A strain of Lactiplantibacillus plantarum YW99 and a preparation method and application of its cell lysate

Abstract

The present invention relates to the field of microorganisms. Specifically, it relates to a Lactiplantibacillus plantarum YW99 and a preparation method and application of its cell lysate. The Lactiplantibacillus plantarum is Lactiplantibacillus plantarum YW99, and its taxonomic name is Lactiplantibacillus plantarum Lactiplantibacillus plantarum , which was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on December 23, 2024, with the deposit number of CGMCC No. 33160. The cell lysate of Lactiplantibacillus plantarum YW99 is prepared by a series of operations after heat-inactivation of Lactiplantibacillus plantarum YW99. It has good safety, will not produce toxic and side effects on the human body, has a certain function of improving the body's immunity, and can show different degrees of antibacterial ability against Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa. It has good application prospects in the fields of food, agriculture, etc.

Description

Technical Field

[0001] The present invention relates to the field of microorganisms, and more specifically, to a Lactiplantibacillus plantarum YW99 and a preparation method and application of its cell lysate. Background Art

[0002] With the improvement of people's living standards, people not only satisfy their basic needs of food and clothing, but also pay increasing attention to health. Pathogenic bacteria are bacteria that cause human diseases. They are tiny in size and can enter the human body through various channels, parasitize, multiply and cause diseases in the human body. Common foodborne pathogenic bacteria mainly include Escherichia coli, Salmonella, and Staphylococcus aureus. They use food as a transmission medium to directly or indirectly contaminate food and water sources, and cause food poisoning or infectious diseases through oral infection. They are also an important cause of food safety problems.

[0003] Existing natural bacteriostatic agents for preventing and eliminating foodborne pathogenic bacteria are relatively limited. In the agricultural field, antibiotics are widely used as agricultural feed additives to prevent animal diseases and promote growth. However, overuse will cause serious problems such as enhanced bacterial drug resistance, posing a potential threat to human health and the ecological environment. Currently, the use of antibiotics has been explicitly prohibited. In the food field, with the increasingly strict global food safety standards, the application of traditional chemically synthesized bacteriostatic agents is restricted. Taking daily staples such as bread and noodles as an example, the commonly used bacteriostatic agent sodium dehydroacetate in the past has been compulsorily required to be replaced due to its potential health risks.

[0004] Natural bacteriostatic agents mainly come from animal, plant, microbial extracts, etc., which are safer for the human body and meet the requirements of green environmental protection. However, the natural antibacterial and bacteriostatic substances available on the market for Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa are still very limited. For example, nisin has certain bacteriostatic effects, but its antibacterial spectrum is relatively narrow, mainly inhibiting Gram-positive bacteria, but not Gram-negative bacteria and yeasts. This not only restricts the high-quality development of various industries, but also cannot meet the increasingly diverse market demands. Therefore, there is an urgent need for a new natural, safe, broad-spectrum antibacterial and highly efficient alternative substance in the fields of feed, food, etc. to promote industrial upgrading. Summary of the Invention

[0005] In view of the deficiencies in the above-mentioned prior art, the present application provides a Lactiplantibacillus plantarum YW99 and a cell lysate of Lactiplantibacillus plantarum YW99.

[0006] The first object of the present invention is to provide a Lactiplantibacillus plantarum, which is Lactiplantibacillus plantarum YW99, and its taxonomic name is Lactiplantibacillus plantarum, it was deposited in the China General Microbiological Culture Collection Center on December 23, 2024, with the deposit number CGMCC No. 33160.

[0007] The second object of the present invention is to provide a cell lysate of Lactiplantibacillus plantarum YW99, which is prepared from Lactiplantibacillus plantarum YW99 with the deposit number CGMCC No. 33160.

