Capsular polysaccharide from bacteroides sailli CSP6 as well as preparation method and application of capsular polysaccharide

Abstract

The invention belongs to the technical field of enteric microorganisms, and particularly relates to capsular polysaccharide derived from bacteroides sailli CSP6 as well as a preparation method and application of the capsular polysaccharide. The monosaccharide composition of the capsular polysaccharide mainly comprises fucose, glucose and glucosamine, the molecular weight is 7.139 * 10 < 5 > Da, the strain is stored in the China Center for Type Culture Collection, and the preservation number is CCTCC NO: M20221993. According to the present invention, the in vitro research results show that the capsular polysaccharide from the Bacteroides sailli CSP6 can provide the alpha-glycosidase inhibition effect; in-vivo animal experiments prove that the capsular polysaccharide can inhibit the increase of postprandial blood sugar and has the effect of reducing blood sugar.

Description

Technical Field

[0001] The present invention belongs to the technical field of intestinal microorganisms, and particularly relates to a capsular polysaccharide derived from Bacteroides salyersii CSP6, and a preparation method and application thereof. Background Art

[0002] Bacteroides spp. are important commensal bacteria in the human intestine (PMID: 33535896). Due to their diverse biological functions, Bacteroides are widely considered a "next-generation probiotic," making them of great research and development value (PMID: 33535896). Bacteroides salyersiae is a rod-shaped, obligate anaerobic bacterium first isolated from a human intestinal clinical specimen in 2004 by YL Song et al. (PMID: 15583282). In 2023, our research team isolated Bacteroides salyersiae CSP6 (PMID: 38419055) from a healthy human fecal sample using systematic culturomics techniques. This strain is currently deposited with the China Center for Type Culture Collection under the accession number CCTCC NO: M 20221993 (China Invention Patent ZL 2023 10238846.8). Previous studies have shown that Bacteroides salierii CSP6 can degrade and produce chondroitin sulfate oligosaccharides and hyaluronic acid oligosaccharides (China Invention Patent ZL202310238846.8; PMID: 38419055). Furthermore, this strain can alleviate dextran sulfate sodium-induced ulcerative colitis by improving intestinal dysbiosis (PMID: 39275234).

[0003] Bacteroides salyersii CSP6 has significant development potential as a new generation of probiotics. However, aside from its anti-enteritis effects, no other pharmacological functions have been identified for this strain. Furthermore, no studies have been reported on the biological activity of its capsular polysaccharide. To address these issues, this study, based on previous systematic in vitro and in vivo activity screening, discovered for the first time that capsular polysaccharides derived from Bacteroides salyersii CSP6 can alleviate postprandial blood glucose elevation by inhibiting α-glucosidase activity, demonstrating broad application prospects in the preparation of drugs and foods that lower postprandial blood glucose. Summary of the Invention

[0004] In view of the deficiencies in the prior art, the present invention provides a capsular polysaccharide derived from Bacteroides salierii CSP6, and a preparation method and application thereof.

[0005] The technical solution of the present invention is: The present invention discloses a capsular polysaccharide derived from Bacteroides salyersiae CSP6, wherein the monosaccharide composition of the capsular polysaccharide is mainly fucose, glucose and glucosamine, and the molar ratios of the three are 51.19%, 11.87% and 11.48%, respectively; The molecular weight of the capsular polysaccharide is 7.139×10 5 Da; The strain is deposited in China Center for Type Culture Collection with a deposit number of CCTCC NO: M20221993. Its classification name is Bacteroides genus, Bacteroides salierii species; its Latin name is: Bacteroides salyersiae CSP6; the deposit date is December 26, 2022, and the deposit address is Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0006] Furthermore, the present invention discloses the use of the capsular polysaccharide in the preparation of a drug for lowering postprandial blood sugar.

[0007] Furthermore, the present invention discloses the use of the capsular polysaccharide in preparing food with blood sugar lowering function.

[0008] The present invention also discloses a drug for lowering postprandial blood sugar, which comprises the capsular polysaccharide and a pharmaceutically acceptable carrier. The drug is in the form of a liquid preparation or a solid preparation.

[0009] Furthermore, the dosage of the drug is 80 mg / kg; the solvent of the liquid preparation is phosphate buffered saline (PBS), pH 7.0-7.4; and the solid preparation is selected from tablets, capsules or granules.

