A fermentation medium for biocontrol trichoderma harzianum using waste by-products and a method for preparing a biocontrol trichoderma harzianum agent

Abstract

The application discloses a fermentation medium of a biocontrol Trichoderma harzianum by using waste by-products and a method for preparing a biocontrol Trichoderma harzianum agent, and belongs to the field of utilization of gibberellic acid production waste, and comprises: 48wt%-52wt% of a plate and frame fungus residue of a gibberellic acid membrane residual liquid, 8wt%-12wt% of corn cob powder, 8wt%-12wt% of glucose, 1wt%-3wt% of urea, 2wt%-6wt% of corn protein powder and 1wt%-3wt% of potassium dihydrogen phosphate, and the rest is water. On the basis of guaranteeing the spore production amount of the Trichoderma harzianum, the production cost of the Trichoderma harzianum agent is remarkably reduced.

Description

Technical Field

[0001] This invention relates to the field of gibberellic acid production waste utilization, and in particular to a biocontrol Trichoderma harzianum fermentation culture medium utilizing waste by-products and a method for preparing biocontrol Trichoderma harzianum agents. Background Technology

[0002] *Metarhizium spp.*, belonging to the family Clavicipitaceae in the order Hypocreales, is an entomopathogenic fungus with significant applications in agricultural biological control. This fungus infects host insects through conidia, which germinate, colonize, and ultimately kill the host under suitable conditions. *Metarhizium anisopliae*, a species of *Trichoderma harzianum*, has been successfully developed into various fungal insecticides and is widely used in organic agriculture, green agriculture, and forestry pest control.

[0003] Currently, the production of Trichoderma harzianum inoculants mainly relies on solid-state fermentation or deep liquid fermentation using artificial culture media. Solid-state fermentation is the main method for producing conidia, with common carbon sources such as rice and maltose, and nitrogen sources such as soybean powder and yeast powder. Although the above methods have certain advantages in terms of spore yield and activity, the raw materials for the culture media are mostly grains or food-grade auxiliary materials, resulting in high production costs and a generally high price for the inoculant, which limits its large-scale promotion in grassroots agriculture.

[0004] Meanwhile, the industrial production of gibberellin generates a large amount of waste byproducts. Gibberellin is mainly produced through microbial fermentation. After the fermentation broth is filtered through a plate and frame filter to remove mycelium, the filtrate is concentrated and purified by membrane separation (such as ceramic membranes, ultrafiltration membranes, etc.). The concentrated membrane residue (concentrate) is then filtered through a plate and frame filter to form gibberellin membrane residue plate and frame bacterial residue (the amount of gibberellin membrane residue plate and frame bacterial residue accounts for about 12-15% of the total weight of the gibberellin fermentation broth). The gibberellin membrane residue plate and frame bacterial residue produced by medium-sized production lines can reach 3,720 tons / year. The gibberellin membrane residue plate and frame bacterial residue is classified as general industrial solid waste. The waste byproducts are rich in organic matter, mycelium, and residual nutrients. If they are not effectively treated, they will not only waste resources but may also cause environmental pollution problems.

[0005] There are existing research reports on the conversion of gibberellin bacterial residue into compound microbial fertilizers. For example, CN108033847A discloses a gibberellin bacterial residue compound microbial fertilizer and its preparation method, and CN112194538A discloses a gibberellin bacterial residue treatment method and a liquid fertilizer containing gibberellin bacterial residue treatment material. In addition, there are also technical reports on the preparation of bio-humic acid by adding Trichoderma harzianum or Trichoderma viride to raw materials such as straw, distiller's grains, fruit pomace, and livestock and poultry manure (e.g., CN104446895A).

[0006] However, the above technologies mainly target the fertilizer utilization of gibberellin residue, or the preparation of microbial fertilizers / humic acid products using different strains such as Trichoderma harzianum and Trichoderma viride. None of them involve technical solutions for preparing highly active biocontrol agents using Trichoderma harzianum as the target strain and gibberellin film residue and plate and frame fungal residue as the only or main culture medium raw materials.

