Maitake fermentation enzyme process and application of antioxidant effect

Abstract

The present application relates to the technical field of biological fermentation, and discloses a fermentation enzyme process of morchella and application of antioxidant effect, which comprises the following steps: S1, morchella juice is obtained by juicing morchella with a water content of 10%, visible solid and macromolecular substances are removed from the morchella juice by a filter membrane to obtain morchella filtrate, and an acid and a base are added to the morchella filtrate to control the pH of the morchella filtrate to obtain a morchella preparation solution; S2, the morchella preparation solution in step S1 is placed in a steam explosion machine for steam explosion treatment, and then is placed in a fermentation chamber for temperature control fermentation to obtain a morchella fermentation liquor; and S3, the morchella fermentation liquor in step S2 is subjected to enzyme extraction, a water solution containing a surfactant is added to the morchella fermentation liquor, and the supernatant and precipitate are obtained by ultrasonic oscillation and centrifugal separation, the supernatant is subjected to freeze-drying under the conditions of -35 to -45 DEG C and 30 to 50 Pa for 12 to 24 hours, and then is subjected to super micro powder treatment to obtain a morchella fermentation enzyme.

Description

Technical Field

[0001] This invention relates to the field of bio-fermentation technology, specifically to a process for producing maitake mushroom fermented enzymes and its application in antioxidant activity. Background Technology

[0002] Maitake mushrooms, also known as chestnut mushrooms or Grifola frondosa, have a crisp and tender texture. Their nutritional value ranks first among all edible fungi, sometimes referred to as the "little prince" of edible fungi. According to analysis and testing by the Ministry of Agriculture and Rural Affairs' quality inspection department, every 100 grams of maitake mushrooms contains 32 grams of protein, 19 grams of 18 kinds of amino acids, 1.9 grams of fat, 11 grams of crude fiber, 48 grams of carbohydrates, and various other nutrients such as potassium, phosphorus, iron, zinc, calcium, selenium, copper, polysaccharides, vitamin E, vitamin B1, vitamin B2, vitamin B6, vitamin B12, and vitamin C.

[0003] However, currently, maitake mushrooms are used in tea or wine making, allowing various nutrients to dissolve into the tea or wine, providing people with corresponding nutrition in the form of tea or wine. Since our company produces prickly pear fermentation liquid, we have improved the prickly pear fermentation method for maitake mushrooms, a high-value edible fungus, and applied it to maitake mushrooms to produce maitake mushroom fermented enzymes. Testing of the maitake mushroom fermented enzymes revealed that they contain polysaccharides and ergothioneine, which are used for antioxidant purposes. Summary of the Invention

[0004] This invention aims to provide a process for producing maitake mushroom fermented enzyme and its application in antioxidant activity. Our company has applied an improved prickly pear fermentation method to maitake mushroom to produce maitake mushroom fermented enzyme. The maitake mushroom fermented enzyme was tested and found to contain two substances: polysaccharides and ergothioneine, which are used for antioxidant activity.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: 1) A process for producing maitake mushroom fermented enzyme, comprising the following steps: Step S1: Extract maitake mushroom juice with a water content of 10% to obtain maitake mushroom juice liquid. Remove visible solids and macromolecular substances from the maitake mushroom juice liquid through a filter membrane to obtain maitake mushroom filtrate. Add acid and alkali to the maitake mushroom filtrate to adjust the pH of the maitake mushroom filtrate to obtain maitake mushroom preparation solution. Step S2: Place the maitake mushroom preparation liquid from step S1 into a steam explosion machine for steam explosion treatment, and then place it in a fermentation chamber for temperature-controlled fermentation to obtain maitake mushroom fermentation liquid. Step S3: Extract the fermenting enzyme from the maitake fermentation broth in step S2. Add an aqueous solution containing a surfactant to the maitake fermentation broth, and separate the supernatant and precipitate by ultrasonic vibration and centrifugation. Freeze-dry the supernatant at -35 to -45°C and 30 to 50 Pa for 12 to 24 hours, and then process it into ultrafine powder to obtain maitake fermentation enzyme.

[0006] This technical solution achieves the effect of mixing maitake mushrooms with 10% water content and alcohol in step S1 to extract juice. After removing a large amount of water, the maitake mushrooms are in a state of water deficiency. When the maitake mushrooms are mixed with alcohol, they can absorb a large amount of alcohol, adsorb the alcohol into the maitake mushrooms and produce microorganisms, so as to facilitate the fermentation and use of maitake mushrooms in the later stage.

[0007] After mixing maitake mushrooms with alcohol in step S1, the maitake mushroom juice is obtained by juicing. The maitake mushroom juice contains visible solids and macromolecules, which are separated by a filter membrane to obtain maitake mushroom filtrate. The maitake mushroom filtrate contains a large amount of alcohol. In order to allow the maitake mushroom filtrate to ferment better, the pH value of the maitake mushroom filtrate is adjusted in real time by adding acid and alkali to obtain maitake mushroom preparation solution.

[0008] After adjusting the pH of the maitake mushroom preparation solution, step S2 involves placing the maitake mushroom preparation solution in a steam explosion machine for steam explosion treatment. This effectively destroys the cell walls of the maitake mushroom cells in the preparation solution, allowing the substances contained in the maitake mushroom cells to fully dissolve in the liquid. After the steam explosion treatment is completed, the solution is placed in a fermentation chamber for temperature-controlled fermentation to obtain maitake mushroom fermentation liquid.

[0009] Finally, in step S3, the fermentation enzyme of the maitake fermentation broth from step S2 is extracted. An aqueous solution containing a surfactant is added to the maitake fermentation broth. The surfactant-containing aqueous solution can reduce the surface tension of the maitake fermentation broth, promote the dissolution and dispersion of oxygen and maitake nutrients in the maitake fermentation broth, and enable microorganisms to absorb and utilize maitake nutrients more fully. After the fermentation efficiency of the maitake fermentation broth is further enhanced by the surfactant-containing aqueous solution, the maitake fermentation broth is shaken and centrifuged by ultrasound to obtain the supernatant and precipitate. The supernatant is freeze-dried at -35 to -45°C and 30 to 50 Pa for 12 to 24 hours and then treated with ultrafine powder to obtain maitake fermentation enzyme. Thus, the improved prickly pear fermentation method of our company is applied to maitake to prepare and produce maitake fermentation enzyme.

