Co-production method for synchronously preparing high-content polysaccharide and beta-glucan

Abstract

The invention discloses a co-production method for synchronously preparing high-content polysaccharide and beta-glucan. The co-production method comprises the following steps: (1) mushroom raw material treatment: crushing raw materials for later use; (2) degreasing treatment: adopting a full-closed solvent circulation workstation, and intelligently proportioning dichloromethane to ethanol according to the ratio of 8: 2 (V / V); drying the material slag; (3) weak base protein removal: carrying out gradient heating on the filter residue obtained in the step (2) by using a weak base aqueous solution, and collecting the filter residue; (4) extracting polysaccharide with boiling water, and collecting filtrate; and (5) fractional alcohol precipitation: precipitating under the ethanol concentration of 70-80%, and drying to obtain a high-content water-soluble polysaccharide product. And further increasing the concentration of ethanol in the supernate to 85-95% of ethanol, precipitating and drying to obtain a high-content beta-glucan product. The invention develops a combined extraction process which can efficiently extract mushroom polysaccharide and beta-glucan at the same time, realizes comprehensive utilization of raw materials, reduces the production cost and can effectively retain the biological activity of the mushroom polysaccharide and beta-glucan.

Description

Technical Field

[0001] This invention relates to the field of natural active ingredient extraction technology, specifically to a method for the co-production of high-content polysaccharides and β-glucan. Background Technology

[0002] With the aging global population and increasing health awareness, the demand for natural, highly effective, and low-toxicity active substances in functional foods, health products, and pharmaceuticals is growing. Mushrooms, as a medicinal and edible resource, are rich in multiple bioactivities, among which mushroom polysaccharides are considered one of the most important functional components. Numerous studies have shown that mushroom polysaccharides possess various physiological functions, including immunomodulation, anti-tumor activity, antioxidant activity, hypoglycemia, hypolipidemia, and gut microbiota regulation, demonstrating great potential in preventing chronic diseases and improving sub-health conditions. Mushroom polysaccharides are a class of water-soluble polysaccharides with glucan as the main structural unit, possessing various bioactivities such as immunomodulation, antioxidant activity, hypoglycemia, and anti-tumor activity, and are widely used in the food, health product, and pharmaceutical fields. β-glucan, as a highly representative functional component of mushroom polysaccharides, possesses a unique β-(1→3) and β-(1→6) glycosidic bond structure, giving it a strong immune-activating effect, and is in high demand in the biopharmaceutical and high-end health product markets. However, traditional extraction methods yield mixed polysaccharides containing impurities such as α-glucan, glycoproteins, and chitin, which are difficult to meet the market's demand for high-purity functional ingredients.

[0003] Against this backdrop, with the continued growth in market demand for natural functional ingredients, the extraction technology of fungal polysaccharides and β-glucan has become a research hotspot. However, existing technologies for the extraction of these two substances have significant shortcomings:

[0004] Single extraction mode leads to resource waste: Traditional processes often adopt a single target component extraction strategy, that is, only extracting mushroom polysaccharides and β-glucan separately. This results in the destruction of another type of active ingredient during the extraction process or loss with waste liquid and waste residue, making it impossible to achieve comprehensive utilization of mushroom raw materials, with a resource utilization rate of less than 30%.

[0005] Low extraction efficiency and high cost: Single extraction processes usually require multiple high-temperature cooking, repeated alcohol precipitation or enzymatic hydrolysis, which not only consumes a lot of energy and is complicated to operate, but also easily leads to the degradation of active ingredients; at the same time, extracting two components separately requires separate investment in equipment, raw materials and manpower, which significantly increases the total production cost and limits large-scale application.

[0006] Poor process compatibility and low retention rate of active ingredients: Most mushroom polysaccharides are water-soluble, while β-glucan is poorly soluble. Current single extraction techniques cannot take into account the solubility characteristics of both. Water-soluble polysaccharide extraction processes easily lead to precipitation and loss of poorly soluble β-glucan, while strong solvent extraction methods for poorly soluble β-glucan will destroy the structural integrity of mushroom polysaccharides. Ultimately, the extraction rate and activity of both components are not ideal.

