Method for separating and purifying active substances in trichoderma citrinoviride fermentation liquor
The fermentation broth of Trichoderma olefin fermentation broth was isolated and purified by ethyl acetate extraction and efficient preparation of liquid chromatography, which solved the problem of complex process and high cost, obtained high-purity antifungal active substances, and promoted in-depth research on the secondary metabolites of Trichoderma olefin olefin.
Patent Information
- Application Number
- CN202410156306.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-02-04
- Publication Date
- 2025-08-22
AI Technical Summary
In the prior art, the separation and purification process of Trichoderma oleracea fermentation broth is complex, costly, and cumbersome, and the functions of its secondary metabolites have not been studied in-depth.
The fermentation broth of Trichoderma vermicelli was separated and purified by ethyl acetate extraction and high-efficiency preparation liquid chromatography. The crude extract was obtained by ethyl acetate extraction. The C18 chromatography column was used and methanol and formic acid water were used as mobile phases for high-efficiency preparation and liquid chromatography separation. Combined with the high-performance liquid chromatography tandem mass spectrometer to detect the purity and analyze the molecular weight, and finally the components with the best inhibition effect were detected by nuclear magnetic hydrogen spectrum.
It realizes the simple separation and purification process, low cost and easy operation, and obtains high-purity antifungal active substances, which are suitable for further research and development of new drugs or pesticides.
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Figure CN120513982A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to a method for separating and purifying a fermentation liquid, in particular to a method for separating and purifying active substances in a fermentation liquid of Trichoderma citriviride. Background Art
[0002] Trichoderma is a common biocontrol strain with a broad spectrum of antagonistic properties, playing an important role in plant biocontrol. The biocontrol mechanisms of Trichoderma include competition, parasitism, antibiotics, and induction of plant resistance. Among them, antibiotics refer to the secretion of certain chemicals during the metabolism of Trichoderma to inhibit the growth of pathogens. Studies have found that Trichoderma harzianum, Trichoderma viride, Trichoderma hooked, and Trichoderma longibrachiatum can all produce substances such as trichoderma, antimicrobial peptides, colloidin, and trichoderma viride. Currently, Trichoderma fungi have been widely used in the prevention and treatment of various plant fungal diseases and are an ideal biocontrol fungus and plant growth promoting fungus. At present, a large number of studies have been conducted on the growth promotion mechanism and biocontrol mechanism of Trichoderma at home and abroad, but due to the differences between strains, the overall theoretical research is still in the stage of continuous improvement, and there are no in-depth reports on the functional research of its secondary metabolites.
[0003] Trichoderma citrinovirideJS84 can grow rapidly at 20-40℃, has strong antagonistic ability and high enzyme activity. Its Trichoderma bio-organic fertilizer can promote crop growth, increase yield and enhance fruit quality, improve soil fertility and soil biological activity, significantly increase the number of total bacteria and fungi in the soil, and reduce the number of Fusarium oxysporum, promoting sustainable soil production and sustainable agricultural development. However, no antagonistic secondary metabolites of Trichoderma citrinoviride have been reported.
[0004] Based on the above, the inventors found that:
[0005] The existing technology has complex processes, higher costs and cumbersome operations.
[0006] Therefore, in view of this, the existing structure was studied and improved, and a method for separating and purifying active substances in Trichoderma viride fermentation broth was proposed to solve the above-mentioned problems. Summary of the Invention
[0007] The present invention provides a method for separating and purifying active substances in Trichoderma citrivirides fermentation broth. Through the steps of extracting the Trichoderma citrivirides fermentation broth and purifying and analyzing the active substances in the Trichoderma citrivirides fermentation broth, the separation and purification process of the Trichoderma citrivirides fermentation broth is simple, low in cost, easy to operate, and the product purity is high.
[0008] In order to solve the above problems, the present invention proposes a technical solution: a method for separating and purifying active substances in Trichoderma viride fermentation broth, including the steps of extracting the Trichoderma viride fermentation broth and purifying and analyzing the active substances in the Trichoderma viride fermentation broth. The Trichoderma viride fermentation broth is extracted with ethyl acetate and concentrated under reduced pressure to obtain a crude extract. The purification and analysis steps use a high-efficiency preparative liquid phase for separation and purification, and methanol and formic acid water are selected in the purification and analysis steps.
[0009] Furthermore, the extraction steps of the Trichoderma virens fermentation broth are as follows:
[0010] Prepare a collection container: Choose a clean container to collect the fermentation liquid. Make sure the container has enough capacity to accommodate the liquid produced during the entire fermentation process.
