A preparation method for improving biological activity of lentinan and application thereof

Abstract

The present application relates to the field of synthetic biology, and specifically discloses a preparation method for improving the biological activity of lentinan and application thereof. The method adopts white shiitake mushroom to perform solid-state fermentation on lentinan, and comprises the following steps: S1, grinding the lentinan into powder, then adding appropriate amount of water to stir uniformly, high-pressure sterilization, and cooling to obtain lentinan culture medium; S2, activating white shiitake mushroom strains and preparing white shiitake mushroom seed liquid, adding the white shiitake mushroom seed liquid into the lentinan culture medium according to a certain inoculation amount to perform solid-state fermentation; S3, taking the fermented lentinan culture medium, performing protease enzymolysis first, then adopting ethanol precipitation, centrifuging to take the precipitate, resolubilizing the precipitate, performing dialysis treatment through a dialysis bag, and freeze-drying the dialyzed solution to obtain lentinan. The preparation method significantly improves the yield and purity of lentinan, and at the same time, the antioxidant, anti-inflammatory and pancreatic lipase clearance abilities are significantly improved, and the method has the effect of lubricating intestines.

Description

Technical Field

[0001] This invention belongs to the field of synthetic biology technology, specifically relating to a preparation method and application for improving the bioactivity of lentinan from shiitake mushroom stems. Background Technology

[0002] The stem of a shiitake mushroom is a byproduct of shiitake mushroom production, accounting for 25%-30% of the mushroom's dry weight. A small portion is used to make mushroom sauce or soup base, while the remainder is typically used as field compost or burned directly, wasting a significant portion of the mushroom stem resource. However, it actually contains the same nutrients as the mushroom cap, both composed of shiitake mycelium and rich in polysaccharides, proteins, amino acids, vitamins, and minerals. Polysaccharides are an important active ingredient in shiitake mushrooms; however, research reports on the extraction and bioactivity of shiitake mushroom polysaccharides are relatively scarce compared to those on shiitake mushroom stem polysaccharides.

[0003] Currently, the extraction of shiitake mushroom stem polysaccharides mainly relies on hot water extraction, with some reports on enzymatic, ultrasonic, and microwave-assisted extraction methods. However, there are few reports on fermentation methods for preparing shiitake mushroom stem polysaccharides. Therefore, how to provide a highly active shiitake mushroom stem polysaccharide to improve the application value of shiitake mushroom stems is a technical problem that researchers urgently need to solve. Summary of the Invention

[0004] The purpose of this invention is to address the shortcomings of the prior art by providing a method for preparing and applying lentinan with improved bioactivity. The method utilizes *Gynostemma pentaphyllum* to prepare lentinan from lentinan via solid-state fermentation, which significantly enhances its antioxidant, anti-inflammatory, and pancreatic lipase-clearing abilities, and also provides intestinal lubrication.

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

[0006] The first objective of this invention is to provide a method for preparing polysaccharides from shiitake stems that enhance their bioactivity, comprising the following steps:

[0007] S1. Preparation of shiitake mushroom stem culture medium

[0008] Grind the shiitake mushroom stems into powder, add an appropriate amount of water and stir well. Sterilize under high pressure and cool to obtain shiitake mushroom stem culture medium.

[0009] S2, Inoculation and Culture

[0010] The white ginseng strain was activated and a white ginseng seed liquid was prepared. The white ginseng seed liquid was added to the shiitake mushroom stem culture medium at a certain inoculation amount for solid-state fermentation.

[0011] S3, Extraction of polysaccharides from shiitake stems

[0012] The fermented shiitake mushroom stem culture medium was first hydrolyzed with protease, then precipitated with ethanol, the precipitate was collected by centrifugation, the precipitate was reconstituted, and then dialyzed through a dialysis bag. The dialyzed solution was then freeze-dried to obtain shiitake mushroom stem polysaccharide.

[0013] Furthermore, in step S1, the particle size of the powder made from the shiitake mushroom stems is 40-50 mesh.

[0014] Furthermore, in step S2, the preparation process of the white ginseng mycelium seed liquid involves inoculating the white ginseng mycelium into CYM medium for activation, inoculating the activated white ginseng mycelium into seed culture liquid in a shake flask for fermentation, removing the mycelium blocks after they form pellets, and homogenizing the seed culture medium and mycelium pellets to obtain the white ginseng mycelium seed liquid.

[0015] Furthermore, the inoculation volume of *Gynostemma pentaphyllum* seed liquid in each 10g *Lentinula edodes* stem culture medium is 10-15mL.

