Lactobacillus gasseri ltg1323 and application thereof

Abstract

The application belongs to the technical field of biotechnology and particularly relates to a lactobacillus gasseri LTG1323 and application thereof.The lactobacillus gasseri has strong abilities of bacteria multiplication and acid production, has strong inhibiting abilities on escherichia coli, staphylococcus aureus, bacillus cereus, shigella flexneri and salmonella typhimurium, has strong abilities of colonization in human intestinal tract, can regulate and optimize microenvironment, is sensitive to 6 common types of antibiotics, has high safety, and the prepared freeze-dried powder has characteristics of high number of viable bacteria and long shelf life.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biotechnology, and particularly relates to a Lactobacillus gasseri LTG1323 and application thereof. BACKGROUND

[0002] Lactobacillus gasseri is a gram-positive bacterium, facultative anaerobic or microaerophilic, and widely symbiotic in the inner bacteria of human oral cavity, gastrointestinal tract, vagina and breast mucosa epithelium. Pursuing nutrition food capable of enhancing immunity and health has become an urgent problem for people, and Lactobacillus gasseri has various health benefits, including maintaining intestinal and vaginal health, producing antibacterial bacteriocin, enhancing immune regulation, antioxidant and obesity control.

[0003] There are about 1000 kinds of symbiotic microorganisms in the intestinal tract of healthy human body, including beneficial probiotic flora, and the most common probiotics are Lactobacillus and Bifidobacterium, but their number gradually decreases with age, and the probiotic strains still existing in the intestinal tract of long-lived old people are particularly valuable. The isolation and application of various probiotic strains are of great significance to the enrichment of probiotic resources and the exploration of the mystery of health and longevity. Therefore, the research group isolated a plurality of Lactobacillus gasseri from the feces of long-lived old people in Guangxi, and screened a Lactobacillus gasseri strain with good probiotic properties through a plurality of experiments.

[0004] Lactobacillus gasseri can inhibit the growth of harmful bacteria by producing lactic acid and other organic acids to reduce the pH of the environment. Nowadays, the bacteriostatic agent commonly used in the food industry is chemical, which is contrary to the concept of green, environmental protection and sustainable development advocated by the state on the one hand, and unscrupulous businessmen use chemical bacteriostatic agents to improve the attractive properties of products such as color, freshness, aroma and taste, which is very harmful to consumers' health, so the use of biological bacteriostatic agent is green and environmentally friendly, and is beneficial to human body, which is the research direction of future bacteriostatic agent. However, there is no related report on Lactobacillus gasseri LTG1323. SUMMARY

[0005] To solve the above problems, the application provides a Lactobacillus gasseri LTG1323, which is preserved in the Guangdong Microbial Digital Culture Collection Center, has a preservation address of China.Guangzhou, a preservation number of GDMCC No:63037, a preservation date of December 04, 2022, and a classification and naming of Lactobacillus gasseri.

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

[0007] The application further provides a bacterial agent, comprising the Lactobacillus gasseri LTG1323.

[0008] Further, the Lactobacillus gasseri LTG1323 can be a living bacterium.

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

[0010] Further, the Lactobacillus gasseri LTG1323 can be a non-living bacterium.

[0011] The application further provides application of the Lactobacillus gasseri LTG1323 in preparation of a medicine for inhibiting Escherichia coli, Staphylococcus aureus, Bacillus cereus, Shigella flexneri and Salmonella typhimurium.

[0012] The application further provides application of the Lactobacillus gasseri LTG1323 in preparation of a feed additive for inhibiting Escherichia coli, Staphylococcus aureus, Bacillus cereus, Shigella flexneri and Salmonella typhimurium.

[0013] The application further provides application of the Lactobacillus gasseri LTG1323 in preparation of a food additive for inhibiting Escherichia coli, Staphylococcus aureus, Bacillus cereus, Shigella flexneri and Salmonella typhimurium.

