Lactobacillus gasseri LTG1323 and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to lactobacillus gasseri LTG1323 and application thereof. The lactobacillus gasseri provided by the invention has relatively strong enrichment ability and acid production ability, has relatively strong inhibition ability on escherichia coli, staphylococcus aureus, bacillus cereus, shigella flexneri and salmonella typhimurium, has strong colonization ability in human intestinal tracts, can regulate and optimize a microenvironment, is sensitive to six common antibiotics, and can be used for preparing a feed additive. And the prepared freeze-dried powder has the characteristics of high viable count, long shelf life and the like.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a Lactobacillus gasseri LTG1323 and an application thereof. Background Art

[0002] Lactobacillus gasseri is a Gram-positive bacterium that is facultatively anaerobic or microaerophilic. It is widely found in the human oral cavity, gastrointestinal tract, vagina and breast milk mucosal epithelium. The pursuit of nutritious foods that can enhance immunity and health has become an urgent concern for people. Lactobacillus gasseri has many health benefits, including maintaining intestinal and vaginal health, producing antibacterial bacteriocins, enhancing immune regulation, anti-oxidation and controlling obesity.

[0003] There are about 1,000 symbiotic microorganisms living in the intestines of healthy people, including probiotics that are beneficial to humans. The most common probiotics are Lactobacillus and Bifidobacterium, but their number gradually decreases with age. The probiotic strains that still exist in the intestines of centenarians are particularly precious. The isolation, screening and application of a variety of probiotic strains are of great significance to the enrichment of probiotic resources and the exploration of the secrets of health and longevity. Therefore, our research group isolated multiple strains of Lactobacillus gasseri from the feces of centenarians in Guangxi, and obtained a strain of Lactobacillus gasseri with good probiotic properties through multiple experiments.

[0004] Lactobacillus gasseri can reduce the pH of the environment by producing organic acids such as lactic acid, thereby inhibiting the growth of harmful bacteria. Nowadays, chemical antibacterial agents are widely used in the food industry. On the one hand, this is contrary to the concept of green, environmental protection, and sustainable development advocated by the country; on the other hand, unscrupulous merchants use chemical antibacterial agents in violation of regulations to improve the attractive properties of products such as color, freshness, fragrance, and taste, which is very detrimental to the health of consumers. Therefore, the use of biological antibacterial agents is both green and environmentally friendly and beneficial to the human body. This is the research direction of antibacterial agents in the future. However, there is no relevant report on Lactobacillus gasseri LTG1323. Summary of the invention

[0005] To solve the above problems, the present invention provides a Lactobacillus gasseri LTG1323, which is deposited in Guangdong Provincial Microbiological Culture Collection Center, with a storage address of Guangzhou, China, a storage number of GDMCC No: 63037, a storage date of December 4, 2022, and a classification name of Lactobacillus gasseri.

[0006] Furthermore, the 16S rDNA complete genome sequence of the Lactobacillus gasseri LTG1323 is shown in SEQ ID No: 1.

[0007] The present invention also provides a bacterial agent, which includes the above-mentioned Lactobacillus gasseri LTG1323.

[0008] Further, the Lactobacillus gasseri LTG1323 may be viable bacteria.

[0009] Further, the Lactobacillus gasseri LTG1323 may be freeze-dried powder.

[0010] Further, the Lactobacillus gasseri LTG1323 may be non-viable bacteria.

[0011] The present invention also provides the application of the above-mentioned Lactobacillus gasseri LTG1323 in the preparation of drugs for inhibiting Escherichia coli, Staphylococcus aureus, Bacillus cereus, Shigella flexneri and Salmonella typhimurium.

[0012] The present invention also provides the application of the above-mentioned Lactobacillus gasseri LTG1323 in the preparation of feed additives for inhibiting Escherichia coli, Staphylococcus aureus, Bacillus cereus, Shigella flexneri and Salmonella typhimurium.

[0013] The present invention also provides the application of the above-mentioned Lactobacillus gasseri LTG1323 in the preparation of food additives for inhibiting Escherichia coli, Staphylococcus aureus, Bacillus cereus, Shigella flexneri and Salmonella typhimurium.

