Lactobacillus plantarum crude enzyme as well as preparation method and application thereof

Abstract

The invention relates to the technical field of microorganisms and functional application, and particularly provides a lactobacillus plantarum crude enzyme as well as a preparation method and application thereof, the lactobacillus plantarum is Lactobacillus plantarum M3 and is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC NO.26065. The lactobacillus plantarum crude enzyme has the advantages that the lactobacillus plantarum crude enzyme can be used for preparing the lactobacillus plantarum crude enzyme; the lactobacillus plantarum crude enzyme is combined with a low-intensity magnetic field to assist biotransformation of the total ginsenoside to obtain a total ginsenoside transformation product, and the content of protopanaxatriol and rare ginsenoside Rh1, Rg3, Rg5, Rh2 and Rk1 in the total ginsenoside can be increased, and the lipid-lowering activity can be improved.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms and functional applications, and particularly provides a crude enzyme of Lactobacillus plantarum, a preparation method thereof, and an application thereof. Background Art

[0002] Ginseng is the dried root and rhizome of Panax ginseng A.Mey. of the genus Panax in the Araliaceae family, and is a perennial herb. Cultivated ginseng is commonly known as "garden ginseng"; ginseng sown and naturally grown in the wild state in the mountains and forests is called "wild ginseng under the forest", commonly known as "seed sea". Ginseng is sweet, slightly bitter, and slightly warm. It belongs to the spleen, lung, heart, and kidney meridians. It has the effects of greatly tonifying qi, restoring pulse and arresting collapse, invigorating the spleen and benefiting the lung, promoting fluid production and nourishing blood, and calming the mind and improving intelligence, and is a well-known traditional precious Chinese medicine.

[0003] Modern pharmacological research has proved that the main active ingredients contained in ginseng are ginsenosides, and at the same time, it also contains other ingredients such as volatile oils, amino acids, polysaccharides, and trace elements. Ginsenosides also widely exist in plants such as American ginseng, notoginseng, and Gynostemma pentaphyllum. Ginsenosides have a steroid skeleton with seventeen carbon atoms on four rings. According to their main structures, they can be roughly divided into two categories: protopanaxadiol (20(S)-protopanaxadiol, PPD) and protopanaxatriol (20(S)-protopanaxatriol, PPT). At the C-3, C-6, and C-20 positions, glycosyl groups are connected in different forms, positions, and numbers to form different ginsenosides. According to the content, ginsenosides are also divided into main ginsenosides and rare ginsenosides. The absorption rate of main ginsenosides in the body is very low, which limits their utilization. More and more studies have shown that in vivo and in vitro, main ginsenosides are converted into small-molecule rare ginsenosides through deglycosylation, which can effectively improve their bioavailability and biological activity. At present, common saponin conversion methods include chemical methods, physical methods, and biological conversion methods. The preparation efficiency of chemical methods and physical methods is low, the cost is high, and the pollution is serious, which is not conducive to large-scale production and preparation of effective saponins. Due to its characteristics of high selectivity, low by-products, and high target yield, biological conversion methods have received more attention in recent years. Summary of the Invention

[0004] In order to solve the above technical problems, the present invention provides a crude enzyme of Lactobacillus plantarum, a preparation method thereof, and an application thereof.

[0005] The present invention is realized as follows: A crude enzyme of Lactobacillus plantarum is provided, and the Lactobacillus plantarum is Lactobacillus plantarum M3, which is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC NO.26065, and the preservation date is November 7, 2022.

[0006] Provided is a method for preparing crude enzyme of Lactobacillus plantarum. The cryopreserved Lactobacillus plantarum M3 is inoculated into the sterilized MRS medium at an inoculation amount of 2%, and cultured in a constant temperature incubator at 37°C. After 16 h, it is inoculated into a new MRS medium at an inoculation amount of 2% for strain subculture. The fourth-generation strain is used for subsequent experiments. The fourth-generation strain is cultured in a 2-L conical flask until the stationary phase, centrifuged at 4°C and 5000 r / min, and the supernatant is filtered through a 0.22-μm microporous membrane and stored at -20°C to obtain the crude enzyme solution of Lactobacillus plantarum, which is freeze-dried after purification to obtain the crude enzyme of Lactobacillus plantarum.

[0007] Provided is an application of the crude enzyme of Lactobacillus plantarum in the fermentation of total ginsenosides to increase the content of protopanaxatriol, ginsenoside Rh 1 、Rg 3 、Rg 5 、Rh 2 、Rk 1 in the product and improve the lipid-lowering activity of the fermentation product.

[0008] Provided is an application method of the crude enzyme of Lactobacillus plantarum, including the following steps:

[0009] 1) Preparation of the crude enzyme solution of Lactobacillus plantarum;

[0010] 2) The ginseng roots and rhizomes are crushed and sieved, 8 times the mass of 75% alcohol is added, and then heated and refluxed at 80 - 90°C for 3 times, with the extraction times being 2 h, 2 h, and 1 h respectively. After filtration, the filtrates are combined to obtain the crude extract of ginseng. The solvent is recovered under reduced pressure using a rotary evaporator and evaporated to dryness to obtain the ethanol extract paste of ginseng;

[0011] 3) Water is added to the ethanol extract paste of ginseng and mixed, and the mixture is heated to completely dissolve the ethanol extract paste of ginseng to obtain an aqueous solution of the ethanol extract paste; then it is extracted three times with equal volume of water-saturated n-butanol, the n-butanol layers are combined, concentrated and dried to obtain total ginsenosides;