[0008] The third object of the present invention is to provide a method for preparing a cell lysate of Lactiplantibacillus plantarum YW99, including: inoculating Lactiplantibacillus plantarum YW99 into a solid MRS medium, culturing at 20°C - 40°C under aerobic conditions for 24h - 48h to obtain an activated YW99 strain; inoculating the activated YW99 strain into a liquid MRS medium at an inoculation amount of 1% - 10%, culturing at 20°C - 40°C under aerobic conditions for 12h - 48h to obtain a YW99 fermentation broth; inactivating the YW99 fermentation broth at a temperature of 80°C - 121°C, centrifuging to obtain the supernatant, and post-treating the supernatant to obtain the cell lysate of Lactiplantibacillus plantarum YW99.

[0009] Further, when the cell lysate of Lactiplantibacillus plantarum YW99 is a solid, the post-treatment adopts at least one of the following operations: (1) Freeze-drying: The pre-freezing temperature is -80°C, the pre-freezing time is 2h, the material thickness is 1.0 cm - 1.5 cm, and the freeze-drying time is 20h - 30h. The obtained solid powder is the YW99 cell lysate; (2) Spray-drying: Under the condition that the solid content is less than 30%, the air intake is 40m 3 / h, the inlet temperature is 75°C, and the outlet temperature is 40°C. The obtained solid powder is the YW99 cell lysate.

[0010] The fourth object of the present invention is to provide the use of Lactiplantibacillus plantarum YW99 and / or the cell lysate of Lactiplantibacillus plantarum YW99 in the preparation of a product with improved body immune function. Further, the above product with improved body immune function is an agricultural product, and the agricultural product is a pet snack or animal feed.

[0011] The fifth object of the present invention is to provide the use of Lactiplantibacillus plantarum YW99 and / or the cell lysate of Lactiplantibacillus plantarum YW99 in the preparation of a product with antibacterial function and / or with anti-corrosion and fresh-keeping function.

[0012] Further, the above antibacterial function is at least: (1) inhibiting the growth of Escherichia coli; (2) inhibiting the growth of Staphylococcus aureus; (3) inhibiting the growth of Pseudomonas aeruginosa.

[0013] Further, the above-mentioned product with antibacterial function is an agricultural product, and the agricultural product is a pet snack or animal feed. Further, the above-mentioned product with anti-corrosion and freshness preservation function can be a food or an agricultural product. The food includes one or more of prefabricated dishes, prefabricated pasta, baked products, and dairy products. The agricultural product is a pet snack or animal feed.

[0014] Compared with the prior art, the present invention has the following advantages:

[0015] (1) The present application provides a Lactiplantibacillus plantarum YW99 strain, which is isolated and screened from Tibetan kefir grains. It is a probiotic, and it has been experimentally confirmed to have a certain effect of improving the body's immunity. It also has a strong antibacterial function against Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa, and has certain application value.

[0016] (2) The present application provides a Lactiplantibacillus plantarum YW99 cell lysate, which is prepared by heat inactivation. The preparation method is simple and efficient. The prepared Lactiplantibacillus plantarum YW99 cell lysate has a good inhibitory effect on the growth of pathogenic bacteria such as Escherichia coli and Staphylococcus aureus. Moreover, this preparation method can increase the concentration of effective substances in the Lactiplantibacillus plantarum YW99 cell lysate and enhance the antibacterial effect of the Lactiplantibacillus plantarum YW99 cell lysate against Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa.

[0017] (3) Based on the function of the Lactiplantibacillus plantarum YW99 cell lysate, the present application applies Lactiplantibacillus plantarum YW99 or the Lactiplantibacillus plantarum YW99 cell lysate to the agricultural field and the food field, providing a natural and highly safe choice for the market. Compared with chemical antibacterial and antifungal drugs and antibiotic agents, it has the characteristics of being natural, safe and non-toxic, and having good biocompatibility, meeting the requirements of green environmental protection, so it has a wider scope of application; compared with traditional probiotic preparations, the Lactiplantibacillus plantarum YW99 cell lysate has no pathogenicity and the metabolite release is more complete. Description of the Drawings

[0018] Figure 1 It is a colony morphology diagram of the YW99 strain in the MRS medium in Example 1 of the present application;

[0019] Figure 2 It is a cell morphology diagram of the YW99 strain in Example 1 of the present application;

[0020] Figure 3 It is a schematic diagram of the comparison of the 16S rDNA sequencing results of the YW99 strain in Example 1 of the present application;

[0021] Figure 4The antibacterial effect of the YW99 cell lysate during the storage of prefabricated dishes in Test Example 3 of this application;

[0022] Figure 5 The antibacterial effect diagram of the YW99 cell lysate of Test Example 1 of this application against Escherichia coli;

[0023] Figure 6 The antibacterial effect diagram of the YW99 cell lysate of Test Example 1 of this application against Staphylococcus aureus;

[0024] Figure 7 The antibacterial effect diagram of the YW99 cell lysate of Test Example 1 of this application against Pseudomonas aeruginosa.

