Tetragenococcus halophilus with inhibitory effect on bacillus cereus and use thereof

Abstract

Provided are a tetragenococcus halophilus with an inhibitory effect on bacillus cereus and a use thereof. The preservation number of the tetragenococcus halophilus is GDMCC 64945. The tetragenococcus halophilus is isolated from fermented bean curd, and colonies of the tetragenococcus halophilus are milky white, small and round on a solid medium; The tetragenococcus halophilus is Gram-positive, and when observed under a microscope, the morphology thereof is mostly diplococci or tetrads, with no chain-like formations. The strain is sensitive to eight antibiotics such as ampicillin, gentamicin and kanamycin, and has no drug resistance gene and virulence gene. A bacterial suspension of the strain has a good inhibitory effect on a bacillus cereus strain isolated from the fermented bean curd, and when the strain is applied to a fermented bean curd fermentation system, the growth of bacillus cereus in the system can be effectively inhibited, and the content of enterotoxin in the system is reduced.

Description

Tetragenococcus halophilus with bacillus cereus inhibiting effect and application TECHNICAL FIELD

[0001] The present application relates to Tetragenococcus halophilus, in particular to a kind of Tetragenococcus halophilus (Tetragenococcus halophilus) LXHMY1 with bacillus cereus inhibiting effect and its application in fermented bean curd fermentation system;Belong to the technical field of microbial application. BACKGROUND

[0002] Bacillus cereus is a common gram-positive bacteria in soil, which is easy to be detected in the environment and various agricultural raw materials, and then enters the food chain. Bacillus cereus can produce diarrhea enterotoxin and emetic toxin, and the main clinical manifestations are diarrhea and vomiting, and some strains can even cause serious eye infection, liver failure and other related diseases. It is generally believed that when the number of bacillus cereus reaches 5-8 Log CFU / g, food poisoning will occur. The spores produced by bacillus cereus have good survival ability under different environmental stress conditions, and have certain tolerance to high temperature, dryness, and some toxic chemicals.

[0003] Fermented bean curd, bean paste, soy sauce and other Chinese traditional fermented soy products are mainly made of soybeans. Open or semi-open pre-fermentation and salted curing are usually used, so microorganisms in the environment have the opportunity to enter the fermentation system. Although high concentration of salt has inhibitory effect on most pathogenic microorganisms, it has limited effect on bacillus cereus and other spore-forming bacteria, which needs to be paid attention to. It has been reported that bacillus cereus has been detected in many traditional fermented foods, and fermented bean curd is prone to exceed the standard of bacillus cereus due to rich nutrition and no sterilization process.

[0004] In view of the above problems existing in traditional fermented foods, food science and technology workers have carried out extensive research. The substances reported so far to inhibit the growth of bacillus cereus include plant extracts, microbial metabolites, biological agents, etc., but these methods still have problems such as complex preparation method, unguaranteed safety, or possible influence on food flavor, which restricts their application in food system. For example, although plant essential oil has good antibacterial effect, it is difficult to apply in fermented food due to its significant influence on product flavor. Therefore, it is of great significance to actively seek new ways to solve the pollution and harm of bacillus cereus in fermented food.

[0005] Chinese invention patent application CN117796403A discloses the application of catechin in inhibiting Bacillus cereus; through double dilution method and agar diffusion method, it is determined that the minimum inhibitory concentration of catechin on Bacillus cereus is 2 mg / mL, and the minimum bactericidal concentration is 4 mg / mL, and it is demonstrated that catechin contained in the extract of Tremella fuciformis is a natural active ingredient for inhibiting the growth of Bacillus cereus. However, the catechin of the technology has the risk of causing sour or astringent taste in fermented food, which may adversely affect the quality and flavor of the fermented food. SUMMARY

[0006] To solve the problems existing in the prior art, the purpose of the present application is to provide a Tetragenococcus halophilus which can inhibit the growth of Bacillus cereus, has simple culture conditions, grows well in a high salt environment, is sensitive to ampicillin, gentamicin, kanamycin, streptomycin, clindamycin, tetracycline, erythromycin and chloramphenicol, and has no drug resistance genes and virulence genes.

[0007] Another purpose of the present application is to provide the application of the Tetragenococcus halophilus in inhibiting Bacillus cereus in a fermented bean curd fermentation system.

[0008] The purpose of the present application is achieved by the following technical solutions:

[0009] A Tetragenococcus halophilus LXHMY1, which was deposited at the Guangdong Microbial Culture Collection Center on August 2, 2024, at the address of 100, Martyrs' Road, Guangzhou, China, in Building 59, 5th Floor, and with the preservation number of GDMCC 64945.

[0010] The present application also protects the application of the Tetragenococcus halophilus in inhibiting Bacillus cereus in a fermented bean curd fermentation system.

[0011] To further achieve the purpose of the present application, preferably, the Tetragenococcus halophilus is inoculated in the sterilized culture medium to obtain a Tetragenococcus halophilus suspension, the Tetragenococcus halophilus suspension is diluted and inoculated into the pre-fermented soybean milk liquid, salt is added, and fermentation is carried out at a temperature of 25-30℃ for 7-28 days.

