Lactobacillus plantarum 5S-5 and application thereof

Abstract

The invention relates to the technical field of food microorganisms, and discloses lactobacillus plantarum 5S-5 and application thereof. The Lactobacillus plantarum 5S-5 is classified and named as Lactiplantibacillus plantarum in Latin, the preservation unit is Guangdong Microbial Culture Collection Center, the preservation address is the 5th floor, No. 59 building, No. 100 Courtyard, Xianlie Middle Road, Guangzhou, the preservation number is GDMCC No: 65678, and the preservation date is December 26, 2024. The lactobacillus plantarum 5S-5 disclosed by the invention has broad-spectrum antibacterial activity and can inhibit the formation of a pathogenic bacteria biological membrane. Besides, the lactobacillus plantarum 5S-5 and methicillin-resistant staphylococcus aureus (MRSA) are co-cultured in food matrix soybean juice, and the result shows that the lactobacillus plantarum 5S-5 still has bacteriostatic activity on the MRSA, so that the lactobacillus plantarum 5S-5 has a good application prospect and has the potential of being researched and developed into a novel food preservative.

Description

Technical Field

[0001] The invention relates to the technical field of food microorganisms, in particular to a strain of Lactobacillus plantarum 5S-5 and application thereof. Background Art

[0002] Lactic acid bacteria are a type of microorganism that can ferment carbohydrates to produce lactic acid, and are currently widely used in the fermented food industry. This type of bacteria can ferment carbohydrates to produce different acids, such as lactic acid, acetic acid, and propionic acid, to create a low pH environment; they can also produce various antimicrobial compounds, such as bacteriocins and terpenes, which together inhibit spoilage pathogens and prolong food preservation. In addition, these bacteria can also produce many metabolites that are beneficial to humans, so they are widely explored and developed into functional probiotic products. They are currently widely used in the fermented food industry. In addition, with people's increasing attention to food quality and health, consumers' demand is more inclined to minimal food processing and the use of chemical preservatives. Therefore, lactic acid bacteria as natural antimicrobial agents are feasible and meet contemporary market needs. Fermented foods usually carry a rich content and variety of bacteria, including a large number of lactic acid bacteria. However, the current development of microbial resources for fermented foods is still insufficient. Therefore, it is of great significance to combine the antibacterial properties of lactic acid bacteria, deeply explore the microbial resources in fermented foods, find natural antimicrobial agents that can replace chemical preservatives, and explore the antibacterial substances and mechanisms therein.

[0003] In recent years, some relevant studies have been conducted on the antibacterial and probiotic properties of lactic acid bacteria metabolites. Behbahani et al. isolated fermented lactobacillus with good anti-yeast activity from fermented dairy products. The organic acids produced by their metabolism reduced the pH value in the growth environment, thereby inhibiting the growth of harmful microorganisms and preventing the spoilage of dairy products. The results of this study indicate the possibility of lactic acid bacteria as a natural and safe preservative to replace chemical preservatives. More studies have shown that metabolites such as bacteriocins produced by lactic acid bacteria can destroy the bacterial cell membrane structure, change the cell membrane permeability, or destroy the biofilm formation to inhibit the growth of pathogens, thereby achieving the effect of preservative and antibacterial. In the food industry, the most famous example is Nisin. Similarly, the production of hydrogen peroxide in the metabolism of some lactic acid bacteria can also make them have good antioxidant activity, limit or inhibit the oxidation of nutrients, and help reduce or eliminate the use of chemical antioxidants in meat processing. In summary, the study of the activity of lactic acid bacteria metabolites has great research value. At the same time, there are relatively few studies on the antibacterial and anti-film mechanisms of drug-resistant pathogens. Strains with broad-spectrum antibacterial activity and potential for application are still to be discovered. This situation has limited the economic use of lactic acid bacteria in controlling biofilm pollution in food processing to a certain extent. In addition, many antimicrobial active substances have high requirements for the action environment. The acidic or high temperature and high pressure environment in the production process will make them susceptible to adverse effects in food processing and weaken the antibacterial effect, which greatly limits the scope and extent of their use in the food industry. Therefore, it is still of great theoretical and application value to continue to develop and explore the microbial resources in fermented foods, especially strains with broad-spectrum antibacterial activity, application prospects, and the ability to produce antibacterial substances with good environmental stability. Summary of the invention

[0004] In view of the above-mentioned deficiencies in the prior art, the present invention provides a strain of Lactobacillus plantarum 5S-5 with antibacterial and probiotic activities and its application, aiming to solve the problem of the lack of more effective and sustainable strategies in the prior art to combat foodborne pathogens.

