Trichoderma fermentation liquor as well as preparation method and application thereof
By preparing and applying Trichoderma fermentation broth, the problem of insufficient secondary metabolites in medicinal plants is solved, the growth promotion of medicinal plants and the improvement of secondary metabolites content is achieved, and the needs of modern agriculture are met.
Patent Information
- Application Number
- CN202510432842.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-08
- Publication Date
- 2025-08-08
AI Technical Summary
The secondary metabolites are insufficient in the planting of existing medicinal plants, the traditional method of using Trichoderma spores is not significant, and the Trichoderma fermentation broth has not been effectively utilized.
Provide a method for preparing a Trichoderma fermentation broth, including culturing Trichoderma spore liquid in PDB culture medium, producing a variety of metabolites such as plant hormones, enzymes and organic acids, and obtaining a fermentation broth by fermentation culture and filtration, for spraying leaves or irrigation root application to promote the growth of medicinal plants.
Significantly increase the biomass and secondary metabolites content of medicinal plants, improve the yield and quality of medicinal plants, and meet the needs of modern agriculture.
Smart Images
Figure CN120442718A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of microbial technology, and in particular to a Trichoderma fermentation liquid, a preparation method and an application thereof. Background Art
[0002] Trichoderma spp. is a filamentous fungus found widely in nature. It is an important functional strain of bio-organic fertilizer, widely distributed in soil, air, litter, and various fermented products. Its fermentation produces plant hormones, which provide trace elements for plant growth, thereby promoting plant growth. Trichoderma has important economic value and is widely used in various aspects of agriculture.
[0003] Asian mint (Mentha arvensis) is a perennial aromatic herb in the genus Mentha, Lamiaceae. Mint is a common Chinese medicinal herb with medicinal properties such as dispelling wind-heat, dispelling wind and swelling, and relieving depression and regulating qi. Artemisia annua L. is an annual herb in the genus Artemisia in the Asteraceae family. Artemisinin, a sesquiterpene lactone oxide containing a peroxide bridge extracted from its aerial parts, is currently the most widely used and most effective antimalarial drug, particularly effective against cerebral malaria and chloroquine-resistant malaria.
[0004] Medicinal plants produce a variety of metabolites through complex metabolic pathways, which are primarily divided into primary and secondary metabolites. Primary metabolites are essential for plant growth and development, including amino acids, lipids, and organic acids. Secondary metabolites are numerous, with thousands, if not tens of thousands, of compounds reported. These compounds primarily include flavonoids, alkaloids, and terpenes. These compounds possess a wealth of biological activities and participate in physiological processes such as cell signaling and regulation of auxin signaling during the growth and development of medicinal plants.
[0005] Secondary metabolites from medicinal plants hold broad application prospects both now and in the future. Currently, artemisinin and menthol are primarily extracted from plants, but their low concentrations in plants, limited by plant supply and extraction efficiency, cannot meet market demand. Excessive use of fertilizers and pesticides to induce growth leads to ecological and environmental problems such as water and soil pollution, pesticide residues, and frequent plant diseases. Therefore, a major bottleneck in the industrialization of secondary metabolites, and a hot topic of research both domestically and internationally, has focused on the key issue of how to effectively increase their concentrations in plants.
[0006] Trichoderma, a beneficial soil bacterium, promotes plant growth and prevents pests and diseases. Trichoderma preparations have been widely used in crop disease prevention (e.g., the publicly available spore root irrigation method), but they have the following limitations in medicinal plant cultivation:
[0007] 1. Traditional application methods (seed treatment, soil irrigation) have no significant effect on the accumulation of secondary metabolites;
[0008] 2. The traditional application method only uses the spores of Trichoderma, and the Trichoderma fermentation liquid is not effectively utilized. Summary of the Invention
[0009] In view of the above-mentioned shortcomings of the prior art, the technical problems to be solved by the present invention are insufficient secondary metabolites produced by existing medicinal plant cultivation and the fact that existing Trichoderma applications are limited to Trichoderma spores. The present invention provides a Trichoderma fermentation liquid, a preparation method, and its application, which can fully utilize Trichoderma resources, significantly increase the biomass of medicinal plants and their active ingredients, and effectively promote the growth of medicinal plants.
