Phytobacterium plantarum YW99 and preparation method and application of cell lysate of phytobacterium plantarum YW99

Abstract

The invention relates to the field of microorganisms, in particular to a plant lactobacillus YW99 strain and a preparation method and application of a cell lysate of the plant lactobacillus YW99 strain. The lactobacillus plantarum is lactobacillus plantarum YW99, the classification name of the lactobacillus plantarum is lactobacillus plantarum, the lactobacillus plantarum is preserved in China General Microbiological Culture Collection Center (CGMCC) on December 23, 2024, and the preservation number of the lactobacillus plantarum is CGMCC No.33160. The lactobacillus plantarum is a lactobacillus plantarum strain. The phytobacterium plantarum YW99 cell lysate is prepared by heating and inactivating phytobacterium plantarum YW99 and then carrying out a series of operations, has good safety, does not generate toxic and side effects on human bodies, has a certain function of improving the immunity of the organism, can show different degrees of bacteriostatic ability to escherichia coli, staphylococcus aureus and pseudomonas aeruginosa, and has a good application prospect. The method has a good application prospect in the fields of food, agriculture and the like.

Description

Technical Field

[0001] The invention relates to the field of microorganisms, and in particular to a strain of Lactobacillus plantarum YW99 and a preparation method and application of a cell lysate thereof. Background Art

[0002] Pathogenic bacteria are bacteria that cause human diseases. They are tiny and can enter the human body through a variety of pathways, parasitize, proliferate and cause diseases in the human body. Common foodborne pathogens include Escherichia coli, Salmonella and Staphylococcus aureus. They use food as a transmission medium, directly or indirectly contaminate food and water sources, and cause food poisoning or infectious diseases through oral infection, which is also an important cause of food safety problems.

[0003] The existing natural antibacterial agents for preventing and eliminating foodborne pathogens are relatively limited. In the agricultural field, antibiotics are widely used as agricultural feed additives to prevent animal diseases and promote growth. However, excessive use can cause serious problems such as increased bacterial resistance, posing a potential threat to human health and the ecological environment. The use of antibiotics has been expressly prohibited. In the food field, with the increasingly stringent global food safety standards, the application of traditional chemical synthetic antibacterial agents is subject to many restrictions. For example, the sodium deoxyacetate antibacterial agent commonly used in daily staple foods such as bread and noodles has been required to be replaced due to its potential health risks.

[0004] Natural antibacterial agents are mainly derived from animal, plant, and microbial extracts, etc. They are safer for the human body and meet green environmental protection requirements. However, the natural antibacterial and antibacterial substances available on the market for Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa are still very limited. For example, although nisin has certain antibacterial effects, its antibacterial spectrum is relatively narrow. It mainly inhibits Gram-positive bacteria, but cannot inhibit Gram-negative bacteria and yeast. This not only restricts the high-quality development of various industries, but also fails to meet the increasingly diversified market needs. Therefore, in the fields of feed, food, etc., there is an urgent need for a new natural, safe, broad-spectrum antibacterial, and efficient alternative substance 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 strain of Lactobacillus plantarum YW99 and a cell lysate of Lactobacillus plantarum YW99.

[0006] The first object of the present invention is to provide a strain of Lactobacillus plantarum, which is Lactobacillus plantarum YW99, classified and named Lactiplantibacillus plantarum , deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on December 23, 2024, with the deposit number CGMCC No.33160.

[0007] The second object of the present invention is to provide a Lactobacillus plantarum YW99 cell lysate, wherein the Lactobacillus plantarum YW99 cell lysate is prepared from Lactobacillus plantarum YW99 with a deposit number of CGMCC No.33160, and the Lactobacillus plantarum YW99 cell lysate at least satisfies the following conditions: (1) at least 10 10 CFU / mL of inactivated Lactobacillus plantarum YW99 bacteria; (2) including metabolites of Lactobacillus plantarum YW99, lactic acid content ≥22mg / 100g, and acetic acid content ≥5mg / 100g; (3) including flavonoids, metabolites of Lactobacillus plantarum YW99, with a flavonoid content ≥52mg / g; (4) including amino acids, metabolites of Lactobacillus plantarum YW99, with an amino acid content ≥70µmol / mL; (5) including polysaccharides, metabolites of Lactobacillus plantarum YW99, with a polysaccharide content ≥700mg / L.

