A capsular polysaccharide derived from Bacteroides saurischia CSP6, its preparation method and application

Abstract

This invention belongs to the field of intestinal microbiome technology, specifically relating to a capsular polysaccharide derived from Bacteroides salorlis CSP6, its preparation method, and its applications. The monosaccharide composition of the capsular polysaccharide is mainly composed of fucose, glucose, and glucosamine, with a molecular weight of 7.139 × 10⁻⁶. 5 The strain described is deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M20221993. In vitro studies have found that the capsular polysaccharide derived from Bacteroides Salieris CSP6 can inhibit α-glucosidase; in vivo animal experiments have demonstrated that this capsular polysaccharide can inhibit the rise in postprandial blood glucose and has a hypoglycemic effect.

Description

Technical Field

[0001] This invention belongs to the field of intestinal microbial technology, specifically relating to a capsular polysaccharide derived from Bacteroides salinalis CSP6, its preparation method, and its application. Background Technology

[0002] Bacteroides spp. are important commensal bacteria in the human gut (PMID: 33535896). Due to their diverse biological functions, Bacteroides are widely considered a "next-generation probiotic," thus possessing significant research and development value (PMID: 33535896). Bacteroides salyersiae is a rod-shaped obligate anaerobic bacterium, first isolated in 2004 by YL Song et al. from clinical specimens derived from the human gut (PMID: 15583282). In 2023, our research team isolated a strain of Bacteroides salyersiae CSP6 (PMID: 38419055) from healthy human fecal samples using systematic culture omics technology. This strain is currently deposited at the China Center for Type Culture Collection (CCTCC), with accession number CCTCC NO: M 20221993 (Chinese Invention Patent ZL 2023 10238846.8). Previous studies have found that Bacteroides salils CSP6 can degrade and prepare chondroitin sulfate oligosaccharides and hyaluronic acid oligosaccharides (Chinese Invention Patent ZL202310238846.8; PMID: 38419055). Furthermore, this strain can alleviate ulcerative colitis induced by sodium dextran sulfate by improving intestinal flora imbalance (PMID: 39275234).

[0003] Bacteroides Salirius CSP6 holds significant development potential as a next-generation probiotic; however, aside from its anti-enteritis activity, no other pharmacological functions have been identified. Furthermore, no research reports have been published regarding the biological activity of its capsular polysaccharide. To address these issues, this study, based on previous systematic in vitro and in vivo activity screening, is the first to discover that capsular polysaccharides derived from Bacteroides Salirius CSP6 can alleviate postprandial hyperglycemia by inhibiting α-glucosidase activity, showing broad application prospects in the preparation of drugs that lower postprandial blood glucose. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a capsular polysaccharide derived from Bacteroides saurischia CSP6, its preparation method, and its applications.

[0005] The technical solution of this invention is as follows:

[0006] This invention discloses a capsular polysaccharide derived from Bacteroides salyersiae CSP6, wherein the monosaccharide composition of the capsular polysaccharide is mainly composed of fucose, glucose and glucosamine, with the molar proportions of the three being 51.19%, 11.87% and 11.48%, respectively.

[0007] The capsular polysaccharide has a molecular weight of 7.139 × 10⁻⁶. 5 Da;

[0008] The strain is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M20221993, and its classification is: *Bacteroides*, *Bacteroides salils*; Latin scientific name: Bacteroides salyersiae CSP6; deposited on December 26, 2022, at Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0009] Furthermore, this invention discloses the application of the above-mentioned capsular polysaccharide in the preparation of a drug for lowering postprandial blood glucose.

[0010] The present invention also discloses a drug for lowering postprandial blood glucose, the drug comprising the above-mentioned capsular polysaccharide and a pharmaceutically acceptable carrier, wherein the dosage form of the drug is a liquid preparation or a solid preparation.

[0011] Furthermore, the dosage of the drug is 80 mg / kg; the solvent of the liquid formulation is phosphate-buffered saline (PBS), pH 7.0-7.4; the solid formulation is selected from tablets, capsules or granules.

