Lactobacillus gasseri strain for alleviating salmonella typhimurium infection and use thereof

Abstract

The application discloses a lactobacillus gasseri for relieving salmonella typhimurium infection and application thereof, and belongs to the technical field of microorganisms. The lactobacillus gasseri CCFM1307 screened by the application has the effect of relieving inflammation caused by salmonella typhimurium infection, specifically embodied in the following aspects: significantly relieving the symptom of weight loss of an individual infected with salmonella typhimurium, significantly relieving pathological damage of an ileum and a colon of the individual infected with salmonella typhimurium, significantly reducing the level of inflammatory cytokines in intestinal tissues of the individual infected with salmonella typhimurium, significantly increasing the content of short-chain fatty acids in feces, and reducing the relative abundance of Bacteroides in feces of the individual infected with salmonella typhimurium. The lactobacillus gasseri has great application prospects in preparation of drugs for preventing, treating and / or adjuvant treating inflammation caused by salmonella typhimurium infection.

Description

TECHNICAL FIELD

[0001] The present application relates to a lactobacillus gasseri strain for relieving salmonella typhimurium infection and application thereof, and belongs to the technical field of microorganisms. BACKGROUND

[0002] In the whole world, salmonella typhimurium is the pathogen causing the most deaths in foodborne diseases, and compared with other common foodborne pathogens, salmonella typhimurium has the greatest impact on human health. The infection of salmonella typhimurium is generally caused by the intake of contaminated animal-derived food. Compared with adults, infants are more susceptible to infection, and enteric infection caused by salmonella is one of the main causes of morbidity and mortality in infants in developing countries. It is estimated that there are about 16 million cases of salmonella typhi infection every year, and the number of deaths is about 200,000 to 600,000 per year. In clinical practice, the infection of salmonella typhimurium can cause symptoms such as headache, listlessness, fever, etc. in the host. After reaching the intestine, salmonella typhimurium contacts with intestinal epithelial cells through flagella and chemotaxis system, activates type III secretion system, and transmits the effector protein produced by salmonella typhimurium to intestinal epithelial cells, so that salmonella typhimurium is internalized to cause inflammatory response and produce various inflammatory cytokines such as IL-8, TNF-α and IL-1β. In addition, lipopolysaccharide and some lipoproteins in the cell wall of salmonella typhimurium can induce a large number of inflammatory responses in the posterior tissue, thereby producing various inflammatory cytokines such as IL-6 and IL-12. At present, the number of multidrug-resistant salmonella typhi isolates with fluoroquinolone resistance is gradually increasing, and salmonella typhi with resistance to ciprofloxacin and cephalosporin has appeared. When treating cats infected with salmonella typhimurium with systemic symptoms, the use of conventional antibiotics can lead to the production of drug-resistant strains, and can prolong the recovery period of cats. Due to drug resistance and possible side effects of antibiotic treatment, probiotics with bacteriostatic effect of bacteriocins are a good alternative to antibiotics.

[0003] Compared with other probiotics, lactobacillus gasseri (Lactobacillus gasseri) has the following advantages: (1) it can produce a variety of bacteriocins with antibacterial activity, and can inhibit the growth of pathogenic bacteria such as salmonella typhimurium, staphylococcus aureus, etc. (2) it can produce a variety of enzymes such as amylase, protease, etc., which can hydrolyze the nutrients in the intestinal tract and provide nutrients for the host. (3) it can produce a variety of short-chain fatty acids such as acetic acid, propionic acid, etc., which can inhibit the growth of pathogenic bacteria and promote the growth of beneficial bacteria in the intestinal tract. (4) it can produce a variety of immunomodulatory factors such as superoxide dismutase, etc., which can enhance the host's immune function and inhibit the growth of pathogenic bacteria. (5) it can produce a variety of probiotic factors such as folic acid, etc., which can promote the growth of the host. Lactobacillus gasseriLactobacillus gasseri is a probiotic naturally existing in the mucosa of the oral cavity, gastrointestinal tract and other mucosa, is one of the dominant species involved in the early colonization of intestinal microorganisms, has been included in the edible list, and can produce multiple bacteriocins such as lactobacillin T, lactobacillin K7B and acidocin B. In the current research, Lactobacillus gasseri SBT2055 producing lactobacillin T can competitively inhibit Campylobacter jejuni, thereby inhibiting the colonization of Campylobacter jejuni in the early growth stage of the natural host chick. Continuous intake of Lactobacillus gasseri CECT5714 and Lactobacillus coryniformis CECT5711 producing bacteriocins can increase the content of butyric acid in the human intestinal tract, promote the secretion of mucosal IgA and reduce the colonization of Salmonella choleraesuis. The recovery rate of patients with bacterial vaginosis who orally take capsules containing Lactobacillus gasseri after receiving clindamycin treatment is 18% higher than that of patients who do not receive it, the vaginal pH is reduced, the bacterial vaginosis-related diagnostic indicators are improved, and the total vaginal lactobacillus content is increased. Therefore, Lactobacillus gasseri producing bacteriocins can help the host maintain intestinal microecological stability, inhibit the infection of pathogens, and alleviate the damage caused by pathogen infection to the host.

