Auricularia auricula polysaccharide prepared through fermentation of eurotium cristatum, method and application of auricularia auricula polysaccharide
Through the fermentation method of Centurosan compost and ultrasonic assisted extraction, combined with a multi-step purification process, the problems of low extraction efficiency and limited yield of black fungus polysaccharides are solved, which significantly improves the extraction efficiency and purity of polysaccharides, enhances biological activity, and is suitable for industrialization Production and application.
Patent Information
- Application Number
- CN202411374990.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-30
- Publication Date
- 2024-12-17
AI Technical Summary
The existing black fungus polysaccharide extraction methods are inefficient, have limited yields, and lack the exploration of new polysaccharides. Most of the traditional methods are physical methods, which fail to effectively change the composition and structure of polysaccharides.
The fermentation method of Centurus Compass fermentation combined with ultrasonic assisted extraction is used to degrade cell walls by using enzymes secreted by the strain, fermentation conditions and multi-step purification process are optimized, and the extraction efficiency and purity of polysaccharides are improved.
It significantly improves the extraction efficiency and purity of black fungus polysaccharides, enhances its biological activity, is suitable for industrial production and widespread application, reduces production costs, and is suitable for the extraction of other edible fungi or plant polysaccharides.
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Figure CN119144678A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of natural product extraction and separation, and specifically relates to a method for obtaining black fungus polysaccharide through a microbial fermentation method. Background Art
[0002] Black fungus is a large edible fungus with rich nutrition and high medicinal value. It is widely used in food, health care products and traditional Chinese medicine. In recent years, black fungus has been cultivated on a large scale in Jilin, Heilongjiang, Henan, Hubei and other provinces, and its output ranks fourth in the world's mushroom production. Black fungus fruiting bodies contain a variety of bioactive substances, among which polysaccharides are an important functional component with multiple activities such as antioxidant, anti-tumor, and immunomodulatory. These functions are related to the structure and properties of black fungus polysaccharides. Therefore, extracting black fungus polysaccharides through different methods and evaluating their structural properties and functions have become a research hotspot.
[0003] The extraction process of black fungus polysaccharide mainly includes the steps of pretreatment of raw materials, extraction and purification of polysaccharide. At present, in order to increase the yield of black fungus polysaccharide, water extraction, enzymolysis and acid-base extraction are usually used to obtain polysaccharide. These methods optimize the extraction scheme of black fungus polysaccharide to a certain extent, but the types of polysaccharides obtained are limited, and most of them are original polysaccharide components in black fungus, lacking the full excavation of new black fungus polysaccharide, because most of these methods are physical methods, and the composition and structure of polysaccharide are not changed. The characteristic of the present invention is to make full use of the crown-like spores, which secretes multiple enzymes including cellulase, pectinase and hemicellulase during metabolism. Due to the specificity of the enzyme, the crown-like spores can degrade pectin, hemicellulose and cellulose, thereby destroying the dense cell wall, so that the polysaccharide in the wall is dissolved from a more sparse structure, thereby obtaining a higher extraction rate; at the same time, due to the degradation of the cell wall by these complex enzymes, new soluble polysaccharides will also be formed. Therefore, the black fungus polysaccharide is fermented by Eurotium cristatum and ultrasonic treatment is combined to realize an efficient, simple and stable black fungus polysaccharide extraction method, improve the yield and biological activity of black fungus polysaccharide, and meet the needs of industrial production and application. In addition, the introduction of microbial fermentation technology is also helpful for the extraction of polysaccharides. The enzymes secreted by microorganisms during the fermentation process can effectively degrade the cell wall and increase the release rate of polysaccharides. In this process, the chemical structure of the substrate can also be changed to improve the bioavailability and biological activity of the substrate. Microbial fermentation has many advantages, such as high specificity, few by-products, simple operation, and environmental friendliness. Summary of the invention
[0004] The present invention innovatively proposes a method for extracting polysaccharides from black fungus fruiting bodies using fermentation of Eurotium cristatum. By optimizing fermentation conditions, introducing ultrasound-assisted extraction, and combining a multi-step purification process, the extraction efficiency and purity of black fungus polysaccharides are significantly improved, and the biological activities of these polysaccharides in terms of anti-oxidation, immunomodulation, and hypoglycemic properties are significantly improved. The development and application of this technology not only provides technical support for the industrial production of black fungus polysaccharides, but also lays the foundation for their wide application in food, medicine, and health products. The method of the present invention is suitable for large-scale production and has the advantages of simple operation and high yield.
