High-activity hericium erinaceus mycelium polysaccharide and preparation method thereof

Abstract

The invention discloses a method for efficiently preparing high-activity hericium erinaceus polysaccharide from waste in the edible mushroom industry, and belongs to the technical field of deep processing of edible mushrooms and recycling of waste. The method comprises the following steps: carrying out steam explosion pretreatment on hericium erinaceus waste mushroom residues, and carrying out synergistic compatibility on the hericium erinaceus waste mushroom residues, tea residues, bean residues and citrus residues which are subjected to specific enzymolysis to prepare a liquid fermentation culture medium; a two-stage control strategy is adopted for submerged fermentation of hericium erinaceus liquid, and hypha proliferation and polysaccharide synthesis conditions are optimized; and after fermentation is finished, extracting and purifying mycelium polysaccharide by adopting a choline chloride-lactic acid eutectic solvent system. According to the method, high-valued utilization of the edible fungus chaff and various agricultural byproducts is achieved, the content of beta-(1-> 3)-D-glucan in the prepared hericium erinaceus polysaccharide is larger than or equal to 55%, the immunocompetence (IC50) reaches 32 micrograms / mL, the hericium erinaceus polysaccharide is remarkably superior to commercially available products, and the hericium erinaceus polysaccharide can be directly used as functional food and medicine raw materials. The process is green and efficient, and has remarkable economic and environmental benefits.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of edible and medicinal mushroom processing and comprehensive utilization of agricultural waste, and particularly relates to a high-activity Hericium erinaceus mycelium polysaccharide and a preparation method thereof. BACKGROUND

[0002] Hericium erinaceus is a kind of precious edible and medicinal mushroom, and its fruiting body and mycelium are rich in bioactive substances such as polysaccharides and polypeptides, and have various pharmacological effects such as enhancing immunity, protecting the gastrointestinal tract, and anti-tumor, and the market demand is increasing. At present, the acquisition of Hericium erinaceus polysaccharide mainly depends on the extraction of fruiting bodies or the direct purchase of mycelium fermentation products.

[0003] Chinese patent CN104277128A discloses a preparation process of Hericium erinaceus fruiting body polysaccharide, which prepares the fruiting body of Hericium erinaceus into a mother liquor, inoculates lactic acid bacteria for fermentation, and then extracts and dries to obtain Hericium erinaceus polysaccharide; however, the traditional fruiting body cultivation period is long (usually several months), is greatly affected by seasons and environmental factors, and the polysaccharide content in the fruiting body is limited, resulting in high production cost. On the other hand, the existing liquid submerged fermentation method for producing mycelium polysaccharide usually uses refined raw materials such as glucose, bran, and soybean meal as culture medium, which has high raw material cost and limits its large-scale industrial application.

[0004] At the same time, in the process of factory cultivation of Hericium erinaceus, a large amount of waste mushroom chaff (also known as mushroom residue) is produced after each harvest of fruiting bodies. These waste mushroom chaff is usually discarded or used only as low-grade fertilizer or fuel, and its value has not been deeply developed, which not only causes resource waste but also may cause environmental pollution problems. In fact, the waste mushroom chaff still contains rich cellulose, hemicellulose, lignin, and unutilized microbial proteins and residual nutrients, and has the potential to be used as a microbial fermentation substrate.

[0005] Therefore, it is of great economic value and environmental significance to develop a technology that can turn waste mushroom chaff into treasure and be used for low-cost and efficient production of high-value Hericium erinaceus polysaccharide. SUMMARY

[0006] In order to solve the problems of the prior art, the present application provides a high-activity Hericium erinaceus mycelium polysaccharide and a preparation method thereof, which uses waste mushroom chaff from Hericium erinaceus cultivation as raw material to produce high-activity Hericium erinaceus mycelium polysaccharide through liquid submerged fermentation technology.

