阅读说明：本技术 一种灰树花菌丝体多肽和β-葡聚糖的联合制备方法 (Combined preparation method of grifola frondosa mycelium polypeptide and beta-glucan ) 是由 冯文娟 蔡颖辉 刘振学 杨传伦 魏圣可 杨朝洋 于 2021-10-14 设计创作，主要内容包括：本发明属于天然产物提取分离技术领域,具体提供了一种灰树花菌丝体多肽和β-葡聚糖的联合制备方法。以发酵灰树花菌丝体为原料,经过乙醇脱脂、干燥、超微粉碎、酶法提取、热碱提取、中和、离心、微滤、超滤脱盐浓缩、喷雾干燥等工序生产灰树花多肽和β-葡聚糖。利用该方法制备的灰树花提取物中多肽和β-葡聚糖的含量高,生产周期短,适合工业化生产。(The invention belongs to the technical field of natural product extraction and separation, and particularly provides a joint preparation method of grifola frondosa mycelium polypeptide and beta-glucan. The fermented Grifola frondosa mycelia are used as raw materials, and processes such as ethanol degreasing, drying, superfine grinding, enzyme extraction, hot alkali extraction, neutralization, centrifugation, microfiltration, ultrafiltration desalination concentration, spray drying and the like are carried out to produce Grifola frondosa polypeptide and beta-glucan. The grifola frondosa extract prepared by the method has high content of polypeptide and beta-glucan, short production period and suitability for industrial production.)

1. A combined preparation method of grifola frondosa mycelium polypeptide and beta-glucan is characterized by comprising the following steps:

(1) adding the fermented grifola frondosa mycelia and ethanol into an extraction container, stirring and degreasing for 12-24 h, centrifuging, drying, and performing superfine grinding;

(2) adding the grifola frondosa superfine powder and water into an extraction container, adding protease, stirring and extracting for 2-6 h, inactivating enzyme for 10min at 100 ℃, cooling, centrifuging, respectively collecting clear liquid and solid, adjusting the pH of the clear liquid to 3.5-4.0 by using hydrochloric acid, centrifuging, and collecting the clear liquid for later use;

(3) adding an alkali solution into the solid, extracting at the temperature of 80-90 ℃ for 1-3 times, combining the extracting solutions after the extraction is finished, neutralizing with acetic acid, centrifuging, and collecting clear liquid;

(4) combining the centrifugal clear liquids in the steps (2) and (3), filtering by adopting a microfiltration membrane with the aperture of 0.2-0.5 mu m, desalting the permeate by using an ultrafiltration membrane with the aperture of 1kD, continuously adding purified water into the trapped liquid to elute and remove salt until the conductivity of the trapped liquid is less than 1mS/cm and the content of soluble solid matters is 2-3%, and stopping ultrafiltration;

(5) spray drying the ultrafiltration retentate to obtain Grifola frondosa extract containing polypeptide and beta-glucan.

2. The combined preparation method of grifola frondosa mycelium polypeptide and beta-glucan according to claim 1, wherein the amount of water used in step (2) is 10-40 times of the weight of grifola frondosa superfine powder.

3. The method for preparing Grifola frondosa mycelium polypeptide and beta-glucan in combination as claimed in claim 1, wherein the protease in step (2) is alkaline protease or neutral protease or a mixture thereof.

4. The combined preparation method of the grifola frondosa mycelium polypeptide and the beta-glucan according to claim 1 or 3, wherein the protease in the step (2) has the enzyme activity of 20 ten thousand U/g, and the addition amount is 0.2-1% of the mass of the grifola frondosa superfine powder.

5. The method for preparing Grifola frondosa mycelium polypeptide and beta-glucan in combination as claimed in claim 3, wherein the pH of the solution is adjusted to 8.5-10.5 by adding potassium hydroxide before using alkaline protease.

6. The method of claim 3, wherein the pH of the mixture of alkaline protease and neutral protease is adjusted to 7.5-9.5 by adding potassium hydroxide before the mixture of alkaline protease and neutral protease is used.