[0007] Environmental pressures are becoming increasingly apparent: the waste liquid and residue produced by traditional single extraction processes still contain a large amount of unused active ingredients and organic matter, which not only wastes resources but also increases the difficulty and cost of subsequent environmental treatment, which is not in line with the industry development trend of green production. Summary of the Invention

[0008] The technical problem to be solved by the present invention is to provide a method for the simultaneous preparation of high-content polysaccharide and β-glucan co-production. The method has a reasonable process route, high extraction efficiency, and good product purity, and can simultaneously prepare high-content polysaccharide products and high-content β-glucan products from the same mushroom raw material.

[0009] The technical solution adopted by the present invention to solve the above-mentioned technical problems is as follows:

[0010] A method for the simultaneous preparation of high-content polysaccharides and β-glucan, comprising the following steps:

[0011] (1) Processing of mushroom raw materials: Crush the mushroom raw materials and set them aside. The screen size is 10 mesh with a 2.0 mm aperture.

[0012] (2) Degreasing treatment: A fully enclosed solvent circulation workstation is used, and the intelligent solvent ratio is: dichloromethane: ethanol = 8:2 (V / V) with real-time correction; the raw material feeding ratio is 1:10 g / mL (material-liquid ratio), and the gradient heating program is: 3℃ / min, raised to 40℃, held for 30min, and then raised to 60℃ and held for 20min; the residue is dried at 40-60℃ for 2-4h.

[0013] (3) Protein removal with weak alkali: The filter residue obtained in step (2) is subjected to gradient heating with a weak alkaline aqueous solution with a pH of 8.0-9.0. The gradient heating program is as follows: heat up to 40℃ at 3℃ / min, hold for 30min, then continue to heat up to 60℃, hold for 20min, discard the filtrate, and collect the filter residue.

[0014] (4) Extracting polysaccharides with boiling water: Extract the filter residue obtained in step (3) with boiling water for 20 minutes and collect the filtrate;

[0015] (5) Fractional alcohol precipitation:

[0016] a. Concentrate the filtrate obtained in step (4) under reduced pressure to 1 / 5 to 1 / 10 of its original volume; b. Add ethanol to the concentrate to a final concentration of 70%-80% (v / v), allow it to stand and precipitate, and centrifuge; c. Collect the precipitate from step (b), and dry it to obtain a high-content water-soluble polysaccharide product; d. Collect the supernatant from step (b), continue to add ethanol to the supernatant to a final concentration of 85%-95% (v / v), allow it to stand and precipitate, and centrifuge; e. Collect the precipitate from step (d), and dry it to obtain a high-content β-glucan product.

[0017] Preferably, in step (1), the raw materials are crushed using a multi-stage turbine crusher.

[0018] Preferably, the mushroom raw material is selected from one or both of Hericium erinaceus and Grifola frondosa.

[0019] Preferably, in step (2), the parameters of the fully enclosed solvent circulation workstation are: mass flow meter accuracy: ±0.5% FS; multi-mode reflux extraction chamber: pressure range: -0.1-0.5MPa; solvent recovery: dichloromethane ≥98% (w / w), ethanol ≥99.5% (v / v).

[0020] Preferably, in step (2), a vacuum ribbon dryer is used for drying at 40-60℃ for 2-4 hours; residual solvent <200ppm.

[0021] Preferably, in step (3), the weakly alkaline aqueous solution is a 0.1% (w / w) sodium bicarbonate solution.

[0022] Preferably, in step (4), the polysaccharide extraction by boiling water is carried out using a dynamic countercurrent ultrasonic extraction system; the extraction parameters are set as follows: precise temperature control at 100℃; microwave-assisted heating at 2450MHz, ≤2kW.

[0023] The innovation of this invention lies in:

[0024] 1. Traditional degreasing methods typically use hexane / petroleum ether, but these non-polar solvents cannot penetrate the chitinous layer of fungi, resulting in high lipid residue (greater than 8%) and low dissolution of active ingredients. This invention employs a composite solvent system for deep cell disruption degreasing. The composite solvent system is formulated with a ratio of dichloromethane to ethanol of 8:2 (V / V). Ethanol breaks the hydrogen bond network of chitin-dextran, allowing dichloromethane to rapidly penetrate the lipid bilayer (4.2 times faster than hexane). This specific ratio of composite polar solvent significantly improves the dissolution rate and speed of active ingredients.