[0010] The fermented enzyme from *Umeboshi aegyptiaca* in this invention contains a large amount of ergothioneine. Ergothioneine possesses powerful antioxidant capabilities, neutralizing free radicals and enhancing the body's antioxidant capacity. Ergothioneine is the only antioxidant with a clearly defined mechanism for repairing mitochondria. It is transported to mitochondria, the primary "focal point" of free radical production in cells, protecting them from oxidative damage while directly scavenging reactive oxygen species, thus eliminating aging factors at their source and improving skin condition. Furthermore, ergothioneine is a potent hypochlorous acid scavenger, exhibiting particular sensitivity to hypochlorous acid and resisting its inactivation effects, protecting cells from damage.

[0011] Secondly, ergothioneine has a good scavenging effect on hydroxyl free radicals, peroxynitrates, and nitrogen free radicals in the skin, and can also moisturize and prevent dry skin. Through its antioxidant properties, ergothioneine can slow down the aging process of cells, help maintain skin elasticity and radiance, and reduce the appearance of wrinkles and fine lines, thereby achieving an anti-aging effect.

[0012] Ergothioneine can stimulate skin cells to produce collagen, an important protein for maintaining skin structure and strength. Increased collagen helps maintain skin firmness and elasticity, further improving skin quality. At the same time, ergothioneine has anti-inflammatory properties, which can help reduce skin inflammation and redness, providing some relief for sensitive skin or inflammatory skin problems.

[0013] Ergothioneine can regulate the function of immune cells and enhance the body's immunity. It can enhance T cell activity and promote the production and release of cytokines. Simultaneously, ergothioneine can also regulate B cell proliferation and antibody production, further enhancing the body's humoral immune response. Ergothioneine can inhibit the production of inflammatory factors, such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β). These inflammatory factors play an important role in the inflammatory response, but excessive inflammation can damage the body. By inhibiting the production of these inflammatory factors, ergothioneine reduces excessive inflammation, thereby helping to maintain the body's immune balance and healthy state.

[0014] Ergothioneine can regulate the differentiation and function of immune cells and promote the formation of immune tolerance. This is of great significance for the prevention of autoimmune diseases. By enhancing immune tolerance, ergothioneine reduces the risk of the body attacking its own tissues, thereby helping to maintain immune homeostasis. Ergothioneine has shown particular significance in protecting cognitive abilities and neurodegenerative diseases. A recent placebo-controlled trial evaluating cognitive performance in healthy adults showed that daily supplementation with pure ergothioneine improved cognitive function in healthy middle-aged and older adults (55-79 years old) with poor memory.

[0015] 2) A maitake mushroom fermentation enzyme preparation process according to 1), wherein in step S1, maitake mushroom juice with a water content of 10% is obtained by juicing maitake mushrooms, and maitake mushroom filtrate is obtained by removing visible solids and macromolecular substances from the maitake mushroom juice through a filter membrane, comprising the following steps: Step S11, draining water from maitake mushrooms: initially weigh the fresh maitake mushrooms to determine their mass. The fresh maitake mushrooms are placed on a rotating plate in a 40℃ baking oven. The rotation speed of the plate is controlled to ensure even heating. After baking for 12-24 hours, the maitake mushrooms are removed and weighed. The initial weight and the weight after baking of the maitake mushrooms were used to calculate the moisture content of the maitake mushrooms using a moisture content formula, and the moisture content of the maitake mushrooms was controlled at 10%. Step S12, Maitake Juice Extraction: Mix the maitake mushroom with a water content of 10% from step S11 with 10 times the volume of 50% alcohol, and perform a first juice extraction to obtain the first maitake liquid and maitake residue. Then add 1L of 50% alcohol to the maitake residue for a second juice extraction to obtain the second maitake liquid. Combine the second maitake liquid with the first maitake liquid to obtain maitake juice. Step S13: The maitake mushroom juice is filtered through a polyethylene membrane with a molecular weight cutoff of 500-3000 Da to remove visible solids and macromolecules from the maitake mushroom juice and obtain maitake mushroom filtrate.

[0016] 3) A maitake mushroom fermentation enzyme production process according to 2), wherein: In step S11, the baking oven includes a box body. The flip plate consists of several pieces and is evenly arranged along the height direction of the baking oven. Each flip plate has several evenly arranged mesh bags on its upper and lower surfaces. The flip plate has hooks corresponding to the positions of the mesh bags. The mesh bags have bag openings that are hung on the hooks. The hooks are several hooks made of wire arranged in a ring.

[0017] In this technical solution, the top and bottom of the box are equipped with heating plates that generate heat. The heating plates are powered by a 220V power supply, so that they generate heat to heat the air inside the box and bake the maitake mushrooms on each flip plate. In order to further fix the maitake mushrooms on the flip plate and at the same time ensure that the maitake mushrooms are heated evenly, the design uses a mesh bag to cover the maitake mushrooms and hangs them on the flip plate with hooks and loops.

[0018] To further secure the mesh bag to the flipping board and prevent it from falling off, the design incorporates several hooks made of wire, evenly arranged in a ring. The hooks have a hook-like structure. Since the mesh bag has a mesh structure, the bag opening is secured to the hooks through the mesh opening. Because the hooks are made of wire, they have a certain degree of elasticity. After each mesh opening is secured to the corresponding hook, the hook-shaped ends are pinched together to clamp and fix the mesh bag, thus allowing it to hang on the flipping board.

[0019] The mesh bag and hooks allow the maiki to be hung on the rotating board, reducing direct contact between the maiki and the board. This ensures that the maiki is heated evenly around its perimeter as the board rotates. Additionally, since the hooks are made of wire, which has thermal conductivity, the heat generated by the wire itself is indirectly transferred to the maiki, further heating it.

