Complex microbial inoculant for regulating intestinal flora in cooperation with plant lactobacillus HNU082, method and application

Abstract

The invention provides a complex microbial inoculant for regulating intestinal flora in cooperation with plant lactobacillus HNU082, and a method and application of the complex microbial inoculant. According to the invention, the intestinal flora is taken as an entry point, two times of intermittent intervention is carried out on the human intestinal tract by using the plant lactobacillus HNU082, a strain synergistically promoted by the plant lactobacillus HNU082 is screened from the local intestinal flora during two times of short-term colonization of the plant lactobacillus HNU082 according to a metagenome sequencing result, and further animal experiments are carried out, so that the application of the plant lactobacillus HNU082 to the human intestinal tract is realized. The synergistic promotion function and mechanism of the lactobacillus plantarum HNU082 for intestinal colonization are deeply explored, and theoretical basis and data support are provided for promoting intestinal colonization of probiotics by regulating intestinal flora.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and relates to a composite bacterial agent, method and application for cooperating with Lactobacillus plantarum HNU082 to regulate intestinal flora. Background Art

[0002] The intestinal microbiome participates in physiological processes such as host nutrient absorption, energy metabolism, and immune regulation, and is an important factor in maintaining host health. Under the intestinal selection pressure jointly shaped by multiple factors such as host immunity, diet, drug use, and microbial competition, the local microbial community has achieved a relatively stable dynamic balance through continuous adaptation and adjustment. When exogenous microorganisms enter the intestine, they face multiple intestinal selection pressures such as nutrient acquisition, niche competition, and host immune response. Their colonization will be affected by multiple limiting factors such as the strain's own genotype diversity, intestinal adaptability, and early growth and reproduction rate. In addition, it is also crucial to establish a good relationship with the local flora in the intestine. The probiotic Lactobacillus plantarum shows great potential in promoting intestinal health, enhancing immunity, and alleviating chronic diseases. Its long-term colonization ability is the key to its sustained and stable efficacy. There may be multiple interactions between probiotic strains and local flora, including mutualism and competition. This interaction relationship will affect the colonization and survival of strains in the intestine, and good interactions may provide the support and environment required by the strains, which helps them survive and reproduce in the intestine. Summary of the invention

[0003] Based on the above, the purpose of the present invention is to provide a composite bacterial agent, method and application for regulating intestinal flora in collaboration with Lactobacillus plantarum HNU082, so as to provide a theoretical basis and data support for promoting intestinal colonization of probiotics by regulating intestinal flora.

[0004] The technical solution adopted by the present invention to achieve the technical purpose is:

[0005] The invention provides a composite bacterial agent for cooperating with Lactobacillus plantarum HNU082 to regulate intestinal flora, comprising Leuconostoc citrinum and Lactobacillus plantarum HNU082.

[0006] Preferably, the Leuconostoc citraceus includes Leuconostoc citraceus ATCC 49370.

[0007] More preferably, the colony forming unit (CFU) of the Leuconostoc citraceus ATCC 49370 is the same as that of Lactobacillus plantarum HNU082.

[0008] The present invention also provides a method for regulating intestinal flora in coordination with Lactobacillus plantarum HNU082, comprising combining Leuconostoc citrinum with Lactobacillus plantarum HNU082 to exert a synergistic effect in vivo.

[0009] Preferably, the above method comprises administering Leuconostoc citrinum and Lactobacillus plantarum HNU082 jointly by gavage.

[0010] More preferably, the Leuconostoc citrea includes Leuconostoc citrea ATCC 49370.

[0011] The present invention further provides the use of Leuconostoc citrifolia in collaboration with Lactobacillus plantarum HNU082 in regulating intestinal flora.

[0012] Preferably, the above application comprises using Leuconostoc citrifolia in combination with Lactobacillus plantarum HNU082 to promote the colonization of Lactobacillus plantarum HNU082 in vivo.

[0013] Preferably, the above application also includes combining Leuconostoc citrinum with Lactobacillus plantarum HNU082 to reduce the disturbance of Lactobacillus plantarum HNU082 strain itself to the local intestinal microbiome.

[0014] Preferably, the above application also includes combining Leuconostoc citrinum with Lactobacillus plantarum HNU082 to increase the content of beneficial metabolites in the intestine; the beneficial metabolites include all-trans retinoic acid, deoxycholic acid, L-arogenate and arachidonic acid.