[0010] To achieve the above objectives, the present invention provides a Trichoderma fermentation broth comprising a variety of metabolites, including plant hormones, enzymes, organic acids, and antibiotics. The broth produces a variety of plant hormones, including indoleacetic acid (IAA), zeatin (ZA), and gibberellin (GA), which can promote plant growth. In PDB medium, after culturing at 28°C and 150 rpm for 5-7 days, the hormone contents range from IAA (15-120 μg / L); GA3 (5-50 μg / L); zeatin (2-25 μg / L), and ABA (10-80 μg / L).
[0011] In a preferred embodiment of the present invention, a method for preparing a Trichoderma fermentation broth is provided, wherein a Trichoderma spore liquid is prepared and activated, a PDB culture medium is used as a substrate, the activated Trichoderma spore liquid is inoculated, and the Trichoderma fermentation broth is obtained after fermentation, culture, and filtration.
[0012] Further, preparing the Trichoderma spore liquid and activating it specifically includes:
[0013] The Trichoderma was spotted on a PDA plate and placed in a constant temperature incubator at 28°C for 5 days. The spores were washed with sterile water and filtered with two layers of sterile gauze. After centrifugation, the spore count was diluted with sterile water to 10. 6 CFU / L, obtain spore liquid.
[0014] Furthermore, the Trichoderma used to prepare the Trichoderma spore liquid includes one or more of Trichoderma harzianum, Trichoderma viride, Trichoderma longibrachiatum, and Trichoderma asperellum.
[0015] Furthermore, the activated Trichoderma spore liquid was inoculated into the PDB culture medium as a substrate, specifically, 1 mL of the activated Trichoderma spore liquid was inoculated into every 100 mL of the culture medium.
[0016] Furthermore, the fermentation was carried out in a shaking incubator at 28°C and 150 r / min for 5 days, and the fermentation broth was obtained after coarse filtration through a double-layer gauze and fine filtration through a 0.22 μm filter membrane.
[0017] In another preferred embodiment of the present invention, a Trichoderma fermentation broth is used to promote the biomass of medicinal plants by spraying it on leaves of medicinal plants.
[0018] In another preferred embodiment of the present invention, a Trichoderma fermentation broth is used in secondary metabolites and applied to mint seedlings by root irrigation.
[0019] Furthermore, the Trichoderma fermentation broth includes Trichoderma fermentation broth stock solution and Trichoderma fermentation broth with different dilution ratios, including Trichoderma fermentation broth diluted 100 to 300 times.
[0020] Technical Effects
[0021] The Trichoderma fermentation liquid, preparation method and application provided by the present invention can well promote the growth of medicinal plants and effectively increase plant yield; the present invention can also promote the increase in the content of secondary metabolites of medicinal plants and improve the quality of medicinal plants, has great application potential in production, and meets the needs of modern agriculture.
[0022] The concept, specific structure and technical effects of the present invention will be further described below in conjunction with the accompanying drawings to fully understand the purpose, characteristics and effects of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] Figure 1 This is the effect of different treatments of Trichoderma fermentation broth on the artemisinin content in a preferred embodiment of the present invention (the values are expressed as the average of 10 independent experiments; the error bars represent the standard deviation);
[0024] Figure 2 This is the effect of different treatments of Trichoderma fermentation broth on the growth rate of artemisinin in a preferred embodiment of the present invention;
[0025] Figure 3 This is the effect of different treatments of Trichoderma fermentation broth on the content of dihydroartemisinic acid in a preferred embodiment of the present invention (the values are expressed as the average of 10 independent experiments; the error bars represent the standard deviation);
[0026] Figure 4 This is the effect of different treatments of Trichoderma fermentation broth on the content of artemisinic acid in a preferred embodiment of the present invention (the values are expressed as the average of 10 independent experiments; the error bars represent the standard deviation);
[0027] Figure 5This is the effect of the Trichoderma agent of a preferred embodiment of the present invention on the biological properties of mint (the values are expressed as the average of 5 independent experiments; the error bars represent the standard deviation);
[0028] Figure 6 This is the effect of a 100-fold diluted Trichoderma fermentation broth on the menthol content according to a preferred embodiment of the present invention (the values are expressed as the average of three independent experiments; the error bars represent the standard deviation). DETAILED DESCRIPTION
[0029] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention more clearly understood, the present invention is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.