[0012] The present invention also discloses a method for producing the above-mentioned capsular polysaccharide, which includes the following steps:

[0013] a. Inoculate Bacteroides Salieri CSP6 into an anaerobic environment containing 1.5 L of BHI liquid medium and incubate at 37°C for 48 hours to achieve a viable bacterial concentration of 9 × 10⁶. 8 CFU / mL;

[0014] b. Centrifuge: run the centrifuge at 8000 xg for 20 minutes, collect the bacterial cells, mix with 500 mL of phenol and 500 mL of distilled water, stir at 60°C for 30 minutes, and let stand overnight;

[0015] c. Take the supernatant and centrifuge it. The centrifuge runs continuously at a centrifugal force of 5000 xg for 20 minutes. Then, use a 3500 Da dialysis bag to dialyze for 24 hours to remove phenol.

[0016] d. After rotary evaporation and concentration, freeze-drying is performed to obtain the capsular polysaccharide.

[0017] Furthermore, the BHI culture medium formula described above is as follows: 10 g / L peptone, 12.5 g / L dehydrated calf brain extract, 5 g / L dehydrated calf heart extract, 5 g / L sodium chloride, 2 g / L glucose, 2.5 g / L disodium hydrogen phosphate, pH 7.4.

[0018] This invention also discloses the application of capsular polysaccharides in the preparation of α-glucosidase inhibitors.

[0019] Advantages and beneficial effects of the present invention:

[0020] The Bacteroides salilus provided by this invention ( Bacteroides salyersiae CSP6-derived capsular polysaccharides can significantly inhibit α-glucosidase activity and have a good effect on reducing postprandial blood glucose. Attached Figure Description

[0021] Figure 1 Growth curve of Bacteroides saurischia CSP6;

[0022] Figure 2 A diagram showing the monosaccharide composition analysis of capsular polysaccharides derived from Bacteroides saurischia CSP6;

[0023] Figure 3 Molecular weight analysis of capsular polysaccharides derived from Bacteroides saurischia CSP6;

[0024] Figure 4 A comparison chart of α-glycosidase inhibitory activities;

[0025] Figure 5 A comparison of postprandial blood glucose changes in mice;

[0026] Figure 6 This is a graph showing the area under the curve (AUC) of postprandial blood glucose levels in mice.

[0027] In this study, * indicates a significant difference compared to the control group (P<0.05); ** indicates an extremely significant difference compared to the control group (P<0.01). The Student t-test was used for statistical analysis. Detailed Implementation

[0028] In a specific embodiment, a capsular polysaccharide derived from Bacteroides salyersiae CSP6 is provided, wherein the monosaccharide composition of the capsular polysaccharide is mainly composed of fucose, glucose, and glucosamine, with the molar proportions of the three being 51.19%, 11.87%, and 11.48%, respectively.

[0029] The capsular polysaccharide has a molecular weight of 7.139 × 10⁻⁶. 5 Da;

[0030] The strain is deposited at the China Center for Type Culture Collection (CCTCC), with accession number CCTCC NO:M20221993, deposit date December 19, 2022, and address China Center for Type Culture Collection, Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0031] The above-mentioned capsular polysaccharides can be used in the preparation of drugs that lower postprandial blood glucose, and can also be used in the preparation of α-glucosidase inhibitors.

[0032] A drug for lowering postprandial blood glucose, the drug comprising the above-mentioned capsular polysaccharide and a pharmaceutically acceptable carrier, the drug being in the form of a liquid or solid dosage form.

[0033] Furthermore, the dosage of the drug is 80 mg / kg; the solvent of the liquid formulation is phosphate-buffered saline (PBS), pH 7.0; and the solid formulation is selected from tablets, capsules, or granules.

[0034] The above-mentioned method for using capsular polysaccharides includes the following steps:

[0035] a. Inoculate Bacteroides Salieri CSP6 into an anaerobic environment containing 1.5 L of BHI liquid medium and incubate at 37°C for 48 hours to achieve a viable bacterial concentration of 9 × 10⁶. 8 CFU / mL;

[0036] b. Centrifuge (8000xg, 20min) to collect the bacterial cells, mix with 500mL phenol and 500mL distilled water, stir at 60℃ for 30 minutes, and let stand overnight;

[0037] c. Take the supernatant liquid and centrifuge (5000 rpm). g Phenol was removed by dialysis using a 3500Da dialysis bag for 24 hours (20 min).