7. The method for preparing grifola frondosa mycelium polypeptide and beta-glucan in combination as claimed in claim 1, wherein the alkali solution in step (3) is potassium hydroxide solution, and the concentration of the alkali solution is 5% to 10% (w/v); the volume of the added alkali solution is 10-30 times (ml/g) of the mass of the original grifola frondosa superfine powder.

8. The method for preparing Grifola frondosa mycelium polypeptide and beta-glucan in combination as claimed in claim 3, wherein the ethanol added in step (1) is more than 75% by weight.

Technical Field

The invention relates to the technical field of natural product extraction and separation, in particular to a combined preparation method of grifola frondosa mycelium polypeptide and beta-glucan.

Background

Grifola frondosa (Grifola frondosa) is a fungus belonging to the genera Grifola, Polyporaceae, Basidiomycetes, Basidiomycotina, Aphyllophorales, Polyporaceae, Grifola, also called Polyporus, Sphaerotheca, Marasma, Lotus flower, etc., and is called Maitake (Maitake) in Japan. Its medicinal action is recorded in "Junzhou" of Japanese saka ran for curing hemorrhoid, and "Weigan, Ping and non-toxic". The "harmonizing spleen and stomach and tranquilizing mind" efficacy is recorded in Shen nong Ben Cao Jing of Dong Han time. The Grifola frondosa mycelia have unique fragrance, and are rich in vitamins, minerals and bioactive substances. The grifola frondosa beta-glucan is grifola frondosa polysaccharide extracted and separated from grifola frondosa mycelia, consists of beta- (1-6) glucan with beta- (1-3) side chains and beta- (1-3) glucan with beta- (1-6) side chains, and can activate various immune cells and release cytokines, improve the immunity of an organism and relieve sub-health symptoms to a certain extent. The grifola frondosa polypeptide is a small molecular peptide obtained by hydrolyzing and separating protease, has the effects of reducing blood pressure, reducing blood fat, resisting fatigue, resisting bacteria, improving immunity, resisting oxidation and the like, and is easier to be absorbed by a human body compared with macromolecular protein.

The existing extraction technologies of the grifola frondosa extract comprise a water extraction method, an alkali extraction method, an enzyme extraction method, an ultrasonic wave auxiliary method, a microwave auxiliary method and the like, wherein the ultrasonic wave auxiliary method and the microwave auxiliary method have high requirements on equipment, high production energy consumption and high production cost. The water extraction method and the enzyme extraction method are used for obtaining the grifola frondosa extract with low content of beta-glucan. The alkali extraction method usually uses sodium hydroxide, the waste water amount is large, and the obtained grifola frondosa extract has high content of beta-glucan but low content of polypeptide. Therefore, how to provide a method for extracting polypeptide and beta-glucan from grifola frondosa mycelia jointly with low energy consumption and high yield of polypeptide and beta-glucan becomes a technical problem to be solved in the field.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a combined preparation method of grifola frondosa mycelium polypeptide and beta-glucan. The invention takes fermented grifola frondosa mycelia as a raw material, and the grifola frondosa polypeptides and beta-glucan are produced by the procedures of degreasing with ethanol, drying, superfine grinding, enzymatic extraction, hot alkali extraction, neutralization, centrifugation, microfiltration, ultrafiltration, desalination and concentration, spray drying and the like. The grifola frondosa extract prepared by the method has high content of polypeptide and beta-glucan, short production period and suitability for industrial production.

The technical scheme of the invention is as follows: according to the method, firstly, ethanol is used for degreasing to destroy cell walls of grifola frondosa mycelia, and then an ultra-micro crushing process is used for physically breaking the walls, so that polypeptide and polysaccharide are more easily extracted when the enzymatic method and the alkali extraction method are combined for extraction in the next step. Performing enzymolysis extraction by adopting protease to obtain an extracting solution with higher content of grifola frondosa polypeptide, then extracting with alkali to obtain an extracting solution with higher content of grifola frondosa beta-glucan, combining the extracting solutions, performing membrane treatment to remove macromolecular substances and micromolecular salts, and performing spray drying to obtain the grifola frondosa extract with high content of polypeptide and beta-glucan. The production alkali adopts potassium hydroxide to replace the traditional sodium hydroxide, and the produced wastewater can be used for developing liquid potash fertilizer.

