Application of polymannuronic acid in the preparation of gastric and / or intestinal flora regulator

Abstract

The present invention belongs to the field of biomedicine and relates to the use of polymannuronic acid in the preparation of a gastric and / or intestinal flora regulator. The polymannuronic acid has an average degree of polymerization of 90 to 100, and after being delivered to the stomach and / or intestines for regulation, the gastric and / or intestinal flora regulator can reduce the abundance of Helicobacter species in feces. Studies have shown that the polymannuronic acid provided by the present invention can effectively reduce the abundance of harmful Helicobacter species, such as Helicobacter, in feces excreted after gastrointestinal regulation.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology and relates to the application of polymannuronic acid in the preparation of a gastric and / or intestinal flora regulator. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance understanding of the overall background of the invention and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to those skilled in the art.

[0003] Harmful bacteria in the gastrointestinal flora can cause gastrointestinal diseases. For example, Helicobacter can cause digestive tract diseases. Current antibiotics have poor specificity. In the process of inhibiting harmful bacteria, they will also inhibit beneficial bacteria, thereby affecting gastrointestinal function. Therefore, it is necessary to provide a preparation that can regulate gastrointestinal flora to reduce harmful bacteria in the stomach and intestines. Summary of the Invention

[0004] In order to address the deficiencies of the prior art, the present invention aims to provide a method for preparing a gastric and / or intestinal flora regulator using polymannuronic acid. Studies have shown that the polymannuronic acid provided by the present invention can effectively reduce the abundance of harmful flora such as Helicobacter spp. in feces after gastrointestinal regulation.

[0005] In order to achieve the above object, the technical solution of the present invention is:

[0006] On the one hand, a polymannuronic acid or a composition with polymannuronic acid as an active ingredient is used in the preparation of a gastric and / or intestinal flora regulator, wherein the average degree of polymerization of the polymannuronic acid is 90-100, and the gastric and / or intestinal flora regulator can reduce the abundance of Helicobacter bacteria in feces after being delivered to the stomach and / or intestine for regulation.

[0007] Furthermore, the dosage of polymannuronic acid was 90-110 mg / kg, which was able to more significantly reduce the abundance of harmful bacterial colonies such as Helicobacter in feces after gastrointestinal conditioning compared to the high-dose administration group (390-410 mg / kg).

[0008] Furthermore, the polymannuronic acid is obtained by hydrolyzing sodium alginate.

[0009] Furthermore, the preparation process of the polymannuronic acid is as follows: the polymannuronic acid is swelled, hydrochloric acid is added, and then hydrolyzed at 95-105° C. The hydrolysis time is 10-15 hours.

[0010] Furthermore, the purification process after hydrolysis is as follows: the hydrolyzed precipitate is dissolved in a NaHCO3 solution, the pH is adjusted to acidic to produce a white flocculent precipitate, and the supernatant is collected after standing and stratification, and the obtained supernatant is subjected to the following process and repeated at least once:

[0011] The supernatant was adjusted to pH 7-8, and a 90-98% ethanol solution was added. After standing for separation, a precipitate was obtained. The precipitate was dissolved with a NaHCO3 solution, and the pH was adjusted to acidic to produce a white flocculent precipitate. The precipitate was removed to obtain a supernatant;

[0012] Anhydrous ethanol was added to the supernatant obtained finally, and the mixture was stirred and mixed for dehydration, and the crude polymannuronic acid was obtained by suction filtration and drying.

[0013] Furthermore, the purification process of the crude polymannuronic acid is as follows: dissolving the crude polymannuronic acid in water, filtering with a 0.45 μm filter membrane, further purifying with an ion exchange chromatography column, and then further purifying with an AKTA protein purification system to obtain pure polymannuronic acid.

[0014] Specifically, the ion exchange chromatography column is Cl - Format Q-Sepharose column.

[0015] Specifically, in further purification using an AKTA protein purification system, mobile phase A is ultrapure water, mobile phase B is a NaCl solution, the upper pressure limit is 0.9 to 1.1 MPa, and the UV detection wavelength is 205 to 215 nm.

[0016] Specifically, in further purification using the AKTA protein purification system, after the column exchange is completed, linear gradient elution is performed with a NaCl solution with a concentration of 0.5 to 2.0 M.

[0017] Specifically, the collected main peak eluate was freeze-dried and concentrated, and then desalted using a Sephadex G-25 column to obtain pure polymannuronic acid.