[0004] Based on the comprehensive analysis of the current research and application status, the bacteriocin-producing Lactobacillus gasseri strains used to alleviate Salmonella typhimurium infection are not rich enough. It is necessary to screen more bacteriocin-producing Lactobacillus gasseri strains with the ability to alleviate Salmonella typhimurium infection in order to cope with individual, regional and diarrhea type differences. SUMMARY

[0005] The present application provides a Lactobacillus gasseri (Lactobacillus gasseri) strain CCFM1307. Lactobacillus gasseri The Lactobacillus gasseri strain CCFM1307 has been deposited at the Guangdong Microbial Culture Collection Center on June 7, 2023, with the accession number GDMCC No: 63533, and the address of the depositary is No. 59, 5th Floor, Building, 100, Martyrs' Road, Guangzhou.

[0006] The present application also provides a composition containing the Lactobacillus gasseri strain CCFM1307.

[0007] In one embodiment, the composition is a drug, a disinfectant or a feed additive.

[0008] The present application also provides a microbial preparation or a starter containing the Lactobacillus gasseri strain CCFM1307.

[0009] In one embodiment, the microbial preparation contains cells and / or cell culture of the Lactobacillus gasseri strain CCFM1307.

[0010] In one embodiment, the viable bacterial count of the Lactobacillus gasseri strain CCFM1307 in the microbial preparation is not less than 1×10 9 CFU / mL or 1×10 9 CFU / g.

[0011] In one embodiment, the preparation method of the fermentation agent is inoculating the Lactobacillus gasseri CCFM1307 into a culture medium, culturing at 37°C for 18 hours to obtain a culture solution; centrifuging the culture solution to obtain bacterial cells; and resuspending the bacterial cells to obtain the fermentation agent.

[0012] In one embodiment, the culture medium is MRS culture medium.

[0013] In one embodiment, the fermentation agent further comprises a cell protective agent.

[0014] The present application also provides use of the Lactobacillus gasseri CCFM1307 in the preparation of a medicament for preventing and / or treating inflammation caused by Salmonella typhimurium infection.

[0015] In one embodiment, the intestinal inflammation is caused by Salmonella typhimurium infection. Salmonella typhimurium ) infection.

[0016] In one embodiment, the prevention and / or treatment of inflammation caused by Salmonella typhimurium infection comprises at least one of the following effects:

[0017] (1) reducing the risk of weight loss in individuals infected with Salmonella typhimurium;

[0018] (2) alleviating pathological damage to the jejunum in individuals infected with Salmonella typhimurium;

[0019] (3) reducing the level of immune factors in intestinal tissue in individuals infected with Salmonella typhimurium;

[0020] (4) increasing the content of short-chain fatty acids in the feces of individuals infected with Salmonella typhimurium.

[0021] In one embodiment, the use further comprises regulating the intestinal flora.

[0022] In one embodiment, the regulation of the intestinal flora comprises reducing the relative abundance of Salmonella typhimurium in the intestinal flora of individuals infected with Salmonella typhimurium. Bacteroides

[0023] In one embodiment, the individual infected with Salmonella typhimurium is a mammal with symptoms of Salmonella typhimurium infection.

[0024] The present application also provides a medicament comprising the above-mentioned Lactobacillus gasseri CCFM1307, a pharmaceutical carrier and / or a pharmaceutical excipient.

[0025] In one embodiment, the dosage form of the medicament includes, but is not limited to, tablets, capsules, lozenges or liquid forms.

[0026] ​In an embodiment, the pharmaceutical carrier is commonly used for preparing pharmaceutical formulations, including but not limited to: lactose, glucose, sucrose, sorbitol, mannitol, starch, acacia, calcium phosphate, alginate, gelatin, calcium, silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, sugar syrup, methyl cellulose, methylparaben, propylparaben, talc, magnesium stearate or mineral oil.

[0027] In an embodiment, the pharmaceutical excipient includes but is not limited to lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents or preservatives.

[0028] The present application also provides use of the Lactobacillus gasseri CCFM1307 in the preparation of health products for regulating intestinal flora.

[0029] In an embodiment, the regulating intestinal flora includes but is not limited to reducing the risk of Bacteroides abnormal increase in relative abundance caused by Salmonella typhimurium infection.

[0030] In an embodiment, the health product further contains prebiotics.

[0031] The present application also provides use of the Lactobacillus gasseri CCFM1307 or microbial preparation containing the Lactobacillus gasseri CCFM1307 in the preparation of food, drink or condiment.

[0032] In an embodiment, the food includes but is not limited to dairy products, bean products, meat products or fruit and vegetable products produced by a fermenting agent containing the Lactobacillus gasseri CCFM1307.

[0033] In an embodiment, the number of viable bacteria of the Lactobacillus gasseri CCFM1307 in the product is not less than 1×10 9 CFU / mL or 1×10 9 CFU / g.

[0034] Beneficial effects:

[0035] The present application screens a Lactobacillus gasseri (Lactobacillus gasseri) Lactobacillus gasseri ) CCFM1307. The Lactobacillus gasseri (Lactobacillus gasseri) Lactobacillus gasseri ) CCFM1307 has the effect of relieving inflammation caused by Salmonella typhimurium infection, which is embodied in:

[0036] (1) reducing the risk of weight loss in individuals infected with Salmonella typhimurium;

[0037] (2) relieving pathological damage to the jejunum in individuals infected with Salmonella typhimurium;

[0038] (3) Reduce the level of immune factors in the intestinal tissue of individuals infected with Salmonella typhimurium;

[0039] (4) Increase the content of short-chain fatty acids in the feces of individuals infected with Salmonella typhimurium.

