Application of Lactiplantibacillus plantarum HNU082 mutant strain in enhancing intestinal colonization ability

Abstract

The invention provides an application of a Lactiplantibacillus plantarum HNU082 mutant strain in the enhancement of the colonization capability of an intestinal tract. The Lactiplantibacillus plantarum HNU082 mutant strain is a mutant strain of Lactiplantibacillus plantarum HNU082. A real-time fluorescent quantitative PCR method finds that the colonization quantity of the mutant strain in the intestinal tract is higher, and the colonization time is longer, so that the mutation site, the basic group after mutation and the mutation type of the mutant strain are analyzed and discriminated through whole genome re-sequencing, and the functional gene of the mutant strain is judged; the influence of the mutant strain on intestinal microbiome ecology, the influence of the mutant strain on a metabolic pathway of a local flora and carbohydrate active enzyme, the relevance with the intestinal flora and the influence of the mutant strain on local flora mutation are further evaluated, the reasons for enhancing the colonization ability of the Lactiplantarum HNU082 mutant strain in the intestinal tract and the influence of the Lactiplantarum HNU082 mutant strain on the local flora in the intestinal tract are comprehensively analyzed, and the influence of the Lactiplantarum HNU082 mutant strain on the local flora in the intestinal tract is further evaluated. Therefore, a feasible domestication strategy is provided for enhancing the colonization ability of the probiotics.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial engineering and relates to application of a Lactiplantibacillus plantarum HNU082 mutant strain in enhancing intestinal colonization ability. Background Art

[0002] Trillions of microorganisms live in the intestines of the body. There is competition for limited resources and advantageous ecological niches between different strains in the intestines. In order to prevent nutritional limitation caused by excessive concentration, there is also internal competition between strains of the same strain. In this dynamic and dense environment, the intestinal flora constantly competes with each other, forms a balance through metabolism and active antagonism, and finally forms a stable bacterial network ecosystem. These microorganisms coordinate with the host and work together on the host's immune, nutritional and metabolic processes. When exogenous microorganisms enter the intestine, facing the resistance of the intestinal flora network, they not only have to seize the micro-ecological niche in the new environment, but also compete for limited nutrients. This is also the main source and concentrated manifestation of intestinal selection pressure on exogenous microorganisms in the intestine. In the long history of biological evolution, genetic variation is absolute, while its stability is relative. The same is true for microorganisms. In different ecological environments, individual adaptive variations are retained and accumulated through environmental selection pressure, and eventually become the main evolutionary direction of the strain. At the same time, due to the characteristics of small genomes and short generation time, microbial genomes change faster and are more adaptable during evolution. Therefore, under the high selection pressure conditions derived from the interaction of complex microbial communities in the intestinal microecological environment, in order to adapt to the high selection pressure environment of the intestine, exogenous microorganisms often join the ranks of intestinal "natives" through spontaneous adaptive mutations, regulation of their own metabolic functions, activation of stress metabolic pathways, and synthesis and release of competitive metabolites, ultimately achieving stable growth and metabolism and achieving the effect of influencing the local microecological environment. In this process, horizontal gene transfer and the microbial genetic variation mediated by it also occur frequently.

[0003] Probiotics are defined as "live microorganisms that produce beneficial effects on the host when ingested in appropriate amounts". Probiotics have been widely used to promote intestinal health and alleviate chronic diseases in humans. Their functions depend on the colonization of surviving probiotics in the intestines. However, current studies have found that probiotics cannot colonize in the intestines continuously. In the intestines of healthy adults, the colonization of probiotics in the intestines only lasts for a few days, and the benefits to the health of the body gradually decrease until they are replenished, which seriously affects their probiotic effects. Therefore, enhancing the colonization of probiotics in the intestines is the key to the long-term probiotic effects of probiotics. Summary of the invention

[0004] Based on the above, the object of the present invention is to provide a use of a Lactiplantibacillus plantarum HNU082 mutant in enhancing intestinal colonization ability.

