CN115109718B

CN115109718B - Enterococcus faecium strain and application thereof

Info

Publication number: CN115109718B
Application number: CN202210583120.3A
Authority: CN
Inventors: 崔红玉; 高玉龙; 李凯; 高立; 祁小乐; 刘长军; 张艳萍; 王笑梅
Original assignee: Harbin Veterinary Research Institute of CAAS
Current assignee: Harbin Veterinary Research Institute of CAAS
Priority date: 2022-05-25
Filing date: 2022-05-25
Publication date: 2023-09-12
Anticipated expiration: 2042-05-25
Also published as: CN115109718A

Abstract

The invention discloses an enterococcus faecium strain and application thereof. The enterococcus faecium strain is named C171 and is preserved in China general microbiological culture Collection center (CGMCC) with a microorganism preservation number of CGMCC No.24533 and a preservation date of 2022, 3 and 15 days. The research shows that the strain has unique dual characteristics of anti-inflammation and anti-infection, has the characteristics of acid resistance, bile salt resistance and the like, and can inhibit death caused by acute inflammatory reaction caused by ultra-virulent virus infection by only orally taking the strain enterococcus faecium C171 strain, thereby reducing the death rate of animals by more than 80 percent. The novel enterococcus faecium strain disclosed by the invention can be used as an additive of a probiotic strain for foods, feeds and medicines, and can be added into the foods, feeds or medicines to realize anti-inflammatory, anti-infection, health care effects and the like on animals, and the natural probiotic strain C171 with strong anti-inflammatory effect and anti-infection effect is expected to be used as an auxiliary therapeutic preparation for various acute inflammations, especially when the animals suffer from acute infections, and the protective effect is better.

Description

Enterococcus faecium strain and application thereof

Technical Field

The invention relates to an enterococcus faecium strain and application of the enterococcus faecium strain in anti-inflammatory and anti-infective preparations, and belongs to the technical field of biology.

Background

Enterococcus faecium (Enterococcus faecium) is one of the important members of the enterococcus genus, most of which is one of the probiotics of the intestinal mucosal surface of any animal. Probiotic Lactic Acid Bacteria (LAB) are normal flora of intestinal, respiratory, genital and skin mucosal systems of humans and animals, and many research results find that lactic acid bacteria and intestinal mucosa together form a protective barrier against invasion of viruses, bacteria etc., and humans increasingly clearly recognize the powerful effects of probiotics on the life health of humans and animals. One of the most exciting scientific advances in recent years is the recognition that commensal microorganisms (our microbiota) play an important role in human physiology, vitamin synthesis, drug metabolism, prevention of infection and disease recovery.

In recent years, the immunoregulation effect of probiotics has been widely focused, the immune system has evolved to maintain the symbiotic relationship of host-microbiota, on the other hand, in order to maintain host immune homeostasis, the intestinal microbiota often plays a non-negligible immunoregulatory role, and the brain-intestinal axis is a bi-directional link between the intestinal microbiota, intestinal barrier and immune system, playing some basic roles in human immunity, metabolism, structure and nervous system. Intestinal flora also has a significant impact on the physiological and psychological health of individuals. Studies have shown that some lactic acid bacteria strains are able to stimulate an immune response in the body, e.g. Lactococcus lactis, lactobacillus paracasei etc. are able to promote IgA production, enhance NK cell activity and enhance specific immune responses by activating macrophages or dendritic cells; the beneficial probiotics can stimulate the organism to release different cytokines to influence the mucosa immune response and the T cell differentiation, so as to carry out immune regulation on the host; the beneficial probiotics can activate dendritic cells of organisms and assist T cell polarization to increase secretion of IFN-gamma and IL-12, so that naive T cells or memory T cells are converted into Th1, and the organisms can improve immunity to realize anti-infection; in addition, the beneficial probiotics can secrete some metabolites in the proliferation process, and some metabolites have anti-inflammatory effect, so that the polarization of Th17 can be weakened, and the differentiation of Treg 1 helper T cells is facilitated. Beneficial probiotics modulate the host immune system by binding their MAMP (e.g., lipoteichoic acid, peptidoglycan, S-layer proteins, and nucleic acids) to PPRS (Toll-like receptor, NOD-like receptor, lectin-type C receptor) expressed by the host intestinal mucosa.

The anti-inflammatory and anti-infective effects of probiotics have been paid attention to in recent years, and have been widely studied and applied, and the previous studies have been mainly focused on the use of probiotics for alleviating inflammation-related diseases such as allergic symptoms, inflammatory bowel diseases and autoimmune diseases. Studies show that after lactobacillus reuteri and lactobacillus casei are orally taken, treg cells are up-regulated for immunoregulation and immune tolerance by activating receptors on bone marrow-derived dendritic cells, and meanwhile, an immune negative regulation cytokine IL-10 is up-regulated, so that the proinflammatory cascade reaction is finally and obviously down-regulated, and the damage of anaphylactic reaction to organisms is reduced; also, studies have shown that a probiotic preparation (l.rhamnoses, b.lactis, and b.longum have the ability to regulate secretion of inflammatory factors, can induce human macrophage line THP1 to produce anti-inflammatory factor IL-10, significantly down-regulate expression of pro-inflammatory factors IL-1 beta and IL-6; lactobacillus rhamnoses B15 and Lactobacillus gasseri M13 inhibit the production of nitric oxide by macrophages and reduce the inflammatory factors IL-1 beta and IL-6 in an experiment of mice infected with influenza A, a strain of Lactobacillus gasseri has been studied to reduce the infiltration of mouse pulmonary pro-inflammatory cytokine IL-6 and significantly reduce the viral titer in a variety of probiotic anti-inflammatory mechanisms, such as by their metabolites mediating anti-inflammatory effects (e.g., short Chain Fatty Acids (SCFA), especially propionic acid, acetic acid and butyric acid), by bifidobacteria, lactobacillus and various symbiotic bacteria which exert anti-inflammatory effects by binding to specific receptors on intestinal epithelial cells and inhibit the production of pro-inflammatory cytokines by neutrophils and macrophages, and the like, and a combination of Lactobacillus casei strain Shiroma (LcS) widely used throughout the world has been studied for its effects on Acute Liver Injury (ALI) and its potential mechanism, and Lactobacillus casei Shiroma has been found to reduce inflammation and metabolic disorders by intestinal microbiota, thereby preventing acute liver injury and inflammatory disorders by oral LA, and inflammatory disorders of the gastrointestinal tract, and human intestinal tract infections by oral administration of Lactobacillus 401 and human tumor cells, and human intestinal tract infection of the human intestinal tract may be reduced by the human intestinal tract tumor cells of human intestinal tract may be reduced by human intestinal tract infection of human intestinal tract infection, IL-17, IL-23 and IL-1β levels.

