Lactobacillus helveticus strain and application thereof

Abstract

The invention discloses a lactobacillus helveticus strain (Lactobacillus helveticus) and an application of the lactobacillus helveticus strain (Lactobacillus helveticus). The lactobacillus helveticus strain is named as C52, and the microbial preservation number of the lactobacillus helveticus strain is CGMCC (China General Microbiological Culture Collection Center) No. 30336. Researches show that the C52 strain has unique anti-inflammatory and anti-infection dual characteristics and also has the characteristics of acid resistance, cholate resistance and the like, and acute or chronic intestinal inflammation reaction caused by diarrhetic virus infection and diarrhea or death caused by the acute or chronic intestinal inflammation reaction can be inhibited only by oral administration of the C52 strain, so that the death rate is reduced. The novel lactobacillus helveticus strain C52 disclosed by the invention can be used as a probiotic strain to be added into feed and medicines, so that the anti-inflammatory and anti-infection capabilities of animals, especially the intestinal inflammation resistance and the intestinal infection resistance, are improved. The lactobacillus helveticus C52 strain with unique anti-inflammatory and anti-infection dual characteristics is expected to be used as an auxiliary treatment preparation for intestinal inflammation and infection, and particularly has a better protection effect when resisting and reducing intestinal acute inflammation, diarrhea and death caused by bacterial or virus infection.

Description

Technical Field

[0001] The present invention relates to a new strain of Lactobacillus helveticus and also to the use of the new strain of Lactobacillus helveticus in preparing anti-inflammatory, especially anti-intestinal inflammation, anti-infection preparations or immunomodulatory preparations. The present invention belongs to the field of biotechnology. Background Art

[0002] Lactobacillus helveticus ( Lactobacillus helveticus ) is one of the important members of the genus Lactobacillus. It belongs to the species of Lactobacillus helveticus. It is a Gram-positive, long rod-shaped, flagella-free, non-spore-forming, rod-shaped bacterium. The colonies are round, milky white, with neat edges. It is chemoheterotrophic, facultatively anaerobic, and does not liquefy gelatin. Lactobacillus helveticus is an obligate homotypic. On MRS agar plates, the colony diameter is 2-3mm or less. When cultured in a medium containing Tween-80 under anaerobic conditions (containing 5% CO2), the colonies are larger and smoother. Lactobacillus helveticus has negative amino acid decarboxylase activity and will not produce biogenic amines that harm the human body; it does not produce gas, mucus, H2S, or H2O2 during fermentation, and has weak nitrate reduction ability; it can ferment carbohydrates including fructose, galactose, glucose, lactose, maltose, mannose, and trehalose, but does not ferment arabinose, cellobiose, esculin, mannitol, melibiose, melezitose, raffinose, rhamnose, sucrose, ribose, xylose, sorbitol, and gluconate, and the fermentation product is DL-lactic acid. It is mainly used to make food yogurt drinks, milk wine, feed additives, etc.

[0003] Lactobacillus helveticus can ferment under anaerobic conditions, mainly producing lactic acid. It produces a variety of protein digestive enzymes, has a strong ability to decompose proteins, and releases biologically active peptides; the optimal growth temperature of Lactobacillus helveticus is 37°C, but it can also grow at lower temperatures. It has a strong adaptability to acidic environments, and artificial gastric juice with a pH value of 3.0 to 3.5 has little effect on Lactobacillus helveticus, and there is no significant difference compared with pH = 7.0 (P>0.05); it also has good bile salt tolerance, and still has 10% bile salt concentration at a bile salt concentration of 5 g / L. 6 The survival rate of CFU / mL indicates that it has good tolerance to bile salts; it also has good salt tolerance.

