Efficient trichoderma solid-state fermentation method

Abstract

The invention belongs to the technical field of microbial fermentation, and particularly relates to an efficient trichoderma solid-state fermentation method. The method comprises the following main fermentation steps: mixing and crushing a solid fermentation culture medium, water and an inorganic salt solution, loading the obtained fermentation culture medium into a tray, controlling the thickness of the culture medium to be 1-3cm, and sterilizing and cooling in a mold starter machine; vacuumizing, starting a fan, and inoculating a liquid strain; performing fermentation culture, introducing sterile air, and spraying mist for humidification; performing drying; and after the fermentation is finished, separating to obtain the trichoderma spore powder. According to the method, the temperature, humidity and ventilation quantity in the trichoderma fermentation process are controlled by using the koji machine equipment, and the whole fermentation process is performed in the equipment, so that the trichoderma fermentation infectious microbe rate is greatly reduced; meanwhile, the inoculation mode is improved, solid inoculation is improved into liquid inoculation, the initial germination rate of trichoderma spores is increased, and the fermentation efficiency of trichoderma is remarkably improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial fermentation, and particularly relates to an efficient solid-state fermentation method for Trichoderma. Background Art

[0002] Trichoderma has important economic significance and is widely applied in various aspects of agriculture, such as preventing plant diseases, promoting plant growth, bioremediating soil and environment, and being used in biological organic fertilizers. In recent years, the research and application of Trichoderma have developed rapidly. Combining research methods such as genetics, molecular biology, biochemistry, and ecology, Trichoderma and its metabolites will surely have a broader application prospect in promoting plant growth, increasing the absorption and utilization rate of nutrients, improving the yield of agricultural crops, and enhancing the defense against diseases. Especially in the context of advocating green agriculture, the large-scale popularization and application of Trichoderma have become an indispensable part of constructing a modern and efficient biological pest control system.

[0003] At present, most Trichoderma uses solid fermentation technology, and the inoculation method mostly uses solid seeds for inoculation. Patent CN109337829A discloses a solid fermentation method for Trichoderma harzianum, a solid fermentation product, a Trichoderma harzianum agent, and its preparation method and application. This solid fermentation method uses a solid fermentation tank for fermentation, and by limiting the process parameters of humidity, turning, and ventilation during the solid fermentation process, it promotes the reproduction of Trichoderma harzianum, reduces the contamination of miscellaneous bacteria, and increases the spore quantity of Trichoderma harzianum. Patent CN 112457997 A discloses a method for solid fermentation of Trichoderma, which includes the following steps: activation of Trichoderma species, preparation of seed fermentation broth, and solid fermentation culture of a porous model. This invention produces Trichoderma spores by solid fermentation of a porous model, uses a honeycomb briquette mold to make the solid matrix into a porous shape, increases the contact area between the unit volume of the solid matrix and air, improves the fermentation conditions, and increases the sporulation amount of solid fermentation of Trichoderma. However, solid seeds have the disadvantages of long preparation time and easy contamination of miscellaneous bacteria during the inoculation process, and the fermentation spore yield and activity in many literatures or patents still need to be improved.

[0004] Therefore, it is of great significance to develop a method that is simple and easy to operate and can significantly increase the yield of Trichoderma fermentation spore powder. Summary of the Invention

[0005] The fermentation method of the present invention is different from the common shallow tray solid fermentation process of Trichoderma. By using a koji-making machine to control the temperature, humidity, and ventilation volume during the Trichoderma fermentation process, the entire fermentation process is carried out inside the equipment, greatly reducing the miscellaneous bacteria rate of Trichoderma fermentation; at the same time, the inoculation method is improved and the components of the fermentation medium are optimized. The solid inoculation is improved to liquid inoculation, increasing the germination rate of Trichoderma spores at the initial stage and significantly improving the fermentation efficiency of Trichoderma.

[0006] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] The present invention provides an efficient solid-state fermentation method for Trichoderma, comprising the following steps:

[0008] Mix the solid fermentation medium with water and an inorganic salt solution, pulverize, load the obtained fermentation medium into a tray, control the thickness of the medium to be 1-3 cm, sterilize in a koji machine, and cool; evacuate, turn on the blower, and inoculate with liquid strain; perform fermentation culture, introduce sterile air, and humidify by spraying; dry; after the fermentation is completed, separate to obtain Trichoderma spore powder.

