A method for breeding pleurotus ostreatus mycelium by taking highland barley as a substrate

Abstract

The present application relates to the technical field of microorganism or culture thereof processing, and particularly relates to a method for breeding pleurotus ostreatus mycelium by using highland barley as a substrate. The method is as follows: highland barley is crushed and sieved, then water is added to prepare highland barley slurry, and a complex enzyme system composed of amylase and cellulase is added to perform liquid enzymolysis to obtain an enzymolysis substrate; lactic acid bacteria strains are inoculated into the enzymolysis substrate to perform solid-state fermentation to obtain a fermentation substrate; the fermentation substrate is subjected to moist heat sterilization treatment to obtain a sterilized substrate; the sterilized substrate is inoculated with pleurotus ostreatus original seeds, and is placed in a culture environment for culture to obtain pleurotus ostreatus mycelium. The scheme breaks the physical barrier of the fiber network of the highland barley epidermis and the starch crystal structure of the endosperm by using the complex enzyme system and lactic acid bacteria fermentation, and the substrate is pre-degraded before the mycelium is inoculated, so that the mycelium can directly absorb free carbon sources, the germination time is shortened, the daily growth rate is improved, the residual sugar rate of the substrate is reduced, and the dry weight biomass of the mycelium is increased.

Description

Technical Field

[0001] This invention relates to the field of microbial or culture processing technology, specifically to a method for propagating oyster mushroom mycelium using barley as a substrate. Background Technology

[0002] In the mycelial propagation process of oyster mushrooms, using highland barley as the basic culture medium is a conventional technique. The specific implementation of this conventional technique is as follows: Highland barley is mechanically crushed, mixed with water to a predetermined moisture content, placed in a cultivation container, and sterilized by high-temperature, high-pressure steam. After cooling, solid oyster mushroom spawn is inoculated onto the substrate surface and placed in a constant temperature and humidity incubation chamber for mycelial cultivation. During this process, the oyster mushroom mycelium relies on its secreted extracellular enzymes to degrade macromolecules in the highland barley particles to obtain carbon and nitrogen sources. The mycelium gradually spreads and grows from the inoculation point into the highland barley substrate until it completely covers the entire cultivation container.

[0003] The aforementioned conventional technical solutions face a core technical problem in practical implementation: the barley epidermis contains a dense fibrous network, and the starch inside its endosperm exhibits a highly crystalline structure. These two components together form a robust physical barrier. Directly crushed barley particles expose extremely limited biodegradable interfaces, making it difficult for the extracellular enzymes secreted by *Pleurotus ostreatus* mycelium in the early stages of growth to directly contact and cleave the starch glycosidic bonds and fiber molecular chains. Consequently, the mycelium cannot obtain sufficient usable carbon sources, resulting in delayed germination, stagnant feeding, and hindered mycelial biomass accumulation. Summary of the Invention

[0004] To address the shortcomings of existing technologies where the dense fiber network of barley substrate and the highly crystalline starch crystal structure form a robust physical barrier, leading to delayed germination of oyster mushroom mycelium, stagnation of feeding, and hindered biomass accumulation, this invention provides a method for propagating oyster mushroom mycelium using barley as a substrate.

[0005] To solve the above-mentioned technical problems, the present invention provides a method for propagating oyster mushroom mycelium using barley as a substrate, comprising the following technical features: S1, crushing and sieving barley to collect barley particles; S2, adding sterile water to the barley particles to prepare barley slurry, adding a complex enzyme system composed of amylase and cellulase to the barley slurry for liquid enzymatic hydrolysis to obtain an enzymatic hydrolysis substrate; S3, inoculating the enzymatic hydrolysis substrate with a lactic acid bacteria strain for solid-state fermentation to obtain a fermentation substrate; S4, subjecting the fermentation substrate to moist heat sterilization to obtain a sterilized substrate; S5, inoculating the sterilized substrate with oyster mushroom spawn and culturing it in a culture environment to obtain oyster mushroom mycelium.

[0006] This method utilizes pre-treatment through enzymatic hydrolysis and fermentation to break down the physical and chemical barriers in the barley substrate that hinder oyster mushroom mycelium from utilizing nutrients. Its core biochemical mechanism is as follows: First, the macroscopic structure of the raw barley is destroyed through pulverization, providing a basic contact interface for subsequent enzymatic hydrolysis. The amylase in the complex enzyme system can target and hydrolyze the α-1,4 and α-1,6 glycosidic bonds of highly crystalline starch in the barley endosperm, destroying the starch's crystal structure and degrading macromolecular starch into soluble small-molecule sugars such as maltose and glucose. The cellulase specifically hydrolyzes the β-1,4-glucan bonds in the dense fibrous network of the barley epidermis, dismantling the rigid cellulose framework. Together, these two enzymes break down the physical barriers of the barley. This significantly improves the release efficiency of available carbon sources in the substrate. The subsequently introduced lactic acid bacteria consume free phenols, phytic acid, and other anti-nutritional factors that inhibit the growth of oyster mushroom mycelia through solid-state fermentation and metabolism, eliminating the chemical inhibition. At the same time, the organic acids produced by their metabolism can further moderately degrade the fiber structure and reduce the pH value of the system, reducing the risk of contamination by other microorganisms in the subsequent process. After eliminating live bacteria and spores in the system through moist heat sterilization, the original oyster mushroom spawn is inoculated. The oyster mushroom mycelia can directly utilize the small molecule carbon sources generated by pre-degradation to germinate and proliferate rapidly, without the need to secrete a large amount of extracellular enzymes to complete substrate degradation in the early stage of growth. This fundamentally solves the technical problems of delayed mycelial germination, stagnant feeding, and hindered biomass accumulation.

