Lactobacillus reuteri hp-b1251 and application thereof

Abstract

The present application belongs to the technical field of microorganisms, and particularly relates to a Limosilactobacillus reuteri HP-B1251 and application thereof. The Limosilactobacillus reuteri HP-B1251 provided by the present application has been preserved in the China Center for Type Culture Collection on August 28, 2024, and the preservation number is CCTCC NO: M 20241872. The strain has good gastric acid tolerance, high temperature tolerance, bile salt tolerance, good acid production capacity, physiological and biochemical capacity suitable for the gastrointestinal tract of the body, and the ability to convert common ginsenosides into rare ginsenosides. At the same time, the biological conversion method has the advantages of strong reaction selectivity, mild conditions, few by-products, simple subsequent treatment process, small environmental pollution, and the like, and is an important way for industrialized production of rare ginsenosides.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of microorganisms, and particularly relates to a strain of Lactobacillus reuteri HP-B1251 and application thereof. BACKGROUND

[0002] Lactobacillus reuteri belongs to the domain of Firmicutes, the class of Bacilli, the order of Lactobacillales, the family of Lactobacillaceae, and the genus of Lactobacillus, and is a kind of peroxidase-negative, gram-positive, non-motile, non-spore-forming, specific heterofermentative bacteria. Research has found that Lactobacillus reuteri has excellent probiotic biological characteristics, has strong acid and bile salt tolerance under simulated gastrointestinal environment conditions, can reach the intestinal tract through the gastrointestinal tract, has obvious cholesterollowering effect, can inhibit harmful bacteria, and plays a role in regulating intestinal flora, and can be added to functional probiotic yogurt or fermented milk drinks.

[0003] Ginseng is a very precious medicinal material in life, and ginsenoside is an important active ingredient of ginseng, which can be generally divided into ginsenoside protopattern and rare ginsenoside. The ginsenoside protopattern can be directly extracted from ginseng, but the rare ginsenoside cannot be directly extracted and can only be obtained by metabolically converting the ginsenoside protopattern through high-tech technology. The ginsenoside protopattern is not easy to be absorbed and utilized, and the rare ginsenoside after metabolism has stronger biological activity and can greatly improve the bioavailability. Compared with the ginsenoside protopattern, the rare ginsenoside has the advantages of smaller molecular weight, more stable structure, faster metabolic absorption, and stronger pharmacological activity, and the rare ginsenoside has stronger anticancer activity than the ginsenoside protopattern. In the human body, the conversion of ginsenoside is mainly through the conversion of ginsenoside by microorganisms in the gastrointestinal tract, but the conversion efficiency is very low, the natural content of rare ginsenoside is very low, and it cannot be simply chemically synthesized.

[0004] Therefore, it is of great significance to find a probiotic bacterium that can safely and efficiently convert rare ginsenoside. SUMMARY

[0005] In order to solve the problems in the prior art, the purpose of the present application is to provide a strain of Lactobacillus reuteri HP-B1251. The Lactobacillus reuteri HP-B1251 involved in the present application has good reproductive capacity, good tolerance to high temperature, excellent acid production capacity, excellent gastric acid tolerance, high tolerance to bile salts, can be relatively adapted to the complex gastrointestinal environment of the body, and has the ability to efficiently convert ordinary ginsenoside to rare ginsenoside in a short time. At the same time, the biological conversion method also has the advantages of strong reaction selectivity, mild conditions, few by-products, simple subsequent treatment process, and small environmental pollution, and is an important way for industrialized production of rare ginsenoside.

[0006] The technical scheme of the present application is:

[0007] The present application provides a strain of Limosilactobacillus reuteri HP-B1251, which has been preserved in the China Center for Type Culture Collection on August 28, 2024, the address of the preservation unit is Wuhan University, Wuhan, China, the preservation number is CCTCC NO: M 20241872, and the taxonomic name is Limosilactobacillus reuteri. The 16S rRNA sequence of the strain is shown in SEQ ID NO: 1, which has been uploaded to the GenBank database with the sequence number PP657605.

[0008] Further, the present application provides an application of the Limosilactobacillus reuteri HP-B1251 in the preparation of rare ginsenosides through fermentation conversion.

[0009] Further, the Limosilactobacillus reuteri HP-B1251 is used for the fermentation conversion of ginsenosides Rf, Rb1, Rb3, Rd, Rg2, Rc into rare ginsenosides F2, Rh4, Rg3, Rh2, Rk2.

[0010] Further, the present application provides a process for the fermentation conversion of rare ginsenosides F2, Rh4, Rg3, Rh2, Rk2 by using the Limosilactobacillus reuteri HP-B1251, which comprises the following steps: culturing the Limosilactobacillus reuteri HP-B1251 to obtain a bacterial liquid, then mixing the bacterial liquid with raw materials containing ginsenosides Rf, Rb1, Rb3, Rd, Rg2, Rc for anaerobic fermentation, so as to convert the ginsenosides Rf, Rb1, Rb3, Rd, Rg2, Rc in the raw materials into rare ginsenosides F2, Rh4, Rg3, Rh2, Rk2, and obtain a fermentation liquid containing rare ginsenosides; then extracting the obtained fermentation liquid containing rare ginsenosides by using a conventional extraction method, and performing high-performance liquid chromatography analysis.