[0005] The technical solution adopted by the present invention to achieve the technical purpose is:

[0006] Application of Lactiplantibacillus plantarum HNU082 mutant strain in enhancing intestinal colonization ability.

[0007] Preferably, compared with the original strain of Lactiplantibacillus plantarum HNU082, the mutant strain of Lactiplantibacillus plantarum HNU082 has a higher number of colonization in the intestine and a longer colonization time.

[0008] Preferably, the mutant genes of the Lactiplantibacillus plantarum HNU082 mutant strain include:

[0009] Gene sequence Gene sequence Gene sequence Gene_3110 SEQ ID NO.1 Gene_1841 SEQ ID NO.11 Gene_2880 SEQ ID NO.21 Gene_2833 SEQ ID NO.2 Gene_289 SEQ ID NO.12 Gene_1614 SEQ ID NO.22 Gene_2832 SEQ ID NO.3 Gene_2824 SEQ ID NO.13 Gene_2889 SEQ ID NO.23 Gene_3080 SEQ ID NO.4 Gene_1438 SEQ ID NO.14 Gene_2852 SEQ ID NO.24 Gene_1512 SEQ ID NO.5 Gene_3079 SEQ ID NO.15 Gene_2831 SEQ ID NO.25 Gene_2942 SEQ ID NO.6 Gene_2834 SEQ ID NO.16 Gene_2901 SEQ ID NO.26 Gene_2232 SEQ ID NO.7 Gene_2862 SEQ ID NO.17 Gene_2851 SEQ ID NO.27 Gene_2850 SEQ ID NO.8 Gene_3111 SEQ ID NO.18 Gene_3081 SEQ ID NO.28 Gene_2814 SEQ ID NO.9 Gene_2808 SEQ ID NO.19 Gene_2829 SEQ ID NO.29 Gene_2906 SEQ ID NO.10 Gene_2899 SEQ ID NO.20 Gene_2908 SEQ ID NO.30 .

[0010] Preferably, the Lactiplantibacillus plantarum HNU082 mutant can significantly upregulate the flavin biosynthesis pathway and unsaturated fatty acid biosynthesis, and downregulate pyrimidine deoxyribonucleoside degradation, pyruvate fermentation to produce acetone, and isopropanol biosynthesis pathways.

[0011] Preferably, the Lactiplantibacillus plantarum HNU082 mutant can significantly improve the carbohydrate metabolism profile of intestinal flora.

[0012] Preferably, compared with the original strain of Lactiplantibacillus plantarum HNU082, the mutant strain of Lactiplantibacillus plantarum HNU082 has fewer bacterial species that interact with local intestinal microorganisms.

[0013] Preferably, the Lactiplantibacillus plantarum HNU082 mutant strain has less ecological and evolutionary impact on the intestinal microbiota than the original strain of Lactiplantibacillus plantarum HNU082.

[0014] The beneficial effects of the present invention are:

[0015] The present invention orally administers Lactiplantibacillus plantarum HNU082 to experimental animals, isolates Lactiplantibacillus plantarum HNU082 mutants from feces, finds that the mutants have a higher colonization number and a longer colonization time in the intestine through a real-time fluorescence quantitative PCR method, and then identifies the mutation site, post-mutation base and mutation type of the mutants through whole genome resequencing analysis, determines the functional genes of the mutants, and further evaluates the effects of the mutants on the ecology of the intestinal microbiome, the effects on the metabolic pathways and carbohydrate-active enzymes of the local flora, the association with the intestinal flora and the effects on the mutations of the local flora, comprehensively analyzes the reasons for the enhanced colonization ability of the Lactiplantibacillus plantarum HNU082 mutants in the intestine, and thus provides a feasible domestication strategy for enhancing the colonization ability of probiotics. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 .Statistics of the number of mutation sites in L. plantarum HNU082 mutant strains isolated from the Lp082 group.

