Lactobacillus reuteri HP-B1251 and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to lactobacillus reuteri HP-B1251 and application thereof. The lactobacillus reuteri HP-B1251 provided by the invention is preserved in the China Center for Type Culture Collection on August 28, 2024, and the preservation number of the lactobacillus reuteri HP-B1251 is CCTCC (China Center for Type Culture Collection) NO: M 20241872. The strain has good gastric acid tolerance, high temperature tolerance, cholate tolerance, good acid production capacity, physiological and biochemical capacity adapting to gastrointestinal tracts of organisms, and capacity of converting common ginsenoside into rare ginsenoside. Meanwhile, the biological conversion method also has the advantages of strong reaction selectivity, mild conditions, few byproducts, simple subsequent treatment process, small environmental pollution and the like, and is an important way for industrial production of the rare ginsenoside.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and specifically relates to a strain of Lactobacillus reuteri HP-B1251 and an application thereof. Background Art

[0002] Lactobacillus reuteri belongs to the Firmicutes, Bacillus, Lactobacilales, Lactobacilaceae, Lactobacillus genus. It is a catalase-negative, Gram-positive, non-motile, non-spore-forming, obligate heterofermentative bacterium. Studies have found that Lactobacillus reuteri has excellent probiotic biological characteristics, has strong acid and bile salt resistance under simulated gastrointestinal environmental conditions, and most of it can pass through the stomach and reach the intestines. It has a significant effect on degrading cholesterol, and can inhibit harmful bacteria and play a role in regulating intestinal flora. It can be added to functional probiotic yogurt or fermented milk drinks.

[0003] Ginseng is an extremely precious medicinal material in life, and ginsenosides are important active ingredients of ginseng, which can generally be divided into prototype ginsenosides and rare ginsenosides. Prototype ginsenosides can be extracted directly from ginseng, but rare ginsenosides cannot be directly extracted and can only be obtained by metabolizing and transforming prototype ginsenosides through high-tech technology. Prototype ginsenosides are not easily absorbed and utilized, while rare ginsenosides after metabolism have stronger biological activity and can greatly improve bioavailability. Compared with prototype ginsenosides, rare ginsenosides have the advantages of smaller molecular weight, more stable structure, faster metabolic absorption, and stronger pharmacological activity. Rare ginsenosides have stronger anti-cancer activity than prototype ginsenosides. In the human body, the transformation of ginsenosides is mainly through the transformation of ginsenosides by microorganisms in the gastrointestinal tract, but the conversion efficiency is extremely low, the natural content of rare ginsenosides is extremely low, and they cannot be simply chemically synthesized.

[0004] Therefore, it is of great significance to discover a probiotic that can safely and efficiently transform rare ginsenosides. Summary of the invention

[0005] In order to solve the problems existing in the prior art, the object of the present invention is to provide a strain of Lactobacillus reuteri HP-B1251. The Lactobacillus reuteri HP-B1251 involved in the present invention has good reproduction ability, good tolerance to high temperature, excellent acid production ability, excellent stomach acid resistance, high tolerance to bile salts, can relatively adapt to the complex gastrointestinal environment of the body, and has the ability to efficiently convert ordinary ginsenosides into rare ginsenosides in a short time. At the same time, the bioconversion method also has the advantages of strong reaction selectivity, mild conditions, few by-products, simple subsequent treatment process, and little environmental pollution, and is an important way for the industrial production of rare ginsenosides.

[0006] The technical solution of the present invention is:

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

[0008] Furthermore, the present invention provides an application of Lactobacillus reuteri HP-B1251 in the fermentation and conversion preparation of rare ginsenosides.

[0009] Furthermore, the Lactobacillus reuteri HP-B1251 is used to ferment ginsenosides Rf, Rb1, Rb3, Rd, Rg2, and Rc to convert them into rare ginsenosides F2, Rh4, Rg3, Rh2, and Rk2.