[0009] In some embodiments, the components of the inorganic salt solution, calculated by mass 100%, are: sucrose 2.0-5.0%, magnesium sulfate 2.5-4.0%, dipotassium hydrogen phosphate 1.5-2.5%, ammonium sulfate 1.0-2.0%, and the rest is water.

[0010] In some embodiments, the water content of the fermentation medium is 55-65 wt%.

[0011] In some embodiments, the liquid medium used for culturing the liquid strain has the following composition: based on 1 L of water, it contains 0.5-1.5 g of glucose, 4.0-6.0 g of corn flour, 0.3-0.5 g of potassium dihydrogen phosphate, 0.1-0.15 g of sodium nitrate, 0.07-0.1 g of ammonium sulfate, and 0.06-0.08 g of sodium chloride.

[0012] In some embodiments, the culturing method of the liquid strain is as follows:

[0013] Inoculate the activated Trichoderma strain into a sterile liquid medium, and perform shaking culture at 25-30 °C and 100-200 rpm for 60-72 h to obtain a liquid strain;

[0014] The inoculation amount of the liquid strain is 30-60 wt% of the fermentation medium.

[0015] In some embodiments, the temperature conditions required for the fermentation culture are controlled as follows: within 0-48 h, the culture temperature is 28-30 °C; within 48-72 h, the culture temperature is 25-30 °C; within 72-168 h, the culture temperature is 25-28 °C.

[0016] In some embodiments, the wind speed conditions required for the fermentation culture are controlled as follows: within 0-24 h, the blower frequency is 0 Hz; within 24-72 h, the blower frequency is 30-40 Hz; within 72-120 h, the blower frequency is 40-45 Hz; within 120-168 h, the blower frequency is 25-35 Hz.

[0017] In some embodiments, the spray conditions required for the fermentation culture are controlled as follows: within 0-12 hours, the spray-on time is 0 minutes; within 12-24 hours, the spray-on time is 4-6 minutes, and the spray-off time is 2-4 minutes; within 24-72 hours, the spray-on time is 7-9 minutes, and the spray-off time is 1-3 minutes; within 72-168 hours, the spray-on time is 6-8 minutes, and the spray-off time is 2-4 minutes, and the spray-on and spray-off operations are performed alternately.

[0018] In some embodiments, the spore powder is separated using a sieve with a mesh size of 100-200 meshes.

[0019] In some embodiments, the solid fermentation medium comprises the following components by weight:

[0020] 70-90 parts of bran, 10-30 parts of sawdust, 0.25-1.5 parts of functionalized silicon dioxide, 0.005-0.018 parts of growth regulator;

[0021] The preparation steps of the functionalized silica are:

[0022] (1) mixing ethyl orthosilicate with water and an organic solvent, adding a catalyst, stirring and hydrolyzing at 40-60° C. for 2-4 hours to form a silica sol;

[0023] (2) adding a solution of polyethyleneimine dropwise to the silica sol obtained in step (1), stirring and reacting at 20-50° C. to obtain a modified silica sol;

[0024] (3) Dissolving the calcium source and the phosphorus source in deionized water to prepare a mixed solution, adding the mixed solution dropwise to the modified silica sol of step (2), adjusting the pH value to 9-11, stirring the reaction at 80-100° C. for 12-24 hours, centrifuging the product, washing, and drying to obtain functionalized silica.

[0025] In some embodiments, the molar ratio of silicon, calcium, and phosphorus in the silicon source, calcium source, and phosphorus source is 1:(0.5-1:1):(0.3-0.6).

[0026] In some embodiments, the mass ratio of tetraethyl orthosilicate to polyethyleneimine is (15-20):1.

[0027] In some embodiments, the growth regulator includes, by weight, 0.001-0.003 parts of indoleacetic acid, 0.001-0.004 parts of 6-benzylaminopurine, 0.001-0.003 parts of gibberellic acid, 0.001-0.003 parts of salicylic acid, and 0.001-0.005 parts of putrescine.

[0028] In some embodiments, the components of the solid fermentation medium further include Bacillus licheniformis.

[0029] In some embodiments, the dosage of Bacillus licheniformis is 1×10 6-7 CFU / g of Trichoderma solid fermentation medium.

[0030] Preferably, the activity of Bacillus licheniformis is 20-100 billion / g.