[0011] Further, the preparation method of the Limosilactobacillus reuteri HP-B1251 bacterial liquid comprises the following steps:

[0012] The single colony of the Limosilactobacillus reuteri HP-B1251 is inoculated into MRS liquid medium, and anaerobic culture is performed at 36-38℃ and 160-200r / min for more than 6h, then inoculation is performed into MRS liquid medium at a inoculation amount of 5-10%, and anaerobic culture is performed at 36-38℃ and 160-200r / min for more than 6h, so as to obtain the Limosilactobacillus reuteri HP-B1251 bacterial liquid.

[0013] Further, the concentration of the Limosilactobacillus reuteri HP-B1251 bacterial liquid is 1.0×109 ~1.0x10 10 CFU / mL.

[0014] Further, the raw material containing ginsenoside Rf, Rb1, Rb3, Rd and Rg2 in the fermentation conversion process of the rare ginsenoside is ginseng powder, and the adding amount of the ginseng powder is 1-5% of the weight of the Lactobacillus reuteri HP-B1251 bacterial liquid.

[0015] Further, in the fermentation conversion process of the rare ginsenoside, the Lactobacillus reuteri HP-B1251 bacterial liquid is uniformly mixed with the ginseng powder, and is anaerobically cultured at 36-38℃ and 160-200r / min for 24-36h.

[0016] Still further, in the fermentation conversion process of the rare ginsenoside, a sugar degree regulator can also be added; the sugar degree regulator is composed of the following components: yeast extract powder 20.1g, sodium acetate 7.5g, peptone 15g, triammonium citrate 4.5g, glucose 30g, potassium phosphate dibasic 3g, magnesium chloride heptahydrate 0.75g, manganese sulfate tetrahydrate 0.375g, Tween-80 1.5mL, cysteine 0.1875g, distilled water 1L and pH is adjusted to 6.8 and sterilized.

[0017] Further, in the fermentation conversion process of the rare ginsenoside, the weight ratio of the Lactobacillus reuteri HP-B1251 bacterial liquid to the sugar degree regulator is 3-8:3, preferably 7:3.

[0018] The application also provides an application of the above-mentioned Lactobacillus reuteri HP-B1251 in the preparation of food and health products containing rare ginsenosides.

[0019] The application also provides an application of the above-mentioned Lactobacillus reuteri HP-B1251 in the preparation of products with enhanced immune function.

[0020] Another technical scheme of the application provides a bacterial agent, which comprises the Lactobacillus reuteri HP-B1251 or the fermentation liquid thereof according to claim 1.

[0021] Further, the viable bacterial count of the Lactobacillus reuteri HP-B1251 in the bacterial agent is 5.0x10 10 ~3.0x10 11 CFU / g.

[0022] Still further, the bacterial agent is a solid bacterial agent or a liquid bacterial agent.

[0023] In the present application, by observing the growth state of Lactobacillus reuteri HP-B1251, evaluating the temperature tolerance ability, acid production ability, acid tolerance ability, bile salt tolerance ability, and the ability of the bacterial liquid and fermentation product to transform ginsenosides, it is found that Lactobacillus reuteri HP-B1251 has strong growth and reproduction ability, strong high-temperature tolerance, strong acid production ability, strong acid tolerance, strong bile salt tolerance, and excellent ginsenoside transformation ability. Finally, the whole genome of Lactobacillus reuteri HP-B1251 is sequenced, providing a basis for further research on Lactobacillus reuteri HP-B1251. Through bioinformatics comparison and analysis, Lactobacillus reuteri HP-B1251 of the present application does not contain resistance genes, drug resistance genes and virulence factors, and is a probiotic with high safety.

[0024] Compared with the prior art, the Lactobacillus reuteri HP-B1251 provided by the present application has the following advantages:

[0025] (1) The Lactobacillus reuteri HP-B1251 screened from fresh cow's milk has strong growth and reproduction ability, good tolerance to high temperature, excellent acid production ability, excellent gastric acid tolerance, and very high tolerance to bile salts, and can adapt to the complex gastrointestinal environment of the body.

[0026] (2) The Lactobacillus reuteri HP-B1251 provided by the present application can efficiently transform common ginsenosides into rare ginsenosides in a short time, and can be used for the production of foods and health products using ginseng as a raw material, to enhance the body's resistance to pathogens, and has good development and application prospects.

