CN103074281A - Enterococcus faecalis FJL19 and application thereof - Google Patents

Abstract

The invention relates to a strain of Enterococcus faecalis (Enterococcus faecalis) FJL19, the preservation number of which is CGMCC No.6995. The invention also provides that the enterococcus faecalis FJL19 has the effect of inhibiting the growth of Escherichia coli in chicken intestines and promoting the growth of chicks. The present invention isolates Enterococcus faecalis FJL19 from the jejunum of caged adult laying hens. The strain grows vigorously, and the number of cultured bacteria can reach 5.1×10 ⁹ cfu/ml in 18 hours, and has antibacterial effect, and can be produced by using the medium Antibacterial protein can reduce the number of intestinal E. coli in chicks; feeding the chicks with the preparation of lactic acid bacteria can improve the growth of the chicks; therefore, Enterococcus faecalis FJL19 has great potential in animal feeding, especially in the development of new feed additives for poultry and the research of antibiotic substitutes. Great social significance and economic value.

Description

A strain of Enterococcus faecalis FJL19 and its application

technical field

The invention belongs to the field of animal microorganisms, and relates to a strain of Enterococcus faecalis FJL19 and its application, in particular to a strain of Enterococcus faecalis FJL19 isolated from chicken intestines and its application. the

Background technique

Lactic acid bacteria are an important class of probiotics and an important class of dominant bacteria in the intestinal tract of animals. As a new type of green animal micro-ecological preparation, lactic acid bacteria preparation has attracted widespread attention in the feed industry because of its non-toxicity, no drug resistance, no residue, and no side effects. the

The use of lactic acid bacteria as probiotics to feed animals is mainly due to the fact that lactic acid bacteria have an inhibitory effect on some spoilage bacteria and harmful bacteria, in addition to their nutritional effect and adhesion to the intestinal tract. First, lactic acid bacteria can produce lactic acid, create an acidic environment to inhibit the growth of harmful bacteria and acid-resistant spoilage bacteria; second, lactic acid bacteria can produce H ₂ O ₂ , activate the catalase-thiocyanate system, inhibit and kill Gram Negative bacteria, catalase-positive bacteria, etc.; third, some lactic acid bacteria can produce small antibacterial proteins or peptides, called bacteriocins, which have antagonistic effects on pathogenic bacteria such as Escherichia coli. Moreover, with lactic acid bacteria producing antibacterial proteins, bacteriocins can be produced, which can replace antibiotics and make animal production safer. Isolation and screening of bacteriocin-producing lactic acid bacteria has become a research hotspot in the development of probiotics.

There are many sources of bacteriocin-producing lactic acid bacteria. Many purchased industrial strains and standard strains can also produce bacteriocins. Lactic acid bacteria can be isolated from the environment, soil, kimchi, and yogurt. However, probiotics are ultimately fed to animals, so compared to strains from other sources, animals are undoubtedly the best source of lactic acid bacteria. Lactic acid bacteria from animal intestines can adapt to the intestinal environment of animals and tolerate the digestion of gastric acid and bile salts of animals, which is undoubtedly the best source of probiotics. It can be seen that isolating and screening lactic acid bacteria capable of producing bacteriocins from animal intestines will be an important research direction for screening probiotics for animals. the

Contents of the invention

The purpose of the present invention is to provide a strain of Enterococcus faecalis (Enterococcus faecalis) FJL19, the preservation number of which is CGMCC No.6995. the

The second object of the present invention is to provide that the above-mentioned Enterococcus faecalis FJL19 has the effect of inhibiting the growth of Escherichia coli in chicken intestines and promoting the growth of chicks. the

The present invention is realized through the following technical solutions:

1. A strain of Enterococcus faecalis FJL19, the preservation number of which is CGMCC No.6995. the

In the present invention, Enterococcus faecalis FJL19 is Gram-positive and spherical in shape. It is a lactic acid bacteria strain isolated from the cecum contents of caged adult laying hens, and is a target strain obtained by measuring and screening properties such as antibacterial protein production. the