The invention relates to a combined preparation method of grifola frondosa mycelium polypeptide and beta-glucan, which specifically comprises the following steps:

(1) adding the fermented grifola frondosa mycelia and ethanol into an extraction container, stirring and degreasing for 12-24 h, centrifuging, drying, and performing superfine grinding;

the ethanol is preferably food grade 95% ethanol; after the ethanol is added, the mass percent of the ethanol is more than 75 percent.

Sieving the Grifola frondosa mycelium powder obtained by micronizing with 200 mesh sieve;

(2) adding the grifola frondosa superfine powder and water into an extraction container, adding protease, stirring and extracting for 2-6 h, inactivating enzyme for 10min at 100 ℃, cooling, centrifuging, respectively collecting clear liquid and solid, adjusting the pH of the clear liquid to 3.5-4.0 by using hydrochloric acid, centrifuging, and collecting the clear liquid for later use;

(3) adding an alkali solution into the solid, extracting for 1-3 times at the temperature of 80-90 ℃ for 2h, combining the extracting solutions after the extraction is finished, neutralizing with acetic acid, centrifuging, collecting clear liquid, and precipitating for processing an organic fertilizer;

(4) combining the centrifugal clear liquids in the steps (2) and (3), filtering by adopting a microfiltration membrane with the aperture of 0.2-0.5 mu m, desalting the permeate by using an ultrafiltration membrane with the aperture of 1kD, continuously adding purified water into the trapped liquid to elute and remove salt until the conductivity of the trapped liquid is less than 1mS/cm and the content of soluble solid matters is 2-3%, and stopping ultrafiltration;

(5) spray drying the ultrafiltration retentate to obtain Grifola frondosa extract containing polypeptide and beta-glucan.

The water consumption in the step (2) is 10-40 times of the mass of the grifola frondosa superfine powder, the protease is alkaline protease or neutral protease or a mixture thereof, the enzyme activity is 20 ten thousand U/g, and the addition amount is 0.2-1% of the mass of the grifola frondosa superfine powder; before using the alkaline protease, adding potassium hydroxide to adjust the pH value to 8.5-10.5; before the mixture of alkaline protease and neutral protease is used, potassium hydroxide is added to adjust the pH value to 7.5-9.5.

In the step (3), the alkali solution is potassium hydroxide solution, and the concentration of the alkali solution is 5-10% (w/v); the volume of the added alkali solution is 10-30 times (ml/g) of the mass of the original grifola frondosa superfine powder.

The invention adopts the combined extraction process of wall breaking, enzyme method, alkaline method and membrane treatment for the first time in the production process of the grifola frondosa extract, mycelium obtained after fermentation of grifola frondosa directly adopts ethanol degreasing to destroy cell walls, the degreased grifola frondosa mycelium adopts an ultramicro crushing process to carry out physical wall breaking after drying, simultaneously, the ethanol degreasing and the ultramicro crushing also lead protein to be denatured, protein molecules are changed into disordered loose extending structures from ordered compact structures, the protein molecules are easier to be hydrolyzed by protease, and polypeptide and polysaccharide are easier to be extracted.

The enzymatic extraction process mainly comprises the steps of obtaining an extracting solution with high content of grifola frondosa polypeptide, and removing macromolecular protein in grifola frondosa mycelia after enzymatic extraction, so that beta-glucan is easier to extract in the alkali extraction process. The alkali extraction process mainly obtains the extract with higher beta-glucan content of the grifola frondosa. Mixing extractive solutions, treating with membrane, removing macromolecular substance and small molecular salt, and spray drying to obtain Grifola frondosa extract containing high content of polypeptide and beta-glucan. The process has better synergistic effect for extracting the beta-glucan, and simultaneously reduces the dosage of alkali, and the combination is the first application in the field and has obvious improvement compared with the prior art.

In addition, the invention adopts potassium hydroxide to replace sodium hydroxide in the alkali extraction process, and can realize the application of the waste water in the liquid fertilizer.

