Preparation method and application of an antifungal epiquinomycin

Abstract

The application provides a preparation method and application of an antifungal epiquickmelin, and relates to the technical field of fermentation.The fruit extract is used as a culture medium component for the first time in the application to ferment Fusarium equiseti to produce epiquickmelin.On the one hand, the application provides a new resource recycling way for the discarded fruit material, and solves the problems of resource waste and environmental pollution.On the other hand, the use of discarded fruit material as a culture medium greatly reduces the production cost of epiquickmelin, and provides technical support for the industrial production of epiquickmelin.In the application, the inhibitory activity of epiquickmelin on seven kinds of plant pathogenic fungi is determined to determine the potential of epiquickmelin as an antifungal pesticide.The experimental results prove that the epiquickmelin produced by Fusarium equiseti has good antifungal activity on plant pathogenic fungi, and is expected to become a substitute for chemical pesticides.

Description

Technical Field

[0001] This application relates to the field of fermentation technology, and in particular to a method for preparing and applying an antifungal agent, ibuprofen. Background Technology

[0002] The control of plant fungal diseases is a global challenge. Historically, chemical pesticides have been widely used to control plant fungal diseases, achieving good results and mitigating significant economic losses. However, the long-term and widespread use of chemical pesticides has led to severe resistance in pathogenic fungi, reducing the control efficacy of existing pesticides. Furthermore, the development of new pesticides cannot keep pace with the evolution of fungal resistance, seriously impacting agricultural production. Equisetin, a microbial secondary metabolite, is a promising "green" antifungal lead compound with a unique structure and low environmental pollution, and has attracted widespread attention as a potential replacement for chemical pesticides. Large-scale production of equisetin and the discovery of its structural analogs have become current research hotspots. However, the current common production method for equisetin is fermentation using commercial PDW medium. The high fermentation cost hinders its industrialization and prevents it from better serving agricultural production. Generally, fermentation feedstock accounts for about 30% of the cost of microbial fermentation, which is crucial for saving overall costs. Therefore, utilizing waste biomass as a fermentation feedstock to produce equisetin is a promising strategy.

[0003] China is the world's largest producer and consumer of fruit. However, it also faces a significant problem of fruit waste. Besides fruit processing and fruit rotting during harvesting and transportation, a large amount of fruit waste also comes from fruit markets and the catering industry. This fruit waste is rich in sugar, cellulose, trace elements, and other components, making it a potentially high-quality fermentation substrate. However, there are currently no reports on using fruit waste as a fermentation material to produce equisetin. Summary of the Invention

[0004] To address the aforementioned problems, the present invention aims to provide a method for converting waste fruit into the high-value antifungal lead compound icariin using a Fusarium equiseti D39 bioreactor, thereby increasing the yield of icariin and disclosing the antibacterial activity of icariin against different bacterial species.

[0005] On one hand, this application provides a culture medium for Fusarium equiseti, which includes fruit.

[0006] Furthermore, the fruit is waste fruit material and / or fruit product scraps; preferably, the fruit includes one or more of apples, watermelons, grapes, peaches, and bananas.

[0007] More preferably, the fruit includes one or more of apples, watermelons, and grapes.

[0008] More preferably, the fruit is a fruit extract; more preferably, the method for preparing the fruit extract includes: soaking and homogenizing the fruit, using water as an extractant, hot-extracting the fruit extract, and filtering and concentrating to obtain the fruit extract.

[0009] Preferably, the sugar content in the fruit extract is 10-50 g / L.

[0010] More preferably, the sugar content in the fruit extract is 20 g / L.

[0011] Preferably, the sugar includes one or more of glucose, fructose, trehalose, maltose, sucrose, rhamnose, sorbitol, mannose, arabinose, galactose, lactose, stachyose, raffinose, and raffinose; preferably, glucose and / or fructose.

[0012] In this application, the types of sugars were screened, and experiments confirmed that the higher the content of fructose and glucose, and the greater the proportion of fructose, the higher the fermentation yield; the greater the proportion of sucrose, the lower the fermentation yield. This indicates that Fusarium equisetifolium has a preference for the absorption and utilization of sugars during the production of itacrylamide. Therefore, the fruits in the culture medium can be fruits containing glucose and / or fructose.

