Lactobacillus plantarum strain as well as extracellular vesicles and application thereof

Abstract

The invention discloses a lactobacillus plantarum strain as well as extracellular vesicles and application thereof. The new lactobacillus plantarum strain is named as C9, and the microbial preservation number of the new lactobacillus plantarum strain is CGMCC (China General Microbiological Culture Collection Center) No.30435. Researches show that the strain has unique anti-inflammatory and anti-infection dual characteristics, and also has the characteristics of acid resistance, cholate resistance and the like. Particularly, the strain can generate extracellular vesicles with very strong immunologic adjuvant activity and immunological enhancement activity, and the extracellular vesicles C9BEVs of the strain can directly activate chicken mucosal epithelium lymphocytes only through nasal dripping and eye dripping, so that mucosal immune response is remarkably enhanced. The lactobacillus plantarum strain C9 and the exovesicles thereof disclosed by the invention can be added into feed or medicines to realize the effects of enhancing the immune response of the organism and protecting health, and are expected to be used as a natural delivery carrier to provide an important tool for developing novel oral vaccines and mucous membrane delivery vaccines.

Description

Technical Field

[0001] The present invention relates to a Lactobacillus plantarum strain and its extracellular vesicles, as well as the use of the Lactobacillus plantarum strain in anti-inflammatory and anti-infective preparations, the use of the new Lactobacillus plantarum strain and its extracellular vesicles in preparing immunopotentiators or vaccine immune adjuvants, the use of preparations for enhancing the body's immune regulatory activity, and the use of the new Lactobacillus plantarum strain and its extracellular vesicles in preparing mucosal immune vaccines or oral immune vaccines. The present invention belongs to the field of biotechnology. Background Art

[0002] Lactobacillus plantarum ( Lactobacillus plantarum ) belongs to the genus Lactobacillus and is a Gram-positive bacterium. It is widely found in nature, such as in soil, on plant surfaces, and in the intestines of humans and livestock. Lactic acid bacteria in mucous membranes such as the oral cavity and intestines of humans and animals are important members of the normal microbial flora, or intestinal flora. Lactobacillus plantarum is present in the human gastrointestinal tract and has a significant positive effect on human health. In the food, feed, and pharmaceutical industries, Lactobacillus plantarum is often added to ferment foods or to antagonize the reproduction of pathogenic microorganisms, maintaining intestinal health, and preventing and treating diseases. Its value is increasingly recognized.

[0003] Lactobacillus plantarum is also a chemoheterotrophic microorganism. It lacks the ability to synthesize a wide range of compounds and requires external nutrient sources for growth and metabolism, such as sugars, amino acids, growth factors, and minerals. The optimal growth temperature for Lactobacillus plantarum is 15-35°C, with some strains thriving at temperatures between 4°C and 15°C. Lactobacillus plantarum is a straight or curved rod, growing singly, sometimes in pairs or chains. Its optimal pH is around 6.0. Lactobacillus plantarum can grow in low pH or low temperatures and has beneficial effects, such as regulating intestinal flora balance and inhibiting the growth of harmful bacteria.

[0004] Lactobacillus plantarum has multiple functions. First, it regulates the balance of the intestinal microbiome. Lactobacillus plantarum produces a variety of metabolites, such as lactic acid and acetic acid, which inhibit the growth of harmful bacteria and promote the proliferation of beneficial bacteria, thereby maintaining a balanced intestinal microbiome. Second, it enhances immunity by stimulating the activity of immune cells, improving the body's immunity and preventing disease. It also has antioxidant properties, scavenging free radicals in the body, which has antioxidant effects and can delay aging. It also lowers cholesterol, preventing cardiovascular disease. It also has hypoglycemic effects. For example, oral administration of Lactobacillus plantarum SS18-5 has been found to control weight, reduce fasting blood glucose, glycated hemoglobin, and insulin levels, and increase liver glycogen levels in a high-fat, streptozotocin-induced type 2 diabetes rat model. Diabetic patients often exhibit low-grade chronic inflammation accompanied by high levels of circulating pro-inflammatory cytokines. Several strains of Lactobacillus plantarum, such as SCS2, have been found to exhibit beneficial hypoglycemic and insulin-regulating effects by reducing MDA and ROS levels and increasing antioxidant enzyme levels in T2DM mice.

[0005] The immune regulation caused by Lactobacillus plantarum has attracted much attention from researchers. Studies have shown that Lactobacillus plantarum can increase the levels of IgA, IgG and IgM on the mucosal surface and in the serum through the bacteria itself or cell wall components, thereby enhancing humoral immunity; promoting the proliferation of T lymphocytes and B lymphocytes, strengthening cellular immunity; enhancing the activity of mononuclear phagocytes (monocytes and macrophages), stimulating the secretion of reactive oxygen species, lysosomal enzymes and monokines, and strengthening nonspecific immune responses. It is reported that Lactobacillus plantarum isolated from fermented foods can increase the level of IgA, IgG and IgM on the mucosal surface and in the serum, thereby enhancing humoral immunity; promoting the proliferation of T lymphocytes and B lymphocytes, strengthening cellular immunity; and enhancing the activity of mononuclear phagocytes (monocytes and macrophages), stimulating the secretion of reactive oxygen species, lysosomal enzymes and monokines, and strengthening nonspecific immune responses. L. plantarumL Lactobacillus plantarum-37 (HK-LP) is a strain that strongly induces IL-12 in mice both in vitro and in vivo. Feeding HK-LP can suppress IgE production, counteracting food allergies induced by the natural diet in mice. It can also inhibit tumor growth and tumor cell transplantation in mice. HK-LP has been shown to induce IL-12 production and promote Th1-type immune responses. Clinical trials have shown that daily intake of HK-LP can enhance adaptive immunity, particularly Th1-related immune function, in healthy adults. Dong et al. found that Lactobacillus plantarum NCIMB 8826 can enhance the activity of lymphocytes, T cells, T cell subsets, and NK cells, while also increasing the activity of cytokines such as IL-1β, IL-6, IL-10, and TNF-α to varying degrees. The immunomodulatory effects of Lactobacillus plantarum are related to the bacteria itself and its metabolites; even dead bacteria can have immunomodulatory effects. As far as we know, there are many types of lactic acid bacteria that can play an immunomodulatory role, such as Lactobacillus acidophilus, Lactobacillus casei, Streptococcus thermophilus, Lactobacillus reuteri, etc.

