Preparation method and application of antifungal illinomycin

Abstract

The invention provides a preparation method and application of antifungal illinomycin, and relates to the technical field of fermentation. According to the invention, the fruit extract is used as a culture medium component for fermenting fusarium equiseti to produce the eosin for the first time. On one hand, a new resource recycling way is provided for waste fruit materials, and resource waste and environmental pollution are solved. On the other hand, waste fruit materials are used as the culture medium, so that the production cost of the iodomycin is greatly reduced, and a technical support is provided for industrial production of the iodomycin. According to the invention, the inhibitory activity of the eosin on seven plant pathogenic fungi is determined, so that the potential of the eosin as an antifungal pesticide is determined. Experimental results prove that the eosin produced by the fusarium equisetum has good plant pathogenic fungus resisting activity and is expected to become a substitute of chemical pesticides.

Description

Technical Field

[0001] This application relates to the field of fermentation technology, and particularly to a method for preparing antifungal equisetin and its application. Background Art

[0002] The control of plant fungal diseases is a worldwide problem. Historically, chemical pesticides have been widely used for the control of plant fungal diseases and achieved good results, saving significant economic losses. However, the long-term and widespread use of chemical pesticides has led to the development of severe drug resistance in pathogenic fungi, reducing the control effect of existing pesticides. Moreover, the R & D speed of new pesticides cannot catch up with the evolution speed of pathogenic fungal drug resistance, which also seriously affects agricultural production. As a "green" antifungal lead compound with a unique structure and low environmental pollution, the microbial secondary metabolite equisetin is expected to replace chemical pesticides, so it has received extensive attention. The large-scale production of equisetin and the exploration of its structural analogs have become the current research hotspots. However, at present, the commonly used production method of equisetin is fermentation using commercial PDW medium, and the high fermentation cost hinders its industrial development and cannot better serve agricultural production. Generally, the fermentation raw materials account for about 30% of the microbial fermentation cost, which is the key to saving the overall cost. Therefore, using waste biomass as a fermentation raw material to produce equisetin is a promising strategy.

[0003] China is the world's largest fruit producer and seller. However, it also faces a huge problem of fruit waste. In addition to the fruit processing industry and the fruits that rot during the harvesting and transportation stages, a large amount of waste fruit also comes from fruit markets and the catering industry. These fruit wastes are rich in sugars, cellulose, trace elements and other components, and can be used as high-quality fermentation substrates. However, there is currently no report on using waste fruit as a fermentation raw material to produce equisetin. Summary of the Invention

[0004] To solve the above problems, the purpose of the present invention is to provide a method for converting waste fruit into a high-value antifungal lead compound equisetin using Fusarium equiseti D39 bioreactor, which improves the yield of equisetin, and discloses the antibacterial ability of equisetin against different strains.

[0005] On the one hand, this application provides a culture medium for Fusarium equiseti, and the culture medium 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 preparation method of the fruit extract includes: soaking and homogenizing the fruit, using water as an extractant, thermally extracting to obtain 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, sorbose, mannose, arabinose, galactose, lactose, stachyose, raffinose, and melibiose; preferably, glucose and / or fructose.

[0012] In this application, the types of sugars were screened. Experiments confirmed that the higher the content of fructose and glucose, and the larger the proportion of fructose, the higher the fermentation yield; the larger the proportion of sucrose, the lower the fermentation yield, indicating that Fusarium equiseti has a preference for the absorption and utilization of sugars during the production of ikarugamycin. Therefore, the fruit in the culture medium can be a fruit containing glucose and / or fructose.

[0013] Moreover, in this application, the starch content in the fruit was also measured. It was found that the higher the starch concentration in the fruit extract, the higher the yield of ikarugamycin produced by Fusarium equiseti fermentation, and the yield of ikarugamycin changes linearly with the starch concentration. Therefore, the fruit in the culture medium can be a fruit with a high starch content or the starch content of the culture medium can be adjusted by adding starch or starch-containing substances externally.

[0014] Furthermore, the culture medium further 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] Among them, the potato extract can be prepared by a conventional method.

