Preparation method and application of mushroom polysaccharide preparation for regulating intestinal flora of obese people
By using the gradient alcohol precipitation method to grade Lentinan, a stable mushroom polysaccharide preparation was prepared, which solved the problem of Lentinan molecular weight degradation, achieved effective regulation of the intestinal flora in obese people, and reduced the risk of obesity.
Patent Information
- Application Number
- CN202510843333.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-23
- Publication Date
- 2025-09-16
AI Technical Summary
Existing technologies make it difficult to degrade the molecular weight of Lentinan to an appropriate range through simple and green methods, resulting in insufficient biological activity or excessive degradation, and unable to effectively regulate the intestinal flora of obese people.
Lentinus edodes polysaccharide was fractionated by gradient alcohol precipitation. By controlling the ethanol concentration and centrifugation steps, Lentinus edodes subfractions of different molecular weights were obtained, and a stable and soluble mushroom polysaccharide preparation was prepared.
It effectively regulates the intestinal flora structure of obese people, inhibits the growth of Fusobacterium, promotes the growth of Firmicutes, reduces the ratio of Firmicutes/Bacteroidetes, reduces energy absorption, reduces the risk of obesity, and provides natural health products.
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Figure CN120643591A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of food and pharmaceutical preparations, and in particular to a preparation method and application of a mushroom polysaccharide preparation for regulating intestinal flora in obese people. Background Art
[0002] Current research suggests that diet-induced obesity may be linked to dysbiosis in the gut microbiome. An unbalanced gut microbiome disrupts energy metabolism, thereby promoting the development of obesity. Studies have found that functionally active factors in food, such as polysaccharides, polyphenols, and alkaloids, can regulate the gut microbiome and improve intestinal health, providing new insights into regulating the gut microbiome.
[0003] Shiitake mushrooms, a fungus with both medicinal and edible properties, are rich in various nutrients and unique flavor components. Their high nutritional value makes them highly sought after in the food and pharmaceutical sectors. Lentinan, in particular, has become a research hotspot in both the food and pharmaceutical industries due to its diverse and important bioactive properties. However, the bioactivity of polysaccharides is closely linked to their chemical composition, chain conformation, and molecular weight. Relative molecular weight is a key factor influencing their bioactivity. For example, lower molecular weight polysaccharides enhance immunomodulatory activity, while higher molecular weight polysaccharides contribute to enhanced anti-tumor activity.
[0004] Degrading high-molecular-weight polysaccharides to low molecular weight through physical, chemical, or enzymatic treatment can significantly enhance their bioactivity. However, when the molecular weight is too low, the polysaccharide may not form a specific structure with biological activity, which will also have a negative effect on the bioactivity of the polysaccharide. Therefore, it is very important to find a simple and green fractionation method to quickly obtain the desired molecular weight of Lentinan. Summary of the Invention
[0005] The present invention aims to overcome the shortcomings of the existing technology and provides a method for preparing and using a mushroom polysaccharide preparation for regulating the intestinal flora of obese people. The mushroom polysaccharide preparation prepared using the present method can effectively improve the bacterial composition of obese people, particularly reducing the presence of Firmicutes and Bacteroidetes, thereby reducing energy absorption in obese people and thus reducing the risk of obesity.
[0006] To achieve the above purpose, the technical solution designed by the present invention is as follows: The present invention provides a method for preparing a mushroom polysaccharide preparation for regulating the intestinal flora of obese people, comprising the following steps: (1) Drying the shiitake mushrooms, crushing them, and sieving them to obtain shiitake mushroom powder; (2) Dissolve the mushroom powder in ethanol solution, sonicate, and centrifuge to obtain precipitate 1; (3) Add water to dissolve the precipitate 1, centrifuge to obtain the supernatant 1, add n-butanol to the supernatant 1 to obtain a rotary evaporation solution; (4) Rotary evaporate the solution, centrifuge to obtain supernatant 2, add anhydrous ethanol to supernatant 2, let it stand, and centrifuge to obtain precipitate 2; (5) Washing the precipitate 2 with anhydrous ethanol and vacuum drying to obtain a powder; (6) Dissolving the powder in water, removing free protein by Sevage method, removing the remaining Sevage reagent by vacuum evaporation, dialyzing the solution to obtain an aqueous phase, and freeze-drying the aqueous phase to obtain water-soluble crude polysaccharide of Lentinus edodes; (7) The crude water-soluble polysaccharide of Lentinus edodes is dissolved in water to obtain a Lentinus edodes polysaccharide solution. Anhydrous ethanol is added to the Lentinus edodes polysaccharide solution to perform alcohol precipitation to obtain a precipitate, which is a Lentinus edodes polysaccharide preparation that regulates the intestinal flora of obese people.