[0027] Biological preservation instructions:

[0028] Lactobacillus reuteri HP-B1251 has been preserved in the China Center for Type Culture Collection on August 28, 2024, the address of the preservation unit is Wuhan University, Wuhan, China, the preservation number is CCTCC NO: M20241872, and the taxonomic name is Lactobacillus reuteri. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 is the growth morphology of Lactobacillus reuteri HP-B1251 strain;

[0030] Figure 2Agarose gel electrophoresis map of PCR amplification product of 16S rRNA of Lactobacillus reuteri HP-B1251, wherein M: 2K plus I DNA marker; 1, 2, 3, 4: PCR amplification product of 16S rRNA of Lactobacillus reuteri HP-B1251;

[0031] Figure 3 Agarose gel electrophoresis map of PCR product of monoclonal bacteria of Lactobacillus reuteri HP-B1251, wherein M: 2K plus I DNA marker; 1: PCR product of monoclonal antibody;

[0032] Figure 4 Genetic evolution tree of Lactobacillus reuteri HP-B1251;

[0033] Figure 5 Color change map of Lactobacillus reuteri HP-B1251 carbohydrate fermentation test; wherein 1-11 are respectively esculin, cellobiose, maltose, mannitol, salicin, sorbitol, sucrose, raffinose, inulin, lactose, 1% sodium hippurate as carbon source;

[0034] Figure 6 High performance liquid chromatography analysis map of biotransformation of ginsenoside by Lactobacillus reuteri HP-B1251, wherein A is ginsenoside blank control (ginseng powder), B is Lactobacillus reuteri HP-B1251 fermented ginseng powder;

[0035] Figure 7 Whole genome map of Lactobacillus reuteri HP-B1251;

[0036] Figure 8 Plasmid 1 map of Lactobacillus reuteri HP-B1251;

[0037] Figure 9 Plasmid 2 map of Lactobacillus reuteri HP-B1251;

[0038] Figure 10 Statistical map of common and unique annotations of general database of coding genes;

[0039] Figure 11 PFAM domain statistical map;

[0040] Figure 12 Classification map of GO annotation results;

[0041] Figure 13 Classification map of KEGG pathway results;

[0042] Figure 14 Classification map of COG results;

[0043] Figure 15 Figure 2 is a distribution map of NR species;

[0044] Figure 16 Figure 3 is a classification map of TCDB results. DETAILED DESCRIPTION

[0045] The present application is further illustrated by the following description of specific embodiments, but the application should not be considered limited thereto, as various modifications or equivalents can be made thereto by those skilled in the art without departing from the scope of the application.

[0046] In the following examples, reagents not specifically mentioned are conventional reagents, which can be purchased from conventional reagent production and sales companies, and the methods used are conventional methods, unless otherwise specified.

[0047] The Limosilactobacillus reuteri HP-B1251 described in the present application is isolated from fresh cow milk (from Heze City, Shandong Province), and the MRS solid medium (model CM188) and MRS liquid medium (model CM187) are purchased from Beijing Land Bridge Technology Co., Ltd.

[0048] Example 1: Isolation, purification and identification of Limosilactobacillus reuteri HP-B1251

[0049] (1) Isolation and purification of Limosilactobacillus reuteri HP-B1251

[0050] This strain is derived from fresh cow milk. The specific method steps for isolating and purifying the strain are as follows: first sterilize the culture dish, bamboo stick, triangular rake, pipette tip, MRS medium and physiological saline. Then pour the MRS medium into the sterilized culture dish to form an MRS solid plate. Then take 1 mL of fresh cow milk, dilute it to a concentration of 10 -5 , 10 -6 , 10 -7 CFU / mL with physiological saline, and use a pipette to transfer the diluent to the MRS solid plate, and use a triangular rake to spread it. After spreading, place it in a 37℃ constant temperature incubator for anaerobic culture. After two days of culture, observe the colony characteristics, select a single colony with typical characteristics, and inoculate it on a new MRS plate with a bamboo stick, and purify it with a triangular rake. Repeat the culture and selection process until a pure culture strain is obtained. Identify the purified strain and determine it as Limosilactobacillus reuteri HP-B1251. Store the pure bacteria under appropriate conditions (such as glycerol, freeze-drying, etc.) for subsequent research and use. The growth morphology of the strain is shown in Figure 1.Figure 1 .

[0051] (2) Identification by full-length sequencing of 16S rRNA from Lactobacillus reuteri HP-B1251

[0052] The purified strain was selected, and the strain DNA fragment was obtained by a modified SDS-alkali lysis method. PCR amplification of the strain's 16S rRNA was performed using 16S primers 27F-PBACS and 1541R-PBACS (as shown in primer sequence I in Table 1). The conditions were: 95℃ pre-denaturation for 10 min, 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 1.5 min, 30 cycles, 72℃ extension for 10 min, and storage at 16℃.

[0053] Table 1 Primer Sequences I

[0054]

[0055] The obtained PCR products were identified by agarose gel electrophoresis, such as... Figure 2 As shown, the gene from the post-gel recovery product was recombined into GB05-dir. After a period of solid-state culture, monoclonal antibodies were picked and amplified by PCR using 16S primers T7-Elong and T7-TermElong (as shown in primer sequence II in Table 2). The conditions were: 95℃ pre-denaturation for 10 min, 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 1.5 min, 30 cycles, 72℃ extension for 10 min, and storage at 16℃. The obtained PCR products were identified by agarose gel electrophoresis. Figure 3 As shown.

[0056] Table 2 Primer Sequences II

[0057] Target Primer sequence T7-Elong 5'-TTAATACGACTCACTATAGGGGAATTG-3' T7-TermElong 5'-TTATGCTAGTTATTGCTCAGCGG-3'

[0058] The PCR products were sent to Suzhou Genewiz Biotechnology Co., Ltd. for sequencing. The nucleotide sequence of the strain's 16S rRNA is shown in SEQ ID NO:1.

