A new type of biological preservative and its application

Abstract

The present invention relates to the technical field of antiseptic and fresh-keeping, and in particular to a novel biological preservative and its application. Specifically, the present invention mixes pectin and extracellular vesicles of Lactobacillus plantarum LPL-1 in proportion to prepare a biological preservative and fresh-keeping agent. The biological preservative and fresh-keeping agent provided by the present invention can effectively inhibit Gram-positive pathogens such as Listeria monocytogenes and Staphylococcus aureus and Gram-negative pathogens such as Pseudomonas; it can be used for food preservation and antiseptic, is a natural biological preservative, and can effectively extend the shelf life. Compared with traditional lactic acid bacteria bacteriocins, the extracellular vesicles produced by Lactobacillus plantarum can be used as a natural carrier of bacteriocin substances, and more bacteriocin substances can be enriched in the extracellular vesicles. The structure and function of the extracellular vesicles are more stable, not affected by external factors, and easier to carry out large-scale industrial production.

Description

Technical Field

[0001] The present invention relates to the technical field of antiseptic and fresh-keeping, and in particular to a novel biological antiseptic and fresh-keeping agent and application thereof. Background Art

[0002] Food safety is not only a critical control point in the food industry but is also closely related to human health. Food spoilage caused by foodborne pathogens is a major factor affecting food safety. Preservatives are the primary means of controlling spoilage caused by foodborne pathogens and spoilage bacteria. Although chemical preservatives are widely used, their safety is a major concern. Biological preservatives are easily degraded by proteases in the human body and offer advantages such as safety, non-toxicity, and a lack of residues. However, their low activity, high cost, and low production of bacteriocins have led to their limited application in food preservation. Therefore, discovering highly active bacteriocins and their producing bacteria, and using molecular biology techniques to develop protein expression platforms to increase the production and antimicrobial activity of biological preservatives and reduce fermentation costs are important research areas in food safety and industry.

[0003] It is well known that lactic acid bacteria and their active metabolites are closely related to human health. They are often intentionally introduced into food products or occur naturally in food, thus imparting the flavor, texture, nutritional, and health characteristics that people desire. Lactic acid bacteria bacteriocins are a class of peptides or precursor peptides with antibacterial activity synthesized and secreted into the environment by lactic acid bacteria during metabolism. They have strong antagonistic properties against the growth of other microorganisms, and their antibacterial properties are not limited to homologous bacteria. The protein properties of bacteriocins allow them to be degraded in the human body, making them non-toxic and residue-free. They are also highly effective, acid-resistant, heat-resistant, and drug-resistant. Most genes are located on plasmids, have a small molecular weight, contain modified amino acids, and have a complex structure. These characteristics not only make lactic acid bacteria bacteriocins a representative natural preservative, but also make them promising materials for molecular genetics, genetic engineering, protein engineering, cosmetics, skin care, and intestinal microbial regulation.

[0004] In recent years, concerns about the safety of live probiotics have led to a growing interest in the beneficial effects of inactive probiotic components on the human body, such as probiotic cell wall components, heat-inactivated probiotic cells, and probiotic metabolites. With the discovery that Gram-positive bacteria can also secrete extracellular vesicles (EVs), increasing interest has been placed on EVs derived from lactic acid bacteria. These EVs contain a variety of lipids, proteins, metabolites, and nucleotides. Furthermore, EVs possess a bimodal lipid membrane structure, effectively protecting encapsulated substances from in vitro and in vivo proteases, DNA / RNA enzymes, and pH. EVs also serve as a natural delivery vehicle for probiotic substances secreted by lactic acid bacteria, with bacteriocins being one important example. By isolating and extracting extracellular vesicles of lactic acid bacteria that produce bacteriocin, the present invention discovered that extracellular vesicles of Lactobacillus plantarum LPL-1 have a wide range of antibacterial properties. Therefore, the present invention develops extracellular vesicles of Lactobacillus plantarum, which are mainly used in preservation and freshness preservation as a natural biological preservative. Summary of the Invention

[0005] The purpose of the present invention is to provide a novel biological preservative and fresh-keeping agent. The preservative and fresh-keeping agent provided by the present invention uses extracellular vesicles of Lactobacillus plantarum LPL-1 as an effective ingredient.