[0005] The method for preparing black fungus polysaccharide by fermenting Eurotium cristatum provided by the present invention has the following beneficial effects:
[0006] 1. The present invention combines microbial fermentation with ultrasonic-assisted extraction technology to significantly improve the extraction efficiency of black fungus polysaccharides. The fermentation process uses enzymes secreted by Eurotium cristatum to pre-degrade the black fungus cell wall and promote the release of polysaccharides; and the ultrasonic treatment further breaks up the cell structure through the cavitation effect and accelerates the dissolution of polysaccharides. Compared with traditional extraction methods, such as water extraction, high-temperature extraction, and high cost of enzymatic hydrolysis, the present invention has high extraction efficiency, low energy consumption, and low cost.
[0007] 2. The multi-step purification process of the present invention includes isoelectric point method protein removal, centrifugal separation, alcohol precipitation and dialysis, which ensures the high purity of polysaccharides. The pH is adjusted to the isoelectric point so that the protein in the solution is precipitated, the ethanol precipitation step effectively concentrates and purifies the polysaccharides, and dialysis removes small molecule impurities. The black fungus polysaccharide finally obtained has a high purity and is suitable for use in food, medicine and health products.
[0008] 3. The extraction method of the present invention has a concise and clear process flow, the equipment used is simple and easy to obtain, the operation process is easy to control, and it is suitable for large-scale production. In addition, after the process parameters are optimized, the conditions in the extraction process are stable and highly repeatable, which can ensure the consistency of product quality in different batches.
[0009] 4. Reduce production costs
[0010] The invention reduces the time and amount of chemical reagents required in the extraction and purification process by optimizing the process flow, thereby reducing the overall production cost.
[0011] 5. Wide range of applications
[0012] The invention is not only suitable for the extraction of black fungus polysaccharide, but also can be extended to the extraction and purification of other edible fungi or plant polysaccharides, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS
[0013] Figure 1 Table of components and contents of black fungus polysaccharides.
[0014] Figure 2 Inhibitory effect of black fungus polysaccharide on α-amylase. Different lowercase letters represent significant differences among the groups at the same polysaccharide concentration (P < 0.05), the same below.
[0015] Figure 3 The scavenging ability of black fungus polysaccharide on DPPH free radicals.
[0016] Figure 4 The scavenging ability of black fungus polysaccharide on hydroxyl radicals.
[0017] Figure 5 Total reducing capacity of black fungus polysaccharides.
[0018] Figure 6 The scavenging ability of black fungus polysaccharide on superoxide anion free radicals.
[0019] Figure 7 ABTS ＋ Free radical scavenging ability.
[0020] Figure 8 Effect of Auricularia auricula polysaccharide on the proliferation of RAW264.7 cells.
[0021] Fig. 9 Effects of Auricularia auriculariae polysaccharides on the phagocytosis of RAW264.7 cells.
[0022] Fig.10 Effects of H2O2 concentration on the survival rate of RAW264.7 cells
[0023] Fig.11 Protective effect of Auricularia auricularia polysaccharide on oxidative damage in RAW264.7 cells.
[0024] Fig.12 Effect of Auricularia auriculariae polysaccharide on cytokine release in RAW264.7 cells. DETAILED DESCRIPTION
[0025] The present invention is further described in detail below in conjunction with specific embodiments. The examples given are only for illustrating the present invention, not for limiting the scope of the present invention.
[0026] The experimental methods in the following examples are conventional methods unless otherwise specified.
[0027] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.