[0012] 4) Citric acid-sodium citrate buffer solution with a pH value of 5 is added to the total ginsenosides, and then heated and stirred until the total ginsenosides are completely dissolved to obtain an aqueous solution of total ginsenosides; then the purified crude enzyme of Lactobacillus plantarum is added to the aqueous solution of total ginsenosides according to the ratio of total ginsenosides: crude enzyme of Lactobacillus plantarum (w / w) = 1:1 - 1:3 for fermentation. The fermentation conditions are: placed in a weak magnetic field of 0.012 - 0.018 T, incubated at a constant temperature of 50 - 54°C, and the fermentation time is 2 - 4 d, thus completing the application of the crude enzyme of Lactobacillus plantarum in the fermentation of total ginsenosides.

[0013] Preferably, the enzyme activity of the crude enzyme of Lactobacillus plantarum is 74 - 95 U / mL.

[0014] Preferably, in the step 2), the method for pulverizing ginseng roots and rhizomes is as follows: slice the ginseng roots and rhizomes, naturally dry them, then dry them in an oven. After pulverizing with a pulverizer and sieving, ginseng powder is obtained; the temperature of the oven is 40 - 75°C, and it is sieved through a 65-mesh sieve.

[0015] Preferably, in the step 2), the ginseng ethanol extract is concentrated under reduced pressure at 60 - 80°C using a rotary evaporator.

[0016] Preferably, in the step 4), the concentration of the aqueous solution of total ginsenosides is 0.03 g / mL.

[0017] Compared with the prior art, the advantages of the present invention are as follows:

[0018] The present invention uses Lactobacillus plantarum M3 (CGMCC NO.26065) to extract crude enzyme, uses the crude enzyme for biotransformation of total ginsenosides, then analyzes the main components of the transformation products of total ginsenosides by ultra-high performance liquid chromatography (UHPLC), and determines the contents of 10 components. Among them, the content of protopanaxatriol (PPT) increases by 10.82 times, and the contents of rare ginsenosides Rh 1 、Rg 3 、Rg 5 、Rh 2 、Rk 1 increase significantly (1.39 - 12.63 times).

[0019] The in vitro lipid-lowering activity of the transformation products was evaluated. The results showed that the products of total ginsenosides biotransformed by the crude enzyme of Lactobacillus plantarum could significantly reduce the intracellular lipid accumulation, and significantly reduce the contents of cholesterol (TC), triglyceride (TG), and low-density lipoprotein cholesterol (LDL-C) in the cells. Among them, the clearance rates of the 450 μg·mL -1 transformation products for TC, TG, and LDL-C reached 30.77%, 53.24%, and 51.54% respectively. And the transformation products could effectively increase the content of high-density lipoprotein cholesterol (HDL-C) in the cells. The content of HDL-C in the treatment group with 450 μg·mL -1 transformation products increased by 29.45%. The crude enzyme of Lactobacillus plantarum and its application described in the present invention are of great significance for the development of new ginseng foods, health products, and drugs. Description of the Drawings

[0020] The following further describes the present invention in detail in conjunction with the drawings and embodiments:

[0021] Figure 1 is the pH value of the biotransformation of the crude enzyme of Lactobacillus plantarum in Example 3;

[0022] Figure 2It is the fermentation temperature for the crude enzyme biotransformation of Lactobacillus plantarum in Example 3;

[0023] Figure 3 It is the substrate concentration for the crude enzyme biotransformation of Lactobacillus plantarum in Example 3;

[0024] Figure 4 It is the fermentation time for the crude enzyme biotransformation of Lactobacillus plantarum in Example 3;

[0025] Figure 5 It is the weak magnetic field intensity for the crude enzyme biotransformation of Lactobacillus plantarum in Example 3;

[0026] Figure 6 It is the liquid chromatography diagram of 10 ginsenoside reference substances in Example 5;

[0027] Figure 7 It is the liquid chromatography diagram of total ginsenosides in Example 5;

[0028] Figure 8 It is the liquid chromatography diagram of the product after the biotransformation of total ginsenosides by the crude enzyme of Lactobacillus plantarum M3 in Example 5;

[0029] Figure 9 It is the liquid chromatography diagram of the product after the biotransformation of total ginsenosides by Lactobacillus plantarum M3 strain in Example 5;

[0030] Figure 10 It is the standard curve of 10 ginsenoside reference substances in Example 5;

[0031] Figure 11 It is the diagram showing the effect of the total ginsenosides prepared in Example 2 and the transformed product of total ginsenosides on the survival rate of L02 cells detected by the MTT method in Example 7; among them, compared with the blank group, *P<0.05 indicates statistical difference, **P<0.01 indicates significant statistical difference, and ***P<0.001 indicates extremely significant statistical difference;

[0032] Figure 12 It is the diagram showing the effect of the total ginsenosides prepared in Example 2 and the transformed product of total ginsenosides on the lipid accumulation in L02 cells incubated with oleic acid detected in Example 7;