[0017] Figure 2 . Quantitative results of original strains and domesticated strains (A) The line graph shows the quantitative results of L. plantarum HNU082 in fecal samples of the two groups after gavage (abscissa: time, ordinate: number of copies of L. plantarum HNU082, error bars: mean ± SD). (B) The bar graph shows the quantitative results of L. plantarum HNU082 in fecal samples of the two groups after gavage (abscissa: time, ordinate: number of copies of L. plantarum HNU082, using t-test, error bars: mean ± SD).

[0018] Figure 3 .The impact of L. plantarum HNU082 on the diversity of local flora in the intestine after oral gavage.

[0019] Figure 4 .Comparison of differential metabolic pathways of local microbiomes in Lp082 group and M-Lp082 group.

[0020] Figure 5 .Comparison of the abundance of carbohydrate hydrolases in the local microbiome of the Lp082 group and the M-Lp082 group.

[0021] Figure 6 .Intestinal association network of L. plantarum HNU082 mutant strain and original strain, (A) Lp082 group, (B) M-Lp082 group.

[0022] Figure 7 .Comparison of the number of mutation sites between the L. plantarum HNU082 mutant strain and the original strain-related species, (A) Lp082 group, (B) M-Lp082 group. DETAILED DESCRIPTION

[0023] In order to explain the present invention more clearly, the present invention is further described in detail below in conjunction with embodiments and with reference to the accompanying drawings. It should be understood by those skilled in the art that the content described below is illustrative rather than restrictive, and should not be used to limit the scope of protection of the present invention.

[0024] Example

[0025] 1. Experimental Materials

[0026] 1.1 Strains and culture media

[0027] The probiotic Lactiplantibacillus plantarum HNU082 is provided by the tropical probiotic lactic acid bacteria resource bank of Hainan University (the deposit number in the Guangdong Microbiological Culture Collection Center is GDMCC NO: 61552), and is also abbreviated as L. plantarum HNU082, or Lp082.

[0028] The formula of MRS medium is as follows: glucose 20g, peptone 10g, beef extract 10g, yeast extract 5g, C 6 H 5 O 7 (NH 4 ) 3 2g, Tween-80 1mL, CH 3 COONa 5g, K 2 HPO 4 2g, MgSO 4 0.58 g, MnSO 4 0.25g, water 1L.

[0029] 1.2 Experimental animals

[0030] This application was approved by the Animal Ethics Committee of Hainan University, and all animal operations were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals of Hainan University.

[0031] SPF grade C57BL / 6J male mice (10 weeks old) were purchased from Hunan Slake Jingda Experimental Animal Co., Ltd. Before the experiment, the mice underwent a two-week adaptation period to adapt to the environment. The day and night cycle was 12 hours under single cage feeding. The feeding temperature was 25±2℃ and the humidity was 55%±5%. The autoclave pad, water and feed were changed once a day. The feed was provided by Jiangsu Collaborative Pharmaceutical Bioengineering Co., Ltd., with basic feed ingredients (80% cereal raw materials, 10% animal protein, and 10% small feed additives).

[0032] 2. Experimental Design

[0033] First, the experimental mice were randomly divided into two groups, a control group (n=6) and a D-Lp082 group (n=6). 8 CFU of the original L. plantarum HNU082 (10 8 CFU L. plantarum HNU082 was resuspended in 300 μL sterile saline and administered by gavage. The daily gavage volume was 300 μL for 7 days. The mice in the control group were gavaged with an equal volume of sterilized 0.85% saline as a control. After gavage, fecal samples were collected for metagenomic sequencing analysis. Fresh fecal samples from the D-Lp082 group were then collected daily. MRS lactic acid bacteria restricted medium mixed with 1280 μg / mL vancomycin and norfloxacin solution were used to dilute and apply the fecal dilution of each mouse every day. The dilution gradient was 10 -3 ~10 -6 , cultured at 37℃ for 48h, and amplified single colonies with specific primers using colony PCR. Specific primers: A7F: 5'-GCCTTGAACTTTGTGCCTGTC-3', A7R: 5'-GGCTTTGCCTGTTGATGCTTA-3'; amplification reaction conditions: 94℃ pre-denaturation for 5min; 94℃ denaturation for 1min, 62℃ annealing for 45s, 72℃ extension for 20s, 26 cycles; 72℃ end extension for 10min. Agarose gel electrophoresis pattern verification. Bands appearing around 250bp and bright bands are considered to be isolated mutants of L. plantarum HNU082 isolated from feces. Isolate strains until no L. plantarum HNU082 mutants can be isolated. Collect all mutant strains for whole genome sequencing to explore their mutation sites.