[0007] Furthermore, in the above technical solution, in step S1, the barley is pulverized using a low-temperature airflow pulverizing device. The particle size distribution range of the barley particles collected after sieving is limited to a first value to a second value. Before entering step S2, the barley particles are placed in a freezing environment to keep the internal moisture of the barley particles in a solid ice crystal state. The solid ice crystals undergo a phase change and generate micro-cracks during the subsequent thawing process.

[0008] In practice, low-temperature airflow pulverization can complete the ultra-fine crushing of barley grains in a low-temperature environment, avoiding the high temperature caused by conventional mechanical pulverization, which leads to starch gelatinization and inactivation of heat-sensitive nutrients in barley. At the same time, limiting the particle size range can ensure the controllability and uniformity of the specific surface area of ​​the particles and the contact area for enzymatic hydrolysis. Freezing treatment causes free water inside the particles to form solid ice crystals. During the thawing phase transition, the volume of the ice crystals changes regularly, which can form a large number of interconnected micro-cracks inside the barley particles. This further expands the contact interface between enzyme molecules and starch and cellulose components inside the barley, improves the efficiency of subsequent enzymatic hydrolysis, and enhances the dismantling effect on the physical barriers of barley.

[0009] Furthermore, in the above technical solution, in step S2, the complex enzyme system is composed of a thermostable α-amylase derived from Aspergillus niger and an endoglucanase derived from Trichoderma reesei, mixed in a first mass ratio. Before being added to the barley pulp, the complex enzyme system is dissolved in an acetate-sodium acetate buffer solution containing calcium ions to form an enzyme solution. The added volume of the enzyme solution is in a first proportional relationship with the mass of the barley pulp.

[0010] In practice, the thermostable α-amylase derived from Aspergillus niger possesses excellent thermal stability and endoglucanase hydrolysis ability, which can efficiently destroy the crystal structure of highly crystalline starch and randomly hydrolyze the glycosidic bonds within starch molecules. The endoglucanase derived from Trichoderma reesei can specifically hydrolyze the amorphous regions of cellulose molecular chains, producing a large amount of cellulose oligosaccharides and new hydrolytic chain ends. The two work together to achieve simultaneous and efficient degradation of starch and cellulose in barley. The acetate-sodium acetate buffer containing calcium ions can provide a stable catalytic microenvironment for enzyme molecules and maintain the stability of the system pH. Calcium ions can act as a metal cofactor of α-amylase, binding to enzyme protein molecules to maintain the stability of their spatial conformation and catalytic active center, preventing enzyme molecules from denaturing and becoming inactive during enzymatic hydrolysis, and ensuring the high efficiency and stability of the enzymatic hydrolysis reaction.

[0011] Furthermore, in the above technical solution, in step S2, an ultrasonic field is introduced into the barley pulp containing the complex enzyme system. The frequency of the ultrasonic field is set to a first frequency value, the power is set to a first power value, the temperature of the liquid enzymatic hydrolysis process is maintained at a first temperature value, and the pH value of the system in the liquid enzymatic hydrolysis process is controlled between a first pH value and a second pH value by adding an acid-base adjusting solution.

[0012] In practice, the cavitation effect of the ultrasonic field can generate a large number of instantaneous high-pressure microbubbles in the barley slurry system. The high-intensity shear force generated by the rupture of microbubbles can further break up the barley particle agglomerates and expand the microporous structure on the particle surface. At the same time, it promotes the diffusion of enzyme molecules into the cracks and micropores inside the barley particles, which greatly increases the contact probability between the enzyme and the substrate and the catalytic reaction rate. By precisely controlling the temperature and pH value of the enzymatic hydrolysis process, the composite enzyme system can always be kept within the optimal catalytic conditions, maximizing the synergistic catalytic efficiency of amylase and cellulase, shortening the enzymatic hydrolysis cycle, and improving the degree of substrate degradation.

[0013] Furthermore, in the above technical solution, in step S3, the lactic acid strain is *Lactobacillus plantarum*, which is activated by slant culture and then propagated by liquid seed culture before being inoculated with the enzymatic hydrolysis substrate to obtain seed liquid. The viable concentration of *Lactobacillus plantarum* in the seed liquid reaches a first concentration value. The seed liquid is applied evenly to the surface and interior of the enzymatic hydrolysis substrate using a spraying device.

