A kind of Lactobacillus plantarum and application thereof

Abstract

The present invention belongs to the field of microorganisms, and specifically relates to a plant lactobacillus and an application thereof. The plant lactobacillus is deposited in the Guangdong Microbial Culture Collection Center, and the deposit number is GDMCC No: 63911. The plant lactobacillus is used to ferment cattle bile, and a large amount of bile salt hydrolase is produced during the fermentation process, which can convert the bound bile acid in the cattle bile into free bile acid, and a high conversion rate can be achieved within 2-3 days, and the utilization value is high.

Description

Technical Field

[0001] The invention belongs to the field of microorganisms, and particularly relates to plant lactobacillus and application thereof. Background Art

[0002] Bile is the main raw material for in vitro cultivation of bezoar. Bile acid is the main organic component in bile. Natural bile is usually mainly conjugated bile acid. Conjugated bile acid cannot co-precipitate with cholesterol, which makes the bile containing conjugated bile acid have a higher viscosity, which is not conducive to the release of bilirubin and calcium ions in the bile, thereby reducing the productivity of in vitro cultivation of bezoar.

[0003] The bound bile acid is converted into free bile acid, which can co-precipitate with cholesterol, thereby reducing bile viscosity, facilitating the release of bilirubin and calcium ions in bile, and thus improving the productivity of in vitro culture of bezoar. To this end, extensive exploration has been carried out in this field, such as:

[0004] The patent document (CN115851497A) discloses a strain of Citrobacter acidophilus CGMCC No.24893, which can tolerate ox bile and has the ability to ferment ox bile to generate free bilirubin and free bile acid. However, the delay period of this Citrobacter acidophilus in ox bile is 40h, and the fermentation cycle is as long as 16 days. In addition, Citrobacter acidophilus is a Gram-negative bacterium, which will produce endotoxins and other toxic factors during the fermentation process. There are certain safety risks when the product is used in medical beauty or pharmaceutical products.

[0005] Patent document (CN115414434A) discloses a method for preparing Arisaema consanguineum slices by fermenting mixed strains, wherein the strains used for fermentation include one or more strains of Aspergillus glaucus, Candida parapsilosis, Penicilliumsteckii, Aspergillus cristatus, and Pseudomonas lundensis, which can convert the less active conjugated bile acid in bile into free bile acid. Under optimal conditions, the conversion rate of conjugated bile acid to free bile acid is only about 15%, and it takes 10 days.

[0006] Non-patent literature: Study on the changes of effective ingredients in bovine bile after fermentation and the mechanism of action in treating ischemic stroke (Pei Yuying, Beijing University of Chinese Medicine, 2022) reported that the conversion rate of conjugated bile acid to free bile acid was about 50% when bovine bile was fermented with Shenqu as the strain, but it took 30 days. Summary of the invention

[0007] In order to overcome the above defects, the present invention provides a plant lactobacillus, which is preserved in Guangdong Province Microbial Culture Collection Center with a preservation number of GDMCC No: 63911, belongs to a safe and effective probiotic strain, promotes the conversion of bound bile acid in the fermentation liquid into free bile acid in the fermentation of bovine bile, and can achieve a high conversion rate within 2-3 days. In addition, the present application provides a bovine bile fermentation method, which has the advantages of mild and simple production process, low production cost, reduced use of flammable and explosive solvents, reduced environmental pollution, safe production process, emission reduction, consumption reduction and environmental protection, product quality safety, etc., and has good economic and social benefits.

[0008] The first aspect of the present invention provides a plant lactobacillus (Lactiplantibacillus plantarum), which has been deposited in the Guangdong Provincial Microbiological Culture Collection Center on October 23, 2023, with a storage address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province, Postal Code: 510070, and the storage number is GDMCCNo:63911.

[0009] The plant lactobacillus described in the present invention is named ZY-002, is derived from the gallbladder of cattle, and is listed in the catalog of safe and edible bacteria published by the National Health Commission.

[0010] Preferably, the nucleotide sequence of 16S rDNA of Lactobacillus plantarum is shown in SEQ ID NO: 1.

[0011] Preferably, the Lactobacillus plantarum has the ability to tolerate ox bile, and further preferably, the Lactobacillus plantarum can survive in pure ox bile for more than 5 days.

[0012] The second aspect of the present invention provides a method for culturing the plant lactobacillus described in the first aspect, wherein the method comprises using a culture medium containing one or more of ox bile, ox bile powder or ox bile salt.

[0013] Preferably, one or more of the ox bile, ox bile powder or ox bile salt is filtered and sterilized;

[0014] Preferably, in the culture medium, the volume fraction of one or more of ox bile, ox bile powder or ox bile salt is at least 40%, preferably any value between 40% and 60%, for example 40%, 45%, 50%, 55% or 60%.