[0004] Studies have shown that Lactobacillus helveticus can alleviate liver damage. Some scholars have reported that Lactobacillus helveticus R0052 can regulate the intestinal microbiome and metabolome of D-galactosamine-treated rats to alleviate liver damage [Wang Q, Lv L, Jiang H, et al. Lactobacillus helveticus R0052 alleviates liver injury by modulatinggut microbiome and metabolome in D-galactosamine-treated rats. Appl MicrobiolBiotechnol. 2019;103(23-24):9673-9686. doi:10.1007 / s00253-019-10211-8]; and it promotes digestive health, lowers intestinal pH by producing lactic acid, inhibits the growth of harmful bacteria, and maintains intestinal microecological balance; Lactobacillus helveticus also has immunomodulatory functions. Studies have shown that Lactobacillus helveticus can enhance the host's immune function, increase antibody production, and improve the host's resistance to infection. Lactobacillus helveticus also has anti-inflammatory effects. For example, Lactobacillus helveticus R0052 can reduce intestinal inflammation and has potential therapeutic effects on inflammatory bowel diseases such as ulcerative colitis. In recent years, it has also been reported that it can treat periodontitis caused by actinomycetes [Jia R, Shi R, Guan D, Wu Y, Qian W. Lactobacillushelveticus Prevents Periodontitis Induced by Aggregatibacteractinomycetemcomitans in Rats by Regulating β-Defensins [retracted in: ComputMath Methods Med. 2023 Sep 27;2023:9816032. doi: 10.1155 / 2023 / 9816032]; Lactobacillus helveticus also has antibacterial activity. By producing antibacterial substances such as organic acids and hydrogen peroxide, Lactobacillus helveticus R0052 has an inhibitory effect on a variety of pathogenic bacteria.

[0005] In 1996, Takano's research found that the content of peptides in its fermented milk products was high, so it had the potential to produce bioactive peptides. Studies have confirmed that Lactobacillus helveticus hydrolyzed milk protein can produce angiotensin converting enzyme inhibitory peptide (ACEIP), which is the lactic acid bacteria with the most reported blood pressure lowering studies. Therefore, Lactobacillus helveticus fermented milk has anti-hypertensive activity. Further studies have shown that the peptides isoleucine-proline-proline and isovaline-proline-proline produced by Lactobacillus helveticus hydrolyzed milk have the ability to inhibit angiotensin converting enzyme activity [Sadat-Mekmene L, Genay M, Atlan D, et al. Original features of cell-envelope proteinases of Lactobacillus helveticus. A review [J]. International Journal of Food Microbiology, 2011, 146 (1): 1-13.]. To date, these two peptides are the most widely studied antihypertensive peptides and have been shown to be effective in both humans and rats [GriffithsMW TellezAM. Lactobacillus herveticus: the proteolytic system [J]. Frontiers in Microbiology, 2013, 4: 1-9].

[0006] Lactobacillus helveticus fermented milk has immunomodulatory functions such as anti-inflammatory and anti-bacterial infection. Generally speaking, these immunomodulatory functions are believed to come from the strain itself [Jia R, Shi R, Guan D, Wu Y, Qian W.Lactobacillus helveticus Prevents Periodontitis Induced by Aggregatibacteractinomycetemcomitans in Rats by Regulating β-Defensins [retracted in: ComputMath Methods Med. 2023 Sep 27;2023:9816032. doi: 10.1155 / 2023 / 9816032.]. Lactobacillus helveticus fermented milk can upregulate IL-6, thereby protecting cells from Salmonella infection. Further studies have shown that peptide fractions from α-lactalbumin and β-lactalbumin play a major protective role. These peptide fractions are non-toxic and their protective effect is dose-dependent. The mechanism may be to stimulate the body's immune regulation and produce pro-inflammatory factor TNF-α [Chen Y, Liu W, Xue J, et al. Angiotensin-converting enzyme inhibitory activity of Lactobacillus helveticus strains from traditional fermented dairy foods and antihypertensive effect offermented milk of strain H9 [J]. Journal of Dairy Science, 2014, 97(11): 6680-6692.].

[0007] Studies have shown that Lactobacillus helveticus also has a psychotropic function. Probiotic preparations of Lactobacillus helveticus and Bifidobacterium can relieve anxiety in rats and psychological stress in human volunteers. Further studies have shown that the host's diet type and whether there is inflammation may significantly change the regulation of Lactobacillus helveticus on anxiety behavior in rats [7, Masafumi M, Naoyuki Y, Toshiaki T, et al. Dentication of an Antihypertensive Peptide from Casein Hydrolysate Produced by a Proteinase from Lactobacillus helveticus CP790 [J]. Journal of Dairy Science, 1996, 79 (8): 1316-1321.].