[0031] The Trichoderma solid fermentation scheme disclosed in the present invention is optimized and designed in multiple dimensions. By adopting shallow pan fermentation, the heat transfer efficiency of fermentation is improved, and the fermentation cycle is shortened to 7 days, showing significant advantages in terms of fermentation efficiency, spore yield, and product quality, which are specifically reflected in the following aspects: 1. Scientific ratio of the nutrient system to enhance the metabolic activity of the bacteria. The liquid strain medium uses a composite carbon source of corn flour + glucose, combined with a phosphate buffer system, effectively promoting the rapid proliferation of hyphae and shortening the seed liquid culture time to 60-72h; the solid fermentation medium is innovatively added with functionalized silica, and its porous structure improves the matrix porosity and oxygen transfer coefficient. At the same time, the functionalized silica realizes the slow release of calcium and phosphorus through polyethyleneimine modification, extending the nutrient supply cycle. 2. Dynamic regulation of environmental parameters to accurately adapt to the growth stage. The temperature gradient control (28-25°C) matches the physiological characteristics of the bacteria. The high temperature in the first 48h promotes the expansion of hyphae, and the subsequent cooling induces spore differentiation, increasing the spore production; while the variable frequency wind speed system realizes the control of the CO2 concentration gradient. Strong ventilation in the middle stage of fermentation makes the CO2 concentration relatively low, improving the spore formation rate, which is improved compared with the traditional fixed wind speed scheme; the spray system uses intermittent humidification to maintain the stability of the matrix water content and avoid the impact of water activity fluctuations on metabolism. 3. Innovative application of functional materials. To improve the fermentation effect of Trichoderma, the present invention synthesizes a kind of functionalized silica. The nanostructured silica is prepared by the sol-gel method and then loaded with calcium and phosphorus sources, continuously providing calcium and phosphorus elements in the later stage of fermentation to improve the spore maturity; and the surface of the silica is functionalized by the amino group of polyethyleneimine and the functional groups related to calcium and phosphorus. The surface functional groups can chemically react with the harmful substance molecules generated during the fermentation process of Trichoderma, form chemical bonds, and firmly adsorb the harmful substances on the silica surface, reducing the harm of harmful substances to the strain. 4. Adding growth regulators to the solid fermentation medium significantly improves the fermentation efficiency and quality of Trichoderma. 5. There is a synergistic effect between Bacillus licheniformis and Trichoderma. Bacillus licheniformis can improve the fermentation microenvironment, inhibit the growth of harmful microorganisms, create favorable conditions for the growth of Trichoderma, and promote the secretion of metabolites of Trichoderma, etc.

[0032] Compared with the prior art, the beneficial effects of the present invention are:

[0033] 1. The fermentation method of the present invention controls the temperature, humidity, and ventilation volume during the fermentation process of Trichoderma. The entire fermentation process is carried out inside the equipment, greatly reducing the contamination rate of Trichoderma fermentation. At the same time, the inoculation method is improved, from solid inoculation to liquid inoculation, which improves the germination rate of Trichoderma spores in the initial stage and significantly improves the fermentation efficiency of Trichoderma.

[0034] 2. The functionalized silica synthesized by the present invention realizes the surface functionalization of silica through a series of chemical reactions. It can provide calcium, phosphorus nutrition and silicon elements for the growth of Trichoderma, improve the air permeability of the culture medium, provide a stable growth environment for Trichoderma, and promote the growth, sporulation and other physiological processes of Trichoderma. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 It is a picture of the fermentation site for Example 1;

[0036] Figure 2 It is a test report of the fermentation product for Example 1;

[0037] Figure 3 It is a picture of the fermentation site for Example 2;

[0038] Figure 4 It is a test report of the fermentation product for Example 2;

[0039] Figure 5 It is a picture of the fermentation site for Example 3;

[0040] Figure 6 It is a test report of the fermentation product for Example 3;

[0041] Figure 7 It is a picture of the fermentation site for Example 4;

[0042] Figure 8 It is a test report of the fermentation product for Example 4;

[0043] Figure 9 It is a picture of the fermentation site for Example 5;

[0044] Figure 10 It is a test report of the fermentation product for Example 5;

[0045] Figure 11 It is a picture of the fermentation site for Example 6;

[0046] Figure 12 It is a test report of the fermentation product for Example 6;

[0047] Figure 13 It is a picture of the fermentation site in the fermentation comparative experiment. DETAILED DESCRIPTION OF THE INVENTION

[0048] The various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation on the present invention, but rather as a more detailed description of certain aspects, features, and implementation schemes of the present invention. It should be understood that the terms described in the present invention are only for describing specific embodiments and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0049] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. Without departing from the scope or spirit of the present invention, various improvements and variations can be made to the specific embodiments of the present invention description, which are obvious to those skilled in the art. Other embodiments obtained from the description of the present invention are obvious to those skilled in the art. The description and examples of the present application are only exemplary.