In conclusion, the combined preparation method of the grifola frondosa mycelium polypeptide and the beta-glucan adopts combined processes of wall breaking, enzyme extraction, alkali extraction, membrane treatment and the like, so that the alkali consumption is greatly reduced, and the yield of the polypeptide and the beta-glucan is improved. The potassium hydroxide is adopted to replace sodium hydroxide, the wastewater contains potassium acetate, and the potassium salt can be applied to the preparation of liquid fertilizer, so that the problem of difficulty in wastewater treatment is solved. The yield of the grifola frondosa extract obtained by the method can reach more than 11%, wherein the polypeptide content is higher than 30%, the beta-glucan content is higher than 60%, and the yield is far higher than that of the prior art.

Detailed Description

The present invention will be described in further detail with reference to the following examples, but it should not be construed that the scope of the above subject matter is limited to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Example 1

(1) Adding the fermented grifola frondosa mycelia and food-grade 95% ethanol into an extraction container, stirring and degreasing for 12-24 h, centrifuging and drying, wherein the final mass percentage of the ethanol is more than 75%. Pulverizing the dried Grifola frondosa mycelia by an ultrafine pulverizer to obtain Grifola frondosa mycelia powder, and sieving with 200 mesh sieve.

(2) Accurately weighing 100g of grifola frondosa superfine powder, adding 3kg of purified water, adding potassium hydroxide to adjust the pH value to 8.5-10.5, adding 0.5g of alkaline protease with enzyme activity of 20 ten thousand U/g, stirring and extracting for 5h, inactivating enzyme at 100 ℃ for 10min, cooling, centrifuging, respectively collecting clear liquid and solid matters, adjusting the pH value of the clear liquid to 3.5-4.0 with hydrochloric acid, centrifuging, and collecting the clear liquid for later use.

(3) Adding 2L of 9% potassium hydroxide solution into the solid, extracting at 85 deg.C for 2 hr for 2 times, mixing extractive solutions, neutralizing with acetic acid, centrifuging, and collecting clear solution.

(4) And (3) combining the centrifugal clear liquids obtained by the enzyme extraction in the step (2) and the alkali extraction in the step (3), filtering by adopting a microfiltration membrane with the aperture of 0.5 mu m, desalting the permeate by using an ultrafiltration membrane with the aperture of 1kD, continuously adding purified water into the trapped liquid to remove salts until the conductivity of the trapped liquid is less than 1mS/cm and the content of soluble solid matters is 2-3%, and stopping ultrafiltration.

(5) Spray drying the ultrafiltration retentate to obtain Grifola frondosa extract containing polypeptide and beta-glucan.

The yield of the Grifola frondosa extract obtained by the method is 12.02% based on the dry weight of Grifola frondosa mycelia, wherein the content of polypeptide is 33.28%, and the content of beta-glucan is 60.5%.

Example 2

(1) Adding the fermented grifola frondosa mycelia and food-grade 95% ethanol into an extraction container, stirring and degreasing for 12-24 h, centrifuging and drying, wherein the final mass percentage of the ethanol is more than 75%. Pulverizing the dried Grifola frondosa mycelia by an ultrafine pulverizer to obtain Grifola frondosa mycelia powder, and sieving with 200 mesh sieve.

(2) Accurately weighing 100g of grifola frondosa superfine powder, adding 2.5kg of purified water, adding 1g of neutral protease with enzyme activity of 20 ten thousand U/g, stirring and extracting for 6h, inactivating enzyme at 100 ℃ for 10min, cooling, centrifuging, respectively collecting clear liquid and solid, regulating the pH of the clear liquid to 3.5-4.0 with hydrochloric acid, centrifuging, and collecting the clear liquid for later use.

(3) Adding 3L of 7% potassium hydroxide solution into the solid, extracting at 85 deg.C for 2 hr for 2 times, mixing extractive solutions, neutralizing with acetic acid, centrifuging, and collecting clear solution.

(4) Combining the centrifugal clear liquid of the enzyme extraction and the alkali extraction, filtering by adopting a microfiltration membrane with the aperture of 0.2 mu m, desalting the permeate by using a 1kD ultrafiltration membrane, continuously adding purified water into the trapped liquid to elute and remove salt until the conductivity of the trapped liquid is less than 1mS/cm, and the content of soluble solid matters is 2-3%, and stopping ultrafiltration.