[0007] Furthermore, in the step (1), the drying temperature is 50-60° C., the product is dried to a constant weight, and the sieve mesh size is 20-120 meshes.
[0008] Furthermore, in step (2), the concentration of the ethanol solution is 95%, the material-liquid ratio of the mushroom powder to the ethanol solution is 1:10-30 g / mL, and the ultrasonic time is 1-3 h.
[0009] Furthermore, in step (3), the water temperature is 80-100°C, the solid-liquid ratio of the precipitate 1 to water is 1:10-40 g / mL, the dissolution time is 1-3 h, and the volume ratio of the supernatant 1 to n-butanol is 1:0.1-0.2.
[0010] Furthermore, in the step (4), the temperature of the rotary evaporation is 60-65°C, and the rotary evaporation is performed until the volume of the solution is 1 / 5 of the volume of the rotary evaporation solution; The volume ratio of supernatant 2 to anhydrous ethanol is 1:4-6.
[0011] Furthermore, in step (6), The material-liquid ratio of powder to water is 1-10:1 mg / mL; Sevage reagent is a chloroform-n-butanol mixed solution, in which the volume ratio of chloroform to n-butanol is 4-5:1; The dialysis temperature is 4~6℃.
[0012] Furthermore, in step (7), the concentration of the lentinan solution is 5-20 mg / mL; Add anhydrous ethanol to a final concentration of 40-80% ethanol in the solution.
[0013] Furthermore, the concentration of the lentinan solution is 10 mg / mL; Anhydrous ethanol was added to the solution until the final concentration of ethanol was 80%.
[0014] The present invention also provides a mushroom polysaccharide preparation prepared by the preparation method for regulating the intestinal flora of obese people.
[0015] The present invention also provides an application of the mushroom polysaccharide preparation in preparing probiotic products or regulating intestinal flora of a population.
[0016] Principle of the present invention: Gradient alcohol precipitation is a method for fractionating polysaccharides, narrowing the molecular weight (MW) distribution. Compared to conventional techniques such as ultrafiltration and chromatography, this method is relatively inexpensive and simple. Polysaccharides form intermolecular hydrogen bonds in aqueous solution, linking and aggregating the polysaccharide molecules. Adding ethanol to a polysaccharide solution initially dehydrates the polysaccharide molecules, strengthening intramolecular hydrogen bonds, and causing conformational changes and aggregation. Different concentrations of ethanol can precipitate polysaccharide fractions of varying molecular weights. In summary, the present invention utilizes gradient alcohol precipitation of crude Lentinus edodes polysaccharides to obtain Lentinus edodes subfractions of desired molecular weights. These fractions not only exhibit good stability and can be used in the production of functional products such as health beverages, but can also effectively improve the microbial structure of obese individuals and increase intestinal microbial diversity, thereby enhancing nutrient absorption and energy metabolism, providing a new approach for alleviating obesity.
[0017] Beneficial effects of the present invention: This invention uses mushrooms as raw materials and aims to obtain different subfractions of lentinan through gradient alcohol precipitation. These subfractions not only exhibit excellent stability and solubility but also effectively regulate the intestinal flora structure of obese individuals, inhibiting the growth of Fusobacteria, promoting the growth of Firmicutes, and reducing the Firmicutes / Bacteroidetes (F / B) population. This reduces energy absorption in obese individuals and thus reduces the risk of obesity. This provides a new direction for functional products and a complete solution for the food industry to develop natural and healthy products that meet modern consumer needs. BRIEF DESCRIPTION OF THE DRAWINGS
[0018] Figure 1 This is the microbial community composition diagram at the post-fermentation level after 48 h; Figure 2 This is the microbial community composition at the genus level after 48 h of fermentation; Figure 3 This is a heat map analysis of the relative abundance of different groups of microorganisms at the genus level. DETAILED DESCRIPTION
[0019] The present invention is further described in detail below with reference to specific embodiments so that those skilled in the art can understand.