[0015] Preferably, the culture medium can be any culture medium suitable for the growth and metabolism of Lactobacillus plantarum in the prior art, preferably MRS culture medium (such as MRS broth culture medium or MRS agar culture medium).

[0016] In a specific embodiment of the present invention, the MRS broth culture medium comprises: peptone, beef extract, yeast extract, triammonium citrate, sodium acetate, magnesium sulfate (MgSO4·7H2O), manganese sulfate (MnSO4·H2O), dipotassium hydrogen phosphate, glucose, and Tween-80.

[0017] In a specific embodiment of the present invention, the MRS agar medium comprises: peptone, beef extract, yeast extract, triammonium citrate, sodium acetate, magnesium sulfate (MgSO4·7H2O), manganese sulfate (MnSO4·H2O), dipotassium hydrogen phosphate, glucose, Tween-80, and agar.

[0018] The third aspect of the present invention provides an application of the Lactobacillus plantarum described in the first aspect, wherein the application comprises one or two of the following groups:

[0019] 1) Application in fermentation of ox bile;

[0020] 2) Application in converting conjugated bile acids into free bile acids.

[0021] Preferably, the converting of conjugated bile acid into free bile acid comprises converting conjugated bile acid in bovine bile into free bile acid.

[0022] A fourth aspect of the present invention provides a method for fermenting bovine bile, wherein the method comprises using a microorganism, wherein the microorganism comprises the Lactobacillus plantarum described in the first aspect.

[0023] Preferably, the method comprises introducing the microorganism into cattle bile.

[0024] Further preferably, the method comprises filtering and sterilizing the bovine bile to obtain sterilized bovine bile.

[0025] Further preferably, the method comprises inoculating the microorganism into a seed culture medium to obtain a microorganism seed solution.

[0026] Further preferably, the method comprises inoculating the microbial seed liquid into the sterilized cow bile to obtain a fermentation system.

[0027] Preferably, the initial pH of the fermentation system is any value or range of 4.5-8.0, more preferably any value or range of 5.5-8.0, and even more preferably any value or range of 6.0-8.0, for example 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5 or 8.0.

[0028] Preferably, the temperature of the fermentation system is any value or range between 20°C and 45°C, further preferably any value or range between 25°C and 40°C, for example, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C or 45°C.

[0029] Preferably, the fermentation time is at least 36 hours, more preferably at least 48 hours, for example 36 hours to 150 hours or more, for example 36, 40, 45, 48, 50, 55, 60, 65, 70, 72, 75, 80, 84, 85, 90, 95, 96, 100, 105, 108, 110, 115, 120, 125, 130, 135, 140, 144 or 150 hours or more.

[0030] In a specific embodiment of the present invention, the method comprises the following steps:

[0031] 1) Preparing sterilized bovine bile: filtering and sterilizing the bovine bile to obtain sterilized bovine bile;

[0032] 2) preparing microbial seed solution: inoculating the activated microorganisms into a seed culture medium and culturing them to the logarithmic growth phase (preferably the late logarithmic growth phase);

[0033] 3) Fermenting cow bile: The microbial seed liquid obtained in 2) is inoculated into the sterilized cow bile obtained in 1) to form a fermentation system, and the initial pH of the fermentation system is adjusted to any value or range of 4.5-8.0, and fermented at any value or range of 20° C. to 45° C. for at least 36 hours.

[0034] Preferably, there is no temporal or spatial order between step 1) and step 2), as long as the sterilized ox bile and the microbial seed solution can be successfully prepared, the order of the two steps has no effect on the result.

[0035] Preferably, the seed culture medium described in 2) can be any culture medium suitable for microbial proliferation in the prior art. It is further preferred that one or more of sterilized ox bile, sterilized ox bile powder or sterilized ox bile salts is added to the seed culture medium. More preferably, in the seed culture medium, the volume fraction of one or more of sterilized ox bile, sterilized ox bile powder or sterilized ox bile salts is at least 40%, preferably any value between 40% and 60%, for example 40%, 45%, 50%, 55% or 60%.

[0036] Preferably, the seed culture medium may be MRS culture medium (eg, MRS broth culture medium or MRS agar culture medium).

[0037] Preferably, the access ratio of the microbial seed liquid accounts for any value or interval of 5%-90 of the volume fraction of the fermentation system, preferably any value or interval of 20%-50%, for example, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90%.

[0038] Preferably, the temperature of seed liquid culture can be any value or range of 20℃-45℃, further preferably any value or range of 25℃-40℃; more preferably any value or range of 30℃-37℃, for example, 20℃, 25℃, 30℃, 35℃, 37℃, 40℃, 42℃ or 45℃.

