Lactobacillus helveticus with high EPS yield and application of lactobacillus helveticus

Abstract

The invention belongs to the technical field of microorganisms and biological medicines, and particularly relates to lactobacillus helveticus with high EPS yield and application of the lactobacillus helveticus. The lactobacillus helveticus with the high EPS yield, provided by the invention, is lactobacillus helveticus LH43 and is preserved in the China Center for Type Culture Collection (CCTCC), and the preservation number is CCTCC NO: M 20242348. Tests prove that the lactobacillus helveticus LH43 can generate high-molecular-weight EPS, and the high-molecular-weight EPS has a better colon improving effect. The lactobacillus helveticus LH43 is utilized to ferment the traditional Chinese medicine gastrodia elata, and by constructing a mouse colitis model, the lactobacillus helveticus LH43 fermented gastrodia elata can significantly improve the colitis treatment effect. Therefore, the invention has important practical guiding significance for scientific and reasonable utilization of gastrodia elata resources, treatment of ulcerative colitis and research and development of medicines.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms and biomedicine, and specifically relates to a strain of Lactobacillus helveticus with high EPS production and application thereof. Background Art

[0002] Ulcerative colitis (UC) is a complex multifactorial disease, usually caused by the interaction of genetics, environmental factors and host immune factors. It is mainly manifested by abdominal pain, diarrhea, bloody stools and weight loss. It is a chronic intermittent inflammatory bowel disease of unknown cause. In recent years, the incidence of ulcerative colitis has continued to increase in various countries, posing a serious threat to human health. Ulcerative colitis is usually incurable, and the use of existing clinical treatment drugs may cause a series of adverse reactions, such as drowsiness, gastrointestinal complications and renal disorders. Therefore, there is an urgent need to develop an effective, safe and suitable alternative for the long-term treatment or prevention of ulcerative colitis.

[0003] Gastrodia elata is the dried tuber of the orchid plant Gastrodia elata. It is one of the earliest and most important traditional herbs used in the history of Chinese medicine. In traditional medicine, Gastrodia elata is often used to treat a variety of diseases, including cerebrovascular disease, hypertension, coronary heart disease, etc. In recent years, with the in-depth study of Gastrodia elata, researchers have found that Gastrodia elata also has multiple biological activities such as anti-inflammatory and antioxidant. In view of the medical benefits of Gastrodia elata, it has become widely accepted as a raw material for health products in many countries.

[0004] At present, Gastrodia elata has been used in the treatment of ulcerative colitis. However, existing Gastrodia elata treatment drugs usually combine Gastrodia elata with a variety of Chinese medicinal materials, which often have the problems of complex formulation and unsatisfactory efficacy. Fermentation is a traditional Chinese medicine processing technology with a long history. With the development of science and technology, its application in traditional Chinese medicine has greatly increased. By fermenting Gastrodia elata, it is expected to enhance the efficacy of Gastrodia elata, change its medicinal properties, and reduce its toxicity. However, existing studies have not yet reported a strain suitable for Gastrodia elata fermentation and can significantly improve the efficacy of ulcerative colitis.

[0005] Lactic acid bacteria exopolysaccharides (EPS) have become a hot topic for extensive research due to their excellent physicochemical properties and physiological activity. Lactic acid bacteria EPS refers to the metabolites produced by lactic acid bacteria during their main metabolic process. It includes capsular polysaccharides attached to the cell wall and mucus polysaccharides secreted outside the cell, and has good biological activity. However, there are many types of existing strains, and the fermentation characteristics and fermentation effects of different strains are different. In addition, strains from different sources have different abilities to produce EPS, which will lead to large differences in the yield and structural characteristics of EPS (monosaccharide composition, number average molecular weight Mn, glycosidic bonds) under different growth and fermentation conditions, which will further affect the physicochemical properties, biological activity and application effects of EPS. At present, no suitable strains that can effectively produce EPS and enhance the efficacy of colitis have been reported in the prior art.

[0006] Therefore, if a new strain with high EPS production and suitable for Gastrodia elata fermentation is screened, especially one that can improve the efficacy of ulcerative colitis after fermentation, it will have important practical guiding significance for the rational use of Gastrodia elata resources and maximizing its medicinal value, as well as the treatment of ulcerative colitis and drug development. Summary of the invention

[0007] In order to overcome the deficiencies in the prior art, the purpose of the present invention is to provide a strain of Lactobacillus helveticus with high EPS production, which can produce high molecular weight lactic acid bacteria exopolysaccharides (EPS). After fermenting the traditional Chinese medicine Gastrodia elata with the strain, the efficacy of ulcerative colitis can be significantly improved, and the strain is suitable for use in the development of drugs for ulcerative colitis.

[0008] In addition, the present invention also aims to provide the use of the above-mentioned Lactobacillus helveticus with high EPS production in the fermentation preparation of a drug for treating ulcerative colitis.

[0009] In addition, the present invention also aims to provide a fermentation product prepared by using the above-mentioned Lactobacillus helveticus with high EPS production.

[0010] In addition, the present invention also aims to provide the use of the above fermentation product in the preparation of a medicine for treating ulcerative colitis.

[0011] In addition, the present invention also aims to provide a medicine for treating ulcerative colitis.

[0012] To achieve the above object, the technical solution adopted by the present invention is: A strain of Lactobacillus helveticus with high EPS production is disclosed. The strain is Lactobacillus helveticus LH43, which is deposited in China Center for Type Culture Collection with a deposit number of CCTCC NO: M 20242348.

[0013] The application of the above-mentioned high-EPS-producing Lactobacillus helveticus in the fermentation preparation of drugs for treating ulcerative colitis.

[0014] A fermented product prepared by using the above-mentioned high-EPS-yielding Lactobacillus helveticus, wherein the fermented product is prepared by fermentation culture with Gastrodia elata as a main fermentation substrate and Lactobacillus helveticus LH43 as a fermentation bacterium.

[0015] As a preferred solution, the method for preparing the fermented product comprises the following steps: The gastrodia elata powder, the carbon source and water are mixed, and then sealed and sterilized to obtain a gastrodia elata mixed liquid; Lactobacillus helveticus LH43 was inoculated into MRS medium, sealed and cultured on a shaker for 20-30 h. After culture, the bacterial concentration was adjusted to (3-5) × 10 8 CFU / mL, bacterial solution was obtained; The bacterial liquid is mixed with the mixed liquid of Gastrodia elata, and then fermented and cultured to obtain the fermented product.

[0016] As a preferred embodiment, the carbon source is one or more of glucose, inulin, and sucrose, more preferably glucose.

[0017] As a preferred solution, the ratio of the gastrodia elata powder, carbon source, and water is (8-12) g: (0.05-0.2) g: (8-12) mL. As a more preferred solution, the ratio of the gastrodia elata powder, carbon source, and water is 10 g: 0.1 g: 10 mL.

[0018] As a preferred solution, the amount of the bacterial liquid is 4v / v% to 6v / v% of the Gastrodia elata mixed liquid, and more preferably 5v / v%.