In summary, the anti-inflammatory effects of many probiotics are mediated through immunomodulation, in particular through balanced control of pro-and anti-inflammatory cytokines, it is becoming increasingly clear that many factors are involved in immunomodulation balance, in particular in the combined regulation of genetic background, host immune response and microbial diversity status, etc.; several studies evaluate the effect of probiotics in the treatment of diseases, prevention and alleviation of inflammation through different study models (cell lines, animal models of colitis, clinical studies). Research data shows that the immunomodulatory effects of various candidate probiotics are different, and the immunomodulatory effects and modes are quite different, for example, different strains have obvious differences on the same inflammation, and the results show that the immunomodulatory characteristics of the probiotics can not be applied until specific and complex detection and characterization are performed. And the exact anti-inflammatory mechanism of most of the applied probiotic strains has not yet been fully elucidated.

Although the initial inflammatory response is critical for clearing the infection, and is necessary to initiate the immune defenses and immune responses, the strong inflammatory response can lead to a Cytokine Storm (Cytokine Storm) which ultimately leads to tissue and organ damage and death. Coronaviruses, including SARS coronavirus, middle east respiratory syndrome coronavirus and novel coronaviruses, infect humans and initiate a strong inflammatory response at the beginning of infection, where cytokine storm and pro-inflammatory cytokine cascades can be observed in severe patients infected with novel coronavirus (COVID-19), which in turn leads to severe injury to the lungs.

For a long time, the treatment of acute inflammatory storms has been to maintain critical organ function mainly by limiting collateral damage caused by the activated immune system, for example by inhibiting the synthesis of pro-inflammatory cytokines and driving lymphocyte death using glucocorticoids and dexamethasone. The probiotics can maintain the dynamic balance of intestinal microorganisms, maintain dominant symbiotic flora in the intestinal microorganisms, and help the organism to regulate the balance between inflammatory response caused by pathogen infection and normal immune function of the organism, so that the whole immune system of the organism benefits, in recent years, the probiotics are reported in the aspect of relieving acute inflammatory response, the serious illness rate and death rate are obviously reduced by supplementing the probiotics, and the specific IgM and IgG aiming at severe acute respiratory syndrome coronavirus 2 (SARS-CoV 2) are increased. However, these reports are all that probiotics play an important anti-inflammatory role as auxiliary factors in the treatment process, if the probiotics are used alone for treatment, whether the probiotics have a better inhibition effect on acute inflammatory storm caused by infection or not, and whether the acute death caused by clinical acute inflammatory storm can be greatly relieved or not, and the research report on the aspect is still lacking at present.

Disclosure of Invention

The invention aims to provide a novel enterococcus faecium strain and application of the strain in anti-inflammation and anti-infection, especially in anti-acute inflammation.

In order to achieve the above purpose, the invention adopts the following technical means:

the strain is separated from the cecum mucosa of a wild healthy chicken in Fenghuangshan county of Heilongjiang province, is inoculated by MRS, cultured, separated and purified, and then is separated to obtain a lactic acid strain which belongs to enterococcus faecium (Enterococcus faecium), is named C171, is named enterococcus faecium (Enterococcus faecium) in a classification mode, is preserved in the common microorganism center of the China Committee for culture Collection of microorganisms, is addressed to the institute of microorganisms in national institute of sciences of North Chen West road No. 1 in the Korean area of Beijing, and has a microorganism preservation number of CGMCC No.24533 and a preservation date of 2022 years, 3 months and 15 days. The microbiological characteristics of the strain are: (1) gram staining is typically positive, and the thallus is medium and large and regular in morphology; aerobic or facultative anaerobes grow well on solid and liquid MRS media; (2) has acid resistance, and can grow well on MRS with pH value of 4.0; (3) has bile salt resistance, and can survive and grow in culture solution containing 50% chicken bile MRS; (4) has unique anti-inflammatory and anti-infective properties; (5) the bacteria culture optimum temperature is 37 ℃, and the bacterial colony on the MRS culture medium is milky white, has neat edges, smooth and moist surface, is raised, is opaque and has a sour and fragrant taste.

Therefore, the invention further provides the application of the enterococcus faecium strain in preparing anti-inflammatory and anti-infection preparations.

Preferably, the anti-inflammatory and anti-infective agents are those that are resistant to acute inflammatory storms, including those that are resistant to acute inflammation and death caused by bacterial or viral infections.

Wherein, preferably, the acute inflammation is caused by a super virulent viral infection.

Furthermore, the invention also provides application of the enterococcus faecium strain in preparing medicines for regulating intestinal flora of animals or promoting growth of animals.

Compared with the prior art, the invention has the beneficial effects that:

the invention separates and purifies a lactobacillus strain from wild healthy chicken cecum mucosa, and the lactobacillus strain is identified to belong to enterococcus faecium (Enterococcus faecium), researches show that the strain has unique anti-inflammatory and anti-infection dual characteristics, and simultaneously has the characteristics of acid resistance, bile salt resistance and the like, and only oral administration of the enterococcus faecium C171 strain can inhibit death caused by acute inflammatory reaction caused by super-virulent virus infection, thereby reducing animal mortality by more than 80%. The novel enterococcus faecium strain disclosed by the invention can be used as an additive for food, feed and medicines, and can be added into food or feed to realize the health care effect of the enterococcus faecium strain on animals. The natural probiotics C171 strain with strong anti-inflammatory effect and anti-infection effect is added into the medicine to be hopeful to be used as auxiliary therapeutic preparations for various acute inflammations, and particularly has better protection effect when animals suffer from acute infection.