[0007] Therefore, developing a method for preparing Trichoderma harzianum inoculants that uses gibberellic acid production waste (membrane residue, plate and frame fungal residue) as the main raw material, is low-cost, has stable process, and high spore activity, can not only realize the resource utilization of waste by-products and reduce the production cost of inoculants, but also provide efficient and environmentally friendly biological pesticides for agricultural pest control, which has important practical significance and industrial value. Summary of the Invention

[0008] To address the aforementioned shortcomings, this invention provides a fermentation culture medium for biocontrol Trichoderma harzianum using waste by-products and a method for preparing biocontrol Trichoderma harzianum agents. While ensuring the spore production of Trichoderma harzianum, this invention significantly reduces the production cost of Trichoderma harzianum agents.

[0009] The technical solution is: a fermentation culture medium for the biocontrol Trichoderma harzianum utilizing waste by-products, comprising: The residue of gibberellic acid film plate and frame bacterial residue is 48wt%~52wt%, corn cob powder is 8wt%~12%, glucose is 8wt%~12%, urea is 1wt%~3%, corn gluten powder is 2wt%~6%, potassium dihydrogen phosphate is 1wt%~3%, and the balance is water.

[0010] Furthermore, the residual liquid from the gibberellic acid film consists of 50 wt% plate and frame bacterial residue, 10 wt% corn cob powder, 10 wt% glucose, 2 wt% urea, 4 wt% corn gluten powder, 2 wt% potassium dihydrogen phosphate, and the remainder is water.

[0011] Furthermore, the residual liquid from the gibberellic acid film consists of 48 wt% plate and frame bacterial residue, 8 wt% corn cob powder, 8 wt% glucose, 1 wt% urea, 1 wt% corn gluten powder, 1 wt% potassium dihydrogen phosphate, and the remainder is water.

[0012] Furthermore, during the fermentation culture of this biocontrol Trichoderma harzianum, the pH was adjusted to 4.5-5.0 using citric acid.

[0013] This invention also provides a method for preparing a biocontrol agent for Trichoderma harzianum using waste by-products.

[0014] A method for preparing a biocontrol agent for Trichoderma harzianum using waste by-products includes the following steps: S1, Trichoderma harzianum slant culture was used to obtain Trichoderma harzianum slant strain; S2, Trichoderma harzianum slant culture was inoculated into primary culture medium to obtain primary culture; S3, the primary seed is inoculated into the secondary seed culture medium to obtain the secondary seed; S4, fermentation culture, includes the following steps: S41, After the above fermentation culture medium is mixed evenly, it is sterilized and cooled; S42, inoculate with secondary seed, mix thoroughly and adjust pH to 4.5-5.0 with citric acid; S43, the inoculated fermentation medium is pressed into fermentation medium blocks using a briquetting machine.

[0015] S44, the fermentation medium blocks are placed on the rack in the culture room and fermented at a temperature of 27℃~28℃ and a humidity of 50%~58% to obtain fermented blocks; S45, after fermentation, is pressed into blocks, dried, crushed, sieved, and the spores are collected to obtain Trichoderma harzianum inoculum. Furthermore, in S1, the culture conditions are: static culture at 28℃ for 8 days; in S2, the culture conditions are: static culture at 28℃ in a 2L Erlenmeyer flask for 8 days; and in S3, the culture conditions are: static culture at 28℃ in a sterilized iron tray for 8 days.

[0016] Furthermore, in S2, the primary culture medium consists of 20 wt% glucose, 10 wt% yeast extract, 20 wt% peptone, and 50 wt% water; in S3, the secondary culture medium consists of 30 wt% rice flour and 80 wt% water.

[0017] Furthermore, in S41, the sterilization conditions are: sterilization at 121℃ for 30 minutes; in S42, the inoculation amount is 1% to 5% of the total fermentation medium.

[0018] Furthermore, in S43, the size of 100 kg of fermentation medium after compression is 10 cm * 6 cm * 3 cm.

[0019] Furthermore, in S44, the fermentation time is 7 days to 15 days; in S45, the crushing conditions are: hammer mill crushing at a speed of 2500 r / min; and sieving by a 60-80 mesh vibrating screen.