[0008] Studies have shown that after mice were gavage-fed with Lactobacillus helveticus, the colonization of related microorganisms in the intestine showed a certain regulation process [Tellez A, Corredig M, Turner PV, et al. A peptidic fraction from milk fermented with Lactobacillus helveticus protects mice against Salmonellanfection [J]. International Dairy Journal, 2011, 21(9): 607-614. Ohland CL, Kish L, Bell H, et al. Effects of Lactobacillus helveticus on murine behavior aredependent on diet and genotype and correlate with iterations in the gut microbiome [J].Psychoneuroendocrinology, 2013, 38(9):1738-1747.], so after 9 days of the experiment, the number of lactic acid bacteria and bifidobacteria increased significantly, and the number of conditional pathogens such as Enterobacteriaceae, Enterococci and Escherichia coli was significantly different from that of the control group. This strongly proves the regulatory effect of probiotics on the intestinal microbial flora of mice, increasing the beneficial microbial flora in the intestine and reducing pathogenic microorganisms.From the appearance observation, the mice in the Lactobacillus helveticus gavage group were large and had normal defecation. These results are consistent with the relevant research results of Bcrnet MF et al. [KANKAANPAA P, SALMINEN SJ, ISOLAURI E, et al. The influence of polyunsaturated fatty acids on probiotic growth and adhesion [J]. FEMS Microbiol Letter, 2001, 194 (2): 149-53. KANKAANPAA P, YANG B, KALLIO H, et al. Effects of polyunsaturated fatty acids in growth medium on lipid composition and on physi-cochemical surface properties of Lactobacilli [J]. Appl Env Microbiol, 2004, 70 (1): 129-136. BERNETM F, BRASSART D, NEESER JR, et al. Lactobacillus acidophilus LA 1 binds to cultured human intestinal cell lines and inhibits cell attachment and cell invasion by enterovirulent bacteria

J

[0009] Studies have also confirmed that Lactobacillus helveticus has a significant regulatory effect on the intestinal flora of mice and plays a significant role in maintaining the balance of animal intestinal microbial flora and the intestinal environment [BERNET MF, BRASSART D, NEESER JR, et al. Adhesion of human bifidobacterial strains to cultured human intestinal epithelial cells and inhibition of enteropathogen-cell interactions [J]. Appl Env Microbiol, 1993, 59 (12): 4121-4128.]; Lactobacillus helveticus hydrolyzes casein to produce antihypertensive peptides, which is its unique fermentation characteristic. Regulating the production of antihypertensive peptides during the fermentation process is a hot topic of research.

[0010] Regulatory recognition of Lactobacillus helveticus: In the food and drug regulations of many countries, Lactobacillus helveticus R0052 is considered safe (GRAS, Generally Recognized As Safe). Lactobacillus helveticus R0052 is isolated from dairy products, Bifidobacterium infantis R0033 is isolated from the intestines of infants, and Bifidobacterium bifidum R0071 is isolated from the intestines of adults. The properties of the three strains of powder are all beige granular powder. Lactobacillus helveticus R0052, Bifidobacterium infantis R0033 and Bifidobacterium bifidum R0071 have been certified as generally recognized as safe (GRAS) substances by the US Food and Drug Administration. Products containing these three strains have been approved for infants and young children in Canada, Poland and Australia. A number of clinical studies on infants and young children conducted at home and abroad have shown that the three strains have good food safety and good tolerance. The Food Safety Law and the New Food Ingredient Safety Review Management Measures stipulate that the review agency shall organize experts to review and approve the safety assessment materials of Lactobacillus helveticus R0052, Bifidobacterium infantis R0033 and Bifidobacterium bifidum R0071 in accordance with legal procedures. The production and use of new food ingredients shall comply with the content of the announcement and relevant food safety regulations. Summary of the invention

[0011] The present invention aims to provide a Lactobacillus helveticus strain and use of the Lactobacillus helveticus strain in anti-inflammatory and anti-infection.