[0014] The present invention has the following beneficial effects:

[0015] (1) The Lactobacillus gasseri LTG1323 of the present invention has inhibitory activity against a variety of pathogenic bacteria and can inhibit the growth of pathogenic bacteria such as Escherichia coli, Staphylococcus aureus, Bacillus cereus, Shigella flexneri and Salmonella typhimurium. Therefore, it can be used alone or in combination with other probiotics for the adjuvant treatment of various bacterial infectious diseases.

[0016] (2) Compared with other Lactobacillus gasseri isolates, the Lactobacillus gasseri LTG1323 screened in the present invention has a higher lactic acid content in the fermentation supernatant, indicating that this strain has high lactic acid production. Description of the Drawings

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required to be used in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0018] Figure 1 It is the Gram staining diagram of LTG1323;

[0019] Figure 2It is the agarose gel electrophoresis pattern of the PCR amplification product of LTG1323 nucleotides;

[0020] Figure 3 It is the phylogenetic tree of LTG1323;

[0021] Figure 4 It is the whole genome map of LTG1323;

[0022] Figure 5 It is the growth curve of LTG1323 strain;

[0023] Figure 6 It is the nuclear magnetic resonance hydrogen spectrum metabolite detection map of the fermentation broth of LTG1323;

[0024] Figure 7 It is the antibacterial circle map of LTG1323 against 5 pathogenic bacteria;

[0025] Figure 8 It is the change in the survival rate of the freeze-dried bacterial powder of LTG1323 stored at different temperatures. Detailed implementation manners

[0026] Now, various exemplary implementation manners of the present invention will be described in detail. In the examples, unless otherwise specified, the methods are all conventional methods, and the reagents, unless otherwise specified, are all conventional commercially available reagents or reagents prepared by conventional methods. This detailed description should not be regarded as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0027] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0028] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0029] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the present invention specification are obvious to those skilled in the art. The present invention specification and examples are merely exemplary.

[0030] Regarding "comprising", "including", "having", "containing", etc. used herein, they are all open-ended terms, meaning including but not limited to.

[0031] The experimental result data in the specific embodiments of the present invention are all expressed as mean ± standard deviation (Means ± SD). Statistical analysis uses one-way analysis of variance [One-way analysis of variance (One-way ANOVA)]. "+" indicates 90% strain positive; "-" indicates 90% strain negative; "d" indicates 11% - 89% strain positive.

[0032] For the experimental methods involved in the embodiments of the present invention, if not otherwise specified, they are all conventional methods used by those skilled in the art; for the experimental materials used, if not otherwise specified, it means they can be obtained from commercial channels.

[0033] Example 1 Screening and Preliminary Identification of Lactobacillus Strains

[0034] 1. Isolation of Lactobacillus

[0035] Weigh 1.0 g of the frozen fecal sample and add it to a test tube containing 9.0 mL of sterile PBS buffer. Perform 10-fold serial dilutions to obtain three dilutions of 10 -6 、10 -7 and 10 -8 . Pour plates with modified MRS solid medium (containing 0.8% CaCO 3 、200 μl / 200 mL 2% X-gal). Spread the sample dilutions on the modified MRS solid medium plates and incubate at 37 °C for 56 h. Pick the single colonies with a calcium dissolution zone or light blue color, and streak-transfer them onto MRS plates. Purify by streaking for 3 generations to obtain suspected Lactobacillus strains.

[0036] 2. Morphological Identification

[0037] Observe the colony morphology, perform Gram staining, and observe the cell morphology of the isolated suspected strains. Refer to the Lactobacillus characteristics section of "Bergey's Manual of Determinative Bacteriology" and "Classification, Identification and Experimental Methods of Lactic Acid Bacteria" to preliminarily screen for suspected Lactobacillus.

[0038] 3. Physiological and Biochemical Identification

[0039] The above suspected strains were respectively subjected to gelatin liquefaction test, nitrate reduction test, litmus milk test, glucose acid production and gas production test, carbohydrate fermentation acid production test, and catalase test. Refer to the Lactobacillus section of "Bergey's Manual of Determinative Bacteriology" and "Classification, Identification and Experimental Methods of Lactic Acid Bacteria" to identify by comparing the experimental results. And the growth of the strains inoculated in modified MRS liquid medium at pH 4.5, pH 7.0 and pH 9.0 was detected, and the growth of the strains in modified MRS liquid medium at 15 °C and 45 °C was also detected. And the purified Lactobacillus single colonies obtained by preliminary identification were inoculated into liquid MRS medium and cultured for 24 h, mixed with 40% glycerol in a ratio of 1:1 and stored at -80 °C for standby.

