TWI417054B - Novel enterococcus faecium ljs-01 and its use for probiotic - Google Patents

Description

Novel Enterococcus faecalis LJS-01 and its probiotic use

This case refers to a lactic acid bacteria isolate Enterococcus faecium LJS-01 and its promotion method for animal health, especially for feeds, food additives and health foods. The invention also has the potential to be an animal sputum ameliorating agent.

The method of using Lactic acid bacteria (LAB) to prolong the shelf life of food has been used for thousands of years. Because the sugar is fermented by lactic acid bacteria, a large amount of lactic acid is produced, which lowers the pH value and inhibits it. Bacteria grow to prevent food spoilage. After long-term research, it is found that the mechanism of lactic acid bacteria inhibiting the growth of other strains, in addition to the production of organic acids such as lactic acid, another antibacterial mechanism, relying on the role of antibacterial peptides, also known as antibacterial peptides Bacteriocins have an activity of inhibiting the growth of pathogenic bacteria.

With the advancement of biotechnology, many research reports indicate the advantages of lactic acid bacteria on the human body, including: (1) alleviating lactose intolerance; (2) improving diarrhea in infants and adults; and (3) regulating body immunity; (4) Improve allergies; (5) Reduce the production of pathogenic bacteria in the digestive tract; (6) Competing with the pathogens for habitats of the digestive tract; (7) Lowering cholesterol; (8) Forming anti-gastrointestinal tumors; (9) Synthesizing folic acid and Group B vitamins; (10) promote mineral absorption and lower blood pressure.

Among the various functions known to lactic acid bacteria, the use of lactic acid bacteria to achieve functions against pathogens, regulation of the immune system, and lowering of cholesterol is being widely used. Since lactic acid bacteria and the bacteriocin produced by them are generally recognized as safe (GRAS), they do not affect humans or other eukaryotes, and have the advantage of promoting health.

So far, it has been known to isolate Enterococcus faecalis that produces bacteriocin from animal feces, and a total of six related reports are Strompfova et al (2008) ⁸ , Du Toit et al (2000) ¹ , Theppangna et al (2007) ⁹ and Poeta et al. (2008) ⁶ mainly use polymerase chain reaction (PCR) technology to detect the antibacterial structural gene to understand the type of bacteriocin gene isolated from Enterococcus faecalis. It is a preliminary study report on Enterococcus faecalis which can produce bacteriocin. In addition, Marekova et al. (2003) 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 carried out Escherichia coli EK13 in piglets. Animal test, the test results confirmed that the EK13 which can produce bacteriocin A can effectively reduce the amount of E. coli in the digestive tract and reduce the blood cholesterol, compared with the control group, whether it is pig serum biochemical value, body weight, food intake and activity There is no difference in health conditions, and it is confirmed that the bacteria belong to GRAS and have the characteristics of probiotics.

However, the above-mentioned urestatin-producing Enterococcus faecalis has not been found to be directly isolated from the intestinal tract of the animal, and is not the same as Enterococcus faecium LJS-01, and can have both anterocin A and enterocin B in the same strain of bacteria. Two bacteriocin genes. Other in vitro and in vivo tests for the analysis of probiotic characteristics such as acid resistance, bile salt resistance, intestinal epithelial cell adsorption and cholesterol degradation are still absent.

The invention provides a lactic acid bacterium, E. faecium isolate E. faecium LJS-01, which has the following characteristics: 1. Firstly, it is separated from the intestinal mucosa of Taiwan native pig; 2. The anti-bacterial structure gene test confirmed that the bacterium has both bacteriocin A and bacteriocin B, which are the broad-spectrum antibacterial genes that can inhibit various pathogens. 3. In vitro and in vivo tests confirmed the original The bacteria have good antibacterial, acid and bile salt-tolerant ability, and can survive in the intestine after passing through the gastrointestinal digestive juice in the animal; 4. After in vitro and in vivo tests, the bacteria have strong adsorption capacity to the intestinal epithelial cells, and then The pathogen has a good competitive elimination effect; 5. It has been proved in vitro and in vivo that the bacteria have a good ability to degrade cholesterol. 6. It has been confirmed by experiments that the bacteria have high temperature resistance.