[0006] Although the antibacterial properties of lactic acid bacteria bacteriocins have been reported in the prior art. However, the lactic acid bacteria bacteriocins obtained by traditional culture have low yields, low antibacterial titers, and short effective antibacterial time. In addition, the traditional lactic acid bacteria bacteriocin extraction process is too expensive, which greatly limits the application of lactic acid bacteria bacteriocins in food preservation and antiseptic treatment. The present invention has found that a method for extracting lactic acid bacteria extracellular vesicles can be used to obtain extracellular vesicles of Lactobacillus plantarum LPL-1 containing lactic acid bacteria bacteriocins, and by adding acetate, sodium acetate and ginger juice, the yield of Lactobacillus plantarum LPL-1 extracellular vesicles is effectively increased, while the antibacterial titer of Lactobacillus plantarum LPL-1 extracellular vesicles is significantly increased.

[0007] The present invention found that different fermentation and centrifugation methods have a greater impact on the antibacterial activity of Lactobacillus plantarum LPL-1 extracellular vesicles. Therefore, in the first aspect of the present invention, a method for preparing Lactobacillus plantarum LPL-1 extracellular vesicles is provided. The OD of Lactobacillus plantarum LPL-1 fermentation liquid is 600 When the pH value is 1.0-2.0, the fermentation broth is centrifuged at 2000-5000g for 5-10 minutes at 1-4°C, and then at 10000-12000g for 10-20 minutes. The supernatant after centrifugation is filtered and then centrifuged at 100000-150000g for 60-90 minutes to obtain extracellular vesicles of Lactobacillus plantarum LPL-1.

[0008] In the preparation method provided by the present invention, the fermentation medium of Lactobacillus plantarum LPL-1 contains glucose, yeast powder, peptone, beef extract, Tween-80, dipotassium hydrogen phosphate, magnesium sulfate, manganese sulfate, diammonium hydrogen citrate, 0.1-0.5g / L acetate, 1-5ml / L ginger juice and distilled water.

[0009] In a second aspect, the present invention seeks to protect an extracellular vesicle of Lactobacillus plantarum LPL-1, which is prepared using the above-mentioned method for preparing extracellular vesicles of Lactobacillus plantarum LPL-1.

[0010] According to the understanding of those skilled in the art, the present invention also seeks protection for the use of the extracellular vesicles of Lactobacillus plantarum LPL-1 prepared by the present invention in the preparation of biological preservatives.

[0011] In a third aspect, the present invention provides a novel biological preservative, which comprises resuspending extracellular vesicles of Lactobacillus plantarum LPL-1 prepared by the above-mentioned preparation method with pectin at a concentration of 10%-20%.

[0012] In the novel biological preservative provided by the present invention, the novel biological preservative is in liquid form; in the novel biological preservative, the content of extracellular vesicles of Lactobacillus plantarum LPL-1 resuspended with pectin is 10 4 -10 10 pcs / ml.

[0013] The present invention also seeks to protect the use of the above-mentioned Lactobacillus plantarum LPL-1 extracellular vesicles or the above-mentioned novel biological preservative in food preservation.

[0014] In the application provided by the present invention, the extracellular vesicles of Lactobacillus plantarum LPL-1 inhibit Gram-positive pathogens and Gram-negative pathogens in food.

[0015] In the application provided by the present invention, the bacteriocin in the extracellular vesicles of Lactobacillus plantarum LPL-1 is not affected by the external environment and proteases in fresh food, which can increase the effective antibacterial time and extend the shelf life of food.

[0016] In the application provided by the present invention, the extracellular vesicles of Lactobacillus plantarum LPL-1 have a stable structure at a pH of 2-10; the extracellular vesicles of Lactobacillus plantarum LPL-1 have an effective shelf life of 28 days at a temperature of 1-4°C.

[0017] The beneficial effects of the present invention are:

[0018] The Lactobacillus plantarum LPL-1 extracellular vesicles prepared by the invention have strong activity and antibacterial ability, and the Lactobacillus plantarum LPL-1 extracellular vesicles obtained by the invention have a stable structure under the condition of pH of 2-10 and a storage time of more than 28 days under the condition of 1-4°C.

[0019] The novel preservative provided by the present invention is safe and non-toxic, has no bad flavor and color, can be directly contacted with the surface of fresh food or added to food, and does not affect the color and flavor of the food.

[0020] Since the bacteriocins in the extracellular vesicles of Lactobacillus plantarum LPL-1 are not affected by the external environment and proteases in fresh food, the novel preservative prepared by the present invention can effectively increase the shelf life of food. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The figure is a comparison of the antibacterial activities at different fermentation degrees in Example 3 of the present invention.

[0022] Figure 2 This is a comparison of antibacterial activity under different centrifugation conditions in Example 4 of the present invention.

