Enzymolysis extraction method for morchella esculenta through liquid submerged fermentation, extract and application

Abstract

The invention discloses a liquid submerged fermentation morchella enzymolysis extraction method, an extract and application, and belongs to the technical field of morchella enzymolysis, and the method comprises the following steps: separating and purifying morchella; activating the strain; liquid fermentation culture: inoculating the seed liquid into a fermentation culture medium; centrifugally separating the fermentation liquor, collecting morchella mycelia, washing with distilled water, and drying for later use; enzymolysis, wherein the mass ratio of cellulase to pectinase to flavourzyme to alkaline protease to neutral protease in a compound enzyme is (2.8-3.1): (1.7-2.2): (0.4-0.6): (0.1-0.3): (0.2-0.4); centrifuging and collecting supernate; concentrating the supernate to obtain a morchella enzymatic hydrolysate concentrate; and drying and crushing the morchella esculenta enzymatic hydrolysate concentrate. According to the method, the yield of the extract, the content of polysaccharide and protein in the extract and the antioxidant activity of the extract can be effectively increased.

Description

Technical Field

[0001] The invention relates to the technical field of Morchella oleracea enzymolysis, and in particular to a liquid submerged fermentation Morchella oleracea enzymolysis extraction method, an extract and an application thereof. Background Art

[0002] Morchella esculenta, also known as Morchella esculenta, belongs to the Ascomycetes, Pestozoales, Morchaceae, and Morchella genus. It is one of the world's most valuable and rare edible fungi. Due to its rich nutritional content and significant medicinal value, morels are considered a "natural, nutritious, and multifunctional" health food. They are rich in several essential amino acids, and their umami properties include free amino acids and 5'-nucleotides. Free amino acids account for 35.67% of the total amino acid content, while 5'-nucleotides make up 21.10% of the total nucleotide content.

[0003] Traditional morel cultivation methods mainly rely on manual operation, which makes it difficult to achieve large-scale industrial production.

[0004] Edible fungus submerged fermentation refers to the process of inoculating the mycelium of edible fungi into a liquid culture medium, and in a deep liquid environment of a closed container such as a fermenter, by controlling conditions such as temperature, pH value, dissolved oxygen, stirring speed, etc., so that the mycelium is allowed to grow, reproduce and metabolize in large quantities in the liquid. In a suitable liquid environment, the mycelium can absorb nutrients such as carbon sources, nitrogen sources, and minerals from the surrounding culture medium, and decompose and transform these substances with the help of its own enzyme system, thereby achieving growth and reproduction, and synthesizing various metabolites at the same time. Liquid submerged fermentation is a method for cultivating edible fungi with the help of modern fermentation technology, and this method plays an important role in edible fungi production and research. For example, patent CN113755341A discloses a method for cultivating morels in liquid fermentation, which obtains a mycelium dry weight of 5.247g / L and an extracellular crude polysaccharide content of 0.587g / L.

[0005] The cell wall structure of Morchella is complex, making it difficult to fully release and utilize the bioactive ingredients within. Traditional water extraction of Morchella powder has a suboptimal extraction yield, and high temperatures can damage the structure of the active ingredients.

[0006] In recent years, enzymatic hydrolysis technology has been widely used in the extraction of active ingredients from edible fungi and plants. It uses cellulase, pectinase, or protease, among other enzymes, to hydrolyze edible fungi or plant materials under appropriate conditions. This hydrolysis of the cell walls facilitates the dissolution of intracellular active ingredients during extraction. Enzymatic hydrolysis-assisted methods not only significantly improve extraction yields, but also operate under mild conditions without disrupting the structure of the active ingredients in the extract. Furthermore, enzymatic hydrolysis can reduce the molecular weight of polysaccharides, resulting in enhanced biological activity. For example, patent CN116548604A discloses a method for preparing Morchella extract using cellulase in conjunction with ultrasound. Applying cellulase enzymatic hydrolysis and ultrasound extraction techniques to the preparation of Morchella extract significantly improves the yield of the extract, increases polysaccharide content, and significantly enhances antioxidant activity.

[0007] However, the existing technology still has the following technical difficulties: the yield of the extract, the polysaccharide and protein content, and the DPPH free radical scavenging activity all have a lot of room for improvement.

[0008] Based on this, the present invention designs a liquid submerged fermentation morel enzymatic extraction method, extract and application to solve the above problems. Summary of the Invention

[0009] In view of the above-mentioned shortcomings of the prior art, the present invention provides a liquid submerged fermentation morel enzymatic extraction method, extract and application.