[0028] Example 1: Fermentation of black fungus polysaccharides
[0029] (1) Raw material preparation
[0030] The dried black fungus fruiting bodies were crushed and passed through an 80-mesh sieve to obtain black fungus powder. 80 g of black fungus powder was weighed and set aside.
[0031] (2) Fermentation treatment
[0032] Mix 80 g of black fungus powder and 400 mL of distilled water at a solid-liquid ratio of 1:5. Place the mixture in an autoclave and sterilize at 121°C for 20 min. After sterilization, cool it to room temperature.
[0033] In a clean bench, add 24 mL of the suspension of Eurotium cristatum to the cooled black fungus mixture. The concentration of the suspension is 1×10 6 CFU / mL. It was placed in a constant temperature incubator at 28°C for fermentation for 6 days.
[0034] (3) Ultrasonic assisted extraction
[0035] After the fermentation is completed, 6400 mL of distilled water is added to the mixture at a solid-liquid ratio of 1:80 and stirred evenly.
[0036] The mixed solution was placed in an ultrasonic processor, the power was selected to be 330 W, the temperature was set to 60°C, and the treatment time was 30 min. The pH of the solution was then adjusted to 10.5 with 0.1 mol / L NaOH solution and stirred on a magnetic stirrer for 1 h.
[0037] (4) Centrifugal concentration to remove protein
[0038] After the treated mixture was balanced, centrifuged at 6796×g for 15 min, and the supernatant was collected and concentrated to 1 / 4 of the original volume (about 1600 mL) in a rotary evaporator.
[0039] The pH of the concentrated supernatant was adjusted to 3.0 and precipitated in a 4°C refrigerator for 24 h. After precipitation, the supernatant was collected by centrifugation at 6796 × g for 15 min.
[0040] (5) Polysaccharide precipitation and purification
[0041] Slowly add 6400 mL of anhydrous ethanol to the supernatant collected by centrifugation, stirring while adding to ensure thorough mixing. The mixture was placed in a 4°C refrigerator for 12 h.
[0042] After standing, the mixture was centrifuged at 6796×g for 15 min to collect the precipitate. Finally, the precipitate was frozen and placed in a freeze dryer for freeze drying to obtain dry black fungus polysaccharide.
[0043] (6) Results
[0044] The final mass of the black fungus polysaccharide product was 6.54 g. According to the yield formula, the yield of black fungus polysaccharide was 8.175% (6.54 g / 80 g × 100%).
[0045] (7) Composition determination
[0046] The total sugar content in polysaccharides was determined by the phenol-sulfuric acid method; the protein content in polysaccharides was determined by the BCA kit method; the uronic acid content in polysaccharides was determined by the sulfuric acid-carbazole method; the polyphenol content in polysaccharides was determined by the folin-phenol colorimetric method; the moisture content in polysaccharides was determined according to GB5009.3-2016; and the ash content in polysaccharides was determined according to GB5009.4-2016.
[0047] The results of the composition determination experiment are as follows Figure 1 As shown, the total sugar content was 62.06±0.31%, the protein content was 7.45±0.12%, the uronic acid content was 27.12±0.69%, and the polyphenol content was 1.07±0.02%.
[0048] Example 2: Unfermented black fungus polysaccharide
[0049] (1) Raw material preparation
[0050] The dried black fungus fruiting bodies were crushed and passed through an 80-mesh sieve to obtain black fungus powder. 40 g of black fungus powder was weighed and set aside.
[0051] (2) Ultrasonic assisted extraction
[0052] Mix 40 g black fungus powder and 200 mL distilled water at a solid-liquid ratio of 1:5. Place the mixture in an autoclave and sterilize at 121°C for 20 min. Add 3200 mL distilled water to the mixture at a solid-liquid ratio of 1:80 and stir evenly.
[0053] The mixed solution was placed in an ultrasonic processor, the power was selected to be 330 W, the temperature was set to 60 °C, and the treatment time was 30 min. The pH of the solution was then adjusted to 10.5 with 0.1 mol / L NaOH solution and stirred on a magnetic stirrer for 1 h.