[0014] The application has the following beneficial effects:

[0015] (1) The Lactobacillus gasseri LTG1323 has inhibitory activity on multiple pathogenic bacteria, and can inhibit the growth of pathogenic bacteria such as Escherichia coli, Staphylococcus aureus, Bacillus cereus, Shigella flexneri and Salmonella typhimurium, and thus can be applied in the auxiliary treatment of various bacterial infection diseases, alone or in combination with other probiotics.

[0016] (2) The Lactobacillus gasseri LTG1323 obtained by screening has higher lactic acid content in the fermentation supernatant than other Lactobacillus gasseri isolates, indicating that the strain has high lactic acid production. BRIEF DESCRIPTION OF DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed in the embodiments. Obviously, the drawings described below are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0018] Figure 1 is a gram staining diagram of LTG1323;

[0019] Figure 2Figure 3 is an agarose gel electrophoresis map of PCR amplification product of LTG1323 nucleotide;

[0020] Figure 3 Figure 4 is a phylogenetic tree of LTG1323;

[0021] Figure 4 Figure 5 is a whole genome map of LTG1323;

[0022] Figure 5 Figure 6 is a growth curve of LTG1323 strain;

[0023] Figure 6 Figure 7 is a nuclear magnetic resonance hydrogen spectrum metabolite detection map of LTG1323 fermentation liquid;

[0024] Figure 7 Figure 8 is an antibacterial circle map of LTG1323 on five pathogenic bacteria;

[0025] Figure 8 Figure 9 is a survival rate change of LTG1323 freeze-dried bacterial powder stored at different temperatures. DETAILED DESCRIPTION

[0026] Various exemplary embodiments of the present application will now be described in detail with reference to the figures. If not specifically stated otherwise, the methods described in the examples are carried out using conventional methods and using reagents that are either commercially available or are prepared by conventional means. This detailed description is not to be taken in a limiting sense, but is made merely for the purpose of describing some aspects, features and embodiments of the present application.

[0027] It should be understood that the terms used herein are merely descriptive, but are not intended to limit the present application. In addition, for numerical ranges in the present application, it should be understood that each intermediate value between the upper limit and the lower limit of the range is specifically disclosed. Each intermediate value within any stated value or stated range, and any other stated value or intermediate value within the stated range, is also included within the present application. The upper and lower limits of these smaller ranges can be independently included or excluded from the ranges.

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, preferred methods and materials are described. All publications mentioned in this specification are herein incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any reference in this specification is not intended as an admission that the reference is prior art, but rather that the reference is part of the state of the art.

[0029] Many modifications and variations of the present application described in the specification are possible without departing from the scope or spirit of the application. Other implementations of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples given are exemplary only.

[0030] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing", or variations thereof, are intended to be open-ended terms that mean inclusion, but not limited to, the listed material or list of materials.

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

[0032] The experimental methods involved in the embodiments of the present application are conventional methods used by those skilled in the art if no special instructions are given; the experimental materials used are commercially available if no special instructions are given.

[0033] Example 1 Screening and preliminary identification of lactobacillus strains

[0034] 1. Isolation of lactobacillus

[0035] Weigh 1.0 g of frozen fecal sample into a test tube containing 9.0 mL of sterile PBS buffer, and perform 10-fold gradient dilution to obtain 10 -6 , 10 -7 , and 10 -8 dilutions. Pour the sample dilutions onto modified MRS solid medium (containing 0.8% CaCO3, 200 μl / 200 mL 2% X-gal) and incubate at 37°C for 56 h. Pick single colonies with calcium-dissolving rings or light blue color and streak them onto MRS plates for 3 generations of streak purification 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 "Berger's Manual of Bacteriology" and the "Classification and Identification of Lactic Acid Bacteria and Experimental Methods" Lactobacillus Characteristics section, and preliminarily screen for suspected lactobacillus.