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

[0017] More preferably, the preparation method of the potato extract includes: cutting the potatoes into pieces, boiling them at 100°C for 20 min, and diluting with deionized water to obtain; more preferably, the material-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] In this application, experiments have proved that watermelon can be used as the best sugar source for the culture medium. However, since Fusarium equiseti requires starch (carbon source) for growth and the yield of ikarugamycin is low under carbon source-free conditions, a rich-starch potato extract is used in combination with a watermelon extract to solve the problem of the lack of carbon source in the watermelon extract, and the optimal combined culture medium for the fermentation of ikarugamycin by Fusarium equiseti is obtained.

[0021] Furthermore, the Fusarium equiseti is Fusarium equiseti D39.

[0022] On the other hand, this application also provides a method for preparing the culture medium, which includes: impregnating and homogenizing fruits, and thermally extracting to obtain fruit extracts, thereby obtaining the Fusarium equiseti culture medium.

[0023] Preferably, the method includes: impregnating and homogenizing fruits, using water as the extractant, thermally extracting to obtain fruit extracts, and filtering and concentrating to obtain fruit extracts.

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

[0025] More preferably, the conditions for thermal extraction include a solid-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: Impregnate and homogenize fruits, and thermally extract to obtain fruit extracts. The conditions for thermal extraction include: a solid-liquid ratio of 1:(1 - 20) (w / v), an extraction temperature of 40°C - 80°C, and an extraction time of 1 - 5 hours;

[0028] Step 2: With a solid-liquid ratio of 100 - 300 g / L, boil potatoes at 80°C - 100°C for 10 min - 30 min to obtain potato extracts;

[0029] Step 3: Mix the fruit extracts and the potato extracts 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, the present 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] In the present application, it is first demonstrated that apple extract is a fermentation raw material with the potential to directly replace PDW medium for the production of equisetin.

[0032] Moreover, in the present application, different fruits are compounded with potatoes, and it is demonstrated that when watermelon extract and grape extract are compounded with potatoes, there is a synergistic effect, which can increase the yield of equisetin to 4 times and 9 times the original, respectively, and is 33.5% and 16.8% higher than that of PDW medium, indicating that the combination of watermelon and potatoes can be used as a high-quality fermentation material to replace PDW medium for the production of equisetin.

[0033] It is found in the present application that the increase in the yield of equisetin is achieved by promoting the expression of key equisetin synthesis genes (eqxS, eqxC, eqxD).

[0034] In addition, it is found in the present application that the products obtained by fermenting Fusarium equiseti with fruits as the culture medium contain equisetin and its various derivatives, which is more conducive to the discovery of natural antibacterial lead compounds.

[0035] On the other hand, the present application also provides a method for producing equisetin and its derivatives, which includes: fermenting Fusarium equiseti with the culture medium to produce equisetin and its derivatives.

[0036] Those skilled in the art can ferment it by using the conventional fermentation method of Fusarium equiseti.

[0037] In a preferred embodiment, the method includes: inoculating a Fusarium equiseti block into the culture medium for fermentation, and the fermentation conditions are 20°C - 42°C, 100 - 200 rpm / min, and fermenting for 7 - 28 days.

[0038] Preferably, the fermentation conditions are 28°C, 180 rpm / min, and fermenting for 14 days.

[0039] On the other hand, the present application also provides the use of equisetin or equisetin and its derivatives prepared by the method in antibacterial.

[0040] Furthermore, the bacterium is a fungus; preferably, the fungus includes Botrytis cinerea Pers. (involved in gray mold of crops such as grapes, tobacco, cucumbers, strawberries, tomatoes, peppers, eggplants, etc.), Fusarium graminearum (involved in wheat scab and Fusarium ear rot of corn), Colletotrichum orbiculare Arx. (involved in anthracnose of melons), Fusarium asiaticum (involved in root rot of crops such as corn, tobacco, soybeans, peppers, tomatoes, green onions, etc. and wheat basal rot), Rhizoctonia cerealis (involved in wheat sharp eyespot), Magnaporthe grisea Barr (involved in rice blast), Phytophthora nicotianae (involved in tobacco black shank), and one or more of them.

[0041] Among them, it is first discovered in this application that equisetin can effectively inhibit Magnaporthe grisea, Fusarium asiaticum, Colletotrichum orbiculare Arx., and Phytophthora nicotianae.

