Modified spirulina platensis polysaccharide with effect of regulating intestinal homeostasis and application of modified spirulina platensis polysaccharide

Abstract

The invention provides modified spirulina polysaccharide with an effect of regulating intestinal steady state and application of the modified spirulina polysaccharide, and belongs to the technical field of biological medicines. The preparation method of the modified spirulina polysaccharide comprises the following steps: mixing spirulina polysaccharide with a hydrochloric acid solution to prepare a polysaccharide solution, then adding sodium selenite and vitamin C, carrying out microwave treatment, and carrying out alcohol precipitation, precipitate collection and drying to obtain the modified spirulina polysaccharide. The modified spirulina polysaccharide disclosed by the invention has a remarkable effect of regulating the steady state of the intestinal tract, and has an outstanding effect on intestinal tract ecological disorder induced by high-fat diet. The modified spirulina polysaccharide and the coptis chinensis extract are combined to prepare products for preventing and treating intestinal ecological imbalance caused by oral administration of antibiotics. The coptis chinensis extract prepared by the invention can inhibit gram-positive pathogenic bacteria and protect gram-negative beneficial bacteria, and cooperates with the modified spirulina polysaccharide to promote colonization of intestinal probiotics (bifidobacterium and lactobacillus) and improve intestinal ecological imbalance.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to a modified spirulina polysaccharide with the function of regulating intestinal homeostasis and its application. Background Technology

[0002] The gut microbiota plays a crucial role in maintaining host health, involving nutrient digestion and absorption, energy metabolism, immune system development and regulation, maintenance of the intestinal barrier function, and defense against pathogen invasion. However, under the continuous influence of modern lifestyles, such as high-fat and high-sugar diets, antibiotic abuse, environmental stress, and irregular sleep patterns, the gut microbiota is highly susceptible to dysbiosis. This dysbiosis is mainly manifested by a significant decrease in the abundance of beneficial symbiotic bacteria, excessive proliferation of potentially pathogenic or opportunistic pathogens, and a reduction in overall microbiota diversity. This imbalance has been widely recognized as a core contributing factor or key promoter of many chronic diseases, including inflammatory bowel disease, obesity, type 2 diabetes, and non-alcoholic fatty liver disease, posing a serious challenge to global public health.

[0003] Current strategies for regulating or correcting gut microbiota dysbiosis mainly include direct probiotic supplementation, providing prebiotics to promote the growth of beneficial bacteria, using synbiotics, fecal microbiota transplantation in extreme cases, and antibiotic treatment for specific infections. However, these methods all have significant limitations or potential risks. For example, the colonization efficiency of probiotics is greatly affected by individual differences and the disruption caused by gastric acid and bile, the number of live bacteria is prone to decay, the effects are unstable, and adverse reactions such as immune activation may occur. Some prebiotics have strain-specific effects, their effects are relatively slow, and excessive intake can easily cause gastrointestinal discomfort such as bloating and diarrhea. Synbiotics attempt to combine the advantages of both, but they are costly and their effects are still limited by the aforementioned constraints. Therefore, there is an urgent need to develop new gut microbiota modulators that are safer, more effective, and have fewer side effects.

[0004] Given the limitations of existing regulatory methods, identifying substances with gut microbiota-regulating activity from natural resources has become an important research direction. Natural polysaccharides have attracted significant attention due to their wide availability, good biocompatibility, high safety, and prebiotic properties. Natural polysaccharides can improve the intestinal environment, enhance intestinal barrier function, and indirectly influence microbiota balance by regulating host immune responses. However, the efficacy of ordinary natural polysaccharides in regulating complex and severe gut microbiota dysbiosis is often limited or unstable. Therefore, structural modification and functional enhancement of natural polysaccharides with a good foundation to improve their ability and efficiency in targeting and regulating dysbiosis has become an inevitable choice and a key breakthrough for overcoming existing bottlenecks and developing next-generation highly effective microecological regulators. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide a modified spirulina polysaccharide with the function of regulating intestinal homeostasis and its application. The modified spirulina polysaccharide of this invention can significantly improve intestinal ecological imbalance.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] A method for preparing modified spirulina polysaccharide with the function of regulating intestinal homeostasis includes: mixing spirulina polysaccharide and hydrochloric acid solution to obtain a polysaccharide solution, mixing it with sodium selenite and vitamin C, treating it with microwave, precipitating it with alcohol, collecting the precipitate, drying it, and obtaining modified spirulina polysaccharide.

[0008] Preferably, the mass ratio of sodium selenite, spirulina polysaccharide and vitamin C is 1:(8-12):(2-3).

[0009] Preferably, the microwave treatment has a power of 300-400W, a temperature of 55-65℃, and a time of 1.5-2.5h.

[0010] This invention provides a modified spirulina polysaccharide obtained according to the above preparation method.