[0025] 2. Traditional Sevag methods for protein removal require large amounts of organic solvents (15-25% of the final volume), while protease methods are costly and suffer from residual enzyme contamination. This invention creatively employs a weak-base controlled-release protein separation technology: defatted bacterial powder is added to a 0.1% sodium bicarbonate solution at 40℃ for 30 min, followed by 60℃ for 20 min, with a gradient temperature increase (40℃ for 30 min releases membrane-bound proteins, 60℃ for 20 min releases intracellular soluble proteins). This weak-base controlled-release protein separation technology not only effectively avoids the destruction of the β-1-3-glucan backbone by strong alkali but also significantly improves the protein removal rate. The protein removal rate of this invention reaches 92.3±1.8% (Bradford method).

[0026] 3. Co-extraction of boiling water extraction and gradient alcohol precipitation: The core innovation of this invention lies in the graded alcohol precipitation of step (4). By precisely controlling the ethanol concentration, the two types of components are accurately separated: at an ethanol concentration of 70-80%, water-soluble components with larger molecular weights and lower solubility are more likely to precipitate out. The ethanol concentration in the supernatant is further increased to 85-95% ethanol. At this point, β-glucan with relatively smaller molecular weights or different structures is precipitated out.

[0027] Compared with the prior art, the beneficial effects of the present invention are:

[0028] (1) This invention develops a combined extraction process that can simultaneously and efficiently extract fungal polysaccharides and β-glucan, realize the comprehensive utilization of raw materials, reduce production costs, and effectively retain the bioactivity of both, which has important economic value and industry application prospects.

[0029] (2) The method of the present invention can simultaneously harvest high-content water-soluble polysaccharide products and high-content β-glucan products; among the high-content water-soluble polysaccharide products, the purity and yield of mushroom polysaccharides are both at a high level, with the purity of mushroom polysaccharides reaching 93% and the yield reaching 98%; among the high-content β-glucan products, the purity and yield of β-glucan are also at a high level, with the purity of β-glucan reaching 87% and the yield reaching 90%.

[0030] (3) The extraction time of polysaccharide extraction by boiling water in this invention is shortened to 20 min, while the traditional process requires 120 min for polysaccharide extraction. Detailed Implementation

[0031] To better understand the content of this invention, further description is provided below with reference to specific embodiments. It should be understood that these embodiments are only for further illustration of the invention and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the description of this invention, those skilled in the art may make some non-essential modifications or adjustments to the invention, which still fall within the protection scope of this invention.

[0032] This invention discloses a method for the simultaneous co-production of high-content polysaccharides and β-glucan, comprising the following steps:

[0033] (1) Processing of mushroom raw materials: The raw materials are crushed by a multi-stage turbine crusher (10 mesh screen, 2.0 mm aperture) and then set aside for use.

[0034] (2) Degreasing treatment: A fully enclosed solvent circulation workstation was used, with intelligent solvent ratio: dichloromethane: ethanol = 8:2 (V / V) with real-time correction; mass flow meter accuracy: ±0.5% FS; multi-mode reflux extraction chamber: pressure range: -0.1-0.5MPa; solvent recovery: dichloromethane ≥98%, ethanol ≥99.5%. The raw material feed ratio was 1:10 g / mL (material-liquid ratio), and the gradient temperature program was: 3℃ / min, raised to 40℃, held for 30min, and then raised to 60℃ and held for 20min. Residue drying: vacuum ribbon dryer at 40-60℃ for 2-4h. Residual solvent <200ppm.

[0035] (3) Protein removal with weak alkali: The filter residue obtained in step (2) is subjected to gradient heating with a weak alkaline aqueous solution with a pH of 8.0-9.0. The gradient heating program is as follows: heat up to 40℃ at 3℃ / min, hold for 30min, then continue to heat up to 60℃, hold for 20min, discard the filtrate (containing protein), and collect the filter residue.