[0059] GAGAGTTTGATCCTGGCTCAGGATGAACGCCGGCGGTGTGCCTAATACATGCAAGTCG

[0060] TACGCACTGGCCCAACTGATTGATGGTGCTTGCACCTGATTGACGATGGATCACCAGT

[0061] GAGTGGCGGACGGGTGAGTAACACGTAGGTAACCTGCCCCGGAGCGGGGGATAACAT

[0062] TTGGAAACAGATGCTAATACCGCATAACAACAAAAGCCACATGGCTTTTGTTTAAAAG

[0063] ATGGCTTTGGCTATCACTCTGGGATGGACCTGCGGTGCATTAGCTAGTTGGTAAGGTA

[0064] ACGGCTTACCAAGGCGATGATGCATAGCCGAGTTGAGAGACTGATCGGCCACAATGG

[0065] AACTGAGACACGGTCCATACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATG

[0066] GGCGCAAGCCTGATGGAGCAACACCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAG

[0067] CTCTGTTGTTGGAGAAGAACGTGCGTGAGAGTAACTGTTCACGCAGTGACGGTATCCA

[0068] ACCAGAAAGTCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAG

[0069] CGTTATCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTGCTTAGGTCTGATGTG

[0070] AAAGCCTTCGGCTTAACCGAAGAAGTGCATCGGAAACCGGGCGACTTGAGTGCAGAA

[0071] GAGGACAGTGGAACTCCATGTGTAGCGGTGGAATGCGTAGATATATGGAAGAACACC

[0072] AGTGGCGAAGGCGGCTGTCTGGTCTGCAACTGACGCTGAGGCTCGAAAGCATGGGTA

[0073] GCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAGTGCTAGGTGTTG

[0074] GAGGGTTTCCGCCCTTCAGTGCCGGAGCTAACGCATTAAGCACTCCGCCTGGGGAGTA

[0075] CGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCA

[0076] TGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATCTTGCGCTAA

[0077] CCTTAGAGATAAGGCGTTCCCTTCGGGGACACAATGACAGGTGGTGCATGGTCGTCGT

[0078] CAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTTACTA

[0079] GTTGCCAGCATTAAGTTGGGCACTCTAGTGAGACTGCCGGTGACAAACCGGAGGAAG

[0080] GTGGGGACGACGTCAGATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAA

[0081] TGGACGGTACAACGAGTCGCAAGCTCGCGAGAGTAAGCTAATCTCTTAAAGCCGTTCT

[0082] CAGTTCGGACTGTAGGCTGCAACTCGCCTACACGAAGTCGGAATCGCTAGTAATCGCG

[0083] GATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCA

[0084] TGGGAGTTTGTAACGCCCAAAGTCGGTGGCCTAACCTTTATGGAGGGAGCCGCCTAAG

[0085] GCGGGACAGATGACTGGGGTGAAGTCGTAACAAGGTAGCCGTAGGAGAACCTGCGGC

[0086] TGGATCACCTCCTT

[0087] The BLAST gene alignment reached 100.00% similarity with Limosilactobacillus reuteri in GenBank, which was a strain of L. reuteri, named HP-B1251, and the 16S rRNA sequence had been uploaded to the GenBank database with the accession number PP657605.

[0088] The genetic phylogenetic tree of L. reuteri HP-B1251 was aligned, and the results are shown in Figure 4 .

[0089] (3) L. reuteri HP-B1251 carbohydrate fermentation experiment

[0090] HBI lactic acid bacteria biochemical identification strips (purchased from Qingdao High-tech Industrial Park Haibo Biotechnology Co., Ltd.) were selected to identify the reaction of L. reuteri HP-B1251 to 11 kinds of carbohydrates. The color change is shown in Figure 5 , wherein 1-11 are esculin, cellobiose, maltose, mannitol, salicin, sorbitol, sucrose, raffinose, inulin, lactose, and 1% sodium hippurate as carbon sources. The results are shown in Table 3. From Table 3, it can be seen that L. reuteri HP-B1251 can utilize, decompose and metabolize maltose, sucrose, raffinose, lactose and other 4 kinds of carbohydrates in the 11 kinds of carbohydrate fermentation experiments.

[0091] Table 3 L. reuteri HP-B1251 carbohydrate fermentation experiment

[0092]

[0093] Example 2 Evaluation of the growth state of L. reuteri HP-B1251 strain

[0094] The single colony of Lactobacillus reuteri HP-B1251 on the MRS solid culture medium was inoculated into fresh sterilized MRS liquid medium with a bamboo stick, and was cultured at 37℃, 100rpm anaerobically for 24h. 1mL of the fermentation liquid was taken by a pipette, centrifuged at 8000rpm for 1min, and the supernatant was discarded, and the bacteria were left, and the wet weight was calculated by weighing. The dry weight was calculated by weighing after drying at 55℃ for 12h, and the test results are shown in Table 4.