[0007] One of the purposes of the present application is to provide a preparation method of high-activity Hericium erinaceus mycelium polysaccharide, which comprises the following steps: (1) The waste mushroom dregs of Hericium erinaceus are treated by steam explosion, dried and crushed; tea dregs, bean dregs and citrus dregs are respectively subjected to targeted enzymatic hydrolysis treatment, dried and crushed; the tea dregs, bean dregs and citrus dregs are subjected to specific enzymatic hydrolysis pretreatment aiming at their anti-nutritional factors or difficult-to-utilize structures, so that balanced nutrients that can be efficiently utilized by the mycelium of Hericium erinaceus are released in the fermentation; (2) The waste mushroom dregs treated in step (1) are mixed with the tea dregs, bean dregs and citrus dregs at a mass ratio of 100:6:8:4, and are prepared into a basic medium with a concentration of 20-50 g / L by adding water, adding inorganic salts, adjusting the pH to 5.5-6.5, and adding sodium nitroprusside as a metabolic regulator; the inorganic salts include KH2PO4 and MgSO4. (3) The spores of Hericium erinaceus are inoculated into the basic medium of step (2) to perform two-stage controlled fermentation: the first stage is controlled to promote mycelium proliferation, and the second stage is controlled to induce polysaccharide synthesis. (4) After the fermentation is completed, the mycelium is separated and subjected to cell wall breaking treatment, and then a eutectic solvent extraction system composed of choline chloride and lactic acid is used to extract polysaccharides, and high-activity Hericium erinaceus polysaccharides are obtained after post-treatment.

[0008] Further, in step (1), the steam explosion treatment is performed under the following conditions: pressure 1.5-2.0 MPa, pressure holding time 60-120 s, and temperature 180-200℃; the pretreated material is dried to a water content of ≤8% and crushed to 300-500 mesh.

[0009] Further, in step (1), the targeted enzymatic hydrolysis treatment is as follows: for tea dregs, tanninase is used, the addition amount is 0.1-0.3% of the mass of the tea dregs, and the enzymatic hydrolysis is performed at pH 6.5-7.5 and 40-50℃ for 0.5-1.5 h; for bean dregs, neutral protease is used, the addition amount is 0.2-0.4% of the mass of the bean dregs, and the enzymatic hydrolysis is performed at pH 6.5-7.5 and 45-55℃ for 1-2 h; for citrus dregs, cellulase and pectinase are used, the mass ratio of cellulase to pectinase is 1: (1-2), the total addition amount is 0.2-0.3% of the mass of the citrus dregs, and the enzymatic hydrolysis is performed at pH 4.5-5.5 and 40-50℃ for 1-2 h.

[0010] Further, in step (2), the addition amount of sodium nitroprusside is 0.01-0.05 g / L; the mass ratio of KH2PO4 to MgSO4 is 1:1.

[0011] Further, in step (3), the two-stage control strategy is specifically: the first stage (0-48 h) controls the temperature 25-27 DEG C, pH 5.8-6.2, and the ventilation amount 0.8-1.2 vvm; the second stage (48-192 h) controls the temperature 21-23 DEG C, pH 5.3-5.7, and the ventilation amount 0.4-0.6 vvm.

[0012] Further, in step (4), the molar ratio of choline chloride to lactic acid in the deep eutectic solvent extraction system is 1:(1-2), the extraction temperature is 50-70 DEG C, and the extraction time is 1-3 h.

[0013] The second object of the present application is to provide a high-activity Hericium erinaceus mycelium polysaccharide. Compared with the prior art, the present application has the following advantages: 1. The fermentation preparation method of the high-activity Hericium erinaceus mycelium polysaccharide uses waste mushroom dregs as the main raw material, cooperatively uses tea dregs, bean dregs, and citrus dregs, three common agricultural and food processing by-products, realizes the simultaneous high-value utilization of multiple waste materials, greatly reduces the production cost, and has significant environmental benefits; specific pretreatment is adopted according to the characteristics of different raw materials, the stubborn structure of the waste mushroom dregs is destroyed by steam explosion, tannase is used for the tea dregs, neutral protease is used for the bean dregs, and cellulase and pectinase are used for the citrus dregs, which makes the specific nutrients released by the enzymes be released more evenly and more easily utilized by the mycelium, and overcomes the problem of nutrient deficiency or inhibitors of a single waste material.

