A kind of green trichoderma fermentation medium, trichoderma agent and preparation method thereof

Abstract

The present invention relates to the technical field of microbial fermentation, and in particular to a fermentation medium of green Trichoderma, a Trichoderma agent and a preparation method thereof. The present invention adopts liquid-solid combined fermentation, solid-state fermentation with millet as the main raw material, supplemented with a quick-acting carbon and nitrogen source and an inorganic salt, ferments for 48 hours, sieves green Trichoderma conidia powder, mixes the conidia powder with a wetting agent, a dispersant and a carrier, etc. to prepare green Trichoderma wettable powder, the preparation has good water solubility, can be used for spraying and drip irrigation of crops, can prevent and treat plant diseases, and has the advantages of safety and high efficiency, no drug residue, no toxic side effects, reducing the use of antibiotics, and improving crop immunity.

Description

Technical Field

[0001] The invention relates to the technical field of microbial fermentation, and in particular to a green trichoderma fermentation medium, a trichoderma agent and a preparation method thereof. Background Art

[0002] Beneficial microorganisms are also called antagonistic microorganisms, or simply biocontrol bacteria. In biological control, biocontrol bacteria reduce the number of pathogens and weaken their virulence by producing various adverse effects on pathogens. There are many types of biocontrol bacteria, and some have been made into various types of biocontrol agents for field disease control. At present, fungi, bacteria, actinomycetes, etc., as important biocontrol bacteria, have been widely used in biological control.

[0003] Trichoderma is an early discovered and most intensively studied biocontrol fungus. It is one of the most abundant biocontrol fungi in the soil. It can control a variety of pathogens that cause soil-borne diseases in the field and has extremely high biocontrol value. A large number of studies have found that Trichoderma can produce metabolites that promote the growth of certain plants, improve the nutrient utilization rate of plants, and also show significant advantages in improving plant stress resistance and improving agricultural chemical pollution. Whether it is the role of Trichoderma in promoting plant growth or the role of Trichoderma in controlling pathogens, it is safe, environmentally friendly and effective. It can solve the problem of pesticide pollution, provide pollution-free and pollution-free green food for humans, and promote ecological environmental balance and sustainable development of agriculture.

[0004] Water-saving irrigation is an irrigation measure that can effectively utilize precipitation and irrigation water. Drip irrigation is currently widely used in my country and has developed rapidly. It is suitable for vegetables, fruit trees, flowers and crops planted in ridges, and has significant economic benefits.

[0005] However, the traditional green Trichoderma solid fermentation process uses corn cobs and bran as the main fermentation raw materials, with a long fermentation cycle and a low number of effective live bacteria. In addition, the koji contains a large amount of cellulose, and the spores are not easy to separate. The separated spore powder contains a large amount of cellulose particles, which is easy to clog the drip irrigation pipe and cannot be used by spraying or drip irrigation. Therefore, it is urgent to provide a green Trichoderma agent with high live bacteria count and good suspension, which can be widely used in water-saving facilities such as sprinkler irrigation and drip irrigation. Summary of the invention

[0006] In view of this, the present invention provides a green Trichoderma agent with high viable count and good suspension and a preparation method thereof.

[0007] The present invention provides a fermentation medium for Trichoderma viride, comprising a liquid fermentation medium and a solid fermentation medium;

[0008] The liquid fermentation medium is composed of corn steep liquor, yeast powder, soybean cake powder, corn powder, glucose and sulfuric acid;

[0009] The liquid fermentation medium consists of millet, water, glucose, yeast powder, ammonium sulfate, magnesium sulfate, calcium carbonate, perilla leaf powder, raw sanguisorba officinalis and ophiopogon japonicus.