[0023] Figure 3 These are the results of extracellular vesicle production under different fermentation agents in Example 5 of the present invention.

[0024] Figure 4 This is the result of the antibacterial ability of extracellular vesicles under different fermentation agents in Example 6 of the present invention.

[0025] Figure 5 This is the antibacterial effect at different extracellular vesicle addition amounts in Example 7 of the present invention.

[0026] Figure 6 This is the antibacterial effect of the extracellular vesicles of Lactobacillus plantarum LPL-1 in Example 7 of the present invention on Gram-positive pathogens such as Listeria monocytogenes and Staphylococcus aureus and Gram-negative pathogens such as Pseudomonas.

[0027] Figure 7 This is the result of the longest antibacterial ability in Example 8 of the present invention.

[0028] Figure 8 These are the results of the antibacterial ability of different pectin concentrations on the new biological preservative in Example 9 of the present invention.

[0029] Figure 9 This is the effect of the new biological preservative in Example 10 of the present invention in preserving fresh pork.

[0030] Figure 10 This is the result of identifying the tolerance of extracellular vesicles of Lactobacillus plantarum to the in vitro environment in Example 11 of the present invention. DETAILED DESCRIPTION

[0031] The following examples are intended to illustrate the present invention but are not intended to limit the scope of the present invention. Without departing from the spirit and substance of the present invention, modifications or substitutions made to the methods, steps or conditions of the present invention are intended to fall within the scope of protection of the present invention.

[0032] Unless otherwise specified, the experimental materials, reagents, instruments, etc. used in the examples of the present invention are all commercially available; unless otherwise specified, all technical means in the examples of the present invention are conventional means well known to those skilled in the art.

[0033] Example 1 Screening of bacteriocin-producing Lactobacillus plantarum

[0034] Lactobacillus plantarum with antibacterial ability was screened from 20 strains of Lactobacillus plantarum in the laboratory, and then 3 strains of Lactobacillus plantarum that secreted extracellular vesicles with antibacterial ability were selected from the Lactobacillus plantarum with antibacterial ability, and one strain of Lactobacillus plantarum LPL-1 was selected for the experiment.

[0035] Example 2 Preparation of Lactobacillus plantarum LPL-1 culture starter

[0036] Preparation of basic culture starter: glucose mass fraction 20 g / L, yeast powder mass fraction 5 g / L, peptone mass fraction 12 g / L, beef extract mass fraction 12 g / L, Tween-80 to starter volume ratio of 1 mL / L, dipotassium hydrogen phosphate mass concentration 2 g / L, magnesium sulfate mass concentration 0.2 g / L, manganese sulfate mass concentration 0.1 g / L, diammonium hydrogen citrate mass concentration 2 g / L, additional additions (sodium acetate mass fraction 0.5 g / L, ginger juice 5 mL) were added to the improved culture starter, the volume was adjusted to 1 L with distilled water, and sterilized at 121°C for 15 min.

[0037] Example 3 Effect of Extracellular Vesicles of Lactobacillus plantarum LPL-1 on the Antibacterial Ability of Extracellular Vesicles of Lactobacillus plantarum LPL-1 When Cultured to Different OD Values

[0038] (1) Fermentation: Lactobacillus plantarum LPL-1 was cultured with an improved culture medium at a rate of 2 × 10 7 CFU / ml inoculation, 37℃, 100rpm, culture to OD 600 = 1.0, 1.5, 1.8, 2.0. Then centrifuge at 2000g for 10 minutes and 10000g for 20 minutes. The supernatant is filtered using a 0.45 μm filter and then centrifuged at 150000g for 90 minutes. Finally, sterile saline is added to resuspend the vesicle pellet. Measure OD 600 The antibacterial ability of extracellular vesicles of Lactobacillus plantarum LPL-1 was determined when pH = 1.0, 1.5, 1.8, and 2.0.

[0039] (2) Plate preparation: Quantitatively spread 6 ml of TSYEB solid culture medium on a 9 cm diameter plate and place it on a horizontal surface to form an agar layer of uniform thickness. 7 CFU / ml Listeria monocytogenes was used as an indicator bacterial solution. 100 μl was taken into a 10 ml warm TSYEB agar test tube, poured into a plate, gently mixed, and placed on a horizontal surface to keep the culture medium thickness uniform.

