CN113621526B - Marine fungus aspergillus versicolor M-7-SW9, mixed source terpenoid and extraction method and application thereof - Google Patents
Marine fungus aspergillus versicolor M-7-SW9, mixed source terpenoid and extraction method and application thereof Download PDFInfo
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Abstract
The invention provides a marine fungus aspergillus versicolor M-7-SW9, a mixed source terpenoid, and an extraction method and application thereof, and relates to the technical field of microbial medicines. The marine fungus Aspergillus versicolor (Aspergillus versicolor) M-7-SW9 provided by the invention has a preservation number of CCTCC NO: m2021454. The mixed source terpenoid with the structure shown in the formula I can be extracted from the marine fungus aspergillus versicolor M-7-SW9, the minimum antibacterial concentration of the mixed source terpenoid for Edwardsiella tarda and vibrio harveyi is 1.0 mug/mL, and the activity of the mixed source terpenoid for Edwardsiella tarda and vibrio harveyi is higher than that of chloramphenicol; provides a new idea for developing natural medicines of marine organism sources and solving the problem of bacterial disease control in the aquaculture process.
Description
Technical Field
The invention belongs to the technical field of microbial medicines, and particularly relates to a marine fungus aspergillus versicolor M-7-SW9, a mixed source terpenoid, and an extraction method and application thereof.
Background
The threat of bacterial diseases in the aquaculture industry is worldwide, almost invading all aquaculture animals, not only causing great economic loss, but also burying hidden danger for the quality safety of the aquatic products. According to the monitoring results of the aquaculture animal diseases, the aquaculture disease species in recent years reach more than 200 species, wherein bacterial diseases account for more than 48%. Among bacterial aquatic animal diseases, vibriosis caused by vibrio bacteria is the most dominant bacterial disease, and the disease has a wide occurrence range and high mortality rate, can cause zoonotic diseases, and is one of the main problems in the field of aquaculture disease control.
In recent years, with the development of aquaculture scale and the large-scale use of traditional antibiotics in aquaculture process, the phenomenon of multiple drug resistance of pathogenic bacteria of aquaculture animals and bacteria in the aquaculture environment is becoming common, and the large-scale use of chemical synthetic drugs brings serious pollution to water and soil and causes direct and potential harm to biological and human health.
Compared with the traditional antibiotics and chemical synthetic drugs used in the aquaculture industry, the marine organism source natural drug has the advantages of high safety, strong pertinence, remarkable activity and environmental friendliness, can solve the problem of drug resistance of most of aquatic disease bacteria to the traditional antibiotics at present, and provides a new thought for solving the problem of bacterial disease control in the existing aquaculture process. For example, chinese patent CN201810906911.9 discloses a diterpenoid compound of the marine fungus tequ mould origin, compound 1 and compound 2, of the following structural formula:
however, the minimum inhibitory concentrations (MIC values) of the above compounds against Edwardsiella tarda and Vibrio harveyi were 8.0. Mu.g/mL, and the inhibitory activity against aquatic disease bacteria was not high enough.
Disclosure of Invention
In view of the above, the invention aims to provide a marine fungus aspergillus omutatus M-7-SW9, a mixed source terpenoid, and an extraction method and application thereof, wherein the mixed source terpenoid extracted from the marine fungus aspergillus omutatus M-7-SW9 has high inhibitory activity on aquatic disease bacteria, in particular on Edwardsiella tarda and vibrio harveyi.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a marine fungus aspergillus versicolor (Aspergillus versicolor) M-7-SW9, wherein the preservation number of the marine fungus aspergillus versicolor M-7-SW9 is CCTCC NO: m2021454.
