Lentinus edodes residue polysaccharide as well as extraction method and application thereof

Abstract

The invention relates to the technical field of biochemical engineering, in particular to lentinus edodes mushroom residue polysaccharide and an extraction method and application thereof, and the extraction method of the lentinus edodes mushroom residue polysaccharide comprises the following steps of smashing, dissolving, ultrasonic treatment, enzymolysis, deproteinization and separation and purification. According to the method disclosed by the invention, the crude lentinus edodes residue polysaccharide is extracted by virtue of ultrasonic synergistic enzymolysis, and meanwhile, deproteinization and separation purification are performed, so that the purity of the lentinus edodes residue polysaccharide is improved, the biological activity of the lentinus edodes residue polysaccharide is reserved, and a reference is provided for deep development and application of the polysaccharide industry.

Description

Technical Field

[0001] The invention relates to the technical field of biochemical engineering, and in particular to shiitake mushroom dregs polysaccharide and an extraction method and application thereof. Background Art

[0002] Shiitake mushroom bran refers to the waste culture medium left after picking shiitake mushrooms, which are grown in the process of shiitake mushroom cultivation using straw, sawdust and other raw materials as substrates. Shiitake mushroom bran contains 4.21% crude protein, 3.36% crude fat, 1.48% starch, 36.53% acid detergent fiber, 49.29% neutral detergent fiber, etc. Due to its special composition and rich nutritional active substances, it can not only be used as animal feed and organic fertilizer, but also as a raw material for the extraction of polysaccharides, enzymes, pigments and other substances, thus realizing the recycling of resources. Polysaccharides have attracted much attention due to their high content and outstanding biological activity, and have good application prospects in agricultural production, medical care and other aspects.

[0003] Traditional methods of extracting polysaccharides, such as hot water extraction, mainly use distilled water as the extraction agent for extraction. It is low-cost and easy to operate, but there is a problem of low polysaccharide purity.

[0004] Therefore, it is very important to develop a polysaccharide extraction process from Lentinus edodes bran with high polysaccharide purity. Summary of the invention

[0005] In order to develop a high-purity shiitake mushroom bran polysaccharide extraction process, the present invention provides a shiitake mushroom bran polysaccharide and an extraction method and application thereof. The present invention utilizes ultrasound-assisted enzymatic hydrolysis to extract shiitake mushroom bran crude polysaccharide, and simultaneously deproteinizes and separates and purifies, thereby not only improving the purity of shiitake mushroom bran polysaccharide, but also retaining the biological activity of shiitake mushroom bran polysaccharide.

[0006] A method for extracting shiitake mushroom husk polysaccharides comprises the following steps: Crushing: crushing and sieving the mushroom bran to obtain the mushroom bran powder; Dissolution: Add shiitake mushroom bran powder into distilled water at a solid-liquid ratio of 1g:10mL~30mL to obtain shiitake mushroom bran solution; Ultrasound: The shiitake mushroom bran solution is ultrasonically extracted for 30min~50min at an ultrasonic power of 250w~350W and an ultrasonic temperature of 50℃~70℃ to destroy the plant cell wall; Enzymatic hydrolysis: After ultrasound, the pH value of the shiitake mushroom bran solution was adjusted to 5.0-6.0, cellulase and pectinase were added, and the total activity of cellulase and pectinase was 800U / g-1000U / g. The enzymatic hydrolysis was carried out at a constant temperature of 45°C-55°C for 40min-60min to improve the extraction efficiency, and a crude extract of shiitake mushroom bran was obtained. The precipitate obtained after alcohol precipitation and centrifugation was crude polysaccharide of shiitake mushroom bran; Deproteinization: removing the protein in the crude polysaccharide from shiitake mushroom husks to obtain deproteinized crude polysaccharide powder from shiitake mushroom husks; Separation and purification: The deproteinized shiitake mushroom bran crude polysaccharide powder is separated, purified and freeze-dried to obtain the purified shiitake mushroom bran polysaccharide powder.

[0007] The present invention utilizes ultrasound-assisted enzymolysis to extract shiitake mushroom chaff polysaccharides, promotes the release of shiitake mushroom chaff polysaccharides from cells, and reduces the interference of impurities; then, the purified shiitake mushroom chaff polysaccharides are obtained by deproteinization and separation and purification, which can not only remove impurities and harmful substances, significantly improve the purity of shiitake mushroom chaff polysaccharides, but also ensure the safety and stability of the product. In addition, the present invention is carried out under mild conditions, avoiding the destruction of the activity of shiitake mushroom chaff polysaccharides by extreme conditions of high temperature, strong acid and strong alkali, and retaining the original biological activity of shiitake mushroom chaff polysaccharides.

[0008] Furthermore, the deproteinization is carried out using the Sevag method, and the concentration of crude polysaccharides from shiitake mushroom bran in the Sevag method is 30 mg / mL to 50 mg / mL.

[0009] Furthermore, DEAE-52 column chromatography is used for separation and purification, and the DEAE-52 column chromatography includes cellulose DEAE-52 pretreatment, column packing equilibration and chromatography liquid collection.

[0010] Furthermore, the cellulose DEAE-52 pretreatment is specifically as follows: soaking the DEAE-52 dry powder in distilled water, discarding the supernatant after sufficient swelling, until the supernatant is clear and transparent, and soaking the lower precipitate in a 10% to 30% ethanol solution by volume for use.

[0011] Furthermore, the column balancing is specifically as follows: first, distilled water is added into the chromatographic column for balancing, a constant flow pump is turned on to observe the uniform outflow of water, the pretreated cellulose DEAE-52 is loaded into the chromatographic column, and bubbles are removed; distilled water is used as the balancing liquid, distilled water is added into the chromatographic column, and the flow rate is controlled so that the chromatographic column reaches balance.