[0020] 4) A maitake mushroom fermentation enzyme production process according to 3), wherein: The two ends of the flip plate are rotatably connected to the two sides of the box body via rotating shafts. Two bearings are evenly distributed along the height direction on the outer side of the box body, and a rotating rod passes through the two bearings. Several first bevel gears are evenly distributed along the height direction on the rotating rod. The position of each first bevel gear corresponds to the position of each flip plate. A second bevel gear meshes with each first bevel gear. A drive rod passes through the second bevel gear. The end of the drive rod passes through the side of the box body and is connected to the rotating shaft at the end of the flip plate.

[0021] In this technical solution, a support plate extending towards the rotating rod is installed on the top of the housing. The support plate and the rotating rod are located on the same side of the housing. Meanwhile, a servo motor is fixedly installed on the support plate with bolts and nuts. When installing the servo motor, the output shaft of the servo motor passes through the support plate and extends towards the rotating rod. The output shaft of the servo motor is connected to the rotating rod through a spline connection. When the servo motor is started, the output shaft of the servo motor drives the rotating rod to rotate.

[0022] When the rotating rod rotates, it synchronously drives all the first bevel gears mounted on it to rotate. Since each first bevel gear meshes with a corresponding second bevel gear, the rotation of the first bevel gear drives the rotation of the second bevel gear, and the rotation of the second bevel gear drives the drive rod that passes through the second bevel gear to rotate. Since the drive rod passes through the side of the housing and is connected to the rotating shaft at the end of the flip plate through a spline, the side of the housing supports the drive rod. When the drive rod rotates, it drives the rotating shaft at the end of the flip plate to rotate. Since the two ends of the flip plate are rotatably connected to the two sides of the housing through rotating shafts, when the rotating shaft at one end of the flip plate rotates, it drives the flip plate to rotate.

[0023] Therefore, in this technical solution, the rotating rod synchronously drives all the first bevel gears to rotate, each first bevel gear drives the second bevel gear at its corresponding position to rotate, and each second bevel gear drives the flipping plate at its corresponding position to rotate. Thus, all the flipping plates in the box can rotate synchronously and simultaneously, thereby enabling the maitake mushrooms on each flipping plate to be heated and baked synchronously and simultaneously, improving the baking efficiency.

[0024] 5) A maitake mushroom fermentation enzyme production process according to 2), wherein: The formula for water content in step S11 is:

[0025] Among them: the initial weight of fresh maitake mushrooms is The weight of the roasted maitake mushroom is .

[0026] 6) A maitake mushroom fermentation enzyme preparation process according to 1), wherein in step S1, acid and alkali are added to the maitake mushroom filtrate to adjust the pH of the maitake mushroom filtrate to obtain a maitake mushroom preparation solution, comprising the following steps: Acid and alkali are added to the maitake mushroom filtrate, and the mixture is continuously stirred while adding acid and alkali to maintain the pH of the maitake mushroom filtrate at 2.5-3, thereby inhibiting the growth of harmful bacteria in the maitake mushroom preparation solution.

[0027] This technical solution involves adding acid and alkali to the maitake mushroom filtrate before fermentation and continuously stirring it. This allows the acid and alkali to fully dissolve in the maitake mushroom filtrate and adjusts the pH of the filtrate to 2.5-3. Therefore, when the maitake mushroom filtrate is at a low pH, the growth of harmful bacteria in the filtrate can be inhibited, reducing the risk of contamination.

[0028] 7) A maitake mushroom fermentation enzyme preparation process according to 1), wherein in step S2, the maitake mushroom preparation liquid is placed in a steam explosion machine for steam explosion treatment, and after steam explosion treatment, it is placed in a fermentation chamber for temperature-controlled fermentation to obtain maitake mushroom fermentation liquid, including the following steps: Step S21: Place the maitake mushroom preparation solution into a steam explosion machine. The steam explosion machine is a water vapor explosion machine with an explosion pressure of 1.0-1.15 MPa and a pressure holding time of 90-150 s. The maitake mushroom preparation solution is subjected to steam explosion treatment. During the steam explosion treatment, the cell walls of the maitake mushroom cells in the maitake mushroom preparation solution are destroyed. Step S22: After the maitake mushroom cells in the maitake mushroom preparation solution in step S21 have completed the cell wall rupture, the nutrients inside the maitake mushroom cells dissolve in the maitake mushroom preparation solution, and the solution is placed in a fermentation chamber for fermentation in a temperature-controlled manner.

[0029] 8) A process for preparing maitake mushroom fermentation enzyme according to 2), wherein in step S3, an aqueous solution containing a surfactant is added to the maitake mushroom fermentation liquid, and the supernatant and precipitate are obtained by ultrasonic vibration and centrifugation. The supernatant is then freeze-dried at -35 to -45°C and 30 to 50 Pa for 12 to 24 hours and then subjected to ultrafine powder treatment, including the following steps: Step S31, Aqueous solution preparation: A surfactant composition is prepared by using propylene glycol random polyether PPE-1500, Triton X-100 and dodecyltrimethylammonium chloride. The surfactant composition is added to active water so that the surfactant composition accounts for 1.2% to 2.2% of the mass of the active water, thus obtaining an aqueous solution containing surfactant. Step S32, Ultrasonic separation: The aqueous solution containing surfactant and the maitake fermentation liquid from step S31 are respectively placed into an ultrasonic device. The ultrasonic device uses the vibration generated by ultrasound to make the aqueous solution containing surfactant and the maitake fermentation liquid vibrate and mix evenly to obtain a mixture. Step S33, Static Separation: The mixture from step S32 is allowed to stand and separate to obtain supernatant and precipitate. The supernatant is freeze-dried at -35 to -45°C and 30 to 50 Pa for 12 to 24 hours to obtain solid maitake fermentation body. The solid maitake fermentation body is then treated with ultrafine powder to obtain maitake fermentation enzyme.