[0012] Preferably, the culture medium of the Tetragenococcus halophilus is MRS broth medium containing 8-10 g / 100 mL NaCl.

[0013] Preferably, the sterilization of the culture medium is carried out at 120-122℃ for 15-20 min.

[0014] Preferably, the inoculation amount of the Tetragenococcus halophilus in the sterilized culture medium is 2-5 vol%.

[0015] Preferably, the inoculation method of the halotolerant Tetragenoccus in the sterilized culture medium is to obtain the halotolerant Tetragenoccus bacterial suspension after culturing at a temperature of 25-30 DEG C for 48-72h.

[0016] Preferably, the pre-fermented soybean liquid contains Bacillus cereus.

[0017] Preferably, the concentration of the diluted halotolerant Tetragenoccus bacterial suspension is 5-6 Log CFU / mL; and the inoculation amount of the diluted halotolerant Tetragenoccus bacterial suspension is 1-3 vol %.

[0018] Preferably, the salt addition is to add 8-12g of salt per 100mL of pre-fermented soybean liquid, and the pre-fermented soybean liquid after salt addition constitutes the fermented bean curd fermentation system.

[0019] The halotolerant Tetragenoccus provided by the application has the advantages and beneficial effects of:

[0020] 1) The halotolerant Tetragenoccus LXHMY1 disclosed in the application has simple culture conditions, a simple preparation method of the bacterium suspension for bacteriostasis, and good application potential in the fermented food industry.

[0021] 2) The halotolerant Tetragenoccus LXHMY1 disclosed in the application grows well in a high-salt environment, and is suitable for the production environment of high-salt products such as fermented bean curd, soybean paste and soy sauce.

[0022] 3) The halotolerant Tetragenoccus LXHMY1 disclosed in the application is sensitive to ampicillin, gentamicin, kanamycin, streptomycin, clindamycin, tetracycline, erythromycin and chloramphenicol, has no drug resistance gene and virulence gene, and has wide application prospects.

[0023] 4) The halotolerant Tetragenoccus LXHMY1 disclosed in the application has a good inhibitory effect on Bacillus cereus separated from fermented bean curd in a solid culture medium, and has potential for preparing related biological agents.

[0024] 5) The halotolerant Tetragenoccus LXHMY1 disclosed in the application can significantly reduce the number of Bacillus cereus in the fermented bean curd fermentation system, reduce the content of enterotoxin in the system, and effectively improve the safety of fermented foods such as fermented bean curd. BRIEF DESCRIPTION OF DRAWINGS

[0025] Fig. 1 is a colony morphology diagram of the halotolerant Tetragenoccus LXHMY1 of the application;

[0026] Fig. 2 is a bacterial body morphology diagram of the halotolerant Tetragenoccus LXHMY1 of the application under a microscope;

[0027] Fig. 3 is a phylogenetic tree of the halotolerant Tetragenoccus LXHMY1 of the application;

[0028] Figure 4 is a genome circle diagram of the salt-tolerant tetragenous Streptococcus LXHMY1 of the present application;

[0029] Figure 5 is the annotation result of the secondary metabolite of the salt-tolerant tetragenous Streptococcus LXHMY1 of the present application in the antiSMASH database;

[0030] Figure 6 is a diagram showing the change in the number of Bacillus cereus in the fermented bean curd fermentation system to which the salt-tolerant tetragenous Streptococcus LXHMY1 of the present application is added in Example 5 of the present application;

[0031] Figure 7 is a diagram showing the change in the content of enterotoxin in the fermented bean curd fermentation system to which the salt-tolerant tetragenous Streptococcus LXHMY1 of the present application is added in Example 6 of the present application. DETAILED DESCRIPTION

[0032] In order to better understand the present application, the present application will be further described in detail below in combination with the accompanying drawings and examples, but the examples do not constitute any limitation on the protection scope of the present application.

[0033] Lactic acid bacteria are common microorganisms in fermented foods, often having good safety and probiotic functions, and some strains can also produce antibacterial substances, including organic acids, lactoperoxidase, diacetyl, bacteriocins and other metabolites. These substances continuously accumulate during fermentation and have a strong antagonistic effect on spoilage or pathogenic bacteria present in fermented foods, which can improve the safety of naturally fermented foods. For example, Lactococcus lactis subsp. lactis can produce bacteriocin Nisin, which has antibacterial effects on Bacillus cereus, Staphylococcus aureus, Listeria monocytogenes and other pathogenic bacteria. In addition, Lactobacillus plantarum, Pediococcus pentosaceus, Lactobacillus reuteri and other lactic acid bacteria have certain inhibitory effects on pathogenic bacteria such as Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa. In addition to the above-mentioned strains, salt-tolerant tetragenous Streptococcus is a lactic acid bacteria widely present in salted fermented foods such as soybean paste and soy sauce. This strain can grow in a high-salt environment and produce acid without gas in a culture environment with glucose as a carbon source. When added to fermented foods, it can promote the generation of alcohols, acids and esters, and has a gain effect of improving the taste and quality of foods. It has been reported that salt-tolerant tetragenous Streptococcus has inhibitory effects on Staphylococcus aureus, Escherichia coli and Listeria monocytogenes, and has a wide application prospect.