[0012] Furthermore, the chromatography liquid is collected specifically as follows: after preparing the crude polysaccharide solution of Lentinus edodes husk and loading the sample, distilled water and NaCl solution are used for concentration gradient elution, and the eluate is collected.

[0013] Furthermore, the crushed mushroom bran powder is sieved through a 70-90 mesh sieve.

[0014] Furthermore, the ultrasonic power is 300w, the ultrasonic time is 50min, and the ultrasonic temperature is 50°C.

[0015] The invention provides a shiitake mushroom husk polysaccharide.

[0016] The invention also provides an application of shiitake mushroom husk polysaccharide in preparing an antioxidant product.

[0017] Compared with the prior art, the present invention has the following beneficial effects: 0. The present invention utilizes ultrasound-assisted enzymolysis to extract shiitake mushroom bran polysaccharides, promotes the release of shiitake mushroom bran polysaccharides from cells, and reduces the interference of impurities and by-products generated in traditional extraction methods; then, purified shiitake mushroom bran polysaccharides are obtained through deproteinization and separation and purification, which can not only further remove impurities and harmful substances, significantly improve the purity of shiitake mushroom bran polysaccharides, but also ensure the safety and stability of the product. Compared with the separate ultrasonic extraction method and enzymolysis extraction method, the present invention utilizes the complementary effect of ultrasound and enzymolysis to reduce the generation of impurities, which is conducive to the extraction of high-purity shiitake mushroom bran polysaccharides. In addition, the present invention is carried out under mild conditions, avoiding the destruction of the activity of shiitake mushroom bran polysaccharides by extreme conditions of high temperature, strong acid and strong alkali, and retaining the original biological activity of shiitake mushroom bran polysaccharides.

[0018] 1. The present invention utilizes ultrasound-assisted enzymolysis to extract shiitake mushroom husk polysaccharides, which significantly shortens the extraction time and improves the extraction efficiency; the shiitake mushroom husk polysaccharides obtained by the present invention have a good molecular structure and a relatively uniform molecular weight distribution; in addition, the in vitro antioxidant activity of shiitake mushroom husk polysaccharides is concentration gradient dependent, and the clearance rate from high to low is: hydroxyl radical>superoxide anion>ABTS + Free radicals>DPPH free radicals, but both were lower than the positive control Vc group. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0020] Figure 1 Glucose standard curve.

[0021] Figure 2 This is the elution curve of LRP on a DEAE-52 cellulose column.

[0022] Figure 3 This is the UV scanning spectrum of LRP.

[0023] Figure 4 This is the ion chromatogram of the standard product.

[0024] Figure 5 This is the LRP ion chromatogram.

[0025] Figure 6Figure 2 shows the standard curve of Pullulan polysaccharide and the LRP chromatogram.

[0026] Figure 7 This is the infrared spectrum of LRP.

[0027] Figure 8 are scanning electron microscope images of LRP; in the figure, A is a scanning electron microscope image of LRP × 4.8k times, with a scale of 2μm; B is a scanning electron microscope image of LRP × 12.31k times, with a scale of 1μm; C is a scanning electron microscope image of LRP × 15.00k times, with a scale of 1μm; D is a scanning electron microscope image of LRP × 25.00k times, with a scale of 400nm.

[0028] Fig. 9 This is the scavenging effect of LRP on DPPH free radicals.

[0029] Fig.10 For LRP to ABTS + Free radical scavenging effect.

[0030] Fig.11 This is the scavenging effect of LRP on superoxide anion free radicals.

[0031] Fig.12 This is the scavenging effect of LRP on hydroxyl radicals. DETAILED DESCRIPTION

[0032] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work belong to the scope of protection of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified, and the materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0033] Experimental reagents and experimental equipment Electric constant temperature blast drying oven: Shanghai Sanfa Scientific Instrument Co., Ltd.; electronic analytical balance: Shanghai Precision Scientific Instrument Co., Ltd.; rotary evaporator: Shanghai Yarong Biochemical Instrument Factory; circulating water vacuum pump: Shanghai Yarong Biochemical Instrument Factory; UV-visible spectrophotometer: Varian Technologies China Co., Ltd.; high-speed centrifuge: Shanghai Lu Xiangyi Centrifugal Instrument Co., Ltd.; ultrasonic cleaning machine: Shenzhen Jiemeng Cleaning Equipment Co., Ltd.; vertical constant temperature culture oscillator: Shanghai Jingshi Experimental Equipment Co., Ltd.; electric constant temperature water bath: Shanghai Yiheng Scientific Instrument Co., Ltd.; freeze dryer: Ningbo Xinzhi Biotechnology Co., Ltd.; constant temperature incubator: Daskart Scientific Experiment Co., Ltd.; ELISA reader: Shanghai Shanpu Biotechnology Co., Ltd.; separation chromatograph: Shanghai Luxi Analytical Instrument Factory Co., Ltd.

[0034] Shiitake mushroom bran: from the Wandong Comprehensive Experimental Station of the New Rural Development Research Institute of Anhui Agricultural University; glucose: Xilong Chemical Co., Ltd.; phenol: Aladdin Shanghai Co., Ltd.; sulfuric acid: Wuxi Zhanwang Chemical Reagent Co., Ltd.; chloroform: Wuxi Zhanwang Chemical Reagent Co., Ltd.; n-butanol: Aladdin Shanghai Co., Ltd.; citric acid: Wuxi Zhanwang Chemical Reagent Co., Ltd.; anhydrous disodium hydrogen phosphate: Shanghai Yuanye Biotechnology Co., Ltd.; anhydrous ethanol: Shanghai Titan Technology Co., Ltd.; sodium chloride: Shanghai Titan Technology Co., Ltd.; cellulase: Shanghai Yuanye Biotechnology Co., Ltd.; pectinase: Shanghai Yuanye Biotechnology Co., Ltd.; ABTS: Shanghai ELISA Biotechnology Co., Ltd.; superoxide anion scavenging ability kit: Shanghai ELISA Biotechnology Co., Ltd.

