Plant lactobacillus with efficient broad-spectrum bacteriostatic activity

Abstract

The invention relates to the field of microorganisms, in particular to a plant lactobacillus and application thereof in bacteriostasis. Specifically, the preservation number of the plant lactobacillus LP06 is CGMCC (China General Microbiological Culture Collection Center) No. 28412. The plant lactobacillus LP06 provided by the invention can be used for inhibiting the growth of debaryomyces hansenii, candida tropicalis and penicillium rochei, and also has an inhibiting effect on super bacteria acinetobacter baumannii, pseudomonas aeruginosa, klebsiella, escherichia coli, staphylococcus aureus and listeria monocytogenes. The plant lactobacillus LP06 provided by the invention has broad-spectrum bacteriostatic ability, and bacteriostatic powder prepared from the bacterium can be used as a biological preservative for corrosion prevention of dairy products such as yoghourt, cheese and the like and other foods.

Description

Technical Field

[0001] The invention belongs to the field of microorganisms, and in particular relates to a strain of Lactobacillus plantarum and its application in antibacterial aspects. Background Art

[0002] Lactic acid bacteria are among the most important microorganisms in the food industry and biotechnology, and are often used as fermentation agents for dairy and plant-derived products. Lactic acid bacteria, especially Lactobacillus species, are recognized by the World Health Organization as having good safety. They not only help improve the taste and texture of food, and have a potential role in promoting human health as probiotics, but also can secrete antibacterial substances, which have a good inhibitory effect on pathogens and microorganisms that cause food spoilage.

[0003] The antibacterial effect of lactic acid bacteria mainly comes from various metabolites produced during its growth and reproduction, mainly including organic acids and protein compounds. The organic acids with antibacterial effects are mainly lactic acid, acetic acid, phenyllactic acid, indoleacetic acid, 2-hydroxy-3-methylbutyric acid and 4-hydroxyphenyllactic acid, etc. They mainly reduce the environmental pH by dissociation or the undissociated organic acids penetrate the microbial cell membrane in a hydrophobic form to enter the cell, affecting the pH in the microorganism, thereby inhibiting the normal growth of the microorganism. Another type of antibacterial substance produced by lactic acid bacteria is mainly bacteriocins, which are protein substances synthesized by lactic acid bacteria through ribosomes and are mainly composed of 20-60 amino acids. Unlike antibiotics, antibiotics are synthesized by microorganisms using primary metabolites through enzymatic reactions, but bacteriocins are usually peptide substances synthesized by coding genes located on plasmids or chromosomal DNA, so their safety and stability are much higher than antibiotics, and they have the advantages of safety, non-toxicity and high stability.

[0004] At present, chemical preservatives such as potassium sorbate and sodium benzoate are still widely used in the food field. There are few food additives with natural antibacterial functions, and the more representative ones are a few such as nisin, natamycin, polylysine, and lysozyme. Lactic acid bacteria use the antagonistic effect of their metabolites on other microorganisms to achieve antibacterial preservation, which can reduce the use of chemical preservatives and improve food safety. In terms of research, the current focus is on the inhibitory effect of antibacterial substances produced by lactic acid bacteria on bacteria such as Escherichia coli, Staphylococcus aureus or Listeria monocytogenes. In the production, processing and storage of food, especially dairy products, they are often contaminated by yeast and mold. However, there is relatively little research on the antibacterial substances of lactic acid bacteria against yeast and mold.

[0005] Therefore, screening lactic acid bacteria that can effectively inhibit the growth of yeast and mold, utilizing the antibacterial substances of lactic acid bacteria, and developing biological preservatives that are safe, efficient, have a wide antibacterial range and high stability are of great significance for the development of green, safe and healthy food.

[0006] In addition, with the development of genome sequencing and analysis technology, it is highly feasible to use genomics to study the functional characteristics of lactic acid bacteria. The existence of antibacterial-related functional genes in the genome of lactic acid bacteria is the fundamental reason for its antibacterial function. For example, genomic analysis can explain the acid production ability of the strain from the genetic level, find the key gene for acid production, and then regulate the gene to promote efficient expression of the gene to increase acid production. For another example, by analyzing the secondary metabolite synthesis gene cluster, the type and synthesis ability of the strain's secondary metabolites can be predicted for application. Analyzing lactic acid bacteria from a genomic perspective is of great significance for functional discovery and improving its antibacterial ability. Summary of the invention

[0007] To overcome the above problems, the present invention provides a strain of Lactobacillus plantarum LP06. The Lactobacillus plantarum LP06 provided by the present invention can inhibit the growth of Debaryomyces hansenii, Candida tropicalis and Penicillium roqueforti, and has an inhibitory effect on Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella, Listeria monocytogenes, Escherichia coli and Staphylococcus aureus. The antibacterial activity of the fermentation broth supernatant of Lactobacillus plantarum LP06 after pepsin treatment is reduced, indicating that protein substances, i.e., bacteriocins, play an important role in antibacterial activity. Genome analysis shows that Lactobacillus plantarum LP06 can secrete Class II bacteriocins, and the predicted types are Plantaricin EF and Plantaricin JK, a complex composed of dipeptides.

[0008] A first aspect of the present invention provides a strain of Lactiplantibacillus plantarum LP06, comprising a 16S rRNA nucleic acid having at least 98% identity to the nucleic acid shown in SEQ ID NO:1.

[0009] In some embodiments, the 16S rRNA nucleic acid is at least 99% identical to the nucleic acid set forth in SEQ ID NO:1.

[0010] In some embodiments, the 16S rRNA nucleic acid has a nucleic acid sequence as shown in SEQ ID NO:1.

[0011] In some embodiments, the Lactobacillus plantarum LP06 has a deposit number of CGMCC No.28412.

[0012] The second aspect of the present invention provides a culture comprising the Lactobacillus plantarum LP06 of the present invention and an optional culture medium.

[0013] In some embodiments, the culture medium includes skim milk and glucose.

[0014] In some embodiments, the weight percentage of the skim milk is 1%-15%. In some embodiments, the weight percentage of the skim milk is 1%-10%. In some embodiments, the weight percentage of the skim milk is 2-7%. In some embodiments, the weight percentage of the skim milk is 4%.

[0015] In some embodiments, the weight percentage of glucose is 0.5%-8%. In some embodiments, the weight percentage of glucose is 0.5%-6%. In some embodiments, the weight percentage of glucose is 1%-3%. In some embodiments, the weight percentage of glucose is 2%.

[0016] In some embodiments, the Lactobacillus plantarum LP06 is a living bacterium.

[0017] In some embodiments, the culture includes bacteriocin Plantaricin EF and bacteriocin Plantaricin JK.

[0018] In some embodiments, the culture includes bacteriocin, bacteriocin Plantaricin EF and bacteriocin Plantaricin JK, wherein the amino acid sequence of the bacteriocin is as shown in SEQ ID NO:4, the bacteriocin Plantaricin EF is composed of plantaricin EF subunit PlnE shown in SEQ ID NO:6 and plantaricin EF subunit PlnF shown in SEQ ID NO:5, and the bacteriocin Plantaricin JK is composed of plantaricin JK subunit PlnJ shown in SEQ ID NO:3 and plantaricin JK subunit PlnK shown in SEQ ID NO:2.

[0019] The third aspect of the present invention provides a culture supernatant, which is the culture supernatant of the second aspect of the present invention.

[0020] The fourth aspect of the present invention provides a composition or an antibacterial agent, comprising the Lactobacillus plantarum LP06 described in the first aspect of the present invention, or the culture described in the second aspect of the present invention, or the culture supernatant described in the third aspect of the present invention.

[0021] In some embodiments, the Lactobacillus plantarum LP06 is a living bacterium.

[0022] In some embodiments, the composition or bacteriostatic agent is a solid bacteriostatic agent or a liquid bacteriostatic agent.

[0023] In some embodiments, the composition or bacteriostatic agent is a solid bacteriostatic agent.

[0024] In some embodiments, the composition or bacteriostatic agent is a bacteriostatic powder.