[0013] Furthermore, this application also determined the starch content in the fruit. The results showed that the higher the starch concentration of the fruit extract, the higher the yield of itacrylamide produced by *Fusarium equisetifolium* fermentation; the itacrylamide yield showed a linear relationship with starch concentration. Therefore, the fruit in the culture medium can be a high-starch fruit, or the starch content of the culture medium can be adjusted by adding starch or starch-containing substances.

[0014] Furthermore, the culture medium also includes potatoes; preferably, the fruit is a fruit extract and the potato is a potato extract; more preferably, the volume ratio of the fruit extract to the potato extract is 0.7-1.1:1.

[0015] Potato extract can be prepared using conventional methods.

[0016] More preferably, the method for preparing the potato extract includes: boiling potatoes at 80℃-100℃ for 10min-30min; more preferably, the ratio of potato to water is 100-300g / L.

[0017] More preferably, the method for preparing the potato extract includes: cutting potatoes into chunks, boiling them at 100°C for 20 minutes, and diluting them with deionized water; more preferably, the material-to-liquid ratio is 200 g / L.

[0018] Furthermore, the sugar content of the culture medium is 10 g / L-50 g / L; and / or, the starch content of the culture medium is 1-20 g / L.

[0019] More preferably, the sugar content of the culture medium is 20 g / L; and / or, the starch content of the culture medium is 8 g / L.

[0020] This application demonstrates through experiments that watermelon can be used as the best sugar source for the culture medium. However, since Fusarium equisetifolium requires starch (carbon source) for growth, the yield of isocyanin is low under carbon source-free conditions. Therefore, potato extract rich in starch is combined with watermelon extract to solve the problem of lack of carbon source in watermelon extract and obtain the best combined culture medium for the fermentation of isocyanin by Fusarium equisetifolium.

[0021] Furthermore, the *Fusarium equisetifolium* is *Fusarium equisetifolium* D39.

[0022] On the other hand, this application also provides a method for preparing the culture medium, the method comprising: soaking and homogenizing the fruit, and then heat-extracting the fruit extract to obtain the Fusarium equisetifolium culture medium.

[0023] Preferably, the method includes: soaking and homogenizing the fruit, using water as an extractant, hot-extracting the fruit extract, and filtering and concentrating to obtain the fruit extract.

[0024] More preferably, the conditions for thermal extraction include a material-to-liquid ratio of 1:(1-20)(w / v), an extraction temperature of 40℃-80℃, and an extraction time of 1-5 hours.

[0025] More preferably, the conditions for thermal extraction include a material-to-liquid ratio of 1:10 (w / v), an extraction temperature of 65°C, and an extraction time of 2 hours.

[0026] In a preferred embodiment, the method for preparing the culture medium includes:

[0027] Step 1: Soak and homogenize the fruit, then heat extract the fruit extract. The heat extraction conditions include: a material-to-liquid ratio of 1:(1-20)(w / v), an extraction temperature of 40℃-80℃, and an extraction time of 1-5 hours.

[0028] Step 2: With a material-to-liquid ratio of 100-300 g / L, boil the potatoes at 80-100℃ for 10-30 minutes to obtain potato extract.

[0029] Step 3: Mix the fruit extract and potato extract at a volume ratio of (0.7-1.1):1, and concentrate the final volume to 45%-55% of the original volume to obtain the culture medium; preferably, 50%.

[0030] On the other hand, this application also provides the use of the culture medium in the production of equisetin and its derivatives and / or in increasing the yield of equisetin.

[0031] This application demonstrates for the first time that apple extract is a fermentation feedstock with the potential to directly replace PDW medium in the production of zebumycin.

[0032] Furthermore, this application also demonstrates that watermelon extract and grape extract have a synergistic effect when combined with potatoes, increasing the yield of erythromycin by 4 times and 9 times respectively, which is 33.5% and 16.8% higher than that of PDW medium. This indicates that the combination of watermelon and potato can be used as a high-quality fermentation material to replace PDW medium for the production of erythromycin.

[0033] This application found that the increased yield of eqxycritin is achieved by promoting the expression of key eqxycritin synthesis genes (eqxS, eqxC, eqxD).

[0034] In addition, this application found that the products obtained by fermenting Fusarium equisetifolium using fruit as a culture medium contain icariin and its various derivatives, which is more conducive to the discovery of natural antibacterial lead compounds.

[0035] On the other hand, this application also provides a method for producing zebumycin and its derivatives, the method comprising: fermenting Fusarium equisetifolium in the culture medium to produce zebumycin and its derivatives.

