CN116925980A - Lactobacillus gasseri strain for relieving salmonella typhimurium infection and application thereof - Google Patents
Lactobacillus gasseri strain for relieving salmonella typhimurium infection and application thereof Download PDFInfo
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- CN116925980A CN116925980A CN202311120721.1A CN202311120721A CN116925980A CN 116925980 A CN116925980 A CN 116925980A CN 202311120721 A CN202311120721 A CN 202311120721A CN 116925980 A CN116925980 A CN 116925980A
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- lactobacillus gasseri
- salmonella typhimurium
- ccfm1307
- mice
- infection
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Abstract
The invention discloses a lactobacillus gasseri strain for relieving salmonella typhimurium infection and application thereof, and belongs to the technical field of microorganisms. The screened lactobacillus gasseri CCFM1307 has the effect of relieving inflammation caused by salmonella typhimurium infection, and is specifically expressed in the following steps: the method has the advantages of remarkably relieving the symptom of weight reduction of salmonella typhimurium infected individuals, remarkably relieving pathological injuries of ileum and colon of salmonella typhimurium infected individuals, remarkably reducing the inflammatory cytokine level in intestinal tissues of salmonella typhimurium infected individuals, remarkably improving the content of short chain fatty acid in feces, and reducing the relative abundance of bacteroides in the feces of salmonella typhimurium infected individuals. Has great application prospect in preparing medicines for preventing, treating and/or assisting in treating inflammation caused by salmonella typhimurium infection.
Description
Technical Field
The invention relates to a lactobacillus gasseri strain for relieving salmonella typhimurium infection and application thereof, belonging to the technical field of microorganisms.
Background
Salmonella typhimurium is the most dying pathogen of food-borne diseases worldwide, and has the greatest effect on human health compared with other common food-borne pathogens. Salmonella typhimurium infection is generally caused by ingestion of contaminated animal-derived foods. Infants are more susceptible to infection than adults. Clinically, infection with Salmonella typhimurium causes symptoms such as headache, listlessness, fever, etc. in the host. After reaching the intestinal tract, salmonella typhimurium contacts with intestinal epithelial cells through flagella and chemotactic system to activate type III secretion system, and effector protein generated by salmonella typhimurium is transferred to the intestinal epithelial cells, so that salmonella typhimurium is internalized to cause inflammatory reaction, and various inflammatory cytokines such as IL-8, TNF-alpha, IL-1 beta and the like are generated. In addition, lipopolysaccharide and certain lipoproteins in the cell wall of Salmonella typhimurium induce a large number of inflammatory responses in the guard tissue, thereby producing a variety of inflammatory cytokines such as IL-6 and IL-12. At present, multi-drug resistant salmonella typhi isolates with fluoroquinolone resistance are increasing, and salmonella typhi with resistance to ciprofloxacin and cephalosporin are emerging. In treating cats with systemic symptoms infected with Salmonella typhimurium, conventional use of antibiotics results in the production of resistant strains and may extend the recovery period of the cat. Because of drug resistance and possible side effects of antibiotic treatment, probiotics with bacteriocin having antibacterial effect can be produced without losing as a good substitute for antibiotics.
Compared with other probiotics, lactobacillus gasseri (Lactobacillus gasseri) is a probiotic naturally existing in mucous membranes of the oral cavity, the gastrointestinal tract and the like, is one of dominant species involved in early colonization of intestinal microorganisms, has been recorded in an edible directory, and can produce various bacteriocins such as lactobacillus gasseri T, lactobacillus gasseri K7B, and acidocin B. In the current research, the lactobacillus gasseri SBT2055 for producing the lactobacillus gasseri T can competitively inhibit campylobacter jejuni, thereby inhibiting the colonization of campylobacter jejuni by a natural host chicken of campylobacter jejuni in an early growth stage. Continuous ingestion of bacteriocin-producing Lactobacillus gasseri CECT5714 and Lactobacillus corynebacterium CECT5711 can increase butyric acid content in human intestinal tract, promote secretion of mucosal layer IgA and reduce colonization of Salmonella cholerae. The recovery rate of the oral administration of the capsule containing lactobacillus gasseri after the clindamycin treatment is 18% higher than that of the oral administration of the capsule containing lactobacillus gasseri, the pH of the vagina is reduced, the relevant diagnostic index of the bacterial vaginitis is improved, and the total lactobacillus content of the vagina is increased. Therefore, the bacteriocin-producing lactobacillus gasseri can help the host to maintain intestinal microecology stable, inhibit pathogen infection and relieve the damage caused by pathogen infection to the host.
The current research application situation of comprehensive analysis is that the bacterial bacteriocin-producing lactobacillus gasseri strain for alleviating salmonella typhimurium infection is not abundant enough, and the screening of more bacterial bacteriocin-producing lactobacillus gasseri strains for alleviating salmonella typhimurium infection is necessary for coping with individual, regional and diarrhea type differences.
