A spore activation conditioner and its use in the germination of spores of fermenting fungi

Abstract

The present application provides a spore activation conditioner and its application in spore germination of fermented fungi. The present application strengthens marine and terrestrial plant-based ingredients from specific culture medium inoculated with Mucor strains, promotes the reproductive performance of spores. Through the implementation of the present application, the spore germination rate of the slant test tube seed inoculation is improved, the mycelium growth period is shortened, and the number of spores of the production strain is proliferated, which is beneficial to large-scale production of food fermentation and stable product quality.

Description

Technical Field

[0001] This invention provides a method for inducing the germination of Mucor spores and promoting high spore production by mycelium in fermentation media using active components from marine and terrestrial plants. Background Technology

[0002] my country has a long history of traditional fermented foods. The types and amounts of microorganisms used in fermentation significantly influence the flavor and nutritional value of these foods. Molds and yeasts are the most common fungi, and fermented bean curd made from Mucor is one of the earliest foods in my country to utilize microorganisms as a fermentation medium. Mucor is a filamentous fungus with a hair-like appearance, lacking rhizoids and stolons. Its hyphae are septate, and sporangiophores arise directly from the mycelium. Reproduction primarily occurs through the direct germination of ascospores. After germination, the hyphae grow and form branched structures, followed by the growth of exophytic hyphae, which eventually form spores again. Therefore, spore germination is crucial. In fact, the fermentation industry often faces low and unstable spore germination rates, making it difficult to obtain stable and high-density inoculants, which to some extent restricts large-scale production applications.

[0003] Application No. 202110393088.8 discloses a method for promoting the germination of arbuscular mycorrhizal fungi spores and mycelial growth. The method involves screening arbuscular mycorrhizal fungi spores by weighing sand containing the fungi and adding it to sterile water, stirring, collecting the material on the sieve surface through a sieve, placing it in a centrifuge tube, and centrifuging. Sucrose solution is added to the precipitate, and after complete resuscitation, it is centrifuged again. The supernatant is then passed through a sieve, and the spores on the sieve surface are transferred to a sterile beaker. For sterilization of the arbuscular mycorrhizal fungi spores, the spores collected in the sterile beaker from step one are poured into a sterile self-made sterilization device, rinsed several times with sterile water, then sterilized by adding sterilization solution, tightening the centrifuge tube cap, shaking, and rinsing with sterile water. The spores are then soaked in an antibiotic solution of streptomycin and gentamicin, rinsed several times with sterile water, and then the spores are selected, inoculated, and cultured. A certain concentration of selenium (supplying in the form of Na2SeO3) is used to promote spore germination and mycelial growth. Application No. 201880074912.X discloses a method for producing dormant fungal structures or organs with improved germination rates. The method includes subjecting the dormant structures or organs to a process comprising heat treatment at 37°C to 65°C followed by a cooling period at 0°C to 36°C. The dormant fungal structures or organs are developing or mature spores. The dormant fungal structures or organs, having undergone the process comprising heat treatment at 37°C to 65°C followed by a cooling period at 0°C to 36°C, exhibit improved germination rates and / or germination efficiency compared to dormant fungal structures or organs not treated in step b). Application No. 201810801893.8 discloses a method for promoting rapid sporulation of Aspergillus flavus. A graphene-doped solid culture medium is used as the culture medium to induce rapid sporulation of Aspergillus flavus mycelium. The advantage of this invention is that it shortens the experimental cycle for sporulation of Aspergillus flavus mycelium, rapidly inducing a large amount of sporulation within 48–72 hours. This quick acquisition of a large number of spores and induction of sporulation in Aspergillus flavus provides researchers with ample experimental materials.

[0004] Based on the above analysis, the problems and shortcomings of the existing technology are as follows:

[0005] (1) Food safety is a prerequisite for fermented foods, and all additives used in the fermentation process must comply with food safety regulations.

[0006] (2) Poor quality of strain seeds will result in poor fermentation. In addition, in actual production, the low spore germination rate is often compensated by increasing the inoculation amount, which will increase production costs.

