Lactic acid bacteria with broad-spectrum antibacterial activity and fermentation method thereof

Abstract

The application discloses a broad-spectrum bacteriostatic lactic acid bacteria and a fermentation method thereof, and belongs to the technical field of microorganisms. The strain has the function of inhibiting the activity of food-borne pathogenic bacteria in a broad spectrum, and is named as plant lactobacillus J50. The plant lactobacillus J50 provided by the application can inhibit both gram-negative bacteria and gram-positive bacteria; the strain does not have common virulence genes of lactic acid bacteria, is not acid-resistant and bile salt-resistant, and the antibacterial substances can be digestively degraded. The fermentation medium and the fermentation condition disclosed by the application can significantly improve the bacteriostatic activity of the fermentation liquor of the strain, and the bacteriostatic activity is 6.6 to 8 times that of the starting condition. The strain enriches the lactic acid bacteria strain resource library and has a wide application prospect in the food, medicine, cosmetic and other industries.

Description

Technical Field

[0001] This invention relates to a broad-spectrum antibacterial lactic acid bacterium and its fermentation method, belonging to the field of microbial technology. Background Technology

[0002] Foodborne pathogens are a major cause of food safety problems. Preservatives are inevitably used in food to inhibit microbial growth. However, the long-term overuse of chemical preservatives and antibiotics has led to varying degrees of drug resistance in foodborne pathogens. Currently, only a few natural food additives, such as nisin, ε-polylactic acid, natamycin, and lysozyme, are approved in my country, limiting their application to varying degrees. Natural antimicrobial peptides produced by lactic acid bacteria, due to their high efficiency, low toxicity, easy degradation, and low likelihood of inducing drug resistance, are considered ideal substitutes or synergists for traditional preservatives and are gradually becoming a research hotspot.

[0003] Lactic acid bacteria are a class of anaerobic or facultative anaerobic Gram-positive cocci or bacilli that do not form spores, are non-motile, and lack cytochrome and catalase activity. They can be isolated from samples such as fermented foods, human intestines, and chicken intestinal mucosa. Long-term research results indicate that most lactic acid bacteria are essential beneficial bacteria with important physiological functions for the human body, including regulating intestinal flora balance, anti-tumor activity, cancer prevention, and prevention of lactose intolerance. Some lactic acid bacteria can also produce bacteriocins, which can inhibit or kill other bacteria, making them potential antibiotic alternatives. Therefore, exploring lactic acid bacteria with inhibitory activity against multidrug-resistant bacteria and foodborne pathogens can not only reduce antibiotic use and mitigate the spread of resistant microorganisms, but also enrich the lactic acid bacteria strain resource bank, further explore the functions of probiotics, and open new avenues for the prevention and control of multidrug-resistant and foodborne pathogens.

[0004] Screening for foodborne lactic acid bacteria with antibacterial properties from fermented foods is an ideal approach. Domestic studies have isolated 38 strains of lactic acid bacteria from sauerkraut and dried cured meat, and screened out one strain with the best antibacterial effect, finding that its fermentation products inhibited various foodborne pathogens. Other researchers have isolated 22 strains of lactic acid bacteria (including Gram-positive and Gram-negative bacteria) from fermented camel milk. In these studies, the inhibition zone diameter of the lactic acid bacteria fermentation broth ranged from 11 to 16 mm. Although the types and sources are numerous and diverse, the antibacterial ability of natural lactic acid bacteria screened from different materials is limited, and the bacteriocin content is low, failing to meet the needs of industrial production. Therefore, strains with good characteristics can be used as a research basis to improve bacteriocin synthesis through other methods.

[0005] Fermentation medium and conditions directly affect the growth and development of bacteriocin-producing microorganisms, influencing bacteriocin production to varying degrees. Certain substances can stimulate or inhibit bacteriocin synthesis. In most cases, researchers optimize fermentation medium and conditions using methods such as single-factor experiments, orthogonal experiments, and response surface methodology to improve bacteriocin yield.

