Enterococcus faecalis strain SKLAN202311L and application thereof

Abstract

The invention relates to the field of probiotics, in particular to an enterococcus faecalis strain SKLAN202311L and application thereof. The strain has a good inhibition effect on diarrhea caused by escherichia coli, salmonella and porcine epidemic diarrhea virus (PEDV); the strain can be colonized in an animal body, and has strong tolerance to artificial cholate, artificial gastric acid and artificial intestinal juice; the feed additive can promote animal growth, regulate intestinal flora, reduce diarrhea rate, improve intestinal immunity and improve intestinal health; the composition has a good prevention and treatment effect on PEDV (porcine epidemic diarrhea virus).

Description

Technical Field

[0001] The invention relates to the field of probiotics, and in particular to an Enterococcus faecalis strain SKLAN202311L and an application thereof. Background Art

[0002] Piglet mortality accounts for 50%-70% of total pig mortality and is one of the major challenges facing the pig industry. Diarrhea in piglets can lead to loss of appetite and severe dehydration, sometimes leading to death without treatment. Although piglets with diarrhea survive, they grow slowly, lose weight, and cause significant economic losses.

[0003] Non-infectious diarrhea in piglets can usually be improved by strengthening feeding management, but infectious diarrhea is usually treated with antimicrobial drugs. However, with the emergence of antimicrobial resistance, the widespread use of antimicrobial drugs in food animal production will make antimicrobial resistance a serious public health problem. Therefore, there is an increasing demand for research and development of alternative therapies to antimicrobial drugs. At present, research on probiotics has become a hot topic. It has been found that probiotics can show great potential in intestinal health and disease treatment. It is a potential alternative to antibiotics.

[0004] Probiotic enterococci are the dominant flora in the pig intestine, especially in healthy pig intestines, where they are often enriched in large quantities. Among them, Enterococcus faecalis can form biofilms on intestinal epithelial cells to protect them from invasion by harmful substances. In addition, Enterococcus faecalis contains a variety of small peptides that can promote the absorption of amino acids and residues and increase the synthesis of tissue proteins. Enterococcus faecalis can also produce antibacterial substances, such as bacteriocins, to inhibit the growth of pathogenic microorganisms in the intestine, improve animal intestinal immunity, regulate animal intestinal flora, reduce animal diarrhea and mortality, improve animal intestinal health, and improve animal growth performance.

[0005] From the perspective of biological classification, "species" is the most basic classification unit, and species can be divided into subspecies, variants (special forms, special pathogenicity, etc.), strains, etc. Therefore, even if they are the same species of Enterococcus faecalis, different strains will have different antibacterial and biochemical properties. For example, Enterococcus faecalis LD33 has no obvious inhibitory effect on the growth of Salmonella and Shigella, but has a strong inhibitory effect on Listeria and Staphylococcus aureus. Enterococcus faecalis NPL-493 has an inhibitory effect on Escherichia coli and Salmonella, but has no inhibitory effect on Staphylococcus aureus.

[0006] More than 90% of the probiotics that can improve diarrhea caused by PEDV in the literature published in the prior art are lactobacilli, and there are few literature reports on the interaction between Enterococcus faecalis and PEDV. In the case of disease, piglets are attacked by foreign pathogens, and the intestinal microenvironment is very different from that in a healthy state. Enterococcus faecalis may not maintain a probiotic effect. For example, a document published in 2020 showed that the enterococci (Enterococcus durans) they isolated had no inhibitory effect on PEDV. Summary of the invention

[0007] The purpose of the present invention is to provide an Enterococcus faecalis strain.

[0008] Another object of the present invention is to provide a bacterial agent comprising the above-mentioned strain.

[0009] Another object of the present invention is to provide the application of the above strain.

[0010] The Enterococcus faecalis strain SKLAN202311L according to the present invention has a deposit number of CGMCC No.28891.

[0011] The bacterial agent according to the present invention comprises the above-mentioned Enterococcus faecalis strain SKLAN202311L.