[0006] Lactobacillus plantarum has an inhibitory effect on Gram-positive and Gram-negative pathogens. It inhibits the growth of pathogens by competing with the pathogens' ability to limit nutrients, regulates the composition of the intestinal microecology, forms a biological barrier, and adjusts the relationship between the flora through the effects of substances such as lactic acid, bacteriocins, and diacetyl produced by its metabolism on other bacteria, maintains and ensures the best dominant combination of flora, and prevents the invasion and colonization of pathogens by antagonizing the growth of pathogens and the adhesion of their toxins. Maragkoudakisa et al. studied the probiotic properties of 29 strains of Lactobacillus in dairy products in vitro and found that all strains had varying degrees of bile salt hydrolysis activity, and the vast majority of strains were tolerant to vancomycin and teicoplanin, but sensitive to tetracycline and chloramphenicol. Among them, L. plantarum ACA-DC 146 can inhibit the adhesion of Escherichia coli and Salmonella to Caco-2 cells and induce the secretion of certain cytokines in human peripheral blood mononuclear cells. Anukam et al. isolated Lactobacillus plantarum from the vagina of healthy Nigerian women, and found that six samples of Lactobacillus plantarum produced large amounts of biosurfactants. The H2O2 produced by the Lactobacillus plantarum also had inhibitory effects on intestinal and vaginal pathogens.

[0007] The determination of the whole genome sequence of Lactobacillus plantarum, combined with functional genomics studies, will help us to comprehensively and systematically understand the structure and composition of the Lactobacillus plantarum genome. Looking at the basic information of the whole genome of Lactobacillus plantarum, we found that Lactobacillus plantarum has the following characteristics: (1) The length of the whole genome is between 2.9 and 3.7 Mb, which is the largest genome among lactic acid bacteria; (2) The G + C percentage is usually around 45%, while the GC% content of Bifidobacterium is as high as about 60%, and the GC% content of Lactobacillus salivarius is as low as about 33%. The important difference between Lactobacillus plantarum and other types of lactobacillus is that the reported Lactobacillus plantarum genomes all contain proteases that do not encode cell wall proteins.

[0008] In summary, Lactobacillus plantarum has unique physiological effects on animals and is poised to dominate the food, feed, and healthcare sectors. With the advancement of science and the deepening of research, insights into the physiological functions of Lactobacillus plantarum are increasing, the scope of Lactobacillus strains used is expanding, and its application areas are also expanding. Among its many application areas, challenges remain: the safety of probiotic Lactobacillus; the inactivation of live Lactobacillus preparations during processing and transportation; the tolerance of lactic acid bacteria to low-pH gastric acid, bile acids, and pancreatic juice to ensure sufficient live bacteria reach the intestines to function; and the selection of appropriate strains and the determination of dosage for specific applications. Further research is needed to determine the effects of carriers on probiotic Lactobacillus; to improve the performance and activity of probiotic Lactobacillus by optimizing culture conditions, and to determine the physiological functions, mechanisms of action, and impact of these strains through clinical trials; to evaluate the regulatory effects of probiotic Lactobacillus on the immune system, cancer, and intestinal microecology; to assess the gene expression patterns of probiotic Lactobacillus in the intestine and their impact on the number and activity of intestinal microorganisms; and to rationally utilize various lactic acid bacterial metabolites and evaluate their specific effects and application scenarios. Each of these areas warrants in-depth research, and the prospects for development and application are immeasurable. Summary of the Invention

[0009] The purpose of the present invention is to provide a new strain of Lactobacillus plantarum and its extracellular vesicles C9BEVs for use in anti-inflammatory and anti-infection; for use in preparing natural immune adjuvant activity or immunopotentiators; for use in preparing excellent mucosal immune preparations; for use in preparing preparations with excellent immunomodulatory properties; and in particular, for use in preparing natural and efficient mucosal delivery carriers.

