A Lactobacillus helveticus strain and its uses

Abstract

The present invention discloses a strain of Lactobacillus helveticus ( Lactobacillus helveticus ), and its uses. The Lactobacillus helveticus strain, named C52, has a microorganism deposit number of CGMCC No. 30336. Research shows that the C52 strain has unique dual anti-inflammatory and anti-infection properties, and also has properties such as acid tolerance and bile salt tolerance. Oral administration of only the C52 strain can inhibit acute or chronic intestinal inflammatory responses caused by diarrheal virus infections and the resulting diarrhea or death, thereby reducing the mortality rate. The novel Lactobacillus helveticus strain C52 disclosed in the present invention can be used as a probiotic strain and added to feeds and drugs 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 anti-inflammatory and anti-infection properties is expected to be used as an adjuvant therapeutic agent for intestinal inflammation and infection, especially when resisting and reducing acute intestinal inflammation, diarrhea, and death caused by bacterial or viral infections, with better protective effects.

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 the preparation of anti-inflammatory agents, especially in the preparation of anti-intestinal inflammation agents, anti-infection agents or immunomodulatory agents. 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 Lactobacillus helveticus, and is a Gram-positive, long rod-shaped, non-flagellated, non-spore-forming, rod-shaped bacterium. The colonies are round, milky white, with neat edges, chemoheterotrophic, facultative anaerobic, and do not liquefy gelatin. Lactobacillus helveticus is an obligate homofermentative bacterium. On MRS agar plates, the colony diameter is 2-3 mm or smaller. Under anaerobic conditions (containing 5% CO2), the colonies are larger and smoother when cultured in a medium containing Tween-80. Lactobacillus helveticus has a negative amino acid decarboxylase activity and does not produce biogenic amines that can harm the human body; it does not produce gas during fermentation, does not produce mucus, does not produce H2S, does not produce H2O2, and has a weak nitrate reduction ability; the carbohydrates it can ferment include fructose, galactose, glucose, lactose, maltose, mannose, and trehalose, and it does not ferment arabinose, cellobiose, esculin, mannitol, melibiose, melezitose, raffinose, rhamnose, sucrose, ribose, xylose, sorbitol, and gluconate. The fermentation product is DL-lactic acid. It is mainly used in the manufacture of 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-digesting enzymes, has a strong protein decomposition ability, and releases bioactive peptide substances; the optimum growth temperature of Lactobacillus helveticus is 37 °C, but it can also grow at lower temperatures. It has a strong adaptability to acidic environments. The effect of artificial gastric juice with a pH value of 3.0-3.5 on Lactobacillus helveticus is small, and there is no significant difference compared with pH = 7.0 (P>0.05); it also has good bile salt tolerance, and there is still a survival amount of 10 6 CFU / mL under the condition of a bile salt concentration of 5 g / L, indicating that it has good tolerance to bile salts; at the same time, it also has good salt tolerance.

[0004] Some studies have shown that Lactobacillus helveticus can alleviate liver injury. Some scholars have reported that Lactobacillus helveticus R0052 can modulate the gut microbiome and metabolome of D-galactosamine-treated rats to reduce liver injury [Wang Q, Lv L, Jiang H, et al. Lactobacillus helveticus R0052 alleviates liver injury by modulating gut microbiome and metabolome in D-galactosamine-treated rats. Appl Microbiol Biotechnol. 2019;103(23-24):9673-9686. doi:10.1007 / s00253-019-10211-8]; and it promotes digestive health by producing lactic acid to lower the intestinal pH value, inhibit the growth of harmful bacteria, and maintain the balance of the intestinal microecology. Lactobacillus helveticus also has immunomodulatory functions. Some studies have shown that Lactobacillus helveticus can enhance the immune function of the host, 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 Actinomyces [Jia R, Shi R, Guan D, Wu Y, Qian W. Lactobacillus helveticus Prevents Periodontitis Induced by Aggregatibacter actinomycetemcomitans in Rats by Regulating β-Defensins [retracted in: Comput Math 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 its fermented dairy products had a high polypeptide content and thus had the potential to produce bioactive peptides. Existing research has confirmed that Lactobacillus helveticus can hydrolyze milk proteins to produce angiotensin-converting enzyme inhibitory peptides (ACEIP), which is the lactic acid bacterium reported the most in blood pressure reduction research. Therefore, Lactobacillus helveticus fermented milk has antihypertensive activity. Further research has shown that the polypeptides isoleucine-proline-proline and isovaline-proline-proline produced by Lactobacillus helveticus hydrolyzing cow's milk have the activity of inhibiting angiotensin-converting enzyme [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.]. So far, these two polypeptides are the most widely studied antihypertensive polypeptides and have been proven to be effective in both humans and rats [Griffiths M W, Tellez A M. Lactobacillus helveticus: the proteolytic system [J]. Frontiers in Microbiology, 2013, 4: 1-9].