[0012] The present invention provides the use of the above-mentioned Enterococcus faecalis strain SKLAN202311L for preparing a bacterial agent for the following purposes:

[0013] (1) Inhibit pathogenic microorganisms;

[0014] (2) Promote animal growth;

[0015] (3) Regulate intestinal flora;

[0016] (4) Improve animal intestinal immunity;

[0017] (5) Improve animal intestinal health;

[0018] (6) Reduce animal diarrhea;

[0019] (7) Prevent and treat porcine epidemic diarrhea.

[0020] The pathogenic microorganisms described in (1) above may be bacteria or viruses.

[0021] Preferably, the pathogenic microorganism is a bacterium, more preferably a Gram-negative bacterium.

[0022] As an embodiment of the present invention, the pathogenic microorganism is Escherichia coli or Salmonella.

[0023] Enterococcus faecalis strain SKLAN202311L was isolated from the colonic digesta of Jinhua pigs at Jinhua Academy of Agricultural Sciences, Wucheng District, Jinhua City, Zhejiang Province.

[0024] The present invention provides a bacterial agent containing the Enterococcus faecalis strain SKLAN202311L.

[0025] The microbial agent of the present invention can be a liquid microbial agent or a solid microbial agent, and can be prepared by conventional technical means and by adding auxiliary materials permitted in the field of microbial preparations.

[0026] The bacterial agent contains Enterococcus faecalis strain SKLAN202311L or bacterial bodies fermented from Enterococcus faecalis strain SKLAN202311L.

[0027] The beneficial effects of the present invention are as follows: the present invention obtains a strain of Enterococcus faecalis SKLAN202311L by isolating it from the colonic chyme of Jinhua pigs, and the strain has a good inhibitory effect on Escherichia coli and Salmonella; it can colonize in the animal body, and has a strong tolerance to artificial bile salts, artificial gastric acid, and artificial intestinal fluid; taking the weight of piglets in the control group and the Enterococcus faecalis group as an indicator, it is found that the strain can effectively improve the growth performance of piglets, and in the feeding test of piglets aged 7-28 days, it is also shown that the strain of Enterococcus faecalis SKLAN202311L can significantly reduce the diarrhea rate of piglets, improve intestinal immunity, and improve intestinal health. In addition, in the feeding test of piglets positive for porcine epidemic diarrhea virus after weaning, the strain of Enterococcus faecalis SKLAN202311L can significantly reduce the diarrhea rate of PEDV-positive piglets and improve the growth performance of infected piglets. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the growth curve of the Enterococcus faecalis strain SKLAN202311L in Example 2 of the present invention;

[0029] Figure 2 This is an oil immersion microscopic examination image of the Enterococcus faecalis strain SKLAN202311L in Example 2 of the present invention after malachite green staining;

[0030] Figure 3 This is a colony morphology diagram of the Enterococcus faecalis strain SKLAN202311L in Example 2 of the present invention;

[0031] Figure 4 This is a graph showing weight changes of the experimental piglets during the entire experimental period in Example 3 of the present invention;

[0032] Figure 5 The weight change of the experimental piglets in Example 3 of the present invention on the 7th day after the start of the experiment;

[0033] Figure 6 The weight change of the experimental piglets in Example 3 of the present invention on the 14th day after the start of the experiment;

[0034] Figure 7 The weight change of the experimental piglets in Example 3 of the present invention on the 21st day after the start of the experiment;

[0035] Figure 8 The weight change of the experimental piglets in Example 3 of the present invention on the 28th day after the start of the experiment;

[0036] Fig. 9 The statistical results of diarrhea rate of piglets in Example 3 of the present invention during the entire test period are shown;

[0037] Fig.10 The results showing the differential microorganisms in piglet feces in Example 3 of the present invention;

[0038] Fig.11 This is a graph showing weight changes of the experimental piglets during the entire experimental period in Example 4 of the present invention;

[0039] Fig.12 The weight change of the experimental piglets in Example 4 of the present invention on the 7th day after the start of the experiment;

[0040] Fig.13 The weight change of the experimental piglets in Example 4 of the present invention on the 14th day after the start of the experiment;

[0041] Fig.14 The weight change of the experimental piglets in Example 4 of the present invention on the 28th day after the start of the experiment;

[0042] Fig.15 The statistical results of diarrhea rate of piglets in Example 4 of the present invention during the entire test period are shown.

