A Trichoderma fermentation agent combination and its application

Abstract

The present invention discloses a Trichoderma fermentation agent combination and its application. It relates to the field of fermentation engineering technology. The present invention provides a Trichoderma fermentation agent combination and related applications. The present invention screens out a defoamer suitable for Trichoderma fermentation. The results show that the most suitable defoamer for fermentation is 0.1% (V / V) B-404, and the fermentation product after adding this defoamer has the best inhibitory effect on three kinds of fungi: pepper white rot fungus, pepper wilt fungus and kiwifruit soft rot fungus. In addition, it was found that adding 3%, 5% or 7% (V / V) tangerine peel essential oil to the culture medium significantly enhanced the antibacterial effect of the fermentation product on the above three pathogenic fungi. In summary, in Trichoderma fermentation production, 0.1% B-404 defoamer and 3%, 5% or 7% tangerine peel essential oil combination can be added as a gain fermentation aid.

Description

Technical Field

[0001] The present invention relates to the technical field of fermentation engineering, and more particularly to a Trichoderma fermentation agent combination and application thereof. Background Art

[0002] Trichoderma spp. are a widely distributed and important class of biocontrol fungi in agriculture. These fungi exhibit significant inhibitory effects against plant pathogens, making them crucial for controlling plant fungal diseases and promoting agricultural production. Industrial production often utilizes liquid fermentation to obtain large quantities of live Trichoderma cells and their secondary metabolites for the preparation of Trichoderma biocontrol agents. However, Trichoderma spp. are prone to producing large amounts of foam during fermentation, which can affect the fermentation process and reduce product quality. Defoamers are commonly used in microbial fermentation processes to remove foam generated during fermentation, but research on defoamers suitable for Trichoderma fermentation is limited.

[0003] There are many types of defoamers available on the market, and there are compatibility issues between the added ingredients and the target fermentation products. The applicable conditions and defoaming effects of different defoamers also vary. Incompatible defoamers not only affect the fermentation growth of the target microorganisms, but also reduce the yield of the final target product. For example, Kochv et al. found that silicone-free defoamers did not have much effect on oxygen transfer, while silicone-containing defoamers had a significant effect on oxygen transfer in the initial stage of fermentation, but the effect gradually weakened as the fermentation continued, until it completely disappeared after 60 to 100 minutes. Adding 0.25% (V / V) defoamer to the fermentation of Saccharomyces cerevisiae increased the volumetric dissolved oxygen transfer coefficient Kla by 90%; however, when the defoamer addition level was less than 0.25% (V / V), Kla actually decreased. Anderssone et al. studied the effects of different concentrations of polyethylene glycol (PEG) 3350 and PEG 600 as defoamers on the synthesis of α-amylase by Bacillus subtilis and Bacillus amyloliquefaciens. They found that the addition of 5% PEG 3350 and 8% PEG 600 doubled the rate of α-amylase synthesis by Bacillus subtilis during fermentation. Addition of only 9% PEG 3350 inhibited α-amylase synthesis, while addition of only 20% (v / v) PEG 600 promoted α-amylase synthesis by Bacillus subtilis but inhibited α-amylase synthesis by Bacillus amyloliquefaciens. Holmesw et al. found that the expression of recombinant Fc fusion proteins in shake flask culture medium by Saccharomyces cerevisiae and Pichia pastoris was affected by the type and concentration of defoamers, as well as the culture medium used.

[0004] At the same time, there are many species under the genus Trichoderma, including green Trichoderma, long-branched Trichoderma, corning Trichoderma, etc., each of which has a different fermentation environment, and the type and amount of defoaming agent added are often confidential. There is currently no report on a universal defoaming agent.

[0005] In addition, in actual production, fermentation aids can be added to the fermentation system to enhance the antibacterial effect of biocontrol bacteria fermentation products. However, there is little research on fermentation aids suitable for Trichoderma fermentation production.

[0006] Therefore, selecting which specific defoaming agent and fermentation aid to optimize the Trichoderma fermentation method is a problem that those skilled in the art urgently need to solve. Summary of the Invention

[0007] In view of this, the present invention provides a Trichoderma fermentation agent combination and its application

[0008] Studying the effects of different defoamers and fermentation aids on the antibacterial effect of biocontrol Trichoderma metabolites, screening suitable defoamers and additive dosages, and determining the optimal addition of fermentation aids and corresponding addition amounts are of great significance for improving the fermentation production efficiency of Trichoderma and reducing the production cost of biocontrol Trichoderma.