[0005] The technical solution of the present invention is as follows:

[0006] In the first aspect, a strain of Lactobacillus plantarum 5S-5 is provided. The Latin classification name of Lactobacillus plantarum 5S-5 is Lactiplantibacillus plantarum. The preservation unit is Guangdong Microbiological Culture Collection Center, the preservation address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, the preservation number is GDMCC No: 65678, and the preservation date is December 26, 2024.

[0007] In a second aspect, a bacterial agent is provided, wherein the bacterial agent includes the Lactobacillus plantarum 5S-5 as described in the first aspect.

[0008] In a third aspect, a fermentation broth is provided, wherein the fermentation broth is obtained by fermenting the Lactobacillus plantarum 5S-5 as described in the first aspect.

[0009] In a fourth aspect, there is provided a use of the Lactobacillus plantarum 5S-5 as described in the first aspect, the bacterial agent as described in the second aspect, or the fermentation broth as described in the third aspect in the preparation of a probiotic product.

[0010] In a fifth aspect, there is provided use of the Lactobacillus plantarum 5S-5 as described in the first aspect, the bacterial agent as described in the second aspect, or the fermentation broth as described in the third aspect in the preparation of an antibacterial product.

[0011] According to a preferred technical solution, the antibacterial product has inhibitory activity against Gram-negative bacteria and Gram-positive bacteria.

[0012] In a preferred technical solution, the Gram-negative bacteria are one or more of Salmonella, Pseudomonas aeruginosa, Acinetobacter baumannii and Escherichia coli.

[0013] In a preferred technical solution, the Gram-positive bacteria are one or more of methicillin-resistant Staphylococcus aureus, Bacillus subtilis, Streptococcus mutans and Listeria monocytogenes.

[0014] In a preferred technical solution, the antibacterial product inhibits the formation of biofilm.

[0015] In a preferred technical solution, the antibacterial product is a food preservative.

[0016] The advantages of the present invention are as follows:

[0017] (1) The present invention isolates a strain of Lactobacillus plantarum 5S-5 with obvious antibacterial activity and probiotic properties from fermented tofu, a traditional fermented food in Guizhou. The strain has a broad-spectrum antibacterial activity, and its fermentation broth has inhibitory activity against both Gram-negative bacteria and Gram-positive bacteria.

[0018] (2) According to whole genome sequencing and analysis, Lactobacillus plantarum 5S-5 was identified as a new strain of Lactobacillus plantarum by comparing its ANI value with that of the reference strain Lactobacillus plantarum KLDS1.0386 to 99.61%. The results of the probiotic property determination of Lactobacillus plantarum 5S-5 showed that most of its properties were comparable to or even better than those of Lactobacillus plantarum L2 isolated from a commercial product (trunature——ADVANCEDDIGESTIVE PROBIOTIC).

[0019] (3) By neutralizing the fermentation supernatant of Lactobacillus plantarum 5S-5, it was found that it still had antibacterial activity against MRSA ATCC 43300. Therefore, it was speculated that in addition to common organic acids such as lactic acid, acetic acid, and propionic acid that inhibit the growth of pathogens by lowering the pH value, Lactobacillus plantarum 5S-5 also has other non-acid-dependent active metabolites.

[0020] (4) By co-culturing Lactobacillus plantarum 5S-5 and MRSAATCC 43300 in transwell culture, it was found that Lactobacillus plantarum 5S-5 had a significant ability to inhibit the growth of pathogens and the activity to inhibit biofilm formation.

[0021] (5) By heating the supernatant of Lactobacillus plantarum 5S-5 at 100°C, it was found that it still had antibacterial activity against MRSA, indicating that its antibacterial active substances have good thermal stability and are more likely to adapt to the high temperature environment in food industry processing and production, and have good application prospects.

[0022] (6) In the food matrix of soy milk, Lactobacillus plantarum 5S-5 and MRSA were co-cultured, and it was found that Lactobacillus plantarum 5S-5 still had antibacterial activity against MRSA, which proved that Lactobacillus plantarum 5S-5 has good application prospects and has the potential to become a new type of food preservative. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The colony plate streaking and evolutionary tree of Lactobacillus plantarum 5S-5 in Example 1 are shown.

[0024] Figure 2 This is a graph showing the antibacterial activity test results of Lactobacillus plantarum 5S-5 in Example 2; wherein 1 is the addition of 5S-5 fermentation broth; 2 is the addition of vancomycin solution as a positive control; 3 is the addition of lactic acid bacteria fermentation broth without antibacterial activity; and 4 is the addition of MRS culture medium as a blank control.