[0019] As a preferred solution, the temperature of the shaking culture is 37±0.5°C; the temperature of the fermentation culture is 37±0.5°C, and the fermentation culture time is 70~80h.

[0020] Application of the fermented product in preparing medicine for treating ulcerative colitis.

[0021] A medicine for treating ulcerative colitis, comprising the above-mentioned Lactobacillus helveticus with high EPS production, or the above-mentioned fermentation product.

[0022] Compared with the prior art, the beneficial effects of the present invention are: The present invention isolates and identifies 46 strains of Lactobacillus helveticus from a variety of dairy products in different regions, further performs whole genome resequencing on the 46 isolated and identified Lactobacillus helveticus strains, and performs detailed assembly and annotation of the sequencing results, adopts comparative genomics and whole genome association analysis methods, deeply analyzes the intrinsic relationship between the genetic evolution of the strains and the molecular weight of EPS, and finally screens out a strain producing high molecular weight EPS from the 46 strains of Lactobacillus helveticus producing EPS, and names it Lactobacillus helveticus LH43, and the microbial classification is named Lactobacillus helveticus LH43. The present invention further deposits Lactobacillus helveticus LH43 in the China Center for Type Culture Collection, with a deposit number of CCTCCNO: M 20242348, a deposit date of October 28, 2024, and a deposit address of Wuhan University.

[0023] Furthermore, the present invention has confirmed through experiments that Lactobacillus helveticus LH43 can produce high molecular weight EPS, and high molecular weight EPS has a better colon improvement effect. The present invention uses Lactobacillus helveticus LH43 to ferment the traditional Chinese medicine Gastrodia elata, and verifies the significant antioxidant activity of the fermented Gastrodia elata through in vivo and in vitro antioxidant tests. Furthermore, the present invention constructs a mouse colitis model and finds that Lactobacillus helveticus LH43 fermented Gastrodia elata can significantly improve the therapeutic effect of colitis. Therefore, the research results of the present invention have important practical guiding significance for the scientific and rational use of Gastrodia elata resources, maximizing its medicinal value, and the treatment of ulcerative colitis and drug development. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The results of the determination of the number of CDs of 46 strains of Lactobacillus helveticus of the present invention are as follows; Figure 2 is a phylogenetic tree of 46 strains of Lactobacillus helveticus of the present invention; Figure 3 The annotation results of the core genes COG of different branches of the present invention; Figure 4 The EPS molecular weight of the isolates assigned to different branches of the present invention; Figure 5 The ANI (A) and DDH (B) results of 46 strains of Lactobacillus helveticus of the present invention; Figure 6 The molecular weight of EPS and the distribution of genes in the EPS gene cluster of the present invention; Figure 7 The average exopolysaccharide molecular weight results of the mutant and wild-type strains of the present invention; Figure 8 The results of scavenging DPPH by the fermentation broths obtained from the three fermented Gastrodia elata of the present invention; Fig. 9The results of scavenging ABTS by the fermentation broths obtained from the three fermented Gastrodia elata of the present invention and the unfermented Gastrodia elata solution; Fig.10 The results of the weight change rate of each group of experimental mice in the present invention are as follows; Fig.11 The DAI scores of the experimental mice in each group in the present invention are shown in FIG. Fig.12 The colon tissue sections of each group of experimental mice in the present invention are stained. DETAILED DESCRIPTION

[0025] The technical scheme and technical effects of the present invention will be clearly and completely described below in conjunction with specific embodiments and drawings. However, it should be understood by those skilled in the art that the embodiments are only used to illustrate the technical scheme of the present invention and should not be regarded as limiting the scope of protection of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the raw materials used are all commonly used in the art, available to the public or commercially available, unless otherwise specified.

[0026] The deposit information involved in the present invention is as follows: Deposit name: Lactobacillus helveticus LH43 ( Lactobacillus helveticus LH43); Deposit number: CCTCC NO: M 20242348; Depository: China Center for Type Culture Collection, Address: Wuhan University, Wuhan, China; Date of preservation: October 28, 2024.

[0027] Among them, in the following examples, the Gastrodia elata used is from a local pharmacy. The preparation method of MRS medium is: take 5.0g of peptone, 10.0g of tryptone, 5.0g of sodium acetate, 2.0g of dipotassium hydrogen phosphate, 20.0g of glucose, 5.0g of yeast powder, 0.25g of manganese sulfate, 0.58g of magnesium sulfate, 2.0g of diammonium hydrogen citrate, 1.0g of Tween-80, 5.0g of beef extract, dissolve it in distilled water and make it to 1L, stir evenly, sterilize it at 121°C for 15min, and obtain MRS medium. In other embodiments, MRS medium can be obtained through commercial channels.

[0028] In the following examples, the shaking culture and fermentation culture are both carried out at conventional cell culture temperatures, which are not particularly limited in the present invention. The rotation speed of the shaking culture is 100-200 rpm, which can be conventionally selected by technicians.

[0029] In the following examples, Lactobacillus helveticus DPC4571 is an existing strain reported in the NCBI database (NCBI number is GCA_000015385.1), which is pre-isolated, identified and preserved by the present invention.

[0030] The 46 strains of Lactobacillus helveticus involved in the following examples are named LH1, LH2, LH3, LH4, LH5, LH6, LH7, LH8, LH9, LH10, ​​LH11, LH12, LH13, LH14, LH15, LH16, LH17, LH18, LH19, LH20, LH21, LH22, LH23, LH24, LH25 H25, LH26, LH29, LH30, LH31, LH32, LH34, LH35, LH36, LH37, LH38, LH39, LH41, LH42, LH43, LH44, LH45, LH46, LH47, LH48, LH49, and LH50 are all isolated from different dairy product samples by the present invention and then identified by 16S rDNA sequencing. The sample sources and identification results of each strain are shown in Table 1.

[0031] Table 1. Strain sources and identification results

[0032]

[0033] Specifically, the following is an example of the Lactobacillus helveticus LH43 that the present invention mainly relates to, and its separation and identification process are described in detail. The Lactobacillus helveticus with other numbers other than Lactobacillus helveticus LH43 is the comparison strain of the screening process of the present invention, and the separation and identification method of the comparison strain is similar to that of Lactobacillus helveticus LH43, and the present invention will not repeat them.

[0034] The isolation and identification process of Lactobacillus helveticus LH43 is as follows: A traditional yogurt sample from Xiahe County, Gannan Tibetan Autonomous Prefecture, was diluted and coated on an MRS solid culture medium. Under suitable temperature and humidity conditions, the colony was allowed to grow and reproduce. Subsequently, a single colony was carefully selected from the coated culture medium for further separation and purification. Multiple streaking cultures were performed to ensure that pure strains of Lactobacillus helveticus were finally obtained. Next, the purified single colony was inoculated into an MRS liquid culture medium for liquid culture. Afterwards, the genomic DNA of the isolated Lactobacillus helveticus was extracted, and its 16S rDNA fragment was amplified using polymerase chain reaction (PCR) technology. The amplified PCR product was sequenced after confirmation by nucleic acid electrophoresis. Finally, the sequencing results were compared with the known Lactobacillus helveticus 16S rDNA sequence for similarity to complete the 16S rDNA gene identification. The comparison results showed that the isolated strain was confirmed to be Lactobacillus helveticus, and the present invention named it Lactobacillus helveticus LH43.

