TW201143631A - Novel Enterococcus faecium LJS-01 and its use as probiotic - Google Patents

Abstract

Enterococcus faecium LJS-01 was isolated from swine intestine that has good antimicrobial activity. The strain inherent acid tolerance, bile salt tolerance, and strong capacity of adhesion on intestinal epithelial cells. The strain exhibited high capacity of colonization when tested in vitro and in vivo. Furthermore, E. faecium LJS-01 has highly cholesterol degrading capacity, and contains enterocin A and enterocin B in the genome. Therefore, this microorganism has potential to replace antibiotics as feed additives, to be used as diarrea treatment agent for animal, and to develop as a health food.

Description

201143631 VI. Description of the invention: [Technical field to which the invention pertains] This case refers to a method for promoting the health of animals by a lactic acid bacteria isolate 5, which is particularly useful for feeds, food additives and health foods. The invention also has the potential to be an animal sputum ameliorating agent. [Prior Art] Humans have been using Lactic acid bacteria (LAB) to ferment the people of Yanxian County for thousands of years, because the sugar is reduced by the lactic acid bacteria and 2 lactic acid to reduce the growth of bacteria. Corrupted. After long-term research, it is found that the milk_inhibiting the growth of other strains, 'in addition to producing organic acids such as lactic acid, another kind of _(4), is the side of 4 j^Mantlbaetenalpeptides), these antifungal peptides are also called pheresins ( Bactenoncs) has an activity of inhibiting the growth of pathogenic bacteria. There have been many research reports pointing out that milk _ is on the body and lactose intolerance; (2) improving the disease of infants and adults; (m body ^ (4) improving allergies; (3) reducing Gastrointestinal pathogens alcohol. (8) The original bacteria compete for the digestive tract; (7) Reduce the gallbladder to promote mineral absorption and lower blood pressure, etc., ,, '() bacteria, species function, use lactic acid bacteria to achieve anti-pathogens = Yuli "The bacteriostats produced by them are generally recognized as safe gnizedasSafe, GRAS) and do not affect humans or other eukaryotes, but have the advantage of promoting health. A total of six related reports on the production of (baCten〇Cm) of Enterococcus faecalis in the feces. Stro呷fova et al. 201143631 (2008) 8, Du Toit et al (2000) 1, Theppangna et al (2007) 9 and P〇eta Et al. (2008) 6 mainly uses the polymerase chain reaction (PCR) technology to detect the antibacterial structural genes, in order to understand the species of the bacteriocin gene isolated from Enterococcus faecalis, so it still belongs to Compared with Enterococcus faecalis which can produce bacteriocin Preliminary research report. Another Marekova et al. (2〇〇3)4 initially performed the purification of bacteriocin A and its characteristics from Enterococcus faecalis EK13 isolated from cow dung, while Strompfova et al. (2006) 7 further Enterococcus faecalis EK13 was tested in piglets for animal experiments. The results confirmed that EK13, which can produce bacteriocin A, can effectively reduce the amount of E. coli in the digestive tract and reduce blood cholesterol, compared with the control group, whether it is the serum biochemical value of pigs. There is no difference in health conditions such as weight, food intake and activity. 'It is confirmed that this strain belongs to GRAS' has the characteristics of probiotics. However, the above-mentioned faecal enterococci can not be found directly from the animal's intestines. It is not the same as the five 'Ljg_〇i, and can have both the antibacterial (enterocin A) and the enterocin B two antibacterial genes in the same strain of bacteria. Other analysis of probiotic characteristics, such as acid and gallbladder resistance In vitro and in vivo tests for the ability of salt, intestinal epithelial cells to adsorb and degrade cholesterol are still absent. [Invention] The present invention provides a lactic acid bacterium, Enterococcus faecalis, translated as The isolate K/^dwmLJS-Ol, the isolate has the following characteristics: 〗 〖Separated from the intestinal mucosa of Taiwan native pigs; 2 by the inhibition of g-protein structural gene test, it is confirmed that the bacterium is rare and possesses bacteriocin A and two kinds of anti-bacterial genes can inhibit a variety of diseases; 3. In vitro and in vivo tests, confirmed that the bacteria have good antibacterial, acid and salt-tolerant ability, can pass in animals After the gastrointestinal digestive juice ^ ^ live in the intestine; 4. In vitro and in vivo tests, confirmed that the bacteria have strong adsorption capacity for intestinal epithelial cells' and thus have a good competitive exclusion effect on disease (four), 5. ς 4 201143631 6. The actual == two bacteria with good degradation of biliary force in the future available for two:: this::::: bacteria £. '-S-01, expected:: application added to animal pharmaceutical composition, It is used in the prevention of 4::: The second anti-bacterial and anti-pathogenic bacteria produced by the drug is completely banned in the EU countries, and the antibiotics are added to the feed in the country. Therefore, it is urgent to seek other methods for yelling health feed additives. . The present invention separates the lactic acid bacteria five/from dwmUS-01' which are inherently resistant to acid, gallbladder I, lactic acid, bacteriostatic and intestinal epithelial cells, and competitive exclusion of pathogenic bacteria. The probiotics for animal health, containing the composition of the lactic acid bacteria of the invention, can be used to solve the deficiency of antibiotics used in conventional animals, and thus can be used as animal feed additives, animal health foods, and can be an improvement agent for animal diarrhea. This case provides a strain of Enterococcus faecalis (the isolate of £·./__ is added as LJS-0) has been deposited in the Hsinchu Food Industry Development Research Institute of Taiwan (F〇〇d Mustry