[0040] The Lactobacillus gasseri CCFM1307 provided by this invention can significantly alleviate inflammation caused by Salmonella typhimurium infection and can be used to prepare drugs for the prevention and treatment of Salmonella typhimurium infection, showing great application potential.

[0041] Preservation of biological materials

[0042] A strain of Lactobacillus gasseri ( Lactobacillus gasseri (CCFM1307, taxonomically named) Lactobacillus gasseri It was deposited on June 7, 2023, at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 63533, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. Attached Figure Description

[0043] Figure 1 : The rate of change in body weight of mice in each group during the modeling period.

[0044] Figure 2 : Pathological damage to ileum and colon tissues in each group.

[0045] Figure 3 Effects of each group on inflammatory cytokines in intestinal tissue; where A: IL-12; B: IL-6; C: IL-17; D: IL-1β.

[0046] Figure 4 Butyric acid content in the feces of mice in each group.

[0047] Figure 5 Isobutyric acid content in feces of mice in each group.

[0048] Figure 6 Valeric acid content in the feces of mice in each group.

[0049] Figure 7 Isovaleric acid content in the feces of mice in each group.

[0050] Figure 8 Differential bacterial species in the feces of mice in each group Bacteroides A schematic diagram of the relative abundance of [elements]. Detailed Implementation

[0051] Technical terms:

[0052] The "culture" referred to in the present application refers to a cell group or growth of microorganisms in a certain time and space, in particular refers to a liquid or solid culture product grown with microorganisms (such as the Lactobacillus gasseri CCFM1307 described in the present application) after artificial inoculation and culture, such as slant culture, fermentation product, etc.

[0053] The "bacterial suspension" referred to in the present application refers to a suspension obtained by dispersing cells of microorganisms (such as the Lactobacillus gasseri CCFM1307 described in the present application) in a solvent (such as water).

[0054] The "prebiotic" referred to in the present application refers to a food ingredient that promotes the growth of probiotics in the intestinal environment, including but not limited to all food ingredients that promote the growth of beneficial bacteria such as bifidobacteria or lactobacilli in the intestine, and / or probiotics. Optionally, it includes oligosaccharides, such as fructose, galactose, mannose; optionally, it includes dietary fiber, such as soluble fiber, soy fiber; optionally, it includes inulin; or a mixture thereof of any two or more of the above.

[0055] The "prevention" referred to in the present application refers to avoiding, minimizing or making it difficult to develop or develop a disease before the onset of the disease by treatment, including preventing Salmonella typhimurium infection, and / or inhibiting the development of or delaying the onset of diseases caused by Salmonella typhimurium infection.

[0056] The "treatment" referred to in the present application refers to preventing, curing, reversing, weakening, reducing, minimizing, inhibiting, stopping and / or stopping one or more clinical symptoms of a disease after the onset of the disease; including all behaviors that inhibit diseases caused by Salmonella typhimurium, and / or alleviate the pathological conditions of diseases caused by Salmonella typhimurium.

[0057] The "composition" referred to in the present application includes products containing Lactobacillus gasseri CCFM1307 in any form of food, medicine, health care product, disinfectant, drinking water additive, feed additive, etc.; wherein the medicine can be prepared according to any method in the prior art using a pharmaceutically acceptable carrier and / or excipient by those skilled in the art; the preparation can be provided in the form of a solution, suspension or emulsion in an oil or water medium, or in the form of an extract, powder, granules, tablets or capsules, and can also contain dispersants or stabilizers.

[0058] Animal models and reagents:

[0059] The C57BL / 6J mice referred to in the following examples were purchased from Zhejiang Weitong Lihua Company, and Salmonella typhimurium (S. typhimurium) was purchased from the National Institute for Food and Drug Control. Salmonella typhimuriumCICC 21483 was obtained from China Industrial Culture Collection Center (CICC); streptomycin involved in the following examples was purchased from Shanghai Generay Biotech Co., Ltd.; ELISA kits for detecting IFN-γ (article number: SBJ-M0594-96T) and IL-12 (article number: SBJ-M0038-96T) involved in the following examples were purchased from Nanjing Sunbiogene Biotech Co., Ltd.; ELISA kits for detecting IL-6 (article number: DY406-05), IL-10 (article number: DY417-05), IL-17 (article number: DY421-05), IL-1β (article number: DY401-05) and TNF-α (article number: DY410-05) involved in the following examples were purchased from R&D Co., Ltd.

[0060] Culture medium:

[0061] MRS solid medium: 10 g / L of proteose peptone, 10 g / L of beef extract, 20 g / L of glucose, 2 g / L of sodium acetate, 5 g / L of yeast powder, 2 g / L of diammonium hydrogen citrate, 2.6 g / L of K2HPO4·3H2O, 0.1 g / L of MgSO4·7H2O, 0.05 g / L of MnSO4, 1 mL / L of Tween 80, 15 g / L of agar, 0.5 g / L of cysteine.