[0035] Lactobacillus helveticus LH43 of the present invention ( Lactobacillus helveticus The 16S rDNA sequence of LH43 is as follows (SEQ ID NO. 1):

[0036] The technical solution of the present invention is described in detail below in conjunction with specific embodiments.

[0037] Example 1

[0038] This embodiment provides a strain of Lactobacillus helveticus with high EPS production, the strain is named Lactobacillus helveticus LH43, and is deposited in China Center for Type Culture Collection with a deposit number of CCTCC NO: M 20242348.

[0039] This embodiment also provides the use of the above-mentioned Lactobacillus helveticus with high EPS production in the fermentation preparation of a drug for treating ulcerative colitis.

[0040] Example 2

[0041] This embodiment provides a fermentation product prepared by using the high-EPS-yielding Lactobacillus helveticus of Embodiment 1. The fermentation product is prepared by fermentation culture using Gastrodia elata as a main fermentation substrate and Lactobacillus helveticus LH43 as a fermentation bacterium.

[0042] The method for preparing the fermented product comprises the following steps: Take 10 g of Gastrodia elata powder, add 10 mL of distilled water and 0.1 g of carbon source (glucose), then seal, place in an autoclave for high pressure sterilization for 20 min, then place in an ultra-clean workbench and cool to room temperature to obtain a Gastrodia elata mixed solution; Lactobacillus helveticus LH43 was inoculated into MRS medium, sealed and cultured in a shaking incubator at 37°C for 24 h, and the bacterial concentration was adjusted to 4 × 10 8 CFU / mL, bacterial solution was obtained; In an ultra-clean workbench, the bacterial solution and the Gastrodia elata mixed solution were stirred and mixed, the amount of the bacterial solution accounting for 5v / v% of the Gastrodia elata mixed solution, and then placed in a constant temperature incubator and fermented at 37°C for 72h to obtain a fermentation product.

[0043] This embodiment also provides the use of the fermentation product in preparing a medicine for treating ulcerative colitis.

[0044] This embodiment also provides a medicine for treating ulcerative colitis, which contains the above-mentioned fermentation product.

[0045] Test Example 1: Screening process of Lactobacillus helveticus LH43 This test example illustrates the screening process of Lactobacillus helveticus LH43 involved in the present invention, which specifically includes the following steps: 1. Genome-wide association analysis 1.1. Extraction of genomic DNA Take out the bacterial suspension of 46 strains of Lactobacillus helveticus preserved in 15% (v / v) glycerol aqueous solution from the refrigerator at -20℃, and then inoculate the bacterial suspension into MRS liquid culture medium at a 2% (v / v) inoculation amount, and culture it in a constant temperature aerobic environment at 37℃ for 24h. After two consecutive generations of subculture, transfer it to 50mL MRS culture medium for expansion culture. Then use TIANamp bacterial DNA kit to extract total bacterial DNA. The concentration and purity of the extracted DNA are detected using a nanodrop spectrophotometer.

[0046] 1.2 Whole genome sequencing, assembly and annotation The Illumina HiSeq high-throughput sequencing platform (Illumina Inc. USA) was used to generate a 400-bp paired-end library for whole-genome sequencing. SOAPdenovo v1.06 software was first used for de novo assembly of paired-end reads. Subsequently, GapCloser (http: / / sourceforge.net / projects / soapdenovo2 / files / GapCloser / ) was used to fill local internal gaps and correct single-base errors, and Glimmer v3.02 software was used to predict putative coding sequences. Finally, the functions of the predicted open reading frames (ORFs) were annotated and interpreted with reference to the RAST 2.0 and COG databases.

[0047] 1.3. Construction of pan-genome-core genome Based on the functional gene prediction results of Prokka software, Roary (v3.6.1) software was used to divide the gene families according to the standard of more than 90% amino acid sequence identity. In this way, a pan-core genome containing 46 strains of Lactobacillus helveticus was successfully constructed.

[0048] 1.4 Construction of SNP phylogenetic tree The maximum distance method (ML) was used to construct an evolutionary tree based on the SNP sites of 46 strains of Lactobacillus helveticus. The phylogenetic tree was constructed using Mega software, and ggtree was used to display the constructed ML tree.

[0049] 1.5. Principal component analysis and kinship Tassel5 (reference TASSEL: software for association mapping of complex traits in diverse samples) was used to perform phylogenetic and principal component analysis on 46 strains of Lactobacillus helveticus.

[0050] 1.6 Calculation of total nucleotide consistency (TNI) and DNA hybridization (DDH) The average nucleotide identity (ANI) and DNA hybridization (DDH) of 46 Lactobacillus helveticus strains were calculated according to the method of Goris et al. (DNA–DNA hybridization values ​​and their relationship to whole-genome sequences similarities). The relationship between species was evaluated using Perl scripts. Cluster heat maps were drawn using TBtool (TBtools: an integrative toolkit developed for interactive analyses of bigbiological data) and Genome-to-Genome Distance Calculator (https: / / ggdc.dsmz.de / home.php) software.

[0051] 1.7. Genomic variation analysis Using the whole genome sequence of Lactobacillus helveticus DPC4571 as a reference, the assembled genome sequences of each strain were compared with the reference sequence one by one using Mummer (v3.20) software. The comparison results can be used to determine all SNP sites that may occur in each strain relative to the reference strain and integrate these sites. The specific base state of each SNP site in the genome of each strain was further examined. After excluding SNPs located in the repetitive region, only sites with clear base states in all strains were retained. Next, the clean data of each strain that had undergone quality control was compared with its corresponding assembled sequence using soap software. By examining the reads coverage and quality value of each SNP site, accurate SNPs were screened out to generate the final accurate SNP set.

[0052] 1.8 Identification of genes in the eps gene cluster BLASTp searches were performed against the predicted genes and reference genes for all studied genomes to identify potential eps genes.

[0053] 1.9. Genome-wide association analysis The present invention uses a genome-wide association analysis method to explore the correlation between SNP sites and the molecular weight of Lactobacillus helveticus EPS, and uses treeWAS software (A phylogenetic method to perform genome-wide association studies in microbes that accounts for population structure andrecombination) to perform an association analysis between the molecular weight of Lactobacillus helveticus EPS and SNP.

[0054] 2. Statistical analysis The present invention conducted three repeated experiments, and the results were expressed as mean ± standard deviation (SD). IBM SPSS Statistics 25.0 software was used for one-way analysis of variance, and the statistical significance level was set at p < 0.05. The R software toolkit was used to complete the Wilcoxon rank sum test and draw relevant charts, and OriginPro 2018 software was used to construct the graphics. The Pearson correlation analysis method was used to explore the correlation between variables.

[0055] 3. Genome-wide association analysis 3.1. Genome characteristics of Lactobacillus helveticus 3.1.1 Genome splicing and assembly The present invention performs whole genome sequencing on 46 Lactobacillus helveticus isolates based on the Illumina HiSeq high-throughput sequencing platform, with an average sequencing depth of 300×, and filtering and selecting high-quality 100× data. The statistical data after genome assembly is shown in Table 2.