[0010] To achieve the above objectives, the present invention is implemented through the following technical solutions: A liquid submerged fermentation morel enzymatic extraction method comprises the following steps: Step 1, separating and purifying the Morchella to obtain the Morchella esculenta strain; Step 2: Activate the bacteria; Step 3: preparing seed solution; Step 4: liquid fermentation culture: the seed liquid is inoculated into the fermentation medium at an inoculum size of 5-20%, the fermentation medium components are: 5-35 g / L konjac flour hydrolysate, 5-15 g / L glucose, 5-15 g / L soy peptone, 1-5 g / L trypsinogen, 1-5 g / L potassium dihydrogen phosphate, 1-5 g / L magnesium sulfate, 0.01-1 g / L vitamin B1, initial pH 5.5-7.5, and culture in a fermentor; and after the start of fermentation, the feed medium is supplemented in batches, and the feed medium components are: glucose and konjac flour hydrolysate; The preparation method of the konjac flour enzymatic hydrolysate comprises the following steps: subjecting the konjac flour to jet liquefaction, plate-and-frame filter pressing for solid-liquid separation, obtaining a liquefied residue, adding 5-8 times the mass of the liquefied residue into 35-45° C. warm water, mixing the mixture uniformly, subjecting the mixture to electron beam irradiation treatment at an intensity of 25-28 kGy for 5-10 minutes, adjusting the pH to 5-6, adding 200-250 U / g of mannanase, carrying out enzymatic hydrolysis for 2-3 hours at a stirring speed of 100-200 r / min, inactivating the enzyme, filtering the mixture, and adjusting the pH to 7 to obtain the obtained product. Step 5, fermentation liquid treatment: after the fermentation is completed, the fermentation liquid is centrifuged, the morel mycelium is collected, washed with distilled water, and dried for later use; Step 6: Enzymolysis: The washed morel mycelium is mixed with a composite enzyme solution at a ratio of 1:10-1:20 (g / mL), and enzymolysis is performed at 40-55°C for 3 hours with a stirring speed of 100-200 r / min; in the composite enzyme, the mass ratio of cellulase: pectinase: flavor protease: alkaline protease: neutral protease is 2.8-3.1:1.7-2.2:0.4-0.6:0.1-0.3:0.2-0.4; after the enzymolysis is completed, the enzyme is inactivated, and then centrifugation is performed to collect the supernatant; Step 7: concentrating the supernatant to obtain a Morchella oleracea enzymatic hydrolysate concentrate; Step 8: Dry and crush the Morchella edodes enzymatic hydrolysis liquid concentrate to obtain a liquid submerged fermentation Morchella edodes enzymatic hydrolysis extract.

[0011] Furthermore, step 2, strain activation, specifically: picking mycelium and inoculating it into PDA slant culture medium, culturing at 20-25°C for 5-7 days to activate the strain; Step 3: Preparation of seed solution, specifically: inoculate the activated bacteria into the seed culture medium, shake and culture at 20-27°C and 150-300 r / min for 5-7 days to obtain the seed solution; the components of the seed culture medium are: 10-30 g / L glucose, 3-8 g / L yeast powder, 1-5 g / L peptone, 0.1-1.2 g / L potassium dihydrogen phosphate, and 0.1-1.0 g / L magnesium sulfate.

[0012] Furthermore, in step 4, the culture temperature is 20-27°C, the stirring speed is 150-300 r / min, the ventilation volume is 0.8-1.2 vvm, and the culture time is 5-12 days.

[0013] Furthermore, in step five, the product is washed with distilled water for 2 to 3 times, dried at 45 to 55° C., and then used for later use.

[0014] Furthermore, in step six, the enzymes are first mixed in a mass ratio of cellulase: pectinase: flavor protease: alkaline protease: neutral protease = 2.8~3.1: 1.7~2.2: 0.4~0.6: 0.1~0.3: 0.2~0.4, and an enzyme solution with an enzyme concentration of 1.0~2.0% (w / v) is prepared using a buffer solution with a pH of 5.5~5.7, and then the washed morel mycelium is mixed with the enzyme solution.

