Preparation method for improving biological activity of lentinus edodes stem polysaccharide and application of lentinus edodes stem polysaccharide

Abstract

The invention relates to the technical field of synthetic biology, and particularly discloses a preparation method for improving the biological activity of lentinus edodes stem polysaccharide and application. The method adopts Schizophyllumcommuneh to perform solid state fermentation on lentinus edodes stems, and comprises the following steps: S1, grinding the lentinus edodes stems into powder, then adding a proper amount of water, uniformly stirring, performing high-pressure sterilization, and cooling to obtain a lentinus edodes stem culture medium; s2, activating a Schizophyllumcommuneh strain, preparing a Schizophyllumcommuneh seed solution, and adding the Schizophyllumcommuneh seed solution into the lentinus edodes stem culture medium according to a certain inoculum size for solid-state fermentation; and S3, taking the fermented lentinus edodes stem culture medium, carrying out protease enzymolysis, carrying out ethanol precipitation, carrying out centrifugation to obtain a precipitate, redissolving the precipitate, carrying out dialysis treatment with a dialysis bag, and freeze-drying the dialyzed solution to obtain the lentinus edodes stem polysaccharide. According to the preparation method, the yield and the purity of the lentinan are remarkably improved, meanwhile, the oxidation resistance, the inflammation resistance and the pancreatic lipase removing capacity are remarkably improved, and the lentinan has the effect of moistening the intestines.

Description

Technical Field

[0001] The invention belongs to the technical field of synthetic biology, and specifically relates to a preparation method and application of improving the biological activity of Lentinus edodes petiole polysaccharide. Background Art

[0002] Shiitake mushroom stalks are byproducts produced during the production of shiitake mushroom products, accounting for 25%-30% of the dry weight of shiitake mushrooms. A small amount of them are made into shiitake mushroom sauce or soup, and the rest are usually used as field compost or directly incinerated, wasting most of the shiitake mushroom stalk resources. But in fact, the nutrients they contain are the same as those of shiitake mushroom caps. They are both composed of shiitake mushroom mycelium and are rich in nutrients such as polysaccharides, proteins, amino acids, vitamins, and mineral elements. Among them, polysaccharides are important active ingredients in shiitake mushrooms. Compared with the extraction and biological activity research of shiitake mushroom polysaccharides, there are fewer reports on the research of shiitake mushroom stalk polysaccharides.

[0003] At present, the extraction of Lentinus edodes stalk polysaccharides is mainly based on hot water extraction, and there are also reports on some enzyme, ultrasound, microwave and other auxiliary extraction methods. There are few reports on the preparation of Lentinus edodes stalk polysaccharides by fermentation. Therefore, how to provide a highly active Lentinus edodes stalk polysaccharide and improve the application value of Lentinus edodes stalks is a technical problem that researchers urgently need to solve. Summary of the invention

[0004] The purpose of the present invention is to address the above-mentioned deficiencies in the prior art and provide a preparation method and application for improving the biological activity of Lentinus edodes stem polysaccharides. Lentinus edodes stem polysaccharides are prepared by solid-state fermentation of Lentinus edodes stems using white ginseng fungus, which significantly improves its antioxidant, anti-inflammatory and pancreatic lipase-scavenging abilities and also has a laxative function.

[0005] To achieve the above object, the present invention adopts the following technical solution:

[0006] The first object of the present invention is to provide a preparation method for improving the biological activity of Lentinus edodes stem polysaccharide, comprising the following steps:

[0007] S1. Preparation of Lentinus edodes stem culture medium

[0008] Grind the mushroom stems into powder, then add appropriate amount of water and stir evenly, sterilize under high pressure, and obtain the mushroom stem culture medium after cooling;

[0009] S2. Inoculation and culture

[0010] Activating the white ginseng fungus strain and preparing a white ginseng fungus seed liquid, and adding the white ginseng fungus seed liquid into the shiitake mushroom stem culture medium according to a certain inoculation amount for solid-state fermentation;

[0011] S3. Extraction of Lentinus edodes stem polysaccharide

[0012] The fermented Lentinus edodes stem culture medium is firstly enzymatically hydrolyzed with protease, then ethanol is used for precipitation, the precipitate is centrifuged, the precipitate is re-dissolved, dialyzed with a dialysis bag, and the dialyzed solution is freeze-dried to obtain Lentinus edodes stem polysaccharide.