[0042] On the other hand, this application also provides the use of ikarugamycin or ikarugamycin and its derivatives prepared by the method in the preparation of pesticides, and the pesticide is an antifungal pesticide.

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

[0044] In a preferred manner, the ikarugamycin can completely inhibit the infection of Botrytis cinerea and has no phytotoxicity to grapes, thus achieving green and safe prevention and control of plant diseases.

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

[0046] In the present invention, extracts of five fruits (apple, banana, grape, peach and watermelon) are first used as culture medium components to ferment Fusarium equiseti for the production of equisetin. On the one hand, it provides a new way for the reuse of waste fruit materials, solving the problems of resource waste and environmental pollution. On the other hand, using waste fruit materials as the culture medium greatly reduces the production cost of equisetin, providing technical support for the industrial production of equisetin.

[0047] Moreover, in the present invention, extracts of five fruits (apple, banana, grape, peach and watermelon) are screened to evaluate whether they can maintain the growth of Fusarium equiseti and the production of equisetin without additional nutrients, providing new materials for the cultivation of Fusarium equiseti and the production process of equisetin.

[0048] In the present invention, fruits are also first compounded with potatoes as the culture medium for Fusarium equiseti, and the optimal culture combination for the production of equisetin is discussed. It is proved that fruits can be used as effective components to increase the yield of equisetin, and they are high-quality fermentation materials for replacing PDW medium to produce equisetin. Finally, transcriptome and qPCR analyses are used to clarify the reasons for the differences in equisetin yields, analyze the main active components of waste fruit extracts and the yield-increasing mechanism, providing a theoretical basis for the large-scale production of equisetin.

[0049] The present invention determines the inhibitory activity of equisetin against seven phytopathogenic fungi to determine its potential as an antifungal pesticide. The experimental results prove that equisetin produced by Fusarium equiseti has good antifungal activity against phytopathogenic fungi and is expected to become a substitute for chemical pesticides. The diversity of equisetin derivatives is analyzed and predicted, providing more lead molecules for the development of natural product pesticides. This will provide technical support and theoretical guidance for the research on the production of new natural product pesticides using waste fruit extracts, and has important social and economic significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] The drawings described herein are used to provide a further understanding of the present application and form a part of the present application. The schematic embodiments of the present application and their descriptions are used to explain the present application and do not constitute an improper limitation of the present application. In the drawings:

[0051] Figure 1 It is a statistical chart of the fermentation yields of equisetin in different fruit extracts;

[0052] Figure 2 It is a statistical chart of the fermentation yields of equisetin in different fruit extracts after adding potato leachate externally;

[0053] Figure 3 It is a statistical chart of the yields of equisetin under different free sugar fermentation conditions;

[0054] Figure 4 Statistical chart of equisetin production at different starch concentrations;

[0055] Figure 5 Node clusters of equisetin in the molecular network, with red representing AWE, green representing WWES, and blue representing PDW;

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

[0057] Figure 7 Expression levels of key equisetin synthesis genes under fructose and sucrose fermentation treatments, with sucrose as the control;

[0058] Figure 8 Control effects of different concentrations of equisetin on Botrytis cinerea. (A) Lesion inhibition rate. (B) Lesion diameter. Detailed implementation manners

[0059] In order to more clearly illustrate the overall concept of the present application, the following will be described in detail by way of examples in conjunction with the accompanying drawings of the specification. In the following description, a large number of specific details are given to provide a more thorough understanding of the present invention. However, it is obvious to those skilled in the art that the present invention can be implemented without one or more of these details. In other examples, in order to avoid confusion with the present invention, some well-known technical features in the art are not described.

[0060] It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present application belongs.

[0061] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular forms are also intended to include the plural forms. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they specify the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0062] Unless otherwise specified, in the following embodiments, reagents or instruments without indicating the manufacturer can be obtained as conventional products through commercial purchase.

[0063] Before further describing the specific embodiments of the present invention, it should be understood that the protection scope of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the embodiments of the present invention are for describing specific embodiments and not for limiting the protection scope of the present invention. The test methods without specifying specific conditions in the following examples are generally carried out under conventional conditions or according to the conditions recommended by each manufacturer.