[0007] (3) Spore germination is affected by a variety of factors, but there is no better way to improve its stability. Summary of the Invention

[0008] This invention provides a method for inducing the germination of *Mucor* spores and promoting high spore production by mycelium in fermentation media using active components from marine and terrestrial plants. Barley (*Hordeum vulgare* L.) is an annual herbaceous plant belonging to the Poaceae family and is one of my country's important cereal crops. Barley leaves (BL), as a plant with both medicinal and edible uses, possess various physiological functions such as lowering blood sugar and lipids, antioxidation, and antitumor effects. Polysaccharides are its main active ingredient and are non-cytotoxic. Marine plants such as seaweed (*Sargassum*) are rich in algal polysaccharides, mannitol, highly unsaturated fatty acids, various natural plant growth regulators such as gibberellins, cytokinins, auxins, abscisic acid, and betaine, among other physiologically active substances. Traditional spore induction methods often involve adding inorganic salt-based culture media, but these media have limited effects on spore germination, mycelial growth, and high-density new spore formation, and some even exhibit cytotoxicity.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] In a first aspect, the present invention provides a spore-activating conditioner, which is prepared according to the following method:

[0011] (1) The activated seed culture of Acinetobacter jumbo is inoculated into the seaweed lysate for fermentation culture until the viscosity of the solution decreases to 25% to 35% of the initial value to obtain seaweed fermentation broth; the seaweed lysate includes seaweed fragments and the following components in mass fractions: 0.25 to 0.45% (preferably 0.035%) (NH4)2SO4, 0.035 to 0.065% (preferably 0.05%) K2HPO4·3H2O, 0.015 to 0.035% (preferably 0.025%) MgSO4, and water is the solvent; wherein, the particle size of the seaweed fragments is less than 1 mm, and the solid content of the seaweed lysate is 12.0 to 15.0% (preferably 13.5%) (w / w); in this invention, "solids" refers to the substance after filtering and draining the fragments, and the solid content refers to the percentage of the mass of the substance after filtering and draining the fragments in the lysate.

[0012] (2) Barley seedlings are soaked in an aqueous solution of an alkaline substance with a mass fraction of 0.10-0.20% (preferably 0.15%), then steamed to kill the green, water is added, and the seedlings are ground and crushed to a particle size of less than 1 mm to obtain a barley seedling crushed liquid with a solid content of 3.5-6.5% (preferably 5%); the alkaline substance contained in the aqueous solution of the alkaline substance is one or a mixture of two or more of sodium carbonate, sodium bicarbonate, calcium oxide, and sodium hydroxide;

[0013] (3) The seaweed fermentation liquid described in step (1) is mixed with the wheat seedling crushing liquid described in step (2) to obtain a mixture. Cellulase and β-glucanase are added, and the mixture is reacted at 50-60°C for 10-14 hours (preferably at 55°C for 12 hours). The resulting bacteria-enzyme coupled reaction solution is sterilized by enzyme inactivation (in one embodiment of the present invention, it is treated at 90°C for 5 minutes, followed by high-temperature sterilization) to obtain the spore activating conditioner. The volume ratio of the seaweed fermentation liquid to the wheat seedling crushing liquid is 1:0.5-2. The amount of cellulase added is 35-65 U / g (preferably 50 U / g) of the dry matter in the mixture, based on its enzyme activity. The amount of β-glucanase added is 5-15 U / g (preferably 10 U / g) of the dry matter in the mixture, based on its enzyme activity. The method for determining the dry matter in the mixture described in this invention is a conventional method in the art. A certain amount of the mixture is dried at 105°C for 2.5 hours and then weighed to obtain the mass of the dry matter, which is used to calculate the mass of the dry matter in the mixture.

[0014] Furthermore, the Acinetobacter juni in the activated seed culture of Acinetobacter juni mentioned in step (1) is Acinetobacter juni. X8 (deposited at China Center for Type Culture Collection, accession number CCTCC No: M209110).

[0015] Furthermore, the activated seed culture of Acinetobacter juni described in step (1) is prepared as follows: Acinetobacter juni is inoculated into seed culture medium and cultured in a constant temperature shaker at 150 r / min and 25℃ for 72 h. During this period, it is transferred to the seed culture medium every 24 h at a volume of 8% to obtain the activated seed culture of Acinetobacter juni. The three transfers are for the purpose of continuous activation of the strain and improving its activity.