[0018] Furthermore, the gastric and / or intestinal flora regulator is used to prepare a medicine.

[0019] Furthermore, the composition is composed of an active ingredient and pharmaceutical excipients. The pharmaceutical excipients include pharmaceutical carriers and / or excipients. The pharmaceutical carriers include, for example, aluminum oxide, serum albumin, phosphate buffer solution, etc. The excipients include, for example, binders, viscosity enhancers, disintegrants, fillers, emulsifiers, etc.

[0020] The beneficial effects of the present invention are:

[0021] The present invention shows through specific experiments that at the phylum level, the low-dose group showed an increasing trend in the B / F value, indicating that low-dose PM is more conducive to the balance of mouse fecal flora. At the genus level, the low-dose group can significantly reduce the abundance of Helicobacter (Helicobacter) flora in the feces of mice 10 and 20 days after PM gavage. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0023] Figure 1 Figure 1 is a diagram showing the separation and purification results of PM by Q-Sepharose ion column in an embodiment of the present invention, wherein a is the elution curve (210 nm) of PM by Q-Sepharose ion column, and b is the monitoring diagram of PM by phenol-sulfuric acid method.

[0024] Figure 2 This is a diagram showing the results of high performance liquid chromatography of PM in an embodiment of the present invention.

[0025] Figure 3 This is the GraPhlAn species composition diagram of the feces of mice gavaged with PM for 10 days in the example of the present invention.

[0026] Figure 4 Figure 1 is a diagram showing the species composition of the fecal flora at the phylum level in mice after PM gavage for 10 days in an embodiment of the present invention; A is the species composition of mouse feces on the 10th day, and B is the difference in species composition of mouse feces on the 10th day. D10 is the control group, L10 is the low-dose group, and H10 is the high-dose group. Compared with the control group: *p<0.05, **p<0.01.

[0027] Figure 5 Figure 1 is a diagram showing the species composition of the family-level flora in the feces of mice after PM gavage for 10 days in an embodiment of the present invention; A is the species composition of mouse feces on the 10th day, and B is the difference in species composition of mouse feces on the 10th day. D10 is the control group, L10 is the low-dose group, and H10 is the high-dose group. Compared with the control group: *p<0.05, **p<0.01.

[0028] Figure 6 Figure 1 is a genus-level bacterial species composition diagram of mouse feces 10 days after PM gavage in an embodiment of the present invention; A is the species composition diagram of mouse feces on the 10th day, B is the difference diagram of mouse feces species composition on the 10th day, D10 is the control group, L10 is the low-dose group, and H10 is the high-dose group; compared with the control group: *p<0.05, **p<0.01.

[0029] Figure 7 This is a correlation network diagram of fecal flora in mice given PM gavage for 10 days in an example of the present invention.

[0030] Figure 8 This is the GraPhlAn species composition diagram of the feces of mice gavaged with PM for 20 days in the examples of the present invention.

[0031] Figure 9 Figure 2 is a diagram showing the species composition of the fecal flora at the phylum level in mice after PM gavage for 20 days in an embodiment of the present invention; A is the species composition of mouse feces on the 20th day, and B is the difference in species composition of mouse feces on the 20th day. D20 is the control group, L20 is the low-dose group, and H20 is the high-dose group. Compared with the control group: *p<0.05, **p<0.01.

[0032] Figure 10 Figure 2 is a diagram showing the species composition of the family-level flora in the feces of mice after PM gavage for 20 days in the examples of the present invention; A is the species composition of mouse feces on the 20th day, and B is the difference in species composition of mouse feces on the 20th day. D20 is the control group, L20 is the low-dose group, and H20 is the high-dose group. Compared with the control group: *p<0.05, **p<0.01.

[0033] Figure 11 Figure 2 is the species composition diagram of the fecal flora at the genus level in mice after PM gavage for 20 days in the examples of the present invention; A is the species composition diagram of mouse feces on the 20th day, B is the difference diagram of species composition of mouse feces on the 20th day, D20 is the control group, L20 is the low-dose group, and H20 is the high-dose group; compared with the control group: *p<0.05, **p<0.01.