[0025] Figure 3 4 is a graph showing the probiotic evaluation results of Lactobacillus plantarum 5S-5 in Example 4; wherein A is the growth curve of Lactobacillus plantarum 5S-5 and Lactobacillus plantarum L2; B is the gastrointestinal tolerance evaluation result; C is the self-aggregation test result; D is the co-aggregation test result; E is the hydrophobicity test result; and F is the test result of biofilm formation.

[0026] Figure 4 This is a graph showing the hemolytic activity evaluation results of Lactobacillus plantarum 5S-5 in Example 5.

[0027] Figure 5 This is a graph showing the antibacterial activity evaluation results of the neutralization supernatant of Lactobacillus plantarum 5S-5 in Example 6.

[0028] Figure 6It is a graph showing the antibacterial activity evaluation results of the fermentation supernatant of Lactobacillus plantarum 5S-5 after heat treatment in Example 7; wherein, A is added with the fermentation supernatant after heat treatment; B is added with the fermentation supernatant without heat treatment; and C is added with MRS culture medium as a blank control.

[0029] Figure 7 1 is a comparative genome analysis result diagram of Lactobacillus plantarum 5S-5 in Example 8; wherein A is the core genes and unique genes of the six strains; B is a phylogenetic tree of gene sequence similarity; and C is a colinearity analysis of the nucleic acid level among the six Lactobacillus plantarum strains.

[0030] Figure 8 This is the sterilization-time curve of the co-culture of Lactobacillus plantarum 5S-5 and MRSA in Example 9.

[0031] Fig. 9 This is a diagram showing the inhibitory effect of co-culturing Lactobacillus plantarum 5S-5 and MRSA on MRSA biofilm formation in Example 10.

[0032] Fig.10 This is the antibacterial curve of Lactobacillus plantarum 5S-5 against MRSA in soy milk in Example 11. DETAILED DESCRIPTION

[0033] The present invention provides a Lactobacillus plantarum 5S-5 and an application thereof. To make the purpose, technical scheme and effect of the present invention clearer and more specific, the present invention is further described in detail below.

[0034] With the continuous improvement of people's living standards, especially the increasing demand for healthy diet quality, health, nutrition and safety have become the core of consumers' attention. Modern people not only pursue the diversity of food and the richness of taste, but also pay more attention to the hygiene and safety of food. Among them, the contamination of food microorganisms is a major threat to food safety. The presence of common foodborne pathogens, such as Salmonella, Campylobacter, Listeria, Enterohemorrhagic Escherichia coli, etc., not only threatens the health of consumers, but may also cause widespread public health crises and affect the sustainable development of the food industry. Therefore, how to improve food hygiene is a global problem to be solved.

[0035] Staphylococcus aureus is a top ten notorious pathogen that is widely present in foods, especially those of animal origin. Illness caused by S. aureus is commonly associated with the consumption of milk as well as sausages, canned meats, salads, prepared foods, and sandwich fillings. This bacterium causes serious infections due to the production of multiple virulence factors and toxins, such as thermonucleases, hyaluronidases, lipases, and hemolysins. Of these, enterotoxins are perhaps the most noteworthy, as these proteins are highly resistant to environmental conditions such as heat, low pH, and proteolytic enzymes. As a result, they can remain active in the digestive tract after ingestion and even after the bacteria have been killed, with high nanogram to low microgram amounts sufficient to cause severe symptoms and even death.

[0036] Another virulence factor that is crucial for the fitness and host infection of S. aureus is the formation of biofilm. This extracellular matrix composed of cells, exopolysaccharides and proteins enables the pathogen to attach to different abiotic and biotic surfaces and is extremely difficult to remove or eradicate. Therefore, biofilm-forming pathogens have long been a major concern for the food industry. More importantly, biofilms, as a protective layer for cells, lead to a significant increase in antimicrobial resistance, up to a thousand times. Currently, the main strategies for disinfecting such foodborne pathogens in the food industry are the use of chemical preservatives, antimicrobial compounds (such as disinfectants), low-dose ultraviolet light and mechanical cleaning. However, these methods cannot prevent their transmission to humans, and some chemical preservatives and disinfectants may have toxic effects. Their overuse may accelerate the emergence of new resistant isolates, posing further threats to human health. Therefore, it is necessary to develop more sustainable and applicable strategies to replace or combine existing methods to combat biofilm-forming foodborne pathogens.