[0050] The Trichoderma used in the present invention, LTR-2, is Trichoderma harzianum Rifai, which is derived from the Institute of Ecology, Shandong Academy of Sciences, and its preservation number in the China General Microbiological Culture Collection Center is CGMCC No. 1498; the number average molecular weight of the polyethyleneimine used is 10,000; the activity of the Bacillus licheniformis used is 100 billion / g, purchased from Xi'an Xianze Biotechnology Co., Ltd.; the diameter of the silica used is 800 nm. The wheat bran and wood chips used are both crushed to 20 mesh.

[0051] Preparation Example 1

[0052] The preparation steps of the functionalized silica are as follows:

[0053] (1) Add 10 mL of tetraethyl orthosilicate and 10 mL of deionized water to 30 mL of anhydrous ethanol in sequence, stir evenly at 200 rpm, and dropwise add 0.5 mL of 25 wt% ammonia water. After the addition is complete, continue to stir and hydrolyze at 40 °C for 4 h to form a silica sol;

[0054] (2) Dropwise add 10 mL of an aqueous solution containing 0.5 g of polyethyleneimine to the silica sol prepared in step (1), and stir and react at 20 °C and 200 rpm for 4 h to obtain a modified silica sol;

[0055] (3) Dissolve 0.0225 mol of calcium nitrate and 0.0135 mol of diammonium hydrogen phosphate in 20 mL of deionized water to prepare a mixed solution. Dropwise add this mixed solution into the modified silica sol obtained in step (2), and adjust the pH value to 9 with 1 mol / L sodium hydroxide solution. After stirring and reacting at 80 °C for 24 h, centrifuge the product, wash it 3 times with deionized water, and dry it to constant weight to obtain functionalized silica.

[0056] Preparation Example 2

[0057] The preparation steps of the functionalized silica are as follows:

[0058] (1) Add 10 mL of tetraethyl orthosilicate and 10 mL of deionized water to 30 mL of absolute ethanol in sequence, stir evenly at 200 rpm, and dropwise add 0.5 mL of 25 wt% ammonia water. After the addition is complete, continue to stir and hydrolyze at 60 °C for 2 h to form a silica sol.

[0059] (2) Dropwise add 10 mL of an aqueous solution containing 0.6 g of polyethyleneimine to the silica sol obtained in step (1), and stir and react at 40 °C and 200 rpm for 2 h to obtain a modified silica sol.

[0060] (3) Dissolve 0.5 mol of calcium nitrate and 0.027 mol of diammonium hydrogen phosphate in 40 mL of deionized water to prepare a mixed solution. Dropwise add this mixed solution into the modified silica sol obtained in step (2), and adjust the pH value to 11 with 1 mol / L sodium hydroxide solution. After stirring and reacting at 100 °C for 12 h, centrifuge the product, wash it 3 times with deionized water, and dry it to constant weight to obtain functionalized silica.

[0061] Preparation Example 3

[0062] The preparation steps of the functionalized silica are as follows:

[0063] (1) Add 10 mL of tetraethyl orthosilicate and 10 mL of deionized water to 30 mL of absolute ethanol in sequence, stir evenly at 200 rpm, and dropwise add 0.5 mL of 25 wt% ammonia water. After the addition is complete, continue to stir and hydrolyze at 50 °C for 3 h to form a silica sol.

[0064] (2) Dropwise add 10 mL of an aqueous solution containing 0.55 g of polyethyleneimine to the silica sol obtained in step (1), and stir and react at 30 °C and 200 rpm for 3 h to obtain a modified silica sol.

[0065] (3) Dissolve 0.036 mol of calcium nitrate and 0.02 mol of diammonium hydrogen phosphate in 30 mL of deionized water to prepare a mixed solution. Dropwise add this mixed solution into the modified silica sol in step (2), adjust the pH value to 10 with 1 mol / L sodium hydroxide solution, stir and react at 90 °C for 18 h, then centrifuge the product, wash it 3 times with deionized water, and dry it to constant weight to obtain functionalized silica.

[0066] Preparation Example 4

[0067] The preparation method of the solid fermentation medium includes the following preparation steps:

[0068] According to the mass parts, pour 70 parts of wheat bran and 10 parts of wood chips into a container, stir at 100 rpm for 30 min; then add 0.25 part of functionalized silica thereto, and continue to stir for 15 min; then add 0.005 part of growth regulator, stir for 20 min, and sterilize to obtain the Trichoderma solid fermentation medium.