[0025] The Lactiplantibacillus plantarum described in the present invention is: Lactiplantibacillus plantarum YW99, with the deposit date of December 23, 2024, deposited with the China General Microbiological Culture Collection Center, and classified and named as Lactiplantibacillus plantarum ( Lactiplantibacillus plantarum ), and the deposit number is CGMCC No. 33160. Detailed implementation manners

[0026] The present invention will be further described below in conjunction with specific embodiments. The following embodiments are only used to more clearly illustrate the technical solutions of the present invention and cannot be used to limit the protection scope of the present invention. Those not described in detail in this patent application of the present invention can be understood as common general knowledge in the art.

[0027] Pathogenic bacteria use various substances as media to directly or indirectly contaminate food, water sources, and people's daily necessities, causing food poisoning or infectious diseases in people, thus affecting human health. However, the existing agents for preventing and eliminating pathogenic bacteria are relatively limited. Antibiotics in the agricultural field, chemical synthetic antibacterial agents in the food field, etc., are gradually subject to more and more strict usage restrictions due to their many negative impacts on people's physical health. Probiotics are widely used in the fields of food, health products, etc. A large number of studies have shown that probiotics have a variety of probiotic functions, such as regulating the intestinal flora, enhancing immunity, promoting nutrient absorption, reducing blood sugar, antioxidant, reducing the risk of diseases, etc. As a preparation of inanimate microorganisms and / or their components beneficial to the health of the host, lactic acid bacteria cell lysates mainly include the metabolites of probiotic cells and the soluble substances produced after the cells are eliminated and decomposed, such as bacteriocins, antibacterial peptides, organic acids, and exopolysaccharides, etc. Moreover, compared with probiotics, they are more stable, have higher safety, are also suitable for some special populations such as newborns and sensitive populations, and are not interfered or inhibited by antibiotics. Therefore, this application aims to use probiotics or lactic acid bacteria cell lysates in multiple industries such as food, health products, and feed, providing a new, natural, safe, broad-spectrum antibacterial, and highly efficient antibacterial and bacteriostatic product alternative idea to promote the upgrading of the industry.

[0028] Unless otherwise specified, the experimental methods in the following examples are all conventional methods; unless otherwise specified, the test materials and culture media used in the following examples are all purchased from commercial channels.

[0029] MRS medium: Glucose 20.0 g / L, peptone 10.0 g / L, beef extract 10.0 g / L, yeast extract 5.0 g / L, sodium acetate 5.0 g / L, diammonium hydrogen citrate 2.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.05 g / L, Tween-80 1.0 g / L, made up to 1,000 mL with distilled water. For solid medium, 2% agar is added. The pH value is 6.2 - 6.4, and it is autoclaved at 115 °C for 15 min.

[0030] Example 1 Isolation, identification and preservation of strain YW99

[0031] 1. Isolation of strain YW99

[0032] A small amount of Tibetan kefir grains and 2 mL of normal saline were thoroughly ground in a sterile mortar to form a homogeneous liquid. According to the 10-fold gradient dilution method, 10 -3 、10 -4 、10 -5 The three gradient homogenates were respectively spread on MRS solid medium and cultured in an incubator at 37 °C for 1 - 2 days. The single colonies with obvious characteristics were picked out and purified by streaking until colonies with consistent morphology and size were isolated. They were numbered and the colony morphology and cell morphology were observed. Then they were inoculated into MRS liquid medium with an inoculation loop and cultured at 37 °C for 48 h, and then preserved with glycerol.