Based on the above characteristics, the lactic acid bacteria E. faecium LJS-01 of the present invention is expected to be available as a health food, animal feed and food additive in the future, and can be used as a probiotic to enhance animal health. Further, the lactic acid bacteria E. faecium LJS-01 of the present invention can also be applied to an animal pharmaceutical composition for the prevention or treatment of infection by pathogenic bacteria.

Since antibiotics for animals that have been added to feed for a long time are closely related to the cause of resistance to pathogenic bacteria, the EU countries have completely banned use, and the country has gradually reduced the use of animal antibiotics in feed. Therefore, it is urgent to seek other methods to promote animal health. Feed additive. The present invention separates the lactic acid bacteria E. faecium LJS-01 which has the innate ability to resist acid, bile salts, produce lactic acid, produce bacteriocin and strong adhesion of intestinal epithelial cells to compete for exclusion of pathogenic bacteria, etc. 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.

The case provides a Enterococcus faecalis (E. faecium) isolates of E. faecium LJS-01, has been deposited at Hsinchu, Taiwan Institute of Food Industry (Food industry Research and Development Institute, FIRDI), register number BCRC 910421.

The object of the present invention is to provide a feed additive comprising an active lactic acid bacteria component lactic acid bacteria isolate E. faecium LJS-01, and an added excipient, prebiotics and the like. The feed additive utilizes its lactic acid bacteria, such as the analysis of the bile salt hydrolysis ability of the fourth embodiment, the cholesterol degradability test of the fifth embodiment, and the acid and bile salt resistance test of the sixth embodiment, showing the ability to withstand acid and bile salts. 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 intestinal epithelial cell line Int-407 adsorption test, the eighth embodiment in vivo ( In vivo ) specific pathogen (SPF) chick intestinal epithelial cell adsorption test, showing that the intestinal epithelium can be passed through the animal The strong adsorption capacity of the tissue presents a competitive exclusion pathogen. The additive is mixed into the feed to be used as a preventive antibacterial agent, and can be used as an anti-formulation agent for enteric pathogens to a certain extent.

Another object of the present invention is to provide an animal pharmaceutical composition comprising the lactic acid bacteria isolate E. faecium LJS-01, and a pharmaceutically acceptable excipient. As the bacteriostatic test of the second embodiment, it was confirmed that LJS-01 exhibited strong bacteriostatic ability against various strains of Listeria , and Staphylococcus aureus and Bacillus cereus also exhibited inhibition. Bacterial capacity. In the ninth embodiment, the improvement of squatting in animals was confirmed, and it was confirmed that oral administration of 1 gram of E. faecium LJS-01 (3x10 ¹⁰ CFU/g) per day can improve the symptoms of diarrhea in animals. It shows that the lactic acid bacteria isolate LJS-01 has strong adsorption capacity to intestinal epithelial cells, and has a good competitive elimination effect on pathogenic bacteria, and can be made into an animal medicine for preventing or treating pathogenic bacteria infection. The pharmaceutical composition can also be added to solid or liquid pharmaceuticals or dietary supplements, health foods, various animal feeds and food additives, and used as a probiotic to enhance animal health. The above-mentioned animal systems include humans as well as animals such as mammals, livestock, laboratory animals, poultry, birds, wild animals, fish and shellfish, including pet animals.

The E. faecium LJS-01 strain of the invention is proliferated by appropriate culture, and the culture solution is concentrated to increase the bacterial number concentration, and then the excipient is added, and then dried by a freeze drying method or a low temperature (lower than 50 ^O C) drying method. . The powder may have a bacterial count of 10 ¹² or more per gram, and may be included in each composition of each of the above-mentioned objects of the invention in accordance with an appropriate formulation. The form of the composition is not limited, and may be in the form of a powder, a liquid, a solid, a granule, a sheet or a capsule.

The invention will be further illustrated by the following examples and the accompanying drawings. The following examples are intended to illustrate the utility of the invention and are not intended to limit the scope of the invention.

First embodiment: E. faecium Separation of LJS-01 isolate

The lactic acid strain E. faecium LJS-01 isolated by the present invention is identified by using the API 20 biochemical analysis kit and 16S ribosomal DNA (rDNA) sequence analysis.

