Method for preparing mushroom cell wall glycosaminoglycan with free radical scavenging activity through compound enzyme method

Abstract

The invention relates to a method for preparing mushroom cell wall glycosaminoglycan with free radical scavenging activity by a compound enzyme method. According to the method, based on the characteristic that the cell wall of the edible fungi is mainly composed of hard and indigestible peptidoglycan, papain, beta-glucanase, pectinase and bromelain are utilized to carry out targeted enzymolysis on peptide bonds and glucosidic bonds of peptidoglycan, and-N + H3 and-COO-of glycosaminoglycan are exposed to increase the activity potential; and separating by using high-concentration ethanol to obtain the mushroom glycosaminoglycan with the free radical scavenging function. The method is suitable for extracting the mushroom cell wall glycosaminoglycan, and has the characteristics of rich raw material sources, high product yield and functional activity of the product. Belongs to the fields of agriculture, health and food.

Description

Technical Field

[0001] This invention discloses a method for separating and preparing aminopolysaccharides from mushroom cell walls using a compound enzyme hydrolysis technique. This method is characterized by abundant raw material sources, high product yield, and functionally active products. It belongs to the fields of agriculture, health care, and food. Background Technology

[0002] Macrobasidiomycetes, such as shiitake mushrooms, lion's mane mushrooms, reishi mushrooms, bamboo fungus, hazel mushrooms, and oyster mushrooms, contain polysaccharides and triterpenoids, possessing nourishing and medicinal functions. Fungal polysaccharides are defined as "large molecular carbohydrates with broad biological activity isolated from the fruiting bodies, mycelia, and fermentation broths of fungi." "Among the fungal polysaccharides studied, their basic building blocks are sugars or uronic acids, including xylose, fucose, rhamnose, mannose, galactose, glucose, arabinose, and glucuronic acid." Currently, the concept of fungal polysaccharides refers to both intracellular and extracellular polysaccharides of fungi, excluding amino acid residue structures. Edible macrobasidiomycetes such as shiitake and hazel mushrooms typically contain less than 10% intracellular polysaccharides by dry weight.

[0003] However, the cell walls of edible fungi such as shiitake and hazel mushrooms account for more than 75% of their dry matter content, exhibiting typical characteristics of fungal cell walls and being difficult for humans to digest and break down. Their main structural components are aminopolysaccharides or peptidoglycans. The backbone of their polysaccharide chains consists of alternating links of N-acetylglucosamine and N-acetylmuramic acid, with side chains of tetrapeptides: L-alanine-D-glutamic acid-L-lysine-D-glutamic acid, cross-linked through five glycine residues to form a hard, difficult-to-digest polymer structure. This polymer structure contains links to glucuronic acid, glucosides, mannosides, arabinosides, chitin, and amino acid residues. This polymer structure is an aminopolysaccharide or peptidoglycan. If its peptide chain and polysaccharide structure can be targeted for degradation, the -N... + H3 terminal or -COO - The terminal portion is considered by our research team to have more unique functional activities, such as an in vitro free radical scavenging rate of 62%-73%.

[0004] Papain is an endopeptidase containing an -SH group. It can hydrolyze the carboxyl terminus of lysine and arginine, and cleave the peptide bonds of leucine or glycine. It exhibits both protease and esterase activities. The optimal reaction temperature for papain is 50-55℃, and the optimal pH is 6-7, which may vary depending on the substrate. The typical addition amount is 0.1-0.3% (1000-3000 u / g protein).

[0005] Bromelain is a thiol-containing protease, and some varieties also contain peroxidase, acid phosphatase, several protein inhibitors, and organic active calcium. Its active site is a thiol group (-SH), enabling it to hydrolyze various proteins, preferentially hydrolyzing peptide chains on the carboxyl side of basic amino acids (e.g., arginine) or aromatic amino acids (e.g., phenylalanine, tyrosine). It has wide applications in the food, pharmaceutical, and biological industries. The optimal reaction temperature is 30-45℃, and the pH is 6-6.8. The optimal conditions vary depending on the substrate, and the optimal addition amount is 0.2-1.0%.

