Trichoderma asperellum and use thereof

Abstract

The application discloses a Trichoderma asperellum and application thereof, and belongs to the technical field of microorganisms, and is named Trichoderma asperellum with a strain number RPT1 and preserved in the China Center for Type Culture Collection with a preservation number CCTCC NO: M 20221879. The application further provides a natural liquid bactericide obtained by removing a bacterium body after fermentation and culture of the Trichoderma asperellum; the natural liquid bactericide is aimed at Escherichia coli and / or Staphylococcus aureus. The natural liquid bactericide has high preparation efficiency, low cost, and easily-obtained raw materials, has a significant killing effect on the Escherichia coli and / or Staphylococcus aureus, and has a popularization and application value in the medical and health fields.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a Trichoderma asperellum and its applications. Background Technology

[0002] Medical health and disease prevention are current focal points of human and societal concern, and surface disinfection of public facilities is a crucial measure to ensure public safety and health. In recent decades, the variety of antibacterial agents developed and used has increased significantly. Commercially available disinfectants mainly include oxidizing agents such as hydrogen peroxide, organic solvent-based agents such as ethanol, cationic agents such as quaternary ammonium salts, and metal- or metal oxide-containing agents such as silver-loaded antibacterial drugs. Among these, oxidizing agents have rapid bactericidal effects but are unstable in storage and irritating; organic solvent-based agents are volatile; metal- or metal oxide-containing agents pose potential cytotoxic risks and may harm individuals with metal allergies; the antibacterial properties of quaternary ammonium salt antibacterial agents are affected by the length of their alkyl chains, with long-chain quaternary ammonium salt antibacterial agents exhibiting poor antibacterial effects, limiting their application; chitosan-based antibacterial agents' antibacterial properties mainly stem from their polycationic structure, but they only exhibit polycationic characteristics and antibacterial properties in acidic solutions, limiting their biomedical applications. Single-component antibacterial agents still suffer from poor bactericidal efficacy and insufficient persistence. While combinations of multiple antibacterial ingredients can improve bactericidal performance, they also pose potential risks of biotoxicity. With increasing public awareness of environmental protection and the rise of green consumption concepts, naturally derived antibacterial agents will become the preferred choice for future disinfection and sterilization products.

[0003] Fungi are an important component of microbial communities, and their secondary metabolites exhibit a range of biological activities, including antibacterial, anti-inflammatory, antitumor, antiviral, and biocontrol abilities against plant pathogens and agricultural pests. Fungi and their secondary metabolites have enormous application potential in the pharmaceutical, food, and chemical industries. Furthermore, the antibacterial substances obtained from fungi are green and environmentally friendly, and will not have negative environmental effects. Commonly used antibacterial drugs such as penicillin are isolated and purified from Penicillium mold; cephalosporin C is derived from Cephalosporium roximatee; in addition, Chinese patent document CN114467978A discloses the application of a prickly pear endophytic fungus and its metabolites in broad-spectrum antibacterial agents. The biological preservation number of this prickly pear endophytic fungus is CGMCC 40110, and its metabolites have inhibitory effects on a variety of bacteria; Chinese patent document CN109082445A discloses the metabolites of a ginkgo endophytic fungus and its application in antibacterial activity. This ginkgo endophytic fungus is Fusarium proliferatum DZHQ1, with preservation number CGMCC No. 14983. After fermentation culture and ethyl acetate extraction, an ethyl acetate extract of the fermentation culture broth is obtained. This extract, after dissolution, can resist Escherichia coli and / or Staphylococcus aureus.

[0004] Trichoderma fungi are widely distributed in nature. Many species, such as *Trichoderma harzianum*, *Trichoderma echinococcus*, and *Trichoderma viride*, exhibit antagonistic effects against various plant pathogens. Trichoderma fungi can effectively prevent plant diseases caused by multiple plant pathogens through mechanisms such as influencing competition, antagonism, and inducing resistance. Currently, commercially available Trichoderma fungal preparations are on the market and widely used for plant disease control. However, reports on the bactericidal activity of Trichoderma fungi and their metabolites are limited. Summary of the Invention

[0005] The purpose of this invention is to provide a Trichoderma acicularis and its application, particularly in the preparation of natural liquid fungicides, thus broadening the range of natural antibacterial agents in the prior art.

[0006] The specific technical solution adopted is as follows:

[0007] A Trichoderma asperellum strain, named Trichoderma asperellum, strain number RPT1, is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M20221879.