[0010] The present invention also discloses a food for lowering postprandial blood sugar, which comprises the capsular polysaccharide and auxiliary materials acceptable in food science.

[0011] Furthermore, the food is a functional food, beverage or health food.

[0012] The present invention also discloses a method for producing the capsular polysaccharide, which comprises the following steps: a. Inoculate Bacteroides salierii CSP6 into 1.5 L of BHI liquid medium in an anaerobic environment and culture at 37°C for 48 hours to achieve a viable bacterial concentration of 9 × 10 8 CFU / mL; b. Centrifuge at 8000 x g for 20 minutes to collect the cells. Mix 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 at 5000 x g for 20 minutes and dialyze using a 3500 Da dialysis bag for 24 hours to remove phenol. d. Concentrating by rotary evaporation and freeze-drying to obtain the capsular polysaccharide.

[0013] Furthermore, the BHI medium described in the above method has a formula of: 10 g / L peptone, 12.5 g / L dehydrated calf brain extract powder, 5 g / L dehydrated buffalo heart extract powder, 5 g / L sodium chloride, 2 g / L glucose, 2.5 g / L disodium hydrogen phosphate, pH 7.4.

[0014] The invention also discloses the application of the capsular polysaccharide in the preparation of α-glucosidase inhibitors.

[0015] Advantages and beneficial effects of the present invention: The present invention provides Bacteroides salyersii ( Bacteroides salyersiae ) CSP6-derived capsular polysaccharide can significantly inhibit the activity of α-glucosidase and has a good effect in lowering postprandial blood sugar. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the growth curve of Bacteroides salyersii CSP6; Figure 2 This is a monosaccharide composition analysis diagram of the capsular polysaccharide derived from Bacteroides salicylicus CSP6; Figure 3 This is a molecular weight analysis diagram of capsular polysaccharide derived from Bacteroides salyersii CSP6; Figure 4 This is a comparison chart of α-glucosidase inhibitory activity; Figure 5 This is a comparison chart of blood sugar changes in mice after a meal; Figure 6 This is an analysis diagram of the area under the curve of postprandial blood glucose in mice; Among them, * indicates a significant difference compared with the control group (P<0.05); ** indicates an extremely significant difference compared with the control group (P<0.01). Student t-test was used for statistical analysis. DETAILED DESCRIPTION

[0017] In a specific embodiment, a capsular polysaccharide derived from Bacteroides salyersiae CSP6, wherein the monosaccharide composition of the capsular polysaccharide is mainly fucose, glucose and glucosamine, with the molar ratios of the three being 51.19%, 11.87% and 11.48%, respectively; The molecular weight of the capsular polysaccharide is 7.139×10 5 Da; The strain is deposited in the China Center for Type Culture Collection, with the deposit number CCTCC NO: M20221993 and the deposit date December 19, 2022. The deposit address is China Center for Type Culture Collection, Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0018] The capsular polysaccharide can be used to prepare drugs for lowering postprandial blood sugar, can also be used to prepare foods with blood sugar lowering function, and can also be used to prepare α-glucosidase inhibitors.

[0019] A drug for lowering postprandial blood sugar, comprising the above-mentioned capsular polysaccharide and a pharmaceutically acceptable carrier, the drug being in the form of a liquid preparation or a solid preparation.

[0020] Furthermore, the dosage of the drug is 80 mg / kg; the solvent of the liquid preparation is phosphate buffered saline (PBS), pH 7.0; and the solid preparation is selected from tablets, capsules or granules.

[0021] A food for lowering postprandial blood sugar, comprising the above-mentioned capsular polysaccharide and excipients acceptable in food science.

[0022] Furthermore, the food is a functional food, beverage or health food.

[0023] The above-mentioned method for preparing capsular polysaccharide comprises the following steps: a. Inoculate Bacteroides salierii CSP6 into 1.5 L of BHI liquid medium in an anaerobic environment and culture at 37°C for 48 hours to achieve a viable bacterial concentration of 9 × 10 8 CFU / mL; b. Collect the cells by centrifugation (8000 x g, 20 min), mix with 500 mL of phenol and 500 mL of distilled water, stir at 60°C for 30 min, and let stand overnight. c. Centrifuge the upper liquid (5000x g , 20 min), and dialyzed for 24 h using a 3500 Da dialysis bag to remove phenol; d. Concentrating by rotary evaporation and freeze-drying to obtain the capsular polysaccharide.