Disclosure of Invention
The invention provides a lactobacillus gasseri (Lactobacillus gasseri) CCFM1307 which is preserved in the microorganism strain collection of Guangdong province in 2023, 6 and 7 days, wherein the preservation number is GDMCC No. 63533, and the preservation address is building 5 of No. 59 of 100 university of Mitsui in Guangzhou City.
The invention also provides a composition containing the lactobacillus gasseri CCFM1307.
In one embodiment, the composition is a food, pharmaceutical, nutraceutical, disinfectant, potable water additive, or feed additive.
The invention also provides a microbial preparation or a starter containing the lactobacillus gasseri CCFM1307.
In one embodiment, the microbial preparation contains cells and/or cell cultures of the lactobacillus gasseri CCFM1307.
In one embodiment, the viable count of Lactobacillus gasseri CCFM1307 in the microbial preparation is not less than 1×10 9 CFU/mL or 1X 10 9 CFU/g。
In one embodiment, the method of preparing the starter is: inoculating the lactobacillus gasseri CCFM1307 into a culture medium, and culturing for 18 hours at 37 ℃ to obtain a culture solution; centrifuging the culture solution to obtain thalli; the bacteria are resuspended to obtain the ferment.
In one embodiment, the medium is MRS medium.
In one embodiment, the fermenting agent further comprises a cytoprotective agent.
The invention also provides application of the lactobacillus gasseri CCFM1307 in preparing a medicament for preventing and/or treating inflammation caused by salmonella typhimurium infection.
In one embodiment, the intestinal inflammation is caused by infection with salmonella typhimurium (Salmonella typhimurium).
In one embodiment, the preventing and/or treating inflammation caused by salmonella typhimurium infection comprises at least one of the following effects:
(1) Reducing the risk of weight loss in Salmonella typhimurium infected individuals;
(2) Relieving pathological damage of jejunum of individual infected by salmonella typhimurium;
(3) Reducing the level of immune factors in intestinal tissue of an individual infected with Salmonella typhimurium;
(4) The content of short chain fatty acid in the feces of the salmonella typhimurium infected individual is improved.
In one embodiment, the use further comprises modulating the intestinal flora.
In one embodiment, the modulating the intestinal flora comprises reducing the relative abundance of bacterioides in the intestinal tract of a salmonella typhimurium infected individual.
In one embodiment, the salmonella typhimurium infected individual is a mammal having symptoms of salmonella typhimurium infection.
The invention also provides a medicine which contains the lactobacillus gasseri CCFM1307, a medicine carrier and/or a pharmaceutic adjuvant.
In one embodiment, the dosage form of the medicament includes, but is not limited to, a tablet, capsule, lozenge, or liquid form.
In one embodiment, the pharmaceutical carrier is a carrier commonly used in the preparation of pharmaceutical formulations including, but not limited to: lactose, dextrose, sucrose, sorbitol, mannitol, starches, acacia, calcium phosphate, alginates, gelatin, calcium, silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate or mineral oil.
In one embodiment, the pharmaceutical excipients include, but are not limited to, lubricants, wetting agents, sweeteners, flavoring agents, emulsifying agents, suspending agents, or preservatives.
The invention also provides application of the lactobacillus gasseri CCFM1307 in preparing health care products for regulating intestinal flora.
In one embodiment, the modulation of intestinal flora includes, but is not limited to, reducing abnormal elevation in the relative abundance of bacteriodes caused by salmonella typhimurium infection.
In one embodiment, the health product further comprises a prebiotic.
The invention also provides application of the lactobacillus gasseri CCFM1307 or the microbial preparation containing the lactobacillus gasseri CCFM1307 in preparing foods, drinks or seasonings.
In one embodiment, the food product includes, but is not limited to, a dairy product, a soy product, a meat product, or a fruit and vegetable product produced from a starter culture comprising the lactobacillus gasseri CCFM1307.
In one embodiment, the viable count of the Lactobacillus gasseri CCFM1307 in the product is not less than 1×10 9 CFU/mL or 1X 10 9 CFU/g。
The beneficial effects are that:
the invention screens out a strain of lactobacillus gasseri (Lactobacillus gasseri) CCFM1307, and the lactobacillus gasseri (Lactobacillus gasseri) CCFM1307 has the effect of relieving inflammation caused by salmonella typhimurium infection, and is specifically expressed in the following steps:
(1) Reducing the risk of weight loss in Salmonella typhimurium infected individuals;
(2) Relieving pathological damage of jejunum of individual infected by salmonella typhimurium;
(3) Reducing the level of immune factors in intestinal tissue of an individual infected with Salmonella typhimurium;
(4) The content of short chain fatty acid in the feces of the salmonella typhimurium infected individual is improved.
The lactobacillus gasseri CCFM1307 provided by the invention can obviously relieve inflammation caused by salmonella typhimurium infection, can be used for preparing medicines for preventing and treating salmonella typhimurium infection, or can be used for producing foods beneficial to intestinal health, such as food additives of probiotic beverages, sour soybean milk, fermented jelly, fermented tea beverages or dairy products (such as yoghurt, cheese products, lactic acid bacteria and milk powder) and the like, and has huge application prospects.