[0008] The *Trichoderma asperellum* strain was isolated from white mica from the Lesser Khingan Mountains in Northeast China. Genomic similarity comparison showed that it was most closely related to *Trichoderma asperellum*, a result consistent with morphological identification. This confirms that the isolated RPT1 strain is *Trichoderma asperellum*, and it was deposited at the China Center for Type Culture Collection (CCTCC) on December 5, 2022, with accession number CCTCC NO: M20221879.

[0009] The ITS sequence of the *Trichoderma hyacinthus* is shown in SEQ ID No. 1.

[0010] The present invention also provides the application of the aforementioned Trichoderma echinosporum in the preparation of a fungicide, wherein the fungicide targets Escherichia coli and / or Staphylococcus aureus.

[0011] The present invention also provides a natural liquid bactericide, obtained by fermenting and culturing the *Trichoderma echinosporum* and then removing the bacterial cells; the natural liquid bactericide targets *Escherichia coli* and / or *Staphylococcus aureus*.

[0012] The specific preparation method of the natural liquid bactericide includes the following steps:

[0013] (1) The activated Trichoderma hyacinth was inoculated into a fermentation medium and fermented to obtain the fermentation stock solution;

[0014] (2) Centrifuge the fermentation liquid to obtain the supernatant, which is the natural liquid bactericide.

[0015] Preferably, the fermentation medium is formulated as follows: potato extract 0-6 g / L, glucose 5-20 g / L, yeast extract 1-5 g / L, peptone 1-5 g / L, potassium dihydrogen phosphate 0-0.5 g / L, chloramphenicol 0-0.1 g / L, with the remainder being artificial seawater or deionized water; more preferably, the pH of the fermentation medium is 6-8.

[0016] The artificial seawater formulation is as follows: sodium chloride 24.53 g / L, magnesium chloride 5.2 g / L, sodium sulfate 4.09 g / L, calcium chloride 1.16 g / L, potassium chloride 0.695 g / L, sodium bicarbonate 0.201 g / L, potassium bromide 0.101 g / L, boric acid 0.027 g / L, strontium chloride 0.025 g / L, sodium fluoride 0.003 g / L, with the balance being deionized water. The solution pH is 8.2 (the pH is adjusted using sodium hydroxide).

[0017] Preferably, the fermentation culture conditions are: dynamic culture in a constant temperature shaker at 25-30℃ for 5-14 days.

[0018] Preferably, the centrifugation conditions are: 10000rpm~12000rpm, and the centrifugation time is 30~40min.

[0019] Further preferably, the fermentation broth is centrifuged at 10,000 rpm for 30 min, and the supernatant is filtered through a 0.22 μm filter membrane to obtain the natural liquid bactericide.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0021] The *Trichoderma echinosporum* strain in this invention, with preservation number CCTCC NO: M 20221879, exhibits significant bactericidal activity against *Escherichia coli* and / or *Staphylococcus aureus* through its fermentation metabolites. This invention utilizes *Trichoderma echinosporum* to ferment and obtain a natural liquid bactericide. This natural liquid bactericide is highly efficient, low-cost, and uses readily available raw materials. It exhibits significant bactericidal activity against *Escherichia coli* and *Staphylococcus aureus*. Furthermore, cell experiments have demonstrated that this natural liquid bactericide is non-toxic and has significant application value in the medical and health fields. Attached Figure Description

[0022] Figure 1 This is a colony diagram of *Trichoderma hygroscopicum* after 3 days of activation.

[0023] Figure 2 This is a colony diagram of *Trichoderma hygroscopicum* after 5 days of activation.

[0024] Figure 3 In the diagrams, 'a' and 'b' are microscopic morphological features of the aforementioned Trichoderma hygroscopicum.

[0025] Figure 4 The diagram shows the effect of Trichoderma hygroscopicum on Escherichia coli, where a is the phosphate buffer control group and b is the natural liquid bactericide experimental group.

[0026] Figure 5 The image shows the effect of *Trichoderma hygroscopicum* on *Staphylococcus aureus*, where a is the phosphate buffer control group and b is the natural liquid bactericide experimental group. Detailed Implementation

[0027] The present invention will be further illustrated below with reference to the embodiments and accompanying drawings. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0028] Isolation and identification of Trichoderma hygroscopicum as described in Example 1

[0029] (1) Fungal isolation and purification

[0030] The *Trichoderma hygroscopica* was isolated from white mica from the Lesser Khingan Mountains in Northeast China. The white mica from the Lesser Khingan Mountains was washed 2-3 times with sterile water, placed in PDB medium, and cultured with shaking at 28°C for 24 hours. 100 μL of the bacterial suspension was evenly spread on PDB medium plates and incubated upside down at 25-28°C for 3-7 days. The colony morphology was observed, and the fungi were isolated and purified according to different colony morphologies.