[0006] Swiss Lactobacillus fermented milk has immunomodulatory functions such as anti-inflammatory and anti-bacterial infection. Generally, these immunomodulatory functions are considered to come from the strain itself [Jia R, Shi R, Guan D, Wu Y, Qian W. Lactobacillus helveticus Prevents Periodontitis Induced by Aggregatibacter actinomycetemcomitans in Rats by Regulating β-Defensins [retracted in: Comput Math Methods Med. 2023 Sep 27;2023:9816032. doi: 10.1155 / 2023 / 9816032.]]. Swiss Lactobacillus fermented milk can upregulate IL-6, thereby protecting cells from Salmonella infection. Further research shows that peptide fractions from α-lactalbumin and β-lactoglobulin 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 the 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 of fermented milk of strain H9 [J]. Journal of Dairy Science, 2014, 97(11):6680-6692.].

[0007] Studies have shown that Lactobacillus helveticus also has mental regulatory functions. 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 the presence or absence of inflammation may significantly alter the regulation of Lactobacillus helveticus on the anxiety behavior of rats [7, Masafumi M, Naoyuki Y, Toshiaki T, et al. Identification 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 external observation, the mice in the Lactobacillus helveticus gavage group were large in size and had normal defecation. These results were consistent with the relevant research results of Bcrnet M F, etc. [KANKAANPAA P, SALMINEN S J, ISOLAURI E, et al. The influence of polyunsaturated fatty acids on probiotic growth and adhesion

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[0009] There are also studies that have confirmed that Lactobacillus helveticus has an obvious regulatory effect on the intestinal flora of mice and plays a significant role in maintaining the balance of the animal intestinal microflora and the intestinal environment [BERNET M F, BRASSART D, NEESER JR, et al. Adhesion of human bifidobacterial strains to cultured human intestinal epithelial cells and inhibition of enteropathogen-cell interactions

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[0010] Regulatory approval of Lactobacillus helveticus: In the food and drug administration regulations of many countries, Lactobacillus helveticus R0052 is considered safe (GRAS, Generally Recognized As Safe). Lactobacillus helveticus R0052 was isolated from dairy products, Bifidobacterium infantis R0033 was isolated from the intestines of infants, and Bifidobacterium bifidum R0071 was isolated from the intestines of adults. The properties of the three strain powders are all beige granular powders. Lactobacillus helveticus R0052, Bifidobacterium infantis R0033, and Bifidobacterium bifidum R0071 have been certified by the US Food and Drug Administration as generally recognized as safe (GRAS) substances. Products containing these three strains have been approved for use in infants and young children in Canada, Poland, and Australia. A number of clinical studies on infants and young children carried out at home and abroad have proved that these three strains have good food safety and good tolerance. The Food Safety Law and the Administrative Measures for the Safety Review of New Food Raw Materials stipulate that the review agency organizes experts to review and pass the safety assessment materials of Lactobacillus helveticus R0052, Bifidobacterium infantis R0033, and Bifidobacterium bifidum R0071 in accordance with the legal procedures. The production and use of new food raw materials shall comply with the content of the announcement and the requirements of food safety-related regulations. Summary of the Invention

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

[0012] To achieve the above object, the present invention adopts the following technical means:

[0013] The strain of the present invention was isolated from the ileal mucosa of a wild and healthy wild boar in the virgin forest of Fuyuan, Jiamusi City, Heilongjiang Province, China. It was obtained after MRS inoculation culture, isolation and purification. After identification, it belongs to Lactobacillus helveticus ( Lactobacillus helveticus ), named C52, and classified as Lactobacillus helveticus ( Lactobacillus helveticus ). It is preserved in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms, located at the Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing. Its microbial preservation number is CGMCC No. 30336, and the preservation date is April 15, 2024. The microbiological characteristics of this strain are as follows: ①Typically Gram-positive, the cells are medium-sized and relatively regular-shaped bacilli; aerobic or facultative anaerobic bacteria, growing well on solid and liquid MRS media; ②It has acid tolerance and can grow well on MRS at pH 4.0; ③It has bile salt tolerance and can survive and grow in the culture medium of MRS containing 50% chicken bile; ④It has unique immune adjuvant activity, anti-inflammatory and anti-infective characteristics; ⑤The optimal temperature for culturing this bacterium is 37°C. The colonies on the MRS medium are milky white, with neat edges, smooth and moist surfaces, raised, opaque medium-sized colonies, and have a sour and fragrant smell.

[0014] 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.

[0015] Among them, preferably, the anti-inflammatory and anti-infective effects refer to anti-intestinal inflammation and anti-intestinal infection.

[0016] Among them, preferably, the anti-inflammatory and anti-infective effects refer to anti-intestinal acute inflammation and cytokine storm, including resistance to and reduction of intestinal acute inflammation, diarrhea, and death caused by bacterial or viral infections.

[0017] Compared with the prior art, the beneficial effects of the present invention are:

[0018] A strain of Lactobacillus helveticus was isolated in the present invention and named C52. Research shows that this strain has unique dual anti-inflammatory and anti-infection properties, and also has properties such as acid resistance and bile salt resistance. Oral administration of only the C52 strain can inhibit acute or chronic intestinal inflammatory responses caused by diarrheal virus infections and the resulting diarrhea or death, thereby reducing the mortality rate. The novel Lactobacillus helveticus strain C52 disclosed in the present invention can be used as a probiotic strain and added to feeds and drugs to improve the anti-inflammatory and anti-infection abilities of animals, especially anti-intestinal inflammation and anti-intestinal infection. The Lactobacillus helveticus strain C52 with unique dual anti-inflammatory and anti-infection properties is expected to be used as an adjuvant therapeutic agent for intestinal inflammation and infection, especially when 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

[0019] Figure 1 Growth of Lactobacillus helveticus strain C52 during aerobic culture on solid medium;

[0020] Figure 2 Growth of Lactobacillus helveticus strain C52 during anaerobic culture on solid medium;

[0021] Figure 3 Detection results of the activation effect of Lactobacillus helveticus strain C52 on the immune signaling pathway of the J774-Dual™ reporter cell line;

[0022] Among them, A shows that Lactobacillus helveticus strain C52 activates the interferon regulatory factor (IRF) signaling pathway in macrophage J774-Dual™ cells; B shows that Lactobacillus helveticus strain C52 activates the NF-κB signaling pathway in macrophage J774-Dual™ cells;

[0023] Figure 4 Detection results of the anti-inflammatory and immune homeostasis effects after the interaction between Lactobacillus helveticus strain C52 and the macrophage HD11 inflammation model;

[0024] Among them, A is the relative transcription level of IL-1β mRNA; B is the relative transcription level of IL-10 mRNA;

[0025] Figure 5 Detection results of the anti-inflammatory effect after the interaction between Lactobacillus helveticus strain C52 and the intestinal mucosal epithelial cell HT29 inflammation model;

[0026] Figure 6 Detection results of the antiviral proliferation characteristics of Lactobacillus helveticus strain C52 in vitro;

[0027] Figure 7Results of the interaction between Lactobacillus helveticus strain C52 and primary chicken bone marrow macrophages, detection of the effect of Lactobacillus helveticus strain C52 on the proliferation activity of primary chicken bone marrow macrophages, and detection of its safety characteristics.