[0043] The Enterococcus faecalis strain SKLAN202311L of the present invention was deposited on November 7, 2023 in the General Microbiology Center of the China Culture Collection Administration (CGMCC for short, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code 100101), and was classified and named Enterococcus faecalis, with a deposit number of CGMCC No. 28891. DETAILED DESCRIPTION

[0044] The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention.

[0045] Example 1 Isolation and Identification of Enterococcus faecalis

[0046] 1. Isolation and identification of Enterococcus faecalis

[0047] Enterococcus faecalis was isolated from the colonic chyme of Jinhua pigs in Jinhua Academy of Agricultural Sciences, Wucheng District, Jinhua City, Zhejiang Province.

[0048] 1. Colonic chyme collection: Collect 1g of colonic chyme from 24 Jinhua piglets, add 24mL of sterile saline containing 20% ​​glycerol, vortex mix, and dispense into sterile cryopreservation tubes, 1mL per tube. After processing, place in dry ice, bring back to the laboratory and store in a -80℃ refrigerator until use.

[0049] 2. Isolation of strains: Take 1g of Jinhua pig colon chyme and mix it thoroughly in 9mL PBS solution, then filter the mixture with a 40μm filter. Take 1mL of the filtrate and culture it in 9mL MRS broth at 37℃ overnight. The next day, mix the MRS broth thoroughly and take 1mL of the broth to make a ten-fold dilution. -1 ~10 -7 The dilutions of these concentrations were spread on MRS solid culture plates and cultured at 37°C overnight. The next day, culture plates with 1-50 single colonies were selected to number each single colony, and half of the single colonies were taken for microscopic examination. Single colonies with initial microscopic examination of enterococcal morphology were streaked on a new MRS culture plate and cultured at 37°C overnight.

[0050] 3. Strain purification and enrichment: Pick a single colony on the streaked plate and streak it again, and culture it at 37°C overnight. Repeat this process 3 times to obtain a single purified strain. Then pick a single colony and culture it in 5mL MRS broth on a shaker at 37°C for two days to fully enrich the strain for 16S rRNA identification, and mix the remaining part with physiological saline containing 50% glycerol and store it in a -80°C refrigerator.

[0051] 4. 16S rRNA identification: The enriched bacterial liquid was amplified by colony PCR using the bacterial universal primer 27F, and then sequenced and identified by 16S rDNA. The 16S rDNA sequences of each strain were compared with the 16S rDNA sequences of all determined bacteria in the database at NCBI. Among them, 4 strains were found to have the highest homology similarity with the 16S rDNA sequence of Enterococcus faecalis ATCC 6055 of the genus Enterococcus. The species type of these 4 strains was determined to be Enterococcus faecalis.

[0052] The microbiological characteristics of Enterococcus faecalis strain SKLAN202311L are as follows:

[0053] (1) Colony morphology: The single colony is round in shape, about 1 to 2 mm in diameter, milky white, opaque, with a smooth surface and regular edges.

[0054] (2) After staining, the bacteria appear oval in shape, without spores, capsules, flagella, and cannot move.

[0055] (3) Growth characteristics: In MRS liquid medium, the cells were cultured in a shaking incubator at 37°C. After 4 hours, the cells entered the logarithmic growth phase and after 14 hours, the cells entered the plateau phase. The maximum number of viable cells was 5.6×10 8 CFU / mL.