The specific steps of screening are as follows:

1. Separation and purification of lactic acid bacteria:

Using aseptic operation, use a glass slide to scrape 1g of the cecum content of cage-grown adult laying hens, put it in a glass test tube filled with 9mL sterile normal saline, shake it well, and then draw 0.5mL of the mixed solution into the glass test tube. In a test tube of 4.5mL sterile normal saline, the dilution is 10 ^-1 , repeat the above process for 10-fold dilution, to a dilution concentration of 10 ^-6 , choose three dilutions of 10 ^-4 , 10 ^-5 , and 10 ^-6 , absorb 0.1mL bacterial liquid and drop it on the MRS medium plate. After the plate is coated, use the anaerobic culture method (5% CO ₂ ) to culture the coated petri dish in a 37°C incubator for 48h. Using the four-partition marking method, use an inoculation loop to pick colonies with different shapes and separate them on the MRS agar medium. Pure culture was carried out on the slant medium, and the subculture was repeated for 3 times, and then stored in a refrigerator at 4°C for later use.

2. Observation of bacterial morphology

Take a clean slide, use an inoculation loop to take a ring of sterile water on the slide, then pick a small amount of bacteria, spread it evenly, and fix it after drying. Gram staining: first stain with ammonium oxalate crystal violet staining solution for 1min, rinse with water, then dropwise add Lushi's iodine solution to cover for 1min, rinse with water, then add 95% ethanol dropwise until the ethanol solution does not appear purple, stop for about 0.5min, Finally, counterstain with safranin staining solution for 1 min, and wash with water. Oil microscope inspection (16×100), select Gram-positive strains with rod-shaped or spherical shape as backup strains for use. the

Gram staining reagent preparation:

① Preparation of ammonium oxalate crystal violet staining solution

Liquid A crystal violet 2g 95% alcohol 20mL

B liquid ammonium oxalate 0.8g distilled water 80mL

Mix liquid A and liquid B, and use it after standing for 48 hours. the

②Lushi's iodine solution

Iodine tablet 1g Potassium iodide 2g Distilled water 300mL

Dissolve potassium iodide in a small amount of water first, then dissolve iodine tablets in potassium iodide solution, and add enough water after the iodine is completely dissolved. the

③95% alcohol

④Safranin counterstain solution

Saffron 2.5g 95% alcohol 100mL

Take the above prepared 10mL of safranin alcohol and mix with 80mL of distilled water. the

3. Screening of bacteriostatic lactic acid bacteria:

(1) Preparation of bacteria liquid: After activating the isolated and purified lactic acid bacteria for 2-3 generations, they were respectively inoculated into fresh MRS liquid medium at an inoculum size of 2% (v/v), and cultured anaerobically at 37°C for 24 hours to make The concentration of the bacterial solution reached 10 ⁸ cfu/mL, and its antibacterial activity was determined. Micrococcus xanthus was inoculated in broth medium, cultured on a shaker at 37°C for 2 generations, and diluted 10 times with sterile water to make the concentration of bacteria liquid reach 10 ⁵ cfu/mL, which was used as indicator bacteria for future use.

(2) Determination of acid production ability: After activating the separated and purified lactic acid bacteria for 2-3 generations, they were inoculated into their respective fresh MRS liquid medium at an inoculum size of 2% (v/v), cultured anaerobically at 37°C, and respectively in At two time points of 0h and 48h, the pH value of 5mL fermentation broth of each experimental strain was measured with a pH meter. The fermentation broth of each test strain was repeated three times, and the average number was taken. The acid-producing ability of lactic acid bacteria was measured by the change of pH value of the fermentation broth of each experimental strain at two time points of 0h and 48h, that is, △pH value. The strains with high acid production ability were selected for the next step of determination. the

(3) Bacteriostasis experiment: The bacteriostasis effect of the isolated and purified lactic acid bacteria was measured by the Oxford cup method. the

