Preparation method of plant lactobacillus Lp-06 strain

Abstract

The preparation method comprises the following steps: S1, chopping Chinese cabbages and cabbages, putting the chopped Chinese cabbages and cabbages into a sterile container, adding saline water to submerge the vegetables, sealing the container, putting the container in a shade place for fermentation, and uncovering and exhausting during fermentation to prepare pickled Chinese cabbages; s2, taking 1.0 mL of pickled Chinese cabbage juice, diluting the pickled Chinese cabbage juice with normal saline, and uniformly coating the MRS culture medium with the fermentation broth; and S3, culturing the MRS culture medium at constant temperature of 37 DEG C for 48 hours, selecting single colonies with different shapes and sizes on a plate, and purifying on the MRS agar culture medium by multiple streaks to obtain a strain of the plant lactobacillus Lp-06. The composition has the advantages of inhibiting bacteria and reducing fat, and solves the problems that in the using process of existing plant lactobacillus, the cholesterol reducing effect is poor, the application of the plant lactobacillus in the aspect of weight losing is influenced, the inhibiting effect on pathogenic bacteria is poor, and the intestinal flora regulation is influenced.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a method for preparing a Lactobacillus plantarum Lp-06 strain. Background Art

[0002] Lactobacillus plantarum is a common lactic acid bacterium found widely in nature, including on plant surfaces and in the intestines of animals. Its ability to ferment carbohydrates to produce lactic acid has led to its widespread use in the food fermentation industry, such as in the production of kimchi, sauerkraut, and yogurt. Lactobacillus plantarum is an important probiotic in the human gastrointestinal tract, contributing to weight loss, regulating lipid metabolism, influencing intestinal flora composition, inhibiting the growth of pathogenic microorganisms, and preventing diarrhea.

[0003] Chinese patent application number 202211396878.2 is a Lactobacillus plantarum and its application. The Lactobacillus plantarum is named: Lactobacillus plantarum YYS-99, with a deposit number of CGMCC No. 25838. The Lactobacillus plantarum YYS-99 provided by the invention can efficiently convert linoleic acid into conjugated linoleic acid, which is biologically active and beneficial to human health. At the same time, the bacterium has good resistance to gastric acid, bile salts and intestinal colonization ability, and can be used to produce fermented products rich in conjugated linoleic acid, or to be used in probiotic products to improve the structure of intestinal flora, play an antioxidant role, lower human cholesterol, resist atherosclerosis, and reduce fat deposition in the body.

[0004] The existing Lactobacillus plantarum has a poor effect on lowering cholesterol during use, which affects the application of Lactobacillus plantarum in weight loss. It also has a poor effect on inhibiting pathogens, which affects the regulation of intestinal flora. Summary of the Invention

[0005] The present invention aims to provide a preparation method of Lactobacillus plantarum Lp-06 strain, which has the advantages of antibacterial and fat-reducing properties, and solves the problems that the existing Lactobacillus plantarum has a poor cholesterol-lowering effect during use, which affects the application of Lactobacillus plantarum in weight loss, and has a poor inhibitory effect on pathogens, which affects the regulation of intestinal flora.

[0006] To achieve the above object, the present invention provides the following technical solution: a method for preparing Lactobacillus plantarum Lp-06 strain, comprising the following steps:

[0007] S1. Chop Chinese cabbage and lettuce into small pieces, put them into a sterile container, add salt water to cover the vegetables, seal the container, and place it in a cool place to ferment. During the fermentation period, open the lid to vent air, thereby making sauerkraut;

[0008] S2. Take 1.0 mL of sauerkraut juice and dilute it with normal saline, then evenly spread the fermentation liquid on MRS medium;

[0009] S3. The MRS culture medium was cultured at a constant temperature of 37° C. for 48 h, single colonies of different morphology and size on the plate were picked, and streaked multiple times on the MRS agar medium for purification to obtain the strain of Lactobacillus plantarum Lp-06.

[0010] As a preferred preparation method of the Lactobacillus plantarum Lp-06 strain of the present invention, Lactobacillus plantarum Lp-06 is deposited in the General Microbiology Center of the China Culture Collection Committee of Microorganisms, with a deposit number of CGMCC No. 29661 and a deposit date of January 18, 2024. The depository address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0011] The invention discloses an application of Lactobacillus plantarum Lp-06 strain. Lactobacillus plantarum Lp-06 is used for inhibiting harmful bacteria, including Escherichia coli, Listeria monocytogenes and Staphylococcus aureus.