[0056] Example 2 Detection of characteristics of Enterococcus faecalis

[0057] 1. Antibacterial ability test

[0058] The pathogen indicator bacteria used were Escherichia coli BW25113 and Salmonella typhimurium ATCC14028, which are two common pathogenic bacteria.

[0059] The antibacterial activity of the strains was determined by the Oxford cup method. Escherichia coli BW25113 and Salmonella typhimurium ATCC14028 were inoculated into LB broth medium and cultured in a 37°C incubator for 12-18 hours. The concentrations of Escherichia coli BW25113 and Salmonella typhimurium ATCC14028 strains were adjusted to 1×108 CFU / mL, take 200 μL of each, evenly spread on LB agar medium, put the sterilized Oxford cup evenly on the LB medium, add 200 μL of activated bacterial solution with a concentration of 1×10 8 CFU / mL of Enterococcus faecalis liquid with MRS broth. Place in a 37°C incubator and culture for 12 hours. Use a vernier caliper to measure the size of the inhibition zone and evaluate the antibacterial effect. The results show (Table 1) that compared with the other three strains of Enterococcus faecalis, Enterococcus faecalis strain SKLAN202311L has a stronger inhibitory effect on Escherichia coli BW25113 and Salmonella typhimurium ATCC14028.

[0060] Table 1 Inhibitory ability of Enterococcus faecalis against pathogens

[0061]

[0062] Note: The above values ​​are the antibacterial diameters, in mm.

[0063] 2. Bile salt tolerance test

[0064] The activated bacterial solution with a concentration of 1×10 8 Take 1 mL of the Enterococcus faecalis liquid with a CFU / mL and inoculate it into 9 mL of sterile saline containing 0.3 g / 100 mL of porcine bile salt. After 3 hours, the number of viable bacteria was determined by the plate spreading method.

[0065] The results showed (Table 2) that at a bile salt concentration of 0.3 g / 100 mL, compared with the other three strains of Enterococcus faecalis, the Enterococcus faecalis strain SKLAN202311L had the best bile salt tolerance, with a survival rate of about 2.74%.

[0066] Table 2 Survival rate of Enterococcus faecalis at 0.3% concentration of strong bile salts (%)

[0067]

[0068] 3. Artificial gastric juice resistance test

[0069] The activated bacterial solution with a concentration of 1×10 8 1 mL of the Enterococcus faecalis bacterial solution with a CFU / mL was inoculated into 9 mL of artificial gastric juice with pH = 1.2, and the number of viable bacteria was determined by the plate spreading method after 1 hour.

[0070] The results showed (Table 3) that after 1 hour in artificial gastric juice with pH = 1.2, compared with the other three strains of Enterococcus faecalis, the Enterococcus faecalis strain SKLAN202311L had the best resistance to artificial gastric juice, with a survival rate of about 8.71%.

[0071] Table 3 Survival rate of Enterococcus faecalis in artificial gastric juice at pH = 1.2%

[0072]

[0073] 4. Artificial intestinal fluid resistance test

[0074] The activated bacterial solution with a concentration of 1×10 8 1 mL of Enterococcus faecalis liquid with a CFU / mL was inoculated into 9 mL of artificial intestinal fluid with pH = 7.0, and the number of viable bacteria was determined by the plate spreading method after 4 hours.

[0075] The results showed (Table 4) that after 4 hours in artificial intestinal fluid, compared with the other three strains of Enterococcus faecalis, the Enterococcus faecalis strain SKLAN202311L had the best resistance to artificial intestinal fluid, with a survival rate of about 42.67%.

[0076] Table 4 Survival rate of Enterococcus faecalis in artificial intestinal fluid (%)

[0077]

[0078] 5. Growth curve determination

[0079] The inoculum concentration was 1×10 8 A certain amount of bacterial solution was taken from the bacterial solution of Enterococcus faecalis SKLAN202311L with a CFU / mL and inoculated into MRS broth medium, which was then placed in a shaker at 37°C. The fermentation broth was measured at OD using an ELISA reader every 2 hours. 600 The result shows ( Figure 1 ), Enterococcus faecalis SKLAN202311L strain began to enter the logarithmic growth phase after 4 hours and entered the plateau phase after 14 hours, with the maximum number of viable bacteria being 5.6×10 8 CFU / mL.