[0010] Furthermore, the process provided by the present invention for fermenting and converting rare ginsenosides F2, Rh4, Rg3, Rh2, and Rk2 using Lactobacillus reuteri HP-B1251 is as follows: Lactobacillus reuteri HP-B1251 is cultured to obtain a bacterial liquid, and then a raw material containing ginsenosides Rf, Rb1, Rb3, Rd, Rg2, and Rc is used as a substrate and mixed with the bacterial liquid of Lactobacillus reuteri HP-B1251 for anaerobic fermentation, that is, the ginsenosides Rf, Rb1, Rb3, Rd, Rg2, and Rc in the raw material are converted into rare ginsenosides F2, Rh4, Rg3, Rh2, and Rk2, so as to obtain a fermentation liquid containing rare ginsenosides; then, the obtained fermentation liquid containing rare ginsenosides is extracted by a conventional extraction method, and analyzed by high performance liquid chromatography.

[0011] Furthermore, the method for preparing the Lactobacillus reuteri HP-B1251 bacterial liquid comprises the following steps:

[0012] A single colony of Lactobacillus reuteri HP-B1251 is inoculated into an MRS liquid culture medium, and anaerobically cultured at 36-38°C and 160-200 r / min for more than 6 hours; then 5-10% of the inoculum amount is inoculated into an MRS liquid culture medium, and anaerobically cultured at 36-38°C and 160-200 r / min for more than 6 hours to obtain a bacterial liquid of Lactobacillus reuteri HP-B1251.

[0013] Furthermore, the concentration of the Lactobacillus reuteri HP-B1251 bacterial solution is 1.0×109 ~1.0×10 10 CFU / mL.

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

[0015] Furthermore, during the fermentation conversion of the rare ginsenosides, the bacterial solution of Lactobacillus reuteri HP-B1251 and ginseng powder are evenly mixed and anaerobically cultured at 36-38° C. and 160-200 r / min for 24-36 hours.

[0016] Furthermore, during the fermentation conversion of the rare ginsenosides, a sugar content regulator can be added; the sugar content regulator is composed of the following components: 20.1 g yeast extract, 7.5 g sodium acetate, 15 g peptone, 4.5 g triammonium citrate, 30 g glucose, 3 g dipotassium hydrogen phosphate, 0.75 g magnesium chloride heptahydrate, 0.375 g manganese sulfate tetrahydrate, 1.5 mL Tween-80, 0.1875 g cysteine, 1 L distilled water and the pH is adjusted to 6.8 for sterilization.

[0017] Furthermore, during the fermentation conversion process of the rare ginsenosides, the weight ratio of the Lactobacillus reuteri HP-B1251 bacterial liquid to the sugar content regulator is 3 to 8:3, preferably 7:3.

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

[0019] The present invention also provides a use of the Lactobacillus reuteri HP-B1251 in preparing a product with immune enhancement function.

[0020] Another technical solution of the present invention is to provide a bacterial agent, wherein the bacterial agent comprises the Lactobacillus reuteri HP-B1251 or its fermentation liquid according to claim 1.

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

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

[0023] In the present invention, by observing the bacterial growth state, evaluating the temperature tolerance, evaluating the acid production, evaluating the acid resistance, evaluating the bile resistance, and evaluating the bacterial liquid and fermented product of Lactobacillus reuteri HP-B1251, the ginsenoside conversion ability is studied, and the analysis shows that the growth and reproduction ability of Lactobacillus reuteri HP-B1251 is strong, and the high temperature tolerance, acid production, acid resistance, bile resistance, and ginsenoside conversion ability are excellent. Finally, the present invention carries out whole genome sequencing to Lactobacillus reuteri HP-B1251, and provides a basis for the in-depth study of follow-up Lactobacillus reuteri HP-B1251. Through bioinformatics comparison analysis, Lactobacillus reuteri HP-B1251 of the present invention does not contain resistance genes, drug resistance genes and virulence factors, and is a probiotic with higher safety.