[0010] In some embodiments, the liquid culture medium consists of water and the following raw materials in percentage by weight:

[0011] Corn syrup 1.5-2.5%, yeast powder 0.5-1.0%, soybean meal powder 0.5-1.0%, corn flour 1.5-2.5%, glucose 0.5-1.5%, magnesium sulfate 0.1-0.2%;

[0012] The sum of all raw materials is 100%;

[0013] The solid culture medium is composed of the following raw materials in parts by weight:

[0014] 100 parts of millet, 40-80 parts of water, 1.0-5.0 parts of glucose, 0.5-2.0 parts of yeast powder, 0.5-1.5 parts of ammonium sulfate, 0.2-0.8 parts of magnesium sulfate, 0.5-1.0 parts of calcium carbonate, 5-10 parts of perilla leaf powder, 2-5 parts of raw sanguisorba officinalis, and 1-2 parts of ophiopogon japonicus.

[0015] In some specific embodiments, in the fermentation medium, the liquid medium and the solid medium are composed of one of the following:

[0016] (1) The liquid culture medium consists of water and the following raw materials in percentage by weight:

[0017] Corn steep liquor 1.5%, yeast powder 1.0%, soybean meal powder 1.0%, corn flour 2.5%, glucose 0.5%, magnesium sulfate 0.1%;

[0018] The sum of all raw materials is 100%;

[0019] The solid culture medium is composed of the following raw materials in parts by weight:

[0020] 100 parts of millet, 80 parts of water, 5.0 parts of glucose, 2.0 parts of yeast powder, 1.5 parts of ammonium sulfate, 0.8 parts of magnesium sulfate, 1.0 parts of calcium carbonate, 5 parts of perilla leaf powder, 2 parts of raw sanguisorba officinalis, and 1 part of ophiopogon japonicus.

[0021] (2) The liquid culture medium consists of water and the following raw materials in percentage by weight:

[0022] Corn steep liquor 1.5%, yeast powder 1.0%, soybean meal powder 1.0%, corn flour 2.5%, glucose 0.5%, magnesium sulfate 0.1%;

[0023] The sum of all raw materials is 100%;

[0024] The solid culture medium is composed of the following raw materials in parts by weight:

[0025] 100 parts of millet; 50 parts of water; 3.0 parts of glucose; 1.5 parts of yeast powder; 1.0 part of ammonium sulfate; 0.5 part of magnesium sulfate; 0.75 part of calcium carbonate; 10 parts of perilla leaf powder; 2 parts of raw sanguisorba officinalis; 2 parts of ophiopogon japonicus.

[0026] (3) The liquid culture medium consists of water and the following raw materials in percentage by weight:

[0027] Corn steep liquor 1.5%, yeast powder 1.0%, soybean meal powder 1.0%, corn flour 2.5%, glucose 0.5%, magnesium sulfate 0.1%;

[0028] The sum of all raw materials is 100%;

[0029] The solid culture medium is composed of the following raw materials in parts by weight:

[0030] 100 parts of millet; 60 parts of water; 4.0 parts of glucose; 1.6 parts of yeast powder; 1.2 parts of ammonium sulfate; 0.6 parts of magnesium sulfate; 0.8 parts of calcium carbonate; 8 parts of perilla leaf powder; 3 parts of raw sanguisorba officinalis; 1.5 parts of ophiopogon japonicus.

[0031] (4) The liquid culture medium is composed of water and the following raw materials in percentage by weight:

[0032] Corn steep liquor 1.5%, yeast powder 1.0%, soybean meal powder 1.0%, corn flour 2.5%, glucose 0.5%, magnesium sulfate 0.1%;

[0033] The sum of all raw materials is 100%;

[0034] The solid culture medium is composed of the following raw materials in parts by weight:

[0035] 100 parts of millet; 40 parts of water; 1.0 part of glucose; 0.5-2.0 parts of yeast powder; 0.5 part of ammonium sulfate; 0.2 part of magnesium sulfate; 0.5 part of calcium carbonate; 6 parts of perilla leaf powder; 4 parts of raw sanguisorba officinalis.