Take 100 μL of the indicator bacteria solution and add it dropwise to the solidified medium, and spread evenly with a coating rod. Under sterile conditions, use sterile tweezers to pick up 4 Oxford cups and put them symmetrically on the plate, then draw 200 μL of lactic acid bacteria fermentation broth into 3 Oxford cups, and add the same amount of MRS culture solution to the other Oxford cup as a control. Incubate at a constant temperature of 37°C for 16-18h. Each lactic acid bacteria fermented liquid was done 2 repetitions, measured the size of its antibacterial zone diameter with a vernier caliper, got the average number, and selected the lactic acid bacteria with a large antibacterial zone. the

In order to screen out the required strains as soon as possible, the preliminary screening adopted the addition and elimination method of two basic indicators of antibacterial and acid production ability. In each indicator, the first place gets 100 points, the second place gets 99 points, and so on, and the last place gets 1 point. The two results of the primary screening test were summed and sorted, and the top 10 strains with higher total scores were selected for further research. the

4. Screening of bacteriocin-producing lactic acid bacteria

The lactic acid bacteria screened in step 2 are carried out in liquid culture, in order to reduce the bacteriostatic effect of acid, adjust the pH value to 6.8 with 1mol/L NaOH solution to the nutrient solution, then carry out the Oxford cup method to measure the bacteriostatic ring, the method is the same as above, select the bacteriostatic Lactobacillus with the largest ring. After further liquid culture, add 0.1mg/L protease to the culture medium to act at 37°C for 2 hours, take the bacterial solution for bacteriostatic test, and screen the strains without bacteriostatic ring as the target lactic acid bacteria producing bacteriostatic protein. the

In the above screening method, the culture medium is MRS medium:

Solid plate or slant medium: tryptone 10g, beef extract 10g, yeast extract 5g, ammonium citrate 2g, glucose 20g, potassium dihydrogen phosphate 2g, sodium acetate 5g, Tween-801g, magnesium sulfate 0.5g, manganese sulfate 0.2 g, agar 20g, distilled water 1000mL, pH6.8. the

Liquid medium: tryptone 10g, beef extract 10g, yeast extract 5g, ammonium citrate 2g, glucose 20g, potassium dihydrogen phosphate 2g, sodium acetate 5g, Tween-801g, magnesium sulfate 0.5g, manganese sulfate 0.2g, distilled water 1000mL, pH6.8. the

Positive effects of adopting the above ^- mentioned technical scheme: the present invention isolates Enterococcus faecalis FJL19 from the jejunum of caged adult laying hens. Bacteria effect, can use the culture medium to produce antibacterial protein, reduce the number of intestinal E. coli in chicks; feed the chicks with preparations prepared by this lactic acid bacteria, can improve the growth of chicks; therefore, Enterococcus faecalis FJL19 is used in animal feeding, especially in new poultry feed additives It has great social significance and economic value in the development and research of antibiotic substitutes.

Description of drawings

Figure 1 is the Gram staining microscopic examination result of Enterococcus faecalis;

Fig. 2 is the antibacterial effect contrast of enterococcus faecalis bacterial liquid;

In the figure, 1 and 2 are the inhibition zones of the target strain, and 3 and 4 are the inhibition zones of other isolated strains;

Fig. 3 is the antibacterial effect of Enterococcus faecalis bacterium liquid after protease treatment;

Among the figure, 1 is the untreated bacterial liquid, and 2, 3 and 4 are respectively proteinase K, trypsin and papain-treated bacterial liquid;

Fig. 4 is the PCR amplification result of Enterococcus faecalis;

In the figure, 1Marker, 2 Lactobacillus plantarum;

Figure 5 is a growth curve graph of Enterococcus faecalis. the

Enterococcus faecalis (Enterococcus faecalis) FJL19 involved in the present invention was preserved in a patent procedure on December 17, 2012 at a depository recognized by the China Patent Office or the International Patent Organization. General Microbiology Center, referred to as CGMCC, address of depository unit: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, deposit number: CGMCC No.6995. the

Detailed ways

Below in conjunction with embodiment, test example technical scheme of the present invention is described further, but should not be interpreted as limitation of the present invention:

The source of biological material in the present invention:

1. Bacterial universal primers were purchased from Shanghai Boya Biotechnology Co., Ltd. the

Example 1

This example is used to illustrate the screening of Enterococcus faecalis FJL19. the

1. Separation and purification of lactic acid bacteria:

Using aseptic operation, use a glass slide to scrape 1g of the cecum content of cage-grown adult laying hens, put it in a glass test tube filled with 9mL sterile normal saline, shake it well, and then draw 0.5mL of the mixed solution into the glass test tube. In a test tube of 4.5mL sterile normal saline, the dilution is 10 ^-1 , repeat the above process for 10-fold dilution, to a dilution concentration of 10 ^-6 , choose three dilutions of 10 ^-4 , 10 ^-5 , and 10 ^-6 , absorb 0.1mL bacterial liquid and drop it on the MRS medium plate. After the plate is coated, use the anaerobic culture method (5% CO ₂ ) to culture the coated petri dish in a 37°C incubator for 48h. Using the four-partition marking method, use an inoculation loop to pick colonies with different shapes and separate them on the MRS agar medium. Pure culture was carried out on the slant medium, and the subculture was repeated for 3 times, and then stored in a refrigerator at 4°C for later use.

2. Observation of bacterial morphology

Take a clean slide, use an inoculation loop to take a ring of sterile water on the slide, then pick a small amount of bacteria, spread it evenly, and fix it after drying. Gram staining: first stain with ammonium oxalate crystal violet staining solution for 1min, rinse with water, then dropwise add Lushi's iodine solution to cover for 1min, rinse with water, then add 95% ethanol dropwise until the ethanol solution does not appear purple, stop for about 0.5min, Finally, counterstain with safranin staining solution for 1 min, and wash with water. Oil microscope inspection (16×100), select Gram-positive strains with rod-shaped or spherical shape as backup strains for use. The result is shown in Figure 1. the

Gram staining reagent preparation:

① Preparation of ammonium oxalate crystal violet staining solution

Liquid A crystal violet 2g 95% alcohol 20mL

B liquid ammonium oxalate 0.8g distilled water 80mL

Mix liquid A and liquid B, and use it after standing for 48 hours. the

②Lushi's iodine solution

Iodine tablet 1g Potassium iodide 2g Distilled water 300mL

Dissolve potassium iodide in a small amount of water first, then dissolve iodine tablets in potassium iodide solution, and add enough water after the iodine is completely dissolved. the

③95% alcohol

④Safranin counterstain solution

Saffron 2.5g 95% alcohol 100mL

Take the above prepared 10mL of safranin alcohol and mix with 80mL of distilled water. the

3. Screening of bacteriostatic lactic acid bacteria:

(1) Preparation of bacteria liquid: After activating the isolated and purified lactic acid bacteria for 2-3 generations, they were respectively inoculated into fresh MRS liquid medium at an inoculum size of 2% (v/v), and cultured anaerobically at 37°C for 24 hours to make The concentration of the bacterial solution reached 10 ⁸ cfu/mL, and its antibacterial activity was determined. Micrococcus xanthus was inoculated in broth medium, cultured on a shaker at 37°C for 2 generations, and diluted 10 times with sterile water to make the concentration of bacteria liquid reach 10 ⁵ cfu/mL, which was used as indicator bacteria for future use.

(2) Determination of acid production ability: After activating the separated and purified lactic acid bacteria for 2-3 generations, they were inoculated into their respective fresh MRS liquid medium at an inoculum size of 2% (v/v), cultured anaerobically at 37°C, and respectively in At two time points of 0h and 48h, the pH value of 5mL fermentation broth of each experimental strain was measured with a pH meter. The fermentation broth of each test strain was repeated three times, and the average number was taken. The acid-producing ability of lactic acid bacteria was measured by the change of pH value of the fermentation broth of each experimental strain at two time points of 0h and 48h, that is, △pH value. The strains with high acid production ability were selected for the next step of determination. the

(3) Bacteriostasis experiment: The bacteriostasis effect of the isolated and purified lactic acid bacteria was measured by the Oxford cup method. the