[0036] (4) Extracting polysaccharides from boiling water: The filter residue obtained in step (3) is extracted using a dynamic countercurrent ultrasonic extraction system (precise temperature control: 100℃, microwave-assisted heating (2450MHz, ≤2kW)), and the extraction time is shortened to 20min (the traditional process requires 120min). The filtrate is then collected.

[0037] (5) Fractional alcohol precipitation: a. Concentrate the filtrate obtained in step (4) under reduced pressure to 1 / 5 to 1 / 10 of its original volume; b. Add ethanol to the concentrate to a final concentration of 70%-80% (v / v), allow to stand and precipitate, and centrifuge; c. Collect the precipitate from step b, dry it to obtain a high-content water-soluble polysaccharide product; d. Collect the supernatant from step b, continue to add ethanol to the supernatant to a final concentration of 85%-95% (v / v), allow to stand and precipitate, and centrifuge; e. Collect the precipitate from step d, dry it to obtain a high-content β-glucan product.

[0038] Example 1:

[0039] 1. Mushroom raw material processing: The lion's mane mushroom is crushed using a multi-stage turbine crusher (10 mesh screen, 2.0mm aperture) and then set aside for use.

[0040] 2. Degreasing treatment: A fully enclosed solvent circulation workstation is used, with intelligent solvent ratio: dichloromethane:ethanol = 8:2 (V / V) with real-time correction; mass flow meter accuracy: ±0.5% FS; multi-mode reflux extraction chamber: pressure range: -0.1-0.5MPa; solvent recovery: dichloromethane ≥98%, ethanol ≥99.5%. The feed ratio is 1:10 g / mL (solid-to-material ratio), with a gradient temperature program: 3℃ / min, reaching 40℃, holding for 30 min, then continuing to increase to 60℃ and holding for 20 min. Residue drying: vacuum ribbon dryer at 40℃ for 2 h. Residual solvent <200ppm.

[0041] 3. Protein removal with weak alkali: The filter residue obtained in step 2 is subjected to a gradient temperature increase using a weakly alkaline aqueous solution with a pH of 8.0. The gradient temperature increase program is as follows: increase the temperature to 40℃ at a rate of 3℃ / min, hold for 30min, then continue to increase the temperature to 60℃, hold for 20min, discard the filtrate (containing protein), and collect the filter residue.

[0042] 4. Extraction of polysaccharides by boiling water: The filter residue obtained in step 3 is subjected to a dynamic countercurrent ultrasonic extraction system (precise temperature control: 100℃, microwave-assisted heating (2450MHz, ≤2kW)) to shorten the extraction time to 20min, and the filtrate is collected.

[0043] 5. Fractional alcohol precipitation:

[0044] a. Concentrate the filtrate obtained in step 4 under reduced pressure to 1 / 5 of its original volume; b. Add ethanol to the concentrate to a final concentration of 70% (v / v), allow to stand and precipitate, then centrifuge; c. Collect the precipitate, dry it to obtain a high-content water-soluble polysaccharide product A1; d. Collect the supernatant, continue to add ethanol to the supernatant to a final concentration of 85% (v / v), allow to stand and precipitate, then centrifuge; e. Collect the precipitate from this step, dry it to obtain a high-content β-glucan product B1.

[0045] Finally, the polysaccharide content was found to be 93%, with a yield of 98%; the β-glucan content was 87%, with a yield of 90%.

[0046] Example 2:

[0047] 1. Mushroom raw material processing: The lion's mane mushroom is crushed using a multi-stage turbine crusher (10 mesh screen, 2.0mm aperture) and then set aside for use.

[0048] 2. Degreasing Treatment: A fully enclosed solvent circulation workstation is used, with intelligent solvent mixing: dichloromethane:ethanol = 8:2 (V / V) with real-time correction; mass flow meter accuracy: ±0.5% FS; multi-mode reflux extraction chamber: pressure range: -0.1-0.5MPa; solvent recovery: dichloromethane ≥98%, ethanol ≥99.5%. The feed ratio is 1:10 g / mL (solid-to-material ratio), with a gradient temperature program: 3℃ / min, reaching 40℃, holding for 30 min, then continuing to increase to 60℃ and holding for 20 min. Residue drying: vacuum ribbon dryer at 40-60℃ for 2-4 hours. Residual solvent <200ppm.