[0006] β-glucanase (β-1,3-1,4-glucanase) is an endonuclease that specifically acts on the 1,3 and 1,4 glycosidic bonds of β-glucan to produce oligosaccharides of 3-5 glucose units and glucose. This product can effectively break down β-glucan in the cell walls of wheat and cereal endosperm, reduce the content of non-starch polysaccharides (NSPs) in feed, and reduce wort viscosity in beer brewing.

[0007] Pectinase is a general term for various enzymes capable of breaking down pectin substances. Type A protopectinase acts on the polygalacturonic acid region (smooth region) of protopectin; type B protopectinase acts on the polysaccharide chains connecting polygalacturonic acid chains to cell wall components (whiskers). Pectin hydrolases can be classified into polygalacturonase, methyl polygalacturonate hydrolase, polyrhamnogalacturonase, arabinogalactanase, galactanase, xylosylgalacturonase, etc., among which polygalacturonase is the most widely used. Pectin esterase is an intracellular cell wall enzyme with high specificity for methyl polygalacturonate, and can also hydrolyze ethyl polygalacturonate, propyl polygalacturonate, and allyl polygalacturonate, but has no effect on methyl polymannuronate. Isoenzymes of pectinase can degrade chitosan to form soluble oligosaccharides. Pectinase can be used to prepare Ginkgo biloba extract, garlic oil concentrate, mushroom concentrate, ginseng extract, angelica extract, licorice extract, auricularia polysaccharide, lentinan, enoki mushroom polysaccharide, and total flavonoids from hawthorn leaves, etc.

[0008] The yield of edible fungal cell wall aminopolysaccharides prepared by enzymatic hydrolysis reached 24%-39%, with a weight-average molecular weight (Mw) of 493,229-85,187 Da and a number-average molecular weight (Mn) of 721-2,429 Da. The dispersion coefficient was relatively large, and the molecular particle size exhibited a multi-peak distribution. The deeply hydrolyzed components of shiitake mushroom cell wall aminopolysaccharides included mannose, ribose, glucuronic acid, glucose, xylose, galactose, arabinose, fucose, and amino-terminal compounds; the deeply hydrolyzed components of hazel mushroom cell wall aminopolysaccharides included mannose, glucuronic acid, glucose, xylose, galactose, arabinose, and amino-terminal compounds.

[0009] By combining β-glucanase, pectinase, papain, and bromelain, the rigid polysaccharide structure and peptide chain cross-linking structure of the aforementioned edible fungi cell walls can be degraded, thereby producing large quantities of aminopolysaccharides derived from edible fungi cell walls. Summary of the Invention

[0010] The purpose of this invention is to provide a method for separating and preparing mushroom cell wall aminopolysaccharides by a compound enzyme hydrolysis method. This method has the advantages of abundant raw material sources, high product yield, and functional activity of the product.

[0011] To achieve the above objectives, the method of the present invention comprises the following technical steps: A. Papain, bromelain, β-glucanase, and pectinase were selected as enzyme combination agents; B. Select shiitake mushrooms, hazel mushrooms, or other edible fungi as raw materials for the production of amino polysaccharides; C. Immerse the raw material from step B in hot water at 80℃-100℃ and wash it 2-15 times to obtain isolated bacterial cells. Dry the isolated bacterial cells at 70℃-85℃, crush them, and pass them through a 120-mesh sieve to obtain the cell wall of edible fungi. D. Add distilled water to the cell wall of edible fungi at a material-to-liquid ratio of 1:40, add enzyme combination agent at a ratio of 1%-5%, adjust the pH to 7, and perform enzymatic hydrolysis at a temperature of 40℃-60℃ and 25kHz-40kHz ultrasound for 30min-24h to obtain cell wall enzymatic hydrolysate. E. Heat the cell wall enzymatic hydrolysate to 85℃-100℃ for 1min-5min to inactivate the enzyme, cool to room temperature, filter with 5-10 layers of gauze, take the filtrate and concentrate it to 1 / 3-1 / 4 of the liquid volume by rotary evaporation to obtain mushroom amino polysaccharide solution. F. Take the mushroom amino polysaccharide solution, add 5 times its volume of 95% ethanol, place it at 4℃ for 12h to settle, continue to centrifuge at 3000-4200r / min for 10min, take the precipitate and freeze dry to obtain mushroom amino polysaccharide; G. In vitro tests were conducted to examine the free radical scavenging rate of mushroom aminopolysaccharides, and mushroom aminopolysaccharides with free radical scavenging activity were obtained. Detailed Implementation

[0012] The following specific implementation examples are used to illustrate the present invention in order to facilitate understanding and operation, but in no way limit the scope of the present invention.