Take 100 μL of the indicator bacteria solution and add it dropwise to the solidified medium, and spread evenly with a coating rod. Under sterile conditions, use sterile tweezers to pick up 4 Oxford cups and put them symmetrically on the plate, then draw 200 μL of lactic acid bacteria fermentation broth into 3 Oxford cups, and add the same amount of MRS culture solution to the other Oxford cup as a control. Incubate at a constant temperature of 37°C for 16-18h. Each lactic acid bacteria fermented liquid was done 2 repetitions, measured the size of its antibacterial zone diameter with a vernier caliper, got the average number, and selected the lactic acid bacteria with a large antibacterial zone. the

In order to screen out the required strains as soon as possible, the preliminary screening adopted the addition and elimination method of two basic indicators of antibacterial and acid production ability. In each indicator, the first place gets 100 points, the second place gets 99 points, and so on, and the last place gets 1 point. The two results of the primary screening test were summed and sorted, and the top 10 strains with higher total scores were selected for further research. the

4. Screening of bacteriocin-producing lactic acid bacteria

The lactic acid bacteria screened in step 2 are carried out in liquid culture, in order to reduce the bacteriostatic effect of acid, adjust the pH value to 6.8 with 1mol/L NaOH solution to the nutrient solution, then carry out the Oxford cup method to measure the bacteriostatic ring, the method is the same as above, select the bacteriostatic Lactobacillus with the largest ring. The result is shown in Figure 2. After further liquid culture, add 0.1mg/L protease to the culture medium to act at 37°C for 2 hours, take the bacterial solution for bacteriostatic test, and screen the strains without bacteriostatic ring as the target lactic acid bacteria producing bacteriostatic protein. The result is shown in Figure 3. the

In the above screening method, the culture medium is MRS medium:

Solid plate or slant medium: tryptone 10g, beef extract 10g, yeast extract 5g, ammonium citrate 2g, glucose 20g, potassium dihydrogen phosphate 2g, sodium acetate 5g, Tween-801g, magnesium sulfate 0.5g, manganese sulfate 0.2 g, agar 20g, distilled water 1000mL, pH6.8. the

Liquid medium: tryptone 10g, beef extract 10g, yeast extract 5g, ammonium citrate 2g, glucose 20g, potassium dihydrogen phosphate 2g, sodium acetate 5g, Tween-801g, magnesium sulfate 0.5g, manganese sulfate 0.2g, distilled water 1000mL, pH6.8. the

Example 2

This example is used to illustrate the identification of Enterococcus faecalis FJL19. the

1. Identification of strain genus

Catalase test: Add 3% H ₂ O ₂ directly to the bacterial lawn on the slope, and observe whether there are bubbles. If bubbles are generated, it is a positive reaction.

Nitrate reduction test: Inoculate the bacteria on the culture medium, incubate at 37°C for 48 hours, add solution A and solution B along the tube wall, and observe. Immediately showing red, orange red is a positive reaction. the

Gelatin liquefaction test: Inoculate the bacteria on the culture medium, incubate at 20°C for 48 hours, and observe. Liquefaction of the inoculum is a positive reaction. the

Indole test: Inoculate the bacteria in the MRS culture medium and incubate at 37°C for 48h. Add 2-3mL of xylene to the culture medium, shake well, let it stand for a while, then add 2mL of indole reagent along the tube wall, if the liquid in the lower layer of xylene turns rose red, it is a positive reaction. the

Hydrogen sulfide test: After the bacteria are inoculated in the MRS culture medium, use sterile tweezers to pick up a lead acetate paper strip and hang it in the inoculation tube. The lower end is close to the surface of the medium without touching the liquid surface. Insert the top end with a cotton plug. Incubate at 37°C for 48h and observe. The paper strip turns black as a positive reaction. the

Through catalase test, nitrate reduction test, gelatin liquefaction test, indole test and hydrogen sulfide test, the results were all negative, indicating that Enterococcus faecalis FJL19 belonged to the genus of lactic acid bacteria. the

