Trichoderma decomposing agent with high cellulase yield, preparation method therefor, and use thereof

Abstract

Provided are a Trichoderma decomposing agent with high cellulase yield, a preparation method therefor, and use thereof. The Trichoderma decomposing agent with high cellulase yield comprises Trichoderma longibrachiatum T6, Trichoderma viride P6, and Trichoderma longibrachiatum Lu3. The enzyme activity is improved by means of co-culturing the Trichoderma strains.

Description

A Trichoderma composting agent with high cellulase production, its preparation method and application Technical Field

[0001] This invention belongs to the field of microbial inoculant technology, and particularly relates to a high-cellulase-producing Trichoderma composting agent, its preparation method, and its application. Background Technology

[0002] my country has extremely rich crop straw resources, with an annual output of nearly 10 9 Of the 12% of agricultural waste in China, 42% is corn straw. Therefore, exploring efficient microbial resources for straw decomposition and developing corresponding straw biological treatment and resource utilization technologies are urgent problems to be solved in the resource utilization of agricultural waste in my country. Lignin, hemicellulose, and cellulose in straw form a three-dimensional cross-linked structure, limiting the bioconversion of organic matter such as hemicellulose and cellulose. In recent years, research on aerobic fermentation of straw has mainly focused on adding exogenous microorganisms to improve the composting effect of aerobic fermentation. Adding a compound microbial system composed of Rhizopus oryzae and Bacillus during the aerobic fermentation of corn straw significantly improved the total nutrients of the fermentation products and the seed germination rate. A composting agent is a microbial agent that can accelerate the decomposition and composting of organic waste (including crop straw, livestock and poultry manure, domestic waste, and urban sludge), converting large organic molecules in straw into small molecules that can be absorbed by plants, and producing a large amount of organic matter, stimulating crop production, and improving soil fertility. Meanwhile, as a microbial preparation capable of composting straw and other waste, cellulase is the main evaluation indicator for whether a composting agent can produce good results. Cellulase is a complex enzyme and a type of biocatalyst. Fibrinogenase A can disrupt plant cell wall tissues, facilitating the hydrolysis and release of enzymes, polysaccharides, and proteins in plant cells, thus accelerating fermentation. Fungi in the genus *Trichoderma* spp. have become a research hotspot due to their synthesis of extracellular cellulases, which exhibit strong activity in the hydrolysis of cellulose. Among the most studied *Trichoderma* fungi are *Trichoderma reesei*, *Trichoderma viride*, *Trichoderma koningii*, and *Trichoderma pseudokoningii*. Summary of the Invention

[0003] To address the aforementioned technical problems, this invention proposes a high-yield cellulase Trichoderma composting agent, its preparation method, and its application. Through co-culture fermentation of Trichoderma strains, the activity of its cellulase is significantly improved compared to that of a single strain, and the optimal culture system is optimized.

[0004] To achieve the above objectives, the present invention provides a Trichoderma composting agent with high cellulase production, wherein the Trichoderma composting agent comprises Trichoderma longibrachiatum T6, Trichoderma viride P6 and Trichoderma longibrachiatum Lu3.

[0005] Preferably, the *Trichoderma longifolia* T6 is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO.13183 and deposit date of November 14, 2016.

[0006] Preferably, the ratio of Trichoderma longifolia T6, Trichoderma viride P6, and Trichoderma longifolia Lu3 in the Trichoderma composting agent is 2:1:1.

[0007] The present invention also provides a method for preparing the Trichoderma composting agent, comprising the following steps:

[0008] Trichoderma longifolia T6, Trichoderma viride P6 and Trichoderma longifolia Lu3 were mixed in a volume ratio of 2:1:1 to obtain a seed mixture. The seed mixture was then inoculated into microcrystalline cellulose medium at a volume fraction of 2% and cultured at 25-27℃, light for 12 h / d, and 180 rpm for 3-4 days to obtain a Trichoderma composting agent.

[0009] The present invention also provides the application of the Trichoderma composting agent in the preparation of high-yield FPA enzyme preparations.

[0010] Compared with the prior art, the present invention has the following advantages and technical effects:

[0011] Based on previous research, this invention uses Trichoderma longifolia T6, Trichoderma viride P6, and Trichoderma longifolia Lu3 as experimental subjects, and studies the effects of initial pH, ambient temperature, and culture days on the produced FPA enzyme. This lays the foundation for the development and utilization of high-yield cellulase Trichoderma composting agents, and prepares a highly efficient Trichoderma composting agent capable of degrading cellulose. Attached Figure Description

[0012] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0013] Figure 1 shows the contour plot and three-dimensional surface plot of the interaction between initial pH and culture days, where a is the contour plot and b is the three-dimensional surface plot;

[0014] Figure 2 shows the contour plot and three-dimensional surface plot of the interaction between temperature and culture days, where a is the contour plot and b is the three-dimensional surface plot;

[0015] Figure 3 shows the contour plot and three-dimensional surface plot of the interaction between initial pH and temperature, where a is the contour plot and b is the three-dimensional surface plot. Detailed Implementation

[0016] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0017] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0018] The description and embodiments of this invention are merely exemplary.