Drawings

FIG. 1 shows the growth characteristics of a strain of a novel strain of enterococcus faecium isolated according to the invention on a solid medium;

FIG. 2 shows the culture characteristics of novel enterococcus strain Enterococcus faecium C171 on X-gal-MRS medium, enterococcus chromogenic medium, MC medium solid medium;

FIG. 3 is a graph showing the interaction of enterococcus faecium strain C171 with a macrophage inflammatory model;

FIG. 4 is the anti-inflammatory and anti-infective properties of enterococcus faecium C171 on chicken primary macrophages;

FIG. 5 shows the anti-inflammatory effect of enterococcus faecium strain C171 by modulating NLRP3 inflammatory small body pathway and inhibiting caspase-1 activity to achieve anti-inflammatory test results;

FIG. 6 shows the results of antiviral proliferation properties of enterococcus faecium strain C171 in vitro;

FIG. 7 shows the animal experiment result that the enterococcus faecium strain C171 can obviously protect the death caused by the infection of the super virulent virus of the chicken, and the protection rate reaches more than 80 percent;

FIG. 8 shows the results of oral enterococcus faecium strain C171 significantly inhibiting acute inflammation in chickens induced by the virulent virus vvIBDV;

FIG. 9 shows the results of oral administration of enterococcus faecium strain C171 to significantly improve the anti-infective effect of chickens in the presence of virulent virus infection;

FIG. 10 shows the results of safety evaluation of enterococcus faecium strain C171, virulence factor (A), drug resistance gene (B) and chick embryo safety (C);

FIG. 11 shows the results of enterococcus faecium strain C171 being able to withstand the conditions of gastrointestinal bile salts (A) and gastric acid (B);

FIG. 12 shows the results of oral enterococcus faecium C171 strain analysis of intestinal flora level top30 flora, A bar graph, B heat graph;

FIG. 13 shows the results of oral enterococcus faecium C171 strain analysis of intestinal flora level top30 flora, A bar graph and B heat graph;

FIG. 14 is an analysis of the small intestine alpha diversity index dilution curve of oral enterococcus faecium strain C171 against intestinal flora; a small intestine alpha diversity related box plot; small intestine beta diversity index analysis;

FIG. 15 is a multivariate statistical analysis of intestinal microbiota of intestinal flora by oral enterococcus faecium strain C171. LEfSe analysis.

Detailed Description

The invention will be further described with reference to specific embodiments, and advantages and features of the invention will become apparent from the description. The embodiments are merely exemplary and do not limit the scope of the invention in any way. It will be understood by those skilled in the art that various changes and substitutions of details and forms of the technical solution of the present invention may be made without departing from the spirit and scope of the present invention, but these changes and substitutions fall within the scope of the present invention.

EXAMPLE 1 isolation and identification of the Strain of the present invention

1. Isolation and identification of strains

The strain is separated from wild healthy chicken cecum mucosa of Fenghuangshan in Wuchang county of Heilongjiang province, grows well on an MRS culture medium, and is obtained by streaking and separation. The microbiological characteristics are: gram staining is typically positive, and the thallus is lactococcus with medium size and regular morphology; aerobic or facultative anaerobes grow well on solid and liquid MRS medium, and the bacteria culture optimum temperature is 37 ℃, and the colony on the MRS medium is milky white, neat in edge, smooth and moist in surface, raised, opaque and medium-sized, and has an acidic flavor.

Bacterial colony characteristics are shown in FIG. 1.

The novel enterococcus faecium strain is named as C171, and is classified as enterococcus faecium (Enterococcus faecium). The microorganism preservation number is CGMCC No.24533, and the preservation date is 2022, 3 and 15.

2. Acid resistance test of strains:

respectively inoculating strain Enterococcus faecium C171 obtained by separation into MRS liquid culture medium with pH value of 3.0,4.0,5.0 and 6.5 according to 10% of the volume of MRS liquid culture medium, setting 3 parallel controls for each acidity gradient, standing at 37deg.C, culturing for 30min, 60min, 90min and 120min, and measuring OD ₆₀₀ nm value. Experiments show that the strain has stronger acid-resistant capability and can grow on MRS with pH of 4.0.

3. Chicken bile resistance test of strains:

inoculating isolate Enterococcus faecium C171 into MRS culture medium containing fresh chicken bile at an inoculum size of 10% of the volume of MRS liquid culture medium, wherein the contents of fresh chicken bile are respectively 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 3 parallel tests are set for each concentration, standing culture is performed at 37deg.C, and bacterial liquid samples cultured for 30min, 60min, 90min, 120min are taken for measuring OD ₆₀₀ The nm value shows that the strain can well grow in a chicken bile culture medium containing 30 percent and has stronger chicken bile resistance.

EXAMPLE 2 culture Properties on solid Medium of the Strain of the present invention

1. Colony characteristics of enterococcus faecium C171 strain on common MRS medium:

the experimental method comprises the following steps: enterococcus faecium C171 was streaked on MRS solid medium, and the colony morphology of C171 was observed.

Experimental results: as shown in FIG. 1, the enterococcus faecium C171 colony is in the form of a medium-sized colony with neat edge, smooth and moist surface, raised surface, opaque, milky white and has a sour and fragrant taste.

2. Colony characteristics of enterococcus faecium C171 strain on X-gal-MRS medium, enterococcus chromogenic medium and MC medium:

the experimental method comprises the following steps: enterococcus faecium C171 was streaked on X-gal-MRS medium, enterococcus chromogenic medium, MC medium, and cultured at 37℃for 48 hours, and the colony characteristics were observed.

Experimental results: as shown in FIG. 2, enterococcus faecium C171 strain was cultured anaerobically on X-gal-MRS with colony morphology similar to that on MRS medium (FIG. 2A); when aerobically cultured on X-gal-MRS, the colony morphology was similar to that on MRS medium, and the color was blue (FIG. 2B) (indicating that enterococcus faecium strain C171 could produce beta-glucosidase, break down the X-gal substrate, making the colony blue); enterococcus faecium strain C171 was cultured aerobically on enterococcus faecium chromogenic medium, and the colony was typically tan-colored (FIG. 2C); after aerobic culture of enterococcus faecium strain C171 on MC medium, transparent rings appear around the colony (FIG. 2D), indicating that enterococcus faecium strain C171 produces acid metabolites and dissolves calcium carbonate.