2. Identification of strain species

Acid and gas production test of fermented sugar: Pick a small amount of bacteria with an inoculation needle and inoculate them into lactose, D-xylose, sucrose, melibiose, raffinose, melezitose, ribitol, sorbitol and mannitol biochemical fermentation tubes, Incubate at 37°C for 48h. Violet in the biochemical tube turns yellow to indicate acid production, which is a positive reaction. the

Growth test at 15°C and 45°C: Pick a few rings of bacteria and inoculate them on the MRS slant medium, culture at 15°C and 45°C for 48 hours, and observe whether the bacteria grow on the slant. the

Arginine hydrolysis test: inoculate the bacteria on the culture medium, incubate at 37°C for 48 hours, observe, red and orange red are positive reactions. the

Hippurate hydrolysis test: Inoculate the bacteria on the culture medium, culture at 37°C for 48 hours, and observe. Red and orange are positive reactions. the

Arginine ammonia production test: inoculate bacteria in medium containing arginine, and culture at 37°C for 48h. Add a few drops of Ganesh's reagent to the culture medium, and when ammonia is produced, an orange or yellow-brown precipitate appears, which is a positive reaction. the

Through hippurate hydrolysis test, arginine hydrolysis test, growth test at 15°C and 45°C, and various sugar fermentation tests, the results were compared with "Common Bacteria System Identification Manual" and "Classification and Identification of Lactic Acid Bacteria". The identification results showed that the strain was Enterococcus faecalis. the

In the above screening method, the preparation of biochemical medium and related reagents is as follows:

Hydrogen sulfide test medium: tryptone 10g; beef extract 3g; yeast extract 5g; NaCl 5g; cysteine 0.4g; glucose 2g; distilled water 1000mL. Adjust the pH to 7.2, and autoclave at 115°C for 20 minutes. the

Lactose, D-xylose, rhamnose, sucrose, melibiose, raffinose, melezitose, ribitol, sorbitol and mannitol sugar fermentation identification biochemical tubes were purchased from Haibo Biological. the

Indole reagent: p-dimethylaminobenzaldehyde 1g, absolute ethanol 95mL, concentrated hydrochloric acid 20mL. the

Arginine solution: 1.5g of L-arginine, 0.05mL of cysteine (1g/mL H ₂ O), 10mL of distilled water, adjust the pH to 7.0, add 3 drops to the PY basic culture medium after sterilization. Nestle's reagent: Dissolve 20g KI in 50mL distilled water, and add _HgI2 small particles to the solution until the solution is saturated (about 32g), then add 460mL water and 134gKOH. Store the supernatant in a dark bottle for later use.

The experimental results of sugar fermentation are shown in Table 1:

Table 1 Sugar Fermentation Test Results

3.16S rDNA sequencing identification

Using a method known to those skilled in the art, use a kit to extract bacterial genomic DNA, reverse transcribe it into cDNA, and use bacterial universal primers to carry out routine PCR amplification of the gene according to the conserved region of the 16S rDNA gene sequence of Lactobacillus.

Upstream primer 5'-AGAGTTTGATCCTGGCTCAG-3'

Downstream primer 5'-ACGGCTACCTTGTTACGACTT3'

The amplification results were detected by electrophoresis. The electrophoresis showed that the bands were clear and single, indicating that the amplification was successful. The results were shown in Figure 4. At the same time, the bands were purified, TA-cloning technology, and sequenced. Enterococcus faecalis. the

Test example 1

This test example is used to illustrate the role of Enterococcus faecalis FJL19. the

1. Preparation of whole lactic acid bacteria liquid preparation

(1) Determination of the fermentation time of lactic acid bacteria: inoculate the activated Lactobacillus as a fermentation seed solution 1% (v/v) in MRS liquid medium, culture on a shaker at 37°C, 170r/min, and every 2h during the first 24h, After 24 hours, samples were taken at 36 and 48 hours. After serially diluting with 0.9% normal saline according to the 10-fold method, take 100 μl of the dilution of 10 ^-4 -10 ^-6 and evenly spread it on the MRS agar medium. Three replicates were performed, and the plates were incubated at 37°C and 5% CO ₂ for 24 hours. Take the dilution value of the number of colonies between 30-300 to calculate the concentration of the bacterial solution, and take samples at the same time, and use the Oxford cup method to measure the antibacterial effect of the bacterial solution at each time point to determine the optimum fermentation time.