[0037] Based on this, an embodiment of the present invention provides a strain of Lactobacillus plantarum 5S-5, the Latin classification name of the Lactobacillus plantarum 5S-5 is Lactiplantibacillus plantarum, the preservation unit is Guangdong Microbial Culture Collection Center, the preservation address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, the preservation number is GDMCC No: 65678, and the preservation date is December 26, 2024.

[0038] The present invention isolates a novel natural probiotic strain 5S-5 with good antibacterial activity from fermented dried bean curd in a local market in Liupanshui, Guizhou. Specifically, the 16S rRNA sequence of strain 5S-5 is shown in SEQ ID NO.1, and it is identified as Lactobacillus plantarum through comparison and analysis. Whole genome sequencing and analysis show that Lactobacillus plantarum 5S-5 has an ANI value of 99.61% compared with the reference strain Lactobacillus plantarum KLDS1.0386, so it is identified as a new strain of Lactobacillus plantarum.

[0039] An embodiment of the present invention provides a bacterial agent, wherein the bacterial agent includes the Lactobacillus plantarum 5S-5 as described above.

[0040] An embodiment of the present invention provides a fermentation broth obtained by fermenting the Lactobacillus plantarum 5S-5 as described above.

[0041] The embodiments of the present invention provide use of the above-mentioned Lactobacillus plantarum 5S-5, the above-mentioned bacterial agent or the above-mentioned fermentation liquid in the preparation of a probiotic product.

[0042] The embodiments of the present invention provide use of the above-mentioned Lactobacillus plantarum 5S-5, the above-mentioned bacterial agent or the above-mentioned fermentation liquid in the preparation of an antibacterial product.

[0043] In one embodiment, the antibacterial product has inhibitory activity against Gram-negative bacteria and Gram-positive bacteria.

[0044] In one embodiment, the Gram-negative bacteria are one or more of Salmonella, Pseudomonas aeruginosa, Acinetobacter baumannii and Escherichia coli.

[0045] In one embodiment, the Gram-positive bacteria are one or more of methicillin-resistant Staphylococcus aureus, Bacillus subtilis, Streptococcus mutans and Listeria monocytogenes.

[0046] In one embodiment, the bacteriostatic product inhibits the formation of biofilm.

[0047] In one embodiment, the antibacterial product is a food preservative.

[0048] The present invention will be further described below by means of specific examples.

[0049] In the embodiment of the present invention, the preparation process of the fermentation liquid, fermentation supernatant and suspension of plant lactobacillus 5S-5 is as follows:

[0050] Fermentation broth of Lactobacillus plantarum 5S-5: A single colony of Lactobacillus plantarum 5S-5 was placed in 5 mL of MRS medium, and cultured at 37° C. and 220 rpm for 24 hours to obtain a fermentation broth of Lactobacillus plantarum 5S-5.

[0051] Fermentation supernatant of Lactobacillus plantarum 5S-5: The fermentation broth of Lactobacillus plantarum 5S-5 was centrifuged at 4000 rpm for 15 min at 4° C., and filtered through a 0.22 μM filter to obtain the fermentation supernatant of Lactobacillus plantarum 5S-5.

[0052] Suspension of Lactobacillus plantarum 5S-5: Take the fermentation broth of Lactobacillus plantarum 5S-5, centrifuge it at 4000 rpm at 4°C for 15 min, remove the supernatant, and resuspend it in the same volume of 1×PBS to obtain a bacterial suspension of Lactobacillus plantarum 5S-5.

[0053] Example 1 Isolation and Identification of Lactobacillus plantarum 5S-5

[0054] Strain 5S-5 was collected from fermented dried tofu in a local market in Liupanshui, Guizhou Province in February 2023, and a single colony was isolated by streaking on a plate. Figure 1 As shown in A.

[0055] DNA of strain 5S-5 was extracted using TIANamp Bacteria DNA Kit (TIANGEN), and the 16s rDNA region of strain 5S-5 was amplified by PCR using forward primer 1492R and reverse primer 27F. The sample was sent for sequencing to obtain its 16s rRNA sequence, as shown in SEQ ID NO.1. A phylogenetic tree was drawn based on the 16S rDNA comparison results, as shown in Figure 1 As shown in B, strain 5S-5 belongs to Lactiplantibacillus plantarum.

[0056] The DNA of strain 5S-5 was extracted using TIANamp Bacteria DNA Kit (TIANGEN), and the sample was sent for sequencing to obtain the whole genome data of strain 5S-5. Whole genome sequencing and analysis showed that the ANI value of strain 5S-5 was 99.61% compared with the reference strain Lactobacillus plantarum KLDS1.0386, so strain 5S-5 was identified as a new strain of Lactobacillus plantarum.