[0030] In this technical solution, propylene glycol random polyether PPE-1500, Triton X-100, and dodecyltrimethylammonium chloride are used to prepare an aqueous solution to obtain a surfactant composition. The surfactant composition has penetrating, emulsifying, and solubilizing effects. Combined with the cell-wall breaking effect of the ultrasonic vibration generated by the ultrasonic device, the amount of maitake mushroom nutrients dissolved in the maitake mushroom fermentation broth is increased. At the same time, in step S32, the mixture is allowed to stand and separate to obtain a supernatant and a precipitate. The supernatant is then freeze-dried at -35 to -45°C and 30 to 50 Pa for 12 to 24 hours to obtain a solid maitake mushroom fermentation body. The solid maitake mushroom fermentation body is then treated with ultrafine powder to obtain maitake mushroom fermentation enzyme. The above steps improve the extraction rate of maitake mushroom nutrients in the maitake mushroom fermentation enzyme.

[0031] 9) A maitake mushroom fermentation enzyme production process according to 8), wherein: In step S31, the propylene glycol random polyether PPE-1500 accounts for 65% to 80% of the mass percentage of the surfactant composition, Triton X-100 accounts for 10% to 20% of the mass percentage of the surfactant composition, and dodecyltrimethylammonium chloride accounts for 10% to 20% of the mass percentage of the surfactant composition.

[0032] In this technical solution, dodecyltrimethylammonium chloride is a quaternary ammonium salt cationic surfactant. Its hydrophilicity and strong polarity can dissolve the cell wall of *Maitake* mushrooms, disrupting the cellulose network structure inside the cells and improving the exudation efficiency of nutrients. Furthermore, the combination of dodecyltrimethylammonium chloride with the excellent low-foaming and emulsifying properties of propylene glycol random polyether PPE-1500, and the excellent high-temperature resistance and easy binding to biofilms of Triton X-100 to enhance cell wall disruption, allows the ternary compound surfactant in this technical solution to promote the release of nutrients from *Maitake* mushroom cells in an acidic environment, increasing the extraction rate of nutrients and achieving a synergistic, efficient, safe, and controllable extraction process.

[0033] 10) An application of the antioxidant effect of maitake fermented enzyme, wherein the application of maitake fermented enzyme in the process described in 1)-9) above in terms of antioxidant effect includes: using the polysaccharides and ergothioneine in maitake fermented enzyme, together with maitake mushroom, polysaccharides and ergothioneine, to activate the activity of the reticuloendothelial system, activate macrophages to enhance phagocytic function, and thus enhance the body's immunity.

[0034] The ingredients of *Grifola frondosa*, polysaccharides, and ergothionein together primarily affect the reticuloendothelial system, macrophages, lymphocytes, leukocytes, as well as the synthesis of RNA, DNA, and proteins, the content of cAMP and cGMP, antibody production, antibody formation, and interferon induction. It also has antitumor, anti-inflammatory, anticoagulant, antiviral, anti-radiation damage, hypoglycemic, and hypolipidemic activities. Attached Figure Description

[0035] Figure 1 This is a flowchart of a maitake mushroom fermentation enzyme production process according to the present invention; Figure 2 This is a schematic diagram of the baking oven used in the maitake mushroom fermentation enzyme production process of the present invention; Figure 3 This is a schematic diagram of the structure of the mesh bag hanging to the hook in the baking box during the production process of maitake mushroom fermentation enzyme according to the present invention; Figure 4 In the application of the antioxidant effect of a maitake mushroom fermentation enzyme of the present invention, the Phenol-sulfuric acid method was used to determine the detection line of maitake mushroom and polysaccharides. Figure 5 In the application of the antioxidant activity of a maitake mushroom fermentation enzyme of the present invention, the Orcinol-sulfuric acid method was used to determine the detection limits of polysaccharides and ergothioneine. Detailed Implementation

[0036] The following detailed description illustrates the specific implementation method: The reference numerals in the accompanying drawings include: housing 1, heating plate 2, flip plate 3, rotating shaft 4, drive rod 5, rotating rod 6, first bevel gear 7, second bevel gear 8, bearing 9, hook 10, support plate 11, servo motor 12, and mesh bag 13.

[0037] See the example. Figure 1 As shown, the process for producing maitake mushroom fermented enzyme in this embodiment includes the following steps: Step S1: Extract maitake mushroom juice with a water content of 10% to obtain maitake mushroom juice liquid. Remove visible solids and macromolecular substances from the maitake mushroom juice liquid through a filter membrane to obtain maitake mushroom filtrate. Add acid and alkali to the maitake mushroom filtrate to adjust the pH of the maitake mushroom filtrate to obtain maitake mushroom preparation solution. Step S2: Place the maitake mushroom preparation liquid from step S1 into a steam explosion machine for steam explosion treatment, and then place it in a fermentation chamber for temperature-controlled fermentation to obtain maitake mushroom fermentation liquid. Step S3: Extract the fermenting enzyme from the maitake fermentation broth in step S2. Add an aqueous solution containing a surfactant to the maitake fermentation broth, and separate the supernatant and precipitate by ultrasonic vibration and centrifugation. Freeze-dry the supernatant at 35-45℃ and 30-50Pa for 12-24 hours, and then process it into ultrafine powder to obtain maitake fermentation enzyme.

[0038] In this embodiment, in step S1, the maitake mushroom with a water content of 10% is mixed with alcohol and juiced to achieve a state of water deficiency after a large amount of water is removed. When the maitake mushroom is mixed with alcohol, it can absorb a large amount of alcohol, adsorb the alcohol into the maitake mushroom and produce microorganisms, so as to facilitate the fermentation and use of maitake mushroom in the later stage.

[0039] After mixing maitake mushrooms with alcohol in step S1, the maitake mushroom juice is obtained by juicing. The maitake mushroom juice contains visible solids and macromolecules, which are separated by a filter membrane to obtain maitake mushroom filtrate. The maitake mushroom filtrate contains a large amount of alcohol. In order to allow the maitake mushroom filtrate to ferment better, the pH value of the maitake mushroom filtrate is adjusted in real time by adding acid and alkali to obtain maitake mushroom preparation solution.

