Lentinan as well as extraction method and application thereof

Abstract

The invention relates to the technical field of biochemical engineering, in particular to lentinan as well as an extraction method and application thereof, and the extraction method of the lentinan comprises the following steps: crushing, dissolving, ultrasonic treatment and enzymolysis. According to the method disclosed by the invention, the lentinan is extracted through ultrasonic synergistic enzymolysis, so that the extraction rate of the lentinan is increased, the biological activity of the lentinan 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 lentinan and an extraction method and application thereof. Background Art

[0002] Shiitake mushrooms, also known as fragrant mushrooms, winter mushrooms, and fragrant mushrooms, belong to the fruiting body of the Agaricaceae family of the Basidiomycetes. Its flesh has a unique aroma and is crisp and tender. It has high nutritional and medicinal value and is an edible and medicinal mushroom. As a major mushroom-growing country, my country's shiitake mushroom industry has huge development potential. In 2022, my country's shiitake mushroom production was about 13.1324 million tons, and the market size of the shiitake mushroom industry was about 104.482 billion yuan, a year-on-year increase of 10.9%. Lentinan , referred to as LNT) is an effective active ingredient extracted from high-quality Lentinus edodes fruiting bodies. The active ingredient is branched β-(1,3)-D-glucan, which has antioxidant, antibacterial, antiviral, antitumor and immunomodulatory effects.

[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 it has the problems of being time-consuming and having a low extraction rate.

[0004] Therefore, it is urgent to develop a Lentinan extraction process with high extraction rate and short extraction time to facilitate the recycling of waste. Summary of the invention

[0005] In order to develop a lentinan extraction process with high extraction rate and short time consumption, the present invention provides a lentinan and its extraction method and application. The present invention uses ultrasound to synergize enzymatic hydrolysis to extract lentinan, thereby improving the extraction rate of lentinan and retaining the biological activity of lentinan.

[0006] A method for extracting lentinan comprises the following steps: Grinding: Grind the mushrooms and sieve to obtain mushroom powder; Dissolution: Add shiitake mushroom powder to distilled water at a ratio of 1g:10mL~30mL to obtain shiitake mushroom solution; Ultrasound: Extract the mushroom solution by ultrasonic method for 30min~50min at an ultrasonic power of 250w~350W and an ultrasonic temperature of 30℃~50℃; Enzymatic hydrolysis: The pH value of the shiitake mushroom solution after ultrasound is adjusted to 4.5-5.5, and cellulase and pectinase are added, with the total enzyme amount added being 200U / g-600U / g. The enzymatic hydrolysis is carried out at a constant temperature of 50℃-60℃ for 20min-40min to obtain an extract, which is then precipitated with alcohol, centrifuged and dried to obtain shiitake mushroom polysaccharide.

[0007] The present invention utilizes ultrasound to coordinate enzymatic hydrolysis to extract lentinan, which promotes the release of lentinan from cells, significantly shortens the extraction time, and improves the extraction efficiency; in addition, the present invention is carried out under mild conditions, avoiding the destruction of the activity of lentinan by extreme conditions of high temperature, strong acid and strong alkali, and retains the original biological activity of lentinan.

[0008] Furthermore, the crushed mushroom powder is sieved through a 70-90 mesh sieve.

[0009] Furthermore, the cellulase and pectinase are added in a mass ratio of 1:1, and the total enzyme amount of the cellulase and pectinase is 600 U / g.

[0010] Furthermore, the ultrasonic power is 50w, the ultrasonic temperature is 40°C, and the ultrasonic time is 30min.

[0011] Furthermore, during the enzymatic hydrolysis, the pH value of the shiitake mushroom solution is 4.5, and the extraction is performed at 60° C. for 40 minutes.

[0012] Furthermore, the shiitake mushroom solution is obtained by adding distilled water to shiitake mushroom powder at a solid-liquid ratio of 1 g:20 mL.

[0013] The invention provides lentinan.

[0014] The present invention also provides an application of Lentinan in the preparation of antioxidant products.

[0015] Compared with the prior art, the present invention has the following beneficial effects: 6. The present invention utilizes ultrasound-assisted enzymatic hydrolysis to extract Lentinan, which further promotes the release of Lentinan from cells, significantly shortens the extraction time, and improves the extraction efficiency; in addition, the method is carried out under mild conditions, avoiding the destruction of the activity of Lentinan by extreme conditions of high temperature, strong acid and strong alkali, and retains the original biological activity of Lentinan.