[0012] In order to achieve the above object, the present invention adopts the following technical means: The strain of the present invention is isolated from the ileum mucosa of wild healthy wild boars in Fuyuan primitive forest, Jiamusi City, Heilongjiang Province, China, and is isolated and cultured by MRS inoculation, separation and purification. It is identified as Lactobacillus helveticus ( Lactobacillus helveticus ), named C52, and classified as Lactobacillus helveticus ( Lactobacillus helveticus ), deposited in the General Microbiology Center of China Microbiological Culture Collection Committee, located at the Institute of Microbiology, Chinese Academy of Sciences, No. 1, Beichen West Road, Chaoyang District, Beijing, with a microbiological deposit number of CGMCC No. 30336 and a deposit date of April 15, 2024. The microbiological characteristics of this strain are: ① Gram staining is typical positive, the bacteria are medium-sized and regular in shape; aerobic or facultative anaerobic bacteria, growing well on solid and liquid MRS medium; ② It has acid resistance and can grow well on MRS with pH 4.0; ③ It has bile resistance and can survive and grow in culture medium containing 50% chicken bile MRS; ④ It has unique immune adjuvant activity, anti-inflammatory and anti-infective properties; ⑤ The optimal culture temperature of the bacteria is 37°C, and the colonies on MRS medium are milky white, with neat edges, smooth and moist surface, bulges, opaque medium-sized colonies, and a sour and fragrant taste.

[0013] Therefore, further, the present invention also proposes the use of the new strain of Lactobacillus helveticus in the preparation of anti-inflammatory preparations, anti-infective preparations, immunomodulatory preparations, and drugs or feeds for promoting animal growth.

[0014] Among them, preferably, the anti-inflammatory and anti-infective effects refer to anti-intestinal inflammation and anti-intestinal infection.

[0015] Among them, preferably, the anti-inflammatory and anti-infection refers to anti-acute intestinal inflammation and inflammatory factor storm, including resisting and reducing acute intestinal inflammation, diarrhea and death caused by bacterial or viral infection.

[0016] Compared with the prior art, the present invention has the following beneficial effects: The present invention has isolated a strain of Lactobacillus helveticus, named C52. Studies have shown that the strain has unique dual characteristics of anti-inflammatory and anti-infection, and also has characteristics such as acid resistance and bile salt resistance. Oral administration of the C52 strain alone can inhibit acute or chronic intestinal inflammatory reactions caused by diarrheal viral infections and the resulting diarrhea or death, thereby reducing mortality. The new strain of Lactobacillus helveticus C52 disclosed in the present invention can be used as a probiotic strain to be added to feed and medicines to improve the anti-inflammatory and anti-infection capabilities of animals, especially anti-intestinal inflammation and anti-intestinal infection. The Lactobacillus helveticus strain C52 with unique dual characteristics of anti-inflammatory and anti-infection is expected to be used as an auxiliary therapeutic preparation for intestinal inflammation and infection, especially in resisting and reducing acute intestinal inflammation, diarrhea and death caused by bacterial or viral infections, and the protective effect is better. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The growth of Lactobacillus helveticus strain C52 in aerobic culture on solid medium; Figure 2 The growth of Lactobacillus helveticus strain C52 in anaerobic culture on solid medium; Figure 3 The results of the test on the activation of the immune signaling pathway of the J774-Dual™ reporter cell line by the Lactobacillus helveticus strain C52. Among them, A is the interferon regulatory factor (IRF) signaling pathway activated by Lactobacillus helveticus strain C52 in macrophage J774-Dual™ cells; B is the NF-κB signaling pathway activated by Lactobacillus helveticus strain C52 in macrophage J774-Dual™ cells; Figure 4 The results of the anti-inflammatory and immune homeostasis tests after the interaction between Lactobacillus helveticus strain C52 and the macrophage HD11 inflammatory model; Among them, A is the relative transcription level of IL-1β mRNA; B is the relative transcription level of IL-10 mRNA; Figure 5 The results of the anti-inflammatory effect test after the interaction between Lactobacillus helveticus strain C52 and the intestinal mucosal epithelial cell HT29 inflammation model; Figure 6 The results are the test results of the antiviral proliferation properties of Lactobacillus helveticus strain C52 in vitro; Figure 7 The results of the interaction between Lactobacillus helveticus strain C52 and primary chicken bone marrow macrophages were tested to determine the effect of Lactobacillus helveticus strain C52 on the proliferation activity of primary chicken bone marrow macrophages and to determine its safety characteristics; Figure 8 These are the safety test results of directly injecting live Lactobacillus helveticus strain C52 into 10-day-old chicken embryos on their growth and development. DETAILED DESCRIPTION

[0018] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, the embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention. Example 1 Isolation and identification of Lactobacillus helveticus strain C52