[0056] Table 2. Genome assembly information of 46 strains of Lactobacillus helveticus

[0057]

[0058] As shown in Table 2, among the 46 strains of Lactobacillus helveticus of the present invention, the genome structure contains an average of 187.89 contigs, and its average N50 value is 21.56±4.15 kb. The genome size of these strains is between 1.83 and 2.42Mb, with an average size of 1.96±0.11Mb. At the same time, the average GC content is 36.57%±0.20%. It is worth noting that the genome assembly quality of Lactobacillus helveticus LH43 is: contigs are 188, N50 is 18.32 kp; the genome characteristics of Lactobacillus helveticus LH43 are: genome size is 19.68Mb; GC is 36.52%; CDs is 2143.

[0059] 3.1.2 Coding gene prediction The present invention uses Prokka (v1.14.6) software to predict the coding genes of the genomes of 46 strains of Lactobacillus helveticus. The prediction results are as follows Figure 1 shown. Figure 1 The results showed that the 46 strains of Lactobacillus helveticus could encode an average of 2081.15±105.75 CDs per strain. Further analysis revealed that the nucleotide sequence lengths in these coding regions were mainly concentrated in the range of 1900-2200 bp, and this part of CDs accounted for 83.43% (79866 / 95733) of the total. CDs with a length of more than 2200 bp accounted for 16.57% (15867 / 95733).

[0060] 3.1.3 Functional annotation of coding genome The RAST online tool was used to annotate the functional genes of 46 strains of Lactobacillus helveticus, and a total of 25 major functional categories were obtained. These major categories cover various biological processes, including amino acid and derivative metabolism (accounting for 12.91%), carbohydrate metabolism (also accounting for 12.91%), and many other functional categories. Among all functional categories, protein metabolism-related genes accounted for the highest proportion, reaching 15.49% of all functional genes. These genes are mainly involved in protein synthesis and degradation processes, such as key functional proteins such as tRNA aminoacylase, ribosomal modification proteins and translation elongation factors, as well as enzymes involved in protein degradation such as proteolytic enzymes, peptidases and proteasome components. In addition, functional genes related to amino acid and derivative metabolism and carbohydrate metabolism also accounted for 12.91% of the total number of annotated genes, respectively, showing the importance of these metabolic pathways in Lactobacillus helveticus.

[0061] In addition, functional gene annotation was also performed using the COG database. The results showed that the largest number of coding genes involved in metabolism (Metabolism) accounted for 43.2% of the 46 genomes, followed by coding genes involved in life activities and signal transduction (Information storage and processing), accounting for 25.5%.

[0062] 3.2 Construction of the pan-core genome According to the classification standard of amino acid consistency exceeding 90%, the core and pan-gene sets of 46 strains of Lactobacillus helveticus are statistically analyzed in detail.Statistical results show that these strains share a stable core gene set comprising 1178 genes, and at the same time, their pan-gene set covers up to 4523 genes.As the number of genomes included in the analysis gradually increases, the scope of the pan-gene set is also continuously expanding.This phenomenon reveals the open characteristics of the Lactobacillus helveticus genome, meaning that more new genes may be found as the research goes deeper.Meanwhile, unlike the pan-gene set, the core gene set does not change significantly with the increase of the number of genomes, and its quantity remains stable basically.Based on this observation, the present invention speculates that these 1178 core genes may constitute the conservative part in the Lactobacillus helveticus genome.In addition, this result of the study also shows that these 46 strains of Lactobacillus helveticus are enough to represent the genome characteristics of the entire Lactobacillus helveticus colony, providing a powerful basis for subsequent functional and evolutionary research.

[0063] After functional annotation of 1,178 core genes, using the COG database, it was found that these genes are mainly involved in two major functions. First, 38.59% of the genes are involved in energy production and conversion, carbohydrate transport and metabolism, amino acid, nucleotide, coenzyme transport and metabolism, lipid transport and metabolism, and inorganic ion transport and metabolism, secondary metabolite biosynthesis, transport and catabolism, as well as phage and transposon functions. On the other hand, 29.41% of the genes are related to translation, ribosome structure and biogenesis, transcription, replication, recombination and repair. It is particularly noteworthy that the number of genes involved in translation and ribosome structure and biogenesis is the largest.

[0064] 3.3 Construction of SNP phylogenetic tree 3.3.1 Genome splicing and assembly The basis for studying population structure and evolutionary relationships is the construction of phylogenetic trees. In order to explore the population structure of Lactobacillus helveticus, this study used the maximum distance method (ML) to construct an evolutionary tree based on the SNP sites of 46 strains. The results are as follows Figure 2 As shown. Figure 2It can be seen that Lactobacillus helveticus is a paraphyletic group, and each strain has evolved from a specific most recent common ancestor (MRCA). The phylogenetic tree of 46 strains of Lactobacillus helveticus can be divided into three obvious branches, namely Clade1, Clade2 and Clade3. Clade1, Clade2 and Clade3 contained 27 strains (LH1, LH2, LH3, LH4, LH5, LH6, LH7, LH8, LH9, LH10, ​​LH18, LH20, LH21, LH23, LH24, LH26, LH29, LH30, LH31, LH35, LH37, LH38, LH42, LH43, LH45, LH46, LH47), 13 strains (LH12, LH14, LH11, LH17, LH22, LH32, LH39, LH15, LH19, LH34, LH44, LH25, LH36) and 6 strains (LH13, LH16, LH41, LH48, LH49, LH50, LH51, LH52, LH53, LH54, LH55, LH56, LH57, LH58, LH59, LH60, LH61, LH62, LH63, LH64, LH65, LH66, LH67, LH68, LH69, LH70, LH71, LH72 The Lactobacillus helveticus LH43 producing high molecular weight EPS provided by the present invention belongs to Clade1.

[0065] 3.3.2. Principal component and kinship analysis The results of principal component analysis (PCA) can reveal the population structure and be used to correct the population hierarchy. Those with close positions indicate close genetic relationships, while those with far positions indicate distant genetic relationships, which can more intuitively reveal the genetic relationships between strains. Based on SNP loci, the principal component analysis method was used to classify the genetic background similarity of 46 strains of Lactobacillus helveticus in three groups (Clade1, Clade2 and Clade3). Through principal component analysis, a total of two principal components (PC1: 36.93%, PC2: 25.37%) were extracted, and the cumulative variance contribution rate was 62.30%, which can better explain the overall changes of the variables. By using a progressive color band to intuitively display the genetic relationship of the 46 strains of Lactobacillus helveticus, it was found that it could divide the 46 strains of Lactobacillus helveticus into three groups, further verifying the clustering results and genetic relationships of the 46 strains of Lactobacillus helveticus.