[0011] The present invention also provides an application of the modified spirulina polysaccharide described above in the preparation of products for preventing and treating intestinal ecological imbalance.

[0012] Preferably, the gut microbiota dysbiosis is gut microbiota dysbiosis induced by a high-fat diet.

[0013] This invention also provides the application of the above-mentioned modified spirulina polysaccharide combined with Coptis chinensis extract in the preparation of products for preventing and treating intestinal ecological imbalance.

[0014] Preferably, the Coptis chinensis extract is obtained by extracting Coptis chinensis from a eutectic solvent aqueous solution and purifying it with a macroporous adsorption resin.

[0015] Preferably, the weight ratio of the modified spirulina polysaccharide to the Coptis chinensis extract is 1:(2-5).

[0016] Preferably, the intestinal dysbiosis is caused by oral antibiotics.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] This invention provides a method for preparing modified spirulina polysaccharides with intestinal homeostasis regulating effects: spirulina polysaccharides are mixed with hydrochloric acid solution to obtain a polysaccharide solution, which is then mixed with sodium selenite and vitamin C. After microwave treatment, the solution is precipitated with alcohol, the precipitate is collected, and dried to obtain the modified spirulina polysaccharides. The modified spirulina polysaccharides of this invention have significant effects on regulating intestinal homeostasis and can be used alone to prepare products for preventing and treating intestinal ecological disorders. They are particularly effective against intestinal ecological disorders induced by a high-fat diet, improving the intestinal flora structure in mice fed a high-fat diet and repairing colonic tissue damage (improving colonic villus breakage and shedding).

[0019] This invention also provides the application of modified spirulina polysaccharide combined with Coptis chinensis extract in the preparation of products for preventing and treating intestinal ecological imbalance. The Coptis chinensis extract, obtained by extracting Coptis chinensis with a low-melting-point solvent aqueous solution and purifying it with macroporous adsorption resin, can inhibit Gram-positive pathogenic bacteria and protect Gram-negative beneficial bacteria. Synergistically with modified spirulina polysaccharide, it promotes the colonization of intestinal probiotics (Bifidobacteria, Lactobacillus), increases the level of beneficial metabolites in the intestine, enhances the barrier function of intestinal epithelial cells, and thus improves intestinal ecological imbalance. Attached Figure Description

[0020] Figure 1 Venn diagrams based on OUTs for different groups.

[0021] Figure 2 The effect of modified Spirulina polysaccharide on the α-diversity of intestinal flora in mice was investigated. a represents the Chao 1 index, b represents the Shannon index, and c represents the Simpson index.

[0022] Note: "*" indicates a significant difference between the control group and the control group (p<0.05), "**" indicates a significant difference between the control group and the control group (p<0.01); "#" indicates a significant difference between the control group and the model group (p<0.05), and "##" indicates a significant difference between the control group and the model group (p<0.01).

[0023] Figure 3 To investigate the effects of modified Spirulina polysaccharide on β-diversity of intestinal flora in mice.

[0024] Figure 4 To investigate the effect of modified spirulina polysaccharide on the phylum level of intestinal flora in mice.

[0025] Figure 5 To investigate the effects of modified spirulina polysaccharide on the genus level of intestinal flora in mice.

[0026] Figure 6 The abundance ratio of Firmicutes to Bacteroidetes is given in different groups.

[0027] Figure 7 It consists of 8 microbial species at the genus level.

[0028] Note: "*" indicates a significant difference between the control group and the control group (p<0.05), "**" indicates a significant difference between the control group and the control group (p<0.01); "#" indicates a significant difference between the control group and the model group (p<0.05), and "##" indicates a significant difference between the control group and the model group (p<0.01).

[0029] Figure 8 The effects of modified spirulina polysaccharide on the histopathology of mouse colon tissue. Detailed Implementation

[0030] This invention provides a method for preparing modified spirulina polysaccharide with the function of regulating intestinal homeostasis, comprising: mixing spirulina polysaccharide and hydrochloric acid solution to obtain a polysaccharide solution, mixing it with sodium selenite and vitamin C, treating it with microwave, precipitating it with alcohol, collecting the precipitate, drying it, and obtaining modified spirulina polysaccharide.