[0054] (3) Centrifugal concentration to remove protein
[0055] After the treated mixture was balanced, centrifuged at 6796×g for 15 min, and the supernatant was collected and concentrated to 1 / 4 of the original volume (about 800 mL) in a rotary evaporator.
[0056] The pH of the concentrated supernatant was adjusted to 3.0 and precipitated in a 4°C refrigerator for 24 h. After precipitation, the supernatant was collected by centrifugation at 6796 × g for 15 min.
[0057] (4) Polysaccharide precipitation and purification
[0058] Slowly add 3200 mL of anhydrous ethanol to the supernatant collected by centrifugation, stirring while adding to ensure thorough mixing. The mixture was placed in a 4°C refrigerator for 12 h.
[0059] After standing, the mixture was centrifuged at 6796×g for 15 min to collect the precipitate. Finally, the precipitate was frozen and placed in a freeze dryer for freeze drying to obtain dry black fungus polysaccharide.
[0060] (5) Results
[0061] The final mass of the black fungus polysaccharide product was 1.755 g. According to the yield formula, the yield of black fungus polysaccharide was 4.39% (1.755 g / 40 g × 100%).
[0062] (6) Composition determination
[0063] The total sugar content in polysaccharides was determined by the phenol-sulfuric acid method; the protein content in polysaccharides was determined by the BCA kit method; the uronic acid content in polysaccharides was determined by the sulfuric acid-carbazole method; the polyphenol content in polysaccharides was determined by the folin-phenol colorimetric method; the moisture content in polysaccharides was determined according to GB5009.3-2016; and the ash content in polysaccharides was determined according to GB5009.4-2016.
[0064] The results of the composition determination experiment are as follows Figure 1 As shown, the total sugar content was 65.43±1.68%, the protein content was 7.25±0.22%, the uronic acid content was 25.06±0.69%, and the polyphenol content was 0.96±0.03%.
[0065] Example 3: Determination of α-amylase inhibition rate
[0066] This example is designed to further describe the hypoglycemic activity of fermented polysaccharides through in vitro hypoglycemic experiments.
[0067] The specific steps are as follows:
[0068] Weigh a certain amount of black fungus polysaccharide sample, prepare polysaccharide sample solutions of different concentrations with 20 mmol / L sodium phosphate buffer (pH 6.9), mix 200 μL of α-amylase solution (1.0 U / mL, dissolved in pH 6.9 sodium phosphate buffer) and 200 μL of polysaccharide solution, record as A1; replace polysaccharide solution with NaCl-containing buffer, record as A0, replace enzyme solution with buffer as background, record as A2, positive control is acarbose, incubate at 37 ℃ for 10 min, add 400 μL of 0.25% starch solution, water bath (37 ℃) for 10 min, then add 1 mL of DNS colorimetric agent to terminate the reaction, add 10 mL of distilled water, and measure the absorbance of background, negative control, and sample at 540 nm. The calculation formula is as follows:
[0069]
[0070] The results of α-amylase inhibition test are as follows Figure 2 As shown in the figure, the experimental results show that the inhibition rate increases with the increase of concentration in the range of 0-2 mg / mL. The fermented black fungus polysaccharide is superior to the unfermented polysaccharide, and there is a significant difference (P < 0.05).
[0071] Example 4: Determination of DPPH free radical scavenging ability
[0072] This example is designed to further describe the antioxidant activity of fermented polysaccharides through in vitro antioxidant experiments.
[0073] The specific steps are as follows:
[0074] Prepare 0.1 mmol / L DPPH solution with anhydrous ethanol and store it in dark place. Adjust OD with anhydrous ethanol before use. 517 nm 1.25±0.05. Take 2.0 mL of black fungus polysaccharide solution in a 10 mL test tube, add 2.0 mL of DPPH solution, place at room temperature and avoid light for 20 min, measure the absorbance at 517 nm and record it as A1. The absorbance of ethanol instead of DPPH is recorded as A2. The absorbance of ethanol instead of sample is recorded as A0. The calculation formula is as follows:
[0075]
[0076] The results of the DPPH free radical scavenging ability experiment are as follows Figure 3 As shown in the figure, the experimental results show that within the concentration range of 0-2 mg / mL, the clearance rate increases with the increase of concentration. The fermented black fungus polysaccharide is superior to the unfermented polysaccharide, and there is a significant difference (P < 0.05).