[0014] 2. The two-stage fermentation control strategy is creatively proposed, the first stage adopts the optimal growth conditions of Hericium erinaceus mycelium to precisely match the demand for maximizing the mycelium biomass in the mycelium growth stage, and the second stage matches the demand for efficiently inducing and accumulating polysaccharides in the product synthesis stage by reducing the temperature and pH value to form a mild stress environment, which significantly improves the conversion efficiency of the biomass to the target product (polysaccharide); a small amount of sodium nitroprusside is added as a metabolic regulator, the addition of sodium nitroprusside is not a simple nutrient supplement, but precisely regulates the metabolic network of Hericium erinaceus mycelium through the released NO, realizes the metabolic flow switching from “promoting growth” to “efficiently synthesizing high-quality polysaccharides”, and is the internal reason for the synchronous significant improvement of the polysaccharide yield and activity (IC 50 value); the deep eutectic solvent is used, which has a unique hydrogen bond effect, realizes high selectivity and high efficiency under mild conditions, and maximizes the preservation of the high activity of the polysaccharide, so that the obtained Hericium erinaceus polysaccharide has high purity, high content of β-(1-3)-D-glucan, and strong immune activity; in the finally obtained Hericium erinaceus polysaccharide, the content of the active core component β-(1→3)-D-glucan is not less than 55%, and the in vitro immune activation activity (IC 50 ) reaches 32 μg / mL, which is significantly better than the same products on the market (IC50 about 45 μg / mL), has the potential to be directly used as raw materials for high-end functional foods and medicines. DETAILED DESCRIPTION

[0015] The scope of the disclosure is not limited to the precise ranges or values recited at the endpoints of the ranges or values, and these ranges or values should be interpreted as including values approximating the recited ranges or values; for ranges of values, the endpoints of the ranges, the endpoints of the ranges and individual points between the endpoints of the ranges, and individual points between the endpoints of the ranges can be combined to form one or more new ranges of values, which should be considered as being specifically disclosed herein.

[0016] The experimental methods in the following examples are all conventional methods, and are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained commercially, unless otherwise specified.

[0017] EXAMPLE The present embodiment provides a preparation method of high-activity Hericium erinaceus mycelium polysaccharide, and the specific steps are as follows: (1) Take 1 kg of Hericium erinaceus waste mushroom chaff, place it in a steam explosion device, and flash explode at 1.8 MPa pressure and 190°C for 90 seconds. Collect the exploded material, dry it at 80°C to a moisture content of 7.5%, and pulverize it to 400 mesh using a super micro pulverizer to obtain waste mushroom chaff ultrafine powder (A).

[0018] Take 60 g of tea dregs, add 0.12 g of tannase (0.2% w / w) and an appropriate amount of water, and enzymatically hydrolyze at 45°C for 1 h. After the enzymatic hydrolysis is completed, inactivate the enzyme at 95°C for 10 min.

[0019] Take 80 g of bean dregs, add 0.24 g of neutral protease (0.3% w / w) and an appropriate amount of water, and enzymatically hydrolyze at 50°C for 1.5 h. After the enzymatic hydrolysis is completed, inactivate the enzyme at 95°C for 10 min.

[0020] Take 40 g of citrus dregs, add 0.05 g of cellulase and 0.05 g of pectinase (total addition amount 0.25% w / w) and an appropriate amount of water, and enzymatically hydrolyze at 45°C for 1 h. After the enzymatic hydrolysis is completed, inactivate the enzyme at 95°C for 10 min.