[0034] The inventors isolated a strain from fermented bean curd, which was gram-positive and mainly in the form of diplococcus and tetracoccus under microscopic observation. Genomic DNA was extracted using a bacterial DNA extraction kit, and PCR amplification was performed. The PCR product was sent to Shenguo Bioengineering (Shanghai) Co., Ltd. for sequencing to obtain a 16S rDNA sequence. Analysis by the BLAST tool of NCBI found that the similarity of the strain to Tetragenococcus halophilus was higher than 99.0%, and therefore, the strain was determined to be Tetragenococcus halophilus. The Tetragenococcus halophilus LXHMY1 was preserved in the Guangdong Microbial Culture Collection Center on August 2, 2024, at an address of 59 Building, 5th Floor, Institute of Guangzhou, 100 Middle Martyrs Road, Guangzhou, with a preservation number of GDMCC 64945.

[0035] It was found through detection that the Tetragenococcus halophilus has simple culture conditions, a simple preparation method of bacterium suspension for antibacterial use, and grows well in a high-salt environment, and is suitable for the production environment of high-salt products such as fermented bean curd, bean paste and soy sauce; the Tetragenococcus halophilus is sensitive to ampicillin, gentamicin, kanamycin, streptomycin, clindamycin, tetracycline, erythromycin and chloramphenicol, and has no drug resistance genes and virulence genes; in particular, the bacterium suspension of the Tetragenococcus halophilus has a good inhibitory effect on Bacillus cereus isolated from fermented bean curd in a solid culture medium, can significantly reduce the number of Bacillus cereus in a fermented bean curd fermentation system, and reduce the content of enterotoxin in the system, thereby effectively improving the safety of fermented foods such as fermented bean curd.

[0036] According to the characteristics of the Tetragenococcus halophilus, the application protects the application of the Tetragenococcus halophilus in the fermented bean product fermentation system. In the specific application, the Tetragenococcus halophilus is inoculated in the sterilized culture medium to obtain a Tetragenococcus halophilus suspension, the Tetragenococcus halophilus suspension is diluted and inoculated into the pre-fermented soybean liquid, salt is added, and the fermentation is carried out at a temperature of 25-30 DEG C for 7-28 days. The culture medium of the Tetragenococcus halophilus is MRS broth medium containing 8-10 g / 100 mL NaCl; the inoculation amount of the Tetragenococcus halophilus in the sterilized culture medium is preferably 2-5 vol%. The inoculation method of the Tetragenococcus halophilus in the sterilized culture medium is to culture at a temperature of 25-30 DEG C for 48-72 h to obtain the Tetragenococcus halophilus suspension. The concentration of the diluted Tetragenococcus halophilus suspension is 5-6 Log CFU / mL; the inoculation amount of the diluted Tetragenococcus halophilus suspension is 1-3 vol%. The salt is added in an amount of 8-12 g per 100 mL of pre-fermented soybean liquid, and the pre-fermented soybean liquid after the addition of salt constitutes the fermented bean product fermentation system.

[0037] In order to verify the effect of the Tetragenococcus halophilus on the inhibition of Bacillus cereus in the fermented bean product fermentation system, the application constructs a "fermented bean product simulation fermentation system", which embodies the production process of fermented bean products such as fermented bean curd, bean paste and soy sauce, and the addition of the Tetragenococcus halophilus single strain is conducive to the determination of its role in the fermented bean product fermentation system. The fermented bean product simulation fermentation system embodies that the Tetragenococcus halophilus can reduce the number of Bacillus cereus and the content of enterotoxin in the production process of fermented bean products. The control group, i.e. the group only added with salt, can reflect the possible Bacillus cereus contamination in the production process of fermented bean products without the addition of the Tetragenococcus halophilus. Specifically, the application constructs a pre-fermented soybean liquid inoculated with Bacillus cereus, and the preparation method of the pre-fermented soybean liquid comprises the following steps:

[0038] 1) Preparation of sterile soybean liquid: select fresh yellow beans with complete particles and no mildew, add 4-6 times the mass of water and 0.5-2 g / 100 mL of baking soda, soak at room temperature for 14-20 h; after soaking, discard the supernatant, wash the yellow beans, and add 8-10 times the mass of double-distilled water for heating and grinding; filter the bean dregs through a 160-200 mesh sieve to obtain fresh soybean liquid; add 5-8 g / 100 mL of sucrose, sterilize at 100-110 DEG C for 20-30 min to obtain sterile soybean liquid;

[0039] 2) Strain activation: inoculate the representative strain of Bacillus cereus into sterilized LB broth medium at an inoculation amount of 2%-5% of the volume of the culture medium to obtain a Bacillus cereus suspension;

[0040] 3) Preparation of pre-fermented soybean milk: The B. cereus bacterial suspension was diluted to a concentration of 5-6 Log CFU / mL, and then inoculated into sterile soybean milk at a volume of 1%-3% of the soybean milk. The mixture was fermented at a temperature of 25-30°C for 6-8 hours to obtain pre-fermented soybean milk.