[0012] As a preferred application of the Lactobacillus plantarum Lp-06 strain of the present invention, Lactobacillus plantarum Lp-06 is used for degrading triglycerides, cholesterol and inhibiting pancreatic lipase.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] 1. The Lactobacillus plantarum Lp-06 in the present invention is isolated from naturally fermented sauerkraut. This strain has the functions of degrading triglycerides and cholesterol and inhibiting pancreatic lipase, and has a certain antibacterial effect on Escherichia coli, Staphylococcus aureus, and Listeria monocytogenes. Therefore, screening out a strain with weight loss, lipid-lowering and antibacterial functions provides high-quality bacterial resources for the probiotics industry, which is of great significance for improving human health. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Schematic diagram of the preparation process of Lactobacillus plantarum Lp-06 strain of the present invention;

[0016] Figure 2 This is a schematic flow chart of the method for preparing fermented fish intestines according to the present invention;

[0017] Figure 3 This is a colony morphology diagram of the Lactobacillus plantarum Lp-06 plate of the present invention;

[0018] Figure 4 This is a Gram staining result of Lactobacillus plantarum Lp-06 of the present invention;

[0019] Figure 5 This is a graph showing the acid resistance of Lactobacillus plantarum Lp-06 of the present invention;

[0020] Figure 6This is a graph showing the alkali resistance of Lactobacillus plantarum Lp-06 of the present invention;

[0021] Figure 7 This is a graph showing the bile salt tolerance of Lactobacillus plantarum Lp-06 of the present invention;

[0022] Figure 8 This is a graph showing the ability of Lactobacillus plantarum Lp-06 of the present invention to tolerate artificial simulated gastrointestinal fluid;

[0023] Figure 9 Explain the cholesterol standard curve for Lactobacillus plantarum Lp-06 of the present invention;

[0024] Figure 10 This is a graph showing the results of an experiment on the inhibition of pathogenic bacteria by the supernatant of the fermentation broth of Lactobacillus plantarum Lp-06 of the present invention;

[0025] Figure 11 This is a comparative diagram showing the effects of Lactobacillus plantarum Lp-06 of the present invention on the food intake of mice fed with high fat and high cholesterol;

[0026] Figure 12 This is a comparative diagram showing the effects of Lactobacillus plantarum Lp-06 of the present invention on total cholesterol in mice fed with high fat and high cholesterol;

[0027] Figure 13 This is a comparative diagram of the effects of Lactobacillus plantarum Lp-06 of the present invention on triglycerides in mice fed with high fat and high cholesterol;

[0028] Figure 14 This is a comparative diagram showing the effects of Lactobacillus plantarum Lp-06 of the present invention on low-density lipoprotein cholesterol in mice fed with high fat and high cholesterol;

[0029] Figure 15 This is the HE staining image of the liver of C57BL / 6J mice of the present invention;

[0030] Figure 16 This is a reference chart for the in vitro triglyceride-lowering ability of Lactobacillus plantarum Lp-06;

[0031] Figure 17 Reference chart for the ability of Lactobacillus plantarum Lp-06 to inhibit pancreatic lipase in vitro;

[0032] Figure 18 This is a reference chart for the in vitro cholesterol-lowering ability of Lactobacillus plantarum Lp-06;

[0033] Figure 19 This is a reference chart for experimental data on the antibacterial ability of Lactobacillus plantarum Lp-06;

[0034] Figure 20 A comparison chart of the antibacterial ability with other probiotic strains;

[0035] Figure 21Figure 2 shows the body weight of three groups of C57BL / 6J mice after eight weeks of feeding.

[0036] Figure 22 This is a diagram showing the changes in the texture of sea bream intestine with different inoculation doses of Lactobacillus plantarum Lp-06. DETAILED DESCRIPTION

[0037] Example 1

[0038] See also Figure 1 A method for preparing a Lactobacillus plantarum Lp-06 strain comprises the following steps:

[0039] S1. Chop Chinese cabbage and lettuce into small pieces, put them into a sterile container, add salt water to cover the vegetables, seal the container, and place it in a cool place to ferment. During the fermentation period, open the lid to vent air, thereby making sauerkraut;

[0040] S2. Take 1.0 mL of sauerkraut juice and dilute it with normal saline, then evenly spread the fermentation liquid on MRS medium;

[0041] S3. The MRS culture medium was cultured at a constant temperature of 37° C. for 48 h, single colonies of different morphology and size on the plate were picked, and streaked multiple times on the MRS agar medium for purification to obtain the strain of Lactobacillus plantarum Lp-06.