[0066] 3.3.3 Analysis of differences in genomic characteristics among different evolutionary branches Changes in genome size and GC content are important characteristics of adaptive evolution of bacteria to adapt to the environment in which they live. To further explore the differences in genomic characteristics between different groups, the genome size, number of CDs and GC content of each strain in the three evolutionary branches were compared and analyzed. The results showed that Clade3 had significant differences in the number of CDs from Clade1 and Clade2 (P<0.05), and there was no significant difference in GC content among the three branches (P>0.05). Clade1 and Clade3 had extremely significant differences in genome size (P<0.01), and Clade2 and Clade3 had significant differences in genome size (P<0.05). In addition, the scatter plot based on strain gene size and CDS number showed that the strain genome size was correlated with its CDS number. Clade3 strains were more concentrated in the upper right corner, that is, the genes were large and the number of CDs was large. According to the characteristics of the adaptive evolution of lactic acid bacteria to the milk environment, it is speculated that Clade3 strains lost fewer functional genes during the process of environmental adaptive evolution.

[0067] Furthermore, in order to explore the differences in genetic diversity among different branches, the present invention performed COG annotation on the core gene sets of the three evolutionary branches. Figure 3 shown. Figure 3 Note: C energy production and conversion, D cell cycle control, cell division, chromosome segmentation, E amino acid transport and metabolism, F nucleotide transport and metabolism, G carbohydrate transport and metabolism, H coenzyme transport and metabolism, I lipid transport and metabolism translation, ribosome structure and biogenesis, K transcription, L replication, recombination and repair, M cell wall / membrane / envelope biogenesis, N cell motility, O post-translational modification, protein turnover, chaperones, P inorganic ion transport and metabolism, Q biosynthesis, transport and catabolism of secondary metabolites, R only general function predictions, S function unknown, T signal transduction mechanism, U intracellular transport, secretion and bladder transport, V defense mechanism, W extracellular structure, X mobile genome: prophage, transposon.

[0068] Figure 3 The results showed that the three core gene sets of Clade1-Clade3 contained 1007, 1201, and 1181 gene families, respectively. The COG annotation results of the core genes of each branch showed that J family genes accounted for the largest proportion of core genes, accounting for an average of 15.16% of the core genes of each branch, with the lowest being 14.39% in Clade3 and the highest being 16.68% in Clade1. Among the genes of each functional classification, the core genes of Clade1 and Clade2 accounted for a large proportion, while the core genes of Clade3 accounted for a small proportion.

[0069] 3.3.4 Analysis of EPS molecular weight in different evolutionary branches In order to test whether the molecular weight of EPS is related to the phylogenetic relationship among isolates, the average molecular weight of the exopolysaccharide of the three clades was calculated. Figure 4 As shown, Figure 4 Kruskal-Wallis test was used to evaluate the differences in EPS molecular weight between groups. ns indicates no significant difference. * indicates significant difference (P < 0.05).

[0070] Depend on Figure 4 It can be seen that the Mn of Clade3 and Clade2 EPS is significantly greater than that of Clade1 (P<0.05), and there is no significant difference in Mn between Clade3 and Clade2 EPS (P>0.05). Therefore, it is speculated that the properties of Lactobacillus helveticus exopolysaccharides are related to the phylogenetic relationship between strains.

[0071] 3.4. Average nucleotide identity and DNA hybridization Average nucleotide identity (ANI) and DNA-DNA hybridization (DDH) are two important indicators for evaluating species relatedness, which can reveal the similarity between two genomes. Generally, when the ANI value of two genomes exceeds 95% and the DDH value exceeds 70%, they are considered to belong to the same species. In the present invention, the ANI and DDH values ​​of 46 strains of Lactobacillus helveticus were calculated and analyzed by heat maps (such as Figure 5 ) are presented visually. Figure 5 The research results showed that the ANI values ​​of all tested strains were over 99% and the DDH values ​​were all greater than 90%, which not only confirmed that these strains all belonged to Lactobacillus helveticus, but also revealed that there was a certain degree of genetic diversity within the Lactobacillus helveticus species.

[0072] 3.5 Analysis of EPS molecular weight and EPS gene cluster In order to study the correlation between EPS gene cluster genes and EPS molecular weight in Lactobacillus helveticus, the present invention conducted a comprehensive analysis of EPS gene clusters of 46 strains. After excluding common genes (>95% presence) and rare genes (<5% presence), the present invention further selected 11 genes [(A) Polysaccharide biosynthesis protein CpsF, (B) Glycosyltransferase, family 28, (C) polysaccharide biosynthesis protein CpsM, (D) polysaccharide polymerase, (E) Alpha-D-GlcNAc alpha-1,2-L-rhamnosyltransferase, (F) Glycosyltransferase, (G) hypothetical protein, (H) UDP-N-acetylglucosamine 2-epimerase, (I) UDP-N-acetylglucosamineLPS N-acetylglucosamine transferase, (J) Transposase, (K) UDP-galactopyranose mutase] and analyzed the molecular weight of EPS. The results are as follows: Figure 6 As shown. Figure 6 It can be seen that Alpha-D-GlcNAc alpha-1,2-L-rhamnosyltransferase and UDP-galactopyranose mutase were negatively correlated with EPS molecular weight (p<0.05), hypothetical protein and UDP-N-acetylglucosamine 2-epimerase were extremely significantly negatively correlated with EPS molecular weight (p<0.01), and other genes had no significant correlation (p>0.05).

[0073] 3.6. Genomic variation analysis Lactobacillus helveticus DPC4571 is the first Lactobacillus helveticus strain to complete whole genome sequencing in the prior art. The present invention selected the genome of DPC4571 as a reference and performed single nucleotide polymorphism (SNP) analysis on 46 strains of Lactobacillus helveticus. The analysis results are shown in Table 3.

[0074] Table 3. Statistics of single base polymorphisms in the genomes of 46 strains of Lactobacillus helveticus

[0075] As shown in Table 3, a total of 43,979 SNP sites were detected in 46 strains. Among them, there were 37,262 SNPs located in the gene coding region, accounting for 84.72% of the total, and 6,717 SNPs located in the intergenic region, accounting for 15.28%. Further analysis of the SNPs in the gene coding region showed that more than half were non-synonymous mutations (nSNPs), with a specific number of 20,109, accounting for 53.97%, and synonymous mutations (sSNPs) of 17,153, accounting for about 46.03%.

[0076] The reference genome was further divided into units of 1000bp, and the distribution of SNPs on the reference genome was plotted. The results showed that SNPs were distributed more or less in individual regions of the reference genome. Among them, there were 6 high-frequency distribution regions with more than 120 SNPs on the reference genome, with a length of 1000bp. The functional annotation of the high-frequency distribution regions of SNPs was performed by comparing the position information on the reference Lactobacillus helveticus DPC4571 genome, and the results are shown in Table 4.

[0077] Table 4. Annotation information of high-frequency mutation regions

[0078] As shown in Table 4, these 6 regions contain 11 genes, of which 8 genes have relevant descriptions in the NR database, and the remaining 3 genes have no relevant descriptions in the NR database. The descriptions of these 8 genes in the NR database are ISLre2-like element ISLhe10 family transposase, ISL3 family transposase, ISL3 family transposase, DUF4422 domain-containing protein, sugar transferase, exopolysaccharide biosynthesis protein, IS256 family transposase, IS256 family transposase. Among them, sugar transferase and exopolysaccharide biosynthesis protein have a greater correlation with the synthesis of EPS.

