Lactobacillus plantarum as well as preparation and application thereof

Abstract

The invention provides lactobacillus plantarum as well as a preparation and application thereof, the lactobacillus plantarum is lactobacillus plantarum WMTY01 and is preserved in the China General Microbiological Culture Collection Center on December 20, 2023, the preservation address is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC No.29360. The lactobacillus plantarum has the advantages that the lactobacillus plantarum is a lactobacillus plantarum strain; the lactobacillus plantarum can produce beta-glucosidase at high yield, ginsenoside is efficiently converted into rare ginsenoside, the conversion efficiency is high, and meanwhile, the safety in the conversion process is guaranteed.

Description

Technical Field

[0001] The invention belongs to the technical field of biological fermentation, and in particular relates to a plant lactobacillus and a preparation and application thereof. Background Art

[0002] Ginseng usually refers to the dried root of Panax ginseng CAMey, which is named after its dried root is spindle-shaped with branches and looks like a human figure. The earliest detailed record of ginseng was in books in the field of medicine, such as "Shenlong Bencao Jing", which recorded that ginseng has the effect of "tonifying the five internal organs, calming the spirit, calming the soul, stopping panic, removing evil spirits, improving eyesight, opening the heart and improving intelligence, and long-term use of ginseng can make the body lighter and prolong life"; "Compendium of Materia Medica" also recorded that it has the effect of "treating all deficiency syndromes of men and women, fever, spontaneous sweating, dizziness, vomiting blood, coughing up blood, bleeding, hematuria, hemorrhage, and various diseases before and after pregnancy". Later, Chinese ancient medical works such as "Tang Materia Medica", "Treatise on Febrile Diseases" and "Famous Doctors' Records" recorded its new effects. Contemporary scholars at home and abroad have also conducted extensive and in-depth research on ginseng, mainly focusing on the chemical structure and biological functions of the ingredients, most of which are concentrated in the field of medicine.

[0003] Ginsenoside is its main active substance. Modern pharmacological research shows that ginsenoside can resist oxidation, inflammation, bacteria, cardiovascular disease, diabetes, cancer, reduce obesity, improve energy and sexual endurance, especially the monomer rare ginsenoside has high antioxidant, anti-inflammatory, antibacterial, cardiovascular disease, diabetes, cancer and other functions, and has a very high utilization value for the human body. Rare ginsenoside Rg3 is a secondary hydrolysis product of protopanaxadiol saponin, which can generally be extracted from ginseng roots, but the content is very small, and its molecular formula is C 42 H 72 O 13 Ginsenoside Rg3 has a strong toxic effect on solid tumor cells and can inhibit the proliferation and metastasis of Lewis lung cancer cells. [1] ; It was also found that 20(S)-Rg3 has a dual regulatory effect on the central nervous system. One is that it can serve as a nutritional factor for the nerve center, and the other is to protect spinal cord neurons by reducing the body's utilization of neurotransmitters and reducing the release of neurotransmitters; Ginsenoside Rg3 also has a hypoglycemic effect in the body. It can not only stimulate the body to secrete insulin and increase the amount of insulin secretion, but also promote AMP (adenosine phosphate protein kinase) phosphorylation, thereby reducing blood sugar levels in the body. [2] .

[0004] Ginsenosides (including Rb1, Rb2, Re, and Rg1) that have been found to exist in nature account for almost 80-90% of all ginsenosides. These ginsenosides are difficult to be absorbed by the human body due to their large size, low cell membrane permeability, and poor bioavailability. However, deglycosylated ginsenosides such as ginsenoside Rg3 obtained by converting these ginsenosides do not exist in natural ginseng, so it is urgent to develop a production method for ginsenoside Rg3.

[0005] [1]Geng L, Fan J, Gao QL, et al. Preliminary study for the roles and mechanisms of 20(R)-ginsenoside Rg3 and PEG-PLGA-Rg3 nanoparticles in the Lewis lung cancer mice[J]. Beijing Da Xue Xue Bao Yi Xue Ban. 2016Jun 18;48(3):496-501.

[0006] [2] Park MW, Ha J, Chung SH. 20(S)-ginsenoside Rg3 enhances glucose-stimulated insulin secretion and activates AMPK. Biol Pharm Bull[J]. 2008Apr; 31(4):748-51. Summary of the invention

[0007] In view of the above shortcomings, the present invention provides a plant lactobacillus and its preparation and application, the plant lactobacillus is plant lactobacillus WMTY01 (Lactobacillus plantarum WMTY01), which has been deposited in the General Microbiological Center of China Microbiological Culture Collection Administration Committee on December 20, 2023, with a deposit address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and a deposit number of CGMCC No. 29360. The plant lactobacillus can produce high β-glucosidase, efficiently convert ginsenosides into rare ginsenosides, with high conversion efficiency, while ensuring safety during the conversion process.

[0008] the term:

[0009] 1. β-Glucosidase: It is a common enzyme in probiotics. It can hydrolyze the glycosidic bonds between aromatic sugar-based atomic groups and is used for the conversion of ginsenosides. It hydrolyzes the sugar groups on the side chains of common saponins into rare saponins with higher medicinal value.