[0034] Then, 10 8 CFU L. plantarum HNU082 original strain and 10 8CFU L. plantarum HNU082 mutant was intragastrically administered to mice in the Lp082 group and the M-Lp082 group (10 8 CFU L. plantarum HNU082 mutants were resuspended in 300 μL sterile saline and administered by gavage, n = 6). The control group mice were gavaged with an equal volume of sterilized 0.85% saline as a control. The daily gavage volume was 300 μL, and the gavage lasted for 7 days. After the gavage, fecal samples were collected for metagenomic sequencing to explore the effects of the original strain and mutant strain on the local flora, and then fresh fecal samples were collected daily for qPCR analysis of the abundance of L. plantarum HNU082 in the intestine.

[0035] 3. Experimental Methods

[0036] 3.1 Microbial DNA extraction, high-throughput metagenomic sequencing and data quality control

[0037] use Total DNA was extracted from stool samples using DNA Stool Mini Kit (Qiagen, Hilden, Germany). To ensure the quality of DNA, 0.8% agarose gel electrophoresis was used to detect the purity and integrity of DNA. NanoDrop 2000 (Novogene Company, Beijing, China) was used to assess DNA concentration. After extracting and testing the DNA of a single bacterium, 0.2 g of DNA from each sample was taken for library preparation. The DNA was randomly interrupted with a Covaris ultrasonic crusher, and the entire library was prepared by repairing the ends, adding A tails and sequencing adapters, purification, PCR amplification, and the like. After the library passed the test, it was sequenced using the Illumina high-throughput sequencing platform NovaSeq 6000. Next, Fastp (version 0.19.7) was used for raw sequencing reads and basic quality control. Sequencing files for single strains were obtained for subsequent analysis.

[0038] Fecal samples were collected after gavage and collected into enzyme-free sterile centrifuge tubes in a sterile environment of a clean bench. After weighing and quantification, 2 volumes of PBS buffer were added and mixed thoroughly for metagenomics research. The QIAGEN fecal extraction and purification kit was used to extract and purify the microbial metagenomic DNA in the samples. The DNA was randomly sheared, and the ends of the fragments were added with A tails, sequencing adapters, purified, PCR amplified, and other steps to complete the library preparation. After accurate quantification using the qPCR method, the second-generation sequencing platform Illumina Hiseq 2500 was used for high-throughput sequence determination, and after sequence preprocessing and data quality control filtering, it was used for subsequent bioinformatics analysis.

[0039] 3.2 qPCR quantitative analysis

[0040] From the 1st to the 10th day after the gavage, DNA was extracted and used for qPCR to quantitatively analyze the content in L. plantarum HNU082.

[0041] The qPCR method for L. plantarum HNU082 was as follows: 94°C pre-denaturation for 20 s; 94°C denaturation for 5 s, 58°C annealing for 35 s, 72°C extension for 50 s, and 40 cycles. The melting curve was set to slowly heat from 60°C to 95°C, and the signal intensity was detected every 0.5°C increase in temperature, and then the melting curve was obtained by continuous fluorescence acquisition. Each sample was performed in triplicate, and the average Cq value of each sample was normalized and converted to copy number according to the standard curve. All qPCR reactions were performed in a 50 μL reaction system, including 1.5 μL DNA template, 1.5 μL upstream primer, 1.5 μL downstream primer, 25 μL Light Cycler 480SYBR Green IMaster, 20.5 μL dd H 2 O. All qPCR samples were performed in triplicate.