[0021] Dry the enzymatically hydrolyzed tea dregs, bean dregs, and citrus dregs slurry at 75°C to a moisture content of ≤8%, and respectively super-micro pulverize them to 400 mesh to obtain ultrafine powders B, C, and D, respectively.

[0022] (2) Weigh 100 g of A powder, 6 g of B powder, 8 g of C powder, and 4 g of D powder, and mix them evenly. Take 11.8 g of the mixed powder and add it to a 1 L conical flask. Add 500 mL of deionized water (concentration of about 23.6 g / L). Then add 0.5 g of KH2PO4, 0.5 g of MgSO4·7H2O, and 0.015 g of sodium nitroprusside (SNP) (final concentration of 0.03 g / L). Adjust the pH to 6.0 with 1 mol / L NaOH solution. Sterilize at 121°C for 20 min.

[0023] (3) Under a sterile operating table, inoculate 10% (v / v) of Hericium erinaceus GDMCC 5.703 liquid seed solution in the logarithmic growth phase into the sterilized medium described above. Place it in a constant temperature shaker for fermentation.

[0024] First stage (0-48 h): control the shaker temperature at 26°C, automatically control the pH at 6.0 by feeding dilute acid / alkali, and control the ventilation of the flask by liquid volume (equivalent to 1 vvm).

[0025] Second stage (48-192 h): adjust the temperature to 22°C, control the pH to 5.5, and reduce the shaker speed (equivalent to ventilation amount of 0.5 vvm). The total fermentation time is 192 hours.

[0026] (4) Centrifuge the fermentation broth at 5000 rpm for 15 min, collect the mycelium, and wash it twice with deionized water. Suspended the wet mycelium in water, and treated it with an ultrasonic cell disruptor (200 W, 20 kHz) for 3 min in an ice bath to break the cell wall.

[0027] Prepare a eutectic solvent composed of choline chloride and lactic acid in a molar ratio of 1:1.5; mix the broken mycelium suspension with DES at a solid-liquid ratio of 1:30 (w / v), and stir in a 60°C water bath for 2 h.

[0028] Filter the hot extract, and concentrate the filtrate under reduced pressure at 60°C to 1 / 5 of the original volume. Add 4 volumes of anhydrous ethanol, and precipitate the polysaccharide at 4°C overnight. Centrifuge to collect the precipitate, wash it with anhydrous ethanol and acetone in turn, and vacuum freeze-dry to obtain white flocculent Hericium erinaceus polysaccharide product.

[0029] Product performance detection: Polysaccharide content and structure: the total polysaccharide yield was 18.5% of the dry weight of mycelium by phenol-sulfuric acid method; through Congo red experiment and nuclear magnetic resonance (NMR) analysis, it was confirmed that the content of β-(1→3)-D-glucan was 57.2%.

[0030] Immune activity assay: the immune activity of polysaccharide was evaluated by detecting the amount of NO released by mouse macrophage RAW264.7 cells; the IC 50 value of polysaccharide prepared in this example was 32 μg / mL, while the IC 50 value of commercially available Hericium erinaceus polysaccharide standard was 45 μg / mL.

[0031] Comparative Example 1 Comparative Example 1 differs from the example in that only waste mycelium (treated by steam explosion) was used as the sole carbon and nitrogen source, and the remaining steps were the same as in Example 1. The final polysaccharide yield was 9.8% of the dry weight of mycelium, and the IC 50 value of the product was 52 μg / mL.

[0032] Comparative Example 2 Comparative Example 2 differs from the example in that only waste mycelium that was not treated by steam explosion was used, and the remaining steps were the same as in Example 1. The final polysaccharide yield was 12.1%, and the IC 50 value was 48 μg / mL.

[0033] Comparative Example 3 The fermentation process of Comparative Example 3 used a single-stage control strategy with constant temperature (24°C), constant pH (5.8), and constant aeration (0.8 vvm), and the remaining steps were the same as in Example 1. The final polysaccharide yield was 15.3%, and the IC 50 value was 40 μg / mL.