[0024] Furthermore, the BHI culture medium described in the above method has a formula of: 10 g / L peptone, 12.5 g / L dehydrated calf brain extract powder, 5 g / L dehydrated ox heart extract powder, 5 g / L sodium chloride, 2 g / L glucose, 2.5 g / L disodium hydrogen phosphate, pH 7.4.

[0025] The technical solutions provided by the present invention are 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.

[0026] Example 1

[0027] This example is a method for preparing capsular polysaccharide derived from Bacteroides salierii CSP6. The main experimental equipment is: DF-101S heat-collecting constant-temperature heating magnetic stirrer (Qingdao Lantern Science and Education Instrument Equipment Co., Ltd.); 7116TR desktop high-speed refrigerated centrifuge (Hunan Hexi Instrument Equipment Co., Ltd.); PD-2 freeze dryer (Shanghai Bilang Instrument Manufacturing Co., Ltd.); RE100-Pro rotary evaporator (Dalong Xingchuang Experimental Instrument (Beijing) Co., Ltd.).

[0028] The preparation and determination method thereof comprises the following steps: 1. First, culture Bacteroides salyersii CSP6: (1) Preparation of culture medium Prepare BHI liquid culture medium with the following ingredients: 10 g / L peptone, 12.5 g / L dehydrated calf brain extract powder, 5 g / L dehydrated ox heart extract powder, 5 g / L sodium chloride, 2 g / L glucose, and 2.5 g / L disodium hydrogen phosphate. The solvent is distilled water with a pH of 7.4. Pour the culture medium into anaerobic vials, fill with nitrogen, seal, and sterilize with high-pressure steam at 121°C for 15 min.

[0029] (2) Strain source Bacteroides salyersii CSP6 was isolated in the laboratory in the early stage (Chinese invention patent: CN 116676207B) and stored in a -80°C refrigerator using 30% glycerol seed solution.

[0030] (3) Strain activation and inoculation culture Bacteroides salyersii CSP6 was taken out of the -80°C freezer and inoculated into a 10 mL anaerobic vial after high-temperature sterilization for activation (the inoculation volume was 1% (v / v), and the anaerobic gas composition in the anaerobic vial was 80% (v / v) N2, 10% (v / v) H2 and 10% (v / v) CO2). After culturing at 37°C for 48 hours, the cells were transferred to a 50 mL anaerobic vial after high-temperature sterilization for expansion culture.

[0031] 2. Growth curve determination of Bacteroides salyersii CSP6 (1) Place the activated Bacteroides salicylicus CSP6 in a 37°C incubator for static culture. After culturing for 12 h, 24 h, 48 h, 72 h, and 96 h, aspirate 200 μL of bacterial solution and measure the OD using a 96-well plate in an enzyme reader. 600nm Take 200 μL of fresh liquid culture medium and measure its absorbance (OD600nm ) as the original control.

[0032] (2) Experimental results and analysis The results are as follows Figure 1 As shown, the concentration of Bacteroides salyersii CSP6 reached a maximum after 24 h of growth at 37°C and stabilized after 48 h.

[0033] 3. Extraction of source capsule polysaccharide: (1) Strain activation and cultivation The activated Bacteroides salierii CSP6 was inoculated into 1.5 L of BHI liquid medium and grown anaerobically for 48 h to a viable bacterial concentration of 9 × 10 8 CFU / mL.

[0034] (2) Extraction of capsular polysaccharides The cultured bacterial solution was centrifuged (8000x g , 20min) to collect the cells, then mix them with 500mL phenol and 500mL distilled water, stir at 60℃ for 30min and let it stand overnight to extract the capsular polysaccharide; the upper liquid was centrifuged (5000x g , 20 min) and dialyzed for 24 h using a 3500Da dialysis bag to remove phenol; finally, the capsular polysaccharide from Bacteroides salyersii CSP6 was obtained by rotary evaporation and freeze-drying.