[0013] Example 1 (1) The fruiting bodies of shiitake mushrooms were immersed in hot water at 100°C and washed 10 times. The supernatant was observed to show no polysaccharide precipitation reaction by ethanol precipitation method. The isolated cells obtained after washing were dried at 85°C, pulverized into powder, and passed through a 120-mesh sieve to obtain the cell wall of shiitake mushrooms. (2) Add the cell wall of shiitake mushroom to distilled water at a ratio of material to liquid of 1:40, add 0.75% papain and 2.0% β-glucanase, adjust the pH to 7, and sonicate at 40 kHz for 30 min to obtain a homogenized solution; (3) Heat the homogenized solution to 55°C for 50 min for enzymatic hydrolysis, then heat it to 100°C for 2 min for enzyme inactivation, filter it with 10 layers of gauze to remove the filter residue, and obtain cell wall enzymatic hydrolysate. (4) The cell wall enzymatic hydrolysate was concentrated by rotary evaporation at 60°C to 1 / 4 of the original volume. Five times its volume of 95% ethanol was added and the mixture was allowed to settle at 4°C for 12 hours. The mixture was then centrifuged at 4200 r / min, 4°C, for 10 minutes. The precipitate was then freeze-dried to obtain shiitake mushroom cell wall amino polysaccharide.

[0014] (5) The DPPH free radical scavenging rate of amino polysaccharides in the cell wall of shiitake mushrooms was >60.00%.

[0015] Example 2 (1) The fruiting bodies of shiitake mushrooms were immersed in hot water at 90°C and washed 5 times. The supernatant was observed to show no polysaccharide precipitation reaction by ethanol precipitation method. The isolated cells obtained after washing were air-dried at 75°C, pulverized into powder, and passed through a 120-mesh sieve to obtain the cell wall of shiitake mushrooms. (2) Add the cell wall of shiitake mushroom to distilled water at a ratio of material to liquid of 1:40, add 0.5% papain, 1.0% pectinase and 2.0% β-glucanase, adjust the pH to 7, and sonicate at 40 kHz for 30 min to obtain a homogenized solution. (3) Heat the homogenized solution to 45°C for 1 hour for enzymatic hydrolysis, and then heat it to 100°C for 2 minutes for enzyme inactivation to obtain cell wall enzymatic hydrolysate; (4) The cell wall enzymatic hydrolysate was concentrated by rotary evaporation at 55°C to 1 / 4 of the original volume. Five times its volume of 95% ethanol was added and the mixture was allowed to settle at 4°C for 12 hours. The mixture was then centrifuged at 3000 r / min at 4°C for 15 minutes. The precipitate was then freeze-dried to obtain shiitake mushroom cell wall amino polysaccharide.

[0016] (5) The DPPH free radical scavenging rate of amino polysaccharides in the cell wall of shiitake mushroom was >65.00%.

[0017] Example 3 The fruiting bodies of hazel mushrooms were immersed in 100℃ hot water and washed 10 times. The supernatant was observed to show no polysaccharide precipitation reaction by ethanol precipitation method. The isolated mycelium obtained after washing was dried at 85℃, pulverized into powder, and passed through a 120-mesh sieve to obtain the cell wall of shiitake mushroom. Add shiitake mushroom cell walls to distilled water at a ratio of material to liquid of 1:40, add 0.75% papain, 0.25% bromelain, 2.0% β-glucanase, and 1.0% pectinase, adjust the pH to 7, and sonicate at 40 kHz for 30 minutes to obtain a homogenized solution. The homogenized solution was heated to 45°C for 3 hours for enzymatic hydrolysis, then heated to 100°C for 2 minutes for enzyme inactivation. The residue was removed by filtration through 10 layers of gauze to obtain the cell wall enzymatic hydrolysate. The cell wall enzymatic hydrolysate was concentrated by rotary evaporation at 60℃ to 1 / 4 of the original volume. Five times its volume of 95% ethanol was added and the mixture was allowed to settle at 4℃ for 12 hours. The mixture was then centrifuged at 4200 r / min, 4℃ for 10 minutes. The precipitate was then freeze-dried to obtain the cell wall amino polysaccharide of *Hazelnut*.

