Application of tobacco stem solid subjected to near-critical water treatment in trichoderma harzianum culture
By treating the tobacco stems with near-critical water and fermenting them with spore suspension, an efficient Trichoderma Harzian fungi agent was prepared, which solved the problem of tobacco waste resource utilization and achieved the improvement of resource recycling and mycelium generation.
Patent Information
- Application Number
- CN202411534591.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-30
- Publication Date
- 2024-12-13
AI Technical Summary
How to use the discarded tobacco tobacco stem resources to cultivate Trichoderma harzian agents, realize resource recycling, and solve the problems of waste and environmental pollution during tobacco cultivation.
The tobacco stem was treated by near-critical water, crushed and mixed with water to react under high temperature and high pressure conditions, and a solid tobacco stem was obtained as a fermentation matrix, and fermented in combination with the spore suspension of Trichoderma harziana spores to prepare the Trichoderma harziana agent. The method includes crushing the tobacco stems and mixing them with water in a certain proportion, treating them at high temperature and high pressure, then sterilizing and inoculating them with the spore suspension, and fermenting them to prepare a bacterial agent.
Recycling of resources has been achieved, and the spore production and mycelium generation of tobacco stems during fermentation is improved, which is better than the treatment of simple tobacco stems or flue-cured tobacco stems. The addition of wheat bran and rice bran is conducive to the production of mycelium, showing that it is close to 140℃. Advantages of critical water-treated tobacco stems in fermentation.
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Figure CN119120224A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of bacterial agent development, and in particular to application of tobacco stem solids treated with near-critical water in culturing Trichoderma harzianum bacterial agent. Background Art
[0002] Tobacco is a crop with high economic value. my country has a large tobacco planting area and a long history of planting. During its planting and processing, a large amount of waste such as tobacco stalks, unsuitable tobacco leaves and tobacco stems is produced, which not only wastes resources but also seriously pollutes the environment. With the development of the tobacco industry, how to give full play to the potential value of these wastes and make comprehensive use of them has become a hot topic for researchers in the tobacco industry and related fields.
[0003] Since it was discovered that Trichoderma has an inhibitory effect on plant pathogens, it has been receiving much attention as an important biological control factor for plant diseases. In recent years, with the successful development of a variety of commercial Trichoderma preparations, the research on the application of Trichoderma in the biological control of plant fungal diseases has developed rapidly, providing an effective exploration path for the practical feasibility of biological control of plant pathogenic fungi. For example, Trichoderma harzianum CGMCC23294 is a strain screened from the rhizosphere soil of tobacco that is highly resistant to parasitic phytophthora and significantly promotes plant growth. In 2021, an experiment was conducted in Lechang City, Shaoguan, Guangdong. The CGMCC23294 bacterial agent was used in plots with severe black shank disease all year round. The tobacco fields in the test area grew vigorously, while the control tobacco fields had a total harvest. The strain was used in 10,000 mu of tobacco-growing areas in Zhen'an and Luonan, Shangluo City, Shaanxi Province in 2021, 15,000 mu of tobacco-growing areas in Zhen'an and Luonan in 2022, and 1,000 mu each in Xuchang, Pingdingshan and Luoyang, Henan Province. The tobacco plants in the experimental group grew vigorously and had strong root systems. The average field control effect of black shank disease exceeded 60%, and tobacco leaf yield increased by 8% to 10%.
[0004] Based on the above situation, the technical problem to be solved by this application is whether waste tobacco can be used to cultivate Trichoderma harzianum, thereby realizing resource recycling. Summary of the invention
[0005] The purpose of the present invention is to provide a method for preparing Trichoderma harzianum agent by solid fermentation of tobacco stems, so as to realize the recycling of resources.
[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:
[0007] The invention provides an application of tobacco stem solids treated with near-critical water in culturing a Trichoderma harzianum agent.
[0008] Preferably, the process of treating the tobacco stems with near-critical water treatment is as follows: crushing the tobacco stems through a 5-15 mesh sieve, mixing with water at a weight ratio of 1:3-5 g / mL, and heating at 120-150°C.、 Pressure 0.2 ～ The reaction was carried out at 0.8 MPa for 12 to 17 minutes, and after the reaction, the solid and liquid were separated, and the solid obtained was the tobacco stem treated with near-critical water.
[0009] The present invention also provides a method for using the tobacco stem solids treated with near-critical water in preparing a Trichoderma harzianum agent, comprising the following steps:
[0010] (1) culturing Trichoderma harzianum to obtain a spore suspension;
[0011] (2) using the tobacco stems treated with the subcritical water as a fermentation substrate and sterilizing the tobacco stems;
[0012] (3) inoculating the spore suspension into a sterilized fermentation matrix to obtain a Trichoderma harzianum agent after fermentation.
[0013] Preferably, the culture in step (1) is to culture the activated Trichoderma harzianum in PDB medium for 3 to 5 days, and the concentration of the spore suspension is 2 to 4×10 9 cfu / mL.
[0014] Preferably, the sterilization temperature in step (2) is 120-122° C., and the sterilization time is 25-35 min.
[0015] Preferably, the spore suspension in step (3) is inoculated at 3-5% of the weight of the dry material, the fermentation temperature is 25-30° C., and the fermentation time is 3-7 days.