[0034] Figure 12 This is a correlation network diagram of fecal flora in mice gavaged with PM for 20 days in an example of the present invention. DETAILED DESCRIPTION

[0035] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0036] Example

[0037] Preparation of PM (polymannuronic acid):

[0038] After 100g of sodium alginate is completely swollen by adding 5000mL of water, concentrated hydrochloric acid is slowly added to the solution to adjust the final concentration to 0.5M. The solution is then subjected to acid hydrolysis at 100°C for 10 hours. After completion of hydrolysis, the precipitate is centrifuged and dissolved in 8% NaHCO₃ solution. The pH is adjusted with dilute HCl, and a large amount of white flocculent precipitate forms as the pH approaches 2.86. The solution is allowed to stand and separate, yielding a yellow supernatant and a lower precipitate. The supernatant is then adjusted to a pH of 7-8 with NaOH solution, and 3 times the solution volume of 95% ethanol is added. The solution is allowed to stand and a white flocculent precipitate is obtained. The precipitate is then centrifuged and dissolved in NaHCO₃ solution, and the pH is adjusted to 2.86 with HCl. The precipitate is then centrifuged and discarded. The supernatant is then subjected to this process twice more. Finally, excess anhydrous ethanol is added, stirred, and dehydrated. The alcohol and water are removed by filtration, and the product is dried at 50°C to obtain a high-purity crude PM product.

[0039] Weigh an appropriate amount of PM crude solid powder obtained by precipitation separation, add pure water to dissolve it, filter it with a 0.45 μm filter membrane, and pass the sample through an ion exchange chromatography column (Q-Sepharose column, 50 cm × 5 cm, Cl - The protein was further purified using the AKTA Protein Purification System (GE, AKTAPure 25L1, USA) as follows: ① Set the mobile phase for pump A to ultrapure water and the mobile phase for pump B to 2.0 M NaCl, with a flow rate of 1.0 mL / min, a pressure limit of 1.0 MPa, and a UV detection wavelength of 210 nm. ② After column exchange, perform a linear gradient elution with NaCl concentrations from 0.5 to 2.0 M. Elution peaks were detected by UV absorption at 210 nm and the phenol-sulfuric acid method. The main peak eluate was collected, lyophilized, concentrated, and desalted on a Sephadex G-25 column to obtain pure PM.

[0040] Purity analysis and molecular weight determination of PM:

[0041] A TSK-gel G4000 PWxl column was installed on a high-performance liquid chromatograph (Agilent, Agilent 1100, USA) according to the manufacturer's instructions, paying attention to the flow rate. The flow rate was adjusted to 0.8 mL / min, and the UV detector was turned on to equilibrate the column with 0.15 M NaCl solution for approximately 1 h. The purified PM and a series of dextran standards of known molecular weight (Mw = 10, 70, and 150 kD) were filtered through a 0.45 μm filter membrane and then passed through a TSK-gel G4000 PWxl column. The column temperature was 40°C, and the injection volume was 20 μL. Elution was performed with 0.15 M NaCl solution. Purity was verified by UV absorption at 210 nm, and the retention times of PM and dextran were measured.

[0042] Experimental animal grouping and drug administration:

[0043] After receiving the mice, they were housed in a mouse house (air-conditioned at 25±1°C, 50±20% relative humidity, and a 12-h light / dark cycle). They were allowed to eat and drink freely throughout the experiment. Drinking water and feed were replaced daily, and bedding was changed every 3 days. Adaptive feeding was performed for the first 7 days, and dosing began on the 8th day, designated as dosing day 0. The mice were examined daily, and all healthy mice were selected for the experiment. The mice were randomly divided into three groups based on body weight: a control group (Control), a low-dose dosing group (PM-L), and a high-dose dosing group (PM-H), with 6 mice in each group.

[0044] Mice in each group were individually numbered and received PM solution orally at a dose of 100 mg / kg and 400 mg / kg, respectively, for 20 consecutive days, once daily at the same time. The drug dosage was adjusted based on body weight. Mice in the control group received an equal volume of normal saline orally. All mice had free access to food and water during the experiment.

[0045] Collection of mouse feces for 16S rRNA sequencing:

[0046] Fresh fecal samples were collected from each mouse on the 10th and 20th day after PM gavage under sterile conditions, and quickly placed in a 1.5 mL sterile EP tube in an ice box and stored in a -40°C ultra-low temperature refrigerator.

[0047] Collection and analysis of intestinal flora 16S rRNA information:

[0048] The 16S rRNA of fecal flora microorganisms of mice in the blank control group, low-dose PM group, and high-dose PM group on days 10 and 20 were obtained, and high-throughput 16S rRNA sequencing of their V3-V4 regions was performed.