Kesearch and Development Institute, FIRDI) 'The registration number is BCRC 910421. The object of the present invention is to provide a feed supplement comprising an active lactic acid bacteria component lactic acid bacteria isolate E. / oecitim LJS-01, and an excipient, probiotic (prebiotics) and the like. The feed additive utilizes its lactic acid bacteria, such as the bile salt hydrolysis ability analysis of the fourth embodiment, the fifth embodiment cholesterol degradation ability test, and the sixth embodiment acid and bile salt resistance test, showing the ability to have acid and bile salt resistance. It can be orally administered to the intestines of animals to play a role in promoting animal health. Another object of the invention is to provide a feed supplement. As in the seventh embodiment, the in vitro (/« Wiro) intestinal epithelial cell line Int-407 adsorption test, the eighth embodiment in vivo 201143631 (/wWvo) no sputum pathogen (SPF) chick intestinal epithelial cell adsorption test, showing The animal's intestinal epithelial tissue has a strong adsorption capacity and appears to compete for pathogens. The additive is mixed into the feed to be used as a preventive antibacterial agent, and can be used as a preventive agent for intestinal pathogens to a certain extent. Another object of the present invention is to provide an animal pharmaceutical composition comprising a lactic acid bacteria isolate V./from LJS-01, and a pharmaceutically acceptable excipient. As the bacteriostatic ability test of the second embodiment, it was confirmed that LJS-01 exhibited strong g-deficiency ability to various strains of Listeria (Zkbr/a) genus, golden yellow (tetra) cocci (ga: foot; _ awrsws) and cactus bacillus (尨c/Y/iAscerezys) also showed antibacterial ability. In the ninth embodiment, in view of the improvement of squatting in animals, it was confirmed that oral administration of 丨克& (10) US-01 (3XHT CFU/g) per sputum can improve the symptoms of diarrhea in animals. The non-lactic acid g isolate US-G1 has strong adsorption capacity for intestinal epithelial cells, and has a good competitive elimination effect on pathogenic bacteria, which can be made into lions or animal medicines for treating diseased egg infections. The pharmaceutical composition can also be added to solid or liquid pharmaceuticals or dietary supplements, health foods, animal feeds and food additives, as a probiotic to enhance animal Wei. The above mentioned animal systems include humans and mammals, livestock, laboratory animals, poultry, birds, wild animals, fish and shellfish, and the like, including pet animals. ϋ明五./(10): The LJS-01 strain is proliferated by appropriate culture, and the culture solution is thickened by the thickening of the nutrient extract to add freshman to the fresh type; the east knot drying method or low temperature (5 (^C below) It is dried by a low-pressure drying method, etc. The powder may have a bacterial count of 10* or more per gram, and may be included in each composition of each of the above-mentioned objects according to an appropriate formulation. The form of the composition is not limited. The invention may be in the form of a powder, a liquid, a solid, a granule, a tablet, a capsule or the like. [Embodiment] The present invention will be further described by way of the following examples and the accompanying drawings. The examples are intended to illustrate the effects of the present invention and to limit the scope of the present invention. Λ 201143631 First Example: Isolation of the ^/eec/iziwLJS-Ol isolate The lactic acid strain of the present invention, isolated from the present invention, The strain was identified by API 20 biochemical: 3⁄4 analysis kit and 16S ribosomal DNA (rDNA) sequence analysis. '(1) Isolation and preservation of lactic acid bacteria strains Disinfect the serosa surface with 75% alcohol, cut the mucosal surface with scissors, and take the intestine 2 cm each of the duodenum, placed in a 5 μml sterile centrifuge tube, and washed with feces and mucus in 0.005 磷酸盐 phosphate buffer pH 7.4 (Phosphate Buffer Solution, pH 7.4). After shaking and washing three times, the tissue was ground with a grinder, thoroughly mixed with 1 〇 ml of phosphate buffer solution and allowed to stand for 10 minutes. ' Take 1 〇〇μ1 supernatant and 900 μl Rogersha selective Lactobacillus agar medium ( Rogosa SLbroth) mixed uniformly '1〇〇μι bacteria solution was applied to R0g0sa Sl plate medium containing 〇5 % carbonate #5 (CaC〇3), 卩 anaerobic culture for hour, each selected with the same colony type A total of 1 bacterium is tested by Gram staining, catalase test, etc., and then cultured in mrs culture medium. The obtained nutrient solution is preserved in two ways: one takes 100_200 μl of the bacterial solution. In the strain preservation solution (including MRSBroth which is %glyCer〇l), 'Place -8 (stored in TC freezer, and the second bacteria solution is added to the appropriate excipients and dispensed in ampoules, freeze-dried, ampoules) Seal ', set _2 〇 ° C below the freezer to save. φ (two) core / α (10) 'wmlJS-O l Strain identification This experiment first identified the strain with the API 20 biochemical analysis kit. The operation steps are as follows: firstly, Gram-positive and catalase-negative cocci are picked up and added to 0.3 ml of sterile water. Uniformly, poured into a Columbia blood agar plate and evaluated for hemolysis in an anaerobic environment at 37 ° C for 18-24 hours. About 5 ml of sterile water was added to the incubator to keep the box moist, and the aluminum crucible on the 2 〇 Strq) StriP was peeled open into the box. The bacteria were added to 2 ml of sterile water to adjust the yield to McFarland Να 4 or higher. First add the bacterial solution to the first ten tests (νρ to 'small 5 tube) (VP to LAP test, add 1 〇〇 of the bacteria solution to the concave part. There will be 201143631 residual liquid (about 0.5 ml) Add to Gp Medium and mix it into the last ten tests (RIB to GLYG. Add sterile mineral oil to the concave of ADH to 。