[0095] Table 4 Strain growth state research of Lactobacillus reuteri HP-B1251

[0096] Culture time (h) Wet weight / (g / mL) Dry weight / (g / mL) 24 4.04 x 10 -2 ±0.0116]]> 3.5 x 10 -3 ±0.0004]]>

[0097] As shown in Table 4, the dry weight of the bacteria obtained by weighing is 3.5x10 -3 ±0.0004g / mL, so it can be seen that Lactobacillus reuteri HP-B1251 has strong reproductive capacity and can reproduce in large quantities.

[0098] Example 3: Evaluation of temperature tolerance of Lactobacillus reuteri HP-B1251

[0099] The single colony of Lactobacillus reuteri HP-B1251 on the MRS solid culture medium was inoculated into fresh sterilized MRS liquid medium with a bamboo stick, and was cultured at 37℃, 100rpm anaerobically for 24h. The bacteria liquid at 37℃, 55℃ and 70℃ was heated for 1min, 2min and 3min respectively, and then diluted to a concentration of 10 -6 and 10 -7 CFU / mL, and then coated on the solid MRS culture medium and cultured anaerobically at 37℃ for 16-20h. The test results are shown in Table 5.

[0100] Table 5 Temperature tolerance research of Lactobacillus reuteri HP-B1251

[0101]

[0102] As shown in Table 5, a large number of live bacteria of Lactobacillus reuteri HP-B1251 of the present application still exist after being heated at 70℃ for 1min, so it can be seen that Lactobacillus reuteri HP-B1251 has good high temperature resistance.

[0103] Example 4: Evaluation of acid production capacity of Lactobacillus reuteri HP-B1251

[0104] The single colony of Lactobacillus reuteri HP-B1251 on the MRS solid culture medium was inoculated into fresh sterilized MRS liquid medium with a bamboo stick, and was cultured at 37℃ under anaerobic condition at 100r / min. The supernatant pH was measured after centrifugation at 3580r / min for 15min at 12h, 24h and 30h, and the test results are shown in Table 6.

[0105] Table 6 Study on the acid-producing capacity of Lactobacillus reuteri HP-B1251

[0106] Culture time (h) pH 12 4.92 24 4.66 30 4.47

[0107] As shown in Table 6, the Lactobacillus reuteri HP-B1251 of the present application can reach a strong acidity at 12h of anaerobic culture, with a pH value of 4.92, and it can be seen that the Lactobacillus reuteri HP-B1251 has strong acid-producing capacity.

[0108] Example 5: Evaluation of the acid tolerance of Lactobacillus reuteri HP-B1251

[0109] An artificial gastric juice was prepared (8.2mL dilute hydrochloric acid, 400mL double distilled water, 5g pepsin, after shaking, add double distilled water to constant volume to 500mL, take a certain amount of solution to adjust pH to 2.64, 3.85, 4.89. Sterilize with 0.44μm filter membrane, and store in a 4℃ refrigerator for standby use). The single colony of Lactobacillus reuteri HP-B1251 on the MRS solid culture medium was inoculated into fresh sterilized MRS liquid medium with a bamboo stick, and was cultured at 37℃ under anaerobic condition at 100rpm for 18-24h. 60mL of bacterial liquid was centrifuged at 3580rpm for 15min, and the supernatant was discarded, and the bacterial body was resuspended with 1.5mL of normal saline. Four centrifuge tubes were taken, and 6mL of artificial gastric juice was added respectively, and then 200μl of resuspended bacterial liquid was added respectively and mixed. At 0min and 150min, dilution was performed to a concentration of 10 -6 and 10 -7 CFU / mL was coated on solid MRS medium, and after culture for 18-24h, the single colonies on the plate were counted and the survival rate was calculated, and the test results are shown in Table 7.

[0110] Table 7 Study on the acid tolerance of Lactobacillus reuteri HP-B1251

[0111]

[0112] As shown in Table 7, the Lactobacillus reuteri HP-B1251 of the present application still has a large number of strains surviving after 150min in the artificial gastric juice with pH 2.64, and therefore it has strong acid tolerance.

[0113] Example 6: Evaluation of bile salt tolerance of Limosilactobacillus reuteri HP-B1251

[0114] The artificial intestinal fluid was prepared (3.4 g of potassium dihydrogen phosphate, 250 mL of double distilled water, and the pH was adjusted to 6.8 with 0.1 mol / L sodium hydroxide solution, 2.5 g of trypsin was added, and after shaking, double distilled water was added to 500 mL, and a certain amount of solution was added to a certain amount of bovine bile salt to prepare artificial intestinal fluid with bile salt concentrations of 0.03%, 0.1%, 0.2%, and 0.3%, respectively. The 0.44 μm filter membrane was used to remove bacteria, and it was stored in the 4 ℃ refrigerator for standby use). The single colony of Limosilactobacillus reuteri HP-B1251 on the MRS solid medium was inoculated into fresh sterilized MRS liquid medium with a bamboo stick, and it was cultured at 37 ℃, 100 rpm, anaerobically for 16-20 h. 60 mL of bacterial solution was centrifuged at 3580 rpm for 15 min, and the supernatant was discarded, and the bacterial body was resuspended with 1.5 mL of normal saline. Four centrifuge tubes were taken, 6 mL of artificial intestinal fluid was added respectively, and then 200 μl of resuspended bacterial solution was added respectively and mixed. At 0 min and 150 min, it was diluted to a concentration of 10 -6 and 10 -7 CFU / mL was coated on the solid MRS medium and cultured for 16-20 h, and the single colonies on the plate were counted and the survival rate was calculated, and the test results are shown in Table 8.