(5) Spray drying the ultrafiltration retentate to obtain Grifola frondosa extract containing polypeptide and beta-glucan.

The yield of Grifola frondosa extract was 11.16% based on dry weight of Grifola frondosa mycelia, wherein the content of polypeptide was 29.05%, and the content of beta-glucan was 64.68%.

Example 3

(1) Adding the fermented grifola frondosa mycelia and food-grade 95% ethanol into an extraction container, stirring and degreasing for 12-24 h, centrifuging and drying, wherein the final mass percentage of the ethanol is more than 75%. Pulverizing the dried Grifola frondosa mycelia by an ultrafine pulverizer to obtain Grifola frondosa mycelia powder, and sieving with 200 mesh sieve.

(2) Accurately weighing 100g of grifola frondosa superfine powder, adding 2kg of purified water, adding potassium hydroxide to adjust the pH value to 7.5-9.5, adding 1.0g of mixed enzyme of alkaline protease and neutral protease with the enzyme activity of 20 ten thousand U/g, mixing the alkaline protease and the neutral protease according to the mass ratio of 1:1, stirring and extracting for 4h, inactivating the enzyme at 100 ℃ for 10min, cooling, centrifuging, respectively collecting clear liquid and solid, adjusting the pH value of the clear liquid to 3.5-4.0 with hydrochloric acid, centrifuging, and collecting the clear liquid for later use.

(3) Adding 2.5L potassium hydroxide solution with mass concentration of 8% into the solid, extracting at 90 deg.C for 2 hr for 2 times, combining the extractive solutions, neutralizing with acetic acid, centrifuging, and collecting the clear solution.

(4) Combining the centrifugal clear liquid of the enzyme extraction and the alkali extraction, filtering by adopting a microfiltration membrane with the aperture of 0.5 mu m, desalting the permeate by using a 1kD ultrafiltration membrane, continuously adding purified water into the trapped liquid to elute and remove salt until the conductivity of the trapped liquid is less than 1mS/cm, and the content of soluble solid matters is 2-3%, and stopping ultrafiltration.

(5) Spray drying the ultrafiltration retentate to obtain Grifola frondosa extract containing polypeptide and beta-glucan.

The yield of Grifola frondosa extract based on dry weight of Grifola frondosa mycelia is 12.25%, wherein the content of polypeptide is 30.47%, and the content of beta-glucan is 62.79%.

Example 4

(1) Adding the fermented grifola frondosa mycelia and food-grade 95% ethanol into an extraction container, stirring and degreasing for 12-24 h, centrifuging and drying, wherein the final mass percentage of the ethanol is more than 75%. Pulverizing the dried Grifola frondosa mycelia by an ultrafine pulverizer to obtain Grifola frondosa mycelia powder, and sieving with 200 mesh sieve.

(2) Accurately weighing 100g of grifola frondosa superfine powder, adding 3kg of purified water, adding potassium hydroxide to adjust the pH value to 8.5-10.5, adding 0.2g of alkaline protease with enzyme activity of 20 ten thousand U/g, stirring and extracting for 3h, inactivating enzyme at 100 ℃ for 10min, cooling, centrifuging, respectively collecting clear liquid and solid matters, adjusting the pH value of the clear liquid to 3.5-4.0 with hydrochloric acid, centrifuging, and collecting the clear liquid for later use.

(3) Adding 3.5L potassium hydroxide solution with mass concentration of 6% into the solid, extracting at 87 deg.C for 2 hr for 1 time, combining the extractive solutions, neutralizing with acetic acid, centrifuging, and collecting the clear solution.

(4) Combining the centrifugal clear liquid of the enzyme extraction and the alkali extraction, filtering by adopting a microfiltration membrane with the aperture of 0.2 mu m, desalting the permeate by using a 1kD ultrafiltration membrane, continuously adding purified water into the trapped liquid to elute and remove salt until the conductivity of the trapped liquid is less than 1mS/cm, and the content of soluble solid matters is 2-3%, and stopping ultrafiltration.

(5) Spray drying the ultrafiltration retentate to obtain Grifola frondosa extract containing polypeptide and beta-glucan.