[0033] The colony morphology is as shown in Figure 1 ; the cell morphology is as shown in Figure 2 It can be clearly seen that YW99 is a bacillus.

[0034] 2. Identification of strain YW99 by physiological and biochemical tests

[0035] Strain YW99 is a G + bacillus and a catalase-negative strain. The API 50 CH identification system of Merieux was used for physiological and biochemical tests to study the catabolism of 48 different fermentable carbohydrates. The changes in the organic acids produced and the pH led to the color change of the indicator, and thus different fermentation effects were observed. The experimental results are shown in Table 1. The carbon sources that strain YW99 can utilize are D-galactose, D-glucose, D-fructose, D-mannose, esculin, maltose, lactose, sucrose, etc.; according to the cell morphology and physiological and biochemical characteristics, referring to "Bergey's Manual of Systematic Bacteriology" and "Classification and Identification of Lactic Acid Bacteria and Experimental Methods", it was preliminarily determined that lactic acid bacteria strain YW99 is Lactiplantibacillus plantarum ( Lactiplantibacillus plantarum).

[0036] Table 1 Results of carbohydrate fermentation test of YW99 strain

[0037]

[0038] In Table 1, +: positive; -: negative

[0039] 3. Identification of YW99 strain by molecular biology techniques

[0040] Genomic DNA of YW99 strain was extracted. The genomic extraction kit was purchased from Sangon Biotech (Shanghai) Co., Ltd. Using the extracted genomic DNA as a template, the 16S rDNA gene fragment of YW99 strain was amplified by PCR with universal primers 27F and 1492R (Table 2), and the PCR amplification product was shown in SEQ ID No.1. The PCR amplification product was sent to Tianjin Genewiz Biotechnology Co., Ltd. for sequencing, and the sequencing results were compared in the Genbank database of NCBI. The comparison results were as Figure 3 shown. It was found that the strain with the highest homology to YW99 strain was Lactiplantibacillus plantarum. Therefore, YW99 strain was identified as Lactiplantibacillus plantarum.

[0041] Table 2 PCR amplification primers of YW99 strain

[0042]

[0043] 4. Preservation of YW99 strain

[0044] The identified YW99 strain was preserved, and the taxonomic name was: Lactiplantibacillus plantarum ( Lactiplantibacillus plantarum ), the strain number was YW99. It was preserved in the China General Microbiological Culture Collection Center on December 23, 2024, with the preservation number CGMCC No. 33160, and the preservation address was No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

[0045] Subsequent examples were the performance research and application research of Lactiplantibacillus plantarum YW99, and all YW99 strains described throughout the article referred to this bacterium.

[0046] Example 2 YW99 cell lysate and preparation method

[0047] The preparation method of Lactiplantibacillus plantarum YW99 cell lysate is as follows: S1. Inoculate the identified Lactiplantibacillus plantarum YW99 in Example 1 into solid MRS medium, and culture it at 20°C - 40°C under aerobic conditions for 24h - 48h to obtain the activated YW99 strain; S2. Inoculate the activated YW99 strain into liquid MRS medium at an inoculation amount of 1% - 10%, and culture it at 20°C - 40°C under aerobic conditions for 12h - 48h to obtain the YW99 fermentation broth; S3. Inactivate the YW99 fermentation broth at a temperature of 80°C - 121°C, centrifuge to obtain the supernatant, and post-treat the supernatant to obtain the Lactiplantibacillus plantarum YW99 cell lysate. The Lactiplantibacillus plantarum YW99 cell lysate can be in liquid or powder solid state. When the Lactiplantibacillus plantarum YW99 cell lysate is in powder solid state, the post-treatment can be freeze-drying or spray-drying; specifically, the pre-freezing temperature for freeze-drying is -80°C, the pre-freezing time is 2h, the material thickness is 1.0 cm - 1.5 cm, and the freeze-drying time is 20h - 30h; or by spray-drying, under the condition that the solid content is less than 30%, the air intake is 40m 3 / h, the inlet temperature is 75°C, and the outlet temperature is 40°C.