Preservation of biological materials
Lactobacillus gasseri (Lactobacillus gasseri) CCFM1307, taxonomic designation Lactobacillus gasseri, was deposited at the Cantonese microorganism strain collection at 6 and 7 days 2023 under the accession number GDMCC No. 63533 and at the accession number Guangzhou Hirship No. 100 university No. 59 building 5.
Drawings
Fig. 1: rate of body weight change during molding in each group of mice.
Fig. 2: the groups had pathological lesions on the ileum and colon tissues.
Fig. 3: effects of groups on inflammatory cytokines of intestinal tissue; wherein A: IL-12; b: IL-6; c: IL-17; d: IL-1 beta.
Fig. 4: butyric acid content in the feces of each group of mice.
Fig. 5: isobutyric acid content in the feces of each group of mice.
Fig. 6: valeric acid content in the feces of each group of mice.
Fig. 7: isovaleric acid content in the feces of each group of mice.
Fig. 8: schematic representation of the relative abundance of differential bacterial species in the feces of each group of mice.
Detailed Description
Technical terms:
the term "culture" as used herein refers to a population or a growth of cells of a microorganism in a certain space over a certain period of time, and particularly refers to a liquid or solid culture product, such as a slant culture, a fermentation product, etc., of a microorganism (e.g., lactobacillus gasseri CCFM 1307) grown after artificial inoculation and culture.
The term "bacterial suspension" as used herein refers to a suspension obtained by dispersing cells of a microorganism (e.g., lactobacillus gasseri CCFM 1307) in a solvent (e.g., water).
"prebiotics" in relation to the present invention refers to food ingredients that promote 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, and/or probiotics in the intestine. Optionally, oligosaccharides such as fructose, galactose, mannose; optionally, dietary fibers, such as soluble fibers, soy fibers; optionally, inulin; or a mixture of any two or more of the above.
The term "preventing" as used herein refers to treating to avoid, minimize or make difficult the onset or progression of a disease prior to the onset of the disease, including preventing Salmonella typhimurium infection, and/or inhibiting the progression of the disease caused by Salmonella typhimurium infection or delaying the onset of the disease.
The term "treatment" as used herein refers to preventing, curing, reversing, attenuating, alleviating, minimizing, inhibiting, arresting, and/or stopping one or more clinical symptoms of a disease after the onset of the disease; including inhibiting all of the behavior of the disease caused by salmonella typhimurium, and/or alleviating the pathological condition of the disease caused by salmonella typhimurium.
The composition comprises a product containing lactobacillus gasseri CCFM1307 in any form of food, medicine, health care product, disinfectant, drinking water additive, feed additive and the like; wherein the pharmaceutical product may be formulated according to the present invention by any method known to those skilled in the art using pharmaceutically acceptable carriers and/or excipients; the formulations may be provided in the form of solutions, suspensions or emulsions in oily or aqueous media, or in the form of extracts, powders, granules, tablets or capsules, and may also contain dispersing or stabilizing agents.
Animal model and reagents:
c57BL/6J mice referred to in the following examples were purchased from Leisha Zhejiang, salmonella typhimurium (Salmonella typhimurium) CICC 21483 obtained from the China industry microbiological collection center CICC; the streptomycin referred to in the examples below was purchased from Shanghai Biotechnology engineering (Shanghai) Inc.; ELISA kits for detection of IFN-gamma (cat# SBJ-M0594-96T) and IL-12 (cat# SBJ-M0038-96T) as referred to in the examples below were purchased from Nanjsen Bei Ga Biotech Co., ltd; ELISA kits for detection of IL-6 (cat# DY 406-05), IL-10 (cat# DY 417-05), IL-17 (cat# DY 421-05), IL-1 beta (cat# DY 401-05) and TNF-alpha (cat# DY 410-05) were purchased from R & D Co., ltd.
Culture medium:
MRS solid medium: 10g/L peptone, 10g/L beef extract, 20g/L glucose, 2g/L sodium acetate, 5g/L yeast powder, 2g/L, K diammonium hydrogen citrate 2 HPO 4 ·3H 2 O 2.6g/L、MgSO 4 ·7H 2 O 0.1g/L、MnSO 4 0.05g/L, tween 80 1mL/L, agar 15g/L, cysteine amino acid salt 0.5g/L.
MRS liquid medium: 10g/L peptone, 10g/L beef extract, 20g/L glucose, 2g/L sodium acetate, 5g/L yeast powder, 2g/L, K diammonium hydrogen citrate 2 HPO 4 ·3H 2 O 2.6g/L、MgSO 4 ·7H 2 O 0.1g/L、MnSO 4 0.05g/L, tween 80 1mL/L, cysteine amino acid salt 0.5g/L.