[0016] Furthermore, the seed culture medium consists of the following components at final concentrations: sodium alginate 5 g / L; (NH4)2SO4 5 g / L; NaCl 20 g / L; K2HPO4·3H2O 2 g / L; MgSO4 1.0 g / L, with water as the solvent and pH 7.5.

[0017] Preferably, the activated seed culture of Acinetobacter jumbo in step (1) is inoculated into the seaweed disruption solution at a volume inoculation rate of 5%.

[0018] In an embodiment of the present invention, the seaweed fragments mentioned in step (1) are kelp (Laminaria japonica) or red algae (Gracilaria lemaneiformis), preferably red algae (Gracilaria lemaneiformis).

[0019] To achieve a seaweed solids content of 12.0–15.0% (preferably 13.5%), this invention employs the method of rehydrating dried seaweed and then crushing it. Washing and other steps to remove sand and gravel before crushing are conventional procedures known to those skilled in the art. Specifically, the seaweed lysate in step (1) is prepared by the following method: after washing the dried seaweed, it is rehydrated to a mass of 3.5 to 4 times the original mass of the dried seaweed, then water is added, and the seaweed is ground to a particle size of less than 1 mm. (NH4)2SO4, K2HPO4·3H2O and MgSO4 are added, and water is added until the solid content is 12.0 to 15.0% (preferably 13.5%). The seaweed is then ultrasonically dispersed to obtain the seaweed lysate. In the seaweed lysate, the final concentration of (NH4)2SO4 is 0.25 to 0.45% (preferably 0.035%), the final concentration of K2HPO4·3H2O is 0.035 to 0.065% (preferably 0.05%), and the final concentration of MgSO4 is 0.015 to 0.035% (preferably 0.025%). In one embodiment of the present invention, the ultrasonic parameters are: time 15s, ultrasonic frequency 40kHz, and power 23w / L. In one embodiment of the present invention, the sterilization is performed at 121℃ for 25min.

[0020] Furthermore, the soaking time of the alkaline substance in the aqueous solution in step (2) is 5 minutes.

[0021] Furthermore, the steam temperature for blanching in step (2) is 130-145°C, and the time is 20 seconds.

[0022] Furthermore, the high-temperature sterilization in step (3) is performed at a temperature of 121°C for 25 minutes.

[0023] The wheat seedling crushing liquid and seaweed fermentation liquid prepared above are mixed in a ratio of 1-2:2-1 and used immediately. If not used within 4 hours, they can be stored for 1-2 days after pasteurization (treatment at 90℃ for 5 minutes).

[0024] Secondly, the present invention provides the application of the above-mentioned spore activating conditioner in the spore generation of fermenting fungi, especially in the spore germination and spore proliferation.

[0025] In one embodiment of the present invention, the fungus is *Mucor* (preferably *Actinomucor elegans*). It should be noted that the fungus of the present invention is not limited to *Mucor*.

[0026] Furthermore, the application is as follows: the spore activating conditioner is mixed with the Mucor slant culture at a mass ratio of 1 to 3:1 (2:1 in one embodiment of the present invention), and activated at 30°C for 60 min to obtain activated spore solution; the activated spore solution is inoculated into wheat bran solid culture medium at a mass inoculation amount of 9% to 15% (preferably 15%), and incubated at 30°C for 48 to 64 h to obtain induced spores.

[0027] Furthermore, the wheat bran solid culture medium is prepared by mixing wheat bran and water in a mass ratio of 1:1.25 and then sterilizing at high temperature.

[0028] Furthermore, before the incubation process, a spore activating conditioner at a mass inoculum of 2-6% is added to the wheat bran solid culture medium. This can slightly further enhance the spore proliferation rate.