[0009] In order to achieve the above object, the present invention adopts the following technical solutions:

[0010] A Trichoderma fermentation agent combination comprises a defoaming agent and a fermentation aid.

[0011] Preferred: defoaming agent: B-404; fermentation aid: tangerine peel essential oil.

[0012] The present invention also provides application of the Trichoderma starter agent combination in Trichoderma fermentation production.

[0013] Preferred: used for producing biocontrol agents.

[0014] Preferably, the method comprises: adding 0.01-0.1% by volume of B-404 to the culture medium for Trichoderma fermentation; and adding 3-11% by volume of tangerine peel essential oil to the fermentation product.

[0015] Preferably: comprising: adding 0.1% by volume of B-404 to the culture medium for Trichoderma fermentation culture;

[0016] When preventing and treating pepper white rot, add tangerine peel essential oil at a volume ratio of 3%, 5%, 7%, 9% or 11% to the fermented product;

[0017] When controlling pepper wilt pathogen, the fermented product is added at a volume ratio of 5%, 9% or 11%;

[0018] When preventing and controlling kiwifruit soft rot, the fermented product is added at a volume ratio of 7%, 9% or 11%.

[0019] The present invention also provides a biocontrol agent prepared based on any of the above applications.

[0020] Preferably, the biocontrol agent inhibits the following pathogens: pepper white rot pathogen, pepper wilt pathogen and kiwifruit soft rot pathogen.

[0021] The present invention also provides the above-mentioned Trichoderma fermentation agent combination, or any of the above-mentioned applications, or the application of the above-mentioned biocontrol agent in agricultural production.

[0022] It can be seen from the above technical solution that, compared with the prior art, the present invention discloses a Trichoderma starter combination and its application, which has the following beneficial effects:

[0023] This study used the antifungal effect on Trichoderma fermentation products as an evaluation indicator for their impact on pathogens. A preliminary screening of defoamers suitable for Trichoderma (strain MHT1134) fermentation was conducted. The results showed that the most suitable defoamer was 0.1% (v / v) B-404. The fermentation products containing this defoamer exhibited the best inhibitory effects against Sclerotium rolfsii, Fusarium oxysporum, and Phomopsis sp., the pathogen causing kiwifruit soft rot.

[0024] Furthermore, it was found that the addition of 3%, 5%, and 7% (V / V) tangerine peel essential oil to the culture medium significantly enhanced the antibacterial effect of the fermentation product against the three aforementioned pathogenic fungi. The addition of humic acid reduced the fermentation liquid's inhibitory ability against pepper white rot and kiwifruit soft rot, and slightly increased its inhibitory effect against pepper wilt. In summary, in Trichoderma (MHT1134 strain) fermentation production, 0.1% B-404 defoamer in combination with 3%, 5%, and 7% tangerine peel essential oil can be added as a fermentation aid. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0026] Figure 1 The accompanying drawings show the effect of the defoaming agent provided by the present invention on the inhibition of three pathogens by the fermentation product of MHT1134.

[0027] Figure 2 The accompanying drawing shows the effect of tangerine peel essential oil and humic acid provided by the present invention on the inhibition of Sclerotium rolfsii of pepper by MHT1134 fermentation product.

[0028] Figure 3 The accompanying drawing shows the effect of tangerine peel essential oil and humic acid provided by the present invention on the inhibition of pepper wilt pathogen by MHT1134 fermentation product.

[0029] Figure 4 The accompanying drawing shows the effect of tangerine peel essential oil and humic acid provided by the present invention on the inhibition of kiwifruit soft rot bacteria by MHT1134 fermentation products. DETAILED DESCRIPTION

[0030] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0031] The embodiment of the present invention discloses a Trichoderma fermentation agent combination and its application

[0032] In the embodiment:

[0033] Bacterial strains, culture media and reagents

[0034] The test strains: Trichoderma hamatum strain MHT1134, pepper wilt pathogen (also known as Fusarium oxysporum), pepper white rot pathogen (also known as Sclerotium solani), kiwifruit soft rot pathogen, are routine experimental materials and were provided by Guizhou Plant Protection Institute.

[0035] PDA medium: Mix 200 g of peeled potatoes with 500 mL of deionized water and boil for 20 minutes. Filter the supernatant, add 20 g of glucose and 17 g of agar powder, mix well, and adjust the volume to 1 L. Sterilize at 121°C for 20 minutes. Cool to 50°C, pour into a Petri dish, and allow to cool until ready for use.