[0049] 3. Protein removal with weak alkali: The filter residue obtained in step 2 is subjected to a gradient temperature increase using a weakly alkaline aqueous solution with a pH of 8.5. The gradient temperature increase program is as follows: increase the temperature to 40℃ at a rate of 3℃ / min, hold for 30min, then continue to increase the temperature to 60℃, hold for 20min, discard the filtrate (containing protein), and collect the filter residue.

[0050] 4. Extraction of polysaccharides by boiling water: The filter residue obtained in step 3 is subjected to a dynamic countercurrent ultrasonic extraction system (precise temperature control: 100℃, microwave-assisted heating (2450MHz, ≤2kW)), which shortens the extraction time to 20min (the traditional process requires 120min), and the filtrate is collected.

[0051] 5. Fractional alcohol precipitation: a. Concentrate the filtrate obtained in step 4 under reduced pressure to 1 / 8 of its original volume; b. Add ethanol to the concentrate to a final concentration of 75% (v / v), allow to stand and precipitate, then centrifuge; c. Collect the precipitate, dry it to obtain a high-content water-soluble polysaccharide product A2; d. Collect the supernatant, continue to add ethanol to the supernatant to a final concentration of 90% (v / v), allow to stand and precipitate, then centrifuge; e. Collect the precipitate from this step, dry it to obtain a high-content β-glucan product B2.

[0052] The test results showed that the mushroom contained 95% polysaccharide with a polysaccharide yield of 98%; and 88% β-glucan with a β-glucan yield of 92%.

[0053] Example 3:

[0054] 1. Mushroom raw material processing: The maitake mushroom raw material is crushed using a multi-stage turbine crusher (10 mesh screen, 2.0mm aperture) and then set aside for use.

[0055] 2. Degreasing Treatment: A fully enclosed solvent circulation workstation is used, with intelligent solvent mixing: dichloromethane:ethanol = 8:2 (V / V) with real-time correction; mass flow meter accuracy: ±0.5% FS; multi-mode reflux extraction chamber: pressure range: -0.1-0.5MPa; solvent recovery: dichloromethane ≥98%, ethanol ≥99.5%. The feed ratio is 1:10 g / mL (solid-to-material ratio), with a gradient temperature program: 3℃ / min, reaching 40℃, holding for 30 min, then continuing to increase to 60℃ and holding for 20 min. Residue drying: vacuum ribbon dryer at 40-60℃ for 2-4 hours. Residual solvent <200ppm.

[0056] 3. Protein removal with weak alkali: The filter residue obtained in step 2 is subjected to a gradient temperature increase using a weakly alkaline aqueous solution with a pH of 9.0. The gradient temperature increase program is as follows: increase the temperature to 40℃ at a rate of 3℃ / min, hold for 30min, then continue to increase the temperature to 60℃, hold for 20min, discard the filtrate (containing protein), and collect the filter residue.

[0057] 4. Extraction of polysaccharides by boiling water: The filter residue obtained in step 3 is subjected to a dynamic countercurrent ultrasonic extraction system (precise temperature control: 100℃, microwave-assisted heating (2450MHz, ≤2kW)) to shorten the extraction time to 20min, and the filtrate is collected.

[0058] 5. Fractional alcohol precipitation: a. Concentrate the filtrate obtained in step 4 under reduced pressure to 1 / 10 of its original volume; b. Add ethanol to the concentrate to a final concentration of 80% (v / v), allow to stand and precipitate, then centrifuge; c. Collect the precipitate, dry it to obtain a high-content water-soluble polysaccharide product A3; d. Collect the supernatant, continue to add ethanol to the supernatant to a final concentration of 95% (v / v), allow to stand and precipitate, then centrifuge; e. Collect the precipitate from this step, dry it to obtain a high-content β-glucan product B3.

[0059] The content of mushroom polysaccharides was found to be 94%, with a polysaccharide yield of 97%, and the content of β-glucan was 86%, with a β-glucan yield of 94%.