[0041] In the following examples and comparative examples:

[0042] 1) MRS broth medium containing 8 g / 100 mL NaCl:

[0043] In 1 L of distilled water, 10.0 g of casein zymopeptone, 2.0 g of ammonium citrate, 10.0 g of beef extract powder, 0.2 g of magnesium sulfate (MgSO4·7H2O), 4.0 g of yeast extract powder, 0.05 g of manganese sulfate (MnSO4·4H2O), 5.0 g of sodium acetate, 2.0 g of potassium phosphate dibasic, 20.0 g of glucose, 1.08 g of Tween-80, and 80 g of sodium chloride (NaCl) were added. The pH was natural, and the mixture was sterilized at 121°C for 15 min. The MRS solid medium containing 8 g / 100 mL NaCl was prepared by adding 2 g / 100 mL of agar powder to the broth medium.

[0044] 2) Observation of colony morphology and cell morphology of the strain

[0045] The strain Tetragenococcus halophilus LXHMY1 was anaerobically cultured on MRS solid medium containing 8 g / 100 mL NaCl using the streak method, and the single colony morphology was observed. The cell morphology was observed under a microscope after Gram staining.

[0046] 3) 16S rDNA sequencing and identification of the strain

[0047] The most commonly used universal bacterial primers 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' and 2492R: 5'-GGTTACCTTGTTACGACTT-3' were used. The PCR reaction system was as follows: 2 μL of DNA template, 2 μL of primer 27F, 2 μL of primer 1492R, 20 μL of 2×PCR Mix, and 24 μL of ultrapure water. The PCR reaction program was as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 46°C for 30 s, and extension at 72°C for 1 min, for 35 cycles; and terminal extension at 72°C for 10 min. The obtained PCR product was sent to Shengong Bioengineering (Shanghai) Co., Ltd. for sequencing. The sequencing results were subjected to homology search using the BLAST tool in NCBI, and a phylogenetic tree was drawn using MEGA 7.0.

[0048] 4) Determination of drug resistance phenotype by micro-broth dilution method

[0049] According to the European Food Safety Authority (EFSA) regulations for drug sensitivity experiments, the drug resistance phenotype experiment was carried out by micro-broth dilution method, and the selected antibiotics were ampicillin, gentamicin, kanamycin, chloramphenicol, erythromycin, clindamycin, tetracycline, chloramphenicol, and the gradient concentrations were 64, 32, 16, 8, 4, 2, 1, 0.5 μg / mL. The halophilic tetragemella stored at -80℃ was activated twice with MRS broth medium containing 8g / 100mL NaCl, and the concentration of bacterial suspension was adjusted to 8LogCFU / mL. 100 μL of gradient concentration of antibiotics and 100 μL of bacterial suspension were added to 96-well plates, and the bacterial suspension containing the highest concentration of antibiotic diluent and MH medium was set as positive control, and the MH medium without bacterial suspension was set as negative control. Incubate at 30℃ for 48h, measure the absorbance at wavelength 625nm, and determine the drug resistance phenotype of different strains according to the minimum inhibitory concentration (MIC) inflection point specified by EFSA. The MIC inflection point of halophilic tetragemella is: ampicillin 4 μg / mL, gentamicin 16 μg / mL, kanamycin 64 μg / mL, streptomycin 64 μg / mL, erythromycin 1 μg / mL, clindamycin 1 μg / mL, tetracycline 8 μg / mL, chloramphenicol 4 μg / mL.

[0050] 5) Whole genome sequencing

[0051] The halophilic tetragemella LXHMY1 was activated twice with MRS broth medium containing 8g / 100mL NaCl, centrifuged at 8000xg for 5min, and the supernatant was discarded. High-quality genomic DNA was extracted, and the purity, concentration and integrity were detected by Nanodrop, Qubit and 0.35% agarose gel electrophoresis. Large fragment DNA was recovered by BluePippin automatic nucleic acid recovery system. SQK-LSK109 ligation kit was used for library construction, including DNA damage repair and end repair, magnetic bead purification, linker ligation and magnetic bead purification, Qubit library quantification, and finally high-quality library DNA was sequenced. The raw data was quality controlled, and low-quality and short-length (<2000bp) reads were filtered. The filtered reads were assembled de novo using Canu v1.5, and the assembly results were corrected by Racon v3.4.3 software. The circularization and adjustment of the starting site were performed by Circlator v1.5.5, and the draft genome after assembly was corrected by Pilon v1.22. The qualified data after assembly can be used for genome component analysis, functional analysis and genome map analysis.

[0052] Genes were predicted by Prodigal v2.6.3, repeat sequences were aligned by RepeatMasker v4.0.5, gene islands were predicted by IslandPath-DIMOB v0.2, and finally, genome circle maps were drawn by Circos v0.66. Drug resistance gene information was annotated by CARD database, virulence genes were annotated by VFDB database, and secondary metabolite information was annotated by antiSMASH database.

[0053] 6) Inhibition of Bacillus cereus isolated from fermented soybeans

[0054] The applicant previously isolated 6 strains of Bacillus cereus from fermented soybeans, numbered LZKSSF4, LZKSSF10, LZKWSF1, LZKWSF10, LZKWSF16, and LZKWSF21.