Example 1: screening and strain identification of lactobacillus gasseri CCFM1307
1. Screening
Fecal samples from the Henan cognac were stored in 30% glycerol and placed in a-80℃freezer. Taking out the sample, sucking 1mL, adding into 9mL of sterile physiological saline (0.85%, w/v), shaking, performing gradient dilution by 10-fold dilution method, sucking 200 μL of diluted solution to 10 -3 、10 -4 、10 -5 The bacterial colony is picked up and purified by streaking, then is cultured for 48 hours at 37 ℃ in MRS liquid culture medium, the bacterial colony is subjected to gram staining and bacterial strain morphology is recorded, gram negative bacterial strains and gram positive cocci in the bacterial colony are removed, gram positive bacillus is selected, the catalase positive bacterial strains are removed after catalase analysis, the catalase negative bacterial strains are reserved, and the bacterial colony is identified as lactobacillus gasseri through 16S rDNA sequencing. And (3) subculturing the obtained lactobacillus gasseri, collecting thalli, placing the thalli in a centrifuge tube, centrifuging at 3000rpm for 10min, washing, repeating for 3 times, and adding the obtained thalli into a matrix protective agent for freezing preservation.
2. Authentication
The genome of the strain is extracted, 16S rDNA is amplified and sequenced (by Huada gene technology Co., ltd.) by taking the genome as a template, and the sequence is subjected to nucleic acid sequence alignment in NCBI, so that the result shows that the strain is lactobacillus gasseri, is preserved in the food biotechnology strain collection of university of Jiangnan, is named as lactobacillus gasseri CCFM1307, and is preserved in the microorganism strain collection of Guangdong province at 6 months and 7 days of 2023, and the preservation number is GDMCC No. 63533.
Example 2: culture of lactobacillus gasseri CCFM1307 and preparation of bacterial suspension
1. Culture and preservation of Lactobacillus gasseri CCFM1307
After lactobacillus gasseri CCFM1307 obtained in example 1 was inoculated into MRS solid medium and cultured in an anaerobic incubator at 37 ℃ for 48 hours, colonies were observed and found to appear off-white, uneven-edged, circular protrusions.
Lactobacillus gasseri (Lactobacillus gasseri) CCFM1307 obtained in example 1 is inoculated into MRS liquid culture medium, anaerobic cultured for 24 hours at 37 ℃, transferred into fresh MRS liquid culture medium, cultured for 24 hours under the same condition, and centrifuged again for 10 minutes after 6000g after 0.9g/100mL physiological saline is used for washing the thallus, thus obtaining the thallus, added into 30% glycerol, frozen at-80 ℃ for standby.
2. The bacterial suspension is prepared as follows
(1) And (3) activating and culturing:
and adopting MRS liquid culture medium, and standing and culturing in an anaerobic workstation at 37 ℃.
Culturing the target: the frozen and preserved thalli are selected, single colony is selected in liquid MRS liquid culture medium, and is subjected to static culture for about 24 hours in an anaerobic workstation at 37 ℃, and lactobacillus gasseri CCFM1307 is activated.
(2) Primary culture:
and adopting MRS liquid culture medium, and standing and culturing in an anaerobic workstation at 37 ℃.
Culturing the target: the activated lactobacillus gasseri CCFM1307 was transferred to MRS broth at 1% inoculum size by volume of the broth for two generations.
(3) Secondary culture:
the bacterial liquid after 3 generations of activation is inoculated into 1L MRS liquid culture medium with an inoculum size of 2% (v/v), and the bacterial liquid is cultured for 24 hours at 37 ℃ in an anaerobic incubator after shaking and mixing. Centrifuging at 8000g/min and 4deg.C for 15min, removing supernatant, washing with sterile physiological saline for 2 times, centrifuging under the same conditions, removing supernatant, and re-suspending with 30% glycerol to obtain the final product. Centrifuging at 6000r/min for 5min, washing twice with sterile physiological saline, re-suspending the bacterial liquid with physiological saline, shaking uniformly, and measuring the initial viable bacteria number by a flat plate pouring method. The viable bacteria were again determined after one week of cryopreservation in 30% glycerol at-80 ℃.
Experimental results: the initial viable count is 7.03X10 9 cfu/mL, viable count after 1 week is 6.5X10 9 cfu/mL, the order of magnitude does not change, which indicates that the fungus liquid can not influence the experiment after freezing and storing, and can be used for animal experiments.
Example 3: lactobacillus gasseri CCFM1307 relieves weight loss in Salmonella typhimurium infected mice
1. Experimental animal
Female SPF grade C57BL/6J mice, 7 week old, were from Experimental animal technology Co., ltd. The mice were housed in polypropylene cages, each group of 8 mice, the cages were filled with food and water, the temperature (22 ℃) and the relative humidity (50.+ -. 10%) were controlled, and the mice were fed with standard feed and any water.
2. Experimental method
(1) Establishment of Salmonella typhimurium (Salmonella typhimurium) CICC 21483 infected mice model
C57BL/6J mice with 7 weeks of age were selected, the adaptation phase was 7 days before the experiment, the intervention phase was 8-17 days, and the modeling phase was 13-17 days. The stomach is filled with 5 multiplied by 10 after the water is cut off for 2 hours every day in the molding period 6 CFU/mL Salmonella typhimurium suspension 0.2mL, drinking water immediately recovered, feed was supplied after 2h, and the blank group was filled with 0.2mL of sterile physiological saline. During the molding period of the mice, the weights of the mice were weighed daily.