[0031] The spirulina polysaccharide of this invention is preferably isolated from *Spirulina platensis*. The preferred method for preparing the spirulina polysaccharide includes: mixing *Spirulina platensis* powder and water at a material-to-liquid ratio of 1:(15-25) g / mL to obtain a polysaccharide solution; ultrasonically treating the solution at 150-250W and 63-68℃ for 13-18 min; stirring at 68-72℃ and 80-120 rpm for 60-80 min; cooling to 25-30℃; centrifuging at 4500-5200 rpm for 8-12 min; concentrating the supernatant to 1 / 2-1 / 4 of its volume; adding solvent (ethyl acetate and n-butanol at a volume ratio of 2-5:1) equal to 1 / 2-1 / 4 of the polysaccharide solution volume; shaking for 30 min; and then... Centrifuge at 500-5200 rpm for 8-12 min, repeat 3-5 times until no protein precipitate is formed. Concentrate the supernatant and add anhydrous ethanol to a volume fraction of 87%-92%. Let stand at 4℃ for 12 h, then centrifuge at 4500-5200 rpm for 8-12 min to obtain a precipitate. Add an ethanol solution with a volume fraction of 87%-92%, let stand at 4℃ for 12 h, then centrifuge at 4500-5200 rpm for 8-12 min to obtain a precipitate. Dissolve the precipitate in deionized water and dialyze at 4℃ using a dialysis bag with a molecular weight cutoff of 3500 Da for 48 h (replace the deionized water every 8 h). Freeze-dry to a water content of 3wt%-5wt% to obtain spirulina polysaccharide.

[0032] The preferred mass ratio of sodium selenite, spirulina polysaccharide and vitamin C in this invention is 1:(8-12):(2-3), and more preferably 1:10:2.5.

[0033] The microwave processing described in this invention has a power of 300-400W, a temperature of 55-65℃, and a time of 1.5-2.5h.

[0034] This invention provides a modified spirulina polysaccharide obtained according to the above preparation method.

[0035] The present invention also provides an application of the modified spirulina polysaccharide described above in the preparation of products for preventing and treating intestinal ecological imbalance, wherein the intestinal ecological imbalance is intestinal ecological imbalance induced by a high-fat diet.

[0036] This invention also provides the application of the above-mentioned modified spirulina polysaccharide combined with Coptis chinensis extract in the preparation of products for preventing and treating intestinal ecological imbalance.

[0037] The Coptis chinensis extract of this invention is preferably obtained by extracting Coptis chinensis with a low-melting-point solvent aqueous solution and purifying it with a macroporous adsorption resin. The preferred preparation method of the Coptis chinensis extract includes: pulverizing Coptis chinensis, passing it through a 100-200 mesh sieve, adding it to a low-melting-point solvent aqueous solution at a material-to-liquid ratio of 1:10-15 g / mL, ultrasonically extracting it at 200-300W and 50-60℃ for 20-40 min, centrifuging it at 4500-5200 rpm for 8-12 min, adsorbing the supernatant onto an AB-8 type macroporous resin at a flow rate of 0.5-1 BV / h, first washing with 3-5 BV of deionized water to remove impurities, then washing with 4-6 BV of 30-50% ethanol solution to remove impurities, and finally eluting with 5-8 BV of 70-80% ethanol solution. The 70-80% ethanol eluent is collected and freeze-dried under vacuum to obtain the Coptis chinensis extract. The eutectic solvent aqueous solution is preferably composed of eutectic solvent and water in a mass ratio of 10:(2-5), more preferably 10:3; the eutectic solvent is preferably composed of choline chloride and lactic acid in a molar ratio of 1:1-3, more preferably 1:2.

[0038] The Coptis chinensis extract obtained by the extraction method of this invention has a significant inhibitory effect on Gram-positive pathogenic bacteria in the intestine, while having a weak inhibitory effect on important Gram-negative beneficial bacteria in the intestine, thus promoting the proliferation of probiotics in the intestine. Antibiotics can severely disrupt the metabolic function of the gut microbiota, leading to a decrease in the production of SCFAs (such as butyric acid and propionic acid). The Coptis chinensis extract can indirectly increase the level of beneficial metabolites (such as butyric acid) by promoting the recovery of beneficial bacteria, thereby enhancing the barrier function of intestinal epithelial cells. This invention uses a green and efficient process of eutectic solvent extraction combined with macroporous adsorption resin purification to prepare a high-purity, high-activity Coptis chinensis extract. The Coptis chinensis extract of this invention effectively prevents and treats intestinal ecological imbalance caused by oral antibiotics by eliminating pathogenic bacteria, relatively protecting beneficial bacteria, repairing the intestinal barrier, and promoting the production of beneficial metabolites.

[0039] The preferred weight ratio of the modified spirulina polysaccharide and Coptis chinensis extract in this invention is 1:(2-5), and more preferably 1:3.

[0040] The intestinal dysbiosis described in this invention is intestinal dysbiosis caused by oral antibiotics.

[0041] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0042] Unless otherwise specified, the following embodiments are all conventional methods.