[0014] In practice, *Lactobacillus plantarum* is a facultative anaerobic bacterium that can stably proliferate in a solid-state fermentation system. Its metabolic process produces enzymes such as phytase and phenolic acid decarboxylase, which efficiently decompose anti-nutritional factors such as phytic acid and free phenols in the enzymatic hydrolysate, eliminating the inhibitory effect of these substances on the mycelial growth of *Pleurotus ostreatus*. At the same time, the lactic acid produced by metabolism can continuously lower the pH value of the system, inhibiting the growth and reproduction of miscellaneous bacteria. By slant activation and liquid expansion to obtain a seed liquid with a high concentration of viable bacteria, and by uniformly applying it by spraying, it is possible to ensure that *Lactobacillus plantarum* is evenly distributed in three dimensions in the enzymatic hydrolysate system, avoiding the problem of insufficient local fermentation and ensuring the uniformity and stability of the solid-state fermentation effect.

[0015] Furthermore, in the above technical solution, in step S3, the fermentation container receiving the enzymatic hydrolysis substrate is made of a composite membrane material composed of a first polymer layer and a second polymer layer. The outer side of the composite membrane material is encapsulated with a gas conditioning membrane. During the solid-state fermentation process, a micro-oxygen mixed gas is periodically introduced into the fermentation container through the gas conditioning membrane. The micro-oxygen mixed gas is composed of nitrogen and oxygen mixed in a first volume ratio.

[0016] In practice, *Lactobacillus plantarum* is a facultative anaerobic bacterium, and its proliferation and metabolism require trace amounts of oxygen to maintain the respiration and cell wall synthesis of the bacteria. The composite membrane material can ensure the airtightness of the fermentation system and prevent the intrusion of external bacteria. Combined with a gas-regulating membrane, it can achieve precise control of the gas environment within the fermentation system. By periodically introducing a micro-oxygen mixed gas with a specific ratio, a stable micro-oxygen environment can be provided for the growth and metabolism of *Lactobacillus plantarum*, avoiding the problems of slow bacterial proliferation and insufficient metabolic activity caused by a strictly anaerobic environment, thereby improving fermentation efficiency and the removal of anti-nutritional factors.

[0017] Furthermore, in the above technical solution, before performing moist heat sterilization in step S4, a mixed protective dry powder containing trehalose and monosodium glutamate is added to the fermentation substrate. The mass ratio of the mixed protective dry powder to the fermentation substrate is a first ratio. After adding the mixed protective dry powder, the fermentation substrate is laid on a porous ceramic tray to form a substrate thin layer of a first thickness.

[0018] In practice, trehalose, a non-reducing disaccharide, can form hydrogen bonds with the hydroxyl groups of biomolecules and soluble sugar nutrients in the matrix under high temperature and high pressure, replacing water molecules to maintain the spatial conformation of the molecules and avoiding the degradation and inactivation of nutrients caused by high temperature. Monosodium glutamate can strengthen the molecular protective membrane structure of trehalose through intermolecular forces. The two work together to reduce the loss of small molecule carbon sources, amino acids and other nutrients generated by pre-degradation in the matrix during moist heat sterilization. By laying the fermentation matrix in a thin layer of a specific thickness on a porous ceramic tray, high temperature steam can quickly and evenly penetrate the matrix system during sterilization, ensuring the thoroughness of sterilization, while avoiding the charring and destruction of nutrients caused by local overheating inside the matrix.

[0019] Furthermore, in the above technical solution, the moist heat sterilization process in step S4 adopts a staged variable temperature and pressure increase process. The staged variable temperature and pressure increase process includes a first gradient heating stage, a second gradient heat preservation stage and a third gradient depressurization stage in sequence. The heating rate of the first gradient heating stage is the first heating rate, and the depressurization rate of the third gradient depressurization stage is set to the first depressurization rate.

[0020] In practice, gradient heating avoids the Maillard reaction that causes browning and inactivation of soluble sugars and amino acids in the substrate due to instantaneous high temperatures. It also ensures a uniform temperature rise inside and outside the substrate system, avoiding the "false pressure" phenomenon where the steam pressure meets the standard but the internal temperature of the substrate is insufficient, thus preventing incomplete sterilization. The gradient heat preservation stage can maintain the set sterilization temperature and pressure for a sufficient time to thoroughly kill miscellaneous bacteria, lactic acid bacteria cells, and spores in the system, ensuring the sterility of the substrate. Gradual depressurization avoids the violent boiling and structural collapse of the water inside the substrate particles caused by instantaneous depressurization, maintaining the loose and porous structure of the substrate. This provides sufficient space and oxygen channels for the subsequent growth and spread of oyster mushroom mycelium, while further reducing the heat loss of nutrients.