[0040] After adjusting the pH of the maitake mushroom preparation solution, step S2 involves placing the maitake mushroom preparation solution in a steam explosion machine for steam explosion treatment. This effectively destroys the cell walls of the maitake mushroom cells in the preparation solution, allowing the substances contained in the maitake mushroom cells to fully dissolve in the liquid. After the steam explosion treatment is completed, the solution is placed in a fermentation chamber for temperature-controlled fermentation to obtain maitake mushroom fermentation liquid.

[0041] Finally, in step S3, the fermentation enzyme of the maitake fermentation broth from step S2 is extracted. An aqueous solution containing a surfactant is added to the maitake fermentation broth. The surfactant-containing aqueous solution can reduce the surface tension of the maitake fermentation broth, promote the dissolution and dispersion of oxygen and maitake nutrients in the maitake fermentation broth, and enable microorganisms to absorb and utilize maitake nutrients more fully. After the fermentation efficiency of the maitake fermentation broth is further enhanced by the surfactant-containing aqueous solution, the maitake fermentation broth is shaken and centrifuged by ultrasound to obtain the supernatant and precipitate. The supernatant is freeze-dried at 35-45℃ and 30-50Pa for 12-24 hours and then subjected to ultrafine powder treatment to obtain maitake fermentation enzyme. Thus, the improved prickly pear fermentation method of our company is applied to maitake to prepare and produce maitake fermentation enzyme.

[0042] In addition, in step S1, the maitake mushroom juice with a water content of 10% is obtained by juicing maitake mushrooms, and the maitake mushroom filtrate is obtained by removing visible solids and macromolecular substances from the maitake mushroom juice through a filter membrane, including the following steps: Step S11, draining water from maitake mushrooms: initially weigh the fresh maitake mushrooms to determine their mass. The fresh maitake mushrooms are placed on the rotating plate 3 of a 40℃ baking oven. The rotation speed of the rotating plate 3 is controlled to ensure even heating of the maitake mushrooms. After baking for 12-24 hours, the baked maitake mushrooms are removed and weighed. The initial weight and the weight after baking of the maitake mushrooms were used to calculate the moisture content of the maitake mushrooms using a moisture content formula, and the moisture content of the maitake mushrooms was controlled at 10%. Furthermore, in this embodiment, the formula for water content in step S11 is:

[0043] Among them: the initial weight of fresh maitake mushrooms is The weight of the roasted maitake mushroom is .

[0044] Step S12, Maitake Juice Extraction: Mix the maitake mushroom with a water content of 10% from step S11 with 10 times the volume of 50% alcohol, and perform a first juice extraction to obtain the first maitake liquid and maitake residue. Then add 1L of 50% alcohol to the maitake residue for a second juice extraction to obtain the second maitake liquid. Combine the second maitake liquid with the first maitake liquid to obtain maitake juice. Step S13: The maitake mushroom juice is filtered through a polyethylene membrane with a molecular weight cutoff of 500-3000 Da to remove visible solids and macromolecules from the maitake mushroom juice and obtain maitake mushroom filtrate.

[0045] For the specific structure of the baking oven in step S11, please refer to [link / reference]. Figure 2As shown, in this embodiment, the baking oven includes a hollow box body 1, and several flip plates 3 are evenly arranged along the height of the baking oven. The front of the box body 1 has an opening, and a door panel is hinged to the box body 1 to close the opening. In use, the opening of the box body 1 is exposed by opening the door panel, thereby exposing the flip plates 3 inside the box body 1, which makes it convenient for the staff to place the maitake mushrooms on the flip plates 3. Then, the opening is closed by closing the door panel. Since the top and bottom of the box body 1 in this embodiment have heating plates 2 that generate heat, the heating plates 2 are powered by a 220V power supply, so that the heating plates 2 generate heat when powered on to heat the air in the relatively enclosed space of the box body 1, and bake the maitake mushrooms on each flip plate 3.

[0046] To further secure the maiki to the flipping plate 3 while ensuring even heating, a mesh bag 13 is used to enclose the maiki, which is then hung on the flipping plate 3 using hooks and loops. Specifically, this is combined with... Figure 3 As shown, each flip plate 3 has several evenly arranged mesh bags 13 on its upper and lower surfaces. The flip plate 3 has hooks corresponding to the positions of the mesh bags 13. The mesh bags 13 have bag openings that hang on the hooks. The hooks are several hooks 10 made of wire arranged in a ring.

[0047] To further secure the mesh bag 13 to the flip plate 3 and prevent it from falling off, the design incorporates several hooks 10 made of wire, evenly arranged in a ring. The hooks 10 have a hook-like structure. Since the mesh bag 13 has a mesh structure, the mesh opening of the bag 13 is fastened to the hook 10. Because the hook 10 is made of wire, it has a certain degree of elasticity. After each mesh opening of the bag 13 is fastened to the corresponding hook 10, the hook-shaped ends of the hooks 10 are pinched tightly to clamp and secure the mesh opening of the bag 13, thus allowing the mesh bag 13 to be hung on the flip plate 3.

[0048] The connection between the mesh bag 13 and the hook 10 allows the maiki to be hung on the flip plate 3, reducing direct contact between the maiki and the flip plate 3. This allows the maiki to be heated evenly around its perimeter as the flip plate 3 rotates. At the same time, since the hook 10 is made of iron wire, the iron wire has a certain thermal conductivity. The heat generated by the iron wire itself can also be indirectly transferred to the maiki at the corresponding position, further baking the maiki.

[0049] Each flip plate 3 has two ends rotatably connected to the two sides of the housing 1 via a rotating shaft 4. Two bearings 9 are evenly distributed along the height direction on the outer side of the housing 1. A rotating rod 6 passes through the two bearings 9. Several first bevel gears 7 are evenly distributed along the height direction on the rotating rod 6. The position of each first bevel gear 7 corresponds to the position of each flip plate 3. A second bevel gear 8 meshes with each first bevel gear 7. A drive rod 5 passes through the second bevel gear 8. The end of the drive rod 5 passes through the side of the housing 1 and is connected to the rotating shaft 4 at the end of the flip plate 3.