[0025] In some embodiments, the composition or antibacterial agent is prepared by a method comprising the following operations:

[0026] 1) inoculating the Lactobacillus plantarum LP06 described in the first aspect of the present invention into a culture medium,

[0027] 2) Fermentation.

[0028] In some embodiments, the culture medium in step 1) comprises skim milk and glucose.

[0029] In some embodiments, the weight percentage of the skim milk is 1%-15%. In some embodiments, the weight percentage of the skim milk is 1%-10%. In some embodiments, the weight percentage of the skim milk is 2-7%. In some embodiments, the weight percentage of the skim milk is 4%.

[0030] In some embodiments, the weight percentage of glucose is 0.5%-8%. In some embodiments, the weight percentage of glucose is 0.5%-6%. In some embodiments, the weight percentage of glucose is 1%-3%. In some embodiments, the weight percentage of glucose is 2%.

[0031] In some embodiments, step 2) has one or both of the following technical features:

[0032] ① The fermentation temperature is 36-38°C,

[0033] ②The fermentation time is 60-90h.

[0034] In some embodiments, the temperature of the fermentation is 37°C.

[0035] In some embodiments, the fermentation time is 65-85 hours. In some embodiments, the fermentation time is 70-80 hours. In some embodiments, the fermentation time is 72 hours.

[0036] The fifth aspect of the present invention provides a method for preparing the composition or antibacterial agent according to the fourth aspect of the present invention, comprising the following steps:

[0037] 1) inoculating the Lactobacillus plantarum LP06 described in the first aspect of the present invention into a culture medium,

[0038] 2) Fermentation.

[0039] In some embodiments, the culture medium in step 1) comprises skim milk and glucose.

[0040] In some embodiments, the weight percentage of the skim milk is 1%-15%. In some embodiments, the weight percentage of the skim milk is 1%-10%. In some embodiments, the weight percentage of the skim milk is 2-7%. In some embodiments, the weight percentage of the skim milk is 4%.

[0041] In some embodiments, the weight percentage of glucose is 0.5%-8%. In some embodiments, the weight percentage of glucose is 0.5%-6%. In some embodiments, the weight percentage of glucose is 1%-3%. In some embodiments, the weight percentage of glucose is 2%.

[0042] In some embodiments, step 2) has one or both of the following technical features:

[0043] ① The fermentation temperature is 36-38°C,

[0044] ②The fermentation time is 60-90h.

[0045] In some embodiments, the temperature of the fermentation is 37°C.

[0046] In some embodiments, the fermentation time is 65-85 hours. In some embodiments, the fermentation time is 70-80 hours. In some embodiments, the fermentation time is 72 hours.

[0047] The sixth aspect of the present invention provides the use of the Lactobacillus plantarum LP06 described in the first aspect of the present invention, the culture described in the second aspect of the present invention, or the culture supernatant described in the third aspect of the present invention in the preparation of an antibacterial product.

[0048] In some embodiments, the bacteriostatic product is a fungistatic and bacteria-static product.

[0049] In some embodiments, the fungus is selected from Candida tropicalis, Debaryomyces hansenii, and Penicillium roqueforti.

[0050] In some embodiments, the bacteria is selected from the group consisting of Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella, Listeria monocytogenes, Escherichia coli, and Staphylococcus aureus.

[0051] The seventh aspect of the present invention provides the use of the Lactobacillus plantarum LP06 described in the first aspect of the present invention, the culture described in the second aspect of the present invention, the culture supernatant described in the third aspect of the present invention, or the composition or antibacterial agent described in the fourth aspect of the present invention in a product.

[0052] In some embodiments, the product is a food or a medicine.

[0053] In some embodiments, the product is a food product.

[0054] In some embodiments, the food product is a dairy product.

[0055] In some embodiments, the dairy product is yogurt, cheese, milk powder, or a milk-containing beverage.

[0056] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0057] The plant lactobacillus LP06 (CGMCC No. 28412) provided by the present invention can be obtained in large quantities by cultivation, for example, by inoculating it into a culture medium and letting it stand for cultivation, a large amount of plant lactobacillus LP06 can be obtained. The culture medium can be a culture medium conventionally used for culturing lactic acid bacteria. In some embodiments, the culture medium is a skim milk glucose culture medium. The culture conditions can be conventional conditions for bacterial culture. In a preferred embodiment, the culture temperature is 37° C.; in another preferred embodiment, the culture time is 72 hours.

[0058] The composition or antibacterial provided by the present invention contains plant lactobacillus LP06 (which can be viable cells) and / or fermentation liquid supernatant. In some embodiments, the composition or antibacterial contains the viable cells of plant lactobacillus, for example, the viable cells of plant lactobacillus are prepared into antibacterial powder, wherein the viable cells of plant lactobacillus can be bred in culture medium and obtain the target product. In some embodiments, the composition or antibacterial contains the fermentation liquid supernatant of plant lactobacillus, for example, when the fermentation ends, the cell is precipitated and centrifuged to obtain the product. In some embodiments, the composition or antibacterial contains the viable cells and fermentation supernatant of plant lactobacillus, for example, the product after the fermentation ends, includes both the viable cells of plant lactobacillus and the fermentation supernatant at the fermentation end point. In some embodiments, the composition or antibacterial is liquid (stock solution or concentrated solution), and in some embodiments, the composition or antibacterial is solid. Those skilled in the art can select suitable form according to the intended purpose. It is understandable that when preparing compositions or antibacterial agents in different forms, auxiliary materials that can stabilize the corresponding forms can be added.

[0059] Beneficial effects achieved by the present invention:

[0060] (1) Provided is a new strain of Lactobacillus plantarum, with a deposit number of CGMCC No.28412.

[0061] (2) The plant lactobacillus LP06 provided by the present invention has a broad-spectrum antibacterial effect, can inhibit the growth of fungi (such as yeast and mold), and has an inhibitory effect on tropical Candida, Debaryomyces hansen, and Penicillium roqueforti; it can also inhibit the growth of bacteria, and has an inhibitory effect on Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella, Listeria monocytogenes, Escherichia coli, and Staphylococcus aureus.

[0062] (3) The Lactobacillus plantarum LP06 provided by the present invention is an edible lactic acid bacterium that can utilize a variety of carbon sources and nitrogen sources and is easy to culture; it is sensitive to a variety of antibiotics, has a low risk of carrying and transferring drug-resistant genes, and has good safety; it has good salt tolerance, and a salt concentration of 1% to 3% has no effect on its growth and antibacterial effect.

[0063] (4) The culture provided by the present invention is suitable for Lactobacillus plantarum LP06 to produce and secrete antibacterial substances, which is not conducive to the growth of spoilage bacteria or pathogenic bacteria.

[0064] (5) The antibacterial agent provided by the present invention can be used as a biological preservative in the field of food such as dairy products, and has a broad-spectrum antibacterial effect.

[0065] Biological Deposit Description

[0066] The Lactobacillus plantarum LP06 provided by the present invention was deposited in the China General Microbiological Culture Collection Center (CGMCC) of the China Microbiological Culture Collection Committee on September 8, 2023, with a deposit number of CGMCC No. 28412, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein:

[0068] Figure 1 Screening results of lactic acid bacteria that effectively inhibit the growth of bacteria and fungi, A: Escherichia coli, B: Staphylococcus aureus, C: Candida tropicalis.

[0069] Figure 2 The identification results of LP06, A: colony morphology of Lactobacillus plantarum LP06, B: bacterial morphology of Lactobacillus plantarum LP06.

[0070] Figure 3 This is the NJ phylogenetic tree of Lactobacillus plantarum LP06 based on the 16SrRNA gene sequence.

[0071] Figure 4 This is the evolutionary tree of Lactobacillus plantarum LP06 based on the gene sequence of housekeeping genes.

[0072] Figure 5 To compare the inhibitory effects of Lactobacillus plantarum and other antibacterial substances using Debaryomyces hansenii as an indicator bacteria, A: Lactobacillus plantarum LP06; B: Nisin; C: Natamycin; D: Potassium sorbate.