[0021] Furthermore, in the above technical solution, in step S5, the oyster mushroom spawn is a composite spawn containing oyster mushroom liquid spawn and a solid carrier. The solid carrier is wheat bran particles with magnetic nanoparticles loaded on the surface. The oyster mushroom liquid spawn is loaded into the internal pores of the solid carrier through a vacuum impregnation device. Before inoculation, the solid carrier loaded with the oyster mushroom liquid spawn is placed above the magnetic field generator and distributed inside the sterilization matrix.

[0022] In practice, wheat bran particles have a rich porous structure and natural carbon and nitrogen nutrients, which can provide initial nutrition and colonization sites for the germination of oyster mushroom spawn. Vacuum impregnation allows the liquid spawn of oyster mushroom to fully penetrate into the internal pores of the wheat bran particles under negative pressure, achieving efficient and stable spawn loading. The magnetic nanoparticles on the surface of the wheat bran particles can generate directional magnetic force under the magnetic field of the magnetic field generator, thereby driving the spawn-loaded wheat bran particles to achieve three-dimensional directional distribution inside the sterilization substrate, completing the multi-point colonization of oyster mushroom spawn inside the substrate. This avoids the problem of mycelium only spreading from the surface to the inside and the long growth cycle caused by traditional surface inoculation, greatly shortening the time for mycelium to fill the substrate and improving the uniformity of mycelial growth.

[0023] Furthermore, in the above technical solution, the cultivation environment in step S5 is equipped with an alternating light-emitting diode (LED) light source matrix and a temperature regulation module. In the initial stage of the cultivation process, the LED light source matrix is ​​in a closed state and the temperature regulation module maintains a first cultivation temperature. In the middle and late stages of the growth process, the LED light source matrix is ​​turned on and emits a light beam of a first wavelength, while the temperature regulation module switches to a second cultivation temperature.

[0024] In practice, the initial stage of oyster mushroom mycelial growth is the spore germination and primary mycelial colonization stage. A dark environment and a suitable first culture temperature can avoid the inhibitory effect of light on spore germination, ensuring rapid mycelial colonization and the formation of a healthy primary mycelial network. The middle and late growth stages are the rapid mycelial proliferation and vigorous secondary metabolism stages. A light beam of a specific wavelength can activate the mycelial proliferation and metabolic pathways by regulating the photoreceptor proteins in the oyster mushroom mycelium, and enhance the secretion activity of extracellular enzymes such as cellulase and laccase in the mycelium. Combined with a suitable second culture temperature, the metabolic activity of the mycelium can be maximized, promoting the rapid accumulation of mycelial biomass and achieving dense encapsulation and complete coverage of the substrate particles by the mycelium.

[0025] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This application utilizes a composite enzyme system composed of amylase and cellulase for liquid enzymatic hydrolysis of highland barley pulp, combined with solid-state fermentation using lactic acid bacteria, thus breaking down the internal physical barriers of highland barley. Amylase and cellulase directly act on the highly crystalline starch crystal structure and dense fiber network, degrading it into small-molecule soluble sugars; the lactic acid bacteria consume free phenols and phytic acid, among other anti-nutritional factors, during fermentation. This treatment method pre-degrades the substrate before inoculating *Pleurotus ostreatus* mycelium, allowing the mycelium to directly contact and absorb free carbon sources after inoculation, shortening mycelial germination time, increasing the average daily growth rate, reducing residual sugar content in the substrate, and increasing the final mycelial dry weight biomass.

[0026] 2. This application utilizes low-temperature airflow pulverization combined with freeze-thaw phase change to generate microscopic cracks, increasing the specific surface area of ​​barley particles and the enzyme contact area; by introducing an ultrasonic field and controlling the pH and temperature of liquid enzymatic hydrolysis in a calcium-ion-containing buffer system, the catalytic efficiency of the composite enzyme system is enhanced; a composite membrane material fermentation container combined with micro-oxygen mixed gas filling provides a stable micro-oxygen solid-state fermentation environment for lactic acid bacteria; before moist heat sterilization, trehalose and monosodium glutamate are added to protect the dry powder, and a staged temperature-pressure-release process is used to reduce the damage of high temperature to pre-degraded nutrients in the substrate; a wheat bran carrier loaded with magnetic nanoparticles is used in combination with vacuum impregnation and magnetic field distribution to achieve three-dimensional directional colonization of oyster mushroom liquid spawn inside the sterilization substrate; combined with the alternating switching of the light-emitting diode light source matrix and temperature regulation module, the metabolic activity of mycelium in the middle and late growth stages is ensured, so that the proportion of mycelium densely encapsulating the substrate particles reaches the level of complete coverage. Detailed Implementation

[0027] The following detailed description, in conjunction with embodiments, comparative examples, and effect test data, further illustrates the specific implementation of the present invention. Those skilled in the art should understand that the following embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the present invention.