Example 3 interaction of enterococcus faecium strain C171 with macrophage inflammatory model, exhibiting potent anti-inflammatory and anti-infective properties

The experimental method comprises the following steps: chicken macrophage HD11s (1×10) ⁶ cells/mL) was inoculated into a 12-well plate, 50MOI C171 was added after cell attachment, and 5% CO at 37 ℃ ₂ Incubating for 24h, and using LPS and Nigericin to make cells produce acute inflammatory reaction, and establishing acute inflammatory model, namely adding LPS with working concentration of 1 μg/mL into each hole, and adding 5% CO at 37deg.C ₂ After 3h of action and 1h of Nigericin at a working concentration of 1. Mu.M was added, the cell pellet was collected and quantified relatively by SYBR Green I fluorescent quantitative PCR, and the primers of Table 1 were used to analyze the transcriptional levels of IL-1. Beta. And IL-10mRNA in the cells. LPS and Nigericin treated cells served as positive controls and untreated cells served as blank controls.

The characteristics of the C171 activated macrophage NF- κB and IRF signaling pathways were examined. J774-Dual ^TM (2.8X105/mL) was inoculated into a 96-well plate, 50MOI of C171 was added, 1. Mu.g/mL of LPS and 10ng/mL of Pam3CSK were used as positive controls, and after 24 hours of stimulation with 5% CO2 at 37℃the supernatant was collected. Then 170. Mu.L of QUANTI-Blue was added to each well in a 96-well plate ^TM ) Then 30 mu L J774-Dual is added ^TM Cell supernatant was then subjected to 5% CO at 37 ℃ ₂ 4h, absorbance at 650nm was measured to reflect NF- κB induced SEAP levels. J774-Dual ^TM cells can detect IRF signal paths simultaneously: J774-Dual ^TM (2.8×10 ⁵ cells/mL) was inoculated into a 96-well plate, 50MOI of C171 was added, and 1. Mu.g/mL of LPS and 1. Mu.g/mL of 2'3' -cGAMP were used as positive controlsAt 37℃5% CO ₂ After 24h of stimulation, the supernatant was collected. 20 mu L J774-Dual was added to an opaque 96-well plate ^TM Cell interaction supernatants were assayed for luciferase expression using a microplate chemiluminescent detector (LB 960).

TABLE 1 primer sequences

Experimental results: as shown in FIGS. 3A-D, the results of the anti-inflammatory and anti-infective effects of enterococcus faecium C171 on macrophages are shown. C171 significantly down-regulated the inflammatory cytokine IL-1β produced by HD11 in the acute inflammation model, with a very significant difference compared to the l+n control group (P <0.0001; fig. 3A). C171 was effective in stimulating HD11 to produce the anti-inflammatory cytokine IL-10, which was very different compared to the L+N control group (P <0.0001; FIG. 3B). C171 was effective in activating NF- κB signaling pathway in macrophage J774-Dual cells, with a very significant difference compared to the placebo (P <0.0001; FIG. 3C); c171 was effective in activating the Interferon Regulatory Factor (IRF) signaling pathway in macrophage J774-Dual cells, with a very significant difference compared to the placebo group (P <0.0001; fig. 3D). The results show that enterococcus faecium C171 has strong anti-inflammatory effect and anti-infective effect.

Example 4 anti-inflammatory and anti-infective Properties of enterococcus faecium C171 on Primary macrophages of chickens

The experimental method comprises the following steps:

1. first, chicken primary bone marrow macrophages were isolated as follows: (1) euthanized 3 week old SPF chickens, and the femur of the chickens was removed in a sterile operating table; (2) cutting the two ends of the femur, and flushing out bone marrow by using a syringe for sucking PBS; (3) bone marrow was collected in a40 μm screen, ground with a 10mL syringe plug, and then the bone marrow on the screen was washed with PBS; (4) the mixture was gently added to an equal volume of Histopaque-1083 separating liquid, centrifuged at 2,000r/min for 25min, and the acceleration was minimized; (5) sucking the middle layer cells, and washing with PBS for 2 times; (6) counting cells, inoculating the cells into a 12-well plate, changing the liquid after 6 hours, and washing off cells which are not adhered to the wall or are semi-adhered to the wall; (7) subsequent experiments were performed after 6d of incubation (medium was changed every 2 d).

2. The effect of C171 on cell proliferation activity was verified using Cell Counting Kit-8 to determine the toxicity of C171 to cells. Chicken primary bone marrow macrophages at 1 x 10 ⁶ 100. Mu.L of cells were seeded into 96-well plates at a concentration of cells/mL, 50MOI of C171 suspension was added, and after 24h incubation, 10. Mu.L of cck-8 solution was added. After incubation at 37℃for 2h, the absorbance at 450nm was determined.

3. Whether C171 had anti-inflammatory effects on chicken primary bone marrow cells was investigated. After cells were inoculated in 12 wells, 50MOI probiotic suspension was added, 5% CO at 37℃ ₂ Incubating for 24h, and using LPS and Nigericin to make cells produce acute inflammatory reaction, and establishing acute inflammatory model, namely adding LPS with working concentration of 1 μg/mL into each hole, and adding 5% CO at 37deg.C ₂ After 3h of action and 1h of Nigericin at a working concentration of 1. Mu.M was added, the cell pellet was collected and analyzed for IL-1. Beta. And IL-10mRNA transcription levels by SYBY Green I fluorescent quantitative PCR according to the primers of Table 2. LPS and Nigericin treated cells served as positive controls and untreated cells served as blank controls.