[0010] 2. p-Nitrophenyl-β-D-glucoside (p-NPG): p-NPG is the enzymatic substrate of β-glucosidase, and one of its reaction products is p-nitrophenol (pNP). The hydrolysis activity of β-glucosidase can be evaluated by directly measuring the absorbance of pNP at a wavelength of 400-420nm using a spectrophotometer.

[0011] 3. Enzyme activity: refers to the ability to hydrolyze substrates. The enzyme activity can be obtained by the amount of substrate consumed.

[0012] 4. API 50 CH: It is a standardized identification system that uses 50 biochemical tests to study the carbohydrate metabolism of microorganisms.

[0013] The technical solution of the present invention includes:

[0014] On the one hand, the present invention provides a Lactobacillus plantarum (Lactobacillus plantarumWMTY01), which is Lactobacillus plantarum WMTY01, which was deposited in the General Microbiology Center of China National Committee for the Preservation of Microorganisms on December 20, 2023, with a preservation address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and a preservation number of CGMCC No. 29360.

[0015] On the other hand, the present invention provides a Lactobacillus plantarum preparation, which comprises one or more of the culture, bacteria, fermentation liquid, fermentation supernatant, fermentation liquid precipitate, and freeze-dried powder of the above-mentioned Lactobacillus plantarum.

[0016] In another aspect, the present invention provides a method for culturing the above-mentioned Lactobacillus plantarum, which comprises inoculating the above-mentioned Lactobacillus plantarum into a culture medium.

[0017] Specifically, the culture medium is MRS agar medium.

[0018] In yet another aspect, the present invention provides use of the above-mentioned Lactobacillus plantarum WMTY01 or Lactobacillus plantarum preparation in the production of β-glucosidase.

[0019] Specifically, the application is to increase the production of β-glucosidase.

[0020] In another aspect, the present invention provides a method for preparing β-glucosidase, comprising inoculating the above-mentioned Lactobacillus plantarum into MRS agar medium.

[0021] In another aspect, the present invention provides use of the above-mentioned Lactobacillus plantarum WMTY01 or Lactobacillus plantarum preparation in the production of ginsenosides.

[0022] Specifically, the ginsenoside is ginsenoside Rg3.

[0023] Specifically, the ginsenoside Rg3 is prepared by fermenting ginseng.

[0024] Preferably, the ginsenoside Rg3 is prepared by fermenting ginseng with a fermentation broth of Lactobacillus plantarum.

[0025] In another aspect, the present invention provides a use of the above-mentioned Lactobacillus plantarum WMTY01 or Lactobacillus plantarum preparation in the preparation of medicines or health products, wherein the medicines or health products are prepared by fermenting ginseng.

[0026] Specifically, the drug is used for anti-oxidation, anti-inflammatory, antibacterial, antiviral, anti-cardiovascular disease, anti-diabetes, anti-central nervous system disease or anti-cancer.

[0027] Preferably, the drug further comprises a pharmaceutically acceptable excipient or carrier.

[0028] Specifically, the health care product is used for anti-oxidation, maintaining cardiovascular health, balancing blood sugar, resisting fatigue or improving energy.

[0029] In another aspect, the present invention provides a method for preparing ginsenoside Rg3, comprising fermenting ginseng using the above-mentioned Lactobacillus plantarum or Lactobacillus plantarum preparation.

[0030] The method comprises the following steps:

[0031] (1) Grind the ginseng roots into powder, pass through a 60-120 mesh sieve to obtain ginseng powder, and add water to prepare a fermentation slurry;

[0032] (2) adding an enzymatic hydrolyzing agent to the fermentation homogenate described in step (1) for enzymatic hydrolysis, and adjusting the pH to obtain an enzymatic hydrolyzate;

[0033] (3) keeping the enzymatic hydrolyzate of step (2) at 60-137° C. for 35-70 min to obtain a sterilized solution;

[0034] (4) The sterilized liquid in step (3) is cooled to 10-30° C., and Lactobacillus plantarum WMTY01 is inoculated into the sterilized liquid. The timing is started from the time of inoculation of the strain, and fermentation is performed to obtain a fermentation liquid.

[0035] Specifically, the weight ratio of the ginseng powder to water in step (1) is 1:1-20.

[0036] Preferably, the weight ratio of the ginseng powder to water in step (1) is 1:5-10.

[0037] Specifically, the weight ratio of the ginseng powder in step (1) to the enzymatic hydrolyzing agent in step (2) is 10-30:1

[0038] Preferably, the weight ratio of the ginseng powder described in step (1) to the enzymatic hydrolyzing agent described in step (2) is 15-20:1.

[0039] Specifically, the enzymatic agent in step (2) includes cellulase and pectinase.

[0040] Preferably, in the enzymatic agent described in step (2), the weight ratio of cellulase to pectinase is 1:1.

[0041] Specifically, the cellulase activity is 10,000-30,000 U / g; the pectinase activity is 20,000-40,000 U / g.

