Production method of Trichoderma harzianum spore powder

Abstract

The present application relates to the field of fermentation engineering technology, and specifically to a method for producing spore powder of Trichoderma harzianum. The production method comprises: activating the conidia of Trichoderma harzianum, and then inoculating and culturing to obtain seed liquid; inoculating the seed liquid into a fermentation matrix for fermentation and culturing; wherein the fermentation matrix comprises coconut bran and nutrient solution, and the nutrient solution comprises: 10% to 20% sucrose, 1% to 3% peptone, 1% to 3% ammonium sulfate, 0.01% to 1% potassium dihydrogen phosphate, and 0.1% to 1% sodium dihydrogen phosphate, based on the total weight of the nutrient solution as 100%; collecting the spore powder of Trichoderma harzianum from the fermentation matrix after fermentation and culturing. The present application is based on a fermentation matrix of coconut bran and nutrient solution, which can increase the yield and vitality of spore powder of Trichoderma harzianum fermentation, and at the same time, the fermentation process can reduce the growth of anaerobic bacteria, save the fermentation cost of Trichoderma harzianum, and the coconut bran residue can be recycled and reused, so it can reduce resource waste.

Description

Technical Field

[0001] The present application belongs to the technical field of fermentation engineering, and specifically relates to a method for producing Trichoderma harzianum spore powder. Background Art

[0002] Trichoderma harzianum Trichoderma harzianum ) belongs to the subphylum Ascomycota, class Hypomycetes, order Combretales, family Combretalae, genus Trichoderma, and is an important type of biocontrol fungi. Trichoderma harzianum mainly exists in the rhizosphere of the soil, and can inhibit the growth or activity of plant pathogenic microorganisms through competition, hyperparasitism, antibiotics, etc., so as to reduce the probability of plant diseases or reduce the severity of the disease. In addition, Trichoderma harzianum can also induce plants to initiate defense responses, effectively improving the stress resistance and yield of crops. At present, its spore powder is made into microbial pesticides and is widely used in agricultural planting, playing an important role in the prevention and treatment of soil-borne diseases.

[0003] The quality of Trichoderma harzianum spore powder determines the control effect of its biological preparation products. At present, pure spore powder can be obtained in large quantities through solid-state fermentation, but the mainstream method uses grain for fermentation, which is costly and has low output. The main substrates selected for solid-state fermentation are mostly rice, soybean powder and corn powder, which are costly and cannot be recycled, causing great waste. Secondly, Trichoderma harzianum needs to consume a lot of oxygen during the growth and spore production process, and the fermentation substrate is more likely to cause hypoxia in some areas due to the small gaps in the stacking, and the ventilation is not smooth, which makes it easy to breed anaerobic bacteria and yeast, increase the bacteria rate of pure spore powder, and then lead to a decrease in the growth rate of Trichoderma harzianum, reducing the output and quality of pure spore powder of Trichoderma harzianum, and the cost of fermentation materials is relatively high. Summary of the invention

[0004] The purpose of the present application is to provide a method for producing Trichoderma harzianum spore powder, aiming to solve the technical problem of how to increase the yield and quality of Trichoderma harzianum spore powder at low cost.

[0005] In order to achieve the above application purpose, the technical solution adopted in this application is as follows:

[0006] A method for producing Trichoderma harzianum spore powder comprises:

[0007] After activating the conidia of Trichoderma harzianum, inoculation and culture are performed to obtain seed liquid;

[0008] The seed liquid is inoculated into a fermentation matrix for fermentation culture; wherein the fermentation matrix comprises coconut bran and a nutrient solution, and based on the total weight of the nutrient solution being 100%, the nutrient solution comprises: 10% to 20% sucrose, 1% to 3% peptone, 1% to 3% ammonium sulfate, 0.01% to 1% potassium dihydrogen phosphate, and 0.1% to 1% sodium dihydrogen phosphate;

[0009] The spore powder of Trichoderma harzianum is collected from the fermentation medium after the fermentation culture.