[0020] Example 1 A method for preparing a mushroom polysaccharide preparation for regulating intestinal flora in obese people comprises the following steps: (1) Using shiitake mushrooms as raw materials, dry them at 50℃ to constant weight, grind them through an 80-mesh sieve, and obtain shiitake mushroom powder.
[0021] (2) Dissolve the mushroom powder in 95% ethanol solution with a solid-liquid ratio of 1:10~30g / mL, ultrasonicate for 1~3h, and centrifuge at 10000×g for 10min to obtain the precipitate 1.
[0022] (3) Dissolve the precipitate 1 in heated water (80-100°C) for 1-3 h at a solid-liquid ratio of 1:10-40 g / mL. Centrifuge at 10,000 × g to obtain the supernatant 1. Add n-butanol at a volume of 1 / 10-1 / 5 of the volume of the supernatant 1 to obtain a rotary evaporation solution.
[0023] (4) The evaporation solution was evaporated at 60°C until the volume was 1 / 5 of the volume of the evaporation solution, and then the insoluble matter was removed by centrifugation. The supernatant 2 was taken, and anhydrous ethanol with a volume 4 to 6 times that of the supernatant 2 was added to the supernatant 2 and allowed to stand overnight. The precipitate 2 was then centrifuged to take out.
[0024] (5) The precipitate was washed three times with anhydrous ethanol and then vacuum-dried to obtain a powder.
[0025] (6) The powder was dissolved in distilled water at a solid-liquid ratio of 1-10:1 mg / mL. Free protein was removed by Sevage method. The remaining Sevage reagent was removed by vacuum evaporation. The solution was dialyzed at 4°C to obtain an aqueous phase. The aqueous phase was freeze-dried to obtain water-soluble crude polysaccharides of Lentinus edodes. The Sevage reagent was a chloroform-n-butanol mixed solution (the volume ratio of chloroform and n-butanol was 4-5:1).
[0026] (7) Dissolve the crude water-soluble polysaccharide of Lentinus edodes in distilled water to obtain a 10 mg / mL Lentinus edodes polysaccharide solution. Slowly add anhydrous ethanol to the Lentinus edodes polysaccharide solution until the final ethanol concentration in the solution is 40-80%. Perform alcohol precipitation. The resulting precipitate is a mushroom polysaccharide preparation for regulating the intestinal flora of obese people.
[0027] Example 2 A method for preparing a mushroom polysaccharide preparation for regulating the intestinal flora of obese people. The preparation method of this embodiment is the same as that of Example 1, with the only difference being that in step (1), the polysaccharide is crushed through a 120-mesh sieve.
[0028] Example 3 A method for preparing a mushroom polysaccharide preparation for regulating intestinal flora in obese people comprises the following steps: (1) Using shiitake mushrooms as raw materials, dry them at 50℃ to constant weight, grind them through a 120-mesh sieve, and obtain shiitake mushroom powder.
[0029] (2) Dissolve the mushroom powder in 95% ethanol solution with a solid-liquid ratio of 1:10 g / mL, sonicate for 1 h, and centrifuge at 10,000 × g for 10 min to obtain the precipitate.
[0030] (3) Dissolve the precipitate 1 in heated water (80-100°C) for 2 h at a solid-liquid ratio of 1:20 g / mL. Centrifuge at 10,000 × g to obtain the supernatant 1. Add n-butanol at a volume of 1 / 10 of the supernatant 1 to obtain a rotary evaporation solution.