[0062] MRS liquid medium: 10 g / L of proteose peptone, 10 g / L of beef extract, 20 g / L of glucose, 2 g / L of sodium acetate, 5 g / L of yeast powder, 2 g / L of diammonium hydrogen citrate, 2.6 g / L of K2HPO4·3H2O, 0.1 g / L of MgSO4·7H2O, 0.05 g / L of MnSO4, 1 mL / L of Tween 80, 0.5 g / L of cysteine.

[0063] Example 1: Screening and strain identification of Lactobacillus gasseri CCFM1307

[0064] 1. Screening

[0065] The sample from Xiaoyi, Henan Province was stored in 30% glycerol at -80°C. 1 mL of the sample was taken and added to 9 mL of sterile normal saline (0.85%, w / v), shaken well, and gradient diluted by 10-fold dilution method. 200 μL of the dilution of 10 -3 ,10 -4 ,10 -5The diluted solution was plated onto MRS solid medium and incubated anaerobically at 37°C for 72 h. Colony morphology was observed and recorded. Colonies were picked, streaked, and purified. Then, the colonies were incubated in MRS liquid medium at 37°C for 48 h. Gram staining was performed on the obtained colonies, and strain morphology was recorded. Gram-negative and Gram-positive cocci were discarded, and Gram-positive bacilli were selected. After catalase analysis, catalase-positive strains were discarded, and catalase-negative strains were retained. All obtained strains were identified as *Lactobacillus gasseri* by 16S rDNA sequencing. The obtained *Lactobacillus gasseri* were passaged, and the bacterial cells were collected and centrifuged at 3000 rpm for 10 min in centrifuge tubes. This process was repeated three times. The obtained bacterial cells were then cryopreserved with a substrate protectant.

[0066] 2. Identification

[0067] The genome of this strain was extracted, and 16S rDNA was amplified and sequenced using the genome as a template (performed by BGI Genomics Co., Ltd.). The sequence was compared with the nucleic acid sequence in NCBI, and the results showed that the strain was Lactobacillus gasseri, which was deposited at the Food Biotechnology Culture Collection Center of Jiangnan University and named Lactobacillus gasseri CCFM1307. It was also deposited at the Guangdong Provincial Microbial Culture Collection Center on June 7, 2023, with the accession number GDMCC No: 63533.

[0068] Example 2: Cultivation of Lactobacillus gasseri CCFM1307 and preparation of bacterial suspension

[0069] 1. Culture and preservation of Lactobacillus gasseri CCFM1307

[0070] The Lactobacillus gasseri CCFM1307 obtained in Example 1 was inoculated into MRS solid medium and cultured in an anaerobic incubator at 37°C for 48 h. After observation, the colonies were found to be grayish-white with rough edges and round convexity.

[0071] The Lactobacillus gasseri obtained in Example 1 ( Lactobacillus gasseri CCFM1307 was inoculated into MRS liquid medium and cultured anaerobically at 37℃ for 24 h. Then, it was transferred to fresh MRS liquid medium and cultured under the same conditions for 24 h. The bacterial cells were centrifuged at 6000g for 15 min, washed with 0.9g / 100mL physiological saline, and centrifuged again at 6000g for 10 min to obtain the bacterial cells. The cells were then added to 30% glycerol and frozen at -80℃ for later use.

[0072] 2. Prepare bacterial suspension as follows:

[0073] (1) Activation culture:

[0074] MRS liquid culture medium was used, and the culture was statically incubated in an anaerobic workstation at 37°C.

[0075] Culture objective: Take cryopreserved bacterial cells, pick single colonies and place them in liquid MRS medium, and incubate statically in an anaerobic workstation at 37°C for about 24 hours to activate Lactobacillus gasseri CCFM1307.

[0076] (2) Primary training:

[0077] MRS liquid culture medium was used, and the culture was statically incubated in an anaerobic workstation at 37°C.

[0078] Culture objective: Transfer activated Lactobacillus gasseri CCFM1307 to MRS liquid medium at an inoculum volume of 1% (by volume of the medium) and passage it for two generations.

[0079] (3) Secondary cultivation:

[0080] The activated bacterial culture, after three generations, was inoculated into 1 L of MRS liquid medium at a 2% (v / v) inoculation rate. After shaking and mixing, it was incubated in an anaerobic incubator at 37°C for 24 h. The culture was centrifuged at 8000 g / min at 4°C for 15 min, the supernatant was discarded, and the culture was washed twice with sterile physiological saline. After centrifugation under the same conditions and discarding of the supernatant, the culture was resuspended in 30% glycerol to obtain the bacterial culture for gavage administration. The culture was centrifuged at 6000 rpm for 5 min, washed twice with sterile physiological saline, resuspended in physiological saline, and shaken thoroughly. The initial viable count was determined using the pour plate method. The culture was then frozen in 30% glycerol at -80°C for one week, and the viable count was determined again.

[0081] Experimental results: The initial viable count was 7.03 × 10⁻⁶. 9 CFU / mL, viable bacterial count after 1 week was 6.5 × 10⁻⁶. 9 The cfu / mL concentration did not change in order of magnitude, indicating that freezing the bacterial culture would not affect the experiment and it can be used for animal experiments.