[0040] The thawed fecal samples were mixed with sterile phosphate buffer solution (PBS), spread on MRS solid medium, and anaerobically cultured at 37 °C. Gram-positive bacteria with smooth surfaces, complete edges, convex circles, shiny, soft and blue colonies were picked and streaked onto MRS solid medium. Gram-positive bacteria that were rod-shaped or spoon-shaped under microscopy, negative in the catalase test and could not grow on MRS solid medium under aerobic conditions were tentatively designated as suspected Lactobacillus gasseri. The picked suspected Lactobacillus gasseri was further streaked and cultured on MRS solid medium, and repeated purification was carried out until the colony morphology was consistent and the cell morphology under microscopy was consistent ( Figure 1 ). A single colony was picked and inoculated into MRS liquid medium, cultured at a constant temperature of 37 °C, and the bacterial solution was preserved and numbered as LTG1323.

[0041] Catalase test, glucose acid production and gas production test, gelatin liquefaction test, nitrate reduction test, litmus milk test, temperature tolerance test, pH test (the results are shown in Table 1), and sugar fermentation test (the results are shown in Table 2) were carried out on LTG1323. According to "Classification, Identification and Experimental Methods of Lactic Acid Bacteria", LTG1323 can be preliminarily identified as Lactobacillus gasseri by the experimental results in Table 1 and Table 2.

[0042] Table 1 Physiological and Biochemical Characteristics of LTG1323

[0043]

[0044] Table 2 Results of Sugar Fermentation Test of LTG1323

[0045]

[0046] Note: "+" indicates that 90% of the strains are positive; "-" indicates that 90% of the strains are negative; "d" indicates that 11% - 89% of the strains are positive.

[0047] 4.16S rDNA Sequence Determination

[0048] Extract the genomic DNA of LTG1323 and use it as a template for polymerase chain reaction amplification. Amplification was carried out using 16S rDNA universal primers, namely the forward primer 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and the reverse primer 1492R (5'–GGCTACCTTGTTACGACTT-3'). After the amplification reaction, the amplification products were subjected to agarose gel electrophoresis, and the electrophoresis results are shown in Figure 2 . There was a bright band at approximately 1500 bp in Figure 2 , indicating successful PCR amplification. The measured 16S rDNA sequence of LTG1323 was aligned using BLAST on NCBI. Then, Poppunk was used to cluster 68 Lactobacillus gasseri strains according to host region, evolutionary relationship, and host, as shown in Figure 3 . According to the clustering results, the 68 Lactobacillus gasseri strains were divided into 3 clusters in terms of evolutionary relationship, and 28 strains were classified by host, mainly from adult feces, human urine, human vagina, infant feces, and human breast milk feces. The strains had a wide geographical origin, covering a total of 11 countries (among them, 28 strains from the United States, 5 strains from South Korea, 5 strains from India, 3 strains from Italy, 3 strains from Canada, 2 strains from Spain, 2 strains from China, 1 strain from Japan, 1 strain from Russia, 1 strain from Bangladesh, and 1 strain from France). There were a total of 5 strains in the same small cluster as Lactobacillus gasseri LTG1323, namely L.gasseri CM2267-MRS2-S780-bin-1, L.gasseri AL3, L.gasseri UMB0056, L.gasseri FR2, and L.gasseri 7135. Among them, AL3 is a strain from Italy and has the closest taxonomic relationship with LTG1323. The slightly more distant strain is UMB0056, which was isolated from human urine in the United States. Among the 5 strains closest to LTG1323, 2 strains were both from human feces, indicating the high enrichment of Lactobacillus gasseri in the intestine and that it is one of the main probiotics in the intestine.

[0049] Example 2 Determination of the complete genome sequence of LTG1323

[0050] 1. Basic characteristics

[0051] Extract the genomic DNA of strain LTG1323 and perform complete genome sequence determination. Figure 4The whole-genome map of LTG1323 was shown. The results indicated that LTG1323 had 1 chromosome and contained 2 plasmids, suggesting that the genes encoded by the plasmids made the traits encoded by the LTG1323 chromosome richer than those of strains with no plasmids or fewer plasmid structures. The chromosomal genome was circular, with the average content of guanine (G) and cytosine (C) being 34.91%. The chromosome size was 5,367,441 bp, containing 5,181 protein-coding genes (CDSs). The total length of the coding regions accounted for 87.75% of the whole genome. There were 81 non-coding RNAs, 17 rRNA operons and 74 tRNAs.