[0033] Figure 13 It is the diagram showing the content level of total cholesterol (TC) in cells intervened by the total ginsenosides prepared in Example 2 and the transformed product of total ginsenosides detected in Example 7; where Control is the blank group; Model is the model group; SIM is the positive control group; TGSL is the total ginsenosides 250 μg·mL -1 group; TGSM is the total ginsenosides 350 μg·mL -1 group; TGSH is the total ginsenosides 450 μg·mL -1Group; TGSTPL is the transformed product of total ginsenosides at 250 μg·mL -1 Group; TGSTPM is the transformed product of total ginsenosides at 350 μg·mL -1 Group; TGSTPH is the transformed product of total ginsenosides at 450 μg·mL -1 Group; Compared with the blank group, # P < 0.05 indicates a statistically significant difference, ## P < 0.01 indicates a significant statistically significant difference, ### P < 0.001 indicates an extremely significant statistically significant difference. Compared with the model group, *P < 0.05 indicates a statistically significant difference, **P < 0.01 indicates a significant statistically significant difference, ***P < 0.001 indicates an extremely significant statistically significant difference;

[0034] Figure 14 It is a graph showing the content levels of total intracellular triglycerides (TG) intervened by the total ginsenosides and the transformed products of total ginsenosides prepared in Example 2 in Example 7; where Control is the blank group; Model is the model group; SIM is the positive control group; TGSL is the total ginsenosides at 250 μg·mL -1 Group; TGSM is the total ginsenosides at 350 μg·mL -1 Group; TGSH is the total ginsenosides at 450 μg·mL -1 Group; TGSTPL is the transformed product of total ginsenosides at 250 μg·mL -1 Group; TGSTPM is the transformed product of total ginsenosides at 350 μg·mL -1 Group; TGSTPH is the transformed product of total ginsenosides at 450 μg·mL -1 Group; Compared with the blank group, # P < 0.05 indicates a statistically significant difference, ## P < 0.01 indicates a significant statistically significant difference, ### P < 0.001 indicates an extremely significant statistically significant difference. Compared with the model group, *P < 0.05 indicates a statistically significant difference, **P < 0.01 indicates a significant statistically significant difference, ***P < 0.001 indicates an extremely significant statistically significant difference;

[0035] Figure 15 It is a graph showing the content levels of intracellular low-density lipoprotein cholesterol (LDL-C) intervened by the total ginsenosides and the transformed products of total ginsenosides prepared in Example 2 in Example 7; where Control is the blank group; Model is the model group; SIM is the positive control group; TGSL is the total ginsenosides at 250 μg·mL -1 Group; TGSM is the total ginsenosides at 350 μg·mL -1 Group; TGSH is the total ginsenosides at 450 μg·mL -1Group; TGSTPL is the total ginsenoside transformation product at 250 μg·mL -1 Group; TGSTPM is the total ginsenoside transformation product at 350 μg·mL -1 Group; TGSTPH is the total ginsenoside transformation product at 450 μg·mL -1 Group; Compared with the blank group, # P < 0.05 indicates a statistically significant difference, ## P < 0.01 indicates a significant statistically significant difference, ### P < 0.001 indicates an extremely significant statistically significant difference. Compared with the model group, *P < 0.05 indicates a statistically significant difference, **P < 0.01 indicates a significant statistically significant difference, ***P < 0.001 indicates an extremely significant statistically significant difference;

[0036] Figure 16 It is a graph showing the content level of intracellular high-density lipoprotein cholesterol (HDL-C) intervened by the total ginsenosides prepared in Example 2 and the total ginsenoside transformation products in Example 7; where Control is the blank group; Model is the model group; SIM is the positive control group; TGSL is the total ginsenosides at 250 μg·mL -1 Group; TGSM is the total ginsenosides at 350 μg·mL -1 Group; TGSH is the total ginsenosides at 450 μg·mL -1 Group; TGSTPL is the total ginsenoside transformation product at 250 μg·mL -1 Group; TGSTPM is the total ginsenoside transformation product at 350 μg·mL -1 Group; TGSTPH is the total ginsenoside transformation product at 450 μg·mL -1 Group; Compared with the blank group, # P < 0.05 indicates a statistically significant difference, ## P < 0.01 indicates a significant statistically significant difference, ### P < 0.001 indicates an extremely significant statistically significant difference. Compared with the model group, *P < 0.05 indicates a statistically significant difference, **P < 0.01 indicates a significant statistically significant difference, ***P < 0.001 indicates an extremely significant statistically significant difference;

[0037] Figure 17 It is a flow cytometry apoptosis scatter plot showing the effect of the total ginsenosides prepared in Example 2 and the total ginsenoside transformation products on cell apoptosis in Example 7;

[0038] Figure 18 It is a graph showing the viable cell content level of the effect of the total ginsenosides prepared in Example 2 and the total ginsenoside transformation products on cell apoptosis in Example 7; where compared with the blank group, # P < 0.05 indicates a statistically significant difference,## P < 0.01 indicates significant statistical difference, ### P < 0.001 indicates extremely significant statistical difference. Compared with the model group, *P < 0.05 indicates statistical difference, **P < 0.01 indicates significant statistical difference, ***P < 0.001 indicates extremely significant statistical difference. Detailed implementation manners

[0039] In order to make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0040] The crude enzyme extracted from Lactobacillus plantarum M3 (deposit number CGMCC NO. 26065) used in the present invention is used for the biotransformation of total ginsenosides in ginseng to increase the content of protopanaxatriol (PPT), rare ginsenosides Rh 1 , Rg 3 , Rg 5 , Rh 2 , Rk 1 content and lipid-lowering activity.