[0073] Figure 6 This is the antibacterial profile analysis of Lactobacillus plantarum LP06, ①A: Escherichia coli, B: Staphylococcus aureus, C: Pseudomonas aeruginosa, D: Klebsiella, E: Acinetobacter baumannii, F: Listeria monocytogenes (i.e. Listeria monocytogenes), G: Debaryomyces hansenii, H: Candida tropicalis, I: Penicillium roqueforti; ② is the diameter of each inhibition zone.

[0074] Figure 7 These are the results of sensitivity of Lactobacillus plantarum LP06 to antibiotics.

[0075] Figure 8 These are the growth curve and acid production curve of Lactobacillus plantarum LP06.

[0076] Fig. 9 This study is to analyze the sensitivity of extracellular metabolites of Lactobacillus plantarum LP06 to pepsin.

[0077] Fig.10 This is the whole genome analysis of Lactobacillus plantarum LP06, A: genome CGView map, B: genome Circos map.

[0078] Fig.11 Secondary structure analysis of bacteriocins secreted by Lactobacillus plantarum LP06, A: bacteriocin encoded by gene2233, B: bacteriocin encoded by gene2234, C: bacteriocin encoded by gene2241, D: bacteriocin encoded by gene2246, E: bacteriocin encoded by gene2247.

[0079] Fig.12 The tertiary structure and Ramachandran plot characteristics of the bacteriocin secreted by Lactobacillus plantarum LP06 obtained by homology modeling, A: bacteriocin encoded by gene2233, B: bacteriocin encoded by gene2234, C: bacteriocin encoded by gene2241, D: bacteriocin encoded by gene2246, E: bacteriocin encoded by gene2247.

[0080] Fig.13 Effects of different salt concentrations on the growth and antibacterial effect of Lactobacillus plantarum LP06. A: LP06 inhibits Debaryomyces hansenii; B: diameter of inhibition zone; C: viable bacterial counts under different salt concentrations; D: OD value under different salt concentrations.

[0081] Fig.14 Optimization of culture medium for preparing antibacterial powder of Lactobacillus plantarum LP06, A: skim milk culture medium supplemented with 2% glucose; B: skim milk culture medium supplemented with 2% glucose and 1% yeast powder; C: skim milk culture medium supplemented with 2% glucose, 1% yeast powder and 1% peptone; D: Inhibitory effect of Lactobacillus plantarum LP06 growing on skim milk supplemented with 2% glucose on Debaryomyces hansenii.

[0082] Fig.15 Prediction and functional interpretation of genes encoding Lactobacillus plantarum LP06. A: GO annotation, including BP, CC, and MF classifications; B: KEGG annotation; C: COG annotation.

[0083] Fig.16 Annotation statistics for predicted carbohydrate-active enzymes in Lactobacillus plantarum LP06.

[0084] Fig.17The arrows in the figure indicate the direction of the reaction, the arrows point to substances produced by this step of the reaction, and the labels next to the arrows are genes encoding the enzymes required for this step of the reaction.

[0085] Fig.18 These are genes related to the pyruvate pathway in Lactobacillus plantarum LP06.

[0086] Fig.19 It is the gene cluster for the synthesis of T3PKS, terpene, and RiPP-like secondary metabolites in the genome of Lactobacillus plantarum LP06. DETAILED DESCRIPTION

[0087] The embodiments of the present invention will be described in detail below in conjunction with the examples, but those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. If no specific conditions are specified in the examples, they are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be obtained commercially.

[0088] Example 1 Screening of broad-spectrum antibacterial lactic acid strains

[0089] 318 lactic acid bacteria were isolated from Tibetan dairy products and screened for antibacterial effects on common pathogens. Among them, Escherichia coli was selected as the indicator bacteria for Gram-negative bacteria, Staphylococcus aureus was selected as the indicator bacteria for Gram-positive bacteria, and Candida tropicalis was selected as the indicator bacteria for fungi.

[0090] The streak-double-layer plate method was selected for screening antibacterial activity. First, the lactic acid bacteria were activated and the lactic acid bacteria in the glycerol tube were picked on solid MRS medium (10 g peptone, 10 g beef extract, 5 g yeast extract, 2 g diammonium citrate, 5 g sodium acetate, 20 g glucose, 80 mL Tween, 0.5 g magnesium sulfate, 0.25 manganese sulfate, pH adjusted to 6.2-6.4, UPH 2 O to 1L), 37°C, culture for 48h. To streak the bottom plate, first use a ruler and a marker to draw two straight lines of 2cm in length and 1.5cm apart on the MRS plate, then pick up the activated lactic acid bacteria colonies and streak along the drawn straight lines. Preparation of the upper culture medium, liquid MRS culture medium (10g peptone, 10g beef extract, 5g yeast extract, 2g diammonium citrate, 5g sodium acetate, 20g glucose, 80mL Tween, 0.5g magnesium sulfate, 0.25 manganese sulfate, adjust pH to 6.2-6.4, add UPH 2The indicator bacteria were cultured in a 1:100 ratio in a semi-solid LB medium (tryptone, 5 g yeast extract, 10 g / L sodium chloride, pH adjusted to 7.0 with 5 mol / L NaOH, and UPH 2 0 to 1L, agar content 0.7%), inoculate indicator bacteria, inoculate indicator fungi into semi-solid PDA medium (Haibo Biotechnology, Qingdao High-tech Industrial Park Haibo Biotechnology Co., Ltd.) cooled to about 45°C at a ratio of 1:200, mix well and pour the upper plate. Incubate in a 37°C incubator for 13 hours, record the antibacterial results, measure the diameter of the antibacterial circle close to the circle, measure the approximate length and width of the irregular antibacterial circle, and finally compare the relative strength of the antibacterial effect by area.

[0091] Through screening, 15 strains with good inhibitory effects on Escherichia coli, Staphylococcus aureus and Candida were obtained, among which LP06 (i.e. 15-1) had the most outstanding inhibitory effect on the fungus Candida tropicalis ( Figure 1 ), as the strain used in this study.

[0092] Example 2 Identification and Deposit of LP06

[0093] 1. Appearance characteristics

[0094] LP06 is round, milky white, and has a short rod shape ( Figure 2 ).

[0095] 2. Genome quality control information

[0096] The insert sequence length of Lactobacillus plantarum LP06 is 515 bp, the read length of the original reads is 150 bp, and the number of paired-end reads in the original sequence data is 3439307×2. The total number of bases in the original data obtained by multiplying the number of original paired-end reads by the length is about 1×10 9 The number of paired-end reads after quality control is 3407835×2, and the number of single-end reads after quality control is 28807. The total number of bases after quality control is about 1×10 9 For details, please see Table 1.

[0097] Table 1 Quality control data statistics of Lactobacillus plantarum LP06

[0098]

[0099] 3. Identification

[0100] The NJ phylogenetic tree based on 16S rRNA gene sequences showed that LP06 formed a monophyletic branch with Lactiplantibacillus plantarum ( Figure 3 ). Through further database comparison, based on 31 housekeeping genes (dnaG, fmr, inic, nusA, pk, pvG, rA, rB, IC, rID, E, IIE, OIK, OL, IIM, OIN, DP, IS, IT, PmA, OB, PSB, DSC, DSE, Psl, rpsJ, rpsK, psM, rpsS, smpB, tsf), 19 strains closest to each other at the species level were selected, and the phylogenetic tree was constructed using the NJ (Neighbor-Joining) method using the MEGA6.0 software. It was determined that LP06 (shown in S15_1) formed a monophyletic branch with Lactobacillus plantarum ( Figure 4 ). After identification, LP06 was found to be a new Lactobacillus plantarum and was named Lactobacillus plantarum LP06.