[0016] 7. The present invention uses ultrasound-assisted enzymatic hydrolysis to extract Lentinan, which can maintain a good molecular structure of Lentinan. At the same time, the extracted Lentinan has a high purity, a relatively uniform molecular weight distribution, and reduces the content of impurities and useless components; the in vitro antioxidant activity of Lentinan is concentration gradient dependent, and the clearance rate from high to low is: hydroxyl free radical>superoxide anion>ABTS + Free radicals>DPPH free radicals, but both were lower than the positive control Vc group.

[0017] 8. The present invention is based on the research of polysaccharide extraction technology to improve the economic conversion efficiency of shiitake mushrooms, providing a reference for the in-depth development and application of the polysaccharide industry; based on the research background of polysaccharides as livestock and poultry feed and their own biological activity, it provides a reference for the development of functional feed additives or their application in the medical field; the components with antioxidant activity are separated and purified, and the structural characteristics of the active ingredients are further explained, providing new ideas for the research of sugar bioengineering. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] 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.

[0019] Figure 1 Glucose standard curve.

[0020] Figure 2 This is the elution curve of LNT-1 on a DEAE-52 cellulose column.

[0021] Figure 3 This is the UV scanning spectrum of LNT-1.

[0022] Figure 4 This is the ion chromatogram of the standard product.

[0023] Figure 5 This is the ion chromatogram of LNT-1.

[0024] Figure 6 Figure 2 shows the standard curve of Pullulan polysaccharide and the chromatogram of LNT-1.

[0025] Figure 7 This is the infrared spectrum of LNT-1.

[0026] Figure 8 is a scanning electron microscope image of LNT-1; in the figure, A is a scanning electron microscope image of LNT-1×500 times, with a scale of 20μm; B is a scanning electron microscope image of LNT-1×5.00k times, with a scale of 2μm; C is a scanning electron microscope image of LNT-1×10.00k times, with a scale of 1μm; D is a scanning electron microscope image of LNT-1×20.00k times, with a scale of 500nm.

[0027] Fig. 9 This is the scavenging effect of LNT-1 on DPPH free radicals.

[0028] Fig.10 LNT-1 to ABTS + Free radical scavenging effect.

[0029] Fig.11 This is the scavenging effect of LNT-1 on superoxide anion free radicals.

[0030] Fig.12 This is the scavenging effect of LNT-1 on hydroxyl free radicals. DETAILED DESCRIPTION

[0031] 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 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.

[0032] 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.

[0033] Shiitake mushrooms: from the Wandong Comprehensive Experimental Station of the Institute of New Rural Development 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.

[0034] Example 1: Lentinan and its extraction method.

[0035] Place the mushrooms in an oven and dry them at 50°C until constant weight. Crush 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.

[0036] Weigh 1.0g of shiitake mushroom powder, add distilled water at a solid-liquid ratio of 1g:20mL, and perform ultrasonic extraction at an ultrasonic power of 250w, an ultrasonic temperature of 40℃, and an ultrasonic time of 30min; after ultrasonication, 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 600U / g. After constant temperature extraction at 60℃ for 40min, inactivate the enzyme at 90℃ for 10min; centrifuge at 5000r / min for 20min, concentrate the supernatant with a 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 with a vacuum freeze dryer to obtain shiitake mushroom polysaccharide.

[0037] 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 5.5 using an acidometer. Cellulase and pectinase were added at a mass ratio of 1:1 with a total enzyme amount of 200 U / g. The extraction was carried out at a constant temperature of 60°C for 40 min.

[0038] 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 5.0 using an acidometer. Cellulase and pectinase were added in a mass ratio of 1:1 with a total enzyme amount of 400 U / g. The mixture was extracted at a constant temperature of 60°C for 20 min.

[0039] 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 4.5 using an acidometer. Cellulase and pectinase were added at a mass ratio of 1:1 with a total enzyme amount of 600 U / g. The extraction was carried out at a constant temperature of 60°C for 30 min.

[0040] Comparative Example 1: Ultrasonic extraction process.

[0041] Weigh 1.0g of shiitake mushroom 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 250w, an ultrasonic temperature of 40℃, and an ultrasonic time of 30min. 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 shiitake mushroom polysaccharide.

[0042] 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 30℃, and an ultrasonic time of 30min.