[0036] The invention provides a green trichoderma conidia powder, which is prepared by fermenting green trichoderma with the liquid fermentation medium and the solid medium in the fermentation medium described in the invention.

[0037] The present invention has no particular limitation on the source of Trichoderma viride, and any Trichoderma viride commonly found in the art may be used. In a specific embodiment of the present invention, Trichoderma viride with a deposit number of CGMCC No. 5044 is used.

[0038] The present invention also provides a method for preparing the viride conidia powder, comprising:

[0039] Step 1: inoculating Trichoderma viride into the liquid fermentation medium in the fermentation medium of the present invention to perform liquid fermentation to obtain a liquid fermentation liquid;

[0040] Step 2: inoculating the liquid fermentation liquid into the solid fermentation medium as a seed liquid for fermentation in a koji pan, drying and sieving.

[0041] In some embodiments, the inoculation amount of the liquid fermentation is 1-8%, specifically 1%, 5% or 8%; the temperature of the liquid fermentation is 28-32°C, the pressure is 0.03-0.05MPa, the stirring speed is 100-160 rpm, the ventilation volume is 0.8-1.2V / V·min, and the time is 24-36 hours.

[0042] In some specific embodiments, the liquid fermentation temperature is 30° C., the pressure is 0.04 MPa, the stirring speed is 100-160 rpm, the ventilation volume is 1 V / V·min, and the time is 30 hours.

[0043] In some embodiments, the inoculation amount of the koji plate fermentation is 5% to 20%, specifically 5%, 8%, 12% or 20%; the temperature of the koji plate fermentation is 28°C-32°C, the humidity is 70-90%, and the time is 48-72 hours.

[0044] In some specific embodiments, the koji plate fermentation is specifically one of the following:

[0045] (1) Fermentation was carried out for 24 hours at a humidity of 70% and a temperature of 28°C until the culture medium was covered with white hyphae and turned over once; the fermentation medium was covered with green spores, and the fermentation was completed.

[0046] (2) Fermentation was carried out at a humidity of 80% and a temperature of 28° C. for 12 hours, and then at a humidity of 80% and a temperature of 32° C. for 24 hours, until the culture medium was covered with white hyphae and turned over once; the fermentation medium was covered with green spores, and the fermentation was completed.

[0047] (3) Ferment at a temperature of 28° C. for 24 hours; then ferment at a humidity of 85% and a temperature of 30° C. for 24 hours; then ferment at a humidity of 85% and a temperature of 30° C. for 24 hours, until the culture medium is covered with white hyphae and turns over once; the fermentation medium is covered with green spores, and the fermentation is completed.

[0048] (4) Cultivate for 24 hours under the conditions of 90% humidity and 32°C, until the culture medium is covered with white hyphae and turns over once; the fermentation medium is covered with green spores, and the fermentation is completed.

[0049] In the present invention, the process of koji plate fermentation also includes a step of turning the koji. Turning the koji is performed when the culture medium is full of white hyphae, and the number of times of turning the koji is one.

[0050] The invention also provides a green Trichoderma agent, comprising the green Trichoderma conidia powder of the invention and auxiliary materials acceptable in pesticides.

[0051] Those skilled in the art can select the auxiliary materials required for preparing different dosage forms, such as one or more of wetting agents, dispersants, penetrants, antifreeze agents, thickeners, synergists, and fillers, and prepare the green Trichoderma conidia powder of the present invention into different dosage forms, such as wettable powders, water-dispersible granules, suspensions or suspoemulsions, etc. according to conventional preparation methods in the art.