[0036] Those skilled in the art can use conventional fermentation methods for Fusarium equisetifolium to ferment it.

[0037] In a preferred embodiment, the method includes: inoculating Fusarium equisetifolium blocks into the culture medium for fermentation, with fermentation conditions of 20℃-42℃, 100-200 rpm / min, and fermentation for 7-28 days.

[0038] Preferably, the fermentation conditions are 28℃, 180rpm / min, and 14 days.

[0039] On the other hand, this application also provides the application of zebumycin or zebumycin and its derivatives prepared by the method in antibacterial applications.

[0040] Further, the fungus is a fungus; preferably, the fungus includes one or more of the following: *Botrytis cinerea* Pers. (involved in gray mold of crops such as grapes, tobacco, cucumbers, strawberries, tomatoes, peppers, and eggplants), *Fusarium graminearum* (involved in wheat scab and corn grain rot), *Colletotrichumorbiculare* Arx. (involved in anthracnose of cucurbits), *Fusarium asiaticum* (involved in root rot of crops such as corn, tobacco, soybeans, peppers, tomatoes, and onions, and wheat stem base rot), *Rhizoctonia cerealis* (involved in wheat sheath blight), *Magnaporthe grisea* Barr (involved in rice blast), and *Phytophthora nicotianae* (involved in tobacco black shank).

[0041] This application is the first to discover that equisetin can effectively inhibit rice blast Magnaporthe grisea, Fusarium asiaticum, Colletotrichum orbiculare Arx., and Phytophthora nicotianae.

[0042] On the other hand, this application also provides the application of zebumycin or zebumycin and its derivatives prepared by the method in the preparation of pesticides, wherein the pesticides are antifungal pesticides.

[0043] Further, the fungus is a fungus; preferably, the fungus includes one or more of the following: Botrytis cinerea Pers., Fusarium graminearum, Colletotrichum orbiculare Arx., Fusarium asiaticum, Rhizoctonia cerealis, Magnaphorthe grisea Barr, and Phytophthora nicotianae.

[0044] In a preferred embodiment, the icariin can completely inhibit Botrytis cinerea infection and has no phytotoxicity to grapes, thereby achieving green and safe plant disease control.

[0045] The present invention has the following beneficial effects:

[0046] This invention is the first to use extracts from five fruits (apple, banana, grape, peach, and watermelon) as a culture medium to ferment Fusarium equisetifolium to produce ikemetrine. On the one hand, this provides a new resource reuse pathway for waste fruit materials, solving the problems of resource waste and environmental pollution. On the other hand, using waste fruit materials as a culture medium significantly reduces the production cost of ikemetrine, providing technical support for the industrial-scale production of ikemetrine.

[0047] Furthermore, this invention screens extracts from five fruits (apple, banana, grape, peach, and watermelon) to evaluate whether they can maintain the growth of Fusarium equisetifolium and the production of isocyanate without the need for additional nutrients, thus providing new materials for the cultivation of Fusarium equisetifolium and the production process of isocyanate.

[0048] This invention also marks the first time that a combination of fruit and potato has been used as a culture medium for *Fusarium equisetifolium*. The optimal culture combination for producing eczemycin was discussed, demonstrating that fruit can act as an active ingredient to increase eczemycin yield and is a high-quality fermentation material to replace PDW medium for eczemycin production. Finally, transcriptomics and qPCR analyses were used to elucidate the reasons for differences in eczemycin yield, and the main active ingredients and yield-enhancing mechanisms of waste fruit extracts were analyzed, providing a theoretical basis for the large-scale production of eczemycin.

[0049] This invention determined the inhibitory activity of isocyanic acid against seven plant pathogenic fungi to identify its potential as an antifungal pesticide. Experimental results demonstrated that isocyanic acid produced by *Fusarium equisetifolium* exhibits good activity against plant pathogenic fungi, showing promise as an alternative to chemical pesticides. Furthermore, the invention analyzed and predicted the diversity of isocyanic acid derivatives, providing more lead molecules for the development of natural product pesticides. This will provide technical support and theoretical guidance for research on producing novel natural product pesticides using waste fruit extracts, and has significant social and economic implications. Attached Figure Description

[0050] The accompanying drawings, which are included to provide a further understanding of this application and form part of this application, illustrate exemplary embodiments and are used to explain this application, but do not constitute an undue limitation of this application. In the drawings:

[0051] Figure 1 A statistical chart showing the fermentation yield of equisetin in different fruit extracts;

[0052] Figure 2 Statistical chart of equisetin fermentation yield in different fruit extracts after exogenous addition of potato extract;

[0053] Figure 3 A statistical graph showing the yield of equisetin under different free sugar fermentation conditions;

[0054] Figure 4 A statistical chart showing equisetin yield at different starch concentrations;

[0055] Figure 5 The nodes of equisetin in the molecular network are shown in red for AWE, green for WWES, and blue for PDW.

