Culture medium for industrially producing trichoderma asperellum TCS007 crude extract and culture process thereof

Abstract

The invention relates to a culture medium formula for industrially producing a trichoderma asperellum TCS007 crude extract, which comprises rice, soybean meal and water, the weight ratio of the rice to the soybean meal is 100: (1-5), and the weight ratio of the rice to the water is 3: (1-3); the initial pH value of the culture medium is 6.0 to 8.0. By adopting the culture medium and culture process conditions provided by the invention, the fermentation production cost can be reduced, the yield of the trichoderma asperellum TCS007 crude extract and the antibacterial effect can be improved, and the requirements of industrial production and market popularization and application can be met.

Description

Technical Field

[0001] The invention relates to the field of biological strain cultivation, in particular to a culture medium for industrial production of a crude extract of Trichoderma aspergillus TCS007, and to optimization of a culture medium formula and fermentation process conditions. Background Art

[0002] Trichoderma asperellum is a new type of biocontrol fungus that can effectively colonize in the rhizosphere of plants. In the process of interacting with plants, it can secrete secondary metabolites with antibacterial and plant growth-promoting functions, regulate the structure of plant roots, and thus improve the efficiency of plant nutrient absorption. Trichoderma asperellum is widely distributed and can survive in adverse conditions such as drought and salinity. There are few related products, and the development prospects are broad.

[0003] Trichoderma asperellum TCS007 was isolated from Antarctic marine sediments, identified and named after mutagenesis and stabilization, and has been deposited in the General Microbiological Center of China Microbiological Culture Collection Administration (CGMCC), with the deposit number CGMCC No.15677, and was first published in patents ZL201810935616.6 and ZL201810936028.4. Trichoderma asperellum TCS 007 grows faster on PDA plates, and it takes about 3 days to grow a 9 cm culture dish at 26°C. The hyphae is moderately thick, felt-like, white and flat in the early stage, and produces crude extracts in dark green concentric circles in the later stage. Clustered crude extracts are formed on CMD plates. The crude extract has opposite stalks, the main branches are tree-like, the phialides are short, the base is thin, the middle is swollen, and the shape is ampelous. The crude extract is 3.4-5.0μm long and 2.8-4.0μm wide, spherical, subspherical or ovoid, with a rough surface and covered with small thorns.

[0004] In previous studies, the research group of the inventor of this application found that the inhibition rate of Trichoderma spinulosum TCS007 on 16 plant pathogenic fungi was

[0005] 56.65%~87.62%, among which the inhibition rate of wheat take-all pathogen, rice blast pathogen, banana leaf spot pathogen, cucumber brown spot pathogen, and pecan dry rot pathogen was greater than 80%. At the same time, a rice culture medium (80g rice + 120mL distilled water) for the production of secondary metabolites of Trichoderma aspergillus TCS007 that can be used for laboratory cultivation was found. The crude extract obtained by ethyl acetate extraction after fermentation had an inhibition rate of 95.8% on rapeseed sclerotinia pathogen (see Zheng Kebin, Antibacterial, Growth-promoting and Stress-resistant Effects of Trichoderma aspergillus TCS007 in Marine Habitats, Master's thesis of Zhejiang A&F University, online publication time 2021-01-16~2021-02-15):

[0006] Based on this, the applicant further studied the culture medium and its culture process for industrial production of conidia of Trichoderma aspergillus TCS007 (see patent CN20241087287.8). The culture medium screened in this patent can reduce the fermentation production cost and increase the number of conidia of Trichoderma aspergillus TCS007. The conidia can meet the requirements of industrial production and market promotion and application. However, in the subsequent test of secondary metabolites produced by Trichoderma aspergillus TCS007, it was found that the crude extract yield and activity produced by wheat bran culture medium were lower than those produced by rice culture medium. Therefore, rice culture medium with higher crude extract yield and activity was selected as the initial culture medium for further experiments in the hope of obtaining a formula and fermentation conditions for high-yield and high-activity crude extracts.

[0007] Culture medium used:

[0008] Wheat bran medium after spore production optimization: 25 g wheat bran, 5 g soybean powder, 0.6 g each of MgS04 and MnS04, 39.7 mL distilled water, initial pH value of 5.0;

[0009] Initial rice culture medium: 72 g rice, 48 mL distilled water, pH 7.0;

[0010] PDA medium: anhydrous glucose 20.0 g / L, potato 200.0 g / L, agar powder 18.0 g / L, distilled water 1 L, pH 7.0;

[0011] However, the secondary metabolites produced by the biocontrol strains during the stable growth period have a certain inhibitory effect on pathogenic fungi, so obtaining the desired target product has strict requirements on the control of the strain growth period. Because microbial fermentation is the basis for obtaining a large number of target products, the components of the fermentation medium and the fermentation conditions (time, temperature, initial pH, inoculation amount and photoperiod time, etc.) are different for the desired target products, and optimizing the conditions can significantly affect the yield of the target product of the strain. In addition to considering the yield of the target product in the microbial fermentation process, the fermentation cost and the activity of the final product should be comprehensively analyzed and evaluated to screen and obtain the best fermentation system suitable for the fermentation and application of the target strain. Based on this, the purpose of optimizing the culture medium and the culture process conditions of the present invention is to improve the yield and antibacterial activity of the crude extract produced by the fermentation of Trichoderma spinulosa TCS007. Summary of the invention

[0012] The technical problem to be solved by the present invention is to screen a culture medium formula that can be used for the industrial production of crude extracts of Trichoderma aspergillus TCS007, and to meet the needs of industrial mass production of crude extracts of Trichoderma aspergillus TCS007 by optimizing fermentation process conditions.