[0035] Embodiment 1: A kind of shiitake mushroom bran polysaccharide and its extraction method.

[0036] Place the mushroom husks in an oven and dry them at 50°C until constant weight. Grind them and pass them through an 80-mesh sieve. Seal them in a ziplock bag and store them in a dry place for later use.

[0037] 1.0 g of shiitake mushroom bran powder was weighed, and distilled water was added at a solid-liquid ratio of 1 g: 20 mL. Ultrasonic extraction was performed at an ultrasonic power of 300 w, an ultrasonic temperature of 50 ° C, and an ultrasonic time of 50 min. After ultrasonication, a buffer solution was added and the pH value was adjusted to 6.0 using an acidometer. Cellulase and pectinase were added at a mass ratio of 1:1, and the total enzyme amount was 1000 U / g. After constant temperature extraction at 45 ° C for 60 min, the enzyme was inactivated at 90 ° C for 10 min. Centrifugation was performed at 5000 r / min for 20 min, the supernatant was concentrated by a rotary evaporator, and 4 times the volume fraction of 95% ethanol solution was added to the concentrate, and it was allowed to stand overnight at 4 ° C. After centrifugation at 5000 r / min for 10 min, the precipitate was collected and dried in a vacuum freeze dryer to obtain shiitake mushroom bran crude polysaccharide.

[0038] Embodiment 2: Embodiment 2 has the same specific steps as Embodiment 1, except that: After ultrasound, a buffer solution was added and the pH value was adjusted to 5.0 using an acidometer. Cellulase and pectinase were added at a mass ratio of 1:1 with a total enzyme amount of 1000 U / g. The extraction was carried out at a constant temperature of 55°C for 50 min.

[0039] Embodiment 3: Embodiment 3 has the same specific steps as Embodiment 1, except that: After ultrasound, a buffer solution was added and the pH value was adjusted to 6.0 using an acidometer. Cellulase and pectinase were added in a mass ratio of 1:1 with a total enzyme amount of 800 U / g. The extraction was carried out at a constant temperature of 55°C for 40 min.

[0040] Embodiment 4: Embodiment 4 has the same specific steps as Embodiment 1, except that: After ultrasound, a buffer solution was added and the pH value was adjusted to 5.5 using an acidometer. Cellulase and pectinase were added at a mass ratio of 1:1 with a total enzyme amount of 800 U / g. The extraction was carried out at a constant temperature of 45°C for 50 min.

[0041] Comparative Example 1: Ultrasonic extraction process.

[0042] Weigh 1.000g of shiitake mushroom bran powder, add distilled water according to the solid-liquid ratio of 1g:20mL, place it in an ultrasonic instrument for extraction with an ultrasonic power of 350w, an ultrasonic temperature of 50℃, and an ultrasonic time of 50min. After the extraction, centrifuge the sample liquid at 5000r / min for 20min, collect the supernatant, filter and separate it with filter paper, and concentrate it on a rotary evaporator for 15min. Add 4 times the volume fraction of 95% ethanol solution to the concentrate, let it stand overnight at 4℃, centrifuge it at 5000r / min for 10min, collect the precipitate, and freeze-dry to obtain crude shiitake mushroom bran polysaccharide.

[0043] Comparative Example 2: The specific steps of Comparative Example 2 are the same as those of Comparative Example 1, except that: Ultrasonic extraction was carried out with an ultrasonic power of 250w, an ultrasonic temperature of 50℃, and an ultrasonic time of 30min.

[0044] Comparative Example 3: The specific steps of Comparative Example 3 are the same as those of Comparative Example 1, except that: Ultrasonic extraction was carried out with an ultrasonic power of 250w, an ultrasonic temperature of 60℃, and an ultrasonic time of 40min.

[0045] Comparative Example 4: The specific steps of Comparative Example 4 are the same as those of Comparative Example 1, except that: Ultrasonic extraction was carried out with an ultrasonic power of 250w, an ultrasonic temperature of 70℃, and an ultrasonic time of 50min.

[0046] Comparative Example 5: The specific steps of Comparative Example 5 are the same as those of Comparative Example 1, except that: Ultrasonic extraction was carried out with an ultrasonic power of 300w, an ultrasonic temperature of 60℃, and an ultrasonic time of 30min.

[0047] Comparative Example 6: The specific steps of Comparative Example 6 are the same as those of Comparative Example 1, except that: Ultrasonic extraction was carried out with an ultrasonic power of 300w, an ultrasonic temperature of 70℃, and an ultrasonic time of 40min.

[0048] Comparative Example 7: The specific steps of Comparative Example 7 are the same as those of Comparative Example 1, except that: Ultrasonic extraction was carried out with an ultrasonic power of 300w, an ultrasonic temperature of 50℃, and an ultrasonic time of 50min.

[0049] Comparative Example 8: The specific steps of Comparative Example 8 are the same as those of Comparative Example 1, except that: Ultrasonic extraction was carried out with an ultrasonic power of 350w, an ultrasonic temperature of 70℃, and an ultrasonic time of 30min.

[0050] Comparative Example 9: The specific steps of Comparative Example 9 are the same as those of Comparative Example 1, except that: Ultrasonic extraction was carried out with an ultrasonic power of 350w, an ultrasonic temperature of 50℃, and an ultrasonic time of 40min.