[0030] Example 1
[0031] The method for preparing fermentation broth by Trichoderma aspergillus GDFS1009 strain comprises the following steps:
[0032] Use PDA as the activation medium, which contains 5g / L potato extract powder, 10g / L peptone, 15g / L glucose, 5g / L sodium chloride, and 15g / L agar, and pour it into the culture dish after sterilization. The preserved Trichoderma aspergillus GDFS1009 strain was streaked onto the PDA medium, cultured at 28°C for 5 days, added with 2mL sterile water, and filtered to prepare a spore suspension. Specifically, Trichoderma aspergillus GDFS1009 was inoculated on the PDA plate, placed in a constant temperature incubator at 28°C for 5 days, the spore liquid was washed with sterile water, and filtered with two layers of sterile gauze. After centrifugation, the spore count was adjusted to 10 with sterile water. 6 CFU / L, and obtain the spore liquid of Trichoderma aspergillus GDFS1009.
[0033] PDB culture medium was weighed, ddH2O was added to dissolve it, and the culture medium was sterilized at high temperature. After cooling, the spore suspension was inoculated at a volume ratio of 1%, that is, 1 mL of activated Trichoderma GDFS1009 spore liquid was inoculated into every 100 mL of culture medium. The culture was shaken at 28°C and 150 r / min for 5 days. After the fermentation was completed, the fermentation liquid was coarsely filtered through a double-layer gauze and finely filtered through a 0.22 μm filter membrane to obtain the Trichoderma fermentation liquid.
[0034] The Trichoderma fermentation broth in this embodiment includes primary metabolites (such as organic acids 0.1-0.5%, sugars 0.5-1.5%), secondary metabolites (antibiotics, enzymes), plant hormones, cell components (mycelium 1.5-3.5%, spores) other active substances (volatile organic matter 0.01-0.1%, exopolysaccharides 0.2-1%) and residual culture medium components.
[0035] After culturing Trichoderma in PDB medium at 28°C and 150 rpm for 5-7 days, a variety of metabolites are produced, which can be detected by HPLC and LC-MS / MS. Excluding residual components in the medium, the major components include bacterial cells, sugars, and organic acids. Trichoderma metabolism produces a variety of plant hormones, which can effectively promote plant growth.
[0036] Example 2
[0037] The fermentation liquid of Trichoderma aspergillus GDFS1009 strain is used to promote the production of secondary metabolites of Artemisia annua by spraying the fermentation liquid and mycelium on leaves in the following steps:
[0038] (1) From the same batch of sown Artemisia annua, 35 seedlings with similar stem length and leaf size were selected for subsequent experiments;
[0039] (2) The fermentation product of Example 1 was diluted 100-fold to obtain a diluted fermentation broth. The diluted fermentation broth, the original fermentation broth, and the ground mycelium were sprayed on leaves, with 5 mL sprayed per seedling. Each treatment was repeated 10 times, and clear water was used as a control.
[0040] (3) On days 3 and 7, the same position of the leaves of Artemisia annua were taken, dried at 37°C, ground, and weighed to obtain 0.1 g. 1.5 mL of methanol was added for ultrasonic extraction. After centrifugation and filtration, the contents of artemisinin, artemisinic acid, and dihydroartemisinic acid were measured by HPCL.
[0041] Investigation records
[0042] The changes in artemisinin content and growth rate in the seedlings treated with spraying were statistically analyzed on days 3 and 7. Figure 1 and Figure 2 It can be seen that the treatment of Trichoderma fermentation broth and mycelium can increase the content of artemisinin, and the effect is significant on the 7th day. Figure 3 Figure 4 , it can be seen that the fermentation broth and mycelium treatment of Trichoderma can also increase the content of artemisinic acid and dihydroartemisinic acid. Intersecting with mycelium, the number of mycelium is small and the steps of crushing and resuspending are required, while the fermentation broth in the embodiment of the present invention is easier to obtain and the operation is also convenient.
[0043] Example 3
[0044] The application of Trichoderma aspergillus GDFS1009 strain in promoting the growth of mint is done by root irrigation, and the steps are as follows:
[0045] (1) After the mint seedlings have taken root, select seedlings with similar stem length and leaf size for subsequent experiments;
[0046] (2) Take the spore count in Example 1 as 10 6CFU / L of Trichoderma aspergillus GDFS1009 suspension was used to water mint at a rate of 10 ml per plant.