[0038] d. After rotary evaporation and concentration, freeze-drying is performed to obtain the capsular polysaccharide.

[0039] Furthermore, the BHI culture medium formula described above is as follows: 10 g / L peptone, 12.5 g / L dehydrated calf brain extract, 5 g / L dehydrated calf heart extract, 5 g / L sodium chloride, 2 g / L glucose, 2.5 g / L disodium hydrogen phosphate, pH 7.4.

[0040] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0041] Example 1

[0042] This embodiment describes a method for preparing capsular polysaccharides derived from Bacteroides sarrillais CSP6. The main experimental equipment includes:

[0043] DF-101S Thermal Collector Type Constant Temperature Heating Magnetic Stirrer (Qingdao Lanten Educational Instrument Equipment Co., Ltd.);

[0044] 7116TR Benchtop High-Speed ​​Refrigerated Centrifuge (Hunan Hexi Instrument Equipment Co., Ltd.);

[0045] PD-2 freeze dryer (Shanghai Bilang Instrument Manufacturing Co., Ltd.);

[0046] RE100-Pro rotary evaporator (Dalongxingchuang Experimental Instruments (Beijing) Co., Ltd.).

[0047] Its preparation and determination methods include the following steps:

[0048] 1. First, culture Bacteroides Salieri CSP6:

[0049] (1) Preparation of culture medium

[0050] Prepare BHI liquid culture medium with the following components: 10 g / L peptone, 12.5 g / L dehydrated calf brain extract, 5 g / L dehydrated calf heart extract, 5 g / L sodium chloride, 2 g / L glucose, and 2.5 g / L disodium hydrogen phosphate. The solvent is distilled water, and the pH value is 7.4. Pour the culture medium into anaerobic vials, purge with nitrogen, seal, and autoclave at 121°C for 15 min.

[0051] (2) Source of strain

[0052] Bacteroides Salieri CSP6 was previously isolated in the laboratory (Chinese Invention Patent: CN 116676207B) and preserved in a -80°C freezer using a 30% glycerol preservation solution.

[0053] (3) Activation and inoculation of strains

[0054] Bacteroides Salieri CSP6, stored at -80℃, was taken out and inoculated into a 10mL anaerobic vial after high-temperature sterilization for activation (inoculation amount was 1% (v / v), and the anaerobic gas composition in the vial was 80% (v / v) N2, 10% (v / v) H2 and 10% (v / v) CO2). After incubation at 37℃ for 48 hours, it was transferred to a 50mL anaerobic vial after high-temperature sterilization for further culture.

[0055] 2. Determination of CSP6 growth curve of Bacteroides salisii.

[0056] (1) Place the activated Bacteroides Salieri CSP6 in a 37℃ incubator for static culture. After 12h, 24h, 48h, 72h, and 96h of culture, take 200μL of bacterial culture and measure the OD using a 96-well plate in an ELISA reader. 600nm Value. Take 200 μL of fresh liquid culture medium and measure its absorbance (OD). 600nm () as the original control.

[0057] (2) Experimental results and analysis

[0058] The results are as follows Figure 1 As shown, Bacteroides salinalis CSP6 reached its maximum concentration after 24 hours of growth at 37°C and tended to stabilize after 48 hours.

[0059] 3. Extraction of source capsular polysaccharides:

[0060] (1) Activation and culture of strains

[0061] The activated Bacteroides Salieri CSP6 was inoculated into 1.5 L of BHI liquid medium and anaerobicly grown for 48 h to achieve a viable cell concentration of 9 × 10⁶. 8 CFU / mL.

[0062] (2) Extraction of capsular polysaccharides

[0063] The cultured bacterial solution was centrifuged (8000x) g Collect bacterial cells (20 min), then mix them with 500 mL phenol and 500 mL distilled water, stir at 60 °C for 30 min and let stand overnight to extract capsular polysaccharides; take the supernatant and centrifuge (5000 x 10⁻⁶). g Phenol was removed by dialysis with a 3500 Da dialysis bag for 24 h; finally, capsular polysaccharide derived from Bacteroides salinalis CSP6 was obtained by rotary evaporation concentration and freeze drying.