[0052] 2. Gene function

[0053] 2.1 NR, Swiss-prot annotation results

[0054] The NR database is a non-redundant protein database created and maintained by NCBI, containing taxonomic information related to metabolic pathways and functions. The Swiss-prot database is a curated protein sequence database that provides high-level annotation results, such as protein functions, domains, post-translational modifications and variations. By aligning the chromosomal genome of LTG1323 with the NR database, a total of 5,356 coding proteins were predicted. By aligning the chromosomal genome of LTG1323 with the Swiss-prot database, 3,732 annotated proteins were obtained.

[0055] 2.2 COG functional gene annotation

[0056] Among all the 5,356 independent coding regions, 1,702 genes could be classified into the functional classification database of the Cluster of Orthologous Group (COG). Excluding the parts with unknown functions (R and S), the genes related to carbohydrate transport and metabolism were the most numerous (208); followed by genes related to translation, ribosomal structure and biogenesis (193); transcription genes (134); cell wall / membrane / envelope biogenesis genes (115); and the expression levels of genes in other categories varied.

[0057] 2.3 KEGG functional gene annotation

[0058] The pathways involved by Lactobacillus gasseri LTG1323 are mainly divided into six categories, namely cell transformation, environmental information processing, human diseases, metabolism, organic systems, and genetic information processing. Among them, the coding genes in metabolism are the most distributed, mainly including amino acid metabolism, nucleotide metabolism, biosynthesis and metabolism of polysaccharides, and metabolism of energy and carbohydrates. The participation of numerous genes in the metabolism of the strain also promotes the good probiotic characteristics of LTG1323. The strain can affect the immune system through metabolites to exert an immunomodulatory function. At the same time, certain metabolites such as lactic acid and bacteriocins can inhibit the growth of pathogenic bacteria, enabling the strain to have a broader application space in industries such as health products and feeds; in environmental information processing, there are also many genes involved in biofilm transport. This enables the LTG1323 strain to maintain the normal morphology of cells by generating a membrane transport system to ensure normal life activities when facing environmental changes, such as changes in osmotic pressure, indicating that Lactobacillus gasseri LTG1323 has strong environmental adaptability; in terms of cell transformation, it is mainly involved in cell transport and catabolism, as well as cell growth and death.

[0059] 2.4 GO functional gene annotation

[0060] There are 2,631 genes of Lactobacillus gasseri LTG1323 annotated to the GO database. In the GO functional classification, the annotated genes are mainly involved in three major categories: biological processes, cellular components and composition, and molecular functions, with a total of 13 functional classifications. Among them, the genes involved in molecular functions are the most numerous, with a total of 6 subcategories and 1,175 genes, accounting for 44.66% of the total number of annotations, mainly including catalytic activity, transport activity, structural molecular activity, transcriptional regulator activity, ATP production activity, etc.; in the biological process classification, it is mainly involved in biological regulation, cell localization, metabolic processes, stimulus response, and cell component biogenesis, etc., accounting for 42.27%; in the cellular component and cell composition functional classification, it is mainly involved in cells and cell parts, accounting for 13.07% of the total number of annotations.

[0061] Example 3 Growth curve and metabolite detection of LTG1323 strain

[0062] 1. Growth curve of LTG1323

[0063] Prepare a clean and sterile 50 mL microbial growth test centrifuge tube in advance, pour 10 mL of culture medium, pick a purified single colony of Lactobacillus gasseri LTG1323 into the culture medium, set the reactor temperature to 37 °C, and continuously test on the machine for 24 h to draw the growth curve of the strain.

[0064] Export the OD 600 value data of the strain cultured continuously for 24 h, and draw the growth curve of the strain as Figure 5 shown. As Figure 5It is known that the strain enters the logarithmic growth phase after 5 hours, while Lactobacillus gasseri LTG1323 reaches the maximum growth at about 18 hours, enters the stable phase after 18 hours, and enters the decay phase after about 23 hours. Therefore, we choose 18 hours as the best time for strain culture and carry out the next experiment.