[0077] Example 5: Determination of Hydroxyl Radical Scavenging Ability
[0078] This example is designed to further describe the antioxidant activity of fermented polysaccharides through in vitro antioxidant experiments.
[0079] The specific steps are as follows:
[0080] Take 1 mL of black fungus polysaccharide solution and place it in a 10 mL test tube, then add 1 mL of 9 mmol / L ferrous sulfate solution, 1 mL of 9 mmol / L salicylic acid ethanol solution and 1 mL of 8.8 mmol / L H2O2 solution in sequence, shake evenly, warm the test tube at 37℃ for 30 min, cool to room temperature, and measure the absorbance at 510 nm as A1. The absorbance of H2O replacing H2O2 is A2. The absorbance of H2O replacing the sample is A0. The calculation formula is as follows:
[0081]
[0082] The results of the hydroxyl radical scavenging ability test are as follows Figure 4 As shown in the figure, the experimental results show that within the concentration range of 0-2 mg / mL, the clearance rate increases with the increase of concentration. The fermented black fungus polysaccharide is superior to the unfermented polysaccharide, and there is a significant difference (P < 0.05).
[0083] Example 6: Determination of total reducing power
[0084] This example is designed to further describe the antioxidant activity of fermented polysaccharides through in vitro antioxidant experiments.
[0085] The specific steps are as follows:
[0086] First, take 1 mL of black fungus polysaccharide solution in a 10 mL centrifuge tube, add 2.5 mL of 0.2 mol / L pH 6.6 PBS buffer and 2.5 mL of 1% potassium ferricyanide solution, mix well, react at 50 ° C for 20 min, and after cooling to room temperature, add 0.5 mL of 10% trichloroacetic acid solution, mix well, centrifuge at 1698 × g for 10 min, take the supernatant, add 0.5 mL of 0.1% FeCl3 solution and 2.5 mL of deionized water, shake and mix well, let stand for 10 min, and measure OD 700 Recorded as A1, and the absorbance value of H2O replacing the sample is recorded as A2. The calculation formula is as follows:
[0087]
[0088] The results of the total reducing power experiment are as follows Figure 5As shown in the figure, the experimental results show that within the concentration range of 0-2 mg / mL, the total reducing power increases with the increase of concentration. The fermented black fungus polysaccharide is superior to the unfermented polysaccharide, and there is a significant difference (P < 0.05).
[0089] Example 7: Determination of superoxide anion free radical scavenging ability
[0090] This example is designed to further describe the antioxidant activity of fermented polysaccharides through in vitro antioxidant experiments.
[0091] The specific steps are as follows:
[0092] Take 1 mL of black fungus polysaccharide solution in a test tube, add 1 mL of 10 mmol / L pyrogallol solution and 5 mL of 50 mmol / L Tris-HCl (pH 8.2), react at 25℃ for 4 min, then add 1 mL of 8 mol / L HCl to terminate the reaction, and measure the OD 320 For A1, H2O replaces pyrogallol OD 320 For A2, H2O replaces the OD of the sample 320 is A0. The calculation formula is as follows:
[0093]
[0094] The results of the superoxide anion free radical scavenging ability experiment are as follows Figure 6 As shown in the figure, the experimental results show that within the concentration range of 0-2 mg / mL, the clearance rate increases with the increase of concentration. The fermented black fungus polysaccharide is superior to the unfermented polysaccharide, and there is a significant difference (P < 0.05).
[0095] Example 8: ABTS ＋ Determination of free radical scavenging ability
[0096] This example is designed to further describe the antioxidant activity of fermented polysaccharides through in vitro antioxidant experiments.