[0035] 4. Determination of the monosaccharide composition of the capsular polysaccharide derived from Bacteroides salyersii CSP6: (1) Monosaccharide composition detection method Monosaccharide composition was determined by high-performance liquid chromatography with pre-column derivatization using an Agilent Eclipse XDB-C18 column; the mobile phase consisted of 0.1 mol / L phosphate buffer (pH 6.7) and acetonitrile (83:17 volume fraction); the column temperature was 30°C; the detection wavelength was 245 nm; the flow rate was 1 mL / min; and the injection volume was 10 μL.

[0036] (2) Experimental results and analysis The results are shown in Table 1 and Figure 2 As shown, the capsular polysaccharide derived from Bacteroides salierii CSP6 is mainly composed of Fuc, Glc and GlcN, and contains Man, GlcN, GlcA, GalA, GalN, Gal, and Xyl.

[0037] Table 1 Monosaccharide composition of capsular polysaccharide from Bacteroides salierii CSP6 monosaccharides Man GlcN Rha GlcA GalA GalN Glc Gal Xyl Fuc Proportion 5.82 % 11.48 % 3.24 % 3.98 % 7.31 % 2.29 % 11.87 % 1.84 % 0.98 % 51.19 % 5. Determine the molecular weight of the capsular polysaccharide derived from Bacteroides salyersii CSP6: (1) Molecular weight detection method Molecular weight was determined using high-performance gel permeation chromatography coupled with multi-angle laser light scattering. The analytical columns were SBHQ 804 and SB HQ 802.5 columns connected in series; the mobile phase consisted of a 0.1 mol / L aqueous sodium sulfate solution; the flow rate was 0.6 mL / min; the column temperature was 35°C; the injection volume was 100 µL; and the detectors were differential refractive index (RI) and multi-angle laser light scattering (MALLS).

[0038] (2) Experimental results and analysis The results are as follows Figure 3 As shown, the molecular weight of the capsular polysaccharide from Bacteroides salierii CSP6 is 7.139×10 5 Da.

[0039] Example 2

[0040] This example demonstrates the effect of the capsular polysaccharide derived from Bacteroides salierii CSP6 prepared in Example 1 in inhibiting α-glucosidase, demonstrating that it can be used to prepare α-glucosidase inhibitors.

[0041] Experimental Materials: Phosphate buffered saline (PBS, pH = 7.0); p-Nitrophenyl-α-pyranoglucopyranoside (PNPG) was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; α-Glucosidase was purchased from Sigma-Aldrich, USA.

[0042] The main experimental equipment was ReadMax 1200 full-wavelength absorbance microplate reader (Shanghai Shanpu Biotechnology Co., Ltd.).

[0043] Experimental methods: Using 2.5 mmol / L PNPG as 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 sample were added to a 96-well plate in sequence. After shaking at 37°C for 15 minutes, 80 μL of 0.2 mol / L Na2CO3 was added to terminate the reaction and the OD was measured. 405nm value.

[0044] A blank control (no sample added, supplemented with PBS) and background absorption (no α-glucosidase added, supplemented with PBS) were set up.

[0045] Inhibition rate (%) = 100% X [A 空白 -(A 样品 A 背录 )] / A 空自, the samples were set to three concentrations: 10 mg / mL, 50 mg / mL and 100 mg / mL.

[0046] Experimental results and analysis: The results are as follows Figure 4 As shown, capsular polysaccharide derived from Bacteroides salyersii CSP6 (10 mg / mL, 50 mg / mL, and 100 mg / mL) all exhibited α-glucosidase inhibitory activity, with significant differences compared to the control group (p < 0.01). Furthermore, the higher the sample concentration, the higher the inhibition rate. These results demonstrate that capsular polysaccharide derived from Bacteroides salyersii CSP6 has α-glucosidase inhibitory activity.

[0047] Example 3

[0048] This example demonstrates the use of the capsular polysaccharide derived from Bacteroides salyersii CSP6 prepared in Example 1 in lowering postprandial blood sugar.

[0049] Experimental Materials: Experimental Animals 10 C57BL / 6J mice (eight-week-old, male) were purchased from Weitonglihua Laboratory Animal Technology Co., Ltd. (Beijing) (certificate number SCXK (Beijing) 2016-0011); Phosphate-buffered saline (PBS, pH=7.0).