[0010] In order to achieve the above object, the present invention adopts the following technical means: The strain of the present invention was isolated from the cecal mucosa of healthy wild boars in the Daxing'anling virgin forest in Heilongjiang Province, China, and was obtained by MRS inoculation, culture, and purification. It was identified as a Lactobacillus plantarum strain and named C9. It was classified as Lactobacillus plantarum ( Lactobacillus plantarum), deposited at the General Microbiology Center of the 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. 30435, and the deposit date is April 25, 2024. This strain is Lactobacillus plantarum C9, a natural strain isolated from the intestinal mucosa of healthy wild pigs. It does not endanger human health, is not an animal or plant pathogen, and does not pollute the environment. Long-term feeding results in animals have shown that it can significantly increase feed intake, and the animals do not show any visible or anatomical adverse changes, and are healthy and active. In vitro experiments have shown that this strain has a strong effect against pathogenic bacteria. In addition, the fermentation broth of this strain has significant antibacterial activity (against pathogens such as Salmonella and Escherichia coli) and strong immunomodulatory activity. The microbiological characteristics of this strain are: ① Gram staining is typically positive, the bacteria are medium to large, and have regular morphology; it is an aerobic or facultative anaerobic bacterium that grows well on solid and liquid MRS culture media; ② it has acid resistance and can grow well on MRS with a pH of 4.0; ③ it has bile salt resistance and can survive and grow in culture medium containing 50% chicken bile MRS; ④ it has unique anti-inflammatory and anti-infection properties; ⑤ the optimal culture temperature of this bacterium is 37°C, and it can grow at 4°C. The colonies on MRS culture medium are milky white, with neat edges, a smooth and moist surface, raised, opaque medium-sized colonies, and a sour and fragrant smell.

[0011] On the basis of the above research, the present invention first proposed a strain of Lactobacillus plantarum, named C9 It 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. 30435, and the deposit date is April 25, 2024.

[0012] Furthermore, the present invention also proposes the use of the Lactobacillus plantarum strain in the following aspects: (1) Use in the preparation of highly effective natural immune adjuvants or immune enhancement preparations; (2) Use in the preparation of health-care or immunity-enhancing feed additives; (3) Use in the preparation of mucosal immune vaccines; (4) Use in the preparation of anti-infective and anti-inflammatory preparations.

[0013] Among them, preferably, the Lactobacillus plantarum strain has the effect of enhancing mucosal immune response and promoting mucosal secretion of specific antibodies.

[0014] Secondly, the present invention also proposes a Lactobacillus plantarum exocyst, wherein the Lactobacillus plantarum exocyst is the Lactobacillus plantarum strain C9 was extracted from the culture medium.

[0015] Wherein, preferably, the Lactobacillus plantarum exosomes are extracted by the following method: (1) Inoculation: according to the Lactobacillus plantarum strain C9 bacterial liquid The volume ratio of the medium to the MRS medium was 1:100, and the cells were activated and cultured at 37°C for 8 h. Then, the cells were inoculated with the MRS medium at a volume ratio of 1:100 and cultured at 37°C for 16 h. (2) Add 10 mL of 10% w / v NaHCO3 solution, 25 mL of 20% w / v glucose solution, and 2 mL of 5 mg / mL cysteine ​​hydrochloride solution to each 1 L of culture medium. Continue culturing for 40 hours and harvest the culture medium. (3) The harvested culture medium was centrifuged at 3000 g / min for 10 minutes to obtain the supernatant; (4) The supernatant was filtered using a 0.45 μm filter membrane and a 750 kD medium-control fiber tangential flow filter to obtain a filtrate; (5) Centrifuge the filtrate at 35,000 g / min for 1 h, then at 120,000 g / min for 1 h; (6) Discard the supernatant and resuspend the lower precipitate with PBS to obtain the extracellular vesicles of Lactobacillus plantarum C9 strain, which are named C9BEVs.

[0016] Furthermore, the present invention also proposes the use of the Lactobacillus plantarum exovesicles in the following aspects: (1) Use in the preparation of highly effective natural immune adjuvants or immune enhancement preparations; (2) Use in the preparation of health-care or immunity-enhancing feed additives; (3) Use in the preparation of mucosal immune vaccines; (4) Use in the preparation of anti-infective and anti-inflammatory preparations.

[0017] Among them, preferably, the Lactobacillus plantarum exosomes have the effect of enhancing mucosal immune response and promoting mucosal secretion of specific antibodies.