[0013] One of the purposes of the present invention is to provide the use of rice and glucose as fermentation substrates in a culture medium for increasing the yield of a crude extract of Trichoderma aspergillus TCS007; preferably, the weight ratio of the rice to the glucose is 100:2-5.

[0014] One of the purposes of the present invention is to provide the use of soybean powder and / or yeast powder and rice as a fermentation substrate in a culture medium for increasing the yield of a crude extract of Trichoderma aspergillus TCS007; preferably, the weight ratio of the rice to the soybean powder and / or yeast powder is 100:1-5. Preferably, the weight ratio of the rice to the soybean powder is 100:1-5; preferably, the weight ratio of the rice to the yeast powder is 100:1-5; preferably, the weight ratio of the rice to the soybean powder and yeast powder combination is 100:1-5.

[0015] One of the purposes of the present invention is to provide the use of sodium chloride and / or anhydrous ferric chloride to inhibit the yield of crude extract of Trichoderma aspergillus TCS007.

[0016] One of the purposes of the present invention is to provide a culture medium for industrial production of a crude extract of Trichoderma aspergillus TCS007, the culture medium comprising rice, soybean powder and water, the weight ratio of the rice to the soybean powder being 100:1-5, the weight ratio of the rice to the water being 3:1-3. Preferably, the initial pH value of the culture medium is 6.0-8.0, preferably, the initial pH value of the culture medium is 7.0-8.0.

[0017] One of the purposes of the present invention is to provide a culture medium for industrial production of crude extract of Trichoderma aspergillus TCS007, a process formula and conditions for producing highly active crude extract of Trichoderma aspergillus TCS007:

[0018] The light cycle of fermentation culture was 12 h light + 12 h dark, 24 h light + 0 h dark;

[0019] and / or, the fermentation culture time is 21-35 days;

[0020] and / or, the fermentation culture temperature is 25-31°C;

[0021] and / or, 1×10 7 The volume and mass ratio of the inoculation amount of Trichoderma spinulosa TCS007 seed solution per mL to the weight of rice in the culture medium is 0.5-2.5 mL:50-200 g.

[0022] Preferably, the fermentation culture light cycle is 12h light + 12h dark;

[0023] Preferably, the fermentation culture time is 25-30 days; particularly preferably, the fermentation culture time is 29 days;

[0024] Preferably, the fermentation temperature is 28°C; 1×10 7 The volume and mass ratio of the inoculation amount of Trichoderma spinulosa TCS007 seed solution and the weight of rice in the culture medium is 1mL:72g.

[0025] One of the purposes of the present invention is to provide a method for optimizing the fermentation conditions of crude extract of Trichoderma aspergillus TCS007, and to optimize the fermentation medium formula and fermentation culture conditions of Trichoderma aspergillus TCS007 by single factor experiment combined with response surface methodology;

[0026] The single factor test includes optimizing the medium single factor and optimizing the fermentation conditions single factor by using a progressive method based on the initial fermentation medium;

[0027] Based on the results of the single factor test, several factors with significant influence are selected, and the experimental design is carried out on the selected factors according to the Placket-Burman Design method;

[0028] Based on the screening results of the key factors of culture medium and culture conditions, several most critical influencing factors were screened out, and the steepest climbing test was designed; the climbing direction and step length of the key factors were determined according to the coefficients of each variable in the model, and the optimal range of the key factors was determined, and the maximum point of the maximum yield of the crude extract of Trichoderma spinulosa TCS007 fermentation obtained was used as the center point of the central combination design experiment;

[0029] The response surface method includes designing a central composite test with several factors and several levels using the Box-Behnken response surface method to optimize key factors, wherein the response factor Y2 is the crude extract of Trichoderma aspergillus TCS007 fermentation, and the test results are analyzed to predict the key factor parameters corresponding to the maximum yield of the crude extract of Trichoderma aspergillus TCS007 fermentation.

[0030] Preferably, the fermentation medium formula is: rice, soybean powder and water;

[0031] Preferably, based on the weight of the rice, the weight percentage of the soybean powder is 3.5% of the weight of the rice, and the amount of water added is 47 / 72 ml / g of the rice;

[0032] Further preferably, the content of rice is 72g, the content of soybean powder is 3.5%, and the amount of water added is 47mL; preferably, the fermentation culture conditions are: the initial pH value is 7.0, the inoculation amount is 1mL (1×10 7 / mL Trichoderma spinulosa TCS007 seed solution), light cycle time was 12h, culture temperature was 28℃, and culture time was 29d.