[0039] A fifth aspect of the present invention provides a method for converting conjugated bile acid into free bile acid, wherein the method comprises using a microorganism, wherein the microorganism comprises the Lactobacillus plantarum described in the first aspect.

[0040] Preferably, the method comprises introducing the microorganism into bovine bile, or into a system containing bound bile acids.

[0041] Preferably, the method comprises filtering and sterilizing the cow bile to obtain sterilized cow bile.

[0042] Preferably, the method comprises filtering and sterilizing the system containing the conjugated bile acid to obtain a sterilized system;

[0043] Preferably, the method comprises inoculating the microorganisms into a seed culture medium to obtain a microorganism seed solution;

[0044] Preferably, the method comprises introducing the microbial seed solution into the sterilized ox bile or sterilization system to obtain a fermentation system.

[0045] Preferably, the fermentation system, fermentation time, fermentation, seed culture medium, access ratio of microorganism seed solution and culture conditions of seed solution are defined as the fourth aspect of the present invention.

[0046] The terms “include” or “comprising” described in the present invention are open-ended expressions, which include the specified components or steps described, and other specified components or steps that will not be substantially affected.

[0047] The term "and / or" of the present invention includes all combinations of items connected by the term, and each combination should be deemed to have been listed separately herein. For example, "A and / or B" includes "A", "A and B" and "B". For another example, "A, B and / or C" includes "A", "B", "C", "A and B", "A and C", "B and C" and "A and B and C".

[0048] Beneficial effects of the present invention:

[0049] 1. The plant lactobacillus ZY-002 of the present invention produces a large amount of bile salt hydrolase during the fermentation of bovine bile, which can convert the bound bile acid in the bovine bile into free bile acid, and can achieve a high conversion rate (more than 60%) in a very short time (2-3 days), and has a high utilization value.

[0050] 2. The plant lactobacillus ZY-002 described in the present invention is an edible grade strain, which replaces pathogenic bacteria or conditional pathogens such as Escherichia coli to ferment cow bile. The fermented product does not contain substances harmful to human health, which is beneficial to ensure the safety of the medical beauty products or pharmaceutical products produced by the product and ensure the health and safety of the people.

[0051] 3. The bile salt hydrolase produced by the fermentation of Lactobacillus plantarum ZY-002 of the present invention can decompose the bound bile salts in bile, and has the ability to utilize the amino acids produced by hydrolysis fermentation during the fermentation process, resulting in a decrease in bile pH. Under acidic conditions, free bile acids and cholesterol co-precipitate; the viscosity of the fermentation liquid decreases, which is beneficial to the freeing of bilirubin and calcium ions in bile, increases the contact probability, and is more likely to form bilirubin calcium.

[0052] 4. The bile salt hydrolase produced by the plant lactobacillus ZY-002 of the present invention during fermentation reduces the concentration of bile salt in bile, which is beneficial to the survival of some microorganisms sensitive to bile salt. Other functional microorganisms can be added during the fermentation process to produce other fermentation products at the same time.

[0053] Strain deposit information:

[0054] Depository: Guangdong Microbiological Culture Collection Center (GDMCC);

[0055] Address of the preservation unit: 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou City, Guangdong Province;

[0056] Deposit number: GDMCC No:63911;

[0057] Deposit date: October 23, 2023;

[0058] Classification name: Lactiplantibacillus plantarum. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] The embodiments of the present invention are described in detail below with reference to the accompanying drawings, wherein:

[0060] Figure 1 : Colony morphology of strain ZY-002 on MRS solid medium;

[0061] Figure 2 : Gram staining microscopic image of strain ZY-002;

[0062] Figure 3 : Growth curve of strain ZY-002 in bovine bile. DETAILED DESCRIPTION

[0063] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0064] Example 1 Isolation, screening and taming of strains for tolerance to bovine bile

[0065] Step 1: Isolation of strains

[0066] Take the bile from the fresh bovine gallbladder containing bezoar, dilute the collected bile sample with physiological saline, evenly spread the bile sample with appropriate dilution multiples on MRS agar medium, culture at 37±1℃ for 24-48h, and observe the morphology of single colonies on the plate (taking the strain of this application as an example, see Figure 1 ), select the colonies that meet the morphology of lactic acid bacteria (the colonies are round, medium-sized, convex, slightly white, moist, and have neat edges). Perform Gram staining and catalase tests on the isolated and purified colonies. Figure 2 ), strains with negative catalase test were preliminarily judged to be lactic acid bacteria.