[0013] Furthermore, in step S1, the mushroom stems are ground into powder to a particle size of 40 to 50 meshes.

[0014] Furthermore, in step S2, the preparation process of the white ginseng fungus seed liquid is to inoculate the white ginseng fungus strain into the CYM culture medium for activation, inoculate the activated white ginseng fungus strain into the seed culture liquid for shake flask fermentation, take out the bacterial blocks after they are balled, homogenize the seed culture medium and the bacterial balls to obtain the white ginseng fungus seed liquid.

[0015] Furthermore, the inoculation amount of the white ginseng fungus seed liquid inoculated into every 10 g of Lentinus edodes stem culture medium is 10 to 15 mL.

[0016] Furthermore, the volume of water added to every 10 g of Lentinus edodes stem culture medium is 1.5 to 2.5 mL.

[0017] Furthermore, the fermentation temperature is 25-27° C., and the fermentation time is 8-10 days.

[0018] Furthermore, in step S3, the protease is a neutral protease.

[0019] The second object of the present invention is to provide Lentinus edodes stipe polysaccharide prepared by the above-mentioned preparation method.

[0020] The third object of the present invention is to provide the use of Lentinus edodes stipe polysaccharide prepared by the above-mentioned preparation method in the preparation of lipid-lowering products.

[0021] The fourth object of the present invention is to provide the use of the Lentinus edodes petiole polysaccharide prepared by the above-mentioned preparation method in the preparation of intestine-lubricating products.

[0022] Compared with the prior art, the technical solution provided by the present invention has the following beneficial effects:

[0023] (1) The method of solid-state fermentation of Lentinus edodes stem substrate using white ginseng fungus significantly improves the yield and purity of Lentinan polysaccharide, which is beneficial to strengthening the development and utilization of Lentinus edodes stem health care and functional food.

[0024] (2) The antioxidant, anti-inflammatory and pancreatic lipase scavenging abilities of the Lentinus edodes stem polysaccharide prepared by solid-state fermentation of Lentinus edodes stem matrix using white ginseng fungus are significantly improved, and the polysaccharide has a laxative effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a result diagram of the present invention investigating the effects of different edible fungi fermentation on the preparation of polysaccharides from Lentinus edodes stems;

[0026] Figure 2 This is a result diagram of the present invention investigating the effect of fermentation days on the preparation of polysaccharides from Lentinus edodes stems;

[0027] Figure 3 This is a result diagram of the present invention investigating the effect of inoculation amount on the preparation of polysaccharides from Lentinus edodes stems;

[0028] Figure 4 This is a result diagram of the present invention investigating the effect of water addition on the preparation of polysaccharides from Lentinus edodes stems;

[0029] Figure 5 A picture of the solid-state fermentation of shiitake mushroom stems by white ginseng fungus provided by the present invention;

[0030] Figure 6 This is a comparison chart of the scavenging effects of WSP and FSP on DPPH free radicals in the present invention;

[0031] Figure 7 This is a comparison diagram of the effects of WSP and FSP on NO secretion in RAW264.7 cells in the present invention;

[0032] Figure 8 This is a graph showing the effect of FSP on small intestinal motility in the present invention. DETAILED DESCRIPTION

[0033] In order to make the purpose, technical scheme and advantages of the present invention clearer, the specific embodiments of the present invention are further described in detail below in conjunction with specific examples and drawings. If no specific technology or conditions are specified in the examples, the technology or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be obtained commercially.

[0034] The biological materials used in the present invention can all be obtained commercially.

[0035] The edible mushrooms used in the invention, such as morel, tuckahoe, stropharia, straw mushroom, shiitake mushroom, ganoderma lucidum, flower face mushroom, white ginseng mushroom, hericium erinaceus and blood ear mushroom, are all provided by the Institute of Applied Fungi of Huazhong Agricultural University.

[0036] The activation of the fermented edible fungi and the preparation of the seed liquid in the present invention are as follows:

[0037] Take a small piece of the bacteria stored in the test tube and inoculate it on the CYM plate. After the mycelium has grown all over the plate, take a square block with a side length of 7.5-8.0mm and inoculate it on the CYM plate for the second activation. Wait until the mycelium has grown all over the plate. Take 8 square blocks with a side length of 7.5-8.0mm and inoculate them into 100mL seed culture solution (250mL conical flask), place them in a full-temperature shaking incubator with a speed of 120r / min and a temperature of 25℃, and take them out after they become balls. Homogenize the seed culture medium and the balls on the clean bench to make a seed solution of the bacteria, and inoculate them into the solid culture medium of the mushroom stem according to a certain inoculation amount for fermentation.