[0056] Figure 6 Transcriptome analysis of Fusarium equisetifolium D39 under fructose and sucrose culture conditions: (A) DEGs volcano plot, red / blue dots indicate DEG upregulation / downregulation; (B) GO enrichment analysis of DEGs; (C) KEGG pathway enrichment of DEGs, x-axis and y-axis represent enrichment factors and pathway terms, respectively, and the significance of DEGs is represented by color and size; (D) Compared with the control, fructose culture affected the major DEGs related to polyketide backbone synthesis in Fusarium equisetifolium D39; (E) Compared with the control, fructose culture affected the major DEGs related to glycolysis / gluconeogenesis pathway in Fusarium equisetifolium D39.

[0057] Figure 7 The expression levels of key equisetin synthesis genes were measured under fructose and sucrose fermentation treatments, with sucrose as a control.

[0058] Figure 8 The control effects of different concentrations of equisetin on grape gray mold. (A) Inhibition rate of lesions. (B) Diameter of lesions. Detailed Implementation

[0059] To more clearly illustrate the overall concept of this application, a detailed description is provided below with reference to the accompanying drawings and embodiments. Numerous specific details are set forth in the following description to provide a more thorough understanding of the invention. However, it will be apparent to those skilled in the art that the invention can be practiced without one or more of these details. In other instances, certain technical features well-known in the art have not been described to avoid confusion with the invention.

[0060] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0061] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to this application. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0062] Unless otherwise specified, in the following embodiments, reagents or instruments whose manufacturers are not indicated are all conventional products that can be purchased commercially.

[0063] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention. Test methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the respective manufacturers.

[0064] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0065] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention all employ conventional techniques in the fields of microbiology, biochemistry, analytical chemistry, cell culture, and related areas.

[0066] Among them, *Fusarium equiseti* D39 (*Fusarium equiseti* GLY27) has been published in Chinese patent application No. 201911110953.2. *Botrytis cinerea* Pers., *Fusarium graminearum*, *Colletotrichum orbiculare* Arx., *Fusarium asiaticum*, *Rhizoctonia cerealis*, *Magnaporthegrisea* Barr, and *Phytophthora nicotianae* are all preserved and provided by the National Germplasm Bank of Agricultural Environmental Microorganisms (Shandong).

[0067] The PDW culture medium formula is: 10 g / L potato extract powder (of which starch content is 8 g / L), 20 g / L glucose, and water added to make up to 1 L.

[0068] In addition, the "water" mentioned in this invention includes any feasible water that can be used in the art, such as deionized water, distilled water, ion-exchanged water, double-distilled water, high-purity water, and purified water.

[0069] In the following examples, unless otherwise specified, % means wt%, i.e., weight percentage.

[0070] 1. Using the Fusarium equisetifolium D39 bioreactor to convert waste fruit into equisetin

[0071] 1.1. Fruit extracts were used to replace PDW medium for culturing Fusarium equisetifolium D39 to produce equisetin.

[0072] (1) Pre-treatment of waste fruit

[0073] Waste apples, bananas, grapes, peaches, and watermelons were soaked and homogenized using a grinder. Free sugars were extracted using distilled water at a material-to-liquid ratio of 1:10 (w / v), at 65℃ for 2 hours, and filtered through gauze. The free sugar concentration of the filtrate was measured and uniformly concentrated to 20 g / L. After sterilization (121℃, 20 min), this filtrate was used as the fermentation substrate for *Fusarium equisetifolium* D39, and compared with commercial PDW medium.