[0043] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0044] The preparation method of the spirulina polysaccharide used in this embodiment of the invention is as follows: Spirulina platensis powder and water are mixed at a material-to-liquid ratio of 1:20 g / mL to obtain a mixture. The mixture is ultrasonically treated at 200W and 65℃ for 15 min, stirred at 70℃ and 100 rpm for 70 min, cooled to 28℃, and centrifuged at 5000 rpm for 10 min. The supernatant is concentrated to 1 / 3, and 1 / 2 volume of solvent (ethyl acetate and n-butanol at a volume ratio of 3:1) is added. The mixture is shaken for 30 min and then centrifuged at 5000 rpm. The process was repeated 4 times for 10 min. After concentrating the supernatant, anhydrous ethanol was added to a volume fraction of 90%. The mixture was allowed to stand at 4 °C for 12 h, and then centrifuged at 5000 rpm for 10 min to obtain a precipitate. A volume fraction of 90% ethanol solution was added, and the mixture was allowed to stand at 4 °C for 12 h, and then centrifuged at 5000 rpm for 10 min to obtain a precipitate. The precipitate was dissolved in deionized water and dialyzed at 4 °C using a dialysis bag with a molecular weight cutoff of 3500 Da for 48 h (with the deionized water replaced every 8 h). The precipitate was then freeze-dried to a water content of 4 wt% to obtain spirulina polysaccharide.

[0045] Example 1

[0046] Preparation of modified spirulina polysaccharide

[0047] 1g of spirulina polysaccharide was mixed with hydrochloric acid solution and stirred at 40℃ and 300rpm for 1.5h to obtain a spirulina polysaccharide solution with pH 4 and a mass fraction of 3%. 0.1g of sodium selenite was added first, and the mixture was stirred at 300rpm for 30min. Then, 0.25g of vitamin C was added and stirring continued for 15min. Nitrogen gas was then introduced, and the mixture was microwaved at 350W, 60℃, and 400rpm for 2h.

[0048] Cool the microwave-treated solution to 25°C, add anhydrous ethanol to 90% by volume, stir at 200 rpm for 10 min, let stand at 4°C for 12 h, centrifuge at 5000 rpm for 10 min, collect the precipitate, add 50 mL of 90% ethanol solution, stir at 200 rpm for 10 min, let stand at 4°C for 12 h, centrifuge at 5000 rpm for 10 min, and collect the precipitate.

[0049] After adding 50 mL of deionized water to the precipitate and mixing, the mixture was transferred to a dialysis bag (molecular weight cutoff 3500 Da) and dialyzed with deionized water at 4 °C for 48 h (the deionized water was changed every 8 h). After dialysis, the solution in the dialysis bag was freeze-dried under vacuum to a water content of 4 wt% to obtain modified spirulina polysaccharide.

[0050] Example 2

[0051] Preparation of modified spirulina polysaccharide

[0052] 0.8 g of spirulina polysaccharide was mixed with hydrochloric acid solution and stirred at 38 °C and 250 rpm for 2 h to obtain a spirulina polysaccharide solution with pH 3.8 and a mass fraction of 2.5%. 0.1 g of sodium selenite was added first, and the mixture was stirred at 250 rpm for 35 min. Then, 0.2 g of vitamin C was added, and the mixture was stirred for another 12 min. Nitrogen gas was then introduced, and the mixture was microwaved at 300 W, 55 °C, and 350 rpm for 2.5 h.

[0053] The microwave-treated solution was cooled to 23°C, anhydrous ethanol was added to a volume fraction of 88%, stirred at 150 rpm for 12 min, allowed to stand at 4°C for 10 h, centrifuged at 4500 rpm for 12 min, the precipitate was collected, 45 mL of 90% ethanol solution was added, stirred at 150 rpm for 12 min, allowed to stand at 4°C for 10 h, centrifuged at 4500 rpm for 12 min, and the precipitate was collected.

[0054] After adding 45 mL of deionized water to the precipitate and mixing, the mixture was transferred to a dialysis bag (molecular weight cutoff 3500 Da) and dialyzed with deionized water at 4 °C for 46 h (the deionized water was changed every 8 h). After dialysis, the solution in the dialysis bag was freeze-dried under vacuum to a water content of 2 wt% to obtain modified spirulina polysaccharide.

[0055] Example 3

[0056] Preparation of modified spirulina polysaccharide

[0057] 1.2 g of spirulina polysaccharide was mixed with hydrochloric acid solution and stirred at 42 °C and 350 rpm for 1 h to obtain a spirulina polysaccharide solution with pH 4.2 and a mass fraction of 4%. 0.1 g of sodium selenite was added first, and the mixture was stirred at 350 rpm for 25 min. Then, 0.3 g of vitamin C was added, and the mixture was stirred for another 12 min. Nitrogen gas was then introduced, and the mixture was microwaved at 400 W, 65 °C, and 450 rpm for 1.5 h.

[0058] The microwave-treated solution was cooled to 27°C, anhydrous ethanol was added to a volume fraction of 92%, stirred at 250 rpm for 8 min, allowed to stand at 4°C for 14 h, centrifuged at 5500 rpm for 8 min, the precipitate was collected, 55 mL of 92% ethanol solution was added, stirred at 250 rpm for 8 min, allowed to stand at 4°C for 14 h, centrifuged at 5500 rpm for 8 min, and the precipitate was collected.