[0018] Compared with the prior art, the present invention has the following beneficial effects: The present invention discloses a new strain of Lactobacillus plantarum and its use. The new strain of Lactobacillus plantarum is named C9 and is deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration, and its microbial deposit number is CGMCC No. 30435. Studies have shown that the strain has unique dual properties of anti-inflammatory and anti-infection, and also has properties such as acid resistance and bile salt resistance. In particular, the strain can produce extracellular vesicles C9BEVs with strong immune adjuvant activity and immune enhancement activity. Only nasal drops or eye drops of the strain's extracellular vesicles C9BEVs can directly activate chicken mucosal intraepithelial lymphocytes and significantly enhance mucosal immune responses; in particular, the strain's extracellular vesicles C9BEVs can be directly used as a highly efficient adjuvant mucosal delivery carrier for inactivated viruses or antigens, and can achieve efficient delivery of inactivated vaccines or antigens to the mucosal immune system through mucosal immunization. By using the strain's extracellular vesicles C9BEVs, new inactivated vaccines or mucosal vaccines of subunit antigens can be obtained. The new strain of Lactobacillus plantarum C9 and its extracellular vesicles disclosed in the present invention can be used as additives for feed and medicine. When added to feed or medicine, they can enhance the body's immune response and health care. They are also expected to serve as natural delivery carriers to provide important tools for the development of new oral vaccines and mucosal delivery vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The aerobic growth of Lactobacillus plantarum C9 strain on Y-MRS, MC and MRS media; Figure 2 The anaerobic growth of Lactobacillus plantarum C9 strain on Y-MRS, MC and MRS media; Figure 3 This is the result of the interaction between Lactobacillus plantarum C9 strain and macrophage inflammation model; Among them, A: IL-1β; B: IL-10; Figure 4 This is the safety test result of Lactobacillus plantarum C9 strain on primary chicken macrophages; Figure 5 The results are the anti-inflammatory effects of Lactobacillus plantarum C9 strain on intestinal epithelial cells HT29; Figure 6 To test the activation effect of Lactobacillus plantarum C9 strain on the immune signaling pathway of J774-Dual™ reporter cell line; Among them, A: interferon regulatory factor (IRF) signaling pathway; B: NF-κB signaling pathway; Figure 7 This is a transmission electron microscopy image of the extracellular vesicles of Lactobacillus plantarum C9 strain; Figure 8 The data for the size characterization of extracellular vesicles of Lactobacillus plantarum C9 strain; Figure 9Zeta potential characterization data of extracellular vesicles of Lactobacillus plantarum C9 strain; Figure 10 The results of the safety evaluation of Lactobacillus plantarum C9 strain injected into chicken embryos are presented; Figure 11 The results of cytokine secretion by Harderian gland lymphocytes (HGLs) in SPF chickens immunized with C9BEVs mucosa (nasal drops and eye drops) were analyzed. Among them, A: IL-1βmRNA level; B: IL-17A mRNA level; C: IL-6 mRNA level; D: IL-23mRNA level; E: IL-4mRNA level; F: IL-10mRNA level; G: TGF-βmRNA level; Figure 12 The results are the detection results of cytokines related to the cellular immune response of Harderian gland lymphocytes (HGLs) after mucosal immunization (nasal drops and eye drops) of SPF chickens with C9BEVs; Among them, A: IFN-βmRNA level; B: IL-28B mRNA level; C: IL-2 mRNA level; D: IL-12p70 mRNA level; E: IFN-γ mRNA level; F: IFNr / IL-4 ratio mRNA level; G: IL-10 mRNAlevel; H: IL-12 / IL-10 ratio; Figure 13 The results are as follows: After C9BEVs were administered to SPF chickens by nasal and eye drops, the interferon regulatory factors that regulate the antiviral innate immune signaling pathway were detected in Harderian gland lymphocytes (HGLs); Among them, A: IRF1 mRNA level; B: IRF7 mRNA level; C: TBK1 mRNA level; D: CCR5 mRNA level; E: CXCR5 mRNA level; Figure 14 The results of the detection of lymphocyte homing receptors in Harderian gland lymphocytes (HGLs) of SPF chickens after nasal and eye drops of C9BEVs were administered; Among them, A: CCR5 / CCR4 ratio; B: CXCR5 mRNA level; Figure 15 The results of the detection of costimulatory molecules highly expressed by DC or APC cells in Harderian gland lymphocytes (HGLs) after nasal and eye drops of C9BEVs in SPF chickens were obtained; Among them, A: CD40 mRNA level; B: CD80 mRNA level; C: TLR4 mRNA level; D: CD40 / CD40L ratio; E: CD40L mRNA level; Figure 16 The results of fluorescence quantitative detection of JAK / STAT signaling pathway (second signaling pathway) signaling molecules that regulate immune cells in Harderian gland lymphocytes (HGLs) after C9BEVs were administered to SPF chickens by nasal and eye drops. Among them, A: STAT1 (Th1, M1 cells) mRNA level; B: STAT3 (Tregs cells) mRNA level; C: STAT5 (Th17 cells) mRNA level; D: STAT1 / STAT3 ratio (Th1 / Th17 ratio); E: STAT1 / STAT5 ratio (Th1 / Tregs ratio); F: STAT1 / STAT6 ratio (Th1 / Th2). DETAILED DESCRIPTION

[0020] 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 merely exemplary and do not limit the scope of the present invention in any way. It should be understood by those skilled in the art that the details and forms of the technical solutions of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, and such modifications and replacements fall within the scope of protection of the present invention. Example 1 Isolation and identification of Lactobacillus plantarum C9 strain

[0021] 1. Isolation and identification of Lactobacillus plantarum C9 strain The strain was isolated from the intestinal mucosa of healthy wild boar in Fuyuan primeval forest, Heilongjiang Province, China. It grew well on MRS medium and was obtained by streaking and identified as Lactobacillus plantarum ( Lactobacillus plantarum ), the strain was named C9 and classified as Lactobacillus plantarum ( Lactobacillus plantarum ). Its microbial deposit number is CGMCC No.30435, and the deposit date is April 25, 2024.

[0022] Its microbiological characteristics are: ① Gram staining is typically positive, the bacteria are medium to large, with regular morphology; it is an aerobic or facultative anaerobic bacterium and grows well on solid and liquid MRS culture media; ② it has acid resistance and can grow well on MRS with a pH of 4.0; ③ it has bile salt resistance and can survive and grow in culture medium containing 50% chicken bile MRS; ④ it has unique anti-inflammatory and anti-infection properties; ⑤ the optimal culture temperature for this bacterium is 37°C, and it can grow at 4°C. The colonies on MRS culture medium are milky white, with neat edges, a smooth and moist surface, raised, opaque medium-sized colonies, and a sour and fragrant smell.

[0023] 2. Cultivation characteristics of the strain of the present invention on solid culture medium 2.1 Colony characteristics of Lactobacillus plantarum C9 strain cultured aerobically on MRS, X-gal-MRS, and MC media: Experimental method: Lactobacillus plantarum C9 strain was streaked onto MRS, X-gal-MRS and MC solid culture media and cultured aerobically at 37℃ for 48 h, and the colony morphology of C9 was observed.