[0038] 3. Physiological and biochemical identification

[0039] The above-mentioned suspected strains were subjected to gelatin liquefaction experiment, nitrate reduction experiment, litmus milk experiment, glucose acid production and gas production experiment, carbohydrate fermentation acid production experiment, catalase experiment, and the results were compared and identified according to the Manual of Bacteriology and the Classification and Identification of Lactic Acid Bacteria and Experimental Methods, Lactobacillus section. The growth of the strains inoculated in pH4.5, pH7.0 and pH9.0 modified MRS liquid medium was detected, and the growth of the strains in 15℃ and 45℃ modified MRS liquid medium was also detected. The preliminary identified purified Lactobacillus single colony was inoculated in liquid MRS medium and cultured for 24h, mixed with 40% glycerol at a ratio of 1:1, and stored at -80℃ for standby.

[0040] The thawed fecal sample was mixed with sterile phosphate buffer solution (PBS) and spread on MRS solid medium, and incubated at 37℃ under anaerobic conditions. The smooth, complete edge, convex, shiny, soft and blue colonies were streaked onto MRS solid medium. The gram-positive bacteria with rod or spoon shape under microscope, negative catalase test and unable to grow on MRS solid medium under aerobic conditions were temporarily identified as suspected Lactobacillus gasseri. The suspected Lactobacillus gasseri was further streaked on MRS solid medium and purified repeatedly until the colony morphology and microscopic morphology were consistent. Figure 1 ) The single colony was inoculated into MRS liquid medium and incubated at 37℃, and the bacterial liquid was stored and numbered as LTG1323.

[0041] The LTG1323 was subjected to catalase test, glucose acid production and gas production test, gelatin liquefaction test, nitrate reduction test, litmus milk test, temperature tolerance test, pH test (results shown in Table 1), and sugar fermentation test (results shown in Table 2). According to the Classification and Identification of Lactic Acid Bacteria and Experimental Methods, the results of Table 1 and Table 2 can preliminarily identify LTG1323 as Lactobacillus gasseri.

[0042] Table 1 Physiological and biochemical characteristics of LTG1323

[0043]

[0044] Table 2 Sugar fermentation test results 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% to 89% of the strains are positive.

[0047] 4.16S rDNA sequence determination

[0048] Genomic DNA was extracted from LTG1323 and used as a template for polymerase chain reaction (PCR) amplification. Universal 16S rDNA primers were used for amplification: forward primer 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and reverse primer 1492R (5'-GGCTACCTTGTTACGACTT-3'). After amplification, the amplified products were subjected to agarose gel electrophoresis. The electrophoresis results are shown below. Figure 2 .exist Figure 2 A bright band was observed at approximately 1500 bp, indicating successful PCR amplification. The obtained LTG132316S rDNA sequence was compared using BLAST on NCBI. Poppunk was then used to cluster the 68 *Lactobacillus gasseri* strains according to host region, evolutionary relationship, and host. Figure 3 Clustering results show that the 68 *Lactobacillus gasseri* strains were divided into 3 clusters based on evolutionary relationships, and 28 strains were identified by host. These strains primarily originated from adult feces, human urine, human vagina, infant feces, and human breast milk / feces. The strains have a wide geographical range, covering a total of 11 countries (USA: 28 strains, South Korea: 5 strains, India: 5 strains, Italy: 3 strains, Canada: 3 strains, Spain: 2 strains, China: 2 strains, Japan: 1 strain, Russia: 1 strain, Bangladesh: 1 strain, and France: 1 strain). Five strains were found in the same cluster as Lactobacillus gasseri LTG1323: L. gasseri CM2267-MRS2-S780-bin-1, L. gasseri AL3, L. gasseri UMB0056, L. gasseri FR2, and L. gasseri 7135. AL3, an Italian strain, is the most closely related to LTG1323. Strain UMB0056, isolated from human urine in the United States, is slightly more distantly related. Two of the five most closely related strains to LTG1323 were derived from human feces, indicating the high bioaccumulation of Lactobacillus gasseri in the gut and its status as one of the major probiotics in the gut.