[0010] The production method of Trichoderma harzianum spore powder provided in the present application uses a unique fermentation matrix for fermentation when activating Trichoderma harzianum conidia and inoculating the obtained seed liquid for fermentation culture. The fermentation matrix includes coconut bran and nutrient solution. Since coconut bran has good air permeability, the hyphae of Trichoderma harzianum can easily obtain oxygen therein, thereby growing and reproducing rapidly. At the same time, combined with the nutrition provided by the nutrient solution components, the yield and vitality of Trichoderma harzianum fermented spore powder can be significantly improved, and the fermentation quality and efficiency can be finally improved. In addition, the fermentation process can reduce the growth of anaerobic bacteria, save the fermentation cost of Trichoderma harzianum, and the coconut bran residue after fermentation can be recycled and reused, thereby reducing resource waste. The method has a good application in the field of industrial production of Trichoderma harzianum spore powder. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0012] Figure 1 This is a result diagram of activation on a PDA plate in the method for producing Trichoderma harzianum spore powder provided in the examples of the present application;

[0013] Figure 2 The seed liquid obtained in the method for producing Trichoderma harzianum spore powder provided in the examples of the present application;

[0014] Figure 3 This is a fermentation diagram of different fermentation substrates in the method for producing Trichoderma harzianum spore powder provided in the examples of the present application;

[0015] Figure 4 It is the Trichoderma harzianum spore powder finally collected in the production method of Trichoderma harzianum spore powder provided in the examples of the present application. DETAILED DESCRIPTION

[0016] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present application more clearly understood, the present application is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0017] In this application, the term "and / or" describes the association relationship of associated objects, indicating that there may be three relationships. For example, A and / or B can mean: A exists alone, A and B exist at the same time, and B exists alone. A and B can be singular or plural. The character " / " generally indicates that the associated objects are in an "or" relationship.

[0018] In this application, "at least one" means one or more, "more than one" means two or more. "At least one of the following" or similar expressions refers to any combination of these items, including any combination of single or plural items.

[0019] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the above-mentioned processes does not mean the order of execution, some or all of the steps can be executed in parallel or sequentially, and the execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0020] The terms used in the embodiments of the present application are only for the purpose of describing specific embodiments, and are not intended to limit the present application. The singular forms "a", "said" and "the" used in the embodiments of the present application and the appended claims are also intended to include plural forms, unless the context clearly indicates other meanings.

[0021] The weight of the relevant components mentioned in the embodiment description of the present application can not only refer to the specific content of each component, but also indicate the proportional relationship between the weights of the components. Therefore, as long as the content of the relevant components is proportionally enlarged or reduced according to the embodiment description of the present application, it is within the scope disclosed in the embodiment description of the present application. Specifically, the mass described in the embodiment description of the present application can be a mass unit known in the chemical industry such as µg, mg, g, kg, etc.

[0022] The terms "first" and "second" are used only for descriptive purposes to distinguish objects such as substances from each other, and should not be understood as indicating or implying relative importance or implicitly indicating the number of indicated technical features. For example, without departing from the scope of the embodiments of the present application, the first XX may also be referred to as the second XX, and similarly, the second XX may also be referred to as the first XX. Thus, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features.

[0023] Solid-state fermentation is a fermentation method in which microorganisms grow on a solid matrix with a certain humidity in the absence or substantial absence of free water. The solid matrix contains three phases, gas, liquid and solid, that is, the porous solid matrix contains water and water-insoluble substances.

[0024] The quality of Trichoderma harzianum spore powder determines the control effect of its biological preparation products. The output and quality of Trichoderma harzianum spore powder obtained by solid-state fermentation need to be improved. Therefore, the present application embodiment develops a method for producing Trichoderma harzianum spore powder by solid-state fermentation using coconut bran, wherein coconut bran is used as a solid matrix in the fermentation matrix, and is matched with a nutrient solution component.