1. Cover with lid and culture; ^ , first interpretation; if necessary, culture for 24 hours and then second interpretation. Except for API20 biochemical analysis, 16S rDNA sequence was performed using pCR method with BSF8 primer P_agaCTtTGATCCTGGCTCAG_3') and BSF1541 primer (5'_AAGGAGGTGATCCAGCCGCA-3') The increase was first. A single colony was taken for 10 minutes (rc for 30 minutes, centrifuged for 4 minutes, 4, 〇〇〇xg for 30 minutes, and then 10 μl of the supernatant containing the chromosome was used as template DNA, and the total reaction volume was 50 μΐ. '29.5 μΐ sterilized deionized water, 5 μΐ i〇x pcR buffer, 2 μΐ deoxyribonucleoside _5,_triphosphate (dNTPs, 2.5 mM), 2.5 μM magnesium sulfide (MgCl2, 50 mM, respectively) ), 3 μΐ Dimercaptosulfoxide (DMS〇, 6%), 1μ1 forward and ReverseprimerS, 2 (^M), 5jLilDNA (l〇ng), and ΐμΐ heat-resistant poly(TaqDNA polymerase). The reaction conditions were 94 ° C. After 2 minutes, the template DNA was made at 94 C for 30 seconds. Denatured '62 ° C, 45 seconds to bind the primer to the template dna, DNA extension at 72 ° C, 1.5 minutes for a total of 35 cycles, then 72 ° C, 1 〇 minutes to fully extend the DNA. Take 5 μ1ρ (The κ product was analyzed by 1% agarose gel electrophoresis to confirm whether the size of the nucleic acid fragment was as expected. If the samples were identical, the amplified PCR product was purified for DNA sequencing and sequence alignment. Figure A, B) 'Sequence ratio The method is to use the results of the sequencing, using the Wasn bioinformatics software to enter the NCBI website and compare it with the sequence registered by GenBank, and take the top five DNA similarity analysis, and confirm that it is all five (4). The analysis and identification of the API 20 biochemical analysis kit and the sequence analysis of 16S ribosome deoxyribonucleic acid (rDNA) can confirm the bacteria, and the strain is named as 5.LJS_〇1. The bacteria is. Example: Antibacterial ability test π will US-ΟΙ culture in Luo A single colony was obtained on the plate medium (R〇g〇sa SL), and then a single colony was cultured to the culture solution for 24 hours. After centrifugation at 201143631, the solution was (10)·Wm LJS-01 with phosphate buffer. After washing, a small amount of phosphate buffer was mixed with auger plus aim US-ΟΙ to form a bacterial solution, and 3 μl of the bacterial solution was taken and inoculated into a lactic acid bacteria plate medium (LCMG, Carrying Medium with Glucose) 'anaerobic culture at 37 ° C 24 After the hour, the semi-solid medium (0.8%) containing various pathogens was covered with an overlay co-culture method, and anaerobic culture was carried out at 37 °C for 18-24 hours to detect the inhibition zone (inhibition zone). The diameter of the diameter, and determine the scope of inhibition (Table 1), according to this strain of the strain of various Listeria (ϋ you η·α) genus such as Listeria (Z 〇 〇 " (4), Grignard Listeria (L., Listeria monocytogenes (ζ, Listeria monocytogenes (1· weM/weW), etc. has strong antibacterial activity against Staphylococcus aureus C^Mv/ococcm·? awret^ ) and Cactus bacillus (5izci7/Mj ceretAS) also have antibacterial ability. Table 1 E.faecium LJS-01 bacteriostatic ability strain AU/ml* Listeria monocytogens ATCC 19111 5120 Listeria grayi ATCC 19120 2560 Listeria innocuaKTCC 33091 2560 Listeria welshiweri ATCC 43547 1280 Staphylococcus aureus 47 10 Bacillus cereus ATCC 49064 10 AU/ml, per The bacteriostatic activity of any unit of arbitrary unit (AU) is defined as 2nx 1,000 μΐ/100 μΐ. Let η be 2x dilution times. Third Embodiment: Antibiotic Sensitivity Test This test was conducted on 16 antibiotics using the Agar Disc Diffusion of the BD BBLTM Sensi-DiscTM Antimicrobial susceptibility Test Discs. Detected to indicate [is sensitive to antibiotics by eciwm LJS-01. The test was first carried out according to the NCCLS specification (Wayne, 2003) 1G for the antibiotic paper ingot (Disc) for the quality control of the antibiotic paper ingot (Disc) by picking & flwrews single colony inoculated in the brain The extract culture medium (BHIBroth) was shaken at 35 ° C for 4 hours, centrifuged at 8,000 x g for 5 minutes, and the supernatant was washed once with phosphate buffer, and then suspended in phosphate buffer. The concentration of the bacterial solution was the same as that of the standard suspension of McFarland No. 0.5 (1.5×l08 CFU/ml), and then the bacterial liquid was taken up with a sterile cotton swab and uniformly coated on Hydrolyzed Casein Agar (MHA), respectively. The antibiotic paper ingot was placed in 35 Ϊ for 24 hours, and the diameter of each antibiotic inhibition ring was measured. The drug resistance was determined according to the Kirby-Bauer method and the BBL specification to confirm the effective concentration and operation of the antibiotic paper ingot. Sex. 5./ From a dwm US-ΟΙ single colony inoculated in the mrs culture medium aseptically' 37 ° C culture overnight, 1% bacterial culture was subcultured in another 3 ml MRS medium, 37 C culture for 4 hours 'bacterial liquid After centrifugation at 8,000 rpm (JA20 rotor, Beckman) for 5 minutes, it was washed twice with the acidate buffer, then the cells were suspended in 2 ml of phosphate buffer' and the bacterial solution was prepared into a suspension of McFarland No. 0.5. For turbid liquid, take 1 ml and 9 ml of 1% MRS agar (45. 〇' quickly and evenly mix and pour into a Petri dish. After solidification, paste various antibiotic paper ingots, put them in an anaerobic tank, and place them overnight. 'The anaerobic culture was further incubated at 37 C for 48 hours' to determine the diameter of each drug inhibition circle, and the drug resistance was determined according to the diffusion susceptibility test and the BBL specification (Table 2). From the results, it was found that the five./aedwrn LJS-01 to ammonia Penicillin (AMPI), amoxicillin (AMC30), bacitracin (B10), clarithromycin (CLR15), erythromycin (E15), purple fungi Gentamicin (GM10), imipenem (imipenem, IPM1〇), neomycin ( Neomycin, N30), novobiocin (novobi〇cin, NB30), penicillin (four), P10) and vancomycin (VA30); ciprofloxacin (CIP5), kanamycin (kanamycin, K3 〇), streptomycin (S10) and sulfamethoxazole-oxime benzylidine (four) such as her tri / / trimethoprim, SXT) are resistant. It was confirmed that this strain is not vancomycin resistance enterococcus (VRE), and it is highly sensitive to most antibiotics that inhibit the synthesis of cell wall of 201143631. Table 2 /flgciMmLJS-Ol against 4 sensitive bismuth antibiotics paper bond AM1 0 AMC30 B10 CIP15 CLR15 E15 GM10 IPM10 suppression ring a 28 37 20 R 27 33 19 57 antibiotic paper sharp K30 N30 NB30 P10 S10 SXT TE30 VA30 suppression ring R 22 25 45 RRI 27 Antibiotic spider: AM10: ampicillin, AMC30: amoxicillin, B10: Bacitracin, CIP5: ciprofloxacin, CLR15: clarithromycin, El5: erythromycin, GM10: gentamicin, IPM10: imipenem, BC30: kanamycin, N30: Lu Neomycin, NB30: novobiocin, P10: penicillin, S10: streptomycin, SXT: sulfamethoxazole/trimethoprim, TE30: tetracycline, VA30: vancomycin. a: inhibition zone (mm) R: drug resistance I: moderate drug resistance Four examples: analysis of bile salt hydrolysis ability According to previous research reports, lactic acid strains with bile salt hydrolysate (BSH) have better ability to degrade cholesterol (Kim Technology d, 2〇〇8 Nguyen et Al., 2007) 3'5 ' Therefore, the present invention first tests five for the expression of LJS_〇1 bile salt hydrolase' and then performs other characteristics analysis.