[0101] 4. Preservation

[0102] Lactobacillus plantarum LP06 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC) on September 8, 2023, with a deposit number of CGMCC No. 28412, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code: 100101. The 16S rRNA sequence of Lactobacillus plantarum LP06 (SEQ ID NO: 1) is as follows:

[0103] GGCTGGTTCCTAAAGGTTACCCCACCGACTTTGGGTGTTACAAACTCTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCGACTTCATGTAGGCGAGT TGCAGCCTACAATCCGAACTGAGAATGGCTTTAAGAGATTAGCTTACTCTCGCGAGTTCGCAACTCGTTGTACCATCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCC

[0104] GGTTTGTCACCGGCAGTCTCACCAGAGTGCCCAACTTAATGC

[0105] TGGCAACTGATAATAAGGGTTGCGCTCGTTGCGGGACTTAAC

[0106] CCAACATCTCACGACACGAGCTGACGACAACCATGCACCACC

[0107] TGTATCCATGTCCCCGAAGGGAACGTCTAATCTCTTAGATTT

[0108] GCATAGTATGTCAAGACCTGGTAAGGTTCTTCCGCGTAGCTTC

[0109] GAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCA

[0110] ATTCCTTTGAGTTTCAGCCTTGCGGCCGTACTCCCCAGGCGG

[0111] AATGCTTAATGCGTTAGCTGCAGCACTGAAGGGCGGAAACC

[0112] CTCCAACACTTAGCATTCATCGTTTACGGTATGGACTACCAG

[0113] GGTATCTAATCCTGTTTGCTACCCATACTTTCGAGCCTCAGC

[0114] GTCAGTTACAGACCAGACAGCCGCCTTCGCCACTGGTGTTCT

[0115] TCCATATATCTACGCATTTCACCGCTACACATGGAGTTCCAC

[0116] TGTCCTCTTCTGCACTCAAGTTTCCCAGTTTCCGATGCACTTC

[0117] TTCGGTTGAGCCGAAGGCTTTCACATCAGACTTAAAAAACCG

[0118] CCTGCGCTCGCTTTACGCCCCAATAAATCCGGACAACGCTTGC

[0119] CACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTG

[0120] GCTTTCTGGTTAAATACCGTCAATACCTGAACAGTTACTCTC

[0121] AGATATGTTCTTCTTTAACAACAGAGTTTTACGAGTCGAAAC

[0122] CCTTCTTCACTCACGCGGCGTTGCTCCATCAGACTTTCGTCC

[0123] ATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTTTGG

[0124] GCCGTGTCTCAGTCCCAATGTGGCCGATTACCCTCTCAGGTC

[0125] GGCTACGTATCATCGCCATGGTGAGCCGTTACCCCACCATCT

[0126] AGCTAATACGCCGCGGGACCATCCAAAAGTGATAGCCGAAG

[0127] CCATCTTTCAAACTCGGACCATGCGGTCCAAGTTGTTATGCG

[0128] GTATTAGCATCTGTTTCCAGGTGTTATCCCCCGCTTCTGGGC

[0129] AGGTTTCCCACGTGTTACTCACCAGTTCGCCACTCACTCAAA

[0130] TGTAAATCATGATGCAAGCACCAATCAATACCAGAGTTCGTT

[0131] CGACTGC。

[0132] Analysis of the antibacterial spectrum of Lactiplantibacillus plantarum LP06 in Example 3

[0133] Yeast is a common contaminating strain in food, especially dairy products. Nisin is widely used in dairy products as a natural preservative. Natamycin and potassium sorbate can widely inhibit the growth of various yeasts. Using Debaryomyces hansenii as an indicator bacteria and nisin, natamycin and potassium sorbate as controls, the antibacterial ability of Lactobacillus plantarum LP06 against yeast was compared by double-layer plate streak method. It was found that the inhibitory effect of Lactobacillus plantarum LP06 on Debaryomyces hansenii was better than that of nisin, natamycin and potassium sorbate ( Figure 5 ).

[0134] In addition, molds and bacteria (such as Listeria monocytogenes) are also common contaminating strains in food, especially dairy products. Using Penicillium roqueforti, Listeria monocytogenes, Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella, Acinetobacter baumannii, Debaryomyces hansenii, and Candida tropicalis as indicator bacteria, the antibacterial ability of Lactobacillus plantarum LP06 against the above-mentioned bacteria was analyzed by the double-layer plate streak method. It was found that Lactobacillus plantarum LP06 has a strong inhibitory effect on super bacteria Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella and Listeria monocytogenes, has a good inhibitory effect on Debaryomyces hansenii, and also has a certain inhibitory effect on Penicillium roqueforti ( Figure 6 ). It can be seen that the Lactobacillus plantarum LP06 in this study has a high efficiency and broad-spectrum antibacterial effect.

[0135] Example 4 Cultivability and safety of Lactobacillus plantarum LP06 as a bacterium producing antibacterial powder

[0136] 1. Analysis of the culturability of Lactobacillus plantarum LP06

[0137] The types of carbon and nitrogen sources that can be used by strains with antibacterial function are very important for obtaining a large number of bacteria and fermentation products, controlling production costs and making subsequent antibacterial powder. Therefore, the carbon and nitrogen source utilization of Lactobacillus plantarum LP06 was analyzed.

[0138] The HBI carbon source detection kit of Qingdao Haibo was selected for detection, and it was found that Lactobacillus plantarum LP06 can use 9 carbohydrates, including esculin, cellobiose, maltose, salicin, sorbitol, sucrose, raffinose, inulin, and lactose, as the sole carbon source for strain growth and reproduction (Table 2-1). At the same time, the utilization of nitrogen sources by Lactobacillus plantarum LP06 was analyzed by supplementing different nitrogen sources in the basic culture medium for bacterial culture and measuring the OD value for analysis. Wherein basal medium (2.0g potassium dihydrogen phosphate, 5.0g sodium acetate anhydrous, 5.0g sodium citrate, 0.2g magnesium sulfate, 0.05g manganese sulfate, 20.0g glucose, 80mL tween, pH=6.6±0.2 is adjusted to add water to 1000mL) without nitrogen source, and the cell does not grow when no nitrogen source is detected, and three kinds of organic nitrogen and four kinds of inorganic nitrogen are added respectively in basal medium, and the utilization of each nitrogen source by the analysis strain is shown that the organic nitrogen that plant lactobacillus is more easily available has yeast powder, beef powder, and tryptone, and organic nitrogen diammonium hydrogen phosphate can also be used to grow, and when urea, ammonium chloride, and ammonium sulfate are the only nitrogen source, it can also grow weakly (table 2-2). It can be seen that the carbon source and nitrogen source that plant lactobacillus can utilize are extensive, and it is easy to cultivate, which is beneficial to subsequent large-scale fermentation production, and the economical carbon and nitrogen source can also be selected according to cost to cultivate plant lactobacillus LP06.

[0139] Table 2-1 Analysis of carbon source utilization of Lactobacillus plantarum LP06

[0140]

[0141] Table 2-2 Analysis of nitrogen source utilization by Lactobacillus plantarum LP06

[0142]

[0143] Note: “-” indicates no growth, “+” indicates weak growth, “++” indicates moderate growth, and “+++” indicates good growth.

[0144] 2. Safety analysis of Lactobacillus plantarum LP06

[0145] There are high safety requirements for microorganisms or products used in the food field. On the one hand, they are required to be edible, and on the other hand, they are expected to be sensitive to antibiotics to prevent them from carrying drug-resistant genes or undergoing horizontal gene transfer that may affect human health.

[0146] Lactobacillus plantarum LP06 is an edible lactic acid bacterium. To further analyze its sensitivity to antibiotics, OXOID drug sensitivity paper was used to analyze the sensitivity of Lactobacillus plantarum LP06 to tetracycline TE 30mcg, ampicillin AMP 10mcg, chloramphenicol C 30μg, penicillin P 10IU, streptomycin S10μg, and erythromycin E15μg. LP06 was cultured to 0.5 McFarland turbidity using MRS liquid culture medium, spread on the surface of the plate, and the drug sensitivity paper was placed on the plate within 15min after inoculation. Inverted culture was performed in a 37℃ incubator, and the diameter of the complete inhibition zone was measured after 18-24h of culture. The results showed that Lactobacillus plantarum LP06 was sensitive to tetracycline, ampicillin, chloramphenicol, and erythromycin, moderately sensitive to penicillin, and only tolerant to streptomycin ( Figure 7 ). It can be seen that Lactobacillus plantarum LP06 is a microorganism with high biological safety.