[0015] Preferably, the above application also includes combining Leuconostoc citrinum with Lactobacillus plantarum HNU082 to reduce the content of metabolites such as protocatechuic acid, hexadecanedioic acid and L-tryptophan in the intestine.

[0016] More preferably, the Leuconostoc citrea includes Leuconostoc citrea ATCC 49370.

[0017] The beneficial effects of the present invention are:

[0018] In the present application, taking the intestinal flora as the starting point, Lactobacillus plantarum HNU082 was used for two intermittent interventions on the human intestine. Through the results of metagenomic sequencing, the bacterial species that synergistically promoted with Lactobacillus plantarum HNU082 during the two short-term colonization periods were screened out from the local intestinal flora. Further through animal experiments, the synergistic promotion function and mechanism of Lactobacillus plantarum HNU082 intestinal colonization were deeply explored, providing a theoretical basis and data support for promoting the intestinal colonization of probiotics by regulating the intestinal flora. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Changes in abundance of Lactobacillus plantarum HNU082 in fecal samples.

[0020] Figure 2 Changes in the abundance of Leuconostoc citrinum in the intestine.

[0021] Figure 3Verification of the in vivo synergistic effect between Leuconostoc citrinum ATCC 49370 and Lactobacillus plantarum HNU082.

[0022] Figure 4 Effects of intragastric administration of Lactobacillus plantarum HNU082 alone or in combination with Leuconostoc citrinum ATCC 49370 on the diversity of intestinal flora.

[0023] Figure 5 Identification of species associated with Lactobacillus plantarum HNU082 within the Lp082 and Lp082+Lc groups.

[0024] Figure 6 Analysis of differential metabolites of Lp082 and Lp082+Lc. DETAILED DESCRIPTION

[0025] 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.

[0026] Example

[0027] 1. Materials and Methods

[0028] 1.1 Strains and culture media

[0029] The probiotic Lactobacillus plantarum HNU082 is provided by the tropical probiotic lactic acid bacteria resource library of Hainan University (detailed in the patent application CN202110619723.X with the application date of 2021-06-03, named a strain of Lactobacillus plantarum HNU082 and its use. It is also abbreviated as Lp082). The representative strain of Leuconostoc citrinum ATCC 49370 was purchased from the Guangdong Microbiological Collection Center. The MRS medium formula is as follows: 20g glucose, 10g peptone, 10g beef extract, 5g yeast extract, 2g C6H5O7(NH4)3, 1mL Tween-80, 5g CH3COONa, 2g K2HPO4, 0.58g MgSO4, 0.25g MnSO4, 1L water.

[0030] 1.2 Experimental design

[0031] After being informed of the experimental plan and details, the 12 volunteers agreed to participate in the follow-up experiment. The study was conducted after obtaining informed consent from all volunteers. The 12 volunteers were divided into two groups: a probiotic intervention group and a placebo group. Six volunteers took 10 8CFU of the original strain of Lactobacillus plantarum HNU082, then stop taking it and continue to collect stool for the isolation of Lactobacillus plantarum HNU082. Then take it again for 10 8 CFU of the original strain of Lactobacillus plantarum HNU082 was collected for 7 days, and stool samples were collected again for isolation and identification of the Lactobacillus plantarum HNU082 strain. At the same time, stool samples were collected for metagenomic sequencing. All participants provided written informed consent to participate in the study.

[0032] 1.3 Gut microbiome DNA extraction and metagenomic deep sequencing

[0033] Fecal samples were collected at each time point and placed in enzyme-free sterile centrifuge tubes in a clean bench. After weighing and quantification, 2 times the volume of PBS buffer was added and mixed thoroughly for metagenomics research.

[0034] The QIAGEN stool extraction and purification kit was used to extract and purify the microbial metagenomic DNA in the sample. The Covaris ultrasonic disruptor was used to randomly break the DNA, and then the ends of the fragments were smoothed and repaired, A-tailed, sequencing adapters were added, purified, and PCR amplified to complete the library preparation. After the library was constructed, Qubit 2.0 was used for preliminary quantification, and then the effective concentration of the library was accurately quantified using the qPCR method to ensure the quality of the library. Finally, the second-generation sequencing platform Illumina Hiseq 2500 was used for high-throughput sequence determination. Sequence preprocessing and data quality control filtering were performed on the sequencing data, and the contamination of the adapters was removed and quality correction was performed. Low-quality sequences and fragments less than 70 bp in length were filtered, and the average quality distribution of the sequencing data was detected to ensure the output of high-quality data. Finally, sequence assembly and splicing were performed to construct a non-redundant sequence set for subsequent bioinformatics analysis.