[0043] 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 40℃, and an ultrasonic time of 40min.

[0044] 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 50℃, and an ultrasonic time of 50min.

[0045] Comparative Example 5: The specific steps of Comparative Example 5 are the same as those of Comparative Example 1, except that: Ultrasonic extraction was performed with an ultrasonic power of 300w, an ultrasonic temperature of 40°C, and an ultrasonic time of 30min.

[0046] 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 50℃, and an ultrasonic time of 40min.

[0047] 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 30℃, and an ultrasonic time of 50min.

[0048] 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 50℃, and an ultrasonic time of 30min.

[0049] 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 30℃, and an ultrasonic time of 40min.

[0050] 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 40℃, and an ultrasonic time of 50min.

[0051] Comparative Example 11: Enzymatic extraction process.

[0052] Weigh 1.0g of shiitake mushroom 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 200U / g. After constant temperature extraction at 40℃ for 20min, inactivate the enzyme 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 polysaccharide.

[0053] 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 5.0 using an acidometer. Cellulase and pectinase were added in a mass ratio of 1:1 with a total enzyme amount of 400 U / g. The mixture was extracted at a constant temperature of 40°C for 30 min.

[0054] Comparative Example 13: The specific steps of Comparative Example 13 are the same as those of Comparative Example 11, except that: Buffer solution was added and the pH value was adjusted to 5.5 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 40°C for 40 min.

[0055] 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 600 U / g. The mixture was extracted at a constant temperature of 45°C for 20 min.

[0056] 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 5.5 using an acidometer. Cellulase and pectinase were added in a mass ratio of 1:1 with a total enzyme amount of 200 U / g. The mixture was extracted at a constant temperature of 45°C for 30 min.

[0057] 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 4.5 using an acidometer. Cellulase and pectinase were added in a mass ratio of 1:1 with a total enzyme amount of 400 U / g. The mixture was extracted at a constant temperature of 45°C for 40 min.

[0058] 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 5.5 using an acidometer. Cellulase and pectinase were added in a mass ratio of 1:1 with a total enzyme amount of 400 U / g. The mixture was extracted at a constant temperature of 50°C for 20 min.

[0059] Comparative Example 18: The specific steps of Comparative Example 18 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 30 min.

[0060] 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 5.0 using an acidometer. Cellulase and pectinase were added in a mass ratio of 1:1 with a total enzyme amount of 200 U / g. The mixture was extracted at a constant temperature of 50°C for 40 min.

[0061] 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 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 50°C for 40 min.

[0062] The above-mentioned Examples 1 to 4 and Comparative Examples 1 to 20 were used to study the method for extracting Lentinan. The relevant test methods and results are as follows: 1. Effects of different extraction methods on the content of Lentinan 1. Test methods The phenol-sulfuric acid colorimetric method was used to determine the content of Lentinan.

[0063] 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.

[0064] Sample content determination: Pipette 1.0 mL of diluted Lentinan extract, follow the above steps, measure the optical density, and calculate the polysaccharide content using the standard curve.

[0065] Calculation of Lentinan extraction rate: Lentinan extraction rate / %=(c×V×N)÷m×100; Where: c is the mass concentration of Lentinan 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.

[0066] 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 polysaccharide concentration 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.

[0067] 2. Test results The results of Example 1 to Example 4 are shown in Table 1 below.

[0068] Table 1 Extraction rate of Lentinan by ultrasound-assisted compound enzyme method The results of Comparative Examples 1 to 10 are shown in Table 2 below.

[0069] Table 2 Ultrasonic extraction rate of Lentinan The results of Comparative Examples 11 to 20 are shown in Table 3 below.

[0070] Table 3 Extraction rate of Lentinan by compound enzyme method From Tables 1 to 3 above, we can see that through the analysis of the maximum extraction rate of Lentinan, the extraction rate of ultrasound-assisted enzymatic hydrolysis is significantly higher than that of the other two methods, which is 28.99%. Therefore, ultrasound-assisted enzymatic hydrolysis was selected to extract Lentinan in large quantities in preparation for subsequent experiments.

[0071] 9. Separation and purification of Lentinan 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.