[0051] Comparative Example 10: The specific steps of Comparative Example 10 are the same as those of Comparative Example 1, except that: Ultrasonic extraction was carried out with an ultrasonic power of 350w, an ultrasonic temperature of 60℃, and an ultrasonic time of 50min.

[0052] Comparative Example 11: Enzymatic extraction process.

[0053] Weigh 1.0g of shiitake mushroom bran powder, add distilled water at a solid-liquid ratio of 1g:20mL, add buffer and adjust the pH value to 4.5 with an acidometer, add cellulase and pectinase at a mass ratio of 1:1, and the total enzyme amount is 1000U / g. After constant temperature extraction at 40℃ for 40min, the enzyme is inactivated at 90℃ for 10min; centrifuge at 5000r / min for 20min, concentrate the supernatant by rotary evaporator, add 4 times the volume fraction of 95% ethanol solution to the concentrate, let it stand overnight at 4℃, centrifuge at 5000r / min for 10min, collect the precipitate, and then dry it in a vacuum freeze dryer to obtain shiitake mushroom bran crude polysaccharide.

[0054] Comparative Example 12: The specific steps of Comparative Example 12 are the same as those of Comparative Example 11, except that: Buffer was added and the pH value was adjusted to 4.0 using an acidometer. Cellulase and pectinase were added in a mass ratio of 1:1 with a total enzyme amount of 600 U / g. The mixture was extracted at a constant temperature of 40°C for 30 min.

[0055] Comparative Example 13: The specific steps of Comparative Example 13 are the same as those of Comparative Example 11, except that: Buffer was added and the pH value was adjusted to 4.5 using an acidometer. Cellulase and pectinase were added in a mass ratio of 1:1 with a total enzyme amount of 800 U / g. The mixture was extracted at a constant temperature of 40°C for 40 min.

[0056] Comparative Example 14: The specific steps of Comparative Example 14 are the same as those of Comparative Example 11, except that: Buffer solution was added and the pH value was adjusted to 5.0 using an acidometer. Cellulase and pectinase were added at a mass ratio of 1:1 with a total enzyme amount of 1000 U / g. The mixture was extracted at a constant temperature of 40°C for 50 min.

[0057] Comparative Example 15: The specific steps of Comparative Example 15 are the same as those of Comparative Example 11, except that: Buffer was added and the pH value was adjusted to 4.5 using an acidometer. Cellulase and pectinase were added in a mass ratio of 1:1 with a total enzyme amount of 1000 U / g. The mixture was extracted at a constant temperature of 45°C for 30 min.

[0058] Comparative Example 16: The specific steps of Comparative Example 16 are the same as those of Comparative Example 11, except that: Buffer was added and the pH value was adjusted to 5.0 using an acidometer. Cellulase and pectinase were added in a mass ratio of 1:1 with a total enzyme amount of 600 U / g. The mixture was extracted at a constant temperature of 45°C for 40 min.

[0059] Comparative Example 17: The specific steps of Comparative Example 17 are the same as those of Comparative Example 11, except that: Buffer was added and the pH value was adjusted to 4.0 using an acidometer. Cellulase and pectinase were added in a mass ratio of 1:1 with a total enzyme amount of 800 U / g. The mixture was extracted at a constant temperature of 45°C for 50 min.

[0060] Comparative Example 18: The specific steps of Comparative Example 18 are the same as those of Comparative Example 11, except that: Buffer solution was added and the pH value was adjusted to 5.0 using an acidometer. Cellulase and pectinase were added at a mass ratio of 1:1 with a total enzyme amount of 800 U / g. The mixture was extracted at a constant temperature of 50°C for 30 min.

[0061] Comparative Example 19: The specific steps of Comparative Example 19 are the same as those of Comparative Example 11, except that: Buffer was added and the pH value was adjusted to 4.0 using an acidometer. Cellulase and pectinase were added in a mass ratio of 1:1 with a total enzyme amount of 1000 U / g. The mixture was extracted at a constant temperature of 50°C for 40 min.

[0062] Comparative Example 20: The specific steps of Comparative Example 20 are the same as those of Comparative Example 11, except that: Buffer was added and the pH value was adjusted to 4.5 using an acidometer. Cellulase and pectinase were added in a mass ratio of 1:1 with a total enzyme amount of 600 U / g. The mixture was extracted at a constant temperature of 50°C for 50 min.

[0063] The above-mentioned Examples 1 to 4 and Comparative Examples 1 to 20 were used to study the method for extracting polysaccharides from shiitake mushroom bran. The relevant test methods and results are as follows: 2. Effects of different extraction methods on the polysaccharide content of Lentinus edodes bran 1. Test methods The phenol-sulfuric acid colorimetric method was used to determine the polysaccharide content in Lentinus edodes bran.

[0064] Preparation of standard curve: accurately weigh 100.00 mg of glucose reference substance dried to constant weight at 103°C, place in a 1L volumetric flask, add appropriate amount of water to dissolve, dilute to scale and shake well to obtain 0.1 mg / mL glucose standard solution. Accurately pipette 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, 1.0 mL, 1.2 mL, 1.4 mL of the above solution into the numbered volumetric flask, add water to 2 mL, accurately add 1 mL of 6% phenol solution, shake well, quickly add 5.0 mL of concentrated sulfuric acid, shake well, color at room temperature for 20 minutes, calibrate the zero point with blank control, and measure its absorbance at 490 nm. Draw the glucose standard curve with absorbance as the ordinate and polysaccharide mass concentration as the abscissa.

[0065] Sample content determination: aspirate 1.0 mL of diluted shiitake mushroom bran polysaccharide extract, follow the above steps, measure the optical density, and calculate the shiitake mushroom bran polysaccharide content using the standard curve.