TABLE 2 primer sequences

Experimental results: FIG. 4 is a graph showing the results of anti-inflammatory and anti-infective effects of C171 on primary macrophages. C171 was non-toxic to chicken primary bone marrow macrophages, and C171 was able to stimulate chicken primary bone marrow cell proliferation, with a very significant difference in C171 compared to the placebo (P < 0.0001). C171 significantly down-regulates the cytokines IL-1 beta (P < 0.01), up-regulates the cytokines IL-10 (P < 0.001) and IFN-gamma (P < 0.0001) produced by chicken primary bone marrow macrophages in an acute inflammation model. The results illustrate: c171 has both anti-inflammatory and anti-infective effects on chicken primary bone marrow macrophages.

Example 5 anti-inflammatory effects of enterococcus faecium strain C171 were achieved by modulating the NLRP3 inflammatory body pathway and inhibiting caspase-1 activity.

The experimental method comprises the following steps:

1. the effect of C171 on HEK293T cell proliferation activity was validated using Cell Counting Kit-8 to determine C171 cytotoxicity. HEK293T cells at 1X 10 ⁶ 100. Mu.L of cells were seeded into 96-well plates at a concentration of cells/mL, 50MOI of C171 suspension was added, and after 24h incubation, 10. Mu.L of cck-8 solution was added. After incubation at 37℃for 2h, the absorbance at 450nm was determined.

2. The anti-inflammatory mechanism of C171 was detected. HEK293T cells were grown at 5X 10 ⁴ The wells were seeded at a concentration of/well in 48 well plates, 100ng iGLuc,10ng pCAGGS-caspase-1,10ng pCAGGS-ASC,12.5ng pCAGGS-NLRP3 were transiently transfected with 1. Mu.L of X-tremeGENE HP DNA Transfection Reagent when the cell density reached 70%, empty plasmid was transfected and iGLuc alone transfected as control group. Probiotic C171 with MOI of 25, 50, 100 was added after 12h and after further co-culture for 12h, cell supernatants were collected and centrifuged at 3000rpm for 5min to remove cell debris. Luciferase activity was measured with PierceTM Gaussia Luciferase Flash Assay Kit according to the manufacturer's instructions. Cell pellet was used for detection of Western blot-related proteins (NLRP 3, ASC, caspase-1p20, pro-IL-1. Beta.).

Experimental results: as shown in fig. 5, C171 was non-toxic to HEK293T cells (fig. 5A) and was able to increase the activity of the cells with a clear dose dependence. The luciferase results show that C171 can significantly inhibit IL-1β secretion, and has dose dependency, and the more obvious the inhibition effect is with the increase of C171, the more significant the difference from the positive control group is (P <0.0001; FIG. 5B). Western blotting results showed that C171 did not affect expression of NLRP3, ASC, pro-IL-1β, but was able to reduce caspase-1p20 expression, and was dose dependent, as seen by gray scale analysis, at 25MOI, group C171 was 0.60 fold of positive control (FIG. 5C); at 50MOI, 0.47 times that of the positive control (FIG. 5C); MOI was 0.29 times that of the positive control at 100 (FIG. 5C). From the above results, it was found that C171 inhibits IL-1β secretion by inhibiting caspase-1 activity, thereby regulating NLRP3 inflammatory small body pathway.

EXAMPLE 6 antiviral proliferation Properties of enterococcus faecium C171 Strain in vitro

The experimental method comprises the following steps: DT40 (1×10) ⁶ Serum-free RPM 1640 per mL) was inoculated into 12-well plates, incubated at 37 ℃ for 30min, infected with 1MOI vvIBDV after cell attachment, and simultaneously added with probiotic C171 with MOI of 25, 50, 100, after 24h of action, 200 μl of cell suspension was collected, the IBDV VP5 copy number was quantitatively determined by absolute assay using Premix Ex Taq (primers of table 3), cell pellet was assayed for cytokines IL-1 β, IL-10, ifn- γ (primers of table 2), and VP2 protein was detected by Western blot.

TABLE 3 primer sequences

Experimental results: as shown in FIG. 6, C171 did not affect the cellular activity of DT40, and was safe and nontoxic to DT40 cells (FIG. 6A). C171 has a remarkable antiviral effect in vitro, and C171 can remarkably reduce the viral copy number of vvIBDV compared with a positive control, has remarkable dose dependency, and has more remarkable inhibition effect with increasing dose of C171. The inhibition was most pronounced at a MOI of 100 for probiotics and DT40 (P <0.0001; FIG. 6B). Western blotting results show that C171 significantly reduced the protein expression of vvIBDV VP2 and that there was a significant dose dependence. The gray scale analysis showed that the expression of VP2 protein gradually decreased with increasing C171 dose, and that the expression level of VP2 was 0.43 times that of positive control when the MOI of the probiotic and DT40 was 100 (FIG. 6C). IL-1β results showed that C171 significantly down-regulated the cytokine IL-1β produced by DT40 cells in the vvIBDV infection model and had a metering dependence, with the most significant differences compared to the positive control group when MOI were 50 and 100 (P <0.0001; FIG. 6D). IL-10 results indicate that C171 is effective in stimulating production of the cytokine IL-10 by DT40 cells, with a very significant difference compared to the positive control group when MOI is 100 (P <0.0001; FIG. 6E). IFN-gamma results showed that C171 was effective in stimulating DT40 cytokine IFN-gamma and was dose dependent, with the most pronounced stimulation when MOI was 100, with very significant differences compared to the positive control group (P <0.0001; FIG. 6F). Taken together, the results show that C171 can reduce viral replication in vitro, reduce the expression of inflammatory factors, and exert anti-infective effects by secreting IFN-gamma.

Example 7 the oral enterococcus faecium C171 strain can obviously protect the death caused by the infection of the super virulent virus of the chicken, and the protection rate is more than 80 percent.