[0115] Table 8: Evaluation of bile salt tolerance of Limosilactobacillus reuteri HP-B1251

[0116]

[0117] As can be seen from Table 8, the survival rate of Limosilactobacillus reuteri HP-B1251 of the present application after 150 min at a bile salt concentration of 0.3% is as high as 112.0%, which indicates that it has high tolerance to bile salt.

[0118] Example 7: Ability of Limosilactobacillus reuteri HP-B1251 to biotransform ginsenosides

[0119] Inoculate a single colony of Lactobacillus reuteri HP-B1251 on MRS solid medium with a bamboo stick into 30 mL of fresh sterilized MRS liquid medium, incubate at 37°C, 180 r / min anaerobically for more than 6 hours. Then inoculate 5% to 10% into 70 mL of fresh sterilized MRS liquid medium, incubate at 37°C, 180 r / min anaerobically for more than 6 hours to obtain Lactobacillus reuteri HP-B1251 bacterial liquid. Add 1.5 g of ginseng powder and 30 mL of sugar regulator (yeast extract powder 20.1 g, sodium acetate 7.5 g, protein peptone 15 g, triammonium citrate 4.5 g, glucose 30 g, potassium phosphate dibasic 3 g, magnesium chloride heptahydrate 0.75 g, manganese sulfate tetrahydrate 0.375 g, Tween-80 1.5 mL, cysteine 0.1875 g, distilled water 1 L, and adjust pH to 6.8 and sterilize) into the Lactobacillus reuteri HP-B1251 bacterial liquid obtained above at the same time, mix well, incubate at 37°C, 180 r / min anaerobically for 24 to 36 hours to obtain a fermentation liquid. Add the fermentation liquid and an extractant (saturated n-butanol solution, ultrapure water:n-butanol = 1:1) into a separating funnel, shake well. Centrifuge the bacterial liquid at 3580 r / min for 10 min, take the supernatant at the top, rotary evaporate at 55°C, dissolve with 20% acetonitrile, and analyze by high performance liquid chromatography (HPLC) with a reverse phase C 18 The gradient elution program is shown in Table 10.

[0120] The column equilibrium system is shown in Table 9:

[0121] Table 9

[0122] 100% acetonitrile 10-15 min 50% acetonitrile, 50% ultrapure water 10-15 min 20% acetonitrile, 80% ultrapure water 20 min

[0123] The gradient elution program is shown in Table 10.

[0124] Table 10

[0125]

[0126] Take the chromatogram at 203 nm wavelength for analysis, and the results are shown in Table 11. Figure 6 Table 11

[0127] Take the types and contents of ginsenosides before and after fermentation (ginseng powder and ginseng powder fermented by Lactobacillus reuteri HP-B1251) as indexes, calculate the content improvement rate of rare ginsenosides, and the test results are shown in Table 11.

[0128] The research results show that the contents of ginsenosides Rf, Rb1, Rb3, Rd, Rg2 and Rc all decrease, and a large number of rare ginsenosides F2, Rh4, Rg3, Rh2 and Rk2 are produced by fermentation and conversion. Therefore, the Lactobacillus reuteri HP-B1251 has strong conversion capacity for rare ginsenosides.

[0129] Table 11 Content improvement rate of rare ginsenosides before and after fermentation

[0130]

[0131]

[0132] Note: Content improvement rate = content of rare ginsenosides after fermentation by Lactobacillus reuteri HP-B1251 / content of rare ginsenosides in ginsenoside sample before fermentation

[0133] Example 8 Whole genome determination of Lactobacillus reuteri HP-B1251

[0134] Based on the Nanopore third-generation sequencing technology and the second-generation sequencing technology platform, the genome of Lactobacillus reuteri HP-B1251 is sequenced and assembled to obtain a complete map of the genome, and a visual whole genome map of the bacteria is attached. Figure 7 The genome size of the bacteria is 2219140 bp, the GC ratio is 39.07%, and the genome is circular. The genome contains 2165 CDS, 69 tRNA and 18 rRNA, and contains two plasmids. The size of plasmid 1 is 19058 bp, the GC ratio is 36.87%, and it is a circular plasmid as shown in Figure 8 The size of plasmid 2 is 7047 bp, the GC ratio is 39.24%, and it is a circular plasmid as shown in Figure 9 .

[0135] Example 9 Database genome function annotation analysis of Lactobacillus reuteri HP-B1251 (1) General database annotation proportion statistics

[0136] In order to obtain comprehensive gene function information, gene function annotation of eight databases is carried out, including: UniProt, KEGG and KEGG Pathway, GO, Pfam, COG, TIGERfams, RefSeq, NR. The predicted gene sequence is subjected to BLAST+ (Version: 2.11.0+) alignment with the functional databases such as COG, KEGG, Uniprot and Refseq, to obtain gene function annotation results. The software hmmer (Version: 3.3.2) is used for function annotation based on the Pfam and TIGERFAM databases. The annotation statistical results are shown in Table 12. The common and unique annotations of the general database gene annotation are shown in Figure 10 , Figure 10 In the table, the left side is the general database gene annotation statistics; the right side is the common and unique statistics, for example, the first column is the common statistics of all general databases, the upper side is the number statistics, and the lower side is the annotation of the common or unique database.