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

[0009] Furthermore, when the cell lysate of Lactobacillus 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 2 hours, the material thickness is 1.0 cm-1.5 cm, the freeze drying time is 20 hours-30 hours, and 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 volume is 40m 3 / h, the inlet temperature was 75°C, the outlet temperature was 40°C, and the obtained solid powder was YW99 cell lysate.

[0010] The fourth object of the present invention is to provide a use of Lactobacillus plantarum YW99 and / or Lactobacillus plantarum YW99 cell lysate in the preparation of a product having the function of improving the immune function of an organism.

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

[0012] Furthermore, the above-mentioned antibacterial function includes at least one of the following: (1) inhibiting the growth of Escherichia coli; (2) inhibiting the growth of Staphylococcus aureus; (3) inhibiting the growth of Pseudomonas aeruginosa.

[0013] Furthermore, the above-mentioned products are foods, and the foods include one or more of pre-prepared dishes, pre-prepared pasta, baked products, and dairy products.

[0014] Furthermore, the above-mentioned products having the function of improving the body's immune function, and the products having the function of inhibiting bacteria and / or having the function of preserving and preserving are agricultural products, and the agricultural products are pet snacks and animal feed.

[0015] Compared with the prior art, the present invention has the following advantages: (1) The present application provides a strain of Lactobacillus plantarum YW99, which is isolated and screened from Tibetan kefir grains. It is a probiotic that has been proven to have a certain effect of improving the body's immunity and has a strong antibacterial function against Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa. It has certain application value in food and agricultural products.

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

[0017] (3) Based on the function of Lactobacillus plantarum YW99 cell lysate, this application applies Lactobacillus plantarum YW99 or Lactobacillus plantarum YW99 cell lysate to the agricultural and food fields, providing the market with a natural and highly safe option for the human body. Compared with chemical antibacterial and antimicrobial drugs and antibiotics, it has the characteristics of being natural, safe and non-toxic, and having good biocompatibility, and meets green environmental protection requirements, so it has a wider range of applications; compared with traditional probiotic preparations, Lactobacillus plantarum YW99 cell lysate is non-pathogenic and its metabolites are released more completely. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a colony morphology diagram of the YW99 strain in Example 1 of the present application on MRS medium; Figure 2 This is a cell morphology diagram of the YW99 strain in Example 1 of the present application; Figure 3This is a schematic diagram of the comparison of the 16S rDNA sequencing results of the YW99 strain in Example 1 of the present application; Figure 4 The antibacterial effect of the YW99 cell lysate pre-prepared dish during storage in Experimental Example 3 of this application; Figure 5 This is a diagram showing the antibacterial effect of the YW99 cell lysate of Experimental Example 1 of this application on Escherichia coli; Figure 6 This is a diagram showing the antibacterial effect of the YW99 cell lysate of Experimental Example 1 of this application on Staphylococcus aureus; Figure 7 This is a diagram showing the antibacterial effect of the YW99 cell lysate of Experimental Example 1 of this application on Pseudomonas aeruginosa;

[0019] The plant lactobacillus of the present invention is: plant lactobacillus YW99, the preservation date is December 23, 2024, the preservation unit is the General Microbiology Center of the China Microbiological Culture Collection Administration, and the classification name is plant lactobacillus ( Lactiplantibacillus plantarum ), the deposit number is CGMCC No.33160. DETAILED DESCRIPTION

[0020] 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 solution of the present invention, and cannot be used to limit the scope of protection of the present invention. Anything not described in detail in the present patent application can be understood as common knowledge in the art.