[0042] Preferably, the cellulase activity is 20,000 U / g; the pectinase activity is 30,000 U / g.

[0043] Specifically, the pH adjuster of the pH in step (2) is sodium bicarbonate.

[0044] Specifically, the pH in step (2) is 5.5-7.0.

[0045] Specifically, the inoculation amount of Lactobacillus plantarum in step (4) is 1%-20%.

[0046] More specifically, the inoculation amount of Lactobacillus plantarum in step (4) is 2%-14%.

[0047] Preferably, the inoculation amount of Lactobacillus plantarum in step (4) is 14%.

[0048] Specifically, the fermentation temperature in step (4) is 25-50°C.

[0049] More specifically, the fermentation temperature in step (4) is 27-32°C.

[0050] Preferably, the fermentation temperature in step (4) is 28°C.

[0051] Specifically, the fermentation time in step (4) is 15-30 days.

[0052] More specifically, the fermentation time in step (4) is 18-27 days.

[0053] Preferably, the fermentation time in step (4) is 25 days.

[0054] The technical effects achieved by the present invention are:

[0055] (1) The present invention provides a Lactobacillus plantarum WMTY01, which has been deposited in the General Microbiological Center of China National Microbiological Culture Collection Administration on December 20, 2023, with the deposit address at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 29360.

[0056] (2) The Lactobacillus plantarum WMTY01 provided by the present invention can produce a high amount of β-glucosidase.

[0057] (3) The Lactobacillus plantarum WMTY01 provided by the present invention can efficiently convert ginsenosides into rare ginsenosides with high conversion efficiency while ensuring the safety of the conversion process.

[0058] Collection Instructions

[0059] Deposited strain: Lactobacillus plantarum WMTY01;

[0060] Classification and nomenclature: Lactobacillus plantarum;

[0061] Deposit number: CGMCC No.29360;

[0062] Deposit date: December 20, 2023;

[0063] Depository: General Microbiology Center, China Microbiological Culture Collection Administration;

[0064] Collection address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 The standard curve of p-nitrophenol.

[0066] Figure 2 The figure shows the growth curve and bacterial activity.

[0067] Figure 3 This is the response surface diagram of bacterial quantity, temperature and Rg3 production.

[0068] Figure 4 This is the response surface diagram of inoculum amount, fermentation time and Rg3 yield.

[0069] Figure 5 This is the response surface diagram of temperature, fermentation time and Rg3 production.

[0070] Figure 6 This is the liquid phase detection diagram of rare ginsenoside Rg3 fermentation liquid. DETAILED DESCRIPTION

[0071] The present invention is described below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention so that the technical solution of the present invention is easier to understand and grasp. The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are all commercially available unless otherwise specified.

[0072] In a specific embodiment of the present invention, the culture medium formula used is as follows:

[0073] 1. MRS medium modified with esculin: 10 g peptone, 8 g beef powder, 4 g yeast powder, 2 g dipotassium hydrogen phosphate, 2 g diammonium hydrogen citrate, 5 g sodium acetate, 0.2 g magnesium sulfate, 0.04 g manganese sulfate, 1 g Tween 80, 3 g esculin, and 1.5 g / L ferric citrate to prepare 1 L of MRS medium modified with esculin.

[0074] 2. MRS liquid culture medium: peptone 10g, beef powder 8g, yeast powder 4g, arbutin 20g, dipotassium hydrogen phosphate 2g, diammonium hydrogen citrate 2g, sodium acetate 5g, magnesium sulfate 0.2g, manganese sulfate 0.04g, Tween 80 1g / L, prepare 1L liquid culture medium.

[0075] 3. MRS agar medium: 15g agar, 10g peptone, 8g beef powder, 4g yeast powder, 20g arbutin, 2g dipotassium hydrogen phosphate, 2g diammonium hydrogen citrate, 5g sodium acetate, 0.2g magnesium sulfate, and 0.04g / L manganese sulfate to prepare 1L solid culture medium.

[0076] In a specific embodiment of the present invention, the strain screening method used is as follows:

[0077] Take 5g of sheep feces sample and dissolve it in a 50mL sterile centrifuge tube with normal saline. Then dilute it four times with normal saline to a concentration of 10% of the initial concentration. -4 Times. Pipette 100μg of diluted sample solution and drop it on MRS agar medium and spread it evenly. After the sample solution penetrates into the medium, seal the culture dish with sealing glue and place it in a 37℃ incubator for inversion culture for 32-46h. Pick a single colony with a morphology similar to that of lactic acid bacteria for continuous streaking and purification culture. Perform Gram staining on the purified strains, observe the morphology of the bacteria under an optical microscope, select strains with morphological characteristics of lactic acid bacteria for further identification, obtain strains 1-15, and store them in a strain library at -80℃.