[0028] Unless otherwise specified, all experimental materials used in the following embodiments and comparative examples of this invention are commercially available biochemical reagents in the art; unless otherwise specified, all experimental equipment used are conventional fermentation engineering and biological culture equipment in the art; all strains used are publicly available standard strains: Lactobacillus plantarum strain CICC20265 preserved by the China Industrial Microbial Culture Collection Center, and Pleurotus ostreatus strain CICC50013; the thermostable α-amylase used is derived from Aspergillus niger with an enzyme activity of 20,000 U / g; the endoglucanase is derived from Trichoderma reesei with an enzyme activity of 10,000 U / g.

[0029] Example 1: This example provides a method for propagating oyster mushroom mycelium using highland barley as a substrate. The specific steps are as follows: Barley Pellet Preparation: The barley grains are pulverized using a low-temperature airflow pulverizer, with the pulverizing chamber temperature controlled at ≤10℃. After pulverization, the barley pellets are passed through a standard inspection sieve to collect those with a particle size distribution of 40-80 mesh (180μm-380μm). The resulting barley pellets are then placed in a -20℃ freezing environment and left to stand for 12 hours to maintain the internal moisture of the pellets in a solid ice crystal state. After removal, the pellets are directly fed into the next process.

[0030] Liquid enzymatic hydrolysis: Sterile water was added to the frozen barley granules at a material-to-liquid ratio of 1:1.5 (m / m), and the mixture was stirred evenly to prepare barley slurry. Thermosensitive α-amylase and endoglucanase were mixed at a mass ratio of 1:2 to form a complex enzyme system. The complex enzyme system was dissolved in a pH 5.5 acetate-sodium acetate buffer solution containing 0.1 mmol / L calcium chloride to prepare an enzyme solution with a total enzyme concentration of 50 mg / mL. The enzyme solution was added to the barley slurry at a volume ratio of 1:10 (mL:g), and the mixture was stirred evenly. An ultrasonic field was introduced, and the ultrasonic frequency was controlled at 28 kHz and the power at 300 W. The temperature of the enzymatic hydrolysis system was maintained at 60℃. The pH value of the system was stabilized between 5.0 and 6.0 by adding dilute hydrochloric acid or sodium hydroxide solution. The enzymatic hydrolysis was carried out at a constant temperature for 2 hours to obtain the enzymatic substrate.

[0031] Solid-state fermentation: After activation of *Lactobacillus plantarum* by MRS slant culture and liquid seed culture, a viable cell concentration of 1×10⁻⁶ was obtained. 9 CFU / mL seed culture was prepared; the seed culture was evenly applied to the surface and interior of the enzymatically hydrolyzed substrate using a spray device, with an inoculation amount of 5% (mL / g) of the substrate mass; the inoculated substrate was then transferred into a composite membrane fermentation vessel composed of a polyethylene layer and a polyamide layer, with a polytetrafluoroethylene microporous gas-regulating membrane sealed on the outside of the composite membrane; during solid-state fermentation, the fermentation temperature was controlled at 30℃, and a micro-oxygen mixed gas was introduced into the fermentation vessel for 10 minutes every 8 hours through the gas-regulating membrane. The micro-oxygen mixed gas was composed of nitrogen and oxygen mixed at a volume ratio of 98:2. The fermentation was carried out at a constant temperature for 24 hours to obtain the fermentation substrate.

[0032] Moist heat sterilization: A mixed protective dry powder consisting of trehalose and monosodium glutamate in a mass ratio of 3:1 was added to the fermentation substrate. The amount of the mixed protective dry powder added was 0.5% (m / m) of the mass of the fermentation substrate. After stirring evenly, the fermentation substrate was spread on a porous ceramic tray to form a substrate layer with a thickness of 2 cm. Moist heat sterilization was carried out using a staged temperature and pressure increase process: In the first gradient temperature increase stage, the temperature was increased from room temperature to 121°C at a rate of 2°C / min, corresponding to a pressure increase to 0.1 MPa; In the second gradient heat preservation stage, the temperature was maintained at 121°C and 0.1 MPa for 20 min; In the third gradient pressure relief stage, the system pressure was reduced from 0.1 MPa to atmospheric pressure at a pressure relief rate of 0.01 MPa / min. After cooling, the sterilized substrate was obtained.

[0033] Inoculation and Cultivation: Preparation of Pleurotus ostreatus composite spawn: 40-mesh wheat bran particles were loaded with 5% Fe3O4 magnetic nanoparticles as a solid carrier. Pleurotus ostreatus liquid spawn (cultured on PD medium, mycelial ball concentration 1×10^6 cells / mL) was mixed with the solid carrier and immersed in a -0.08MPa vacuum environment for 30 min to ensure the liquid spawn was fully loaded into the internal pores of the solid carrier, thus obtaining the composite spawn. The composite spawn was inoculated at 10% (m / m) of the sterilized substrate mass. During inoculation, the solid carrier loaded with the Pleurotus ostreatus liquid spawn was placed in a 0.2T magnetic field. The inoculation is evenly distributed inside the sterilized substrate above the container. After inoculation, the substrate is transferred to the culture environment for cultivation: In the initial stage of cultivation (0-5 days after inoculation), the LED light source matrix is ​​turned off, and the temperature regulation module maintains the cultivation temperature at 25℃ and the relative humidity at 65%. In the middle and late stages of cultivation and growth (6 days after inoculation until the mycelium fully fills the substrate), the LED light source matrix is ​​turned on to emit red light with a wavelength of 660nm. The photoperiod is 12h light / 12h dark. At the same time, the temperature regulation module is switched to 22℃ and the relative humidity is maintained at 65%. Cultivation continues until the mycelium completely fills the substrate, and oyster mushroom mycelium is obtained.