[0074] (2) Fermentation process and equisetin quantification

[0075] Fusarium equisetifolium D39 was fermented in different fruit extracts. 5 mm diameter mycelial blocks were cut from the edge of 3-day-old Fusarium equisetifolium colonies and transferred to 500 mL Erlenmeyer flasks containing 200 mL of fruit extract. Each group was repeated three times. The above systems were incubated at 28 °C and 180 rpm / min for 14 days. After 14 days, the fermentation products were processed. The mycelia were filtered through gauze. The filtrate was extracted three times with an equal volume of ethyl acetate, and the extract was obtained by vacuum evaporation. The broken mycelia were soaked in methanol and dichloromethane (1:1 [v / v]), ultrasonically extracted at 40 Hz for 15 min, the organic phase was evaporated to dryness under vacuum, dissolved in 100 mL of distilled water, and extracted three times with 2 times the volume of ethyl acetate. The ethyl acetate phase was then evaporated to dryness to obtain the mycelial extract. The filtrate extract and the mycelial extract were combined to obtain the total extract sample.

[0076] A series of solutions containing different concentrations of equisetin were prepared and analyzed using high-performance liquid chromatography (HPLC). A calibration curve was constructed by plotting the peak area (y) versus concentration (x) of equisetin. Calibration curve: y = 15.843x + 0.1263, R0 2 =0.997, applicable concentration range: 0.1-1.1 mg / mL. The equisetin content in each sample was quantitatively determined using this calibration curve.

[0077] (3) Differences in equisetin yield when different fruit extracts are used as culture media

[0078] Table 1 shows the equisetin yields produced by *Fusarium equisetifolium* D39 when cultured in different fruit extracts. The equisetin was obtained from Table 1. Figure 1 .

[0079] Table 1. Fermentation yield of ikepimic acid in different fruit extracts

[0080]

[0081] Note: “a”, “b”, “c”, “d”, “e”, “f” represent equisetin production from high to low, respectively.

[0082] As shown in Table 1 and Figure 1 As shown, the yield of equisetin produced by *Fusarium equisetifolium* D39 varied significantly when cultured in different fruit extracts. Apple extract yielded 20.1 mg / L of equisetin, comparable to the yield of PDW (25.8 mg / L), a commonly used culture medium for fungal fermentation, indicating that apple extract is a potential direct substitute for PDW in equisetin production. Except for apple extract, the yields of other fruits were lower than the control and cannot be directly used as fermentation feedstocks.

[0083] 1.2 A combination of fruit extracts and potato extracts was used to replace PDW medium for culturing Fusarium equisetifolium D39 to produce equisetin.

[0084] (1) Pretreatment of fermentation raw materials

[0085] Cut 200g of potatoes into small pieces, put them in a pot and boil for 20 minutes to obtain an extract. Dilute the extract with deionized water to 1L to obtain potato extract. Add apple extract, banana extract, grape extract, peach extract and watermelon extract with a sugar content of 20g obtained by the method in 1.1(1) to the above potato extract. Concentrate the final volume to 1L, sterilize and use it for fermentation of Fusarium equisetifolium D39, and compare it with commercial PDW medium.

[0086] (2) The fermentation process and equisetin quantification are the same as in 1.1(2).

[0087] (3) Equisetin yield variation

[0088] Table 2 shows the equisetin yields of Fusarium equisetifolium D39 cultured in different media. The equisetin production was obtained from Table 2. Figure 2 .

[0089] Table 2. Yields of ikepimic acid in different fruit extracts after exogenous addition of potato extract.

[0090]

[0091]

[0092] Note: “a”, “b”, “c”, “d”, “e”, “f” represent equisetin production from high to low, respectively.

[0093] As shown in Table 2 and Figure 2 As shown, the fermentation yield of all groups increased after the exogenous addition of potato extract. Specifically, the yield of equisetin in watermelon extract and grape extract increased by 4-fold and 9-fold, respectively, reaching 38.2 mg / L and 33.6 mg / L, which were 33.5% and 16.8% higher than those in PDW medium, respectively. This indicates that the combination of watermelon and potato can serve as a high-quality fermentation material to replace PDW medium for equisetin production.

[0094] 2. Main active components in fruit extracts and their effects on equisetin yield

[0095] 2.1 Analysis of free sugar components in fruit extracts and the effect of different sugars on equisetin yield

[0096] (1) Determination of free sugar components in fruit extracts

[0097] Standard substances (glucose, fructose, trehalose, maltose, sucrose, rhamnose, sorbitol, mannose, arabinose, galactose, lactose, stachyose, raffinose, and raffinose) were dissolved in water to prepare solutions with final concentrations of 0.5, 1, 5, 10, 20, 30, 40, and 50 μg / mL. Calibration curves were constructed by plotting the relationship between the peak area (y) and concentration (x) of free sugars (as shown in Table 3). The soluble sugars in the samples were quantitatively determined using the calibration curves. Since the sugar content in the potato extract was negligible, the sample used here was the fruit extract prepared in section 1.1.