[0059] After adding 55 mL of deionized water to the precipitate and mixing, the mixture was transferred to a dialysis bag (molecular weight cutoff 3500 Da) and dialyzed with deionized water at 4 °C for 50 h (the deionized water was changed every 8 h). After dialysis, the solution in the dialysis bag was freeze-dried under vacuum to a water content of 5 wt% to obtain modified spirulina polysaccharide.

[0060] Example 4

[0061] Gut ecological imbalance composition

[0062] (1) Preparation of Coptis chinensis extract

[0063] Coptis chinensis was pulverized and passed through a 150-mesh sieve. A eutectic solvent aqueous solution was added at a material-to-liquid ratio of 1:12 g / mL. The mixture was ultrasonically extracted at 250 W and 55 °C for 30 min, followed by centrifugation at 5000 rpm for 10 min. The supernatant was adsorbed onto AB-8 macroporous resin at a flow rate of 0.8 BV / h. The supernatant was first eluted with 4 BV of deionized water to remove impurities, then with 5 BV of 40% ethanol solution, and finally with 6 BV of 75% ethanol solution. The 75% ethanol eluent was collected and freeze-dried under vacuum until the water content reached 3 wt%, yielding the Coptis chinensis extract.

[0064] The eutectic solvent aqueous solution is composed of eutectic solvent and water in a mass ratio of 10:3, and the eutectic solvent is composed of choline chloride and lactic acid in a molar ratio of 1:2.

[0065] (2) Preparation of the composition

[0066] The modified spirulina polysaccharide from Example 1 and the extract of Coptis chinensis were mixed at a mass ratio of 1:3 to obtain a composition for intestinal ecological imbalance.

[0067] Example 5

[0068] Gut ecological imbalance composition

[0069] (1) Preparation of Coptis chinensis extract

[0070] Coptis chinensis was pulverized and passed through a 100-mesh sieve. A eutectic solvent aqueous solution was added at a material-to-liquid ratio of 1:10 g / mL. The mixture was ultrasonically extracted at 200W and 50℃ for 40 min, followed by centrifugation at 4500 rpm for 12 min. The supernatant was adsorbed onto AB-8 macroporous resin at a flow rate of 0.5 BV / h. The supernatant was first eluted with 3 BV of deionized water to remove impurities, then with 4 BV of 30% ethanol solution, and finally with 5 BV of 70% ethanol solution. The 70% ethanol eluent was collected and freeze-dried under vacuum until the water content reached 3 wt%, yielding the Coptis chinensis extract.

[0071] The eutectic solvent aqueous solution is composed of eutectic solvent and water in a mass ratio of 10:2, and the eutectic solvent is composed of choline chloride and lactic acid in a molar ratio of 1:1.

[0072] (2) Preparation of the composition

[0073] The modified spirulina polysaccharide from Example 1 and the extract of Coptis chinensis were mixed at a mass ratio of 1:2 to obtain a composition for intestinal ecological imbalance.

[0074] Example 6

[0075] Gut ecological imbalance composition

[0076] (1) Preparation of Coptis chinensis extract

[0077] Coptis chinensis was pulverized and passed through a 200-mesh sieve. A eutectic solvent aqueous solution was added at a material-to-liquid ratio of 1:15 g / mL. The mixture was ultrasonically extracted at 300 W and 60 °C for 20 min, followed by centrifugation at 5500 rpm for 8 min. The supernatant was adsorbed onto AB-8 macroporous resin at a flow rate of 1 BV / h. It was then eluted with 5 BV of deionized water to remove impurities, followed by 6 BV of 50% ethanol solution, and finally eluted with 8 BV of 80% ethanol solution. The 80% ethanol eluent was collected and freeze-dried under vacuum until the water content reached 4 wt%, yielding the Coptis chinensis extract.

[0078] The eutectic solvent aqueous solution is composed of eutectic solvent and water in a mass ratio of 10:5, and the eutectic solvent is composed of choline chloride and lactic acid in a molar ratio of 1:3.

[0079] (2) Preparation of the composition

[0080] The modified spirulina polysaccharide from Example 1 and the extract of Coptis chinensis were mixed at a mass ratio of 1:5 to obtain a composition for intestinal ecological imbalance.

[0081] Comparative Example 1

[0082] The specific implementation method differs from Example 4 in that the Coptis chinensis extract is discarded and step (1) is deleted.

[0083] Comparative Example 2

[0084] The specific implementation method differs from Example 4 in that the modified spirulina polysaccharide is omitted.

[0085] Comparative Example 3

[0086] The specific implementation method differs from Example 4 in that the eutectic solvent aqueous solution in step (1) is replaced with a 75% ethanol solution by volume.

[0087] Comparative Example 4

[0088] The specific implementation method differs from Example 4 in that the mass ratio of modified spirulina polysaccharide and Coptis chinensis extract in Example 1 is 1:0.5.