[0055] The halophilic Tetragenococcus LXHMY1 was activated twice in succession in MRS broth medium containing 8 g / 100 mL NaCl, and the Bacillus cereus was activated twice in succession in LB broth medium, both adjusted to a bacterial suspension concentration of 8 Log CFU / mL. The Bacillus cereus bacterial suspension was inoculated at an inoculum of 1 vol% in MRS solid medium, and a hole was punched in the solid medium plate using the Oxford cup method, 200 μL of the Tetragenococcus bacterial suspension was added, and three replicates were set up. After 48 h of incubation, the diameter of the inhibition zone was measured using a vernier caliper.

[0056] 7) Fermented soybean simulation system

[0057] Fresh yellow beans with intact particles and no mold were selected, 4-6 times the mass of water and 0.5-2 g / 100 mL of baking soda were added, and soaked at room temperature for 14-20 h. After soaking, the supernatant was discarded, the yellow beans were washed, and 8-10 times the mass of double-distilled water was added for heating and pulping. The bean dregs were filtered out through a 160-200 mesh sieve to obtain fresh soybean milk. 5-8 g / 100 mL of sucrose was added, sterilized at 100-110°C for 20-30 min to obtain sterile soybean milk. The representative strain of Bacillus cereus was inoculated into sterilized LB broth medium at an inoculum of 2-5% of the volume of the medium to obtain a Bacillus cereus bacterial suspension. The Bacillus cereus bacterial suspension was inoculated into the sterile soybean milk, the bacterial suspension concentration was adjusted to 5-6 Log CFU / mL, the inoculum was 1-3 vol%, and the pre-fermented soybean milk was obtained after fermentation at 30°C for 6-8 h. Tetragenococcus bacterial suspension with a concentration of 5-6 Log CFU / mL was inoculated into the pre-fermented soybean milk at an inoculum of 1-3 vol%, and 8-12 g / 100 mL of salt was added to form a fermented soybean simulation system, with a sample containing only 8-12 g / 100 mL of salt as a control group. Sealed and post-fermented at 25-30°C for 7-28 days.

[0058] 8) Bacillus cereus count

[0059] The method for counting Bacillus cereus in the simulated fermentation system of fermented bean curd refers to GB 4789.14-2014 Food Safety National Standard Food Microbiological Examination Bacillus cereus Examination.

[0060] 9) Detection of enterotoxin content

[0061] Enterotoxin is one of the common toxins in the fermentation system, which is derived from Bacillus cereus. Detecting the content of enterotoxin in the fermentation system can further understand the safety of fermented food. The content of enterotoxin is detected by double antibody one-step sandwich enzyme-linked immunosorbent assay (ELISA), and the specific operation refers to the Bacillus cereus enterotoxin ELISA kit manual.

[0062] Example 1: Screening and identification of Tetragenococcus halophilus

[0063] (1) Isolation and purification of strains

[0064] Take 5 g of fermented bean curd sample, add 45 mL of sterile physiological saline, and grind and stir thoroughly in a mortar. Take 1 mL of the grinding liquid and dilute it in 9 mL of sterile physiological saline. Take 3 appropriate gradient dilutions and coat them on MRS solid medium containing 8 g / 100 mL NaCl, and incubate at 30°C for 48 h. Select single colonies with different morphologies and further purify them by plate streaking method. Repeat this process 2 more times until single colonies with uniform morphology are obtained on the same plate. Purified single colonies are cultured in MRS liquid medium containing 8 g / 100 mL NaCl for 48 h, and preserved in a -80°C freezer with glycerol.

[0065] (2) Observation of colony and cell morphology

[0066] Tetragenococcus halophilus LXHMY1 presents white, small and round colonies on MRS solid medium containing 8 g / 100 mL NaCl (see Figure 1). Gram staining is positive, and under microscopic observation, the morphology is mostly diplococcus and tetragenus, without chains (see Figure 2).

[0067] (3) 16S rDNA sequencing identification

[0068] Genomic DNA was extracted using a bacterial DNA extraction kit, PCR amplification was performed, the PCR product was sent to Shenguo Bioengineering (Shanghai) Co., Ltd. for sequencing, and the 16S rDNA sequence was obtained. Analysis by BLAST tool of NCBI found that the strain had a similarity higher than 99.0% with Tetragenococcus halophilus NBRC 12172, Tetragenococcus halophilus DSW01, Tetragenococcus halophilus JCM 5888, Tetragenococcus halophilus GZH2-28, and Tetragenococcus halophilus JCM20247. Therefore, LXHMY1 was determined to be Tetragenococcus halophilus, and a phylogenetic tree was constructed (see Figure 3).

[0069] The Tetragenococcus halophilus LXHMY1 was preserved in the Guangdong Microbial Culture Collection Center on August 2, 2024, at an address of 5th Floor, Building 59, 100, Martyrs' Road, Guangzhou, with a preservation number of GDMCC 64945.