[0034] Comparative Example 4 The polysaccharide extraction step in Comparative Example 4 used the traditional hot water extraction method (100°C, 3 h, repeated 3 times), and the remaining steps were the same as in Example 1. The polysaccharide yield was 14.0%, and the product had a darker color, with a β-(1→3)-D-glucan content of 49.5%.

[0035] The above examples and comparative examples show that the complete integrated technical solution of the present application has a significant advantage of synergistic effect in improving polysaccharide yield, product purity, and biological activity.

[0036] The above only describes examples of the present application and does not limit the patent scope of the present application. Any equivalent structure or equivalent process transformation using the content of the present application specification, or direct or indirect application in other related technical fields, is also included in the patent protection scope of the present application.

Claims

1. A method for preparing highly active Hericium erinaceus mycelial polysaccharide, characterized in that, Includes the following steps: (1) The waste mushroom residue of Hericium erinaceus was steam-exploded, dried and pulverized; tea residue, soybean residue and citrus residue were enzymatically hydrolyzed, dried and pulverized respectively; (2) The waste fungus after step (1) is mixed with tea residue, soybean residue and citrus residue in a mass ratio of 100:6:8:4, and water is added to prepare a basic culture medium with a concentration of 20-50 g / L. Inorganic salts are added, the pH is adjusted to 5.5-6.5, and sodium nitroprusside is added as a metabolic regulator. (3) Inoculate the Hericium erinaceus strain into the basic culture medium of step (2) and carry out two-stage distributed controlled fermentation; (4) After fermentation, the mycelium was separated, and after cell wall breaking, the polysaccharide was extracted using a low eutectic solvent extraction system composed of choline chloride and lactic acid. After post-treatment, highly active Hericium erinaceus polysaccharide was obtained.

2. The method for preparing highly active Hericium erinaceus mycelial polysaccharide according to claim 1, characterized in that, In step (1), the steam explosion treatment pressure is 1.5-2.0 MPa, the pressure holding time is 60-120 s, and the temperature is 180-200℃; the pulverized material is dried to a moisture content of ≤8%.

3. The method for preparing highly active Hericium erinaceus mycelial polysaccharide according to claim 1, characterized in that, In step (1), tanninase is used on tea residue at a concentration of 0.1-0.3% of the tea residue mass, and the enzyme is hydrolyzed at pH 6.5-7.5 and 40-50℃ for 0.5-1.5 h; neutral protease is used on soybean residue at a concentration of 0.2-0.4% of the soybean residue mass, and the enzyme is hydrolyzed at pH 6.5-7.5 and 45-55℃ for 1-2 h; cellulase and pectinase are used on citrus residue at a mass ratio of 1:(1~2), with a total addition amount of 0.2-0.3% of the citrus residue mass, and the enzyme is hydrolyzed at pH 4.5-5.5 and 40-50℃ for 1-2 h.

4. The method for preparing highly active Hericium erinaceus mycelial polysaccharide according to claim 1, characterized in that, In step (2), the amount of sodium nitroprusside added is 0.01-0.05 g / L.

5. The method for preparing highly active Hericium erinaceus mycelial polysaccharide according to claim 1, characterized in that, In step (3), the two-stage control strategy is as follows: the first stage fermentation time is 0-48h, the controlled temperature is 25-27℃, pH is 5.8-6.2, and the aeration rate is 0.8-1.2 vvm; the second stage fermentation time is 48-192h, the controlled temperature is 21-23℃, pH is 5.3-5.7, and the aeration rate is 0.4-0.6 vvm.

6. The method for preparing highly active Hericium erinaceus mycelial polysaccharide according to claim 1, characterized in that, In step (4), the molar ratio of choline chloride to lactic acid in the eutectic solvent extraction system is 1:(1-2), the extraction temperature is 50-70℃, and the extraction time is 1-3 h.

7. A highly active Hericium erinaceus mycelium polysaccharide prepared by the preparation method according to any one of claims 1-6.