[0097] The specific steps are as follows:
[0098] First, prepare 7 mmol / L ABTS solution and 2.45 mmol / L K2S2O8 solution, mix them in equal proportions, and react them at 4℃ in the dark for 12-16 hours before use. Dilute with anhydrous ethanol to OD 734 0.7±0.02. Take 0.5 mL of black fungus polysaccharide solution in a test tube, add 4.5 mL of diluted ABTS stock solution, react at 30℃ for 10 min, and measure OD 734 For A1. H2O replaces ABTS OD 734A2. H2O replaces the OD of the sample 734 is A0. The calculation formula is as follows:
[0099]
[0100] ABTS ＋ The free radical scavenging ability test results are as follows Figure 7 As shown in the figure, the experimental results show that within the concentration range of 0-2 mg / mL, the clearance rate increases with the increase of concentration. The fermented black fungus polysaccharide is better than the unfermented polysaccharide, and there is a significant difference at a concentration of 1.2 mg / mL (P < 0.05).
[0101] Example 9: Effect of Auricularia auricula polysaccharide on the proliferation of RAW264.7 cells
[0102] This example is designed to further describe the immune activity of fermented polysaccharides through cell experiments.
[0103] The specific steps are as follows:
[0104] RAW264.7 cells were cultured to the logarithmic growth phase, counted by hemocytometer, and the cell suspension was diluted with DMEM medium to a cell concentration of 2×10 5 cells / mL, 100 μL of cell suspension was taken with a pipette and placed in 60 wells in the inner circle of the 96-well plate, and 100 μL of PBS buffer was added to each well in the outer circle of the well plate (to prevent water loss in the inner circle wells and achieve moisturizing effect), and the 96-well plate was placed in a 37°C, 5% CO2 incubator for 24 h. The 96-well plate was taken out, the supernatant was discarded, and after washing with PBS, 100 μL of black fungus polysaccharide of different concentrations (12.5, 25, 50, 100, 200, 400, 800 μg / mL) was added. Three replicate wells were set for each of the above groups. Continue to culture in a 37°C, 5% CO2 incubator for 24 h, then add 100 μL of 10% CCK8 solution to each well, place it in a 37°C, 5% CO2 incubator for 1-4 h, and measure OD with an enzyme marker. 450 nm The cell survival rate was calculated according to the following formula.
[0105]
[0106] Where: A0—OD value of the well containing cells, culture medium, CCK-8 solution, and no sample;
[0107] A1—OD value of the well containing culture medium, CCK-8 solution, and no cells;
[0108] A2—OD values of wells containing cells, culture medium, CCK-8 solution, and samples.
[0109] The experimental results of the effect of black fungus polysaccharide on the proliferation of RAW264.7 cells are as follows Figure 8 As shown in the results, the cell survival rate increased with the concentration in the range of 12.5-800 μg / mL. The fermented black fungus polysaccharide was superior to the unfermented polysaccharide, and there was a significant difference (P < 0.05).
[0110] Example 10: Effect of Auricularia auricula polysaccharide on phagocytosis of RAW264.7 cells
[0111] This example is designed to further describe the immune activity of fermented polysaccharides through cell experiments.
[0112] The specific steps are as follows:
[0113] Treat RAW264.7 cells according to the grouping of the proliferation experiment and culture them. Discard the supernatant, wash with PBS, add 100 μL of 0.075% neutral red solution to each well, and place in the incubator for 1 h. Take the supernatant for later use and wash with PBS several times. Add 100 μL of cell lysis buffer (0.01% acetic acid and anhydrous ethanol mixed in equal volumes). Incubate at room temperature overnight and measure OD 470 nm And calculate the phagocytic index.
[0114]
[0115] The results of the experiment on the phagocytic effect of black fungus polysaccharide on RAW264.7 cells are as follows Fig. 9 As shown in the results, the phagocytic index increased with the concentration in the range of 12.5-800 μg / mL. The fermented black fungus polysaccharide was superior to the unfermented polysaccharide, and there was a significant difference (P < 0.05).