[0024] Experimental results: Figure 1 Lactobacillus plantarum C9 can grow aerobically on MRS, X-gal-MRS, and MC solid media. Its colonies are medium-sized, opaque, and milky white with neat edges, a smooth, moist surface, and a tart, fragrant aroma. Aerobically cultured on X-gal-MRS, the colonies are similar in morphology to those on MRS, but light blue (indicating that Lactobacillus plantarum C9 produces β-glucosidase, which degrades the X-gal substrate and gives the colonies their blue color). Aerobically cultured on MC media produces a transparent ring around the colonies, indicating that Lactobacillus plantarum C9 produces acidic metabolites that dissolve calcium carbonate.

[0025] 2.2 Colony characteristics of Lactobacillus plantarum C9 strain cultured anaerobically on MRS, X-gal-MRS, and MC media: Experimental method: Lactobacillus plantarum C9 strain was streaked onto MRS, X-gal-MRS and MC solid culture media and cultured anaerobically at 37℃ for 48 h, and the colony morphology of C9 was observed.

[0026] Experimental results: Figure 2 , Lactobacillus plantarum C9 strain can grow on MRS, X-gal-MRS and MC solid media under anaerobic culture conditions. And when Lactobacillus plantarum C9 strain is cultured anaerobically on X-gal-MRS, the colony morphology is similar to that on MRS medium ( Figure 2 ). Example 2 Lactobacillus plantarum C9 strain interacts with macrophage inflammation model, exhibiting strong anti-inflammatory and anti-infective properties

[0027] Experimental methods: I. Establishment of cell model: According to the method provided in the literature (Saresella et al., 2020; Suzuki et al., 2020; Yazdi and Drexler, 2013), chicken macrophages (HD11) were modified and cultured in culture medium (DMEM medium containing 10% FBS, 2 mM L-glutamine, 150 μg / mL gentamicin, 100 mg / mL normocin, and 100 U / mL penicillin-streptomycin) at 37°C and 5% CO2. They were serially passaged until the cells were stable. When the cells adhered to the wall and grew to 70%-80% confluence, they were digested with 0.25% trypsin-EDTA and centrifuged at 500 g for 10 min at room temperature. The supernatant was discarded and the cell concentration was adjusted to 1×10 6 Cells were seeded in 12-well plates at a concentration of 1 μg / mL. After incubation at 37°C, 5% CO₂ for 24 hours, lipopolysaccharide (LPS) at a working concentration of 1 μg / mL was added for 3 hours at 37°C, 5% CO₂, and then nigericin at a working concentration of 1 μM was added for 1 hour to establish an acute inflammatory cell model. Untreated cells served as blank controls (blank controls), cells treated with the corresponding working concentrations of LPS and nigericin served as positive controls (L+N), cells treated with 50 μL of PBS served as negative controls (PBS), and cells treated with 50 μL of MRS liquid medium served as medium controls (MRS). Cell pellets were then harvested and RNA was extracted using RNAiso Plus. Reverse transcription to cDNA was used as a template for quantification of IL-1β and IL-10 mRNA levels using the SYBY Green I fluorescent quantitative PCR kit with the primers listed in Table 1.

[0028] II. Detection of the antibacterial effect of Lactobacillus plantarum C9 against HD11 cells: Live and inactivated Lactobacillus plantarum C9 cell suspensions at a 50 MOI (multiplicity of infection; ratios of Lactobacillus plantarum C9 to cells were 1:10, 1:20, 1:50, and 1:100) and 50 μL of cell lysate were added to HD11 cells grown as monolayers in 12-well plates. After stimulation at 37°C, 5% CO₂ for 24 hours, an acute inflammatory cell model and a control group were established. RNA was extracted from the harvested cell pellet using RNAisoPlus and reverse-transcribed into cDNA, which was then used as a template for quantitative PCR analysis of Il-1β and Il-10 mRNA levels in the cells using the primers listed in Table 1 using the SYBY Green I fluorescent quantitative PCR kit.

[0029]

[0030] Experimental results: Figure 3 The results show that Lactobacillus plantarum C9 strain can significantly downregulate the inflammatory cytokine IL-1β produced by HD11 in the acute inflammation model, and the difference is extremely significant compared with the L+N control group (P<0.0001; Figure 3 A). C9 can effectively stimulate HD11 to produce the anti-inflammatory cytokine IL-10, and the difference is extremely significant compared with the L+N control group (P<0.0001; Figure 3 B). Example 3 Effect of Lactobacillus plantarum C9 strain on the proliferation activity of primary chicken macrophages

[0031] Experimental method: The Cell Counting Kit-8 (CCK-8) was used to test the effect of Lactobacillus plantarum C9 on the proliferation of chicken primary macrophages to determine whether the Lactobacillus plantarum C9 strain was toxic to the cells. 6 Cells were inoculated at a concentration of 100 cells / mL in a 96-well plate and cultured at 37°C in 5% CO₂ for 12 hours. A 50 MOI suspension of Lactobacillus plantarum C9 was then added to the cells. After incubation for 12 hours, 10 µL of CCK-8 solution was added. After incubation at 37°C for 2 hours, the absorbance at OD450 nm was measured using a microplate spectrophotometer, and cell viability was calculated using statistical methods. Cells treated with Con A (concanavalin A) + PMA (propylene glycol methyl ether acetate) or LPS (lipopolysaccharide) served as positive controls, while untreated cells served as blank controls.