[0021] Pathogenic bacteria use various substances as transmission media, directly or indirectly contaminate food, water sources, and people's daily necessities, causing food poisoning or infectious diseases, thereby affecting human health. However, the existing agents for preventing and eliminating pathogenic bacteria are relatively limited. Antibiotics in the agricultural field and chemical synthetic antibacterial agents in the food field are gradually subject to increasingly stringent restrictions on their use due to their many negative effects on people's 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 intestinal flora, enhancing immunity, promoting nutrient absorption, lowering blood sugar, anti-oxidation, and reducing disease risks. Lactic acid bacteria cell lysate is a preparation of inanimate microorganisms and / or their components that are beneficial to host health. It mainly includes metabolites of probiotic bacteria, as well as soluble substances produced after the bacteria are eliminated and decomposed, such as bacteriocins, antimicrobial peptides, organic acids, and extracellular polysaccharides. Compared with probiotics, it is more stable and safer, and is also suitable for some special populations such as newborns and sensitive populations, and is not interfered with 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, feed, etc., to provide a new, natural, safe, broad-spectrum antibacterial, and highly effective alternative to antibacterial and antimicrobial products, and to promote industrial upgrading.

[0022] Unless otherwise specified, the experimental methods in the following examples are all conventional methods; the experimental materials and culture media used in the following examples are all purchased from commercial channels unless otherwise specified.

[0023] 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, 1.0 g / L Tween-80, distilled water to 1,000 mL, solid culture medium with 2% agar, pH 6.2-6.4, autoclaved at 115°C for 15 min.

[0024] Example 1 Isolation, identification and preservation of YW99 strain 1. Isolation of YW99 strain A small amount of Tibetan kefir grains was thoroughly ground with 2 mL of saline in a sterile mortar to form a uniform liquid. -3 , 10 -4 , 10 -5The three gradient homogenates were spread on MRS solid medium and cultured in a 37°C incubator for 1-2 days. Single colonies with obvious characteristics were picked out and purified by streaking until colonies with consistent morphology and size were isolated, numbered, and the colony morphology and cell morphology were observed. They were inoculated into MRS liquid medium with an inoculation loop, cultured at 37°C for 48 hours, and stored in glycerol.

[0025] Colony morphology Figure 1 As shown; cell morphology Figure 2 As shown, it can be clearly seen that YW99 is a bacillus.

[0026] 2. Physiological and biochemical tests of YW99 strain YW99 strain is G + Bacillus, a catalase-negative bacteria, was subjected to physiological and biochemical tests using the Merri API 50 CH identification system to study the catabolism of 48 different fermentable carbohydrates. The changes in the organic acids produced and the pH caused the indicator to change color, and different fermentation effects were observed. The experimental results are shown in Table 1. The carbon sources that the YW99 strain can utilize are D-galactose, D-glucose, D-fructose, D-mannose, esculin, maltose, lactose, sucrose, etc.; based on cell morphology and physiological and biochemical characteristics, with reference to the Bergey's Manual of Bacterial Identification and the Classification and Identification of Lactic Acid Bacteria and Experimental Methods, the lactic acid bacteria YW99 strain was preliminarily identified as Lactobacillus plantarum ( Lactiplantibacillus plantarum ).

[0027] Table 1 Carbohydrate fermentation test results of strain YW99

[0028] In Table 1, +: positive; -: negative 3. Molecular biological identification of YW99 strain The genomic DNA of YW99 strain was extracted, and the genomic extraction kit was purchased from Shanghai Biotech Co., Ltd. The 16SrDNA gene fragment of YW99 strain was amplified by PCR using the universal primers 27F and 1492R (Table 2). The PCR amplification product was shown in SEQ ID No. 1. The PCR amplification product was sent to Tianjin Jinweizhi Biotechnology Co., Ltd. for sequencing, and the sequencing results were compared with the Genbank database of NCBI. The comparison results are shown in Figure 3 As shown, it was found that the strain with the highest homology to the YW99 strain was Lactobacillus plantarum. Therefore, the YW99 strain was identified as Lactobacillus plantarum.

[0029] Table 2 PCR amplification primers for YW99 strain

[0030] 4. Storage of YW99 strain The identified YW99 strain was preserved and classified as: Lactobacillus plantarum ( Lactiplantibacillus plantarum ), strain number is YW99, which was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on December 23, 2024, with the deposit number CGMCC No.33160, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

[0031] The following examples are performance and application studies of Lactobacillus plantarum YW99, and the YW99 strain mentioned throughout the article refers to this strain.