[0029] Compared with existing technologies, the beneficial effects of this invention are mainly reflected in the following aspects: This invention strengthens marine and terrestrial plant-based ingredients in a specific culture medium inoculated with Mucor strains, promoting spore reproduction. Through the implementation of this invention, the spore germination rate of slant tube seed inoculation is improved, the mycelial growth period is shortened, and the number of spores of the production strain is increased, which is beneficial for large-scale food fermentation production and stable product quality. Attached Figure Description

[0030] Figure 1 This is a technical roadmap of the present invention. Detailed Implementation

[0031] In this invention, "solids" refers to the substance remaining after filtering and draining the crushed material. The solids content refers to the percentage of the mass of the filtered and drained material in the crushing liquid. Before adding water to adjust the solids content, a sample can be taken to test the solids concentration before adding water, and the mass of solids in the overall sample can be calculated to determine the required water volume. The method for determining the dry matter in the mixture described in this invention is a conventional method in the art. A certain amount of the mixture is dried at 105°C for 2.5 hours and then weighed to obtain the mass of dry matter, which is used to calculate the mass of dry matter in the mixture. Before adding the enzyme solution, a sample can be taken to test the concentration of dry matter, and the mass of solids in the overall mixture can be calculated to determine the required amount of enzyme.

[0032] Example 1:

[0033] Dried kelp (Laminaria japonica, containing 16.6% alginate, 15.1% mannitol, and 8.0% crude fiber, etc.) with a moisture content of 18.5% (w / w) was soaked in water for 12 hours, increasing its weight to 3.5-4 times. After washing to remove mud and impurities, the kelp was drained and ground in a colloid mill with twice its weight of water until the particle size was below 1 mm. Then, (NH₄)₂SO₄, 0.05% K₂HPO₄·3H₂O, and 0.025% MgSO₄ were added to a final concentration, and water was added to adjust the kelp solids content to 13.5% (w / w). The mixture was then ultrasonically treated for 15 seconds (ultrasonic frequency 40 kHz, 23 w / L) and sterilized at 121°C for 25 minutes. It was then set aside for later use.

[0034] Barley seedlings (Hordeum vulgare L., dry basis containing 20.9% crude fiber, 0.5% total flavonoids, 0.3% chlorophyll, 12.0% protein, etc., with a moisture content of about 85%), 15cm long, were soaked in 0.15% Na2CO3 solution for 5 minutes, then blanched with superheated steam at 135℃ for 20 seconds, ground with water until the particle size was less than 1mm, the solid content of the barley seedlings was about 5%, and then pasteurized (85℃, 5 minutes).

[0035] Acinetobacter juni. X8 (China Center for Type Culture Collection, accession number CCTCC No: M 209110) was inoculated into seed culture medium. The seed culture medium consisted of 1000 mL distilled water with 5 g sodium alginate, 5 g (NH4)2SO4, 20 g NaCl, 2 g K2HPO4·3H2O, and 1.0 g MgSO4, adjusted to pH 7.5, and sterilized at 121℃ for 20 min. Under aseptic conditions, the culture was incubated in a shaker at 150 rpm and 25℃. Subculture was performed every 24 h, with an inoculum size of 8% each time, for a total of three subcultures. The total incubation period was 72 h to obtain activated seed culture.

[0036] The Acinetobacter juncea solution was inoculated into the sterilized kelp material at a concentration of 5% (v / v) and fermented at a constant temperature of 32°C and 180 rpm for 64 h in a shaker. Through the action of alginate lyase secreted by Acinetobacter juncea (the alginate lyase concentration in the solution at the end of fermentation was 2.0 U / mL), the viscosity of the seaweed fermentation broth decreased to 30% of its initial value.

[0037] The wheatgrass treatment solution and the above-mentioned seaweed fermentation broth were mixed at a ratio of 1:2 (v / v). Cellulase (20000 U / g, DSM (Jiangsu) Biotechnology Co., Ltd.) and β-glucanase (50000 U / g, Zhengzhou Tianshun Food Additives Co., Ltd.) were added at amounts of 50 U / g of dry matter and 10 U / g of dry matter, respectively. The mixture was reacted at 55°C for 12 hours. At this point, the solution contained 8.7 mg / mL oligosaccharides, 1.4 mg / mL β-glucan, and 7.2 mg / mL reducing sugars.

[0038] The above bacterial-enzyme coupled reaction solution was heated to 90℃ and kept at that temperature for 5 minutes to inactivate the enzyme and sterilize it. After cooling, it was bottled and sterilized at 121℃ for 25 minutes to obtain the spore activation conditioner.