[0042] Lactobacillus plantarum Lp-06 is deposited in the General Microbiology Center of the China Culture Collection Committee of Microorganisms, with the deposit number CGMCC No. 29661 and the deposit date of January 18, 2024. The address of the depository institution is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0043] Lactobacillus plantarum Lp-06 was isolated from naturally fermented sauerkraut. This strain has the function of degrading triglycerides and cholesterol, inhibiting pancreatic lipase, and has a certain antibacterial effect on Escherichia coli, Staphylococcus aureus, and Listeria monocytogenes. Therefore, screening out strains with weight loss, lipid-lowering and antibacterial functions provides high-quality bacterial resources for the probiotics industry, which is of great significance to improving human health.

[0044] Example 2

[0045] The invention discloses an application of Lactobacillus plantarum Lp-06 strain. Lactobacillus plantarum Lp-06 is used for inhibiting harmful bacteria, including Escherichia coli, Listeria monocytogenes and Staphylococcus aureus.

[0046] Furthermore, Lactobacillus plantarum Lp-06 was used to degrade triglycerides, cholesterol and inhibit pancreatic lipase.

[0047] Example 3

[0048] See also Figure 2A method for preparing fermented fish intestines using Lactobacillus plantarum Lp-06 strain comprises the following steps:

[0049] S1, preparing surimi, putting fish meat into an electric meat grinder and mincing it to prepare surimi;

[0050] S2, mixing and stirring, putting the minced fish into a mixing device, adding salt, sugar, soy sauce, cooking wine, pepper, monosodium glutamate, starch and water to the minced fish, and the mixing device stirs the raw materials into mixed meat;

[0051] S3, activating Lactobacillus plantarum, preparing Lactobacillus plantarum Lp-06 into an activated Lactobacillus plantarum Lp-06 bacterial suspension, and then evenly inoculating the prepared Lactobacillus plantarum Lp-06 bacterial suspension into the mixed minced meat;

[0052] S4, stuffing the mixed minced meat into sausage using a sausage stuffer, and keeping a moderate tightness, and tying it tightly with cotton thread after stuffing to prepare fish sausage;

[0053] S5. Fermentation of fish intestines: Seal the prepared fish intestines and place them in a constant temperature incubator for fermentation. After fermentation, place the fish intestines in water and steam them for maturation.

[0054] Furthermore, the electric meat grinder in S1 includes a body, a motor, a transmission mechanism, a hopper, a pushing and cutting structure, and a discharge screen plate. The processed fish meat enters the body through the hopper, and the motor drives the pushing and cutting structure to rotate through the transmission mechanism. The pushing and cutting structure rotates inside the body to chop the fish meat, thereby breaking the fish meat into fish paste, and pushing the fish paste to be discharged through the discharge screen plate.

[0055] Furthermore, by weight, S2 uses 100 parts of surimi, 2 parts of salt, 2.4 parts of sugar, 0.8 parts of soy sauce, 2.4 parts of cooking wine, 0.3 parts of pepper, 0.8 parts of MSG, 8.3 parts of starch, and 30 parts of water.

[0056] Furthermore, the stirring time of the mixing device in S2 is 5 minutes.

[0057] Furthermore, S3's Lactobacillus plantarum Lp-06 was deposited in the General Microbiology Center of the China Culture Collection Committee of Microorganisms, with the deposit number CGMCC No. 29661, the deposit date being January 18, 2024, and the address of the depository institution being No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0058] Furthermore, when activating Lactobacillus plantarum Lp-06 in S3, Lactobacillus plantarum Lp-06 is activated, and then the activated Lactobacillus plantarum Lp-06 is inoculated into the culture medium, and then Lactobacillus plantarum Lp-06 is cultured. After the culture is completed, the strains are separated and collected, and the strains are mixed with sterile physiological saline to prepare a Lactobacillus plantarum Lp-06 bacterial suspension.

[0059] Furthermore, the ratio of Lactobacillus plantarum Lp-06 to the mixed minced meat in S3 is 0.01-0.05:1.

[0060] Furthermore, Lactobacillus plantarum Lp-06 in S3 is used to inhibit harmful bacteria, including Escherichia coli, Listeria monocytogenes and Staphylococcus aureus.

[0061] Furthermore, Lactobacillus plantarum Lp-06 in S3 was used to degrade triglycerides, cholesterol and inhibit pancreatic lipase.