[0048] This example provides a YW99 cell lysate and a specific preparation method:

[0049] S1. Inoculate Lactiplantibacillus plantarum YW99 into solid MRS medium, and culture it at 30°C under aerobic conditions for 36h to obtain the activated YW99 strain;

[0050] S2. Inoculate the activated YW99 strain into liquid MRS medium at an inoculation amount of 4%, and culture it at 30°C under aerobic conditions for 36h to obtain the YW99 fermentation broth;

[0051] S3. Inactivate the YW99 fermentation broth at 80°C for 15 min, centrifuge at 8000 rpm / min for 15 min to obtain the supernatant, and freeze-dry the supernatant to obtain the Lactiplantibacillus plantarum YW99 cell lysate. The pre-freezing temperature for freeze-drying is -80°C, the pre-freezing time is 2h, the material thickness is 1.0 cm, and the freeze-drying time is 20h.

[0052] The obtained YW99 cell lysate has a pH of 4.0, and the inactivated cell concentration of Lactiplantibacillus plantarum YW99 is 10 10 CFU / mL. Component detection shows that the YW99 cell lysate contains metabolites such as lactic acid, acetic acid, flavonoids, amino acids, and polysaccharides. The content detection results of the main metabolites are shown in Table 3.

[0053] Table 3 Component content in YW99 cell lysate

[0054]

[0055] In other specific embodiments, the contents of the bacterial cells and metabolites of the YW99 cell lysate both satisfy at least: inactivated Lactiplantibacillus plantarum YW99 cells ≥ 10 10 CFU / mL, lactic acid ≥ 22 mg / 100 g, acetic acid ≥ 5 mg / 100 g, flavonoids ≥ 52 mg / g, amino acids ≥ 70 μmol / mL, polysaccharides ≥ 700 mg / L. In some other specific embodiments, the above-mentioned metabolites may be contained alone or simultaneously in the YW99 cell lysate to meet different application requirements.

[0056] Example 3 Application of YW99 cell lysate in enhancing body immunity

[0057] Lactiplantibacillus plantarum YW99 and / or the YW99 cell lysate of Lactiplantibacillus plantarum have certain applications in enhancing body immunity. In some specific embodiments, Lactiplantibacillus plantarum YW99 and / or the YW99 cell lysate of Lactiplantibacillus plantarum can be used to prepare products with enhanced body immune function; in some specific embodiments, the products can be agricultural products, such as adding Lactiplantibacillus plantarum YW99 and / or the YW99 cell lysate to pet snacks and animal feeds to enhance the immunity of small rodent pets, poultry, or large livestock such as cattle and horses.

[0058] Test Example 1 Performance determination of YW99 cell lysate in enhancing body immune function

[0059] This embodiment provides an application of the YW99 cell lysate in agricultural products, specifically involving using the YW99 cell lysate prepared in Example 2 as an additive to assist animal feed, and simultaneously observing its effect on the immune function of animals.

[0060] Forty 7-week-old SPF male BALB / c mice were used in the experiment, and an immunosuppression model of mice was established by intraperitoneal injection of cyclophosphamide (CTX). After a 7-day adaptation period, the BALB / c mice were randomly divided into 4 groups (the normal diet group as the blank control, the model group, the positive group, and the test group), with 10 mice in each group. The mice in the normal diet group, the model group, the positive group, and the test group were intraperitoneally injected with 80 mg / kg of CTX on days 1-3, 18, and 22 of the experiment, and the normal diet group was given an equal amount of physiological saline. From days 4-28, the positive group was continuously intragastrically administered levamisole hydrochloride (100 mg / kg) at 0.2 mL, and the test group continuously intragastrically administered the YW99 cell lysate to the mice at a dose of 100 mg / kg of feed.