[0025] Coconut pith is a byproduct or waste of coconut processing. It is a pure natural organic medium that falls off during the processing of coconut shell fibers. Coconut pith contains a large number of plant fibers with a porous structure. It has a fluffy texture and high toughness. It is a plant cultivation medium with excellent air permeability and water retention.

[0026] A large amount of cellulase is released during the growth and reproduction of Trichoderma harzianum, and this is used to decompose macromolecular organic substances such as crude fiber and lignin, and convert them into absorbable and usable small molecules. Coconut chaff is used as a solid matrix for the growth of Trichoderma harzianum, which can provide a large amount of cellulose as a nutritional basis, and a nutrient solution with nutrient elements such as a quick-acting carbon source and a nitrogen source during the fermentation process can accelerate the growth and reproduction of Trichoderma harzianum, reduce the time required for fermentation, and greatly improve the yield and quality of Trichoderma harzianum spore powder. In addition, fermentation is carried out using a fermentation matrix of coconut chaff and nutrient solution, which can be recycled and reused after use, reducing the processing of organic waste and reducing the production cost of spore powder. The specific technical scheme of the embodiment of the present application is as follows.

[0027] The present application embodiment provides a method for producing Trichoderma harzianum spore powder. The specific steps of the method for producing Trichoderma harzianum spore powder in the present application embodiment include:

[0028] S01: activating the conidia of Trichoderma harzianum and then inoculating and culturing them to obtain seed liquid;

[0029] S02: inoculating the seed liquid into a fermentation matrix for fermentation culture; wherein the fermentation matrix includes coconut bran and nutrient solution, and the nutrient solution includes: 10% to 20% sucrose, 1% to 3% peptone, 1% to 3% ammonium sulfate, 0.01% to 1% potassium dihydrogen phosphate and 0.1% to 1% sodium dihydrogen phosphate, based on the total weight of the nutrient solution as 100%;

[0030] S01: collecting Trichoderma harzianum spore powder from the fermentation medium after fermentation culture.

[0031] The production method of Trichoderma harzianum spore powder provided in the embodiment of the present application uses a unique fermentation matrix for fermentation when activating Trichoderma harzianum conidia and inoculating the obtained seed liquid for fermentation culture. The fermentation matrix includes coconut bran and nutrient solution. Since coconut bran has good air permeability, the hyphae of Trichoderma harzianum can easily obtain oxygen therein, thereby growing and reproducing rapidly. At the same time, combined with the nutrition provided by the nutrient solution components, the yield and vitality of Trichoderma harzianum fermented spore powder can be significantly improved, and ultimately the fermentation quality and efficiency can be improved. In addition, the fermentation process can reduce the growth of anaerobic bacteria, save the fermentation cost of Trichoderma harzianum, and the coconut bran residue after fermentation can be recycled and reused, thereby reducing resource waste. The method has a good application in the field of industrial production of Trichoderma harzianum spore powder.

[0032] It should be noted that the fermentation matrix in the embodiments of the present application can be understood as a culture medium for fermentation, which contains a solid phase and a liquid phase, with coconut bran as a solid matrix and a liquid nutrient solution, thereby forming a fermentation matrix that is conducive to the production of Trichoderma harzianum spore powder. The fermentation process can be understood as solid-state fermentation.

[0033] Step S01 is a process of activating the conidia of Trichoderma harzianum and culturing them into seed liquid.

[0034] In some embodiments, the step of activating the conidia of Trichoderma harzianum includes: inoculating the conidia of Trichoderma harzianum on a potato dextrose agar (PDA) medium plate and culturing at 24-28° C. for 2-4 days. This process can activate the conidia of Trichoderma harzianum well and make them viable. Specifically, 0.1 g of conidia of Trichoderma harzianum can be inoculated on a PDA medium plate for activation and cultured in a 26° C. incubator for 3 days.