[0035] 1.4 Gut microbiome species abundance and functional annotation

[0036] 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.

[0037] 1.5 Synergistic bacterial species identification between intestinal microbiome and Lactobacillus plantarum HNU082

[0038] By using the abundance change (Delta) as the criterion, the bacterial species with the same change trend as Lactobacillus plantarum HNU082 in both oral gavages were counted. Specifically, during the two interventions of Lactobacillus plantarum HNU082, the abundance change of the bacterial species was greater than zero compared with the previous time point, and after the second intervention, the abundance change of the bacterial species was greater than the abundance change of the bacterial species in the first intervention, which was defined as a synergistic bacterial species. On the contrary, after the first intervention of Lactobacillus plantarum HNU082, the abundance change of the bacterial species was less than zero compared with the previous time point. At the same time, calibration was performed by excluding the bacterial species with the same abundance trend in the fecal samples of the control group. Then, the synergistic bacterial species that were positively correlated with the abundance of Lactobacillus plantarum HNU082 were screened by Spearman correlation.

[0039] 1.6 Verification of the synergistic effect of Leuconostoc citrinum ATCC 49370 and Lactobacillus plantarum HNU082 in vivo

[0040] The representative strain of Leuconostoc citreum ATCC 49370 was purchased from Guangdong Microbiological Collection Center. First, in order to ensure the accuracy of the quantitative results of Lactobacillus plantarum HNU082 during co-culture, the specificity of the primers of Lactobacillus plantarum HNU082 was verified for Leuconostoc citreum ATCC 49370, and the primer sequences were (Lp082_F: 5'-GCCTTGAACTTTGTGCCTGTC-3'; Lp082_R: 5'-GGCTTTGCCTGTTGATGCTTA-3'). Leuconostoc citreum ATCC 49370 was cultured according to the culture instructions. After the strain was activated, 2% of the bacterial solution was subcultured in 5 mL sterilized MRS medium and cultured in a constant temperature incubator at 37°C for 48 hours. DNA of Leuconostoc citrinum ATCC 49370 was extracted by colony PCR method, and then PCR amplification was performed. The amplification system was: 50μL reaction system, including 1μL DNA template, 1μL upstream primer, 1μL downstream primer, 20μL Taq Mix, 27μL dd H2O. PCR 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. There were no bands in agarose gel electrophoresis, which proved that the primers of Lactobacillus plantarum HNU082 had good specificity and could be used for absolute quantification of Lactobacillus plantarum HNU082 in feces.

[0041] Animal experiments were carried out (C57BL / 6J mice, 10 weeks old, Hunan Slake Jingda Experimental Animal Co., Ltd.). The experiment was divided into two groups. The Lp082 group was gavaged with Lactobacillus plantarum HNU082 (10 8CFU), Lp082+Lc group was intragastrically administered with Lactobacillus plantarum HNU082 mixed with Leuconostoc citrinum ATCC 49370 (10 8 CFU+10 8 Fecal samples were collected daily to extract DNA for quantitative analysis of Lactobacillus plantarum HNU082 using qPCR.

[0042] 1.7 Quantitative analysis of Lactobacillus plantarum HNU082 in fecal samples

[0043] Fecal samples of mice were collected daily after feeding was stopped, and the content of Lactobacillus plantarum HNU082 in the feces was determined by qPCR. 100 mg of feces was taken from each sample in a 2 mL centrifuge tube, 1.5 mL of TE buffer was added, and the fecal sample was homogenized in a magnetic homogenizer for 60 seconds to completely mix the fecal sample. Then, the cell wall was broken by liquid nitrogen freeze-thaw method, and DNA was dissolved in buffer. Then, the sample was purified by PCR according to the instructions. DNA was extracted from stool samples using the DNAStool Mini Kit.

[0044] At the same time, the genomic DNA of Lactobacillus plantarum HNU082 was extracted, purified by a purification kit, and the DNA purity and concentration were detected by Nanodrop through the OD 260 / 280 ratio. The purified DNA sample was used as the stock solution for ten-fold serial dilution to 8 concentrations for the preparation of the standard curve, and the copy number was calculated according to the length of the amplified fragment and the DNA concentration. Using the fecal DNA of each sample as a template, a 50 μL system was selected for qPCR amplification, including 1.5 μL DNA template, 1.5 μL upstream primer, 1.5 μL downstream primer, 25 μL SYBR PCR Mix and 20.5 μL dd H2O. The upstream primer selected the forward primer of Lactobacillus plantarum HNU082: A7F (5'-GCCTTGAACTTTGTGCCTGTC-3'), and the downstream primer selected the reverse primer of Lactobacillus plantarum HNU082 A7R (5'-GGCTTTGCCTGTTGATGCTTA-3').