[0032] Example 2 YW99 cell lysate and preparation method The preparation method of Lactobacillus plantarum YW99 cell lysate is as follows: S1, inoculating the Lactobacillus plantarum YW99 identified in Example 1 into a solid MRS culture medium, and culturing it at 20°C-40°C and aerobic conditions for 24h-48h to obtain an activated strain of YW99; S2, inoculating the activated strain of YW99 into a liquid MRS culture medium at an inoculum amount of 1%-10%, and culturing it at 20°C-40°C and aerobic conditions for 12h-48h to obtain a YW99 fermentation liquid; S3, placing the YW99 fermentation liquid at a temperature of 80°C-121°C to inactivate , centrifuge to obtain the supernatant, post-treat the supernatant to obtain Lactobacillus plantarum YW99 cell lysate, the Lactobacillus plantarum YW99 cell lysate can be in liquid or powdered solid state, when the Lactobacillus plantarum YW99 cell lysate is in powdered solid state, the post-treatment can be freeze drying or spray drying; specifically, the pre-freezing temperature of freeze drying is -80°C, the pre-freezing time is 2h, the material thickness is 1.0cm-1.5cm, and the freeze drying time is 20h-30h; or spray drying is adopted, under the condition that the solid content is less than 30%, the air intake volume is 40m 3 / h, inlet temperature is 75℃, outlet temperature is 40℃.

[0033] This example provides a YW99 cell lysate and a specific preparation method: S1. Inoculate Lactobacillus plantarum YW99 into solid MRS medium and culture at 30° C. under aerobic conditions for 36 h to obtain an activated strain of YW99; S2, inoculating the activated strain YW99 into liquid MRS medium at an inoculum rate of 4%, and culturing at 30° C. under aerobic conditions for 36 h to obtain YW99 fermentation broth; S3. Inactivate the YW99 fermentation broth at 80°C for 15 min, centrifuge at 8000 rpm / min for 15 min, take the supernatant, and freeze-dry the supernatant to obtain the Lactobacillus plantarum YW99 cell lysate. The pre-freezing temperature for freeze-drying is -80°C, the pre-freezing time is 2 h, the material thickness is 1.0 cm, and the freeze-drying time is 20 h.

[0034] The pH of the YW99 cell lysate obtained was 4.0, and the concentration of inactivated Lactobacillus plantarum YW99 cells was 10 10 CFU / mL, and component detection revealed that YW99 cell lysate contained 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.

[0035] Table 3 Content of components in YW99 cell lysate

[0036] In other specific embodiments, the content of YW99 cell lysate and metabolite content at least meets the following requirements: inactivated Lactobacillus plantarum YW99 cells ≥ 10 10 CFU / mL, lactic acid ≥22mg / 100g, acetic acid ≥5mg / 100g, flavonoids ≥52mg / g, amino acids ≥70µmol / mL, polysaccharides ≥700mg / L. In other specific embodiments, the YW99 cell lysate may also contain the above metabolites alone or simultaneously to meet different application requirements.

[0037] Example 3 Application of YW99 cell lysate in improving body immunity Lactobacillus plantarum YW99 and / or Lactobacillus plantarum YW99 cell lysate have certain applications in improving the body's immunity. In some embodiments, Lactobacillus plantarum YW99 and / or Lactobacillus plantarum YW99 cell lysate can be used to prepare products that improve the body's immune function; in some embodiments, the product can be an agricultural product, such as adding Lactobacillus plantarum YW99 and / or YW99 cell lysate to pet snacks and animal feed to improve the immunity of small pets such as rodents, poultry, or large livestock such as cattle and horses.

[0038] Experimental Example 1 Determination of the performance of YW99 cell lysate in improving the body's immune function This embodiment provides an application of a YW99 cell lysate in agricultural products, specifically involving using the YW99 cell lysate prepared in Example 2 as an additive to animal feed, while observing its effect on the immune function of the animal.