[0147] Example 5 Analysis of antibacterial functional substances of Lactobacillus plantarum LP06

[0148] 1. Growth and acid production of Lactobacillus plantarum LP06

[0149] Organic acids have a certain inhibitory effect on the growth of microorganisms. Lactobacillus will secrete some organic acids during the growth process, which will cause the pH of the environment to decrease, thereby inhibiting the normal growth of microorganisms. After activating 2-3 generations of Lactobacillus plantarum LP06 stored at -80℃, inoculate 2% of the bacteria into 10mL MRS liquid culture medium and measure the OD at different times. 630 and pH value, record the data, with the culture time as the horizontal axis and the OD 630 The value was used as the vertical axis to draw the growth curve and pH change curve. The lag phase of Lactobacillus plantarum LP06 was 0-2h, it started to grow after 2h, entered the growth stable phase after 14h, and began to die after 30h ( Figure 8 A). Lactobacillus plantarum LP06 began to produce acid at 2 hours, and the acidity of the culture fluid dropped sharply at 4 hours. At 12 hours, the acidity basically reached a stable period, and the lowest pH could reach 4.0 ( Figure 8 B). The change in acidity is basically consistent with the growth trend of Lactobacillus plantarum LP06. It can be seen that Lactobacillus plantarum LP06 can significantly reduce the acidity of its surroundings during its growth, which plays an important role in inhibiting the growth of other microorganisms.

[0150] 2. Bacteriocins secreted by Lactobacillus plantarum LP06

[0151] a) Analysis of bacteriocin production by Lactobacillus plantarum LP06

[0152] Bacteriocins are polypeptides secreted by microorganisms that are sensitive to proteases, which can inactivate bacteriocins. Different types of bacteriocins have different sensitive proteases. According to existing research reports, most bacteriocins are sensitive to pepsin. Therefore, this study selected pepsin to treat the supernatant of the fermentation broth of Lactobacillus plantarum, and analyzed the difference in the antibacterial effect of the fermentation broth supernatant before and after treatment. If bacteriocins exist in the supernatant, the antibacterial effect of the fermentation broth supernatant will be reduced after pepsin treatment, so as to preliminarily identify whether Lactobacillus plantarum secretes bacteriocins.

[0153] Using Listeria monocytogenes as the indicator bacteria, the difference in the antibacterial effect of the fermentation supernatant of Lactobacillus plantarum LP06 before and after pepsin treatment was compared and analyzed. Fig. 9 In the left figure, No. 1 is the inhibition zone of the fermentation supernatant of Lactobacillus plantarum LP06, No. 2 is the inhibition zone after the fermentation supernatant of Lactobacillus plantarum LP06 was treated at 37°C for 30 minutes and 90°C for 5 minutes, and No. 3 is the inhibition zone after the fermentation supernatant of Lactobacillus plantarum LP06 was treated with pepsin to a final concentration of 100μg / mL and treated at 37°C for 30 minutes and 90°C for 5 minutes. The results show that the inhibitory effect of the fermentation supernatant of Lactobacillus plantarum LP06 on Listeria was weakened after being treated with pepsin, and the diameter of the inhibition zone was reduced, with a reduction rate of 38.1% ( Fig. 9 ). The above results show that Lactobacillus plantarum LP06 can secrete bacteriocin and has a good bacteriocin production ability.

[0154] b) Verification of bacteriocin production by Lactobacillus plantarum LP06

[0155] To further verify that Lactobacillus plantarum LP06 can secrete bacteriocins, the genome of Lactobacillus plantarum LP06 was analyzed by whole genome sequencing. The gene results were compared with NR, Swiss-Prot, Pfam, COG, GO, KEGG, and BAGEL4 databases to obtain functional annotation information in the databases. The results showed that the chromosome genome size of Lactobacillus plantarum LP06 was 3291183bp, containing 3133 coding genes with a GC content of 44.38%, 63 tRNAs, 3 rRNAs ( Fig.10 ).

[0156] Antismash software was used to predict the secondary metabolite synthesis gene clusters of the samples, and it was found that Lactobacillus plantarum LP06 had genes encoding bacteriocins (Table 3-1). It was predicted that Lactobacillus plantarum LP06 could secrete class II bacteriocins, the main types of which were plantaricin EF and plantaricin JK, a complex composed of dipeptides. Among them, gene2233 encoded PlnK (SEQ ID NO: 2), gene2234 encoded PlnJ (SEQ ID NO: 3), gene2241 encoded bacteriocin (SEQ ID NO: 4), gene2246 encoded PlnF (SEQ ID NO: 5), and gene2247 encoded PlnE (SEQ ID NO: 6) (Table 3-2).

[0157] Table 3-1 Genes encoding bacteriocins in Lactobacillus plantarum LP06

[0158]

[0159] Table 3-2 Bacteriocin sequence list encoded by Lactobacillus plantarum LP06

[0160]

[0161] c) Results of physicochemical properties analysis of Lactobacillus plantarum LP06 bacteriocin

[0162] The total number of amino acids (SEQ ID NO: 2) of the bacteriocin encoded by Lactobacillus plantarum LP06 gene2233 is 57, the relative molecular mass is 6.24 kDa, the isoelectric point is 9.52, the number of positively charged residues is 10, the number of negatively charged residues is 7, and the molecular formula is C 273 H 443 N 81 O 84 S 1 ; The instability index (II) is 50.01. Since an index less than 40 indicates a stable protein and greater than 40 indicates an unstable protein, bacteriocin gene2233 is an unstable protein; the fat coefficient is 80.53, and the overall average hydrophilicity (GRAVY) is -0.479, which is a negative value, so bacteriocin gene2233 is a hydrophilic protein (Table 4-1).

[0163] The total number of amino acids (SEQ ID NO: 3) of the bacteriocin encoded by Lactobacillus plantarum LP06 gene2234 is 55, the relative molecular mass is 6.07 kDa, the isoelectric point is 9.99, the number of positively charged residues is 9, the number of negatively charged residues is 5, and the molecular formula is C 271 H 427 N 79 O 76 S2 ; The instability index (II) is 42.19. Since an index less than 40 indicates a stable protein and greater than 40 indicates an unstable protein, bacteriocin gene2234 is an unstable protein; the fat coefficient is 78.00, and the overall average hydrophilicity (GRAVY) is -0.291, which is a negative value, so bacteriocin gene2234 is a hydrophilic protein (Table 4-1).

[0164] The total number of amino acids (SEQ ID NO: 4) of the bacteriocin encoded by Lactobacillus plantarum LP06 gene2241 is 48, the relative molecular mass is 5.46 kDa, the isoelectric point is 10.48; the number of positively charged residues is 11, the number of negatively charged residues is 1; the molecular formula is C 247 H 413 N 67 O 63 S 4 ; The instability index (II) is 13.82. Since an index less than 40 indicates a stable protein and greater than 40 indicates an unstable protein, bacteriocin gene2241 is a stable protein; the fat coefficient is 75.21, and the overall average hydrophilicity (GRAVY) is -0.475, which is a negative value, so bacteriocin gene2241 is a hydrophilic protein (Table 4-2).

[0165] The total number of amino acids (SEQ ID NO: 5) of the bacteriocin encoded by Lactobacillus plantarum LP06 gene2246 is 52, the relative molecular mass is 5.73 kDa, the isoelectric point is 10.52, the number of positively charged residues is 8, the number of negatively charged residues is 3, and the molecular formula is C 259 H 406 N 78 O 68 S 1 ; The instability index (II) is 12.73. Since an index less than 40 indicates a stable protein and greater than 40 indicates an unstable protein, bacteriocin gene2246 is a stable protein; the fat coefficient is 90.00, and the overall average hydrophilicity (GRAVY) is -0.021, which is a negative value, so bacteriocin gene2246 is a hydrophilic protein (Table 4-2).