[0038] CYM medium: peptone 2g, yeast extract powder 2g, MgSO4·7H2O 0.5g, KH2PO4 0.6g, K2HPO4·3H2O 1g, glucose 22g, agar 20g, distilled water 1L, natural pH, high temperature and high pressure sterilization at 121℃ for 20min.

[0039] Seed culture medium: peptone 2g, yeast extract powder 2g, MgSO4·7H2O 0.5g, KH2PO4 0.6g, K2HPO4·3H2O 1g, glucose 22g, distilled water 1L, natural pH, high temperature and high pressure sterilization at 121℃ for 20min.

[0040] The method for purifying the extractant of Lentinus edodes polysaccharide in the present invention is as follows:

[0041] Take a certain amount of sample, add distilled water at a solid-liquid ratio of 1:20 (g / mL), add 0.4% neutral protease (BR, 100u / mg, Bacillus subtilis), and perform enzymolysis in a water bath at pH 7.0 and temperature 50℃ for 1h; then the water temperature is raised to 90℃ and treated for 2h. Centrifuge the enzymatic solution at 4000r / min and 20℃ for 15min, take the supernatant, add 4 times the volume of 95% ethanol, mix well, and precipitate at room temperature for 12h, and centrifuge to obtain the precipitate using the same method as above. Add an appropriate amount of distilled water to re-dissolve the precipitate, add the solution to a 8000-14000 dialysis bag, put distilled water in the dialysis device for 48h dialysis, and replace the distilled water every 6h. The dialyzed solution is freeze-dried to obtain a water-soluble polysaccharide (hereinafter referred to as polysaccharide).

[0042] Calculation of water-soluble polysaccharide yield:

[0043]

[0044] During the research, the inventor accidentally discovered that different edible fungi have different fermentation abilities on shiitake mushroom stems. A certain amount of Morchella, Poria, Stropharia, Volvariella, Shiitake, Ganoderma, Flower Face Mushroom, White Ginseng Mushroom, Hericium erinaceus, and Blood Ear Mushroom were inoculated into the shiitake mushroom stems. It was found that Morchella, Poria, Stropharia, and Flower Face Mushroom could not grow mycelium on the shiitake mushroom stems, while the remaining species could grow within 12 to 44 days, indicating that different edible fungi have great differences in their ability to grow on the solid fermentation medium of shiitake mushroom stems. Figure 1 As shown in the results, after fermentation and extraction, only the yield after fermentation by white ginseng fungus and Hericium erinaceus can be higher than that of the blank group (Lentinus edodes stem polysaccharide prepared without fermentation, hereinafter referred to as WSP), and the yield of polysaccharide (hereinafter referred to as FSP) produced by solid-state fermentation of Lentinus edodes stem matrix by white ginseng fungus is as high as 17.20%, which is 23.74% higher than 13.90% of the blank group. This is because white ginseng fungus has good adaptability, efficient metabolic capacity and superior growth characteristics, which makes it show higher polysaccharide synthesis ability and yield in the solid-state fermentation process. Therefore, white ginseng fungus was selected as the edible and medicinal fungus strain for subsequent solid-state fermentation of Lentinus edodes stem matrix to produce polysaccharides.

[0045] Determination of total sugar:

[0046] The phenol-sulfuric acid method was used to prepare a glucose standard solution with a concentration of 0.1 mg / mL. 0 mL, 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, and 1.0 mL of the standard solution were placed in a test tube, and distilled water was added to make the total volume 2.0 mL. Then 1.0 mL of 5% phenol was added to each tube and shaken well. 5.0 mL of concentrated sulfuric acid was added quickly within 20 seconds, and the mixture was shaken well (tube by tube). After color development at room temperature for 30 minutes, the absorbance value was measured at 490 nm to prepare a standard curve.

[0047] Accurately prepare a polysaccharide sample solution of a certain concentration, determine its absorbance value according to the above method, compare it with the standard curve, and calculate the total sugar content of the sample.