[0039] The experiment used 40 7-week-old SPF male BALB / c mice, and intraperitoneal injection of cyclophosphamide (CTX) was used to establish an immunosuppressive model in mice. After a 7-day adaptation period, the BALB / c mice were randomly divided into 4 groups (normal feed group as blank control, model group, positive group, and test group), with 10 mice in each group. The mice in the normal feed group, model group, positive group, and test group were intraperitoneally injected with 80 mg / kg of CTX on days 1-3, 18, and 22 of the experiment, and the normal feed group was given an equal amount of normal saline. On days 4-28, the positive group was continuously gavaged with 0.2 mL of levamisole hydrochloride (100 mg / kg), and the test group was continuously gavaged with YW99 cell lysate at a rate of 100 mg / kg of feed.

[0040] After 28 days, the thymus index and spleen index of mice were tested and it was found that compared with the blank group (thymus index 1.63mg / g±0.30mg / g), the thymus index of mice in the model group was significantly decreased (0.77mg / g±0.10mg / g), and the spleen index showed a significant increase (6.34mg / g±0.33mg / g); compared with the model group, both the positive group and the test group had recovered thymus index, which were 1.43mg / g±0.15mg / g and 1.45mg / g±0.35mg / g, respectively, and the thymus index of the post-test group was significantly higher than that of the model group; compared with the model group (6.34mg / g±0.33mg / g), the spleen index of the positive group and the test group decreased, which were decreased by 19.9% ​​and 9.1%, respectively, which were significant effects (p<0.01). The above results indicate that YW99 cell lysate can improve the abnormal thymus index and spleen index of mice induced by CTX to a certain extent, and has an immunoprotective effect on CTX-treated mice.

[0041] After 28 days, the spleen lymphocyte index of mice was detected and it was found that the experimental group had an effect on the proliferation of spleen T lymphocytes. The results showed that compared with the blank group (1.0±0.15), the stimulation index of the model group (0.54±0.03) was significantly reduced, down 0.46±0.12. After intervention with YW99 cell lysate, the stimulation index of T lymphocytes was significantly increased, which was 75.5% higher than that of the model group. The results showed that YW99 cell lysate can promote the proliferation and differentiation of T lymphocytes. The effect on the proliferation of spleen B lymphocytes was also found that compared with the blank group (1.0±0.11), the stimulation index of the model group (0.54±0.04) was extremely significantly reduced, down 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 showed that YW99 cell lysate can promote the proliferation and differentiation of spleen B lymphocytes.

[0042] In summary, YW99 cell lysate has significant immune activation effect on splenic lymphocytes, and can effectively promote the proliferation of splenic lymphocytes, thereby participating in the body's more extensive cellular immune response activities.

[0043] Example 4 Application of YW99 cell lysate in the preparation of antiseptic and fresh-keeping products Lactobacillus plantarum YW99 and / or Lactobacillus plantarum YW99 cell lysate have certain applications in the preparation of products with antibacterial function and / or antiseptic and fresh-keeping function. In some specific embodiments, Lactobacillus plantarum YW99 and / or Lactobacillus plantarum YW99 cell lysate can be used to be directly or indirectly added to food to inhibit the growth of foodborne pathogenic bacteria Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa, such as for preparing food, including but not limited to pre-prepared dishes, pre-prepared pasta, fresh meat, baked pastries, fruits and vegetables, and dairy products; in other specific embodiments, Lactobacillus plantarum YW99 and / or YW99 cell lysate can also be added to agricultural products, such as pet snacks, animal feed, etc., to improve the ability of agricultural products to inhibit foodborne pathogenic bacteria Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa.

[0044] The present embodiment provides an application of YW99 cell lysate in food, specifically involving the application of the YW99 cell lysate prepared in Example 2 to prepared aquatic products, aiming to explore the inhibitory effect of YW99 cell lysate on microorganisms during food storage. This embodiment is implemented in accordance with GB19295-2021 and SB / T10648 national standards and industry standards.