[0089] Comparative Example 5

[0090] The specific implementation method differs from Example 4 in that the mass ratio of modified spirulina polysaccharide and Coptis chinensis extract in Example 1 is 1:7.

[0091] Experimental Example 1

[0092] Animal experiments on how modified polysaccharides improve gut microbiota dysbiosis induced by a high-fat diet

[0093] (1) Animal experimental design

[0094] Five-week-old male C57BL / 6J mice (20±2g, SPF grade) were purchased from Beijing Vital River Biotechnology Co., Ltd. The animals were housed under controlled conditions: temperature (25±2℃), humidity (50±10%), and a 12-hour light / dark cycle.

[0095] After a one-week adaptation period, eight mice were randomly assigned to the normal control group (NC group) and fed 10% of the normal diet, while the remaining mice were fed 60% of the high-fat diet.

[0096] Five weeks later, all mice on the high-fat diet were fasted for 12 hours and then intraperitoneally injected with streptozotocin solution at 100 mg / kg bw in 0.1 M citrate buffer (pH 4.5). The NC group received an equal volume of citrate buffer. Fasting blood glucose levels were measured 72 hours after injection. Mice with fasting blood glucose levels exceeding 11.1 mmol / L were randomly divided into three groups: the model control group (MC group, treated with 0.9% saline), the high-dose SPP group (SPPH group, 400 mg / kg bw SPP), and the high-dose SeSPP group (SeSPPH group, 400 mg / kg bw SeSPP). SeSPP was the modified polysaccharide from Example 1.

[0097] (2) High-throughput sequencing of 16S rRNA from mouse feces

[0098] DNA was extracted from fecal samples using a DNA extraction kit. DNA concentration was detected using the dsDNAHS Assay Kit. Using 20-50 ng of DNA as a template, PCR primers 338F and 806R were used to amplify two highly variable regions (V3-V4) on prokaryotic 16S rDNA. Additionally, adapters with indices were added to the ends of the PCR products of the 16S rDNA for NGS sequencing. Finally, the PCR product library was analyzed by 1.5% agarose gel electrophoresis to detect the target fragment at 600 bp. Library concentration was detected using a microplate reader (Tecan, Infinite 200Pro), and paired-end sequencing was performed using an Illumina MiSeq / Novaseq instrument on PE250 / FE300. After quality filtering, the final sequences were used for OTU clustering. Sequence clustering was performed using VSEARCH (1.9.6) (sequence similarity set at 97%), and the 16S rRNA reference database for alignment was Silva 138. Then, the RDP classifier Bayesian algorithm was used to perform taxonomic analysis on representative sequences of OTUs, and the community composition of each sample was statistically analyzed at different species classification levels.

[0099] High-throughput 16S rRNA sequencing was used to further investigate the effects of modified Spirulina polysaccharide on the structure and composition of the gut microbiota in mice induced by a high-fat diet. 46276, 56457, 52892, 51880, and 57847 valid sequences were obtained from the NC, MC, SPPH, and SeSPPH groups, respectively, with average sequence lengths of 440, 441, 440, 437, and 440, respectively. Venn diagram results showed ( Figure 1The MC group had 471 operational taxonomic units (OTUs), significantly higher than the NC group's 432 OTUs. This indicates that a high-fat diet significantly alters the structure and diversity of the mouse gut microbiota. Notably, the number of OTUs increased to 486 and 487 after intervention with spirulina polysaccharide and modified spirulina polysaccharide, respectively, suggesting that these two polysaccharides can regulate the composition of the gut microbiota. This increase in OTUs may be related to the two polysaccharides promoting the proliferation of specific beneficial bacteria.

[0100] The Chao1 index, Shannon index, and Simpson index were used to assess species diversity, such as... Figure 2 As shown, the Chao1 index, Shannon index, and Simpson index of the MC group, SPPH group, and SeSPPH group were all greater than those of the NC group. These results indicate that compared with the NC group mice, the diversity and richness of the gut microbiota in the MC group, SPPH group, and SeSPPH group mice were significantly altered. However, intervention with SPP and SeSPP failed to significantly reverse the diversity and richness of the gut microbiota in high-fat diet mice. β-diversity analysis assesses the differences between microbial communities, representing the differences between every two samples in the community. Principal coordinate analysis results show ( Figure 3 The NC group clustered in different regions compared to other groups, indicating that the gut microbiota of mice on a high-fat diet had been disrupted, and the intervention of spirulina polysaccharide and modified spirulina polysaccharide tended to force them to move closer to the NC group.

[0101] Based on the sequencing classification annotation results, the changes in the gut microbiota of each group of mice at the phylum level were analyzed. Figure 4 The distribution of the top 10 most abundant bacterial communities at the phylum level is shown. At the phylum level, Firmicutes and Bacteroidetes were absolutely dominant in all experimental groups, with their combined relative abundance remaining around 90%. After intervention with Spirulina polysaccharides and modified Spirulina polysaccharides, the relative abundance of Actinobacteriah and Deferribacterota in mice approached that of the NC group, indicating that the two polysaccharides can improve high-fat diet-induced gut microbiota dysbiosis by regulating the abundance of specific phyla. Simultaneously, intervention with Spirulina polysaccharides and modified Spirulina polysaccharides also reversed the Firmicutes / Bacteroidetes ratio.