[0052] The present invention adopts liquid-solid combined fermentation, solid-state fermentation with millet as the main raw material, supplemented with a quick-acting carbon and nitrogen source and an inorganic salt, and compared with the traditional process, the viable bacteria count and suspension phase of the green Trichoderma conidia powder obtained by fermentation through a specific fermentation medium and a fermentation process are significantly improved. The green Trichoderma conidia powder obtained by the present invention is mixed with a wetting agent, a dispersant, a carrier, etc. to prepare a green Trichoderma wettable powder, which has good water solubility, can be used for spraying and drip irrigation of crops, can prevent and treat plant diseases, and has the advantages of safety and high efficiency, no drug residue, no toxic side effects, reducing the use of antibiotics, and improving crop immunity. DETAILED DESCRIPTION

[0053] The present invention provides a fermentation medium of green Trichoderma, a Trichoderma agent and a preparation method thereof. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application of this article without departing from the content, spirit and scope of the present invention to realize and apply the technology of the present invention.

[0054] The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0055] The present invention will be further described below in conjunction with embodiments:

[0056] Example 1

[0057] The viride strain was cultured to produce a concentration of 5×10 8 cfu / mL of spore suspension; the spore suspension is inoculated into a liquid fermentation medium for fermentation, wherein the liquid fermentation medium comprises the following components: 1.5% corn steep liquor, 1.0% yeast powder, 1.0% bean cake powder, 2.5% corn flour, 0.5% glucose, 0.1% magnesium sulfate, natural pH value, and sterilized at 121°C for 30 minutes.

[0058] The inoculation amount is 5%, the fermentation temperature is 28-32°C, the pressure is 0.03-0.05MPa, the stirring speed is 100-160 rpm, the ventilation volume is 0.8-1.2V / V·min, and the culture is carried out for 24-36 hours to prepare liquid seeds.

[0059] The liquid seed liquid was inoculated into the solid fermentation medium for koji fermentation. The solid fermentation medium included the following components in unit weight: 100 parts millet; 80 parts water; 5.0 parts glucose; 2.0 parts yeast powder; 1.5 parts ammonium sulfate; 0.8 parts magnesium sulfate; 1.0 parts calcium carbonate; 5 parts perilla leaf powder; 2 parts raw sanguisorba officinalis; 1 part ophiopogon japonicus. Sterilize at 121°C for 60 minutes, turning the medium every 5 minutes for 1 minute each time.

[0060] Each plate of culture medium is loaded with 2 kg, 3 cm thick, inoculated with 5%, fermented at 28°C, 70% humidity, and cultured for 24 hours; the culture medium is full of white mycelium and turned over once. After 72 hours of culture, the fermentation medium is full of green spores, and the fermentation is finished. After drying, the spore powder is screened with a 100-mesh sieve to obtain an effective live bacteria count of 33.2 billion / gram.

[0061] Example 2

[0062] The viride strain was cultured to produce a concentration of 5×10 8 cfu / mL of spore suspension; the spore suspension is inoculated into a liquid fermentation medium for fermentation, wherein the liquid fermentation medium comprises the following components: 1.5% corn steep liquor, 1.0% yeast powder, 1.0% bean cake powder, 2.5% corn flour, 0.5% glucose, 0.1% magnesium sulfate, natural pH value, and sterilized at 121°C for 30 minutes.

[0063] The inoculation amount is 5%, the fermentation temperature is 28-32°C, the pressure is 0.03-0.05MPa, the stirring speed is 100-160 rpm, the ventilation volume is 0.8-1.2V / V·min, and the culture is carried out for 24-36 hours to prepare liquid seeds.

[0064] The liquid seed liquid is inoculated into a solid fermentation medium for fermentation. The solid fermentation medium includes the following components in unit weight: 100 millet; 50 water; 3.0 glucose; 1.5 yeast powder; 1.0 ammonium sulfate; 0.5 magnesium sulfate; 0.75 calcium carbonate; 10 perilla leaf powder; 2 raw sanguisorba officinalis; 2 ophiopogon japonicus. Sterilize at 121°C for 60 minutes, turning over once every 5 minutes for 1 minute each time.