[0078] Take 5g of cow feces sample and dissolve it in a 50mL sterile centrifuge tube with normal saline. Then dilute it four times with normal saline to a concentration of 10% of the initial concentration. -4Times. Pipette 100μg of diluted sample solution and drop it on MRS agar medium and spread it evenly. After the sample solution penetrates into the medium, seal the culture dish with sealing glue and place it in a 37℃ incubator for inversion culture for 32-46h. Pick a single colony with a morphology similar to that of lactic acid bacteria for continuous streaking and purification culture. Perform Gram staining on the purified strains, observe the morphology of the bacteria under an optical microscope, select strains with morphological characteristics of lactic acid bacteria for further identification, obtain strains No. 16-30, and store them in the strain library at -80℃.

[0079] Take 5g of child feces sample and dissolve it in a 50mL sterile centrifuge tube with saline. Then dilute it 4 times with saline to a concentration of 10 times the initial concentration. -4 Times. Pipette 100μg of diluted sample solution and drop it on MRS agar medium and spread it evenly. After the sample solution penetrates into the medium, seal the culture dish with sealing glue and place it in a 37℃ incubator for inversion culture for 32-46h. Pick a single colony with a morphology similar to that of lactic acid bacteria for continuous streaking and purification culture. Perform Gram staining on the purified strains, observe the morphology of the bacteria under an optical microscope, select strains with morphological characteristics of lactic acid bacteria for further identification, obtain strains No. 31-50, and store them in the strain library at -80℃.

[0080] Basic Experimental Example 1 Determination of β-glucosidase activity

[0081] (1) Determination of p-nitrophenol concentration:

[0082] The hydrolysis activity of β-glucosidase was evaluated by ultraviolet spectrophotometer. The specific determination method was as follows: 7 dry and clean test tubes were taken, numbered 0, 1, 2, 3, 4, 5, and 6, and 0, 0.1, 0.2, 0.3, 0.4, 0.5, and 0.6 mL of p-nitrophenol standard solution were accurately weighed, 1, 0.9, 0.8, 0.7, 0.6, 0.5, and 0.4 mL of acetic acid-sodium acetate buffer were added, and 2 mL of 0.5 mol / L sodium carbonate solution was added and mixed thoroughly. The absorbance value was measured at 410 nm using an ultraviolet spectrophotometer, and a standard curve was prepared with p-nitrophenol concentration as the X-axis and absorbance as the Y-axis. Figure 1 .

[0083] A regression equation was established based on the standard curve to calculate the corresponding pNP concentration, and the regression equation was y=0.1496x+0.0151;

[0084] Where Y represents absorbance in AU, and x represents the concentration of p-nitrophenol in mmol / L.

[0085] (2) Calculation of β-glucosidase activity

[0086] Take 1mL of bacterial solution and centrifuge it at 10000rpm for 5min. The supernatant obtained is the crude enzyme solution. Take 0.1mL of crude enzyme solution in a test tube, add 0.2mL of 5mmol / L p-PNG solution and 0.7mL of acetic acid-sodium acetate buffer with pH 5.0, and react in a 50℃ water bath for 30min. Then immediately add 2.0mL of 0.5mol / L sodium carbonate solution to terminate the reaction. Add 3.0mL of distilled water and vortex mix. Add the crude enzyme solution inactivated by heating as the blank control group, and use a UV spectrophotometer to measure its absorbance at 410nm. Calculate the enzyme activity based on its absorbance.

[0087] The enzymatic activity of β-glucosidase was calculated according to the formula U = CV / tV′;

[0088] Wherein, U represents the enzyme activity of β-glucosidase, in U / mL; C represents the concentration of pNP, in the value on the p-nitrophenol-optical density curve; V represents the total volume of the reaction solution, in mL; t represents the reaction time, in min; V′ represents the volume of the enzyme crude solution, in mL.

[0089] Basic Experimental Example 2 Determination of saponin content:

[0090] The saponin content was determined by high performance liquid chromatography (HPLC), and the HPLC parameters were as follows:

[0091] Chromatographic column: C18 (5μm 4.6mm×250mm). Mobile phase A is acetonitrile; mobile phase B is distilled water. Gradient elution conditions are 0-10min mobile phase A 30-44%; 10-25min mobile phase A 44-50%; 25-40min mobile phase A 50-100%; 40-60min mobile phase A 30%, using C18 (250mm×4.6mm, 5μm) chromatographic column, flow rate 1.0mL / min, detection wavelength 203nm, column temperature set to 30℃.

[0092] Example 1 Determination of β-glucosidase production capacity

[0093] Strain No. 1-50 was obtained from the strain library, and the ability of strain No. 1-50 to produce β-glucosidase lactic acid bacteria was determined using MRS medium modified with esculin. The results are shown in Table 1.

[0094] Table 11-50 strain's ability to produce β-glucosidase

[0095]

[0096]

[0097]

[0098] The above results show that 36% (18 / 50) of the strains can secrete β-glucosidase lactic acid bacteria, making the MRS medium modified by aesculin black. The strains that can secrete β-glucosidase lactic acid bacteria are: No. 1-5, No. 11, No. 18, No. 22, No. 24, No. 32, No. 34-35, No. 37, No. 39, No. 41, No. 45, No. 48.