[0020] Compared with the prior art, the inventive principle and beneficial effects of this invention are as follows: This invention achieves sporulation of Trichoderma hominis by organically combining gibberellic acid film residue plate and frame fungal residue with other components, under conditions of extremely low spore yield when using gibberellic acid plate and frame fungal residue alone.

[0021] This invention significantly reduces the production cost of Trichoderma harzianum inoculants while ensuring the spore production of Trichoderma harzianum. The proportion of gibberellic acid film residue in plate and frame inoculum residue can reach more than 50%. Detailed Implementation

[0022] As used in this article: "Prepared from" is synonymous with "comprising". The terms "comprising", "including", "having", "containing", or any other variations thereof as used herein are intended to cover non-exclusive inclusion. For example, a composition, step, method, article, or apparatus that includes the listed elements is not necessarily limited to those elements, but may include other elements not expressly listed or elements inherent to such composition, step, method, article, or apparatus.

[0023] The conjunction "composed of..." excludes any unspecified elements, steps, or components. If used in a claim, this phrase makes the claim closed, excluding materials other than those described, except for associated conventional impurities. When the phrase "composed of..." appears in a clause of the body of a claim rather than immediately following it, it limits only the elements described in that clause; other elements are not excluded from the claim as a whole.

[0024] When a quantity, concentration, or other value or parameter is expressed as a range, a preferred range, or a range defined by a series of upper and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pair of any upper or preferred value with any lower or preferred value, regardless of whether the range is disclosed individually. For example, when the range “1–5” is disclosed, the described range should be interpreted as including ranges “1–4”, “1–3”, “1–2”, “1–2 and 4–5”, “1–3 and 5”, etc. When numerical ranges are described herein, unless otherwise stated, the range is intended to include its endpoints and all integers and fractions within that range.

[0025] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0026] Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be construed as limiting the scope of this application. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0027] In these embodiments, unless otherwise specified, the portions and percentages are all by weight.

[0028] "Parts by mass" refers to the basic unit of measurement that expresses the mass ratio of multiple components. One part can represent any unit mass, such as 1g or 2.689g. If we say that component A has "a" parts by mass and component B has "b" parts by mass, it means the ratio of the mass of component A to the mass of component B is a:b. Alternatively, it can mean that the mass of component A is aK and the mass of component B is bK (K is any number representing a multiplier). It is important to understand that, unlike the number of parts by mass, the sum of the mass parts of all components is not limited to 100 parts.

[0029] "And / or" is used to indicate that one or both of the described situations may occur, for example, A and / or B includes (A and B) and (A or B).

[0030] In this invention, the method for determining sporulation yield is as follows: Weigh the compressed culture medium block (m1), then collect the spores. Weigh all the collected conidial powder (m2). Weigh 1.0 g of conidial powder and add it to a 100.0 mL sterile Erlenmeyer flask containing 0.01% Tween-80 water. Place the flask in a shaker at 5°C and 200 rpm for 30 min. Then, observe and count the conidia (x) under an optical microscope using a hemocytometer. Calculate the number of conidia per gram of culture medium using the following formula: Y = m2 × x / m1 Where Y: number of conidia per gram of culture medium; x: number of conidia per 1.0 gram of conidial powder; m1: weight of the culture medium block; m2: weight of the collected conidial powder.

[0031] Example 1: Preparation of secondary seeds The preparation of secondary seeds includes the following steps: S1, Slant culture of strain: After thawing the glycerol tubes of Trichoderma harzianum, inoculate them onto the eggplant slant of potato dextrose agar medium and incubate them at 28°C for 8 days to obtain the Trichoderma harzianum slant strain.

[0032] S2, Primary culture: Wash the above-prepared Trichoderma harzianum slant culture with sterile water, inoculate it into the primary culture medium, and incubate it statically in a 2L Erlenmeyer flask at 28℃ for 8 days to obtain the primary culture. The inoculum size is 8%.

[0033] The primary culture medium consisted of 20 wt% glucose, 10 wt% yeast extract, 20 wt% peptone, and 50 wt% water.