[0016] Furthermore, the volume of water added to each 10g of shiitake mushroom stem culture medium is 1.5~2.5 mL.

[0017] Furthermore, the fermentation temperature is 25~27℃, and the fermentation time is 8~10 days.

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

[0019] A second objective of this invention is to provide lentinan prepared by the above-described preparation method.

[0020] A third objective of this invention is to provide the application of the lentinan prepared by the above-described preparation method in the preparation of lipid-lowering products.

[0021] The fourth objective of this invention is to provide the application of the lentinan prepared by the above-described preparation method in the preparation of laxative products.

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

[0023] (1) The method of solid-state fermentation of shiitake mushroom stem substrate by white ginseng fungus in this invention significantly improves the yield and purity of shiitake polysaccharides, which is conducive to strengthening the development and utilization of health care and functional foods of shiitake mushroom stem.

[0024] (2) The polysaccharide of shiitake mushroom stem prepared by solid-state fermentation of shiitake mushroom stem substrate by white ginseng fungus in this invention has significantly improved antioxidant, anti-inflammatory and pancreatic lipase scavenging abilities, and also has a laxative effect. Attached Figure Description

[0025] Figure 1 The figure shows the results of this invention investigating the effects of different edible fungi fermentation on the preparation of polysaccharides from shiitake mushroom stems;

[0026] Figure 2 The figure shows the results of this invention's investigation into the effect of fermentation days on the preparation of polysaccharides from shiitake mushroom stems;

[0027] Figure 3 The figure shows the results of this invention's investigation into the effect of inoculum amount on the preparation of polysaccharides from shiitake stems;

[0028] Figure 4 The figure shows the results of this invention's investigation into the effect of water addition on the preparation of polysaccharides from shiitake mushroom stems;

[0029] Figure 5 Image of the stem of a shiitake mushroom fermented with white ginseng fungus, provided for this invention;

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

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

[0032] Figure 8 This is a graph showing the effect of FSP on the small intestinal propulsion rate in this invention. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of this invention clearer, the specific embodiments and accompanying drawings are described in further detail below. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0034] All biological materials used in this invention are commercially available.

[0035] The edible fungi used in this invention, including morel, poria cocos, king oyster mushroom, straw mushroom, shiitake mushroom, Ganoderma lucidum, variegated mushroom, white ginseng mushroom, lion's mane mushroom, and blood ear fungus, were all provided by the Institute of Applied Fungi, Huazhong Agricultural University.

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

[0037] Take a small piece of the mycelium stored in a test tube and inoculate it onto a CYM plate. After the mycelium has covered the entire plate, take a square mycelium block with a side length of 7.5-8.0 mm and inoculate it onto the CYM plate for a second activation until the mycelium has covered the plate. Take eight square mycelium blocks with a side length of 7.5-8.0 mm and inoculate them into 100 mL of seed culture medium (250 mL Erlenmeyer flask). Place them in a full-temperature shaking incubator at 120 r / min and 25 ℃. After the mycelium blocks form pellets, remove them and homogenize the seed culture medium and mycelium pellets on a clean bench to prepare a mycelium seed solution. Inoculate this solution into the solid culture medium of shiitake mushroom stems at a certain inoculation rate for fermentation.

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

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

[0040] The purification method for extracting lentinan from shiitake stems in this invention is as follows:

[0041] A certain amount of sample was taken and added to distilled water at a material-to-liquid ratio of 1:20 (g / mL). 0.4% neutral protease (BR, 100u / mg, Bacillus subtilis) was added, and enzymatic hydrolysis was performed in a water bath at pH 7.0 and 50℃ for 1 h. The water temperature was then raised to 90℃ and treated for 2 h. The hydrolysate was centrifuged at 4000 r / min and 20℃ for 15 min. The supernatant was collected, and 4 volumes of 95% ethanol were added. After mixing, the solution was precipitated at room temperature for 12 h. The precipitate was collected by centrifugation using the same method described above. The precipitate was reconstituted with an appropriate amount of distilled water. The solution was then added to an 8000-14000 dialysis bag, and dialyzed in a dialysis apparatus with distilled water for 48 h, changing the distilled water every 6 h. The dialyzed solution was freeze-dried to obtain the water-soluble polysaccharide (hereinafter referred to as polysaccharide).

[0042] Calculation of water-soluble polysaccharide yield:

[0043] .