[0033] Beneficial technical effects of the technical solution of the present invention

[0034] The present invention provides for the first time a culture medium formula that can be used for industrial production of a crude extract of Trichoderma aspergillus TCS007, and finds that glucose and rice can be used as fermentation substrates in a culture medium for increasing the yield of a crude extract of Trichoderma aspergillus TCS007; finds that soybean powder and / or yeast powder and rice can be used as fermentation substrates in a culture medium for increasing the yield of a crude extract of Trichoderma aspergillus TCS007; and sodium chloride and / or anhydrous ferric chloride have the use of inhibiting the yield of a crude extract of Trichoderma aspergillus TCS007.

[0035] The present invention verifies the optimized culture process for the first time by combining a single factor experiment with a response surface optimization experiment and a steepest climbing test model. The fermentation medium formula is as follows: the rice content is 72g, the soybean powder content is 3.5%, and the water addition amount is 47mL; the fermentation culture conditions are as follows: the initial pH value is 7.0, the light cycle time is 12h, the culture temperature is 28°C, the culture time is 29d, and the inoculum amount is 1mL (1×10 7 The method can not only reduce the fermentation production cost, but also increase the yield of crude extract of Trichoderma aspergillus TCS007, especially significantly improve the antibacterial activity, and can be used for the industrial production and popularization of crude extract of Trichoderma aspergillus TCS007.

[0036] Based on 72g of rice, under the culture conditions of the initial fermentation medium, the yield of the crude extract of Trichoderma aspergillus TCS007 obtained by culture and fermentation is 0.9g; after optimization by the response surface method, the yield of the crude extract of Trichoderma aspergillus TCS007 obtained by culture and fermentation is 1.21g, which is increased by 34.44%. Further optimization is performed by the shallow tray fermentation method, and the yield of the crude extract of Trichoderma aspergillus TCS007 obtained by culture and fermentation using the fermentation medium and fermentation culture process provided by the present invention reaches 1.45g; compared with before optimization, it shows a better optimization effect and can meet the industrialization requirements.

[0037] The present invention firstly finds that under the optimized fermentation medium and culture conditions, the antibacterial activity of the crude extract of Trichoderma aspergillus TCS007 on rapeseed sclerotia is significantly improved compared with the crude extract of Trichoderma aspergillus TCS007 before optimization. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 Effects of different carbon sources on the yield of crude extract of Trichoderma aspergillus TCS007;

[0039] Figure 2 Effects of different nitrogen sources on the yield of crude extract of Trichoderma aculeatus TCS007;

[0040] Figure 3 Effects of different glucose contents on the yield of crude extract of Trichoderma aspergillus TCS007;

[0041] Figure 4 Effects of different soybean powder contents on the yield of crude extract of Trichoderma aspergillus TCS007;

[0042] Figure 5 Effects of different inorganic salts on the yield of crude extract of Trichoderma acanthosporum TCS007;

[0043] Figure 6 Effects of different water addition amounts on the yield of crude extract of Trichoderma aspergillus TCS007;

[0044] Figure 7 Effects of different inoculation amounts on the yield of crude extract of Trichoderma aspergillus TCS007;

[0045] Figure 8 Effects of different initial pH values ​​on the yield of crude extract of Trichoderma aspergillus TCS007;

[0046] Fig. 9 Effects of different culture times on the yield of crude extract of Trichoderma aspergillus TCS007;

[0047] Fig.10 Effects of different culture temperatures on the yield of crude extracts of Trichoderma aspergillus TCS007;

[0048] Fig.11Effects of different photoperiods on the yield of crude extracts of Trichoderma aspergillus TCS007;

[0049] Fig.12 The corresponding contour map and surface map of the effect of water addition and culture time on the crude extract yield of Trichoderma aspergillus TCS007;

[0050] Fig.13 The corresponding contour map and surface map of the effect of culture time and soybean powder on the crude extract yield of Trichoderma aspergillus TCS007;

[0051] Fig.14 The corresponding contour map and surface map of the effect of water addition and soybean powder on the yield of crude extract of Trichoderma aspergillus TCS007;

[0052] Fig.15 Schematic diagram of Trichoderma aspergillus TCS007 fermentation under the fermentation conditions of the initial fermentation medium;

[0053] Fig.16 Schematic diagram of shallow tray fermentation of Trichoderma aspergillus TCS007 under the fermentation conditions of the optimal culture medium provided by the present invention; DETAILED DESCRIPTION

[0054] The initial test culture medium selected for studying the technical solution of the present invention is as follows:

[0055] Patent CN20241087287.8 Wheat bran medium after spore production optimization: 25g wheat bran, 5g soybean powder, 0.6g each of MgS04 and MnS04, 39.7mL distilled water, initial pH value of 5.0;

[0056] PDA medium: anhydrous glucose 20.0 g / L, potato 200.0 g / L, agar powder 18.0 g / L, distilled water 1L, pH 7.0;

[0057] Initial fermentation medium and fermentation process conditions: 72 g rice, 48 mL distilled water, culture conditions were 28 ° C dark culture for 21 days, initial pH 7.0, inoculation size 1×10 7 / mL Trichoderma spinulosa TCS007.