[0019] 1. Isolation and identification of strains The strain was isolated from the ileal mucosa of healthy wild boars in Fuyuan primitive forest, Jiamusi City, Heilongjiang Province, China. It was isolated and cultured by MRS inoculation, separation and purification. It was identified as Lactobacillus helveticus ( Lactobacillus helveticus ), named C52, deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration, located at the Institute of Microbiology, Chinese Academy of Sciences, No. 1, Beichen West Road, Chaoyang District, Beijing, and its microbiological deposit number is CGMCC No.30336, and the deposit date is April 15, 2024. Its microbiological characteristics are: ① Gram staining is typical positive, the bacteria are medium-sized and regular in shape; aerobic or facultative anaerobic bacteria, growing well on solid and liquid MRS medium; ② It has acid resistance and can grow well on MRS with pH 4.0; ③ It has bile resistance and can survive and grow in culture medium containing 50% chicken bile MRS; ④ It has unique immune adjuvant activity, anti-inflammatory and anti-infective properties; ⑤ The optimal temperature for the culture of the bacteria is 37°C, and the colonies on the MRS medium are milky white, with neat edges, smooth and moist surfaces, bulges, opaque medium-sized colonies, and a sour and fragrant taste.

[0020] Colony characteristics of strains Figure 1 As shown in the left picture.

[0021] 2. Acid resistance test of Lactobacillus helveticus strain C52: The isolated Lactobacillus helveticus strain C52 was inoculated into MRS liquid culture medium with pH values ​​of 3.0, 4.0, 5.0, and 6.5 at an inoculum volume of 10% of the volume of the MRS liquid culture medium. Three parallel controls were set for each acidity gradient and cultured at 37°C. The bacterial liquid samples were taken at 30 min, 60 min, 90 min, and 120 min of culture to determine the OD 600 The experiment showed that Lactobacillus helveticus strain C52 had strong acid resistance and could grow on MRS at pH 4.0.

[0022] 3. Chicken bile resistance test of Lactobacillus helveticus strain C52: The isolated Lactobacillus helveticus strain C52 was inoculated into MRS medium containing fresh chicken bile at 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, and 90% of the volume of MRS liquid culture medium at an inoculum volume of 10%; three parallel tests were set for each concentration, and the culture was statically cultured at 37°C. The bacterial liquid samples were taken after 30 min, 60 min, 90 min, and 120 min of culture to determine the OD 600 nm value. The results showed that Lactobacillus helveticus strain C52 could grow well in the medium containing 30% chicken bile and had a strong resistance to chicken bile.

[0023] 4. Aerobic and anaerobic culture characteristics of Lactobacillus helveticus strain C52 on solid culture medium Experimental method: Lactobacillus helveticus strain C52 was streaked on X-gal-MRS, ordinary MRS and MC solid culture media, and observed after aerobic and anaerobic culture for 48 hours.

[0024] Experimental results: Figure 1 , Lactobacillus helveticus strain C52 grew well in X-gal-MRS, ordinary MRS and MC solid culture media for 48 hours. The colony morphology was neatly edged, smooth and moist, slightly raised, opaque, medium to large colonies, milky white, and had a sour and fragrant taste. When aerobically cultured on X-gal-MRS, the colony morphology was similar to that on MRS culture medium, and the color was blue (indicating that Lactobacillus helveticus strain C52 can produce β-glucosidase, decompose X-gal substrate, and make the colony light blue); after aerobic culture of Lactobacillus helveticus strain C52 on MC culture medium, no obvious transparent ring appeared around the colony.

[0025] like Figure 2 , Lactobacillus helveticus C52 strain grew well in X-gal-MRS, ordinary MRS and MC solid culture media after anaerobic culture for 48 hours. The colony morphology was neatly edged, smooth and moist surface, slightly raised, opaque, medium to large colonies, milky white, and had a sour and fragrant taste. When cultured aerobically on X-gal-MRS, the colony morphology was similar to that on MRS medium; after aerobic culture of Lactobacillus helveticus C52 strain on MC medium, no obvious transparent ring appeared around the colony. Example 2 Detection of interaction between Lactobacillus helveticus strain C52 and J774-Dual™ reporter cell line to activate immune signaling pathways