[0045] The prepared aquatic food is a semi-finished fish meat, and the preparation process specifically includes: S1. Pretreatment of fresh fish: Select fresh fish such as silver carp and crucian carp, slaughter and clean them until the blood and impurities on the surface of the fish are cleaned, cut into pieces for later use, and each piece weighs 20g-30g; S2, cooking processing: ginger, onion, black pepper, cooking wine, and water are prepared into a marinating liquid in a certain proportion, the fish pieces are immersed in the marinating liquid, and marinated at room temperature for 30 minutes to 60 minutes; the marinated fish pieces are immersed in oil for frying, the frying temperature is 150° C. to 180° C., and the frying time is 110 seconds to 130 seconds; S3. Deodorizing and seasoning: weigh bean paste, star anise powder, soy sauce, soybean oil, salt, monosodium glutamate, pepper, ginger, cinnamon, oyster sauce, and soy sauce in a certain proportion, add oil first, and when the temperature of the oil reaches a certain level, add onion, ginger, and pepper, add the remaining ingredients, add appropriate amount of water, heat to boil, and prepare a sauce; when the temperature of the sauce drops below 60°C, add YW99 cell lysate in an amount of 5% of the weight of the sauce to the seasoning, stir evenly, and put the fried fish pieces into the sauce for seasoning; S4. Vacuum packaging: put the processed fish pieces and sauce into food-grade high-temperature cooking bags at a rate of 100 g per bag, vacuum seal the bags, and store them at 4°C after microwave sterilization.

[0046] The microbial content of semi-finished fish meat at different times was tested, and the sauce without YW99 cell lysate was used as the control group. The results are as follows Figure 4 As shown in the figure, the number of colonies on the frozen fish pieces added with YW99 cell lysate was much smaller, indicating that YW99 cell lysate can effectively reduce the number of total bacteria in food, especially after 20 days of storage, the total number of colonies was 2.5*10 2 cfu / g, compared with the control group, it can effectively reduce the content of microorganisms, extend the storage period of the product, and improve the quality and safety of the product.

[0047] Test Example 2 Determination of antibacterial properties of YW99 cell lysate In this test example, the antibacterial performance of the YW99 cell lysate prepared in Example 2 was determined.

[0048] Take activated Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa respectively, and prepare a concentration of 1×10 8 CFU / mL of bacterial liquid, 100 μL of each bacterial liquid was spread on the corresponding solid culture medium, and holes were punched on the solid culture medium with a puncher with an inner diameter of 6 mm, with 4 or 5 holes per dish, of which 2 wells were used as positive control (+: 50 μg / mL ampicillin) and negative control (-: saline water). The YW99 cell lysate prepared in Example 2 was punched into the wells at a content of 100 μL per well, and cultured at a certain temperature for a period of time, and the diameter of the inhibition zone was measured with a vernier caliper. The analysis results are as follows.

[0049] 1. Detection of the inhibitory ability of YW99 cell lysate against Escherichia coli by the inhibition zone method The solid culture medium is LB agar medium: peptone 10.0g / L, yeast extract powder 5.0g / L, sodium chloride 5.0g / L, distilled water to 1000mL, pH value 7.0, autoclaved at 121℃ for 20min. Each dish has 5 wells and cultured at 37℃ for 16h-18h. The results are as follows Figure 5As shown, the diameter of the inhibition zone was 2.3 cm ± 0.2 cm, indicating that the YW99 cell lysate had an inhibitory effect on Escherichia coli.

[0050] 2. Detection of the inhibitory ability of YW99 cell lysate against Staphylococcus aureus by the inhibition zone method The solid culture medium was LB agar medium, with 5 wells per dish, and cultured at 37°C for 16h-18h. Figure 6 As shown, the diameter of the inhibition zone was 1.9 cm ± 0.2 cm, indicating that the YW99 cell lysate had an inhibitory effect on Staphylococcus aureus.

[0051] 3. Detection of the inhibitory ability of YW99 cell lysate against Pseudomonas aeruginosa by the inhibition zone method The solid culture medium was LB agar medium, with 5 wells per dish, and cultured at 37°C for 16h-18h. Figure 7 As 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.

[0052] Test Example 3 Determination of Minimum Inhibitory Concentration (MIC) of YW99 Cell Lysate In this test example, the minimum inhibitory concentration (MIC) of the YW99 cell lysate prepared in Example 2 was determined.