[0049] Example 2: LTG1323 whole genome sequencing

[0050] 1. Basic characteristics

[0051] Genomic DNA was extracted from strain LTG1323 and its whole genome was sequenced. Figure 4The whole genome map of LTG1323 is shown, and the results show that LTG1323 has one chromosome and contains two plasmids, indicating that the genes encoded by the plasmids make the traits encoded by the chromosome of LTG1323 more abundant than those of strains without plasmids or with fewer plasmids. The chromosomal genome is circular, and the average content of guanine (G) and cytosine (C) is 34.91%. The size of the chromosome is 5,367,441 bp, containing 5181 protein coding genes (CDSs), and the total length of the coding region accounts for 87.75% of the whole genome. There are 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, which contains metabolic pathways and function-related taxonomic information. The Swiss-prot database is a selected protein sequence database that provides high-level annotation results, such as protein function, domain, post-translational modification and variation information. The LTG1323 chromosomal genome was compared with the NR database, and a total of 5356 protein coding genes were predicted. The LTG1323 chromosomal genome was compared with the Swiss-prot database, and 3732 annotated proteins were obtained.

[0055] 2.2 COG functional gene annotation

[0056] Among all 5356 independent coding regions, 1702 genes can be classified into the Cluster of Orthologous Group (COG) functional classification database. Among them, except for the unknown function part (R and S), the most are genes of the Carbohydrante Transport and Metabolism class (208); followed by genes of the Translation, ribosomal structure and biogenesis class (193); genes of the Transcription class (134); genes of the Cell wall / membrane / envelope biogenesis class (115); and the expression levels of genes of other classes vary.

[0057] 2.3 KEGG functional gene annotation

[0058] The pathways in which L. gasseri LTG1323 is involved are mainly divided into six categories, namely cell transformation, environmental information processing, human disease, metabolism, organic system, and genetic information processing. Among them, the coding genes in the metabolism aspect are the most distributed, mainly including amino acid metabolism, nucleotide metabolism, polysaccharide biosynthesis and metabolism, and energy and carbohydrate metabolism. The participation of numerous genes in strain metabolism also promotes the good probiotic characteristics of LTG1323. The strain can affect the immune system to play an immune regulation function through metabolic products. At the same time, some metabolic products such as lactic acid and bacteriocin can inhibit the growth of pathogenic bacteria, so that the strain has a wider application space in health products, feed and other industries. In the environmental information processing, there are also many related genes involved in biofilm transport, which enables the LTG1323 strain to maintain the normal form of cells through the generation of a membrane transport system to ensure normal life activities when facing environmental changes such as osmotic pressure changes, indicating that L. gasseri LTG1323 has strong environmental adaptability. In the cell transformation aspect, it mainly participates in cell transport and catabolism, as well as cell growth and death.

[0059] 2.4 GO functional gene annotation

[0060] L. gasseri LTG1323 has 2631 genes annotated to the GO database. In the GO functional classification, the annotated genes are mainly involved in three categories: biological process, cell component and composition, and molecular function, a total of 13 functional classifications. Among them, the genes involved in molecular function are the most, a total of 6 subcategories and 1175 genes, accounting for 44.66% of the total number of annotations. They mainly include catalytic activity, transport activity, structural molecular activity, transcription regulator activity, ATP production activity, etc. In the biological process classification, they mainly participate in biological regulation, cell localization, metabolic process, stimulus response, and cell composition biological occurrence, accounting for 42.27%. In the cell component and cell composition functional classification, they mainly participate 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] A clean and sterile 50 mL microbial growth test centrifuge tube was prepared in advance, 10 mL of culture solution was poured into it, and L. gasseri LTG1323 single colony after purification was picked into the culture solution. The reactor temperature was set to 37°C, and the machine was continuously tested for 24 h. The growth curve of the strain was drawn.