[0028] Figure 8 Results of the safety detection of the growth and development of chicken embryos by directly injecting live Lactobacillus helveticus strain C52 into 10-day-old chicken embryos. Detailed implementation manners

[0029] The present invention will be further described below in conjunction with specific embodiments. The advantages and features of the present invention will become clearer with the description. However, the embodiments are only exemplary and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that without departing from the spirit and scope of the present invention, modifications or substitutions can be made to the details and forms of the technical solutions of the present invention, but these modifications and substitutions all fall within the protection scope of the present invention.

[0030] Example 1 Isolation and identification of Lactobacillus helveticus strain C52

[0031] 1. Isolation and identification of the strain

[0032] This strain was isolated from the ileal mucosa of a wild and healthy wild boar in the virgin forest of Fuyuan, Jiamusi City, Heilongjiang Province, China. After inoculation and culture on MRS, isolation and purification were carried out, and it was identified as Lactobacillus helveticus ( Lactobacillus helveticus ), named C52, and deposited in the China General Microbiological Culture Collection Center, 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. Its microbiological characteristics are as follows: ①Typical Gram-positive staining, medium-sized cells, regular-shaped bacilli; aerobic or facultative anaerobic bacteria, growing well on solid and liquid MRS media; ②Having acid tolerance, being able to grow well on MRS at pH 4.0; ③Having bile salt tolerance, being able to survive and grow in the culture medium of MRS containing 50% chicken bile; ④Having unique immune adjuvant activity, anti-inflammatory and anti-infective characteristics; ⑤The optimal temperature for culturing this bacterium is 37°C. The colonies on the MRS medium are milky white, with neat edges, smooth and moist surfaces, raised, opaque medium-sized colonies, and have an acid fragrance.

[0033] The colony characteristics of the strain are as shown in Figure 1 the left figure.

[0034] 2. Acid tolerance test of Lactobacillus helveticus strain C52:

[0035] The isolated Lactobacillus helveticus strain C52 was inoculated into MRS liquid media with pH values of 3.0, 4.0, 5.0, and 6.5 at an inoculum size of 10% of the volume of the MRS liquid medium. Three parallel controls were set for each acidity gradient, and the cultures were statically incubated at 37°C. Bacterial liquid samples were taken at 30 min, 60 min, 90 min, and 120 min of incubation to measure the OD 600 nm value. The experiment showed that Lactobacillus helveticus strain C52 has strong acid resistance and can grow on MRS at pH 4.0.

[0036] 3. Bile tolerance test of Lactobacillus helveticus strain C52:

[0037] The isolated Lactobacillus helveticus strain C52 was inoculated into MRS media containing 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, and 90% fresh chicken bile at an inoculum size of 10% of the volume of the MRS liquid medium. Three parallel tests were set for each concentration, and the cultures were statically incubated at 37°C. Bacterial liquid samples were taken at 30 min, 60 min, 90 min, and 120 min of incubation to measure the OD 600 nm value. The results showed that Lactobacillus helveticus strain C52 can grow well in the medium containing 30% chicken bile and has strong chicken bile resistance.

[0038] 4. Aerobic and anaerobic culture characteristics of Lactobacillus helveticus strain C52 on solid media

[0039] Experimental method: Lactobacillus helveticus strain C52 was streaked on X-gal-MRS, ordinary MRS, and MC solid media and observed after 48 h of aerobic and anaerobic culture respectively.

[0040] Experimental results: As Figure 1 , Lactobacillus helveticus strain C52 grew well after 48 h of aerobic culture on X-gal-MRS, ordinary MRS, and MC solid media. The colony morphology was medium to large, with a neat edge, smooth and moist surface, slightly raised, and opaque. The colonies were milky white and had an acid fragrance. When aerobically cultured on X-gal-MRS, the colony morphology was similar to that on MRS medium, and the color was blue (indicating that Lactobacillus helveticus strain C52 can produce β-glucosidase to decompose the X-gal substrate, making the colonies light blue); after aerobic culture of Lactobacillus helveticus strain C52 on MC medium, there was no obvious transparent ring around the colonies.