The yield of Grifola frondosa extract was 11.85% based on dry weight of Grifola frondosa mycelia, wherein the content of polypeptide was 35.42%, and the content of beta-glucan was 58.06%.

Example 5

(1) Adding the fermented grifola frondosa mycelia and food-grade 95% ethanol into an extraction container, stirring and degreasing for 12-24 h, centrifuging and drying, wherein the final mass percentage of the ethanol is more than 75%. Pulverizing the dried Grifola frondosa mycelia by an ultrafine pulverizer to obtain Grifola frondosa mycelia powder, and sieving with 200 mesh sieve.

(2) Accurately weighing 100g of grifola frondosa superfine powder, adding 4kg of purified water, adding 1g of neutral protease with 20 ten thousand U/g of enzyme activity, stirring and extracting for 5.5h, inactivating enzyme at 100 ℃ for 10min, cooling, centrifuging, respectively collecting clear liquid and solid, regulating the pH of the clear liquid to 3.5-4.0 with hydrochloric acid, centrifuging, and collecting the clear liquid for later use.

(3) Adding 1L of 10% potassium hydroxide solution into the solid, extracting at 90 deg.C for 2 hr for 3 times, mixing extractive solutions, neutralizing with acetic acid, centrifuging, and collecting clear solution.

(4) Combining the centrifugal clear liquid of the enzyme extraction and the alkali extraction, filtering by adopting a microfiltration membrane with the aperture of 0.5 mu m, desalting the permeate by using a 1kD ultrafiltration membrane, continuously adding purified water into the trapped liquid to elute and remove salt until the conductivity of the trapped liquid is less than 1mS/cm, and the content of soluble solid matters is 2-3%, and stopping ultrafiltration.

(5) Spray drying the ultrafiltration retentate to obtain Grifola frondosa extract containing polypeptide and beta-glucan.

The yield of Grifola frondosa extract was 12.18% based on dry weight of Grifola frondosa mycelia, wherein the content of polypeptide was 28.86%, and the content of beta-glucan was 65.11%.

Comparative example 1

Accurately weighing 100g of Grifola frondosa superfine powder, adding 2L of 10% potassium hydroxide solution, extracting at 85 deg.C for 2 hr for 2 times, mixing extractive solutions, neutralizing with acetic acid, centrifuging, and collecting clear liquid. Filtering with a microfiltration membrane with the pore diameter of 0.5 mu m, desalting the permeate with a 1kD ultrafiltration membrane, continuously adding purified water into the retentate to elute and remove the salt until the conductivity of the retentate is less than 1mS/cm and the content of soluble solids is 2-3%, and spray-drying the ultrafiltration retentate to obtain the grifola frondosa extract containing the polypeptide and the beta-glucan. The yield of the grifola frondosa extract is 4.68%, wherein the polypeptide content is 8.72%, and the beta-glucan content is 83.41%.

Comparative example 2

Accurately weighing 100g of grifola frondosa superfine powder, adding 4L of purified water, adding potassium hydroxide to adjust the pH value to 8.5-10.5, adding 0.3g of alkaline protease with enzyme activity of 20 ten thousand U/g, stirring and extracting for 5h, inactivating enzyme at 100 ℃ for 10min, cooling, centrifuging, respectively collecting clear liquid and solid matters, adjusting the pH value of the clear liquid to 3.5-4.0 by hydrochloric acid, centrifuging, collecting the clear liquid, filtering the clear liquid by adopting a microfiltration membrane with the pore diameter of 0.2 mu m, desalting permeate by adopting an ultrafiltration membrane of 1kD, continuously adding purified water into trapped liquid to elute and remove salt until the conductivity of the trapped liquid is less than 1mS/cm and the content of soluble solid matters is 2-3%, and spray-drying the trapped liquid of ultrafiltration to obtain the grifola extract containing polypeptide and beta-glucan. The yield of the grifola frondosa extract is 4.06%, wherein the polypeptide content is 76.85%, and the beta-glucan content is 10.26%.

Therefore, the combined preparation method of the grifola frondosa mycelium polypeptide and the beta-glucan, which is adopted by the invention, has great improvement on the yield of the grifola frondosa extract and better popularization and application values.