First, the five./izecmm US-ΟΙ was continuously activated three times, and then it was incubated on a 5 _ plate (MRS agar plate) with a sterile toothpick.

The ill is 3 mm aseptic paper, and then the paper containing lactic acid bacteria is placed, ίηΓΛ (taurodeoxycholic acid s〇H minus 7; 丨1a3^g/1 gasification 32 of MRS _ culture dish, by -nn. Ancient _ 4' to determine the diameter of the white precipitate ring around the sterile paper (the actual bacteria have a noisy surface, the dance _ axis has a very high potential. 201143631 Fifth embodiment: cholesterol degradation test

In this test, LJS-01 was cultured in MRS containing water-soluble cholesterol and mrs containing 0.4% bile salt and water-soluble cholesterol. After 24 and 48 hours, the supernatant was obtained by centrifugation, and the supernatant was measured. The cholesterol content was evaluated to determine the ability of the five-/fledwm US-ΟΙ to degrade cholesterol. The test was carried out in accordance with the method described by Gmiland et al. (1985) 2. Take it first! Ml supernatant to a clean test tube, add 3 ml of 95% ethanol and 2 ml of 50 〇 / 〇 potassium hydroxide, after shaking, put it in a 60 C water bath for 15 minutes, take it out and cool at room temperature. 10 ml is burnt, add 3 mi of sterile water after the shock, completely shake for about 1 minute, let stand for 15 minutes at room temperature, then take 1 ml from the organic layer and put it in another clean tube at 60 ° Dry under nitrogen with C, add 2 ml of o-phthalaldehyde reagent (1 ml glacial acetic acid containing mg5 mg ^-phthalaldehycie), mix and let stand for 10 minutes, then slowly add 1 Concentrated sulfuric acid, after shaking, is allowed to stand for ίο minutes, and finally absorbance is detected at 55Qnm with a spectrophotometer at wavelength 〇r). In addition, the test takes different concentrations of water-soluble cholesterol (lml containing hydrazine, 1 〇, 2 〇, 3 [40, 50 pg) according to the above method for the determination of standard curve, as the basis for the conversion of absorbance and g degree, The results (Fig. 3) confirmed that the bacteria were cultured in MRS solution containing cholesterol (Chlo.) or in cholesterol (10).