[0064] When the embodiments give a numerical range, it should be understood that unless otherwise stated in the present invention, any value between the two endpoints of each numerical range and either of the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the art of this technology. In addition to the specific methods, devices, and materials used in the embodiments, according to the knowledge of those skilled in the art of this technology and the description of the present invention, any methods, devices, and materials similar to or equivalent to the methods, devices, and materials described in the embodiments of the present invention can also be used to implement the present invention.

[0065] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt the conventional technologies in the fields of microbiology, biochemistry, analytical chemistry, cell culture, and related fields in this technical field.

[0066] Among them, Fusarium equiseti D39 (Fusarium equiseti GLY27) has been disclosed in the Chinese patent document with the application number 201911110953.2. Botrytis cinerea Pers., Fusarium graminearum, Colletotrichum orbiculare Arx., Fusarium asiaticum, Rhizoctonia cerealis, Magnaporthe grisea Barr, and Phytophthora nicotianae are all preserved and provided by the National Agricultural Environmental Microbial Germplasm Resource Bank (Shandong).

[0067] The formula of the PDW medium is: 10 g / L of potato leaching powder (with a starch content of 8 g / L), 20 g / L of glucose, and the volume is made up to 1 L with water.

[0068] In addition, the "water" mentioned in the present 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, if there is no other special description, % represents wt%, that is, weight percentage.

[0070] 1 Conversion of waste fruits into equisetin using Fusarium equiseti D39 bioreactor

[0071] 1.1 Culturing Fusarium equiseti D39 with fruit extract instead of PDW medium to produce equisetin

[0072] (1) Pretreatment of waste fruits

[0073] Soak and homogenize waste apples, waste bananas, waste grapes, waste peaches, and waste watermelons separately using a grinder. Extract the free sugars in them with distilled water, with a material-liquid ratio of 1:10 (w / v), an extraction temperature of 65 °C, an extraction time of 2 hours, and filter with gauze to obtain the filtrate. Measure the free sugar concentration of the filtrate and concentrate it uniformly to 20 g / L. After sterilization (121 °C, 20 min), it is used as the fermentation substrate for Fusarium equiseti D39 and compared with the commercial PDW medium.

[0074] (2) Fermentation process and equisetin quantification

[0075] Fermenting Fusarium equiseti D39 in different fruit extracts. Mycelial blocks with a diameter of 5 mm were cut from the edge of a 3-day-old Fusarium equiseti colony and transferred to a 500 mL conical flask containing 200 mL of fruit extract, with each group repeated three times. The above system was incubated at 28 °C and 180 rpm / min for 14 days. After 14 days, the fermentation products were processed. The mycelium was filtered through gauze. The filtrate was extracted three times with an equal volume of ethyl acetate, and the filtrate extract was obtained by vacuum evaporation. The crushed mycelium was soaked in methanol and dichloromethane (1:1 [v / v]), ultrasonic extracted at 40 Hz for 15 min, the organic phase was evaporated to dryness under vacuum, dissolved in 100 mL of distilled water, extracted three times with 2 volumes of ethyl acetate, and the ethyl acetate phase was evaporated to dryness to obtain the mycelium extract. The filtrate extract and the mycelium extract were combined to obtain the total extract sample.

[0076] Prepare a series of solutions containing different concentrations of equisetin and analyze them using a high-performance liquid chromatograph. By plotting the relationship between the peak area (y) and concentration (x) of equisetin, a calibration curve was constructed. Calibration curve: y = 15.843x + 0.1263, R 2 = 0.997, and the applicable concentration range is 0.1 - 1.1 mg / mL. This calibration curve was used to quantitatively determine the content of equisetin in each sample.

[0077] (3) Yield differences of equisetin fermented with different fruit extracts as the medium

[0078] The yields of equisetin produced by Fusarium equiseti D39 when cultured in different fruit extracts are shown in Table 1, which was made from Table 1 Figure 1 .

[0079] Table 1 Fermentation yields of equisetin in different fruit extracts

[0080]

[0081] Note: "a", "b", "c", "d", "e", "f" represent the equisetin yields from high to low in sequence.