[0034] Example 2: The only difference between this example and Example 1 is that in step 2, the mass ratio of the thermostable α-amylase to the endoglucanase in the complex enzyme system is 1:1. All other conditions and operating steps are completely consistent with Example 1.

[0035] Example 3: The only difference between this example and Example 1 is that in step 2, the mass ratio of the thermostable α-amylase to the endoglucanase in the complex enzyme system is 2:1. All other conditions and operating steps are completely consistent with Example 1.

[0036] Example 4: The only difference between this example and Example 1 is that in step 3, the viable bacterial concentration of Lactobacillus plantarum seed liquid is 5×10^8 CFU / mL, and the other conditions and operation steps are completely consistent with Example 1.

[0037] Example 5: The only difference between this example and Example 1 is that in step 3, the volume ratio of nitrogen to oxygen in the micro-oxygen mixture is 95:5. All other conditions and operating steps are completely consistent with Example 1.

[0038] Example 6: The only difference between this example and Example 1 is that in step 4, the amount of mixed protective dry powder added is 0.3% (m / m) of the fermentation substrate mass. All other conditions and operating steps are completely consistent with Example 1.

[0039] Example 7: The only difference between this example and Example 1 is that in step 4, the amount of mixed protective dry powder added is 0.8% (m / m) of the fermentation substrate mass. All other conditions and operating steps are completely consistent with Example 1.

[0040] Example 8: The only difference between this example and Example 1 is that in step 5, the wavelength of the light beam emitted by the LED light source matrix is ​​450nm. All other conditions and operating steps are completely the same as in Example 1.

[0041] Example 9: The only difference between this example and Example 1 is that in step 1, the particle size distribution of the collected barley grains is 20 mesh to 40 mesh (380 μm to 830 μm). All other conditions and operating steps are completely consistent with Example 1.

[0042] Example 10: The formulation of this example is completely the same as that of Example 1. The only difference is that in step 2, the system temperature of the liquid enzymatic hydrolysis process is maintained at 55°C. All other conditions and operation steps are completely the same as those of Example 1.

[0043] Example 11: The formula of this example is completely the same as that of Example 1. The only difference is that in step 4, the heating rate of the first gradient heating stage of moist heat sterilization is 3℃ / min. All other conditions and operation steps are completely the same as those of Example 1.

[0044] Example 12: The formula of this example is completely the same as that of Example 1. The only difference is that in step 5, the culture temperature in the initial stage of culture is maintained at 24°C. All other conditions and operation steps are completely the same as those in Example 1.

[0045] Comparative Example 1: The only difference between this comparative example and Example 1 is that in step 2, the complex enzyme system composed of amylase and cellulase is not added. All other conditions and operating steps are completely consistent with Example 1.

[0046] Comparative Example 2: This comparative example uses the existing conventional technology described in the background art. The specific steps are as follows: the barley grain is crushed using conventional mechanical crushing equipment and passed through a 40-mesh standard sieve. Sterile water is added to the crushed barley powder to adjust the moisture content to 60%. The mixture is then placed in a glass culture bottle and sterilized by high-pressure steam at 121°C and 0.1 MPa for 30 minutes. After cooling, solid oyster mushroom spawn is inoculated on the substrate surface. The inoculation amount is the same as in Example 1. The mixture is then placed in a constant temperature and humidity culture room at 25°C and 65% relative humidity and cultured until the mycelium fully colonizes the substrate. The remaining culture environment parameters are the same as in Example 1.

[0047] Comparative Example 3: The only difference between this comparative example and Example 1 is that in step 2, the pH value of the system in the liquid enzymatic hydrolysis process is controlled between 3.0 and 4.0 by acid-base adjustment solution. All other conditions and operating steps are completely consistent with Example 1.

[0048] Comparative Example 4: The only difference between this comparative example and Example 1 is that the solid-state fermentation treatment in step 3 is omitted, and the enzymatic hydrolysate obtained in step 2 is directly fed into step 4 for wet heat sterilization. All other conditions and operating steps are completely consistent with Example 1.