[0065] 2. Detection of metabolically active substances in LTG1323 strain

[0066] Take the fermentation solution in the stable period for metabolite extraction. Mix 1 mL of the fermentation solution in the stable period with 1 mL of the metabolite extraction solution. The metabolite extraction solution is composed of NaH 2 PO 4 -K 2 HPO 4 Buffer and acetonitrile were mixed in a ratio of 1:1 (v / v). The mixture was treated with an ultrasonic ice bath to disrupt the cells. Then, the mixture was stored frozen at -20 °C for 2 h to extract the metabolites. After that, the thawed samples were centrifuged (12000×g, 4 °C, 15 min) to obtain the supernatant containing the metabolites. The supernatant was then transferred to a rotary evaporator to remove water and acetonitrile. Each sample was dissolved in 700 μL of tritiated water containing 0.01% TSP, and after centrifugation, 600 μL of the supernatant was transferred to an NMR tube for testing. The samples should be stored at 4 °C until use, and the storage time before NMR testing should not exceed 24 h. All samples were measured by a standard Bruker NOESY pulse sequence. The acquisition parameters were as follows: number of scans (64), spectral width (20 ppm), relaxation delay (2 s), number of sampling points (65536), sampling time (3.277 s), mixing time (0.1 s), and FID resolution (0.245).

[0067] The H NMR metabolite spectrum of LTG1323 strain is shown in Figure 6 shown.

[0068] Example 4: Simulation of gastrointestinal adverse environment and adhesion of LTG1323 strain

[0069] In order for probiotics to colonize in the intestine, they must first be able to tolerate the low pH gastric acid environment, so the evaluation of acid resistance and pepsin resistance is an important indicator for probiotic screening. By measuring these indicators, the survival ability of the selected strains after gastric digestion can be determined.

[0070] 1. LTG1323 simulated gastrointestinal transit experiment

[0071] Centrifuge the overnight culture of the test bacterial solution (4000 g, 20 min) to collect the bacterial cells, wash them twice with sterile normal saline, and resuspend the bacterial cells in 10 mL of artificial gastric juice with a pH of 2.5 (0.2 g / 100 mL of NaCl, 0.32 g / 100 mL of pepsin, adjust the pH to 2.5 with 1 mol / L HCl, filter and sterilize for later use). Incubate on a shaker at 37°C and 200 rpm for 120 min, centrifuge (4000 g, 20 min) to collect the bacterial cells, and resuspend the bacterial cells in 10 mL of artificial intestinal juice with a pH of 8.0 ( 2 KH 4 PO

[0072] 0.68 g / 100 mL, 1 g / 100 mL of trypsin, 0.3 g / 100 mL of bile salt, pH 8.0), and incubate on a shaker at 37°C and 200 rpm for 120 min. Detect the viable bacterial count of the samples before and after treatment, and the results are shown in Table 3.

[0073]

[0074] 2. Determination of auto - aggregation ability

[0075] For the determination of auto - aggregation ability, with slight modification according to the reference, centrifuge the overnight culture of the test bacterial solution (4000 g, 20 min), wash it twice with normal saline, and resuspend the bacterial cells in PBS buffer to make the viable bacterial count 10 8 CFU / mL. Vortex the bacterial suspension (5 mL) for 10 s, let it stand at room temperature for 5 h, and determine the auto - aggregation ability. During this period, take the supernatant every 1 h and measure the absorbance at 600 nm. The auto - aggregation value (%) is expressed by the following formula:

[0076]

[0077] Among them, A t represents the absorbance values at time t of 1 h, 2 h, 3 h, 4 h, and 5 h respectively,

[0078] A 0 represents the absorbance value at time 0.

[0079] 3. Detection of surface hydrophobicity

[0080] For the detection of surface hydrophobicity, centrifuge the overnight culture of the test bacterial solution (4000 g, 20 min), wash it twice with normal saline, and resuspend it in 0.1 mol / L KNO 3 (pH 6.2) to make the viable bacterial count concentration 10 8 CFU / mL, and measure the absorbance A of the sample at 600 nm 0Add 0.1 mL of xylene solvent to 3 mL of bacterial solution, let it stand at room temperature for 10 min, vortex the two-phase system for 2 min, let it stand at room temperature for 20 min, take the aqueous phase, and measure the absorbance at 600 nm (A 1 ). The adhesion percentage (%) of bacteria to the solvent is expressed by the following formula:

[0081]

[0082] Table 4 Auto-aggregation rate and hydrophobicity rate of LTG1323

[0083]

[0084] Example 5 Antibiotic susceptibility

[0085] If the screened probiotic is not sensitive to a certain antibiotic, it means that it carries the corresponding resistance gene. If this probiotic colonizes in the human intestine, the resistance gene it carries has the potential risk of being implanted into other microbial cells in some way. Therefore, the higher the sensitivity of the screened probiotic to antibiotics, the better. Of course, some bacteria have natural resistance to certain antibiotics, which does not come from the transmission of other strains.