[0067] These isolated colonies were inoculated in MRS broth medium and cultured at 37°C for 24 hours. Then, the bacterial solution was added to sterilized and filtered bovine bile at a volume ratio of 5%, and cultured at 37°C for 96 hours. Then, 10 μL of the culture was diluted and spread on MRS medium and cultured at 37°C for 72 hours. The number of colonies was calculated, and strains with a colony number greater than 1000 were selected as bovine bile-tolerant strains. A total of 56 bovine bile-tolerant lactic acid bacteria were screened for subsequent screening.

[0068] The screened and purified strains can be stored on MRS slants at 4°C for a short term, or in 30% glycerol cryovials at -80°C for long term storage.

[0069] The formula of MRS broth medium is as follows: peptone 10.0 g / L, beef extract 10.0 g / L, yeast extract 5.0 g / L, triammonium citrate 2.0 g / L, sodium acetate 5.0 g / L, magnesium sulfate (MgSO4·7H2O) 0.1 g / L, manganese sulfate (MnSO4·H2O) 0.05 g / L, dipotassium hydrogen phosphate 2.0 g / L, glucose 20.0 g / L, Tween-80 1 mL / L, pH value 5.7±0.2.

[0070] MRS agar medium formula ingredients: peptone 10.0 g / L, beef extract 10.0 g / L, yeast extract 5.0 g / L, triammonium citrate 2.0 g / L, sodium acetate 5.0 g / L, magnesium sulfate (MgSO4·7H2O) 0.1 g / L, manganese sulfate (MnSO4·H2O) 0.05 g / L, dipotassium hydrogen phosphate 2.0 g / L, glucose 20.0 g / L, Tween-80 1 mL / L, agar 15 g / L, pH 6.2±0.2.

[0071] Step 2: Screening of Bezoar Transformation Strains

[0072] Step 1 obtained 56 strains that were preliminarily judged to be lactic acid bacteria and tolerant to ox bile (see Table 1 below). After activating these strains three times, 4 ml of the bacterial solution was inoculated into 16 ml of MRS medium containing 50% sterilized ox bile, and a control group of 4 ml of MRS medium added with 16 ml of MRS medium containing 50% sterilized ox bile was set up, and cultured at 37 ° C for 96 h.

[0073] At the end of fermentation, take an appropriate amount of ox bile and fermented ox bile, add 4 times the volume of methanol-acetonitrile extract (methanol: acetonitrile = 1:1, containing 0.1% formic acid) respectively, vortex thoroughly, ultrasonicate in an ice water bath for 10 minutes, and then centrifuge at 14000g for 5 minutes at 4°C, and take the supernatant for detection.

[0074] The HPLC-ELSD method was used to detect the bound bile acid and free bile acid in the fermented bovine bile. The specific detection method was as follows: a Waters Symmetryshield C18 column (4.6 mm × 250 mm, 5 μm) was used, acetonitrile was used as the mobile phase A, and 0.2% formic acid solution (containing 10 mmol·L -1 Ammonium acetate) was used as mobile phase B, gradient elution (0-5 min, 2% A→35% A; 5-12 min, 35% A→37% A; 12-23 min, 37% A; 23-45 min, 37% A→50% A), flow rate 1.0 mL min -1, column temperature 35°C; evaporative light detector detection, drift tube temperature 102°C, nitrogen 1.9 mL·min -1 ;

[0075] The conversion rate of bound bile acid in fermented bovine bile to free bile acid was calculated by the formula (C1-C2) / C1*100% (in the conversion system, after bound bile acid contacts bile salt hydrolase, the consumed part will be converted into free bile acid and amino acids), where C1 is the bound bile acid content in bovine bile before fermentation, and C2 is the bound bile acid content in bovine bile after fermentation. Eight strains with conversion rates higher than 50% were obtained.

[0076] The preparation method of the MRS medium containing 50% sterilized ox bile is as follows: weigh each component according to the MRS formula, fully dissolve it with an appropriate amount of distilled water, slowly add 50 mL of sterilized and filtered ox bile to the solution, and distilled water to make the volume to 100 mL to prepare a 50% ox bile-MRS medium.

[0077] Table 1

[0078]

[0079] Step 3: Adaptation of strains to ox bile tolerance

[0080] The strains with the ability to transform bezoar obtained above were further domesticated for tolerance to ox bile. The liquid culture medium for domesticating lactic acid bacteria tolerant to ox bile was prepared based on MRS by adding an appropriate amount of ox bile, and the formula was as follows: relative to a volume of 1000 mL, it contained 10.0 g / L of peptone, 10.0 g / L of beef extract, 5.0 g / L of yeast extract, 2.0 g / L of triammonium citrate, 5.0 g / L of sodium acetate, 0.1 g / L of magnesium sulfate (MgSO4·7H2O), 0.05 g / L of manganese sulfate (MnSO4·H2O), 2.0 g / L of dipotassium hydrogen phosphate, 20.0 g / L of glucose, and 100 g / L of Tween-80. 1mL / L, stir and dissolve an appropriate amount of distilled water, on this basis, add 100mL, 200mL, 300mL, 400mL, 500mL, 600mL, 700mL, 800mL of sterilized and filtered ox bile respectively, make up to 1000ml with distilled water or purified water, prepare ox bile acclimation medium with ox bile addition of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% (volume fraction), adjust the pH to 6.2±0.2, and sterilize at 121℃ for 20 minutes before use.