[0049] After gene extraction from each sample, 30 ng of RNA samples of acceptable quality were collected and used with the appropriate primers to prepare a PCR reaction system. PCR amplification was then performed according to the pre-set parameters. The PCR amplification product was purified using Agencourt AMPure XP magnetic beads, and the purified sample was dissolved in elution buffer to complete library construction. The library fragment range and concentration were tested using the Agilent 2100 Bioanalyzer Biochip Analysis System. Qualified libraries were sequenced on the HiSeq platform based on their insert size. Finally, high-throughput sequencing of the V3-V4 region was performed using the extracted 16S rRNA.

[0050] After sequencing is completed, the raw sequencing data is processed as follows to obtain Clean Data:

[0051] (1) Set a 25bp window. If the average quality value of the window is lower than 20, the back-end bases are truncated from the window. If the read length after truncation is less than 75% of the original read length, the entire sequence is removed.

[0052] (2) Remove adapter-contaminated reads (the default overlap between the adapter sequence and the read sequence is 15 bp, which is set to 15 bp and the allowed mismatch number is 3);

[0053] (3) remove reads containing N;

[0054] (4) Remove low-complexity reads (by default, reads with a continuous base length of ≥10 are considered low-complexity reads). Samples are distinguished based on barcodes and primers, and the number of mismatches allowed in the alignment of barcode sequences and sequencing reads is 0 bp.

[0055] Gene sequence splicing was performed using FLASH (Fast Length Adjustment of Shortreads, v1.2.11) software. Paired reads obtained from double-end sequencing were assembled into a single sequence using overlapping relationships to obtain tags for the hypervariable regions. The splicing conditions were as follows:

[0056] (1) Minimum matching length 15 bp;

[0057] (2) The allowed mismatch rate in the overlapping area is 0.1.

[0058] Clustering result statistics: OTUs were generated based on 97% sequence similarity. The spliced ​​tags were clustered into OTUs using USEARCH (v7.0.1090) software. The method is as follows:

[0059] (1) UPARSE was used to cluster the OTUs at 97% similarity to obtain representative sequences;

[0060] (2) Chimeras generated by PCR amplification were removed from OTU representative sequences using UCHIME (v4.2.40) software;

[0061] (3) Use the usearch_global method to align all tags back to the OTU representative sequence to obtain the abundance statistics of the OTU for each sample.

[0062] Results and Analysis:

[0063] Preparation and purification of PM:

[0064] 100g of sodium alginate was initially purified by acid degradation and three pH fractions to obtain 27.5g of PM crude product with a yield of 27.5%. The PM crude product was then passed through a Q-Sepharose column, and the elution curve and phenol-sulfuric acid method were used to monitor the elution components. Figure 1 As shown, the 210 nm UV elution curve for PM separation reveals a major elution peak after anion exchange column separation, with other components present in minimal amounts. The phenol-sulfuric acid method analysis curve generally agrees with the 210 nm elution curve. The symmetry of this peak indicates relatively uniform charge. The major fractions were collected, desalted, lyophilized, and then tested for purity by high-performance liquid chromatography.

[0065] Purity analysis and molecular weight determination of PM:

[0066] The retention times of PM and dextran were measured on a high-performance liquid chromatograph (Agilent, Agilent 1100, USA) using a TSK-gel G4000 PWxl column. Based on the HPLC chromatogram results, the retention time (t) was regressed against the logarithm of the molecular weight of the dextran standard (log Mw) to obtain the linear regression equation:

[0067] log Mw=-0.4482t+9.2931,R 2 =0.9992.

[0068] The results of HPLC analysis of the purified PM were as follows: Figure 2 As shown, the purified PM is essentially a symmetrical single peak, indicating high purity. The retention time is 11.305 min, and substituting this into the equation yields Mw(PM) = 16.83 kDa. Calculated from the molecular weight of mannuronic acid, the average degree of polymerization of PM is 95.

[0069] Effects of PM on Alpha Diversity of Fecal Microbiota in Mice:

[0070] Alpha diversity refers to the biodiversity within a specific region or ecosystem and is a comprehensive indicator of richness and evenness. When studying changes in microbial community structure, alpha diversity is often used to reflect the abundance and diversity of microbial communities. Commonly used indices include the Chao, ACE, Shannon, and Simpson indices. Community richness indices primarily include the Chao and Ace indices. Community diversity indices include the Shannon and Simpson indices.