[0011] Ensure sterile conditions: Before collecting the fermentation broth, ensure that the working environment and collection container are sterile. This can be achieved by using ultraviolet light on the work surface, spraying disinfectant, etc.
[0012] Collect the fermentation broth: Generally, the fermentation broth of Trichoderma virens will naturally flow out of the culture medium or accumulate at the bottom. Use gauze to filter and transfer the fermentation broth to a collection container.
[0013] Close the container: After collecting the fermentation liquid, immediately close the container to prevent external contamination. You can use a sterile stopper or aluminum foil to seal the container mouth;
[0014] Further processing: If further processing is required to separate and purify the target substance in the fermentation broth, the collected fermentation broth can be pretreated, such as solid-liquid separation and impurity removal.
[0015] Furthermore, the steps for purifying and analyzing the active substances in the Trichoderma viride fermentation broth are as follows:
[0016] S1. Extracting the fermentation broth of Trichoderma citriviride with the deposit number of CGMCC NO.17466 with ethyl acetate and concentrating under reduced pressure to obtain a crude extract;
[0017] S2, re-dissolving the crude extract obtained in step S1 with chromatographic methanol;
[0018] S3, filtering the sample in step S2 through a 0.22 μm filter membrane, and separating and purifying it using a high-efficiency preparative liquid phase;
[0019] S4. In step S3, the high performance preparative liquid chromatography column is a C18 column 5 μm (19×250 mm);
[0020] S5, high performance preparative liquid chromatography conditions in step S3;
[0021] S6. Methanol and 0.1% formic acid water in step S5, with a flow rate of 7 ml / min, UV detection wavelengths of 254 nm and 363 nm, and a column temperature of 30°C.
[0022] S7. The purity and molecular weight of each component of S7 obtained in step S6 were tested by high performance liquid chromatography tandem mass spectrometry.
[0023] S8. Detecting the inhibitory effects of the five components with higher purity in step S7 on Fusarium oxysporum.
[0024] S9. The component with the best inhibitory effect in step S8 is subjected to nuclear magnetic resonance (NMR) spectroscopy, and its structure is obtained by comparison with literature data.
[0025] Due to the adoption of the above technical solution, the beneficial effects of the method for separating and purifying active substances in Trichoderma viride fermentation broth of the present invention are as follows:
[0026] 1. Simple process: This method adopts the steps of ethyl acetate extraction and high-performance preparative liquid chromatography, which is relatively simple to operate and easy to implement and master.
[0027] 2. Low cost: Common reagents such as ethyl acetate and methanol used are relatively low in cost, which can reduce the cost of the separation and purification process.
[0028] 3. Easy to operate: The operating steps in the method are clear and simple, easy to operate, and do not require complex equipment and conditions, making it more convenient for experimenters to operate.
[0029] 4. High Product Purity: Using separation and purification techniques such as high-performance preparative liquid chromatography, the target active substances in the Trichoderma viride fermentation broth can be effectively isolated and purified. High-purity products have improved activity and stability, making them suitable for further applied research.
[0030] 5. Good extraction effect of antifungal active substances: After the extraction and separation and purification steps of this method, the target substance with high antifungal activity can be obtained. This is of great significance for the further research and development of new drugs or pesticides with fungal inhibitory effects.
[0031] In summary, the method for separating and purifying the fermentation broth of Trichoderma citriodora has the beneficial effects of simple process, low cost, easy operation and high product purity, which helps to improve the separation and purification efficiency and quality of antifungal active substances in the fermentation broth of Trichoderma citriodora. BRIEF DESCRIPTION OF THE DRAWINGS
[0032] Figure 1 It is the material structure with the best antibacterial effect.
[0033] Figure 2Figure 5 is a high performance preparative liquid chromatogram. Figures AG and AG are seven compounds separated from the crude fermentation broth extract by high performance preparative liquid chromatography.
[0034] Figure 3 Chromatographic purity test of 7 purified compounds. DETAILED DESCRIPTION
[0035] The present invention is further described in detail below with reference to examples. A method for separating and purifying active substances in a fermentation broth of Trichoderma viride, comprising the steps of extracting the fermentation broth of Trichoderma viride and purifying and analyzing the active substances in the fermentation broth of Trichoderma viride, wherein the fermentation broth of Trichoderma viride is extracted with ethyl acetate and concentrated under reduced pressure to obtain a crude extract, wherein the purification and analysis steps use a high-efficiency preparative liquid phase for separation and purification, wherein methanol, formic acid, and aqueous solution are used in the purification and analysis steps.