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

[0025] (1) The Lactobacillus reuteri HP-B1251 obtained by screening fresh milk of the present invention has strong growth and reproduction ability, good tolerance to high temperature, excellent acid production ability, excellent stomach acid resistance, extremely high tolerance to bile salts, and can relatively adapt to the complex gastrointestinal environment of the body.

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

[0027] Description of biological deposit:

[0028] Limosilactobacillus reuteri HP-B1251 was deposited in the China Center for Type Culture Collection on August 28, 2024. The depository address is Wuhan University, Wuhan, China. The deposit number is CCTCCNO: M20241872, and the taxonomic name is Limosilactobacillus reuteri. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0030] Figure 2The agarose gel electrophoresis diagram of the PCR product obtained by PCR amplification reaction of Lactobacillus reuteri HP-B1251 16S rRNA, wherein M: 2K plus II DNA marker; 1, 2, 3, 4: Lactobacillus reuteri HP-B1251 16S rRNA PCR amplification products;

[0031] Figure 3 The figure is an agarose gel electrophoresis diagram of the PCR product of the monoclonal bacterium Lactobacillus reuteri HP-B1251, where M: 2Kplus II DNA marker; 1: monoclonal antibody PCR product;

[0032] Figure 4 is the genetic evolution tree of Lactobacillus reuteri HP-B1251;

[0033] Figure 5 This is a color change diagram of the carbohydrate fermentation test of Lactobacillus reuteri HP-B1251; 1-11 use esculin, cellobiose, maltose, mannitol, salicin, sorbitol, sucrose, raffinose, inulin, lactose, and 1% sodium hippurate as carbon sources respectively;

[0034] Figure 6 The figure is a high performance liquid chromatography analysis diagram of the biotransformation of ginsenosides by Lactobacillus reuteri HP-B1251, wherein A is a ginsenoside blank control (ginseng powder), and B is ginseng powder fermented by Lactobacillus reuteri HP-B1251;

[0035] Figure 7 is the complete genome map of Lactobacillus reuteri HP-B1251;

[0036] Figure 8 is the plasmid 1 map of Lactobacillus reuteri HP-B1251;

[0037] Fig. 9 is the plasmid 2 map of Lactobacillus reuteri HP-B1251;

[0038] Fig.10 Statistical graphs of common and unique annotations for the ENCODE gene general database;

[0039] Fig.11 It is the statistical diagram of PFAM domain;

[0040] Fig.12 This is a classification diagram of GO annotation results;

[0041] Fig.13 This is the classification diagram of KEGG pathway results;

[0042] Fig.14 This is the classification diagram of COG results;

[0043] Fig.15 This is the NR species distribution map;

[0044] Fig.16 This is the classification diagram of TCDB results. DETAILED DESCRIPTION

[0045] The present invention is further illustrated below through the description of specific implementation methods, but this is not a limitation of the present invention. Those skilled in the art can make various modifications or improvements based on the basic idea of ​​the present invention, but as long as they do not deviate from the basic idea of ​​the present invention, they are all within the protection scope of the present invention.

[0046] In the following examples, reagents not specially described are conventional reagents and can be purchased from conventional reagent production and sales companies. The methods used are all prior arts unless otherwise specified.

[0047] The Lactobacillus reuteri HP-B1251 described in the present invention is isolated from fresh milk (from Heze City, Shandong Province), and the MRS solid culture medium (model CM188) and the MRS liquid culture medium (model CM187) are both purchased from Beijing Luqiao Technology Co., Ltd.