[0137] From Table 12 and Figure 10 It can be seen that the Limosilactobacillus reuteri HP-B1251 of the present application contains more gene fragments, which can obtain comprehensive gene function information in various general databases, and can obtain more gene function information, which provides a certain basis for the research of each gene of the strain.

[0138] Table 12: Encoding gene annotation result statistics

[0139]

[0140]

[0141] (2) Pfam domain classification

[0142] The Pfam database is a homologous protein family database, which is a collection of protein domain families, including more than 16,000 protein families. Proteins are generally composed of one or more functional regions, which are usually called domains. Domains appear in different combinations in different proteins, forming the diversity of proteins, and identifying the domains in the protein is of great significance to understand the function of the protein.

[0143] Based on the annotation of the Pfam domain, the genes annotated for each domain are statistically summarized, and the top 20 domains with the most annotations are plotted and displayed, and the results are shown in Figure 11 The horizontal coordinate in the figure is the name of the protein family, and the vertical coordinate is the number of genes aligned to the protein family.

[0144] (3) Gene Ontology classification

[0145] Gene Ontology (GO) is an international standardized gene function classification system, which provides a set of dynamically updated controlled vocabulary to comprehensively describe the attributes of genes and gene products in organisms.

[0146] The present application obtains GOslim classification by simplifying GO (Gene Ontology) annotation information, and selects the top 20 GOslim secondary classifications with the most annotations in each classification after statistically summarizing the functions of genes from three aspects of cell components, molecular functions and biological processes. The result is shown in Figure 12 , wherein the horizontal coordinate is the GO classification content, and the vertical coordinate is the number of genes. Figure 12 The gene enrichment of each secondary function under the background of all genes is shown in Figure 12 , which reflects the position of each secondary function under this background. From , the functions of genes and gene products in Limosilactobacillus reuteri HP-B1251 and the number of genes showing the functions are shown, which shows the strength of each function and provides a research direction for subsequent in-depth research.

[0147] (4) KEGG Pathway classification

[0148] KEGG (Kyoto Encyclopedia of Genes and Genomes) is a major public database for systematically analyzing the metabolic pathways of gene products in cells and the functions of these gene products. KEGG can be used to further study the complex behaviors of genes in biology.

[0149] After KEGG annotation of genes, the genes are classified according to the KEGG metabolic pathways they participate in, and the result is shown in Figure 13 , wherein the horizontal coordinate is the number of genes annotated under the Pathway classification; and the vertical coordinate is the Pathway classification, and different colors represent different large classifications. Figure 13 The metabolic pathways of each gene product in Limosilactobacillus reuteri HP-B1251 in cells are shown in

[0150] (5) COG classification

[0151] COG (Cluster of Orthologous Groups of proteins) is a database for orthologous classification of gene products, each COG protein is assumed to come from an ancestral protein, and the COG database is constructed based on the coding proteins of bacteria, algae and eukaryotes with complete genomes, the phylogenetic relationship, the Unigene and the COG database are compared, the possible function of the gene is predicted and the function classification statistics is carried out, and the gene function distribution characteristics of the species are known from a macroscopic point of view. COG is divided into 26 groups.

[0152] The application classifies the COG annotated genes according to the group of COG, and the result is shown in Figure 14 The horizontal coordinate in the figure is the classification content of COG, and the vertical coordinate is the number of genes. In different functional classes, how many genes are occupied reflects the metabolic or physiological bias under the corresponding period and environment, and scientific explanation can be made in combination with the distribution of the research object in each functional class.

[0153] (6) Uniprot annotation

[0154] UniProt Archive is a comprehensive non-redundant database that contains all major, public protein sequence databases. Proteins can exist in different databases and can have multiple versions in the same database.

[0155] The number of genes of the Limosilactobacillus reuteri HP-B1251 whole genome annotated to the Uniprot database is 2162, accounting for 99.86%.

[0156] (7) Refseq annotation

[0157] The Refseq (Reference Sequences) database is a database that collects annotated non-redundant transcripts, proteins and genome sequences, is a database corrected by NCBI and other organizations, uses the terms defined by the Human Gene Nomenclature Committee, and includes official gene symbols and optional symbols. RefSeq sequences are non-redundant databases screened by NCBI, and generally have high reliability.

[0158] The number of genes of the Lactobacillus casei HP-B1142 whole genome annotated to the Refseq database is 2154, accounting for 99.49%.

[0159] (8) NR annotation

[0160] NR database: Non-redundant protein amino acid sequence database, including SwissProt, PIR (Protein Information Resource), PRF (Protein Research Foundation), PDB (Protein Data Bank) protein database non-redundant data, and protein data translated from CDS data of GenBank and RefSeq.