[0016] The invention also provides a Trichoderma harzianum agent obtained by the application method.
[0017] The invention also provides an application of the Trichoderma harzianum agent in preventing and controlling tobacco pathogenic bacteria.
[0018] The present invention utilizes near-critical water to treat tobacco stems for fermenting Trichoderma harzianum, and uses tobacco stems, treated tobacco stems, wheat bran, rice bran and the like as fermentation substrates. From the perspective of five-day fermentation, simple tobacco stem and post-roasting tobacco treatment are not conducive to the production of mycelium, while a culture medium with wheat bran and rice bran added is conducive to the production of mycelium, and tobacco stems treated with 140°C near-critical water are superior to other treatments; from the perspective of spore production, tobacco stems treated with 140°C near-critical water are more conducive to fermentation. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Figure 1 This is a column chart of spore production in each group of comparative example 1 on the fifth day of fermentation. DETAILED DESCRIPTION
[0020] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.
[0021] Example 1
[0022] A method for preparing a Trichoderma harzianum agent comprises the following steps:
[0023] S1: After the activation of the plate, Trichoderma harzianum was inoculated into PDB medium and cultured in the dark at 28°C for 4 days to obtain a spore suspension for use. The concentration of the spore suspension was 3×10 9 cfu / mL; PDB medium formula: 200g potato, 20g glucose and 1000mL distilled water.
[0024] S2: The tobacco stems are crushed and passed through a 10-mesh sieve, and placed in a high-temperature and high-pressure reactor with water at a weight ratio of 1:4 g / mL. The reaction is carried out at 0.4 Pa and 140°C for 14 minutes. After the reaction, the solid and liquid are separated, and the solid obtained is the tobacco stems treated with near-critical water at 140°C.
[0025] S3: The tobacco stems treated with near-critical water at 140°C were used as fermentation substrates. After sterilization at 121°C for 30 minutes, spore suspensions were added at 4% of the weight of the dry material, and fermented in a light-shielded incubator at 28°C for 5 days to obtain a Trichoderma harzianum agent.
[0026] Comparative Example 1
[0027] Different culture medium treatment groups were set according to Table 1. Example 1 was group T2. Untreated tobacco stems, roasted tobacco, wheat bran and rice bran were all sieved through a 10-mesh sieve. 50 g of each treatment was put into a 500 mL conical flask and evenly spread on the bottom of the conical flask with a thickness of 2 cm. The fermentation matrix was adjusted with sterile water, stirred evenly, and sterilized in a high-pressure steam sterilizer at 121° C. for 30 min.
[0028] Each treatment was repeated 3 times, and the spore suspension was inoculated at a rate of 4% of the weight of the dry material; the mixture was placed in a 28°C light-shielded incubator for fermentation for 5 days.
[0029] The results are as follows Figure 1 As shown in the figure, from the five-day fermentation situation, the simple tobacco stem (CK1) and cured tobacco (T1) treatments are not conducive to the production of mycelium, but the culture medium with added wheat bran and rice bran (T3, T5) is conducive to the production of mycelium, and the tobacco stem treated with 140℃ near-critical water (T2) is better than other treatments; from the perspective of spore production, the tobacco stem treated with 140℃ near-critical water is more conducive to fermentation.
[0030] Table 1 Culture medium formula for different treatment groups
[0031]
[0032] The above is only a preferred embodiment 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 principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.
Claims
1. Application of tobacco stem solids treated with near-critical water in the cultivation of Trichoderma harzianum.
2. The use according to claim 1, characterized in that: The treatment process of the tobacco stems treated with near-critical water is as follows: the tobacco stems are crushed through a 5-15 mesh sieve, mixed with water at a weight ratio of 1:3-5 g / mL, reacted at 120-180° C. and a pressure of 0.2-0.8 MPa for 12-17 minutes, and then the solid-liquid separation is performed to obtain the solid tobacco stems treated with near-critical water.
3. A method for using the near-critical water treated tobacco stem solids according to any one of claims 1 to 2 in the preparation of Trichoderma harzianum inoculum, characterized in that: The steps include: (1) culturing Trichoderma harzianum to obtain a spore suspension; (2) using the tobacco stems treated with the subcritical water as a fermentation substrate and sterilizing the tobacco stems; (3) inoculating the spore suspension into a sterilized fermentation matrix to obtain a Trichoderma harzianum agent after fermentation.
4. The method according to claim 3, characterized in that The culture in step (1) is to culture the activated Trichoderma harzianum in PDB medium for 3 to 5 days, and the concentration of the spore suspension is 2 to 4×10 9 cfu / mL.
5. The method according to claim 4, characterized in that The sterilization temperature in step (2) is 120-122° C., and the sterilization time is 25-35 min.
6. The method according to claim 5, characterized in that In step (3), the spore suspension is inoculated at 3-5% of the weight of the dry material, the fermentation temperature is 25-30° C., and the fermentation time is 3-7 days.
7. A Trichoderma harzianum agent obtained by the application method according to any one of claims 3 to 6.
8. Use of the Trichoderma harzianum agent according to claim 7 in preventing and controlling tobacco pathogens.