[0082] Example 3: Lactobacillus gasseri CCFM1307 alleviates weight loss in mice infected with Salmonella typhimurium.

[0083] 1. Laboratory animals

[0084] Seven-week-old female SPF-grade C57BL / 6J mice were obtained from Zhejiang Vital River Laboratory Animal Technology Co., Ltd., China. Mice were housed in polypropylene cages, eight mice per group, with food and water provided in the cages. Temperature (22℃) and relative humidity (50±10%) were controlled. Mice were fed standard feed and any amount of water.

[0085] 2. Experimental Methods

[0086] (1) Salmonella Typhimurium ( Salmonella typhimurium Establishment of a CICC 21483-infected mouse model

[0087] C57BL / 6J mice aged 7 weeks were selected, and the 7 days before the experiment were the adaptation period, the 8-17 days were the intervention period, and the 13-17 days were the modeling period. After 2 h of fasting and water deprivation, 5x10 6 0.2 mL of Salmonella typhimurium bacterial suspension with a CFU / mL of 0.2 mL, immediately restore drinking water, 2 h later feed, the blank group was given 0.2 mL of sterile normal saline by gavage. During the modeling period, the mice were weighed every day.

[0088] (2) Experimental grouping and administration

[0089] The mice were divided into a blank group, a model group, and a CCFM1307 group.

[0090] Before gavage, the bacterial solution was taken out from -80℃, centrifuged at 6000 r / min for 5 min, washed twice with sterile normal saline, and resuspended in normal saline, with a concentration of 5x10 9 CFU / mL.

[0091] During the intervention period, the blank group and the model group were given 0.2 mL of sterile normal saline by gavage every day for a total of 10 days. The CCFM1307 group was given Lactobacillus gasseri CCFM1307 bacterial suspension by gavage, and the mice in each group were fed with standard feed during the experiment.

[0092] In clinical practice, infection with non-typhoid Salmonella can cause symptoms such as headache, listlessness, fever, and in existing research, it has been found that mice infected with Salmonella typhimurium experience a sustained decrease in body weight. During the entire modeling period, the body weight of the model group mice decreased by 11.4%, and the body weight of the blank group and the CCFM1307 group decreased by 7.1% and 7.26%, respectively, which was significantly different from the model group (p<0.05) Figure 1 Therefore, Lactobacillus gasseri CCFM1307 has a good effect on restoring the decrease in body weight of mice infected with Salmonella typhimurium.

[0093] Example 4: Lactobacillus gasseri CCFM1307 improves ileum and colon tissue damage in mice infected with Salmonella typhimurium

[0094] The method for constructing the animal model is the same as in Example 3. After the experiment, the mice are sacrificed, and the ileum and colon of the mice are taken and soaked in 4% (v / v) paraformaldehyde for 24 h to obtain fixed intestinal tissues. The fixed intestinal tissues are sequentially dehydrated, transparentized, and impregnated with wax, and then the tissues are embedded in wax blocks using a Leica paraffin embedding machine to obtain wax blocks with embedded intestinal tissues. The specific steps of dehydration, transparentization, and impregnation with wax are as follows: (1) dehydration: the fixed tissues are sequentially dehydrated in 70%, 80%, and 90% (v / v) gradient ethanol solutions for 30 min each, and then placed in 95% and 100% (v / v) alcohol solutions for 20 min each time for 2 times; (2) transparentization: the tissues are first placed in an alcohol and xylene mixed solution with a volume ratio of 1:1 for 15 min, and then placed in xylene I and xylene II for 15 min each; (3) impregnation with wax: the tissue samples are placed in 62°C paraffin I and paraffin II liquids for 30 min each.

[0095] The wax block embedding the intestinal tissue is sliced by a Leica manual rotary microtome, and the thickness of the slice is 5 μm, to obtain an intestinal tissue slice; the intestinal tissue slice is subjected to spreading and fishing, baking, hematoxylin staining, differentiation, rinsing, eosin re-staining, dehydration, transparency, and mounting, to obtain an H&E slice; wherein, the specific operations of spreading and fishing, baking, hematoxylin staining, differentiation, rinsing, eosin re-staining, dehydration, transparency, and mounting are as follows: (1) spreading and fishing: the slice is placed in a 42℃ constant temperature water bath for spreading, and then carefully fished out with a glass slide; (2) baking: the slice is placed in a 60℃ oven overnight for baking; (3) hematoxylin staining: the slice is first hydrated (i.e., first placed in xylene I and xylene II for 5 min each, and then placed in 100%, 95%, 90%, 80% and 70% (v / v) gradient alcohol solutions for 5 min each, and finally placed in distilled water for 3 min), then dyed (i.e., the slice is placed in a hematoxylin staining solution for about 20 s), and finally washed (i.e., the slice is washed with tap water for about 30 min); (4) differentiation: the slice is placed in a 1% (v / v) hydrochloric acid ethanol solution for 7 s for decolorization; (5) rinsing: the slice is washed with tap water for about 20 min; (6) re-staining: the slice is immersed in an eosin staining solution and immediately taken out; (7) dehydration: the slice is first placed in 95% (v / v) ethanol solution I, 95% (v / v) ethanol solution II, and 70% (v / v) ethanol solution, and immediately taken out after each placement, then immersed in 80% (v / v) ethanol solution for 50 s, and finally immersed in 100% (v / v) ethanol for 2 min; (8) transparency: the slice is first immersed in an equal volume mixture of ethanol and xylene for 1 min, and then immersed in xylene I and xylene II for 2 min each; (9) mounting: the slice is mounted with neutral balsam. The prepared H&E intestinal slice is scanned by a Pannoramic MIDI digital slice scanner, and photographed to observe the damage of the mouse intestinal tissue and perform pathological scoring, and the results are shown in Figure 2 .