[0034] S3, Secondary culture: The above-cultured primary culture is inoculated into the secondary culture medium at an inoculation rate of 1% by weight, and then cultured in a sterilized iron tray at 28°C for 8 days to obtain the secondary culture.

[0035] The secondary culture medium consisted of 30 wt% rice flour and 80 wt% water.

[0036] Example 2 A method for preparing a Trichoderma harzianum inoculum includes the following steps: S4, fermentation culture, includes the following steps: S41. After mixing 100 kg of fermentation medium evenly with a winnowing machine, sterilize at 121℃ for 30 min. After sterilization, wait for the fermentation medium to cool down.

[0037] S42, then the secondary seed from Example 1 is introduced. The amount of secondary seed introduced is 1% of the total amount of fermentation medium. The secondary seed is mixed evenly with the fermentation medium using a winnowing machine, and the pH is adjusted to 4.5 with citric acid.

[0038] S43, use a briquetting machine to compress the inoculated fermentation medium into briquetting blocks with a length, width, and height of 10cm*6cm*3cm.

[0039] S44: The fermentation medium blocks were placed on a rack in the culture room and fermented for 14 days at a temperature of 28℃ and a humidity of 55% to obtain fermented blocks.

[0040] S45, after fermentation and pressing, the blocks are naturally air-dried, crushed by a hammer mill (2500 r / min), and then sieved with a 70 mesh vibrating screen to collect spores and obtain Trichoderma harzianum inoculum.

[0041] The fermentation medium for fermentation culture was: 50wt% gibberellic acid film residue, 10wt% corn cob powder, 10wt% glucose, 2wt% urea, 4wt% corn gluten meal, 2wt% potassium dihydrogen phosphate, and 22wt% water.

[0042] The sporulation rate was measured, and the results are shown in Table 1 below.

[0043] Example 3 The difference compared to Example 2 is as follows: The fermentation medium for fermentation culture consists of the following raw materials in the following mass percentages: 48 wt% gibberellic acid film residue, 8 wt% corn cob powder, 8 wt% glucose, 1 wt% urea, 1 wt% corn gluten powder, 1 wt% potassium dihydrogen phosphate, and the remainder is water.

[0044] Example 4 The difference compared to Example 2 is as follows: The fermentation medium for fermentation culture was: 50wt% gibberellic acid film residue, 12wt% corn cob powder, 12wt% glucose, 3wt% urea, 5wt% corn gluten meal, 3wt% potassium dihydrogen phosphate, and the remainder was water.

[0045] Comparative Example 1 The difference compared to Example 2 is as follows: The fermentation medium for fermentation culture was: 78 wt% gibberellic acid film residue, plate and frame bacterial residue, and 22 wt% water.

[0046] Comparative Example 2 The difference compared to Example 2 is as follows: The fermentation medium for fermentation culture was: 27.9 wt% corn cob powder, 27.9 wt% glucose, 5.6 wt% urea, 11.1 wt% corn gluten meal, 5.6 wt% potassium dihydrogen phosphate, and 22 wt% water.

[0047] Comparative Example 3 The difference compared to Example 2 is as follows: The fermentation medium for fermentation culture was: sawdust 27.9 wt%, glucose 27.9 wt%, urea 5.6 wt%, corn gluten meal 11.1 wt%, potassium dihydrogen phosphate 5.6 wt%, and water 22 wt%.

[0048] Comparative Example 4 The difference compared to Example 2 is as follows: The fermentation medium for fermentation culture is: 50wt% gibberellic acid film residue, 10wt% sawdust, 10wt% glucose, 2wt% urea, 4wt% corn gluten meal, 2wt% citric acid, 2wt% potassium dihydrogen phosphate, and 22wt% water.

[0049] Comparative Example 5 The difference compared to Example 2 is as follows: In S42, malic acid is used to replace citric acid, and the pH is adjusted to 4.5.

[0050] Comparative Example 6 The difference compared to Example 2 is as follows: In S42, the pH is not adjusted (i.e., the pH is around 7).

[0051] Comparative Example 7 The difference compared to Example 2 is as follows: In the fermentation medium used for fermentation culture, the glucose in Example 1 was replaced with the same amount of sucrose.