[0072] Take 10mL of 40mg / mL Lentinan solution, mix the Lentinan solution with Sevag reagent at a volume ratio of 4:1, place it on a magnetic stirrer and stir rapidly for 20min, centrifuge it at 3850r / min for 15min, the solution is divided into three layers, the upper layer is Lentinan sample solution, the middle layer is denatured protein, and the lower layer is organic solvent. Collect the upper Lentinan sample solution, repeat the above operation until no protein layer appears in the middle after centrifugation. Collect the deproteinized Lentinan solution obtained by rotary evaporation to remove the organic solvent, then add anhydrous ethanol to precipitate overnight, centrifuge, and freeze-dry to obtain Lentinan dry powder.

[0073] 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.

[0074] 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.

[0075] 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.

[0076] Chromatographic liquid collection: 4g of deproteinized Lentinan was prepared into a solution with a concentration of 40mg / mL. 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 to ensure a flow rate of 1.5mL / min. The outlet was collected with an automatic collector, and the eluent was collected in different tubes and gradients, with each tube collecting about 5mL. The absorbance of each tube of eluent was measured by the phenol-sulfuric acid method, and an elution curve was drawn, in which the tube number was the horizontal coordinate and the absorbance and concentration gradient were the vertical coordinates. According to the elution curve, the eluent under the main peak was selected for merging and concentration, and freeze-drying was performed to obtain purified Lentinan, which was enriched and collected multiple times.

[0077] 2. Test results like Figure 2As shown in the figure, after elution with distilled water and 0.10mol / L, 0.20mol / L, and 0.30mol / L NaCl solutions, a polysaccharide component was collected in the water-washed part, showing a single symmetrical peak, indicating that it was a single component with uniform molecular weight distribution, and it was named LNT-1. Since the polysaccharide component eluted from the salt-washed part was very small and had no significance for collection and research, it was ignored.

[0078] After dialysis, concentration and freeze-drying, purified LNT-1 polysaccharide was obtained. After multiple enrichments, purified Lentinan components were collected.

[0079] 10. Structural characterization of Lentinan 1. Purity Identification of Lentinan 1.1 Test results Purity identification of Lentinan: Prepare a single component Lentinan into a 0.1 mg / mL aqueous solution, and scan the wavelength range of 190nm~400nm using an ultraviolet spectrophotometer to determine whether there is an obvious protein characteristic absorption peak in the range of 260nm~280nm.

[0080] 1.2 Test results Depend on Figure 3 It can be seen that the purified Lentinan has no absorption at 260nm, indicating that the sample basically does not contain protein and nucleic acid and has a high purity.

[0081] 2. Monosaccharide composition analysis 2.1 Test methods Monosaccharide composition analysis: Take a clean chromatographic bottle, weigh 15 mg of Lentinan 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 2 to 3 times. Add sterile water to dissolve and transfer to the chromatographic bottle for testing. The Thermo ICS5000+ ion chromatography system is used to introduce the sample into the chromatographic column through the injector, and the mobile phase brings the sample into the chromatographic column for separation. The detector detects the signal for reading. Among them, mobile phase A: H2O mobile phase B: 0.1M NaOH.

[0082] 2.2 Test results The ion chromatograms of the monosaccharide mixed standard and Lentinan are shown in Figure 2. Figure 4 , Figure 5 As shown, the peak at 30 min corresponds to the solvent peak of NaOH.

[0083] As can be seen from the figure, the separation of different monosaccharides is good, and the monosaccharide composition of purified LNT-1 is composed of galactose and glucose. According to the molar concentration and peak area of ​​the monosaccharide mixed standard, the molar mass percentage of each monosaccharide in purified LNT-1 is 2.61:97.39, among which glucose is the main monosaccharide component of purified LNT-1.

[0084] 3. Molecular weight determination 3.1 Test methods Molecular weight determination: The standard polysaccharides of 1044000Da, 348000Da, 107000Da, 49400Da, 21900Da, 6400Da, 4320Da, 3480Da, 2250Da, and 1560Da were prepared into 2mg / mL aqueous solutions, shaken and dissolved for 2h, and filtered through a 0.45μm water system microporous filter membrane for use. Prepare 0.1M sodium nitrate aqueous solution as the mobile phase, and set the flow rate to 1mL / min. Connect the PL aquagel-OH Mixed-M chromatographic column to the chromatograph and run the instrument. The chromatographic conditions are as follows: the detector is a differential refractive index detector, the test temperature is 40℃, the injection volume is 100μL, and the standard and sample are transferred to a 2mL injection bottle. Linear regression was performed with the horizontal axis corresponding to the retention time and the vertical axis corresponding to the concentration response. The relative molecular weight distribution and distribution range of the polysaccharide in the sample were calculated based on the standard curve of pullulan polysaccharide.