[0098] Table 3 Retention times and calibration curves of various standard substances

[0099]

[0100] (2) The effect of carbohydrates on equisetin production

[0101] Fusarium equisetin D39 was fermented using equal amounts of glucose, fructose, sucrose, maltose, and raffinose instead of the sugars in PDW medium (mainly composed of potato extract and glucose) (20 g / L). Each treatment was repeated three times. The yield of equisetin under different free sugar fermentation conditions was determined by HPLC, and the fermentation and determination methods were the same as in 1.1(2).

[0102] (3) Equisetin yield is related to the sugar content composition of fruit extracts

[0103] The results of the determination of 2.1(1) are shown in Table 4. Table 4 shows that the free sugar components of the five fruit extracts differed greatly, and the main components were fructose, glucose and sucrose.

[0104] Table 4. Free sugar content (%) in fruit extracts

[0105]

[0106] Results are expressed as mean ± standard deviation (SD) (n=3)

[0107] A comprehensive analysis of Tables 4 and 2 shows that after the addition of potato extract, the higher the content of fructose and glucose in the total free sugars of each extract, the higher the proportion of fructose and the higher the fermentation yield; the higher the proportion of sucrose, the lower the fermentation yield. This indicates that Fusarium equisetin has a preference for the absorption and utilization of sugars during its equisetin production process.

[0108] (4) Fusarium equisetifolium D39 prefers fructose.

[0109] 2.1(2) The yield statistics of equisetin produced by fermentation of Fusarium equisetifolium D39 are shown in Table 5. The data in Table 5 were plotted to obtain the yield statistics. Figure 3 .

[0110] Table 5. Effects of different free sugars on equisetin yield

[0111]

[0112] Note: “a”, “b”, “c”, “d”, “e”, “f” represent equisetin production from high to low, respectively.

[0113] Table 5 and Figure 3 The results showed that fructose mainly promoted the production of equisetin, with a yield of 31.8 mg / L, followed by glucose at 25.9 mg / L, while sucrose had the lowest yield at 8.6 mg / L, which was only 0.3 times that of fructose.

[0114] 2.2 Starch content in fruit extracts and its effect on equisetin yield

[0115] (1) The starch content of apple extract, banana extract, grape extract, peach extract and watermelon extract in 1.1(1) with a free sugar content of 20 g / L was detected.

[0116] (2) Fusarium equisetin D39 was fermented using starch concentration gradients (3, 6, 9, 12 g / L), with each group repeated 3 times. The yield of equisetin was measured. The fermentation method and determination method were the same as in 1.1(2).

[0117] (3) Starch can promote the production of equisetin.

[0118] The results of 2.2(1) are shown in Table 6.

[0119] Table 6. Starch content in different fruit extracts

[0120]

[0121] Note: Parts that can be ignored are indicated by an asterisk [*].

[0122] Based on the combined results in Tables 1 and 6, it can be seen that the higher the starch content in the fruit extract, the higher the yield of equisetin.

[0123] The results of 2.2(2) are shown in Table 7. A graph can be plotted from Table 7 to obtain the results. Figure 4 .

[0124] Table 7. Effect of starch concentration on equisetin yield

[0125]

[0126] From Table 7 and Figure 4 The results showed that the higher the starch concentration of the fruit extract, the higher the equisetin production from Fusarium equisetifolium fermentation, and the equisetin production changed linearly with starch concentration.

[0127] 3. Structural diversity of equisetin under optimal fermentation combination conditions

[0128] (1) The diversity of equisetin derivatives of watermelon extract (WWES) and apple extract (AWE) with 8 g / L starch (starch content in PDW medium) added (since apple extract contains starch, starch was not added separately here) and Fusarium equisetin D39 cultured in PDW medium was analyzed by MS / MS-based molecular network technology.

[0129] (2) Waste fruit extracts stimulated the production of equisetin derivatives.