[0035] In some embodiments, the step of inoculating and culturing the activated conidia on the plate to obtain the seed solution includes: inoculating the activated Trichoderma harzianum conidia into the seed culture solution, and culturing at 24-28° C. in a shaking table for 40-50 hours to obtain the seed solution. The seed solution is a solution containing the strain of Trichoderma harzianum solid-state fermentation.

[0036] Wherein, based on the total weight of the seed culture solution as 100%, the seed culture solution includes: 1%~3% sucrose, 1%~3% peptone, 0.01%~0.5% ammonium sulfate, 0.0001%~0.01% potassium dihydrogen phosphate, 0.01%~0.5% yeast powder and 0.01%~0.5% wheat bran. Wherein, the preparation method of the seed culture solution can be to weigh the components in mass ratio and then fully dissolve them in water. Carrying out the inoculation culture in the above process in the above seed culture solution can obtain a seed solution with good vitality for subsequent fermentation, and the spore concentration in the seed solution can reach: 10 7 ~10 8 Spores / mL.

[0037] In some embodiments, the seed culture solution includes the following components: 1.5% sucrose, 1.5% peptone, 0.05% ammonium sulfate, 0.001% potassium dihydrogen phosphate, 0.1% yeast powder, and 0.1% wheat bran. The activated Trichoderma harzianum conidia are inoculated into the above seed culture solution, and cultured in a shaker at 26°C and 180 r / min for 48 hours before use.

[0038] Step S02 is a process for producing Trichoderma harzianum spore powder by fermenting Trichoderma harzianum.

[0039] The fermentation substrate used in the fermentation process includes coconut bran and nutrient solution.

[0040] The coconut bran includes desalted coconut bran. Specifically, the pH range of the desalted coconut bran is 6.2-7.5.

[0041] Wherein, based on the total weight of the nutrient solution as 100%, the nutrient solution includes: 10% to 20% sucrose, 1% to 3% peptone, 1% to 3% ammonium sulfate, 0.01% to 1% potassium dihydrogen phosphate and 0.1% to 1% sodium dihydrogen phosphate. For example, the nutrient solution includes the following components: 15% sucrose, 1.5% peptone, 1% ammonium sulfate, 0.05% potassium dihydrogen phosphate, and 0.5% sodium dihydrogen phosphate. The nutrient solution can be prepared by weighing the components in mass ratio and then fully dissolving them in water.

[0042] In some embodiments, the weight ratio of coconut bran to nutrient solution is 1-1.5:1-1.5.

[0043] Furthermore, water can be added to adjust the concentration according to actual needs, for example, coconut bran, nutrient solution and purified water can be mixed evenly in a ratio of 1-1.5:1-1.5:1; the mixing process can include: first mixing the nutrient solution with coconut bran after preparing it, and then adding purified water to replenish the water after the mixing is completed.

[0044] The mixed fermentation substrate is sterilized. For example, the fermentation substrate is placed in a propylene polyethylene bag for sterilization and standby. The specification of the propylene polyethylene bag for packaging is 18 cm wide, 36 cm long, 0.05 mm thick, and 9.5 cm in diameter.

[0045] In some embodiments, the seed solution is inoculated into the fermentation substrate for fermentation culture according to the seed solution accounting for 8-12% of the weight of the fermentation substrate. Exemplarily, the seed solution is added to the fermentation substrate for solid fermentation according to the inoculation amount of 10% (w / w).

[0046] In some embodiments, the fermentation culture conditions include: a temperature of 24-28°C; a time of 8-12 days; and a humidity (i.e., relative humidity) of 75-85%. Specifically, ventilation fermentation can be performed. Through the above fermentation process, not only the spore powder of Trichoderma harzianum is increased, but also its vitality is increased.

[0047] For example, the seed solution is added to the fermentation matrix at an inoculation amount of 10% (w / w), and placed in a ventilated incubator at 26°C and 80% humidity for fermentation for 10 days.

[0048] S03 is the process of collecting Trichoderma harzianum spore powder.