As shown in Figure 5, the abscissa in the figure represents the time, and the ordinate represents the number of viable bacteria. The number of viable bacteria of Enterococcus faecalis increases slowly within 10 hours after inoculation, and then shows a state of rapid increase after 10 hours, and reaches the level at 18 hours. The number of viable bacteria reached more than 10 ⁹ , which reached the highest value at this time. However, the number of viable bacteria had dropped to 10 ⁸ at 36 hours, and showed a gradual decline. The main reason is that the consumption of nutrients in the medium and the accumulation of harmful metabolites lead to changes in the growth environment of the bacteria, thereby reducing the number of viable bacteria. It can be seen from the growth curve that the number of viable bacteria of Enterococcus faecalis is maintained at the order of 10 ⁹ at 16-20 hours, and is relatively stable, so the time period of 18 hours is selected to harvest the bacteria.

(2) Preparation of whole bacterial solution: After activating the test strain for 2-3 generations, transfer it to the optimized liquid medium at an inoculum amount of 1% (v/v), culture it on a shaker at 37°C and 170r/min for 18h, Centrifuge at 3500 rpm for 30 min at 4°C, suspend the cells with a certain volume of supernatant, and store at 4°C for later use. the

2. The application effect of lactic acid bacteria preparation in chick feeding

(1) Test materials: the above-mentioned whole bacteria liquid preparation, the number of viable bacteria is 5.1×10 ⁹ cfu/mL (number of bacteria); the antibiotic is 4% flavomycin.

(2) Experimental animals and experimental design: Select 240 1-day-old healthy AA broilers, half male and half female, with an average body weight of 40.55±0.67g, and randomly divide them into 4 groups, Group I (control group, basal diet), Group Ⅱ (antibiotic group, basal diet + 0.01% flavomycin), Ⅲ (whole-bacteria solution group, basal diet + 0.5% whole-bacteria solution), group Ⅳ (whole-bacteria solution group, basic day grain + 1% whole bacterial liquid preparation), 5 repetitions in each group, 12 chickens in each repetition, and the test period was 28 days. the

(3) Measuring indicators:

A. Production performance index: Weigh the fasting weight of each repetition (fasting for 12 hours) at 8:00 am on the first day, the 15th day, and the 29th day of the test respectively, and accurately record the feed intake of each repetition. The average daily gain (ADG), average daily feed intake (ADFI) and feed conversion ratio (FCR) of chickens in each group were calculated. The results are shown in Table 2:

Table 2 Effects of lactic acid bacteria preparations on daily gain and feed-to-weight ratio of chicks

Note: Compared with peers, different lowercase letters indicate significant differences (p<0.05), different uppercase letters indicate extremely significant differences (p<0.01), and those with no marks or the same letters indicate no significant differences (p>0.05). the

The test results showed that from 1 to 14 days and 15 to 28 days, the average daily gain of broilers in the 1.0% lactic acid bacteria preparation group was significantly higher than that in the control group (p<0.05), and the average daily gain in the 0.5% lactic acid bacteria preparation group was slightly higher than that in the control group, but The difference was not significant (p>0.05), the average daily weight gain between the preparation groups was not significant (p>0.05), from 1 to 14 days, the average daily weight gain was not significantly different between the antibiotic group and other treatments (p>0.05); 15 On day 28, the average daily weight gain of the antibiotic group was significantly higher than that of the control group (p<0.05), but there was no significant difference with other treatment groups (p>0.05). During the whole test period, the average daily weight gain of the lactic acid bacteria preparation group was significantly higher than that of the control group (p<0.05), the 1.0% preparation group was significantly higher than the 0.5% preparation group (p<0.05), and the average daily weight gain of the antibiotic group was significantly higher than that of the control group group and 0.5% lactic acid bacteria preparation group (p<0.05), and there was no significant difference with 1% lactic acid bacteria preparation group (p>0.05). the