[0079] 3.7. Association analysis between the molecular weight of EPS and SNP loci Given that there is no significant correlation between the eps gene cluster and EPS molecular weight, in order to further explore the potential relationship between the genetic evolution of Lactobacillus helveticus and its EPS molecular weight, the SNP site data of 46 strains of Lactobacillus helveticus were successfully obtained using genomic variation analysis. Subsequently, the treeWAS software was used for genome-wide association analysis to reveal functional genes related to EPS molecular weight, thus providing a strong basis for understanding the complex relationship between the genetic characteristics of Lactobacillus helveticus and EPS molecular weight.

[0080] The correlation between the genes containing the common SNPs of 46 strains of Lactobacillus helveticus and the molecular weight of EPS was analyzed by GWAS. Among them, based on the relationship between the molecular weight of EPS and the fermentation performance of the strain, three representative strains were selected from 46 strains of Lactobacillus helveticus producing EPS: LH43 (producing high molecular weight EPS), LH13 (producing medium molecular weight EPS) and LH23 (producing low molecular weight EPS). The molecular weights of EPS produced by these three strains were significantly different, with the number average molecular weights (Mn) of 5.21×10 6 Da, 2.39×10 6 Da and 3.76×10 5 Among the 46 strains of Lactobacillus helveticus, the EPS molecular weight of Lactobacillus helveticus LH43 was the highest, reaching 5.21×10 6 Da.

[0081] Then, le-3 (i.e., p<0.005) was used as the screening threshold to first screen out all positive sites that affect EPS molecular weight. Then, among these positive sites, those sites that belong to missense mutations were further screened. After such double screening, a set of 51 positive sites was finally obtained, and the results are shown in Table 5.

[0082] Table 5. GWAS analysis results of SNP-based genotypes and EPS molecular weight of Lactobacillus helveticus

[0083]

[0084] In Table 5, the key SNP site mutations that affect the molecular weight of Lactobacillus helveticus EPS are located on the gene encoding exopolysaccharide synthetic protein. Each positive site not only has significant statistical significance (p < 0.005), but also involves missense mutations, which may have a direct effect on the molecular weight of EPS. These 51 positive sites are threonine-tRNA ligase, Is3-like element ISLhe6 family transposase, RNA-guided endonuclease TnpB family protein, IS3 family transposase, amino acid permease, exopolysaccharide biosynthesis protein, RNA-guided endonuclease TnpB family protein, 1-acyltriglyceride 3-phosphate acyltransferase, hypothetical protein, glucose-6-phosphate dehydrogenase, glucose-6-phosphate dehydrogenase, glucose-6-phosphate dehydrogenase, bifunctional oligoribonuclease / PAP phosphatase NrnA, protein containing transporter substrate binding domain, ABC transporter ATP binding protein / permease, glycerate kinase, Glycerate kinase, Isoleucine-tRNA ligase, Hypothetical protein, DNA-binding transcriptional regulator, Hypothetical protein, Phosphoenolpyruvate carboxylase, α / β hydrolase, IS256 family transposase, GIY-YIG nuclease family protein, 1-acyltriglyceride 3-phosphate acyltransferase, Cyclopropane-fatty-acylphospholipid synthase family protein, Cyclopropane-fatty-acylphospholipid synthase family protein, M1 family metallopeptidase, Multispecies: DUF4479 and tRNA-binding domain-containing protein, Zinc ribbon domain-containing protein, Zinc ribbon domain-containing protein, AAA family ATPase, AAA family ATPase, NAD (P) H-dependent oxidoreductase, TetR / AcrR family transcriptional regulator, RNA-guided endonuclease TnpB family protein, RNA-guided endonuclease TnpB family protein, ABC transporter ATP-binding protein / permease, hypothetical protein, hypothetical protein, copper-translocating P-type ATPase, ABC transporter ATP-binding protein / permease, cyclopropane-fatty-acylphospholipid synthase family protein, cyclopropane-fatty-acylphospholipid synthase family protein, threonine-tRNA ligase, hypothetical protein, hypothetical protein, amino acid permease, copper-translocating P-type ATPase, copper-translocating P-type ATPase.

[0085] Furthermore, the present invention discovered a SNP site located on the gene encoding the exopolysaccharide biosynthesis protein, specifically, the base of the LHV_RS08910 gene at position 1775405 of the reference genome mutated from C to T. This mutation caused the 341st codon of the LHV_RS08910 gene to change from G to A, thereby causing the serine (Ser) at position 114 in the protein encoded by the gene to change to aspartic acid (Asn). In view of the key role of exopolysaccharide biosynthesis protein in EPS synthesis and that the gene is in a high-frequency mutation region, it is speculated that this SNP site has a significant correlation with the molecular weight of EPS. In order to further verify this correlation, the present invention calculated the average exopolysaccharide molecular weight of the mutant type and the wild type strain, and the results are as follows. Figure 7 As shown. Figure 7 As shown, the Wilcoxon test showed that the EPS molecular weight of the 1775405-C strain was significantly higher than that of the 1775405-T strain, indicating that the 1775405-C strain had a higher EPS molecular weight.

[0086] As can be seen from the above content, in order to further explore the genes closely related to the molecular weight of Lactobacillus helveticus EPS, the present invention adopts the whole genome association analysis (GWAS) method and analyzes based on single nucleotide polymorphism (SNP) site data. After strict screening thresholds and missense mutation analysis, 51 positive sites significantly associated with EPS molecular weight were successfully identified. It is worth noting that one of the SNP sites is located on the key gene encoding the exopolysaccharide biosynthesis protein, and the gene is in a high-frequency mutation region. This finding strongly suggests that there is a significant correlation between the SNP site and the molecular weight of EPS. In order to verify this correlation, the Wilcoxon test was further used. The results showed that when this specific site mutated, the molecular weight of EPS produced by Lactobacillus helveticus decreased significantly. This finding has important practical significance, indicating that strains with 1775405-C type genes have the potential to produce high molecular weight EPS. This discovery not only provides a new perspective for understanding the molecular mechanism of EPS synthesis, but also lays a solid foundation for the rapid screening and cultivation of excellent strains producing high molecular weight EPS in the future.

[0087] As can be seen, there are significant differences in the molecular weight of EPS between different strains. The present invention, by obtaining the whole genome draft of 46 strains of Lactobacillus helveticus, is divided into 3 evolutionary branches based on the SNP phylogenetic evolutionary tree, and the genetic difference of the same evolutionary branch is small, the evolutionary distance is close, and the genome size difference of different evolutionary branches is significant. A total of 43979 SNP sites were identified in 46 strains of bacteria, of which 84.72% of SNPs were located in the gene coding region, and 15.28% of SNPs were located in the intergenic region. There are 6 high-frequency mutation regions, including 11 genes. Comparative genome analysis found that the genes encoding hypothetical protein, UDP-N-acetylglucosamine 2-epimerase, Alpha-D-GlcNAcalpha-1,2-L-rhamnosyltransferase and UDP-galactopyranose mutase in the EPS gene cluster were significantly negatively correlated with the molecular weight of Lactobacillus helveticus EPS. The results of the correlation analysis between the SNP site and the EPS molecular weight showed that a SNP site located in the protein encoding the biosynthesis of exopolysaccharides was significantly correlated with the EPS molecular weight. The Wilcoxon test further confirmed that when the specific site was mutated, the molecular weight of the EPS produced by Lactobacillus helveticus would be significantly reduced. Therefore, by the method of the present invention, a strain of Lactobacillus helveticus LH43 producing high molecular weight EPS was screened and obtained.