[0040] (3) Antibacterial experiment: Use sterile tweezers to gently place the Oxford cup on the plate, and add 10% extracellular vesicles of Lactobacillus plantarum LPL-1 from the above group into it. 10 The cells were then placed in a 4°C refrigerator for approximately 5 hours, and then transferred to a 37°C incubator to observe the appearance of an inhibition zone. After the inhibition zone is formed, it was measured with a vernier caliper to an accuracy of 0.01 mm.

[0041] The results show (such as Figure 1 ):with OD 600 =1.0 group, OD 600 =1.5 and 1.8 groups showed significant increase in antibacterial activity, but at OD 600 =2.0, the antibacterial activity decreased. This showed that the antibacterial ability of Lactobacillus plantarum LPL-1 extracellular vesicles was strongest when the OD value was 1.8.

[0042] Example 4 Effects of different centrifugation conditions on the antibacterial ability of extracellular vesicles of Lactobacillus plantarum LPL-1

[0043] (1) Centrifugation: Lactobacillus plantarum LPL-1 was cultured with the modified culture starter at 2×10 7 CFU / ml inoculation, 37℃, 100rpm, culture to OD 600=1.8. At 4°C, the fermentation broth was divided into the following groups for centrifugation: Group A was centrifuged at 2000g for 10 minutes and 10000g for 20 minutes, the supernatant was filtered through a 0.45μm filter, and then centrifuged at 100000g for 90 minutes; Group B was centrifuged at 5000g for 5 minutes and 12000g for 10 minutes, the supernatant was filtered through a 0.45μm filter, and then centrifuged at 100000g for 90 minutes; Group C was centrifuged at 2000g for 10 minutes and 10000g for 20 minutes, the supernatant was filtered through a 0.45μm filter, and then centrifuged at 100000g for 90 minutes. The supernatant of group D was filtered using a 0.45 μm filter and then centrifuged at 150,000 g for 90 minutes; group E was centrifuged at 2,000 g for 10 minutes and 10,000 g for 20 minutes, the supernatant was filtered using a 0.45 μm filter and then centrifuged at 150,000 g for 60 minutes; finally, sterile saline was added to resuspend the vesicle pellet.

[0044] (2) Antibacterial test: According to the above inhibition zone test, add 10 10 The antibacterial ability of extracellular vesicles of Lactobacillus plantarum LPL-1 was determined by counting cells / ml.

[0045] The results show (such as Figure 2 ): Compared with the five groups, the antibacterial activity of extracellular vesicles of Lactobacillus plantarum LPL-1 was the highest under the centrifugation conditions of group C, and different centrifugation speeds and centrifugation times had a significant effect on the antibacterial activity of extracellular vesicles of Lactobacillus plantarum LPL-1.

[0046] Example 5 Effect of adding sodium acetate and ginger juice to the improved culture starter on the extracellular vesicle production of Lactobacillus plantarum LPL-1

[0047] (1) The vesicles were quantitatively analyzed using the NanoSight NS300 nanoparticle analyzer.

[0048] (2) The basic culture starter was used as the blank control (NPs) group, and the improved culture starter was added with 0.5 g / L sodium acetate + 0, 1, 5, and 10 ml / L ginger juice to culture Lactobacillus plantarum LPL-1, and the extracellular vesicles were extracted using the above-mentioned optimal centrifugation method.

[0049] The results show (such as Figure 3): Compared with the blank control group (NPs), the addition of sodium acetate (0.5 g / L) alone had no effect on the extracellular vesicle production of Lactobacillus plantarum LPL-1, but the addition of sodium acetate (0.5 g / L) + ginger juice could significantly increase the extracellular vesicle production of Lactobacillus plantarum LPL-1. The addition of 5 ml of ginger juice increased the extracellular vesicle production of Lactobacillus plantarum LPL-1 to a maximum of 173.3%.

[0050] Example 6 Effect of adding sodium acetate and ginger juice to the improved culture starter on the antibacterial ability of extracellular vesicles of Lactobacillus plantarum LPL-1

[0051] 1. The basic culture starter was used as the blank control (NPs) group. Ginger juice 5 ml / L + sodium acetate 0, 0.1, 0.5, and 1.0 g / L were added to the improved culture starter to culture Lactobacillus plantarum LPL-1, and extracellular vesicles were extracted using the above-mentioned optimal centrifugation method.

[0052] (2) Antibacterial test: According to the above-mentioned antibacterial zone test, 10 10 The antibacterial ability of extracellular vesicles of Lactobacillus plantarum LPL-1 was determined by counting cells / ml.