[0039] In a sterilized 250mL Erlenmeyer flask containing glass beads, under aseptic conditions, a spore activating conditioner at a mass ratio of 2:1 was mixed with Mucor slant culture. After the mold spores were activated at 30℃ for 60 minutes, the spore germination rate increased by 17%.

[0040] In a solid medium of wheat bran (Triticum sativum) that had been sterilized at 121℃ for 25 min (wheat bran:water = 1:1.25), 15% of the culture medium mass of activated spores from a germination treatment was inoculated. Simultaneously, 6% of the wheat bran solid medium mass of spore activating conditioner was added, and the mixture was shaken to distribute evenly. After solid-state incubation at 30℃ for 56 h, a *Mucor* spawn for fermentation production was obtained. Compared with the control (without spore activating conditioner treatment), the *Mucor* spawn showed robust mycelium and spore proliferation of 27%.

[0041] Example 2:

[0042] Seaweed and wheatgrass were treated, and Acinetobacter jumbo was inoculated for fermentation, as in Example 1. Through the action of alginate lyase secreted by Acinetobacter jumbo (the alginate lyase concentration in the solution at the end of fermentation was 1.5 U / mL), the viscosity of the seaweed fermentation broth decreased to 35% of its initial value.

[0043] The wheat seedling treatment solution and the above-mentioned seaweed fermentation broth were mixed at a 1:1 ratio. Cellulase and β-glucanase were added at amounts of 50 U / g of liquid per dry matter and 10 U / g of liquid per dry matter, respectively. The mixture was reacted at 55℃ for 12 hours. At this point, the solution contained 5.6 mg / mL oligosaccharides, 0.96 mg / mL β-glucan, and 7.4 mg / mL reducing sugars.

[0044] The above-mentioned bacterial-enzyme coupling reaction solution was heated to 90°C and kept at that temperature for 5 minutes to inactivate the enzyme and sterilize it. After cooling, it was bottled and sterilized at high temperature to obtain the spore activation conditioner.

[0045] In a sterilized 250mL Erlenmeyer flask containing glass beads, under aseptic conditions, a spore activating conditioner at a mass ratio of 2:1 was mixed with Mucor slant culture. After conditioning and activation at 30℃ for 60 minutes, the spore germination rate increased by 12%.

[0046] In a sterilized wheat bran solid medium (wheat bran:water = 1:1.25, sterilized at 121℃ for 25 min), germination-treated spore liquid was inoculated at 9% of the medium mass. Simultaneously, 4% spore activating conditioner was added, and the mixture was shaken to distribute evenly. The mixture was then incubated at 30℃ for 64 h to obtain *Mucor* spawn for fermentation production. Compared to the control (without spore activating conditioner treatment), the *Mucor* spawn culture showed robust mycelia and a spore proliferation rate of 16%.

[0047] Example 3:

[0048] Seaweed and wheatgrass were treated, and Acinetobacter jumbo was inoculated for fermentation, as in Example 1. Through the action of alginate lyase secreted by Acinetobacter jumbo (the alginate lyase concentration in the solution at the end of fermentation was 1.7 U / mL), the viscosity of the seaweed fermentation broth decreased to 32% of its initial value.

[0049] The wheatgrass treatment solution and the above-mentioned seaweed fermentation broth were mixed at a ratio of 2:1. Cellulase and β-glucanase were added at amounts of 50 U / g of liquid per dry matter and 10 U / g of liquid per dry matter, respectively, and the mixture was reacted at 55℃ for 12 hours. At this point, the solution contained 3.3 mg / mL oligosaccharides, 1.3 mg / mL β-glucan, and 9.1 mg / mL reducing sugars.

[0050] The above-mentioned bacterial-enzyme coupling reaction solution was heated to 90°C and kept at that temperature for 5 minutes to inactivate the enzyme and sterilize it. After cooling, it was bottled and sterilized at high temperature to obtain the spore activation conditioner.

[0051] In a sterilized 250mL Erlenmeyer flask containing glass beads, under aseptic conditions, a spore activating conditioner at a mass ratio of 2:1 was mixed with Mucor slant culture. After conditioning and activation at 30℃ for 60 minutes, the spore germination rate increased by 18%.