[0060] Comparative Example 1: (Basically the same as Example 1, except that step 5 is fractional alcohol precipitation, which is a one-step precipitation)

[0061] 1. Mushroom raw material processing: The lion's mane mushroom is crushed using a multi-stage turbine crusher (10 mesh screen, 2.0mm aperture) and then set aside for use.

[0062] 2. Degreasing treatment: A fully enclosed solvent circulation workstation is used, with intelligent solvent ratio: dichloromethane:ethanol = 8:2 (V / V) with real-time correction; mass flow meter accuracy: ±0.5% FS; multi-mode reflux extraction chamber: pressure range: -0.1-0.5MPa; solvent recovery: dichloromethane ≥98%, ethanol ≥99.5%. The feed ratio is 1:10 g / mL (solid-to-material ratio), with a gradient temperature program: 3℃ / min, reaching 40℃, holding for 30 min, then continuing to increase to 60℃ and holding for 20 min. Residue drying: vacuum ribbon dryer at 40℃ for 2 h. Residual solvent <200ppm.

[0063] 3. Protein removal with weak alkali: The filter residue obtained in step 2 is subjected to a gradient temperature increase using a weakly alkaline aqueous solution with a pH of 8.0. The gradient temperature increase program is as follows: increase the temperature to 40℃ at a rate of 3℃ / min, hold for 30min, then continue to increase the temperature to 60℃, hold for 20min, discard the filtrate (containing protein), and collect the filter residue.

[0064] 4. Extraction of polysaccharides by boiling water: The filter residue obtained in step 3 is subjected to a dynamic countercurrent ultrasonic extraction system (precise temperature control: 100℃, microwave-assisted heating (2450MHz, ≤2kW)) to shorten the extraction time to 20min, and the filtrate is collected.

[0065] 5. Fractional alcohol precipitation: a. Concentrate the filtrate obtained in step 4 under reduced pressure to 1 / 5 of its original volume; b. Add ethanol to the concentrate to a final concentration of 70% (v / v), allow to stand to precipitate, and centrifuge; c. Collect the precipitate, dry it to obtain a high-content water-soluble polysaccharide product C1; d. Collect the supernatant, dry it to obtain a high-content β-glucan product D1.

[0066] Finally, the polysaccharide content was found to be 93%, with a yield of 98%; the β-glucan content was 50%, with a yield of 90%.

[0067] Comparative Example 2: Compared with Example 1 (all other conditions are the same, the main difference is that petroleum ether was used for degreasing in step 2)

[0068] 1. Mushroom raw material processing: The raw material lion's mane mushroom is crushed using a multi-stage turbine crusher (10 mesh screen, 2.0mm aperture) and then set aside for use.

[0069] 2. Degreasing treatment: A fully enclosed solvent circulation workstation is used, with petroleum ether as the intelligent solvent; a multi-mode reflux extraction chamber with a pressure range of -0.1-0.5 MPa; solvent recovery: petroleum ether ≥98%. The feed ratio is 1:10 g / mL (material-to-liquid ratio), with a gradient temperature program of 3℃ / min, reaching 45℃, holding for 30 min, then continuing to increase the temperature to 60℃ and holding for 20 min. Residue drying: vacuum ribbon dryer at 40℃ for 2 h. Residual solvent <200 ppm.

[0070] 3. Protein removal with weak alkali: The filter residue obtained in step 2 is subjected to a gradient temperature increase using a weakly alkaline aqueous solution with a pH of 8.0. The gradient temperature increase program is as follows: increase the temperature to 40℃ at a rate of 3℃ / min, hold for 30min, then continue to increase the temperature to 60℃, hold for 20min, discard the filtrate (containing protein), and collect the filter residue.

[0071] 4. Extraction of polysaccharides by boiling water: The filter residue obtained in step 3 is subjected to a dynamic countercurrent ultrasonic extraction system (precise temperature control: 100℃, microwave-assisted heating (2450MHz, ≤2kW)) to shorten the extraction time to 20min, and the filtrate is collected.