[0057] Lactobacillus plantarum 5S-5 was deposited in the Guangdong Provincial Microbiological Culture Collection Center (GDMCC) on December 26, 2024. The deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the deposit number is GDMCC No: 65678.

[0058] Example 2 Antibacterial activity of Lactobacillus plantarum 5S-5 against methicillin-resistant Staphylococcus aureus (MRSA)

[0059] The antibacterial activity of Lactobacillus plantarum 5S-5 against MRSA was evaluated by agar diffusion method. Lactobacillus plantarum 5S-5 was cultured in MRS medium at 37°C for 24 h. MRSA was inoculated on LB agar plates at a final concentration of 1×10 6CFU / mL. After the culture medium solidified, a hole (8 mm in diameter) was punched on the agar and 100 μL of the fermentation broth of Lactobacillus plantarum 5S-5 was added. The same volume of vancomycin (50 μg / mL) was used as the positive control, and MRS culture medium was used as the negative control. After incubation at 37°C overnight, the diameter of the inhibition zone was measured. The results are as follows: Figure 2 As shown. Figure 2 It can be seen that the fermentation broth of Lactobacillus plantarum 5S-5 has obvious antibacterial activity against MRSA.

[0060] Example 3 Determination of the antimicrobial spectrum of Lactobacillus plantarum 5S-5

[0061] The antimicrobial spectrum of Lactobacillus plantarum 5S-5 was obtained by agar diffusion method. Methicillin-resistant Staphylococcus aureus ATCC 433300, Bacillus subtilis 168, Streptococcus mutans ATCC 700610, Listeria monocytogenes ATCC 19115, Salmonella ATCC 14028, Pseudomonas aeruginosa ATCC 15692, Pseudomonas aeruginosa ATCC 10145, Acinetobacter baumannii ATCC 19606 and Escherichia coli ATCC 25922 were used as indicator bacteria. Referring to Example 2, the fermentation liquid of Lactobacillus plantarum 5S-5 was added, and the diameter of the inhibition zone was measured after incubation at 37° C. The antibacterial effect of Lactobacillus plantarum 5S-5 was evaluated based on the size of the inhibition zone against different pathogens. The results are shown in Table 1.

[0062] Table 1 Antimicrobial spectrum of Lactobacillus plantarum 5S-5

[0063]

[0064] As shown in Table 1, Lactobacillus plantarum 5S-5 has a broad-spectrum antibacterial activity and has good antibacterial activity against both Gram-negative bacteria and Gram-positive bacteria tested.

[0065] Example 4 Probiotic Evaluation of Lactobacillus plantarum 5S-5

[0066] (1) Determination of growth curve and acid production capacity

[0067] The initial concentration was 1×10 6 CFU / mL of Lactobacillus plantarum 5S-5 was inoculated into MRS medium and incubated at 37°C for 18 h. The OD was measured every 2 h using a microplate reader. 600 The pH value was measured at 24h. The commercial strain Lactobacillus plantarum L2 was used as the positive control (the same below).

[0068] Lactobacillus plantarum 5S-5 grew well and its acid production capacity was comparable to that of commercial L2 (see Figure 3 (A).

[0069] (2) Gastrointestinal tolerance assessment

[0070] To evaluate the strain's tolerance to the gastrointestinal environment, a suspension of Lactobacillus plantarum 5S-5 was added to simulated gastric fluid and simulated intestinal fluid at a volume ratio of 1:9. Simulated gastric fluid was prepared by adding 0.35% (w / v) pepsin and 0.2% (w / v) NaCl to MRS medium at pH 2.0. Simulated intestinal fluid was prepared by adding 0.3% (w / v) ox bile salt to MRS medium adjusted to pH 7.0. After incubation at 37°C for 3 hours, the number of viable bacteria on the plate was counted. The results are expressed as a percentage of survival (%):

[0071] Survival rate percentage (%) = N t / N0*100%;

[0072] Where N0 and N t Represent the viable bacterial counts at 0h and 3h, respectively.

[0073] The gastrointestinal tolerance of Lactobacillus plantarum 5S-5 is comparable to that of the commercial strain Lactobacillus plantarum L2 (see Figure 3 (middle B).