[0015] Furthermore, in step six, a single enzyme solution of 1.0-2.0% (w / v) of cellulase, pectinase, flavor protease, alkaline protease, and neutral protease is prepared using a buffer solution with a pH of 5.5-5.7, and then the five single enzyme solutions are mixed with Morchella mycelium at a ratio of 1:15 (g / mL); wherein the mass ratio of cellulase: pectinase: flavor protease: alkaline protease: neutral protease is 3:2:0.5:0.2:0.3.

[0016] Furthermore, in step seven, the supernatant is concentrated under reduced pressure at 55° C. and -0.1 MPa to 1 / 10 of the original volume to obtain a Morchella oleracea hydrolysate concentrate; in step eight, the Morchella oleracea hydrolysate concentrate is transferred to a culture dish, placed under vacuum drying and crushing at 60° C. and -0.1 MPa to obtain a liquid deep fermentation Morchella oleracea hydrolysate extract.

[0017] Furthermore, in step six, after the enzymatic hydrolysis is completed, the enzymatic hydrolysis solution is heated at 90-100° C. for 10-15 minutes to inactivate the enzyme.

[0018] In order to better achieve the purpose of the present invention, the present invention also provides a liquid submerged fermentation Morchella enzymatic hydrolysis extract prepared according to the preparation method.

[0019] In order to better achieve the purpose of the present invention, the present invention also provides a use of the liquid submerged fermentation Morchella enzymatic hydrolysis extract in the preparation of food or medicine.

[0020] Compared with the prior art, the present invention has the following beneficial effects: the present invention adopts an optimized process to prepare konjac flour enzymatic hydrolysate in the fermentation medium and the feed medium in the cultivation stage, and then combines it with the optimized complex enzyme ratio in the enzymatic hydrolysis stage, which can synergistically increase the yield of the extract and the polysaccharide and protein content in the extract. DETAILED DESCRIPTION

[0021] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0022] Example 1: A method for enzymatic extraction of Morchella edulis by submerged fermentation in liquid, comprising the following steps: Step 1: Isolating and purifying the Morchella to obtain Morchella strains; Experimental strain: Morchella edulis (isolated and purified in the laboratory, identified as Morchella edulis); Step 2: Bacteria activation; An appropriate amount of mycelium was selected from the preserved Morchella strain, inoculated into a PDA slant culture medium, and cultured at 20°C for 7 days to activate the strain.

[0023] Step 3: seed solution preparation; The activated strain was inoculated into seed culture medium (10 g / L glucose, 8 g / L yeast powder, 1 g / L peptone, 1.2 g / L potassium dihydrogen phosphate, 0.1 g / L magnesium sulfate, natural pH), and cultured with shaking at 27°C and 150 r / min for 7 days to obtain seed solution.

[0024] Step 4: liquid fermentation culture; A 5% inoculum of seed liquid was inoculated into a fermentation medium (5 g / L konjac flour hydrolysate, 15 g / L glucose, 5 g / L soy peptone, 5 g / L trypsinogen, 1 g / L potassium dihydrogen phosphate, 5 g / L magnesium sulfate, 0.01 g / L vitamin B1, initial pH 7.5). Cultures were performed in a fermentor at 20°C, 300 rpm, and 0.8 vvm for 12 days. Feed medium (10 g / L glucose and 30 g / L konjac flour hydrolysate) was added in batches 48 hours after the start of fermentation. Residual sugar levels were monitored every 24 hours and replenished when the concentration dropped below 2 g / L.

[0025] The preparation method of konjac flour enzymatic hydrolysate is as follows: konjac flour is subjected to jet liquefaction, plate and frame filtration for solid-liquid separation, and a liquefied residue is obtained; warm water (45°C) 5 times the mass of the liquefied residue is added, mixed evenly, and then subjected to electron beam irradiation treatment with an intensity of 25 kGy for 10 minutes; then the pH is adjusted to 5; mannanase is added in an amount of 200 U / g; enzymatic hydrolysis is carried out for 3 hours at a stirring speed of 100 r / min; after the enzyme is inactivated, it is filtered and the pH is adjusted to 7 to obtain the product.

[0026] Step 5: fermentation broth treatment; After the fermentation is completed, the fermentation liquid is centrifuged and the Morchella mycelium is collected, washed twice with distilled water, and dried at 45°C for later use.

[0027] Step 6: Enzymatic hydrolysis; Preparation of enzyme solution: Select complex enzymes, including cellulase, pectinase, flavor protease, alkaline protease, and neutral protease, and mix them in the mass ratio of cellulase: pectinase: flavor protease: alkaline protease: neutral protease = 3.1:1.7:0.6:0.1:0.4, and use a buffer solution with a pH of 5.5 to prepare an enzyme solution with an enzyme concentration of 1.0% (w / v).