[0042] 3.3 Mutation sites and gene annotation

[0043] In order to compare the mutation sites carried by the L. plantarum HNU082 mutant strain, we aligned all the reads obtained by resequencing with the genome of the original L. plantarum HNU082 and annotated the genomic functions of L. plantarum HNU082 using Prodigal software. The complete genome sequence of the original strain of Lactobacillus plantarum HNU082 was used as the reference genome to construct a reference database using Bowtie2 software. The sequencing reads of each sample were compared with the reference genome database constructed by the original strain of Lactobacillus plantarum HNU082, and the bam file of each isolated strain sample was obtained after sorting and conversion using SAMtools software. Finally, the InStrain (v1.0.0) software (https: / / github.com / MrOlm / inStrain) program was executed on the bam file of each sample, and the parameters called inStrain profile*.sorted.bamref.fasta-c 100-f 0.49-o*.profile-pg ref_genes.fna were used to screen out SNVs with a coverage of less than 100 and a mutation frequency of less than 0.49 detected in the sample, and finally the SNVs information of each isolated strain was obtained. At the same time, if InStrain calculates that the average nucleotide identity (ANI) between all reads in the sample and the aligned reference genome is less than 96%, the isolated strain sample will be discarded and not used for subsequent analysis.

[0044] 3.4 Gut microbial abundance and functional annotation

[0045] After obtaining high-quality sequencing reads of the fecal sample metagenomics, the species and abundance in the feces were annotated using MetaPhlAn3 software. HUMAnN3 (The HMP unified metabolic analysis network) software was used to complete the annotation of functional genes and metabolic pathways. The MetaCyc database was used as the metabolic pathway annotation database, and the DIAMOND (v2.0.15) software was used for gene family annotation to obtain the annotation results by comparing with the UniRef90 database.

[0046] 3.5 Annotation of gut microbial carbohydrate functional abundance

[0047] MEGAHIT software was used to assemble high-quality metagenomic sequencing reads of each fecal sample and spliced ​​into contigs with a minimum length of 1500 bp. Metagenemark software was then used to predict genes in these contigs to obtain the predicted gene DNA sequences in each sample. Finally, dbCAN2 software was used to compare these gene sequences with the carbohydrate active enzymes (CAZyme) database to annotate the glycoside hydrolase gene family information encoded in each sample. At the same time, the predicted gene DNA sequences in all samples were merged, and the sequences were compared with each other using CD-HIT software (https: / / github.com / weizhongli / cdhit) software to obtain the de-redundant gene sequences as reference genome sequences. Bowtie2-build was then used to build a reference database, and SAMtools software (https: / / github.com / samtools / samtools) was used to compare metagenomic sequencing reads with the database constructed by annotated carbohydrate functional genes to obtain the abundance information of each carbohydrate function in each sample. Finally, the merged data was converted into relative abundance for subsequent analysis.

[0048] 3.6 Analysis of the association between L. plantarum HNU082 and intestinal flora

[0049] Metagenomic sequencing was performed on the two groups of fecal samples, and after annotating the abundance of microorganisms, the original strain of Lactobacillus plantarum HNU082 and the intestinal domesticated strain were analyzed for association with the intestinal flora according to the method in Section 3.3.8. Based on the abundance matrix of microbial species, the correlation between microorganisms was calculated by Spearman correlation analysis, and the threshold of ±0.4 was used to retain the microorganisms with correlation coefficients greater than 0.4 or less than -0.4 with Lactobacillus plantarum HNU082 for association analysis.