[0052] In a sterilized wheat bran solid medium (wheat bran:water = 1:1.25, sterilized at 121℃ for 25 min), germination-treated activated spore solution was inoculated at 9% of the medium mass. Simultaneously, 6% spore activating conditioner was added, and the mixture was shaken to distribute evenly. The mixture was then incubated at 30℃ for 48 h to obtain *Mucor* spawn for fermentation production. Compared to the control (without spore activating conditioner treatment), the *Mucor* spawn culture showed robust mycelia and a 21% spore proliferation.

[0053] Example 4:

[0054] The procedures for treating seaweed and wheat seedlings, inoculating and fermenting Acinetobacter jumbo, performing bacterial-enzyme coupling reaction, preparing spore activating conditioner, and inoculating and germinating Mucor slant seeds are the same as in Example 1.

[0055] In a sterilized wheat bran solid medium (wheat bran:water = 1:1.25, sterilized at 121℃ for 25 min), germination-treated activated spore liquid was inoculated at a rate of 15% of the medium mass. At the same time, 2% spore activation conditioner was added, and the mixture was shaken to distribute it evenly. The mixture was then incubated at 30℃ for 50 h to obtain the Rhizopus inoculum for fermentation production.

[0056] (3) The mycelium after the above treatment was robust, and the spores of Mucor multiplied by 7% compared with the control sample (treated without spore activating conditioner).

[0057] Example 5:

[0058] Specific plan:

[0059] Using the red algae Gracilaria lemaneiformis as raw material (dry material contains about 60% polysaccharides and crude fiber, 18.9% protein), the material was soaked in water for 10-12 hours, increasing its weight by 3.5-4 times. After washing to remove sand and impurities, the material was ground with water in a colloid mill until the particle size was less than 1 mm. Then, 0.035% (NH4)2SO4, 0.05% K2HPO4·3H2O, and 0.025% MgSO4 were added to the final concentration, and water was added to adjust the seaweed solids content to 13.5±0.5% (w / w). The material was then ultrasonically treated for 15 seconds (ultrasonic frequency 40 kHz, 23 w / L) and sterilized.

[0060] The treatment of wheat seedlings and the inoculation and culture of Acinetobacter jung were the same as in Example 1.

[0061] The viscosity of the seaweed fermentation broth decreased to 31% of its initial value due to the action of alginate lyase secreted by Acinetobacter juncea (the alginate lyase concentration in the solution at the end of fermentation was 1.9 U / mL).

[0062] (3) Mix the wheatgrass treatment solution with the above-mentioned red algae Gracilaria fermentation broth at a 1:1 ratio, add cellulase and β-glucanase at amounts of 50 U / g of liquid per dry matter and 10 U / g of liquid per dry matter, respectively, and react at 55℃ for 12 h. At this time, the solution contains 7.8 mg / mL oligosaccharides, 1.1 mg / mL β-glucan, and 7.5 mg / mL reducing sugar.

[0063] (4) The above bacterial-enzyme coupling reaction solution is heated to 90°C and kept warm for 5 minutes to inactivate the enzyme and sterilize. After cooling, it is bottled and sterilized at high temperature to obtain the spore activation conditioner.

[0064] (5) Spore germination: In a sterilized 250mL Erlenmeyer flask containing glass beads, under aseptic conditions, a spore activating conditioner with a mass ratio of 2:1 was mixed with Mucor slant culture. After conditioning and activation at 30℃ for 60min, the spore germination rate increased by 14%.

[0065] In a sterilized wheat bran solid medium (wheat bran:water = 1:1.25, sterilized at 121℃ for 25 min), germination-treated activated spore solution was inoculated at 15% of the medium mass. Simultaneously, 4% spore activating conditioner was added, and the mixture was shaken to distribute evenly. The mixture was then incubated at 30℃ for 64 h to obtain *Mucor* spawn for fermentation production. Compared to the control (without spore activating conditioner), the *Mucor* spawn culture showed robust mycelia and spore proliferation of 18%.