[0061] After 28 days, the thymus index and spleen index of mice were measured and found that: compared with the blank group (thymus index 1.63 mg / g ± 0.30 mg / g), the thymus index of the model group mice decreased significantly (0.77 mg / g ± 0.10 mg / g), and at the same time, the spleen index showed a significant increase (6.34 mg / g ± 0.33 mg / g); compared with the model group, the thymus index of both the positive group and the experimental group recovered, being 1.43 mg / g ± 0.15 mg / g and 1.45 mg / g ± 0.35 mg / g respectively, and the thymus index of the latter experimental group was significantly higher than that of the model group; compared with the model group (6.34 mg / g ± 0.33 mg / g), the spleen indexes of the positive group and the experimental group both decreased, by 19.9% and 9.1% respectively, both being significant effects (p < 0.01). The above results indicate that the YW99 cell lysate can, to a certain extent, improve the abnormalities of the thymus index and spleen index of mice induced by CTX, and has an immune protective effect on mice treated with CTX.

[0062] After 28 days, the spleen lymphocyte index of mice was measured and found that: the results of the effect of the experimental group on the proliferation of splenic T lymphocytes showed that compared with the stimulation index of the blank group (1.0 ± 0.15), the stimulation index of the model group (0.54 ± 0.03) decreased significantly, by 0.46 ± 0.12. After intervention with the YW99 cell lysate, the stimulation index of T lymphocytes increased significantly, increasing by 75.5% compared with the model group. The results indicate that the YW99 cell lysate can promote the proliferation and differentiation of T lymphocytes. The effect on the proliferation of splenic B lymphocytes was also found: compared with the blank group (1.0 ± 0.11), the stimulation index of the model group (0.54 ± 0.04) decreased extremely significantly, by 0.46 ± 0.12 (p < 0.01); compared with the model group, the stimulation index of the experimental group increased by 52.4% compared with the model group. The results indicate that the YW99 cell lysate can promote the proliferation and differentiation of splenic B lymphocytes.

[0063] In summary, the YW99 cell lysate has a significant immune activation effect on spleen lymphocytes, can effectively promote the proliferation process of spleen lymphocytes, and thus participates in the more extensive cellular immune response activities of the body.

[0064] Example 4 Application of YW99 cell lysate in the preparation of anti-corrosion and freshness-preserving products

[0065] Lactiplantibacillus plantarum YW99 and / or the cell lysate of Lactiplantibacillus plantarum YW99 have certain applications in the preparation of products with antibacterial function and / or preservation function. In some other specific embodiments, Lactiplantibacillus plantarum YW99 and / or the YW99 cell lysate can also be added to agricultural products, such as pet snacks, animal feeds, etc., to improve the inhibitory ability of agricultural products against foodborne pathogenic bacteria Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa.

[0066] This example provides an application of the YW99 cell lysate in food, specifically involving applying the YW99 cell lysate prepared in Example 2 to prefabricated aquatic food products, aiming to explore the inhibitory effect of the YW99 cell lysate on microorganisms during food storage. This example is carried out in accordance with the national standards and industry standards of GB19295—2021 and SB / T10648.

[0067] The prefabricated aquatic food product is a semi-finished fish meat, and the preparation process specifically includes:

[0068] S1. Fresh fish pretreatment: Select fresh fish such as silver carp and crucian carp, slaughter them and wash them until the blood and impurities on the fish body surface are washed clean, cut them into pieces for standby, and each piece weighs 20g - 30g;

[0069] S2. Cooking process: Prepare a pickling solution by mixing ginger, scallions, black pepper powder, cooking wine, and water in a certain proportion, immerse the fish pieces in the pickling solution, and marinate them at room temperature for 30min - 60min; Immerse the marinated fish pieces in oil for frying, with the frying temperature at 150℃ - 180℃ and the frying time at 110s - 130s;

[0070] S3. Debittering and flavoring: Weigh the broad bean paste, star anise powder, soy sauce, soybean oil, salt, monosodium glutamate, Chinese prickly ash, ginger, cinnamon, oyster sauce, and soy sauce in a certain proportion. First, add the oil, and when the oil temperature reaches a certain level, add scallions, ginger, and Chinese prickly ash, then add the remaining ingredients, add an appropriate amount of water, and heat to boiling to prepare the flavoring sauce; When the temperature of the flavoring sauce drops below 60℃, add the YW99 cell lysate to the flavoring agent in an amount of 5% of the mass of the flavoring sauce, stir well, and put the fried fish pieces into the flavoring sauce for flavoring;

[0071] S4. Vacuum packaging: Put the processed fish pieces and flavoring sauce into food-grade high-temperature cooking bags at 100g per bag for vacuum sealing, and store them at 4℃ after microwave sterilization.