The BRS salt MRS towel exhibited 70% and 63% cholesterol degradation after 48 hrs. In addition, we also applied the laying hens to perform the test of the ability of the g to degrade serum cholesterol in the body (9) (4). First, the chickens were divided into the control group (c〇_igr〇up cg). =perimental gn)up, EG) each 64, in the trial to analyze the serum biliary content, the experimental _ oral method to the forest bacteria (4 χΐ〇 〇〇=Fn/day/only), Lai group was given oral skim milk powder (1 g/day/only), and stopped after 7 days. Blood was collected on the third, seventh, and fourth days. Changes in alcohol content (Table 3). The results showed that the experimental group chickens had a serum biliary value of 7 days after the continuation of the bacteria, and compared with the control group or before the experiment, ^ 12 201143631 had about 21% significant cholesterol lowering effect, but stopped giving the bacteria 7 In the future, there is no difference in serum cholesterol levels between the two groups. It is speculated that the bacteria must maintain a high concentration of bacteria in the intestinal tract of the chicken to have the ability to lower cholesterol. Table 2 shows the effect of oral administration of Wuji • pulse LJS-01 on serum cholesterol concentration. (mg/dL) dav 0 day 7 day 14 CGa EGb CG EG CG EG Serum Cholesterol a . / 130±21c 138±19 135±14 109±7.0 145 7.5 138 士 8.4 Control group (n=6)·Experimental group (n=6). c (mean soil SD) Sixth Example · Acid and Bile Salt Resistance Test First, in the acid resistance test, five LJS-01 was inoculated into MRS Broth and cultured at 37 ° C for 24 hours to activate. After that, 1 μ of the bacterial solution was added, and 9 ml of phosphate buffered saline (PBS) having pH 7.4 (control), pH 2.0, and pH 3.0 was added. Mix the bacterial solution with each sulphate buffer solution and place it in a constant temperature incubator at 37 ° C. After each hour, 1 hour, and 3 hours, remove 1 ml of the bacterial solution and 10 times with phosphate buffer. Serial dilutions were made by uniformly mixing 1 ml of each dilution with 1 〇/〇mjg agar '37 C anaerobic culture for 48 hours' to calculate the acid resistance (Table 4). In addition, in the bile salt test, first change the five plus w us_〇1 to 1〇ml MRS broth containing 0.3% (w/v) bovine bile (oxgall), culture at 37 ° C, and in At 3, 12, and 24 hours, 1 μ of the bacterial liquid was taken out, and the phosphate buffer solution was used for 1 doubling. Dilute the column, mix 1 ml of each dilution and i % agar evenly, and calculate the number of colonies by 37 ° (anaerobic culture for 48 hours) to evaluate its ability to resist bile salts (Table 5). After incubation for 3 hours under conditions of pH 2.0 and pH 7.4, the results obtained were approximately the same for the number of bacteria surviving (CFU/ml), and were comparable to the number of survivals at the start (〇 hours), indicating no survival and no gastric acid. At the same time, the strain was cultured in MRS medium containing 0.3% bovine bile at 3T>C, and the number of mycelial production (CFU/ml) in the culture for 3 hours 13 201143631 was comparable to that of the control group without bovine bile. The number of colonies generated in 24 hours was compared with the control group, and it was decreased within the logarithm, indicating that it had a good ability to bile salts. It was confirmed that the strain had excellent acid and bile salt-resistant properties. Table 4 shows gchm LJS-01 Acid resistance pH logarithmic colony number (CFU/ml) logarithm 0 hrs 1 hrs 3 hrs Control pH 7.4 PBS 1Q.05±0.10 10.05±0.10 10.Q5±0.10 LJS-01 PH2.0 PBS 1Q.20 ±0.10 10.15±0.13 10.03±Q.05 pH3.0 PBS 10.15±0.15 10.06±0.Q5 10.24 Soil 0_07 Table V E .faeciumUS-Ql salt resistance ability colony number (CFU/ml) logarithm 3 hrs 12 hrs 24 hrs MRS 9.60 soil 0.51 1Q.13±0.09 10.15±0.13 MRS-Bile 9.28±0.50 9.23±0.16 9.21±0.65 Seven Examples: In vitro (J/iv ό) Winning Epithelial Cell Line lnt-407 Adsorption Test Intestinal epithelial cell line (Intestine 407, ATCC: CCL-6) was cultured in BME containing 10% fetal bovine serum (Basal medium Eagle) In Earle's BSS) growth medium, placed at 37 ° C, grown to 8% in a 5% CO 2 incubator. After washing the cells with phosphate buffer, add a small amount of trypsin-ethylenediaminetetraacetic acid mixture (Trypsin -EDTA) After suspending the cells and removing Trypsin-EDTA by low-speed centrifugation, the cells were re-cultured in a 24-well culture dish in an antibiotic-free growth medium with a number of 104 per well, and placed at 37 ° C. The cells were grown to monolayer cells in a 5% CO 2 incubator. Before the adsorption experiment, the test strains V. ATCC 6057 and LJS-01 were washed twice with the acid buffer buffer and centrifuged at 5,000 x g for 5 minutes. Suspended in BME at a concentration of lxl 〇 8 CFU/ml. After removing the supernatant from the single-layer cultured Intestine 407, add 1 μμ of the test strain to each well, and carry out the adsorption reaction for 2 hours at 37 ° C in a 5% CO 2 incubator. Shake the plate gently every 15 201143631 minutes. To make the adsorption uniform, and each strain to be tested is subjected to 2 repetitions. After the adsorption reaction, the supernatant was removed and washed twice with phosphate buffer, and the following quantitative methods were carried out: 1. Gram's stain: The washed cells were fixed with 10% neutral formalin for 30 minutes. After that, it was washed 4 times with a citrate buffer. Gram staining was performed on the cells in the culture and the strain to be tested. Using the microscope to enlarge, 〇〇〇 随意 randomly select 20 fields of view, calculate the number of lactic acid bacteria adsorbed in int_4〇7, the average number of LAB on each cell exceeds 40 LAB, which means that the LAB has good adsorption capacity. The experiment confirmed that the above-mentioned five LJS-01 averaged more than 4 bacteria per cell, which was effective for spring adsorption on Int-407, indicating good adsorption capacity (fourth figure, €). 1 PCR semi-quantitative method: Add the cleaned 24-well plate to 1 〇〇... Trypsin-EDTA to suspend all cells, and transfer the contents of each well to a 5 ml centrifuge tube. In the DNA extraction reagent set, the contents of the centrifuge tube are subjected to DNA extraction according to the operation procedure of the bacterial sample, and are dissolved in an equal volume of sterile water. Coca BSF8 (5^-AGAGTTTGATCCTGGCTCAG-3〇, Primer BSF1541 (5'-AAGGAGGTGATCCAGCCGCA-3'), 2 mM dNTP, Taq polymerase buffer, D&9 with sample DNA specificity The polymerase, 1 tonne <:12 in a 21.21111 centrifuge tube, in the polymerase chain reactor (PE 9700) 'make its temperature change: 94. 〇, 5 minutes, then run 15-25 cycles 94C, 1 minute, 56. (:, 30 seconds, 72. (:, 1 minute. After the PCR reaction, the semi-quantitative analysis was carried out by DNA electrophoresis in the number of saturated unresolved rings. The result of DNA electrophoresis In the analysis software (AlphaEase FC software), the optical density of the expected part of the PCR product is quantified, and the measured value of the test strain/standard strain is used as a reference for the adsorption capacity (fifth image), The measured value of Shi theory is set to 100%, which is equal to !, and the measured value of the Kawasaki strain Wujiakou·Wear US-ΟΙ is relatively 140%', which is equal to 丨4. The result shows that five plus LJS-01 to Int-407 The adsorption capacity of cells is 1.4 times or more of L _· or _·dirty ATCC 6057. 15 201143631 Eighth embodiment: in vivo (JlIvMSPF Intestinal epithelial cell adsorption test Through the above experiments, strains with antibacterial, degraded cholesterol and in vitro intestinal epithelial cell line int. 407 have been selected to have a good adsorption capacity of 5.. 顚 · face LJS-01, for evaluation of US-ΟΙ strain in animals In the digestive tract of the body, the bile-salt and acid-resistant and intestinal epithelial cells absorb the force 'Cheka (9) called the animal experiment, and E LJS 01 is administered orally to the specific pathogen-free (Qi Shenhua pathigen j7ree, chicken, and collected Fecal and intestinal mucosa, analysis of the number of lactic acid bacteria in the intestine, and evaluation of its probiotic characteristics. The experimental procedure is as follows: Prepare eggs without specific pathogens (SPF), and degenerate chickens without specific pathogens after about 21 days. The chickens were divided into four groups, which were the control group, the five LJS 01 were administered, the five were administered, the ecz was called ATCC 6057, and the group such as /ζναη17a was administered. The date of hatching of the SPF chicks was the third day. , on the first day, the first day and the second day, respectively, 1〇8 cFu/mi/only five/LJS_〇1, five, (10) ATCC 6057 and such as "Cao Ru 17a (Table 6) On the 7th and 7th day, each of the two chicks was sacrificed to collect the jejunum, ileum and cecum valley. Each lg 'suspensed the contents of the intestines in a sterile citrate buffer, and allowed to stand for 30 minutes at room temperature. The supernatant was taken and serially diluted 10 times, and diluted with 1 〇〇μ1 of each dilution ratio. After the growth of the lactic acid bacteria colonies was added to the Rogosa SL plate medium supplemented with appropriate antibiotics, the amount of bacteria (CFU/ml) was calculated (Table 7). In addition, 'collect the jejunum, ileum and cecal tissue 3 cm each, and use 70% alcohol mouth syrup · membrane surface' to boil the intestine and place it in a 5 〇 ml centrifuge tube, add 1 〇m| sterilized phosphate buffer Slightly oscillate for 10-20 seconds. After repeating this three times, the cleaned intestinal tract is placed on the plastic wrap. 'The intestinal mucosa is scraped off with a slide. The scraped intestinal mucosa is smeared on another 50 mL centrifuge tube, also at 1 〇mi. The disc culture buffer was sterilized to suspend the intestinal mucosa tissue, and the pellet was allowed to stand at room temperature for 3 minutes. The supernatant was taken and serially diluted 10 times. 1 〇〇μΐ dilution of each dilution was applied to Rogosa SL plate medium supplemented with appropriate antibiotics. After the growth of each lactic acid bacteria colony, the amount of bacteria (CFU/ml) was calculated. Seven). 201143631 On the seventh day, the growth of the empty 'back, cecal contents and intestinal mucosal tissues of each group of SPF chicks was sacrificed. It was found that in the intestinal helper-like analysis, there was no such lactic acid bacteria in the chicken back and cecum segments. The growth of the strain 'test group in the jejunum k, except for the five. / 顚 (four) ATCC 6057 can not grow, Wu Jia she us (1) and 厶 · "ώναπ (10) 17a can grow. In the ileum and cecum, all three can grow. In addition, in the sampling of intestinal mucosa, from Z and Z. such as / ^ π. (10) 17a can be grown in the three segments of the intestines such as empty, ileum and cecum. It should be noted that in the jejunum, the growth of the tile is (10)·(10)LJ, which is better than that of the 厶-Yang Cong 17a 'displayed five·/fiber·(10)us_〇1 even on the seventh day after oral administration· φ intestinal mucosa still has Good colonization capacity (Table 7). Second, probiotic g takes more oral route's and stays in the digestive tract to be able to send it. The time of staying in the oral cavity is very short, so the oral influencing factors can rule out the stability of the 2 strains in the digestive tract. The in vivo test of 5 SPF chicks in the intestinal bile salt is based on living body digestion i; whether the refractory strain with the inhibitory activity can survive. As shown in Table 6, the more measurable _ number is the _ strain in the digestive tract, the thin female white net is not in the month of the genus Enterococcus cocci five. / aecii / m LJS-01 on acid and spring " Bacterial bacteria and good adsorption capacity for intestinal epithelial cells

Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 7 is administered for collection and collection - fecal manure feces # sacrifice 2 small sacrifice 2 chicken chick 17 201143631 Table 7 No specific pathogen (SPF) small The concentration of lactic acid bacteria in the intestinal contents and intestinal mucosa of chickens after oral administration of Wu./fledwmLJS-01 lactic acid bacteria L. salivarius 17a E.faecium ATCC 6057 E.faecium LJS-01 Content jejunum 0 2.5x106* 3·7χ106 0 0 1.6x107 5·5χ106 1.9x107 Content ileum 0 1.2x107 4.2x107 2.4x107 5.2x106 3.2x107 3.4x107 1.8x107 Content cecum 0 6.0x108 3.6x107 1.0x107 2·3χ108 4.6x108 2.3χ108 8·1χ107 Mucosal tissue jejunum 0 1·3χ1〇6 2.7χ105 0 0 7·8χ106 1·2χ107 3.0χ107 Mucosal tissue ileum 0 1·4χ106 3.3χ1〇7 2.6χ1〇5 2.5χ106 2.6χ107 3.6χ107 1.3χ107 Mucosal tissue cecum 0 6·8χ108 3.7χ107 6.32 Χ1〇6 3.4χ105 4·1χ108 1.1x10® 2·0χ108 *: Colony Forning Unit/ml (CFU/ml) Ninth Example: Improvement of animal snoring This trial was conducted in cooperation with a central animal hospital. / I ^ c ^ mUS-Ol suffering