[0032] The results are as follows Figure 4As shown, the results showed that Lactobacillus plantarum C9 had no toxicity to chicken primary bone marrow macrophages, C9 had no effect on the proliferation of chicken primary bone marrow cells, and there was no significant difference between C9 and the blank control (P<0.05). Example 4 Anti-inflammatory Effects of Lactobacillus plantarum C9 on Intestinal Epithelial Cells HT29

[0033] To investigate the anti-inflammatory effects of Lactobacillus plantarum C9 on intestinal epithelial cells HT29, cells were seeded in 12-well plates and incubated at 50 MOI of Lactobacillus plantarum C9. The cells were then incubated at 37°C, 5% CO₂ for 24 hours. LPS and nigericin were then used to induce an acute inflammatory response in the cells to establish an acute inflammation model. LPS at a working concentration of 1 μg / mL was added to each well for 3 hours at 37°C, 5% CO₂, followed by nigericin at a working concentration of 1 μM for 1 hour. Cell pellets were then collected and analyzed for IL-1β transcription using SYBY Green I quantitative PCR using the primers listed in Table 1. Cells treated with LPS and nigericin served as positive controls, while untreated cells served as blank controls.

[0034] The results show that if Figure 5 As shown in the results, under the inflammatory model, Lactobacillus plantarum C9 can significantly reduce the expression of IL-1β and has anti-inflammatory immunomodulatory effects. Example 5 Detection of the Activation Effect of Lactobacillus plantarum C9 Strain on the Immune Signaling Pathway of the J774-Dual™ Reporter Cell Line

[0035] J774-Dual™ cells are derived from the murine macrophage-like cell line J774.1 and stably incorporate two inducible reporter genes. Activation of the NF-κB pathway is assessed by SEAP activity in the supernatant, while activation of the interferon regulatory factor (IRF) pathway is assessed by Lucia luciferase activity in the supernatant. J774-Dual™ cells were passaged three times in growth medium (DMEM supplemented with 10% heat-inactivated FBS, 100 mg / mL normocin, and 1% penicillin-streptomycin). After stabilization, the cells were passaged three times in selection medium (growth medium supplemented with 5 μg / mL blasticidin and 100 μg / L zeocin). The cells were digested with 0.25% trypsin-EDTA and centrifuged at 500 g for 10 minutes at room temperature. The supernatant was discarded, and the J774-Dual™ cells were adjusted to a cell density of 2.8 × 10 cells / mL in growth medium. 5Cells were plated in a 96-well plate at 400 μL of lysate. A 50 MOI suspension of live Lactobacillus plantarum C9 cells, a 50 μL suspension of inactivated cells, and 50 μL of cell lysate were added to the corresponding wells. Untreated cells served as blank controls, while cells treated with 1 μg / mL LPS, 10 ng / mL Pam3CSK, and 1 μg / mL 2',3'-cGAMP served as positive controls. After stimulation at 37°C and 5% CO₂ for 24 hours, the supernatant was collected.

[0036] Add 170 μL of QUANTI-Blue™ to each well of a 96-well plate, followed by 30 μL of supernatant from J774-Dual™ cells stimulated with Lactobacillus plantarum C9 strain. Incubate at 37°C, 5% CO2 for 4 hours. Measure the absorbance at OD650 nm using a microplate reader. Higher absorbance values ​​indicate stronger activation of the NF-κB signaling pathway. The interferon (IRF) signaling pathway assay follows: add 20 μL of supernatant from J774-Dual™ cells stimulated with Lactobacillus plantarum C9 strain to a 96-well white (opaque) or black plate. Detect Lucia luciferase expression using a microplate chemiluminescence reader (LB 960). Higher relative unit (RLU) values ​​indicate stronger activation of the IRF signaling pathway.

[0037] The results are as follows Figure 6 As shown in the results, the Lactobacillus plantarum C9 strain can effectively activate the NF-κB signaling pathway of macrophage J774-Dual™ cells, and the difference is extremely significant compared with the blank control (P<0.0001; Figure 6 C9 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<0.0001; Figure 6 A. The results show that Lactobacillus plantarum C9 has strong anti-inflammatory and anti-infective effects. Example 6 Extraction and Characterization of Extracellular Vesicles of Lactobacillus plantarum C9 Strain

[0038] The extraction of extracellular vesicles from Lactobacillus plantarum C9 strain was carried out according to the following steps: (1) Inoculation: according to the Lactobacillus plantarum strain C9 bacterial liquid Inoculate MRS medium (50 mL centrifuge tube) at a volume ratio of 1:100 with MRS medium, activate and culture at 37°C for 8 h, then inoculate 3 L of MRS medium at a volume ratio of 1:100, and continue culture at 37°C for 16 h. (2) Add 10 mL of 10% w / v NaHCO3 solution, 25 mL of 20% w / v glucose solution, and 2 mL of 5 mg / mL cysteine ​​hydrochloride solution to each 1 L of culture medium. Continue culturing for 40 hours and harvest the culture medium. (3) The harvested culture medium was centrifuged twice using a 500 mL centrifuge bucket at 375 mL / bucket and 3000 g / min for 10 minutes to obtain the supernatant; (4) The supernatant was filtered using a 0.45 μm filter membrane and a 750 kD medium-control fiber tangential flow filter to obtain a filtrate; (5) Centrifuge the filtrate at 35,000 g / min for 1 h, then at 120,000 g / min for 1 h; (6) Discard the supernatant and resuspend the lower precipitate with PBS to obtain the extracellular vesicles of Lactobacillus plantarum C9 strain, which are named C9BEVs.