[0130] The results of the diversity analysis are as follows Figure 5 As shown, compared to 10 nodes in the PDW treatment, the samples treated in WWES and AWE contained 17 and 12 nodes in the equisetin cluster, respectively. Furthermore, five products at m / z 372.216, 360.216, 404.243, 407.29, and 420.238 were found only in WWES and AWE, suggesting that these two culture conditions are more suitable for producing equisetin analogs, potentially containing more antifungal analogs, and are more conducive to the discovery of natural antibacterial lead compounds.

[0131] 4. Mechanism by which fructose promotes equisetin production

[0132] (1) To investigate the mechanism by which fructose enhances equisetin production, Fusarium equisetifolium D39 was cultured for 7 days in 8 g / L starch with 20 g / L fructose or sucrose added. The collected mycelia were washed three times with phosphate buffer and frozen in liquid nitrogen for RNA-Seq analysis. The control group (CK) was sucrose.

[0133] (2) qRT-PCR verification

[0134] The expression levels of key biosynthetic genes (eqxS, eqxC, eqxD) of equisetin under fructose and sucrose treatments were detected by qRT-PCR, further elucidating the reasons for the differences in equisetin yield under different free sugar fermentation conditions.

[0135] (3) Production enhancement mechanism

[0136] Compared with sucrose treatment, a total of 1198 degrees were found in the fructose treatment group, of which 505 were upregulated and 693 were downregulated. Figure 6 A) All degs were annotated in both the Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Figure 6 B and Figure 6 C). The most important enriched GO term was "ribonucleoprotein complex biogenesis," followed by "rRNA processing," "cellular component biogenesis," and "monocarboxylic acid metabolism." Highly enriched KEGG pathways included "pyruvate metabolism" (Map00620), "glycolysis / gluconeogenesis" (Map00010), "ribosome biogenesis in eukaryotes" (Map03008), "glycine, serine, and threonine metabolism" (Map00260), and "alanine, aspartate, and glutamate metabolism" (Map00250). Previous studies have shown that these pathways are involved in energy and material metabolism as well as cell proliferation.

[0137] Compared with sucrose treatment, fructose treatment significantly upregulated the expression levels of genes related to the glycolysis pathway in Fusarium equisetifolium D39. Figure 6 Furthermore, compared to sucrose treatment, the expression levels of polyketide synthase-related genes (A6789), SAM-dependent methyltransferase-related genes (A1115), and N-methyltransferase-related genes (A6788) in *Fusarium equisetifolium* D39 were significantly upregulated under fructose treatment. Figure 6 D). This indicates that fructose promotes equisetin synthesis by enhancing the bioactivity of key enzymes involved in the biosynthetic pathway.

[0138] and Figure 7 The results demonstrated that fructose stimulated the expression of key genes involved in equisetin synthesis.

[0139] 5. Studies on the in vitro and in vivo antifungal activity of Equisetin

[0140] (1) Test strains

[0141] *Botrytis cinerea* Pers., *Fusarium graminearum*, *Colletotrichum orbiculare* Arx., *Fusarium asiaticum*, *Rhizoctonia cerealis*, *Magnaporthe grisea* Barr., and *Phytophthoranicotianae* were all cultured using a universal method to obtain mycelial discs.

[0142] (2) Experimental Design

[0143] Equisetin dissolved in DMSO (prepared by the method described in 1 or 2) was added to PDA and oat agar (OA) media (0.5% DMSO [vol / vol]) at final concentrations of 0, 2.5, 5, 10, 20, 40, and 60 μg / mL, respectively. Five-mm diameter mycelial discs were cut from the edge of colonies grown for 3 days and inoculated into the center of each medium (OA for *Phytophthora tobaccois*, PDA for other fungi), then incubated at 28°C for 3 days. Each treatment was repeated three times, with carbendazim or metalaxyl as positive controls. Hyphae diameter was determined using the cross-cross method, and the inhibition rate was calculated as [(Rr) / R-0.5]×100%, where R and r are the mean diameters of the control and treated colonies, respectively. EC50 was obtained using a toxicity regression equation. 50 value.