[0071] The Chao index estimates the number of OTUs in a sample using the Chao1 algorithm. Chao1 is commonly used in ecology to estimate the total number of species. The Ace index estimates the number of OTUs in a sample. Larger Chao and Ace values ​​indicate a greater number of OTUs in the sample and a greater sample richness.

[0072] The Shannon index is one of the indices used to estimate the diversity of microorganisms in a sample. A larger Shannon value indicates a higher sample diversity. The Simpson index reflects the position and role of dominant species in a sample. If a sample has a higher proportion of dominant species and a lower proportion of other non-dominant species, a higher Simpson index indicates a lower sample diversity. This index is negatively correlated with other diversity indices.

[0073] Coverage refers to the coverage of each sample library. A higher value indicates a lower probability that a sequence in the sample was not called. This index reflects whether the sequencing results are representative of the sample. A coverage closer to 1 indicates that the experimental results more accurately reflect the sample.

[0074] In conjunction with the examples, various diversity index analyses were performed on the effects of PM on the Alpha diversity of the mouse intestinal flora based on the rRNA data obtained for each sample.

[0075] Table 1 Alpha diversity analysis of fecal flora in each group of mice on day 10

[0076]

[0077] Table 2 Alpha diversity analysis of fecal flora in each group of mice on day 20

[0078]

[0079] Compared with the control group: *p<0.05.

[0080] As shown in Tables 1 and 2, the coverage values ​​for all groups were close to 1, indicating that the results accurately reflect the sample conditions. On the 10th day of PM gavage, the Chao index, Ace index, Shannon index, and Simpson index of the high- and low-dose groups were not significantly different from those of the control group (Table 1). After 20 days of PM gavage, the Chao and Ace indices of both the low- and high-dose groups were higher than those of the control group. However, there was no significant difference between the low-dose group and the control group, while the Ace index of the high-dose group was significantly different from that of the control group, indicating that the high-dose group increased the abundance of the fecal microbiota in mice. The Shannon index of both the low- and high-dose groups was not significantly different from that of the control group, while the Simpson index was significantly lower than that of the control group (Table 2). Therefore, the effect of PM on the intestinal microbiota is time-dependent. There was no significant effect on the abundance and diversity of the fecal microbiota at 10 days of PM gavage. As the duration of PM gavage increases, the main effect is on the diversity of the fecal microbiota.

[0081] Effects of PM gavage for 10 days on the species composition of fecal flora in mice:

[0082] from Figure 3 It can be seen that after 10 days of PM gavage, the feces of mice in each group mainly contained bacteria from four phyla: Firmicutes, Bacteroidetes, Proteobacteria, and Deferribacteres. The dominant bacterial groups in the feces after 10 days of PM gavage mainly included two:

[0083] (1) Bacteroidetes → Bacteroidia → Bacteroidales → Prevotellaceae → Prevotella;

[0084] (2) Bacteroidetes → Bacteroidia → Bacteroidales → Bacteroidaceae → Bacteroides.

[0085] The B / F ratios for the control group were 1.9343, the low-dose group was 2.0403, and the high-dose group was 1.6333. This suggests that after 10 days of oral administration of low-dose PM, no microbial imbalance occurred in the low-dose group, while some imbalance did occur in the high-dose group.

[0086] At the phylum level, there were no significant changes in mouse feces. Figure 4 .

[0087] At the family level, the dominant bacterial groups in the feces of the control group, the low-dose group, and the high-dose group after PM gavage for 10 days were Prevotellaceae, Lachnospiraceae, and Bacteroidaceae (see Figure 5 A). A differential analysis of the bacterial communities with higher abundance showed that compared with the control group, both the high-dose and low-dose groups could significantly reduce the abundance of the Acidaminococcaceae family and increase the abundance of the Lactobacillus family. The low-dose group also significantly reduced the abundance of the Prevotellaceae and Helicobacteraceae families and increased the abundance of the Porphyromonadaceae family. The high-dose group had the effect of reducing the abundance of the Erysipelotrichaceae family and increasing the abundance of the Enterobacteriaceae family (see Figure 5 B).

[0088] At the genus level, the dominant bacterial groups in the feces of the control group, low-dose group, and high-dose group were Prevotella, Bacteroides, and Alloprevotella 10 days after PM gavage (see Figure 6 A). A differential analysis of the bacterial communities with higher abundance showed that compared with the control group, both the high-dose and low-dose groups could significantly reduce the abundance of Prevotella and Phascolarctobacterium, and increase the abundance of Lactobacillus. The low-dose group also significantly reduced the abundance of Clostridium_XIVa and Helicobacter, and increased the abundance of Bacteroides, Barnesiella, and Blautia (see Figure 6 B).