[0081] The solid culture medium for domesticating lactic acid bacteria tolerant to ox bile is prepared by adding 15 g / L agar to the above liquid culture medium formula, adjusting the pH to 6.2±0.2, and sterilizing at 121°C for 20 minutes before use.

[0082] After activating the above-mentioned strain with ox bile transformation ability three times, the bacterial liquid was inoculated in an acclimation medium containing 10% ox bile-MRS at an inoculum volume ratio of 5%, and cultured at 37°C until turbidity was observed. Then, 100 μL of the culture was spread on an MRS solid acclimation medium containing 10% ox bile. After culture at 37°C for 48 hours, a single colony with good growth was selected and preserved for later use, and inoculated into an acclimation medium containing 20% ​​ox bile. After culture at 37°C until turbidity was observed, 100 μL of the culture was spread on an MRS solid acclimation medium containing 20% ​​ox bile. After culture at 37°C for 48 hours, a single colony with good growth was selected and preserved for later use; the above steps were repeated, and the culture was spread on an acclimation medium with a volume fraction increasing by 10% in sequence (30%, 40%, 50%, 60%, 70%, the process is consistent with the above process and omitted), and the strain was acclimated to survive and reproduce in 80% ox bile-MRS medium. After domestication, 5 strains of lactic acid bacteria were obtained that could survive and reproduce in 80% ox bile-MRS medium, and were numbered L29, L48, L51, L137, and L189.

[0083] The five lactic acid bacteria strains were inoculated into 5 ml of MRS medium containing 60% ox bile, and cultured at 37 °C for 48 h. Then, 5 ml of the bacterial solution was inoculated into 95 ml of sterilized ox bile and cultured at 80 rpm. Samples were taken at different time points to measure the OD 600 , with OD 600 The growth curve was drawn with the value as the ordinate and the culture time as the abscissa. It was found that L137 survived in ox bile for more than 120 hours, with a stagnation period from 0 to 2 hours, and then entered the late logarithmic growth period. When the OD was 0.04 at 24 hours, the growth rate was 0.01%. 600 It reaches a maximum of 1.26, and then slowly decreases to around 1.1 ( Figure 3 Among the five lactic acid bacteria investigated, L137 had the shortest delay period, the highest growth rate and the highest bacterial concentration in bovine bile, and had good growth performance and bile tolerance (see Table 2 below for the results).

[0084] Table 2

[0085]

[0086]

[0087] Example 2 Strain Identification

[0088] The strain L137 obtained in Example 1, which has the ability to hydrolyze bound bile acid in bile into free bile acid and can tolerate bovine bile, was selected and named ZY-002 and identified.

[0089] 1. Identification of colony morphology: strain ZY-002 was inoculated on MRS solid medium and cultured at 37°C for 24 hours. The colony surface was convex and round, smooth, dense and white. Under the microscope, the bacteria were rod-shaped, thick and fat, regularly arranged, and without spores.

[0090] 2. Molecular identification of strains: The 16S rDNA sequence of the ZY-002 sample was compared on NCBI (https: / / blast.ncbi.nlm.nih.gov) and found that the sample had a homology of 99.86% with Lactiplantibacillus plantarum and 99.71% with Lactiplantibacillus pentosus and Lactiplantibacillus argentoratensis. Comparison on EZBioCloud (https: / / www.ezbiocloud.net) found that the homology with Lactiplantibacillus paraplantarum reached 99.71%; identification of the specific gene recA found that the homology between the sample and the model strain Lactiplantibacillus plantarum reached 99.46%. Combined with the results of morphological and 16S rDNA sequence comparison, ZY-002 was determined to be Lactiplantibacillus plantarum. Deposited in Guangdong Provincial Microbiological Culture Collection Center, deposit number: GDMCCNo: 63911, deposit date: October 23, 2023, address: 5th Floor, Laboratory Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province.

[0091] The 16s rDNA sequencing sequence of ZY-002 is shown in SEQ ID NO: 1.

