Compound Macrolactin A as well as separation method and application thereof

Abstract

The invention relates to the technical field of bioengineering, in particular to a compound Macrolactin A as well as a separation method and application thereof. The compound Macrolactin A disclosed by the invention has the activity of preventing and treating aphids, the compound Macrolactin A is generated by a bacterial strain of bacillus amyloliquefaciens YJNbs21.10, and the preservation number of the bacterial strain of the bacillus amyloliquefaciens YJNbs21.10 is CGMCC (China General Microbiological Culture Collection Center) No.25798. The compound Macrolactin A provided by the invention has a very strong capability of controlling aphids; the invention further provides a separation method of the compound, the separation method is simple in step, and the obtained compound is high in purity.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and in particular to a compound Macrolactin A and a separation method and application thereof. Background Art

[0002] Aphids are major pests in plant cultivation or crops. A total of 10 families and about 4,400 species have been found. Due to the small size of aphids, the large number of crops they harm, their wide range, their fast reproduction rate, and the difficulty in eradicating them, the leaves of plants curl up, the growth of plants gradually stagnates, and even the whole plant wilts and dies. At present, the control technology of aphids mainly uses chemical agents. However, the negative impact of chemical agents is becoming increasingly prominent. The excessive use of pesticides has caused environmental pollution and has adverse effects on the health of humans and other organisms. Minimizing the use of pesticides in the production of agricultural products and making pest control harmless to the environment have become common demands of the public worldwide.

[0003] There have been many studies on alternative strategies for pest control. For example, patent CN2020104683120 discloses a method for controlling brown citrus aphids by combining RNAi with Beauveria bassiana, and discloses the control effect of Beauveria bassiana on pea aphids, peach aphids, or brown citrus aphids. MacrolactinA is a natural macrolide product containing a 24-membered lactone ring isolated from natural microorganisms. It contains three independent diene structural elements. The compound has a complex structure and a difficult preparation process. It has been found to have antibacterial activity, but there are no reports on the control of aphids by MacrolactinA.

[0004] Therefore, it is particularly important to develop a compound Macrolactin A that has aphid control activity and a simple preparation method. Summary of the invention

[0005] The object of the present invention is to provide a compound Macrolactin A, which has strong aphid-controlling ability; the present invention also provides a method for separating the compound, the separation method has simple steps, and the obtained compound has high purity.

[0006] In a first aspect, the present invention provides a compound MacrolactinA, which has the activity of preventing and controlling aphids. The compound MacrolactinA is produced by Bacillus amyloliquefaciens YJNbs21.10 strain. The Bacillus amyloliquefaciens YJNbs21.10 strain is deposited in the General Microbiological Center of China National Microbiological Culture Collection Administration, and the deposit number of the Bacillus amyloliquefaciens YJNbs21.10 strain is CGMCC No.25798.

[0007] In the present invention, the Bacillus amyloliquefaciens YJNbs21.10 strain has been deposited on September 26, 2022. For information on the Bacillus amyloliquefaciens YJNbs21.10 strain, please refer to the patent with application number CN202211743742.4 and patent name "A Bacillus amyloliquefaciens and its fermentation method and application".

[0008] Preferably, the compound Macrolactin A is isolated from the fermentation broth of Bacillus amyloliquefaciens YJNbs21.10 in saline-alkali soil.

[0009] Preferably, the compound MacrolactinA has a structure as shown in formula (I):

[0010]

[0011] In a second aspect, a method for separating the compound Macrolactin A according to the present invention is provided, wherein the separation method comprises the following steps:

[0012] The Bacillus amyloliquefaciens YJNbs21.10 strain was activated in PDA medium;

[0013] The activated YJNbs21.10 strain was inoculated into the first culture medium for fermentation to obtain a first-level seed solution; wherein the fermentation conditions were as follows: fermentation time 72 h, fermentation speed 150 rpm, fermentation temperature 28°C;

[0014] The first-level seed liquid is inoculated into the second culture medium, and fermented to obtain the second-level seed liquid; wherein the fermentation conditions are: fermentation time 72h, fermentation speed 150rpm, fermentation temperature 28°C;

[0015] The secondary seed liquid is centrifuged in a high-speed centrifuge and filtered through a microporous filter membrane to obtain a filtrate;

[0016] The filtrate was extracted with ethyl acetate and then rotary evaporated to obtain an organic phase extract, which was then chromatographed on a silica gel column to obtain seven components;

[0017] The seven components were tested for activity against aphids.