[0050] Main experimental equipment: GA-3 blood glucose meter (Sinocare Biosensor Co., Ltd.).

[0051] Experimental methods: Ten mice were randomly divided into two groups: a control group (n=5) and a drug-treated group (BS group, n=5). All mice were housed at approximately 23°C with a 12-hour light-dark cycle and free access to standard chow and water. After a one-week acclimation period, drug administration began.

[0052] After fasting for 10 hours at night, the mice in the control group were gavage-administered with 150 μL of PBS, and the mice in the BS group were gavage-administered with 150 μL of capsular polysaccharide derived from Bacteroides salicylica CSP6 (80 mg / kg, dissolved in 150 μL of PBS solution). After fasting for another 1 hour, the initial blood glucose was measured, and then food was given. The postprandial blood glucose of the mice was continuously measured at various time points within 120 minutes (15 min, 30 min, 45 min, 60 min, 90 min, and 120 min).

[0053] Experimental results and analysis: The results are as follows Figure 5 、 Figure 6As shown in Table 2, oral administration of capsular polysaccharide derived from Bacteroides salierii CSP6 significantly slowed the rise in postprandial blood glucose in mice. Blood glucose levels in the BS group were significantly lower than those in the control group at 30 and 45 minutes after feeding (p < 0.01); and 30 minutes after feeding, blood glucose levels in the BS group were significantly lower than those in the control group (p < 0.05).

[0054] Table 2 Capsular polysaccharide from Bacteroides salierii CSP6 reduces postprandial blood glucose in mice

[0055] The capsular polysaccharide derived from Bacteroides salierii CSP6 can be applied to foods for lowering postprandial blood sugar.

[0056] Foods prepared from capsular polysaccharides derived from Bacteroides salyersii CSP6 do not require specific dosage forms and can be prepared using conventional food dosage forms in the art, typically solid or liquid preparations. When the food is a liquid preparation, the drug is obtained by dissolving the capsular polysaccharide derived from Bacteroides salyersii CSP6 in a suitable solvent. When preparing the food, acceptable excipients and additives are used, and there are no specific restrictions on the types and amounts of these excipients and additives. The food can be a functional food, beverage, or health food.

[0057] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A capsular polysaccharide derived from Bacteroides salyersii CSP6, characterized in that: The monosaccharide composition of the capsular polysaccharide is mainly fucose, glucose and glucosamine, with the molar ratios of the three being 51.19%, 11.87% and 11.48% respectively; The molecular weight of the capsular polysaccharide is 7.139×10 5 Da; The strain is deposited in China Center for Type Culture Collection with a deposit number of CCTCC NO: M20221993.

2. Use of the capsular polysaccharide according to claim 1 in the preparation of a medicament for lowering postprandial blood sugar.

3. Use of the capsular polysaccharide according to claim 1 in preparing food with blood sugar lowering function.

4. A drug for lowering postprandial blood sugar, characterized in that: The drug comprises the capsular polysaccharide according to claim 1 and a pharmaceutically acceptable carrier, wherein the drug is in the form of a liquid preparation or a solid preparation.

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

6. A food for lowering postprandial blood sugar, characterized in that: The invention comprises the capsular polysaccharide according to claim 1 and excipients acceptable in food science.

7. The food according to claim 6, characterized in that The food is a functional food, a beverage or a health food.

8. A method for preparing the capsular polysaccharide according to claim 1, characterized in that: The following steps are involved: a. Inoculate Bacteroides salierii CSP6 into 1.5 L of BHI liquid medium in an anaerobic environment and culture at 37°C for 48 hours to achieve a viable bacterial concentration of 9 × 10 8 CFU / mL; b. Collect the cells after centrifugation, mix 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 for 24 hours using a 3500 Da dialysis bag to remove phenol; d. Concentrating by rotary evaporation and freeze-drying to obtain the capsular polysaccharide.

9. The method according to claim 8, characterized in that The BHI culture medium formula is: 10 g / L peptone, 12.5 g / L dehydrated calf brain extract powder, 5 g / L dehydrated ox heart extract powder, 5 g / L sodium chloride, 2 g / L glucose, 2.5 g / L disodium hydrogen phosphate, pH 7.

4.

10. Use of the capsular polysaccharide according to claim 1 in the preparation of an α-glucosidase inhibitor.

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