[0074] (3) Evaluation of self-cohesion and co-cohesion ability

[0075] Good adhesion ability means that probiotics can form a compact and stable biofilm during growth, which is conducive to their colonization in the intestine. In addition, in the processing of functional foods, the good polymerization ability of lactic acid bacteria can also improve the quality and stability of food. Among them, coagulation ability, hydrophobicity and biofilm formation are usually important indicators to characterize its adhesion ability. Take the fermentation broth of Lactobacillus plantarum 5S-5 and centrifuge it at 4000rpm for 15min, remove the supernatant, and resuspend it in PBS to obtain a bacterial suspension of Lactobacillus plantarum 5S-5 (OD 600 The self-aggregation ability of the strain was evaluated at 0 h and 4 h by measuring the absorbance at 600 nm.

[0076] The percentage of self-aggregation is calculated as follows:

[0077] Self-aggregation (%) = [1-(A t / A0)]*100;

[0078] Where A0 and A t Represents OD at 0h and 4h respectively 600 .

[0079] To determine the coaggregation ability, a suspension of Lactobacillus plantarum 5S-5 was prepared as described above and mixed with the same volume of MRSA suspension at OD 600 Mix in the range of 0.8 to 1). Incubate at 37°C for 4 h, and measure OD at 0 h and 4 h. 600 The formula for calculating the co-aggregation rate is the same as that for calculating the self-aggregation rate.

[0080] The self-aggregation and co-aggregation abilities of Lactobacillus plantarum 5S-5 are comparable to those of the commercial strain Lactobacillus plantarum L2 (see Figure 3 C and D).

[0081] (4) Hydrophobicity evaluation

[0082] Xylene, petroleum ether and chloroform were selected as hydrophobic organic solvents. A suspension of Lactobacillus plantarum 5S-5 was prepared as described above, and then mixed with an organic solvent at a ratio of 3:1 and incubated at 37°C for 4 hours.

[0083] Monitoring OD 600 The value is recorded as At. The hydrophobicity percentage is calculated as follows:

[0084] Hydrophobicity (%) = (1-A t / A0)×100%;

[0085] Where A0 and A t Represents OD at 0h and 4h respectively 600 .

[0086] The hydrophobicity of Lactobacillus plantarum 5S-5 is basically equivalent to that of the commercial strain Lactobacillus plantarum L2 (see Figure 3 In (E), when petroleum ether is used as the organic solvent, the hydrophobicity of 5S-5 is significantly higher than that of L2.

[0087] (5) Evaluation of biofilm formation ability

[0088] The formation of biofilm provides a protective barrier for lactic acid bacteria, especially under stress conditions, which can enhance their adaptability. The crystal violet staining method was used to determine the biofilm content. The overnight lactic acid bacteria fermentation broth (1%, v / v) was inoculated into MRS medium and incubated in a 96-well plate at 37°C for 24 hours. After incubation, the bacterial solution was removed and washed with saline. The 96-well plate was then placed at 65°C to fix the cells. The biofilm was stained with 0.1% (w / v) crystal violet for 5 minutes, and the excess dye was washed with PBS. Dry at 37°C for 20 minutes, and the stained crystal violet was dissolved with 30% (w / v) acetic acid solution. The absorbance was measured at 570nm.

[0089] The biofilm formation ability of Lactobacillus plantarum 5S-5 was significantly higher than that of the commercial strain Lactobacillus plantarum L2 (see Figure 3 Middle F).

[0090] Example 5 Safety Evaluation of Lactobacillus plantarum 5S-5

[0091] (1) Hemolytic evaluation

[0092] To evaluate hemolytic activity, plant lactobacillus 5S-5 was cultured on Columbia agar plates containing 5% (v / v) sheep blood at 37°C for 48 h. Activity was considered positive when a clear hemolytic ring (β-hemolysis) was observed around the colonies. Activity was considered negative when green or brown discoloration (α-hemolysis) or no hemolytic ring (γ-hemolysis) appeared in the culture medium.

[0093] MRSA is known to be β-hemolytic, with a transparent hemolytic ring around the colonies. However, there is no similar hemolytic ring around the colonies of Lactobacillus plantarum 5S-5, which is negative and a safe strain (see Figure 4 ).

[0094] (2) Antibiotic sensitivity test

[0095] The minimum inhibitory concentration (MIC, μg / mL) of vancomycin (Van), streptomycin (Str), chloramphenicol (Chl), tetracycline (Tet), erythromycin (Ery), and kanamycin (Kan) was determined by microbroth dilution. Briefly, broth dilutions of each antibiotic were prepared in 96-well plates using MRS broth. Lactobacillus plantarum 5S-5 was cultured overnight and appropriately diluted to 10 6 CFU / mL concentration. The diluted bacterial solution was inoculated into a 96-well plate. After incubation at 37°C for 24 hours, the turbidity of each well was observed. According to the definition of the European Food Safety Authority (EFSA), the minimum inhibitory concentration is considered to be the first concentration at which no growth is observed. When the MIC value is higher than the breakpoint, the strain is considered to be resistant.