[0069] The functionalized silica used is obtained from Preparation Example 1; the growth regulator used includes 0.001 part of indoleacetic acid, 0.001 part of 6-benzylaminopurine, 0.001 part of gibberellic acid, 0.001 part of salicylic acid, and 0.001 part of putrescine, and their contents in the Trichoderma solid fermentation medium are 0.001 part, 0.001 part, 0.001 part, 0.001 part, and 0.001 part respectively.

[0070] Preparation Example 5

[0071] The preparation method of the solid fermentation medium includes the following preparation steps:

[0072] According to the mass parts, pour 90 parts of wheat bran and 30 parts of wood chips into a container, stir at 200 rpm for 15 min; then add 1.5 parts of functionalized silica thereto, and continue to stir for 10 min; then add 0.015 part of growth regulator, stir for 15 min, and sterilize to obtain the Trichoderma solid fermentation medium.

[0073] The functionalized silica used is obtained from Preparation Example 2; the growth regulator used includes 0.003 part of indoleacetic acid, 0.004 part of 6-benzylaminopurine, 0.003 part of gibberellic acid, 0.003 part of salicylic acid, and 0.005 part of putrescine.

[0074] Preparation Example 6

[0075] The preparation method of the solid fermentation medium includes the following preparation steps:

[0076] By mass fraction, 80 parts of wheat bran and 20 parts of wood chips are poured into a container and stirred at 150 rpm for 25 min; then 1 part of functionalized silica is added thereto and stirring is continued for 10 min; then 0.015 part of a growth regulator is added and stirring is carried out for 20 min, followed by sterilization to obtain a solid fermentation medium for Trichoderma.

[0077] The functionalized silica used is obtained from Preparation Example 3; the growth regulator used includes 0.002 part of indoleacetic acid, 0.002 part of 6-benzylaminopurine, 0.002 part of gibberellic acid, 0.002 part of salicylic acid, and 0.003 part of putrescine.

[0078] Preparation Example 7

[0079] A method for preparing a solid fermentation medium, the specific implementation manner is substantially the same as that of Preparation Example 6, except that Bacillus licheniformis is further added after sterilization, and the dosage thereof is 1×10 7 CFU / g of the solid fermentation medium for Trichoderma.

[0080] Preparation Example 8

[0081] A method for preparing a solid fermentation medium, the specific implementation manner is substantially the same as that of Preparation Example 6, except that functionalized silica is not added.

[0082] Preparation Example 9

[0083] A method for preparing a solid fermentation medium, the specific implementation manner is substantially the same as that of Preparation Example 6, except that the growth regulator is not added.

[0084] Example 1

[0085] An efficient solid-state fermentation method for Trichoderma, comprising the following steps:

[0086] In a mixer, 200 kg of the solid fermentation medium is mixed and pulverized with 220 kg of water and 20 L of an inorganic salt solution, the obtained fermentation medium is loaded into a tray, the thickness of the medium is controlled to be 1 cm, sterilization is carried out in a koji-making machine and then cooling; vacuum is pumped, the blower is turned on, 40 L of liquid strain is inoculated, and the inoculation time is 2.5 h; fermentation culture is carried out in the koji-making machine, sterile air is introduced, and humidification is carried out by spraying; drying; after the fermentation is completed, separation is carried out with a 120-mesh sieve to obtain Trichoderma spore powder.

[0087] The koji-making machine includes two fermentation tanks, No. 1 and No. 2, and other fermentation parameters are substantially the same, except for the following culture and fermentation parameters:

[0088] Tank No. 1

[0089]

[0090] Tank No. 2

[0091]

[0092] The cultivation method of the liquid spawn used is as follows:

[0093] 1 cm 2 The activated Trichoderma strain is inoculated into 1 L of sterile liquid medium and cultured with shaking at 30 °C and 100 rpm for 60 h to obtain liquid spawn;

[0094] The composition of the liquid medium used for culturing the liquid spawn is: 0.5 g of glucose, 4.0 g of corn flour, 0.3 g of potassium dihydrogen phosphate, 0.1 g of sodium nitrate, 0.07 g of ammonium sulfate, 0.06 g of sodium chloride, and 1 L of water.

[0095] The components of the inorganic salt solution used, calculated as 100% by mass, are: 2.0% of sucrose, 2.5% of magnesium sulfate, 1.5% of dipotassium hydrogen phosphate, 1.0% of ammonium sulfate, and the rest is water.