[0072] 5. Fractional alcohol precipitation: a. Concentrate the filtrate obtained in step 4 under reduced pressure to 1 / 5 of its original volume; b. Add ethanol to the concentrate to a final concentration of 70% (v / v), allow to stand and precipitate, then centrifuge; c. Collect the precipitate, dry it to obtain high-content water-soluble polysaccharide product C2; d. Collect the supernatant, continue to add ethanol to the supernatant to a final concentration of 85% (v / v), allow to stand and precipitate, then centrifuge; e. Collect the precipitate from this step, dry it to obtain high-content β-glucan product D2.

[0073] Finally, the polysaccharide content was found to be 85%, with a yield of 95%; the β-glucan content was 75%, with a yield of 85%.

[0074] Comparative Example 3: Compared with Example 1 (the main difference is the second step, where protein is extracted directly with alkaline water; everything else remains the same).

[0075] 1. Mushroom raw material processing: The raw material lion's mane mushroom is crushed using a multi-stage turbine crusher (10 mesh screen, 2.0mm aperture) and then set aside for use.

[0076] 2. Degreasing Process: Employs a fully enclosed solvent circulation workstation with intelligent solvent mixing: dichloromethane:ethanol = 8:2 (V / V) with real-time correction; mass flow meter accuracy: ±0.5% FS; multi-mode reflux extraction chamber: pressure range: -0.1-0.5MPa; solvent recovery: dichloromethane ≥98%, ethanol ≥99.5%. Raw material feed ratio is 1:10 g / mL (material-to-liquid ratio); gradient temperature program: 3℃ / min, reaching 40℃, holding for 30 min, then continuing to increase to 60℃ and holding for 20 min. Residue drying: vacuum ribbon dryer at 40℃ for 2 h. Residual solvent <200ppm.

[0077] Protein removal with alkaline water at pH 12: The filter residue obtained in step 2 is subjected to a gradient temperature increase using an alkaline aqueous solution with a pH of 12. The gradient temperature increase program is as follows: increase the temperature to 40°C at a rate of 3°C / min, hold for 30 min, then continue to increase the temperature to 60°C, hold for 20 min, discard the filtrate (containing protein), and collect the filter residue.

[0078] 4. Extraction of polysaccharides by boiling water: The filter residue obtained in step 3 is subjected to a dynamic countercurrent ultrasonic extraction system (precise temperature control: 100℃, microwave-assisted heating (2450MHz, ≤2kW)) to shorten the extraction time to 20min, and the filtrate is collected.

[0079] 5. Fractional alcohol precipitation: a. Concentrate the filtrate obtained in step 4 under reduced pressure to 1 / 5 of its original volume; b. Add ethanol to the concentrate to a final concentration of 70% (v / v), allow to stand and precipitate, then centrifuge; c. Collect the precipitate, dry it to obtain high-content water-soluble polysaccharide product C3; d. Collect the supernatant, continue to add ethanol to the supernatant to a final concentration of 85% (v / v), allow to stand and precipitate, then centrifuge; e. Collect the precipitate from this step, dry it to obtain high-content β-glucan product D3.

[0080] Finally, the polysaccharide content was found to be 50%, with a yield of 60%; the β-glucan content was 40%, with a yield of 50%.

[0081] Table 1. Polysaccharide purity and β-glucan purity in extracts obtained by different processing methods

[0082] Polysaccharide purity / % Polysaccharide yield / % β-glucan purity / % β-glucan yield / % Comparison parameters Example 1 93 98 87 90 Comparative Example 1 93 98 50 90 Step 5, one-step ethanol precipitation Comparative Example 2 85 95 75 85 Petroleum ether degreasing Comparative Example 3 50 60 40 50 pH 12 alkaline water for protein removal

[0083] As shown in Table 1, Example 1 of the present invention can simultaneously harvest a high-content water-soluble polysaccharide product A1 and a high-content β-glucan product B1. In the high-content water-soluble polysaccharide product A1, the purity and yield of mushroom polysaccharides both reach a high level, with a purity of 93% and a yield of 98%. In the high-content β-glucan product B1, the purity and yield of β-glucan also reach a high level, with a purity of 87% and a yield of 90%.

[0084] In Comparative Example 1, the polysaccharide purity (93%) and polysaccharide yield (98%) were consistent with those of Example 1, indicating that one-step alcohol precipitation had no significant effect on the purity and yield of the polysaccharide. However, in Comparative Example 2, the polysaccharide purity decreased to 85%, and the yield slightly decreased to 95%. In Comparative Example 3, the polysaccharide purity plummeted to 50%, and the yield decreased to 60%, both significantly lower than those of Example 1.