[0018] In the present invention, the fermentation stock solution (secondary seed solution) is centrifuged in a high-speed refrigerated centrifuge (10000r / min, 4°C) three times to obtain bacterial cells and supernatant, respectively. The supernatant is then filtered through a 0.22μm diameter microporous filter membrane to obtain a filtrate without sterile cells.

[0019] The filtrate was extracted with ethyl acetate and then rotary evaporated to obtain an organic phase extract, which was then chromatographed on a silica gel column to obtain seven components;

[0020] In the present invention, after the filtrate is extracted with ethyl acetate for three times, the filtrate is rotary evaporated in a rotary evaporator (45°C) to volatilize the solvent to obtain an organic phase extract; after the obtained extract is dissolved in ethyl acetate, it is fully stirred and mixed with 200-300 mesh silica gel powder, and placed in a fume hood to dry; after the solvent is completely volatilized, a silica gel column (silica gel powder 200-300 mesh) is loaded by a dry method, and petroleum ether, ethyl acetate, and methanol are used in sequence for gradient elution (the gradient ratio of two adjacent eluents is 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, and 0:1), and the eluents are continuously combined after TLC inspection to finally obtain seven components.

[0021] The seven components were tested for activity against aphids.

[0022] Preferably, the testing of the seven components for aphid control activity specifically includes: configuring the seven components into solutions of the same concentration for aphid control activity testing, calculating the corrected control effect, comparing the aphid control activity of each component, purifying and identifying the component with the highest aphid control activity, and obtaining the compound Macrolactin A.

[0023] Preferably, the aphid control activity test of the seven components specifically includes: taking 10 mg of each component and dissolving it in 190 μL DMSO as a mother solution; dissolving 20 μL of the mother solution in 0.1% Tween aqueous solution to prepare a spray, and taking 1.8 mL 0.1% Tween aqueous solution + 20 μL DMSO as a negative control and then conducting an aphid control activity test.

[0024] In the present invention, the calculation formula of the corrected prevention effect is as follows:

[0025]

[0026] Among them, the medicines applied before and after application refer to spray agents.

[0027] Preferably, the first culture medium and the second culture medium have the same components, namely: 10 g peptone, 3 g beef extract, 5 g NaCl, and 1000 mL water.

[0028] In a third aspect, the invention provides the use of the compound Macrolactin A in the preparation of a biological pesticide for controlling aphids.

[0029] In the present invention, the pesticide applied before and after the application of the pesticide refers to the biological pesticide for controlling aphids.

[0030] Preferably, the concentration of the aphid control biological pesticide is 500 ppm.

[0031] In a fourth aspect, there is provided the use of the compound MacrolactinA described in the present invention in the preparation of a biological pesticide for controlling cereal aphids.

[0032] In the present invention, the pesticide applied before and after the application of the pesticide refers to the biological pesticide for controlling cereal aphids.

[0033] Preferably, the concentration of the biological pesticide for controlling cereal aphids is 500 ppm.

[0034] In a fifth aspect, the present invention provides a compound MacrolactinA, which has the activity of preventing and controlling cereal aphids. The compound MacrolactinA is produced by Bacillus amyloliquefaciens YJNbs21.10 strain. The Bacillus amyloliquefaciens YJNbs21.10 strain is deposited in the General Microbiological Center of China National Microbiological Culture Collection Administration, and the deposit number of the Bacillus amyloliquefaciens YJNbs21.10 strain is CGMCC No.25798.