(1) Isolation and preservation of lactic acid bacteria strains

Intestines of the pigs were firstly disinfected on the serosa surface by 75% alcohol. After cutting the mucosal surface with scissors, the intestines and duodenum were taken 2 cm each and placed in a 50 ml sterile centrifuge tube to 0.005 M phosphate buffer pH7. .4 (Phosphate Buffer Solution, pH 7.4) (referred to as phosphate buffer) Wash off the feces and mucus, wash it three times with shaking and then grind the tissue with a grinder, mix thoroughly with 10 ml of phosphate buffer solution and let stand 10 Minutes, 100 μl of the supernatant was mixed with 900 μl of Rogersa selective Lactobacillus agar medium (Rogosa SL broth), and 100 μl of the bacterial solution was applied to Rogosa SL plate medium containing 0.5% calcium carbonate (CaCO ₃ ). Anaerobic culture was carried out at 37 °C for 48 hours. A total of 10 bacteria with the same colony type were selected and tested by Gram staining, catalase test, etc., and then cultured in MRS medium. Two ways to save: one 100-200 μl of the bacterial solution is placed in the strain preservation solution (MRS Broth containing 25% glycerol), placed in a -80 ° C freezer, and the second bacteria solution is added to the appropriate shape The agent is divided into ampoules, freeze-dried, sealed in an ampoule, set at -20 °C Freezer storage.

(II) Identification of E. faecium LJS-01 strain

In this experiment, the strain identification of the API 20 biochemical analysis kit was first carried out. The operation steps were as follows: firstly, the Gram-positive and catalase-reactive cocci were picked up, and a single colony was picked up and added to 0.3 ml of sterile water to mix evenly. In a Columbia blood agar plate, it was cultured in an anaerobic environment at 37 ° C for 18-24 hours to evaluate its hemolysis ability.

About 5 ml of sterile water was added to the incubator to maintain the humidity in the box, and the aluminum foil on the API 20 Strep strip was peeled open into the box. The bacteria were added to 2 ml of sterile water to adjust the concentration to above McFarland No. 4. The bacterial solution was first added to the small test tubes of the first ten tests (VP to ADH ) (about 100 μl of the bacterial solution added to the concave portion of the VP to LAP test). The remaining bacterial solution (about 0.5 ml) was added to glucose-peptone medium (GP Medium), mixed uniformly, and added to the last ten tests ( RIB to GLYG ). Sterile mineral oil was added to the pits of ADH to GLYG . Cover the lid and incubate at 37 ° C for 4 hours, first interpretation; if necessary, culture for 24 hours and then a second interpretation.

In addition to the API20 biochemical analysis, the 16S rDNA sequence was amplified by PCR using BSF8 primer (5'-AGAGTTTGATCCTGGCTCAG-3') and BSF1541 primer (5'-AAGGAGGTGATCCAGCCGCA-3'). First, a single colony was taken at 100 ° C for 30 minutes, centrifuged at 13,000 xg for 30 minutes at 4 ° C, and then 10 μl of the supernatant containing the chromosome was used as template DNA. The total volume of the reaction was 50 μl, respectively, and 29.5 μl was sterilized. Ionized water, 5 μl of 10x PCR buffer, 2 μl of deoxyribonucleoside-5'-triphosphate (dNTPs, 2.5 mM), 2.5 μl of magnesium chloride (MgCl ₂ , 50 mM), 3 μl of dimethyl sulfoxide (DMSO, 6%), 1 μl forward and 1 μl reverse primers (20 μM), 5 μl DNA (10 ng) and 1 μl of thermostable polymerase (Taq DNA polymerase). The reaction conditions were 94 ° C. After 2 minutes, the template DNA was denatured at 94 ° C for 30 seconds, and the primer was conjugated to the template DNA at 62 ° C for 45 seconds. DNA extension was carried out at 72 ° C for 1.5 minutes for a total of 35 cycles. The DNA was sufficiently extended by reacting at 72 ° C for 10 minutes. 5 μl of the PCR 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 (Fig. A, B). The sequence alignment method is the result of sequencing. The blastn bioinformatics software is input into the NCBI website and compared with the sequence registered by GenBank. The top five DNA similarity analysis is confirmed to be E. faecium .

The above analysis using the API 20 biochemical analysis kit and the sequence analysis of 16S ribosomal DNA (rDNA) confirmed that the strain was E. faecium and the strain was named E. faecium LJS-01.