[0028] Enzymatic hydrolysis: The washed Morchella mycelium was mixed with the enzyme solution at a ratio of 1:10 (g / mL), and enzymatic hydrolysis was performed at 40°C for 3 h with a stirring speed of 200 r / min.

[0029] Termination of enzymatic hydrolysis: After the enzymatic hydrolysis is completed, the enzymatic hydrolyzate is heated at 100°C for 10 minutes to inactivate the enzyme, and then centrifuged to collect the supernatant.

[0030] Step 7: Concentration; The supernatant was concentrated under reduced pressure at 55°C and -0.1 MPa to 1 / 10 of the original volume to obtain a Morchella oleracea hydrolysate concentrate; Step 8: Drying and crushing; The Morchella edulis enzymatic hydrolysate concentrate was transferred into a clean petri dish, and vacuum dried and crushed under the conditions of 60°C and -0.1MPa.

[0031] The dry weight of the obtained mycelium was 31.01 g, the dry weight of the enzymatic hydrolysis extract was 25.54 g, and the yield was 82.4%.

[0032] Example 2: A method for enzymatic extraction of Morchella edulis by submerged fermentation in liquid, comprising the following steps: Step 1: Isolating and purifying the Morchella to obtain Morchella strains; Experimental strain: Morchella edulis (isolated and purified in the laboratory, identified as Morchella edulis); Step 2: Bacteria activation; An appropriate amount of mycelium was selected from the preserved Morchella strain, inoculated into a PDA slant culture medium, and cultured at 25°C for 5 days to activate the strain.

[0033] Step 3: seed solution preparation; The activated strain was inoculated into seed culture medium (30 g / L glucose, 3 g / L yeast powder, 5 g / L peptone, 0.1 g / L potassium dihydrogen phosphate, 1.0 g / L magnesium sulfate, natural pH), and cultured with shaking at 20°C and 300 r / min for 5 days to obtain seed solution.

[0034] Step 4: liquid fermentation culture; The seed solution was inoculated at a 20% inoculum rate into a fermentation medium (35 g / L konjac flour hydrolysate, 5 g / L glucose, 15 g / L soy peptone, 1 g / L trypsinogen, 5 g / L potassium dihydrogen phosphate, 1 g / L magnesium sulfate, 1 g / L vitamin B1, initial pH 5.5). Cultures were performed in a fermentor at 27°C, with a stirring rate of 150 rpm and an aeration rate of 1.2 v / min for 5 days. Feed medium (40 g / L glucose and 10 g / L konjac flour hydrolysate) was added in batches 48 hours after the start of fermentation. Residual sugar levels were monitored every 24 hours and replenished when the concentration dropped below 2 g / L.

[0035] The preparation method of konjac flour enzymatic hydrolysate is as follows: konjac flour is subjected to jet liquefaction, plate and frame filtration for solid-liquid separation, and a liquefied residue is obtained; warm water (35°C) 8 times the mass of the liquefied residue is added, and after mixing evenly, it is subjected to electron beam irradiation treatment with an intensity of 28 kGy for 5 minutes; then the pH is adjusted to 6; mannanase is added in an amount of 280 U / g, and enzymatic hydrolysis is carried out for 2 hours at a stirring speed of 200 r / min. After the enzyme is inactivated, it is filtered and the pH is adjusted to 7 to obtain the product.

[0036] Step 5: fermentation broth treatment; After the fermentation is completed, the fermentation liquid is centrifuged and the Morchella mycelium is collected, washed three times with distilled water, and dried at 55°C for later use.

[0037] Step 6: Enzymatic hydrolysis; Preparation of enzyme solution: Select complex enzymes, including cellulase, pectinase, flavor protease, alkaline protease, and neutral protease, and mix them in a mass ratio of cellulase: pectinase: flavor protease: alkaline protease: neutral protease = 2.8:2.2:0.4:0.3:0.2. Use a buffer solution with a pH of 5.7 to prepare an enzyme solution with an enzyme concentration of 2.0% (w / v).

[0038] Enzymatic hydrolysis: The washed morel mycelium was mixed with the enzyme solution at a ratio of 1:20 (g / mL), and enzymatic hydrolysis was performed at 55°C for 3 hours with a stirring speed of 100 r / min.