[0166] The total number of amino acids (SEQ ID NO: 6) of the bacteriocin encoded by Lactobacillus plantarum LP06 gene2247 is 56, the relative molecular mass is 6.19 kDa, the isoelectric point is 11.11; the number of positively charged residues is 11, the number of negatively charged residues is 2; the molecular formula is C 279 H 460 N 84 O 73 S 1; The instability index (II) is 19.85. Since the index is less than 40, it is a stable protein, and greater than 40, it is an unstable protein, so bacteriocin gene2247 is a stable protein; the fat coefficient is 95.71, and the overall average hydrophilicity (GRAVY) is -0.196, which is a negative value, so bacteriocin gene2247 is a hydrophilic protein (Table 4-3)

[0167] Table 4-1 Analysis of amino acid composition of bacteriocin of Lactobacillus plantarum LP06

[0168]

[0169]

[0170] Table 4-2 Analysis of amino acid composition of bacteriocin of Lactobacillus plantarum LP06

[0171]

[0172]

[0173] Table 4-3 Amino acid composition analysis of bacteriocin of Lactobacillus plantarum LP06

[0174]

[0175] d) Secondary and tertiary structure analysis results of Lactobacillus plantarum LP06 bacteriocin

[0176] In the bacteriocin encoded by gene2233 in Lactobacillus plantarum LP06, 29 amino acids are involved in α-helix structure, accounting for 50.88%, 10 amino acids are involved in chain extension, accounting for 17.54%, 3 amino acids are involved in β-turn, accounting for 5.26%, and 15 amino acids are involved in random coiling, accounting for 26.32% ( Fig.11 A). Among the bacteriocins encoded by gene2234, 27 amino acids participate in the α-helix structure, accounting for 49.09%, 4 amino acids participate in the extended chain, accounting for 7.27%, 9 amino acids participate in the β-turn, accounting for 16.36%, and 15 amino acids participate in the random coil, accounting for 27.27% ( Fig.11 B).

[0177] In the bacteriocin encoded by gene2241 in Lactobacillus plantarum LP06, 34 amino acids are involved in α-helix structure, accounting for 70.83%, 3 amino acids are involved in chain extension, accounting for 6.25%, 4 amino acids are involved in β-turn, accounting for 8.33%, and 7 amino acids are involved in random coiling, accounting for 14.58% ( Fig.11 C).

[0178] Among the bacteriocins encoded by gene2246, 28 amino acids participate in the α-helix structure, accounting for 53.85%, 9 amino acids participate in the extended chain, accounting for 17.31%, 4 amino acids participate in the β-turn, accounting for 7.69%, and 11 amino acids participate in the random coil, accounting for 21.15% ( Fig.11 D). Among the bacteriocins encoded by gene2247, 36 amino acids participate in the α-helix structure, accounting for 64.29%, 8 amino acids participate in the β-turn, accounting for 14.29%, and 12 amino acids participate in the random coil, accounting for 21.43% ( Fig.11 E).

[0179] The Ramachandran plot shows that the amino acids of bacteriocin gene2233, bacteriocin gene2234, bacteriocin gene2241, bacteriocin gene2246, and bacteriocin gene2247 fall in the dark area, accounting for more than 90% ( Fig.12 ), so the tertiary structure obtained by homology modeling is reasonable.

[0180] Example 6 Analysis of the antibacterial ability of Lactobacillus plantarum LP06 at different salt concentrations

[0181] Considering that some dairy products such as cheese and other foods are salted during production, the salt tolerance and antibacterial ability of Lactobacillus plantarum LP06 obtained in this study under different salt concentrations were analyzed. Lactobacillus plantarum LP06 was inoculated into liquid MRS medium containing 0%, 1%, 2%, and 3% NaCl at a ratio of 2%, and its OD value was measured after culturing at 37°C for 48 hours. There was no significant change in the OD value of the bacteria under different salt concentrations ( Fig.13 D) It proves that salt concentration of 1% to 3% has no effect on the growth of Lactobacillus plantarum LP06.

[0182] To further analyze whether different salt concentrations have an effect on the number of viable Lactobacillus plantarum, the bacterial solution cultured under different NaCl concentrations was taken by the drop count method and diluted to 10 -4 , 10 -5 5 μL of the dilution solution was taken for spot counting, and 3 groups were performed in parallel at each dilution factor. The two rows in the upper left corner are the bacterial solution diluted to 10 at 0% NaCl concentration. -4 , 10 -5 The two rows in the upper right corner show the bacterial solution diluted to 10% NaCl concentration. -4 , 10 -5 The two rows in the lower left corner show the bacterial solution diluted to 10% NaCl concentration. -4 , 10 -5 The two rows in the lower right corner show the bacterial solution diluted to 10% NaCl concentration.-4 , 10 -5 The colony growth after that showed that there was no significant difference in the number of viable bacteria of Lactobacillus plantarum in different NaCl concentrations ( Fig.13 C), which again proves that the salt concentration commonly used in cheese making (1% to 3%) has no effect on the growth of Lactobacillus plantarum LP06.

[0183] Using the double-layer plate streak method, two 2 cm long lines with a spacing of 1.5 cm were streaked on MRS agar plates containing 0%, 1%, and 3% NaCl, respectively. After culturing at 37°C for 48 hours, the upper plate containing Debaryomyces hansenii was poured. The differences in the inhibition zones of Lactobacillus plantarum LP06 in MRS agar plates containing different NaCl concentrations were compared. There was no significant difference in the inhibition zone of Lactobacillus plantarum LP06 against Debaryomyces hansenii on the medium with 1%-3% NaCl ( Fig.13 A, B). It was demonstrated that the maximum salt concentration (3%) required for cheese making had no effect on the antibacterial ability of Lactobacillus plantarum LP06.

[0184] Example 7 Optimization of culture medium for producing antibacterial powder from Lactobacillus plantarum LP06

[0185] As a biological agent used for food preservation, antibacterial powder is first required to be safe and edible as a production culture medium. Secondly, the ingredients in the culture medium are not conducive to the growth of spoilage bacteria or pathogens, but are conducive to the secretion of more antibacterial substances by antibacterial microorganisms.

[0186] The plant lactobacillus LP06 obtained in this study was isolated from dairy products, so skim milk was selected as the basic culture medium, and the effects of three culture medium formulas on the production of antibacterial substances by plant lactobacillus LP06 were compared. The first formula was 4% skim milk powder, 2% glucose, UPH 2 O water to 100mL; the second formula is 4% skim milk powder, 2% glucose, 1% yeast powder, UPH 2 O water to 100 mL; the third formula is 4% skim milk powder, 2% glucose, 1% yeast powder, 1% tryptone, UPH 2 O water to 100mL. The antibacterial ability of Lactobacillus plantarum LP06 was analyzed by double-layer plate streak method. Two lines of 2cm in length and 1.5cm in interval were drawn on the plate, LP06 was inoculated, and cultured at 37℃ for 48h. The upper plate containing Penicillium roqueforti was poured. When yeast powder and tryptone were added to the skim milk-based culture medium, it was more conducive to the growth of Penicillium roqueforti ( Fig.14 B, C), the medium with only glucose can make Lactobacillus plantarum LP06 produce antibacterial substances. Penicillium roqueforti grows weakly on the plate streaked with Lactobacillus plantarum LP06 and produces less pigment ( Fig.14A), indicating that Lactobacillus plantarum LP06 has a relatively good antibacterial effect on skim milk and glucose plates.

[0187] In order to verify the effect of the optimal skim milk plus glucose on the antibacterial effect of Lactobacillus plantarum LP06, Debaryomyces hansenii was selected as the indicator bacteria. The results showed that Lactobacillus plantarum LP06 had a significant inhibitory effect on Debaryomyces hansenii on the optimal culture medium ( Fig.14 D) Therefore, 4% skim milk and 2% glucose can be used as the culture medium formula for preparing antibacterial powder of Bacillus plantarum LP06.

[0188] Example 8 Genome characteristics and prediction of Lactobacillus plantarum LP06

[0189] 1. Genomic characteristics

[0190] The genome length of Lactobacillus plantarum LP06 is 3.29Mbp, and the G+C content is 44.38%. The prediction shows that Lactobacillus plantarum encodes a total of 3133 genes, encoding 63 tRNAs, 3 rRNAs and 45 sRNAs. In addition, other gene components were predicted, including 9 gene islands, 6 prophages and 2 potential CRISPR sequences (Table 5).