[0096] The solid fermentation medium used is obtained from Preparation Example 4.

[0097] Example 2

[0098] An efficient solid-state fermentation method of Trichoderma includes the following steps:

[0099] In a mixer, 200 kg of solid fermentation medium is mixed and pulverized with 200 kg of water and 30 L of inorganic salt solution. The obtained fermentation medium is loaded into trays, and the thickness of the medium is controlled to be 3 cm. It is sterilized and cooled in a koji-making machine; evacuated, the blower is turned on, and 60 L of liquid spawn is inoculated, and the inoculation time is 3.5 h; it is fermented and cultured in the koji-making machine, sterile air is introduced, and it is spray humidified; dried; after the fermentation is completed, it is separated with a 150-mesh sieve to obtain Trichoderma spore powder.

[0100] The koji-making machine includes two fermentation tanks, No. 1 and No. 2, and other fermentation parameters are roughly the same, except for the following culture and fermentation parameters:

[0101] Tank No. 1

[0102]

[0103] Tank No. 2

[0104]

[0105]

[0106] The cultivation method of the liquid spawn used is as follows:

[0107] 1 cm 2The activated Trichoderma strain was inoculated into 1 L of sterile liquid medium and cultured with shaking at 25 °C and 200 rpm for 72 h to obtain liquid spawn;

[0108] The liquid medium used for culturing the liquid spawn had the following composition: 1.5 g of glucose, 6.0 g of corn flour, 0.5 g of potassium dihydrogen phosphate, 0.15 g of sodium nitrate, 0.1 g of ammonium sulfate, 0.08 g of sodium chloride, and 1 L of water.

[0109] The components of the inorganic salt solution, calculated as 100% by mass, were: 2.0% sucrose, 2.5% magnesium sulfate, 1.5% dipotassium hydrogen phosphate, 1.0% ammonium sulfate, and the rest was water.

[0110] The solid fermentation medium used was obtained from Preparation Example 5.

[0111] Example 3

[0112] An efficient solid-state fermentation method for Trichoderma includes the following steps:

[0113] In a mixer, 200 kg of solid fermentation medium was mixed with 200 kg of water and 25 L of inorganic salt solution, pulverized, and the obtained fermentation medium was placed in trays, with the thickness of the medium controlled at 2 cm. It was sterilized and cooled in a koji-making machine; vacuum was drawn, the blower was turned on, and 60 L of liquid spawn was inoculated, with the inoculation time being 3 h; it was fermented and cultured in the koji-making machine, sterile air was introduced, and humidification was carried out by spraying; it was dried; after the fermentation was completed, it was separated with a 120-mesh sieve to obtain Trichoderma spore powder.

[0114] The koji-making machine includes two fermentation tanks, No. 1 and No. 2. Other fermentation parameters are roughly the same, with the following differences in the culture and fermentation parameters:

[0115] Tank No. 1

[0116]

[0117]

[0118] Tank No. 2

[0119]

[0120] The culturing method of the liquid spawn used was as follows:

[0121] 1 cm 2 The activated Trichoderma strain was inoculated into 1 L of sterile liquid medium and cultured with shaking at 25 °C and 200 rpm for 72 h to obtain liquid spawn;

[0122] The liquid medium used for culturing the liquid spawn is composed of: 1 g of glucose, 5 g of corn flour, 0.4 g of potassium dihydrogen phosphate, 0.12 g of sodium nitrate, 0.8 g of ammonium sulfate, 0.7 g of sodium chloride, and 1 L of water.

[0123] The components of the inorganic salt solution used are, by mass 100%: 2.0% of sucrose, 2.5% of magnesium sulfate, 1.5% of dipotassium hydrogen phosphate, 1.0% of ammonium sulfate, and the rest is water.

[0124] The solid fermentation medium used is obtained from Preparation Example 6.

[0125] Example 4

[0126] An efficient solid-state fermentation method of Trichoderma, the specific implementation method is the same as that of Example 3, the difference is that the fermentation parameters of the koji machine culture are:

[0127] Tank No. 1

[0128]

[0129] Tank No. 2

[0130]

[0131]

[0132] The solid fermentation medium used is obtained from Preparation Example 7.

[0133] Example 5

[0134] An efficient solid-state fermentation method of Trichoderma, the specific implementation method is the same as that of Example 3, the difference is that the solid fermentation medium used is obtained from Preparation Example 8.