[0035] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages:

[0036] (1) The present invention separates the natural product macrolide antibiotic A (Macrolactin A) from Bacillus amyloliquefaciens YJNbs21.10. The method obtains Macrolactin A through microbial liquid fermentation, effectively reduces production costs, makes up for the difficulties and disadvantages of the compound synthesis process, and reduces production costs. In addition, the YJNbs21.10 fermentation broth also has a variety of active ingredients, which provides the possibility for the development of other microbial metabolite resources.

[0037] (2) The steps for isolating Macrolactin A in the present invention are simple, and the obtained compound has high purity. Compared with chemical synthesis of the substance, the present invention saves costs and reduces the difficulty of obtaining it, thereby greatly reducing the production cost of the compound.

[0038] (3) Macrolactin A in the present invention is separated from natural non-toxic microorganisms, and the activation, fermentation and separation processes are all harmless to nature and organisms.

[0039] (4) At present, aphid control is mainly carried out by chemical pesticides, which have high chemical pesticide residues and strong resistance, causing serious harm to humans and the natural environment. The present invention prepares and verifies the biological activity of the microbial natural product MacrolactinA for controlling aphids for the first time.

[0040] (5) The present invention is the first to prepare and verify the biological activity of the microbial natural product Macrolactin A in controlling the cereal aphid, providing a new idea for the development of natural, non-toxic and sustainable biological pesticides. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to more clearly illustrate the technical solution of the present invention, the drawings involved in the description of the embodiments will be briefly introduced below. Obviously, the drawings in the description are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0042] Figure 1 This is a diagram of the liquid fermentation process.

[0043] Figure 2 It is a column chromatography process.

[0044] Figure 3 Compound Macrolactin A was precipitated into crystals.

[0045] Figure 4 This is the nuclear magnetic resonance hydrogen spectrum of the compound MacrolactinA.

[0046] Figure 5 This is the carbon NMR spectrum of the compound Macrolactin A.

[0047] Figure 6 This is the high-resolution mass spectrum of the compound Macrolactin A; a is the positive ion; b is the negative ion.

[0048] Figure 7 It is the structural formula of the compound MacrolactinA. DETAILED DESCRIPTION

[0049] The technical scheme of the present invention is described below in conjunction with the examples, but the present invention is not limited to the following examples. The experimental methods and detection methods described in each example are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0050] Example 1

[0051] The Bacillus amyloliquefaciens YJNbs21.10 strain was inoculated on PDA medium for activation.

[0052] 10g of peptone, 3g of beef extract, and 5g of NaCl were dissolved in 1L of water, sterilized (sterilization conditions were 121°C, 20min), and cooled to room temperature to obtain a liquid fermentation medium (first medium). The activated Bacillus amyloliquefaciens YJNbs21.10 strain was inoculated on the above medium, and the first-level seed liquid was obtained after shaking flask fermentation for 72h (fermentation speed 150rpm, fermentation temperature 28°C).

[0053] The first-stage seed solution was then inoculated into the second culture medium (the culture medium used was the same as that used to prepare the first-stage seed solution) at an inoculum volume of 5%, and the second-stage seed solution was obtained after 72 hours of shake flask fermentation (fermentation speed 150 rpm, fermentation temperature 28°C). Figure 1 shown.

[0054] Example 2

[0055] Prepare the components.

[0056] The fermentation stock solution was centrifuged in a high-speed refrigerated centrifuge (10000r / min, 4°C) three times to obtain the bacteria and the supernatant respectively. The supernatant was then filtered through a microporous filter membrane with a diameter of 0.22μm to obtain a filtrate free of sterile cells. The filtrate was extracted three times with equal volumes of ethyl acetate, and the solvent was evaporated in a rotary evaporator (45°C) to obtain an extract. After the obtained extract was dissolved in ethyl acetate, it was fully stirred and mixed with silica gel powder (200-300 mesh), and placed in a fume hood to dry. After all the solvents were evaporated, the silica gel column (silica gel powder 200-300 mesh) was loaded by dry method, and gradient elution was performed with petroleum ether, ethyl acetate, and methanol in turn (the gradient ratios of two adjacent eluents were 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 0:1), and the eluents were continuously merged after TLC inspection to obtain seven components. The silica gel column chromatography process is as follows Figure 2 After column chromatography, all solvents evaporated and Macrolactin A crystals were as shown in Figure 3 shown.