[0089] Ten days after oral administration of high- and low-dose PM, both high- and low-dose groups showed some changes in the composition of the colonic microbiota. At the phylum level, the low-dose group showed an increasing trend in the B / F ratio, while the high-dose group showed a decreasing trend, indicating that low-dose PM was more conducive to the balance of the mouse fecal microbiota. However, high and low doses had no significant effect on microbial abundance. At the genus level, differential analysis of highly abundant microbial groups revealed that, compared with the control group, at 10 days, both high- and low-dose groups had less overall impact on the mouse fecal microbiota. Both groups significantly reduced the abundance of Prevotella and Phascolarctobacterium and increased the abundance of Lactobacillus. Reducing the abundance of Prevotella is generally beneficial to the intestinal flora, and increasing the abundance of Lactobacillus is also beneficial to the balance of fecal intestinal flora. However, Phascolarctobacterium, a beneficial intestinal flora that produces short-chain fatty acids, has a reduced abundance, which is not conducive to the balance of intestinal flora. The low-dose group also significantly reduced the abundance of Clostridium_XIVa and Helicobacter, and increased the abundance of Bacteroides, Barnesiella, and Blautia. A decrease in the abundance of Clostridium_XIVa and Helicobacter bacteria is beneficial to the intestinal microbiota. Barnesiella and Blautia are both potentially beneficial intestinal bacteria, so the abundance of Bacteroides, Barnesiella, and Blautia is beneficial. Therefore, after 10 days of PM gavage, the high and low dose groups had little effect on the fecal microbiota of mice, but the low dose group had a more pronounced regulatory effect on the intestinal microbiota composition, surpassing the high dose group.

[0090] R (v3.4.1) and Cytoscape software were used to construct a correlation network diagram of fecal microbiota in mice after PM gavage for 10 days. Figure 7The interactions among fecal microbiota are complex, with a rich and diverse bacterial population, and minimal differences in abundance between species. Five species, Mucispirillum schaedleri, Anaerotruncus colihominis, Clostridium lactatifermentans, Clostridium hylemonae, and Clostridium leptum, showed strong positive correlations with each other. Seven species, Bacteroides vulgatus, Barnesiella intestinihominis, Paraprevotella clara, Blautia schinkii, Allobaculum stercoricanis, Phascolarctobacterium succinatutens, and Bacteroides sartorii, all showed positive correlations. Most bacteria exhibited negative correlations with Prevotella dentalis and Alloprevotella rava. No significant inhibitory effects were observed against the harmful bacteria Escherichia and Helicobacter rodentium, and the differences between positively and negatively correlated bacteria were similar. In general, PM did not show significant effects on fecal flora after 10 days of oral administration.

[0091] Effects of PM gavage for 20 days on the species composition of fecal flora in mice:

[0092] from Figure 8 It can be seen that after 20 days of PM gavage, the feces of mice in each group mainly contained bacteria from five phyla, namely Firmicutes, Bacteroidetes, Proteobacteria, Tenericutes, and Deferribacteres. The dominant bacterial groups in the feces mainly include three:

[0093] (1) Bacteroidetes → Bacteroidia → Bacteroidales → Prevotellaceae → Prevotella;

[0094] (2) Firmicutes → Clostridia → Clostridiales → Lachnospiraceae → Clostridium_XIVa;

[0095] (3) Proteobacteria → Epdilonproteobacteria → Campylobacterales → Helicobacteraceae → Helicobacter.

[0096] The B / F ratio was 0.8234 in the control group, 0.2267 in the low-dose group, and 0.5162 in the high-dose group. This suggests that both high- and low-dose PM gavages can lead to an imbalance in fecal flora after 20 days.

[0097] At the phylum level, compared with the control group, PM gavage for 20 days reduced the abundance of Bacteroidetes and Candidatus_Saccharibacteria in both the high-dose and low-dose groups. The low-dose group also increased the abundance of Firmicutes, while the high-dose group increased the abundance of Tenericutes. There were significant differences between the high- and low-dose groups for Proteobacteria. The low-dose group reduced the abundance of Proteobacteria, while the high-dose group increased the abundance of Proteobacteria (see Figure 9 ).