[0096] The results are shown in Table 2. Lactobacillus plantarum 5S-5 showed sensitivity to vancomycin and kanamycin. Previous studies have shown that Lactobacillus plantarum has natural resistance to vancomycin, and this resistance of lactic acid bacteria is not easily transferred to other species. In addition, Lactobacillus plantarum 5S-5 showed resistance to kanamycin (aminoglycoside antibiotic), which is considered to be chromosomally encoded and non-transferable.

[0097] Table 2 Antibiotic sensitivity test of Lactobacillus plantarum 5S-5

[0098] Strain Cut-off(μg / mL) MIC (μg / mL) Van not required >128 Str not required >128 Ery 1 <0.25 Tet 32 32 Chl 8 8 Kan 64 >128

[0099] Example 6 Evaluation of the antimicrobial activity of the neutralized supernatant of Lactobacillus plantarum 5S-5

[0100] The antibacterial activity of the strains was evaluated using the microplate method under neutral conditions. The fermentation supernatant of Lactobacillus plantarum 5S-5 was adjusted to pH 7.0 with NaOH (1 M) and filtered through a 0.22 μM filter to obtain a neutralized fermentation supernatant. MRS medium was also adjusted to pH 7.0 as a control. MRSA was cultured overnight (at a concentration of approximately 10 8 CFU / mL). In a 96-well plate, 150 μL LB medium (containing 20 μL overnight cultured MRSA) and 100 μL neutralized fermentation supernatant were added to each well and incubated at 37°C. The OD of each well was monitored during the culture process. 600 , in order to evaluate the activity of the neutralized fermentation supernatant against pathogens. The experimental results showed that the neutralized fermentation supernatant still had significant antibacterial activity compared with the negative control group (MRS medium) (see Figure 5 ).

[0101] Example 7 Evaluation of antimicrobial activity of the fermentation supernatant of Lactobacillus plantarum 5S-5 after heat treatment

[0102] The fermentation supernatant of Lactobacillus plantarum 5S-5 was heated at 100° C. for 5 minutes, and the antibacterial activity test was performed according to the agar well diffusion method in Example 2.

[0103] The fermentation supernatant of Lactobacillus plantarum 5S-5 still had antibacterial activity after heat treatment, and there was no significant difference compared with the untreated fermentation supernatant, indicating that the antibacterial active substances were heat stable (see Figure 6 ).

[0104] Example 8 Comparative genomics analysis of Lactobacillus plantarum 5S-5

[0105] ANI was used to compare 5S-5 with the reference strains Lactobacillus plantarum WCFS1, Lactobacillus plantarum JCM1149, Lactobacillus plantarum NCU116, Lactobacillus plantarum JDM1, and Lactobacillus plantarum ZJ316 to detect structural differences, enabling comparative genomic colinearity analysis to elucidate the relationship between mutations and strain evolution (see Figure 7 ).

[0106] Example 9 Evaluation of the inhibitory activity of Lactobacillus plantarum 5S-5 against MRSA in the co-culture system

[0107] 5S-5 and MRSA were cultured to the logarithmic growth phase and mixed in a 6-well plate (Transwell) at a ratio of 1:1. The viable count method was used to monitor the survival rate of MRSA at 37°C for 24 hours. MRS culture medium was used instead of 5S-5 fermentation broth as a negative control. The bactericidal kinetic curve was drawn with time as the x-axis and the log10 value of CFU as the y-axis.

[0108] In the Transwell co-culture system, Lactobacillus plantarum 5S-5 and MRSA only exchanged metabolites. The experimental results showed that in this co-culture system, Lactobacillus plantarum 5S-5 had obvious antibacterial activity against MRSA (see Figure 8 ).

[0109] Example 10 Evaluation of the inhibitory activity of Lactobacillus plantarum 5S-5 on MRSA biofilm formation in the co-culture system 5S-5 and MRSA were cultured to the logarithmic growth phase and mixed in a 6-well plate (Transwell) at a ratio of 1:1 and cultured at 37° C. for 24 h. The biofilm was quantified according to the method of Example 4 (5).

[0110] The experimental results showed that in this co-culture system, Lactobacillus plantarum 5S-5 had a significant inhibitory activity against MRSA biofilm formation (see Fig. 9 ).