[0026] Experimental method: J774-Dual™ cells are derived from the mouse macrophage-like cell line J774.1, which is stably integrated with two inducible reporter genes. The activation of the NF-κB pathway is reflected by detecting the activity of secretory alkaline phosphatase (SEAP) in the supernatant; the activation of the interferon regulatory factor (IRF) pathway is reflected by detecting the activity of Lucia luciferase in the supernatant. J774-Dual™ was passaged three times in growth medium (DMEM, 10% heat-inactivated fetal bovine serum (FBS), 100 mg / mL Normocin, 1% penicillin-streptomycin). After the cells were stable, they were passaged three times in selection medium (growth medium, 5 μg / mL Blasticidin, 100 μg / L Zeocin). The cells were digested with 0.25% trypsin-EDTA, centrifuged horizontally at 500 g for 10 min at room temperature, and the supernatant was discarded. The cell concentration of J774-Dual™ was adjusted to 2.8×10 5 / mL, inoculated in a 96-well plate, and added 50 MOI of Lactobacillus helveticus strain C52 suspension, inactivated Lactobacillus helveticus strain C52 suspension and 50 μL of Lactobacillus helveticus strain C52 lysate to the corresponding cell wells. The cells without any treatment were used as blank control, and the cells added with working concentrations of 1 μg / mL LPS, 10 ng / mL Pam3CSK, and 1 μg / mL 2',3'-cGAMP were used as positive controls. After stimulation at 37 ℃ and 5% CO2 for 24 hours, the supernatant was collected.

[0027] 170 μL QUANTI-Blue™ was added to each well of a 96-well plate, followed by 30 μL of the cell supernatant of J774-Dual™ stimulated by Lactobacillus helveticus strain C52, and the plate was placed at 37°C and 5% CO2 for 4 h. The absorbance at OD650 nm was measured using a microplate reader. The greater the absorbance, the stronger the activation ability of the NF-κB signaling pathway. The specific steps for detecting the interferon IRF signaling pathway are as follows: 20 μL of the cell supernatant of J774-Dual™ stimulated by Lactobacillus helveticus strain C52 was added to a 96-well white (opaque) or black plate, and the expression of Lucia luciferase was detected using a microplate chemiluminescence detector (LB 960). The greater the RLU value detected, the stronger the activation ability of the IRF signaling pathway.

[0028] Experimental results: Lactobacillus helveticus strain C52 can effectively activate the interferon regulatory factor (IRF) signaling pathway in macrophage J774-Dual™ cells, and the difference is extremely significant compared with the blank control group (P<0.01; Figure 3A); Lactobacillus helveticus strain C52 can effectively activate the NF-κB signaling pathway in macrophage J774-Dual™ cells, and the difference is significant compared with the blank control group (P<0.05; Figure 3 B). The above results show that Lactobacillus helveticus strain C52 has strong anti-inflammatory and anti-infective effects. Example 3 Detection of the strong anti-inflammatory and anti-infective properties of Lactobacillus helveticus strain C52 interacting with the macrophage inflammation model

[0029] Experimental method: Chicken macrophage HD11s (1 × 10 6 cells / mL) were inoculated in a 12-well plate. After the cells adhered to the wall, 50MOI of Lactobacillus helveticus strain C52 was added and incubated at 37 ˚C 5 % CO2 for 24 h. Lipopolysaccharide (LPS, L) and Nigericin (Nigericin, N) were used to induce acute inflammatory response in the cells to establish an acute inflammatory model, that is, LPS with a working concentration of 1μg / mL was added to each well and incubated at 37 ˚C 5 % CO2 for 3 h. Nigericin with a working concentration of 1μM was then added for 1 h. Cell pellets were collected and quantified by SYBY Green Ⅰ fluorescence quantitative PCR. The primers in Table 1 were used to analyze the transcription levels of IL-1β and IL-10 mRNA in the cells. Cells treated with LPS and Nigericin were used as positive controls (L+N), and cells without any treatment were used as blank controls.