[0064] 4. The monosaccharide composition of the capsular polysaccharide derived from Bacteroides Salieris CSP6 was determined:

[0065] (1) Method for detecting monosaccharide composition

[0066] Monosaccharide composition was determined by pre-column derivatization high-performance liquid chromatography (HPLC) using PMP. The chromatographic column was an Agilent Eclipse XDB-C18; the mobile phase was 0.1 mol / L phosphate buffer (pH 6.7)-acetonitrile (83:17 v / v); the column temperature was 30°C; the UV detector was used at a detection wavelength of 245 nm; the flow rate was 1 mL / min; and the injection volume was 10 μL.

[0067] (2) Experimental results and analysis

[0068] The results are shown in Table 1 and Figure 2 As shown, the capsular polysaccharide derived from Bacteroides Salieris CSP6 is mainly composed of Fuc, Glc, and GlcN, and contains Man, GlcN, GlcA, GalA, GalN, Gal, and Xyl.

[0069] Table 1. Monosaccharide composition of capsular polysaccharides from Bacteroides sauris CSP6

[0070] Monosaccharides Man GlcN Rha GlcA GalA GalN Glc Gal Xyl Fuc percentage 5.82 % 11.48 % 3.24 % 3.98 % 7.31 % 2.29 % 11.87 % 1.84 % 0.98 % 51.19 %

[0071] 5. The molecular weight of the capsular polysaccharide derived from Bacteroides Salieris CSP6 was determined:

[0072] (1) Molecular weight detection method

[0073] Molecular weight was determined using high-performance gel permeation chromatography coupled with multi-angle laser scattering (MALLS). Analytical columns: SBHQ 804 column and SBHQ 802.5 column in series; mobile phase: 0.1 mol / L Na2SO4 aqueous solution; flow rate: 0.6 mL / min; column temperature: 35°C; injection volume: 100 µL; detectors: differential detector (RI) and multi-angle laser scattering detector (MALLS).

[0074] (2) Experimental results and analysis

[0075] The results are as follows Figure 3 As shown, the molecular weight of the capsular polysaccharide derived from Bacteroides Salieris CSP6 is 7.139 × 10⁻⁶. 5 Da.

[0076] Example 2

[0077] This example demonstrates the role of the capsular polysaccharide derived from Bacteroides Salieris CSP6 prepared in Example 1 in inhibiting α-glucosidase, proving that it can be applied to the preparation of α-glucosidase inhibitors.

[0078] Experimental materials:

[0079] Phosphate-buffered saline (PBS, pH=7.0);

[0080] p-Nitrophenyl-α-glucopyranoside (PNPG) was purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0081] α-Glucosidase was purchased from Sigma-Aldrich, USA.

[0082] Main experimental equipment: ReadMax 1200 full-wavelength microplate reader (Shanghai Flash Spectrum Biotechnology Co., Ltd.).

[0083] Experimental methods:

[0084] Using 2.5 mmol / L PNPG as the substrate, 20 μL of PBS (0.1 mol / L, pH = 6.7), 20 μL of PNPG, 20 μL of α-glucosidase (0.2 U / mL), and 20 μL of the sample were successively added to a 96-well plate. After reacting in a shaker at 37 °C for 15 minutes, 80 μL of 0.2 mol / L Na2CO3 was added to terminate the reaction, and the OD 405nm value was measured.

[0085] A blank control (without adding the sample, supplemented with PBS) and a background absorption control (without adding α-glucosidase, supplemented with PBS) were set up.

[0086] Inhibition rate (%) = 100% X [A 空白 - (A 样品 A 背录 )] / A 空自 , and the sample was set at three concentrations: 10 mg / mL, 50 mg / mL, and 100 mg / mL.

[0087] Experimental results and analysis:

[0088] The results were as Figure 4 shown that the capsular polysaccharide derived from Bacteroides sartorii CSP6 (10 mg / mL, 50 mg / mL, and 100 mg / mL) all had α-glucosidase inhibitory activity, and there were significant differences compared with the control group (p < 0.01). In addition, the higher the sample concentration, the higher the inhibition rate. The above results indicate that the capsular polysaccharide derived from Bacteroides sartorii CSP6 has α-glucosidase inhibitory activity.