[0098] At the family level, 20 days after PM gavage, the dominant bacterial communities in the feces of mice were Lachnospiraceae and Prevotellaceae in the control group, Lachnospiraceae, Prevotellaceae, Ruminococcaceae, and Lactobacillaceae in the low-dose group, and Lachnospiraceae, Prevotellaceae, Ruminococcaceae, and Helicobacteraceae in the high-dose group. Compared with the control group, both the high- and low-dose groups significantly reduced the abundance of Prevotellaceae and Porphyromonadaceae and increased the abundance of Ruminococcaceae. The low-dose group also increased the abundance of Lachnospiraceae and decreased the abundance of Bacteroidaceae. The high-dose group also decreased the abundance of Lactobacillaceae and increased the abundance of Desulfovibrionaceae. The abundance of Helicobacteraceae was different between the high and low doses. The low dose decreased the abundance of Helicobacteraceae, while the high dose increased it (see Figure 10 ).

[0099] At the genus level, 20 days after PM gavage, the dominant bacterial species in the feces of mice was Prevotella in the control group, Clostridium_XIVa and Lactobacillus in the low-dose group, and Prevotella, Helicobacter, and Clostridium_IV in the high-dose group. Compared with the control group, both the high- and low-dose groups significantly reduced the abundance of Prevotella and Blautia, while increasing the abundance of Clostridium_IV and Anaerotruncus. The low-dose group also significantly increased the abundance of Clostridium_XIVa and decreased the abundance of Bacterioides. The high-dose group increased the abundance of Desulfovibrio bacteria and decreased the abundance of Lactobacillus bacteria. For Helicobacter, the high-dose and low-dose groups showed significant differences. The low-dose group significantly decreased the abundance of Helicobacter bacteria, while the high-dose group significantly increased the abundance of Helicobacter bacteria (see Figure 11 ).

[0100] After 20 days of oral administration of high- and low-dose PM, both high- and low-dose groups showed some changes in the composition of the fecal microbiota. At the phylum level, both high- and low-dose groups showed a decreasing trend in B / F ratios, with the decrease being greater in the low-dose group. This suggests that both high- and low-dose PM may induce a transient imbalance in the fecal microbiota of mice. Of note, compared with the B / F ratios after 10 days of PM oral administration, the B / F ratios in all groups were significantly lower, indicating that the transient imbalance in the fecal microbiota of mice was not solely caused by PM oral administration. Furthermore, a systematic review has shown that this relationship between the B / F ratio and intestinal microbiota imbalance is not always consistent. Compared with the control group, both high- and low-dose groups significantly reduced the abundance of Prevotella and Blautia and increased the abundance of Clostridium IV and Anaerotruncus. A proper reduction in the abundance of Prevotella is beneficial to the balance of intestinal flora, but a decrease in the abundance of Blautia is not conducive to the balance of intestinal flora. Anaerobic Anaerobic Bacteria are related to metabolism. Abnormal lipid metabolism will cause their content to increase, and their increased abundance is not favorable. The high-dose group has the effect of increasing the abundance of Desulfovibrio flora. Desulfovibrio is toxic to the intestinal epithelium, causing gastrointestinal diseases, and is a harmful intestinal bacterium. For Helicobacter, the high and low dose groups showed obvious differences. The low dose group had the effect of significantly reducing its flora abundance, and the high dose group had the effect of significantly increasing its flora abundance. This shows that the low dose group has a better effect than the high dose group. Therefore, 20 days after PM gavage, although the high- and low-dose groups may cause a temporary imbalance in the fecal flora, according to the analysis results, the low-dose group can still regulate the downregulation of related pathogens and the upregulation of probiotics, and the effect is better than that of the high-dose group.

[0101] R (v3.4.1) and Cytoscape software were used to construct a correlation network diagram of fecal microbiota in mice after PM gavage for 20 days. Figure 12It can be seen that the interactions between fecal microbiota are more complex than those in fecal microbiota after 10 days of gavage, with a richer and more diverse bacterial species, and smaller differences in abundance between species: Blautia schinkii, Phascolarctobacterium succinatutens, Clostridium hylemonae, and Bacteroides sartorii all have positive correlations; Mycoplasma microti, Helicobacter rodentium, Clostridium methylpentosum, Clostridium lactatifermentans, Clostridium aldenense, Mucispirillum schaedleri, Pseudoflavonifractor capillosus, and Anaerotruncus colihominis all have positive correlations. Most bacteria have negative correlations with Escherichia, Clostridium bolteae, and Clostridium glycyrrhizinilyticum, and the beneficial bacterium Lactobacillus apodemi also has a strong negative correlation with most bacteria. The number and negative correlation of negatively correlated bacteria with the harmful bacteria Escherichia increased compared with the 10th day after oral administration. In general, the correlation between fecal microbiota became more complex after 20 days of PM oral administration, and the effect on the harmful bacteria Escherichia gradually increased.