[0036] PDB liquid medium: Mix 200 g peeled potatoes with 500 mL of water, boil for 20 minutes, filter, remove the supernatant, add 20 g of glucose, dissolve thoroughly, and adjust the volume to 1 L. Sterilize at 121°C for 20 minutes and cool until ready to use.

[0037] Defoaming agents: Six defoaming agents of three types were purchased from Zhonglian Federation Company for use in the experiment. See Table 1 for details.

[0038] Fermentation aids: Tangerine peel essential oil (which has anti-inflammatory, anti-cancer, and antioxidant medicinal values ​​in the medical field, and also has certain insecticidal and anthelmintic effects), humic acid (which has the effect of improving soil and increasing crop stress resistance, and because of its good adsorption properties, it can absorb a large amount of microbial agents, which can prevent the loss of microbial agents in the soil, and contains rich organic matter and trace elements, which can promote the growth and reproduction of microorganisms). The present invention uses the above materials as fermentation aids to explore their effects on Trichoderma fermentation.

[0039] Table 1 Defoamers used in the test

[0040]

[0041] Data processing

[0042] ① The growth inhibition rate was calculated using the following formula:

[0043]

[0044] Where: D0 is the mycelial growth diameter of the control group; D is the mycelial growth diameter of the treatment group.

[0045] ② Use SPSS software for data analysis

[0046] Example 1

[0047] Effects of Defoamers on the Growth of Trichoderma

[0048] At concentrations of 0.01%, 0.05%, and 0.1% (V / V), 10 μL, 50 μL, and 100 μL of defoamer were added to freshly sterilized PDA culture medium cooled to approximately 50°C. After thorough mixing, Trichoderma culture plates were prepared. A PDA culture plate without defoamer was used as a control. A piece of MHT1134 bacterial cake (Φ=5 mm) was inoculated in the center of the Trichoderma culture plate, mycelium side facing down. This was repeated three times for each defoamer concentration. After inoculation, the plate was cultured in a 28°C incubator for 3 days. The colony diameter was measured and the growth inhibition rate was calculated.

[0049] Effects of Defoamers on the Antibacterial Effect of Trichoderma Metabolites

[0050] Prepare PDB liquid culture medium. Add the selected defoamers at the corresponding concentrations to the culture medium, using three bottles of each dose, inoculating two MHT1134 bacterial cakes per bottle. A blank control was performed without defoamer. Each sample was cultured three times. Incubate at 28°C in a shaker at 150 rpm for 5 days, observing the culture medium daily for foaming.

[0051] After 5 days of shake flask culture, the mycelium was filtered through filter paper and the filtrate was centrifuged at 7500 rpm for 3 minutes. The filtrate was then filtered through a 0.45 μm bacterial filter and added to polydimethylsiloxane (PDA) medium at a ratio of 1:10 (v / v). PDA medium without Trichoderma culture filtrate and PDA medium without defoamer served as controls. The culture was inoculated with pepper wilt pathogen, kiwifruit soft rot pathogen, and pepper white rot pathogen. The culture was incubated at 28°C for 3 days. Colony diameters were measured and growth inhibition rates were calculated.

[0052] The experimental results show that:

[0053] It can be found from Table 2 that the addition of 0.1% and 0.01% B-404, 0.1% B-402 and 0.05% B-400 defoamers has a significant growth-promoting effect on the growth of the MHT1134 strain, and is significantly better than other defoamers, so these four defoamers were selected for in-depth study.

[0054] Table 2 Effect of defoamers on the growth of Trichoderma MHT1134

[0055]

[0056]

[0057] Note: Lowercase letters refer to the comparison between different concentrations of the same defoamer, and uppercase letters refer to the comparison between different defoamers at the same concentration, P<0.05.

[0058] Effect of defoaming agents on the antibacterial effect of MHT1134 fermentation products

[0059] from Figure 1 It was found that adding 0.1% B-404 defoamer significantly inhibited the growth of pepper Sclerotium rolfsii with MHT1134 compared to other defoamers. Adding 0.05% B-400, 0.1% B-402, and 0.01% B-404 had the least inhibitory effect on kiwifruit soft rot. Overall, adding 0.1% B-404 defoamer had a strong inhibitory effect on all three pathogens and was significantly superior to other defoamers.

[0060] Example 2

[0061] Based on the experiment of Example 1, the effect of fermentation aids on the antibacterial effect of Trichoderma metabolites was studied.