[0064] The OD 600 value data of the strain cultured for 24 h continuously was exported, and the growth curve of the strain was drawn as shown in Figure 5 . Figure 5The growth curve of Lactobacillus gasseri LTG1323 is shown in Figure 1. The strain entered the logarithmic phase after 5h, reached the maximum value of growth at about 18h, and entered the stationary phase after 18h, and entered the decline phase after about 23h. Therefore, we selected 18h as the optimal time for the strain culture for the next experiment.

[0065] 2. Metabolic active substance detection of LTG1323 strain

[0066] The fermentation solution in the stationary phase was used for metabolite extraction. 1 mL of the fermentation solution in the stationary phase and 1 mL of the metabolite extraction solution were mixed thoroughly. The metabolite extraction solution was mixed by NaH2PO4-K2HPO4 buffer and acetonitrile at a ratio of 1:1 (v / v). The mixture was treated with ultrasonic ice bath to break the cells. Then, the mixture was stored at -20°C for 2 hours to extract metabolites. After that, the thawed sample was centrifuged (12000 x g, 4°C, 15min) to obtain the supernatant containing metabolites. Then the supernatant was 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 supernatant was transferred to an NMR tube for testing. The sample should be stored at 4°C for standby, and the storage time should not exceed 24 hours before nuclear magnetic resonance test. All samples were measured by standard Bruker NOESY pulse sequence. The acquisition parameters are as follows: number of scans (64), spectral width (20ppm), relaxation delay (2s), number of sampling points (65536), sampling time (3.277s), mixing time (0.1s), FID resolution (0.245).

[0067] The nuclear magnetic resonance hydrogen spectrum metabolite spectrum of the LTG1323 strain is shown in Figure 2. Figure 6

[0068] Example 4 Simulated gastrointestinal reverse environment and adhesion of LTG1323 strain

[0069] Probiotics colonize in the intestinal tract, and first of all, they can tolerate the low-pH gastric acid environment, so the evaluation of acid tolerance and pepsin resistance is an important indicator for screening probiotics. Determining these indicators can determine the survival ability of the screened strain after gastric digestion.

[0070] 1. Simulated gastrointestinal transport experiment of LTG1323

[0071] ​The bacteria to be tested were centrifuged (4000g, 20 min) from the overnight culture solution, washed twice with sterilized physiological saline, and resuspended in 10 mL of artificial gastric juice (NaCl 0.2 g / 100 mL, pepsin 0.32 g / 100 mL, pH 2.5 adjusted with 1 mol / L HCl, sterilized by filtration and stored for use) with a pH of 2.5. The bacteria were cultured at 37°C for 120 min on a 200 rpm shaker, centrifuged (4000g, 20 min) to collect the bacteria, resuspended in 10 mL of artificial intestinal juice (KH2PO4 0.68 g / 100 mL, trypsin 1 g / 100 mL, bile salt 0.3 g / 100 mL, pH 8.0) with a pH of 8.0, and cultured at 37°C for 120 min on a 200 rpm shaker. The number of viable bacteria in the sample before and after treatment was detected, and the results are shown in Table 3.

[0072] Table 3 Results of the tolerance of LTG1323 to simulated digestive juice (action time 240 min)

[0073]

[0074] 2. Self-aggregation ability determination

[0075] The self-aggregation ability determination was slightly modified according to the reference. The bacteria to be tested were centrifuged (4000g, 20 min) from the overnight culture solution, washed twice with physiological saline, and resuspended in PBS buffer to make the number of viable bacteria 10 8 CFU / mL. The bacterial suspension (5 mL) was vortexed for 10 s and left to stand at room temperature for 5 h, and the self-aggregation ability was determined. The supernatant was taken every hour during this period, and the absorbance was measured at 600 nm. The self-aggregation value (%) was represented by the following formula:

[0076]

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

[0078] A0represents the absorbance at time 0.