There is the ability to improve the secret of the disease. First, after recording the medical records of the animals with the separation, the mother's day is given orally. (10) us_〇1 (3xl〇1G CF is good. The results show that except for a long-term sputum, it takes 5 days to return to normal, and the test results confirm this. Lactic acid bacteria have a good effect on animals with sputum (Table 8).

18 201143631 1444 1 year Male sausage 3 squat normal 1440 April Male VIP 2 squatting normal Tenth embodiment: Five-year-old LJS_01 high-temperature resistance test in the feed process Because the feed in the process, whether it is granulation or mixing, often produces high temperature, Therefore, it is usually used at 50 ° C for 30 minutes as a test condition for whether the lactic acid bacteria have high temperature resistance. In the present invention, after /aedwm LJS-01 was cultured overnight, it was found that the amount of bacteria was 2 18χ1〇9 CFU/m, and the bacterial liquid was divided into two parts, one part was placed at 5〇〇c for 3 minutes, and the other was placed. The part was placed at 37 ° C for 30 minutes, and the last five measured was edwmUS-01 viable count number = similar, about 5xl 〇 9 CFU / ml. It is confirmed that the five us_〇1 Φ of the present invention has the ability to withstand two temperatures, the process does not affect its activity, and can be used as a feed additive and has a high number of viable cells. The above is only the preferred embodiment of the present invention, and the scope of the present invention is not limited thereto, and the equivalent structural changes of the present specification and the illustrated contents are all included in the same reason. Within the scope of the present invention, Chen Ming is incorporated. [Simplified description of 囷] Figure 1 Sequence analysis of 16S rDNA fragment of LJS-01 (A) V. Sequence of 16S rDNA fragment of ec/ww LJS-01 Sequence of 16S rDNA fragment of LJS-01 sequenced by BSF8 primer Library sequence alignment Figure 2 / aedwm LJS-01 bile salt hydrolysis ability Figure 25. ec/wm LJS-01 cholesterol (Cliolestrol, Cho.) degradation ability containing water-soluble cholesterol MRS (LJS-01 + cho.) MRS containing 〇·4% bile salts and water-soluble cholesterol (LJS-01+cho.+bile) Figure iv./amwmLJS-01 adsorption activity on Int-407 cell line (A) control group (B) V. (10) Cong ATCC 6057 (C) E. faecium 19 201143631 LJS-01 Figure V5. Adsorption activity of eciwmLJS-01 on Int-407 cell line (A) by PCR semi-quantitative analysis

Lane Μ: 1 kb DNA marker.