[0085] 3.2 Test results Table 4 Relative molecular weight of Lentinan The relative molecular weight of LNT-1 was determined by gel permeation chromatography, and the relative molecular weight distribution and distribution range of the polysaccharide 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: LNT-1 =9.173489-0.314669x, R²=0.994867.

[0086] Depend on Figure 6 It can be seen that LNT-1 has a polysaccharide characteristic peak at 17min~20min, and the weight average molecular weight is 1.536×10 3 Da; As can be seen from Table 4, the polydispersity coefficients are 1.03924. The closer the polydispersity coefficient is to 1, the more uniform the molecular weight distribution is.

[0087] 4. Infrared spectroscopy 4.1 Test methods The potassium bromide tablet test method was used. 1 mg of Lentinan 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 ~500cm -1 , scanning times 32 times, resolution 4cm-1 The infrared spectrum of Lentinan obtained is shown in Figure 7 .

[0088] 4.2 Test results Depend on Figure 7 It can be seen that at 3000cm -1 ~3750cm -1 There is a broad band in region LNT-1 at 3427.97 cm -1 There is an absorption peak at 1409.53cm, which is caused by the stretching vibration of OH in Lentinan; -1 There is a CH angle vibration absorption peak at 1253.31cm -1 The peak of CO stretching vibration on the ring is 1147.23cm -1 The CC skeleton stretching vibration absorption peak on the ring is 1076.40cm -1 It is indicated that Lentinan is pyranose, 937.74cm -1 It is an asymmetric pyran ring stretching vibration, indicating that Lentinan contains β-glycosidic bonds between sugar units.

[0089] 5. Scanning Electron Microscope Analysis 5.1 Test methods 10 mg of Lentinan sample was glued to the sample stage with conductive glue, and gold was sprayed for 45 seconds using ETD-2000M sputtering coating instrument, with a gold spraying current of 10 mA. Then, the sample morphology was photographed using a scanning electron microscope at different magnifications, with an accelerating voltage of 10 kV. Figure 8

[0090] 5.2 Test results like Figure 8 As shown, LNT-1 exhibits a fuller elliptical shape and a smooth surface.

[0091] 11. Evaluation of the antioxidant activity of Lentinan 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.

[0092] 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:

[0093] DPPH free radical scavenging rate / %=[1-(A1-A2) / A0]×100%.

[0094] 1.2 Test results like Fig. 9 It can be seen that within the concentration range of 1 mg / mL~6 mg / mL, the scavenging rate of LNT-1 on DPPH free radicals showed a positive correlation with the increase in the mass concentration of Lentinan. When it reached 6 mg / mL, the scavenging ability of DPPH free radicals was the strongest, which was 50.33%, indicating that LNT-1 can provide a hydrogen donor for scavenging DPPH. Although the scavenging ability was lower than that of the positive control Vc, it had a certain scavenging ability at high concentrations.

[0095] 2. ABTS free radical scavenging ability 2.1 Test methods Follow the instructions of the ABTS kit. Accurately weigh 100 mg of pure Lentinan and dissolve it in 1 mL of distilled water to obtain a 100.0 mg / mL Lentinan solution as the mother solution. Dilute the mother solution to obtain reaction solutions with 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. 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:

[0096] ABTS free radical scavenging rate / %=(A 空白 -A测定 ) / A 空白 ×100%.

[0097] A 空白 : The absorbance of the blank group of Lentinus edodes husk polysaccharide sample was replaced by deionized water; A 测定 : Absorbance of the experimental group containing Lentinus edodes bran polysaccharide samples.

[0098] 2.2 Test results like Fig.10 As shown in the figure, within the concentration range of 1 mg / mL to 6 mg / mL, LNT-1 has a strong affinity for ABTS + The free radical scavenging rate gradually increased with the increase of Lentinan mass concentration, showing a significant positive correlation and a significant dose-effect relationship. + The free radical scavenging ability was the strongest, at 51.53%. Although the scavenging ability was lower than that of the positive control Vc, it had a certain scavenging ability at high concentrations.