[0041] Example 1 Isolation and purification of the crude enzyme of Lactobacillus plantarum of the present invention

[0042] I. Isolation of the crude enzyme of Lactobacillus plantarum

[0043] The laboratory cryopreserved Lactobacillus plantarum strain M3 was inoculated into the sterilized MRS medium at an inoculation amount of 2%, and cultured in a constant temperature incubator at 37°C. After 16 hours, it was inoculated into a new MRS medium at an inoculation amount of 2% for strain subculture, and the fourth-generation strain was used for subsequent experiments. The fourth-generation Lactobacillus plantarum strain was cultured in a 2L conical flask until the stationary phase (12 hours), centrifuged at 4°C and 5000 r / min, and the supernatant was filtered through a 0.22 μm microporous filter membrane and stored at -20°C to obtain the crude enzyme solution.

[0044] II. Purification of the crude enzyme solution

[0045] 35% saturated ammonium sulfate was slowly added to the crude enzyme solution. After the ammonium sulfate was completely dissolved, stirring was continued for half an hour, and it was left to stand overnight at 4°C. After removing the precipitate, ammonium sulfate was continuously added to 80% saturation, and it was left to stand overnight at 4°C. Centrifuged at 4°C and 10,000 r / min for 10 minutes, the supernatant was discarded, and the precipitate was dissolved in a deionized water buffer solution (citric acid - sodium citrate) with a pH of 5 and dialyzed at 4°C for 3 days until no precipitate was detected by barium chloride. After lyophilization, the purified crude enzyme was obtained.

[0046] III. Standard curve of enzyme activity

[0047] Determination of the standard curve of p-nitrophenol (pNP): Weigh 0.0070 g of pNP, dissolve and make up the volume to 50 mL in a volumetric flask. The concentration of pNP is 1 mmol / L. Respectively pipette 0.0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6 mL of 1 mmol / L pNP, add a certain amount of 1 mol / L sodium carbonate solution to make the final volume 10 mL, and prepare standard solutions with concentrations of 0, 10, 20, 30, 40, 50, 60 μmol / L respectively. Then pipette 150 μL of the pNP standard solution into a 96-well plate, and measure the absorbance at 400 nm with an enzyme-labeling instrument.

[0048] Table 1 Standard curve of enzyme activity

[0049]

[0050] IV. Determination of enzyme activity

[0051] Using 4-nitrophenyl-β-D-glucoside (pNPG) as the substrate, take 250 μL of the enzyme solution, add 500 μL of pre-warmed pNPG (5 mmol / L) for 3 min, mix well by pipetting, and incubate at 50 °C for 10 min. Add 1 mL of 1 mol / L Na 2 CO 3 , quench the reaction, observe the color change, measure the absorbance at 400 nm with an enzyme-labeling instrument, substitute it into the formula, and calculate the enzyme activity. One unit of enzyme activity (U) is defined as the amount of enzyme that produces 1 nmol of pNP (p-nitrophenol) per minute in 1 mL of the enzyme solution under the experimental conditions.

[0052] Enzyme activity calculation formula:

[0053]

[0054] U: β-glucosidase activity (U / mL)

[0055] X: The concentration of p-nitrophenol obtained by substituting the absorbance value into the enzyme activity standard curve (μmol / L);

[0056] V 1 : The total volume of the reaction system (mL);

[0057] N: Dilution factor;

[0058] t: Reaction time (min);

[0059] V 2 : The volume of the enzyme in the reaction system (mL).

[0060] The enzyme activity is 74 - 95 U / mL. The crude enzyme extraction rate is 1.16 g / g for the bacterial sludge; 2.42 g / L for the bacterial liquid.

[0061] Example 2

[0062] The method for converting crude enzyme of Lactobacillus plantarum to increase the contents of protopanaxatriol (PPT), rare ginsenosides Rh 1 , Rg 3 , Rg 5 , Rh 2 , Rk 1 and lipid-lowering activity in total ginsenosides of ginseng is as follows:

[0063] I. Preparation of total ginsenosides of ginseng

[0064] The ginseng roots and rhizomes were crushed (passed through a No. 4 sieve), and then mixed with 8 times the volume of 75% alcohol, and heated under reflux at 80 - 90 °C for 3 times, with the extraction times being 2 h, 2 h, and 1 h respectively. After filtration, the filtrates were combined to obtain the ethanol extract of ginseng. The solvent was recovered under reduced pressure and evaporated to dryness to obtain the ethanol extract paste of ginseng. Water was added to the ethanol extract paste of ginseng and mixed, and the mixture was heated to completely dissolve the crude extract paste to obtain an aqueous solution of the crude extract paste; then it was extracted three times with an equal volume of water-saturated n-butanol, the n-butanol layers were combined, concentrated and dried to obtain total ginsenosides of ginseng;

[0065] II. Biotransformation of total ginsenosides of ginseng by crude enzyme of Lactobacillus plantarum

[0066] A citric acid - sodium citrate buffer solution with a suitable pH value of 5 was added to the total ginsenosides of ginseng, and then heated and stirred until the total ginsenosides of ginseng were completely dissolved to obtain an aqueous solution of total ginsenosides of ginseng; then the purified crude enzyme of Lactobacillus plantarum was added to the aqueous solution of total ginsenosides of ginseng according to the ratio of total ginsenosides of ginseng:crude enzyme of Lactobacillus plantarum (w / w) = 1:1 - 1:3 for fermentation. The fermentation conditions were: the sample was placed in a weak magnetic field of 0.012 - 0.018 T, incubated at a constant temperature of 50 - 54 °C, and the fermentation time was 2 - 4 d, thus realizing the application in the fermentation of total ginsenosides of ginseng; among them, the enzyme activity of the crude enzyme of Lactobacillus plantarum was 74 - 95 U / mL.