From 15 to 28 days, the feed-to-meat ratio of the 1.0% lactic acid bacteria preparation group was significantly lower than that of the control group (p<0.05), and there was no significant difference between the antibiotic group and the 0.5% preparation group (p>0.05). There was no significant difference in feed-to-meat ratio between groups (p>0.05). During the whole test period, the feed-to-meat ratio of the preparation group was significantly lower than that of the control group (p<0.05), the feed-to-meat ratio of the 1.0% preparation group was significantly lower than that of the 0.5% preparation group (p<0.05), and the feed-to-meat ratio of the antibiotic group was significantly lower than that of the control group group (p<0.05), there was no significant difference with other treatment groups (p>0.05). the

Overall, both lactic acid bacteria preparations and antibiotics can improve the growth performance of broilers, and the 1.0% preparation group is better than the 0.5% preparation group and antibiotics group. the

B. The number of cecal flora: On the 15th and 29th day of the test, a test chicken with a weight close to the average weight was randomly taken from each repetition, sacrificed by bloodletting from the jugular vein, dissected immediately, and taken out after ligating the cecum with sterile cotton thread, under aseptic conditions Weigh 0.5 g of the cecal content, dilute it 10 times, and determine the number of lactic acid bacteria and E. coli in the cecum by the plate coating method. The results are shown in Table 3:

Table 3 Effect of lactic acid bacteria preparations on the number of lactic acid bacteria and E. coli in chick cecum (log ₁₀ cfu/g)

Note: Compared with peers, different lowercase letters indicate significant differences (p<0.05), different uppercase letters indicate extremely significant differences (p<0.01), and those with no marks or the same letters indicate no significant differences (p>0.05). the

At 2w, the number of lactic acid bacteria in the cecum of broilers in the 1.0% lactic acid bacteria preparation group was significantly higher than that in the control group and the antibiotic group (p<0.05), but compared with the 0.5% lactic acid bacteria preparation group, the difference was not significant (p>0.05); 0.5% The number of lactic acid bacteria in the lactic acid bacteria preparation group was significantly higher than that in the antibiotic group (p<0.05), but there was no significant difference compared with the control group (p>0.05); the number of lactic acid bacteria in the control group was significantly higher than that in the antibiotic group (p<0.05). The number of E. coli in the 1.0% lactic acid bacteria preparation group was significantly lower than that of all other treatment groups (p<0.05); the number of E. coli in the 0.5% preparation group was not significantly different from that in the control group (p>0.05), and the number of E. coli in the antibiotic group was significantly lower than that in the control group. There was no significant difference in the number of Escherichia coli between the control group (p<0.05), the 0.5% preparation group and the antibiotic group (p>0.05). the

At 4w, the number of lactic acid bacteria in the 1% and 0.5% lactic acid bacteria preparation groups was significantly higher than that in the antibiotic group (p<0.05), except for the antibiotic group, there was no significant difference in the number of lactic acid bacteria among the other groups (p>0.05); 0.5% and 1.0% lactic acid bacteria The number of Escherichia coli in the preparation group was significantly lower than that in other treatment groups (p<0.05). With the increase of lactic acid bacteria preparation, the number of lactic acid bacteria in the cecum increased, and the number of Escherichia coli decreased, which indicated that lactic acid bacteria can optimize the structure of intestinal flora. the

In summary, the strain can significantly inhibit the number of Escherichia coli in the intestinal tract of chickens, and at the same time promote the growth of chicks. It can replace traditional antibiotics, reduce the application of antibiotics in animal feeding, and greatly improve biological safety. the

Claims (2)

1. A strain of Enterococcus faecalis FJL19, the preservation number of which is CGMCC No.6995. 2. The Enterococcus faecalis FJL19 according to claim 1 has the effect of inhibiting the growth of Escherichia coli in the chicken intestine and promoting the growth of chicks.

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