[0039] Termination of enzymatic hydrolysis: After the enzymatic hydrolysis is completed, the enzymatic hydrolyzate is heated at 90°C for 15 minutes to inactivate the enzyme, and then centrifuged to collect the supernatant.

[0040] Step 7: Concentration; The supernatant was concentrated under reduced pressure at 55°C and -0.1 MPa to 1 / 10 of the original volume to obtain a Morchella oleracea hydrolysate concentrate; Step 8: Drying and crushing; The Morchella edulis enzymatic hydrolysate concentrate was transferred into a clean petri dish, and vacuum dried and crushed under the conditions of 60°C and -0.1MPa.

[0041] The dry weight of the obtained mycelium was 30.22 g, the dry weight of the enzymatic hydrolysis extract was 24.41 g, and the yield was 80.8%.

[0042] Example 3: A method for enzymatic extraction of Morchella edulis by submerged fermentation in liquid, comprising the following steps: Step 1: Isolating and purifying the Morchella to obtain Morchella strains; Experimental strain: Morchella edulis (isolated and purified in the laboratory, identified as Morchella edulis); Step 2: Bacteria activation; An appropriate amount of mycelium was selected from the preserved Morchella strain, inoculated into a PDA slant culture medium, and cultured at 23°C for 6 days to activate the strain.

[0043] Step 3: seed solution preparation; The activated strain was inoculated into seed culture medium (20 g / L glucose, 5 g / L yeast powder, 3 g / L peptone, 1 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate, natural pH), and cultured with shaking at 25°C and 170 r / min for 6 days to obtain seed solution.

[0044] Step 4: liquid fermentation culture; The seed liquid was inoculated into the fermentation medium (30 g / L konjac flour hydrolysate, 10 g / L glucose, 10 g / L soy peptone, 4.21 g / L trypsinogen, 2 g / L potassium dihydrogen phosphate, 1 g / L magnesium sulfate, 0.1 g / L vitamin B1, initial pH 6.5) at a 10% inoculum rate and cultured in a fermenter at 25°C, a stirring speed of 200 r / min, and a ventilation volume of 1.1 vvm for 10 days. Feed medium (15 g / L glucose, 15 g / L konjac flour hydrolysate) was supplemented in batches 48 hours after the start of fermentation. The residual sugar was monitored every 24 hours, and the feed medium was supplemented when the concentration dropped below 2 g / L.

[0045] The preparation method of konjac flour enzymatic hydrolysate is as follows: konjac flour is subjected to jet liquefaction, plate and frame filtration for solid-liquid separation, and a liquefied residue is obtained; warm water (42°C) 7 times the mass of the liquefied residue is added, mixed evenly, and then subjected to electron beam irradiation treatment with an intensity of 26 kGy for 6 minutes; then the pH is adjusted to 5.5; mannanase is added in an amount of 250 U / g; enzymatic hydrolysis is carried out for 2.5 hours at a stirring speed of 180 r / min; after inactivation of the enzyme, it is filtered and the pH is adjusted to 7 to obtain the product.

[0046] Step 5: fermentation broth treatment; After the fermentation is completed, the fermentation liquid is centrifuged and the Morchella mycelium is collected, washed twice with distilled water, and dried at 50°C for later use.

[0047] Step 6: Enzymatic hydrolysis; Preparation of enzyme solution: Select complex enzymes, including cellulase, pectinase, flavor protease, alkaline protease, and neutral protease, and use a buffer solution with a pH of 5.6 to prepare 1.2% (w / v) single enzyme solution of cellulase, pectinase, flavor protease, alkaline protease, and neutral protease.

[0048] Enzymatic hydrolysis: The five single enzyme solutions (cellulase: pectinase: flavor protease: alkaline protease: neutral protease in a mass ratio of 3:2:0.5:0.2:0.3) were mixed with Morchella mycelium at a ratio of 1:15 (g / mL) and subjected to enzymatic hydrolysis at 45°C for 3 h with a stirring speed of 120 r / min.

[0049] Termination of enzymatic hydrolysis: After the enzymatic hydrolysis is completed, the enzymatic hydrolyzate is heated at 95°C for 12 minutes to inactivate the enzyme, and then centrifuged to collect the supernatant.

[0050] Step 7: Concentration; The supernatant was concentrated under reduced pressure at 55°C and -0.1 MPa to 1 / 10 of the original volume to obtain a Morchella oleracea hydrolysate concentrate; Step 8: Drying and crushing; The Morchella edulis enzymatic hydrolysate concentrate was transferred into a clean petri dish, and vacuum dried and crushed under the conditions of 60°C and -0.1MPa.