[0006] Todorov et al. isolated *Lactobacillus plantarum* ST31 from dough. The optimal temperature was 30 °C, and the optimal pH was 6.0. Using MRS medium as a base, the addition of biotin, pantothenic acid, and thiamine promoted bacteriocin synthesis, with the highest synthesis reaching 6400 AU / mL. Glycerol significantly inhibited bacteriocin synthesis. Abo-Amer et al. screened *Lactobacillus acidophilus* AA11 from Egyptian cheese. Single-factor analysis determined the optimal temperature for bacteriocin synthesis to be 30 °C, and the optimal pH to be 6.5. Using M17 medium as a base and 4% yeast extract as the nitrogen source, the bacteriocin synthesis reached 7492 AU / mL, a four-fold increase compared to the control. KH₂PO₄, CaCl₂, and NH₄PO₄ inhibited bacteriocin synthesis, while MnSO₄ increased it. The addition of vitamins did not increase bacteriocin synthesis. Jie Kang et al. studied the synthesis and gene expression of bacteriocins produced by Lactobacillus paracasei HD1.7. The results showed that with increasing acetic acid pressure, the production of bacteriocins increased, and the synthesis rate also increased.

[0007] Different lactic acid bacteria have different requirements for the types and concentrations of nutrients. Currently, the optimization of bacteriocin fermentation media is mostly based on M17, MRS, and TGE media. The optimal fermentation conditions and fermentation media are strain-specific. If suitable media and culture conditions are found, the yield of bacteriocin can be increased many times over, which is reflected in the increased antibacterial ability of the fermentation broth, which facilitates subsequent research and production practice.

[0008] Existing lactic acid bacteria technologies exhibit relatively weak antibacterial capabilities. Their inhibition zones against common pathogenic bacteria such as Bacillus, Salmonella, Staphylococcus aureus, Escherichia coli, Vibrio parahaemolyticus, and Cronobacter sakazakii are less than 16 mm in diameter, and the types of bacteria they inhibit are limited. They also show no inhibitory effect against Aspergillus oryzae and yeast. Therefore, identifying lactic acid bacteria strains with highly effective antibacterial capabilities is of great significance for their industrial application. Summary of the Invention

[0009] To address the above-mentioned problems, the purpose of this invention is to provide a lactic acid bacteria strain that broadly inhibits foodborne pathogens and its fermentation method.

[0010] To achieve the above objectives, the technical solution adopted by this invention is as follows: a lactic acid bacteria strain with broad-spectrum inhibitory function against foodborne pathogens, named *Lactobacillus plantarum* J50 (… Lactiplantibacillus plantarumJ50 was deposited on December 23, 2022 at the China Center for Type Culture Collection (Wuhan University), address: Wuhan University, Wuhan, China, accession number: CCTCC NO: M 20222055.

[0011] This invention screens lactic acid bacteria with antibacterial activity in fermented soybean paste, obtaining a strain of lactic acid bacteria with strong inhibitory effects against various foodborne pathogens (including Gram-positive and Gram-negative bacteria). After 16S RNA identification, it was named *Lactobacillus plantarum* J50. Lactiplantibacillus plantarum J50). Experiments have shown that this strain is intolerant to the gastrointestinal environment, and the antibacterial substances released into the extracellular space during fermentation can be inactivated by protease digestion. Furthermore, no virulence genes have been detected, making it a safe food-grade lactic acid bacteria.

[0012] The present invention also provides a microbial preparation containing the aforementioned *Lactobacillus plantarum* J50.

[0013] In one embodiment, the viable count of *Lactobacillus plantarum* J50 in the microbial preparation is ≥ 2.9 × 10⁻⁶. 9 CFU / mL In one embodiment, the microbial preparation further comprises excipients and / or carriers; the excipients include, but are not limited to, glycerol; the carriers include, but are not limited to, diatomaceous earth.

[0014] The present invention also provides an antibacterial agent containing the aforementioned *Lactobacillus plantarum* J50 or the aforementioned microbial preparation.