[0089] Example 3

[0090] This example demonstrates the application of the capsular polysaccharide derived from Bacteroides sartorii CSP6 prepared in Example 1 in reducing postprandial blood glucose.

[0091] Experimental materials:

[0092] 10 C57BL / 6J mice (8 weeks old, male), purchased from Vital River Laboratory Animal Technology Co., Ltd. (Beijing) (certificate number SCXK (Beijing) 2016 - 0011);

[0093] Phosphate buffer solution (PBS, pH = 7.0).

[0094] Main experimental equipment: GA-3 type blood glucose meter (San诺 Biochemical Sensing Co., Ltd.).

[0095] Experimental method:

[0096] Ten mice were randomly divided into two groups: a control group (Control, n=5) and a treatment group (BS, n=5). All mice were housed in an environment with a temperature of approximately 23°C, a 12-hour light cycle, and free access to standard feed and water. After a one-week acclimatization period, the mice were given the drug.

[0097] After fasting for 10 hours at night, mice in the Control group were administered 150 μL of PBS by gavage, while mice in the BS group were administered 150 μL of capsular polysaccharide derived from Bacteroides Salieris CSP6 (80 mg / kg, dissolved in 150 μL of PBS solution) by gavage. After fasting for another hour, initial blood glucose was measured. Subsequently, mice were fed, and postprandial blood glucose was measured at various time points within 120 minutes (15 min, 30 min, 45 min, 60 min, 90 min, 120 min).

[0098] Experimental Results and Analysis:

[0099] The results are as follows Figure 5 , Figure 6 As shown in Table 2, gavage administration of Bacteroides Salirius CSP6-derived capsular polysaccharide significantly slowed the rise in postprandial blood glucose in mice. At 30 and 45 minutes after feeding, the blood glucose levels in the BS group were significantly lower than those in the Control group (p<0.01); at 30 minutes after feeding, the blood glucose levels in the BS group were significantly lower than those in the Control group (p<0.05).

[0100] Table 2. Capsular polysaccharides derived from Bacteroides salils CSP6 reduced postprandial blood glucose in mice.

[0101]

[0102] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A capsular polysaccharide derived from Bacteroides saurischia CSP6, characterized in that, The monosaccharide composition of the capsular polysaccharide is mainly composed of fucose, glucose, and glucosamine, with molar proportions of 51.19%, 11.87%, and 11.48%, respectively. The capsular polysaccharide has a molecular weight of 7.139 × 10⁻⁶. 5 Da; The strain is deposited at the China Center for Type Culture Collection, with accession number CCTCC NO:M20221993; The method for preparing the capsular polysaccharide includes the following steps: a. Inoculate Bacteroides Salieri CSP6 into an anaerobic environment containing 1.5 L of BHI liquid medium and incubate at 37°C for 48 hours to achieve a viable bacterial concentration of 9 × 10⁶. 8 CFU / mL; b. After centrifugation, collect the bacterial cells, mix them with 500 mL of phenol and 500 mL of distilled water, stir at 60°C for 30 minutes, and let stand overnight; c. Centrifuge the supernatant and dialyze it for 24 hours using a 3500 Da dialysis bag to remove phenol; d. After rotary evaporation and concentration, freeze-drying is performed to obtain the capsular polysaccharide.

2. The use of the capsular polysaccharide according to claim 1 in the preparation of a drug for lowering postprandial blood glucose.

3. A drug for lowering postprandial blood glucose, characterized in that, The drug comprises the capsular polysaccharide of claim 1 and a pharmaceutically acceptable carrier, wherein the dosage form of the drug is a liquid or solid formulation.

4. The drug according to claim 3, characterized in that, The dosage of the drug is 80 mg / kg; The solvent for the liquid formulation is phosphate buffer, pH 7.0; the solid formulation is selected from tablets, capsules or granules.

5. The capsular polysaccharide derived from Bacteroides Salieris CSP6 as described in claim 1, characterized in that, The BHI culture medium formula is as follows: 10 g / L peptone, 12.5 g / L dehydrated calf brain extract, 5 g / L dehydrated calf heart extract, 5 g / L sodium chloride, 2 g / L glucose, 2.5 g / L disodium hydrogen phosphate, pH 7.4.

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