[0048] Example 1 Isolation, Purification and Identification of Lactobacillus reuteri HP-B1251

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

[0050] This strain is derived from fresh milk. The specific method and steps for isolating and purifying the strain are as follows: first sterilize the culture dish, bamboo stick, triangular rake, pipette tip, MRS culture medium, and saline. Then pour the MRS culture medium into the sterilized culture dish to form an MRS solid plate. Then take 1 mL of fresh milk and dilute it with saline to a concentration of 10 -5 , 10 -6 , 10 -7 CFU / mL, use a pipette to transfer the dilutions to the MRS solid plates respectively, and spread them with a triangular rake. After spreading, place them in a 37°C constant temperature incubator for anaerobic culture. After two days of culture, observe the characteristics of the colonies, select single colonies with typical characteristics, and use bamboo sticks to inoculate them onto new MRS plates for three-zone purification culture. Repeat the culture and selection process until a pure culture strain is obtained. The purified strain was identified and determined to be Lactobacillus reuteri (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 the figure below. Figure 1 .

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

[0052] The purified strains were picked and the DNA fragments of the strains were obtained by the modified SDS-alkaline lysis method. The 16S rRNA of the strains was amplified by PCR using the 16S primers 27F-PBACS and 1541R-PBACS (as shown in the primer sequence I in Table 1), and the conditions were: 95°C pre-denaturation for 10 min, 95°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 1.5 min, 30 cycles, 72°C extension for 10 min, and 16°C storage.

[0053] Table 1 Primer sequence I

[0054]

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

[0056] Table 2 Primer sequence II

[0057] Target Primer sequences T7-Elong 5′-TTAATACGACTCACTATAGGGGAATTG-3′ T7-TermElong 5′-TTATGCTAGTTATTGCTCAGCGG-3′

[0058] The PCR product was sent to Suzhou Jinweizhi Biotechnology Co., Ltd. for sequencing. The nucleotide sequence of the 16S rRNA of the strain 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] BLAST gene comparison showed that the similarity with Limosilactobacillus reuteri in GenBank was 100.00%, indicating that it was a strain of Lactobacillus reuteri, named HP-B1251. The 16S rRNA sequence had been uploaded to the GenBank database, and the receiving sequence number was PP657605.

[0088] The genetic evolution tree was used to compare Lactobacillus reuteri HP-B1251. Figure 4 shown.

[0089] (3) Carbohydrate fermentation experiment with Lactobacillus reuteri HP-B1251

[0090] HBI lactic acid bacteria biochemical identification strips (purchased from Qingdao Hi-Tech Industrial Park Haibo Biotechnology Co., Ltd.) were used to identify the response of Lactobacillus reuteri HP-B1251 to 11 carbohydrates. Figure 5 As shown in Table 3, among which, 1-11 respectively used esculin, cellobiose, maltose, mannitol, salicin, sorbitol, sucrose, raffinose, inulin, lactose, and 1% sodium hippurate as carbon sources. The results are shown in Table 3. It can be seen from Table 3 that Lactobacillus reuteri HP-B1251 can utilize, decompose and metabolize 4 carbohydrates, including maltose, sucrose, raffinose, and lactose, in the 11 carbohydrate fermentation tests.

[0091] Table 3 Carbohydrate fermentation experiment of Lactobacillus reuteri HP-B1251

[0092]

[0093] Example 2 Evaluation of the growth status of Lactobacillus reuteri HP-B1251

[0094] Use a bamboo stick to inoculate a single colony of Lactobacillus reuteri HP-B1251 on MRS solid medium into fresh sterilized MRS liquid medium, and culture anaerobically at 37°C and 100rpm for 24h. Use a pipette to draw 1mL of fermentation liquid, centrifuge at 8000rpm for 1min, discard the supernatant, keep the bacteria, weigh and calculate the wet weight. Dry at 55°C for 12h and weigh again to calculate the dry weight. The test results are shown in Table 4.

[0095] Table 4 Study on the growth status of Lactobacillus reuteri HP-B1251

[0096] Cultivation time (h) Wet weight / (g / mL) Dry weight / (g / mL) 24 <![CDATA[4.04×10 -2 ±0.0116]]> <![CDATA[3.5×10 -3 ±0.0004]]>

[0097] As shown in Table 4, the dry weight of the bacteria was 3.5×10 -3 ±0.0004g / mL, which shows that Lactobacillus reuteri HP-B1251 has a strong reproductive ability and can reproduce in large quantities.