[0015] The present invention also provides a modified MRS culture medium for enhancing the antibacterial activity of *Lactobacillus plantarum* J50, comprising 8-12 g / L tryptone, 8-12 g / L beef meal, 2-6 g / L yeast extract, 6-10 g / L glycerol, 1-3 g / L dipotassium hydrogen phosphate heptahydrate, and 1-3 g / L triammonium citrate.

[0016] The present invention also provides a method for culturing and fermenting the plant lactobacillus J50, comprising the following steps: inoculating the plant lactobacillus J50 seed culture into MRS medium and culturing it at 37°C and 180 rpm for 24 h; inoculating the obtained strain into MRS medium or modified MRS medium at a volume ratio of 1 / 10 and culturing it at 37°C and 180 rpm for at least 24 h.

[0017] In one embodiment, the microbial preparation further includes excipients and / or carriers, wherein the excipients include, but are not limited to, glycerol; and the carriers include, but are not limited to, diatomaceous earth.

[0018] The present invention also provides the application of the plant lactobacillus J50, microbial preparations or antibacterial agents in pharmaceutical or non-pharmaceutical products, the non-pharmaceutical products including cosmetics or feed additives.

[0019] Beneficial effects: The *Lactobacillus plantarum* J50 provided by this invention has a good antibacterial effect against a variety of foodborne pathogens, inhibiting both Gram-negative and Gram-positive bacteria. The *Lactobacillus plantarum* J50 does not have virulence genes, and the antibacterial substances it produces can be digested and inactivated by proteases, and it is not tolerant to the gastrointestinal environment.

[0020] The modified MRS medium provided by this invention is suitable for *Lactobacillus plantarum* J50. Fermentation of lactic acid bacteria using the modified MRS medium yields a 2.0 × 10⁻⁶ ppm higher concentration than standard MRS medium. 9 The fermentation broth obtained from CFU (Continuous Fumed Lactobacillus plantarum) J50 showed a 6.6-8 fold increase in antibacterial activity. Inhibition zones against Bacillus, Salmonella, and Staphylococcus aureus reached over 40 mm; against Escherichia coli, the inhibition zone reached 39.63 mm; against Vibrio parahaemolyticus, the inhibition zone reached 37.07 mm; and against Cronobacter sakazakii, the inhibition zone reached 30.41 mm. It also exhibited good antibacterial properties against Aspergillus oryzae, Pilsneriaceae, and Saccharomyces cerevisiae. While enriching the lactic acid bacteria strain resource bank, it also has broad application prospects in pharmaceuticals, cosmetics, and feed additives.

[0021] Preservation of biological materials The plant lactobacillus J50 described in this invention ( Lactiplantibacillus plantarum J50 was deposited on December 23, 2022, at the China Center for Type Culture Collection (Wuhan University), address: Wuhan University, Wuhan, China, accession number: CCTCC NO: M 20222055. Attached Figure Description

[0022] Figure 1 Evolutionary relationship tree of Lactobacillus plantarum J50.

[0023] Figure 2 PCR electrophoresis bands of the virulence gene in *Lactobacillus plantarum* J50; 1: 2000 Da DNA marker; 2: 16S rRNA; 3: ace 4: efaAfs 5: cylLs 6: gelE 7: asa1 .

[0024] Figure 3 Antibacterial spectrum of fermentation broth of Lactobacillus plantarum J50. Detailed Implementation

[0025] The culture media involved in the following examples are as follows: Table 1. Basic MRS Culture Medium

[0026] Table 2 Modified MRS medium

[0027] *The culture medium was adjusted to pH 6.5 using 2M HCl and 2M NaOH before sterilization.

[0028] The components of the simulated gastric juice and simulated pancreatic juice involved in the following embodiments are as follows: Simulated gastric fluid: Dissolve 0.35% (w / v) porcine pepsin in 1×PBS buffer and adjust the pH to 2.0±0.1 with 1M HCl.

[0029] Simulated pancreatic juice: 1×PBS buffer containing 1.1% (w / v) NaHCO3 and 0.1% (w / v) trypsin, with the pH adjusted to 8.0 ± 0.1 using 1M NaOH.