[0051] The dry weight of the obtained mycelium was 34.82 g, the dry weight of the enzymatic hydrolysis extract was 29.96 g, and the yield was 86.0%.

[0052] Comparative Example 1: Different from Example 1, in step 4, the preparation method of konjac flour enzymatic hydrolyzate is as follows: add konjac flour to 7 times the mass of warm water (42°C), mix evenly, adjust the pH to 5.5, add mannanase in an amount of 5000U / g, enzymolysis for 6h, stirring at a speed of 180r / min, filter after inactivating the enzyme and adjust the pH to 7, to obtain.

[0053] The dry weight of the obtained mycelium was 18.46 g, and the dry weight of the enzymatic hydrolysis extract was 14.62 g, with a yield of 79.2%.

[0054] Comparative Example 2: Unlike Example 1, in step 4, the konjac flour hydrolysate in the fermentation medium and the feed medium was replaced with a corn flour hydrolysate. The corn flour hydrolysate was prepared by jet liquefaction of the corn flour, solid-liquid separation by plate and frame filtration, and a liquefied residue was obtained. Warm water (42°C) with a mass 7 times that of the liquefied residue was added, mixed evenly, and then irradiated with an electron beam at an intensity of 26 kGy for 6 minutes. The pH was then adjusted to 4.5, and cellulase (100 U / g) was added and hydrolyzed for 1 hour. Subsequently, a protease (100 U / g) was added and hydrolyzed for 1 hour. Glucoamylase (100 U / g) and pullulanase (100 U / g) were then added and hydrolyzed for 2 hours.

[0055] The dry weight of the obtained mycelium was 28.63 g, the dry weight of the enzymatic hydrolysis extract was 23.14 g, and the yield was 80.8%.

[0056] Comparative Example 3: The difference from Example 1 is that in step 6, the mass ratio of cellulase: pectinase: flavor protease: alkaline protease: neutral protease in the complex enzyme is 1:1:1:1:1.

[0057] The dry weight of the obtained mycelium was 20.77 g, the dry weight of the enzymatic hydrolysis extract was 12.50 g, and the yield was 60.2%.

[0058] Comparative Example 4: The difference from Example 1 is that in step 6, the mass ratio of cellulase: pectinase: flavor protease: alkaline protease: neutral protease in the complex enzyme is 4:3:0.2:0.05:0.1.

[0059] The dry weight of the obtained mycelium was 23.57 g, the dry weight of the enzymatic hydrolysis extract was 15.81 g, and the yield was 67.1%.

[0060] Comparative Example 5: Unlike Example 1, in step 4, the konjac flour enzymatic hydrolyzate was prepared by adding 7 times the mass of konjac flour to warm water (42°C), mixing uniformly, adjusting the pH to 5.5, adding 5000 U / g of mannanase, and performing enzymatic hydrolysis for 6 hours at a stirring speed of 180 r / min. After inactivation of the enzyme, filtering, and adjusting the pH to 7 were performed. In step 6, the mass ratio of cellulase: pectinase: flavor protease: alkaline protease: neutral protease in the complex enzyme was 1:1:1:1:1.

[0061] The dry weight of the obtained mycelium was 29.17 g, the dry weight of the enzymatic hydrolysis extract was 21.26 g, and the yield was 72.9%.

[0062] Experimental Example: The dry weight of Morchella mycelia and the dry weight of the enzymatic hydrolysis extract in Examples 1 to 3 and Comparative Examples 1 to 5 were determined, and the yield of the Morchella extract was calculated; the polysaccharide content, antioxidant activity, and protein content in the Morchella enzymatic hydrolysis extract were determined.

[0063] 1) Determination of polysaccharide content: According to the test method of Part 1 of the 2020 edition of the Chinese Pharmacopoeia (i.e., the phenol-sulfuric acid method for measuring polysaccharide content). The polysaccharide content of Morchella extract is calculated as follows: .

[0064] 2) Determination of mycelium dry weight: After liquid fermentation, the fermentation liquid was filtered through four layers of gauze. The mycelium filtered onto the gauze was washed three times with distilled water and freeze-dried in a freeze dryer until its mass remained unchanged. The mass was then weighed and the mycelium dry weight was calculated.