[0057] Example 3

[0058] The 7 components obtained in Example 2 were tested for their biological activity against aphids.

[0059] The bioassay activity test includes an experimental group (all 7 components are configured as reagents with a concentration of 500ppm) and a control group (0.1% Tween solution). The specific operation is: take 10mg of each component and dissolve it in 190μL DMSO as a mother solution. Dissolve 20μL of the mother solution in a 0.1% Tween aqueous solution to prepare the spray used in the test (concentration is 500ppm). Use 1.8mL 0.1% Tween aqueous solution + 20μLDMSO as a negative control. Raise the aphid on wheat pots, and the temperature is controlled at around 25°C. After centralized breeding, 20 aphid nymphs were transplanted on each pot for product activity testing. During the test, three parallel tests were set up for each group, and a manual sprayer was used to spray the medicine once so that the front and back of the leaves were evenly covered with the liquid. The specific test results are shown in Table 1 below:

[0060] Table 1 Biological activity effect of active products in controlling aphids

[0061]

[0062]

[0063] After filtering, concentrating and separating the filtrate of strain YJNbs21.10 by silica gel column chromatography, seven components were obtained. The spray toxicity of each component to the cereal aphid at a concentration of 500 mg / L was tested (Table 1). As shown in Table 1, some components have no control effect on the cereal aphid (component 1 and component 4). The control effect of component 5 and component 7 does not have a lasting period, and large-scale reproduction occurs 72 hours after application. The corrected control effects of component 2 and component 7 at 24h, 48h and 72h of application were 5.21%, 25.00%, 26.09% (component 2) and 26.04%, 28.33%, 20.29% (component 7), respectively, indicating that both components have weak aphid control activity. The corrected control effects of component 6 on three days were 39.59%, 68.33% and 74.64%, respectively, with good control activity.

[0064] The test results of the embodiments of the present invention show that the present invention is the first to prepare and verify the biological activity of the microbial natural product MacrolactinA in controlling aphids; further, the present invention is the first to prepare and verify the biological activity of the microbial natural product MacrolactinA in controlling cereal aphids; the present invention provides a new idea for the development of natural, non-toxic and sustainable biological pesticides.

[0065] Example 4

[0066] The product with the highest aphid control activity in Example 3 was identified and analyzed.

[0067] By performing nuclear magnetic resonance analysis on component 6, the molecular structure of the product can be analyzed, thereby determining the chemical structure of the product.

[0068] Figure 4 , Figure 5 It is the hydrogen nuclear magnetic resonance spectrum and the carbon nuclear magnetic resonance spectrum. Figure 6 This is the high-resolution mass spectrum of Macrolactin A. Figure 7 It is the structural formula of component 6 (compound Macrolactin A).

[0069] according to Figure 4 , Figure 5 , Figure 6 The molecular formula of the compound is determined to be C 24 H 35 O5(Obsd425.2293(M++Na),C 24 H 34 O5Na,Calc 425.2310). According to Figure 4 Proton spectrum analysis of the structural unit of the compound, combined with Figure 5 middle 13 C NMR analysis showed that the compound contained 1 ester carbonyl (δ165.4), 12 methine carbons (δ141.8, 138.6, 135.0, 133.9, 131.9, 129.6, 129.3, 128.9, 128.7, 126.5, 123.7, 116.9), belonging to 6 double bonds, 4 oxygen carbons (δ70.1, 70.1, 69.2, 68.6), 6 methylene carbons (δ40.5, 39.6, 34.3, 33.9, 30.9 and 23.4 respectively), and 1 methyl group (18.9ppm). Figure 4 , Figure 5 , Figure 6 By comparing with the published data of macrolide antibiotic A, the compound was identified as macrolide antibiotic A (Macrolactin A).