[0062] Furthermore, when the fish intestines were fermented in S5, the fermentation was carried out at 37°C for 16 h.

[0063] The present invention uses Lactobacillus plantarum Lp-06 to prepare fermented fish intestines, which can ferment sugars into lactic acid and other organic acids, lower the pH value, and thus give the fermented fish intestines a unique sour taste. In addition, ester and alcohol compounds may be produced during the fermentation process, further enriching the aroma and taste of the product. By producing lactic acid to lower the pH value, the growth of pathogens and spoilage bacteria is inhibited, effectively extending the shelf life of the fermented fish intestines. Metabolites produced during the fermentation process can help change the texture of the fish intestines, making them softer and having a better taste. Lactic acid bacteria can also participate in protein modification, affecting the elasticity and chewiness of the final product.

[0064] Example 4

[0065] See also Figure 3-Figure 4 The screening and identification method of Lactobacillus plantarum Lp-06 comprises the following steps:

[0066] S1. Isolation and purification of strains. Chop the Chinese cabbage and Chinese cabbage into small pieces and put them into a sterile container. Add salt water to cover the vegetables and seal it. Place it in a cool place for fermentation. During the fermentation period, open the lid to vent air. Prepare sauerkraut. Take 1.0 mL of sauerkraut juice and dilute it with normal saline. Take 100 μL of the dilution gradient of 10 -2 -10 -7 The fermentation broth was evenly spread on MRS medium and cultured at 37°C for 48 hours. Single colonies of different shapes and sizes on the plate were picked and streaked multiple times on MRS agar medium for purification until they were confirmed to be pure strains.

[0067] S2. Microscopic examination: Pick a single colony and inoculate it into MRS broth medium. After culturing for 24 hours, perform Gram staining. Smear the test bacterial solution. After Gram staining, observe the cell morphology and arrangement under an optical microscope, record it, and take photos for preservation.

[0068] S3. Physiological and biochemical experiments were used to identify and screen bacterial strains using hydrogen peroxide test, indole test and carbohydrate test. The isolated and purified strain was milky white, with a convex middle and a smooth and moist surface, with a diameter of 1.47±0.31mm. The strain was Gram-positive, and the cells were short rod-shaped and arranged in pairs. Its physicochemical characteristics were negative in hydrogen peroxide test, positive indole reaction, and positive in esculin hydrolysis test. It can ferment cellobiose, maltose, mannitol, salicin, sorbitol, sucrose, raffinose, inulin and lactose.

[0069] S4. Molecular biological identification: The strain was expanded and cultured, and the bacterial solution was centrifuged to obtain the bacteria, which were then sequenced. The sequencing was completed by Nanjing Paisonno Gene Technology Co., Ltd. The DNA sequence of Lactobacillus plantarum Lp-06 after sequencing was:

[0070]

[0071] Example 5

[0072] See also Figure 5-Figure 8 The tolerance test method of Lactobacillus plantarum Lp-06 comprises the following steps:

[0073] S1. Acid resistance test: The strain was activated in MRS medium at 37°C for two generations and then a bacterial suspension was prepared. The pH value of the MRS medium was adjusted to 5.0, 4.0, 3.0, 2.0 and 1.0 using 4.0 mol / L hydrochloric acid (HCl). The bacterial cells were then resuspended in these culture media with different pH values. After culturing at 37°C for 24 hours, the bacterial suspension was taken and the OD600nm value was measured. Three parallel experiments were set, and the OD value of the MRS medium with a pH of 6.0 was used as the control. The survival rate was calculated as follows: survival rate = Nt / N0, where Nt is the OD value of the MRS medium with pH values ​​of 1.0-5.0, and N0 is the OD value of the MRS medium at a pH of 6.0.

[0074] The pH value of gastric juice of a normal person is 0.9-1.8 when fasting. After taking food, the pH value of gastric juice will change to about 1.8-5.0. Therefore, this experiment selected pH value 1-5 to test the strain's tolerance to acid. The results are as follows Figure 5 As shown by Figure 5 It can be seen that Lactobacillus plantarum Lp-06 has poor tolerance under pH 1.0 conditions. After 24 hours of treatment, the survival rate is only 7.61%. At pH 4.0, it has strong tolerance. After 24 hours of treatment, the survival rate reaches 79.22%, indicating that the bacteria has a strong ability to tolerate acidic conditions.