[0144] Equisetin was dissolved in 0.5% DMSO [v / v] solution to prepare aqueous solutions with final concentrations of 0, 6, 12, and 18 μg / mL, respectively, to verify its in vivo antibacterial effect. Grapes of similar weight and uniform shape were selected, surface-sterilized with 75% ethanol, rinsed with sterile water, and air-dried. A sterile toothpick was used to pierce the grape at its maximum circumference, and the grape was immersed in the equisetin aqueous solution for 5 minutes before air-drying. Subsequently, a 2 mm diameter Botrytis cinerea cake was inoculated at the piercing site of each grape. All treated samples were incubated at 25℃ for 3 days, with sterile water containing 0.5% DMSO solution as a control. Ten grapes were used in each treatment group, with three replicates. The diameter of lesions was determined using the cross-hatching method, and the inhibition rate was calculated as [(Rr) / R-0.2]×100%, where R and r are the average diameters of the control and treated lesions, respectively.

[0145] (3) Experimental Results

[0146] The antibacterial effects of Equisetin against *Botrytis cinerea* Pers., *Fusarium graminearum*, *Colletotrichum orbiculare* Arx., *Fusarium asiaticum*, *Rhizoctonia cerealis*, *Magnaporthe grisea* Barr., and *Phytophthora nicotianae* are recorded in Table 8.

[0147] Table 8. EC50 of Equisetin against seven plant pathogenic fungi 50 (μg / mL)

[0148]

[0149] Table 8 shows that equisetin possesses broad-spectrum activity against plant pathogenic fungi, exhibiting significant inhibitory effects against a variety of common pathogenic fungi. This application is the first to discover that equisetin can effectively inhibit *Magnaporthegrisea*, *Fusarium asiaticum*, *Colletotrichum orbiculare* Arx., and *Phytophthora nicotianae*.

[0150] The results of the in vivo antibacterial effect experiment are shown in Table 9. A graph was plotted from Table 9 to obtain... Figure 8 .

[0151] Table 9. Preventive effects of different concentrations of iclamycin on fruit fungal diseases.

[0152]

[0153] From Table 9 and Figure 8 As shown, at concentrations of 6 μg / mL and 12 μg / mL, equisetin exhibited inhibitory effects of 27.7% and 67.8% on Botrytis cinerea infection, respectively. At a concentration of 18 μg / mL, equisetin completely inhibited Botrytis cinerea infection without causing phytotoxicity to grapes. Figure 8 B) The results showed that equisetin is a natural antifungal pesticide with broad application prospects.

[0154] The above description is merely an embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principle of this application should be included within the scope of the claims of this application.

Claims

1. A type of *Fusarium equisetifolium* ( Fusarium equiseti The application of GLY27 culture medium in the production of zebumycin and / or in increasing zebumycin yield is characterized by, The culture medium includes a fruit extract, wherein the fruit extract is an apple extract; The method for preparing the fruit extract includes: soaking and homogenizing the fruit, and then hot-extracting the fruit extract. The hot extraction conditions include: using distilled water as the extraction solvent, a material-to-liquid ratio of 1:(1-20) w / v, an extraction temperature of 40 ℃-80 ℃, and an extraction time of 1-5 hours. Alternatively, the culture medium includes fruit extract and potato extract, wherein the fruit extract is one of apple extract, watermelon extract, and grape extract; the preparation method of the fruit extract includes: soaking and homogenizing the fruit, and then hot-extracting the fruit extract; the hot extraction conditions include: using distilled water as the extraction solvent, a solid-liquid ratio of 1:(1-20) w / v, an extraction temperature of 40 ℃-80 ℃, and an extraction time of 1-5 hours; the preparation method of the potato extract includes: using deionized water as the extraction solvent, a solid-liquid ratio of 100-300 g / L, and boiling the potato at 80 ℃-100 ℃ for 10 min-30 min to obtain the potato extract; Using the aforementioned *Fusarium equisetifolium* ( Fusarium equiseti GLY27 medium is used for fermentation of *Fusarium equisetifolium* to produce erythromycin and / or increase erythromycin yield, wherein *Fusarium equisetifolium* is *Fusarium equisetifolium* (… Fusarium equiseti )GLY27.

2. The application according to claim 1, characterized in that, The volume ratio of the fruit extract to the potato extract is 0.7-1.1:

1.

3. The application according to claim 1, characterized in that, The sugar content of the culture medium is 10 g / L-50 g / L; and / or the starch content of the culture medium is 1-20 g / L.

4. A method for producing ibuprofen, characterized in that, The method includes: fermenting *Fusarium equisetifolium* to produce erythromycin using the culture medium described in any one of claims 1-3, wherein *Fusarium equisetifolium* is *Fusarium equisetifolium* (…). Fusarium equiseti )GLY27.

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