[0096] The mouse ileum and colon tissues infected with Salmonella typhimurium exhibit inflammatory cell infiltration and other characteristics, which can serve as an indicator of the damage caused by Salmonella typhimurium infection to some extent. Figure 2 It is shown that, compared with the blank group, the ileal tissue of the model group mice has villus shedding, and there is a large area of inflammatory cell infiltration in the ileum and colon, and Lactobacillus gasseri CCFM1307 can well alleviate this symptom and significantly reduce the pathological score.

[0097] Example 5: Lactobacillus gasseri CCFM1307 reduces the level of inflammatory factors in the ileum of mice infected with Salmonella typhimurium

[0098] The C57BL / 6J mice are grouped, modeled, and treated in the same manner as in Example 3.

[0099] After the mice were sacrificed on day 18, the ileum of the mice was homogenized, and tissue supernatant was obtained after centrifugation at 4000 x g for 15 min. The IL-12 level in the ileum of mice infected with Salmonella typhimurium was detected by enzyme-linked immunosorbent assay (ELISA). As shown in Figure 3 the IL-12 level in the ileum tissue of the model group was 3.40 pg / mg, which was significantly increased by Salmonella typhimurium infection (p<0.05) compared with the blank group (1.84 pg / mg), and was 1.85 times that of the blank group. Compared with the model group, Lactobacillus gasseri CCFM1307 treatment significantly reduced the IL-12 level in the ileum of mice to 2.39 pg / mg (29.71% lower than the model group).

[0100] Example 6: Lactobacillus gasseri CCFM1307 reduces the level of inflammatory factors in the colon of mice infected with Salmonella typhimurium

[0101] After the mice were sacrificed on day 18, the colon of the mice was homogenized, and tissue supernatant was obtained after centrifugation at 4000 x g for 15 min. The IL-6, IL-17, and IL-1β levels in the colon of mice infected with Salmonella typhimurium were detected by enzyme-linked immunosorbent assay (ELISA).

[0102] As shown in Figure 3 the IL-6 level in the colon tissue of the model group mice was 99.51 pg / mg, which was significantly higher than that of the blank group (56.62 pg / mg); compared with the model group mice, the IL-6 level in the colon tissue of the CCFM1307 group mice was significantly reduced to 58.98 pg / mg, a decrease of 40.80%. The IL-17 level in the colon tissue of the model group mice was 99.65 pg / mg, which was significantly higher than that of the blank group (65.38 pg / mg); compared with the model group mice, the IL-17 level in the colon tissue of the CCFM1307 group mice was significantly reduced to 64.30 pg / mg, a decrease of 35.47%. The IL-1β level in the colon tissue of the model group mice was 182.53 pg / mg, which was significantly higher than that of the blank group (123.16 pg / mg); compared with the model group mice, the IL-6 level in the colon tissue of the CCFM1307 group mice was significantly reduced to 111.87 pg / mg, a decrease of 38.71%.

[0103] The above results show that the inflammatory factor level of the model group mice is increased compared with the blank group, and Lactobacillus gasseri CCFM1307 can well alleviate the inflammation of the colon caused by Salmonella typhimurium infection.

[0104] Example 7: Lactobacillus gasseri CCFM1307 increases the content of short-chain fatty acids in feces of mice infected with Salmonella typhimurium

[0105] The C57BL / 6J mice were grouped, modeled and treated as in Example 3.

[0106] After the end of the experiment, the mouse feces were collected and placed in liquid nitrogen, then transferred to a -80°C refrigerator, and before the detection of the content of short-chain fatty acids, the feces were taken out, vacuum freeze-dried, accurately weighed 0.05 g of the freeze-dried feces sample, dissolved in 0.5 mL of saturated sodium chloride solution, soaked for 30 min, homogenized by a tissue homogenizer, added with 0.02 mL of 10% sulfuric acid, oscillated for 30 s, accurately added with 1 mL of ether solution to the feces solution in a fume hood, oscillated for 30 s, and centrifuged for 15 min (8000 g, 4°C), and the supernatant was transferred to a centrifuge tube containing 0.25 g of anhydrous sodium sulfate, oscillated uniformly, centrifuged for 15 min (8000 g, 4°C), and the supernatant was taken to a GC-MS volume flask, and the content of short-chain fatty acids was detected by GC-MS.