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

[0099] Use a bamboo stick to inoculate a single colony of Lactobacillus reuteri HP-B1251 on MRS solid medium into fresh sterilized MRS liquid medium and culture at 37°C, 100r / min for 24h. Heat the bacterial solution at 37°C, 55°C, and 70°C for 1min, 2min, and 3min respectively, and dilute the bacterial solution to a concentration of 10 -6 and 10 -7 CFU / mL, and then spread on solid MRS medium and cultured anaerobically at 37°C for 16-20 hours. The test results are shown in Table 5.

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

[0101]

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

[0103] Example 4: Evaluation of the Acid Production Capacity of Lactobacillus reuteri HP-B1251

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

[0105] Table 6 Study on the acid production ability of Lactobacillus reuteri HP-B1251

[0106] Cultivation 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 invention can reach strong acidity after anaerobic culture for 12 hours, with a pH value of 4.92, which shows that the Lactobacillus reuteri HP-B1251 has a strong acid-producing ability.

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

[0109] Prepare artificial gastric juice (8.2mL dilute hydrochloric acid, 400mL double distilled water, 5g pepsin, shake well and add double distilled water to make up to 500mL, take a certain amount of solution to adjust the pH to 2.64, 3.85, and 4.89. Sterilize with a 0.44μm filter membrane and store in a 4℃ refrigerator for later use). Use a bamboo stick to inoculate a single colony of Lactobacillus reuteri HP-B1251 on MRS solid culture medium into freshly sterilized MRS liquid culture medium, and culture anaerobically at 37℃, 100rpm for 18-24h. Take 60mL of bacterial solution and centrifuge at 3580rpm for 15min, pour off the supernatant, and resuspend the bacteria with 1.5mL of normal saline. Take 4 centrifuge tubes, add 6mL of artificial gastric juice to each, and then add 200μl of resuspended bacterial solution to each and mix well. Dilute to a concentration of 10 at 0min and 150min. -6 and 10 -7 The CFU / mL was plated on solid MRS medium and cultured for 18-24 hours, and then the single colonies on the plate were counted and the survival rate was calculated. The test results are shown in Table 7.

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

[0111]

[0112] As shown in Table 7, a large number of strains of Lactobacillus reuteri HP-B1251 of the present invention still survived in the artificial gastric juice of pH 2.64 for 150 minutes, so it has a strong acid resistance.

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

[0114] Prepare artificial intestinal fluid (3.4g potassium dihydrogen phosphate, 250mL double distilled water, adjust pH to 6.8 with 0.1mol / L sodium hydroxide solution, add 2.5g trypsin, shake well, add double distilled water to 500mL, take a certain amount of solution and add a certain amount of ox bile salt to prepare artificial intestinal fluid with bile salt concentrations of 0.03%, 0.1%, 0.2%, and 0.3%, respectively. Sterilize with a 0.44μm filter membrane and store in a 4℃ refrigerator for later use). Use a bamboo stick to inoculate a single colony of Lactobacillus reuteri HP-B1251 on MRS solid culture medium into freshly sterilized MRS liquid culture medium, and culture anaerobically at 37℃, 100rpm for 16-20h. Take 60mL of bacterial solution and centrifuge at 3580rpm for 15min, pour off the supernatant, and resuspend the bacteria with 1.5mL of saline. Take 4 centrifuge tubes, add 6 mL of artificial intestinal fluid to each tube, and then add 200 μl of resuspended bacterial solution to mix well. Dilute to a concentration of 10 at 0 min and 150 min. -6 and 10 -7 The CFU / mL was plated on solid MRS medium and cultured for 16 to 20 hours, and then the single colonies on the plate were counted and the survival rate was calculated. The test results are shown in Table 8.