[0047] The application of Trichoderma aspergillus GDFS1009 strain in promoting the production of secondary metabolites in mint is achieved by spraying the fermentation liquid on the leaves. The steps are as follows:
[0048] (1) After the mint seedlings have taken root, select a batch of 6 large seedlings with an average stem length of 30 cm and 6 small seedlings with an average stem length of 10 cm;
[0049] (2) Take the fermentation product of Example 1, dilute it 100 times, and spray it on the leaves. Spray 5 mL per seedling in 3 replicates of each of the large and small seedlings. Use clear water as a control.
[0050] (3) On days 3 and 7, 1.5-2 g of mint leaves from the same position were collected, and three seedlings were mixed into one sample. After grinding with liquid nitrogen, 10 times the volume of anhydrous ethanol was added. Ultrasonic extraction was performed, and the supernatant was collected after centrifugation. Anhydrous NaSO4 was added overnight, and the menthol content was measured by LC-MS.
[0051] Investigation records
[0052] After 20 days, the biological characteristics of the seedlings treated with root irrigation were counted, and the stem length, root length and root weight were measured. Figure 5 It can be seen that the stem length, root length and root weight of the Trichoderma treatment were better than those of the water treatment.
[0053] The secondary metabolite content of the sprayed seedlings was counted on days 3 and 7. Figure 6 It can be seen that Trichoderma fermentation broth can increase the menthol content, and the effect on large seedlings is more significant.
[0054] The Trichoderma fermentation liquid, preparation method and application provided by the present invention can well promote the growth of medicinal plants and effectively increase plant yield; the present invention can also promote the increase in the content of secondary metabolites of medicinal plants and improve the quality of medicinal plants, has great application potential in production, and meets the needs of modern agriculture.
[0055] The above describes in detail the preferred embodiments of the present invention. It should be understood that those skilled in the art can make numerous modifications and variations based on the concepts of the present invention without inventive effort. Therefore, any technical solutions that can be derived by those skilled in the art through logical analysis, reasoning, or limited experimentation based on the concepts of the present invention and the prior art should be within the scope of protection defined by the claims.
Claims
1. A Trichoderma fermentation broth, characterized in that It includes a variety of metabolites, including plant hormones, enzymes, organic acids, antibiotics, etc.; among them, plant hormones include 15-120μg / L IAA, 5-50μg / L GA3, 2-25μg / L zeatin and 10-80μg / L ABA.
2. A method for preparing a Trichoderma fermentation broth, characterized in that: The Trichoderma spore liquid is prepared and activated, and the activated Trichoderma spore liquid is inoculated into a PDB culture medium as a substrate, and the Trichoderma fermentation liquid is obtained after fermentation, cultivation, and filtration.
3. The method for preparing a Trichoderma fermentation broth as claimed in claim 2, wherein: The method comprises the following steps: preparing a Trichoderma spore solution and activating the solution, specifically comprising: The Trichoderma was spotted on a PDA plate and placed in a constant temperature incubator at 28°C for 5 days. The spores were washed with sterile water and filtered with two layers of sterile gauze. After centrifugation, the spore count was diluted with sterile water to 10. 6 CFU / L, obtain spore liquid.
4. The method for preparing a Trichoderma fermentation broth as claimed in claim 2, wherein: The Trichoderma used to prepare the Trichoderma spore liquid includes one or more of Trichoderma harzianum, Trichoderma viride, Trichoderma longibrachiatum and Trichoderma asperellum.
5. The method for preparing a Trichoderma fermentation broth as claimed in claim 2, wherein: The activated Trichoderma spore liquid was inoculated into the PDB culture medium as a substrate, specifically, 1 mL of the activated Trichoderma spore liquid was inoculated into every 100 mL of the culture medium.
6. The method for preparing a Trichoderma fermentation broth according to claim 5, wherein: The fermentation was carried out in a shaking incubator at 28°C and 150 r / min for 5 days. The fermentation broth was coarsely filtered through a double-layer gauze and then finely filtered through a 0.22 μm filter membrane to obtain the fermentation broth.
7. An application of Trichoderma fermentation broth in promoting the biomass of medicinal plants and increasing the content of secondary metabolites, characterized in that: Spray on the leaves of medicinal plants.
8. The use according to claim 7, characterized in that The Trichoderma fermentation liquid includes the original Trichoderma fermentation liquid and the Trichoderma fermentation liquid with different dilution ratios, including the Trichoderma fermentation liquid diluted 100 to 300 times.