[0030] The following examples describe *Lactobacillus plantarum* J50 ( Lactiplantibacillus plantarum J50 was deposited in December 2022 at the China Center for Type Culture Collection (Wuhan University), address: Wuhan University, Wuhan, China, accession number: CCTCC NO: M 20222055.

[0031] Example 1: Screening and identification of *Lactobacillus plantarum* I. Preliminary Screening of Lactic Acid Bacteria Doubanjiang (fermented broad bean paste) from Chuxiong Yunquan Sauce Garden Co., Ltd. was collected as a sample and diluted 10-fold with sterile physiological saline. Sterile glass beads were added, and the sample was placed in a shaker at 37℃ and 180 rpm for 3 hours to ensure thorough dispersion. The dispersion was serially diluted 10-fold and spread onto MRS agar medium supplemented with 50 mg / L nystatin. A thin layer of MRS medium supplemented with 50 mg / L nystatin and 1% calcium carbonate powder was then poured on top to isolate some oxygen and allow the calcium carbonate powder to settle on the colony surface. After incubation at 37℃ for 48 hours, typical colonies with obvious calcium dissolution zones were observed and picked from the plates by stab. Single colonies were obtained by streaking for two generations and subjected to Gram staining microscopy and catalase activity test. Gram-positive bacteria and catalase-negative bacteria were selected as the lactic acid bacteria obtained from the initial screening. The bacterial suspension was mixed with 40% glycerol at a ratio of 1:1 and stored at -80℃.

[0032] In this experiment, a total of 106 suspected lactic acid bacteria strains were obtained in the initial screening.

[0033] II. Screening of Antibacterial Lactic Acid Bacteria (1) Preparation of antibacterial supernatant: The lactic acid bacteria obtained from the initial screening were continuously activated for 2 generations using MRS medium. After culturing for 24 hours to the stationary phase, they were inoculated at a ratio of 1 / 10 for fermentation. After culturing at 37℃ and 180 rpm for 24 hours, the supernatant was collected by centrifugation at 10000×g. The pH of the supernatant was adjusted to 4.0 using 2M NaOH and 2M HCl. (2) Preparation of antibacterial indicator plates: Agar is divided into two layers. The lower layer is a 15g / L agar aqueous solution, poured into plates with a uniform thickness of about 1.5mm (about 10mL for a 9mm diameter petri dish). After cooling and solidification, an 8mm Oxford cup is placed on top using sterile forceps, and slight pressure is applied to stabilize it. The upper layer is 15g / L agar LB medium. When cooled to about 45℃, 0.5% of the indicator bacterial solution that has been cultured overnight to OD=0.8 is added. E. coli , S. aureus Shake well, pour a second layer of agar on top of the lower agar, ensuring a uniform thickness of about 1.5 mm. After cooling and solidification, remove the Oxford cup with sterile tweezers to complete the preparation of the antibacterial indicator plate.

[0034] (3) Agar diffusion method: Take 100 μL of the prepared antibacterial supernatant and slowly add it dropwise into the agar wells of the antibacterial indicator plate. Incubate at 37℃ for 12 h, observe and measure the size of the inhibition zone, and calculate the antibacterial activity according to the following formula: (Equation 1) Results: Among the 106 suspected lactic acid bacteria strains obtained in the initial screening, 66 strains showed antibacterial effects in their fermentation broth. Of these, 16 strains showed inhibitory effects against only one of *Escherichia coli* or *Staphylococcus aureus*, and were therefore discarded. Finally, 50 lactic acid bacteria strains were obtained that exhibited inhibitory effects against both Gram-positive and Gram-negative bacteria. After three rounds of comparison, strain J50, with the highest sum of antibacterial activity (AU) against *Escherichia coli* and *Staphylococcus aureus*, was selected.