[0115] Table 8 Study on bile salt tolerance of Lactobacillus reuteri HP-B1251

[0116]

[0117] As shown in Table 8, the survival rate of Lactobacillus reuteri HP-B1251 of the present invention is still as high as 112.0% after 150 minutes when the bile salt concentration is 0.3%, indicating that it has a high tolerance to bile salts.

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

[0119] A single colony of Lactobacillus reuteri HP-B1251 on MRS solid culture medium was inoculated into 30 mL of freshly sterilized MRS liquid culture medium using a bamboo stick and cultured anaerobically at 37°C and 180 rpm for more than 6 hours. Then, inoculate 70 mL of freshly sterilized MRS liquid culture medium with an inoculum amount of 5% to 10%, and culture anaerobically at 37° C. and 180 r / min for more than 6 h to obtain a Lactobacillus reuteri HP-B1251 bacterial liquid. Add 1.5 g of ginseng powder and 30 mL of a sugar content regulator (20.1 g of yeast extract powder, 7.5 g of sodium acetate, 15 g of peptone, 4.5 g of triammonium citrate, 30 g of glucose, 3 g of dipotassium hydrogen phosphate, 0.75 g of magnesium chloride heptahydrate, 0.375 g of manganese sulfate tetrahydrate, 1.5 mL of Tween-80, 0.1875 g of cysteine, 1 L of distilled water and sterilize by adjusting the pH to 6.8) to the Lactobacillus reuteri HP-B1251 bacterial liquid obtained by the above culture, mix well, and culture anaerobically at 37° C. and 180 r / min for 24 h to 36 h to obtain a fermentation liquid. Take a separatory funnel, add the fermentation liquid and the extractant (saturated n-butanol solution, ultrapure water: n-butanol = 1:1), shake and mix. Centrifuge the bacterial liquid at 3580r / min for 10min, take the supernatant on the top, evaporate it at 55℃, dissolve it in 20% acetonitrile, and pass it through a reverse phase C 18 HPLC analysis was performed using a high performance liquid chromatograph.

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

[0121] Table 9

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

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

[0124] Table 10

[0125]

[0126] With 20% acetonitrile as mobile phase, 10 μL was injected and the chromatogram at 203 nm was analyzed. The results are as follows: Figure 6 As shown, Figure A is a ginsenoside blank control (ginseng powder), and Figure B is a ginseng powder fermented by Lactobacillus reuteri HP-B1251.

[0127] The types and contents of saponins before and after fermentation (ginseng powder and fermented with Lactobacillus reuteri HP-B1251) were used as indicators to calculate the increase in the content of rare ginsenosides. The test results are shown in Table 11.

[0128] The results show that the contents of ginsenosides Rf, Rb1, Rb3, Rd, Rg2 and Rc are all decreased, and a large amount of new rare ginsenosides F2, Rh4, Rg3, Rh2 and Rk2 are produced by fermentation conversion. It can be seen that the Lactobacillus reuteri HP-B1251 of the present invention has a strong conversion ability for rare ginsenosides.

[0129] Table 11 Rare ginsenoside content increase ratio before and after fermentation

[0130]

[0131]

[0132] Note: Content enhancement ratio = rare ginsenoside content after fermentation with Lactobacillus reuteri HP-B1251 / rare ginsenoside content in ginsenoside sample before fermentation

[0133] Example 8 Determination of the whole genome of Lactobacillus reuteri HP-B1251

[0134] The genome of Lactobacillus reuteri HP-B1251 was obtained by sequencing and assembling the genome based on Nanopore third-generation sequencing technology and second-generation sequencing technology platform. Figure 7 The visualized whole genome map of the bacterium is shown in Figure 1. The genome size of the bacterium is 2219140bp, the GC ratio is 39.07%, and it is a circular gene. Its genome contains 2165 CDS, 69 tRNAs and 18 rRNAs, and contains two plasmids. Plasmid 1 is 19058bp in size, with a GC ratio of 36.87%, and is a circular plasmid. Figure 8 As shown; Plasmid 2 is 7047 bp in size, with a GC ratio of 39.24%, and is a circular plasmid. Fig. 9 shown.