[0041] As Figure 2, Lactobacillus helveticus strain C52 grew well after anaerobic culture on X-gal-MRS, normal MRS, and MC solid media for 48 h. The colony morphology was medium-sized to large, with regular edges, smooth and moist surfaces, slightly raised, opaque, milky white colonies, and had a sour and fragrant smell. When aerobically cultured on X-gal-MRS, the colony morphology was similar to that on the MRS medium; after aerobic culture of Lactobacillus helveticus strain C52 on the MC medium, no obvious transparent ring appeared around the colonies.

[0042] Example 2 Detection of the interaction between Lactobacillus helveticus strain C52 and the J774-Dual™ reporter cell line and activation of the immune signaling pathway

[0043] Experimental method: J774-Dual™ cells are derived from the mouse macrophage-like cell line J774.1 and are stably integrated with two inducible reporter genes. The activation of the NF-κB pathway is reflected by detecting the activity of secreted 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 the growth medium (DMEM, 10% heat-inactivated fetal bovine serum (FBS), 100 mg / mL Normocin, 1% penicillin-streptomycin). After the cell state was stable, the cells were passaged three times in the selection medium (growth medium, 5 μg / mL Blasticidin, 100 μg / L Zeocin). The cells were digested with 0.25% trypsin-EDTA, centrifuged at 500 g for 10 min at room temperature, the supernatant was discarded, and the cell concentration of J774-Dual™ was adjusted to 2.8×10 5 cells / mL with the growth medium and inoculated into 96-well plates. A suspension of Lactobacillus helveticus strain C52 at 50 MOI, an inactivated suspension of Lactobacillus helveticus strain C52, and 50 μL of a lysate of Lactobacillus helveticus strain C52 were added to the corresponding cell wells. Cells without any treatment were used as the blank control (Blank control), and cells with the working concentrations of 1 μg / mL LPS, 10 ng / mL Pam3CSK, and 1 μg / mL 2',3'-cGAMP were used as the positive control. After stimulation at 37 °C and 5% CO2 for 24 h, the supernatant was collected.

[0044] Add 170 μL of QUANTI - Blue™ to each well of a 96 - well plate, then add 30 μL of the cell supernatant of Lactobacillus helveticus strain C52 stimulating J774 - Dual™. Incubate at 37 °C and 5% CO2 for 4 h, and measure the absorbance at OD650 nm using a microplate reader. The greater the absorbance value, the stronger the activation ability of the NF - κB signaling pathway. The specific steps for detecting the interferon IRF signaling pathway are as follows: Add 20 μL of the cell supernatant of Lactobacillus helveticus strain C52 stimulating J774 - Dual™ to a 96 - well white (opaque) or black plate, and use a microplate chemiluminescence detector (LB 960) to detect the expression of Lucia luciferase. The greater the detected RLU value, the stronger the activation ability of the IRF signaling pathway.

[0045] 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 (P < 0.05; Figure 3 B). The above results indicate that Lactobacillus helveticus strain C52 has a strong anti - inflammatory effect and at the same time has an anti - infectious effect.

[0046] Example 3 Detection of the interaction between Lactobacillus helveticus strain C52 and the macrophage inflammation model, showing strong anti - inflammatory and anti - infectious characteristics

[0047] Experimental method: Chicken macrophages HD11s (1 × 10 6 cells / mL) were seeded in 12 - well plates. After the cells adhered, 50 MOI of Lactobacillus helveticus strain C52 was added, and incubated at 37 °C and 5% CO2 for 24 h. An acute inflammation model was established by using lipopolysaccharide (LPS, L) and nigericin (N) to cause an acute inflammatory response in the cells. That is, add LPS with a working concentration of 1 μg / mL to each well, act at 37 °C and 5% CO2 for 3 h, then add nigericin with a working concentration of 1 μM and act for 1 h. After that, collect the cell pellet and perform relative quantification by SYBY Green Ⅰ fluorescence quantitative PCR, and use the primers in Table 1 to analyze the transcriptional levels of IL - 1β and IL - 10 mRNA in the cells. The cells treated with LPS and nigericin were used as the positive control (L + N), and the untreated cells were used as the blank control.