[0079] 3. Surface hydrophobicity detection

[0080] The surface hydrophobicity detection was performed by centrifuging (4000g, 20 min) the bacteria to be tested from the overnight culture solution, washing twice with physiological saline, and resuspending in 0.1 mol / L KNO3 (pH 6.2) to make the number of viable bacteria 10 8CFU / mL, the absorbance A0 of the sample was measured at 600 nm. 1 mL of xylene solvent was added to 3 mL of bacterial solution, and the two-phase system was vortexed for 2 min at room temperature, and then left to stand for 20 min at room temperature. The water phase was taken, and the absorbance at 600 nm (A1) was measured. The adhesion percentage (%) of the bacteria to the solvent was expressed by the following formula:

[0081]

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

[0083]

[0084] Example 5 Antibiotic sensitivity

[0085] If the screened probiotic bacteria are not sensitive to a certain antibiotic, it means that it carries the corresponding resistance gene. If such probiotic bacteria colonize in the human intestinal tract, the resistance gene it carries has the potential danger of being implanted into other microbial cells in some way, so the higher the sensitivity of the screened probiotic bacteria to antibiotics is, the better, of course, some bacteria have natural resistance to certain antibiotics, not from the spread of other strains.

[0086] The present application selects six common antibiotics for drug sensitivity test. LTG1323 is cultured overnight, and the bacterial solution is spread on MRS agar medium, and then antibiotic paper (erythromycin, chloramphenicol, compound sulfamethoxazole, ampicillin, cefazolin, norfloxacin) is covered on the agar medium, which is first pre-cultured and diffused at 4°C for 2 h, and then transferred to 37°C for incubation for 48 h, and the inhibition diameter is measured, and the sensitivity (S), intermediate (I) and resistance (R) are judged according to the Clinical and Laboratory Standards Institute (CLSI) provisions. The results are shown in Table 6.

[0087] Except that the standard of compound sulfamethoxazole is intermediate, LTG1323 is sensitive to erythromycin, chloramphenicol, ampicillin, cefazolin and norfloxacin, which shows that LTG1323 has relatively safe use value.

[0088] Table 6 Drug sensitivity of strain LTG1323 to six common antibiotics

[0089]

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

[0091] E. coli ATCC2522, Staphylococcus aureus, Bacillus cereus, Shigella flexneri and Salmonella typhimurium were used as indicator bacteria, which were from the laboratory or China Center for Type Culture Collection. E. coli ATCC2522, Staphylococcus aureus, Bacillus cereus, Shigella flexneri and Salmonella typhimurium were inoculated into LB liquid broth medium, and incubated at 37°C overnight, and then subcultured for 3-5 generations to stabilize the properties. LTG1323 was inoculated into MRS liquid medium, and incubated to the stationary phase, and then centrifuged to obtain the supernatant. The five pathogenic bacteria after liquid expansion to the same concentration were diluted by 10 times gradient, and 100 μL was coated on LB broth agar plates. Two oxford cups were placed equidistantly on each plate, and 200 μL of LTG1323 culture supernatant was injected into each cup, and each pathogenic bacteria was parallelly tested for three times. After incubation at 4°C for 2 h and at 37°C for 36 h, the size of the inhibition zone was measured, and a typical inhibition zone diagram is shown in FIG. 5. -1 Figure 7 The size of the inhibition zone of the culture supernatant of the strain on the five pathogenic bacteria is shown in Table 5, and the results show that LTG1323 has obvious inhibition ability on the five pathogenic bacteria.

[0092] Table 5 Size of inhibition zone of culture supernatant of strain on five pathogenic bacteria

[0093]

[0094] Example 7 Application of LTG1323 in the fields of food, food supplements, health products, pharmaceuticals and the like

[0095] LTG1323 can be directly used for fermentation of dairy products or mutual fermentation of dairy products with traditional fermentation agents, or can be added in the form of freeze-dried powder to food, feed, pharmaceuticals, etc. to regulate the intestinal flora of humans and animals and improve the immunity of the body. LTG1323 can also be consumed in a non-living form.