[0082] As shown in Table 1 and Figure 1 shown, the yields of equisetin produced by Fusarium equiseti D39 when cultured in different fruit extracts are significantly different. The yield of equisetin obtained using apple extract as the medium is 20.1 mg / L, which is comparable to the yield (25.8 mg / L) of the commonly used medium PDW for fungal fermentation, indicating that apple extract is a potential fermentation raw material that can directly replace PDW medium for the production of equisetin. Except for apple extract, the yields of other fruits are lower than the control and cannot be directly used as fermentation raw materials.

[0083] 1.2 Substitute the combination of fruit extract and potato extract for PDW medium to culture Fusarium equiseti D39 to produce equisetin

[0084] (1) Pretreatment of fermentation raw materials

[0085] Cut the potatoes (200 g) into small pieces, put them into a pot and boil for 20 min to obtain the leaching solution, dilute it to 1 L with deionized water to obtain the potato extract. Add the apple extract, banana extract, grape extract, peach extract, and watermelon extract with a sugar content of 20 g prepared by the method in 1.1(1) to the above potato extract, and re-concentrate the final volume to 1 L. After sterilization, it is used for the fermentation of Fusarium equiseti D39 and compared with the commercial PDW medium.

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

[0087] (3) Difference in equisetin yield

[0088] The equisetin yields produced by Fusarium equiseti D39 when cultured in different media are shown in Table 2, and are made from Table 2 Figure 2 .

[0089] Table 2 Yields of iketomycin in different fruit extracts after exogenous addition of potato leachate

[0090]

[0091]

[0092] Note: "a", "b", "c", "d", "e", "f" represent the equisetin yields from high to low in sequence.

[0093] As shown in Table 2 and Figure 2 shown, after exogenous addition of potato leachate, the fermentation yields of each group are increased. Among them, the yields of equisetin in watermelon extract and grape extract are increased by 4 times and 9 times respectively, reaching 38.2 mg / L and 33.6 mg / L, which are 33.5% and 16.8% higher than the PDW medium respectively. It shows that the combination of watermelon and potato can be used as a high-quality fermentation material to replace the PDW medium for producing equisetin.

[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 effects of different sugars on equisetin yield

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

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

[0098] Table 3 Retention times and calibration curves of each standard substance

[0099]

[0100] (2) Effects of sugars on equisetin production

[0101] Replace the sugar (20 g / L) in the PDW medium (the main components are potato extract and glucose) with equal amounts of glucose, fructose, sucrose, maltose, and melibiose, and ferment Fusarium equiseti D39. Each treatment was repeated three times. The equisetin production under different free sugar fermentation conditions was detected by HPLC, and the fermentation method and determination method were the same as in 1.1(2).

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

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

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

[0105]

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

[0107] Combined with the comprehensive analysis of Table 4 and Table 2, it can be obtained that after adding potato extract externally, among the total free sugars of each extract, the higher the content of fructose and glucose, and the larger the proportion of fructose, the higher the fermentation yield; the larger the proportion of sucrose, the lower the fermentation yield, indicating that Fusarium equiseti has a preference for the absorption and utilization of sugars during the production of equisetin.

[0108] (4) Fusarium equiseti D39 prefers fructose

[0109] 2.1(2) The production statistics of equisetin by fermentation of Fusarium equiseti D39 are shown in Table 5. Plotting the data in Table 5 gives Figure 3 .

[0110] Table 5 Effects of different free sugars on the production of equisetin

[0111]

[0112] Note: "a", "b", "c", "d", "e", "f" represent the equisetin production from high to low in sequence.

[0113] Table 5 and Figure 3 The results show 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 the yield of sucrose was the lowest, at 8.6 mg / L, only 0.3 times that of fructose.

[0114] 2.2 Starch content in fruit extracts and the effect of starch content on the production of equisetin

[0115] (1) The starch contents 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 were detected respectively.

[0116] (2) Fusarium equiseti D39 was fermented using a starch concentration gradient (3, 6, 9, 12 g / L), with each group repeated 3 times, and the production of equisetin was detected. 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 contents in different fruit extracts

[0120]

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

[0122] Combining the results of Table 1 and Table 6, it can be seen that the higher the starch content in the fruit extract, the higher the production of equisetin.