[0049] In some embodiments, after the fermentation is completed, the step of collecting the spore powder of Trichoderma harzianum from the fermentation substrate after fermentation culture comprises: washing the fermentation substrate after fermentation culture with Tween-80 solution, then filtering to obtain a spore suspension, centrifuging the spore suspension, and freeze-drying the precipitate to obtain the spore powder of Trichoderma harzianum.

[0050] In some embodiments, the Tween-80 solution is a polysorbate 80 solution, wherein the mass concentration of polysorbate 80 may be 0.01-0.05%, for example, a 0.02% Tween-80 solution may be selected.

[0051] In some embodiments, the spore suspension centrifugation process can be placing the spore suspension in a centrifuge at 5000-7000 r / min for 4-6 minutes, and discarding the supernatant to obtain a precipitate.

[0052] In some embodiments, the precipitate is freeze-dried at a temperature of -70 to -20°C for 20 to 26 hours.

[0053] In some embodiments, the process of collecting spore powder of Trichoderma harzianum includes: after the fermentation and spore production of Trichoderma harzianum is completed, 0.02% Tween-80 is mixed with it to wash out the spores, and the spore suspension is filtered with a double-layer gauze, which is placed in a centrifuge for centrifugation at 6000 r / min for 5 minutes, the supernatant is discarded, the precipitate is taken, and the precipitate is freeze-dried for 24 hours and then packaged.

[0054] In some embodiments, the germination rate of the finally collected Trichoderma harzianum spore powder is ≥97%, and the moisture content is ≤5%.

[0055] The following describes the invention in conjunction with specific embodiments.

[0056] Example 1

[0057] A method for producing Trichoderma harzianum spore powder comprises the following steps:

[0058] Step 1: After activating the conidia of Trichoderma harzianum, inoculate and culture to obtain seed liquid; the details are as follows:

[0059] Take 0.1 g of Trichoderma harzianum conidia and inoculate them on PDA plates for activation. Incubate them in a 26°C incubator for 3 days (the results are shown in Table 2). Figure 1 As shown). Then, the conidia of Trichoderma harzianum on the PDA plate were inoculated into the seed culture solution of Trichoderma harzianum; wherein, the seed culture solution of Trichoderma harzianum includes: 1.5% sucrose, 1.5% peptone, 0.05% ammonium sulfate, 0.001% potassium dihydrogen phosphate, 0.1% yeast powder, and 0.1% wheat bran. Then, the culture was carried out in a shaker at 26°C and 180 r / min for 48 hours to obtain the seed solution for use (the results are shown in Figure 2 as shown).

[0060] Step 2: Inoculate the seed solution obtained in step 1 into the fermentation matrix for fermentation culture; the details are as follows:

[0061] Preparation of fermentation substrate: Mix coconut bran, nutrient solution and purified water in a weight ratio of 1.5:1.5:1, and put 500g of the mixed fermentation substrate into a propylene polyethylene bag (specification: 18 cm wide, 36 cm long, 0.05 mm thick, 9.5 cm in diameter) for sterilization. The coconut bran is desalted coconut bran with a pH value of 6.5; the nutrient solution includes 15% sucrose, 1.5% peptone, 1% ammonium sulfate, 0.05% potassium dihydrogen phosphate, and 0.5% sodium dihydrogen phosphate.

[0062] The seed liquid obtained in step 1 is added to the sterilized fermentation matrix in an amount of 9% by weight of the fermentation matrix for solid fermentation; the specific conditions include: placing in a ventilated incubator at 26°C and 80% humidity for 10 days, wherein the effect of the fermentation matrix is ​​as follows: Figure 3 As shown in A.

[0063] Step 3: After 10 days of fermentation, the substrate was mixed with 0.02% Tween-80 solution to wash out the spores, and the spore suspension was filtered through double gauze to obtain a spore suspension. The spore suspension was centrifuged in a centrifuge at 6000 r / min for 5 minutes, the supernatant was discarded, and the precipitate was freeze-dried at -40°C for 24 hours to obtain the spore powder of Trichoderma harzianum (such as Figure 4 Finally, the packaging is completed.