[0058] Test methods

[0059] Activation of Trichoderma aspergillus TCS007: Take 10 μL of Trichoderma aspergillus TCS007 bacterial solution from the thawed glycerol tube, spread it on a PDA plate, and incubate it inverted at 28°C in the dark for 5 days. Then, cut the bacterial cake with a sterile borer of 8 mm in diameter under a sterile environment, and then place the bacterial cake in the center of the PDA plate with a sterile inoculation needle. Incubate it inverted at 28°C in the dark for 5 days and set aside.

[0060] Preparation of Trichoderma aspergillus TCS007 seed solution: Under sterile conditions, the PDA culture medium of Trichoderma aspergillus TCS007 grown to 2 / 3 of the diameter of the culture dish was rinsed with sterile water, and then the hyphae were filtered out with sterile gauze to obtain the Trichoderma aspergillus TCS007 spore suspension. The spore content was determined under an optical microscope using the hemocytometer method, and the spore concentration was adjusted to 1x10 with sterile water. 7 / mL seed solution for later use.

[0061] Fermentation method of Trichoderma aspergillus TCS007: Weigh 72g of rice, pour into a 300mL conical flask, add 48mL of distilled water with a pH value of 7.0, stir thoroughly, seal and sterilize for 60min, then add 1mL of seed liquid in a sterile environment, and finally place in a 28℃ mold incubator for dark culture for 21d.

[0062] Preparation and yield calculation of the crude extract of Trichoderma aspergillus TCS007: The fermentation product of Trichoderma aspergillus TCS007 was mutually dissolved and extracted with equal volumes of ethyl acetate three times, and the extraction was fully stirred and filtered after standing for 24 hours. The obtained extract was then filtered through a 0.22 μm microporous filter membrane for 3 times, and anhydrous sodium sulfate was added to the obtained extract for drying. After that, it was concentrated and evaporated to dryness under reduced pressure using a rotary evaporator, and then dissolved in dichloromethane and methanol and collected in a centrifuge tube. After the solvent evaporates, the crude extract of Trichoderma aspergillus TCS007 is obtained, which is weighed for later use.

[0063] Shallow plate fermentation method: weigh 216g of rice, pour into a 500mL conical flask, add 141mL of distilled water with a pH value of 7.0, stir thoroughly, seal and sterilize for 60min, then inoculate 3mL of Trichoderma aspergillus TCS007 seed liquid under a sterile environment, and finally place it in a 28℃ mold incubator for dark culture for 7d, then transfer it to a shallow plate (21cm×33cm×4cm) under sterile conditions, seal it with plastic wrap, and then make holes evenly on the top with a fine needle.

[0064] Determination of the antibacterial activity of the crude extract of Trichoderma spinulosum TCS007: The organic phase extract obtained was prepared with sterile water and DMF into a 10% mother liquor and then diluted into aqueous solutions of different concentrations. The boscalid aqueous solution was used as a positive control, and 6 mL was taken and added to 54 mL PDA culture medium, with 4 replicates for each treatment. The method of inoculating rapeseed sclerotinia and the culture conditions were the same as above. When the mycelium diameter of the solvent control exceeded 2 / 3 of the culture dish, the solvent control was used as the control group, and the inhibition rate was calculated. The statistical data was processed with SPSS software to obtain the toxicity regression equation and calculate the EC 50 value.

[0065] Single factor optimization of culture medium of Trichoderma aspergillus TCS007

[0066] Recipe Optimization

[0067] Based on the initial fermentation medium, the single factor optimization of the medium components was carried out using the 1% fast-acting carbon and nitrogen source screening method and the progressive method. The experimental conditions of each group of medium formula were set as follows:

[0068] (1) With the added carbon source as the only variable, 1.0% (the added weight is the percentage of the rice weight) of sucrose, sucrose, fructose, glucose, α-lactose, and mannitol were added respectively, for a total of 5 treatment groups, and other fermentation parameters remained unchanged.

[0069] (2) With the added nitrogen source as the only variable, 1.0% (the added weight is the percentage of the rice weight) of urea, peptone, soybean powder, ammonium sulfate, and yeast powder were added respectively, for a total of 5 treatment groups, and other fermentation parameters remained unchanged.

[0070] (3) With the addition of inorganic salts as the only variable, 1.0% (the added weight is the percentage of the rice weight) of anhydrous magnesium sulfate, sodium chloride, anhydrous calcium chloride, anhydrous manganese sulfate, and anhydrous ferric chloride were added respectively, and no addition of inorganic salts was used as the control. Other fermentation parameters remained unchanged, for a total of 6 treatment groups. Each treatment group of the above experiment was repeated 3 times.

[0071] Optimization of culture conditions

[0072] Based on the initial fermentation medium, the single factor optimization fermentation conditions were set as follows:

[0073] (1) The water content in the initial fermentation medium was removed and the amount of water added was used as the only variable. 24, 36, 48, 60, and 72 mL of distilled water were added, respectively. Other fermentation parameters remained unchanged, with a total of 5 treatment groups.

[0074] (2) The inoculum size in the initial fermentation medium was removed and the inoculum size was used as the only variable. 0.5, 1, 1.5, 2, and 2.5 mL of seed liquid were added respectively. Other fermentation parameters remained unchanged, with a total of 5 treatment groups.