[0123] The results of 2.2(2) are shown in Table 7. Plotting the data in Table 7 gives Figure 4 .

[0124] Table 7 Effects of starch concentration on the production of equisetin

[0125]

[0126] From Table 7 and Figure 4 As can be seen from the results, the higher the starch concentration of the fruit extract, the higher the production of equisetin by Fusarium equiseti, and the yield of equisetin changes linearly with the starch concentration.

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

[0128] (1) The diversity of equisetin derivatives of Fusarium equiseti D39 cultured in watermelon extract (WWES) and apple extract (AWE) (no additional starch is added here because the apple extract contains starch) with 8 g / L starch (starch content in PDW medium) and PDW medium was analyzed using 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 Figure 5 shown. Compared with the 10 nodes treated with PDW, the samples treated with WWES and AWE contained 17 and 12 nodes respectively in the equisetin cluster. In addition, 5 products with m / z 372.216, 360.216, 404.243, 407.29 and 420.238 were only found in WWES and AWE, indicating that these two culture conditions are more suitable for the production of equisetin analogs, may contain more antifungal analogs, and are more conducive to the discovery of natural antibacterial lead compounds.

[0131] 4. Mechanism of fructose promoting equisetin production

[0132] (1) To explore the mechanism by which fructose increases the yield of equisetin, Fusarium equiseti D39 was cultured for 7 days under the conditions of adding 20 g / L fructose or sucrose to 8 g / L starch. The collected mycelia were washed 3 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 of equisetin (eqxS, eqxC, eqxD) under fructose and sucrose treatments were detected by qRT-PCR to further clarify the reasons for the difference in equisetin yield under different free sugar fermentation conditions.

[0135] (3) Yield improvement mechanism

[0136] Compared with the sucrose treatment, a total of 1198 differentially expressed genes (DEGs) were found in the fructose treatment group, among which 505 were up-regulated and 693 were down-regulated. Figure 6 A), and all DEGs were annotated in Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Figure 6 B and Figure 6 C). Among them, the most important enriched GO term was "ribonucleoprotein complex biogenesis", followed by "rRNA processing", "cellular component biogenesis", and "monocarboxylic acid metabolic process". 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 related to energy and material metabolism and cell proliferation.

[0137] Compared with the sucrose treatment, fructose treatment significantly up-regulated the expression levels of genes related to the glycolysis pathway in Fusarium equiseti D39. Figure 6 E), in addition, compared with the sucrose treatment, the expression levels of genes related to polyketide synthase (A6789), SAM-dependent methyltransferase-encoding gene (A1115), and N-methyltransferase-related encoding gene (A6788) in Fusarium equiseti D39 under fructose treatment were significantly up-regulated. Figure 6 D). It is indicated that fructose promotes the synthesis of equisetin by enhancing the biological activities of key enzymes involved in the biosynthetic pathway.

[0138] And Figure 7 The results demonstrated that fructose stimulated the expression of key equisetin synthetic genes.

[0139] 5. Study on the antifungal activities of equisetin in vitro and in vivo

[0140] (1) Test strains

[0141] Botrytis cinerea Pers., Fusarium graminearum, Colletotrichum orbiculare Arx., Fusarium asiaticum, Rhizoctonia cerealis, Magnaporthe grisea Barr, Phytophthora nicotianae. All strains were cultured by a general method to obtain fungal discs.

[0142] (2) Experimental design

[0143] Equisetin (which can be prepared by the methods described in 1 or 2) dissolved in DMSO was added to PDA and oatmeal agar (OA) media (0.5% DMSO [vol / vol]) at final concentrations of 0, 2.5, 5, 10, 20, 40, and 60 μg / mL, respectively. Fungal discs with a diameter of 5 mm were cut from the edge of the 3-day-old colonies and inoculated at the center of each medium (OA for Phytophthora nicotianae and PDA for other fungi), and then incubated at 28 °C for 3 days. Each treatment was repeated three times, with carbendazim or metalaxyl as the positive control. The mycelial diameter was measured by the cross method, and the inhibition rate was calculated as follows: inhibition rate = [(R - r) / R - 0.5] × 100%, where R and r are the average diameters of the control colony and the treated colony, respectively. The EC 50 value was obtained using the toxicity regression equation.