[0049] Test method: To assess the technical effects claimed in this invention, the following test indicators were set. All groups used the exact same detection method and parallel experimental setup, with three parallel samples in each group. The results were averaged: Mycelial germination time: After inoculation, mycelial germination time was recorded by microscopic observation every 2 hours at more than 50% of the inoculation sites. Daily average growth rate of mycelium: The cross-crossing method was used to measure the diameter of mycelial colonies daily and calculate the daily average radial growth rate during the culture period. Final residual sugar content in the substrate: After the culture was completed, the content of remaining reducing sugar in the substrate was determined by the 3,5-dinitrosalicylic acid method (DNS method), and the residual sugar content was calculated. Mycelial dry weight biomass: After the culture is completed, the mycelium is separated from the substrate, dried at 65℃ to constant weight, and the mycelial dry weight corresponding to 100g of dry substrate is weighed. Hyphae-to-substrate particle encapsulation rate: After the culture was completed, 100 substrate particles were randomly selected and observed under a stereomicroscope. The percentage of particles completely and densely encapsulated by hyphae was counted.

[0050] Test results: Table 1 Performance test results of each embodiment and comparative example ; Results analysis: The core technical solution of this invention has achieved unexpected and significant technical effects. Example 1, as the preferred embodiment, shows that its mycelial germination time is only 18 hours, a 75% reduction compared to Comparative Example 2; the average daily growth rate of mycelium reaches 4.2 mm / d, a 425% increase compared to Comparative Example 2; the final residual sugar rate of the substrate is as low as 1.2%, an 88.2% reduction compared to Comparative Example 2; the mycelial dry weight biomass reaches 12.8 g / 100g dry substrate, a 412% increase compared to Comparative Example 2; and the mycelium's encapsulation rate of substrate particles reaches 100%. These results directly demonstrate that this invention, through its core solution of liquid enzymatic hydrolysis combined with solid-state fermentation of lactic acid bacteria, completely breaks down the physical barrier formed by the dense fiber network and highly crystalline starch in the barley substrate, while simultaneously eliminating the chemical inhibition of anti-nutritional factors. This fundamentally solves the core defects of existing technologies, such as delayed mycelial germination, stagnant feeding, and hindered biomass accumulation, achieving significant technical progress.

[0051] The technical solution of this invention has broad applicability and robustness. Examples 2-9, based on the core solution, made gradient adjustments to key parameters such as the compound enzyme ratio, strain concentration, fermentation gas ratio, protective agent dosage, light source wavelength, and barley particle size. Although the indicators of all groups were slightly lower than those of the optimal Example 1, they were still significantly better than all comparative examples, achieving a reduction of more than 50% in mycelial germination time and an increase of more than 300% in growth rate. This demonstrates that the parameter range defined by this invention has good applicability and can be adapted to different production scenarios and raw material conditions. Examples 10-12, with the formula completely unchanged, only made reasonable adjustments to process parameters such as enzymatic hydrolysis temperature, sterilization heating rate, and culture temperature. All indicators were close to those of Example 1, maintaining excellent levels. This demonstrates that the preparation method of this invention has good universality and process tolerance, and can stably achieve the expected technical effects within the reasonable parameter fluctuation range of industrial production.

[0052] The necessity and synergistic effect of the various technical features of this invention were verified in reverse. Comparative Example 1 omitted the core technical feature of the complex enzyme system. The physical barriers of starch and fiber in barley were not effectively degraded, leading to a significant extension of mycelial germination time and a sharp deterioration in growth rate, biomass, and other indicators. This proves that pre-degradation by the complex enzyme system is a fundamental and necessary condition for achieving the technical effects of this invention. Comparative Example 3 deviated the enzymatic hydrolysis pH value from the range defined in this invention, causing the complex enzyme system to lose its optimal catalytic environment and catalytic activity, resulting in a significant decrease in enzymatic hydrolysis effect. The technical effect was similar to Comparative Example 1, proving that the enzymatic hydrolysis process parameters defined in this invention are key to ensuring the pre-degradation effect. Comparative Example 4 omitted the lactic acid bacteria solid-state fermentation step. Although the enzymatic hydrolysis step was retained and the residual sugar rate of the substrate was low, anti-nutritional factors such as phytic acid and free phenols were not removed, and mycelial growth was still significantly inhibited. All indicators were significantly worse than in Example 1, proving that lactic acid bacteria solid-state fermentation is an indispensable technical link in this invention, forming a synergistic effect with the enzymatic hydrolysis step to jointly achieve the optimal mycelial growth effect.

[0053] The preferred technical features of this invention further enhance the technical effect. Example 1, through preferred technical means such as low-temperature airflow pulverization combined with freezing phase change pore creation, ultrasonic field-enhanced enzymatic hydrolysis, micro-aerobic environment-controlled solid-state fermentation, protective agent combined with gradient sterilization to reduce nutrient loss, magnetic carrier for three-dimensional directional inoculation, and photothermal synergistic regulation of mycelial growth, achieves optimal levels of various indicators, proving that the preferred scheme of this invention can further amplify the core technical effect and provide the optimal technical path for industrial production.