[0072] Detect the microbial content of the semi-finished fish meat at different times, using the sauce without adding the YW99 cell lysate as the control group, and the results are as Figure 4As shown, there are fewer colony counts on the frozen fish blocks added with YW99 cell lysate, indicating that YW99 cell lysate can effectively reduce the total number of bacteria in food. Especially after 20 days of storage, the total number of colonies is 2.5*10 2 cfu / g, which can effectively reduce the microbial content compared to the control group, extend the storage period of the product, and improve the quality and safety of the product.

[0073] Test Example 2 Determination of the antibacterial performance of YW99 cell lysate

[0074] In this test example, the antibacterial performance of the YW99 cell lysate prepared in Example 2 was determined.

[0075] Activated Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa were separately taken and prepared into bacterial suspensions with a concentration of 1×10 8 CFU / mL. 100 μL of each bacterial suspension was spread on the corresponding solid medium. A punch with an inner diameter of 6 mm was used to punch holes on the solid medium, with 4 or 5 holes per petri dish. Among them, 2 holes were used as positive controls (+: 50 μg / mL ampicillin) and negative controls (-: physiological saline). The YW99 cell lysate prepared in Example 2 was injected into the holes at a content of 100 μL per hole, cultured at a certain temperature for a period of time, and the diameter of the antibacterial zone was measured with a vernier caliper. The analysis results are as follows.

[0076] 1. Detection of the inhibitory ability of YW99 cell lysate against Escherichia coli by the antibacterial zone method

[0077] The solid medium was LB agar medium: peptone 10.0 g / L, yeast extract powder 5.0 g / L, sodium chloride 5.0 g / L, made up to 1000 mL with distilled water, pH value 7.0, autoclaved at 121 °C for 20 min. There were 5 holes per petri dish and cultured at 37 °C for 16 h - 18 h. The results are as Figure 5 shown. The diameter of the antibacterial zone was 2.3 cm ± 0.2 cm, indicating that YW99 cell lysate has an inhibitory effect on Escherichia coli.

[0078] 2. Detection of the inhibitory ability of YW99 cell lysate against Staphylococcus aureus by the antibacterial zone method

[0079] The solid medium was LB agar medium, with 5 holes per petri dish, and cultured at 37 °C for 16 h - 18 h. The results are as Figure 6 shown. The diameter of the antibacterial zone was 1.9 cm ± 0.2 cm, indicating that YW99 cell lysate has an inhibitory effect on Staphylococcus aureus.

[0080] 3. Detection of the inhibitory ability of YW99 cell lysate against Pseudomonas aeruginosa by the antibacterial zone method

[0081] The solid medium was LB agar medium, with 5 wells per petri dish, and cultured at 37 °C for 16 h - 18 h. The results were as Figure 7 shown. The diameter of the inhibition zone was 1.5 cm ± 0.06 cm, indicating that the YW99 cell lysate had an inhibitory effect on Pseudomonas aeruginosa.

[0082] Test Example 3 Determination of the minimum inhibitory concentration (MIC) of YW99 cell lysate

[0083] In this test example, the minimum inhibitory concentration (MIC) of the YW99 cell lysate prepared in Example 2 was determined.

[0084] The microbroth dilution method was adopted and carried out in accordance with the "National Health Industry Standard of the People's Republic of China" (WS / T 639 - 2018). Bacterial suspensions of Escherichia coli (ATCC8739) and Staphylococcus aureus (ATCC6538) with a concentration of 1×10 6 CFU / mL were added to a 96 - well plate at an addition amount of 100 μL / well, and the medium containing 0.85% normal saline was used as the control group. The experimental groups were as follows: the undiluted YW99 cell lysate prepared in Example 2; the YW99 cell lysate prepared in Example 2 was successively diluted with 0.85% sterile normal saline to concentrations of 1:10 (V / V), 1:20 (V / V), and 1:40 (V / V). The incubation conditions were incubation at 25 °C - 40 °C for 16 h - 24 h. After the incubation, the turbidity of the liquid in the 96 - well plate was observed to determine the lowest concentration at which the lactic acid bacteria cell lysate inhibited the visible growth of pathogenic bacteria, which was the MIC.