[0107] As shown in Figure 4~7 After the mice were infected with Salmonella typhimurium, the contents of butyric acid, isobutyric acid, valeric acid and isovaleric acid in the feces of the model group mice were all reduced to a certain extent compared with the blank group, wherein the content of butyric acid was 5.88 μmol / g, the content of isobutyric acid was 2.67 μmol / g, the content of valeric acid was 2.01 μmol / g, and the content of isovaleric acid was 2.38 μmol / g, which were respectively decreased by 36.57%, 32.23%, 37.58% and 35.15% (p<0.05) compared with the blank group. However, after the mice were orally administered with Lactobacillus gasseri CCFM1307, the contents of butyric acid, isobutyric acid, valeric acid and isovaleric acid were respectively 8.02 μmol / g, 3.70 μmol / g, 2.89 μmol / g and 3.25 μmol / g, which were respectively increased by 36.39%, 38.58%, 43.78% and 36.55% (p<0.05) compared with the model group. Short-chain fatty acids can reduce the pH value of the intestinal tract, promote the absorption of calcium and magnesium ions in the intestinal tract, and inhibit the infection of harmful bacteria.

[0108] Example 8: Lactobacillus gasseri CCFM1307 reduces the relative abundance of Salmonella typhimurium in the feces of mice infected with Salmonella typhimurium Bacteroides Relative abundance

[0109] The C57BL / 6J mice were grouped, modeled and treated as in Example 3.

[0110] After the end of the experiment, the genomic DNA in the feces was extracted, the V3-V4 region was specifically PCR-amplified, 16S rDNA sequencing was performed, and the change of the fecal flora was analyzed. As shown in Figure 8, the results showed that compared with the blank group, the relative abundance of Salmonella typhimurium in the feces of infected mice Bacteroides significantly increased (p<0.05). CCFM1307 can significantly reduce the relative abundance (p<0.05) of Salmonella typhimurium in the feces of infected mice. Bacteroides

[0111] The above results show that Lactobacillus gasseri CCFM1307 can effectively improve the symptoms of weight loss and intestinal tissue damage in mice. By regulating inflammatory factors IL-12, IFN-γ, IL-6, IL-17 and IL-1β, it can alleviate the inflammation caused by Salmonella typhimurium infection. By increasing the content of short-chain fatty acids to reduce the pH value of the intestine, inhibit the invasion of harmful bacteria, stimulate intestinal peristalsis, and regulate intestinal flora to maintain intestinal microecological homeostasis.

[0112] Example 9: Lactobacillus gasseri CCFM1307 for preparing a medicament

[0113] Lactobacillus gasseri CCFM1307 screened in Example 1 can inhibit Salmonella typhimurium (S. typhimurium) Salmonella typhimurium ), and can prevent and treat diseases caused by Salmonella typhimurium infection, and is used as an active ingredient to prepare bacteriostatic and / or bactericidal products, and drugs for preventing / treating Salmonella typhimurium infection.

[0114] The drug contains Lactobacillus gasseri CCFM1307 and a pharmaceutically acceptable carrier, and the dosage form of the drug can be any one of solution, powder, tablet, capsule, suspension, emulsion.

[0115] Taking the drug in the form of tablets as an example, the specific preparation process is as follows:

[0116] Pick up the single colony of Lactobacillus gasseri (L. gasseri) Lactobacillus gasseri ) CCFM1307 obtained in Example 1 and inoculate it into MRS liquid medium, and incubate it at 37°C for 16 hours to obtain a bacterial concentration of 1×10 7 ​The activation solution was prepared at CFU / mL. The activation solution was inoculated into MRS liquid medium at a 1% (v / v) inoculation rate and cultured at 37°C for 16 h to obtain the primary seed culture. The primary seed culture was inoculated into MRS liquid medium at a 1% (v / v) inoculation rate and cultured at 37°C for 16 h to obtain the secondary seed culture. The secondary seed culture was inoculated into MRS liquid medium at a 1% (v / v) inoculation rate and cultured at 37°C for 16 h to obtain the bacterial culture. The bacterial culture was centrifuged at 6000 g for 15 min, and the precipitate was collected. The precipitate was washed twice with PBS buffer at pH 7.4, and then centrifuged again at 6000 g for 10 min to obtain the bacterial cells. The Lactobacillus gasseri bacterial cells were resuspended in a protective solution containing 130 g / L skim milk, 20 g / L trehalose, and 20 g / L sucrose to a cell concentration of 1×10⁻⁶. 9 CFU / mL was used to obtain Lactobacillus gasseri bacterial solution; the Lactobacillus gasseri bacterial solution was freeze-dried to obtain Lactobacillus gasseri powder; 2% stearic acid (by weight of Lactobacillus gasseri powder) as a lubricant and 3% sodium carboxymethyl cellulose (CMC-Na) as a binder were added to the Lactobacillus gasseri powder and then compressed into tablets to obtain tablets.

[0117] The tablets prepared by the above method were administered orally to mice infected with Salmonella typhimurium at a dose of 1 g / mouse daily for 10 consecutive days. This effectively relieved the inflammatory symptoms caused by Salmonella typhimurium and had excellent effects in preventing and / or treating inflammation caused by Salmonella typhimurium.