[0111] Example 11 Test on the inhibitory activity of Lactobacillus plantarum 5S-5 against MRSA in soy milk

[0112] MRSA and Lactobacillus plantarum 5S-5 were inoculated simultaneously in soy milk (the initial inoculation concentration was about 10 3 CFU / mL) and incubated at 37°C for 5 days. Soy milk inoculated with MRSA alone was used as a control. The viable count of MRSA in the single culture system and the co-culture system was measured every day, and the change in pH was monitored at the same time.

[0113] In the soy milk co-culture system, Lactobacillus plantarum 5S-5 still had significant inhibitory activity against MRSA. After the second day, the number of viable MRSA cells decreased significantly (from 10 9 CFU / mL decreased to 103 CFU / mL), revealing that Lactobacillus plantarum 5S-5 has good application prospects and has the potential to be developed into a new type of food preservative (see Fig.10 ).

[0114] It should be understood that the application of the present invention is not limited to the above examples. For ordinary technicians in this field, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to the present invention.

[0115] The nucleotide sequence involved in the present invention is as follows:

[0116] SEQ ID NO.1:

[0117] TGCAGTCGAACGAACTCTGGTATTGATTGGTGCTTGCATCATGATTTACATTTGAGTGAGTGGCGAACTGGTGAGTAACACGTGGGAAACCTGCCCAGAAGCGGGGGATAACACCTGGAAACAGATGCTAATACCGCATAACAACTTGGACCGCATGGTCCGAGCTTGAAAGATG GCTTCGGCTATCACTTTTGGATGGTCCCGCGGCGTATTAGCTAGATGGTGGGGTAACGGCTCACCATGGCAATGATACGTAGCCGACCTGAGAGGGTAATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGAAA GTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAACTCTGTTGTTAAAGAAGAACATATCTGAGAGTAACTGTTCAGGTATTGACGGTATTTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGA TTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCTTCGGCTCAACCGAAGAAGTGCATCGGAAACTGGGAAACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGC

[0118] TGTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGTATGGGTAGCAAACAG

[0119] GATTAGATACCCTGGTAGTCCATACCGTAAACGATGAATGCTAAGTGTTGG

[0120] AGGGTTTCCGCCCTTCAGTGCTGCAGCTAACGCATTAAGCATTCCGCCTGG

[0121] GGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCA

[0122] CAAGCGGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACC

[0123] AGGTCTTGACATACTATGCAAATCTAAGAGATTAGACGTTCCCTTCGGGGA

[0124] CATGGATACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTG

[0125] GGTTAAGTCCCGCAACGAGCGCAACCCTTATTATCAGTTGCCAGCATTAAG

[0126] TTGGGCACTCTGGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGA

[0127] TGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAA

[0128] TGGATGGTACAACGAGTTGCGAACTCGCGAGAGTAAGCTAATCTCTTAAA

[0129] GCCATTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAA

[0130] TCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTT

[0131] GTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCAAAGTCGGTG

[0132] GGGTAACCTTTTAGGAACCAGCCGCCTAA。

Claims

1. A strain of Lactobacillus plantarum 5S-5, characterized in that The Latin classification name of the plant lactobacillus 5S-5 is Lactiplantibacillus plantarum, the preservation unit is Guangdong Microbial Culture Collection Center, the preservation address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, the preservation number is GDMCC No: 65678, and the preservation date is December 26, 2024.

2. A bacterial agent, characterized in that The bacterial agent includes the Lactobacillus plantarum 5S-5 as described in claim 1.

3. A fermentation broth, characterized in that: The fermentation broth is obtained by fermenting the Lactobacillus plantarum 5S-5 as claimed in claim 1.

4. Use of the plant lactobacillus 5S-5 according to claim 1, the bacterial agent according to claim 2 or the fermentation liquid according to claim 3 in the preparation of a probiotic product.

5. Use of the plant lactobacillus 5S-5 according to claim 1, the bacterial agent according to claim 2 or the fermentation liquid according to claim 3 in the preparation of an antibacterial product.

6. The use according to claim 5, characterized in that: The antibacterial product has inhibitory activity against Gram-negative bacteria and Gram-positive bacteria.

7. The use according to claim 6, characterized in that: The Gram-negative bacteria are one or more of Salmonella, Pseudomonas aeruginosa, Acinetobacter baumannii and Escherichia coli.

8. The use according to claim 6, characterized in that: The Gram-positive bacteria are one or more of methicillin-resistant Staphylococcus aureus, Bacillus subtilis, Streptococcus mutans and Listeria monocytogenes.

9. The use according to claim 5, characterized in that: The antibacterial product inhibits the formation of biofilm.

10. The use according to claim 5, characterized in that: The antibacterial product is a food preservative.

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