The experimental method comprises the following steps:

1. animal experiment scheme: SPF chickens were purchased from the laboratory animal center of Harbin veterinary research institute, national academy of agricultural sciences and fed into a negative pressure filtered air isolator. 40 SPF chickens at 18 days of age were randomly divided into three groups: blank control group (15), oral C171 group (15), challenge control group (10). Group C171 for oral administration: after C171 was resuspended in PBS, 1mL, 2X 10 per day per chicken ⁹ CUF/mL was directly infused into the mouth for 7 consecutive days. Seven days later, 5 chickens were killed in each of the oral group C171 and the blank. Except for the blank control group, the remaining 20 SPF chickens with 21 days age were challenged with Chinese vvIBDV reference strain HLJ0504 (vvIBDV-HLJ 0504) by nasal drop and eye drop, the challenge dose was 5LD 50/chicken 200ul, and the onset was observed daily. Wherein group C171 was orally administered, and after detoxification, C171 was orally administered again for 7 days. And observing the disease condition of the chickens every day, killing all chickens after seven days of toxin attack, and performing section inspection to calculate the survival rate.

2. The bursa and body weight of each group of chickens were determined, and the bursa index (BBIX) of each group of chickens was calculated according to the following formula: BBIX = bursa weight ratio of test group chickens/average bursa weight ratio of placebo group chickens; bursa weight ratio = (bursa weight/body weight) ×100%, with BBIX less than 0.70, is considered atrophy.

3. ELISA detects IFN-gamma content in serum 1 week after oral administration.

4. The copy number of IBDV in bursa of Fabricius was detected by means of fluorescence quantification.

Experimental results:

as shown in FIG. 7, C171 was orally administered to chickens, then SPF chickens were infected with vvIBDV-HLJ0504 (FIG. 7A), and the anti-inflammatory, anti-infective effects of C171 were examined in animals. Serum ELISA results showed that after C171 oral administration, IFN-gamma production was induced in the body, which was very different from that of the Blank group (P <0.0001; FIG. 7B). The challenge results showed that the survival rate of IBDV group SPF chickens was 20% and that of C171+ IBDV group SPF chickens was 80% after one week challenge with vv-IBDV-HLJ0504, which was a 4-fold increase compared to IBDV groups (FIG. 7C). The results of the section showed that the BBIX of both the C171+ IBDV group and the IBDV group-surviving chickens were less than 0.7, with no significant difference (P >0.05; FIG. 7D), and both were significantly lower than the Blank group (P <0.0001; FIG. 7D). We also examined the replication capacity of the virus in vivo and the fluorescent quantitation showed that the viral copy number in the C171+ IBDV group bursa was significantly lower than that of IBDV group (p <0.01; FIG. 7E). Taken together, the results indicate that C171 can inhibit viral replication in vivo, reducing mortality.

Example 8 oral administration of enterococcus faecium strain C171 significantly inhibited acute inflammation in chickens caused by the virulent virus vvIBDV.

The experimental method comprises the following steps:

1. the animal protocol was the same as in example 7. A portion of bursa of Fabricius was taken from each group of chickens, fixed with 10% neutral buffered formalin, stained with hematoxylin and eosin, and subjected to further histopathological examination.

2. The expression of HMGB1 protein in bursa of Fabricius, spleen and intestinal tract was examined by Western blot.

3. ELISA detects the content of IL-1 beta and IL-10 in serum after 1 week after toxin attack.

4. The transcription level of IL-1 beta and IL-10 in bursa of Fabricius is detected by a fluorescence quantification method.

Experimental results:

as shown in FIG. 8, to investigate whether IBDV can alleviate damage to bursa of Fabricius after oral administration of C171, chicken bursa pathological section observations showed that histopathological damage occurred in C171+ IBDV group (FIG. 8A-a), follicular atrophy, extensive lymphocyte necrosis, reduction, and interstitial proliferation in bursa; IBDV group (FIGS. 8A-b) showed more severe histopathological lesions than C171+ IBDV group, severe atrophy of the bursa of Fabricius follicles, extensive lymphocyte necrosis, reduction, interstitial proliferation, formation of a crypt in the center of the follicles, and necrotic cell masses; both the Blank group (FIGS. 8A-C) and the C171 group (FIGS. 8A-d) were normal, and no clinical symptoms or microscopic lesions were found (FIG. 8A). To verify whether C171 can reduce the acute inflammatory response of the body, inflammatory cytokines in serum and tissues and HMGB1 expression in bursa of fabricius, spleen, intestinal tract were examined. Western blotting results show that the expression level of HMGB1 protein of IBDV group in bursa of Fabricius, spleen and intestinal tract is higher than that of C171+ IBDV group (FIG. 8B). The content of IL-1 beta and IL-10 in serum is detected, and ELISA results show that the concentration of C171+ IBDV group in the serum is extremely lower than that of IBDV group (p <0.001; FIG. 8C), and has no obvious difference from Blank group (p >0.05; FIG. 8C); the concentration of IL-10 in serum C171+ IBDV group was significantly higher than IBDV group (p <0.0001; FIG. 8D) and Blank group (p <0.0001; FIG. 8D). The detection of the mRNA transcription level of IL-1 beta and IL-10 in bursa of Fabricius shows that the mRNA transcription level of IL-1 beta in bursa of Fabricius can be down-regulated after C171 is orally taken, and the C171+ IBDV group is extremely lower than the IBDV group (p <0.001; FIG. 8E) and has no obvious difference from the Blank group (p >0.05; FIG. 8E); the mRNA transcription level of IL-10 in bursa of Fabricius was up-regulated after oral administration of C171, and the C171+ IBDV group was significantly different from the IBDV group, blank group (p <0.05; FIG. 8F). Taken together, the results show that C171 can inhibit the production of IL-1 beta, promote the expression of IL-10 and reduce the inflammatory reaction of the organism.

Example 9 oral administration of enterococcus faecium strain C171 can significantly improve the anti-infective effect of chickens when infected with a super virulent virus.

The experimental method comprises the following steps:

1. the animal protocol was the same as in example 7. Peripheral Blood Mononuclear Cells (PBMCs) were isolated from fresh anticoagulants and their T cell subsets were examined. Three anticoagulated blood specimens taken orally for C171 weeks and 1 week after toxicity attack were randomly collected, and PBMCs were isolated using a chicken peripheral blood lymphocyte separation kit. The ratio of CD3+ lymphocytes, CD4+ lymphocytes and CD8+ lymphocytes was analyzed by flow cytometry after diluting R-physoerythrin/Cyanine 5 (SPRD) labeled anti-chicken CD3 antibody, fluorescein Isothiocyanate (FITC) labeled anti-chicken CD4 antibody and R-Phycoerythrin (PE) labeled anti-chicken CD8 antibody in PBS containing 5% FBS, staining the PBMCs according to the dye to cell concentration ratio provided in the specification, staining the PBMCs on a shaker at 4℃for 30min, washing the PBMCs three times with PBS containing 5% FBS.