[0075] (3) The initial pH value in the initial fermentation medium was removed, and the initial pH value was used as the only variable. The pH values ​​of distilled water were adjusted to 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0 and 11.0 respectively with 1 mol / L dilute sulfuric acid and 1 mol / L sodium hydroxide. Other fermentation parameters remained unchanged. There were 9 treatment groups in total.

[0076] (4) The culture time was removed from the initial fermentation medium conditions and the culture time was used as the only variable, which was set to 7, 14, 21, 28, 35 and 42 days. Other fermentation parameters remained unchanged, for a total of 6 treatments.

[0077] (5) The culture temperature was removed from the initial fermentation medium conditions and the culture temperature was used as the only variable. The culture medium was cultured in the dark in an incubator at 22, 25, 28, 31, and 34 °C, respectively. Other fermentation parameters remained unchanged, for a total of 5 treatments.

[0078] (6) The light cycle in the initial fermentation medium conditions was removed, and the light cycle of the incubator was adjusted to 24 h light, 8 h light + 16 h dark, 12 h light + 12 h dark, 6 h light + 18 h dark, and 24 h dark, respectively, while other fermentation parameters remained unchanged, for a total of 5 treatments. Each treatment in the above experiment was repeated 3 times.

[0079] Response Surface Optimization Design

[0080] Plackett-Burman Design

[0081] Table 1 Placket-Burman design variables and levels

[0082]

[0083] Based on the results of single factor experiment, the effects of fermentation medium and fermentation conditions on the crude extract yield of Trichoderma aspergillus TCS007 were comprehensively considered, and 8 factors with significant influence were selected. According to the Placket-Burman Design method, the software Design Expert 10 was used to design the experiment for the selected 8 factors. The crude extract yield of TCS007 was taken as the response quantity. The significance of the influence of each factor on the crude extract yield of the strain was determined from the 8 factors A1-culture time (7-42d), B1-initial pH (3.0-11.0), C1-temperature (22-34℃), D1-photoperiod (0-24h light), E1-water addition (24-72mL), F1-inoculation amount (0.5-2.5mL), G1-glucose (1.0%-5.0%) and H1-soybean powder (1.0%-5.0%), and the key factors were screened. Each factor in the experiment took 2 levels, namely the high level "1" and the low level "-1" (Table 1).

[0084] Steepest climbing design

[0085] Based on the screening results of the key factors of culture medium and culture conditions, the three most critical influencing factors were selected from the above factors, and the steepest climbing test was designed. The climbing direction and step length of the key factors were determined according to the coefficients of each variable in the model, and the optimal range of the key factors was determined. The highest point of crude extract yield of Trichoderma spinulosa TCS007 obtained by this was taken as the center point of the central combination design experiment.

[0086] Key Factors Central Basis Test

[0087] Table 2 Box-Behnken design variables and levels

[0088]

[0089] The Box-Behnken response surface methodology (BBD) was used to design a 3-factor 3-level central composite experiment (Table 2) to optimize the key factors. The response factor Y2 was the spore production of Trichoderma aspergillus TCS007. The experimental results were analyzed to predict the key factor parameters corresponding to the maximum yield of the crude extract of Trichoderma aspergillus TCS007.

[0090] Data Statistical Analysis

[0091] SPSS26 software was used for single-factor experiments to perform single-factor analysis of variance (ANOVA), and the results of ANOVA were subjected to multiple tests and (P<0.05), and the data were plotted using Graphpad prism 9 software. Design-Expert 10 software was used for relevant data analysis of response surface method experimental design.

[0092] Test results

[0093] Depend on Figure 1 It can be seen that 1% (the added weight accounts for the percentage of rice weight) sucrose, fructose, glucose, α-lactose, and mannitol were added to the initial fermentation medium, and other conditions were the same. No carbon source was added as CK control. After dark culture for 21 days, the crude extract yields of Trichoderma aculeatus TCS007 were tested to be 1.07g, 1.10g, 1.13g, 1g, 0.99g, and 0.91g, respectively. Compared with the CK treatment, the addition of sucrose, fructose, glucose, α-lactose and mannitol can increase the crude extract yield of Trichoderma aculeatus TCS007, among which glucose increased the most.

[0094] Depend on Figure 2 It can be seen that 1% (the added weight accounts for the percentage of rice weight) urea, peptone, soybean powder, ammonium sulfate, and yeast powder were added to the initial fermentation medium, and no nitrogen source was added as the CK control. Other conditions were the same. After 21 days of dark culture, the crude extract yields of Trichoderma aculeatus TCS007 were tested to be 0.77g, 1.20g, 1.25g, 0.78g, 1.25g, and 0.89g, respectively. Compared with the CK treatment, the addition of peptone, soybean powder, and yeast powder can increase the crude extract yield of Trichoderma aculeatus TCS007, among which soybean powder and yeast powder have the highest increase, while other treatments reduce the crude extract yield of Trichoderma aculeatus TCS007.