[0135] Example 6

[0136] An efficient solid-state fermentation method of Trichoderma, the specific implementation method is the same as that of Example 3, the difference is that the solid fermentation medium used is obtained from Preparation Example 9.

[0137] Performance Test

[0138] 1. The spore powder yields obtained in Examples 1-6 are shown in Table 1, the fermentation site diagrams and the activity detection reports are shown in the attached drawings.

[0139] Table 1

[0140]

[0141] From Table 1 and the attached Figures 1-6 It can be seen that in Examples 1-3, the fermentation media obtained from Preparation Examples 4-6 were used for the fermentation of Trichoderma spore powder, and the spore powder yields were relatively high; from Table 1 and Figures 7-12It can be seen that due to the addition of a certain amount of Bacillus licheniformis in the fermentation medium of Preparation Example 7 used in Example 4, the yield of the obtained spore powder is further improved. Compared with Example 3, due to the lack of functionalized silica / growth regulator in the fermentation media used in Examples 5 and 6 respectively, the yield of the spore powder obtained by fermentation has decreased to varying degrees.

[0142] 2. Fermentation comparison experiments

[0143] 2.1 Rotary sterilizer + koji-making machine scheme

[0144] 2.1.1 Rotary sterilizer batching: 200 kg of solid fermentation medium (Preparation Example 6) + 200 kg of water (July 6, 2024). 2.1.2 Rotary sterilizer sterilization: At 121 °C, after sterilization for 30 min, start to cool to room temperature (July 6, 2024).

[0145] 2.1.3 Rotary sterilizer inoculation: Inoculation amount of 60 kg of liquid strain (prepared according to the method described in Example 3). After inoculation, rotate and mix evenly for 1 h (July 6, 2024).

[0146] 2.1.4 Rotary sterilizer material loading: After inoculation, the rotary sterilizer is cultured for 12 h, then start to discharge and load into the koji-making machine for culture (No. 1 koji-making tank, July 7, 2024). 2.2

[0148] Mix 200 kg of solid fermentation medium (Preparation Example 6) + 200 kg of water, then start to load into the koji-making machine. After sterilization and cooling are completed, inoculate 60 kg of liquid strain (prepared according to the method described in Example 3) (No. 2 koji-making tank, July 7, 2024). 2.3

[0150] Mix 200 kg of solid fermentation medium (Preparation Example 8) + 200 kg of water, then start to load into the koji-making machine. After sterilization and cooling are completed, inoculate 60 kg of liquid strain (prepared according to the method described in Example 3) (No. 3 koji-making tank, July 7, 2024). 2.4

[0152] Mix 200 kg of solid fermentation medium (Preparation Example 9) + 200 kg of water, then start to load into the koji-making machine. After sterilization and cooling are completed, inoculate 60 kg of liquid strain (prepared according to the method described in Example 3) (No. 4 koji-making tank, July 7, 2024).

[0153] The thickness of the culture medium for the four schemes is 2 cm;

[0154] The fermentation parameters for the koji-making machine culture of the four schemes are all:

[0155]

[0156]

[0157] Other experimental operations and parameters can be referred to in Example 3.

[0158] 3 Experimental results

[0159] In the plan of the digester + koji starter machine with 2.1, a total of 30 kg of spore powder was collected, and the number of effective viable bacteria was 22 billion cfu / g; in the plan of the koji starter machine with 2.2, a total of 46 kg of spore powder was collected, and the number of effective viable bacteria was 51 billion cfu / g. It can also be seen Figure 13 the fermentation effects of the two fermenters; in the plan of the koji starter machine with 2.3, a total of 40 kg of spore powder was collected, and the number of effective viable bacteria was 45 billion cfu / g; in the plan of the koji starter machine with 2.4, a total of 42 kg of spore powder was collected, and the number of effective viable bacteria was 41 billion cfu / g.

[0160] Thus, it can be seen that the technical solution of the present invention can significantly improve the yield and activity of the spore powder in the fermentation of Trichoderma.

[0161] The above is only a preferred embodiment of the present invention, and does not impose any form of limitation on this application. Although this application is disclosed as above with a preferred embodiment, it is not intended to limit this application. Any person skilled in the relevant art can make some changes or modifications within the scope of the technical solution of this application by using the disclosed technical content, which is equivalent to equivalent implementation cases. However, as long as it does not depart from the technical solution content of the present invention, any simple modification, equivalent change and modification made to the above embodiments based on the technical essence of the present invention still fall within the scope of the technical solution.