[0099] Example 2 Determination of β-glucosidase activity

[0100] Accurately weigh p-nitrobenzene-β-D-glucoside (p-NPG) and prepare it into a 5 mol / L solution with sodium acetate buffer solution. Centrifuge No. 1-5, No. 11, No. 18, No. 22, No. 24, No. 32, No. 34-35, No. 37, No. 39, No. 41, No. 45, No. 48 bacterial liquid at 5000rpm for 5 minutes, take 100 μL of supernatant, add 700 μL of sodium acetate buffer, and mix with 2mL of p-NPG solution (preheated at 37℃ for 10min) for 10min after 37℃ constant temperature water bath, add 2mL of Na2CO3 (1mol / L) solution to terminate the reaction, and cool to room temperature at room temperature to obtain a reaction solution. Use the crude enzyme solution inactivated by heating as a blank control, and use a spectrophotometer to measure the ultraviolet absorbance of the reaction solution at a wavelength of 405nm. According to the method described in the basic experimental example, the enzymatic activity of β-glucosidase was calculated. The results are shown in Table 2:

[0101] Table 2 Enzyme activity of β-glucosidase

[0102]

[0103] The above results show that the enzyme activities of strains 1, 5 and 45 are relatively high, with the highest being 0.80U / mL.

[0104] Example 3 Ginseng fermentation test

[0105] Ginseng was pre-fermented using strains No. 1, No. 5, and No. 45, and the fermentation steps were as follows:

[0106] (1) Grind the ginseng roots and pass them through a 90-mesh sieve to obtain ginseng powder. Take 12 g of ginseng powder and add 78 mL of water to prepare a fermentation slurry;

[0107] (2) adding 0.6 g of an enzymatic agent to the fermentation homogenate, wherein the enzyme preparation includes 0.3 g of cellulase and 0.3 g of pectinase, wherein the enzymatic activity of the cellulase is 20,000 U / g and the enzymatic activity of the pectinase is 30,000 U / g, performing enzymolysis at 60° C. for 120 min, and adjusting the pH to 6.5 to obtain an enzymatic solution;

[0108] (3) The enzymatic hydrolyzate is kept at 90°C for 60 min to obtain a sterilized solution;

[0109] (4) cooling the sterilized liquid to room temperature of 25° C., adding 10% plant lactobacillus No. 1, No. 5, and No. 45 to 90 mL of the sterilized liquid, respectively, starting from the time of inoculation of the strains, and fermenting at a fermentation temperature of 37° C. for 21 days to obtain a fermentation liquid;

[0110] The HPLC method described in Basic Experimental Example 2 was used to determine the rare saponin content of the above sterilized liquid and fermented liquid. The results are shown in Table 3.

[0111] Table 3 Ginsenoside transformation after 21 days of fermentation

[0112]

[0113] The results showed that after 21 days of transformation, the content of rare ginsenoside Rg3 in strain No. 1 increased from 0.03 mg / mL to 0.20 mg / mL, an increase of 566.67%; while the corresponding Rb1 content decreased from 4.80 mg / mL before fermentation to 4.61 mg / mL.

[0114] Example 4 Identification of WMTY01 strain

[0115] After identification, strain No. 1 was determined to be Lactobacillus plantarum and was officially named Lactobacillus plantarum WMTY01. The bacteria was deposited in the General Microbiology Center of China Culture Collection Administration on December 20, 2023. The deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 29360.

[0116] Example 5 Utilization ability of Lactobacillus plantarum WMTY01 on 48 carbohydrates

[0117] API 50CH was used to measure the ability of plant lactobacillus WMTY01 to utilize carbohydrates. When different substances in the substrate available to bacteria are the sole carbon source, if the bacteria can utilize or grow, the color changes, which is indicated by positive (+), and the color does not change, which is indicated by negative (-). The specific operation is as follows: WMTY01 is cultured in MRS liquid medium for 24 hours for activation, and a suspension of two McFarland turbidities is prepared. The suspension is added to 50 microbiological tubes on the API reagent strip, and the ability of plant lactobacillus WMTY01 to utilize carbohydrates is measured. The measurement results are shown in Table 4.

[0118] Table 4 WMTY01's ability to utilize 48 carbohydrates

[0119]

[0120] Example 6 Growth curve and bacterial activity of Lactobacillus plantarum WMTY01

[0121] Lactobacillus plantarum WMTY01 was cultured in MRS liquid medium, and samples of the growing Lactobacillus plantarum were taken every 2 hours for the following determinations:

[0122] (1) Dilute the bacterial solution to 10 -5 , 10 -6 , 10 -7 Three concentration gradients were spread on solid MRS medium and the colony growth was observed after 48 hours; (2) the solution was diluted to the visible range of the UV spectrophotometer (within the concentration range of the standard curve) and its absorbance was measured. Figure 2 shown.

[0123] The results showed that Lactobacillus plantarum WMTY01 entered the logarithmic growth phase after 6 hours of growth, the bacterial body increased rapidly, and reached the stable growth phase after 18 hours, at which time the number of live bacteria remained relatively stable.