[0044] During their research, the inventors unexpectedly discovered that different edible fungi have varying fermentation abilities on shiitake mushroom stems. Certain amounts of seed cultures of morel, poria cocos, king oyster mushroom, straw mushroom, shiitake mushroom, reishi mushroom, variegated mushroom, white ginseng mushroom, lion's mane mushroom, and blood ear fungus were inoculated into shiitake mushroom stems. It was found that morel, poria cocos, king oyster mushroom, and variegated mushroom failed to grow mycelium on the stems, while the other strains could fully colonize within 12-44 days. This indicates that different edible fungi have significantly different growth abilities on the solid-state fermentation medium for shiitake mushroom stems. Figure 1 As shown, after fermentation and extraction, only the yields obtained from fermentation with *Mycorrhiza uralensis* and *Hericium erinaceus* were higher than those from the control group (unfermented shiitake mushroom stem polysaccharide, hereinafter referred to as WSP). Furthermore, the yield of polysaccharide produced by solid-state fermentation of shiitake mushroom stem substrate with *Mycorrhiza uralensis* (hereinafter referred to as FSP) reached 17.20%, a 23.74% increase compared to the 13.90% of the control group. This is because *Mycorrhiza uralensis* possesses good adaptability, efficient metabolic capacity, and superior growth characteristics, resulting in high polysaccharide synthesis capacity and yield during solid-state fermentation. Therefore, *Mycorrhiza uralensis* was selected as the edible and medicinal fungal strain for subsequent solid-state fermentation of shiitake mushroom stem substrate to produce polysaccharides.

[0045] Determination of total sugar:

[0046] The phenol-sulfuric acid method was used. Specifically, a 0.1 mg / mL glucose standard solution was prepared. 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 test tubes, and distilled water was added to bring the total volume to 2.0 mL. Then, 1.0 mL of 5% phenol was added to each tube, and the mixture was shaken well. Within 20 seconds, 5.0 mL of concentrated sulfuric acid was rapidly added, and the mixture was shaken well (operating one tube at a time). The mixture was then incubated at room temperature for 30 min, and the absorbance was measured at 490 nm to create 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 method for preparing polysaccharides from shiitake mushroom stems with enhanced bioactivity. The specific steps are as follows:

[0050] S1. Preparation of shiitake mushroom stem culture medium

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

[0052] S2, Inoculation and Culture

[0053] A small piece of *Pleurotus ostreatus* spawn stored in a test tube was inoculated onto a CYM plate. After the mycelium had completely covered the plate, a square mycelial block with a side length of 7.5–8.0 mm was inoculated onto the CYM plate for a second activation until the mycelium had completely covered the plate. Eight square mycelial blocks with a side length of 7.5–8.0 mm were then inoculated into 100 mL of seed culture medium (250 mL Erlenmeyer flask) and placed in a full-temperature shaking incubator at 120 r / min and 25 °C. After the mycelial blocks formed pellets, they were removed and homogenized with the seed culture medium and mycelial pellets on a clean bench to prepare a seed culture solution. This seed culture solution was then inoculated into solid culture medium containing *Lentinula edodes* stems at a specific inoculation rate for solid-state fermentation. The inoculation rate was 10 mL of *Pleurotus ostreatus* seed culture solution per 10 g of *Lentinula edodes* stem medium. The solid-state fermentation temperature was 25 °C, and the fermentation time was observed at 4, 6, 8, 10, and 12 days.

[0054] S3, Extraction of polysaccharides from shiitake stems

[0055] A certain amount of solid-state fermented shiitake mushroom stem culture medium sample was taken and added to distilled water at a material-to-liquid ratio of 1:20 (g / mL). 0.4% neutral protease was added, and enzymatic hydrolysis was performed in a water bath at pH 7.0 and 50℃ for 1 h. The water temperature was then raised to 90℃ and treated for 2 h. The hydrolysate was centrifuged at 4000 r / min and 20℃ for 15 min, and the supernatant was collected. Four volumes of 95% ethanol were added, mixed well, and precipitated at room temperature for 12 h. The precipitate was collected by centrifugation using the same method. An appropriate amount of distilled water was added to reconstitute the precipitate, and the solution was added to an 8000-14000 dialysis bag. Dialysis was performed in a dialysis apparatus with distilled water for 48 h, with the distilled water changed every 6 h. The dialysis solution was freeze-dried to obtain the water-soluble polysaccharide (hereinafter referred to as polysaccharide).