[0075] S2. Alkali resistance test results: The strain was activated in MRS medium at 37°C for two generations and then a bacterial suspension was prepared. The suspension was resuspended in MRS medium with pH values ​​of 11.0, 10.0, 9.0, 8.0, and 7.0. The suspension was then cultured at 37°C for 24 h and the OD was measured. 600 nm value, set 3 parallels, take the OD value of MRS medium with pH 6.0 as the control, calculate the survival rate, that is, survival rate = Nt / N0, where: Nt is the OD value of MRS medium with pH values ​​of 7.0-11.0, N0 is the OD value of MRS medium with pH 6.0;

[0076] In this experiment, pH 7-11 was selected to test the strain's tolerance to alkali. Figure 6 As shown. Figure 6It can be seen that the survival rate of Lactobacillus plantarum Lp-06 gradually increased as the pH value tended to be neutral, but after culturing at a pH of 11.0 for 24 hours, its survival rate was still 74.798%, indicating that the bacteria has a strong ability to tolerate alkaline conditions.

[0077] S3. Results of bile salt tolerance test: After activation and passage for two generations, Lactobacillus plantarum Lp-06 strain was inoculated into MRS medium containing 0.1%, 0.3%, 0.5%, 0.7%, and 0.9% bile salt concentrations at a 3% inoculum volume. After incubation at 37°C for 24 h, the bacterial solution was taken and the OD was measured. 600 nm value, set 3 parallels, and use the OD value of MRS medium without bile salts as the control to calculate the survival rate of the strain; that is, survival rate = Nt / N0, where: Nt is the number of viable bacteria in MRS medium containing different concentrations of bile salts, and N0 is the number of viable bacteria in MRS medium without bile salts;

[0078] The bile salt concentration in the small intestine of most human bodies is about 0.3% (w / v). Probiotics need to tolerate bile salts in the intestine to play a probiotic role in the small intestine. By selecting 5 different mass fractions of bile salts (0.1%, 0.3%, 0.5%, 0.7%, 0.9%) to treat Lactobacillus plantarum Lp-06 strain for 24 hours (the strain cultured without bile salts was used as the control), the results are as follows Figure 7 As shown. Figure 7 It can be seen that with the increase of bile salt concentration, the survival rate of the strain gradually decreased. After 24 hours of treatment with 0.3% bile salt group, the survival rate was still 62.52%, which shows that Lactobacillus plantarum Lp-06 has good bile salt tolerance.

[0079] S4. Results of the simulated gastrointestinal fluid tolerance test: Lactobacillus plantarum Lp-06 strain was activated and passaged for two generations in MRS medium at 37°C. A bacterial suspension was prepared and resuspended in simulated gastric fluid. The suspension was treated at 37°C for 3 h, centrifuged and the supernatant was discarded. The sludge was collected and washed three times with sterile saline. The sludge precipitate was collected aseptically, and saline was added at a ratio of 1:5. The mixture was mixed evenly, diluted in a gradient, and then the colony count was performed.

[0080] Then, 1.0 g of bacterial sludge after 3 h of gastric juice treatment was taken and added with simulated bile. After treatment at 37°C for 20 min, the supernatant was discarded after centrifugation. The bacterial sludge was taken and washed three times with sterile saline. The precipitate was collected aseptically and added with normal saline at a ratio of 1:5. The mixture was evenly mixed, and the mixture was diluted stepwise for colony counting.

[0081] Next, 1.0 g of bacterial sludge from the bile treatment for 20 minutes was added to simulated intestinal fluid and treated at 37°C for 4 hours. The supernatant was centrifuged and discarded. The bacterial sludge was then washed three times with sterile saline. The precipitate was collected aseptically and mixed with saline at a ratio of 1:5. The mixture was diluted serially and the colonies were counted.

[0082] Survival rate of artificial gastrointestinal fluid and bile = Nt / N0, where: Nt is the number of viable bacteria after treatment with artificial simulated gastric fluid, bile, and intestinal fluid, and N0 is the number of viable bacteria before treatment;