Second embodiment: antibacterial ability test

E. faecium LJS-01 was cultured on Rogsa plate medium (Rogosa SL) to obtain a single colony, and then a single colony was cultured to MRS medium for 24 hours. After centrifugation, E. faecium LJS-01 was phosphate buffered. After washing, a small amount of phosphate buffer was mixed with E. faecium LJS-01 to form a bacterial solution, and 3 μl of the bacterial solution was taken, and inoculated into a lactic acid bacteria plate medium (LCMG, Lactobacillus Carrying Medium with Glucose), and anaerobic culture was carried out at 37 ° C for 24 hours. Thereafter, a semi-solid medium (0.8%) containing various pathogenic bacteria was covered thereon by an overlay co-culture method, and anaerobic culture was carried out at 37 ° C for 18-24 hours to detect an inhibition zone. The size of the diameter, and determine the scope of inhibition (Table 1), according to this strain against the species of Listeria genus such as L. monocytogenes , L. grayi L. innocua , L. welshiweri , etc. have strong antibacterial activity, and also have antibacterial activity against Staphylococcus aureus and Bacillus cereus . ability.

Third Embodiment: Antibiotic Sensitivity Test

In this test, 16 kinds of antibiotics were detected by Agar Disc Diffusion of BD BBL ^TM Sensi-Disc ^TM Antimicrobial susceptibility Test Discs to indicate E. faecium LJS-01 pair Sensitivity of antibiotics.

The test was first carried out according to the NCCLS specification (Wayne, 2003) ¹⁰ for S. aureus ATCC 25923 for quality control of antibiotic ingots (Disc) by picking S. aureus single colony inoculated into brain heart leaching BHI Broth, cultured at 35 ° C for 4 hours, centrifuged at 8,000 xg for 5 minutes, removed the supernatant, washed with phosphate buffer once, and then suspended the bacteria in phosphate buffer to prepare the bacteria. The liquid concentration was the same as that of McFarland No. 0.5 (1.5×10 ⁸ CFU/ml) standard suspension, and then the bacterial liquid was taken up with a sterile cotton swab and uniformly coated on hydrolyzed casein agar (Miller-Hinton agar, MHA), respectively. Antibiotic paper ingots were placed and cultured at 35 ° C for 24 hours. The diameter of each antibiotic inhibition ring was measured. The drug resistance was determined by the Kirby-Bauer method and the BBL specification to confirm the effective concentration and operation of the antibiotic paper ingot. The correctness.

The E. faecium LJS-01 single colony was inoculated in an aseptic manner in MRS culture medium, and cultured overnight at 37 ° C, 1% bacterial solution was subcultured in another 3 ml MRS culture medium, and cultured at 37 ° C for 4 hours, and the bacterial solution was 8,000 rpm. (JA20 rotor, Beckman) After centrifugation for 5 minutes, it was washed twice with phosphate 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. 1 ml and 9 ml 1% MRS agar (45 ° C), mix quickly and evenly, then pour into the Petri dish. After solidification, paste all kinds of antibiotic paper ingots, put them in an anaerobic tank, place them at 4 ° C overnight, then The anaerobic culture was carried out at 37 ° C for 48 hours, and the diameter of each drug inhibition zone was measured. The drug resistance was determined according to the diffusion susceptibility test and the BBL specification (Table 2). From the results, it was found that E. faecium LJS-01 against ampicillin (AM10), ampocillin (AMC30), bacitracin (B10), clarithromycin (CLR15), red Erythromycin (E15), gentamicin (GM10), imipenem (IPM10), neomycin (N30), novobiocin (NB30), penicillin (penicillin, P10) and vancomycin (VA30) sensitive; for ciprofloxacin (CIP5), kanamycin (K30), streptomycin (S10) and sulfamethoxazole-trimethoprim Pyridine (sulfamethoxazole/trimethoprim, SXT) is resistant. It was confirmed that this strain is not vancomycin resistance enterococcus (VRE), and it is highly sensitive to most antibiotics that inhibit bacterial cell wall synthesis.

Fourth embodiment: analysis of bile salt hydrolysis ability

According to previous research reports, lactic acid strains with bile salts hydrolase (BSH) have better ability to degrade cholesterol (Kim et. al., 2008, Nguyen et. al., 2007) ^3,5 Therefore, the present invention first tests the expression amount of E. faecium LJS-01 bile salt hydrolase, and then performs other characteristics analysis.