[0095] Depend on Figure 3It can be seen that 0%, 1%, 2%, 3%, 4%, and 5% glucose were added to the initial fermentation medium, and no glucose was added as the CK control. Other conditions were the same. After 21 days of dark culture, the crude extract yields of Trichoderma spinulosa TCS007 were tested to be 0.92g, 0.97g, 1.01g, 1.06g, 1.02g, and 1g, respectively. Compared with the CK treatment, the addition of 3% glucose increased the crude extract yield to the highest level.

[0096] Depend on Figure 4 It can be seen that 0%, 1%, 2%, 3%, 4%, and 5% soybean powder were added to the initial fermentation medium, and no soybean powder was added as the CK control. Other conditions were the same. After 21 days of dark culture, the crude extract yields of Trichoderma spinulosa TCS007 were 0.9g, 1.28g, 1.3g, 1.38g, 1.28g, and 1.27g, respectively. Compared with the CK treatment, the addition of 3% glucose increased the crude extract yield to the highest level.

[0097] Depend on Figure 5 It can be seen that 1% (the added weight accounts for the percentage of rice weight) of anhydrous magnesium sulfate, sodium chloride, anhydrous calcium chloride, anhydrous magnesium sulfate, and anhydrous ferric chloride were added to the initial fermentation medium, and no inorganic salt was added as CK control. Other conditions were the same. After dark culture for 21 days, the crude extract yields of Trichoderma aculeatus TCS007 were tested to be 0.89g, 0.51g, 0.77g, 0.9g, 0.35g, and 0.9g, respectively. Compared with the CK treatment, the addition of the above-mentioned inorganic salts could not improve the crude extract yield of Trichoderma aculeatus TCS007.

[0098] Depend on Figure 6 It can be seen that 24mL, 36mL, 48mL, 60mL, and 72mL of distilled water were added to the initial fermentation medium, and other conditions were the same. After 21 days of dark culture, the crude extract yields of Trichoderma aculeatus TCS007 were tested to be 0.93g, 1.09g, 1.32g, 1.19g, and 0.83g, respectively. When the amount of water added exceeded 48mL, the fermentation substrate was easy to stick together during the growth of mycelium, reducing the space gap, resulting in a decrease in air content and insufficient dissolved oxygen, which ultimately restricted the growth of Trichoderma aculeatus TCS007. Therefore, the optimal amount of water added is 48mL.

[0099] Depend on Figure 7 Analysis shows that the inoculation size was set to 0.5mL, 1mL, 1.5mL, 2mL, and 2.5mL, respectively. Other conditions were the same. After 21 days of dark culture, the crude extract yields of Trichoderma aspergillus TCS007 were tested to be 0.93g, 1.03g, 1g, 0.92g, and 0.88g, respectively. When the inoculation size was 1mL, the crude extract yield of Trichoderma aspergillus TCS007 was the largest.

[0100] Depend on Figure 8 It can be seen that the initial pH values ​​were set to 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0 and 11.0, respectively. Other conditions were the same. After 21 days of dark culture, the crude extract yields of Trichoderma aspergillus TCS007 were tested to be 0.88g, 0.88g, 0.9g, 0.95g, 1.06g, 1.05g, 0.94g, 0.9g and 0.83g, respectively. When the initial pH was 7.0, the crude extract yield of Trichoderma aspergillus TCS007 was the largest.

[0101] Fermentation culture time screening Fig. 9 It can be seen that the culture time was set to 7, 14, 21, 28, 35, and 42 days, and other conditions were the same. Dark culture was performed. After testing, the crude extract yields of Trichoderma aspergillus TCS007 were 0.36g, 0.54g, 0.93g, 1.34g, 1.24g, and 0.67g, respectively. When the culture time was 28d, the crude extract yield of Trichoderma aspergillus TCS007 was the largest.

[0102] See the culture temperature screening Fig.10 It can be seen that when the culture temperature was set to 22, 25, 28, 31 and 34°C, and other conditions were the same, after 21 days of dark culture, the crude extract yields of Trichoderma aspergillus TCS 007 were tested to be 0.86g, 0.88g, 0.96g, 0.94g and 0.93g, respectively. When the temperature was 28°C, the crude extract yield of Trichoderma aspergillus TCS007 was the largest.

[0103] Photoperiod screening by Fig.11 It can be seen that the light cycle was set to 24 light + 0 darkness, 18 light + 6 darkness, 12 light + 12 darkness, 6 light + 18 darkness, 0 light + 24 darkness, and other conditions were the same. After 21 days of cultivation, the crude extract yields of Trichoderma aspergillus TCS007 were tested to be 0.98g, 1.01g, 1.28g, 1.22g, and 1.17g, respectively. When the light cycle was 12 light + 12 darkness, the crude extract yield of Trichoderma aspergillus TCS007 was the largest.

[0104] Table 3 PB test design and test results of key fermentation factors

[0105]

[0106]

[0107] Through the analysis of the results of the single factor test, the factors of the PB test were determined. According to the PB test design, each factor was designed with two levels of "-1" and "1". The crude extract yield of Trichoderma aculeatus TCS007 was used as the response value to determine the influence of each factor on the crude extract produced by fermentation. Three factors with more significant effects were selected for the next step of the steepest climbing test, thereby determining the center point of the BBD test. Finally, the crude extract yield of Trichoderma aculeatus TCS007 was used as the response value, combined with the center point obtained by the steepest climbing test, 3 factors and 3 levels were designed, a total of 17 groups of experiments, and the results were analyzed and processed. First, the PB test was designed according to the Design expert 10.0 software (Table 3), with a total of 12 groups of treatments, and 3 parallels in each group.