[0067] Example 3 Single-factor experiment on the biotransformation of total ginsenosides of ginseng by crude enzyme of Lactobacillus plantarum

[0068] Through single-factor experiments, the optimal transformation process for the biotransformation of total ginsenosides of ginseng by the crude enzyme of Lactobacillus plantarum in Example 2 was determined. Ultra-high performance liquid chromatography was used to analyze total ginsenosides of ginseng and their transformation products, and taking the total peak area of the chromatographic peaks of protopanaxatriol (PPT) and rare ginsenosides Rg 3 , Rg 5 , Rh 1 , Rh 2 , Rk 1 whose contents increased during the transformation of total ginsenosides of ginseng as the standard, single-factor experiments were carried out under different transformation conditions, and the results are as Figures 1 - 5 shown.

[0069] The crude enzyme of Lactobacillus plantarum converts total ginsenosides under the condition of pH value ranging from 4.0 to 6.5. As the conversion pH value increases, the peak area shows a trend of first increasing and then decreasing. When the conversion pH value is 5.0, the peak area is the highest, reaching 8766653 AU.

[0070] The crude enzyme of Lactobacillus plantarum converts total ginsenosides under the condition of temperature ranging from 48 to 56 °C. As the conversion temperature increases, the peak area also shows a trend of first increasing and then decreasing. When the conversion temperature is 50 °C, the peak area is the highest, reaching 9280057 AU.

[0071] The ratio of total ginsenosides to the crude enzyme of Lactobacillus plantarum in the conversion system is changed, and total ginsenosides are converted under the conditions of the ratio of total ginsenosides to the crude enzyme of Lactobacillus plantarum being 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, and 4:1 respectively. As the content of the crude enzyme of Lactobacillus plantarum in the conversion system decreases, the peak area of the conversion product first increases and then decreases. When the ratio of total ginsenosides to the crude enzyme of Lactobacillus plantarum is 1:2, the peak area of the conversion product is the largest, reaching 9324165 AU.

[0072] The crude enzyme of Lactobacillus plantarum converts total ginsenosides for 1 - 5 days under the conditions of a conversion temperature of 50 °C and a conversion pH value of 5.0. As the conversion time increases, the peak area of the conversion product gradually increases. After the conversion time reaches 3 days, the peak area of the conversion product tends to be stable. And when the conversion time is 3 days, the peak area is the highest, reaching 8865280 AU.

[0073] During the biotransformation of total ginsenosides by the crude enzyme of Lactobacillus plantarum, a weak magnetic field with an intensity of 0.006 - 0.018 T is applied for auxiliary conversion. As the intensity of the weak magnetic field increases, the peak area of the conversion product first increases and then decreases. When the intensity of the weak magnetic field is 0.015 T, the peak area of the conversion product is the highest, reaching 11527023 AU.

[0074] Based on the results of the above single - factor experiments, the optimal conversion process for the biotransformation of total ginsenosides by the crude enzyme of Lactobacillus plantarum is determined as follows: under the conditions of pH 5.0, the ratio of total ginsenosides to the crude enzyme of Lactobacillus plantarum (w / w) = 1:2, place the sample in a weak magnetic field of 0.015 T, and carry out constant - temperature conversion at 50 °C for 3 days.

[0075] Example 4: Biotransformation of total ginsenosides by Lactobacillus plantarum strains

[0076] This example aims to explore the ability of the crude enzyme of Lactobacillus plantarum to transform total ginsenosides. The biotransformation of total ginsenosides by Lactobacillus plantarum strains was used as a control group to compare the differences in catalytic efficiency and transformation efficiency between the two. The Lactobacillus plantarum strain M3 stored in the laboratory was inoculated into the sterilized MRS medium at an inoculation amount of 2 - 5%, and cultured in a constant temperature incubator at 37°C. After 16 - 24 hours, it was inoculated into a new MRS medium at an inoculation amount of 2 - 5% for strain subculture. The fourth-generation strain was used for subsequent experiments. The strain was inoculated into a mixed medium prepared by mixing an aqueous solution of total ginsenosides and MRS medium at a ratio of 1:4 at an inoculation amount of 2 - 5%, where the content of total ginsenosides was 0.01 - 0.06 g / mL, and cultured at a constant temperature of 37°C for 2 - 4 days to obtain the transformation product of total ginsenosides transformed by Lactobacillus plantarum strains.

[0077] Example 5 Analysis of the Main Components in the Transformation Product of Total Ginsenosides