[0124] Example 7 Preparation of rare ginsenoside Rg3 fermentation broth

[0125] 1. Select a number of ginseng roots without scars on the surface, put them into an ultrafine grinder for powdering, and sieve the obtained powder through a 120-mesh sieve to obtain ginseng powder; take 12g of ginseng powder, add 74mL of water and mix evenly to prepare a fermentation slurry;

[0126] 2. After the fermentation slurry is stirred evenly, add 0.3g of cellulase and 0.3g of pectinase (the activity of cellulase is 20,000 U / g; the activity of pectinase is 30,000 U / g). After stirring at 60°C for 120 minutes, adjust the pH to 6.5 with sodium bicarbonate to obtain an enzymatic solution;

[0127] 3. The enzymatic hydrolysate is kept at 90°C for 60 minutes to obtain a sterilized solution;

[0128] 4. Cooling the sterilized liquid to room temperature of 25° C., inoculating Lactobacillus plantarum WMTY01 into the sterilized liquid, starting the timing from the inoculation of the strain, and fermenting to obtain fermentation liquid 1-17;

[0129] The amount of Lactobacillus plantarum WMTY01 inoculated into the fermentation liquid 1-17, the fermentation temperature, and the fermentation time are shown in Table 5:

[0130] Table 5 Fermentation broth 1-17 preparation method

[0131] Serial number Inoculation amount (%) Temperature(℃) Fermentation time (d) Fermentation broth 1 6 32 27 Fermentation broth 2 10 37 21 Fermentation broth 3 6 32 21 Fermentation broth 4 6 27 24 Fermentation broth 5 10 37 27 Fermentation broth 6 14 32 21 Fermentation broth 7 10 27 21 Fermentation broth 8 6 37 24 Fermentation broth 9 10 32 24 Fermentation broth 10 14 37 24 Fermentation broth 11 10 32 24 Fermentation broth 12 10 27 27 Fermentation broth 13 10 32 24 Fermentation broth 14 10 32 24 Fermentation broth 15 10 32 24 Fermentation broth 16 14 32 27 Fermentation broth 17 14 27 24

[0132] 5. Determine the content of ginsenoside Rg3 in fermentation broth 1-17. The specific steps are as follows:

[0133] Place the fermentation broth in a centrifuge and centrifuge at 10000rpm for 5min. Use a pipette to draw 5.0mL of the supernatant of fermentation broth 1-17 into a clean test tube A, add 5.0mL of water-saturated n-butanol, mix thoroughly, ultrasonicate for 10min, let stand for 20min, and take the n-butanol phase into test tube B. Repeat the above operation, i.e. extract ginsenosides with saturated n-butanol three times, so that the saponins in the fermentation supernatant are extracted as completely as possible. Combine the n-butanol phases, evaporate to dryness under reduced pressure, add chromatographic methanol to dissolve the saponins on the wall, make up to 5.0mL, which is the sample, and store at 4°C. The HPLC method described in Basic Experimental Example 2 (1) was used to determine the content of rare ginsenoside Rg3. The results are shown in Table 6:

[0134] Table 6 Content of rare ginsenoside Rg3 in fermentation broth 1-17

[0135] Serial number Inoculation amount (%) Temperature(℃) Fermentation time (d) Rg3 yield (mg / mL) Fermentation broth 1 6 32 27 0.29 Fermentation broth 2 10 37 21 0.35 Fermentation broth 3 6 32 21 0.25 Fermentation broth 4 6 27 24 0.29 Fermentation broth 5 10 37 27 0.27 Fermentation broth 6 14 32 21 0.33 Fermentation broth 7 10 27 21 0.28 Fermentation broth 8 6 37 24 0.28 Fermentation broth 9 10 32 24 0.42 Fermentation broth 10 14 37 24 0.38 Fermentation broth 11 10 32 24 0.4 Fermentation broth 12 10 27 27 0.38 Fermentation broth 13 10 32 24 0.43 Fermentation broth 14 10 32 24 0.43 Fermentation broth 15 10 32 24 0.42 Fermentation broth 16 14 32 27 0.38 Fermentation broth 17 14 27 24 0.43

[0136] (1) Establishment of regression model:

[0137] Design Expert 10.0 software was used to investigate the effects of inoculation amount, fermentation temperature and fermentation time on ginsenoside Rg3. The data in Table 6 were subjected to regression analysis to obtain a quadratic polynomial regression equation. A response surface diagram was drawn based on the quadratic polynomial regression equation, as shown in Figure 6. Figure 3-Figure 5 As shown. The quadratic multinomial regression equation is:

[0138] Y=0.42+0.05125A-0.0125B+0.01375C-0.01AB+0.0025AC-0.045BC-0.04125A 2 -0.03375B 2 -0.06625C 2 ; R of the model 2 The value is 0.9723, and the correction coefficient R 2 The Adj value is 0.9368; where Y represents the yield of ginsenoside Rg3 (mg / mL); A represents the inoculation amount (%); B represents the fermentation temperature (℃); and C represents the fermentation time (d).