[0031] (4) The evaporation solution was evaporated at 60°C until the volume was 1 / 5 of the volume of the evaporation solution, and then the insoluble matter was removed by centrifugation. The supernatant 2 was taken, and anhydrous ethanol with a volume 4 to 6 times that of the supernatant 2 was added to the supernatant 2 and allowed to stand overnight. The precipitate 2 was then centrifuged to take out.
[0032] (5) The precipitate was washed three times with anhydrous ethanol and then vacuum-dried to obtain a powder.
[0033] (6) The powder was dissolved in distilled water at a solid-liquid ratio of 5:1 mg / mL. Free protein was removed by Sevage method. The remaining Sevage reagent was removed by vacuum evaporation. The solution was dialyzed at 4°C to obtain an aqueous phase. The aqueous phase was freeze-dried to obtain water-soluble crude polysaccharides of Lentinus edodes.
[0034] (7) Dissolve the crude water-soluble polysaccharide of Lentinus edodes in distilled water to obtain a 10 mg / mL Lentinus edodes polysaccharide solution. Slowly add anhydrous ethanol to the Lentinus edodes polysaccharide solution until the final ethanol concentration in the solution is 40-80%. Perform alcohol precipitation. The resulting precipitate is a mushroom polysaccharide preparation for regulating the intestinal flora of obese people.
[0035] Example 4 A method for preparing a mushroom polysaccharide preparation for regulating the intestinal flora of obese people. The preparation method of this embodiment is the same as that of Example 3, with the only difference being that in step (4), the volume of anhydrous ethanol added to the supernatant 2 is 4 times the volume of the supernatant 2.
[0036] Example 5 A method for preparing a mushroom polysaccharide preparation for regulating intestinal flora in obese people comprises the following steps: (1) Using shiitake mushrooms as raw materials, dry them at 50℃ to constant weight, grind them through a 120-mesh sieve, and obtain shiitake mushroom powder.
[0037] (2) Dissolve the mushroom powder in 95% ethanol solution with a solid-liquid ratio of 1:10 g / mL, sonicate for 1 h, and centrifuge at 10,000 × g for 10 min to obtain the precipitate.
[0038] (3) Dissolve the precipitate 1 in heated water (80-100°C) for 2 h at a solid-liquid ratio of 1:20 g / mL. Centrifuge at 10,000 × g to obtain the supernatant 1. Add n-butanol at a volume of 1 / 10 of the supernatant 1 to obtain a rotary evaporation solution.
[0039] (4) The rotary evaporation solution was evaporated at 60°C to a volume of 1 / 5 of the volume of the rotary evaporation solution, and then the insoluble matter was removed by centrifugation. The supernatant 2 was taken, and anhydrous ethanol with a volume 4 times that of the supernatant 2 was added to the supernatant 2 and allowed to stand overnight. The precipitate 2 was then centrifuged to take out.
[0040] (5) The precipitate was washed three times with anhydrous ethanol and then vacuum-dried to obtain a powder.
[0041] (6) The powder was dissolved in distilled water at a solid-liquid ratio of 5:1 mg / mL. Free protein was removed by Sevage method. The remaining Sevage reagent was removed by vacuum evaporation. The solution was dialyzed at 4°C to obtain an aqueous phase. The aqueous phase was freeze-dried to obtain water-soluble crude polysaccharides of Lentinus edodes.
[0042] (7) Dissolve the crude water-soluble polysaccharide of Lentinus edodes in distilled water to obtain a 10 mg / mL Lentinus edodes polysaccharide solution. Slowly add anhydrous ethanol to the Lentinus edodes polysaccharide solution until the final ethanol concentration in the solution reaches 40%. Perform alcohol precipitation. The resulting precipitate is a mushroom polysaccharide preparation for regulating the intestinal flora of obese people, named WSL-40.
[0043] Example 6 A method for preparing a mushroom polysaccharide preparation for regulating intestinal flora in obese people comprises the following steps: (1) Using shiitake mushrooms as raw materials, dry them at 50℃ to constant weight, grind them through a 120-mesh sieve, and obtain shiitake mushroom powder.