[0039] Characterization of Extracellular Vesicles of Lactobacillus plantarum C9 Strain: C9BEVs protein quantification: UV protein quantification: 278 μg / mL C9BEVs appearance: Figure 7 shown.

[0040] C9BEVs size: Figure 8 As shown, the average size of extracellular vesicles of Lactobacillus plantarum C9 strain is 814.4 nm.

[0041] C9BEVs potential: Figure 9 As shown, the Zeta potential of the extracellular vesicles of Lactobacillus plantarum C9 strain is -19.3 mV. Example 7 Safety Evaluation of Lactobacillus plantarum C9 Strain

[0042] Experimental Method: For the safety test of chicken embryos, three 10-day-old chicken embryos were removed from a 37°C incubator and observed on an egg candling device. The air chamber and embryo position were marked with a pencil, and the area with fewer blood vessels on the chorioallantoic membrane was marked. The air chamber eggshell was disinfected with iodine, then deiodinated with 75% alcohol. A small hole was drilled at the marked area with a steel needle. A 12-hour culture of Lactobacillus plantarum C9 strain (1×108 CFU / mL) was drawn with a 1ml syringe. The needle was inserted into the hole and injected through the chorioallantoic membrane into the allantoic cavity. 1×10 C9 bacteria were injected. 9 Each chicken embryo was sealed with paraffin and incubated in a 37°C incubator for 11 days; air chamber inoculation: the operation was the same as above, the inoculation site was the air chamber, and 5 10-day-old chicken embryos were inoculated each time, of which 5 10-day-old chicken embryos were not inoculated and kept as controls. The development status of the chicken embryos was observed daily until they hatched.

[0043] The results are as follows Figure 10As shown in the figure, a total of five 10-day-old chicken embryos in the C9 group and the blank control group were successfully hatched. PCR amplification using Lactobacillus plantarum C9 DNA as a template and primers for virulence factor genes and drug resistance genes revealed no bands, indicating that Lactobacillus plantarum C9 lacks these virulence and drug resistance genes. In summary, Lactobacillus plantarum C9 is considered safe. Example 8 Activation of the mucosal system by oral administration of extracellular vesicles (C9BEVs) of Lactobacillus plantarum strain C9

[0044] Objective: To investigate the effects of C9 extracellular vesicles on mucosal immune cells in SPF white-feather broiler chickens through mucosal administration.

[0045] Six 21-day-old SPF white broiler chickens were used for the study. They were divided into two groups: an experimental group (three chickens) received 200 μl of the extracellular vesicle solution via mucosal administration (eye drops); and a control group (three chickens) received 200 μl of PBS solution orally. Blood sampling: 5 ml of blood was collected 24, 48, and 72 hours after administration for isolation of peripheral blood mononuclear cells (PBMCs). Endpoint analysis: All chickens were humanely sacrificed at 72 hours, and tracheal intraepithelial lymphocytes (IELs) and Harderian gland lymphocytes (HGLs) were isolated for further analysis.

[0046] Isolation of Harderian Gland Lymphocytes (HGLs): Sample collection: First, obtain the sample from the lower part of the chicken's eye. Tissue processing: The collected tonsil tissue is finely minced under sterile conditions and then treated with an appropriate buffer to release lymphocytes. Cell separation: Next, lymphocytes are separated using density gradient centrifugation. This step can help separate lymphocytes and remove dead cells and other tissue debris. Cell washing: The separated cells need to be washed with sterile saline or buffer to remove residual density gradient medium. Cell counting and activity assessment: Count the number of cells using a hemocytometer or an automatic cell counter, and exclude dead cells by staining to assess cell activity and purity.

[0047] Verification of the activation of mucosal immunity by nasal and eye drops of C9BEVs: 1. Resuspend the isolated HGLs and IELs in 1640 medium containing 10% FBS, and keep the cell density at 2*10 6 cell / ml; 2. Incubate at 37°C in 5% CO2 for 24 h; 3. Centrifuge at 300g for 5 min, remove the supernatant, and freeze the cells at -80°C until further use; 4. Extract cellular RNA; 5. Detect related molecules by fluorescence quantitative detection using primers shown in Table 2.

[0048]

[0049]

[0050] The results are as follows Figure 11 As shown, the results show that C9BEVs can significantly activate Harderian gland lymphocytes (HGLs), and the secretion of cytokines by Harderian gland lymphocytes (HGLs) shows that C9BEVs activate immune regulatory cells and maintain immune activation in an appropriate steady state. After C9BEVs were dripped into the nose and eyes of SPF chickens, Harderian gland lymphocytes HGLs were extracted from the chicken eyes, and the expression of immune cell CK in HGLs was detected by fluorescence quantitative analysis. The expression of inflammatory cytokines IL-1β, IL-17, IL-6, and IL-23 in HGLs of the immunized group was extremely significantly higher than that in the control group ( Figure 11 AD); and immune homeostasis and immune negative regulatory cytokines IL-10, IL-4, TGF-β were also significantly higher than those in the control group ( Figure 11 The results showed that C9BEVs nasal and eye drops directly activated the immune response of chicken Harderian gland lymphocytes (HGLs) and also activated immune regulatory cells, maintaining an appropriate steady state of immune activation.

[0051] like Figure 12 The results show that after C9BEVs were administered to SPF chickens via nasal and ocular administration, Harderian gland lymphocytes (HGLs) were extracted from the eyes. Fluorescence quantitative analysis of immune cell CK expression in HGLs revealed significantly higher Th1 CKs: IFN-β, IL-28B, IL-2, IL-12p70, and IFN-γ compared to the control group. Furthermore, the IFNr / IL-4 ratio and IL-12 / IL-10 ratio were significantly higher than those in the control group. These results suggest that C9BEVs administered via nasal and ocular administration activate immune responses, particularly those that benefit anti-infective immunity.