(2) Experimental grouping and administration
Divided into blank, model, and CCFM1307 groups.
Before stomach irrigation, the bacterial liquid is taken out from the temperature of minus 80 ℃ and centrifuged for 5min at 6000r/min, and then the bacterial liquid is washed twice with sterile physiological saline, and the bacterial liquid is resuspended with physiological saline with the concentration of 5 multiplied by 10 9 CFU/mL。
Dry expectation: the blank and model groups were perfused with 0.2mL of sterile saline daily for 10 days throughout the intervention period. CCFM1307 group the Lactobacillus gasseri CCFM1307 bacterial suspension, and each group of mice was fed standard feed during the test period.
Clinically, infection with Salmonella typhimurium causes symptoms such as headache, listlessness, fever and the like in a host, and the phenomenon that the weight of a mouse is continuously reduced after infection with Salmonella typhimurium is found in the prior researches. The mice in the model group had a weight loss of 11.4% during the entire molding period, and the blank group and the CCFM1307 group had a weight loss of 7.1% and 7.26%, respectively, with a significant difference (p < 0.05) compared to the model group (FIG. 1). Thus, lactobacillus gasseri CCFM1307 has a good effect in recovering the symptoms of weight loss in salmonella typhimurium infected mice.
Example 4: lactobacillus gasseri CCFM1307 improves ileum and colon tissue damage in Salmonella typhimurium infected mice
The animal model was constructed in the same manner as in example 3, after the end of the experiment, mice were sacrificed, and the ileum and colon of the mice were taken and immersed in 4% (v/v) paraformaldehyde for 24 hours to obtain a fixed intestinal tissue; sequentially dehydrating, transparentizing and waxing the fixed intestinal tissues, and embedding the tissues into the wax blocks by using a lycra paraffin embedding machine to obtain wax blocks embedded with the intestinal tissues; the method comprises the following specific steps of dehydration, transparency and wax dipping: (1) dehydration: dehydrating the fixed tissue by sequentially passing through 70%, 80% and 90% (v/v) ethanol solutions, each gradient being 30min, and then placing into 95% and 100% (v/v) ethanol solutions for 2 times each for 20min; (2) transparent: firstly, putting the tissue into a mixed solution of alcohol and dimethylbenzene in an equal volume ratio for 15min, and then putting the tissue into dimethylbenzene I and dimethylbenzene II for 15min respectively; (3) wax dipping: the tissue samples were placed in paraffin I and paraffin II liquids at 62℃for 30min each.
Slicing the wax block embedded with the intestinal tissue by using a lycra manual rotary slicing machine, wherein the slicing thickness is 5 mu m, and obtaining the intestinal tissue slice; the intestinal tissue slice is subjected to spreading and pulling, baking, hematoxylin staining, differentiation, rinsing, eosin counterstain, dehydration, transparency and sealing to obtain an H & E slice; the specific operations of spreading and pulling up, baking, hematoxylin staining, differentiation, rinsing, eosin counterstaining, dehydration, transparency and sealing are as follows: (1) spreading and fishing: placing the slices in a constant-temperature water bath at 42 ℃ for spreading, and carefully taking out the slices with a glass slide; (2) baking the slices: placing the slices in a 60 ℃ oven for overnight baking; (3) hematoxylin staining: firstly hydrating the slice (namely firstly placing the slice in xylene I and xylene II for 5min respectively, then sequentially placing the slice in 100%, 95%, 90%, 80% and 70% (v/v) gradient alcohol solutions for 5min respectively, finally placing the slice in distilled water for 3 min), then dyeing (namely placing the slice in hematoxylin staining solution for about 20 s), and finally washing the slice with water (namely washing the slice with tap water for about 30 min); (4) differentiation: placing the slices into 1% (v/v) ethanol hydrochloride solution for 7s, and performing color fading; (5) rinsing: washing the slices with tap water for about 20min; (6) counterstaining: immersing the slice into eosin staining solution, and immediately taking out; (7) dehydration: the slice is firstly put into 95% (v/v) ethanol solution I, 95% (v/v) ethanol solution II and 70% (v/v) ethanol solution in turn, taken out immediately after being put into the slice, then immersed into 80% (v/v) ethanol solution for 50s, and finally immersed into 100% (v/v) ethanol for 2min; (8) transparent: immersing the slice into a mixed solution of ethanol and dimethylbenzene in an equal volume ratio for 1min, and immersing the slice into dimethylbenzene I and dimethylbenzene II for 2min respectively; (9) sealing piece: the slices were sealed with neutral gum. The H & E intestinal section prepared was scanned by a Pannolic MIDI digital section scanner, and the intestinal tissue injury condition of the mice was observed and pathological scoring was performed by photographing, and the results are shown in FIG. 2.