After three consecutive activations of E. faecium LJS-01, they were inoculated onto a MRS agar plate with a sterile toothpick. After overnight incubation at 37 ° C, a piece of sterile filter paper with a pore size of 3 mm was covered, and then the lactic acid bacteria were contained. The filter paper was placed on a MRS agar plate containing 0.5% taurodeoxycholic acid sodium salt (TDCA; Sigma) and 0.37 g/l calcium chloride, and cultured at 37 ° C for 72 hours to determine the sterility. The diameter of the white precipitate ring around the filter paper (Fig. 2), with a distinct white precipitated ring indicating strong BSH activity. From the results, it can be confirmed that the bacteria have the activity of BSH, and there is a high potential for degrading cholesterol.

Fifth Embodiment: Cholesterol Degradation Ability Test

In this test, E. faecium 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 remaining cholesterol content was used to assess the ability of E. faecium LJS-01 to degrade cholesterol. The test was carried out in accordance with the method described by Gilliland et al. (1985) ² . First, take 1 ml of the supernatant into a clean tube, add 3 ml of 95% ethanol and 2 ml of 50% potassium hydroxide. After shaking, put it in a 60 ° C water bath for 15 minutes, remove it at room temperature, cool it and add it. 10 ml of n-hexane, add 3 ml of sterile water after shaking, shake well 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 ° C. dry nitrogen, was added 2 ml of o-phthalaldehyde (o -phthalaldehyde) reagent (1 ml glacial acetic acid containing 0.5 mg o -phthalaldehyde), after standing for 10 minutes, then slowly 1 ml of concentrated sulfuric acid, dried After shaking for 10 minutes, the absorbance was detected by a spectrophotometer at a wavelength of OD ₅₅₀ nm.

In addition, the test takes different concentrations of water-soluble cholesterol (1 ml containing 0,10,20,30,40,50 μg) according to the above method to determine the standard curve, as the basis for the conversion of absorbance and concentration, from the results (the first Three figures) confirmed that the bacteria were cultured in MRS solution containing cholesterol (Chlot., Chlo.) or in MRS containing cholesterol and 0.4% bile salt, 70% and 63% of cholesterol after 48 hrs. Degradability.

In addition, we also apply the in vivo test of laying hens to reduce the cholesterol in the serum. First, the chickens were divided into control group (Control group, CG) and experimental group (EG), respectively. Blood samples were collected before the test, and the experimental group was given orally (4 x 10 ¹⁰ CFU/g/day/only), the control group was given skim milk powder (1 g/day/only) by oral administration, and stopped after 7 days of continuous administration. Blood was collected on days 0, 7, and 14 to determine serum cholesterol levels. Analysis of changes in serum cholesterol levels (Table 3). The results showed that the experimental group chickens had a serum cholesterol level of about 21% after 7 days of continuous administration of the bacteria, compared with the control group or before the experiment, but after stopping the administration of the bacteria for 7 days, the two groups of serum cholesterol There is no difference in value. 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.

Sixth embodiment: acid and bile salt resistance test

First, in the acid resistance test, E. faecium LJS-01 was inoculated into MRS Broth, and after incubation at 37 ° C for 24 hours, 1 ml of the bacterial solution was taken and added to pH 7.4 (control), pH 2.0, pH 3.0, respectively. Ml phosphate buffer (PBS). The bacterial solution was mixed with each phosphate buffer solution, placed in a 37 ° C constant temperature incubator, and treated for 0 hours, 1 hour, and 3 hours, respectively, and 1 ml of the bacterial solution was taken out, and 10-fold serial dilution was performed with phosphate buffer. The dilutions of 1 ml of each dilution were uniformly mixed with 1% MRS agar, and anaerobic culture was carried out at 37 ° C for 48 hours, and the number of colonies was counted to evaluate the acid resistance (Table 4).

In addition, in the bile salt test, E. faecium LJS-01 was first changed to 10 ml of MRS broth containing 0.3% (w/v) bovine bile (oxgall), cultured at 37 ° C, and at 3, 12, 24 respectively. In the hour, take 1 ml of the bacterial solution, serially dilute with 10 times of phosphate buffer, mix 1 ml of each dilution and 1% MRS agar, anaerobic culture for 48 hours at 37 °C, calculate the number of colonies to evaluate the resistance. The ability of bile salts (Table 5).