[0085] The β-glucan purity (50%) in Comparative Example 1 was significantly lower than that in Example 1 (87%), while the β-glucan purity (75%) in Comparative Example 2 was also lower than that in Example 1. The β-glucan purity (40%) and yield (50%) in Comparative Example 3 were both significantly lower than those in Example 1.

[0086] The detection methods for polysaccharides and β-glucan are described in the table below:

[0087] Table 2

[0088] Detection methods polysaccharides Phenol-sulfuric acid process β-glucan reagent kit method

[0089] The foregoing description is not intended to limit the invention, nor is the invention limited to the examples given. Any changes, modifications, additions, or substitutions made by those skilled in the art within the scope of the invention should also be considered within the protection scope of the invention.

Claims

1. A method for the simultaneous preparation of high-content polysaccharides and β-glucan, characterized in that, Includes the following steps: (1) Processing of mushroom raw materials: Crush the mushroom raw materials and set them aside. The screen size is 10 mesh with a 2.0 mm aperture. (2) Degreasing treatment: A fully enclosed solvent circulation workstation is used, and the intelligent solvent ratio is: dichloromethane: ethanol = 8:2 (V / V) with real-time correction; the raw material feeding ratio is 1:10 g / mL (material-liquid ratio), and the gradient heating program is: 3℃ / min to 40℃, hold for 30min, then continue to heat to 60℃ and hold for 20min; the residue is dried at 40-60℃ for 2-4h. (3) Protein removal with weak alkali: The filter residue obtained in step (2) is subjected to gradient heating with a weak alkaline aqueous solution with a pH of 8.0-9.

0. The gradient heating program is as follows: heat up to 40℃ at 3℃ / min, hold for 30min, then continue to heat up to 60℃, hold for 20min, discard the filtrate, and collect the filter residue. (4) Extracting polysaccharides with boiling water: Extract the filter residue obtained in step (3) with boiling water for 20 minutes and collect the filtrate; (5) Fractional alcohol precipitation: a. Concentrate the filtrate obtained in step (4) under reduced pressure to 1 / 5 to 1 / 10 of its original volume; b. Add ethanol to the concentrate to a final concentration of 70%-80% (v / v), allow it to stand and precipitate, and centrifuge; c. Collect the precipitate from step (b), and dry it to obtain a high-content water-soluble polysaccharide product; d. Collect the supernatant from step (b), continue to add ethanol to the supernatant to a final concentration of 85%-95% (v / v), allow it to stand and precipitate, and centrifuge; e. Collect the precipitate from step (d), and dry it to obtain a high-content β-glucan product.

2. The method for simultaneous preparation of high-content polysaccharides and β-glucan as described in claim 1, characterized in that, In step (1), the raw materials are crushed using a multi-stage turbine crusher.

3. The method for simultaneous preparation of high-content polysaccharides and β-glucan as described in claim 2, characterized in that, In step (2), the parameters of the fully enclosed solvent circulation workstation are as follows: mass flow meter accuracy: ±0.5% FS; multi-mode reflux extraction chamber: pressure range: -0.1-0.5MPa; solvent recovery: dichloromethane ≥98%, ethanol ≥99.5%.

4. The method for simultaneous preparation of high-content polysaccharides and β-glucan as described in claim 3, characterized in that, In step (2), a vacuum ribbon dryer is used for drying at 40-60℃ for 2-4 hours; residual solvent <200ppm.

5. The method for simultaneous preparation of high-content polysaccharides and β-glucan as described in claim 4, characterized in that, In step (3), the weakly alkaline aqueous solution is a 0.1% sodium bicarbonate solution.

6. The method for simultaneous preparation of high-content polysaccharides and β-glucan as described in claim 5, characterized in that, In step (4), the polysaccharide extraction by boiling water is carried out using a dynamic countercurrent ultrasonic extraction system; the extraction parameters are set as follows: precise temperature control at 100℃; microwave-assisted heating at 2450MHz, ≤2kW.