[0078] The main components in the transformation product of total ginsenosides (the total ginsenosides obtained in Step 1 of Example 2) were detected by UPLC. An American Agilent InfinityLab Poroshell 120SB-AQ (2.7 μm, 3.0 × 100 mm; P / N: 685975-314(T)) chromatographic column was used, with gradient elution of acetonitrile (A) - water (B) (see Table 2), a flow rate of 0.3 mL / min, a column temperature of 30°C, a detection wavelength of 203 nm, and an injection volume of 2 μL. Figure 8 This is the liquid chromatogram of the transformation product of total ginsenosides obtained by the biotransformation of total ginsenosides by the crude enzyme of Lactobacillus plantarum. By comparison Figure 7 It can be seen that after the transformation by the crude enzyme of Lactobacillus plantarum (CGMCC NO.26065), the components in the transformation product have changed significantly compared with those before transformation. According to Figure 6 The liquid chromatograms of 10 ginsenoside reference substances were analyzed to determine the ginsenoside components whose contents changed during the transformation of total ginsenosides. Compared with total ginsenosides, the transformation product showed a significant decrease at the peak positions of Rg 1 , Re, Rb 1 , Rd, and a significant increase at the peak positions of PPT, Rh 1 , Rg 3 , Rg 5 , Rh 2 , Rk 1 . The results show that the crude enzyme transformation of Lactobacillus plantarum converts ginsenosides Rg 1 , Re, Rb 1 , Rd in total ginsenosides into protopanaxatriol (PPT) and rare ginsenosides Rh 1 , Rg 3 , Rg 5, Rh 2 , Rk 1 . However, within the same conversion time, the conversion effect of Lactobacillus plantarum strains is not significant. Figure 9 It is the liquid chromatogram of the conversion product of total ginsenosides after using Lactobacillus plantarum strains to convert total ginsenosides. Comparing Figure 7 It can be seen that after 2 - 4 days of conversion, the changes in various ginsenoside monomers in total ginsenosides are not significant, and there is no obvious increase or decrease in the chromatographic peaks in the liquid chromatogram. Compared with Figure 8 Figure 9 In the original protopanaxatriol (PPT) and rare ginsenoside Rh 1 , Rg 3 , Rg 5 , Rh 2 , Rk 1 the peak heights and peak areas at the peak positions are significantly lower, and there is a large difference from Figure 8 . This indicates that within the same conversion time, the conversion ability of Lactobacillus plantarum strains is relatively poor, and the crude enzyme of Lactobacillus plantarum shows higher conversion efficiency and catalytic efficacy for total ginsenosides, and can significantly shorten the conversion time required. Therefore, extracting the crude enzyme from Lactobacillus plantarum for the bioconversion of total ginsenosides can effectively improve its conversion ability and conversion efficiency.

[0079] Table 2 Elution conditions of the liquid chromatographic mobile phase

[0080]

[0081] Respectively take ginsenoside Rg 1 , Re, Rh 1 , Rb 1 , Rd, Rg 3 , PPT, Rk 1 , Rg 5 and Rh 2 reference substances, and respectively add methanol to make solutions containing 2.35 mg, 0.55 mg, 2.00 mg, 7.75 mg, 2.00 mg, 4.50 mg, 3.00 mg, 2.50 mg, 0.50 mg and 0.85 mg per 1 mL. Qualitative and quantitative analysis of the ginsenoside reference substances is carried out according to the same liquid phase method above. Taking the peak area as the ordinate and the standard substance content (mg / mL) as the abscissa, a standard curve is drawn (such as Figure 10 ), and the linear equations of 10 reference substances are obtained. They show good linearity within their respective concentration ranges, and the results are shown in Table 3.

[0082] Table 3 Linear regression equations of ginsenoside reference substances

[0083] ​

[0084] The contents of the main components in total ginsenosides of Panax ginseng before and after conversion by the crude enzyme of Lactobacillus plantarum were determined according to the above liquid-phase method, and the results are shown in Table 4. Among them, the content of protopanaxatriol (PPT) increased by 10.82 times, and the contents of rare ginsenosides Rh 1 , Rg 3 , Rg 5 , Rh 2 , Rk 1 increased significantly (1.39 - 12.63 times). According to the changes in the contents of the main components in total ginsenosides of Panax ginseng before and after conversion by the crude enzyme of Lactobacillus plantarum, it was analyzed that the biotransformation pathway of total ginsenosides of Panax ginseng by the crude enzyme of Lactobacillus plantarum was mainly divided into two types: protopanaxatriol (PPT)-type ginsenosides: Re → Rg 1 → Rh 1 → PPT; protopanaxadiol (PPD)-type ginsenosides: Rb 1 → Rd → Rg 3 → Rg 5 / Rk 1 / Rh 2 . Among them, protopanaxatriol (PPT) and rare ginsenoside Rh 1 are the main products of the conversion of protopanaxatriol (PPT)-type ginsenosides; rare ginsenosides Rg 3 , Rg 5 , Rh 2 , Rk 1 are the main products of the conversion of protopanaxadiol (PPD)-type ginsenosides.

[0085] Table 4 Contents and ratios of ginsenosides in total ginsenosides of Panax ginseng before and after conversion

[0086]

[0087] Example 6 Influence of weak magnetic field on biotransformation of total ginsenosides of Panax ginseng

[0088] Table 5 Contents of conversion products of total ginsenosides of Panax ginseng under different weak magnetic field intensities

[0089]

[0090]