[0031] The formula for PDB medium is: 5 g / L potato extract powder, 20 g / L glucose, 0.1 g / L chloramphenicol, and the remainder is deionized water. The preparation method is as follows: Weigh 5 g of potato extract powder, add it to 500 mL of deionized water, stir evenly at room temperature, and after it dissolves, add 20 g of glucose, continue to stir evenly at room temperature, and after it dissolves, add 0.1 g of chloramphenicol, stir evenly, and then add deionized water to 1000 mL. Then place it in an autoclave and sterilize at 121°C for 15 min, and finally cool to room temperature.

[0032] The formula for PDA medium is: 5 g / L potato extract powder, 20 g / L glucose, 15 g / L agarose, and 0.1 g / L chloramphenicol. The preparation method is as follows: Weigh 5 g of potato extract powder, add it to 500 mL of deionized water, stir evenly at room temperature until dissolved, then add 20 g of glucose, continue stirring evenly at room temperature until dissolved, then add 0.1 g of chloramphenicol, and after dissolving, add agarose powder, stir evenly, and then add deionized water to 1000 mL. Then place it in an autoclave and sterilize at 121℃ for 15 min. After cooling to 50–60℃, pour it into culture plates and cool to room temperature to obtain the final product.

[0033] (2) Identification of the aforementioned Trichoderma echinosporum

[0034] Colony morphology characteristics: The *Trichoderma echinosporum* species grows rapidly on PDA medium, covering the entire culture plate in about 3-5 days. Short, fluffy mycelia are produced on the colony surface, initially white in color. After 5-8 days of growth, the colonies turn green. The conidiophores are slender, and the conidia are green, spherical, and nearly spherical. The colony images of *Trichoderma echinosporum* after 3 and 5 days of activation are shown below. Figure 1 and 2 As shown, the microscopic morphological features are as follows: Figure 3 As shown, Figure 3 In the image, 'a' represents a microscopic image of a white bacterial colony. Figure 3 b in the image represents the microscopic morphology of a green bacterial colony.

[0035] Molecular biological identification: PCR amplification was performed using universal primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') to determine the ITS sequence of the strain. The sequence was then compared using the BLAST program on NCBI (https: / / wwwjicbi.nlm.nih.gov / ). The results showed that the strain was *Trichoderma asperellum*. Combined with morphological and cultural characteristics, strain RPT1 was identified as *Trichoderma asperellum*. The ITS sequence of *Trichoderma asperellum* is shown in SEQ ID No. 1 and was deposited at the China Center for Type Culture Collection on December 5, 2022, with accession number CCTCC NO: M20221879.

[0036] Example 2: Preparation of a natural liquid fungicide using the aforementioned Trichoderma echinosporum.

[0037] (1) Activation of bacterial strain: A small amount of mycelium was scraped from the PDA medium using an inoculation loop and placed in the PDB medium. The culture was carried out at 25-28℃ with shaking at 120-180 rpm for 24 hours to obtain the bacterial solution.

[0038] (2) Preparation of fermentation medium: Weigh 10g of glucose, 5g of yeast extract and 1g of peptone and add them to 500mL of deionized water. Make up the volume to 1000mL with deionized water. Stir magnetically to mix thoroughly. Adjust the pH to 6-8 with citric acid or sodium hydroxide. Then place it in a high-pressure steam sterilizer and sterilize at 121℃ for 15min. Finally, cool to room temperature.

[0039] (3) Inoculate the bacterial solution from step (1) into the fermentation medium prepared in step (2), with an inoculation amount of 10-20%, and repeatedly blow and agitate to ensure that the fungi are evenly dispersed in the fermentation medium; then place the liquid medium after inoculation into an incubator and shake and ferment for 5 days at 25-28℃ to obtain the fermentation stock solution.

[0040] (4) After centrifuging the fermentation stock solution obtained in step (3) at 10000-12000 rpm for 30 min, take the supernatant and filter the supernatant through a filter membrane with a pore size of 0.22 μm to obtain a natural liquid bactericide.

[0041] Example 3: Preparation of a natural liquid fungicide using the aforementioned Trichoderma echinosporum.