[0044] (2) Dissolve the mushroom powder in 95% ethanol solution with a solid-liquid ratio of 1:10 g / mL, sonicate for 1 h, and centrifuge at 10,000 × g for 10 min to obtain the precipitate.
[0045] (3) Dissolve the precipitate 1 in heated water (80-100°C) for 2 h at a solid-liquid ratio of 1:20 g / mL. Centrifuge at 10,000 × g to obtain the supernatant 1. Add n-butanol at a volume of 1 / 10 of the supernatant 1 to obtain a rotary evaporation solution.
[0046] (4) The rotary evaporation solution was evaporated at 60°C to a volume of 1 / 5 of the volume of the rotary evaporation solution, and then the insoluble matter was removed by centrifugation. The supernatant 2 was taken, and anhydrous ethanol with a volume 4 times that of the supernatant 2 was added to the supernatant 2 and allowed to stand overnight. The precipitate 2 was then centrifuged to take out.
[0047] (5) The precipitate was washed three times with anhydrous ethanol and then vacuum-dried to obtain a powder.
[0048] (6) The powder was dissolved in distilled water at a solid-liquid ratio of 5:1 mg / mL. Free protein was removed by Sevage method. The remaining Sevage reagent was removed by vacuum evaporation. The solution was dialyzed at 4°C to obtain an aqueous phase. The aqueous phase was freeze-dried to obtain water-soluble crude polysaccharides of Lentinus edodes.
[0049] (7) Dissolve the crude water-soluble polysaccharide of Lentinus edodes in distilled water to obtain a 10 mg / mL Lentinus edodes polysaccharide solution. Slowly add anhydrous ethanol to the Lentinus edodes polysaccharide solution until the final ethanol concentration in the solution reaches 60%. Perform alcohol precipitation. The resulting precipitate is a mushroom polysaccharide preparation for regulating the intestinal flora of obese people, named WSL-60.
[0050] Example 7 A method for preparing a mushroom polysaccharide preparation for regulating intestinal flora in obese people comprises the following steps: (1) Using shiitake mushrooms as raw materials, dry them at 50℃ to constant weight, grind them through a 120-mesh sieve, and obtain shiitake mushroom powder.
[0051] (2) Dissolve the mushroom powder in 95% ethanol solution with a solid-liquid ratio of 1:10 g / mL, sonicate for 1 h, and centrifuge at 10,000 × g for 10 min to obtain the precipitate.
[0052] (3) Dissolve the precipitate 1 in heated water (80-100°C) for 2 h at a solid-liquid ratio of 1:20 g / mL. Centrifuge at 10,000 × g to obtain the supernatant 1. Add n-butanol at a volume of 1 / 10 of the supernatant 1 to obtain a rotary evaporation solution.
[0053] (4) The rotary evaporation solution was evaporated at 60°C to a volume of 1 / 5 of the volume of the rotary evaporation solution, and then the insoluble matter was removed by centrifugation. The supernatant 2 was taken, and anhydrous ethanol with a volume 4 times that of the supernatant 2 was added to the supernatant 2 and allowed to stand overnight. The precipitate 2 was then centrifuged to take out.
[0054] (5) The precipitate was washed three times with anhydrous ethanol and then vacuum-dried to obtain a powder.
[0055] (6) The powder was dissolved in distilled water at a solid-liquid ratio of 5:1 mg / mL. Free protein was removed by Sevage method. The remaining Sevage reagent was removed by vacuum evaporation. The solution was dialyzed at 4°C to obtain an aqueous phase. The aqueous phase was freeze-dried to obtain water-soluble crude polysaccharides of Lentinus edodes.
[0056] (7) Dissolve the crude water-soluble polysaccharide of Lentinus edodes in distilled water to obtain a 10 mg / mL Lentinus edodes polysaccharide solution. Slowly add anhydrous ethanol to the Lentinus edodes polysaccharide solution until the final ethanol concentration in the solution reaches 80%. Perform alcohol precipitation. The resulting precipitate is a mushroom polysaccharide preparation for regulating the intestinal flora of obese people, named WSL-80.