[0091] According to the above liquid chromatography analysis method, the contents of the main components of total ginsenosides of Panax ginseng after conversion by the crude enzyme of Lactobacillus plantarum under different weak magnetic field intensities were determined, and the results are shown in Table 5. When the weak magnetic field intensity was 0.015 T, the conversion effect of total ginsenosides of Panax ginseng was the best, and the protopanaxatriol (PPT) and rare ginsenosides Rh 1 , Rg 3 , Rg 5 , Rh2 , Rk 1 The total content reaches 2.23 mg / mL, which is 1.57 times that of the control group without weak magnetic field application. At the same time, the contents of ginsenoside Rg 1 , Re, Rd, Rb 1 also decreased to varying degrees compared with the control group without weak magnetic field application. This indicates that the weak magnetic field can significantly promote the enzymatic conversion of total ginsenosides, and the direction of promoting the conversion of total ginsenosides is consistent with the two bioconversion pathways of total ginsenosides described in Example 5. Specifically, after applying a weak magnetic field with an intensity of 0.015 T, the contents of protopanaxatriol (PPT) and rare ginsenosides Rh 1 , Rg 3 , Rg 5 in the conversion products of total ginsenosides increased significantly, rising to 1.56 - 2.51 times that of the control group without weak magnetic field application, while the contents of ginsenoside Rg 1 and Rd decreased significantly. This shows that the weak magnetic field mainly promotes some conversion processes in the bioconversion pathway of total ginsenosides, namely Rg 1 →Rh 1 →PPT and Rd→Rg 3 →Rg 5 . And with the increase of the weak magnetic field intensity, the total contents of protopanaxatriol (PPT) and rare ginsenosides Rh 1 , Rg 3 , Rg 5 , Rh 2 , Rk 1 in the conversion products of total ginsenosides showed a trend of first increasing and then decreasing. When the weak magnetic field intensity was lower than 0.015 T, the weak magnetic field promoted the enzymatic conversion of total ginsenosides and effectively increased the content of rare ginsenosides in the conversion products. However, when the weak magnetic field intensity reached 0.018 T, the content of rare ginsenosides in the conversion products decreased significantly, lower than that of the control group without adding weak magnetic field. This indicates that a stronger weak magnetic field may interfere with the enzymatic conversion, inhibit the activity of crude enzymes, and even cause enzyme inactivation, thereby hindering the bioconversion process of total ginsenosides.

[0092] Example 7 Evaluation of the lipid-lowering activity of the conversion products of total ginsenosides

[0093] (1) Preparation of total ginsenosides: The method was prepared by the preparation method in Step 1 of Example 2 and stored at 4 °C for later use.

[0094] (2) Preparation of the products of the conversion of total ginsenosides by crude enzymes of Lactobacillus plantarum: Prepared by the preparation method in Step 2 of Example 1 and stored at 4 °C for later use.

[0095] (3) The MTT method was used to detect the effect of the conversion products of total ginsenosides on the survival rate of L02 cells.

[0096] Culture L02 cells until the logarithmic growth phase, pour out the culture medium in the culture flask, wash with PBS, add complete medium, pipette the cells with trypsin, transfer them to a centrifuge tube for centrifugation, and count under an inverted microscope. Dilute the cell concentration to 5×10 4 cells / mL. Transfer the diluted cell suspension into a 96-well plate, add 100 μL to each well, and place the 96-well culture plate in a cell culture incubator at 37 °C containing 5% CO 2 for 24 h. After the cells adhere after 24 h of culture, aspirate the culture medium, add different concentration drug treatment groups diluted with basal medium. The normal control group only adds basal medium, and continue to culture in a cell culture incubator at 37 °C containing 5% CO 2 for 24 h. After 24 h of drug treatment, carefully aspirate the culture medium in the well plate, add 100 μL of 0.5 mg / mL MTT to each well, and continue to culture in the incubator for 4 h. Carefully aspirate the liquid in the 96-well plate, add 150 μL of DMSO to each well, oscillate in an enzyme-linked immunosorbent assay (ELISA) reader for 10 min, and then measure the absorbance at 490 nm. Calculation of relative cell viability:

[0097] Relative cell viability (%) = (absorbance of experimental group - absorbance of normal control group) / (absorbance of control group - absorbance of normal control group) × 100%

[0098] Figure 11 This is the effect of the total ginsenoside transformation product prepared in Example 2 on the viability of L02 cells detected by the MTT method. As Figure 11 shown (compared with the blank control group, *P < 0.05 indicates statistical significance, **P < 0.01 indicates significant statistical significance, ***P < 0.001 indicates extremely significant statistical significance), in vitro cell experiments, within the concentration range of 0 - 600 μg / mL, the viability of L02 cells shows a trend of first increasing and then decreasing, and the cell viability exceeds 90%. Therefore, within the concentration range of 0 - 600 μg / mL, the total ginsenoside transformation product prepared in Example 2 has no obvious toxicity to L02 cells.

[0099] (4) Observe the effect of the total ginsenoside transformation product on lipid accumulation in L02 cells by Oil Red O staining method.

[0100] Establish a cell lipid accumulation model by inducing L02 cells with oleic acid, and observe the intervention of high, medium, and low doses of the total ginsenoside transformation product by Oil Red O fat staining method. From Figure 12It can be seen that total ginsenosides and the transformation products of total ginsenosides both have certain effects on lipid accumulation in L02 cells incubated with oleic acid. Compared with total ginsenosides at the same concentration, the lipid accumulation in L02 cells incubated with the transformation products of total ginsenosides is relatively less, and with the increase of the drug concentration, the lipid accumulation in cells also significantly decreases. Thus, it can be seen that both of them can reduce lipid accumulation in L02 cells.

[0101] (5) Determine the content levels of total cholesterol (TC), total triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) in cells.

[0102] Figures 13 - 16 They are respectively the graphs of determining the content levels of TC, TG, LDL-C, and HDL-C in cells. The results show that the fermentation product of total ginsenosides transformed by the crude enzyme of Lactobacillus plantarum can significantly reduce the lipid accumulation phenomenon in cells, and significantly reduce the contents of TC and TG in cells. Among them, the clearance rates of the transformation products of total ginsenosides at 450 μg·mL -1 for TC and TG reach 30.77% and 53.24% respectively. The content of LDL-C in L02 cells incubated with oleic acid is reduced by 51.54% by the transformation products of total ginsenosides, and the growth rate of the content of HDL-C can reach 29.45%.