[0030]

[0031] Experimental results: Figure 4 The results show the anti-inflammatory and anti-infective effects of Lactobacillus helveticus strain C52 on macrophages. Lactobacillus helveticus strain C52 can significantly downregulate the inflammatory cytokine IL-1β produced by HD11 in the acute inflammatory model, and the difference is extremely significant compared with the L+N control group (P<0.0001; Figure 4 A). Lactobacillus helveticus C52 can effectively stimulate HD11 to produce the anti-inflammatory cytokine IL-10, which is significantly different from the L+N control group (P<0.0001; Figure 4 B) The results showed that Lactobacillus helveticus strain C52 has strong anti-inflammatory effects, as well as anti-infection and immune homeostasis effects. Example 4 Immunomodulatory and anti-inflammatory effects of Lactobacillus helveticus strain C52 on intestinal epithelial cells

[0032] Experimental method: Intestinal epithelial cell line HT29 (1 × 10 6 cells / mL) were inoculated in a 12-well plate. After the cells adhered to the wall, 50 MOI of Lactobacillus helveticus strain C52 was added and incubated at 37 ˚C 5 % CO2 for 24 h. LPS and Nigericin were used to induce acute inflammatory response in the cells to establish an acute inflammation model, that is, LPS with a working concentration of 1 μg / mL was added to each well and incubated at 37 ˚C 5 % CO2 for 3 h, and then Nigericin with a working concentration of 1 μM was added for 1 h. The cell pellet was collected and quantified by SYBY Green Ⅰ fluorescence quantitative PCR. The primers were used to analyze the transcription levels of IL-1β and IL-10 mRNA in the cells. Cells treated with LPS and Nigericin were used as positive controls, and cells without any treatment were used as blank controls.

[0033] Experimental results: Figure 5 The results are shown in Figure 2, which show the anti-inflammatory effect of Lactobacillus helveticus strain C52 on intestinal epithelial cells. Lactobacillus helveticus strain C52 can significantly downregulate the inflammatory cytokine IL-1β produced by HT29 in the acute inflammation model, and the difference is extremely significant compared with the L+N control group (P<0.0001; Figure 5 ). The Lactobacillus helveticus strain C52 stimulated HT29 to produce the anti-inflammatory cytokine IL-1β, which was not significantly different from the blank control group (P > 0.05; Figure 5 ). The results showed that Lactobacillus helveticus strain C52 had a strong anti-inflammatory effect. Example 5 Detection of the antiviral proliferation properties of Lactobacillus helveticus strain C52 in vitro

[0034] Experimental method: Chicken lymphoma DT40 cells (1×10 6 / mL) were inoculated in a 12-well plate with serum-free RPM 1640 and cultured at 37 ℃ for 30 min. After the cells adhered, they were infected with 1MOI of IBDV (SHG19), and Lactobacillus helveticus strain C52 with MOIs of 25, 50, and 100 were added at the same time. After 24 h of the three-in-one reaction, 200μL of the cell suspension was collected and the IBDV VP5 copy number was quantitatively determined by absolute analysis using Premix Ex Taq (primers in Table 2), the cell pellets were used to detect cytokines IL-1β, IL-10, IFN-γ (primers in Table 1), and the VP2 protein was detected by Western blot.

[0035]

[0036] Experimental results: Lactobacillus helveticus strain C52 does not affect the cell activity of DT40 and is safe and non-toxic to DT40 cells. Lactobacillus helveticus strain C52 has a significant antiviral effect in vitro. Compared with the positive control, Lactobacillus helveticus strain C52 can significantly reduce the viral copy number of vvIBDV, and there is a significant dose-dependency. As the dose of Lactobacillus helveticus strain C52 increases, the inhibitory effect becomes more obvious. When the MOI of Lactobacillus helveticus strain C52 and DT40 is 100, the inhibitory effect is most significant (P<0.0001). Western blotting results show that Figure 6 As shown in the figure, Lactobacillus helveticus strain C52 can significantly reduce the protein expression of vvIBDVVP2, and there is a clear dose dependence. Grayscale analysis shows that as the dose of Lactobacillus helveticus strain C52 increases, the expression of VP2 protein gradually decreases. When the MOI of Lactobacillus helveticus strain C52 is 100, the expression of VP2 is the lowest, which is 0.43 times that of the positive control ( Figure 6 ). The above results show that Lactobacillus helveticus strain C52 can significantly inhibit viral replication and reduce viral load in vitro, and has obvious antiviral and anti-infection effects. Example 6. Safety characteristics of Lactobacillus helveticus strain C52 on the proliferation activity of primary chicken macrophages

[0037] Experimental methods: First, isolate primary bone marrow macrophages from chickens. The isolation steps are as follows: ① Euthanize 3-week-old SPF chickens and take chicken femurs on a sterile operating table; ② Cut both ends of the femur and flush out the bone marrow with a syringe filled with PBS; ③ Collect the bone marrow into a 40 μM filter, grind it with a 10 mL syringe plug, and then flush the bone marrow off the filter with PBS; ④ Gently add the mixture to an equal volume of Histopaque-1083 separation solution, centrifuge at 2,000 r / min for 25 min, and adjust the acceleration to the minimum; ⑤ Aspirate the middle layer cells and wash them twice with PBS; ⑥ Count the cells and inoculate them into a 12-well plate. After 6 hours, change the medium to wash away the non-adherent or semi-adherent cells; ⑦ Culture for 6 days (change the culture medium every 2 days) and then conduct subsequent experiments.