2. ELISA detects the content of IL-12, IL-2, IL-4 and IFN-gamma in serum after 1 week after toxin attack.

3. Peripheral Blood Mononuclear Cells (PBMCs) were isolated from fresh anticoagulants and tested for proliferative activity. T cell proliferation activity was detected by concanavalin A (ConA) and Phorbol 12-myristate 13-acetate (PMA) -stimulated PBMCs. Chicken peripheral blood mononuclear cells were isolated using chicken peripheral blood mononuclear cell isolation kit (TBD, china). PBMCs were diluted to 1X 10 with RPMI 1640 medium containing 10% fetal bovine serum and penicillin-streptomycin ⁶ cells/mL. 100. Mu.L of PBMCs were added to each well of a 96-well plate. Then 5. Mu.g/mL ConA and 100ng/mL PMA were added to each well and incubated in a cell incubator at 37℃for 48h. The absorbance at 450nm was then measured after the addition of 10. Mu. LCCK-8 for 4 hours.

Experimental results: as shown in FIG. 9, the anti-infective mechanism of C171 in vivo was explored, and T cell subtypes in PBMCs were analyzed by flow cytometry. Flow results indicated that the cd8+ T cell content was elevated after C171 weeks of oral administration (fig. 9A) compared to Blank group, and cd8+/CD 4T cell ratio of C171 group was 0.192,Blank group and 0.157 (fig. 9B); after one week of challenge v-IBDV-HLJ0504, the CD8+ T cell content of both IBDV group and C171+ IBDV group was increased (FIG. 9A), and the CD8+/CD 4T cell ratio of IBDV group and C171+ IBDV group was 0.259 and 0.546, respectively (FIG. 9B). Wherein the ratio of CD8+/CD4+ T cells in the C171+ IBDV group surviving chickens is highest, significantly higher than in IBDV groups (p <0.05; FIG. 9B); c171 The group was not significantly different from the Blank group (p >0.05; FIG. 9B). Since CD8+T cells can induce the organism to produce high levels of IFN-gamma, CD4+T cells can induce the organism to produce high levels of IL-4, we have examined the content of IFN-gamma, IL-4 in serum again. ELISA results showed that the concentration of IFN-. Gamma.in serum C171+ IBDV group was significantly higher than IBDV group (p <0.0001; FIG. 9C) and Blank group (p <0.0001; FIG. 9C). The concentration of IL-4 in serum C171+ IBDV group was significantly lower than IBDV group (p <0.0001; FIG. 9D) and significantly higher than Blank group (p <0.0001; FIG. 9D). This demonstrates that the oral administration of C171 can induce the body to produce high-level CD 8T cells, promote IFN-gamma secretion and improve the anti-infective ability of the body. To explore the differences in T cell proliferation activity of each group after challenge, we stimulated PBMCs one week after challenge with cona+pma and examined T cell proliferation activity in each group of chicken PBMCs. The results showed that the PBMCs proliferation activity of C171+ IBDV group was significantly higher than that of IBDV group (p <0.01; FIG. 9E) and Blank group (p <0.0001; FIG. 9E). The cytokine can stimulate the proliferation of T cells, and the content of the T cell growth factors IL-12, IL-2 and IL-23 in serum is detected. ELISA results showed that the concentration of IL-12 in serum C171+ IBDV group was significantly higher than that of IBDV group and Blank group (p <0.0001; FIG. 9F). The concentration of IL-2 in serum C171+ IBDV group was significantly higher than IBDV group and Blank group (p <0.0001; FIG. 9G). The concentration of IL-23 in serum C171+ IBDV group is significantly higher than that of IBDV group and Blank group (p <0.0001; FIG. 9H), and interleukin-23 (Interleukin-23; IL-23), consisting of the 40kD subunits of p19 and IL-12, is secreted by activated DCs. p19 has a structure similar to the 35kD subunits of IL-6, G-CSF and IL-12 and can promote proliferation of CD45RO+ memory T cells and production of gamma interferon. The results show that the oral administration of the C171 can obviously improve the proportion of CD 8T cells in PBMCs, promote the secretion of IFN-gamma by organisms, improve the proliferation activity of T cells and facilitate the improvement of anti-infection capability of the organisms.

Example 10 safety evaluation of enterococcus faecium strain C171.

The experimental method comprises the following steps: the safety detection of chick embryo, taking 3 chick embryos of 10 days old out of the incubator at 37 ℃, irradiating the chick embryos on an egg candler, drawing the positions of the air chamber and the embryo by using a pencil, and marking the places with less chorioallantoic membrane blood vessels. Sterilizing the air chamber eggshell with iodine, deiodinating with 75% alcohol, and drilling a small hole on the mark with steel needle. Sucking and culturing 12h bacterial liquid by a 1ml syringe, penetrating into the hole by a needle, entering the allantoic cavity through the chorioallantoic membrane, and injecting 0.1ml; sealing the holes by paraffin, and incubating for 11 days in an incubator at 37 ℃; air chamber inoculation: the inoculation part is an air chamber, and 3 chick embryos of 10 days old are inoculated.

Detecting virulence factors and drug resistance genes, placing 200 mu L of C171 strain subjected to stationary culture for 6h at 37 ℃ into a 1.5ml centrifuge tube, taking an isolated colony, centrifuging for min at 12000g, and discarding the supernatant. 200ul of Insta Gene solution was added to the precipitate and incubated at 56℃for 15-30 minutes in a metal bath. Vortex for 10s, put into metal bath at 100deg.C for 10min, take out and put into ice, centrifuge for 5min at 12000g, aspirate 200 μl supernatant into new centrifuge tube for subsequent PCR detection.