[0019] Materials used in this invention: PDA culture medium: 200g potato, 20g glucose, 15g agar, 1L water.

[0020] Microcrystalline cellulose culture medium: 10g microcrystalline cellulose, 0.3g urea, 0.75g peptone, 1.4g (NH4)2SO4, 2g KH2PO4, 0.06g MgSO4, 0.06g CaCl2, trace elements (CoCl2·6H2O 0.0037g, FeSO4·7H2O 0.005g, ZnSO4·7H2O 0.0014g, MnSO4·H2O 0.0016g), 2mL Tween 80, pH 6.

[0021] Trichoderma longifolia strain T6 (T6) is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO.13183 and deposit date of November 14, 2016; Trichoderma viride strain P6 (P6) and Trichoderma longifolia strain Lu3 (Lu3) were provided by the Plant Pathology Laboratory of the College of Plant Protection, Gansu Agricultural University.

[0022] Example 1

[0023] (1) Preparation of Trichoderma spore suspension

[0024] Trichoderma longicornis strain T6, Trichoderma viride strain P6, and Trichoderma longicornis strain Lu3 were inoculated into PDA medium and placed in a constant temperature incubator at 28℃ with light for 12 h / d for 7 days. After incubation, 5 mL of sterile water was added to each strain, and all conidia were transferred into sterile water to obtain stock solutions of conidial suspensions. The conidia were counted and diluted to achieve a spore concentration of 1×107 cfu / mL, resulting in T6 spore suspension, P6 spore suspension, and Lu3 spore suspension for later use.

[0025] (2) Preparation of Trichoderma strain seed liquid

[0026] Dispense 100 mL of PDB medium into each bottle, sterilize at 121 °C for 30 min, and inoculate T6 spore suspension, P6 spore suspension and Lu3 spore suspension respectively at a volume fraction of 2%. Place them in a constant temperature shaker at 28 °C, light for 12 h / d, and culture at 180 rpm for 2 days to obtain T6 seed solution, P6 seed solution and Lu3 seed solution for later use.

[0027] (3) Preparation of Trichoderma composting agent

[0028] Dispense 100 mL of microcrystalline cellulose medium and sterilize at 121 °C for 30 min. Mix T6 seed solution, P6 seed solution and Lu3 seed solution at a volume ratio of 2:1:1 to obtain seed mixture. Then inoculate the seed mixture into microcrystalline cellulose medium at a volume fraction of 2% and place it in a constant temperature shaker at 25 °C. Incubate at 180 rpm for 4 days under light for 12 h / d to obtain Trichoderma composting agent.

[0029] (4) Determine FPA enzyme activity

[0030] After centrifuging the Trichoderma composting agent at 8000 rpm for 10 min, the supernatant was collected as the crude enzyme solution, and its FPA enzyme activity was measured. After 6 repeated measurements, the initial average FPA enzyme activity of the Trichoderma composting agent was 134.26 U / mL.

[0031] Example 2

[0032] (1) Preparation of Trichoderma spore suspension

[0033] Trichoderma longicornis strain T6, Trichoderma viride strain P6, and Trichoderma longicornis strain Lu3 were inoculated into PDA medium and incubated at 28°C with 12 h / d light for 7 days. After incubation, 5 mL of sterile water was added to each strain, and all conidia were transferred into sterile water to obtain stock solutions of conidia suspensions. The conidia were counted and diluted to achieve a concentration of 1 × 10⁻⁶ spores. 7 CFU / mL was used to obtain T6 spore suspension, P6 spore suspension and Lu3 spore suspension for later use.

[0034] (2) Preparation of Trichoderma strain seed liquid

[0035] Dispense 100 mL of PDB medium into each bottle, sterilize at 121 °C for 30 min, and inoculate T6 spore suspension, P6 spore suspension and Lu3 spore suspension respectively at a volume fraction of 2%. Place them in a constant temperature shaker at 28 °C, light for 12 h / d, and culture at 180 rpm for 2 days to obtain T6 seed solution, P6 seed solution and Lu3 seed solution for later use.

[0036] (3) Preparation of Trichoderma composting agent

[0037] The pH of the microcrystalline cellulose medium was adjusted to 5.92, and then dispensed into 100 mL portions. The medium was sterilized at 121°C for 30 min. The T6 seed solution, P6 seed solution, and Lu3 seed solution were mixed at a volume ratio of 2:1:1 to obtain a seed mixture. The seed mixture was then inoculated into the microcrystalline cellulose medium at a volume fraction of 2% and placed in a constant temperature shaker at 26.95°C. The mixture was then incubated at 180 rpm for 3.59 days under light for 12 h / d to obtain the Trichoderma composting agent.

[0038] (4) Determine FPA enzyme activity

[0039] After centrifuging the Trichoderma composting agent at 8000 rpm for 10 min, the supernatant was collected as the crude enzyme solution, and its FPA enzyme activity was measured. The initial FPA enzyme activity was 171.13 U / mL.