[0099] 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 set the lentinan solutions with mass concentrations of 1mg / mL, 2mg / mL, 3mg / mL, 4mg / mL, 5mg / mL, and 6mg / mL as the assay groups. Add 10μL of reagent 1 and 40μL of reagent 2 to the test tubes of the control group with 25μL of distilled water and the assay group without sample solution, mix thoroughly, and react at 25℃ for 1min. Then accurately pipette 25μL of the lentinan solution of serial concentrations and 50μL of reagent 3 into the test tubes, mix thoroughly, react at 37℃ for 30min, and then continue to add 50μL of reagent 4 and reagent 5. Mix thoroughly, color at 37℃ for 20min, pipette 220μL into a 96-well plate, and measure the absorbance of the control tube and the assay tube at 530nm, which are recorded as control tube A3 and assay tube A4 respectively. Vc is the positive control, and three groups are parallel for each concentration, and the results are averaged. Calculate the clearance rate according to the following formula:

[0100] Superoxide anion clearance rate / %=(A3-A4) / A3×100%.

[0101] 3.2 Test results like Fig.11As shown in the figure, within the concentration range of 1 mg / mL to 6 mg / mL, the scavenging rate of LNT-1 on superoxide anion free radicals gradually increased with the increase of Lentinan mass concentration, showing a positive linear relationship. When reaching 6 mg / mL, LNT-1 had the strongest scavenging ability on superoxide anion free radicals, which was 62.06%, and had a certain scavenging ability at high concentrations.

[0102] 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 Lentinan and dissolve it in 1mL of distilled water to obtain 100.0mg / mL Lentinan 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 Lentinan solution of different concentrations into sequential test tubes, and add 1mL9mmol / 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 lentinan 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 lentinan at 510nm was measured by UV 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:

[0103] Hydroxyl free radical scavenging rate / %=(A5-A6) / A5×100%.

[0104] A5: The absorbance of the blank group of Lentinan sample was replaced by deionized water.

[0105] A6: Absorbance of the experimental group containing 1 mL of Lentinan sample.

[0106] 4.2 Test results like Fig.12 As shown in the figure, within the concentration range of 1 mg / mL to 6 mg / mL, the scavenging rate of LNT-1 on hydroxyl radicals gradually increased with the increase of the mass concentration of Lentinan, showing a positive linear relationship. The scavenging rate of LNT-1 on hydroxyl radicals at different concentrations increased slowly, but was close to the positive control Vc. At a concentration of 6 mg / mL, the highest scavenging rate was 79.79%, while the scavenging rate of Vc was 85.74%, indicating that LNT-1 had a certain scavenging effect on hydroxyl radicals.

[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 Lentinan, characterized in that: The steps include: Grinding: Grind the mushrooms and sieve to obtain mushroom powder; Dissolution: Add shiitake mushroom powder to distilled water at a ratio of 1g:10mL~30mL to obtain shiitake mushroom solution; Ultrasound: Extract the mushroom solution by ultrasonic method for 30min~50min at an ultrasonic power of 250w~350W and an ultrasonic temperature of 30℃~50℃; Enzymatic hydrolysis: The pH value of the shiitake mushroom solution after ultrasound is adjusted to 4.5-5.5, and cellulase and pectinase are added. The total enzyme amount of cellulase and pectinase is 200U / g-600U / g. The enzymatic hydrolysis is carried out at a constant temperature of 50℃-60℃ for 20min-40min to obtain an extract. After alcohol precipitation and centrifugation, the shiitake mushroom polysaccharide is dried.

2. The method for extracting lentinan according to claim 1, characterized in that: The crushed mushroom powder is passed through a 70-90 mesh sieve.

3. The method for extracting lentinan according to claim 1, characterized in that: The cellulase and pectinase are added in a mass ratio of 1:1, and the total enzyme amount of the cellulase and pectinase is 600 U / g.

4. The method for extracting lentinan according to claim 1, characterized in that: The ultrasonic power is 50w, the ultrasonic temperature is 40°C, and the ultrasonic time is 30min.

5. The method for extracting lentinan according to claim 1, characterized in that: The pH value of the shiitake mushroom solution during the enzymatic hydrolysis was 4.5, and the extraction was performed at 60° C. for 40 minutes.

6. The method for extracting lentinan according to claim 1, characterized in that: The shiitake mushroom solution is obtained by adding distilled water to shiitake mushroom powder at a solid-liquid ratio of 1g:20mL.

7. Lentinan prepared by the method for extracting Lentinan according to any one of claims 1 to 6.

8. Use of the lentinan according to claim 7 in the preparation of antioxidant products.

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