[0045] qPCR reaction conditions: 94℃ pre-denaturation for 30s; 94℃ denaturation for 10s, 62℃ annealing for 30s, 72℃ extension for 30s, 34 cycles; then slowly heated from 60℃ to 95℃, the signal intensity was detected every 0.5℃ increase in temperature, and the melting curve was obtained by continuous fluorescence acquisition. Each sample was run in triplicate as a parallel, and the average Cq value of each sample was normalized and converted to copy number according to the standard curve.

[0046] 2. Results

[0047] 2.1 Analysis of the abundance of probiotic Lactobacillus plantarum HNU082 in fecal samples

[0048] The relative abundance of Lactobacillus plantarum HNU082 was higher after the second dose than after the first dose ( Figure 1 ). This suggests that there may be bacteria in the human intestinal flora that promote the colonization of Lactobacillus plantarum HNU082 in the intestine and respond quickly after the second intervention, making it easier for probiotics to colonize and expand their abundance during the second intervention, thereby increasing their relative advantage in the microecological system.

[0049] 2.2 Synergistic bacterial species identification between intestinal microbiome and Lactobacillus plantarum HNU082

[0050] The abundance of intestinal microorganisms in the population was annotated, and the species that may have a synergistic relationship with Lactobacillus plantarum HNU082 were identified. The abundance increased after Lactobacillus plantarum HNU082 entered the intestine, and the second abundance change was greater than the first abundance change. The correlation coefficient with Lactobacillus plantarum HNU082 was calculated by Spearman correlation analysis, as shown in Table 1 below.

[0051] Table 1 Correlation between intestinal microbial species and Lactobacillus plantarum HNU082

[0052]

[0053]

[0054] Among them, the correlation between Leuconostoc citreum and Lactobacillus plantarum HNU082 was 0.56, the highest correlation among all the screened and retained strains, and the abundance changes at different time points were consistent with the abundance changes of Lactobacillus plantarum HNU082 in the intestine. A line graph was drawn based on the abundance changes of Leuconostoc citreum at different time points during the experimental stage ( Figure 2 2.3 Analysis of the synergistic effect of Leuconostoc citrinum ATCC 49370 and Lactobacillus plantarum HNU082 in vivo

[0055] To verify the synergistic effect of Leuconostoc citrinum ATCC 49370 on Lactobacillus plantarum HNU082 in the intestine, mice were divided into two groups, one group was gavaged with Lactobacillus plantarum HNU082 alone, and the other group was gavaged with Lactobacillus plantarum HNU082 and Leuconostoc citrinum ATCC 49370 at the same time. The absolute quantification of Lactobacillus plantarum HNU082 in fecal samples during gavage and after feeding was performed by qPCR. In addition, to ensure that the gavage dose of the two groups of mice was consistent, the absolute quantification of Lactobacillus plantarum HNU082 in the gavage samples was also performed ( Figure 3A). The results showed that under the condition of intragastric administration of the same dose of Lactobacillus plantarum HNU082, supplementary intragastric administration of Leuconostoc citrinum ATCC 49370 significantly increased the content of Lactobacillus plantarum HNU082 in the intestine. On the second day of intragastric administration, the abundance of Lactobacillus plantarum HNU082 in the intestine of mice increased by 2.57 times compared with intragastric administration alone ( Figure 3 B). This demonstrates the synergistic effect of Leuconostoc citrinum ATCC 49370 and Lactobacillus plantarum HNU082 in the host intestine.

[0056] 2.4 Analysis of the synergistic promotion mechanism between Leuconostoc citrinum ATCC 49370 and Lactobacillus plantarum HNU082

[0057] The fecal samples on the 0th and 2nd day of gavage were annotated for species, and the diversity and structural changes of the intestinal microbiome were analyzed. From the Shannon and Simpson indexes, gavage of Lactobacillus plantarum HNU082 alone and combined with Leuconostoc citrinum ATCC 49370 increased the Alpha diversity of the intestinal microbiome, and the combined gavage had a smaller increase in the Alpha diversity index ( Figure 4 A). The Bray-Curtis distance was used to evaluate the effect of the microbiome structure. The results showed that the normal distribution curve representing the combined gavage was closer to the left, indicating that the composition of the intestinal flora between the 0th day and the 2nd day of combined gavage was more similar and the distance was closer, indicating that combined gavage may have a smaller effect on the change of the flora structure ( Figure 4 B and C).