[0102] At the genus level, there were also significant differences in the gut microbiota composition between the NC and MC groups of mice. Figure 5Compared to the NC group, the abundance of *Faecalibaculum*, *Muribaculaceae*, and *Lactobacillus* in the gut of mice in the MC group was significantly reduced, while the abundance of *Lachnospiraceae*, *Blautia*, and *Colidextribacter* was significantly increased. Intervention with *Spirulina* polysaccharide and modified *Spirulina* polysaccharide improved the abundance of all genera. Furthermore, at the genus level, the abundance of *Faecalibaculum*, *Coriobacteriance_UCG_002*, *Lactobacillus*, and *Muribaculaceae* was significantly reduced in the MC group, while the relative abundance of *f_Lachnospiraceae_Unclassified*, *Alloprevotella*, *Bacteroides*, and *Colidextribacter* increased. *Faecalibaculum* and *Lactobacillus* are considered to play an important role in maintaining gut health, while *Alloprevotella* has been reported to be a pathogen associated with inflammation and toxin production. At the genus level, the two polysaccharides downregulated the levels of Alloprevotella, Bacteroides, Coridextribacter, and f_Lachnospiraceae_Unclassified, and upregulated the levels of Coriobacteriaceae_UCG_002, Faecalibaculum, Lactobacillus, and Muribaculaceae, indicating that the two polysaccharides can improve the gut microbiota structure to some extent. Figure 7 This indicates that the two polysaccharides can regulate gut microbiota dysbiosis induced by a high-fat diet by inhibiting the proliferation of harmful bacteria and promoting the growth of beneficial bacteria.

[0103] (3) Histological observation of mouse colon

[0104] A small portion of the colon from each group of isolated mice was excised from the same location, rinsed with ice-cold saline, blotted dry with filter paper, and then fixed in 10% formalin solution at room temperature. After 72 hours, the tissue was rinsed with running water, followed by graded dehydration with ethanol, clearing with xylene, paraffin embedding, dewaxing to anhydrous, routine hematoxylin-eosin staining, dehydration, clearing, and mounting. The resulting tissue sections were observed under an optical microscope and photographed at 20x magnification. Figure 8 As shown.

[0105] Figure 8The results showed that the colonic villi in the NC group mice were neatly arranged, long, dense, and intact; compared with the NC group, the colonic villi in the MC group mice were shorter, broken, and detached, with larger gaps between the villi. After intervention with spirulina polysaccharide and modified spirulina polysaccharide, the colonic villi in mice became longer and more neatly arranged, and the morphology of the colonic villi was somewhat alleviated. This indicates that the two polysaccharides can effectively improve intestinal damage caused by a high-fat diet and maintain the small intestinal mucosal barrier and villi morphology.

[0106] Experimental Example 3

[0107] Animal experiments on the improvement of intestinal ecology dysbiosis caused by oral antibiotics by the composition

[0108] Five-week-old male C57BL / 6J mice (20±2g, SPF grade) were purchased from Beijing Vital River Biotechnology Co., Ltd. The animals were housed under controlled conditions: temperature (25±2℃), humidity (50±10%), and a 12-hour light / dark cycle.

[0109] After a one-week adaptation period, the animals were divided into 8 groups, with 8 animals in each group: blank control group, model group, and experimental group (Example 4, Comparative Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 4 and Comparative Example 5).

[0110] Except for the blank control group, the other groups were given antibiotic saline dispersion by gavage at 8:00 am and 8:00 pm (dose of lincomycin hydrochloride 0.4 g / kg·bw·time and cefadroxil 0.4 g / kg·bw·time) for 5 consecutive days. The blank control group was given an equal volume of saline by gavage.

[0111] 24 hours after the last oral administration of antibiotics, the experimental group was given the corresponding combination at a dose of 200 mg / kg, while the blank control group and the model group were given an equal volume of physiological saline. The administration was performed once a day for 7 consecutive days.

[0112] Twenty-four hours after the last gavage, mice were euthanized by cervical dislocation and immediately dissected aseptically in a laminar flow hood. The intact intestinal tissue was removed, and the ileocecal junction was separated and longitudinally incised with sterile surgical scissors. The contents of the ileocecal junction were collected by rinsing with a sterile inoculation loop or sterile saline and immediately placed in a pre-filled container of 2 mL of sterile, pre-reduced phosphate buffer (pH 7.2), and accurately weighed. Subsequently, the mixture was vigorously vortexed for 3 minutes to thoroughly mix and form a homogeneous fecal suspension.