[0052] like Figure 13 As shown, the results showed that after C9BEVs were nasally and ocularly administered to SPF chickens, the expression of interferon regulatory factors IRF1 / 7, TBK1 molecules, and lymphocyte homing chemokine receptors CCR5 and CXCR5 in Harderian gland lymphocytes HGLs that regulate the antiviral innate immune signaling pathway was significantly higher than that in the control group ( Figure 13 AE). The results showed that C9BEVs directly activated Th1 immune cells in chicken Harderian gland lymphocytes (HGLs) by nasal and eye drops.

[0053] like Figure 14As shown in the results, after C9BEVs were nasally and ocularly administered to SPF chickens, the detection results of lymphocyte homing chemokine receptors in Harderian gland lymphocytes (HGLs) showed that the CCR5 / CCR4 ratio and CXCR3 / CCR4 ratio were significantly higher than those in the control group ( Figure 14 AB). The results showed that C9BEVs directly activated Th1 immune cells in chicken Harderian gland lymphocytes (HGLs) by nasal and eye drops.

[0054] like Figure 15 The results showed that after C9BEVs were nasally and ocularly administered to SPF chickens, the high expression of costimulatory molecules CD40, CD80 / 86, TLR4 and T cell costimulatory molecule CD40L in DC or APC cells in Harderian gland lymphocytes HGLs was detected, and the CD40 / CD40L ratio was also significantly higher than that in the control group ( Figure 15 AE). The results showed that C9BEVs significantly activated antigen-presenting cells and T cells in HGLs.

[0055] like Figure 16 As shown, the results showed that after C9BEVs were nasally and eye-dropped in SPF chickens, the fluorescence quantitative detection results of the JAK / STAT signaling pathway (second signaling pathway) signaling molecules that regulate immune cells in Harderian gland lymphocytes HGLs showed that the related signaling molecules STAT1 (Th1, M1 cells), STAT5 (Tregs cells), and STAT3 (Th17 cells) that activate immune cells; the STAT1 / STAT3 ratio (Th1 / Th17 ratio) and STAT1 / STAT5 ratio (Th1 / Tregs ratio) were extremely significantly higher than those in the control group ( Figure 16 AE); STAT1 / STAT6 ratio (Th1 / Th2) was significantly higher than that in the control group ( Figure 16 F). The above results show that C9BEVs comprehensively and significantly activated HGLs lymphocytes and extremely significantly activated the anti-infection cellular immune response.

Claims

1. A strain of Lactobacillus plantarum ( Lactobacillus plantarum ) strain, named C9 It is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, located at the Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing. Its microorganism deposit number is CGMCC No.30435, and the deposit date is April 25, 2024.

2. The use of the plant lactobacillus strain according to claim 1 in the following aspects: (1) Use in the preparation of highly effective natural immune adjuvants or immune enhancing preparations; (2) Use in the preparation of health-care or immune-enhancing feed additives; (3) Use in the preparation of mucosal immune vaccines; (4) Use in the preparation of anti-infective and anti-inflammatory preparations.

3. The use according to claim 2, characterized in that The plant lactobacillus strain has the functions of enhancing mucosal immune response and promoting mucosal secretion of specific antibodies.

4. A Lactobacillus plantarum exovesicle, characterized in that The Lactobacillus plantarum exosome is obtained from the Lactobacillus plantarum strain according to claim 1. C9 was extracted from the culture medium.

5. The Lactobacillus plantarum exovesicle according to claim 4, characterized in that The Lactobacillus plantarum exocyst is extracted by the following method: (1) Inoculation: according to the strain of Lactobacillus plantarum C9 bacterial liquid The volume ratio of the medium to the MRS medium was 1:100, and the medium was inoculated with MRS medium, and the culture was activated at 37°C for 8 hours. Then, the medium was inoculated with MRS medium at a volume ratio of 1:100, and the culture was continued at 37°C for 16 hours; (2) Add 10 mL of 10% w / v NaHCO3 solution, 25 mL of 20% w / v glucose solution, and 2 mL of 5 mg / mL cysteine ​​hydrochloride solution to each 1 L of culture medium. Continue culturing for 40 hours and harvest the culture medium. (3) The harvested culture medium was centrifuged at 3000 g / min for 10 min to obtain the supernatant; (4) The supernatant was filtered using a 0.45 μm filter membrane and a medium control fiber tangential flow 750 KD filter to obtain a filtrate; (5) The filtrate was centrifuged at 35,000 g / min for 1 h, and then at 120,000 g / min for 1 h; (6) Discard the supernatant and resuspend the precipitate in PBS to obtain the extracellular vesicles of Lactobacillus plantarum C9 strain, which are named C9BEVs.

6. Use of the Lactobacillus plantarum exovesicles according to claim 4 or 5 in the following aspects: (1) Use in the preparation of highly effective natural immune adjuvants or immune enhancing preparations; (2) Use in the preparation of health-care or immunity-enhancing feed additives; (3) Use in the preparation of mucosal immune vaccines; (4) Use in the preparation of anti-infection and anti-inflammatory preparations.

7. The use according to claim 6, characterized in that The Lactobacillus plantarum exovesicles have the functions of enhancing mucosal immune response and promoting mucosal secretion of specific antibodies.

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