[0086] Six common antibiotics were selected for the susceptibility test in the present invention. LTG1323 was cultured overnight, and the bacterial solution was spread on MRS agar medium. Then, antibiotic discs (erythromycin, chloramphenicol, co-trimoxazole, ampicillin, cefazolin, norfloxacin) were covered on the agar medium. First, pre-culture and diffusion were carried out at 4 °C for 2 h, and then transferred to 37 °C for incubation for 48 h. The inhibition diameter was measured, and sensitive (S), intermediate (I), and resistant (R) were judged with reference to the terms of the Clinical and Laboratory Standards Institute (CLSI). The results are shown in Table 6.

[0087] Except that the standard for co-trimoxazole is intermediate, LTG1323 is sensitive to erythromycin, chloramphenicol, ampicillin, cefazolin, and norfloxacin, indicating that LTG1323 has relatively safe application value.

[0088] Table 6 Susceptibility of strain LTG1323 to 6 common antibiotics

[0089]

[0090] Example 6 Evaluation of the antibacterial ability of LTG1323 strain

[0091] Using Escherichia coli ATCC2522, Staphylococcus aureus, Bacillus cereus, Shigella flexneri, and Salmonella typhimurium as indicator bacteria, these 5 kinds of bacteria are from our laboratory or the China Center for Medical Bacterial Culture Collection. Escherichia coli ATCC2522, Staphylococcus aureus, Bacillus cereus, Shigella flexneri, and Salmonella typhimurium were respectively inoculated into liquid LB broth medium and cultured overnight at 37°C, and passed 3 - 5 generations until the traits were stable. LTG1323 was inoculated into MRS liquid medium, and after culturing to the plateau phase, the supernatant was obtained by centrifugation. The 5 kinds of pathogenic bacteria after liquid expansion to the same concentration were diluted at a 10-fold gradient to obtain 10 -1 -fold dilutions. 100 μL was taken and spread on LB broth agar plates. Two Oxford cups were placed equidistantly on each plate, and 200 μL of the LTG1323 culture supernatant was injected into each cup. Each kind of pathogenic bacteria was in parallel for 3 times. After culturing at 4°C for 2 h and then at 37°C for 36 h, the size of the inhibition zone was measured. A typical inhibition zone diagram is shown in Figure 7 . The measurement results of the inhibition zone size are shown in Table 5, and the results show that LTG1323 has obvious inhibitory ability against the five pathogenic bacteria.

[0092] Table 5 The inhibition zone size of the supernatant of the strain culture solution against 5 kinds of pathogenic bacteria

[0093]

[0094] Example 7 Application of LTG1323 in the fields of food, food supplements, health products, drugs, etc.

[0095] LTG1323 can be directly used for fermented dairy products or co-fermented with traditional starters for fermented dairy products, or added to food, feed, and drugs in the form of freeze-dried powder to regulate the intestinal flora of humans and animals and improve the body's immunity. LTG1323 can also be consumed in a non-living form.

[0096] One of the preferences is to prepare fermented milk using Lactobacillus gasseri LTG1323. The production method of Lactobacillus gasseri LTG1323 fermented milk is as follows: The raw milk powder is reconstituted with water at a ratio of 10%. The milk powder, granulated sugar, and purified water are mixed according to the experimental design ratio, heated and stirred to dissolve, and reconstituted milk is prepared. The raw milk is preheated to 50 - 60°C and homogenized at 20 - 25 MPa for 5 min. Subsequently, the raw milk is sterilized at 90°C for 15 min. After sterilization, the raw milk is cooled to 40 - 45°C, and Lactobacillus gasseri LTG1323 is inoculated under aseptic conditions. Ferment at 40 - 42°C for 6 - 8 h to make the raw milk reach a solidified state; refrigerate and store at 2 - 6°C for 24 h for after-ripening.