[0108] Table 4 Analysis of variance of PB experimental design for key fermentation factors

[0109]

[0110] As shown in Tables 3 and 4, the P value of this model is 0.0380 (P<0.05) when using Design Expert 10 for regression analysis, which proves that this model is significant and statistically significant. The effect evaluation of the test results is shown in Table 2. The cultivation time, soybean powder content and water addition are the key influencing factors, and the influence is from large to small in the order of cultivation time, soybean powder content and water addition.

[0111] Table 5 Design and results of the fermentation steepest ramp test

[0112]

[0113] As shown in Table 5, when the culture time was 32 days, the amount of water added was 56 mL and the soybean powder content was 4%, the crude extract yield of Trichoderma aspergillus TCS007 was the highest, which was 1.1 g, so this point was selected as the center point of the response surface test factor level.

[0114] Table 6 Fermentation BBD test design and results

[0115]

[0116]

[0117] Table 7 Regression analysis of fermentation BBD experimental design

[0118]

[0119]

[0120] According to the results of the steepest climbing test, the value ranges of the three key factors of the Box-Behnken test design were determined. The yield data of the crude extract of Trichoderma spinulosa TCS007 were fitted by quadratic polynomial regression, and the regression equation model was Y2=1.16-0.21A-0.032B-0.051C+0.050AB+0.033AC+0.020BC-0.35A 2 -0.071B 2 -0.054C 2 , R 2 =91.80%, corrected R 2 =81.26% (Table 6). The model was subjected to variance analysis and difference significance test, and the P value of this model was 0.0047 (P<0.01), proving that this model was significant and statistically significant, and the equation lack of fit term was 0.8291>0.05, indicating that its lack of fit term was not significant, the model was relatively stable, and it could be well predicted (Table 7).

[0121] The response surface plot obtained by Design Expert10 analysis revealed the effects of the interaction between the three key factors of water addition, culture time and soybean powder on the yield of crude extract of Trichoderma aspergillus TCS007. Fig.12 This reflects the interactive relationship between water addition and culture time on the yield of crude extract from Trichoderma spinulosa TCS007 culture medium. Fig.13 This reflects the interactive relationship between culture time and soybean powder content on the yield of crude extract of Trichoderma aspergillus TCS007 culture medium. Fig.14 It reflects the interactive relationship between the amount of water added and the content of soybean powder on the crude extract yield of Trichoderma aspergillus TCS007 culture medium.

[0122] Using the software Design Expert10 to analyze the data and solve the regression equation, it was found that this model had a maximum value. The actual values ​​of the three factors were obtained by conversion, namely, the amount of water added was 47 mL, the culture time was 29 days, and the soybean powder content was 3.5%. The maximum response value of the crude extract yield of Trichoderma spinulosa TCS007 was predicted to be 1.22 g.

[0123] In order to verify the accuracy of the model prediction, three repeated experiments were carried out according to the optimal predicted conditions. The results showed that the average yield of the crude extract of Trichoderma aspergillus TCS007 was 1.21 g, and the actual value was close to the predicted value (P<0.05), indicating that the constructed model is reliable and can accurately predict the true value.

[0124] In summary, after single factor optimization test and response surface model optimization, the optimal culture medium fermentation conditions for the yield of crude extract of Trichoderma spinulosa TCS007 provided by the present invention are: rice content of 72g, soybean powder content of 3.5%, water addition amount of 47mL, initial pH value of 7.0, inoculation amount of 1mL (1×107 / mL), light cycle time was 12h, culture temperature was 28℃, and culture time was 29d.

[0125] Under the fermentation conditions of the initial fermentation medium (72 g rice + 48 mL distilled water), Trichoderma spinulosum TCS007 was fermented as Fig.15 As shown in the figure, the crude extract yield of Trichoderma aspergillus TCS007 was 0.9 g / 72 g rice; after optimization by response surface methodology and fermentation under the optimal culture medium and fermentation conditions, the crude extract yield of Trichoderma aspergillus TCS007 was 1.21 g / 72 g rice, which was increased by 34.44%; further fermentation by shallow tray fermentation under the optimal culture medium and fermentation conditions, as shown in the figure, Fig.16 As shown, using the optimized post-fermentation medium and fermentation culture process provided by the present invention, the yield of the crude extract of Trichoderma aspergillus TCS007 obtained by culture and fermentation reached 1.45g / 72g rice.

[0126] Antibacterial activity assay

[0127] The experimental agents were crude extract of Trichoderma aspergillus TCS007 (before optimization) under the culture conditions of the initial fermentation medium, crude extract of Trichoderma aspergillus TCS007 (after optimization) optimized by single factor optimization test and response surface model, and boscalid. The antibacterial activity of the agents was determined by the toxic medium method and the cross method. The statistical data was processed by SPSS software to obtain the toxicity regression equation and calculate the EC 50 value.