[0102] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. Use of polymannuronic acid or a composition containing polymannuronic acid as an active ingredient in the preparation of a gastric and / or intestinal flora regulator, characterized in that: The average degree of polymerization of the polymannuronic acid is 90-100, and the gastric and / or intestinal flora regulator can reduce the abundance of Helicobacter bacteria in feces after being delivered to the stomach and / or intestine for regulation; The gastric and / or intestinal flora regulator is used for preparing medicine.

2. Use of the polymannuronic acid or a composition containing polymannuronic acid as an active ingredient as claimed in claim 1 in the preparation of a gastric and / or intestinal flora regulator, characterized in that: The polymannuronic acid is obtained by hydrolyzing sodium alginate.

3. Use of the polymannuronic acid or a composition containing polymannuronic acid as an active ingredient as claimed in claim 2 in the preparation of a gastric and / or intestinal flora regulator, characterized in that: The preparation process of the polymannuronic acid is as follows: the polymannuronic acid is swelled, hydrochloric acid is added, and then hydrolyzed at 95-105° C. for 10-15 hours.

4. Use of the polymannuronic acid or a composition containing polymannuronic acid as an active ingredient as claimed in claim 2 in the preparation of a gastric and / or intestinal flora regulator, characterized in that: The purification process after hydrolysis is as follows: the hydrolyzed precipitate is dissolved in a NaHCO3 solution, the pH is adjusted to acidic to produce a white flocculent precipitate, and the supernatant is collected after standing and stratification. The obtained supernatant is subjected to the following process and the following process is repeated at least once: The pH of the supernatant was adjusted to 7-8, and a 90-98% ethanol solution was added. After standing for stratification, a precipitate was obtained. The precipitate was dissolved with a NaHCO3 solution, and the pH was adjusted to acidic to produce a white flocculent precipitate. The precipitate was removed to obtain a supernatant; Anhydrous ethanol was added to the supernatant obtained finally, and the mixture was stirred and mixed for dehydration, and the mixture was filtered and dried to obtain a crude polymannuronic acid product.

5. Use of the polymannuronic acid or a composition containing polymannuronic acid as an active ingredient as claimed in claim 4 in the preparation of a gastric and / or intestinal flora regulator, characterized in that: The purification process of crude polymannuronic acid is as follows: the crude polymannuronic acid is dissolved in water, filtered using a 0.45 μm filter membrane, further purified using an ion exchange chromatography column, and then further purified using an AKTA protein purification system to obtain pure polymannuronic acid.

6. Use of the polymannuronic acid or a composition containing polymannuronic acid as an active ingredient as claimed in claim 5 in the preparation of a gastric and / or intestinal flora regulator, characterized in that: The ion exchange chromatography column is Cl - Format Q-Sepharose column.

7. Use of the polymannuronic acid or a composition containing polymannuronic acid as an active ingredient as claimed in claim 5 in the preparation of a gastric and / or intestinal flora regulator, characterized in that: In further purification using the AKTA protein purification system, mobile phase A was ultrapure water, mobile phase B was NaCl solution, the upper pressure limit was 0.9–1.1 MPa, and the UV detection wavelength was 205–215 nm; Alternatively, for further purification using the AKTA protein purification system, after column exchange, perform linear gradient elution with a 0.5-2.0 M NaCl solution.

8. Use of the polymannuronic acid or a composition containing polymannuronic acid as an active ingredient as claimed in claim 5 in the preparation of a gastric and / or intestinal flora regulator, characterized in that: The collected main peak eluate was freeze-dried and concentrated, and then desalted using a Sephadex G-25 column to obtain pure polymannuronic acid.

9. Use of the polymannuronic acid or a composition containing polymannuronic acid as an active ingredient as claimed in claim 1 in the preparation of a gastric and / or intestinal flora regulator, characterized in that: The composition consists of active ingredients and pharmaceutical excipients.

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