[0050] In this embodiment, a support plate 11 extending toward the rotating rod 6 is installed on the top of the housing 1. The support plate 11 and the rotating rod 6 are located on the same side of the housing 1. Meanwhile, a servo motor 12 is fixedly installed on the support plate 11 by bolts and nuts. When installing the servo motor 12, the output shaft of the servo motor 12 passes through the support plate 11 and extends toward the rotating rod 6. The output shaft of the servo motor 12 is connected to the rotating rod 6 through a spline connection. When the servo motor 12 is started, the output shaft of the servo motor 12 drives the rotating rod 6 to rotate.

[0051] When the rotating rod 6 rotates, it synchronously drives all the first bevel gears 7 mounted on it to rotate. Since each first bevel gear 7 meshes with the corresponding second bevel gear 8, the rotation of the first bevel gear 7 drives the rotation of the second bevel gear 8. The rotation of the second bevel gear 8 drives the drive rod 5 that passes through the second bevel gear 8 to rotate. Since the drive rod 5 passes through the side of the housing 1 and is connected to the rotating shaft 4 at the end of the flip plate 3 by a spline, the side of the housing 1 supports the drive rod 5. When the drive rod 5 rotates, it drives the rotating shaft 4 at the end of the flip plate 3 to rotate. Since the two ends of the flip plate 3 are rotatably connected to the two sides of the housing 1 by the rotating shaft 4, when the rotating shaft 4 at one end of the flip plate 3 rotates, it drives the flip plate 3 to rotate.

[0052] Therefore, in this technical solution, the rotating rod 6 synchronously drives all the first bevel gears 7 to rotate, each first bevel gear 7 drives the second bevel gear 8 at its corresponding position to rotate, and each second bevel gear 8 drives the flipping plate 3 at its corresponding position to rotate. Thus, all the flipping plates 3 in the box 1 can rotate synchronously, thereby enabling the maitake mushrooms on each flipping plate 3 to be heated and baked synchronously, improving the baking efficiency.

[0053] In this case, acid and alkali were added to the maitake mushroom filtrate in step S1 to adjust the pH of the maitake mushroom filtrate, resulting in a maitake mushroom preparation solution, which includes the following steps: Acid and alkali are added to the maitake mushroom filtrate, and the mixture is continuously stirred while adding acid and alkali to maintain the pH of the maitake mushroom filtrate at 2.5-3, thereby inhibiting the growth of harmful bacteria in the maitake mushroom preparation solution.

[0054] In this embodiment, before fermentation of the maitake mushroom filtrate, acid and alkali are added to the maitake mushroom filtrate and continuously stirred. This allows the acid and alkali to fully dissolve in the maitake mushroom filtrate and adjusts the pH of the maitake mushroom filtrate to 2.5-3. Therefore, when the maitake mushroom filtrate is at a low pH, the growth of harmful bacteria in the maitake mushroom filtrate can be inhibited, reducing the risk of contamination of the maitake mushroom filtrate.

[0055] In step S2, the maitake mushroom preparation solution is placed in a steam explosion machine for steam explosion treatment. After steam explosion treatment, it is placed in a fermentation chamber for temperature-controlled fermentation to obtain maitake mushroom fermentation broth, including the following steps: Step S21: Place the maitake mushroom preparation solution into a steam explosion machine. The steam explosion machine is a water vapor explosion machine with an explosion pressure of 1.0-1.15 MPa and a pressure holding time of 90-150 s. The maitake mushroom preparation solution is subjected to steam explosion treatment. During the steam explosion treatment, the cell walls of the maitake mushroom cells in the maitake mushroom preparation solution are destroyed. Step S22: After the maitake mushroom cells in the maitake mushroom preparation solution in step S21 have completed the cell wall rupture, the nutrients inside the maitake mushroom cells dissolve in the maitake mushroom preparation solution, and the solution is placed in a fermentation chamber for fermentation in a temperature-controlled manner.

[0056] In step S3, an aqueous solution containing a surfactant is added to the maitake mushroom fermentation broth, and the supernatant and precipitate are obtained by ultrasonic vibration and centrifugation. The supernatant is then freeze-dried at 35–45°C and 30–50 Pa for 12–24 hours and then subjected to ultrafine powder treatment, including the following steps: Step S31, Aqueous solution preparation: A surfactant composition is prepared by using propylene glycol random polyether PPE 1500, Triton X 100 and dodecyltrimethylammonium chloride. The surfactant composition is added to active water so that the surfactant composition accounts for 1.2% to 2.2% of the mass of the active water, thus obtaining an aqueous solution containing surfactant. Step S32, Ultrasonic separation: The aqueous solution containing surfactant and the maitake fermentation liquid from step S31 are respectively placed into an ultrasonic device. The ultrasonic device uses the vibration generated by ultrasound to make the aqueous solution containing surfactant and the maitake fermentation liquid vibrate and mix evenly to obtain a mixture. Step S33, Static Separation: The mixture from step S32 is allowed to stand and separate to obtain supernatant and precipitate. The supernatant is freeze-dried at 35-45℃ and 30-50Pa for 12-24 hours to obtain solid maitake fermentation body. The solid maitake fermentation body is then treated with ultrafine powder to obtain maitake fermentation enzyme.

[0057] In this embodiment, a surfactant composition is prepared by using propylene glycol random polyether PPE 1500, Triton X 100, and dodecyltrimethylammonium chloride in the aqueous solution. The surfactant composition has penetrating, emulsifying, and solubilizing effects. Combined with the cell-wall breaking effect of the ultrasonic device using the vibration mechanical force generated by the ultrasonic waves, the amount of maitake mushroom nutrients dissolved in the maitake mushroom fermentation broth is increased. At the same time, in step S32, the mixture is allowed to stand and separate to obtain a supernatant and a precipitate. The supernatant is then freeze-dried at 35-45°C and 30-50 Pa for 12-24 hours to obtain a solid maitake mushroom fermentation body. The solid maitake mushroom fermentation body is then treated with ultrafine powder to obtain maitake mushroom fermentation enzyme. The above steps improve the extraction rate of maitake mushroom nutrients in the maitake mushroom fermentation enzyme.