[0064] Comparative Example 1

[0065] A method for producing Trichoderma harzianum spore powder is different from that of Example 1 in that the fermentation substrate is different.

[0066] Specifically, the fermentation substrate of this comparative example is mixed at a weight ratio of rice: wheat bran: nutrient solution = 4:1:5, wherein the nutrient solution is the same as that in Example 1. The other steps and parameters are the same as those in Specific Example 1. The effect of the fermentation substrate after fermentation is as follows Figure 3As shown in B.

[0067] Comparative Example 2

[0068] A method for producing Trichoderma harzianum spore powder is different from that of Example 1 in that the fermentation substrate is different.

[0069] Specifically, the fermentation matrix of this comparative example is mixed at a weight ratio of bamboo powder: wheat bran: nutrient solution = 4:1:5, wherein the nutrient solution is the same as that in Example 1. The other steps and parameters are the same as those in Specific Example 1. The effect of the fermentation matrix after fermentation is as follows Figure 3 As shown in C.

[0070] Comparative Example 3

[0071] A method for producing Trichoderma harzianum spore powder is different from that of Example 1 in that the nutrient solution in the fermentation matrix is ​​different.

[0072] The nutrient solution of this comparative example is the same as that of Example 1 except that the sucrose in Example 1 is replaced by glucose.

[0073] Comparative Example 4

[0074] A method for producing Trichoderma harzianum spore powder is different from that of Example 1 in that the nutrient solution in the fermentation matrix is ​​different.

[0075] The nutrient solution of this comparative example has the sucrose in Example 1 replaced by lactose, and the other aspects are the same as those of Example 1.

[0076] Comparative Example 5

[0077] A method for producing Trichoderma harzianum spore powder is different from that of Example 1 in that the nutrient solution in the fermentation matrix is ​​different.

[0078] The nutrient solution of this comparative example deletes the ammonium sulfate in Example 1, and adds 0.2% magnesium sulfate and 0.0025% ferrous sulfate, and the rest is the same as Example 1.

[0079] Effect test

[0080] The yield and activity of the spore powder of Trichoderma harzianum collected in the embodiments and comparative examples were tested, and the results are shown in Table 1.

[0081] in,

[0082] (1) Amount of spore powder produced per kg of fermentation substrate:

[0083] It can be calculated according to the following formula.

[0084] ;

[0085] (2) Spore powder mass (i.e. the spore content per unit mass of spore powder):

[0086] The test procedure is to weigh 1 g of the fermented Trichoderma harzianum spore powder and mix it evenly with 100 mL of 0.02% Tween-80 solution, and use a hemocytometer to determine the number of spores. The spore content per unit mass of spore powder can be calculated according to the following formula.

[0087] ;

[0088]

[0089] (3) Spore germination rate:

[0090] The test procedure is to weigh 10 mg of the fermented Trichoderma harzianum spore powder and inoculate it into 50 mL of potato dextrose water (PDB) medium (i.e., spore germination solution). After sufficient shaking and mixing, it is placed in a shaker at 26°C and 180 r / min for 12 h. The number of germinated and ungerminated spores is recorded using a hemocytometer (spore expansion is twice that of the original spore or spore budding is considered germination), and the spore germination rate is calculated according to the following formula.

[0091] ;

[0092] Table 1

[0093]

[0094] The fermentation in Example 1 only takes 10 days, which significantly shortens the fermentation time and improves the production efficiency of Trichoderma harzianum spore powder.

[0095] According to the market price of solid substrate: rice 3 yuan / kg, bamboo powder 2 yuan / kg, wheat bran 2.5 yuan / kg, coconut bran 1.7 yuan / kg. Calculated based on the cost of solid fermentation substrate alone: ​​one ton of solid substrate (rice and wheat bran) in Comparative Example 1 requires about 4000-4500 yuan, one ton of solid substrate (bamboo powder and wheat bran) in Comparative Example 2 requires about 2000-2400 yuan, and one ton of coconut bran in Example 1 only requires 1700 yuan. This material can be reused repeatedly, and the cost of use is lower as the number of uses increases.