[0096] As one of the preferred embodiments, fermented milk is prepared by using Lactobacillus gasseri LTG1323. The preparation method of Lactobacillus gasseri LTG1323 fermented milk is as follows: raw milk powder is reconstituted with water at a ratio of 10%, and the reconstituted milk is prepared by mixing the raw milk powder, white granulated sugar and purified water according to the experimental design, heating and stirring to dissolve. The raw milk is preheated to 50-60°C, and homogenized at 20-25 MPa for 5 min. Then, 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 sterile conditions. The raw milk is fermented at 40-42°C for 6-8 h to achieve coagulation, and then stored at 2-6°C for 24 h to mature.

[0097] ​As a second preferred method, Lactobacillus gasseri LTG1323 was used to prepare lyophilized powder. The fermentation broth of LTG1323 cultured to the logarithmic growth phase was centrifuged at 4°C, the supernatant was discarded, and Lactobacillus gasseri sludge was obtained. The sludge was then resuspended in 1 / 10 volume of sterile physiological saline from the original fermentation broth and centrifuged again to obtain sludge. The optimized Lactobacillus gasseri lyophilization protectant (9.98% skim milk powder, 8.18% sucrose, 3.94% glycerol, 0.91% L-cysteine) was mixed with the sludge at a ratio of 10:1, and frozen at -80°C for 4 hours. After lyophilization, the LTG1323 lyophilized powder was obtained. Under this protectant, the lyophilized survival rate of the strain reached (83.30±0.092)%, which is 2.42 times that before optimization. Compared with relevant reports at home and abroad, the lyophilized survival rate of Lactobacillus gasseri reached the highest value to date.

[0098] Under dry and sterile conditions, 2.0g of bacterial powder was placed in an aluminum foil bag and vacuum-sealed. The sealed freeze-dried bacterial powder samples were placed in environments at -80℃, -20℃, and -4℃, respectively. The viable bacterial count was measured every 7 days, and the survival rate was calculated. The measurements were repeated for 2 months. Figure 8 It can be seen that when the bacterial powder is stored under different temperature conditions, low-temperature storage is better than room-temperature storage, with the optimized group ( Figure 8 B) and the control group ( Figure 8 A) All strains showed optimal storage performance at -20℃, with higher survival rates than the other three storage temperatures. Comparison revealed that the optimized composite protectant reduced the probability of frost damage during storage. In the control group, the survival rate decreased at a faster rate, indicating that the protection effect of a single protectant was weak. With the optimized composite protectant, the freeze-drying survival rate of the strain decreased more gradually under all four temperature conditions, which to some extent extended the shelf life of *Lactobacillus gasseri* LTG1323 freeze-dried products and ensured high biological activity of the strain. Therefore, the optimized composite protectant not only improved the freeze-drying survival rate of *Lactobacillus gasseri* LTG1323 powder but also enhanced the stability of the strain during storage.

[0099] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. Lactobacillus gasseri LTG1323, characterized in that, The Lactobacillus gasseri LTG1323 is preserved in Guangdong Microbial Culture Collection Center, and the preservation number is GDMCC No: 63037, and the preservation date is December 4, 2022.

2. An inoculant characterized in that, The Lactobacillus gasseri LTG1323 as claimed in claim 1.

3. The bacterial agent of claim 2, wherein The Lactobacillus gasseri LTG1323 is a live bacterium.

4. The bacterial agent of claim 2, wherein The Lactobacillus gasseri LTG1323 is a freeze-dried powder.

5. The bacterial agent of claim 2, wherein The Lactobacillus gasseri LTG1323 is a non-live bacterium.

6. The use of the Lactobacillus gasseri LTG1323 as claimed in claim 1 in the preparation of a medicine for inhibiting Escherichia coli, Staphylococcus aureus, Bacillus cereus, Shigella flexneri and Salmonella typhimurium.

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

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

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