[0048]

[0049] Experimental results: As Figure 4As shown, the results of the anti-inflammatory and anti-infective effects of Lactobacillus helveticus strain C52 on macrophages are presented. Lactobacillus helveticus strain C52 can significantly downregulate the inflammatory cytokine IL-1β produced by HD11 in the acute inflammation model, showing a highly significant difference 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, showing a highly significant difference compared with the L+N control group (P<0.0001; Figure 4 B). The results indicate that Lactobacillus helveticus strain C52 has a strong anti-inflammatory effect, as well as anti-infective and immune homeostasis effects.

[0050] Example 4 Immunomodulatory and anti-inflammatory effects of Lactobacillus helveticus strain C52 on intestinal epithelial cells

[0051] Experimental method: The intestinal epithelial cell line HT29 (1 × 10 6 cells / mL) was seeded in 12-well plates. After the cells adhered, 50 MOI of Lactobacillus helveticus strain C52 was added, and the cells were incubated at 37 °C in 5% CO2 for 24 h. An acute inflammation model was established by inducing an acute inflammatory response in the cells using LPS and Nigericin. That is, LPS with a working concentration of 1 μg / mL was added to each well and incubated at 37 °C in 5% CO2 for 3 h, and then Nigericin with a working concentration of 1 μM was added and incubated for 1 h. After that, the cell pellets were collected and relative quantification was performed by SYBY Green Ⅰ fluorescence quantitative PCR, and primers were used to analyze the transcriptional levels of IL-1β and IL-10 mRNA in the cells. The cells treated with LPS and Nigericin were used as the positive control, and the untreated cells were used as the blank control.

[0052] Experimental results: As Figure 5 shown, the results of the anti-inflammatory effect of Lactobacillus helveticus strain C52 on intestinal epithelial cells are presented. Lactobacillus helveticus strain C52 can significantly downregulate the inflammatory cytokine IL-1β produced by HT29 in the acute inflammation model, showing a highly significant difference compared with the L+N control group (P<0.0001; Figure 5 ). Lactobacillus helveticus strain C52 stimulated HT29 to produce the anti-inflammatory cytokine IL-1β, showing no significant difference compared with the blank control group (P > 0.05; Figure 5 ). The results indicate that Lactobacillus helveticus strain C52 has a strong anti-inflammatory effect.

[0053] Example 5 Detection of antiviral proliferation characteristics of Lactobacillus helveticus strain C52 in vitro

[0054] Experimental method: Chicken lymphoma cells DT40 (1×10 6Inoculate in a 12-well plate with serum-free RPMI 1640 at a density of (number / mL), incubate at 37 °C for 30 min. After the cells adhere, infect them with 1 MOI of IBDV (SHG19), and simultaneously add Lactobacillus helveticus strain C52 with MOIs of 25, 50, and 100 respectively. After the three act on the cells for 24 h, collect 200 μL of cell suspension, use Premix Ex Taq to quantitatively determine the copy number of IBDV VP5 by absolute analysis method (Table 2 primers), detect cytokines IL-1β, IL-10, IFN-γ in the cell precipitate (Table 1 primers), and detect VP2 protein by Western blot.

[0055]

[0056] Experimental results: Lactobacillus helveticus strain C52 does not affect the cell viability of DT40 and is safe and non-toxic to DT40 cells. Lactobacillus helveticus strain C52 has an obvious antiviral effect in vitro. Compared with the positive control, Lactobacillus helveticus strain C52 can significantly reduce the virus copy number of vvIBDV, and there is an obvious dose-dependence. 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 the most significant (P < 0.0001). The Western blotting results show that, as Figure 6 shown, Lactobacillus helveticus strain C52 can significantly reduce the protein expression of vvIBDV VP2, and there is an obvious dose-dependence. Gray-scale 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 level of VP2 is the lowest, which is 0.43 times that of the positive control ( Figure 6 ). Comprehensive above results indicate that Lactobacillus helveticus strain C52 can significantly inhibit virus replication and reduce virus load in vitro, and has obvious antiviral and anti-infection effects.