[0065] Each plate of culture medium has a capacity of 2.5 kg, a thickness of 4 cm, an inoculation amount of 8%, a fermentation temperature of 28°C for 0-12 hours, 32°C after 12 hours, a humidity of 80%, and a culture of 24 hours; the culture medium is full of white mycelium and turned over once. After 60 hours of culture, the fermentation medium is full of green spores, and the fermentation is finished. After drying, the spore powder is screened with a 100-mesh sieve to obtain an effective number of live bacteria of 46.5 billion / gram.

[0066] Example 3

[0067] The viride strain was cultured to produce a concentration of 5×10 8cfu / mL of spore suspension; the spore suspension is inoculated into a liquid fermentation medium for fermentation, wherein the liquid fermentation medium comprises the following components: 1.5% corn steep liquor, 1.0% yeast powder, 1.0% bean cake powder, 2.5% corn flour, 0.5% glucose, 0.1% magnesium sulfate, natural pH value, and sterilized at 121°C for 30 minutes.

[0068] The inoculation amount is 5%, the fermentation temperature is 28-32°C, the pressure is 0.03-0.05MPa, the stirring speed is 100-160 rpm, the ventilation volume is 0.8-1.2V / V·min, and the culture is carried out for 24-36 hours to prepare liquid seeds.

[0069] The liquid seed liquid is inoculated into a solid fermentation medium for fermentation. The solid fermentation medium includes the following components in unit weight: 100 parts millet; 60 parts water; 4.0 parts glucose; 1.6 parts yeast powder; 1.2 parts ammonium sulfate; 0.6 parts magnesium sulfate; 0.8 parts calcium carbonate; 8 parts perilla leaf powder; 3 parts raw sanguisorba officinalis; 1.5 parts ophiopogon japonicus. Sterilize at 121°C for 60 minutes, turning over every 5 minutes for 1 minute each time.

[0070] Each plate of culture medium has a capacity of 2.0 kg, a thickness of 3 cm, an inoculation amount of 12%, a fermentation temperature of 28°C for 0-24 hours, 30°C after 24 hours, a humidity of 85%, and a culture of 24 hours; the culture medium is full of white mycelium and turned over once. After 72 hours of culture, the fermentation medium is full of green spores, and the fermentation is finished. After drying, the spore powder is screened with a 100-mesh sieve to obtain an effective number of live bacteria of 43.2 billion / gram.

[0071] Example 4

[0072] The viride strain was cultured to produce a concentration of 5×10 8 cfu / mL of spore suspension; the spore suspension is inoculated into a liquid fermentation medium for fermentation, wherein the liquid fermentation medium comprises the following components: 1.5% corn steep liquor, 1.0% yeast powder, 1.0% bean cake powder, 2.5% corn flour, 0.5% glucose, 0.1% magnesium sulfate, natural pH value, and sterilized at 121°C for 30 minutes.

[0073] The inoculation amount is 5%, the fermentation temperature is 28-32°C, the pressure is 0.03-0.05MPa, the stirring speed is 100-160 rpm, the ventilation volume is 0.8-1.2V / V·min, and the culture is carried out for 24-36 hours to prepare liquid seeds.

[0074] The liquid seed liquid is inoculated into a solid fermentation medium for fermentation. The solid fermentation medium includes the following components in unit weight: millet 100; water 40; glucose 1.0; yeast powder 0.5-2.0; ammonium sulfate 0.5; magnesium sulfate 0.2; calcium carbonate 0.5; perilla leaf powder 6; raw sanguisorba officinalis 4; ophiopogon japonicus 2. Sterilize at 121°C for 60 minutes, turning over once every 5 minutes, each time for 1 minute.

[0075] Each plate of culture medium is loaded with 3 kg, 5 cm thick, inoculated with 20%, fermented at 32°C and 90% humidity; after 24 hours of culture, the culture medium is covered with white mycelium and turned over once. After 48 hours of culture, the fermentation medium is covered with green spores, and the fermentation is completed. After drying, the spore powder is screened with a 100-mesh sieve to obtain an effective live bacteria count of 39.4 billion / gram.