[0097] Second, preferably, freeze-dried powder is prepared using Lactobacillus gasseri LTG1323. The LTG1323 fermentation broth cultured to the logarithmic growth phase is centrifuged at 4°C, the supernatant is removed, and the Lactobacillus gasseri sludge is obtained. Then, the sludge is resuspended with sterile normal saline at 1 / 10 volume of the original fermentation broth and centrifuged again to obtain the sludge. The optimized cryoprotectant for Lactobacillus gasseri freeze-dried products (skim milk powder 9.98%, sucrose 8.18%, glycerol 3.94%, L-cysteine 0.91%) is mixed with the sludge at a ratio of 10:1, placed in a -80°C refrigerator and frozen for 4 h, then taken out and freeze-dried to obtain the LTG1323 freeze-dried bacterial powder. Under this cryoprotectant, the freeze-dried survival rate of the strain reached (83.30 ± 0.092)% after vacuum freeze-drying, which is 2.42 times that before optimization. Compared with relevant reports at home and abroad, the freeze-dried survival rate of Lactobacillus gasseri reached the highest value so far.

[0098] Under dry and sterile conditions, 2.0 g of the bacterial powder is filled into an aluminum foil bag and vacuum-sealed. The packaged freeze-dried bacterial powder samples are placed in environments at -80°C, -20°C, -4°C, and room temperature respectively. The viable count of the bacterial powder is measured every 7 days, and the survival rate is calculated, and the measurement is continued for 2 months. According to Figure 8 it can be seen that when the bacterial powder is stored under different temperature conditions, the low-temperature storage effect is better than that at room temperature. Among them, the optimized group ( Figure 8 B) and the control group ( Figure 8 A) both have the best storage effect at -20°C, and the survival rate is higher than the other 3 storage temperatures. After comparison, it is found that the optimized compound cryoprotectant can reduce the probability of frostbite of the strain during storage. In the control group, the rate of decline in its survival rate is relatively large, indicating that the use of a single cryoprotectant has a weak protective effect on the strain. Using the optimized compound cryoprotectant under 4 temperature conditions, the decline of the freeze-dried survival rate of the strain is gentle, which can extend the shelf life of the Lactobacillus gasseri LTG1323 freeze-dried product to a certain extent and ensure a high biological activity of the strain. Therefore, the optimized group of the compound cryoprotectant can not only improve the freeze-dried survival rate of the Lactobacillus gasseri LTG1323 bacterial powder, but also improve the stability of the strain during storage.

[0099] The above-described embodiments are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A Lactobacillus gasseri LTG1323, characterized in that The Lactobacillus gasseri LTG1323 is deposited in the Guangdong Provincial Microbiological Culture Collection Center with a deposit number of GDMCC No: 63037 and a deposit date of December 4, 2022.

2. A Lactobacillus gasseri LTG1323 according to claim 1, characterized in that The 16S rDNA complete genome sequence of the Lactobacillus gasseri LTG1323 is shown in SEQ ID No:

1.

3. A bacterial agent, characterized in that: It comprises the Lactobacillus gasseri LTG1323 as described in claim 1 or 2.

4. The bacterial agent according to claim 3, characterized in that The Lactobacillus gasseri LTG1323 is a living bacterium.

5. The bacterial agent according to claim 3, characterized in that The Lactobacillus gasseri LTG1323 is a freeze-dried powder.

6. The bacterial agent according to claim 3, characterized in that The Lactobacillus gasseri LTG1323 is a non-living bacterium.

7. Use of Lactobacillus gasseri LTG1323 as claimed in claim 1 or 2 in the preparation of drugs for inhibiting Escherichia coli, Staphylococcus aureus, Bacillus cereus, Shigella flexneri and Salmonella typhimurium.

8. Use of Lactobacillus gasseri LTG1323 as claimed in claim 1 or 2 in the preparation of a feed additive for inhibiting Escherichia coli, Staphylococcus aureus, Bacillus cereus, Shigella flexneri and Salmonella typhimurium.

9. Use of Lactobacillus gasseri LTG1323 as claimed in claim 1 or 2 in the preparation of a food additive for inhibiting Escherichia coli, Staphylococcus aureus, Bacillus cereus, Shigella flexneri and Salmonella typhimurium.

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