[0038] Second, use Cell Counting Kit-8 to verify the effect of Lactobacillus helveticus strain C52 on cell proliferation activity and determine the toxicity of Lactobacillus helveticus strain C52 to cells. Chicken primary bone marrow macrophages were cultured at 1 × 10 6100 µL of cells were inoculated into a 96-well plate at a concentration of cells / mL, and a 50 MOI suspension of Lactobacillus helveticus strain C52 was added. After incubation for 24 h, 10 µL of cck-8 solution was added. After incubation at 37°C for 2 h, the absorbance at 450 nm was measured. Cells treated with Con A (concanavalin A) + PMA (propylene glycol methyl ether acetate) or LPS were used as positive controls, and cells without any treatment were used as blank controls.

[0039] Experimental results: Figure 7 As shown, Lactobacillus helveticus strain C52 has no toxicity to primary chicken bone marrow macrophages, and C52 can stimulate the proliferation of primary chicken bone marrow cells. The difference between Lactobacillus helveticus strain C52 and the blank control is extremely significant (P<0.01). The results show that Lactobacillus helveticus strain C52 has no toxicity to primary chicken bone marrow macrophages, and Lactobacillus helveticus has safety characteristics. Example 7 Testing of the safety characteristics of chicken embryos injected with Lactobacillus helveticus strain C52

[0040] Experimental method: Chicken embryo safety test, 3 10-day-old chicken embryos were taken out from the 37℃ incubator, the embryos were observed on the egg candling device, the air chamber and the embryo position were drawn with a pencil, and the area with fewer blood vessels on the chorioallantoic membrane was marked. The air chamber eggshell was disinfected with iodine, and then deiodinated with 75% alcohol, and a small hole was drilled at the mark with a steel needle. A 1ml syringe was used to absorb the bacterial liquid of the helveticus strain C52 cultured for 12 hours, the needle was inserted into the hole, and 0.1ml was injected into the allantoic cavity through the chorioallantoic membrane; the amount of Lactobacillus helveticus strain C52 injected was 1×10 9 CFU per chicken embryo, seal the holes with paraffin and incubate in a 37℃ incubator for 11 days; air chamber inoculation: the operation is the same as above, the inoculation site is the air chamber, and 5 10-day-old chicken embryos are inoculated each time, of which 5 10-day-old chicken embryos are not inoculated and kept as controls, and the development status of the chicken embryos is observed daily until hatching.

[0041] Experimental results: Figure 8 As shown, a total of 5 10-day-old chicken embryos in the Lactobacillus helveticus strain C52 group and the blank control group were successfully hatched ( Figure 8 The results showed that the injection of Lactobacillus helveticus strain C52 had no effect on the growth and development of chicken embryos, and the results showed that Lactobacillus helveticus strain C52 was safe for chicken embryos.

Claims

1. A strain of Lactobacillus helveticus ( Lactobacillus helveticus ) strain, named C52, is deposited in the General Microbiology Center of China Culture Collection Administration, located at the Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing. Its microbial deposit number is CGMCC No.30336, and the deposit date is April 15, 2024.

2. Use of the Lactobacillus helveticus strain according to claim 1 in the preparation of anti-inflammatory preparations, anti-infective preparations, immunomodulatory preparations, and medicines or feeds for promoting animal growth.

3. The use according to claim 2, characterized in that The anti-inflammatory and anti-infective effects refer to anti-intestinal inflammation and anti-intestinal infection.

4. The use according to claim 3, characterized in that The anti-inflammatory and anti-infection effects refer to resistance to acute intestinal inflammation and inflammatory factor storm, including resistance to and reduction of acute intestinal inflammation, diarrhea and death caused by bacterial or viral infection.

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