[0057] Example 6. Safety characteristics of Lactobacillus helveticus strain C52 on the proliferation activity of chicken primary macrophages

[0058] Experimental method:

[0059] I. First, isolate primary chicken bone marrow macrophages. The isolation steps are as follows: ① Euthanize 3-week-old SPF chickens, and take the chicken femurs in a sterile operating table; ② Cut both ends of the femurs, 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 plunger, and then wash the bone marrow on the filter with PBS; ④ Gently add the mixture onto an equal volume of Histopaque-1083 separation liquid, centrifuge at 2,000 r / min for 25 min, and adjust the acceleration to the minimum; ⑤ Aspirate the cells in the middle layer and wash them twice with PBS; ⑥ After counting the cells, inoculate them into a 12-well plate, change the medium 6 h later, and wash away the non-adherent or semi-adherent cells; ⑦ After culturing for 6 d (change the culture medium every 2 d), conduct subsequent experiments.

[0060] II. Use Cell Counting Kit-8 to verify the effect of Lactobacillus helveticus strain C52 on cell proliferation activity to determine the toxicity of Lactobacillus helveticus strain C52 to cells. Inoculate 100 μL of chicken primary bone marrow macrophages at a concentration of 1 × 10 6 cells / mL into a 96-well plate, add 50 MOI of Lactobacillus helveticus strain C52 suspension, after incubating for 24 h, add 10 μL of cck-8 solution. After incubating at 37 °C for 2 h, measure the absorbance at 450 nm. Use cells treated with Con A (Concanavalin A) + PMA (Propylene glycol methyl ether acetate) or LPS as positive controls, and cells without any treatment as blank controls.

[0061] Experimental results: As Figure 7 shown, Lactobacillus helveticus strain C52 is non-toxic to chicken primary bone marrow macrophages, and C52 can stimulate the proliferation of chicken primary bone marrow cells. There is a highly significant difference between Lactobacillus helveticus strain C52 and the blank control (P < 0.01). The results indicate that Lactobacillus helveticus strain C52 is non-toxic to chicken primary bone marrow macrophages, and Lactobacillus helveticus has safety characteristics.

[0062] Example 7 Detection of the safety characteristics of injecting Lactobacillus helveticus strain C52 into chicken embryos

[0063] Experimental method: For the safety detection of chicken embryos, take out 3 10-day-old chicken embryos from a 37 °C incubator, view the egg embryos under an egg candler, use a pencil to mark the air chamber and the embryo position, and make a mark at a place where there are fewer chorioallantoic membrane blood vessels. Disinfect the eggshell of the air chamber with iodine tincture, then deiodize it with 75% alcohol, and drill a small hole at the marked place with a steel needle. Use a 1 ml syringe to aspirate the bacterial solution of Lactobacillus helveticus strain C52 cultured for 12 h, insert the needle into the hole, enter the allantoic cavity through the chorioallantoic membrane, and inject 0.1 ml; the inoculum of Lactobacillus helveticus strain C52 is 1 × 10 9CFU per chicken embryo. After sealing the holes with paraffin, incubate in an incubator at 37°C for 11 days; Inoculation in the air chamber: The operation is the same as above. The inoculation site is the air chamber. Inoculate 5 10-day-old chicken embryos each, and leave 5 10-day-old chicken embryos uninoculated as controls. Observe the development status of the chicken embryos daily until they hatch.

[0064] Experimental results: As Figure 8 shown, all 5 10-day-old chicken embryos in the Lactobacillus helveticus strain C52 group and the blank control group hatched successfully ( Figure 8 ). The results indicate that injecting Lactobacillus helveticus strain C52 has no effect on the growth and development of chicken embryos, and the results show that Lactobacillus helveticus strain C52 is 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 accession 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 and medicines or feeds for promoting animal growth.

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