[0065] 3) Determination of the antioxidant activity of Morchella extract by DPPH method: Morchella extract was prepared into 1-5 g / L aqueous solution of the extract to be tested with deionized water. 1 mL of the aqueous solution of the extract to be tested was taken in a test tube, and 3 mL of 0.1 mmol / L DPPH solution (2,2-biphenyl-1-picrylhydrazyl, prepared in 95% ethanol, prepared immediately before use) was added. The solution was shaken and allowed to react at room temperature in the dark for 20 min. The absorbance (As) was measured at a wavelength of 517 nm using 95% ethanol as a reference. In the blank group, 1 mL of deionized water was used instead of the aqueous solution of the extract to be tested, and the absorbance (Ab) at 517 nm was measured. In the control group, 1 mL of the aqueous solution of the extract to be tested was taken in a test tube, and 3 mL of 95% ethanol was added and shaken. The absorbance (Ac) at 517 nm was measured. The DPPH free radical scavenging rate was calculated according to the following formula: .

[0066] 4) Protein content determination: Refer to GB 5009.5-2016. The protein content of Morchella extract is calculated as follows: .

[0067] 5) The calculation formula for the yield of Morchella extract is: .

[0068] The measurement results are shown in Tables 1 and 2.

[0069] Table 1

[0070] Table 2

[0071] As can be seen from Tables 1 and 2, in Examples 1 to 3 of the present invention, the seed liquid is inoculated into the fermentation medium for liquid fermentation culture, and the konjac flour hydrolysate is added to the fermentation medium and the feed medium, which can effectively increase the yield of the extract, the polysaccharide and protein contents in the extract, and the antioxidant activity of the extract.

[0072] It can be seen from the measurement results of Example 3, Comparative Example 1 and Comparative Example 2 that the use of konjac flour enzymatic hydrolysate and a specific preparation method can effectively increase the yield of the extract, the polysaccharide and protein contents in the extract, and the antioxidant activity of the extract.

[0073] The results of Example 3, Comparative Example 3, and Comparative Example 4 indicate that the optimal mass ratio of cellulase: pectinase: flavor protease: alkaline protease: neutral protease in the complex enzyme is 2.8-3.1:1.7-2.2:0.4-0.6:0.1-0.3:0.2-0.4. Enzymatic hydrolysis of Morchella mycelia using the complex enzyme within this range significantly increases the yield of the extract, significantly increases the polysaccharide and protein contents in the extract, and significantly enhances the antioxidant activity of the extract.

[0074] It can be seen from the measurement results of Example 3, Comparative Example 1, Comparative Example 3 and Comparative Example 5 that the konjac flour hydrolysate is prepared by adopting an optimized process in the fermentation medium and the feed medium in the cultivation stage of the present invention, and then combined with the optimized complex enzyme ratio in the enzymatic hydrolysis stage, the yield of the extract and the polysaccharide and protein contents in the extract can be synergistically increased.

[0075] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements will not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A liquid submerged fermentation enzymatic extraction method for Morchella, characterized in that: The following steps are involved: Step 1, separating and purifying the Morchella to obtain the Morchella esculenta strain; Step 2: Activate the bacteria; Step 3: preparing seed solution; Step 4: liquid fermentation culture: the seed liquid is inoculated into the fermentation medium at an inoculum size of 5-20%, the fermentation medium components are: 5-35 g / L konjac flour hydrolysate, 5-15 g / L glucose, 5-15 g / L soy peptone, 1-5 g / L trypsinogen, 1-5 g / L potassium dihydrogen phosphate, 1-5 g / L magnesium sulfate, 0.01-1 g / L vitamin B1, initial pH 5.5-7.5, and culture in a fermentor; and after the start of fermentation, the feed medium is supplemented in batches, and the feed medium components are: glucose and konjac flour hydrolysate; The preparation method of konjac flour enzymatic hydrolysate comprises the following steps: subjecting konjac flour to jet liquefaction, plate-and-frame filter pressing for solid-liquid separation, obtaining a liquefied residue, adding 5-8 times the mass of the liquefied residue into 35-45° C. warm water, mixing the mixture uniformly, subjecting the mixture to electron beam irradiation treatment at an intensity of 25-28 kGy for 5-10 minutes, adjusting the pH to 5-6, adding mannanase in an amount of 200-250 U / g, carrying out enzymatic hydrolysis for 2-3 hours at a stirring speed of 100-200 r / min, inactivating the enzyme, filtering the mixture, and adjusting the pH to neutral, thereby obtaining the enzymatic hydrolysate; Step 5, fermentation liquid treatment: after the fermentation is completed, the fermentation liquid is centrifuged, the morel mycelium is collected, washed with distilled water, and dried for later use; Step 6, enzymatic hydrolysis: the washed morel mycelium is mixed with a composite enzyme solution at a ratio of 1:10-1:20 g / mL, and enzymatic hydrolysis is carried out at 40-55°C for 3 hours with a stirring speed of 100-200 r / min; in the composite enzyme, the mass ratio of cellulase: pectinase: flavor protease: alkaline protease: neutral protease is 2.8-3.1:1.7-2.2:0.4-0.6:0.1-0.3:0.2-0.4; after the enzymatic hydrolysis is completed, the enzyme is inactivated, and then centrifugation is performed to collect the supernatant; Step 7: concentrating the supernatant to obtain a Morchella oleracea enzymatic hydrolysate concentrate; Step 8: Dry and crush the Morchella edodes enzymatic hydrolysis liquid concentrate to obtain a liquid submerged fermentation Morchella edodes enzymatic hydrolysis extract.