[0085] Through the MIC experiment, the minimum inhibitory concentrations of the YW99 cell lysate against different pathogenic bacteria were obtained. The results showed that compared with the growth control group, the minimum inhibitory concentrations of the YW99 cell lysate against Escherichia coli and Staphylococcus aureus were 0.05 g / mL and 0.05 g / mL, respectively.

[0086] Based on the ideal embodiments of the present invention as inspiration, through the above description, relevant personnel can completely make various changes and modifications without departing from the technical idea of this invention. The technical scope of this invention is not limited to the content in the specification, and the technical scope must be determined according to the scope of the claims.

Claims

1. A Lactobacillus plantarum, characterized in that, The Lactiplantibacillus plantarum is Lactiplantibacillus plantarum YW99, and its taxonomic name is Lactiplantibacillus plantarum , which was deposited in the China General Microbiological Culture Collection Center on December 23, 2024, with the deposit number of CGMCC No. 33160.

2. A method for preparing a cell lysate of Lactiplantibacillus plantarum YW99, characterized in that, Comprising: Inoculating Lactiplantibacillus plantarum YW99 with the preservation number of CGMCC No. 33160 described in claim 1 into a solid MRS medium, and culturing at 20°C - 40°C under aerobic conditions for 24h - 48h to obtain an activated strain of YW99; Inoculating the activated strain of YW99 into a liquid MRS medium at an inoculation amount of 1% - 10%, and culturing at 20°C - 40°C under aerobic conditions for 12h - 48h to obtain a fermentation broth of YW99; Inactivating the fermentation broth of YW99 at a temperature of 80°C - 121°C, centrifuging to obtain the supernatant, and post-treating the supernatant to obtain a cell lysate of Lactiplantibacillus plantarum YW99.

3. The preparation method according to claim 2, wherein, When the cell lysate of Lactiplantibacillus plantarum YW99 is a solid, the post-treatment adopts at least one of the following operations: (1) Freeze-drying: The pre-freezing temperature is -80°C, the pre-freezing time is 2h, the material thickness is 1.0 cm - 1.5 cm, and the freeze-drying time is 20h - 30h. The obtained solid powder is the cell lysate of YW99; (2) Spray drying: Under the condition that the solid content is less than 30%, the intake air volume is 40 m 3 / h, the inlet temperature is 75 °C, and the outlet temperature is 40 °C. The obtained solid powder is the YW99 cell lysate.

4. A cell lysate of Lactiplantibacillus plantarum YW99 prepared by the preparation method described in claim 2 or 3.

5. Use of Lactobacillus plantarum YW99 as claimed in claim 1 and / or the cell lysate of Lactobacillus plantarum YW99 as claimed in claim 4 in the preparation of a product with enhanced immune function of the body, characterized in that, The product is an agricultural product, and the agricultural product is a pet snack or animal feed.

6. Use of the Lactiplantibacillus plantarum YW99 described in claim 1 and / or the cell lysate of Lactiplantibacillus plantarum YW99 described in claim 4 in the preparation of a product with antibacterial function, wherein the antibacterial function is to inhibit the growth of Escherichia coli, Staphylococcus aureus or Pseudomonas aeruginosa.

7. The application according to claim 6, characterized in that The product is an agricultural product, and the agricultural product is a pet snack, animal feed.

8. Use of the Lactiplantibacillus plantarum YW99 described in claim 1 and / or the cell lysate of Lactiplantibacillus plantarum YW99 described in claim 4 in the preparation of a product with anti-corrosion and preservation function, and the product is one of the following: (1) Food, and the food is prefabricated dishes, prefabricated pasta, baked products or dairy products; (2) Agricultural products, and the agricultural products are pet snacks or animal feeds.

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