[0056] refer to Figure 2 Different fermentation days affect the FSP yield. As the fermentation days increase, the FSP yield rises continuously, peaking on day 8. Afterward, the FSP yield tends to level off with further fermentation days. It is speculated that this is because in the early stages of fermentation, the mycelium of *Gynostemma pentaphyllum* continuously accumulates metabolic products, leading to a gradual increase in FSP yield. As fermentation time extends, the mycelial biomass gradually increases, but the nutrients in the culture medium are insufficient to maintain the normal physiological activities of the mycelium, affecting the synthesis of new substances and causing the FSP yield to decrease.

[0057] Example 2

[0058] This embodiment provides a method for preparing polysaccharides from shiitake mushroom stems with enhanced bioactivity. The specific steps are basically the same as in Example 1, except that the inoculation amount was 2 mL, 5 mL, 10 mL, 15 mL, 20 mL, and 25 mL per 10 g shiitake mushroom stem culture medium; the fermentation time was 8 days.

[0059] refer to Figure 3 Different inoculum sizes have a significant impact on FSP yield. As the inoculum size increases, the FSP content also gradually increases. When the inoculum size is low, the mycelial growth rate is slow and the distribution is uneven. When the inoculum size increases to 10 mL, the polysaccharide yield is the highest, and then the FSP yield gradually decreases with further increases in the inoculum size. Furthermore, excessive inoculum size can lead to bacterial aggregation and the formation of fruiting bodies, which is not conducive to the spread and growth of mycelium.

[0060] Example 3

[0061] This embodiment provides a method for preparing polysaccharides from shiitake mushroom stems with enhanced bioactivity. The specific steps are basically the same as in Example 1, except that the amount of water added is examined: 10 mL of *Pleurotus ostreatus* inoculum is inoculated into 10 g of shiitake mushroom stem substrate, and the water addition ratios 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 solid-state fermentation systems, the amount of water added affects the growth and metabolism of mycelium. Too little water will cause the mycelium to lack water and fail to grow evenly on the shiitake mushroom stems; too much water will reduce aeration, preventing the mycelium from obtaining sufficient oxygen. Therefore, the amount of water added is crucial for the healthy growth of mycelium. As the amount added increases, the yield increases significantly, reaching its highest point at a ratio of 1:1.5. Further increasing the amount added leads to a gradual decrease in yield.

[0063] To investigate the optimal preparation conditions for FSP yield, the inventors conducted orthogonal experimental research.

[0064] The inventors discovered that fermentation days, inoculum size, and moisture content all have a certain impact on FSP yield. An orthogonal experiment with three factors and three levels was conducted to explore the optimal preparation conditions for 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 in orthogonal experiments

[0066]

[0067] Table 2. Results of the orthogonal experiment

[0068]

[0069] Based on the FSP yield in Table 2, a range analysis was performed on the three factors: fermentation days, inoculum size, and water addition. The range analysis results in Table 2 show that the influence of the three factors on the FSP preparation process, from largest to smallest, is: water content > fermentation days > inoculum size. This indicates that water addition has the greatest impact. The optimal scheme is A2B2C1, that is, the optimal preparation conditions using 10 g of shiitake mushroom stems as a substrate are: fermentation days 9 days, inoculum size 12.5 mL, and water addition ratio 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 using the sulfuric acid-phenol method. The polysaccharide contents of WSP and FSP were 64.67%±3.96% and 75.40%±1.81%, respectively. Therefore, the optimized fermentation preparation process not only improved the polysaccharide yield but also its purity. Please refer to [reference needed]. Figure 5 Image of a shiitake mushroom stem fermented with white ginseng.

[0070] To better illustrate the high bioactivity of the lentinan provided by this invention, the inventors conducted the following research.

[0071] Example 4

[0072] Research on antioxidant activity.

[0073] Accurately prepare polysaccharide sample solutions of specific concentrations. Pipette 100 µL of each concentration into a well of the enzyme-labeled enzyme. Add 100 µL of 0.2 mmol / L DPP-anhydrous ethanol solution (Vsample:VDPP-anhydrous ethanol = 1:1; volume can be increased proportionally). Mix thoroughly with a pipette and react in the dark for 10 min. Measure the absorbance of the reaction system at 517 nm. The absorbance value was measured using anhydrous ethanol instead of DPPH solution. The absorbance value was measured using distilled water instead of the sample. .