[0070] Example 2: Antibiotic resistance

[0071] The drug resistance phenotype detection results of Tetragenococcus halophilus LXHMY1 were as follows: ampicillin 2 μg / mL, gentamicin 8 μg / mL, kanamycin 32 μg / mL, streptomycin 32 μg / mL, erythromycin 0.5 μg / mL, clindamycin 0.5 μg / mL, tetracycline 4 μg / mL, chloramphenicol 4 μg / mL. The detection values were all less than or equal to the inflection point value specified by EFSA, so LXHMY1 was sensitive to the 8 kinds of antibiotics detected.

[0072] Example 3: Whole genome information analysis

[0073] (1) Basic information

[0074] The genome size of Tetragenococcus halophilus LXHMY1 was 2450789 bp (circular chromosome), and there was no plasmid. The whole genome contained 2348 coding genes, with a total predicted gene sequence length of 2097015 bp, and 15 and 62 predicted rRNA genes and tRNA genes, respectively. The strain predicted 14 gene islands with a total length of 146180 bp and an average length of 10441. The LXHMY1 genome circle diagram was drawn using the assembled and predicted genomic information (see Figure 4).

[0075] In Fig. 4, the first circle from outside to inside is genome size marker, each scale is 5 kb; the second and third circles are genes of positive and negative strands of the genome, different colors are different COG classifications; the fourth circle is repeat sequences; the fifth circle is tRNA (blue) and rRNA (purple); the sixth circle is GC content, light yellow and blue respectively indicate parts higher and lower than the average GC content of the genome, the peak indicates the difference size; the seventh circle is GC-skew, dark gray indicates G content is greater than C, and red is the opposite.

[0076] (2) Drug resistance gene analysis

[0077] Comparing the whole genome information to the CARD database, no related antibiotic resistance genes were found.

[0078] (3) Virulence gene analysis

[0079] Comparing the whole genome information to the VFDB database, setting sequence similarity ≥ 85%, sequence coverage ≥ 85%, and Evalue < 0.05 as the screening standard, no related virulence genes were found.

[0080] (4) Secondary metabolite analysis

[0081] Uploading the whole genome sequence to the antiSMASH database, one secondary metabolite Terpene, i.e., terpene, was annotated (see Fig. 5). In Fig. 5, the first line is the terpene gene cluster annotated in LXHMY1, and the second to fifth lines are the terpene gene clusters annotated in Tetragenococcus halophilus MJ4, Tetragenococcus halophilus YJ1, Tetragenococcus halophilus NBRC 12172, and Tetragenococcus halophilus LMG 26042, with similarities of 85%, 82%, 85%, and 84%, respectively. It can be considered to some extent that the expression material of the gene cluster is terpene.

[0082] Example 4: Inhibition of Bacillus cereus representative strain isolated from fermented soybean

[0083] It was measured that the bacteriostatic circle data of Tetragenococcus halophilus LXHMY1 on Bacillus cereus was shown in Table 1.

[0084] Table 1 Bacteriostatic experiment results

[0085] As shown in Table 1, the halophilic tetragemus LXHMY1 has a significant bacteriostatic effect on the bacillus cereus isolated from fermented soybeans, and the maximum bacteriostatic circle diameter can reach 19.60±0.75 mm, and the blank control is 8.03 mm (i.e. the diameter of the oxford cup).

[0086] Comparative Example 1

[0087] The halophilic tetragemus LYL1001 also isolated from fermented soybeans was activated twice in succession in MRS broth medium containing 8 g / 100 mL NaCl, and the bacteriostatic experiment was performed according to the method in Example 4, and three parallels were set. The experimental results showed that the bacteriostatic circle diameters of the strain on the bacillus cereus isolated from fermented soybeans were all 8.03 mm (i.e. the diameter of the oxford cup), i.e. no bacteriostatic effect appeared.

[0088] Enterotoxin is one of the toxins produced by bacillus cereus. In order to conveniently verify the effect of the halophilic tetragemus on inhibiting bacillus cereus in the fermented soybean fermentation system, the fermented soybean simulation fermentation system was constructed in Examples 5 and 6, which reflects the production process of fermented soybean, soybean paste, soy sauce and other fermented soy products. The addition of bacillus cereus in the pre-fermentation period can reflect the possible bacillus cereus pollution in the actual production of fermented soy products. The control group only adding salt and the experimental group adding salt and halophilic tetragemus in the post-fermentation period can reflect the inhibition of halophilic tetragemus on bacillus cereus in the fermentation system.

[0089] Example 5: Test of the inhibition of bacillus cereus in the fermented soybean simulation fermentation system

[0090] Construction of the fermented soybean simulation fermentation system: Fresh yellow beans with complete particles and no mildew were selected, 4 times the mass of water and 0.5 g / 100 mL of baking soda were added, and soaked at room temperature for 14 h. After soaking, the supernatant was discarded, the yellow beans were washed, and 8 times the mass of double distilled water was added for heating and pulping. The bean dregs were filtered out through a 180-mesh screen to obtain fresh soybean milk liquid. 5 g / 100 mL of sucrose was added, and the sterile soybean milk liquid was obtained by sterilizing at 100℃ for 20 min. The representative strain of bacillus cereus was inoculated in the sterilized LB broth medium, and the inoculation amount was 2% of the volume of the medium to obtain the bacillus cereus bacterial suspension. The bacillus cereus bacterial suspension was inoculated into the sterile soybean milk liquid, and the concentration of the bacillus cereus bacterial suspension was adjusted to 5 Log CFU / mL, and the inoculation amount was 1 vol%. The pre-fermentation soybean milk liquid was obtained by fermentation at 30℃ for 6 h. The halophilic tetragemus bacterial suspension with a concentration of 5 Log CFU / mL was inoculated in the pre-fermentation soybean milk liquid, and the inoculation amount was 1 vol%. 8 g / 100 mL of salt was further added to construct the fermented soybean simulation fermentation system, and a sample only adding 8 g / 100 mL of salt was set as a control group. Sealed, post-fermented at 30℃ for 7-28 days.