As a result, it was revealed that the strain was cultured under the conditions of pH 2.0 and pH 7.4 for 3 hours, and the results obtained were approximately the same as the number of bacteria survival (CFU/ml), and were comparable to the initial (0 hour) survival number, indicating survival. Not affected by stomach acid. At the same time, the strain was cultured in MRS medium containing 0.3% bovine bile at 37 ° C, and the number of colonies produced in the culture for 3 hours (CFU/ml) was equivalent to the control group without bovine bile, and the number of colonies generated in the culture for 24 hours was The control group was compared, and the decrease was within 1.0 of the logarithm, indicating that the bile salt resistance was excellent. This strain was confirmed to have excellent acid and bile salt-tolerant properties.

Seventh embodiment: in vitro ( In vitro Adsorption test of intestinal epithelial cell line Int-407

The intestinal epithelial cell line (Intestine 407, ATCC: CCL-6) was cultured in BME (Basal medium Eagle in Earle's BSS) growth medium containing 10% fetal calf serum, and placed in a 37 ° C, 5% CO ₂ incubator. Grow to eight full. After washing the cells with phosphate buffer, add a small amount of Trypsin-EDTA to suspend the cells, and after removing the Trypsin-EDTA by low-speed centrifugation, the cells are re-cultured in antibiotic-free growth medium. The cells were cultured in a 24-well culture dish, and the number of cells was 10 ⁴ per well. After standing at 37 ° C, the cells were grown to monolayer cells in a 5% CO ₂ incubator. Before the adsorption experiment, the test strains E. faecium ATCC 6057 and E. faecium LJS-01 were washed twice with phosphate buffer, centrifuged at 5,000 x g for 5 minutes, and resuspended in BME at a concentration of 1×. 10 ⁸ CFU/ml. After removing the supernatant from the single-layer cultured Intestine 407, add 100 μl of the test strain to each well, and carry out the adsorption reaction in a 37 ° C 5% CO ₂ incubator for 2 hours. Gently shake the plate every 15 minutes to allow adsorption. Evenly, each strain to be tested was subjected to 2 repetitions. After the adsorption reaction, the supernatant was removed, and washed twice with a phosphate buffer, and the following quantitative method was carried out.

1. Gram's stain: After washing, the cells are fixed with 10% neutral formalin for 30 minutes, and then washed 4 times with phosphate buffer to grind the cells in the culture and the strain to be tested. dyeing. Using a microscope to zoom in 1,000 times and randomly selecting 20 fields of view, the number of lactic acid bacteria adsorbed on Int-407 was calculated, and an average of more than 40 LABs per cell indicates that the LAB has good adsorption capacity. The experiment confirmed that the E. faecium LJS-01 of the present invention has an average adsorption capacity of more than 40 bacteria per cell on Int-407, indicating good adsorption capacity (Fig. 4, A, B, C).

2. Semi-quantitative PCR: The cleaned 24-well plate was added to 100 μl of Trypsin-EDTA to suspend all cells, and the contents of each well were transferred to a 1.5 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. The sample DNA is Primer BSF8 (5'-AGAGTTTGATCCTGGCTCAG-3'), Primer BSF1541 (5'-AAGGAGGTGATCCAGCCGCA-3'), 2 mM dNTP, Taq polymerase buffer, Taq polymerase, which are to be tested for cell specificity. 1 mM MgCl ₂ in a 0.2 ml centrifuge tube, in a polymerase chain reactor (PE 9700), the temperature was changed to: 94 ° C, 5 minutes, and then run 15-25 cycles of 94 ° C, 1 minute, 56 °C, 30 seconds, 72 ° C, 1 minute. The PCR-reacted solution was subjected to semi-quantitative analysis by DNA electrophoresis at a number of cycles that had not yet reached saturation. As a result of DNA electrophoresis, the optical density of the expected part of PCR was quantified by AlphaEase FC software, and the measured value of the strain to be tested/the measured value of the standard strain was used as a reference for adsorption capacity (fifth). The measured value of L. casei Shirota is set to 100%, which is equal to 1, and the measured value of the strain E. faecium LJS-01 to be tested is relatively 140%, which is equal to 1.4. The results show that the adsorption capacity of E. faecium LJS-01 to Int-407 cells is 1.4 times or more of L. casei Shirota or E. faecium ATCC 6057.