[0054] In summary, this invention, through the core technical solution of pre-degradation by a compound enzyme system combined with solid-state fermentation of lactic acid bacteria, along with various optimized process steps, effectively solves the core defects in the prior art, achieves significant technical effects that cannot be expected by those skilled in the art, and possesses outstanding substantive features and significant progress.

Claims

1. A method for propagating oyster mushroom mycelium using barley as a substrate, characterized in that, Includes the following steps: S1. Crush and sieve the barley to collect the barley grains; S2. Sterile water is added to the barley granules to prepare barley slurry. A complex enzyme system consisting of amylase and cellulase is added to the barley slurry for liquid enzymatic hydrolysis to obtain the enzymatic hydrolysis substrate. S3. Inoculate the enzymatic hydrolysis substrate with lactic acid bacteria and carry out solid-state fermentation to obtain a fermentation substrate; S4. Perform wet heat sterilization on the fermentation substrate to obtain a sterilized substrate; S5. Inoculate the sterilized substrate with oyster mushroom spawn and place it in a culture environment for cultivation to obtain oyster mushroom mycelium.

2. The method according to claim 1, characterized in that, In step S1, the barley is pulverized using a low-temperature airflow pulverizer. The particle size distribution range of the barley particles collected after sieving is limited to a first value to a second value. Before entering step S2, the barley particles are placed in a freezing environment to keep the internal moisture of the barley particles in a solid ice crystal state. The solid ice crystals undergo a phase change and generate micro-cracks during the subsequent thawing process.

3. The method according to claim 1, characterized in that, In step S2, the complex enzyme system is composed of a thermostable α-amylase derived from Aspergillus niger and an endoglucanase derived from Trichoderma reesei, mixed in a first mass ratio. Before being added to the barley pulp, the complex enzyme system is dissolved in an acetate-sodium acetate buffer solution containing calcium ions to form an enzyme solution. The added volume of the enzyme solution is in a first proportional relationship with the mass of the barley pulp.

4. The method according to claim 3, characterized in that, In step S2, an ultrasonic field is introduced into the barley pulp containing the complex enzyme system. The frequency of the ultrasonic field is set to a first frequency value, and the power is set to a first power value. The temperature of the liquid enzymatic hydrolysis process is maintained at a first temperature value, and the pH value of the system in the liquid enzymatic hydrolysis process is controlled between a first pH value and a second pH value by adding an acid-base adjusting solution.

5. The method according to claim 1, characterized in that, In step S3, the lactic acid strain is Lactobacillus plantarum. Before being inoculated with the enzymatic hydrolysis substrate, Lactobacillus plantarum is sequentially activated by slant culture and expanded by liquid seed culture to obtain seed liquid. The viable concentration of Lactobacillus plantarum in the seed liquid reaches a first concentration value. The seed liquid is uniformly applied to the surface and interior of the enzymatic hydrolysis substrate using a spraying device.

6. The method according to claim 5, characterized in that, In step S3, the fermentation container that receives the enzymatic hydrolysis substrate is made of a composite membrane material consisting of a first polymer layer and a second polymer layer. The outer side of the composite membrane material is encapsulated with a gas conditioning membrane. During the solid-state fermentation process, a micro-oxygen mixed gas is periodically introduced into the fermentation container through the gas conditioning membrane. The micro-oxygen mixed gas is composed of nitrogen and oxygen mixed in a first volume ratio.

7. The method according to claim 1, characterized in that, Before performing moist heat sterilization in step S4, a mixed protective dry powder containing trehalose and monosodium glutamate is added to the fermentation substrate. The mass ratio of the mixed protective dry powder to the fermentation substrate is a first ratio. After adding the mixed protective dry powder, the fermentation substrate is laid on a porous ceramic tray to form a substrate thin layer of a first thickness.

8. The method according to claim 7, characterized in that, The moist heat sterilization process in step S4 adopts a staged variable temperature and pressure increase process, which includes a first gradient heating stage, a second gradient heat preservation stage and a third gradient depressurization stage in sequence. The heating rate of the first gradient heating stage is the first heating rate, and the depressurization rate of the third gradient depressurization stage is set to the first depressurization rate.

9. The method according to claim 1, characterized in that, In step S5, the oyster mushroom spawn is a composite spawn containing oyster mushroom liquid spawn and a solid carrier. The solid carrier is wheat bran particles with magnetic nanoparticles loaded on the surface. The oyster mushroom liquid spawn is loaded into the internal pores of the solid carrier through a vacuum impregnation device. Before inoculation, the solid carrier loaded with the oyster mushroom liquid spawn is placed above the magnetic field generator and distributed inside the sterilization matrix.

10. The method according to claim 9, characterized in that, The cultivation environment in step S5 is equipped with an alternating LED light source matrix and a temperature regulation module. In the initial stage of the cultivation process, the LED light source matrix is ​​in the off state and the temperature regulation module maintains the first cultivation temperature. During the later stages of growth in the cultivation process, the light-emitting diode light source matrix is ​​turned on and emits a light beam of the first wavelength, while the temperature regulation module switches to the second cultivation temperature.