[0048] Example 1

[0049] This embodiment provides a preparation method for improving the biological activity of Lentinus edodes stem polysaccharide. The specific steps are as follows:

[0050] S1. Preparation of Lentinus edodes stem culture medium

[0051] The commercially available dried shiitake edodes stems were ground into powder with a particle size of 40 mesh. 10 g of shiitake edodes stem powder and 25 mL of distilled water were placed in a fermentation tank (150 mL), stirred evenly, and sterilized at 121°C under high pressure for 20 min. After cooling, the shiitake edodes stem culture medium was obtained.

[0052] S2. Inoculation and culture

[0053] A small piece of white ginseng fungus stored in a test tube was inoculated on the CYM plate. After the mycelium grew all over the plate, a square fungus block with a side length of 7.5-8.0 mm was inoculated on the CYM plate for the second activation, and the mycelium grew all over the plate. Eight square fungus blocks with a side length of 7.5-8.0 mm were inoculated into 100 mL seed culture solution (250 mL conical flask), and placed in a full-temperature shaking incubator with a speed of 120 r / min and a temperature of 25 ° C for cultivation. After the fungus blocks were balled, they were taken out, and the seed culture medium and the fungus balls were homogenized on the clean bench to make the fungus seed liquid. According to a certain inoculation amount, it was inoculated into the solid culture medium of Lentinus edodes stems for solid fermentation. Among them, the inoculation amount of white ginseng fungus seed liquid inoculated in every 10g Lentinus edodes stem culture medium was 10 mL, the temperature of solid fermentation was 25 ° C, and the fermentation time was 4d, 6d, 8d, 10d, and 12d.

[0054] S3. Extraction of Lentinus edodes stem polysaccharide

[0055] Take a certain amount of solid-state fermented Lentinus edodes stalk culture medium sample, add distilled water at a material-liquid ratio of 1:20 (g / mL), add 0.4% neutral protease, and perform enzymolysis in a water bath at pH 7.0 and temperature 50℃ for 1h; then the water temperature is raised to 90℃ and treated for 2h. Centrifuge the enzymolysis solution at 4000r / min and 20℃ for 15min, take the supernatant, add 4 times the volume of 95% ethanol, mix well, and precipitate at room temperature for 12h, and centrifuge to obtain the precipitate using the same method as above. Add appropriate amount of distilled water to re-dissolve the precipitate, add the solution to a 8000-14000 dialysis bag, put distilled water in the dialysis device for 48h dialysis, and replace the distilled water every 6h. The dialyzed solution is freeze-dried to obtain water-soluble polysaccharides (hereinafter referred to as polysaccharides).

[0056] refer to Figure 2 Different fermentation days will affect the FSP yield. With the increase of fermentation days, the FSP yield continues to increase, reaching a peak on the 8th day of fermentation, and then with the increase of fermentation days, the FSP yield tends to be flat. It is speculated that the reason is that in the early stage of fermentation, the mycelium of white ginseng fungus continuously accumulates metabolites, which gradually increases the FSP yield. With the extension of fermentation time, the mycelium biomass gradually increases, and the nutrients in the culture medium are not enough to maintain the normal physiological activities of the mycelium, which affects the synthesis of new substances and reduces the FSP yield.

[0057] Example 2

[0058] This embodiment provides a preparation method for improving the biological activity of Lentinus edodes stem polysaccharide. The specific steps are basically the same as in Example 1, and the inoculation amount is examined: the inoculation amount of white ginseng seed liquid in 10g Lentinus edodes stem culture medium is 2mL, 5mL, 10mL, 15mL, 20mL, and 25mL; the fermentation time is 8d.

[0059] refer to Figure 3 Different inoculation amounts will have a relatively large impact on the FSP yield. As the inoculation amount continues to increase, the FSP content is also gradually increasing. When the inoculation amount is low, the mycelium grows slowly and is unevenly distributed. When the inoculation amount increases to 10mL, the polysaccharide yield is the highest, and then as the inoculation amount increases, the FSP yield gradually decreases. And when the inoculation amount is too large, it will cause the bacterial liquid to aggregate and form fruiting bodies, which is not conducive to the spread and growth of the mycelium.