[0057] Example 8 Determination of Molecular Weight of Lentinan in Different Subfractions Molecular weight was determined using SEC-MALLS-RI tandem analysis. Samples were WSL-40, WSL-60, and WSL-80 from Examples 5–8, filtered through a 0.45 μm filter membrane before injection. The column oven temperature was 35°C, the injection volume was 600 μL, the flow rate was 0.4 mL / min, and the refractive index (dn / dc) was 0.146. Normalization was performed using dextran with a Mw of 40,000. Data processing and graphical analysis were performed using ASTRA software (Version 6.1.1.84).
[0058] The results are shown in Table 1. The polydispersity coefficient (Mw / Mn) can be used to determine the uniformity of the molecular weight distribution within a sample. The closer the value is to 1, the more uniform the molecular weight distribution. The molecular weight distribution of WSL-40 and WSL-80 is relatively uniform, while the molecular weight distribution of WSL-60 is relatively dispersed.
[0059] Table 1 Molecular weight of Lentinan from different subfractions Example 9 In vitro fermentation test of mushroom polysaccharide preparations for regulating intestinal flora in obese people The fermentation medium formula was as follows: 4.5 g / L yeast extract, 3.0 g / L tryptone, 3.0 g / L peptone, 0.5 g / L mucin, 0.4 g / L porcine bile salt, 0.8 g / L L-cystine hydrochloride, 4.5 g / L NaCl, 2.5 g / L KCl, 0.45 g / L MgCl2·6H2O, 0.2 g / L CaCl2, 0.4 g / L KH2PO4, 0.05 g / L hemin, 2 mg / L vitamin K1, 1 mL / L Tween 80, and 100 μL 1% resazurin. The initial pH was adjusted to 6.5.
[0060] Fresh feces were collected from four healthy-weight volunteers (two men and two women with a BMI of 18.5 to 23.9 and ages 20 to 25) and four obese-weight volunteers (two men and two women with a BMI of 28 or higher and ages 20 to 25). All volunteers had no history of antibiotic or probiotic treatment for at least three months. The volunteers excreted feces onto sterile medical pads to minimize contamination of the fecal samples by urine, toilet bowl walls, etc. A sterile spoon was used to scoop an equal amount of feces from the middle inner part of the sample and placed in a sterile preservation tube for low-temperature storage at -80°C. The fecal samples from the four healthy-weight volunteers were mixed in equal amounts to obtain a mixed feces sample for healthy-weight individuals, and the fecal samples from the four obese volunteers were mixed in equal amounts to obtain a mixed feces sample for obese individuals.
[0061] In a laminar flow hood, 4 g of mixed feces from healthy individuals and 4 g of mixed feces from obese individuals (equal amounts from each donor) were weighed and thoroughly mixed with 36 mL of sterile PBS buffer (0.1 M, pH 7.4) to obtain fecal slurry suspensions. The fecal slurry suspensions were centrifuged at 500 rpm for 5 minutes, and insoluble particles were discarded to obtain fecal slurries from healthy individuals and obese individuals, respectively.
[0062] 100 mg of each of WSL-40, WSL-60, WSL-80, and inulin (positive control) were dissolved in 27 mL of autoclaved fermentation medium. A blank control was also used, using fermentation medium without an additional carbon source. 3.0 mL of 10% fecal slurry from healthy individuals and 3.0 mL of fecal slurry from obese individuals were added to the fermentation medium, respectively. The mixture was then fermented at 37°C under an anaerobic environment for 48 hours. Three replicates were run in three groups, each with three replicates. The experimental groups were set up as follows: (1) Fecal slurry from healthy weight individuals was added to the fermentation medium containing WSL-40, which was experimental group 1 (A_LEP40); (2) Fecal slurry from healthy weight people was added to the fermentation medium containing WSL-60, which was experimental group 2 (A_LEP60); (3) Fecal slurry from healthy weight people was added to the fermentation medium containing WSL-80, which was experimental group 3 (A_LEP80); (4) Fecal slurry from obese people was added to the fermentation medium containing WSL-40, which was experimental group 4 (B_LEP40); (5) Fecal slurry from obese people was added to the fermentation medium containing WSL-60, which was experimental group 5 (B_LEP60); (6) Fecal slurry from obese people was added to the fermentation medium containing WSL-80, which was experimental group 6 (B_LEP80); (7) Fecal slurry from healthy weight people was added to the fermentation medium containing inulin, which served as control group 1 (A_Inulin); (8) Fecal slurry from obese people was added to the fermentation medium containing inulin, which served as control group 2 (B_Inulin); (9) Fecal slurry from healthy weight people was added to the fermentation medium, which was blank group 1 (A_Blank); (10) Fecal slurry from obese people was added to the fermentation medium, which was the blank group 2 (B_Blank).