[0052] Table 1 The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A biocontrol Trichoderma harzianum fermentation culture medium utilizing waste by-products, characterized in that, include: The residue of gibberellic acid film plate and frame bacterial residue is 48wt%~52wt%, corn cob powder is 8wt%~12%, glucose is 8wt%~12%, urea is 1wt%~3%, corn gluten powder is 2wt%~6%, potassium dihydrogen phosphate is 1wt%~3%, and the balance is water.

2. The biocontrol Trichoderma harzianum fermentation culture medium utilizing waste by-products according to claim 1, characterized in that, The mixture consists of 50 wt% gibberellic acid film residue, 10 wt% corn cob powder, 10 wt% glucose, 2 wt% urea, 4 wt% corn gluten powder, 2 wt% potassium dihydrogen phosphate, and the remainder is water.

3. The biocontrol Trichoderma harzianum fermentation culture medium utilizing waste by-products according to claim 1, characterized in that, The residue of gibberellic acid film plate and frame bacterial residue is 48wt%, corn cob powder is 8wt%, glucose is 8wt%, urea is 1wt%, corn gluten powder is 1wt%, potassium dihydrogen phosphate is 1wt%, and the balance is water.

4. The biocontrol Trichoderma harzianum fermentation culture medium utilizing waste by-products according to claim 1, characterized in that, When fermenting the biocontrol Trichoderma harzianum on the fermentation medium, the pH is adjusted to 4.5-5.0 with citric acid.

5. A method for preparing a biocontrol agent for Trichoderma harzianum using waste by-products, characterized in that, Includes the following steps: S1, Trichoderma harzianum slant culture was used to obtain Trichoderma harzianum slant strain; S2, Trichoderma harzianum slant culture was inoculated into primary culture medium to obtain primary culture; S3, the primary seed is inoculated into the secondary seed culture medium to obtain the secondary seed; S4, fermentation culture, includes the following steps: S41, after the fermentation medium according to any one of claims 1-3 is mixed evenly, it is sterilized and cooled; S42, inoculate with secondary seed, mix thoroughly and adjust pH to 4.5-5.0 with citric acid; S43, the inoculated fermentation medium is pressed into fermentation medium blocks using a briquetting machine; S44, the fermentation medium blocks are placed on the rack in the culture room and fermented at a temperature of 27℃~28℃ and a humidity of 50%~58% to obtain fermented blocks; S45, after fermentation, is pressed into blocks, dried, crushed, sieved, and spores are collected to obtain Trichoderma harzianum inoculum.

6. The method for preparing biocontrol Trichoderma harzianum agent using waste by-products according to claim 5, characterized in that, In S1, the culture conditions are: static culture at 28℃ for 8 days; in S2, the culture conditions are: static culture at 28℃ in a 2L Erlenmeyer flask for 8 days; in S3, the culture conditions are: static culture at 28℃ in a sterilized iron tray for 8 days.

7. The method for preparing a biocontrol agent for Trichoderma harzianum using waste by-products according to claim 5, characterized in that, In S2, the primary culture medium consists of 20 wt% glucose, 10 wt% yeast extract, 20 wt% peptone, and 50 wt% water; in S3, the secondary culture medium consists of 30 wt% rice flour and 80 wt% water.

8. The method for preparing a biocontrol agent for Trichoderma harzianum using waste by-products according to claim 5, characterized in that, In S41, the sterilization conditions are: sterilization at 121℃ for 30 minutes; in S42, the inoculation amount is 1% to 5% of the total fermentation medium.

9. The method for preparing a biocontrol agent for Trichoderma harzianum using waste by-products according to claim 4, characterized in that, In S43, the size of 100 kg of fermentation medium after compression is 10 cm * 6 cm * 3 cm.

10. The method for preparing a biocontrol agent for Trichoderma harzianum using waste by-products according to claim 4, characterized in that, In S44, the fermentation time is 7 days to 15 days; in S45, the crushing conditions are: hammer mill crushing at a speed of 2500 r / min; and sieving by a 60-80 mesh vibrating screen.

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