The salmonella typhimurium infection has the characteristics of inflammatory cell infiltration and the like of the ileum and colon tissues of the mice, and can be used as an index for reflecting the injury degree caused by salmonella typhimurium infection to a certain extent. Figure 2 shows that model mice had a large area of inflammatory cell infiltration in both the ileum and colon, with the ileum tissue villus shedding compared to the blank, and that lactobacillus gasseri CCFM1307 can well alleviate this symptom and the pathology score is significantly reduced.
Example 5: lactobacillus gasseri CCFM1307 reduces inflammatory factor levels in the ileum of Salmonella typhimurium infected mice
The grouping, modeling and processing method of the C57BL/6J mice are the same as in example 3.
After the mice were sacrificed on day 18, the ileum of the mice was homogenized and centrifuged at 4000 Xg for 15min to obtain a tissue supernatant, which was ELISA-detected for IL-12 levels in the ileum of Salmonella typhimurium infected mice. As shown in FIG. 3, the IL-12 level in the ileum tissue of the modeling group was 3.40pg/mg, and Salmonella typhimurium infection significantly increased the pro-inflammatory cytokine IL-12 (p < 0.05) compared to the blank group (1.84 pg/mg), 1.85-fold over the blank group. Compared to the model group, the treatment with lactobacillus gasseri CCFM1307 significantly increased the ileal IL-12 level of the mice to 2.39pg/mg (29.71% reduction in the model group).
Example 6: lactobacillus gasseri CCFM1307 reduces the inflammatory factor level in the colon of Salmonella typhimurium infected mice C57BL/6J mice grouping, modeling and treatment methods were as in example 3.
After the mice were sacrificed on day 18, the colon of the mice was homogenized, centrifuged at 4000 Xg for 15min to obtain tissue supernatants, and ELISA was used to detect IL-6, IL-17 and IL-1β levels in the colon of Salmonella typhimurium infected mice.
As shown in FIG. 3, IL-6 levels in colon tissue of model mice were 99.51pg/mg, which was significantly elevated compared to the blank (56.62 pg/mg); compared with the model mice, the IL-6 level in colon tissue of CCFM1307 mice is obviously reduced to 58.98pg/mg, which is reduced by 40.80 percent. IL-17 levels in colon tissue of model mice were 99.65pg/mg, significantly elevated compared to the blank (65.38 pg/mg); compared with the model mice, the IL-17 level in colon tissue of CCFM1307 mice is obviously reduced to 64.30pg/mg, which is reduced by 35.47%. IL-1β levels 182.53pg/mg in colon tissue of model mice were significantly elevated compared to the blank (123.16 pg/mg); compared with the model mice, the IL-6 level in colon tissue of CCFM1307 mice is obviously reduced to 111.87pg/mg, which is reduced by 38.71%.
The results show that the inflammatory factor level of the mice in the model group is increased compared with that of the mice in the blank group, and the lactobacillus gasseri CCFM1307 can well relieve colon inflammation caused by salmonella typhimurium infection.
Example 7: lactobacillus gasseri CCFM1307 improves short chain fatty acid content in mouse feces infected with Salmonella typhimurium C57BL/6J mice grouping, modeling and treatment methods are the same as in example 3.
After the experiment is finished, collecting the mouse feces, placing the mouse feces in liquid nitrogen, transferring the liquid nitrogen to a refrigerator at the temperature of minus 80 ℃, taking out the mouse feces before the detection of the content of the short-chain fatty acid, carrying out vacuum freeze-drying, accurately weighing 0.05g of the freeze-dried feces sample, dissolving the sample in 0.5mL of saturated sodium chloride solution, soaking the sample for 30min, homogenizing the sample by a tissue homogenizer, adding 0.02mL of 10% sulfuric acid, oscillating for 30s, accurately adding 1mL of diethyl ether solution into the feces solution in a fume hood, oscillating for 30s, centrifuging for 15min (8000 g and 4 ℃), transferring the supernatant to a centrifuge tube containing 0.25g of anhydrous sodium sulfate, oscillating uniformly, centrifuging for 15min (8000 g and 4 ℃), taking the supernatant to a gas quality volumetric flask, and detecting the content of the short-chain fatty acid by GC-MS.
As shown in FIGS. 4 to 7, after mice were infected with Salmonella typhimurium, the levels of butyric acid, isobutyric acid, valeric acid and isovaleric acid in the feces of the mice in the model group were reduced to some extent as compared with the blank group, wherein the butyric acid level in the model group was 5.88. Mu. Mol/g, the isobutyric acid level was 2.67. Mu. Mol/g, the valeric acid level was 2.01. Mu. Mol/g, and the isovaleric acid level was 2.38. Mu. Mol/g, which were reduced by 36.57%, 32.23%, 37.58% and 35.15% (p < 0.05), respectively, as compared with the blank group. However, after perfusion with Lactobacillus gasseri CCFM1307, the contents of butyric acid, isobutyric acid, valeric acid and isovaleric acid were 8.02. Mu. Mol/g, 3.70. Mu. Mol/g, 2.89. Mu. Mol/g and 3.25. Mu. Mol/g, respectively, which were increased by 36.39%, 38.58%, 43.78% and 36.55% compared to the building block (p < 0.05). The short chain fatty acid can reduce pH value of intestinal tract, promote absorption of calcium and magnesium ions in intestinal tract, and inhibit infection of harmful bacteria.