[0191] Table 5 Basic information of the genome of Lactobacillus plantarum LP06

[0192]

[0193] 2. Prediction of coding genes and interpretation of their functions

[0194] GO (Gene Ontology) is a classification system used to describe and annotate three types of gene functions, including: cellular component (CC), molecular function (MF) and biological process (BP). The results of GO annotation can reflect the relationship between genes and corresponding functions. The total number of coding genes of Lactobacillus plantarum LP06 annotated with GO is 2353 ( Fig.15 A), where the three major branches of GO show that in the BP category, the number of genes related to phosphorylation is the largest (102); in the CC category, the number of genes related to membrane components is the largest (745); in the MF category, the number of genes related to ATP synthesis is the largest (102).

[0195] KEGG (Kyoto Encyclopedia of Genes and Genomes) integrates data from genomes, chemical molecules, and biochemical systems, and can be used to systematically analyze the metabolic pathways of gene products and compounds in cells and the functions of these gene products. KEGG annotation showed that the coding genes of Lactobacillus plantarum LP06 were enriched in the KEGG pathway, with a total of 2012 genes annotated ( Fig.15 B), the largest number of genes were related to carbohydrate metabolism (1375).

[0196] COG (cluster of orthologous group) relies on matching unknown sequences with known proteins and annotating them as a certain type of COG, thereby inferring the biological function of the sequence. COG was used to annotate protein-encoding genes in the genome of Lactobacillus plantarum LP06. The results showed that there were 2433 functional categories that could be classified into COG ( Fig.15 C).

[0197] Example 9 Analysis of Carbohydrate Synthase Genes in the Genome of Lactobacillus plantarum LP06

[0198] Carbohydrates play an important role in many biological functions. By studying carbohydrate-related enzymes, a lot of meaningful biological information can be obtained. The antibacterial ability of lactic acid bacteria is closely related to the type and content of organic acids. The production of organic acids is inseparable from carbohydrate metabolism. Therefore, the analysis of carbohydrate-active enzyme-related genes is of great significance for revealing the differences in the production ability of organic acids in different lactic acid bacteria. The Carbohydrate Active Enzyme Database (CAZy, http: / / www.cazy.org / ) is a professional database about enzymes that synthesize or decompose complex carbohydrates and sugar complexes. According to the similarity of amino acid sequences in the protein domain, carbohydrate-active enzymes from different species can be divided into six major protein families: glycoside hydrolases (GHs), glycosyltransferases (GTs), polysaccharide lyases (PLs), carbohydrate esterases (CEs), carbohydrate-binding modules (CBMs), and auxiliary oxidoreductases (AAs).

[0199] LP06 has a total of 59 genes encoding glycoside hydrolases (GHs). The reason is that LP06 is a plant lactobacillus, and the carbohydrates it can utilize in its growth environment are grains, which shows its metabolic adaptability. There are 32 genes annotated to encode glycosyltransferases (GTs), which is less than glycoside hydrolases (GHs). There are 18 genes encoding carbohydrate esterases (CEs). Compared with GHs and GTs, the number of genes encoding CEs in the LP06 genome is relatively small. The small number of genes encoding CEs may be because lactic acid bacteria mainly grow and survive by fermenting carbohydrates such as lactose, and do not need a large number of CEs to degrade other carbohydrates. There are only 10 genes encoding auxiliary oxidoreductases (AAs) in LP06, which may be that auxiliary oxidoreductases (AAs) are usually involved in the electron transfer process in oxidative respiration, used to transfer electrons and produce three-way substrates to generate energy. Lactic acid bacteria are a type of Gram-positive facultative anaerobic bacteria. In an oxygen-free environment, lactic acid bacteria mainly produce energy through lactic acid fermentation and do not need oxidative respiration. Under such environmental influences, the demand for auxiliary oxidoreductases (AAs) is weak, so there are relatively few genes encoding auxiliary oxidoreductases (AAs) in their genomes ( Fig.16 ).

[0200] Example 10 Analysis of metabolic pathways for organic acid synthesis by Lactobacillus plantarum LP06

[0201] Starting from the typical organic acid metabolic pathways in lactic acid bacteria, we specifically analyze the genetic reasons why the strains produce certain organic acids. We mainly analyze the glycolysis pathway and the pyruvate pathway, and the organic acids analyzed include lactic acid (Lactate), acetic acid (Acetate), formic acid (Formate), fumaric acid (Fumarate), oxaloacetate (Oxaloacetate), and malic acid (Malate). These substances can inhibit the growth of microorganisms such as bacteria and fungi by affecting the metabolism, growth, and reproduction of microbial cells, and analyze the antibacterial mechanism of the strains. Fig.17 The process of the strain using glucose to produce organic acids through the glycolysis pathway and the pyruvate pathway is demonstrated. Pyruvate is the key intermediate metabolite in the production of organic acids in these two metabolic pathways. Other organic acids can be obtained through the catalytic reaction of pyruvate by various enzymes. It can be seen that the production and utilization of pyruvate affects the acid production capacity of the strain.

[0202] Example 11 Analysis of acid production by Lactobacillus plantarum LP06 via the pyruvate pathway

[0203] According to the annotation of KEGG database, the genome of Lactobacillus plantarum LP06 contains genes that completely encode key enzymes required for the production of related organic acids (Table 6). In lactic acid bacteria, the poxL gene encodes pyruvate oxidase (pyruvate oxidase [EC: 1.2.3.3]), which catalyzes pyruvate to produce acetyl phosphate, which is then catalyzed by acetate kinase to produce acetate. It can be inferred that Lactobacillus plantarum LP06 has the ability to produce formic acid, oxaloacetic acid, fumaric acid, and malic acid through the pyruvate pathway. There are 6 poxL genes on the genome of Lactobacillus plantarum 172-1, which has a strong ability to metabolize pyruvate during growth ( Fig.18 ).

[0204] Table 6 Related genes or gene clusters encoding antibacterial substances

[0205]

[0206]

[0207]

[0208] Example 12 Analysis of gene clusters for the synthesis of secondary metabolites of Lactobacillus plantarum LP06

[0209] The secondary metabolites secreted by lactic acid bacteria are another reason for their antibacterial ability. Studying the differences in the synthetic gene clusters of secondary metabolites of different lactic acid bacteria from the genome is of great significance for revealing the antibacterial mechanism of each strain. The synthetic gene clusters of secondary metabolites of samples can be predicted by antismash software.

[0210] The LP06 genome was used to predict the secondary metabolite synthesis gene clusters, including polyketide compounds (T3PKS), ribosome synthesis and post-translational modification peptides (RiPP-like), cyclic-lactone-autoinducer, Lanthipeptide-class IV, and terpene. Different strains will express different secondary metabolites to adapt to different survival pressures due to different growth environments. Only four secondary metabolite synthesis gene clusters, T3PKS, RiPP-like, cyclic-lactone-autoinducer, and terpene, were predicted in Lactobacillus plantarum LP06. The starting site of the cyclic-lactone-autoinducer gene cluster on the scaffold is about 197 kb, and the ending site of the gene cluster on the scaffold is 218 kb; the starting site of the ribosome synthesis and post-translational modification peptide (RiPP-like) gene cluster on the scaffold is about 91 kb, and the ending site of the gene cluster on the scaffold is 103 kb; the starting site of the polyketide compound (T3PKS) gene cluster on the scaffold is about 1 bp, and the ending site of the gene cluster on the scaffold is 26 kb; the starting site of the terpene gene cluster on the scaffold is about 98 kb, and the ending site of the gene cluster on the scaffold is 110 kb (Table 7).

[0211] Table 7 Analysis of gene clusters for secondary metabolite synthesis

[0212]

[0213]

[0214] 1. T3PKS Analysis of Lactobacillus plantarum LP06

[0215] Type III polyketide synthase is a class of enzymes that catalyze the synthesis of polyketides and belongs to the polyketide synthase (PKS) family. Type III polyketide synthase plays an important role in the biosynthesis of natural products, such as the synthesis of antibiotics and plant secondary metabolites. The study of type III polyketide synthase is of great significance for revealing the biosynthesis process of natural products and synthesizing new drugs and compounds. In recent years, scientists have successfully synthesized a variety of biologically active compounds using the characteristics of type III polyketide synthase, providing new ideas and methods for drug research and development.