[0035] III. Gene Identification of Strains DNA from strain J50 was obtained using a genomic DNA extraction kit and used as a template for PCR amplification. Universal primers (27F: 5'-agagtttgatcctggctcag-3' for the forward primer and 1492R: 5'-ctacggctaccttgttacga-3' for the reverse primer) were used to amplify the 16S rDNA sequence of each bacterium via polymerase chain reaction (PCR). After electrophoresis detection of the amplification products, they were sent to Wuxi Tianlin Biotechnology Co., Ltd. for sequencing. The sequencing results were compared with sequences in the NCBI database for homology analysis. Based on the results, the strain was named: Lactiplantibacillus plantarumJ50, Chinese name: *Lactobacillus plantarum* J50, and a phylogenetic tree was constructed (maximum likelihood method), as shown in the attached figure. Figure 1 .

[0036] Example 2 Characteristics of *Lactobacillus plantarum* J50 I. Virulence gene testing Common virulence genes of *Lactobacillus plantarum* J50 were detected using the corresponding primers (see Table 1). ace , efaAfs , cylLs , gelE and asa1 PCR amplification was performed, followed by nucleic acid gel electrophoresis testing, such as... Figure 2 As shown, the results indicate that *Lactobacillus plantarum* J50 does not possess the common virulence genes of the aforementioned lactic acid bacteria.

[0037] Table 3 Primer sequences for virulence genes

[0038] II. Simulated Gastric and Pancreatic Juice Tolerance Test Glycerol cryopreservation solution of *Lactobacillus plantarum* J50 was added to MRS medium at a ratio of 1 / 10 (v / v) and anaerobically cultured at 37°C and 180 rpm for 12 h, followed by overnight culture under the same conditions.

[0039] Simulated gastric fluid test: After centrifugation, the bacterial cells from the overnight culture were collected, washed twice with PBS, and resuspended in an equal volume of simulated gastric fluid. The mixture was incubated at 37°C, and the viable cell count was determined using the plate count method at 0 h and 3 h to calculate the survival rate (%). The separated supernatant was mixed with an equal volume of simulated gastric fluid and placed in a shaker at 37°C and 180 rpm. The antibacterial activity was measured at 0 h and 3 h (with Staphylococcus aureus as the indicator bacterium).

[0040] Simulated pancreatic juice test: After centrifugation, the bacterial cells treated with simulated gastric juice were suspended in an equal volume of simulated pancreatic juice and incubated at 37°C for 24 hours. The viable bacterial count was determined by plate counting at 0 hours and 24 hours, and the survival rate (%) was calculated. The supernatant after treatment with simulated gastric juice was mixed with an equal volume of simulated pancreatic juice and placed in a shaker at 37°C and 180 rpm. The antibacterial performance was determined after 24 hours (Staphylococcus aureus was used as an indicator bacterium).

[0041] The results are shown in the table below: Table 4. Simulated gastric and pancreatic juice treatments of lactic acid bacteria and fermentation broth

[0042] It is known that Lactobacillus plantarum J50 is sensitive to simulated gastric and pancreatic juices, the bacteria themselves are not tolerant to simulated digestive environments, and the fermentation products are also decomposed by endogenous digestive enzymes.

[0043] III. Antibacterial spectrum Fermentation conditions for Lactobacillus plantarum J50: Glycerol cryopreservation solution of *Lactobacillus plantarum* J50 was added to MRS medium at a ratio of 1 / 10 (v / v) and anaerobically cultured at 37°C and 180 rpm for 24 h. The resulting bacterial suspension was then continuously expanded at the same inoculum size for at least two generations. After expansion, the expanded bacterial suspension (CFU ≈ 2.9 × 10⁻⁶) was diluted at a ratio of 1 / 10 (v / v) to obtain a further amount of the culture. 9 The samples were inoculated into MRS medium and modified MRS medium, respectively, and anaerobic fermented under the same conditions for 24 h until CFU ≈ 2.0 × 10⁻⁶. 9 After fermentation, the mixture was centrifuged at 6000×g for 5 minutes, and the supernatant was filtered through a 0.22μm filter membrane to obtain cell-free fermentation broth.