Example 8: lactobacillus gasseri CCFM1307 reduces the relative abundance of Bactoides in Salmonella typhimurium infected mouse faeces
The grouping, modeling and processing method of the C57BL/6J mice are the same as in example 3.
After the test is finished, extracting genome DNA in the feces, carrying out specific PCR amplification on the V3-V4 region, sequencing the 16S rDNA, and analyzing the change of the feces flora. As shown in fig. 8, the results demonstrate a significant increase in the relative abundance of bacterioides in the feces of salmonella typhimurium infected mice compared to the blank (p < 0.05). CCFM1307 can significantly reduce the relative abundance of Bactroids (p < 0.05) in Salmonella typhimurium infected mice faeces.
The results show that the lactobacillus gasseri CCFM1307 can effectively improve the weight loss and intestinal tissue injury symptoms of mice. The inflammation caused by salmonella typhimurium infection is relieved by modulating inflammatory factors IL-12, IFN-gamma, IL-6, IL-17 and IL-1 beta. The pH value of the intestinal canal is reduced by improving the content of short chain fatty acid, the infection of harmful bacteria is inhibited, the intestinal canal peristalsis is stimulated, and the intestinal canal micro-ecological steady state is maintained by regulating the intestinal canal flora.
Example 9: lactobacillus gasseri CCFM1307 for preparing medicament
The lactobacillus gasseri CCFM1307 screened in example 1 can inhibit salmonella typhimurium (Salmonella typhimurium) and prevent and treat diseases caused by salmonella typhimurium infection, and is used as an active ingredient for preparing antibacterial and/or bactericidal products and medicines for preventing/treating salmonella typhimurium infection.
The medicine comprises lactobacillus gasseri CCFM1307 and a pharmaceutically acceptable carrier, and the dosage form of the medicine can be any one of solution, powder, tablet, capsule, suspension and emulsion.
Taking a tablet form as an example, the preparation process is as follows:
picking single colony of Lactobacillus gasseri (Lactobacillus gasseri) CCFM1307 obtained in example 1, inoculating into MRS liquid culture medium, culturing at 37deg.C for 16 hr to obtain strain with concentration of 1×10 7 CFU/mL of activating solution; inoculating the activating solution into MRS liquid culture medium according to the inoculum size of 1% (v/v), and culturing at 37 ℃ for 16 hours to obtain first-stage seed solution; inoculating the first-level seed liquid into MRS liquid culture medium according to an inoculum size of 1% (v/v), and culturing at 37 ℃ for 16 hours to obtain a second-level seed liquid; inoculating the secondary seed solution into MRS liquid culture medium according to the inoculum size of 1% (v/v), and culturing at 37 ℃ for 16h to obtain bacterial solution; centrifuging 6000g of bacterial liquid for 15min, and collecting precipitate; washing the precipitate with PBS buffer solution with pH of 7.4 twice, and centrifuging 6000g for 10min again to obtain thallus; lactobacillus gasseri is treated with a protectant solution comprising 130g/L skim milk, 20g/L trehalose and 20g/L sucroseThe cells were resuspended to a cell concentration of 1X 10 9 CFU/mL, obtaining lactobacillus gasseri bacterial solution; freeze-drying the lactobacillus gasseri bacterial liquid to obtain lactobacillus gasseri bacterial powder; adding stearic acid accounting for 2 percent of the total weight of the lactobacillus gasseri powder as a lubricant and sodium carboxymethylcellulose (CMC-Na) accounting for 3 percent of the total weight of the lactobacillus gasseri powder as an adhesive into the lactobacillus gasseri powder, and tabletting to obtain tablets.
The tablet prepared by the method is used for daily administration of salmonella typhimurium to mice at a dosage of 1 g/mouse, and the continuous 10 days can effectively relieve inflammation symptoms caused by salmonella typhimurium, and has excellent effects on preventing and/or treating inflammation caused by salmonella typhimurium.
Example 10: lactobacillus gasseri CCFM1307 for preparing bacterial powder
The lactobacillus gasseri CCFM1307 can be used for preparing bacterial powder, and the specific preparation process of the bacterial powder is as follows:
picking single colony of Lactobacillus gasseri (Lactobacillus gasseri) CCFM1307 obtained in example 1, inoculating into MRS liquid culture medium, culturing at 37deg.C for 16 hr to obtain strain with concentration of 1×10 7 CFU/mL of activating solution; inoculating the activating solution into MRS liquid culture medium according to the inoculum size of 1% (v/v), and culturing at 37 ℃ for 16 hours to obtain first-stage seed solution; inoculating the first-level seed liquid into MRS liquid culture medium according to an inoculum size of 1% (v/v), and culturing at 37 ℃ for 16 hours to obtain a second-level seed liquid; inoculating the secondary seed solution into MRS liquid culture medium according to the inoculum size of 1% (v/v), and culturing at 37 ℃ for 16h to obtain bacterial solution; centrifuging 6000g of bacterial liquid for 15min, and collecting precipitate; washing the precipitate with PBS buffer solution with pH of 7.4 twice, and centrifuging 6000g for 10min again to obtain thallus; lactobacillus gasseri strain was resuspended to a cell concentration of 1X 10 with a protectant solution containing 130g/L skim milk, 20g/L trehalose and 20g/L sucrose 9 CFU/mL, obtaining lactobacillus gasseri bacterial solution; and freeze-drying the lactobacillus gasseri bacterial liquid to obtain bacterial powder.