50. Mu.L of PCR reaction system, 25. Mu.L of Premix Taq enzyme (Ex Taq2.0 plus), 1. Mu.L of each of the upstream and downstream primers (10. Mu. Mol/L), 1.5. Mu.L of template, and ddH ₂ O21.5. Mu.L. The PCR reaction procedure is that the mixture is pre-denatured for 5min at 95 ℃; denaturation at 95℃for 1min, annealing 45s, elongation at 72℃for 1min30s,35 cycles; extending at 72℃for 10min. The PCR products were detected by 1.0% agarose gel electrophoresis. The primers and annealing temperatures are shown in Table 4.

TABLE 4 virulence factor, drug resistance gene primer sequence and annealing temperature

Experimental results: as shown in FIG. 10, 6 10 day old chick embryos of group C171 and the blank group were successfully hatched (FIG. 10A). PCR amplification was performed using enterococcus faecium C171 DNA as a template, and virulence factor gene and drug-resistant gene primers, wherein the virulence factor gene FsrA had a bright band, and Hyl, bopD, cylB, cylA, scm, cylM, sprE, gelE, asal was not seen in the target band (FIG. 10B); no target band was seen for the drug resistance gene TetM, mefA, TEM, aac, ermB, vanA (FIG. 10C). In conclusion, enterococcus faecium C171 has better safety.

Example 11 acid and bile salt resistant probiotic properties of enterococcus faecium strain C171.

The experimental method comprises the following steps: to the fresh MRS culture solution, 1mol/L HCl was added to adjust the pH of the culture solution to 3.0,4.0,5.0. 0.4%,0.6%,0.8%,1.0%,2.0%,3.0% and 4.0% (w/v) of bovine bile salt was added to fresh MRS broth. 180 mu L of prepared MRS culture solution with different pH values or culture solution containing bile salts with different concentrations is added into each well of a sterile 96-well plate, then 20 mu L of enterococcus faecium C171 strain solution cultured for 12 hours is inoculated into each well, and the culture is carried out in a bacterial incubator at 37 ℃. The growth state of probiotics per hour was recorded at 630nm using a microplate reader.

Experimental results: enterococcus faecium C171 has the best proliferation activity when the mass fraction of bile salt is 0.2%, and gradually decreases with the increase of the mass fraction of bile salt, but still proliferates when the concentration is 2.0%. FIG. 11A shows that the probiotic strain C171 has a high bile salt tolerance; the probiotics C171 can grow in MRS culture medium with pH value of 3-6, OD ₆₃₀ The nm value decreased significantly with decreasing pH, but still grew, indicating that C171 had stronger acid resistance (fig. 11B). In summary, strain C171 is resistant to the intestinal bile salt environment and can successfully pass through the acidic environment of the gastrointestinal tract and survive in the gastrointestinal tract.

Example 12 effect of oral enterococcus faecium strain C171 on intestinal flora of SPF chickens.

The experimental method comprises the following steps: as in example 7, the intestinal contents were collected after one week of oral administration of enterococcus faecium C171 strain, at least 3 replicates of each sample were stored in a-80℃refrigerator and sent to Shanghai European company for 16S diversity analysis.

Experimental results: analyzing the results of the bacterial flora of the genus level top30 species according to the histogram analysis and the heat map of the community structure: after C171 was orally administered, the intestinal flora structure was unchanged and the abundance of species composition was altered compared to that in the control group. Among the first 30 bacteria, beneficial bacteria such as lactobacillus, citrobacter, parabacteroides, fecal bacteria, enterococcus and clostridium are more abundant and more abundant (fig. 12A and 12B); whereas burkholderia-carbanilla-pamburkholderia decreased, weissella decreased, pantoea decreased, sphingomonas decreased, romiboutsia abundance decreased. Burkholderia-Carbaroreflex subgenera-Parabarkhold, romboutsia, lysobacterium, sediminibacillus, shigella, and Brevibacterium, and Arthrobacter were transiently abundant in the control group (FIGS. 12A, 12B).

Analyzing species level top30 species according to the colony structure histogram and the heat map, and obtaining the following results: after C171 oral administration, the intestinal flora structure was unchanged, the abundance of species composition was changed, lactobacillus plantarum, weissella antrum, bacteroides simplex, enterococcus durans, bifidobacterium longum, lactobacillus johnsonii increased, while fungorum burkholderia decreased, weissella antrum decreased (fig. 13A, 13B). Pantoea agglomerans, romboustia_ilealis_g __ Romboustia, bifidobacterium pseudolongum, E.coli, E.rhamnosus, E.reuteri, fungarm Burkholderia, jejuni Prevotella, any species of the genus helicobacter, bacteroides vulgaris, risk Ralstonia, bacteroides acidophilus, any species of the genus Lysobacter, eubacterium contorted, sediminibacter sp.G Sediminutus, du Leini bacteria are more abundant (FIGS. 13A, 13B).

The analysis of the small intestine alpha diversity index dilution curve of the oral enterococcus faecium C171 strain to the intestinal flora shows that the small intestine alpha diversity becomes gentle along with dilution, and the alpha diversity is representative of the flora; the alpha diversity related box diagram of the small intestine increases the diversity of oral administration C171; small intestine β diversity index analysis, there were differences between groups (fig. 14).

Fig. 15 shows that the analysis of intestinal microbiota variant LEfSe of enterococcus faecium strain C171 for intestinal flora shows that the results show a significant increase in lactobacillus, enterococcus, etc. after C171 is orally administered.

Claims

1. Enterococcus faecium (Enterococcus faecium) strain, named C171, is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) and is addressed to the national institute of microbiology (CGMCC No. 24533) in national institute of sciences, north China, which is the Korean, with a preservation date of 2022, 3 months and 15 days.

2. Use of the enterococcus faecium strain according to claim 1 for the preparation of an anti-inflammatory and/or anti-infective formulation.

3. The use according to claim 2, wherein said anti-inflammatory and anti-infective agents are against acute inflammatory storms, including against acute inflammation and death caused by bacterial or viral infections.

4. The use according to claim 3, wherein the acute inflammation is caused by a virulent viral infection.

5. Use of the enterococcus faecium strain of claim 1 in the manufacture of a medicament for modulating intestinal flora in an animal or promoting growth in an animal.