[0092] SEQ ID NO: 1:

[0093] AACGAACTTCCTGGTATTGATTGGTGCTTGCATCATGATTTACATTTGAGTGAGTGGCGAACTGGT

[0094] GAGTAACACGTGGGAAACCTGCCCAGAAGCGGGGGATAACACCTGGAAACAGATGCTAATACCG

[0095] CATAACAACTTGGACCGCATGGTCCGAGCTTGAAAGATGGCTTCGGCTATCACTTTTGGATGGTC

[0096] CCGCGGCGTATTAGCTAGATGGTGGGGTAACGGCTCACCATGGCAATGATACGTAGCCGACCTGA

[0097] GAGGGTAATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGG

[0098] AATCTTCCACAATGGACGAAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGGTTTCGGCT

[0099] CGTAAAACTCTGTTGTTAAAGAAGAACATATCTGAGAGTAACTGTTCAGGTATTGACGGTATTTAA

[0100] CCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCC

[0101] GGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCTTCGGCTCA

[0102] ACCGAAGAAGTGCATCGGAAACTGGGAAACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGT

[0103] GTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGGCTGTCTGGTCTGTA

[0104] ACTGACGCTGAGGCTCGAAAGTATGGGTAGCAAACAGATTAGATACCCTGGTAGTCCATACCGTA

[0105] AACGATGAATGCTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCTAACGCATTAAGCATT

[0106] CCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCG

[0107] GTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATACTATGCAA

[0108] ATCTAAGAGATTAGACGTTCCCTTCGGGGACATGGATACAGGTGGTGCATGGTTGTCGTCAGCTC

[0109] GTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTATCAGTTGCCAGCATTA

[0110] AGTTGGGCACTCTGGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATC

[0111] ATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAACGAGTTGCGAACTC

[0112] GCGAGAGTAAGCTAATCTCTTAAAGCCATTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATG

[0113] AAGTCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACA

[0114] CACCGCCCGTCACACCATGAGAGTTTGTAACA

[0115] Detection of bile salt hydrolase activity of strain ZY - 002 in Example 3

[0116] Bile salt hydrolase (BSH) can convert the conjugated bile acid in bovine bile into free bile acid, but not all Lactobacillus plantarum has BSH activity. For example, Rajesh Kumar et al. screened 33 strains of Lactobacillus plantarum in 2012, of which only 14 were BSH-positive strains (see non-patent literature: Bile Salt Hydrolase (Bsh) Activity Screening of Lactobacilli: In Vitro Selection of Indigenous Lactobacillus Strains with Potential Bile Salt Hydrolysing and Cholesterol-Lowering Ability). Further, the bile salt hydrolase activity of ZY-002 was detected by two methods.

[0117] 1. Detect bile salt hydrolase activity using the calcium ion precipitation method.

[0118] The bile salt hydrolase produced by BSH-positive strains will hydrolyze the bound bile salts in the culture medium to form free bile salts. Under slightly acidic conditions, the free bile salts are easily co-precipitated with calcium chloride, forming a white opaque precipitation circle around the filter paper.

[0119] The culture medium for BSH activity detection by calcium precipitation method is an MRS solid culture medium containing 0.37 g / L calcium chloride, 2 g / L sodium thioglycolate and 5 g / L sodium deoxytaurocholate (TDCA), wherein TDCA is first filtered through a 0.22 μm filter membrane and added after the culture medium is sterilized and slightly cooled.

[0120] After Lactobacillus plantarum ZY-002 was activated three times, the bacterial solution was inoculated into MRS broth at a 1% inoculation volume and cultured at 37°C until the stable period. Then 10 μL of the bacterial solution was dropped on a sterile filter paper, and the filter paper was attached to a bile salt-MRS plate and cultured at 37°C for 120 hours. After observation, an obvious calcium precipitation circle was observed around the filter paper, indicating that ZY-002 had bile salt hydrolase activity.

[0121] 2. The bile salt hydrolase activity was detected by the ninhydrin method.

[0122] Bile salt hydrolase can hydrolyze the bound bile salts in bile into free bile acids and free amino acids. The free amino acids can be detected by ninhydrin, thereby reflecting the activity of bile salt hydrolase.

[0123] After Lactobacillus plantarum ZY-002 was activated 3 times, the bacterial solution was inoculated into MRS broth medium (containing 3g / LTDCA) at 1% inoculum, cultured at 37°C until the stable period, 1.0ml of culture solution was taken, centrifuged at 4000g for 5min, the supernatant was taken, and an equal volume of 15% trichloroacetic acid was added, mixed evenly, centrifuged at 14000g for 10min, and the supernatant was taken. 100μL of supernatant was taken, mixed with 1900μL of ninhydrin colorimetric solution, shaken and mixed, boiled in water bath for 15min, and blue-purple was observed after cooling, indicating that bile salt hydrolase was positive.

[0124] Example 4 Effect of fermentation time on the conversion rate of bound bile acid to free bile acid in fermentation of bovine bile by ZY-002

[0125] Pretreatment of cow bile: obtain fresh cow bile, filter and sterilize it to remove impurities and bacteria, and set aside.