[0050] 3.7 Annotation of mutation sites in the genome of intestinal-associated bacterial species

[0051] The strains directly related to Lactobacillus plantarum HNU082 in the Lp082 group and Lp082-M group were identified, and the complete representative strain genomes were downloaded as reference genomes by searching the National Center for Biotechnology Information (NCBI) database. All strain genomes were merged as the overall reference genome, and the reference database of intestinal-associated bacterial genomes was constructed using Bowtie2 software. The metagenomic sequencing reads of fecal samples at different time points were compared with the reference genome database of intestinal-associated bacterial species to obtain the bam files of fecal samples at each time point. Finally, the Instrain v1.0.0 software was executed to obtain the SNVs of each isolated strain sample. The fecal samples before gavage were used as the baseline. At the same time, in order to avoid the influence of gavage operation and intervention time, the mutation sites of the fecal samples of the control group were annotated for correction, and the SNVs information of intestinal-associated strains in the probiotic acclimation group was obtained.

[0052] 3.8 Statistical analysis

[0053] Statistical analysis was performed using R software and GraphPad Prism 8. Data are presented as mean ± standard deviation (SD). Statistical comparisons were performed using a two-tailed t-test. P < 0.05 was considered statistically significant (*p < 0.05, **p < 0.01).

[0054] IV. Results

[0055] 4.1 Number of mutations in L. plantarum HNU082 mutants in the Lp082 group

[0056] We resequenced the genomes of the L. plantarum HNU082 mutants isolated from different mice in the Lp082 group, compared the genomes with the original strains, and annotated the single nucleotide mutation sites. The number of mutation sites of the L. plantarum HNU082 mutants isolated from different mice was counted, and the number of mutation sites of the L. plantarum HNU082 mutants in each mouse was about 600, with no significant difference between them ( Figure 1 ). This indicates that the same mouse strain, diet, and environment shaped similar intestinal selection pressures, and L. plantarum HNU082 was subject to similar intestinal selection pressures among these different mice, showing the same evolutionary trend.

[0057] 4.2 Quantitative results of L. plantarum HNU082 in mouse intestine

[0058] To investigate the colonization ability and duration of the original strain of L. plantarum HNU082 and the mutant strain of L. plantarum HNU082 in the mouse intestine, we collected fecal samples of the mice continuously after oral administration and performed absolute quantification of the content of L. plantarum HNU082 in the feces. 8 CFU mL -1 plantarumHNU082, but the quantitative results showed that the number of L. plantarum HNU082 mutants colonized in the intestine was higher, and the difference could even be an order of magnitude ( Figure 2 A, B). As time went by, the content of L.plantarum HNU082 in the intestines of both groups showed a trend of gradual decrease, but the content of L.plantarum HNU082 mutant strains in the intestines was still higher than that of L.plantarum HNU082 original strains. After 1, 2, 3, 4, and 5 days after feeding cessation, the content of L.plantarum HNU082 in the intestines was significantly different between the two groups. And after feeding cessation, the colonization time of L.plantarum HNU082 mutant strains in the mouse intestines was also longer than that of L.plantarum HNU082 original strains. This may be because the adaptive mutation of L.plantarum HNU082 has enhanced its colonization ability and colonization time in the mouse intestines.

[0059] 4.3 Enrichment of mutation sites and functional gene annotation of L. plantarum HNU082 mutants

[0060] We identified the mutation sites, bases after mutation, and mutation types that were present in all L. plantarum HNU082 mutants. In the same gene, not only synonymous mutations but also a large number of non-synonymous mutations occurred, encoding amino acid changes (Table 1). These mutation sites in L. plantarum HNU082 mutants may be the reason for their enhanced ability to colonize in the intestine.

[0061] Table 1 Base information and mutation type of L. plantarum HNU082 mutation site

[0062]

[0063]

[0064]

[0065]

[0066]

[0067]

[0068]

[0069] 4.4 Functional gene annotation of L. plantarum HNU082 mutant

[0070] We annotated the functions of the genes at the mutation sites in all L. plantarum HNU082 mutants and screened the genes enriched in all strains (Table 2). The mutations in these genes may be the cause of the enhanced colonization ability of L. plantarum HNU082 mutants, including catalysis of deoxyribonucleotide biosynthesis, glycoside hydrolase, peptidoglycan hydrolase, potassium ion transport, ABC transporter, etc.