The tablet prepared by the method is used for daily administration of salmonella typhimurium to mice at a dosage of 1 g/mouse, and the continuous 10 days can effectively relieve inflammation symptoms caused by salmonella typhimurium, and has excellent effects on preventing and/or treating inflammation caused by salmonella typhimurium.
Example 11: lactobacillus gasseri CCFM1307 for preparing fermented food
The lactobacillus gasseri CCFM1307 or the lactobacillus gasseri CCFM 1307-containing bacterial powder prepared in example 10 is used for preparing fermented food or fermented drink.
The raw materials used for preparing the fermented beverage may be selected from: mixing one or more of animal milk, plant milk, fruit and vegetable juice, and cereal juice.
The raw materials used for preparing the fermented food product may be selected from: animal milk, plant milk, fruit and vegetable raw materials, cereal powder or cereal mixture, and meat product raw materials.
Example 12: lactobacillus gasseri CCFM1307 for preparing health products
The lactobacillus gasseri CCFM1307 or the fungus powder containing the lactobacillus gasseri CCFM1307 prepared in the embodiment 10 is used for preparing health care products. Optionally, the health care product is prepared by compounding lactobacillus gasseri CCFM1307 and prebiotics. The prebiotics include, but are not limited to, all food ingredients that promote the growth of beneficial bacteria, such as bifidobacteria or lactobacilli, and/or probiotics in the intestine, such as oligosaccharides, dietary fibers, bacterial powders, and the like.
Alternatively, the oligosaccharide is selected from fructose, galactose or mannose; the dietary fiber is selected from soluble fiber or soybean fiber.
While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (14)
1. Lactobacillus gasseri (Lactobacillus gasseri) CCFM1307, deposited at the Cantonese microorganism strain collection at month 7 of 2023 under accession number GDMCC No:63533.
2. a composition comprising the lactobacillus gasseri CCFM1307 of claim 1.
3. The composition of claim 2, wherein the composition is a food, pharmaceutical, nutraceutical, disinfectant, potable water additive, or feed additive.
4. A composition according to claim 3, wherein the pharmaceutical product comprises the lactobacillus gasseri CCFM1307, together with a pharmaceutical carrier and/or a pharmaceutical adjuvant.
5. A microbial preparation comprising the lactobacillus gasseri CCFM1307 of claim 1.
6. The microbial preparation according to claim 5, characterized in that it contains cells and/or cell cultures of the lactobacillus gasseri CCFM1307.
7. The microbial preparation according to claim 5 or 6, wherein the viable count of lactobacillus gasseri CCFM1307 in the microbial preparation is not less than 1×10 9 CFU/g or 1X 10 9 CFU/mL。
8. A starter culture comprising the lactobacillus gasseri CCFM1307 of claim 1.
9. Use of lactobacillus gasseri CCFM1307 according to claim 1 for the manufacture of a medicament for the prevention and/or treatment of inflammation caused by salmonella typhimurium infection.
10. The use according to claim 9, characterized in that the use comprises at least one of the following actions:
(1) Reducing the risk of weight loss in Salmonella typhimurium infected individuals;
(2) Relieving pathological damage of jejunum of individual infected by salmonella typhimurium;
(3) Reducing the level of immune factors in intestinal tissue of an individual infected with Salmonella typhimurium;
(4) The content of short chain fatty acid in the feces of the salmonella typhimurium infected individual is improved.
11. The use according to claim 9 or 10, further comprising modulating the intestinal flora.
12. Use of lactobacillus gasseri CCFM1307 according to claim 1 for the preparation of a health product for the regulation of intestinal flora.
13. The use according to claim 12, wherein the health product further comprises a prebiotic.
14. Use of a lactobacillus gasseri CCFM1307 according to claim 1 or a microbial preparation according to any of claims 5 to 7 for the preparation of a fermented food, beverage or condiment.
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| CN202311120721.1A CN116925980A (en) | 2023-08-31 | 2023-08-31 | Lactobacillus gasseri strain for relieving salmonella typhimurium infection and application thereof |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN118109375A (en) * | 2024-04-30 | 2024-05-31 | 善恩康生物科技（苏州）有限公司 | Lactobacillus gasseri JM6 and application thereof in vaginitis |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN118109375A (en) * | 2024-04-30 | 2024-05-31 | 善恩康生物科技（苏州）有限公司 | Lactobacillus gasseri JM6 and application thereof in vaginitis |
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