[0116] Example 11: Protective effect of black fungus polysaccharide on oxidative damage of RAW264.7 cells
[0117] This example is designed to further describe the immune activity of fermented polysaccharides through cell experiments.
[0118] The specific steps are as follows:
[0119] RAW264.7 cells were intervened with H2O2 at concentrations of 100, 200, 400, 600, 800, 1000, and 1200 μmol / L, and the cell survival rate was determined. The survival rate was used to determine whether the oxidative damage model was successfully established. The concentrations of the fungus fermentation broth were selected to be 12.5, 25, 50, 100, 200, 400, and 800 μg / mL, and the H2O2 concentration was selected to be 400 μmol / L according to the results of the oxidation model experiment. 5After culturing RAW264.7 cells with a concentration of 10 cells / mL for 24 h, the cells were intervened with different concentrations of Auricularia auricularia fermentation broth and H2O2, and the cell survival rate was determined according to the proliferation experiment method.
[0120] The experimental results of the protective effect of black fungus polysaccharide on oxidative damage of RAW264.7 cells are as follows Fig.10 , Fig.11 As shown in the results, the cell survival rate increased with the concentration in the range of 12.5-800 μg / mL. The fermented black fungus polysaccharide was superior to the unfermented polysaccharide, and there was a significant difference at a concentration of 400 μg / mL (P<0.05).
[0121] Example 12: Effect of Auricularia auricula polysaccharide on the release of cytokines in RAW264.7 cells
[0122] This example is designed to further describe the immune activity of fermented polysaccharides through cell experiments.
[0123] ELISA kits were used to detect the release of IL-10, IL-1β, IL-6, and TNF-α in the cell supernatant.
[0124] The experimental results of the effect of black fungus polysaccharide on the release of IL-10 in RAW264.7 cells are as follows Fig.12 As shown in the figure, the experimental results show that the release amount increases with the concentration in the range of 12.5-800 μg / mL. The fermented black fungus polysaccharide is better than the unfermented polysaccharide, and there are significant differences when the concentration is 100-800 μg / mL (P<0.05).
[0125] The experimental results of the effect of black fungus polysaccharide on the release of IL-1β in RAW264.7 cells are as follows Fig.12 As shown in the figure, the experimental results show that the release amount increases with the concentration in the range of 12.5~800 μg / mL. The fermented black fungus polysaccharide is better than the unfermented polysaccharide, with no significant difference (P<0.05).
[0126] The experimental results of the effect of black fungus polysaccharide on the release of IL-6 in RAW264.7 cells are as follows Fig.12 As shown in the figure, the experimental results show that the release amount increases with the concentration in the range of 12.5~800 μg / mL. The fermented black fungus polysaccharide is better than the unfermented polysaccharide, and there is a significant difference at the concentration of 100 and 800 μg / mL (P<0.05).
[0127] The experimental results of the effect of black fungus polysaccharide on the release of TNF-α in RAW264.7 cells are as follows Fig.12As shown in the figure, the experimental results show that the release amount increases with the concentration in the range of 12.5~800 μg / mL. The fermented black fungus polysaccharide is better than the unfermented polysaccharide, and there are significant differences at concentrations of 100, 200, and 400 μg / mL (P<0.05).
[0128] The above contents are further detailed descriptions of the present invention in combination with specific preferred embodiments, and it cannot be determined that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the technical field to which the present invention belongs, several simple deductions or substitutions can be made without departing from the concept of the present invention, which should be regarded as falling within the protection scope of the present invention.