[0066] Calculation of the extraction rate of polysaccharides from shiitake mushroom bran: extraction rate of polysaccharides from shiitake mushroom bran / %=(c×V×N)÷m×100; Where: c is the mass concentration of shiitake mushroom bran polysaccharide calculated by the standard curve; V is the volume of the extract; N is the dilution multiple; m is the mass of the sample dry powder.

[0067] Use glucose as the standard, glucose concentration as the horizontal axis, and absorbance obtained according to the above operation as the vertical axis to draw the corresponding standard curve. Figure 1 As shown, the regression equation is: y=0.0507x-0.0283 (R²=0.9981). This result shows that the concentration of shiitake mushroom husk polysaccharide has a good linear relationship in the range of 2.5μg / mL~17.5μg / mL. In the formula: y is the absorbance, and x is the sample concentration.

[0068] 2. Test results The results of Example 1 to Example 4 are shown in Table 1 below.

[0069] Table 1 Extraction rate of polysaccharides from Lentinus edodes husk by ultrasound-assisted composite enzyme method The results of Comparative Examples 1 to 10 are shown in Table 2 below.

[0070] Table 2 Ultrasonic extraction rate of polysaccharides from Lentinus edodes husk The results of Comparative Examples 11 to 20 are shown in Table 3 below.

[0071] Table 3 Extraction rate of polysaccharides from Lentinus edodes husk by compound enzyme method From Tables 1 to 3 above, we can see that through the analysis of the maximum extraction rate of polysaccharides from Lentinus edodes husks, the extraction rate of ultrasound-assisted enzymatic hydrolysis is significantly higher than that of the other two methods, which is 20.96%. Therefore, ultrasound-assisted enzymatic hydrolysis was selected to extract a large amount of crude polysaccharides from Lentinus edodes husks in preparation for subsequent experiments.

[0072] 3. Separation and purification of crude polysaccharides from shiitake mushroom waste 1. Test methods Preparation of Sevag reagent: Prepare 100 mL of Sevag reagent in advance by mixing chloroform and n-butanol in a volume ratio of 4:1. Mix well and place in a brown bottle for later use.

[0073] Take 10mL of 40mg / mL shiitake champignon ...

[0074] Cellulose DEAE-52 pretreatment: Take 10g of DEAE-52 dry powder, stir vigorously with 20mL of distilled water and soak for 20min. After full swelling, absorb excess supernatant to remove impurities. Repeat the above operation five times until the supernatant is clear and transparent, and soak in 20% ethanol solution for later use.

[0075] Wet column packing: clean the column and fix it with an iron stand to ensure its vertical stability; place a thin layer of cotton wool at the bottom of the column to prevent cellulose loss during the subsequent column packing process; first add 1 / 3 of the column volume of distilled water to the column, turn on the constant flow pump and observe the even outflow of water; load 20mL of treated DEAE-52 cellulose into the column. The principle of column packing is to load the column in small amounts and multiple times, and compact the cellulose after it settles naturally at the bottom of the column. If bubbles appear, tap the column gently with an ear suction bulb to remove them. Note that the entire column packing process should ensure wetting, and the filler should not be dry.

[0076] Equilibration of chromatography column: Use distilled water as equilibration liquid, add distilled water into the chromatography column, open the valve, control the flow rate of equilibration liquid at 0.75mL / min, and the chromatography column reaches equilibrium.

[0077] Collection of chromatographic liquid: 4g of deproteinized shiitake mushroom chaff crude polysaccharide was prepared into a 40mg / mL solution, and after wet loading, it was stabilized with distilled water and allowed to stand; gradient elution was performed with distilled water and 0.1mol / L, 0.2mol / L, and 0.3mol / L NaCl solutions, ensuring a flow rate of 1.5mL / min, and the outlet was collected with an automatic collector, and the eluate was collected in different tubes and gradients, with each tube collecting about 5mL. The absorbance of each tube of eluate was measured by the phenol-sulfuric acid method, and an elution curve was drawn, in which the elution curve used the tube number as the horizontal coordinate and the absorbance and concentration gradient as the vertical coordinate; the eluate under the main peak was selected according to the elution curve for merging and concentration, and freeze-drying was performed to obtain purified shiitake mushroom chaff polysaccharide, and multiple enrichment and collection were performed.

[0078] 2. Test results like Figure 2 As shown in the figure, two elution powders were obtained by elution with distilled water and 0.10mol / L, 0.20mol / L, and 0.30mol / L NaCl solutions. The first peak was the water elution rod, named LRP-1; the second peak was the 0.2mol / L NaCl elution peak, named LRP-2. The remaining elution peaks were small in peak value and had no significance for collection and research, so they were ignored.

[0079] After dialysis, concentration and freeze-drying, purified LRP-1 and LRP-2 polysaccharides were obtained. After multiple enrichments, the purified shiitake mushroom husk polysaccharide components were collected. Since the actual collection rate of LRP-1 was relatively low, subsequent research was mainly based on LRP-2, which was uniformly named LRP.

[0080] 3. Structural Characterization of Lentinula edodes Mushroom Bran Polysaccharides 1. Identification of the purity of shiitake mushroom bran polysaccharides 1.1 Test results A single component of Lentinus edodes bran polysaccharide was prepared into a 0.1 mg / mL aqueous solution, and a band scan was performed at 190 nm to 400 nm using an ultraviolet spectrophotometer to determine whether there was an obvious protein characteristic absorption peak in the range of 260 nm to 280 nm.

[0081] 1.2 Test results Depend on Figure 3 It can be seen that the purified shiitake mushroom bran polysaccharide has no absorption at 280nm, indicating that the sample basically does not contain protein and nucleic acid and has a high purity.