[0060] Example 3

[0061] This embodiment provides a preparation method for improving the biological activity of Lentinus edodes stem polysaccharide. The specific steps are basically the same as in Example 1, and the amount of water added is examined: 10 mL of white ginseng bacteria is inoculated into 10 g of Lentinus edodes stem substrate, and the water addition amounts are 1:1, 1:1.5, 1:2.5, 1:3.5, 1:4.5, and 1:5.5 (g / mL) respectively; the fermentation time is 8 days.

[0062] refer to Figure 4 In the solid fermentation system, the amount of water added will affect the growth and metabolism of mycelium. Too low water content will cause mycelium to lack water and cannot grow evenly on the mushroom stem; too high water content will lead to reduced air permeability, so that mycelium cannot get enough oxygen. Therefore, the amount of water added is the key to the good growth of mycelium. With the increase of the amount of water added, the yield rate increases significantly, and the yield rate is the highest at 1:1.5. If the amount of water added continues to increase, the yield rate will gradually decrease.

[0063] In order to explore the optimal preparation conditions for FSP yield, the inventors conducted an orthogonal experimental study.

[0064] The inventors found in the study that fermentation days, inoculation amount and water content all have a certain influence on the FSP yield. An orthogonal experiment with three factors and three levels was used to explore the optimal preparation conditions for the FSP yield. The factor level table is shown in Table 1, and the experimental results are shown in Table 2.

[0065] Table 1. Experimental factors and levels of orthogonal experiment

[0066]

[0067] Table 2. Orthogonal test results

[0068]

[0069] According to the FSP yield in Table 2, the three factors of fermentation days, inoculation amount and water addition were analyzed by range analysis. From the results of the range analysis in Table 2, it can be seen that the influence of the three factors on the FSP preparation process is from large to small: water content> fermentation days> inoculation amount, indicating that the influence of water addition is the greatest. The optimal solution is A2B2C1, that is, the optimal preparation conditions with 10g of shiitake mushroom stem as the matrix are fermentation days 9d, inoculation amount 12.5mL, and water addition amount 1:1.5 (g / mL). The polysaccharide yield extracted under the optimal preparation conditions is 16.80%±0.60%. The polysaccharide content of WSP and FSP was detected by the sulfuric acid phenol method. The polysaccharide content of WSP and FSP was 64.67%±3.96% and 75.40%±1.81%, respectively. It can be seen that the optimized fermentation preparation process not only improves the yield of polysaccharides, but also improves its purity. Please refer to Figure 5 This is a picture of shiitake mushroom stems fermented with white ginseng fungus in the solid state.

[0070] In order to better illustrate the high biological activity of Lentinus edodes petiole polysaccharide provided by the present invention, the inventors conducted the following research.

[0071] Example 4

[0072] Study on antioxidant activity.

[0073] Accurately prepare polysaccharide sample solutions of a certain concentration, pipette 100 μL of polysaccharide sample solutions of different concentrations into the enzyme-labeled wells, add 100 μL of 0.2 mmoL / L DPP-anhydrous ethanol solution (V sample: V DPP-anhydrous ethanol = 1:1, the volume can be expanded in equal proportion), mix with a pipette, and react in the dark for 10 minutes. Measure the absorbance value A of the reaction system at a wavelength of 517 nm. i , use anhydrous ethanol instead of DPPH solution to measure the absorbance value A j , use distilled water instead of the sample to measure the absorbance value A0.

[0074] The calculation formula of clearance rate is:

[0075]

[0076] WSP and FSP with a concentration of 1-5 mg / mL were selected to conduct experiments on DPPH free radical scavenging ability. Figure 6 , WSP and FSP both have a certain ability to scavenge DPPH free radicals, and the scavenging effect changes with the concentration, showing a good dose-effect relationship. Specifically, the IC 50 The value is 2.445 mg / mL, and the IC 50The value is 3.354 mg / mL; at the same concentration, the scavenging ability of FSP is significantly higher than that of WSP. At 5 mg / mL, the scavenging rate of FSP is 93.03%, while that of WSP is 67.27%. The effect of FSP is significantly better than that of WSP.

[0077] Example 5

[0078] Study on anti-inflammatory activity.