[0080] 6. Morphology and colony observation

[0081] The bacteria were smeared on a glass slide and dried, then stained with 1% malachite green for 3 minutes. After staining, the slide was slowly rinsed with deionized water to wash off the excess staining solution. After drying the slide, it was placed under a microscope for observation and photographing. The results are shown in ( Figure 2 ).

[0082] Use an inoculation loop to dip a loop of bacterial liquid and streak it on MRS agar medium. After culturing at 37℃ for 24 hours, observe the colony morphology. The results show that ( Figure 3 ).

[0083] Example 3 Effect of Enterococcus faecalis SKLAN202311L on the intestinal health of piglets

[0084] 1. Experimental Methods

[0085] Twelve healthy sows (3-4 years old, 5-6 litters) and their farrowing piglets from a farm in Tianjin were selected. Before weaning, all sows with suckling piglets were housed in closed farrowing rooms and provided with commercial feed and free drinking water. The pigpen environment was kept clean, ventilated, and disinfected regularly.

[0086] All sows were randomly divided into a control group (Con) and a bacterial liquid feeding group (Ef). Each group consisted of 6 sows, and 6 piglets were selected from each litter. The initial weight of the piglets in the two groups was similar (initial BW = 1.32 ± 0.09 kg), and they were weaned at 21 days of age. In the Con group, piglets were fed commercial creep feed (ND) from 7 days of age and allowed to eat freely until the end of the experiment at 28 days of age; in the Ef group, piglets were fed with an additional feed containing 5×10 10 The piglets were fed commercial feed of the same brand as the Enterococcus faecalis strain SKLAN202311L with CFU / kg, and the piglets were allowed to eat freely until the end of the experiment at the age of 28 days. During the experiment, the piglet weight was measured on days 7, 14, 21, and 28, the piglet status was observed and recorded, the piglet diarrhea rate was counted, and feces were collected.

[0087] 2. Testing indicators

[0088] 1. During the experiment, the diarrhea situation of piglets was counted every day.

[0089] 2. Measure the piglet weight on days 7, 14, 21, and 28.

[0090] 3. Collect piglet feces on the 28th day.

[0091] 3. Experimental Results

[0092] 1. Piglet weight: The weekly growth changes of piglets during the entire experiment are as follows: Figure 4 As shown, there was no significant difference in piglet weight between the two groups on day 7 ( Figure 5 ); on the 14th day, although there was no significant difference in the weight of piglets between the Ef group and the Con group, the weight of piglets in the Ef group was higher than that in the Con group ( Figure 6 ); on day 21, the weight of piglets in the Ef group was significantly higher than that in the Con group ( Figure 7 ); on day 28, the weight of piglets in the Ef group was significantly higher than that in the Con group ( Figure 8 ).

[0093] 2. Diarrhea rate: Fig. 9 As shown, the diarrhea rate of piglets in the Ef group was significantly lower than that in the Con group.

[0094] 3. Microbiome: e.g. Fig.10 As shown, the beneficial microorganisms in the feces of the Ef group piglets were significantly higher than those in the Con group. Example 4 Feeding Enterococcus faecalis SKLAN202311L to weaned piglets positive for porcine epidemic diarrhea virus

[0095] 1. Experimental Methods

[0096] 180 PEDV-positive piglets after weaning from a PEDV (epidemic diarrhea virus)-positive farm in Jinhua were selected. After weaning, every 10 piglets were housed in separate pens and provided with commercial feed and free drinking water. The pigpen environment was kept clean, ventilated, and disinfected regularly.