[0082] 2. Monosaccharide composition analysis 2.1 Test methods Take a clean chromatographic bottle, weigh 15 mg of shiitake mushroom bran polysaccharide sample, add 1 mL of 2MTFA solution, and heat at 121°C for 2 hours. Pass nitrogen, blow dry, add 99.99% methanol to wash, blow dry again, and repeat the methanol washing 3 times. Add sterile water to dissolve and transfer to a chromatographic bottle for testing. Using the Thermo ICS5000+ ion chromatography system, the sample is introduced into the chromatographic column through the injector, the mobile phase brings the sample into the chromatographic column for separation, and the detector detects the signal for reading. Among them, mobile phase A: H2O mobile phase B: 0.1M NaOH.

[0083] The ion chromatograms of the monosaccharide mixed standard and shiitake mushroom bran polysaccharide are shown in Figure 2. Figure 4 , Figure 5 As shown, the peak at 30 min corresponds to the solvent peak of NaOH.

[0084] 2.2 Test results As can be seen from the figure, the separation of different monosaccharides is good, and the purified LRP is mainly composed of rhamnose, arabinose, galactose, glucose, xylose, mannose, and galacturonic acid. According to the molar concentration and peak area of ​​the monosaccharide mixed standard, the molar mass percentage of each monosaccharide in the purified LRP is 4.91:15.32:4.69:44.29:20.98:6.7:3.11, among which glucose and xylose are the main monosaccharide components of the purified LRP.

[0085] 3. Molecular weight determination 3.1 Test methods The standard products of 1044000Da, 348000Da, 107000Da, 49400Da, 21900Da, 6400Da, 4320Da, 3480Da, 2250Da, and 1560Da were prepared into 2mg / mL aqueous solution, shaken and dissolved for 2h, and filtered through a 0.45μm water system microporous filter membrane for standby use. Prepare 0.1M sodium nitrate aqueous solution as the mobile phase, and set the flow rate to 1mL / min. The PL aquagel-OH Mixed-M chromatographic column was connected to the chromatograph and the instrument was operated. The chromatographic conditions were as follows: the detector was a differential refractive index detector, the test temperature was 40℃, the injection volume was 100μL, and the standard and sample were transferred to a 2mL injection bottle. The horizontal axis corresponded to the retention time and the vertical axis corresponded to the concentration response for linear regression. The relative molecular weight distribution and distribution range of polysaccharides in the sample were calculated according to the standard curve of pullulan polysaccharide.

[0086] Table 4 Relative molecular weight of Lentinus edodes husk polysaccharides The relative molecular weight of LRP was determined by gel permeation chromatography, and the relative molecular weight distribution and distribution range of shiitake mushroom bran polysaccharides in the sample were obtained according to the standard curve of pullulan polysaccharide. Pullulan polysaccharides of different molecular weights were used as standard products, and linear regression was performed with the horizontal axis corresponding to the retention time and the vertical axis corresponding to the concentration response to obtain the calibration curve equation: LRP =9.236855-0.378171x, R²=0.992632.

[0087] 3.2 Test results Depend on Figure 6 It can be seen that LRP has a polysaccharide characteristic peak at 10min~18min, and the weight average molecular weight is 1.904×10 4 Da; As can be seen from Table 4, the polydispersity coefficient is 2.80992. The closer the polydispersity coefficient is to 1, the more uniform the molecular weight distribution is.

[0088] 4. Infrared spectroscopy 4.1 Test methods The potassium bromide tablet test method was used. 1 mg of Lentinus edodes husk polysaccharide powder sample and 200 mg of pure KBr were ground evenly, placed in a mold, and pressed into a transparent sheet on a hydraulic press. The sample was placed in an infrared spectrometer for testing. The wave number range was 4000 cm -1 Up to 500cm -1 , scanning times 32 times, resolution 4cm -1 The infrared spectrum of the obtained Lentinus edodes husk polysaccharide is shown in Figure 7 .

[0089] 4.2 Test results Depend on Figure 7 It can be seen that LRP is 3403.74 cm -1 The absorption peak at 2930.19 cm -1 The micro-absorption band at 1667.81 cm-1 may be caused by asymmetric CH stretching vibration, which represents the typical groups of polysaccharides, hydroxyl and alkyl. -1 There is a strong absorption peak near 1200cm -1 ~1000 cm -1 The absorption peak at 1075.45 cm indicates the vibration of the pyran ring of CO stretching. -1 This indicates that the polysaccharide of Lentinus edodes bran contains pyranose ring; 939.07 cm -1 The absorption peak is the β-glycosidic bond.

[0090] 5. Scanning Electron Microscope Analysis 5.1 Test methods 10 mg of shiitake mushroom husk polysaccharide sample was glued to the sample stage with conductive glue, and gold was sprayed for 45 seconds using an ETD-2000M sputtering coater at 10 mA. Then, a scanning electron microscope was used to select different magnifications to photograph the sample morphology. The accelerating voltage was 10 kV when photographing the morphology. The obtained shiitake mushroom husk polysaccharide scanning electron microscope image is shown in Figure 8 .

[0091] 5.2 Test results like Figure 8 As shown in Figure 2, LRP has an irregular cluster structure composed of stacked fragmented particles, indicating that the small intermolecular force of LRP may contribute to the formation of its loose structure; after magnification of Figures C and D, irregular flake-like protrusions can be observed on the surface of LRP.

[0092] 4. Evaluation of the antioxidant activity of Lentinus edodes bran polysaccharides in vitro 1. DPPH free radical scavenging ability 1.1 Test methods Accurately weigh 15.8 mg DPPH, dissolve it in anhydrous ethanol and make up to volume in a 100 mL volumetric flask to obtain a 0.4 mmol / L DPPH solution. Store it at 4°C away from light until use. Make sure to prepare it before use.