[0079] NO 2- Standard curve determination: accurately weigh 69 mg NaNO2, dissolve it in ultrapure water and dilute it to a 1L volumetric flask to prepare a 1mM standard solution, then dilute it to 0, 5, 10, 20, 40, 60, 80, 100μM, take 100μL of the above concentrations of NaNO2 solution in the ELISA plate, add 100μL of Griess reagent, react at room temperature in the dark for 5 minutes, and measure its absorbance at 540nm on the ELISA instrument. 2- The standard curve is drawn with concentration as the horizontal axis and absorbance value as the vertical axis.

[0080] Cell treatment and NO content determination: 1×10 cells in logarithmic growth phase were taken 6 Cells were inoculated with 1 μg / mL of 1 μg / mL LPS in a 24-well plate and cultured in a constant temperature incubator at 37°C and 5% CO2 for 24 hours to allow the cells to adhere to the wall and grow. The supernatant was then discarded and 500 μL of sample solutions of different concentrations were added. The control group was added with 500 μL of phenol red-free DMEM basal medium and the model control group was added with 500 μL of 1 μg / mL LPS. Three parallels were set up for each group. After continuing to culture for 24 hours, the cell supernatant was carefully collected in a centrifuge tube. After centrifugation at 2500 r / min and 4°C for 5 minutes, 100 μL was taken in a 96-well plate, and then 100 μL of Griess reagent was added. The reaction was carried out at room temperature in the dark for 5 minutes. The absorbance was measured at 540 nm on an enzyme reader and the nitrite (NO) in the supernatant was calculated according to the standard curve. 2- )content.

[0081] After the cells were cultured for 24 hours and adhered to the wall, the culture medium was carefully aspirated, and different concentrations of drug groups and LPS modeling groups diluted with phenol red-free basal medium were added. 500 μL of sample and LPS mixture was added to each well and cultured in an incubator for 24 hours. The supernatant was transferred to the corresponding centrifuge tube and centrifuged at 2500 r / min for 5 minutes. 100 μL was taken in a 96-well plate, and then 100 μL of Griess reagent was added. The reaction was carried out at room temperature in the dark for 5 minutes, and the absorbance was measured at 540 nm on an ELISA reader. The absorbance was brought into the standard curve to calculate the NO content in the cell culture supernatant.

[0082] WSP and FSP with concentrations of 12.5, 25, and 50 μg / mL were selected for the experiment. Figure 7 The NO concentration produced by the blank group cells was low, and the NO concentration produced by the modeling group treatment was significantly increased, indicating that the RAW264.7 inflammation model was successfully established under the stimulation of LSP. In the co-culture mode, both WSP and FSP inhibited the secretion of NO, and the NO content was lower in the FSP treatment.

[0083] Example 6

[0084] Study on hypolipidemic activity.

[0085] Take 12 100mL Erlenmeyer flasks (divided into 4 groups, 3 flasks in each group), add 5mL of 0.025mol / L phosphate buffer and 4mL of polyvinyl alcohol olive oil emulsion to each flask, and place them in a 40℃ water bath for 5min.

[0086] (1) Add 1 mL of pancreatic lipase solution (2 mg / mL) to each of the three Erlenmeyer flasks, start timing from the time the enzyme solution is added, and continue to keep warm for 30 minutes; immediately add 15 mL of 95% ethanol to each of the flasks to stop the enzyme action; then add 3 drops of phenolphthalein indicator and titrate with 0.05 mol / L sodium hydroxide until the solution turns pink, and record the amount of sodium hydroxide consumed as V1. Take another 3 Erlenmeyer flasks as blank controls, add the enzyme solution of the blank controls after the reaction is terminated, and record the amount of sodium hydroxide consumed as V0.

[0087] (2) Take 3 Erlenmeyer flasks, add 1 mL of sample solution first, then add 1 mL of lipase, and then proceed as in (1), recording the amount of sodium hydroxide consumed as V2. Take another 3 Erlenmeyer flasks as blank controls, add the enzyme solution of the blank controls after the reaction is terminated, and record the amount of sodium hydroxide consumed as V3.

[0088] Inhibition rate calculation formula:

[0089]

[0090] WSP and FSP with a concentration of 5 mg / mL were selected for the experiment of pancreatic lipase inhibition rate. The inhibition rate of WSP on pancreatic lipase was 41.39% ± 8.02%, and the inhibition rate of FSP was 67.44% ± 4.46%, and the activity effect was significantly improved. FSP not only showed a higher inhibitory effect, but also outperformed most of the samples in the study, indicating that it has stronger activity in inhibiting pancreatic lipase.