[0044] To test the antibacterial spectrum of *Lactobacillus plantarum* J50 fermentation broth, the indicator bacteria range was broadened to include molds, yeasts, and other foodborne pathogens. The antibacterial performance of fermentation broths on MRS medium and modified MRS medium was compared. The preparation of the antibacterial indicator plates and the inhibition zone test were performed according to the method described in Example 1, but appropriate culture media were selected for each indicator bacteria: *Aspergillus oryzae* was treated with PDA medium, yeast with YPD medium, *Vibrio parahaemolyticus* with LB medium containing 3.5% NaCl, and other bacteria with LB medium.

[0045] The experimental results are shown in Table 5. It can be seen that after fermentation using the modified MRS medium, the antibacterial ability of the fermentation broth of *Lactobacillus plantarum* J50 was significantly improved, with the improvement (calculated in AU) ranging from 6.6 times to 8 times. Inhibition of *Aspergillus oryzae*, *Pilsneriaceae*, and *Saccharomyces cerevisiae* was also observed.

[0046] Table 5 Comparison of antibacterial activity of fermentation broths from two different culture media

[0047] Comparative Example 1 For specific implementation methods, refer to Example 2, except that the concentration of glycerol in the modified MRS medium is changed. Finally, the antibacterial effect of the fermentation broth on Staphylococcus aureus is detected by the aforementioned agar diffusion method, as shown in Table 6.

[0048] Table 6. Antibacterial activity of modified MRS media with different glycerol concentrations

[0049] Comparative Example 2 The specific implementation method is as follows: Preparation of 50mM sodium acetate buffer: Weigh 2.550g glacial acetic acid and 1.027g sodium acetate trihydrate, dissolve in 950mL deionized water, adjust the pH to 4.0 using 1M HCl and 1M NaOH, and bring the volume to 1L.

[0050] Preparation of Nisin Z solution: Accurately weigh 0.8000 g of Nisin Z solid (available from Macklin), dissolve it in 50 mM sodium acetate buffer, and bring the volume to 50 mL to prepare a 16 mg / mL Nisin Z stock solution. Then, dilute with 50 mM sodium acetate buffer to the corresponding concentrations according to Table 7.

[0051] Table 7. Preparation of Nisin Z solutions of different concentrations

[0052] Finally, using Staphylococcus aureus as the indicator bacterium, the antibacterial effect of different concentrations of Nisin Z solution was tested using the agar diffusion method described in Example 1, as shown in Table 8. The inhibition zone diameter (D) - concentration (C) conforms to the equation D = 1.8418lnC + 13.668, (R 2 =0.9839).

[0053] Table 8. Antibacterial activity of Nisin Z solutions at different concentrations

[0054] Nisin Z is a legal food preservative. According to the national standard GB 1886.231-2016, its maximum permitted concentration in food is 0.2-0.5 g / L. A comparison of Tables 8 and 5 clearly shows that the fermentation broth of *Lactobacillus plantarum* J50 obtained using modified MRS medium exhibits significantly greater antibacterial activity against *Staphylococcus aureus* than a 16 mg / mL Nisin Z solution, demonstrating excellent antibacterial performance and proving to be an effective antibacterial method.

[0055] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. *Lactobacillus plantarum* ( Lactiplantibacillus plantarum The cell-free fermentation broth of J50 is characterized by, The seed culture of *Lactobacillus plantarum* J50 was inoculated into a modified MRS medium for fermentation. The fermentation product was centrifuged and the supernatant was collected. The supernatant was then filtered through a 0.22 μm filter membrane. The modified MRS medium contains 10 g / L tryptone, 5 g / L beef meal, 4 g / L yeast extract, 8 g / L glycerol, 2 g / L dipotassium hydrogen phosphate heptahydrate and 2 g / L triammonium citrate. The plant lactobacillus J50 was deposited at the China Center for Type Culture Collection on December 23, 2022, with accession number CCTCC NO: M 20222055.

2. The cell-free fermentation broth according to claim 1, characterized in that, Fermentation conditions: 35-40℃, 150-200rpm.

3. The use of the cell-free fermentation broth according to claim 1 or 2 as an antibacterial agent in the preparation of pharmaceutical products or cosmetics.

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