[0093] Sequentially pipette 1.0mL of sample solution of 1mg / mL~6mg / mL into a test tube, add 0.5mL of 0.4mmol / L DPPH anhydrous ethanol aqueous solution, and then add 2.0mL of water. Mix the reaction system and react in the dark at 30℃ for 30min, and use anhydrous ethanol as blank to measure its absorbance A1 at 517nm; blank control: 0.5mL anhydrous ethanol, 2mL water and 1mL sample solution of different concentrations are mixed, placed in the dark for 30min, and its absorbance A2 is measured at 517nm; negative control: 0.5mL of 0.4mmol / L DPPH anhydrous ethanol solution is added to 3mL of pure water and mixed, placed in the dark for 30min, and its absorbance A0 is measured at 517nm. Vc is the positive control. Three parallels are made for each test tube, and the results are averaged. Calculate the clearance rate according to the following formula:

[0094] DPPH free radical scavenging rate (%) = [1-(A1-A2) / A0] × 100%.

[0095] 1.2 Test results like Fig. 9It can be seen that within the concentration range of 1 mg / mL~6 mg / mL, the scavenging rate of LRP for DPPH free radicals showed a positive correlation with the increase in the mass concentration of Lentinus edodes bran polysaccharides. When it reached 6 mg / mL, the scavenging ability for DPPH free radicals was the strongest, which was 28.75%, indicating that LRP can provide a hydrogen donor for scavenging DPPH. Although the scavenging ability of each sample was lower than that of the positive control Vc, it had a certain scavenging ability at high concentrations.

[0096] 2. ABTS free radical scavenging ability 2.1 Test methods Follow the instructions of the ABTS kit. Accurately weigh 100 mg of pure shiitake mushroom bran polysaccharide and dissolve it in 1 mL of distilled water to obtain 100.0 mg / mL of shiitake mushroom bran polysaccharide solution as the mother solution. Dilute the mother solution to obtain reaction solutions with final concentrations of 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, and 6 mg / mL, respectively. Preheat the microplate reader for 30 minutes and adjust the wavelength to 734 nm. Prepare the working solution before use: take a bottle of reagent 2, add 11 mL of reagent 1, shake and mix for 20 minutes, let it stand, and take the supernatant for use. Add 10 μL of extract and 190 μL of working solution to the EP tube of the blank group, add 10 μL of sample solution and 190 μL of working solution to the EP tube of the measurement group, mix thoroughly, and measure the absorbance at 734 nm within 10 minutes. Make three parallels for each test tube and take the average of the results. Calculate the clearance rate according to the following formula:

[0097] ABTS free radical scavenging rate (%) = (A 空白 -A 测定 ) / A 空白 ×100%.

[0098] A 空白 : The absorbance of the blank group of the sample with Vc replacing the shiitake mushroom bran polysaccharide; A 测定 : Absorbance of the experimental group containing Lentinus edodes bran polysaccharide samples.

[0099] 2.2 Test results like Fig.10 As shown in the figure, within the concentration range of 1 mg / mL to 6 mg / mL, LRP has a significant effect on ABTS. + The free radical scavenging rate gradually increased with the increase of the concentration of shiitake mushroom husk polysaccharide, showing a significant positive correlation and a significant dose-effect relationship. + The free radical scavenging ability was the strongest, at 50.16%. Although the scavenging ability of each sample was lower than that of the positive control Vc, it had a certain scavenging ability at high concentrations.

[0100] 3. Superoxide anion free radical scavenging ability 3.1 Test methods Follow the instructions of the superoxide anion free radical kit. Set distilled water as the control group, and the shiitake mushroom chaff polysaccharide solution with mass concentrations of 1mg / mL, 2mg / mL, 3mg / mL, 4mg / mL, 5mg / mL, and 6mg / mL as the test group. Add 10μL of reagent 1 and 40μL of reagent 2 to the test tubes of the control group with 25μL distilled water and the test group without sample solution, mix well, and react at 25℃ for 1min. Then accurately pipette 25μL of shiitake mushroom chaff polysaccharide solution and 50μL of reagent 3 of serial concentration into the test tube, mix well, react at 37℃ for 30min, and then continue to add 50μL of reagent 4 and reagent 5. Mix well, color at 37℃ for 20min, pipette 220μL into a 96-well plate, and measure the absorbance of the control tube and the test tube at 530nm, which are recorded as control tube A3 and test tube A4 respectively. Vc is a positive control, and three groups of parallel groups are made for each concentration, and the results are averaged. The clearance rate was calculated according to the following formula:

[0101] Superoxide anion clearance rate (%) = (A3-A4) / A3×100%.

[0102] 3.2 Test results like Fig.11 As shown in the figure, within the concentration range of 1 mg / mL to 6 mg / mL, the scavenging rate of LRP for superoxide anion radicals gradually increased with the increase of the mass concentration of Lentinus edodes husk polysaccharide, showing a positive linear correlation. When it reached 6 mg / mL, LRP had the strongest scavenging ability for superoxide anion radicals, which was 53.17%. Although the scavenging ability of LRP was lower than that of the positive control Vc, it had a certain scavenging ability at high concentrations.