[0083] Gastric juice contains mucus, gastric acid, pepsin, etc. The premise for probiotics to exert their probiotic effects is that they can survive in the stomach, that is, they need to have the ability to tolerate the gastric acid environment and resist pepsin. Usually, food stays in the stomach for 3 hours, so this study selected simulated gastric juice to treat Lactobacillus plantarum Lp-06 bacterial liquid for 3 hours. After digestion in the stomach, food enters the intestine. The small intestine environment is weakly alkaline and contains trypsin and bile. The number of viable probiotics in the intestine reaches 10 6 cfu / mL or above to play a probiotic role, so probiotics need to tolerate the weak alkaline environment in the intestine and have the ability to resist trypsin and bile. Food generally stays in the small intestine for 3-8 hours, so this experiment selected simulated intestinal fluid to treat Lactobacillus plantarum Lp-06 bacterial solution for 4 hours. The results are as follows Figure 8 As shown by Figure 8 It can be seen that after 3 hours of gastric juice treatment, the survival rate of Lactobacillus plantarum Lp-06 was 82.30%, and the number of viable bacteria reached 14.93±0.01lg (cfu / g); after 20 minutes of bile treatment, the survival rate was 75.98%, and the number of viable bacteria reached 13.79±0.05lg (cfu / g); after 4 hours of intestinal juice treatment, the survival rate was still 59.85%, and the number of viable bacteria reached 10.86±0.12lg (cfu / g). It can be seen that Lactobacillus plantarum Lp-06 has a strong ability to tolerate artificial simulated gastrointestinal juice and bile.

[0084] Example 6

[0085] The in vitro lipid-lowering ability test method of Lactobacillus plantarum Lp-06 comprises the following steps:

[0086] S1. Determination of triglyceride-lowering ability of Lactobacillus plantarum Lp-06

[0087] Prepare triglyceride culture medium: 2% polyvinyl alcohol aqueous solution and vegetable oil are mixed in a volume ratio of 3:1, and after ultrasonic treatment, the mixture is evenly mixed to form a vegetable oil emulsion. The above liquid is added to MRS culture medium at a ratio of 5%, the pH is adjusted to 6.5±0.2, and sterilized for later use. Then, Lactobacillus plantarum Lp-06, which has been activated and passaged twice, is inoculated into the triglyceride liquid culture medium at an inoculum size of 3%, and the culture is statically cultured at 37°C for 24 hours. The triglyceride content in the supernatant is determined using a triglyceride kit, and the triglyceride removal rate is calculated using the following formula;

[0088] Triglyceride removal rate = (1-A1 / A0) × 100%, where A1 is the triglyceride content of the supernatant of the fermentation strain, and A0 is the triglyceride content of the supernatant of the uninoculated strain;

[0089] Reference for in vitro triglyceride-lowering ability of Lactobacillus plantarum Lp-06 Figure 16 The triglyceride removal rate of Lactobacillus plantarum Lp-06 was 45.03%.

[0090] S2, when determining the inhibition rate of pancreatic lipase of Lactobacillus plantarum Lp-06, first prepare a 50mmol / L solution of p-nitrophenyl palmitate with isopropyl alcohol, then add ultrapure water to dilute to 10mmol / L, prepare a pancreatic lipase solution with a concentration of 50mg / mL, mix the sample with the pancreatic lipase solution in a 1:1 ratio, and immediately add the same volume of p-nitrophenyl palmitate solution as the above mixed solution. After incubation at 37°C for 2h, quickly place in a 100°C water bath for 5min to terminate the reaction. The reaction solution after termination is centrifuged at 6000r / min for 5min, and the supernatant is measured for absorbance at a wavelength of 405nm;

[0091] The enzyme activity inhibition rate formula is as follows: Inhibition rate (%) = (1-(As-An) / (Ac-A0)) × 100

[0092] , where As is the absorbance of the sample group, An is the absorbance of the sample control group, Ac is the absorbance of the blank group; A0 is the absorbance of the blank control group;

[0093] Reference for the ability of Lactobacillus plantarum Lp-06 to inhibit pancreatic lipase in vitro Figure 17 The pancreatic lipase inhibition rate of Lactobacillus plantarum Lp-06 was 57.42%.

[0094] S3. Draw a cholesterol standard curve. Prepare cholesterol standard solutions with mass concentrations of 2, 1.25, 0.625, 0.3125, 0.15625, and 0.078 μmol / mL. Measure their absorbance at 500 nm using a total cholesterol content detection kit. Draw a cholesterol content standard curve with absorbance as the vertical axis and cholesterol mass concentration as the horizontal axis. The results are shown in the figure. Figure 9As shown, the standard curve has a good linear relationship and can be used to determine the cholesterol content in the sample.