[0091] Example 7

[0092] Studies on laxative activity.

[0093] Animal model: The blank group and model group were given 10mL / (kg·bw) distilled water by gavage, the positive group was given 4g / (kg·bw) of Ma Ren Wan by gavage, and the experimental group was given a certain dose of sample by gavage, and the mice were fed for 12 days. After 24 hours of fasting but not water restriction, the intestinal peristalsis of mice was inhibited by gavage of 50mg / (kg·bw) of compound diphenoxylate solution, and the constipation model of mice was established.

[0094] Small intestinal (ink) propulsion rate: After 11 days of feeding, fasting for 24 hours but not water, and 30 minutes after modeling, mice were gavaged with an equal amount of ink (semi-solid paste) containing the corresponding test substance, and then killed by cervical dislocation 25 minutes after the administration of ink. Referring to the method of Chu Chunxia et al. (2018), the intestines of mice were dissected from the pylorus to the cecum, and the "total length of the small intestine" was measured in a straight line without traction, and the length from the pylorus to the front of the ink was the "ink propulsion length". The intestinal (ink) propulsion rate of mice was calculated according to the following formula:

[0095]

[0096] 300 mg / (kg·bw) FSP was selected for the experiment of small intestinal propulsion rate. Figure 8 , it was observed that the effect of FSP was comparable to that of the positive drug Ma Ren Wan, and both significantly promoted the peristalsis of the small intestine of mice. And the dosage of FSP was much lower than that of the positive drug, but its effect was close to that of the blank group. This shows that FSP has good activity and effect in promoting small intestinal peristalsis, and its relatively low-dose application may provide a more economical and efficient option.

[0097] In the absence of conflict, the above embodiments and features in the embodiments may be combined with each other.

[0098] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for improving the biological activity of Lentinus edodes stem polysaccharide, characterized in that: Solid-state fermentation of shiitake mushroom stems is carried out using white ginseng bacteria, comprising the following steps: S1. Preparation of Lentinus edodes stem culture medium Grind the mushroom stems into powder, then add appropriate amount of water and stir evenly, sterilize under high pressure, and obtain the mushroom stem culture medium after cooling; S2. Inoculation and culture Activating the white ginseng fungus strain and preparing a white ginseng fungus seed liquid, and adding the white ginseng fungus seed liquid into the shiitake mushroom stem culture medium according to a certain inoculation amount for solid-state fermentation; S3. Extraction of Lentinus edodes stem polysaccharide The fermented Lentinus edodes stem culture medium is firstly subjected to protease hydrolysis, then ethanol precipitation is adopted, the precipitate is obtained by centrifugation, the precipitate is redissolved, dialyzed through a dialysis bag, and the dialyzed solution is freeze-dried to obtain Lentinus edodes stem polysaccharide.

2. The preparation method according to claim 1, characterized in that In step S1, the mushroom stems are ground into powder with a particle size of 40 to 50 meshes.

3. The preparation method according to claim 1, characterized in that: In step S2, the preparation process of the white ginseng fungus seed liquid is to inoculate the white ginseng fungus strain into the CYM culture medium for activation, inoculate the activated white ginseng fungus strain into the seed culture liquid for shake flask fermentation, take out the fungus block after it is balled, homogenize the seed culture medium and the fungus ball to obtain the white ginseng fungus seed liquid.

4. The preparation method according to claim 3, characterized in that: The inoculation amount of the white ginseng fungus seed liquid in every 10g of Lentinus edodes stem culture medium is 10-15mL.

5. The preparation method according to claim 4, characterized in that: The volume of water added to every 10g of Lentinus edodes stem culture medium is 1.5-2.5mL.

6. The preparation method according to claim 5, characterized in that: The fermentation temperature is 25-27°C and the fermentation time is 8-10 days.

7. The preparation method according to claim 1, characterized in that: In step S3, the protease is a neutral protease.

8. Lentinus edodes petiole polysaccharide prepared by the preparation method according to any one of claims 1 to 7.

9. Use of Lentinus edodes stipe polysaccharide prepared by the preparation method according to any one of claims 1 to 7 in the preparation of lipid-lowering products.

10. Use of Lentinus edodes stipe polysaccharide prepared by the preparation method according to any one of claims 1 to 7 in the preparation of intestinal moisturizing products.

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