[0040] Experimental Example 1

[0041] I. Response Surface Optimization of Trichoderma Composting Agent Preparation Process

[0042] 1. Response surface methodology for significant impact factors

[0043] Based on the single-factor experiment and Box-Benhnken design principle, response surface methodology was conducted using Design Expert 8.0 software. Initial pH, culture time and light were selected as independent variables, and a total of 17 center combinations with 3 factors and 3 levels were designed for response surface methodology (Table 2). Sporulation yield was used as the response value, and the center value experiment was repeated 5 times. The design and experimental analysis results are shown in Tables 1 and 2.

[0044] Table 1. Factor Level Design in the Box-Behnken Experiment

[0045] ;

[0046] Table 2 Optimization design of Trichoderma composting agent culture conditions

[0047] ;

[0048] 2. Analysis of variance for the regression model

[0049] Based on the multiple regression fitting analysis, the regression equation model between the significant influencing factors initial pH (A), culture days (B), and temperature (C) and the total cellulase activity of the Trichoderma composting agent is as follows:

[0050] Y=159.22-5.58A+6.69B-10.08C+0.74AB+3.49AC+0.86BC-73.92A 2 -54.56B 2 -70.24C 2 In the equation, Y represents the predicted activity of cellulase in the Trichoderma composting agent.

[0051] Analysis of variance of the predictive regression model showed that the regression level was significant. The interactions of initial pH (A), culture days (B), and temperature (C), as well as the interactions of initial pH (A) and culture days (B), initial pH (A) and temperature (C), and culture days (B) and temperature (C), had the most significant effects on the cellulase activity of the *Trichoderma* composting agent (P < 0.0001). Meanwhile, the R² and Pred R² of the regression model equations showed good correlation with Adj R², indicating that the predictive regression model equations had a good fit and could predict the optimal fermentation conditions and optimal FPA enzyme activity of the *Trichoderma* composting agent. Furthermore, among the three significant factors—temperature, initial pH, and culture days—fermentation temperature had the most significant effect on the FPA enzyme activity of the *Trichoderma* composting agent, followed by initial pH and culture days. In addition, the optimal culture conditions and optimal FPA enzyme activity were predicted based on the regression model. The results showed that the optimal culture conditions for Trichoderma composting agent were: initial pH 5.92, culture days 3.59 days, temperature 26.95℃, and the optimal enzyme activity was predicted to be 159.90 U / mL.

[0052] 3. Analysis of the interaction of significant influencing factors

[0053] Two-dimensional contour plot analysis results show that the contour plots of A (initial pH)-B (culture days) (Figure 1a and Figure 1b), B (culture days)-C (temperature) (Figure 2a and Figure 2b), and A (initial pH)-C (temperature) (Figure 3a and Figure 3b) are close to elliptical, indicating a significant interaction between the two factors. 3D surface plot analysis results show that the three-dimensional plots of A (initial pH)-B (culture days) (Figure 1) and A (initial pH)-C (temperature) (Figure 3) have a large slope, indicating a strong interaction and a highly significant impact on the activity of the Trichoderma composting agent FPA enzyme. B (culture days)-C (temperature) also has a significant impact on the activity of the Trichoderma composting agent FPA enzyme (Figure 2).

[0054] 4. Validation of optimal fermentation conditions based on response surface methodology

[0055] The results of the optimal fermentation condition validation showed that the predicted value of the optimal FPA enzyme activity of the Trichoderma composting agent, 159.90 U / mL, was basically consistent with the measured value, proving that the established prediction model is reliable. When the initial pH of the Trichoderma composting agent was 5.92, the culture period was 3.59 days, and the temperature was 26.95℃, the measured FPA enzyme activity was 171.13 U / mL. Compared with the unoptimized fermentation conditions of the Trichoderma composting agent, the FPA enzyme activity increased by 27.46% after optimization.

[0056] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

A Trichoderma composting agent with high cellulase production, characterized in that, The Trichoderma composting agent includes Trichoderma longibrachiatum T6, Trichoderma viride P6, and Trichoderma longibrachiatum Lu3. According to claim 1, the Trichoderma composting agent is characterized in that, The *Trichoderma longifolia* T6 was deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO.13183 and deposit date of November 14, 2016. According to claim 1, the Trichoderma composting agent is characterized in that, The ratio of Trichoderma longifolia T6, Trichoderma viride P6, and Trichoderma longifolia Lu3 in the Trichoderma composting agent is 2:1:

1. The method for preparing the Trichoderma composting agent according to any one of claims 1 to 3 is characterized in that, Includes the following steps: Trichoderma longifolia T6, Trichoderma viride P6 and Trichoderma longifolia Lu3 were mixed in a volume ratio of 2:1:1 to obtain a seed mixture. The seed mixture was then inoculated into microcrystalline cellulose medium at a volume fraction of 2% and cultured at 25-27℃, light for 12 h / d, and 180 rpm for 3-4 days to obtain a Trichoderma composting agent. The application of the Trichoderma composting agent as described in any one of claims 1 to 3 in the preparation of high-yield FPA enzyme preparations.

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