Eighth embodiment: the body ( In vivo Adsorption test of SPF chicken intestinal epithelial cells

Through the above experiments, E. faecium LJS-01, which has antibacterial, degraded cholesterol and in vitro intestinal epithelial cell line int-407, has been screened for evaluation of E. faecium LJS-01 strain in the digestive tract of animals. Salt and acid and intestinal epithelial cell adsorption capacity, using in vivo animal experiments, E. faecium LJS-01 was orally administered to Specific Pathogen Free (SPF) chicks, and feces and intestines were collected. The mucosa was analyzed for the amount of lactic acid bacteria remaining in the intestine, and the probiotic characteristics were evaluated. The experimental procedure is as follows: Prepare eggs without specific pathogens (SPF), and hatch specific chickens can be hatched after about 21 days. The chickens were divided into four groups, which were the control group, the E. faecium LJS-01, the E. faecium ATCC 6057, and the L. salivarius 17a group. The test was carried out on the 0th day of the SPF chick hatching date, and 10 ⁸ CFU/ml/ E. faecium LJS-01, E. faecium ATCC 6057 and L were administered on the 0th day, the 1st day and the 2nd day respectively. Salivarius 17a (Table 6).

On the 3rd and 7th day, two chicks were sacrificed, and 1 g of each of the jejunum, ileum and cecal contents were collected, and the intestinal contents were suspended in a sterilized phosphate buffer solution, and allowed to stand at room temperature for 30 minutes. The supernatant was taken and serially diluted 10 times. 100 μl of each diluted 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 (Table 7). .

In addition, collect 3 cm of jejunum, ileum and cecal tissue, spray the serosa surface with 70% alcohol, cut the intestine and place it in a 50 ml centrifuge tube, and add 10 ml of sterile phosphate buffer to gently oscillate 10-20 second. After repeating this three times, the cleaned intestinal lining was placed on the plastic wrap, the intestinal mucosa was scraped off with a slide, and the scraped intestinal mucosa was immersed in another 50 mL centrifuge tube, and the same was sterilized by 10 ml. Phosphate buffer was used to suspend intestinal mucosa tissue, and the pellet was allowed to stand at room temperature for 30 minutes. The supernatant was taken and serially diluted 10 times. 100 μl of each dilution ratio 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 (Table 7). .

From the seventh day, the growth of the empty, cecal, cecal contents and intestinal mucosal tissues of the SPF chicks in each group was sacrificed. It was found that in the sampling analysis of the intestinal contents, the control chickens were empty, back, There was no growth of the above lactic acid bacteria strain in the cecum segment. The experimental group was in the jejunum segment, except E. faecium ATCC 6057 could not grow, E. faecium LJS-01 and L. salivarius 17a could grow. In the ileum and cecum, all three strains can be grown. In addition, in the sampling of intestinal mucosa, E. faecium LJS-01 and L. salivarius 17a can be grown in three segments of the intestine, such as empty, ileum and cecum. It is worth noting that the growth of E. faecium LJS-01 in the jejunum is superior to that of L. salivarius 17a, indicating that E. faecium LJS-01 has good colonization ability even on the jejunal mucosa on the seventh day after oral administration ( Table VII).

Animals give probiotics more oral route, while staying in the digestive tract can play a role, because the bacteria stay in the mouth for a short time, so the oral factors can be ruled out, the stability of the probiotic strain in the digestive tract needs to consider the stomach pH and the effects of intestinal bile salts. According to the present invention, in the in vivo test of SPF chickens, it is tested whether the lactic acid bacteria strain having antibacterial ability can survive in the living digestive tract environment. As shown in Table 6, the samples of intestinal contents were collected. The more the number of bacteria can be measured, the better the colonization ability of the strain in the digestive tract, and the more the number of bacteria in the intestinal mucosa sample, the more capable of adsorbing the intestinal tract. good. The test results show that the E. faecium LJS-01 of the present invention has good tolerance to acid and bile salts, and has good adsorption capacity to intestinal epithelial cells, and can be used as a probiotic for promoting animal health.