[0036] The extraction steps of the Trichoderma viride fermentation broth are as follows:
[0037] a. Prepare a collection container: Choose a clean container to collect the fermentation liquid. Make sure the container has enough capacity to accommodate the liquid produced during the entire fermentation process;
[0038] b. Ensure sterile conditions: Before collecting the fermentation broth, ensure that the working environment and collection container are in a sterile state. This can be achieved by using ultraviolet light on the work surface, spraying disinfectant, etc.
[0039] c. Collect the fermentation broth: Generally, the fermentation broth of Trichoderma virens will naturally flow out of the culture medium or accumulate at the bottom. Use gauze to filter and transfer the fermentation broth to a collection container;
[0040] d. Close the container: After collecting the fermentation liquid, immediately close the container to prevent external contamination. You can use a sterile stopper or aluminum foil to seal the container mouth;
[0041] e. Further processing: If further processing is required to separate and purify the target substance in the fermentation broth, the collected fermentation broth can be pretreated, such as solid-liquid separation and impurity removal.
[0042] The steps for purifying and analyzing the active substances in the Trichoderma viride fermentation broth are as follows:
[0043] S1. Extracting the fermentation broth of Trichoderma citriviride with the deposit number of CGMCC NO.17466 with ethyl acetate and concentrating under reduced pressure to obtain a crude extract;
[0044] S2, re-dissolving the crude extract obtained in step S1 with chromatographic methanol;
[0045] S3, filtering the sample in step S2 through a 0.22 μm filter membrane, and separating and purifying it using a high-efficiency preparative liquid phase;
[0046] S4. In step S3, the high performance preparative liquid chromatography column is a C18 column 5 μm (19×250 mm);
[0047] S5, high performance preparative liquid chromatography conditions in step S3;
[0048] S6. The methanol and 0.1% formic acid water in step S5 were separated at a flow rate of 7 ml / min, with UV detection wavelengths of 254 nm and 363 nm, and a column temperature of 30° C., wherein mobile phase A was methanol and mobile phase B was 0.1% formic acid water.
[0049] Table 1 Liquid phase separation method for preparation of Trichoderma viride metabolites
[0050] Time (min) Flow rate (ml / min) ％A ％B 0 7 15 85 30 7 35 65 105 7 65 35 125 7 75 25 150 7 70 30 170 7 80 20
[0051] S7. The purity and molecular weight of each component of S7 obtained in step S6 were tested by high performance liquid chromatography tandem mass spectrometry.
[0052] S8. Detecting the inhibitory effects of the five components with higher purity in step S7 on Fusarium oxysporum.
[0053] S9, the component with the best inhibitory effect in step S8 is detected by nuclear magnetic hydrogen spectrum, and the structure is obtained by comparison with literature data. Figure 1 .
[0054] Table 1
[0055]
[0056] Example 1, fermentation and extraction of Trichoderma citriodora
[0057] The JS84 strain of Trichoderma virens stored in a -80°C refrigerator was inoculated onto a PDA medium plate for activation. After culturing at 28°C for 2 days, fresh mycelium was picked for transfer and cultured at 28°C for 7-10 days to produce spores. The spores on the PDA medium were then washed with sterile water, the spore suspension was collected by filtration, and the concentration of the spore suspension was diluted to 1×10 6 mL-1. The diluted spore suspension was inoculated into 400 mL of PDB medium at a ratio of 1:100 (i.e., 4 ml) and incubated at 28°C, 140 rpm in a shaker for 6 days. After fermentation, the fermentation broth was filtered and collected for material extraction. The filtered fermentation broth was extracted with ethyl acetate at a ratio of 1:1, repeated three times, and the crude extract was obtained by rotary evaporation.
[0058] Example 2: Separation of crude extracts of Trichoderma citriodora by high performance preparative liquid chromatography
[0059] The crude extract was redissolved in methanol, filtered through a 0.22 μm filter membrane, and purified by high performance preparative liquid chromatography to collect 7 major compounds. Figure 2 It can be seen that the separation effects of the seven compounds are basically pure.
[0060] Example 3: Purity detection and molecular weight analysis after HPLC separation
[0061] The purity and molecular weight of the seven components separated by HPLC were analyzed by Agilent 6530Q-TOF L C / MS. Figure 3 It can be seen that the purity of samples A and C is not high, while the other samples are relatively pure; the molecular weights of samples B, D, E, F, and G are 206.0943, 192.0786, 248.1049, 496.2097, and 512.2046, respectively.