Lanes 1_4: J5./izecrnm 58, respectively. edww US-01, V. ecz'wm ATCC 6〇57 and Z/. ciwez· Shirota and other experimental treatment groups of Int-407 cell line β-actin gene The product was used as a quantitative control group.

Lanes 5-8: 5, 5, 5, ec/MW LJS-01, 5, faecium ATCC 6057 L. casei Shirota 16S rDNA ° Lane 9: Int-407 cell line β-actin as a negative control group. (B) The adsorption capacity results were found to be five. / from dwm LJS-01 is 厶c like ^ shirota and ATCC 6057 is 1.4 times or more. [Main component symbol description] None. 20 201143631 References: 1. du Toit, M., Franz, CM, Dicks, LM and Holzapfel, WH (2000) Preliminary characterization of bacteriocins produced by Enterococcus faecium and Enterococcus faecalis isolated from pig faeces. J. Appl. Microbiol. : 482-494. 2. Gilliland, SE, Nelson, CR and Maxwell, C. (1985) Assimilation of cholesterol by Lactobacillus acidophilus. Appl. Environ. Microbiol. 49: 377-381. 3. Kim, Y., Whang, JY, Whang, KY, Oh, S. and Kim, SH (2008) Characterization of the cholesterol-reducing activity in a cell-free supernatant of Lactobacillus acidophilus ATCC 43121. Biosci. Biotechnol. Biochem. 72: 1483-1490. Marekova, M., Laukova, A., DeVuyst, L., Skaugen, M. and Nes, IF (2003) Partial characterization of bacteriocins produced by ^ environmental strain Enterococcus faecium EK13. J. Appl. Microbiol. 94: 523-530 5. Nguyen, TD, Kang, JH and Lee, MS (2007) Characterization of Lactobacillus plantarum PH04, a potential probiotic bacterium Int. J. Food Microbiol. 113: 358-361. 6. Poeta, P., Igrejas, G., Costa, D., Sargo, R., Rodrigues, J. and Torres, C. (2008) Virulence factors and bacteriocins in faecal 21 201143631 enterococci of wild boars. J. Basic Microbiol. 48: 385-392. 7. Strompfova, V., Marcindkova, M., Simonova, M., Gancarcikova, S., Jonecova , Z., Scirankova, L., Koscovd, J., Buleca, V.,

Cobanova, K. and Laukova, A. (2006) Enterococcus faecium EK13—an enterocin a-produdng strain with probiotic character and its effect in piglets. Anaerobe 12: 242-248.

8. Strompfova, V., Laukova, A., Simonova, M. and Marcinakova, M. (2008) Occurrence of the structural enterocin A, P, B, L50B genes in enterococci of different origin. Vet. Microbiol. 132: 293 -301. 9. Theppangna, W., Murase, T., Tokumaru, N., Chikumi, H., Shimizu, E. and Otsuki, K. (2007) Screening of the enterocin genes and antimicrobial activity against pathogenic bacteria in Enterococcus Strains obtained from different origins. J. Vet. Med. Sci. 69: 1235-1239.

10. Wayne, P. A. (2003) Performance standards for antimicrobial disk susceptibility tests. Approved standard 8th ed. NCCLS document M2-A8. National Committee for Clinical Laboratory Standards. 22

Claims (1)

201143631 VII. Patent application scope: 1. A strain of Enterococcus faecalis (five er () ci> cc (10) is eciWW2) 5. For eciwm US-01 ' deposited at Taiwan Hsinchu Food Industry Development Research Institute (Food industry Research and Development Institute, FIRDI), registration number BCRC 910421. 2. For the isolate of Patent Application No. 1, the isolate has the properties of acid resistance, bile salt resistance, adsorption capacity of digestive tract epithelial cells, degradation of cholesterol and antibacterial properties, and has resistance to 5 (TC temperature properties. a feed additive comprising the Lactobacillus acid bacterium isolate No. 1 of the patent application scope 5./aedwm LJS-01. 4. The feed additive of claim 3, which provides animal use 5 · As for the feed additive of the third paragraph of the patent application, the lactic acid bacteria can have strong adsorption capacity in the intestinal epithelial tissue of the animal to eliminate the action of the competitive pathogen. 6. A dietary supplement, the composition includes the scope of the patent application. Lactic acid bacteria isolate £· /aecz.ww LJS-01. 7. If the dietary supplement of claim 6 is for human use, 8. If the dietary supplement of claim 6 is applied, Lactic acid bacteria can strongly adsorb the intestinal epithelial tissue of animals to eliminate the role of competitive pathogens. • 9· An animal and pharmaceutical composition for preventing or treating pathogenic infections, including the scope of application for patents The lactic acid bacteria isolate tt LJS-01, and excipients acceptable for pharmaceutical use.