[0139] The results show that the coefficients of the quadratic terms in the model are all negative, indicating that the parabola of the model opens downward and has a maximum point. 2 The value is 0.9723, and the correction coefficient R 2The Adj value is 0.9368, indicating that the regression model can explain 97.23% of the response value changes, indicating that the model has a high degree of fit and can better predict the relationship between fermentation conditions and the yield of rare ginsenoside Rg3. The absolute value of the first-order coefficient of the regression equation determines the order of influence of each factor on the response value. In this experiment, the order of influence of each factor on the response value is: inoculation amount (A)> fermentation time (C)> fermentation temperature (B).

[0140] (2) Regression model analysis

[0141] The obtained regression model was further analyzed using Design Expert 10.0 software. The results are shown in Table 7:

[0142] Table 7 Regression model analysis

[0143] source sum of squares Degrees of Freedom Mean Square F-number P-value Significance Model 0.066 9 7.324E-003 27.34 0.0001 Significant A-Inoculation amount 0.021 1 0.021 78.45 <0.0001 B-Fermentation temperature 1.250E-003 1 1.250E-003 4.67 0.0676 C-Fermentation time 1.513E-003 1 1.513E-003 5.65 0.0492 AB 4.000E-004 1 4.000E-004 1.49 0.2612 AC 2.500E-005 1 2.500E-005 0.093 0.7689 BC 8.100E-003 1 8.100E-003 30.24 0.0009 <![CDATA[A 2 ]]> 7.164E-003 1 7.164E-003 26.75 0.0013 <![CDATA[B 2 ]]> 4.796E-003 1 4.796E-003 17.91 0.0039 <![CDATA[C 2 ]]> 0.018 1 0.018 68.99 <0.0001 Residual 1.875E-003 7 2.679E-004 Lack of Fit 1.275E-003 3 4.250E-004 2.83 0.1701 Not significant Pure Error 6.000E-004 4 1.500E-004 sum 0.068 16

[0144] The results showed that the P value of the model was less than 0.0001, the model was extremely significant, and the P value of the lack of fit term was 0.1701 (greater than 0.05), indicating that the model was established effectively. Based on the above regression model analysis and the results of predicting the effect of fermentation conditions on the yield of rare ginsenoside Rg3, the regression model coefficient significance results showed that the linear term of inoculation amount had an extremely significant effect on the regression model of rare ginsenoside Rg3 yield (P<0.0001), the linear term of fermentation time had a significant effect on the regression model of rare ginsenoside Rg3 yield (P<0.05), and the linear term of fermentation temperature had no significant effect on the regression model of rare ginsenoside Rg3 yield (P>0.05). The quadratic term of factor C had an extremely significant effect on the model (P<0.0001), the quadratic terms of factors A and B had a significant effect on the model (P<0.05), the interaction term of factors BC had a significant effect (P<0.05), and the interaction terms of the remaining factors had no significant effect (P>0.05). According to the regression model and response surface, the yield of rare ginsenoside Rg3 was the highest when the inoculation amount was 14%, the fermentation temperature was 28℃, and the fermentation time was 25 days.

[0145] (3) Repeat the experiment

[0146] S1. Select a number of ginseng roots without scars on the surface, put them into an ultrafine grinder for powdering, and sieve the obtained powder through a 120-mesh sieve to obtain ginseng powder; take 12g of ginseng powder, add 74mL of water and mix evenly to prepare a fermentation slurry;

[0147] S2. After the fermentation slurry is stirred evenly, 0.3 g of cellulase and 0.3 g of pectinase (the activity of cellulase is 20,000 U / g; the activity of pectinase is 30,000 U / g) are added. After stirring at 60°C for 120 min, sodium bicarbonate is used to adjust the pH to 6.5 to obtain an enzymatic solution;

[0148] S3, keeping the enzymatic hydrolyzate at 90°C for 60 minutes to obtain a sterilized solution;

[0149] S4. Cool the sterilized liquid to room temperature 25°C, inoculate 14% Lactobacillus plantarum WMTY01 into 86 mL of the sterilized liquid, start timing from the inoculation of the bacteria, control the temperature at 28°C, and ferment for 25 days to obtain rare ginsenoside Rg3 fermentation liquid.

[0150] S5. Place the fermentation broth in a centrifuge and centrifuge at 10000rpm for 5min. Use a pipette to draw 5.0mL of the supernatant of the fermentation broth 1-17 into a clean test tube A, add 5.0mL of water-saturated n-butanol, mix thoroughly, sonicate for 10min, let stand for 20min, and take the n-butanol phase into test tube B. Repeat the above operation, i.e. extract ginsenosides with saturated n-butanol three times, so that the saponins in the fermentation supernatant are extracted as completely as possible. Combine the n-butanol phases, evaporate to dryness under reduced pressure, add chromatographic methanol to dissolve the saponins on the wall, and make the volume to 5.0mL. Use the HPLC method described in =Basic Experimental Example 2 (1) to determine the content of rare ginsenoside Rg3. The liquid phase detection diagram of rare ginsenoside Rg3 fermentation broth is shown in FIG. Figure 6 .

[0151] The measurement results showed that the content of rare ginsenosides was 0.44 mg / mL, which was increased by 1366.67% compared with Example 3 without optimized conditions.