[0091] At 0h and 6h of pre-fermentation, and 0, 7, 14, 21, 28 days of post-fermentation, samples were taken from the fermented bean curd simulation system, and the number of B. cereus was detected according to GB 4789.14-2014 Food safety national standard Food microbiological examination B. cereus detection. The specific quantity change is shown in FIG. 7 (Note: the "0" in the vertical coordinate is the actual B. cereus count value, with CFU / mL as the unit; the rest of the values in the vertical coordinate are the logarithmic values of the number of B. cereus, with LogCFU / mL as the unit), QJ0 and QJ6 are 0h and 6h of pre-fermentation respectively, and HJ0, HJ7, HJ14, HJ21, HJ28 are 0, 7, 14, 21, 28 days of post-fermentation respectively.

[0092] Compared with QJ0, the number of B. cereus in the fermented bean curd simulation system after 6 hours of pre-fermentation (QJ6) reached 1.11 x 10 7 CFU / mL. At 0-21 days of post-fermentation, the number of B. cereus in the control group showed a gradual downward trend, which was 2.53 x 10 5 CFU / mL at 7 days of post-fermentation, 1.33 x 10 4 CFU / mL at 14 days of post-fermentation, and completely decreased to zero at 21 days of post-fermentation. Compared with the control group, the number of B. cereus in the experimental group added with T. halophilus had already decreased to 0 at 7 days of post-fermentation, and was not detected at 14, 21, and 28 days of post-fermentation, indicating that T. halophilus had a significant inhibitory effect on B. cereus in the fermented bean curd simulation system.

[0093] Example 6: Effect on the content of enterotoxin in the fermented bean curd simulation system

[0094] Construction of the fermented bean curd simulation system: Fresh soybeans with complete particles and no mildew were selected, 6 times the mass of water and 1 g / 100 mL of baking soda were added, and soaked at room temperature for 16 h. After soaking, the supernatant was discarded, the soybeans were washed, and 8 times the mass of double-distilled water was added for heating and pulping. The soybean dregs were filtered out through a 180-mesh sieve to obtain fresh soybean milk liquid. 5 g / 100 mL of sucrose was added, and the sterile soybean milk liquid was obtained by sterilizing at 100°C for 25 min. The representative strain of B. cereus was inoculated into the sterilized LB broth medium, and the inoculation amount was 4% of the volume of the medium to obtain a B. cereus bacterial suspension. The B. cereus bacterial suspension was inoculated into the sterile soybean milk liquid, the concentration of the B. cereus bacterial suspension was adjusted to 6 LogCFU / mL, the inoculation amount was 2 vol%, and the pre-fermentation soybean milk liquid was obtained by fermentation at 30°C for 6 h. The T. halophilus bacterial suspension with a concentration of 6 LogCFU / mL was inoculated into the pre-fermented soybean milk liquid, the inoculation amount was 2 vol%, and 10 g / 100 mL of salt was further added to constitute the fermented bean curd simulation system, and a sample with only 10 g / 100 mL of salt added was set as the control group. Sealed, and post-fermented at 30°C for 7-28 days.

[0095] At 0h and 6h of pre-fermentation, 0, 7, 14, 21, 28 days of post-fermentation, samples were taken from the fermented bean curd simulation system, and the amount of enterotoxin in the system was detected by ELISA method. The specific operation refers to the Bacillus cereus enterotoxin ELISA detection kit manual. The specific content change is shown in Figure 8, QJ0 and QJ6 are 0h and 6h of pre-fermentation respectively, and HJ0, HJ7, HJ14, HJ21 and HJ28 are 0, 7, 14, 21 and 28 days of post-fermentation respectively.

[0096] After 6 hours of pre-fermentation (QJ6), due to the rapid reproduction of Bacillus cereus (Example 5), the content of enterotoxin rapidly increased to 0.308 ng / mL. In the control group, the content of enterotoxin showed a downward trend during the post-fermentation period, and decreased to 0.049 ng / mL at 7 days of post-fermentation, to 0.032 ng / mL at 14 days of post-fermentation, and was not detected at 21 days of post-fermentation and 28 days of post-fermentation. Compared with the control group, the experimental group added with Tetragenococcus halophilus had a significant decrease in the content of enterotoxin to 0.019 ng / mL at 7 days of post-fermentation; at 14 days, 21 days and 28 days of post-fermentation, the enterotoxin could not be detected, indicating that Tetragenococcus halophilus could effectively inhibit Bacillus cereus and reduce the content of enterotoxin produced by Bacillus cereus.