[0088] Therefore, the present invention deeply analyzes the relationship between the potential genetic evolution law of Lactobacillus helveticus and the molecular weight of EPS of the strain from the genome level, which is of vital importance for screening and developing high-quality starter strains. In addition, the present invention performs whole genome resequencing on 46 strains of Lactobacillus helveticus, and by carefully assembling and annotating the sequencing results, using comparative genomics and whole genome association analysis methods, deeply analyzes the intrinsic relationship between the genetic evolution of these strains and the molecular weight of EPS, which can provide strong support for the screening and development of excellent starter strains.

[0089] The following test examples mainly illustrate the application effect of Lactobacillus helveticus LH43 fermented with Gastrodia elata obtained by the present invention. Experimental Example 2: Changes in Antioxidant Properties of Gastrodia Elata Before and After Fermentation This test example mainly investigates the antioxidant properties before and after numbing fermentation. During the test, the fermentation effects of Lactobacillus helveticus LH43 (producing high molecular weight EPS), Lactobacillus helveticus LH13 (producing medium molecular weight EPS), and Lactobacillus helveticus LH23 (producing low molecular weight EPS) were compared. Specifically, according to the preparation method of the fermented product described in Example 2, Lactobacillus helveticus LH13 and Lactobacillus helveticus LH23 were used to replace Lactobacillus helveticus LH43, respectively, to prepare the corresponding fermented products for subsequent tests.

[0090] The DPPH free radical scavenging ability is to determine the scavenging ability of the fermented product of Gastrodia elata fermented with Lactobacillus helveticus LH43 (producing high molecular weight EPS) in Example 2 of the present invention (referred to as GFTM), the fermented product of Gastrodia elata fermented with Lactobacillus helveticus LH13 (producing medium molecular weight EPS) prepared by the present invention (referred to as ZFTM), the fermented product of Gastrodia elata fermented with Lactobacillus helveticus LH23 (producing low molecular weight EPS) (referred to as DFTM), and the unfermented Gastrodia elata mixed solution (TM) on DPPH. During the test, 2 mL of three kinds of Gastrodia elata fermented products with gradient concentrations and unfermented Gastrodia elata solution were uniformly mixed with 1 mL of 0.2 mM DPPH solution, and distilled water was used as a blank control at room temperature and in the dark. The OD 517nm The absorption values ​​were measured at 4 °C and the clearance rates and inhibitory concentrations (IC 50 ).

[0091] Clearance rate (%) = [A1−(A2− A3)] / A1. Wherein, A1: absorbance of blank group; A2: absorbance of unfermented Gastrodia elata solution and Gastrodia elata solution fermented with Lactobacillus helveticus producing EPS of different molecular weights; A3: absorbance of background group.

[0092] In addition, the ABTS free radical scavenging ability was determined by the same test objects as above, and the scavenging ability of Gastrodia elata fermentation products (GFTM, ZFTM, DFTM) and unfermented Gastrodia elata solution (TM) on ABTS was determined. During the test, a mixed solution of ABTS and potassium persulfate was prepared with deionized water and diluted to a working solution before use, and the OD was controlled at 734nm The absorbance at OD was about 0.70. Take 0.2 mL of fermented and unfermented Gastrodia elata solutions of gradient concentrations and add them to 5.8 mL of the prepared working solution. Use distilled water as a control. 734nm The absorption value was measured under low temperature and the clearance rate and inhibition concentration (IC) of Gastrodia elata before and after fermentation were calculated. 50 ).

[0093] Clearance rate (%) = [A1 − (A2 − A3)] / A1. Wherein, A1: absorbance of blank group; A2: absorbance of unfermented Gastrodia elata solution and Gastrodia elata solution fermented with Lactobacillus helveticus producing EPS of different molecular weights; A3: absorbance of background group.

[0094] The results of the DPPH free radical scavenging ability and the ABTS free radical scavenging ability were as follows: Figure 8 and Fig. 9 shown.

[0095] Depend on Figure 8 It can be seen that the Gastrodia elata solutions of the DFTM group, ZFTM group and GFTM group showed strong DPPH scavenging ability, and the Gastrodia elata fermented with Lactobacillus helveticus LH43 showed more significant DPPH scavenging ability ( P <0.05).

[0096] In addition, if Fig. 9 As shown, the test results of ABTS are similar to the test results of DPPH capacity, indicating that the use of LH43 in the present invention to ferment Gastrodia elata can significantly improve the antioxidant effect of Gastrodia elata.

[0097] Experimental Example 3: Animal Experiment to Verify the Efficacy of Colitis Construction of ulcerative colitis mouse model: In this experiment, dextran sodium sulfate (DSS) was used to construct an ulcerative colitis mouse model: 3.5 g of DSS powder was weighed on a scale to prepare a 3.5 wt% DSS aqueous solution. ICR mice were allowed to drink water freely for 7 days, and fresh DSS solution was replaced every other day. After one week of feeding, the drinking water was stopped until the end of the experiment.

[0098] Experimental animal grouping and administration: SPF male ICR mice (purchased from Harbin Medical University), 5 weeks old, weighing 30±2g, were adaptively fed before the start of the experiment. They were randomly divided into 5 groups (n=8 in each group), namely, a blank control group (NC group), a DSS model group (DSS group), a fermentation group after DSS+Lactobacillus helveticus LH23 fermented Gastrodia elata (DFTM group), a fermentation group after DSS+Lactobacillus helveticus LH13 fermented Gastrodia elata (ZFTM group) and a fermentation group after DSS+Lactobacillus helveticus LH43 fermented Gastrodia elata (GFTM group). Among them, when Lactobacillus helveticus LH23, Lactobacillus helveticus LH13, and Lactobacillus helveticus LH43 fermented Gastrodia elata to prepare fermentation products, only the strain type was changed, and the other process parameters were all carried out with reference to Example 2.

[0099] Three days before the experiment, each group drank drinking water; seven days later, except for the control group, all experimental groups drank 3.5% DSS solution, the DFTM group was gavaged with Gastrodia elata solution fermented by Lactobacillus helveticus LH23, the ZFTM group was gavaged with Gastrodia elata solution fermented by Lactobacillus helveticus LH13, and the GFTM group was gavaged with Gastrodia elata solution fermented by Lactobacillus helveticus LH43.