[0053] The results show (such as Figure 4 Compared with the blank control (NPs) group, the addition of 5 ml / L ginger juice and 0.1 and 0.5 g / L sodium acetate significantly enhanced the antibacterial activity of Lactobacillus plantarum LPL-1 extracellular vesicles. However, the antibacterial activity of Lactobacillus plantarum LPL-1 extracellular vesicles was limited when 1.0 g / L sodium acetate was added.

[0054] Example 7 Identification of the Maximum and Minimum Antibacterial Capacity of Extracellular Vesicles of Lactobacillus plantarum Obtained by the Optimal Culture Starter Culture and the Optimal Centrifugation Method

[0055] 1. Sterile saline was used as blank control (Control) group, and 10 4 , 10 6 , 10 8 , 10 10 The antibacterial ability was identified at the concentration of 100mg / ml.

[0056] (1) Antibacterial experiment: fresh culture 10 7 CFU / ml (OD 600 =1) of Listeria monocytogenes as an indicator bacterial solution, different concentrations of Lactobacillus plantarum LPL-1 extracellular vesicles were added, and cultured at 37°C. The OD values ​​were measured every 2 hours. 600 Value determination.

[0057] The results show (such as Figure 5):Adding extracellular vesicles of Lactobacillus plantarum LPL-1 can significantly reduce the OD of the indicator bacteria solution 600 Value, where 10 8 and 10 10 The antibacterial effect was most obvious at a concentration of 10 8 and 10 10 There was no significant difference in the concentrations of cells / ml.

[0058] 2. Inhibitory effect of extracellular vesicles of Lactobacillus plantarum LPL-1 on Gram-negative and Gram-positive bacteria

[0059] The results show (such as Figure 6 ):Exocellular vesicles of Lactobacillus plantarum LPL-1 have significant antibacterial effects on Gram-positive pathogens such as Listeria monocytogenes and Staphylococcus aureus and Gram-negative pathogens such as Pseudomonas.

[0060] Example 8 Identification of the Longest Effective Antibacterial Time of Extracellular Vesicles of Lactobacillus Plantarum at 4°C

[0061] At 4 °C, the extracellular vesicles of Lactobacillus plantarum were placed for 0, 21, 28, 35, and 42 days, and 10 10 The antibacterial ability of extracellular vesicles of Lactobacillus plantarum LPL-1 was determined by counting cells / ml.

[0062] The results show (such as Figure 7 ): Compared with the initial 0-day group, the antibacterial ability of Lactobacillus plantarum extracellular vesicles began to decline significantly after 28 days at 4°C, with 42 days being the longest antibacterial time.

[0063] Example 9 Determination of the antibacterial ability of different pectin concentrations on the new biological preservative

[0064] 1. Combining the above-mentioned optimal culture starter ratio and the optimal centrifugation method to culture Lactobacillus plantarum LPL-1 and obtain Lactobacillus plantarum LPL-1 extracellular vesicles, resuspend Lactobacillus plantarum LPL-1 extracellular vesicles at pectin concentrations of 1%, 5%, 10%, and 20% for 10 min. 8 / ml, a new type of biological preservative is prepared, and then it is packaged.

[0065] 2. The refrigerated quality of rainbow trout fillets was tested using a new type of biological preservative made with different pectin concentrations.

[0066] (1) Fresh rainbow trout fillets were treated with the novel biological preservatives and then packaged in trays at 40 g / portion. The packages were stored in a refrigerator at 4°C. After storage for one week, the fillets were made into a meat paste in the tray packaging and mixed evenly, and then microbial indicators were tested.

[0067] The results show (such as Figure 8): Compared with the 1% pectin group, the 10% pectin group could significantly inhibit the bacterial colony count in rainbow trout, and the 20% pectin group had a more significant antibacterial effect.

[0068] In summary, the new preservative is 10% pectin containing extracellular vesicles of Lactobacillus plantarum LPL-1 10 8 pcs / ml.

[0069] Example 10 Application of a Novel Biological Preservative in the Preservation of Fresh Pork

[0070] 1. Processing of meat samples

[0071] Fresh pork was cut into small pieces of approximately 25g. The meat was immersed in 10% pectin for 15 seconds as a blank control (control) and in a novel biopreservative (P-NPs) for 15 seconds as an experimental group. The meat was removed and placed on a clean plate, drained for 5 minutes, then placed on a polyester tray, sealed with PE plastic wrap, labeled, and refrigerated at (4±0.5)°C for microbial testing.