[0058] According to the Spearman correlation coefficient, after screening and P value correction, the interacting bacteria species between the Lp082 group and the Lp082+Lc group and the intestinal local microbiome were identified as follows: Figure 5 As shown. The results showed that in the Lp082 group, there were more species interacting with the local intestinal bacteria in Lactobacillus plantarum HNU082, and a total of 16 species fluctuated significantly in abundance and were associated with Lactobacillus plantarum HNU082. In the Lp082+Lc group, statistical analysis showed that only 8 species interacted with Lactobacillus plantarum HNU082, and only one species, Lachnospiraceae bacterium 3_1, was negatively correlated with Lactobacillus plantarum HNU082 in abundance. This is also consistent with the results of changes in the Beta diversity of the intestinal microbiome. The combined intragastric administration of Lactobacillus plantarum HNU082 and Leuconostoc lemon ATCC 49370 had less disturbance to the local flora.

[0059] Finally, we compared the effects of Lactobacillus plantarum HNU082 alone and the combined use of Lactobacillus plantarum HNU082 and Leuconostoc citrinum ATCC 49370 on the levels of intestinal microbial metabolites. By comparing the metabolites with significant differences between the two groups, it was found that the combined use of Lactobacillus plantarum HNU082 and Leuconostoc citrinum ATCC 49370 increased the levels of metabolites such as all-trans retinoic acid, deoxycholic acid, L-arogenate and arachidonic acid in the intestine compared with gavage of Lactobacillus plantarum HNU082 alone, and reduced the levels of metabolites such as protocatechuic acid, hexadecanedioic acid and L-tryptophan ( Figure 6 ). The elevation of all-trans retinoic acid and the degradation of L-tryptophan may help regulate intestinal immune function and enhance the beneficial effects of Lactobacillus plantarum HNU082 on the host intestinal health.

[0060] Leuconostoc citrinum ATCC 49370 significantly promoted the colonization of Lactobacillus plantarum HNU082 in vivo, which may be achieved by affecting the ecological environment of the intestinal flora. Combined oral administration weakened the disturbance of the Lactobacillus plantarum HNU082 strain itself on the intestinal microbiome, which may have reduced the direct competition with some bacterial species, thus showing a new coexistence relationship in the association network, thereby promoting the growth and reproduction of Lactobacillus plantarum HNU082 in the intestine, increasing the content of beneficial metabolites, and achieving a promoting effect on host health.

[0061] 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. A composite bacterial agent that synergizes with Lactobacillus plantarum HNU082 to regulate intestinal flora, including Leuconostoc citrinum and Lactobacillus plantarum HNU082.

2. The composite bacterial agent according to claim 1, characterized in that The lemon limonene includes lemon limonene ATCC 49370.

3. A method for synergistically regulating intestinal flora with Lactobacillus plantarum HNU082, comprising combining Leuconostoc citrinum with Lactobacillus plantarum HNU082 to exert a synergistic effect in vivo.

4. The method according to claim 3, characterized in that It includes oral administration of Leuconostoc citrinum and Lactobacillus plantarum HNU082.

5. Application of Leuconostoc citrinum and Lactobacillus plantarum HNU082 in regulating intestinal flora.

6. The use according to claim 5, characterized in that: It includes combining Leuconostoc citrinum with Lactobacillus plantarum HNU082 to promote the colonization of Lactobacillus plantarum HNU082 in vivo.

7. The use according to claim 6, characterized in that: It also includes the combination of Leuconostoc citrinum and Lactobacillus plantarum HNU082 to weaken the disturbance of Lactobacillus plantarum HNU082 strain itself to the local intestinal microbiome.

8. The use according to claim 7, characterized in that: It also includes the combined use of Leuconostoc lemon and Lactobacillus plantarum HNU082 to increase the content of beneficial metabolites in the intestine; the beneficial metabolites include all-trans retinoic acid, deoxycholic acid, L-arogenate and arachidonic acid.

9. The use according to claim 8, characterized in that: It also includes the combination of Leuconostoc citrinum and Lactobacillus plantarum HNU082 to reduce the levels of metabolites protocatechuic acid, hexadecanedioic acid and L-tryptophan in the intestine.

10. The use according to any one of claims 5 to 9, characterized in that: The lemon limonene includes lemon limonene ATCC 49370.

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