[0103] 4. Hydroxyl free radical scavenging ability 4.1 Test methods Accurately prepare 9mmol / L ferrous sulfate solution, 9mmol / L salicylic acid-ethanol solution, and 8.8mmol / L hydrogen peroxide solution, and store at room temperature for use. Accurately weigh 100mg of pure shiitake mushroom bran polysaccharide and dissolve it in 1mL of distilled water to obtain 100.0mg / mL shiitake mushroom bran polysaccharide solution as the mother solution. Dilute the mother solution to obtain reaction solutions with concentrations of 1mg / mL, 2mg / mL, 3mg / mL, 4mg / mL, 5mg / mL, and 6mg / mL, respectively. Sequentially transfer 1.0mL of shiitake mushroom bran polysaccharide solutions of different concentrations into sequential test tubes, and add 1mL 9mmol / L ferrous sulfate solution, 1mL 9mmol / L salicylic acid-ethanol solution, and 1mL 8.8mmol / L hydrogen peroxide solution, respectively. After the reaction system is fully mixed, it is water bathed in a 37℃ water bath for 30min, and then centrifuged at 1200r / min for 6min in a centrifuge, and the supernatant is retained. Deionized water was used to replace the shiitake mushroom husk polysaccharide sample as a blank group, and deionized water was used as a control group to adjust the zero. The absorbance of each concentration gradient of shiitake mushroom husk polysaccharide at 510nm was measured by ultraviolet spectrophotometer, and Vc was used as a positive control. Three parallels were made for each test tube, and the results were averaged. The clearance rate was calculated according to the following formula:

[0104] Hydroxyl free radical scavenging rate (%) = (A5-A6) / A5×100%.

[0105] A5: Absorbance of blank group with deionized water replacing the shiitake mushroom bran polysaccharide sample; A6: Absorbance of the experimental group containing 1 mL of Lentinus edodes bran polysaccharide sample.

[0106] 4.2 Test results like Fig.12 As shown, in the concentration range of 1 mg / mL to 6 mg / mL, the scavenging rate of LRP for hydroxyl radicals gradually increased with the increase of the mass concentration of shiitake mushroom bran polysaccharide, showing a positive linear relationship. At a concentration of 6 mg / mL, the scavenging rate of LRP for hydroxyl radicals also reached a maximum of 69.82%. Although the scavenging ability of LRP was lower than that of the positive control Vc, it had a certain scavenging ability within the concentration gradient.

[0107] Although preferred embodiments of the present invention have been described, additional changes and modifications may occur to these embodiments once those skilled in the art are aware of the basic inventive concepts.

[0108] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A method for extracting polysaccharides from shiitake mushroom bran, characterized in that: The steps include: Crushing: crushing and sieving the mushroom bran to obtain the mushroom bran powder; Dissolution: Add shiitake mushroom bran powder into distilled water at a solid-liquid ratio of 1g:10mL~30mL to obtain shiitake mushroom bran solution; Ultrasound: The shiitake mushroom bran solution is ultrasonically extracted for 30min~50min at an ultrasonic power of 250w~350W and an ultrasonic temperature of 50℃~70℃ to destroy the plant cell wall; Enzymatic hydrolysis: After ultrasound, the pH value of the shiitake mushroom bran solution was adjusted to 5.0-6.0, cellulase and pectinase were added, and the total activity of cellulase and pectinase was 800U / g-1000U / g. The enzymatic hydrolysis was carried out at a constant temperature of 45°C-55°C for 40min-60min to improve the extraction efficiency, and the shiitake mushroom bran crude polysaccharide extract was obtained. The precipitate obtained after alcohol precipitation and centrifugation was the shiitake mushroom bran crude polysaccharide; Deproteinization: removing the protein in the crude polysaccharide from shiitake mushroom husks to obtain deproteinized crude polysaccharide powder from shiitake mushroom husks; Separation and purification: The deproteinized shiitake mushroom bran crude polysaccharide powder is separated, purified and dried to obtain the purified shiitake mushroom bran polysaccharide powder.

2. The method for extracting shiitake mushroom husk polysaccharides according to claim 1, wherein: The deproteinization is performed using the Sevag method, wherein the concentration of the crude polysaccharide from shiitake mushroom husks in the Sevag method is 30 mg / mL to 50 mg / mL.

3. The method for extracting shiitake mushroom husk polysaccharides according to claim 1, wherein: The separation and purification uses DEAE-52 column chromatography, which includes DEAE-52 cellulose pretreatment, column packing equilibration and chromatography liquid collection.

4. The method for extracting shiitake mushroom husk polysaccharides according to claim 3, wherein: The cellulose DEAE-52 pretreatment is specifically as follows: soaking the DEAE-52 dry powder in distilled water, discarding the supernatant after sufficient swelling, until the supernatant is clear and transparent, and soaking the lower precipitate in a 10% to 30% ethanol solution by volume for later use.

5. The method for extracting polysaccharides from shiitake mushroom waste according to claim 3, wherein: The column balancing method comprises the following steps: firstly adding distilled water to the chromatographic column for balancing, loading the pretreated cellulose DEAE-52 into the chromatographic column, and removing bubbles; using distilled water as the balancing liquid, adding distilled water to the chromatographic column, and controlling the flow rate so that the chromatographic column reaches equilibrium.

6. The method for extracting polysaccharides from Lentinus edodes husks according to claim 3, characterized in that: The chromatography liquid collection is specifically as follows: after preparing the crude polysaccharide solution of Lentinus edodes husk and loading the sample, distilled water and NaCl solution are used for concentration gradient elution, and the eluate is collected.

7. The method for extracting polysaccharides from shiitake mushroom waste according to claim 1, characterized in that: The crushed mushroom bran powder is passed through a 70-90 mesh sieve.

8. The method for extracting polysaccharides from shiitake mushroom waste according to claim 1, characterized in that: The ultrasonic power is 300w, the ultrasonic time is 50min, and the ultrasonic temperature is 50°C.

9. Lentinus edodes bran polysaccharide prepared by the method for extracting Lentinus edodes bran polysaccharide according to any one of claims 1 to 8.

10. Use of the shiitake mushroom bran polysaccharide according to claim 9 in the preparation of antioxidant products.

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