[0063] Fermentation products were collected after 0, 6, 12, 24, and 48 hours, bacterial genomic DNA was extracted from the fermentation products, and the 16S rRNA gene V3-V4 variable region was amplified. In the β-diversity study, weighted and unweighted unifrac distances were used to measure the differences in species diversity between samples.
[0064] Fusobacteria are mainly concentrated in the human mouth and colon, where they may cause inflammation and disease. Firmicutes, which include many probiotics such as butyrate-producing bacteria and lactic acid bacteria, are closely related to the metabolism of obese patients. Figure 1 As shown, WSL-40, WSL-60, WSL-80, and inulin all effectively inhibited the growth of Fusobacteria and promoted the growth of Firmicutes. Furthermore, WSL-40, WSL-60, and WSL-80 promoted the growth of Bacteroidetes, with their relative abundance increasing by 0.9%, 10.1%, and 10.3%, respectively, in the healthy weight group and by 6.4%, 12.4%, and 12.8%, respectively, in the obese group compared to the control group. A reduction in the F / B ratio (the ratio of Firmicutes to Bacteroidetes) may reduce energy absorption and help mitigate the risk of obesity. The F / B ratios of A_LEP60 and A_LEP80 were effectively reduced to 1.57 and 1.45, respectively, compared to 1.84 in the control group. In obese individuals, the F / B ratios of B_LEP40, B_LEP60, and B_LEP60 were 2.15, 1.04, and 0.93, respectively, compared to 4.82 in the control group, indicating a significant reduction in F / B ratios (p<0.05). Compared to healthy weight individuals, the three fractions of Lentinus edodes water-soluble polysaccharides were more effective in regulating the F / B ratio in the gut microbiota of obese individuals. Among them, the lower molecular weight WSL-80 had the greatest effect on reducing the F / B ratio.
[0065] like Figure 2As shown, the blank groups of healthy and obese people were mainly composed of Bacteroides and Fusobacterium ( Fusobacterium ), Enterococcus spp. ( Enterococcus ) and Klebsiella spp. ( Klebsiella ) composition. Bifidobacterium spp. ( Bifidobacterium ) has multiple physiological functions, such as enhancing intestinal immunity, anti-tumor and reducing intestinal pathogen infection. It is also the main producer of acetic acid and has potential benefits for intestinal diseases and obesity. WSL-40, WSL-60, WSL-80 and inulin all promote the growth of Bifidobacterium in human intestinal microorganisms. In both populations, the LEP80 group showed a higher abundance of Bifidobacterium than the LEP40 and LEP60 groups. Parabacteroides ( Parabacteroides ) helps digest high-fiber diets and dominates the intestinal tract. This commensal bacterium has demonstrated significant regulatory effects on the host mucosal immune system, effectively alleviating inflammatory responses and actively participating in amino acid metabolism. Notably, Parabacteroides possesses multiple polysaccharide utilization sites, displaying similar polysaccharide degradation potential as Bacteroides, and is capable of efficiently degrading a variety of complex polysaccharides. This ability may give it an advantage in competition with other microorganisms.