[0103] (6) Use a flow cytometer to determine the effect of the transformation products of total ginsenosides in Example 2 on the apoptosis of L02 cells.

[0104] As Figures 17 - 18 shown, compared with the model group, the percentage of viable cells in the blank control group is relatively high, reaching 82.8%, and the apoptosis rate is low. The difference is significantly changed and has statistical significance (P < 0.001); Simvastatin, total ginsenosides, and the transformation products of total ginsenosides can all increase the percentage of viable cells in L02 cells and effectively inhibit cell apoptosis, and the difference has statistical significance (P < 0.05). The results show that the percentage of viable cells in L02 cells incubated with the transformation products of total ginsenosides transformed by the crude enzyme of Lactobacillus plantarum for 24 h is 71.5%, while the percentages of viable cells in L02 cells incubated with total ginsenosides before transformation and the positive control simvastatin for 24 h are only 65.3% and 66.3% respectively. This indicates that the total ginsenosides transformed by the crude enzyme of Lactobacillus plantarum have a stronger inhibitory effect on cell apoptosis than total ginsenosides before transformation and the positive control simvastatin, and it can better inhibit the apoptosis of L02 cells.

[0105] The above has described the embodiments of the present invention in detail with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made without departing from the gist of the present invention within the knowledge scope of those of ordinary skill in the art.

Claims

1. A crude Lactobacillus plantarum enzyme, characterized in that The plant lactobacillus is Lactobacillus plantarum M3, which is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, with a deposit number of CGMCC NO.26065.

2. A method for preparing a crude Lactobacillus plantarum enzyme, characterized in that: The frozen Lactobacillus plantarum M3 is inoculated into a sterilized MRS medium at a 2% inoculation amount, and cultured in a constant temperature incubator at 37° C. After 16 hours, the inoculation amount is inoculated into a new MRS medium at a 2% inoculation amount, and the strain is subcultured. The fourth-generation strain is used for subsequent experiments, and the fourth-generation strain is placed in a 2L conical flask and cultured to a stable period, centrifuged at 4° C. and 5000 r / min, and the supernatant is filtered through a 0.22 μm microporous filter membrane and then stored at -20° C. to obtain a crude Lactobacillus plantarum enzyme solution, which is purified and freeze-dried to obtain a crude Lactobacillus plantarum enzyme.

3. The application of the crude Lactobacillus plantarum enzyme according to claim 1, characterized in that: Used in the fermentation of total ginsenosides of ginseng to increase the content of protopanaxatriol, ginsenosides Rh1, Rg3, Rg5, Rh2, and Rk1 in the product and to improve the lipid-lowering activity of the fermentation product.

4. A method for applying the crude Lactobacillus plantarum enzyme according to claim 1, characterized in that: The following steps are involved: 1) Preparation of crude Lactobacillus plantarum enzyme solution; 2) Ginseng roots and rhizomes are crushed and sieved, 8 times the amount of 75% alcohol by weight is added, heated, and refluxed for extraction at 80-90° C. for 3 times, with extraction times of 2 h, 2 h, and 1 h, respectively, filtered, and the filtrates are combined to obtain a crude ginseng extract, and the solvent is recovered under reduced pressure using a rotary evaporator, evaporated to dryness, and a ginseng alcohol extract is obtained; 3) adding water to the ginseng alcohol extract and mixing, heating the mixture to completely dissolve the ginseng alcohol extract to obtain an alcohol extract aqueous solution; then extracting three times with an equal volume of water-saturated n-butanol, combining the n-butanol layers, concentrating and drying, and obtaining ginseng total saponins; 4) adding a citric acid-sodium citrate buffer solution with a pH value of 5 to the total ginsenosides, and then heating and stirring until the total ginsenosides are completely dissolved to obtain a total ginsenosides aqueous solution; then adding the purified Lactobacillus plantarum crude enzyme to the total ginsenosides aqueous solution in a ratio of ginsenosides: Lactobacillus plantarum crude enzyme (w / w) = 1:1 to 1:3 for fermentation, and the fermentation conditions are: placing in a weak magnetic field of 0.012 to 0.018T, culturing at a constant temperature of 50 to 54°C, and fermenting for 2 to 4 days, thus completing the application of the Lactobacillus plantarum crude enzyme in the fermentation of total ginsenosides.

5. The application method of the crude Lactobacillus plantarum enzyme according to claim 4, characterized in that: The crude enzyme activity of Lactobacillus plantarum is 74-95U / mL.

6. The application method of the crude Lactobacillus plantarum enzyme according to claim 4, characterized in that: In the step 2), the ginseng root and rhizome are crushed by slicing the ginseng root and rhizome, drying them naturally in the sun and then drying them in an oven, crushing them in a grinder and sieving them to obtain ginseng powder; wherein the oven temperature is 40-75° C. and the powder is sieved through a 65-mesh sieve.

7. The application method of the crude Lactobacillus plantarum enzyme according to claim 4, characterized in that: In the step 2), the ginseng alcohol extract is concentrated under reduced pressure at 60-80° C. using a rotary evaporator.

8. The application method of the crude Lactobacillus plantarum enzyme according to claim 4, characterized in that: In the step 4), the concentration of the ginsenosides aqueous solution is 0.03 g / mL.

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