Claims

1. An efficient solid-state fermentation method for Trichoderma, characterized in that, It includes the following steps: Mix the solid fermentation medium with water and an inorganic salt solution, crush them, place the obtained fermentation medium in a tray, control the thickness of the medium to be 1 - 3 cm, sterilize it in a koji-making machine, and cool it; evacuate, turn on the blower, and inoculate with liquid strains; conduct fermentation culture, introduce sterile air, and spray for humidification; dry; after the fermentation ends, separate to obtain Trichoderma spore powder.

2. The solid-state fermentation method of Trichoderma according to claim 1, characterized in that The components of the inorganic salt solution, calculated by mass 100%, are as follows: sucrose 2.0 - 5.0%, magnesium sulfate 2.5 - 4.0%, dipotassium hydrogen phosphate 1.5 - 2.5%, ammonium sulfate 1.0 - 2.0%, and the rest is water.

3. The solid-state fermentation method of Trichoderma according to claim 1, characterized in that, The water content of the fermentation medium is 55 - 65 wt%.

4. The solid-state fermentation method of Trichoderma according to claim 1, characterized in that The liquid medium used for the fermentation culture of the liquid strain has the following composition: based on 1 L of water, it contains 0.5 - 1.5 g of glucose, 4.0 - 6.0 g of corn flour, 0.3 - 0.5 g of potassium dihydrogen phosphate, 0.1 - 0.15 g of sodium nitrate, 0.07 - 0.1 g of ammonium sulfate, and 0.06 - 0.08 g of sodium chloride.

5. The solid-state fermentation method of Trichoderma according to claim 1, characterized in that, The culture method of the liquid strain is as follows: Inoculate the activated Trichoderma into a sterile liquid medium, and shake-culture at 25 - 30 °C and 100 - 200 rpm for 60 - 72 h to obtain the liquid strain; The inoculation amount of the liquid strain is 30 - 60 wt% of the fermentation medium.

6. The solid-state fermentation method of Trichoderma according to claim 1, characterized in that, The temperature conditions required for the fermentation culture are controlled as follows: within 0 - 48 h, the culture temperature is 28 - 30 °C; within 48 - 72 h, the culture temperature is 25 - 30 °C; within 72 - 168 h, the culture temperature is 25 - 28 °C.

7. The solid-state fermentation method of Trichoderma according to claim 1, characterized in that, The wind speed conditions required for the fermentation culture are controlled as follows: within 0 - 24 h, the blower frequency is 0 Hz; within 24 - 72 h, the blower frequency is 30 - 40 Hz; within 72 - 120 h, the blower frequency is 40 - 45 Hz; within 120 - 168 h, the blower frequency is 25 - 35 Hz.

8. The solid-state fermentation method of Trichoderma according to claim 1, characterized in that, The spray conditions required for the fermentation culture are controlled as follows: within 0 - 12 h, the spray-on time is 0 min; within 12 - 24 h, the spray-on time is 4 - 6 min, and the spray-off time is 2 - 4 min; within 24 - 72 h, the spray-on time is 7 - 9 min, and the spray-off time is 1 - 3 min; within 72 - 168 h, the spray-on time is 6 - 8 min, and the spray-off time is 2 - 4 min, and the spray-on and spray-off operations are alternated.

9. The solid-state fermentation method of Trichoderma according to claim 1, characterized in that The sieve mesh number used for separating the spore powder is 100 - 200 meshes.

10. The solid-state fermentation method of Trichoderma according to claim 1, characterized in that The solid fermentation medium, calculated by mass parts, includes the following components: 70 - 90 parts of bran, 10 - 30 parts of wood chips, 0.25 - 1.5 parts of functionalized silica, 0.005 - 0.018 parts of growth regulator; The preparation steps of the functionalized silica are as follows: (1) Mix tetraethyl orthosilicate with water and an organic solvent, add a catalyst, and stir and hydrolyze at 40 - 60 °C for 2 - 4 h to form silica sol; (2) Dropwise add a solution of polyethyleneimine to the silica sol prepared in step (1), and stir and react at 20 - 50 °C to obtain modified silica sol; (3) Dissolve the calcium source and phosphorus source in deionized water to prepare a mixed solution, and drop this mixed solution into the modified silica sol obtained in step (2). Adjust the pH value to 9 - 11, and stir and react at 80 - 100 °C for 12 - 24 h. Then, centrifuge, wash, and dry the product to obtain functionalized silica.

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