Ninth Embodiment: Improvement of animal snoring

This trial, in collaboration with a central animal hospital, evaluated the ability of E. faecium LJS-01 to improve animals with snoring. The test first recorded the medical records of the animals with squats, and orally administered 1 gram of E. faecium LJS-01 (3x10 ¹⁰ CFU/g/head) daily, and recorded the improvement of the lower jaw daily. The results showed that except for a long-term treatment, the 5-day course of treatment returned to normal after 2 to 3 days of treatment. The results confirmed that the lactic acid bacteria had a good effect on the animals with sputum (Table 8).

Tenth embodiment: in the feed process E. faecium LJS-01 high temperature resistance test

Since the feed often generates high temperature in the process of granulation or stirring, it is usually used at 50 ° C for 30 minutes as a test condition for whether the lactic acid bacteria have high temperature resistance. After culturing E. faecium LJS-01 of the present invention, it is known that the amount of bacteria is 2.18× ^{10 9} CFU/ml, and the bacterial liquid is divided into two parts, one part is placed at 50 ° C for 30 minutes, and the other part is placed at 50 ° C for 30 minutes. At 37 ° C for 30 minutes, the final E. faecium LJS-01 viable count was similar, about 5x10 ⁹ CFU/ml. It is confirmed that the E. faecium LJS-01 of the present invention has the ability to withstand high temperature, the process does not affect its activity, and can be used as a feed additive and has a high number of viable cells.

However, the above description 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. Within the scope of the invention, it is given to Chen Ming.

references:

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Figure I. Sequence analysis of 16S rDNA fragment of faecium LJS-01 (A) E. Sequence of 16S rDNA fragment of faecium LJS-01 sequenced by BSF8 primer (B) E. 16S rDNA fragment sequence and gene of faecium LJS-01 Library sequence alignment

Figure II E. Faculty of faecium LJS-01

Figure III E. Faculty of faecium LJS-01 (Cholestrol, Cho.) Degradation ability MRS (LJS-01+cho.) containing water-soluble cholesterol containing 0.4% bile salt and water-soluble cholesterol MRS (LJS-01+cho.+ Bile)

Figure 4 E. Adsorption activity of faecium LJS-01 on Int-407 cell line (A) Control group (B) E. faecium ATCC 6057(C) E. faecium LJS-01

Figure 5 E. Adsorption activity of faecium LJS-01 on Int-407 cell line (A) Lane M: 1 kb DNA marker was analyzed by PCR semi-quantitative method. Lanes 1-4: Int-407 cell line β-actin gene product of the experimental treatment group of E. faecium 58, E. faecium LJS-01, E. faecium ATCC 6057 and L. casei Shirota, respectively, as a quantitative control group. Lanes 5-8: 16S rDNA of E. faecium 58, E. faecium LJS-01, E. faecium ATCC 6057 and L. casei Shirota, respectively. Lane 9: β-actin of the Int-407 cell line served as a negative control group. (B) Adsorption capacity results show that E. faecium LJS-01 is 1.4 times or more of L. casei Shirota and E. faecium ATCC 6057.

Claims (10)

An Enterococcus faecium isolate E.faecium LJS-01, deposited in the Food Industry Research and Development Institute (FIRDI), Taiwan, under the accession number BCRC 910421; the isolate is acid and resistant Bile salt, digestive tract epithelial cell adsorption capacity, degradation of cholesterol and antibacterial properties, and has a temperature resistance of 50 ° C. A feed additive comprising the lactic acid bacteria isolate E. faecium LJS-01 of claim 1 of the patent application. For example, the feed additive of claim 2 is provided for use in animals. For example, in the feed additive of claim 2, the animal includes animals such as mammals, livestock, laboratory animals, poultry, birds, wild animals, fish and shellfish, including pet animals. For example, in the feed additive of claim 3, 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. A dietary supplement comprising the lactic acid bacteria isolate E. faecium LJS-01 of claim 1 of the patent application. For example, the dietary supplement of claim 6 is for human use. For example, the dietary supplement of claim 6 of the patent scope, the lactic acid bacteria can have strong adsorption capacity in the intestinal epithelial tissue of the animal to eliminate the role of competitive pathogenic bacteria. An animal pharmaceutical composition for preventing or treating a pathogenic infection, which comprises the lactic acid bacteria isolate E. faecium LJS-01 of claim 1 and a pharmaceutical acceptable excipient. The composition of claim 9 is characterized in that the animal includes humans as well as pet animals such as mammals, livestock, laboratory animals, poultry, birds, wild animals, fish and shellfish.