[0097] According to the results of Example 2, another strain of Enterococcus faecalis SKLAN202311M, whose stress resistance (capable of inhibiting Escherichia coli and Salmonella, and strong tolerance to artificial bile salts, artificial gastric acid, and artificial intestinal fluid) was second only to Enterococcus faecalis SKLAN202311L, was selected for testing. All piglets were randomly divided into a control group (Con), an Enterococcus faecalis SKLAN202311L bacterial solution feeding group (L), and an Enterococcus faecalis SKLAN202311M bacterial solution feeding group (M). There were 60 piglets in each group, 6 pens in each group, and 10 piglets in each pen. The initial body weights of the three groups of piglets were similar (initial BW = 7.06 ± 0.51 kg). Con group: Starting from the first day of the experiment, the piglets were fed commercial creep feed (ND) and allowed to eat freely until the end of the experiment on the 28th day; M group: Starting from the first day of the experiment, the piglets were fed with an additional feed containing 5×10 9 CFU / kg Enterococcus faecalis strain SKLAN202311M, and allowed them to eat freely until the end of the experiment on the 28th day; Group L: From the first day of the experiment, piglets were fed with an additional commercial feed containing 5×10 9 The piglets were fed commercial feed of the same brand as that of Enterococcus faecalis strain SKLAN202311L with CFU / kg, and the piglets were allowed to eat freely until the end of the experiment on day 28. During the experiment, the weight of the piglets was measured on days 7, 14, and 28, the status of the piglets was observed and recorded, and the diarrhea rate of the piglets was calculated.

[0098] 2. Testing indicators

[0099] 1. During the experiment, the diarrhea situation of piglets was counted every day.

[0100] 2. Measure the piglet weight on days 7, 14, and 28.

[0101] 3. During the experiment, the feed intake of piglets was counted every day.

[0102] 3. Experimental Results

[0103] 1. Piglet weight: The weekly growth changes of piglets during the entire experiment are as follows: Fig.11 As shown, there was no significant difference in piglet weight among the three groups on day 7 ( Fig.12 ); on the 14th day, although there was no significant difference in the weight of piglets in the L group, M group and Con group, the weight of piglets in the L group was higher than that in the M group and Con group ( Fig.13 ); On day 28, the weight of piglets in group L was significantly higher than that in groups M and Con ( Fig.14 ).

[0104] 2. Diarrhea rate: Fig.15 As shown, the diarrhea rate of piglets in group L was significantly lower than that in group M and group Con.

[0105] The results showed that Enterococcus faecalis SKLAN202311L had a significant improvement on the diarrhea and growth performance of PEDV-positive piglets, but Enterococcus faecalis SKLAN202311M had no significant improvement effect. Therefore, Enterococcus faecalis SKLAN202311L has the ability to inhibit PEDV that other Enterococci do not have.

[0106] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. Enterococcus faecalis strain SKLAN202311L, characterized in that The deposit number of the Enterococcus faecalis is CGMCC No.28891.

2. A bacterial agent comprising the Enterococcus faecalis strain SKLAN202311L according to claim 1.

3. The bacterial agent according to claim 2, characterized in that The bacterial agent includes the bacterial liquid obtained after fermentation of the Enterococcus faecalis strain SKLAN202311L.

4. Use of the Enterococcus faecalis strain SKLAN202311L according to claim 1 for preparing a bacterial agent for any of the following purposes: (1) Inhibit pathogenic microorganisms; (2) Promote animal growth; (3) Improve animal intestinal immunity; (4) Improve animal intestinal health; (5) Reduce animal diarrhea; (6) Regulate intestinal flora; (7) Prevent and treat diarrhea caused by porcine epidemic diarrhea virus.

5. The use according to claim 4, characterized in that: In the use (1), the pathogenic microorganism is a bacterium.

6. The use according to claim 5, characterized in that: The pathogenic microorganism is a Gram-negative bacterium.

7. The use according to claim 6, characterized in that: The pathogenic microorganism is Escherichia coli or Salmonella.

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