[0074] Clearance rate calculation formula:

[0075]

[0076] Experiments were conducted to assess the DPPH free radical scavenging capabilities of WSP and FSP at concentrations ranging from 1 to 5 mg / mL. (Reference) Figure 6 Both WSP and FSP possess a certain ability to scavenge DPPH free radicals, and their scavenging effect is consistent with changes in concentration, exhibiting a good dose-response relationship. Specifically, the IC50 of FSP... 50 The value is 2.445 mg / mL, and the IC50 of WSP is... 50The value was 3.354 mg / mL; at the same concentration, the scavenging ability of FSP was significantly higher than that of WSP. At 5 mg / mL, the scavenging rate of FSP was 93.03%, while that of WSP was 67.27%, and FSP was significantly better than WSP.

[0077] Example 5

[0078] Research on anti-inflammatory activity.

[0079] NO 2- Standard curve determination: Accurately weigh 69 mg NaNO2, dissolve it in ultrapure water, and dilute to a 1 L volumetric flask to prepare a 1 mM standard solution. Then, dilute this solution to 0, 5, 10, 20, 40, 60, 80, and 100 µM, respectively. Take 100 µL of each NaNO2 solution and place it in an ELISA plate, add 100 µL of Griess reagent, and react at room temperature in the dark for 5 min. Measure the absorbance at 540 nm using an ELISA reader. (NO3) 2- A standard curve was plotted with concentration on the x-axis and absorbance on the y-axis.

[0080] Cell treatment and NO content determination: Logarithmic growth phase cells were treated with 1×10⁻⁶ cells. 6 Cells were seeded at a density of 1 / mL in 24-well plates and cultured at 37°C with 5% CO2 for 24 h to allow for cell adhesion and growth. The supernatant was then discarded, and 500 µL of different concentrations of sample solutions were added. The control group received 500 µL of phenol red-free DMEM basal medium, while the model control group received 500 µL of 1 µg / mL LPS. Each group was tested in triplicate. After another 24 h of culture, the cell supernatant was carefully collected into centrifuge tubes and centrifuged at 2500 r / min at 4°C for 5 min. 100 µL of the centrifuge solution was then transferred to 96-well plates, and 100 µL of Griess reagent was added. The plates were incubated at room temperature in the dark for 5 min. The absorbance was measured at 540 nm using a microplate reader, and the nitrite (NO3) content in the supernatant was calculated based on the standard curve. 2- )content.

[0081] After cell culture for 24 h and cell adhesion, the culture medium was carefully aspirated, and different concentrations of the drug group and the LPS modeling group diluted with phenol red-free basal medium were added. 500 µL of the sample-LPS mixture was added to each well, and the cells were incubated for 24 h. The supernatant was transferred to the corresponding centrifuge tubes and centrifuged at 2500 r / min for 5 min. 100 µL of the supernatant was then added to a 96-well plate, followed by the addition of 100 µL of Griess reagent. The plate was incubated at room temperature in the dark for 5 min, and the absorbance was measured at 540 nm using a microplate reader. The absorbance was then used to calculate the NO content in the cell culture supernatant using a standard curve.

[0082] Experiments were conducted using WSP and FSP at concentrations of 12.5, 25, and 50 μg / mL. (Reference) Figure 7 The blank control group produced lower NO concentrations, while the modeling group showed significantly higher NO concentrations, indicating that the RAW264.7 inflammation model was successfully established under LSP stimulation. In co-culture mode, both WSP and FSP inhibited NO secretion, with FSP treatment showing even lower NO levels.

[0083] Example 6

[0084] Research on lipid-lowering activity.

[0085] Take 12 100mL Erlenmeyer flasks (divided into 4 groups of 3), add 5mL of 0.025mol / L phosphate buffer and 4mL of polyvinyl alcohol olive oil emulsion to each flask, and incubate 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 incubation for 30 min. Immediately after removing the flasks, add 15 mL of 95% ethanol to each flask to stop the enzyme activity. Then add 3 drops of phenolphthalein indicator and titrate with 0.05 mol / L sodium hydroxide until the solution turns pink. Record the amount of sodium hydroxide consumed. Three additional Erlenmeyer flasks were used as blank controls. The enzyme solution for the blank controls was added after the reaction was terminated, and the amount of sodium hydroxide consumed was recorded. .

[0087] (2) Take 3 Erlenmeyer flasks, first add 1 mL of sample solution, then add 1 mL of lipase, and then proceed as in (1). Record the amount of sodium hydroxide consumed. Three additional Erlenmeyer flasks were used as blank controls. The enzyme solution for the blank controls was added after the reaction was terminated, and the amount of sodium hydroxide consumed was recorded. .