[0097] As can be seen from the above examples, the Tetragenococcus halophilus LXHMY1 isolated from fermented bean curd has an inhibitory effect on Bacillus cereus, has good growth in a high-salt environment, has good safety (Examples 2 and 3), has a good inhibitory effect on Bacillus cereus isolated from fermented bean curd (Examples 4 and Comparative Example 1), can significantly reduce the number of Bacillus cereus in the fermented bean curd fermentation system (Example 5), and can reduce the content of enterotoxin in the system (Example 6), thereby effectively improving the safety of fermented foods such as fermented bean curd.

[0098] Chinese invention patent application CN 118325758 A discloses a culture of Tetragenococcus halophilus and its use. The Tetragenococcus halophilus is used for fermenting soybean paste, which significantly reduces the content of tyrosine in the mash, thereby inhibiting the generation of white spots during the fermentation of soybean paste, does not produce biological amines, and can produce rich organic acids to improve the flavor of the mash. Unlike the Tetragenococcus halophilus involved in Chinese invention patent application CN 118325758 A, the Tetragenococcus halophilus of the present application is sensitive to ampicillin, gentamicin, kanamycin, streptomycin, clindamycin, tetracycline, erythromycin and chloramphenicol, has no drug resistance genes and virulence genes, and has a significant growth inhibitory effect on Bacillus cereus isolated from fermented bean curd, which can significantly reduce the number of Bacillus cereus in the fermented bean curd fermentation system.

[0099] Chinese invention patent application CN117796403A utilizes catechin to inhibit Bacillus cereus, and tests that the minimum bacteriostatic concentration of catechin on Bacillus cereus is 2 mg / mL, and the minimum bactericidal concentration is 4 mg / mL. But catechin is usually extracted from tea leaves, and the prior art proves that the increase of the content of catechin will increase the turbidity and bitterness of tea soup, which means that it has a greater risk of adversely affecting food quality; and the concentration of epicatechin in tea leaves is significantly reduced under salt treatment, so the high salt environment of the fermentation system may have a negative impact on the bacteriostatic effect of catechin. The halophilic tetragenococcus in the application is isolated from fermented bean curd, and the halophilic tetragenococcus is one of the dominant bacteria in the post-fermentation period of fermented food, and is more suitable for inhibiting bacteria in fermented food and will not affect the quality of fermented food.

[0100] The present application is not limited by the above-mentioned embodiments, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application shall be equivalent to the alternative means, and all shall be included in the protection scope of the present application.

Claims

1. A Tetragenococcus halophilus characterized in that, It is preserved as GDMCC 64945.

2. The application of Tetragenococcus halophilus in inhibiting Bacillus cereus in fermented bean curd fermentation system according to claim 1.

3. The use of Tetragenococcus halophilus according to claim 2 for inhibiting Bacillus cereus in a fermented bean curd system, characterized in that, Inoculate Tetragenococcus halophilus in sterilized culture medium to obtain Tetragenococcus halophilus bacterial suspension, inoculate the diluted Tetragenococcus halophilus bacterial suspension into pre-fermented soybean milk liquid, add salt, and ferment at 25-30℃ for 7-28 days.

4. The Tetragenococcus halophilus for use in a sufu fermentation system for inhibiting Bacillus cereus according to claim 3, wherein, The culture medium of Tetragenococcus halophilus is MRS broth medium containing 8-10 g / 100 mL NaCl.

5. The use of Tetragenococcus halophilus according to claim 3 for inhibiting Bacillus cereus in a fermented bean curd system, characterized in that, The sterilization of the culture medium is at 120-122℃ for 15-20 min.

6. The use of Tetragenococcus halophilus according to claim 3 for inhibiting Bacillus cereus in a fermented bean curd system, characterized in that, The inoculation amount of Tetragenococcus halophilus in the sterilized culture medium is 2-5 vol%.

7. The use of Tetragenococcus halophilus according to claim 3 for inhibiting Bacillus cereus in a fermented bean curd system, characterized in that, The inoculation method of Tetragenococcus halophilus in the sterilized culture medium is to culture at 25-30℃ for 48-72 h to obtain Tetragenococcus halophilus bacterial suspension.

8. The use of Tetragenococcus halophilus according to claim 3 for inhibiting Bacillus cereus in a fermented bean curd system, characterized in that, The pre-fermented soybean milk liquid contains Bacillus cereus.

9. The use of Tetragenococcus halophilus according to claim 3 for inhibiting Bacillus cereus in a fermented bean curd system, characterized in that, The concentration of the diluted Tetragenococcus halophilus bacterial suspension is 5-6 Log CFU / mL, and the inoculation amount of the diluted Tetragenococcus halophilus bacterial suspension is 1-3 vol%.

10. The use of Tetragenococcus halophilus according to claim 3 for inhibiting Bacillus cereus in a fermented soybean cheese fermentation system, characterized in that, The salt is added at 8-12 g per 100 mL of pre-fermented soybean milk liquid, and the pre-fermented soybean milk liquid with salt constitutes the fermented bean curd fermentation system.

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