[0126] Preparation of seed solution: Weigh each component according to the MRS formula, fully dissolve with appropriate amount of distilled water, slowly add 10 mL of the above-mentioned pretreated ox bile to the solution, dilute to 100 mL with distilled water, prepare 50% ox bile-MRS medium, inoculate ZY-002 into 50% ox bile-MRS medium, and culture at 37°C to the late logarithmic growth stage;

[0127] Effect of fermentation time on the conversion rate of conjugated bile acid to free bile acid when ZY-002 fermented bovine bile: The seed liquid of the above-mentioned strain was inoculated into sterile bovine bile culture medium at a ratio of 20 mL of culture per 80 mL of sterile bovine bile, and the culture was carried out at 37°C and 80 rpm. Samples were taken at 36 h, 48 h, 60 h, 72 h, 84 h, 96 h, 108 h, and 120 h, and the conjugated bile acid content of bovine bile before fermentation and after different fermentation times was detected, and the conversion rate of conjugated bile acid to free bile acid was calculated.

[0128] Table 3 Effect of fermentation time on bile acid conversion rate during ZY-002 fermentation of bovine bile

[0129]

[0130] The results are shown in Table 3. Compared with unfermented bovine bile, the free bile acid content of bovine bile fermented by ZY-002 was increased at each sampling time investigated. When fermented at 37°C for 48 hours, the conversion rate of conjugated bile acid to free bile acid could reach 65%, and then the conversion rate fluctuated between 59% and 65%, indicating that the conversion of conjugated bile acid in bovine bile could reach a higher level when fermented for 48 hours. The preferred fermentation time for subsequent experiments is 48 hours.

[0131] Example 6 Effect of initial pH on the conversion rate of bound bile acid to free bile acid in ZY-002 fermentation of bovine bile

[0132] Pretreatment of cattle bile: obtain fresh cattle bile, filter and sterilize it to remove impurities and bacteria, and adjust the pH to 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, and 8.0 respectively for use.

[0133] Preparation of seed solution: Weigh each component according to the MRS formula, fully dissolve with appropriate amount of distilled water, slowly add 10 mL of the above-mentioned pretreated ox bile to the solution, dilute to 100 mL with distilled water, prepare 50% ox bile-MRS medium, inoculate ZY-002 into 50% ox bile-MRS medium, and culture at 37°C to the late logarithmic growth stage;

[0134] Effect of the initial pH of the fermentation system on the conversion rate of conjugated bile acids to free bile acids of ZY-002 during the fermentation of bovine bile: The seed liquid of the above-mentioned strain was inoculated into sterile bovine bile culture medium with pH values ​​of 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, and 8.0, respectively, at a ratio of 20 mL of culture per 80 mL of sterile bovine bile. The culture was carried out at 37°C and 80 rpm for 60 h to detect the conjugated bile acid content before and after bovine bile fermentation.

[0135] As shown in Table 4, when the pH of ZY-002 was 4.5-8.0, the conjugated bile acids in the fermented bovine bile at each pH investigated could be converted into free bile acids, among which the conversion rate of conjugated bile acids in the fermented bovine bile into free bile acids was the highest at pH 6.5, which was 65%.

[0136] Table 4 Effect of initial pH of fermentation system on bile acid conversion rate during ZY-002 fermentation of bovine bile

[0137]

[0138]

[0139] Example 7 Effect of fermentation temperature on the conversion rate of bound bile acid to free bile acid in ZY-002 fermentation of bovine bile

[0140] Pretreatment of cow bile: obtain fresh cow bile, filter and sterilize it to remove impurities and bacteria, and set aside.

[0141] Preparation of seed solution: Weigh each component according to the MRS formula, fully dissolve with appropriate amount of distilled water, slowly add 50 mL of the above-mentioned pretreated ox bile to the solution, dilute to 100 mL with distilled water, prepare 50% ox bile-MRS medium, inoculate ZY-002 into 50% ox bile-MRS medium, and culture at 37°C to the late logarithmic growth stage;

[0142] Effect of fermentation temperature on the conversion rate of conjugated bile acid to free bile acid of ZY-002 when fermenting bovine bile: The seed liquid of the above strain was inoculated into sterile bovine bile culture medium at a ratio of 20 mL of culture per 80 mL of sterile bovine bile, and then cultured at 25°C, 30°C, 37°C and 40°C at 80 rpm for 60 h. The free bile acid content in the bovine bile fermentation broth was detected, and the conversion rate of conjugated bile acid to free bile acid was calculated.