Claims

1. A spore-activating conditioner, characterized in that... The spore-activating conditioner was prepared according to the following method: (1) The activated seed culture of Acinetobacter juncea is inoculated into the seaweed lysate for fermentation culture until the viscosity of the solution decreases to 25% to 35% of the initial value to obtain seaweed fermentation broth; the seaweed lysate includes seaweed fragments and the following components in mass fractions: 0.25 to 0.45% (NH4)2SO4, 0.035 to 0.065% K2HPO4·3H2O, 0.015 to 0.035% MgSO4, and water is the solvent; wherein the particle size of the seaweed fragments is less than 1 mm, and the solid content of the seaweed lysate is 12.0 to 15.0% (w / w); (2) After soaking in an aqueous solution of an alkaline substance with a mass fraction of 0.10-0.20%, barley seedlings are steam-killed, water is added, and they are ground and crushed to a particle size of less than 1 mm to obtain a barley seedling crushed liquid with a solid content of 3.5-6.5%; the alkaline substance contained in the aqueous solution of the alkaline substance is one or a mixture of two or more of sodium carbonate, sodium bicarbonate, calcium oxide, and sodium hydroxide; (3) Mix the seaweed fermentation liquid from step (1) with the wheat seedling crushing liquid from step (2) to obtain a mixture. Add cellulase and β-glucanase and react at 50-60℃ for 10-14 hours. Sterilize the resulting bacteria-enzyme coupling reaction solution to obtain the spore activating conditioner. The volume ratio of the seaweed fermentation liquid to the wheat seedling crushing liquid is 1:0.5-2. The amount of cellulase added is 35-65 U / g of the dry matter in the mixture, based on its enzyme activity. The amount of β-glucanase added is 5-15 U / g of the dry matter in the mixture, based on its enzyme activity.

2. The spore-activating conditioner as described in claim 1, characterized in that: The Acinetobacter juni in the activated seed culture of Acinetobacter juni mentioned in step (1) is Acinetobacter juni. X8.

3. The spore-activating conditioner as described in claim 1, characterized in that: The activated seed culture of Acinetobacter juni. in step (1) is prepared as follows: Acinetobacter juni. is inoculated into seed culture medium and cultured in a constant temperature shaker at 150 r / min and 25℃ for 72 h. During this period, it is transferred to seed culture medium at a volume of 8% every 24 h to obtain the activated seed culture of Acinetobacter juni. The seed culture medium consists of the following components at the following final concentrations: sodium alginate 5 g / L; (NH4)2SO4 5 g / L; NaCl 20 g / L; K2HPO4·3H2O 2 g / L; MgSO4 1.0 g / L, with water as the solvent and pH 7.

5.

4. The spore-activating conditioner as described in claim 1, characterized in that: The seaweed fragments mentioned in step (1) are kelp (Laminaria japonica) or red algae Gracilaria lemaneiformis.

5. The spore-activating conditioner as described in claim 1, characterized in that: The seaweed lysate in step (1) is prepared by the following method: dried seaweed is washed and rehydrated to a mass of 3.5 to 4 times the original mass of dried seaweed. After removal, water is added, and the seaweed is ground to a particle size of less than 1 mm. (NH4)2SO4, K2HPO4·3H2O and MgSO4 are added, and water is added until the solid content is 12.0 to 15.0%. The seaweed is then ultrasonically dispersed to obtain the seaweed lysate. In the seaweed lysate, the final concentration of (NH4)2SO4 is 0.25 to 0.45%, the final concentration of K2HPO4·3H2O is 0.035 to 0.065%, and the final concentration of MgSO4 is 0.015 to 0.035%.

6. The spore-activating conditioner as described in claim 1, characterized in that: The soaking time of the alkaline substance in the aqueous solution in step (2) is 5 minutes; The steam temperature for blanching in step (2) is 130-145℃ and the time is 20s.

7. The use of the spore activating conditioner as described in any one of claims 1-6 in the spore induction of fermenting fungi.

8. The application as described in claim 7, characterized in that: The fungus is Mucor.

9. The application as described in claim 7, characterized in that... The application is as follows: the spore activating conditioner is mixed with the Mucor slant culture at a mass ratio of 1 to 3:1, and activated at 30°C for 60 min to obtain activated spore solution; the activated spore solution is inoculated into wheat bran solid culture medium at a mass inoculation amount of 9% to 15%, and incubated at 30°C for 48 to 64 h to obtain induced spores.

10. The application as described in claim 9, characterized in that: Before the incubation, a spore activation conditioner with an inoculation amount of 2-6% is added to the wheat bran solid culture medium.

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