[0062] The selected defoamer (0.1% B-404) was added to PDB liquid medium, inoculated with two MHT1134 bacterial cakes, and cultured on a shaker for 5 days. The filtrate was added to PDA medium at a 1:10 (v / v) ratio. Tangerine peel essential oil was mixed at ratios of 1%, 3%, 5%, 7%, 9%, and 11% (v / v), and humic acid at ratios of 6%, 8%, and 10% (w / v) to prepare the culture medium. Fusarium wilt, kiwifruit soft rot, and Sclerotium rolfsii were inoculated in the center of the medium with the mycelium facing downward. After inoculation, the culture was cultured in a 28°C incubator. Colony diameters were measured on days 3, 5, and 7, and the growth inhibition rate was calculated.

[0063] The experimental results show that:

[0064] After adding tangerine peel essential oil to the MHT1134 fermentation broth, the antibacterial ability against the three pathogens was significantly improved. Figure 2 、 Figure 3 and Figure 4 As shown. The addition of 3%, 5%, 7%, 9% and 11% tangerine peel essential oil to the MHT1134 fermentation broth improved the inhibitory effect on pepper white rot fungus, and was significantly better than the addition of 1% tangerine peel essential oil on the fifth and seventh days. The inhibitory effect of 5%, 9% and 11% tangerine peel essential oil on pepper wilt fungus was significantly enhanced compared with CK on the third, fifth and seventh days. The inhibitory effect of 7%, 9% and 11% tangerine peel essential oil on kiwifruit soft rot fungus was significantly enhanced compared with CK on the third, fifth and seventh days. At the same time, the addition of humic acid significantly reduced the inhibitory ability of MHT1134 fermentation broth on pepper white rot fungus and kiwifruit soft rot fungus. On the third day after inoculation, the addition of 6%, 8% and 10% humic acid to the MHT1134 fermentation broth had a certain promoting effect on the inhibition rate of pepper wilt fungus compared with CK.

[0065] By comparing the effects of various defoamers on the growth of Trichoderma spp. MHT1134, a preliminary screening using three concentration gradients was conducted. B-404 (0.1%), B-402 (0.1%), and B-400 (0.05%) were initially selected as suitable defoamers for Trichoderma spp. MHT1134 fermentation production. Furthermore, antibacterial experiments were performed on the fermentation products obtained by simulated shake flask fermentation, and the optimal defoamer was B-404, added at a concentration of 0.1%. Based on this, fermentation adjuvant screening was conducted. Considering both antibacterial efficacy and economic cost, it was recommended that 3% tangerine peel essential oil be added to the fermentation liquid of MHT1134 strain as an adjuvant when used to control pepper white rot, 5% tangerine peel essential oil when used to control pepper wilt, and 7% tangerine peel essential oil when used to control kiwifruit soft rot.

[0066] The examples explored the effects of different defoamers and fermentation aids on the growth of Trichoderma and the antibacterial effect of fermentation products, and preliminarily screened a starter culture combination suitable for Trichoderma fermentation production, providing valuable data reference for the improvement of Trichoderma fermentation production process.

[0067] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0068] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Trichoderma fermentation agent combination in hooked Trichoderma ( Trichoderma hamatum ) Application of MHT1134 in the production of biocontrol agents by fermentation, characterized in that: The Trichoderma fermentation agent combination is composed of defoamer B-404 and tangerine peel essential oil; The application comprises adding 0.01 to 0.1% by volume of defoamer B-404 to the Trichoderma culture medium for Trichoderma fermentation; and adding 3 to 11% by volume of tangerine peel essential oil to the fermentation product; The biocontrol agent inhibits the following pathogens: pepper white rot, pepper wilt and kiwifruit soft rot; The defoamer B-404 was purchased from Zhonglian Federal Corporation.

2. The use according to claim 1, characterized in that include: 0.1% by volume of B-404 was added to the culture medium for Trichoderma fermentation; as well as When preventing and treating pepper white rot, the fermented product is added with 3%, 5%, 7%, 9% or 11% by volume of tangerine peel essential oil; When preventing and controlling pepper wilt pathogen, the fermented product is added with 5%, 9% or 11% by volume of tangerine peel essential oil; or When preventing and controlling kiwifruit soft rot pathogens, the fermented product is added with 7%, 9% or 11% by volume of tangerine peel essential oil.

3. A biocontrol agent prepared based on the application according to any one of claims 1 to 2.

4. Use of the biocontrol agent according to claim 3 in agricultural production, wherein the use in agricultural production is to inhibit the following pathogens: pepper white rot, pepper wilt and kiwifruit soft rot.

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