[0076] Example 5

[0077] 15 parts of the green Trichoderma spore powder prepared in Example 4 were added to a mixer, and then 5 parts of sodium lauryl sulfate, 4 parts of sodium lignin sulfonate, 60 parts of modified starch and 16 parts of talcum powder were added in sequence, and a uniform mixture was obtained after mixing for 0.5 to 1 hour, that is, a wettable powder with good dispersibility and suspension was obtained. The suspension rate of the wettable powder was 75%, and the wetting time was 84 seconds.

[0078] Example 6

[0079] 20 parts of the green Trichoderma spore powder prepared in Example 1 were added to a mixer, and then 8 parts of sodium lauryl sulfate, 6 parts of sodium lignin sulfonate, 50 parts of modified starch and 16 parts of talcum powder were added in sequence, and a uniform mixture was obtained after mixing for 0.5 to 1 hour, that is, a wettable powder with good dispersibility and suspension was obtained. The suspension rate of the wettable powder was 86%, and the wetting time was 76 seconds.

[0080] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A fermentation medium combination for Trichoderma viride, characterized in that: Including liquid fermentation medium and solid fermentation medium; The liquid fermentation medium consists of water and the following raw materials in percentage by mass: Corn syrup 1.5-2.5%, yeast powder 0.5-1.0%, soybean meal powder 0.5-1.0%, corn flour 1.5-2.5%, glucose 0.5-1.5%, magnesium sulfate 0.1-0.2%; The sum of water and various raw materials is 100%; The solid fermentation medium is composed of the following raw materials in parts by weight: 100 parts of millet, 40-80 parts of water, 1.0-5.0 parts of glucose, 0.5-2.0 parts of yeast powder, 0.5-1.5 parts of ammonium sulfate, 0.2-0.8 parts of magnesium sulfate, 0.5-1.0 parts of calcium carbonate, 5-10 parts of perilla leaf powder, 2-5 parts of raw sanguisorba officinalis, and 1-2 parts of ophiopogon japonicus.

2. The fermentation medium combination according to claim 1, characterized in that: The liquid fermentation medium consists of water and the following raw materials in percentage by mass: Corn steep liquor 1.5%, yeast powder 1.0%, soybean cake powder 1.0%, corn flour 2.5%, glucose 0.5%, magnesium sulfate 0.1%; The sum of water and various raw materials is 100%; The solid fermentation medium is composed of the following raw materials in parts by weight: 100 parts of millet; 50 parts of water; 3.0 parts of glucose; 1.5 parts of yeast powder; 1.0 part of ammonium sulfate; 0.5 part of magnesium sulfate; 0.75 part of calcium carbonate; 10 parts of perilla leaf powder; 2 parts of raw sanguisorba officinalis; 2 parts of ophiopogon japonicus.

3. A method for preparing conidia powder of Trichoderma viride, characterized in that: include: Step 1: inoculating Trichoderma viride with a deposit number of CGMCC No. 5044 into a liquid fermentation medium in the fermentation medium combination according to any one of claims 1 to 2 for liquid fermentation to obtain a liquid fermentation liquid; Step 2: inoculating the liquid fermentation liquid into the solid fermentation medium as a seed liquid for fermentation in a koji pan, drying and sieving.

4. The preparation method according to claim 3, characterized in that: The inoculation amount of the liquid fermentation is 1-8%; the temperature of the liquid fermentation is 28-32°C, the pressure is 0.03-0.05MPa, the stirring speed is 100-160 rpm, the ventilation volume is 0.8-1.2V / V·min, and the time is 24-36 hours.

5. The preparation method according to claim 3, characterized in that: The inoculation amount of the koji plate fermentation is 5%-20%; the temperature of the koji plate fermentation is 28°C-32°C, the humidity is 70-90%, and the time is 48-72 hours.

6. The preparation method according to claim 3, characterized in that: The koji plate fermentation process also includes a koji turning step, and the number of times of turning the koji is 1 time.