[0088] Inhibition rate calculation formula:

[0089]

[0090] The inhibition rates of pancreatic lipase were tested using WSP and FSP at concentrations of 5 mg / mL. WSP showed an inhibition rate of 41.39% ± 8.02% against pancreatic lipase, while FSP showed an inhibition rate of 67.44% ± 4.46%, demonstrating significantly improved activity. FSP not only exhibited a higher inhibitory effect but also outperformed most samples in other studies, indicating its stronger activity in inhibiting pancreatic lipase.

[0091] Example 7

[0092] Research on its laxative activity.

[0093] Animal model: Mice in the blank control group and model group were administered 10 mL / (kg·bw) of distilled water by gavage, the positive control group was administered 4 g / (kg·bw) of hemp seed pill by gavage, and the experimental group was administered a certain dose of the sample by gavage. The mice were to be fed for 12 days. After 24 hours of fasting but with free access to water, intestinal peristalsis in mice was inhibited by gavage with a solution of 50 mg / (kg·bw) of compound diphenoxylate, thus establishing a mouse constipation model.

[0094] Small intestinal (ink) propulsion rate: After 11 days of feeding, mice were fasted for 24 hours but allowed free access to water. 30 minutes after modeling, mice were gavage with an equal volume of ink (semi-solid paste) containing the corresponding test substance. They were euthanized by cervical dislocation 25 minutes after ink administration. Following the method of Chu Chunxia et al. (2018), the intestine from the pylorus to the cecum was dissected and the "total length of the small intestine" was measured while the intestine was laid flat in a straight line without traction. The distance from the pylorus to the ink protrusion edge was defined as the "ink propulsion length." The intestinal (ink) propulsion rate of the mice was calculated using the following formula:

[0095] .

[0096] An experiment was conducted to determine the small intestinal propulsion rate using 300 mg / (kg·bw) of FSP. (Reference) Figure 8 The study observed that the effect of FSP was comparable to that of the positive control drug, Ma Ren Wan, both significantly promoting intestinal peristalsis in mice. Furthermore, the dosage of FSP was much lower than that of the positive control drug, yet its effect was close to that of the control group. This indicates that FSP has good activity and efficacy in promoting small intestinal peristalsis, and its relatively low-dose application may provide a more economical and efficient option.

[0097] Where there is no conflict, the above embodiments and features described herein can 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 principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing polysaccharides from shiitake mushroom stems to enhance their bioactivity, characterized in that, Solid-state fermentation of shiitake mushroom stems using white ginseng includes the following steps: S1. Preparation of shiitake mushroom stem culture medium Grind the shiitake mushroom stems into powder, add an appropriate amount of water and stir well. Sterilize under high pressure and cool to obtain shiitake mushroom stem culture medium. S2, Inoculation and Culture The white ginseng strain was activated and a white ginseng seed liquid was prepared. The white ginseng seed liquid was added to the shiitake mushroom stem culture medium at a certain inoculation amount for solid-state fermentation. S3, Extraction of polysaccharides from shiitake stems The fermented shiitake mushroom stem culture medium was first subjected to enzymatic hydrolysis with protease, then precipitated with ethanol, the precipitate was collected by centrifugation, the precipitate was reconstituted, and then dialyzed through a dialysis bag. The dialyzed solution was then freeze-dried to obtain shiitake mushroom stem polysaccharide.

2. The preparation method according to claim 1, characterized in that, In step S1, the particle size of the powder made from the shiitake mushroom stems is 40-50 mesh.

3. The preparation method according to claim 1, characterized in that, In step S2, the preparation process of the white ginseng mycelium seed liquid involves inoculating the white ginseng mycelium into CYM medium for activation, inoculating the activated white ginseng mycelium into seed culture medium for shake-flask fermentation, removing the mycelium blocks after they form pellets, and homogenizing the seed culture medium and mycelium pellets to obtain the white ginseng mycelium seed liquid.

4. The preparation method according to claim 3, characterized in that, The inoculation volume of *Gynostemma pentaphyllum* seed liquid in each 10g *Lentinula edodes* stem culture medium is 10-15mL.

5. The preparation method according to claim 4, characterized in that, The volume of water added to each 1g of shiitake mushroom stem culture medium is 1.5~2.5 mL.

6. The preparation method according to claim 5, characterized in that, The fermentation temperature is 25~27℃, 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. A lentinan prepared by the method described in any one of claims 1-7.

9. The application of lentinan prepared by the preparation method according to any one of claims 1-7 in the preparation of lipid-lowering products.

10. The application of lentinan prepared by the preparation method according to any one of claims 1-7 in the preparation of laxative products.

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