[0135] Example 9 Functional Annotation Analysis of the HP-B1251 Database Genome of Lactobacillus reuteri (1) Statistics of the Annotation Ratio of General Databases

[0136] In order to obtain comprehensive gene function information, the present invention has performed gene function annotation of eight major databases, including: UniProt, KEGG and KEGG Pathway, GO, Pfam, COG, TIGERfams, RefSeq, and NR. The predicted gene sequence was compared with functional databases such as COG, KEGG, Uniprot, and Refseq by BLAST+ (Version: 2.11.0+) to obtain the gene function annotation results. The software hmmer (Version: 3.3.2) was used to perform functional 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 coding gene database are as follows Fig.10 As shown, Fig.10 In the figure, the left side shows the gene annotation statistics of the general database; the right side shows the common and unique statistics. For example, the first column shows the common statistics of all general databases, the top part shows the number statistics, and the bottom part shows the annotations of the common or unique databases.

[0137] From Table 12 and Fig.10 It can be seen that the more gene fragments contained in the Lactobacillus reuteri HP-B1251 of the present invention can obtain comprehensive gene function information in major general databases, and the gene function information that can be obtained is relatively large, which provides a certain basis for the research on each gene of the strain.

[0138] Table 12 Statistics of coding gene annotation results

[0139]

[0140]

[0141] (2) Pfam domain classification

[0142] The Pfam database is a homologous protein family database, 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. Identifying domains in proteins is important for understanding the function of proteins.

[0143] Based on the annotation of Pfam domains, the present invention statistically summarizes the genes annotated in each domain, and displays the top 20 domains with the most annotations. The results are as follows: Fig.11 As shown in the figure, the horizontal axis is the name of the protein family, and the vertical axis is the number of genes mapped to the protein family.

[0144] (3) Gene Ontology Classification

[0145] Gene Ontology (GO) is an internationally standardized gene function classification system that provides a set of dynamically updated controlled vocabularies to comprehensively describe the properties of genes and gene products in organisms.

[0146] The present invention simplifies GO (Gene Ontology) annotation information to obtain GOslim classification, summarizes and statistics the functions of genes from three aspects: cell components, molecular functions, and biological processes, and selects the top 20 GOslim secondary classifications with the most annotations in each classification for mapping. The results are shown in the figure. Fig.12 As shown in the figure, the horizontal axis is the content of each GO classification, and the vertical axis is the number of genes. Fig.12 The figure shows the gene enrichment of each secondary function of GO in the context of all genes, reflecting the status of each secondary function in this context; Fig.12 The functions of genes and gene products in Lactobacillus reuteri HP-B1251 and the number of genes showing this function can be concluded, 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 that systematically analyzes the metabolic pathways of gene products in cells and the functions of these gene products. KEGG can be used to further study the complex biological behaviors of genes.

[0149] After KEGG annotation of the genes, the present invention classified them according to the KEGG metabolic pathways in which they participated. Fig.13 As shown in the figure, the horizontal axis is the number of genes annotated under the Pathway classification; the vertical axis is the Pathway classification, and different colors represent different major classifications; Fig.13 The metabolic pathways of each gene product of Limosilactobacillus reuteri HP-B1251 in the cell are shown in FIG.

[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 be derived from an ancestral protein. The COG database is constructed based on the coding proteins and phylogenetic relationships of bacteria, algae, and eukaryotes with complete genomes. Unigene and COG databases are compared to predict the possible functions of genes and perform functional classification statistics on them, so as to understand the gene function distribution characteristics of the species from a macro perspective. COG is divided into 26 groups.

[0152] The present invention classifies the COG annotated genes according to the COG group. Fig.14 As shown in the figure, the horizontal axis is the content of each COG classification, and the vertical axis is the number of genes. In different functional categories, the number of genes reflects the metabolism or physiological bias in the corresponding period and environment, and can be scientifically explained in combination with the distribution of the research object in each functional category.