[0161] The NR species distribution map of the Limosilactobacillus reuteri HP-B1251 according to the present application is shown in FIG. 1, wherein different colors represent different species. Figure 15

[0162] (9) TIGRFAMs annotation

[0163] TIGRFAMs is a database composed of multiple sequence alignment, hidden Markov model (HMM) and related information, which is a member of interPro, used for protein sequence classification and supporting automatic annotation of proteins (mostly prokaryotes). From v10.0, TIGRFAMs models use HMMER 3, which has excellent search speed and sensitivity.

[0164] The number of genes of the Limosilactobacillus reuteri HP-B1251 genome annotated to the Tigrfams database according to the present application is 1159, accounting for 53.53%.

[0165] Example 10: Limosilactobacillus reuteri HP-B1251 specific database annotation

[0166] The present application is personalized analysis of Limosilactobacillus reuteri HP-B1251, and specific database annotation thereof, including ARDB database annotation, carbohydrate enzyme annotation based on CAZy database, pathogen and host interaction analysis, drug resistance gene annotation, cytochrome P450 annotation, VFDB annotation, signal peptide prediction, TCDB transporter annotation.

[0167] ​The whole genome of the Limosilactobacillus reuteri HP-B1251 is annotated by a special database, and it is found that: no resistance genes are annotated in the ARDB database; no completely matched gene sequences are annotated in the pathogen and host interaction database; no drug resistance genes are annotated in the CARD database; no completely matched gene sequences are annotated in cytochrome P450 annotation; no completely matched virulence factors are annotated in the VFDB database; the results of carbohydrate enzyme annotation are shown in Table 13; the results of signal peptide prediction of the whole genome of the Limosilactobacillus reuteri HP-B1251 are shown in Table 14, and the results of transmembrane protein and secreted protein prediction are shown in Table 15; the results of TCDB membrane transport protein annotation are shown in Table 16, and the horizontal coordinate of the figure is the classification content of TCDB, and the vertical coordinate is the number of genes. Figure 16

[0168] Table 13 CAZy annotation

[0169]

[0170]

[0171] Table 14 Signal peptide prediction statistics table

[0172] Protein type Number Protein with information peptide 99

[0173] Table 15 Transmembrane protein and secreted protein prediction statistics table

[0174] Protein type Number Transmembrane protein 479 Secreted protein 30

[0175] Based on the above data, it is shown that the Limosilactobacillus reuteri HP-B1251 has strong enzyme activity, protein expression effect and good edibility, and can be safely applied to the preparation of related food and health products, and has good development and application prospect.

[0176] The above examples only exemplarily illustrate the principles and effects of the present application, and are not used to limit the present application. Any person skilled in the art can modify or change the above examples without departing from the spirit and scope of the present application. Therefore, all equivalent modifications or changes completed by those skilled in the art without departing from the spirit and technical thought disclosed by the present application should be covered by the claims of the present application.​

Claims

1. A Lactobacillus reuteri (L. reuteri) strain HP-B1251, characterized in that, Limosilactobacillus reuteri It has been deposited in China Center for Type Culture Collection on August 28, 2024, and the preservation number is CCTCC NO: M 20241872. ​ 2. The use of Lactobacillus reuteri HP-B1251 according to claim 1 in the fermentation conversion preparation of rare ginsenosides.

3. Use according to claim 2, characterized in that, The Lactobacillus reuteri HP-B1251 is used for the fermentation conversion of ginsenosides Rf, Rb1, Rb3, Rd, Rg2, Rc to rare ginsenosides F2, Rh4, Rg3, Rh2, Rk2.

4. Use according to claim 2, characterized in that, The fermentation conversion process of the rare ginsenosides is as follows: the Lactobacillus reuteri HP-B1251 is cultured to obtain a bacterial liquid, then the raw material containing ginsenosides Rf, Rb1, Rb3, Rd, Rg2, Rc is used as a substrate, mixed with the Lactobacillus reuteri HP-B1251 bacterial liquid for anaerobic fermentation, and the ginsenosides Rf, Rb1, Rb3, Rd, Rg2, Rc in the raw material are converted to rare ginsenosides F2, Rh4, Rg3, Rh2, Rk2.

5. Use according to claim 4, characterized in that, The concentration of the Lactobacillus reuteri HP-B1251 bacterial solution in the fermentation conversion process of the rare ginsenoside is 1.0×10 9 ~1.0×10 10 CFU / mL.

6. Use according to claim 4, characterized in that, The raw material containing ginsenosides Rf, Rb1, Rb3, Rd, Rg2, Rc in the fermentation conversion process of the rare ginsenosides is ginseng powder, and the addition amount of the ginseng powder is 1-5% of the weight of the Lactobacillus reuteri HP-B1251 bacterial liquid.

7. The use of Lactobacillus reuteri HP-B1251 according to claim 1 in the preparation of food containing rare ginsenosides.

8. An inoculant characterized in that, The bacterial agent comprises the Lactobacillus reuteri HP-B1251 according to claim 1 or a fermentation liquid thereof.

9. The bacterial agent of claim 8, characterized in that, The viable cell number of Lactobacillus reuteri HP-B1251 in the bacterial agent is 5.0 x 10 10 3.0 x 10 11 CFU / g.

10. The bacterial agent of claim 8, characterized in that, The bacterial agent is a solid bacterial agent or a liquid bacterial agent.

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