[0216] The core biosynthetic gene of all predicted secondary metabolite biosynthetic gene clusters encodes hydroxymethylglutaryl-CoA synthase (EC:2.3.3.10), which is sensitive to feedback substrate inhibition by acetoacetyl-CoA and is also a metabolite of fatty acid catabolism, with the potential to block the growth of pathogenic microorganisms during food fermentation.

[0217] The T3PKS secondary metabolite synthesis gene cluster predicted in the LP06 genome contains 29 genes. There are genes encoding the transport part of the transport protein and biosynthetic genes. This shows that LP06 can both synthesize secondary metabolites and transport the secondary metabolites produced ( Fig.19 ).

[0218] 2. Terpene Analysis of Lactobacillus plantarum LP06

[0219] During the fermentation process, lactic acid bacteria produce some terpenes, which have antibacterial, antioxidant, and anti-inflammatory effects. They can enhance human immunity and regulate intestinal flora. Common terpenes produced by lactic acid bacteria include β-sitosterol, β-carotene, γ-sitosterol, etc. These substances can be ingested by eating foods containing lactic acid bacteria, which is beneficial to health. In Lactobacillus plantarum LP06, there is a regulatory gene perR, which encodes a transcriptional repressor protein ( Fig.19 ).

[0220] 3. RiPP-like analysis of Lactobacillus plantarum

[0221] RiPP-like secondary metabolite biosynthesis gene clusters in lactic acid bacteria are a group of genes encoding enzymes and regulatory elements involved in the synthesis of a certain RiPP-like secondary metabolite. These gene clusters usually include promoters, structural genes, and regulatory genes, which work together to synthesize and regulate the production of the final product.

[0222] RiPPs (modified peptide products) are a class of natural products, which are characterized by chemical modification of peptides by modifying enzymes to form products with biological activity. The RiPP-like secondary metabolite synthesis gene clusters found in lactic acid bacteria may be involved in different biological activities, such as antibacterial and anti-tumor, and have potential application prospects. Studying the RiPP-like secondary metabolite synthesis gene clusters in lactic acid bacteria not only helps to gain a deeper understanding of the biosynthetic ability and metabolic pathways of lactic acid bacteria, but also helps to discover and develop new bioactive products with medicinal or industrial application value.

[0223] LP06 predicted that the core biosynthetic gene blpAg of the RiPP-like secondary metabolite synthesis gene cluster was annotated as encoding a protein related to peptide modification or transport (peptide cleavage / export ABCtransporter). The gene encoding plant lactobacillus E / F was annotated in the secondary metabolite synthesis gene cluster in LP06. Plant lactobacillus E / F belongs to the second class of bacteriocins. This bacteriocin has strong antibacterial activity only when two polypeptides are present at the same time. The antibacterial activity of a certain peptide alone is weak. The genes encoding plant lactobacillus E and F in LP06 exist adjacent to each other ( Fig.19 ).

[0224] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, ordinary technicians in the field should understand that the specific implementation methods of the present invention can still be modified or some technical features can be replaced by equivalents without departing from the spirit of the technical solution of the present invention, which should be included in the scope of the technical solution for protection of the present invention.

Claims

1. A strain of Lactiplantibacillus plantarum LP06 comprising a 16S rRNA nucleic acid having at least 98% identity to the nucleic acid shown in SEQ ID NO:

1.

2. The Lactobacillus plantarum LP06 of claim 1, wherein the 16S rRNA nucleic acid has at least 99% identity to the nucleic acid shown in SEQ ID NO:

1.

3. The Lactobacillus plantarum LP06 of claim 2, wherein the 16S rRNA nucleic acid has a nucleic acid sequence as shown in SEQ ID NO:

1.

4. The Lactobacillus plantarum LP06 according to any one of claims 1 to 3, which has a deposit number of CGMCC No. 28412.

5. A culture comprising the Lactobacillus plantarum LP06 according to any one of claims 1 to 4 and an optional culture medium; Preferably, the culture medium comprises skim milk and glucose, preferably the weight percentage of the skim milk is 1%-15% (e.g., 1%-10%, 2-7%, 4%), and preferably the weight percentage of the glucose is 0.5%-8% (e.g., 0.5%-6%, 1%-3%, 2%); Preferably, the Lactobacillus plantarum LP06 is a living bacterium; Preferably, the culture comprises bacteriocin Plantaricin EF and bacteriocin Plantaricin JK; Preferably, the culture comprises bacteriocin, bacteriocin Plantaricin EF and bacteriocin Plantaricin JK, wherein, The amino acid sequence of the bacteriocin is shown in SEQ ID NO:4, the bacteriocin Plantaricin EF is composed of plantaricin EF subunit PlnE shown in SEQ ID NO:6 and plantaricin EF subunit PlnF shown in SEQ ID NO:5, and the bacteriocin Plantaricin JK is composed of plantaricin JK subunit PlnJ shown in SEQ ID NO:3 and plantaricin JK subunit PlnK shown in SEQ ID NO:

2. A culture supernatant, which is the culture supernatant according to claim 5.

7. A composition or antibacterial agent, comprising the Lactobacillus plantarum LP06 (preferably viable bacteria) according to any one of claims 1 to 4, or the culture according to claim 5, or the culture supernatant according to claim 6; Preferably, the composition or bacteriostatic agent is a solid bacteriostatic agent or a liquid bacteriostatic agent, preferably a solid bacteriostatic agent (such as bacteriostatic powder).

8. The composition or antibacterial agent according to claim 7, which is prepared by a method comprising the following operations: 1) inoculating the Lactobacillus plantarum LP06 according to any one of claims 1 to 4 into a culture medium, 2) Fermentation; Preferably, the culture medium in step 1) comprises skim milk and glucose, preferably the weight percentage of the skim milk is 1%-15% (e.g., 1%-10%, 2-7%, 4%), and preferably the weight percentage of the glucose is 0.5%-8% (e.g., 0.5%-6%, 1%-3%, 2%); Preferably, step 2) has one or two of the following technical features: ① The fermentation temperature is 36-38°C (e.g. 37°C), ② The fermentation time is 60-90h (e.g. 65-85h, 70-80h, 72h).

9. A method for preparing the composition or antibacterial agent according to claim 7 or 8, comprising the following steps: 1) inoculating the Lactobacillus plantarum LP06 according to any one of claims 1 to 4 into a culture medium, 2) Fermentation; Preferably, the culture medium in step 1) comprises skim milk and glucose, preferably the weight percentage of the skim milk is 1%-15% (e.g., 1%-10%, 2-7%, 4%), and preferably the weight percentage of the glucose is 0.5%-8% (e.g., 0.5%-6%, 1%-3%, 2%); Preferably, step 2) has one or two of the following technical features: ① The fermentation temperature is 36-38°C (e.g. 37°C), ② The fermentation time is 60-90h (e.g. 65-85h, 70-80h, 72h).

10. Use of the Lactobacillus plantarum LP06 according to any one of claims 1 to 4, the culture according to claim 5, or the culture supernatant according to claim 6 in the preparation of an antibacterial product, preferably, in the preparation of a product for inhibiting fungi and bacteria; Preferably, the fungus is selected from Candida tropicalis, Debaryomyces hansenii and Penicillium roqueforti; Preferably, the bacteria is selected from the group consisting of Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella, Listeria monocytogenes, Escherichia coli and Staphylococcus aureus.

11. Use of Lactobacillus plantarum LP06 according to any one of claims 1 to 4, the culture according to claim 5, the culture supernatant according to claim 6, or the composition or antibacterial agent according to claim 7 or 8 in a product (e.g., food, medicine), preferably the product is food, preferably the food is a dairy product (e.g., yogurt, cheese, milk powder, milk-containing beverage).

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