[0153] (6) Uniprot annotation

[0154] UniProt Archive is a comprehensive, non-redundant database that contains protein sequences from all major, publicly available databases. Since proteins may exist in different databases and there may be multiple versions in the same database,

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

[0156] (7) Refseq annotation

[0157] The Refseq (Reference Sequences) database is a non-redundant transcript, protein and genome sequence database that has been annotated. It is a database that has been corrected by NCBI and other organizations. It 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 are generally more credible.

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

[0159] (8) NR Notes

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

[0161] The NR species distribution diagram of Lactobacillus reuteri HP-B1251 of the present invention is as follows: Fig.15 As shown, different colors in the figure represent different species.

[0162] (9)TIGRFAMs annotation

[0163] TIGRFAMs is a database consisting of multiple sequence alignments, hidden Markov models (HMMs) and related information. It is a member of interPro for protein sequence classification and supports automatic annotation of (mostly prokaryotic) proteins. Starting from v10.0, the TIGRFAMs model uses HMMER 3, which has excellent search speed and sensitivity.

[0164] The number of genes annotated to the Tigrfams database of the whole genome of Lactobacillus reuteri HP-B1251 described in the present invention is 1159, accounting for 53.53%.

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

[0166] The present invention is a personalized analysis of Lactobacillus reuteri HP-B1251, and a proprietary database annotation is performed on it, including ARDB database annotation, carbohydrate enzyme annotation based on CAZy database, pathogen-host interaction analysis, drug resistance gene annotation, cytochrome P450 annotation, VFDB annotation, signal peptide prediction, and TCDB transporter annotation.

[0167] After the whole genome of Lactobacillus reuteri HP-B1251 described in the present invention was annotated with a proprietary database, it was found that: no resistance gene results were annotated in the ARDB database; no completely matching gene sequences were annotated in the pathogen-host interaction database; no drug resistance genes were annotated in the CARD database; no completely matching gene sequences were annotated for cytochrome P450; no completely matching virulence factors were annotated in the VFDB database; the carbohydrate enzyme annotation results are shown in Table 13; the signal peptide prediction results of the whole genome of Lactobacillus reuteri HP-B1251 are shown in Table 14, and the transmembrane protein and secretory protein prediction results are shown in Table 15; the TCDB membrane transport protein annotation results are shown in Table 16. Fig.16 As shown in the figure, the horizontal axis is the content of each classification of TCDB, and the vertical axis is the number of genes.

[0168] Table 13 CAZy annotation

[0169]

[0170]

[0171] Table 14 Signal peptide prediction statistics

[0172] Protein Type quantity Protein containing information peptide 99

[0173] Table 15 Prediction statistics of transmembrane proteins and secretory proteins

[0174] Protein Type quantity Transmembrane proteins 479 Secretory proteins 30

[0175] Based on the above data, it can be shown that Lactobacillus reuteri HP-B1251 has strong enzyme activity, protein expression function and good edibility, can be safely used in the production of related foods and health products, and has good development and application prospects.

[0176] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. A strain of Lactobacillus reuteri HP-B1251, characterized in that: It was deposited in the China Center for Type Culture Collection on August 28, 2024, with the deposit number being CCTCC NO:M 20241872.

2. Use of Lactobacillus reuteri HP-B1251 according to claim 1 in fermentation and conversion to prepare rare ginsenosides.

3. The use according to claim 2, characterized in that: The Lactobacillus reuteri HP-B1251 is used for fermenting ginsenosides Rf, Rb1, Rb3, Rd, Rg2, and Rc into rare ginsenosides F2, Rh4, Rg3, Rh2, and Rk2.

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

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

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

7. Use of the Lactobacillus reuteri HP-B1251 according to claim 1 in preparing food and health products containing rare ginsenosides.

8. A bacterial agent, characterized in that The bacterial agent includes the Lactobacillus reuteri HP-B1251 or its fermentation liquid according to claim 1.

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

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

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