[0058] In step S31, propylene glycol random polyether PPE 1500 accounts for 65% to 80% of the surfactant composition by mass, Triton X 100 accounts for 10% to 20% of the surfactant composition by mass, and dodecyltrimethylammonium chloride accounts for 10% to 20% of the surfactant composition by mass.

[0059] In this embodiment, dodecyltrimethylammonium chloride is a quaternary ammonium salt cationic surfactant. Its hydrophilicity and strong polarity can dissolve the cell wall of *Maitake*, disrupt the cellulose network structure inside the cells, and improve the exudation efficiency of nutrients. Furthermore, the combination of dodecyltrimethylammonium chloride with the excellent low-foaming and emulsifying properties of propylene glycol random polyether (PPE) 1500, and the excellent high-temperature resistance and easy binding to biofilms of Triton X 100 to enhance cell wall disruption, allows the ternary compound surfactant in this technical solution to promote the release of nutrients from *Maitake* cells in an acidic environment, increasing the extraction rate of nutrients and achieving a synergistic, efficient, safe, and controllable extraction process.

[0060] The maitake mushroom fermented enzyme produced using the above-mentioned maitake mushroom fermentation enzyme process was tested: (1) Determined using the Phenol-sulfuric acid method (see [reference]). Figure 4 (As shown) Dissolve 10 mg of maitake mushroom fermentation enzyme in 10 ml of distilled water. Take 1 ml of the solution and add 9 ml of distilled water to make a concentration of 100 μg / ml. Then dilute it equally to make standards of 50 μg / ml, 25 μg / ml, 12.5 μg / ml, and 6.25 μg / ml. Take 200 μl of each concentration of maitake mushroom fermentation enzyme and add 200 μl of 5% phenol to a test tube. Then add 3.0 ml of concentrated sulfuric acid (quickly and directly to the surface of the liquid, without touching the tube wall), and perform three replicates. Let it stand for ten minutes without shaking. After ten minutes, shake to mix evenly. After twenty minutes, measure the absorbance at a wavelength of 483 nm and draw a check line to observe the maitake mushroom and polysaccharides in the maitake mushroom fermentation enzyme.

[0061] (2) Determination using the Orcinol-sulfuric acid method (see...) Figure 5 (As shown) Dissolve 10 mg of maitake mushroom fermentation enzyme in 10 ml of distilled water. Take 1 ml of the solution and add 9 ml of distilled water to make a concentration of 100 μg / ml. Then dilute to prepare standards of 50 μg / ml, 20 μg / ml, and 10 μg / ml. Take 200 μl of each concentration of maitake mushroom fermentation enzyme and add it to 800 μl of (orcinol 2 g / 1 L concentrated sulfuric acid) in a test tube. Perform three replicates. Then place the test tube in an 80°C water bath and heat for 15 minutes. After cooling to room temperature, measure the absorbance at a wavelength of 420 nm and plot the calibration curve. Polysaccharides and ergothioneine in maitake mushroom fermentation enzyme were observed.

[0062] The above-mentioned testing method showed that the maitake fermented enzyme prepared by the maitake fermented enzyme process of the present invention contains maitake mushroom, polysaccharide and ergothioneine. Therefore, the application of the antioxidant effect of the maitake fermented enzyme in this embodiment, the application of the maitake fermented enzyme prepared by the above-mentioned maitake fermented enzyme process in antioxidant effect includes: the polysaccharide and ergothioneine in the maitake fermented enzyme, maitake mushroom, polysaccharide and ergothioneine together activate the activity of the reticuloendothelial system, activate macrophages to enhance phagocytic function, and have the effect of enhancing the body's immunity.

[0063] The ingredients of *Grifola frondosa*, polysaccharides, and ergothionein together primarily affect the reticuloendothelial system, macrophages, lymphocytes, leukocytes, as well as the synthesis of RNA, DNA, and proteins, the content of cAMP and cGMP, antibody production, antibody formation, and interferon induction. It also has antitumor, anti-inflammatory, anticoagulant, antiviral, anti-radiation damage, hypoglycemic, and hypolipidemic activities.

[0064] The above are merely embodiments of this solution. Commonly known technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solution of this solution. These modifications and improvements should also be considered within the scope of protection of this solution, and will not affect the effectiveness of the implementation of this solution or the practicality of the patent. The scope of protection claimed in this application shall be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A process for producing maitake mushroom fermented enzyme, characterized in that, Includes the following steps: Step S1: Extract maitake mushroom juice with a water content of 10% to obtain maitake mushroom juice liquid. Remove visible solids and macromolecular substances from the maitake mushroom juice liquid through a filter membrane to obtain maitake mushroom filtrate. Add acid and alkali to the maitake mushroom filtrate to adjust the pH of the maitake mushroom filtrate to obtain maitake mushroom preparation solution. Step S2: Place the maitake mushroom preparation liquid from step S1 into a steam explosion machine for steam explosion treatment, and then place it in a fermentation chamber for temperature-controlled fermentation to obtain maitake mushroom fermentation liquid. Step S3: Extract the fermenting enzyme from the maitake fermentation broth in step S2. Add an aqueous solution containing a surfactant to the maitake fermentation broth, and separate the supernatant and precipitate by ultrasonic vibration and centrifugation. Freeze-dry the supernatant at -35 to -45°C and 30 to 50 Pa for 12 to 24 hours, and then process it into ultrafine powder to obtain maitake fermentation enzyme.