Claims
1. A method and application of preparing black fungus polysaccharide by fermenting Eurotium cristatum, characterized in that: The following steps are involved: (1) grinding black fungus fruiting bodies and passing through an 80-mesh sieve to obtain black fungus powder; (2) Take a certain amount of black fungus powder, add distilled water at a material-liquid ratio of 1:2 to 1:10, place it in an autoclave at 121°C for 15 to 30 min, cool it to room temperature, and add 5% (v / w) suspension of Eurotium cristatum (1×10 6 CFU / mL), fermented at 28℃ for 2-10 days; (3) After fermentation, distilled water was added at a material-liquid ratio of 1:20 to 1:100, and the mixture was stirred evenly. The mixture was ultrasonically treated at 200 to 400 W and 40 to 80 °C for 20 to 40 min, and the pH was adjusted to 9 to 11 with 0.1 mol / L NaOH solution. The mixture was magnetically stirred for 0.5 to 2 h, and then centrifuged for 10 to 30 min (6796 × g), and the supernatant was collected. (4) Concentrate the supernatant to 1 / 4 to 1 / 8 of the original volume, adjust the pH to 2.0 to 5.0 with 0.1 mol / L HCl solution, cool in a 4°C refrigerator for 12 to 36 h, and then centrifuge for 10 to 30 min (6796 × g) to collect the supernatant; (5) Slowly add anhydrous ethanol to the supernatant, mix in a ratio of 1:4, place in a 4°C refrigerator for 8-20 h, centrifuge for 15 min (6796 × g), and collect the precipitate; (6) freeze-drying the precipitate to obtain a black fungus polysaccharide product.
2. The method according to claim 1, characterized in that The strain used in the fermentation method is Eurotium cristatum, which has the ability to produce protease, cellulase and pectinase.
3. The method according to claim 1, characterized in that The black fungus powder has a particle size of 80 meshes, and distilled water is added according to a material-liquid ratio of 1:2-1:
10. The sterilized black fungus material has moderate humidity and is suitable for aerobic fermentation of Eurotium cristatum.
4. The method according to claim 1, characterized in that: The inoculation ratio of the suspension of Eurotium cristatum was 5% (v / w) of the mass of the black fungus mixture, and the concentration of the suspension was 1×10 6 CFU / mL, and the fermentation time was 6 days.
5. The method according to claim 1, characterized in that After 6 days of fermentation, the fermentation material sample obtained was added with distilled water at a material-liquid ratio of 1:20 to 1:100, stirred evenly, and ultrasonically treated at 200-400W and 40-80℃ for 20-40min to further destroy the cell wall; then the pH was adjusted to 9-11 with 0.1mol / L NaOH solution, and magnetic stirring was performed for 0.5-2h to completely release the cell contents; then centrifuged for 10-30min (6796×g) to collect the supernatant.
6. The method according to claim 1, characterized in that The supernatant obtained in the previous step was concentrated to 1 / 4 to 1 / 8 of the original volume by rotary evaporator under reduced pressure, and the pH was adjusted to 2.0 to 5.0 with 0.1 mol / L HCl solution. The solution was placed in a 4°C refrigerator for 12 to 36 hours to precipitate protein, and centrifuged for 10 to 30 minutes (6796 × g) to collect the supernatant.
7. The method according to claim 1, characterized in that The ethanol precipitation step is performed at 4°C to reduce the solubility of the polysaccharide, thereby effectively precipitating the polysaccharide.
8. The method according to claim 1, characterized in that When the polysaccharide concentration was in the range of 0-2 mg / mL, the inhibition rate of α-amylase, the scavenging rate of DPPH free radical, the scavenging rate of hydroxyl free radical, the scavenging rate of superoxide anion free radical, and the scavenging rate of ABTS + The free radical scavenging rate and total reducing power increased with the increase of concentration. The fermented black fungus polysaccharide was superior to the unfermented polysaccharide, and there was a significant difference (P<0.05).
9. The method according to claim 1, characterized in that: When the concentration of black fungus polysaccharide was in the range of 12.5-800 μg / mL, the survival rate of RAW264.7 cells, phagocytic index, and survival rate of RAW264.7 cells with oxidative damage increased with the increase of concentration. The fermented black fungus polysaccharide was superior to the unfermented polysaccharide, and the difference was significant (P < 0.05).
10. The method according to claim 1, characterized in that When the concentration of black fungus polysaccharide was in the range of 12.5-800 μg / mL, the release of IL-10, IL-1β, IL-6 and TNF-α of RAW264.7 cells increased with the increase of concentration. The fermented black fungus polysaccharide was superior to the unfermented polysaccharide (P<0.05).