[0128] Table 8 Toxicity of crude ethyl acetate extract of Trichoderma spinulosum TCS007 to Sclerotinia sclerotiorum

[0129]

[0130] Note: Test sample 1 is the crude extract obtained by fermentation of the optimized fermentation medium of the present invention; Test sample 2 is the crude extract obtained by fermentation of the initial fermentation medium under initial conditions; Test sample 3 is the crude extract obtained by fermentation of the wheat bran medium after spore production optimization provided in patent CN202410872878.8.

[0131] As shown in Table 8, the antibacterial activity of the crude extract EC obtained by fermentation of the fermentation medium optimized by the present invention against rapeseed sclerotia is 50 0.510 μg mL -1 The crude extract EC was obtained by fermentation under the initial conditions compared with the initial fermentation medium before optimization. 50 17.907 μg·mL -1 The activity of crude extracts produced by the optimized wheat bran culture medium and culture conditions in patent CN202410872878.8 (EC 5022.212 μg·mL -1 ).

[0132] The above specific implementation methods further illustrate the purpose, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above are only specific implementation methods of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made on the basis of the technical solutions of the present invention should be included in the scope of protection of the present invention.

Claims

1. Application of rice and glucose as fermentation substrates in culture medium for increasing the yield of crude extract of Trichoderma aspergillus TCS007.

2. The use according to claim 1, characterized in that: The weight ratio of the rice to the glucose is 100:2-5.

3. Application of soybean powder and / or yeast powder and rice as fermentation substrate in culture medium for increasing the yield of crude extract of Trichoderma aspergillus TCS007.

4. The use according to claim 3, characterized in that: The weight ratio of the rice to soybean powder and / or yeast powder is 100:1-5.

5. Use of sodium chloride and / or anhydrous ferric chloride to inhibit the yield of crude extract of Trichoderma spinulosa TCS007.

6. A culture medium for industrial production of a crude extract of Trichoderma aspergillus TCS007, characterized in that: The culture medium comprises soybean powder, rice and water, the weight ratio of the rice to the soybean powder is 100:1-5, and the weight ratio of the rice to the water is 3:1-3; preferably, the initial pH value of the culture medium is 6.0-8.0, preferably, the initial pH value of the culture medium is 7.0-8.

0.

7. A process for producing a crude extract of Trichoderma aspergillus TCS007 using the culture medium according to claim 6, characterized in that: 1×10 7 The volume and mass ratio of the inoculum amount of Trichoderma spinulosa TCS007 seed solution per mL to the weight of rice in the culture medium is 0.5-2.5 mL: 50-200 g; Preferably, the fermentation culture light cycle is 12h light + 12h dark - 24h light + 0h dark; preferably, the fermentation culture time is 21-35d; Preferably, the fermentation culture temperature is 25-31°C.

8. The process according to claim 7, characterized in that 1×10 7 The volume-to-mass ratio of the inoculum amount of Trichoderma spinulosa TCS007 seed solution to the weight of rice in the culture medium is 1 mL:72 g; Preferably, the fermentation culture light cycle is 12h light + 12h dark; Preferably, the fermentation culture time is 25-30 days; particularly preferably, the fermentation culture time is 29 days; preferably, the fermentation culture temperature is 28°C.

9. A method for optimizing the fermentation conditions of crude extracts of Trichoderma aspergillus TCS007, characterized in that: The fermentation medium formula and fermentation conditions of Trichoderma spinulosa TCS007 were optimized by single factor experiment combined with response surface methodology. The single factor test includes optimizing the medium single factor and optimizing the fermentation conditions single factor by using a progressive method based on the initial fermentation medium; Based on the results of single factor test, several factors with significant influence were selected, and the experimental design was carried out for the selected factors according to the Placket-Burman Design method; based on the screening results of key factors of culture medium and culture conditions, several most critical influencing factors were screened out, and the steepest climbing test was designed; the climbing direction and step length of the key factors were determined according to the coefficients of each variable in the model, and the optimal range of the key factors was determined, and the maximum point of the maximum yield of the crude extract of Trichoderma spinulosa TCS007 fermentation obtained was taken as the center point of the central combination design experiment; The response surface method includes designing a central composite test with several factors and several levels using the Box-Behnken response surface method to optimize key factors, wherein the response factor Y2 is the crude extract of Trichoderma aspergillus TCS007 fermentation, and the test results are analyzed to predict the key factor parameters corresponding to the maximum yield of the crude extract of Trichoderma aspergillus TCS007 fermentation.

10. The method according to claim 9, characterized in that The fermentation medium formula is: rice, soybean powder and water; preferably, based on the weight of rice, the weight percentage of soybean powder is 3.5% of the weight of rice, and the amount of water added is 47 / 72mL / g of rice; further preferably, the content of rice is 72g, the content of soybean powder is 3.5%, and the amount of water added is 47mL; Preferably, the fermentation culture conditions are: initial pH value is 7.0, inoculum volume is 1 mL (1×10 7 / mL Trichoderma spinulosa TCS007 seed solution), light cycle time was 12h, culture temperature was 28℃, and culture time was 29d.

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