[0095] S4. Determination of the cholesterol-lowering ability of Lactobacillus plantarum Lp-06: Lactobacillus plantarum Lp-06 activated and passaged twice was inoculated into MRS-CHOL medium at a 3% (V / V) inoculum size, cultured at 37°C for 24 hours, and then centrifuged at 13,400 × g for 10 minutes. Culture fluid of the uninoculated Lactobacillus plantarum Lp-06 strain was used as a blank control. The cholesterol concentration in the culture medium was determined using a total cholesterol content detection kit. The cholesterol mass concentration was calculated according to the regression equation of the standard curve, and the cholesterol removal rate was calculated using the formula;

[0096] That is, cholesterol removal rate = (1-C1 / C0) × 100%, where C1 is the mass concentration of cholesterol in the supernatant after the strain is inoculated with MRS-CHOL and cultured for 24 hours, and C0 is the mass concentration of cholesterol in the culture medium without inoculation;

[0097] Reference for in vitro cholesterol-lowering ability of Lactobacillus plantarum Lp-06 Figure 18 The cholesterol degradation rate of Lactobacillus plantarum Lp-06 was 47.66%.

[0098] Example 7

[0099] The antibacterial activity test method of Lactobacillus plantarum Lp-06 comprises the following steps:

[0100] S1. Antibacterial test results: After activating the indicator bacteria Escherichia coli, Listeria monocytogenes, and Staphylococcus aureus, they were inoculated into LB liquid culture medium and cultured at 37°C for 24 hours. The bacterial suspension was prepared for use. After activation of Lactobacillus plantarum Lp-06, it was inoculated into liquid MRS culture medium and cultured at 37°C for 24 hours. It was centrifuged at 8000×g for 5 minutes, and the supernatant was collected. The Oxford cup agar diffusion method was used to spread the plates with Escherichia coli, Listeria monocytogenes, and Staphylococcus aureus as indicator bacteria. Sterile saline was used as the blank control. Four Oxford cups were evenly placed on each plate. 200μL of fermentation supernatant was added to three Oxford cups, and 200μL of sterile saline was added to one Oxford cup. The plates were cultured at 37°C for 24 hours. Observe whether there is an inhibition zone around the bottom of the Oxford cup, measure the diameter of the inhibition zone, and take the average value.

[0101] Reference for experimental data on the antibacterial ability of Lactobacillus plantarum Lp-06 Figure 19 ,from Figure 19 and Figure 10It can be seen that inhibition zones appeared in the culture dishes, and the diameters of the inhibition zones all reached above 14.0 mm, indicating that Lactobacillus plantarum Lp-06 had an inhibitory effect on the growth of Escherichia coli, Staphylococcus aureus and Listeria monocytogenes. The diameters of the inhibition zones were 14.17±0.26 mm, 16.41±0.48 mm and 15.75±0.65 mm, respectively, indicating that Lactobacillus plantarum Lp-06 had obvious antibacterial effect and strong antibacterial ability.

[0102] S5. Comparison of antibacterial effects with common strains. Figure 20 To compare the antibacterial ability with other probiotic strains, Figure 20 It can be seen that the inhibitory effect of Lactobacillus plantarum Lp-06 on Escherichia coli, Staphylococcus aureus and Listeria monocytogenes is significantly higher than that of other common probiotic strains, indicating that Lactobacillus plantarum Lp-06 is an excellent strain with strong antibacterial function.

[0103] Example 8

[0104] The test method for the effect of Lactobacillus plantarum Lp-06 on food intake, body weight, serum total cholesterol, triglycerides, and low-density lipoprotein cholesterol in mice fed a high-fat and high-cholesterol diet comprises the following steps:

[0105] S1. Drug treatment: Eighteen C57BL / 6J mice (half male and half female) were randomly divided into three groups after adaptive feeding for one week: a control group, a high-fat and high-cholesterol model group, and a Lactobacillus plantarum Lp-06 treatment group. The mice were fed with a normal diet, a high-fat and high-cholesterol diet, and a high-fat and high-cholesterol diet plus Lactobacillus plantarum Lp-06, respectively. After eight weeks of feeding, serum biochemical indicators were tested, including TG, TC, and LDL-C; and liver morphology was observed by HE staining.

[0106] like Figure 11 As shown in Figure 2, there was no significant difference in food intake between the high-fat and high-cholesterol model group and the Lactobacillus plantarum Lp-06 treatment group. Figure 12-14 As shown in the results, after 8 weeks of feeding, the serum low-density lipoprotein cholesterol, triglyceride, and total cholesterol levels of the mice were measured. The serum TG, LDL, and TC levels of the mice in the high-fat and high-cholesterol model group were significantly higher than those in the control group, and there was a significant difference. After treatment with Lactobacillus plantarum Lp-06, the serum TG, TC, and LDL-C levels were significantly lower than those in the high-fat and high-cholesterol model group, and there was a significant difference.