[0152] Example 8 Preparation of rare ginsenoside Rg3 fermentation broth by different methods

[0153] The fermentation broth 18-24 was prepared by the following method:

[0154] 1. Select a number of ginseng roots without scars on the surface, put them into an ultrafine grinder for powdering, and sieve the obtained powder through a 120-mesh sieve to obtain ginseng powder; take 12g of ginseng powder, add 74mL of water and mix evenly to prepare a fermentation slurry;

[0155] 2. After the fermentation slurry is stirred evenly, add 0.3g of cellulase and 0.3g of pectinase (the activity of cellulase is 20,000 U / g; the activity of pectinase is 30,000 U / g). After stirring at 60°C for 120 minutes, adjust the pH to 6.5 with sodium bicarbonate to obtain an enzymatic solution;

[0156] 3. Keep the enzymatic solution at 90°C for 60 minutes to obtain a sterilized solution;

[0157] 4. Cool the sterilized liquid to room temperature 25°C, inoculate the bacteria into the sterilized liquid, start timing from the inoculation of the bacteria, and ferment to obtain fermentation liquid 18-24;

[0158] The strains inoculated into the fermentation broth 18-24, the amount of the inoculated strains, the fermentation temperature, and the fermentation time are shown in Table 8, and the measurement results are shown in Table 9.

[0159] Table 8 Preparation method of fermentation broth 18-24

[0160] Serial number Inoculated strains Inoculation amount (%) Temperature(℃) Fermentation time (d) Fermentation broth 18 WMTY01 14 28 25 Fermentation broth 19 WMTY01 20 28 25 Fermentation broth 20 WMTY01 14 28 30 Fermentation broth 21 WMTY01 14 28 15 Fermentation broth 22 WMTY01 14 35 25 Fermentation broth 23 WMTY01 14 20 25 Fermentation broth 24 Strain 5 14 28 25

[0161] Table 9 Content of rare ginsenoside Rg3 in fermentation broth 18-24

[0162] Serial number Rg3 yield (mg / mL) Fermentation broth 18 0.45 Fermentation broth 19 0.40 Fermentation broth 20 0.44 Fermentation broth 21 0.37 Fermentation broth 22 0.31 Fermentation broth 23 0.22 Fermentation broth 24 0.09

[0163] The above results show that the content of rare ginsenoside Rg3 in the fermentation broth prepared according to the technical solution of the present invention is the highest.

[0164] The above detailed description is a specific description of one of the feasible embodiments of the present invention, and the embodiment is not intended to limit the scope of the present invention. It should be pointed out that any equivalent implementation or change that does not deviate from the present invention should be included in the scope of the technical solution of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached requirements.

Claims

1. A Lactobacillus plantarum, characterized in that The plant lactobacillus is plant lactobacillus WMTY01, and its deposit number is CGMCC No.29360.

2. A plant lactobacillus preparation, characterized in that The plant lactobacillus preparation comprises one or more of the culture, bacterial cells, fermentation liquid, fermentation supernatant, fermentation liquid precipitation and freeze-dried powder of the plant lactobacillus according to claim 1.

3. The method for cultivating plant lactobacillus according to claim 1, wherein The culture method comprises inoculating the Lactobacillus plantarum according to claim 1 into a culture medium.

4. The culture method according to claim 3, characterized in that The culture medium is MRS culture medium.

5. Use of the plant lactobacillus according to claim 1 or the plant lactobacillus preparation according to claim 2 in the production of β-glucosidase.

6. A method for preparing β-glucosidase, characterized in that: The preparation method comprises culturing the plant lactobacillus according to claim 1.

7. The preparation method according to claim 6, characterized in that: The preparation method comprises inoculating the plant lactobacillus described in claim 1 into an MRS agar medium.

8. Use of the plant lactobacillus according to claim 1 or the plant lactobacillus preparation according to claim 2 in producing ginsenosides.

9. The use according to claim 8, characterized in that: The ginsenoside is ginsenoside Rg3.

10. The use according to claim 9, characterized in that: The ginsenosides are prepared by fermenting ginseng.

11. The use according to claim 10, characterized in that: The fermentation comprises using a fermentation broth, and the fermentation broth is the fermentation broth of the plant lactobacillus according to claim 1.

12. Use of the plant lactobacillus according to claim 1 or the plant lactobacillus preparation according to claim 2 in the preparation of medicines or health foods, characterized in that: Prepared by fermenting ginseng.

13. The use according to claim 11, characterized in that: The medicine is used for anti-oxidation, anti-inflammation, anti-bacteria, anti-virus, anti-cardiovascular disease, anti-diabetes, anti-central system disease or anti-cancer.

14. The use according to claim 13, characterized in that The medicine also includes pharmaceutically acceptable excipients or carriers.

15. The use according to claim 11, characterized in that: The health food is used for anti-oxidation, maintaining cardiovascular health, balancing blood sugar or relieving physical fatigue.

16. A method for preparing ginsenoside Rg3, characterized in that: The method comprises fermenting ginseng using the plant lactobacillus according to claim 1 or the plant lactobacillus preparation according to claim 2.

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