgcggtgcat tagctagttg gtaaggtaac ggcttaccaa 300 ggcgatgatg catagccgag ttgagagact gatcggccac aatggaactg agacacggtc 360 catactccta cgggaggcag cagtagggaa tcttccacaa tgggcgcaag cctgatggag 420 caacaccgcg tgagtgaaga agggtttcgg ctcgtaaagc tctgttgttg gagaagaacg 480 tgcgtgagag caactgttca cgcagtgacg gtatccaacc agaaagtcac ggctaactac 540 gtgccagcag ccacggtcat acgtaggtgg caagcgttat ccggatttat tgggcgtaaa 600 gcgagggcag gcggttgctt aggtctgatg tgaaagcctt cggcttaacc gaagaagtgc 660 atcggaaacc gggcgacttg agtgcagaag aggacagtgg aactccatgt gtagcggtgg 720 aatgcgtaga tatatggaag tacaccagtg gcgaaggcgg ctgtctggtc tgcaactgac 780 gctgaggctc gaaagcatgg gtagcgaaca ggattagata ccctggttgt ccatgccgta 840 aacgatgagt gctaagtgtt ggagggtttc cgcccttcag tgccggagct aacgcattaa 900 gcactccgcc tggggagtac gaccgcaagg ttgaaactca aaggaattga cggcggcccg 960 cacaagctat ggagcatgtg gtttaattcg aagttacgcg aagaacctta ccaggtcttg 1020 acatcttgcg ctaaccttag agataaggcg ttccattcgg ggacgtaatg acaggtggtg 1080 catggtcgtc gtcagctcgt gtcgtgagat gttgggttaa gtcccgcaac gagcgcaacc 1140 cttgttacta gttgccagca ttaagttggg cactctagtg agactgccgg tgacaaaccg 1200 gaggaaggtg gggacgacgt cagatcatca tgccccttat gacctgggct acacacgtgc 1260 tacaatggac ggtacaacga gtcgcaagct cgcgagagta agctaatctc ttaaagccgt 1320 tctcagttcg gactgtaggc tgcaactcgc ctacacgaag tcggaatcgc tagtaatcgc 1380 ggatcagcat gccgcggtga atacgttacc gggcgttgta cacaccgccc gtcacaccat 1440 gggagtttgt aacgcccaaa gtcggtggcc taacctttat ggagggagcc gcctaaggcg 1500 ggacagatga ctggggtgaa gtcgtaacaa ggtagccgta ggagaacctg cggctggatc 1560 acctcctttc t 1571  

Claims (4)

Lactobacillus reuteri HY 25101 having inhibitory activity against swine pandemic diarrheal virus infection and growth inhibition of harmful microorganisms. Livestock feed additives comprising a Lactobacillus reuteri HY 25101 of claim 1. A method of preventing or treating swine epidemic diarrheal disease by inhibiting the infection of swine epidemic diarrheal virus using Lactobacillus reuteri HY 25101 of claim 1. Lactobacillus of claim 1 ( Lactobacillus) reuteri ) A method for preventing or treating diarrhea caused by the microorganisms by inhibiting the growth of harmful pathogenic microorganisms in the intestine of animals using HY 25101.

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