[0107] After 8 weeks of feeding, the weight of mice in the high-fat, high-cholesterol model group was significantly higher than that of the control group. However, at 4 weeks, the weight gain of mice in the Lactobacillus plantarum Lp-06-treated group slowed significantly, showing a significant difference compared to the high-fat, high-cholesterol model group. After 6 weeks, the weight of mice in the Lactobacillus plantarum Lp-06-treated group was significantly lower than that of the high-fat, high-cholesterol model group. After 8 weeks, the weight of mice in the Lactobacillus plantarum Lp-06-treated group was basically the same as that of the control group, with no significant difference. Therefore, Lactobacillus plantarum Lp-06 treatment can effectively reduce the weight of mice with high-fat diet-induced obesity. Figure 21 , Figure 21 Weight table for three groups of C57BL / 6J mice fed for eight weeks;

[0108] like Figure 15 As shown in the figure, compared with the control group mice, HE staining of the livers of high-fat and high-cholesterol fed mice showed disordered arrangement of liver cells, displacement of cell nuclei, small lipid droplets in the cytoplasm, and fatty degeneration of the liver. However, after being fed with Lactobacillus plantarum Lp-06, the liver cells of obese mice were neatly arranged, the cell nuclei were uniform in color, no obvious fat vacuoles were observed, and fatty degeneration of the liver was significantly improved.

[0109] Example 9

[0110] Changes in the texture of sea bream intestine with different inoculation amounts of Lactobacillus plantarum Lp-06, reference Figure 22 The results of the changes in the texture of sea bream intestines with different inoculation amounts of Lactobacillus plantarum Lp-06 are as follows: Figure 22 As shown in the figure, with the continuous increase of the strain inoculation amount, the tissue structure of the fermented fish intestine changed. The larger the inoculation amount, the harder the fish intestine increased significantly. This is because the low pH will cause the structure of the protein in the fish intestine to change, the myofibril to contract, and the hardness to increase. The elasticity and cohesion of the fish intestine will first increase and then decrease with the increase of the inoculation amount, indicating that the inoculation of lactic acid bacteria can make the fish intestine firm and elastic, and the cross-section will be smooth and not loose. However, when the inoculation amount is too high, the pH value of the internal environment of the fish intestine will decrease more quickly. Too low a pH value will cause excessive denaturation of muscle protein in the fish intestine, destroy the normal cross-linking between proteins, reduce its ability to form gel, and reduce the water retention of muscle protein, causing the fish intestine to lose water. The adhesiveness of the fish intestine increased significantly with the increase of the inoculation amount, and the chewiness first increased significantly and then decreased. The texture changes of the fish intestine group inoculated with Lactobacillus plantarum Lp-06 were higher than those of the natural fermentation group, indicating that inoculated fermentation can better improve the texture of fish intestine than natural fermentation.

[0111] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing a Lactobacillus plantarum Lp-06 strain, characterized in that: The following steps are involved: S1. Chop Chinese cabbage and lettuce into small pieces, put them into a sterile container, add salt water to cover the vegetables, seal the container, and place it in a cool place to ferment. During the fermentation period, open the lid to vent air, thereby making sauerkraut; S2. Take 1.0 mL of sauerkraut juice and dilute it with normal saline, then evenly spread the fermentation liquid on MRS medium; S3. The MRS culture medium was cultured at a constant temperature of 37° C. for 48 h, single colonies of different morphology and size on the plate were picked, and streaked multiple times on the MRS agar medium for purification to obtain the strain of Lactobacillus plantarum Lp-06.

2. The method for preparing a Lactobacillus plantarum Lp-06 strain according to claim 1, wherein: Lactobacillus plantarum Lp-06 is deposited in the General Microbiology Center of the China Culture Collection Committee of Microorganisms, with the deposit number CGMCC No. 29661 and the deposit date of January 18, 2024. The address of the depository institution is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

3. An application of a Lactobacillus plantarum Lp-06 strain, applicable to the preparation method of a Lactobacillus plantarum Lp-06 strain according to any one of claims 1-2, characterized in that: Lactobacillus plantarum Lp-06 is used to inhibit harmful bacteria, including Escherichia coli, Listeria monocytogenes and Staphylococcus aureus.

4. The use of a Lactobacillus plantarum Lp-06 strain according to claim 3, wherein: Lactobacillus plantarum Lp-06 is used to degrade triglycerides, cholesterol and inhibit pancreatic lipase.

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