[0118] Example 10: Lactobacillus gasseri CCFM1307 used to prepare bacterial powder

[0119] Lactobacillus gasseri CCFM1307 can be used to prepare bacterial powder. The specific preparation process for the bacterial powder is as follows:

[0120] Pick the Lactobacillus gasseri obtained in Example 1 ( Lactobacillus gasseri A single colony of CCFM1307 was inoculated into MRS liquid medium and cultured at 37°C for 16 h, yielding a bacterial concentration on the order of 1×10⁻⁶. 7CFU / mL; the activation liquid was inoculated into MRS liquid medium at an inoculation amount of 1% (v / v) and cultured at 37°C for 16 h to obtain a first-stage seed liquid; the first-stage seed liquid was inoculated into MRS liquid medium at an inoculation amount of 1% (v / v) and cultured at 37°C for 16 h to obtain a second-stage seed liquid; the second-stage seed liquid was inoculated into MRS liquid medium at an inoculation amount of 1% (v / v) and cultured at 37°C for 16 h to obtain a bacterial liquid; the bacterial liquid was centrifuged at 6000 g for 15 min to collect the precipitate; the precipitate was washed twice with PBS buffer having a pH of 7.4 and then centrifuged at 6000 g for 10 min to obtain bacterial bodies; the bacterial bodies of L. gasseri were resuspended in a protective agent solution containing 130 g / L skim milk, 20 g / L trehalose and 20 g / L sucrose to a cell concentration of 1 x 10 9 CFU / mL, to obtain a L. gasseri bacterial liquid; the L. gasseri bacterial liquid was freeze-dried to obtain a bacterial powder.

[0121] The tablets prepared by the above method were administered to mice infected with S. typhimurium at a dose of 1 g per mouse per day for 10 consecutive days, which effectively alleviated the inflammation symptoms caused by S. typhimurium and had an excellent effect on preventing and / or treating inflammation caused by S. typhimurium.

[0122] Example 11: L. gasseri CCFM1307 for preparing fermented food

[0123] L. gasseri CCFM1307 or the bacterial powder containing L. gasseri CCFM1307 prepared in Example 10 was used to prepare fermented food or fermented drink.

[0124] The raw materials for preparing the fermented drink can be selected from the group consisting of one or more of animal milk, plant milk, fruit and vegetable juice, and grain juice.

[0125] The raw materials for preparing the fermented food can be selected from the group consisting of animal milk, plant milk, fruit and vegetable raw materials, grain powder or grain mixture, and meat product raw materials.

[0126] Example 12: L. gasseri CCFM1307 for preparing health products

[0127] L. gasseri CCFM1307 or the bacterial powder containing L. gasseri CCFM1307 prepared in Example 10 was used to prepare health products. Alternatively, the health products were prepared by compounding L. gasseri CCFM1307 and prebiotics. The prebiotics included, but were not limited to, all food ingredients that promote the growth of beneficial bacteria such as bifidobacteria or lactobacilli in the intestines, and / or probiotics, such as oligosaccharides, dietary fibers, bacterial powders, etc.

[0128] Optionally, the oligosaccharide is selected from fructose, galactose or mannose; and the dietary fiber is selected from soluble fiber or soy fiber.

[0129] While the application has been disclosed by reference to the preferred embodiments thereof, a worker skilled in the art will recognize that changes can be made in form and detail without departing from the spirit and the scope of the application. Accordingly, the proper scope of the application is to be determined by reference to the appended claims.

Claims

1. A strain of Lactobacillus gasseri ( Lactobacillus gasseri CCFM1307 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on June 7, 2023, with accession number GDMCC No: 63533.

2. Composition comprising Lactobacillus gasseri CCFM1307 according to claim 1, characterized in that, The composition is a pharmaceutical product, a disinfectant or a feed additive.

3. The composition of claim 2, wherein, The pharmaceutical product contains the Lactobacillus gasseri CCFM1307, a pharmaceutical carrier and / or a pharmaceutical adjuvant.

4. A microbial preparation containing the Lactobacillus gasseri CCFM1307 of claim 1.

5. The microbial preparation according to claim 4, characterized in that, The microbial preparation contains cells and / or cell culture of the Lactobacillus gasseri CCFM1307.

6. The microbial preparation according to claim 4 or 5, characterized in that, The viable cell number of Lactobacillus gasseri CCFM1307 in the microbial preparation is not less than 1 x 10 9 CFU / g or 1 x 10 9 CFU / mL.

7. A fermenting agent containing the Lactobacillus gasseri CCFM1307 of claim 1.

8. Use of the Lactobacillus gasseri CCFM1307 of claim 1 in the preparation of a medicament for preventing and / or treating inflammation caused by Salmonella typhimurium infection.

9. Use according to claim 8, characterized in that, The use includes at least one of the following effects: (1) reducing the risk of weight loss in an individual infected with Salmonella typhimurium; (2) alleviating pathological damage to the jejunum in an individual infected with Salmonella typhimurium; (3) reducing the level of immune factors in the intestinal tissue of an individual infected with Salmonella typhimurium; (4) increasing the content of short-chain fatty acids in the feces of an individual infected with Salmonella typhimurium.

10. Use according to claim 8 or 9, characterized in that, The use also includes regulating intestinal flora.

11. Use of the Lactobacillus gasseri CCFM1307 of claim 1 in the preparation of a health product for helping to regulate intestinal flora.

12. Use according to claim 11, characterized in that, The health product also contains a prebiotic.

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