[0066] Figure 3 The heatmap in Figure 2 shows the relative abundance of the top 35 species-level microorganisms in different groups. In healthy-weight individuals, the five dominant species in the control group were Fusobacterium, Bacteroides, Parabacteroides, Klebsiella, and Erysipelothrix. WSL-40, WSL-60, and WSL-80 increased the relative abundance of Weissella, some Bacteroides, Bacteroides microti, and Bifidobacterium. Lactococcus gasseri, Enterococcus faecium, Fusobacterium ulcerans, and Actinomyces dominated in obese individuals. WSL-40, WSL-60, and WSL-80 increased the relative abundance of Bacteroides acidophilus, Bacteroides monothecae, Bacteroides thetaiotaomicron, Parabacteroides, Clostridium butyricum, and Phascolarctobacterium.
[0067] Although the above embodiments have been described in detail, they are only a part of the embodiments of the present invention, not all of them. People can also obtain other embodiments based on this embodiment without inventiveness, and these embodiments all fall within the scope of protection of the present invention.
Claims
1. A method for preparing a mushroom polysaccharide preparation for regulating intestinal flora in obese people, characterized by: The following steps are involved: (1) Drying the shiitake mushrooms, crushing them, and sieving them to obtain shiitake mushroom powder; (2) Dissolve the mushroom powder in ethanol solution, sonicate, and centrifuge to obtain precipitate 1; (3) Add water to dissolve the precipitate 1, centrifuge to obtain the supernatant 1, add n-butanol to the supernatant 1 to obtain a rotary evaporation solution; (4) Rotary evaporate the solution, centrifuge to obtain supernatant 2, add anhydrous ethanol to supernatant 2, let it stand, and centrifuge to obtain precipitate 2; (5) Washing the precipitate 2 with anhydrous ethanol and vacuum drying to obtain a powder; (6) Dissolving the powder in water, removing free protein by the Sevage method, removing the Sevage reagent by vacuum evaporation, dialyzing the solution to obtain an aqueous phase, and freeze-drying the aqueous phase to obtain water-soluble crude polysaccharides of Lentinus edodes; (7) The crude water-soluble polysaccharide of Lentinus edodes is dissolved in water to obtain a Lentinus edodes polysaccharide solution. Anhydrous ethanol is added to the Lentinus edodes polysaccharide solution to perform alcohol precipitation to obtain a precipitate, which is a Lentinus edodes polysaccharide preparation that regulates the intestinal flora of obese people.
2. The preparation method according to claim 1, wherein: In the step (1), the drying temperature is 50-60°C, the product is dried to a constant weight, and the sieve mesh size is 20-120 meshes.
3. The preparation method according to claim 1, wherein: In the step (2), the concentration of the ethanol solution is 95%, the material-liquid ratio of the mushroom powder to the ethanol solution is 1:10-30 g / mL, and the ultrasonic time is 1-3 h.
4. The preparation method according to claim 1, wherein: In the step (3), the water temperature is 80-100° C., the solid-liquid ratio of the precipitate 1 to water is 1:10-40 g / mL, the dissolution time is 1-3 h, and the volume ratio of the supernatant 1 to n-butanol is 1:0.1-0.
2.
5. The preparation method according to claim 1, wherein: In the step (4), the temperature of the rotary evaporation is 60-65°C, and the rotary evaporation is performed until the volume of the solution is 1 / 5 of the volume of the rotary evaporation solution; The volume ratio of supernatant 2 to anhydrous ethanol is 1:4-6.
6. The preparation method according to claim 1, wherein: In step (6), the material-liquid ratio of powder to water is 1-10:1 mg / mL; Sevage reagent is a chloroform-n-butanol mixed solution, in which the volume ratio of chloroform to n-butanol is 4-5:1; The dialysis temperature is 4~6℃.
7. The preparation method according to claim 1, wherein: In step (7), the concentration of the lentinan solution is 5-20 mg / mL; Add anhydrous ethanol to a final concentration of 40-80% ethanol in the solution.
8. The preparation method according to claim 7, characterized in that: The concentration of the lentinan solution is 10 mg / mL; Anhydrous ethanol was added to the solution until the final concentration of ethanol was 80%.
9. A mushroom polysaccharide preparation for regulating intestinal flora in obese people prepared by the preparation method according to claim 1.
10. Use of the mushroom polysaccharide preparation according to claim 9 in preparing probiotic products or regulating intestinal flora of humans.