[0113] Perform a 10-fold serial dilution of the fecal suspension. Take nine test tubes and add 9 mL of sterile saline or PBS to each. Add 1 mL of the homogeneous suspension to the first 9 mL dilution and mix well to obtain the 10-fold serial dilution. -1 Dilution. Add 1 mL from this tube to the next dilution tube, mix well, and the result is 10:10.-2 Dilution. And so on, usually diluted to 10⁻⁸ to ensure countable colonies (ideally 30-300 colonies per plate).

[0114] Bifidobacteria were cultured in BS selective medium with the addition of mupirocin (50 μg / mL) and gentamicin sulfate (50 μg / mL) antibiotics to inhibit the growth of other bacteria. The culture was then placed in an anaerobic chamber and strictly anaerobic at 37°C for 72 h.

[0115] Lactobacillus was cultured on LBS agar medium at 37°C for 48 hours.

[0116] After the culture is completed, select plates with 30-300 single colonies for counting.

[0117] Colony identification: Bifidobacterium colonies are typically milky white, round, raised, with a smooth, moist surface and regular edges. Lactobacillus colonies on LBS plates are typically milky white or pale yellow, round, opaque small colonies.

[0118] Colony count is expressed as the logarithm of colony-forming units per gram of ileocecal contents (lg). CFU / g) indicates that the specific results are shown in Table 1.

[0119] Bacterial count (CFU / g) = Plate colony count × Dilution factor / (Inoculation volume × Sample weight / Total volume of diluent).

[0120] Table 1. Logarithmic values ​​of colony-forming units in different groups of mice.

[0121] Group <![CDATA[Bifidobacterium (lg CFU / g)]]> <![CDATA[Lactobacillus (lg CFU / g)]]> Blank control group 9.21 8.82 Model group 7.59 7.19 Example 4 group 9.07 8.73 Comparative Example 1 8.02 7.68 Comparative Example 2 8.24 7.90 Comparative Example 3 Groups 8.38 8.16 Comparative Example 4 Groups 8.50 8.25 Comparative Example 5 Groups 8.66 8.37

[0122] Table 1 shows that the number of Bifidobacteria and Lactobacilli in the ileocecal intestinal tract was significantly lower in the model group compared to the blank control group, indicating that the mouse model of antibiotic-induced intestinal flora imbalance was successfully established. The experimental groups showed varying degrees of increase in the number of Bifidobacteria and Lactobacilli compared to the model group, with the best results observed in Example 4. A comparison between Example 4 and Comparative Examples 1-2 shows that modified Spirulina polysaccharide and Coptis chinensis extract have a synergistic effect; their combined use significantly promotes the colonization of probiotics in the intestine, thereby improving antibiotic-induced intestinal flora imbalance. A comparison between Example 4 and Comparative Example 3 shows that extraction with a eutectic solvent is more effective than ethanol solution in increasing the number of Bifidobacteria and Lactobacilli in the intestine. A comparison between Example 4 and Comparative Examples 4-5 shows that an appropriate mass ratio of modified Spirulina polysaccharide to Coptis chinensis extract is more beneficial in improving intestinal ecological imbalance caused by oral antibiotics.

[0123] 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 method for preparing modified spirulina polysaccharide with the function of regulating intestinal homeostasis, characterized in that, include: Spirulina polysaccharide was mixed with hydrochloric acid solution to obtain a polysaccharide solution, which was then mixed with sodium selenite and vitamin C. After microwave treatment, the solution was precipitated with alcohol, the precipitate was collected, and dried to obtain modified spirulina polysaccharide.

2. The preparation method according to claim 1, characterized in that, The mass ratio of sodium selenite, spirulina polysaccharide and vitamin C is 1:(8-12):(2-3).

3. The preparation method according to claim 1, characterized in that, The microwave processing power is 300-400W, the temperature is 55-65℃, and the time is 1.5-2.5h.

4. A modified spirulina polysaccharide obtained by the preparation method according to any one of claims 1-3.

5. The application of the modified spirulina polysaccharide according to claim 4 in the preparation of products for preventing and treating intestinal ecological imbalance.

6. The application according to claim 5, characterized in that, The gut dysbiosis mentioned is a gut dysbiosis induced by a high-fat diet.

7. The application of the modified spirulina polysaccharide of claim 4 combined with Coptis chinensis extract in the preparation of products for preventing and treating intestinal ecological imbalance.

8. The application according to claim 7, characterized in that, The Coptis chinensis extract was obtained by extracting Coptis chinensis from a eutectic solvent aqueous solution and purifying it with a macroporous adsorption resin.

9. The application according to claim 8, characterized in that, The weight ratio of the modified spirulina polysaccharide to the Coptis chinensis extract is 1:(2-5).

10. The application according to any one of claims 7-9, characterized in that, The gut dysbiosis mentioned refers to gut dysbiosis caused by oral antibiotics.

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