[0144] Equisetin was dissolved in a 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 with similar weights and uniform shapes were surface-sterilized with 75% ethanol, then rinsed with sterile water and air-dried. The grapes were punctured with a sterilized toothpick at the maximum circumference, soaked in the equisetin aqueous solution for 5 minutes, and then air-dried. Subsequently, fungal discs with a diameter of 2 mm of Botrytis cinerea were inoculated at the puncture site of each grape. All treated samples were incubated in an incubator at 25 °C for 3 days, with sterile water containing a 0.5% DMSO solution as the control. There were 10 grapes in each treatment group, and the experiment was repeated 3 times. The lesion diameter was measured by the cross method, and the antibacterial rate was calculated as follows: antibacterial rate = [(R - r) / R - 0.2] × 100%, where R and r are the average diameters of the control lesion and the treated lesion, respectively.

[0145] (3) Experimental results

[0146] The antibacterial effect experiments 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 as follows.

[0147] Table 8 EC of Equisetin against Seven Kinds of Phytopathogenic Fungi 50 (μg / mL)

[0148]

[0149] The results in Table 8 prove that Equisetin has broad-spectrum antifungal activity against phytopathogenic fungi and has significant inhibitory effects on a variety of common pathogenic fungi. In this application, it is first discovered that Equisetin can effectively inhibit Magnaporthe grisea, Fusarium asiaticum, Colletotrichum orbiculare Arx., and Phytophthora nicotianae.

[0150] The results of the in-vivo antibacterial effect experiments are shown in Table 9. Plotting from Table 9 gives Figure 8 .

[0151] Table 9 Preventive Effects of Different Concentrations of Equisetin on Fruit Fungal Diseases

[0152]

[0153] As shown in Table 9 and Figure 8 shown, at concentrations of 6 μg / mL and 12 μg / mL, the inhibitory effects of Equisetin on the infection of Botrytis cinerea are 27.7% and 67.8% respectively. When the concentration is 18 μg / mL, Equisetin can completely inhibit the infection of Botrytis cinerea without phytotoxicity to grapes ( Figure 8 B), and the results show that Equisetin is a natural antifungal pesticide with broad application prospects.

[0154] The above are only examples of this application and do not limit this application. For those skilled in the art, various changes and modifications can be made to this application. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of this application shall be included within the scope of the claims of this application.

Claims

1. A culture medium for Fusarium equiseti, characterized in that: The culture medium includes fruit.

2. The culture medium according to claim 1, characterized in that The fruit is waste fruit material and / or leftover fruit product material; preferably, the fruit includes one or more of apples, watermelons, grapes, peaches and bananas.

3. The culture medium according to claim 1, characterized in that The culture medium further comprises 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.

4. The culture medium 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.

5. The culture medium according to claim 1, characterized in that The Fusarium equisetifolia is Fusarium equisetifolia D39 。 6. The method for preparing the culture medium according to any one of claims 1 to 5, characterized in that: The method comprises: soaking and homogenizing the fruit, and performing heat extraction to obtain the fruit extract, namely, obtaining the Fusarium equisetum culture medium.

7. Use of the culture medium according to any one of claims 1 to 5 in producing equisetin and its derivatives and / or increasing the yield of equisetin.

8. A method for producing isoflurane and its derivatives, characterized in that: The method comprises: using the culture medium as claimed in any one of claims 1 to 5 to ferment Fusarium equiseti to produce erythromycin and its derivatives.

9. Use of irinotecan or irinotecan prepared by the method of claim 8 and its derivatives in antibacterial treatment, wherein preferably, the fungus is a fungus; more preferably, the fungus includes one or more of Botrytis cinerea Pers., Fusarium graminearum, Colletotrichum orbiculare Arx., Fusarium asiaticum, Rhizoctonia cerealis, Magnaporthe grisea Barr, and Phytophthora nicotianae.

10. Use of irinotecan or irinotecan prepared by the method of claim 8 in the preparation of pesticides, characterized in that: The pesticide is an antifungal pesticide.

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