2. The liquid submerged fermentation morel enzymatic extraction method according to claim 1, wherein Step 2: Activate the strain, specifically: pick the mycelium and inoculate it into PDA slant culture medium, and culture it at 20-25℃ for 5-7 days to activate the strain; Step 3: Preparation of seed solution, specifically: inoculate the activated bacteria into the seed culture medium, shake and culture at 20-27°C and 150-300 r / min for 5-7 days to obtain the seed solution; the components of the seed culture medium are: 10-30 g / L glucose, 3-8 g / L yeast powder, 1-5 g / L peptone, 0.1-1.2 g / L potassium dihydrogen phosphate, and 0.1-1.0 g / L magnesium sulfate.

3. The liquid submerged fermentation morel enzymatic extraction method according to claim 1, wherein In step 4, the culture temperature is 20-27° C., the stirring speed is 150-300 r / min, the ventilation volume is 0.8-1.2 vvm, and the culture time is 5-12 days.

4. The liquid submerged fermentation morel enzymatic extraction method according to claim 1, wherein In step 5, wash with distilled water 2 to 3 times, dry at 45 to 55°C and set aside.

5. The liquid submerged fermentation morel enzymatic extraction method according to claim 1, characterized in that, In step six, cellulase: pectinase: flavor protease: alkaline protease: neutral protease are mixed in a mass ratio of 2.8-3.1: 1.7-2.2: 0.4-0.6: 0.1-0.3: 0.2-0.4, and an enzyme solution with an enzyme concentration of 1.0-2.0% is prepared using a buffer solution with a pH of 5.5-5.

7. The washed morel mycelium is then mixed with the enzyme solution.

6. The liquid submerged fermentation enzymatic extraction method of Morchella according to claim 1, characterized in that: In step six, cellulase, pectinase, flavor protease, alkaline protease, and neutral protease are first prepared into 1.0-2.0% single enzyme solutions using a buffer solution with a pH of 5.5-5.7, and then the five single enzyme solutions are mixed with Morchella mycelium at a ratio of 1:15 g / mL; wherein the mass ratio of cellulase: pectinase: flavor protease: alkaline protease: neutral protease is 3:2:0.5:0.2:0.

3.

7. The liquid submerged fermentation enzymatic extraction method of Morchella according to claim 1, characterized in that: Step 7: Concentrate the supernatant under reduced pressure at 55° C. and −0.1 MPa to 1 / 10 of the original volume to obtain a Morchella oleracea hydrolysate concentrate; Step 8: Transfer the Morchella oleracea hydrolysate concentrate into a culture dish, vacuum dry and crush it at 60° C. and −0.1 MPa to obtain a liquid submerged fermentation Morchella oleracea hydrolysate extract.

8. The liquid submerged fermentation enzymatic hydrolysis and extraction method of Morchella according to claim 1, characterized in that: In step six, after the enzymatic hydrolysis is completed, the enzymatic hydrolyzate is heated at 90-100° C. for 10-15 minutes to inactivate the enzyme.

9. A liquid submerged fermentation enzymatic hydrolysis extract of Morchella edulis prepared according to the preparation method according to any one of claims 1 to 8.

10. Use of the liquid submerged fermentation Morchella enzymatic hydrolysis extract according to claim 9 in the preparation of food or medicine.

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