[0042] In this embodiment, the preparation method of the natural liquid bactericide differs from that in Example 2 only in that: in step (2), 10g of glucose, 5g of yeast extract powder and 1g of peptone are weighed and added to 500mL of artificial seawater in sequence, and the volume is adjusted to 1000mL with artificial seawater. After being thoroughly mixed by magnetic stirring, the pH value of the solution is adjusted to 6-8 using citric acid or sodium hydroxide.

[0043] Example 4: Preparation of a natural liquid fungicide using the aforementioned Trichoderma acicularis.

[0044] In this embodiment, the preparation method of the natural liquid bactericide differs from that in Example 2 only in that: in step (2), 20g of glucose, 1g of yeast extract powder and 5g of peptone are weighed and added to 500mL of deionized water in sequence, and the volume is adjusted to 1000mL with deionized water. After being thoroughly mixed by magnetic stirring, the pH value of the solution is adjusted to 6-8 using citric acid or sodium hydroxide.

[0045] Sample Analysis

[0046] The bactericidal performance of the natural liquid bactericides in Examples 2-4 was tested using Escherichia coli and Staphylococcus aureus. Phosphate buffer and the natural liquid bactericides from Examples 2-4 were first sterilized by irradiation under a UV lamp for 30 minutes. Bacterial suspensions of the test strains in the logarithmic growth phase were taken, and the OD value was adjusted to 0.1. 100 μL of the bacterial suspension was diluted to 10 mL with phosphate buffer (control group) and the bactericides from Examples 2-4 (experimental group), respectively, with three parallel samples. After incubation at 37°C for 6 hours, the samples were plated, and the colony count was calculated to verify the antibacterial activity of the bactericides in Examples 2-4.

[0047] Experimental results show that the natural liquid bactericide obtained in Example 2 exhibited inhibitory effects on both *Escherichia coli* and *Staphylococcus aureus* after 6 hours of interaction. Figure 4 and Figure 5 As shown. Among them, Figure 4 In the diagram, 'a' represents the E. coli colony diagram of the control group. Figure 4 In the diagram, b represents the Escherichia coli colony diagram of the experimental group. Calculations show that the natural liquid bactericide obtained in Example 2 can achieve a bactericidal rate of over 99% against Escherichia coli. Figure 5 In the diagram, 'a' represents the Staphylococcus aureus colony diagram of the control group. Figure 5 In the diagram, b represents the Staphylococcus aureus colony diagram of the experimental group. Calculations show that the natural liquid bactericide obtained in Example 2 can achieve a bactericidal rate of over 90% against Staphylococcus aureus.

[0048] The antibacterial activity of the bactericides in Examples 2-4 is shown in Table 1:

[0049] Table 1. Antibacterial activity of the bactericides in Examples 2-4

[0050]

[0051] The above embodiments provide a detailed description of the technical solution of the present invention. It should be understood that the above descriptions are only specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, additions, or similar substitutions made within the scope of the principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A natural liquid bactericide, characterized in that, The product is obtained by fermenting and culturing *Trichoderma echinosporum* and then removing the bacterial cells; the natural liquid bactericide targets *Escherichia coli* and *Staphylococcus aureus*; the *Trichoderma echinosporum* is named *Trichoderma echinosporum* (…). Trichoderma asperellum The specimen, RPT1, is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20221879. The preparation method of the natural liquid bactericide includes the following steps: (1) The activated Trichoderma hyacinth was inoculated into a fermentation medium and fermented to obtain the fermentation stock solution; (2) Centrifuge the fermentation broth under the following conditions: 10,000 rpm to 12,000 rpm for 30 to 40 minutes to obtain the supernatant, which is the natural liquid bactericide. Weigh 10g of glucose, 5g of yeast extract, and 1g of peptone and add them sequentially to 500mL of deionized water. Make up the volume to 1000mL with deionized water, stir magnetically to mix thoroughly, and then adjust the pH to 6-8 using citric acid or sodium hydroxide. Then place it in an autoclave and sterilize at 121℃ for 15min. Finally, cool to room temperature to obtain the fermentation medium.

2. The natural liquid bactericide according to claim 1, characterized in that, The fermentation culture conditions are: dynamic culture in a constant temperature shaker at 25-30℃ for 5-14 days.

3. The natural liquid bactericide according to claim 1, characterized in that, The supernatant was filtered through a membrane to obtain the natural liquid bactericide.

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