[0096] If the consumption cost of seed liquid and nutrient solution is added for calculation, one ton of prepared solid-liquid fermentation matrix in comparative example 1 requires about 2,500 yuan, one ton of prepared solid-liquid fermentation matrix in comparative example 2 requires about 2,000 yuan, and one ton of prepared solid-liquid fermentation matrix in embodiment 1 only requires about 1,300 yuan. Coconut bran is cheap and widely available, and can be used repeatedly for fermentation countless times. The more times it is used, the closer its cost is to 700 yuan per ton. When large-scale fermentation is carried out, using coconut bran as a solid matrix can greatly reduce the production cost of Trichoderma harzianum spore powder.

[0097] Combination Figure 3As shown in Table 1, the bottom and interior of Comparative Example 1 using rice and Comparative Example 2 using bamboo powder are not fully fermented due to insufficient ventilation, resulting in material waste, and they are easy to breed bacteria and yeast, which limits the growth of Trichoderma harzianum. The quality of the spore powder produced is poor, and the bacterial content per gram of spore powder is low. After fermentation with coconut bran in Example 1, the Trichoderma harzianum fermentation bag turns green all over, and the spore content of the spore powder finally obtained exceeds that of rice and bamboo powder by more than 40%. In addition, it can be seen from the results in Table 1 that the use of coconut bran and the nutrient solution formula of the embodiment of the present application, that is, the spore powder output and quality of the fermentation output of Example 1 are significantly better than those of Comparative Examples 3 to 5, indicating that coconut bran as a solid fermentation matrix and the nutrient solution formula of the embodiment have excellent spore production effects.

[0098] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present application should be included in the protection scope of the present application.

Claims

1. A method for producing Trichoderma harzianum spore powder, characterized in that: The following steps are involved: Step 1: After activating the conidia of Trichoderma harzianum, inoculate and culture to obtain a seed solution, specifically as follows: take 0.1 g of conidia of Trichoderma harzianum and inoculate it on a PDA plate for activation, culture it in a 26°C incubator for 3 days, and then inoculate the conidia of Trichoderma harzianum on the PDA plate into the seed culture solution of Trichoderma harzianum; The Trichoderma harzianum seed culture solution comprises: 1.5% sucrose, 1.5% peptone, 0.05% ammonium sulfate, 0.001% potassium dihydrogen phosphate, 0.1% yeast powder and 0.1% wheat bran, and then cultured in a shaking incubator at 26° C. and 180 r / min for 48 hours to obtain a seed solution for standby use; Step 2: Inoculate the seed liquid obtained in step 1 into the fermentation matrix for fermentation culture, as follows: Preparation of fermentation matrix: Evenly mix coconut bran, nutrient solution and purified water in a weight ratio of 1.5:1.5:1, and put 500 g of the mixed fermentation matrix into a propylene polyethylene bag for sterilization for standby use, wherein the coconut bran is desalted coconut bran with a pH value of 6.5; the nutrient solution comprises 15% sucrose, 1.5% peptone, 1% ammonium sulfate, 0.05% potassium dihydrogen phosphate and 0.5% sodium dihydrogen phosphate, and the seed liquid obtained in step 1 is added in an amount of 9% by weight of the fermentation matrix, and the seed liquid is added to the sterilized fermentation matrix for solid fermentation, and the specific conditions include: placing in a ventilated incubator at 26°C and 80% humidity for 10 days; Step 3: After 10 days of fermentation, the substrate was mixed with 0.02% Tween-80 solution to wash out the spores, and the spore suspension was filtered with double gauze to obtain a spore suspension. The spore suspension was placed in a centrifuge at 6000 r / min for 5 minutes, the supernatant was discarded, and the precipitate was taken and freeze-dried at -40°C for 24 hours to obtain the spore powder of Trichoderma harzianum.

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