[0053] The microbroth dilution method was used in accordance with the "Health Industry Standard of the People's Republic of China" (WS / T 639-2018). 6 CFU / mL of Escherichia coli (ATCC8739) and Staphylococcus aureus (ATCC6538) were added to a 96-well plate at an addition amount of 100 μL / well, and a culture medium containing 0.85% saline was used as the control group. The experimental groups were: the YW99 cell lysate prepared in Example 2; the YW99 cell lysate prepared in Example 2 was diluted with 0.85% sterile saline to a concentration of 1:10 (V / V), 1:20 (V / V), and 1:40 (V / V). The incubation conditions were incubated at 25°C-40°C for 16h-24h. After the incubation, the turbidity of the liquid in the 96-well plate was observed to determine the minimum concentration of lactic acid bacteria cell lysate that inhibited the visible growth of pathogenic bacteria as MIC.

[0054] The minimum inhibitory concentration of YW99 cell lysate against different pathogenic bacteria was obtained by MIC experiment. The results showed that compared with the growth control group, the minimum inhibitory concentrations of YW99 cell lysate against Escherichia coli and Staphylococcus aureus were 0.05g / mL and 0.05g / mL, respectively.

[0055] The above is based on the ideal embodiment of the present invention. Through the above description, relevant personnel can make various changes and modifications without departing from the technical concept of the present invention. The technical scope of the present 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 strain of Lactobacillus plantarum, characterized in that The plant lactobacillus is plant lactobacillus YW99, which is classified and named Lactiplantibacillus plantarum , deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on December 23, 2024, with the deposit number CGMCC No.33160.

2. A Lactobacillus plantarum YW99 cell lysate, characterized in that: The Lactobacillus plantarum YW99 cell lysate is prepared from the Lactobacillus plantarum YW99 with a deposit number of CGMCC No. 33160, and the Lactobacillus plantarum YW99 cell lysate satisfies at least one of the following conditions: (1) At least 10 10 CFU / mL of inactivated Lactobacillus plantarum YW99 bacteria; (2) Lactic acid and acetic acid, metabolites of Lactobacillus plantarum YW99, wherein the lactic acid content is ≥22 mg / 100 g and the acetic acid content is ≥5 mg / 100 g; (3) Flavonoids comprising metabolites of Lactobacillus plantarum YW99, wherein the content of the flavonoids is ≥ 52 mg / g; (4) Amino acids comprising metabolites of Lactobacillus plantarum YW99, wherein the amino acid content is ≥ 70 µmol / mL; (5) A polysaccharide comprising a metabolite of Lactobacillus plantarum YW99, wherein the content of the polysaccharide is ≥ 700 mg / L.

3. A method for preparing a Lactobacillus plantarum YW99 cell lysate, characterized in that: include: Inoculate Lactobacillus plantarum YW99 in a solid MRS medium, and culture at 20°C-40°C under aerobic conditions for 24h-48h to obtain an activated strain of YW99; The activated strain YW99 is inoculated into a liquid MRS medium at an inoculation rate of 1%-10%, and cultured at 20° C.-40° C. under aerobic conditions for 12 h-48 h to obtain a YW99 fermentation broth; The YW99 fermentation broth is placed at 80° C.-121° C. for inactivation, the supernatant is obtained by centrifugation, and the supernatant is post-treated to obtain Lactobacillus plantarum YW99 cell lysate.

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

5. Use of the Lactobacillus plantarum YW99 according to claim 1 and / or the cell lysate of Lactobacillus plantarum YW99 according to claim 2 in the preparation of a product capable of improving the immune function of an organism.

6. Use of the Lactobacillus plantarum YW99 according to claim 1 and / or the cell lysate of Lactobacillus plantarum YW99 according to claim 2 in preparing products with antibacterial function and / or antiseptic and fresh-keeping function.

7. The use according to claim 6, characterized in that: The antibacterial function includes at least one of the following: (1) Inhibit the growth of Escherichia coli; (2) Inhibit the growth of Staphylococcus aureus; (3) Inhibit the growth of Pseudomonas aeruginosa.

8. The use according to claim 6, characterized in that: The product is food, which includes one or more of pre-prepared dishes, pre-prepared pasta, baked products, and dairy products.

9. The use according to claim 5 or 6, characterized in that: The products are agricultural products, and the agricultural products are pet snacks and animal feed.

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