[0071] Table 2 Mutated genes of L. plantarum HNU082 mutant

[0072]

[0073]

[0074]

[0075]

[0076]

[0077]

[0078]

[0079]

[0080]

[0081]

[0082]

[0083]

[0084]

[0085]

[0086]

[0087] 4.5 Effects of L. plantarum HNU082 mutants on local bacterial diversity

[0088] The effects of intervention of the original strain and mutant strains of L. plantarum HNU082 on the ecology of the intestinal microbiome were evaluated by alpha diversity. Figure 3 As shown, the results showed that after intervention with the original strain of L. plantarum HNU082, the Alpha diversity index increased with gavage, as manifested by an increase in the Chao1 index (Mann-Whitney test, P<0.05), while after intervention with the mutant strain of L. plantarum HNU082, there was no significant effect on the overall diversity of the intestinal flora.

[0089] 4.6 Effects of L. plantarum HNU082 mutants on metabolic pathways of local flora

[0090] Based on the results of metabolic pathway abundance annotation in the MetaCyc database, it was found that the metabolic profile characteristics of the intestinal local flora were different between the L. plantarum HNU082 mutant and the original strain after intervention, and there were 18 differential metabolic pathways between the two groups. Figure 4 As shown in the figure, the differential metabolic pathways mainly include stachyose degradation pathway, glucose and xylose degradation pathway, sucrose degradation pathway, unsaturated fatty acid biosynthesis pathway, L-ascorbic acid degradation pathway, hexanol fermentation to produce lactic acid, formate, ethanol and acetate pathway, carbohydrate degradation and glycogen biosynthesis, pyruvate fermentation to produce propionic acid pathway and flavin biosynthesis pathway. Compared with the original strain, the L. plantarum HNU082 mutant strain significantly upregulated the flavin biosynthesis pathway and unsaturated fatty acid biosynthesis, and downregulated the degradation of pyrimidine deoxyribonucleosides, pyruvate fermentation to produce acetone and isopropanol biosynthesis pathways. The functional changes caused by the adaptive mutation of the L. plantarum HNU082 mutant strain have different regulatory effects on the metabolic profile of the intestinal flora from the original strain.

[0091] 4.7 Effects of L. plantarum HNU082 mutants on carbohydrate-active enzymes in local flora

[0092] The carbohydrate metabolism potential of the intestinal flora after intervention with the original strain and mutant strain of L. plantarum HNU082 was evaluated, and a carbohydrate metabolism feature matrix of the mouse intestinal flora was constructed based on the CAZyme database. The differential carbohydrate metabolic pathways of the two groups were compared, such as Figure 5As shown in the results, a total of 54 carbohydrate metabolic pathways were found to be significantly different, including glycosidases that hydrolyze various types of monosaccharides (such as galactose, glucose, mannose and fucose, etc.), and the abundance of 45 metabolic pathways such as α-glucosidase, β-L-arabinosidase, α-mannosidase, α-galactosidase, α-L-rhamnosidase, and α-L-fucosidase in the M-Lp082 group was significantly higher than that in the Lp082 group. The intervention of L. plantarum HNU082 mutant significantly improved the carbohydrate metabolic profile of intestinal flora, indicating that the intervention of L. plantarum HNU082 mutant caused changes in the metabolic activity of intestinal microbial communities and enhanced the ability to degrade carbohydrates in the intestine, which may have a positive impact on the health of the host, such as promoting the absorption and digestion of nutrients and maintaining the balance of intestinal microecology.