[0070] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. Professionals and technicians in this industry should fully understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention. The scope of protection of the present invention is defined by the attached claims and their equivalents.

[0071] It should be understood that the disclosed invention is not limited only to the specific method, scheme and material of description, because these all can change.It should also be understood that the terminology used herein is only for the purpose of describing specific embodiment scheme, rather than being intended to limit the scope of the present invention, and the scope of the present invention is only limited to the appended claims.

[0072] Those skilled in the art will also recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are also intended to be encompassed by the appended claims.

Claims

1. The compound Macrolactin A, characterized in that The compound Macrolactin A has the activity of preventing and controlling aphids. The compound Macrolactin A is produced by the Bacillus amyloliquefaciens YJNbs21.10 strain. The Bacillus amyloliquefaciens YJNbs21.10 strain is preserved in the General Microbiological Center of the China Microbiological Culture Collection Administration. The preservation number of the Bacillus amyloliquefaciens YJNbs21.10 strain is CGMCC No.25798.

2. The compound Macrolactin A according to claim 1, characterized in that The compound MacrolactinA has a structure as shown in formula (I):

3. The method for separating the compound Macrolactin A according to claim 1, characterized in that: The separation method comprises the following steps: The Bacillus amyloliquefaciens YJNbs21.10 strain was activated in PDA medium; The activated YJNbs21.10 strain is inoculated into the first culture medium for fermentation to obtain a first-level seed solution; Inoculating the first-level seed solution into the second culture medium, fermenting and culturing, and obtaining the second-level seed solution; The secondary seed liquid is centrifuged in a high-speed centrifuge and filtered through a microporous filter membrane to obtain a filtrate; The filtrate was extracted with ethyl acetate and then rotary evaporated to obtain an organic phase extract, which was then chromatographed on a silica gel column to obtain seven components; The seven components were tested for activity against aphids.

4. The separation method according to claim 3, characterized in that The seven components are tested for aphid control activity, specifically comprising: After the seven components were prepared into solutions of the same concentration for aphid control activity testing, the corrected control effect was calculated, the aphid control activity of each component was compared, and the component with the highest aphid control activity was purified and identified to obtain the compound Macrolactin A.

5. The separation method according to claim 3, characterized in that The seven components are tested for aphid control activity, specifically comprising: 10 mg of each component was dissolved in 190 μL DMSO as the mother solution; 20 μL of the mother solution was dissolved in 0.1% Tween aqueous solution to prepare the spray, 1.8 mL 0.1% Tween aqueous solution + 20 μL DMSO was used as the negative control, and then the activity of controlling aphids was tested.

6. The separation method according to claim 3, characterized in that The first culture medium and the second culture medium have the same components, namely: 10 g peptone, 3 g beef extract, 5 g NaCl, and 1000 mL water.

7. The separation method according to claim 3, characterized in that The conditions of the two fermentation cultures were the same, namely, fermentation time 72 h, fermentation speed 150 rpm, and fermentation temperature 28°C.

8. The method according to claim 3, characterized in that The obtained extract was dissolved in ethyl acetate, fully stirred and mixed with 200-300 mesh silica gel powder, and placed in a fume hood to dry; after the solvent was completely evaporated, it was loaded onto a silica gel column by a dry method, and gradient eluted with petroleum ether, ethyl acetate, and methanol in sequence. The eluents were continuously combined after TLC inspection, and finally seven components were obtained.

9. Use of the compound Macrolactin A according to claim 1 or 2 in the preparation of a biopesticide for controlling aphids.

10. Use of the compound Macrolactin A according to claim 1 or 2 in the preparation of a biological pesticide for controlling cereal aphids.

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