[0100] Experimental sampling and sample processing: On the last day of the experiment, the mice were fasted for 12 hours, and the blood of mice in different treatment groups was taken. After being placed at room temperature for 2 hours, the blood was centrifuged at 4000r / min for 10 minutes in a pre-cooled centrifuge at 4°C. The supernatant was taken out and stored in a refrigerator at -20°C. The mice were then killed by cervical dislocation, and their colorectum (ileocecal to anus) was quickly dissected and laid flat on a dissection board to measure its length. 0.5cm of the colon was cut from different groups, rinsed with pre-cooled PBS solution, and placed in 4% paraformaldehyde for making pathological sections.

[0101] Daily Disease Activity Index (DAI) score of mice: During the experiment, the weight changes of mice in all groups were recorded daily, the activity and mental state of mice in each group, the fecal morphology, and fecal occult blood were observed, and the above three indicators were used for comprehensive evaluation to perform DAI scoring. After the experiment, the DAI scores of mice in each group during the experiment were compared to evaluate the severity of colitis in mice and the effect of drug treatment. The scoring criteria are shown in Table 6.

[0102] Table 6. DAI scoring details

[0103] Colon tissue pathological analysis: The colons of each group were dissected using surgical scissors and fixed with 4% paraformaldehyde solution to prepare pathological tissue sections, and then stained with hematoxylin and eosin (HE) to observe the pathological changes of colon tissue under an optical microscope.

[0104] The following are the test results of the above test.

[0105] (i) Effects of three kinds of Gastrodia elata fermentation products on body weight and DAI index of colitis mice In order to verify whether fermented Gastrodia elata has a therapeutic effect on DSS-induced colitis in mice, the present invention first induced a colitis model, observed its therapeutic effect on colitis, and monitored the weight of each group of mice every day during the experiment. In DSS-induced colitis, because the intestinal function of mice is destroyed, it usually leads to weight loss, so weight loss, blood in stool and softened stool are all clinical symptoms that can be directly seen. The weight of mice and the disease activity index (DAI) score can be used to evaluate the condition of mouse colitis.

[0106] Fig.10The results are the changes in the weight of mice. Fig.10 It can be seen that compared with the DSS group, the DZTM group, the ZFTM group and the GFTM group can all improve the weight loss of mice. Among them, the therapeutic effect of the GFTM group is significantly better than that of the other two groups (P < 0.05), which shows that the present invention uses Lactobacillus helveticus LH43 to ferment Gastrodia elata, which can significantly improve the weight loss of mice and enhance the efficacy of colitis.

[0107] The DAI index is often used to determine the progression of colitis. An increase in the DAI index indicates severe intestinal damage. Fig.11 shown. Fig.11 The recording process showed that the mice in the DSS group had a significant decrease in weight, loose and unformed feces, and severe symptoms of blood in the feces, which led to a higher DAI score, but the treatment of the DFTM group, ZFTM group, and GFAM group significantly inhibited the trend of increased DAI scores. The test results based on body weight and DAI showed that the three groups of fermented Gastrodia elata solutions all had a certain therapeutic effect on DSS-induced colitis in mice, and the relief effect of GFTM was more significant, with significant differences from the other groups (P < 0.05). This result shows that the present invention uses Lactobacillus helveticus LH43 to ferment Gastrodia elata, which can effectively improve the intestinal protection effect.

[0108] (ii) Effects of three kinds of Gastrodia elata fermentation products on colonic histopathology in colitis mice After the above colitis model was successfully established, the colon of each group of mice was sampled to prepare pathological sections and stained with HE method. The results are as follows Fig.12 shown.

[0109] Depend on Fig.12 It can be seen that the colon mucosal tissue of mice in the DSS group was severely damaged, lacking glands, crypts and goblet cells, and there was a large amount of inflammatory factor overflow. After intervention with DFTM, ZFTM and GFTM, the damage to the colon can be alleviated to varying degrees. The improvement of the colon mucosa in the GFTM group was more obvious, the goblet cells were arranged in an orderly manner, the number of glands was angular, and the overall structural damage was slight. Therefore, compared with the DSS group, the symptoms of colitis in mice in the three fermented Gastrodia groups were alleviated, and all histopathological scores were significantly reduced, but the therapeutic effect of the GFTM group was better than that of the other groups. It is shown that the present invention uses Lactobacillus helveticus LH43 to ferment Gastrodia, which can significantly improve the lesions of the colon mucosal tissue.

[0110] In summary, the present invention explores the effects of fermenting Gastrodia elata with Lactobacillus helveticus LH43 producing high molecular weight EPS, Lactobacillus helveticus LH13 producing medium molecular weight EPS, and Lactobacillus helveticus LH23 producing low molecular weight EPS on its antioxidant activity and colitis in mice, and finds that fermenting Gastrodia elata with Lactobacillus helveticus LH43 (producing high molecular weight EPS) not only shows better antioxidant activity, but also makes colitis mice show higher body weight, lower DAI score and better colon section test results. Therefore, Gastrodia elata obtained by fermentation with Lactobacillus helveticus LH43 has better colitis treatment effect, can significantly improve the efficacy of ulcerative colitis, and has good application prospects in the field of drug development for ulcerative colitis.

[0111] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A strain of Lactobacillus helveticus that produces high EPS, characterized in that: The high-EPS-producing Lactobacillus helveticus is Lactobacillus helveticus LH43, which is deposited in China Center for Type Culture Collection with a deposit number of CCTCC NO: M 20242348.

2. Use of the high-EPS-producing Lactobacillus helveticus as claimed in claim 1 in fermenting and preparing a drug for treating ulcerative colitis.

3. A fermented product prepared by using the high-EPS-producing Lactobacillus helveticus according to claim 1, characterized in that: The fermented product is prepared by fermentation culture with Gastrodia elata as the main fermentation substrate and Lactobacillus helveticus LH43 as the fermentation bacteria.

4. The fermentation product according to claim 3, characterized in that The method for preparing the fermented product comprises the following steps: The gastrodia elata powder, the carbon source and water are mixed, and then sealed and sterilized to obtain a gastrodia elata mixed liquid; Lactobacillus helveticus LH43 was inoculated into MRS medium, sealed and cultured on a shaker for 20-30 h. After culture, the bacterial concentration was adjusted to (3-5) × 10 8 CFU / mL, bacterial solution was obtained; The bacterial liquid is mixed with the Gastrodia elata mixed liquid, and then fermented and cultured to obtain the fermented product.

5. The fermentation product according to claim 4, characterized in that The carbon source is one or more of glucose, inulin and sucrose.

6. The fermentation product according to claim 4, characterized in that The dosage ratio of the gastrodia elata powder, the carbon source and the water is (8-12) g: (0.05-0.2) g: (8-12) mL.

7. The fermentation product according to claim 4, characterized in that The dosage of the bacterial liquid is 4v / v%~6v / v% of the Gastrodia elata mixed liquid.

8. The fermentation product according to claim 4, characterized in that The shaking culture temperature is 37±0.5°C; the fermentation culture temperature is 37±0.5°C, and the fermentation culture time is 70-80h.

9. Use of the fermentation product according to any one of claims 3 to 8 in the preparation of a medicament for treating ulcerative colitis.

10. A drug for treating ulcerative colitis, characterized in that: The method comprises the high-EPS-producing Lactobacillus helveticus according to claim 1, or the fermentation product according to any one of claims 3 to 8.

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