[0062] Example 4: Detection of the inhibitory effects of five high-purity compounds on Fusarium oxysporum
[0063] Five compounds were added to a 96-well plate containing PDB medium at concentrations of 60 ppm, 80 ppm, 100 ppm, and 120 ppm, and each well was inoculated with Fusarium oxysporum. A medium without compound was inoculated in the same manner as a positive control, and a blank medium was used as a negative control. Each treatment was repeated three times.
[0064] The mixture was placed in a 28°C incubator for 24 hours, and the OD value was measured using a microplate reader to determine the growth of the pathogen. As shown in Table 1, compound G had the lowest MIC value, indicating the most significant inhibitory effect against Fusarium oxysporum.
[0065] Example 5: Structural identification and data comparison of the component with the best antibacterial effect
[0066] The NMR spectra of component 1 with the lowest MIC value were measured using a Burker 500 MHz nuclear magnetic resonance spectrometer. The obtained data are as follows: 1 HNMR (500 MHz, Chloroform-d) δ7.57 (ddd, J = 15.0, 9.1, 4.0 Hz, OH), 7.39 (d, J = 15.0 Hz, OH), 7.33 (dd, J = 14.7, 11.0 Hz, OH), 6.41 (d, J = 14.9 Hz, OH), 6.14 (dd, J = 14.8, 7.1 Hz, OH), 4.78 (s, OH), 4.49 (s, 0H), 3.77 (s, OH), 1.90 (dd, J = 16.2, 6.1 Hz, 1H). The obtained data were compared with the literature, and the structure of the compound was finally determined.
[0067] The above description of the present invention and its embodiments is non-limiting. In short, if a person skilled in the art is inspired by the above description and designs a similar structure and embodiment to the technical solution without departing from the purpose of the present invention, they should fall within the scope of protection of the present invention.
Claims
1. A method for separating and purifying active substances in Trichoderma viride fermentation broth, characterized in that: The invention comprises the steps of extracting the fermentation broth of Trichoderma viride and purifying and analyzing the active substances in the fermentation broth of Trichoderma viride. The fermentation broth of Trichoderma viride is extracted with ethyl acetate and concentrated under reduced pressure to obtain a crude extract. The purification and analysis steps of the active substances in the fermentation broth of Trichoderma viride adopt high-efficiency preparative liquid phase for separation and purification. Methanol and formic acid water are used in the purification and analysis steps.
2. The method for separating and purifying active substances in a Trichoderma citriviride fermentation broth according to claim 1, wherein: The extraction steps of the Trichoderma viride fermentation broth are as follows: a. Prepare a collection container: Choose a clean container to collect the fermentation liquid. Make sure the container has sufficient capacity to accommodate the liquid produced during the entire fermentation process. b. Ensure sterile conditions: Before collecting the fermentation broth, ensure that the working environment and collection container are in a sterile state. This can be achieved by using ultraviolet light on the work surface, spraying disinfectant, etc. c. Collect the fermentation broth: Generally, the fermentation broth of Trichoderma virens will naturally flow out of the culture medium or accumulate at the bottom. Use gauze to filter and transfer the fermentation broth to a collection container; d. Close the container: After collecting the fermentation liquid, immediately close the container to prevent external contamination. You can use a sterile stopper or aluminum foil to seal the container mouth; e. Further processing: If further processing is required to separate and purify the target substance in the fermentation broth, the collected fermentation broth can be pretreated, such as solid-liquid separation and impurity removal.
3. The method for separating and purifying active substances in a Trichoderma citriviride fermentation broth according to claim 1, wherein: The steps for purifying and analyzing the active substances in the Trichoderma viride fermentation broth are as follows: S1. Extracting the fermentation broth of Trichoderma citriviride with the deposit number of CGMCC NO.17466 with ethyl acetate and concentrating under reduced pressure to obtain a crude extract; S2, re-dissolving the crude extract obtained in step S1 with chromatographic methanol; S3, filtering the sample in step S2 through a 0.22 μm filter membrane, and separating and purifying it using a high-efficiency preparative liquid phase; S4. In step S3, the high performance preparative liquid chromatography column is a C18 column 5 μm (19×250 mm); S5, high performance preparative liquid chromatography conditions in step S3; S6. Methanol and 0.1% formic acid water in step S5, with a flow rate of 7 ml / min, UV detection wavelengths of 254 nm and 363 nm, and a column temperature of 30°C. S7. The purity and molecular weight of each component of S7 obtained in step S6 were tested by high performance liquid chromatography tandem mass spectrometry. S8. Detecting the inhibitory effects of the five components with higher purity in step S7 on Fusarium oxysporum. S9. The component with the best inhibitory effect in step S8 is subjected to nuclear magnetic resonance (NMR) spectroscopy, and its structure is obtained by comparison with literature data.