[0018] The DPPH free radical scavenging rate of aminopolysaccharides in the cell wall of *Hazelnut* was >50.00%.

Claims

1. A method for preparing mushroom cell wall aminopolysaccharides with free radical scavenging activity using a compound enzyme method, characterized in that, The following technical steps are included: A. Papain, bromelain, β-glucanase, and pectinase were selected as enzyme combination agents; B. Select shiitake mushrooms, hazel mushrooms, or other edible fungi as raw materials for the production of amino polysaccharides; C. Immerse the raw material from step B in hot water and wash it several times to obtain isolated bacterial cells. Dry the isolated bacterial cells with hot air, crush them, and sieve them to obtain the cell walls of edible fungi. D. Distilled water is added to the cell wall of edible fungi at a certain material-to-liquid ratio, and enzyme combination agent is added at a certain percentage. The pH value and temperature range are adjusted, and the mixture is homogenized at a certain ultrasonic frequency for several tens of minutes. After enzymatic hydrolysis for a period of time, cell wall enzymatic hydrolysate is obtained. E. Heat the cell wall enzymatic hydrolysate to 85℃-100℃ for 1min-5min to inactivate the enzyme, cool to room temperature, filter with 5-10 layers of gauze, take the filtrate and concentrate it to 1 / 3-1 / 4 of the liquid volume by rotary evaporation to obtain mushroom amino polysaccharide solution. F. Take the mushroom amino polysaccharide solution, add several times its volume of 95% ethanol, place it at 4℃ for 12h to settle, continue to centrifuge at a certain speed for 10min, take the precipitate and freeze dry to obtain mushroom amino polysaccharide. G. In vitro tests were conducted to examine the free radical scavenging rate of mushroom aminopolysaccharides, and mushroom aminopolysaccharides with free radical scavenging activity were obtained.

2. As described in step A of claim 1, the type and dosage of the selected enzyme combination agent are adjusted based on the type of raw material, the preparation temperature and sedimentation conditions of the mushroom cell wall amino polysaccharide, and the free radical scavenging rate test value.

3. As described in step B of claim 1, other edible fungi selected should have similar morphological characteristics to shiitake mushrooms and hazel mushrooms, and have equivalent or higher potential for preparing free radical scavenging active mushroom amino polysaccharides.

4. As described in step C of claim 1, the purpose is to remove intracellular and extracellular polysaccharides and other soluble impurities, with a hot water temperature of 80℃-100℃, 2-15 times, an air drying temperature of 70℃-85℃, and a sieve mesh size of 80-200 mesh.

5. As described in step D of claim 1, the purpose is to enzymatically hydrolyze the cell walls of edible fungi, with a material-to-liquid ratio of 1:10-1:40, an enzyme combination agent ratio of 1%-5%, a pH value of 6-7, a temperature range of 40℃-60℃, an ultrasonic frequency of 25kHz-40kHz, a homogenization time of 10min-40min, and an enzymatic hydrolysis time of 2h-24h.

6. As described in step E of claim 1, the purpose of which is to remove residual enzymes, step E may be omitted for the functional purposes of the product.

7. As described in step F of claim 1, the purpose is to use a higher concentration of ethanol to compete for the -N group of the aminopolysaccharide. + H3 and -COO - The water around the -OH group of the polysaccharide structure is removed, thus precipitating and separating the aminopolysaccharide. The volume ratio of ethanol added is 5 to 10 times, and the centrifugation speed is 3000 to 4200 r / min.