[0143] The results are shown in Table 5. Compared with unfermented bovine bile, the bile acid content of ZY-002 was increased at each fermentation temperature investigated. Among them, when fermented at 37°C for 60 hours, the conversion rate of bound bile acid to free bile acid was the highest, reaching 62%.

[0144] Table 5 Effect of fermentation temperature on bile acid conversion rate in bovine bile fermentation

[0145]

[0146] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0147] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A Lactobacillus plantarum ( Lactiplantibacillus plantarum ), characterized in that, The plant lactobacillus was deposited in the Guangdong Provincial Microbiological Culture Collection Center on October 23, 2023, with the collection number GDMCC No: 63911.

2. A method for culturing plant lactobacillus according to claim 1, characterized in that: The culture method comprises using a culture medium, wherein the culture medium contains one or more of ox bile, ox bile powder or ox bile salt.

3. The culture method according to claim 2, characterized in that One or more of the ox bile, ox bile powder or ox bile salt is filtered and sterilized.

4. The culture method according to claim 2 or 3, characterized in that: In the culture medium, the volume fraction of ox bile is at least 40%.

5. The culture method according to claim 4, characterized in that In the culture medium, the volume fraction of ox bile is 40%-60%.

6. An application of Lactobacillus plantarum according to claim 1, characterized in that: The applications include one or both of the following groups: 1) Application in fermentation of cow bile; 2) Application in converting conjugated bile acids into free bile acids.

7. A method for fermenting bovine bile, characterized in that: The method comprises using microorganisms and introducing the microorganisms into cattle bile; the microorganisms comprise the plant lactobacillus as claimed in claim 1.

8. The method according to claim 7, characterized in that The method comprises filtering and sterilizing the cow bile to obtain sterilized cow bile, and inoculating the microorganisms into a seed culture medium to obtain a microorganism seed liquid.

9. The method according to claim 8, characterized in that The method comprises introducing the above-mentioned microbial seed liquid into the above-mentioned sterilized ox bile to obtain a fermentation system.

10. The method according to claim 9, characterized in that The initial pH of the fermentation system is 4.5-8.

0.

11. The method according to claim 9, characterized in that The temperature of the fermentation system is 20°C to 45°C.

12. The method according to claim 11, characterized in that The temperature of the fermentation system is 25°C-40°C.

13. The method according to any one of claims 7 to 12, characterized in that: The fermentation time is at least 36 hours.

14. The method according to claim 13, characterized in that The fermentation time is at least 48 hours.

15. The method according to claim 8, characterized in that The seed culture medium is a culture medium suitable for microorganisms.

16. The method according to claim 15, characterized in that The culture medium contains one or more of ox bile, ox bile powder or ox bile salt.

17. The method according to claim 16, characterized in that One or more of the ox bile, ox bile powder or ox bile salt is filtered and sterilized.

18. The method according to claim 16, characterized in that In the culture medium, the volume fraction of ox bile is at least 40%.

19. The method according to claim 18, characterized in that In the culture medium, the volume fraction of ox bile is 40%-60%.

20. A method for converting conjugated bile acid into free bile acid, characterized in that: The method comprises using microorganisms, introducing the microorganisms into cow bile, or a system containing bound bile acid for fermentation; the microorganisms comprise the plant lactobacillus described in claim 1.

21. The method according to claim 20, characterized in that The method comprises filtering and sterilizing cattle bile to obtain sterilized cattle bile, or filtering and sterilizing a system containing conjugated bile acid to obtain a sterilized system; The method comprises inoculating microorganisms into a seed culture medium to obtain a microorganism seed solution.

22. The method according to claim 21, characterized in that The method comprises the steps of inoculating the above-mentioned microbial seed solution into the above-mentioned sterilized ox bile or sterilization system to obtain a fermentation system.

23. The method according to claim 22, characterized in that The initial pH of the fermentation system is 4.5-8.

0.

24. The method according to claim 22, characterized in that The temperature of the fermentation system is 20°C to 45°C.

25. The method according to claim 24, characterized in that The temperature of the fermentation system is 25°C-40°C.

26. The method according to any one of claims 20-25, characterized in that: The fermentation time is at least 36 hours.

27. The method according to claim 26, characterized in that The fermentation time is at least 48 hours.

28. The method according to claim 21, characterized in that The seed culture medium is a culture medium suitable for microorganisms.

29. The method according to claim 28, characterized in that The culture medium contains one or more of ox bile, ox bile powder or ox bile salt.

30. The method according to claim 29, characterized in that One or more of the ox bile, ox bile powder or ox bile salt is filtered and sterilized.

31. The method according to claim 29, characterized in that In the culture medium, the volume fraction of ox bile is at least 40%.

32. The method according to claim 31, characterized in that In the culture medium, the volume fraction of ox bile is 40%-60%.

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