[0072] 2. Determination of total bacterial colony count

[0073] The total bacterial count refers to the total number of bacterial colonies in 1 gram or 1 mL of a processed animal food sample, cultured under specific conditions. It is primarily used to determine the degree of food contamination. It can also be used to observe the dynamics of bacterial growth in food, allowing for hygienic evaluation of samples.

[0074] my country's current food hygiene standards have not yet established bacterial indicators for meat. The general recommended standard for the total colony count of meat quality hygiene indicators is: Grade 1 fresh: ≤5×10 4 cfu / g; secondary fresh: 5×10 4 -5×10 6 cfu / g; deterioration: >5×10 6 cfu / g.

[0075] The determination method can refer to GB 4789.2-2008.

[0076] The results show (such as Figure 9 ): As the storage time prolonged, the total bacterial count in the Control group exceeded 10 on the 8th day. 7 According to the standard of total bacteria count in meat, the control group was close to becoming spoiled meat on the 8th day, and exceeded 10 on the 12th day. 8, completely spoiled. The total bacterial count in the chilled meat treated with the novel biological preservative (P-NPs) was significantly lower than that in the control group. The P-NPs group extended its shelf life to approximately the 20th day, but completely spoiled by the 24th day. This demonstrates that the novel biological preservative effectively inhibits microbial growth in chilled meat and extends its shelf life.

[0077] Example 11 Identification of the tolerance of Lactobacillus plantarum extracellular vesicles to in vitro environments

[0078] 1. Extracellular vesicles of Lactobacillus plantarum LPL-1 were treated with the following conditions:

[0079] A: Treat in hydrochloric acid at 37°C, pH 2.2 for 30 minutes;

[0080] B: sodium hydroxide treatment at 37°C, pH = 10 for 30 minutes;

[0081] C: Trypsin treatment at 37°C for 30 minutes;

[0082] After the above treatment, the pellet was centrifuged at 150,000 g for 90 minutes at 4°C and then resuspended in sterile saline. Untreated extracellular vesicles of Lactobacillus plantarum LPL-1 were used as blank control (NPs) group for inhibition zone experiment.

[0083] The results show (such as Figure 10 ): Compared with the NPs group, there was no difference among groups A, B, and C, which proved that the extracellular vesicles of Lactobacillus plantarum LPL-1 were not affected by proteases, acids, and alkalis, and its unique dual-mode layer structure could effectively protect the internal bacteriocins.

[0084] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. A method for preparing extracellular vesicles of Lactobacillus plantarum LPL-1, characterized in that: OD of Lactobacillus plantarum LPL-1 fermentation broth 600 When the pH reaches 1.0-2.0, the fermentation broth is centrifuged at 2000-5000 g for 5-10 minutes at 4° C., and then at 8000-12000 g for 5-20 minutes. The supernatant after centrifugation is filtered and then centrifuged at 100000-150000 g for 60-90 minutes to obtain extracellular vesicles of Lactobacillus plantarum LPL-1. The culture starter of Lactobacillus plantarum LPL-1 contains 20 g / L glucose, 5 g / L yeast powder, 12 g / L peptone, 12 g / L beef extract, 1 mL / L Tween-80, 2 g / L dipotassium hydrogen phosphate, 0.2 g / L magnesium sulfate, 0.1 g / L manganese sulfate, 2 g / L diammonium hydrogen citrate, 0.1-0.5 g / L sodium acetate, 5 ml / L ginger juice and distilled water.

2. Application of the preparation method of the extracellular vesicles of Lactobacillus plantarum LPL-1 according to claim 1 in the preparation of a biological preservative.

3. The application according to claim 2, characterized in that: The biological preservative and fresh-keeping agent uses 10%-20% concentration of pectin to resuspend the extracellular vesicles of Lactobacillus plantarum LPL-1 prepared by the preparation method of claim 1.

4. The use according to claim 3, characterized in that: The biological preservative is in liquid form; in the biological preservative, the content of extracellular vesicles of Lactobacillus plantarum LPL-1 resuspended in pectin is 10 4 -10 10 pcs / ml.

5. Application of the method for preparing the extracellular vesicles of Lactobacillus plantarum LPL-1 according to claim 1 in food preservation and fresh-keeping.

6. The use according to claim 5, characterized in that: The extracellular vesicles of Lactobacillus plantarum LPL-1 prepared by the preparation method described in claim 1 inhibit Gram-positive pathogens and Gram-negative pathogens in food; the Gram-positive pathogens are Listeria monocytogenes and / or Staphylococcus aureus; the Gram-negative pathogens are Pseudomonas.

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