[0093] 4.8 Analysis of the association between L. plantarum HNU082 mutants and intestinal flora

[0094] Correlation analysis was used to identify species that competed or cooperated with the original strain and mutant strain of L. plantarum HNU082 in the intestine, such as Figure 6 As shown in the figure, the mutant strain and the original strain had association networks of different complexities in the intestine. After statistical analysis, there were 23 bacterial species that directly interacted with the original strain of L. plantarum HNU082, and the bacterial species that had competitive relationships in the intestine included Lachnospiraceae bacterium 3_1, Bifidobacterium pseudolongum, Oscillibacter sp 1_3, Anaerotruncus sp G3_2012, Alistipes indistinctus, Parasutterella excrementihominis, Proteobacteria bacterium CAG_139, Lachnospiraceae bacterium 3_2, Eubacterium plexicaudatum, Lachnospiraceaebacterium M18_1, Enterorhabdus caecimuris, Acutalibacter muris, Dorea sp 5_2, Faecalibaculum rodentium( Figure 6A), while the mutant strain of L. plantarum HNU082 only interacted with 16 local bacterial species after entering the intestine, among which the negatively correlated species were Lachnospiraceae bacterium COE1, Firmicutes bacterium ASF500, Lactobacillus reuteri, Helicobacter ganmani, Lachnospiraceae bacterium A4, Mucispirillum schaedleri, Clostridium sp ASF356, Alistipes indistinctus, Bacteroides vulgatus, Lachnospiraceae bacterium 3_2, Anaerotruncus sp G3_2012 ( Figure 6 B). After intervention with the L. plantarum HNU082 mutant, the number of bacterial species that interacted with native intestinal microorganisms was reduced, indicating that it may have less impact on the native intestinal microbiome.

[0095] 4.9 Effect of L. plantarum HNU082 mutant on local bacterial flora

[0096] The number of SNVs in the genome of L. plantarum HNU082 mutant strain and the original strain were compared and analyzed. Figure 7 As shown in the figure, the number of SNVs in the native bacterial species directly related to L. plantarum HNU082 was found to be lower in the M-Lp082 group compared with the Lp082 group, indicating that the L. plantarum HNU082 mutant strain had less ecological and evolutionary impact on the intestinal microbiota. The lower coevolution of this native bacterial community indicates that it is less competitively pressured by probiotics, which may help the L. plantarum HNU082 mutant strain to colonize in the intestine.

[0097] Obviously, the above embodiments of the present invention are merely examples to more clearly illustrate the present invention, and are not limitations on the implementation methods of the present invention. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is impossible to list all the implementation methods here. All obvious changes or modifications derived from the technical solution of the present invention are still within the protection scope of the present invention.

Claims

1. Application of Lactiplantibacillus plantarum HNU082 mutant strain in enhancing intestinal colonization ability.

2. The use according to claim 1, characterized in that: Compared with the original strain of Lactiplantibacillus plantarum HNU082, the mutant strain of Lactiplantibacillus plantarum HNU082 has a higher number of colonization in the intestine and a longer colonization time.

3. The use according to claim 1, characterized in that: Mutant gene of the Lactiplantibacillus plantarum HNU082 mutant strain include: 。 4. The use according to claim 3, characterized in that: The mutation site base information and mutation type of the mutant gene are:

5. The use according to claim 1, characterized in that: The Lactiplantibacillus plantarum HNU082 mutant strain can significantly upregulate the flavin biosynthesis pathway and the unsaturated fatty acid biosynthesis, and downregulate the pyrimidine deoxyribonucleoside degradation, the pyruvate fermentation to produce acetone, and the isopropanol biosynthesis pathway.

6. The use according to claim 1, characterized in that: The Lactiplantibacillus plantarum HNU082 mutant strain can significantly improve the carbohydrate metabolism profile of intestinal flora.

7. The use according to claim 1, characterized in that: Compared with the original strain of Lactiplantibacillus plantarum HNU082, the mutant strain of Lactiplantibacillus plantarum HNU082 has fewer bacterial species that interact with local intestinal microorganisms.

8. The use according to claim 1, characterized in that: The Lactiplantibacillus plantarum HNU082 mutant has less impact on the ecology and evolution of the gut microbiota.

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