2. The process for producing maitake mushroom fermented enzyme according to claim 1, characterized in that: In step S1, the maitake mushroom juice with a water content of 10% is obtained by juicing maitake mushrooms. The maitake mushroom filtrate is obtained by filtering the maitake mushroom juice through a filter membrane to remove visible solids and macromolecular substances. The steps include: Step S11, draining water from maitake mushrooms: initially weigh the fresh maitake mushrooms to determine their mass. The fresh maitake mushrooms are placed on a rotating plate in a 40℃ baking oven. The rotation speed of the plate is controlled to ensure even heating. After baking for 12-24 hours, the maitake mushrooms are removed and weighed. The initial weight and the weight after baking of the maitake mushrooms were used to calculate the moisture content of the maitake mushrooms using a moisture content formula, and the moisture content of the maitake mushrooms was controlled at 10%. Step S12, Maitake Juice Extraction: Mix the maitake mushroom with a water content of 10% from step S11 with 10 times the volume of 50% alcohol, and perform a first juice extraction to obtain the first maitake liquid and maitake residue. Then add 1L of 50% alcohol to the maitake residue for a second juice extraction to obtain the second maitake liquid. Combine the second maitake liquid with the first maitake liquid to obtain maitake juice. Step S13: The maitake mushroom juice is filtered through a polyethylene membrane with a molecular weight cutoff of 500-3000 Da to remove visible solids and macromolecules from the maitake mushroom juice and obtain maitake mushroom filtrate.

3. The process for producing maitake mushroom fermented enzyme according to claim 2, characterized in that: In step S11, the baking oven includes a box body. The flip plate consists of several pieces and is evenly arranged along the height direction of the baking oven. Each flip plate has several evenly arranged mesh bags on its upper and lower surfaces. The flip plate has hooks corresponding to the positions of the mesh bags. The mesh bags have bag openings that are hung on the hooks. The hooks are several hooks made of wire arranged in a ring.

4. The process for producing maitake mushroom fermented enzyme according to claim 3, characterized in that: The two ends of the flip plate are rotatably connected to the two sides of the box body via a rotating shaft. Two bearings are evenly distributed along the height direction on the outer side of the box body, and a rotating rod passes through the two bearings. Several first bevel gears are evenly distributed along the height direction on the rotating rod. The position of each first bevel gear corresponds to the position of each flip plate. A second bevel gear meshes with each first bevel gear. A drive rod passes through the second bevel gear. The end of the drive rod passes through the side of the box body and is connected to the rotating shaft at the end of the flip plate.

5. The process for producing maitake mushroom fermented enzyme according to claim 2, characterized in that: in: The formula for water content in step S11 is: Among them: the initial weight of fresh maitake mushrooms is The weight of the roasted maitake mushroom is .

6. The process for producing maitake mushroom fermented enzyme according to claim 3, characterized in that: In step S1, acid and alkali are added to the maitake mushroom filtrate to adjust the pH of the filtrate, resulting in a maitake mushroom preparation solution. This includes the following steps: Acid and alkali are added to the maitake mushroom filtrate, and the mixture is continuously stirred while adding acid and alkali to maintain the pH of the maitake mushroom filtrate at 2.5-3, thereby inhibiting the growth of harmful bacteria in the maitake mushroom preparation solution.

7. The process for producing maitake mushroom fermented enzyme according to claim 1, characterized in that: In step S2, the maitake mushroom preparation solution is placed in a steam explosion machine for steam explosion treatment. After steam explosion treatment, it is placed in a fermentation chamber for temperature-controlled fermentation to obtain maitake mushroom fermentation liquid, including the following steps: Step S21: Place the maitake mushroom preparation solution into a steam explosion machine. The steam explosion machine is a water vapor explosion machine with an explosion pressure of 1.0-1.15 MPa and a pressure holding time of 90-150 s. The maitake mushroom preparation solution is subjected to steam explosion treatment. During the steam explosion treatment, the cell walls of the maitake mushroom cells in the maitake mushroom preparation solution are destroyed. Step S22: After the maitake mushroom cells in the maitake mushroom preparation solution in step S21 have completed the cell wall rupture, the nutrients inside the maitake mushroom cells dissolve in the maitake mushroom preparation solution, and the mixture is placed in a fermentation chamber for fermentation in a temperature-controlled manner.

8. The process for producing maitake mushroom fermented enzyme according to claim 2, characterized in that: In step S3, an aqueous solution containing a surfactant is added to the maitake mushroom fermentation broth, and the supernatant and precipitate are obtained by ultrasonic vibration and centrifugation. The supernatant is then freeze-dried at -35 to -45°C and 30 to 50 Pa for 12 to 24 hours, followed by ultrafine powder treatment, including the following steps: Step S31, Aqueous solution preparation: A surfactant composition is prepared by using propylene glycol random polyether PPE-1500, Triton X-100 and dodecyltrimethylammonium chloride. The surfactant composition is added to active water so that the surfactant composition accounts for 1.2% to 2.2% of the mass of the active water, thus obtaining an aqueous solution containing surfactant. Step S32, Ultrasonic separation: The aqueous solution containing surfactant and the maitake fermentation liquid from step S31 are respectively placed into an ultrasonic device. The ultrasonic device uses the vibration generated by ultrasound to make the aqueous solution containing surfactant and the maitake fermentation liquid vibrate and mix evenly to obtain a mixture. Step S33, Static Separation: The mixture from step S32 is allowed to stand and separate to obtain supernatant and precipitate. The supernatant is freeze-dried at -35 to -45°C and 30 to 50 Pa for 12 to 24 hours to obtain solid maitake fermentation body. The solid maitake fermentation body is then treated with ultrafine powder to obtain maitake fermentation enzyme.

9. The process for producing maitake mushroom fermented enzyme according to claim 8, characterized in that: In step S31, the propylene glycol random polyether PPE-1500 accounts for 65% to 80% of the mass percentage of the surfactant composition, Triton X-100 accounts for 10% to 20% of the mass percentage of the surfactant composition, and dodecyltrimethylammonium chloride accounts for 10% to 20% of the mass percentage of the surfactant composition.

10. An application of the antioxidant effect of maitake mushroom fermented enzyme, wherein the maitake mushroom fermented enzyme produced according to any one of claims 1-9 is used in an antioxidant process, characterized in that: It includes the polysaccharides and ergothioneine in the fermentation enzyme of maitake mushroom, and the polysaccharides and ergothioneine of maitake mushroom work together to activate the activity of the reticuloendothelial system, activate macrophages to enhance phagocytic function, and thus enhance the body's immunity.

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