The invention provides a mixed source terpenoid, which has a structure shown in a formula I:
the invention provides an extraction method of the mixed source terpenoid, which comprises the following steps:
(1) Fermenting and culturing the marine fungus Aspergillus versicolor M-7-SW9 in a PDB liquid culture medium, and extracting the obtained fermentation product by using a polar organic solvent to obtain a fermentation crude extract;
(2) Separating the fermented crude extract by normal phase silica gel column chromatography to obtain an eluting component; the elution mode of the normal phase silica gel column chromatographic separation is gradient elution, and the eluent adopted by the gradient elution is low-polarity solvent-high-polarity solvent; the volume ratio of the low-polarity solvent to the high-polarity solvent in the low-polarity solvent-high-polarity solvent is 50-0: 1, a step of;
(3) Separating the eluted component by reverse phase silica gel column chromatography to obtain a crude product; the elution mode of reversed-phase silica gel column chromatographic separation is gradient elution, the eluent adopted by the gradient elution is water-alcohol solvent, and the volume ratio of water to alcohol in the water-alcohol solvent is 4-0: 1, a step of;
(4) Purifying the crude product to obtain a mixed source terpenoid with a structure shown in a formula I; the purification comprises gel column chromatography and thin layer chromatography which are sequentially carried out, or high performance liquid chromatography purification;
the eluent adopted by the gel column chromatography is methylene dichloride-alcohol solvent; the volume ratio of the dichloromethane to the alcohol in the dichloromethane-alcohol solvent is 2-0: 1, a step of;
the developing agent adopted by the thin layer chromatography is a petroleum ether-ethyl acetate mixed solvent, and the volume ratio of petroleum ether to ethyl acetate in the petroleum ether-ethyl acetate mixed solvent is 2:1;
the mobile phase of the high performance liquid chromatography purification is a water-methanol mixed solvent, and the volume ratio of methanol to water in the water-methanol mixed solvent is 1:1.
Preferably, the PDB liquid culture medium uses water solvent, including 200-300 g/L of potato powder and/or wheat powder, 20-30 g/L of glucose, 5-8 g/L of peptone, 3-5 g/L of yeast extract powder and 35-40 g/L of sodium chloride.
Preferably, the temperature of the fermentation culture is 25-30 ℃ and the time is 7-14 days.
Preferably, the polar organic solvent includes one or more of dichloromethane, ethyl acetate and lower alcohol.
Preferably, the extraction temperature is 20-40 ℃ and the extraction time is 3-7 days.
The invention provides the application of the mixed source terpenoid in the technical scheme or the mixed source terpenoid obtained by the extraction method in the technical scheme in preparing the aquatic disease resistant bacterial medicine.
The invention provides a marine fungus aspergillus versicolor (Aspergillus versicolor) M-7-SW9, which is characterized in that the preservation number of the marine fungus aspergillus versicolor M-7-SW9 is CCTCC NO: m2021454. The mixed source terpenoid with the structure shown in the formula I can be extracted from the marine fungus aspergillus versicolor M-7-SW9, and has high inhibitory activity on aquatic disease bacteria, especially on Edwardsiella tarda and vibrio harveyi.
The invention provides a mixed source terpenoid, which has a structure shown in a formula I. The mixed source terpenoid provided by the invention has high inhibitory activity on aquatic disease bacteria, especially on Edwardsiella tarda and Vibrio harveyi; moreover, compared with the traditional antibiotics and chemical synthetic drugs used in the aquaculture industry, the mixed source terpenoid provided by the invention is a marine organism source natural drug, and provides a new thought for developing the marine organism source natural drug to solve the problem of bacterial disease control in the aquaculture process. As shown by the test results of examples, the minimum inhibitory concentration (MIC value) of the mixed source terpenoid provided by the invention on Edwardsiella tarda and Vibrio harveyi is 1.0 mug/mL; MIC values of chloramphenicol for Edwardsiella tarda are 2.3 μg/mL, respectively, and MIC values for Vibrio harveyi are 2.2 μg/mL; the mixed source terpenoid provided by the invention has higher inhibition activity on Edwardsiella tarda and Vibrio harveyi than chloramphenicol, compound 1 and compound 2.
The invention provides an extraction method of the mixed source terpenoid. The extraction method provided by the invention is simple to operate, low in cost and suitable for large-scale production.
Biological preservation information
The marine fungus Aspergillus versicolor (Aspergillus versicolor) M-7-SW9 has a preservation date of 2021, 04 and 25, a preservation place of China center for type culture Collection, a specific address of China university of Wuhan, and a preservation number of CCTCCNO: m2021454.
Detailed Description
The invention provides a marine fungus aspergillus versicolor (Aspergillus versicolor) M-7-SW9, wherein the preservation number of the marine fungus aspergillus versicolor M-7-SW9 is CCTCC NO: m2021454. In the invention, the marine fungus aspergillus versicolor M-7-SW9 is separated and collected from a seawater sample; the seawater sample is preferably a seawater sample collected at the position of 0.5-3 m of the water depth of the sea area near the island of the urban-raising equine in the smoke-stage of Shandong province. In the present invention, the method for separating the marine fungus Aspergillus versicolor M-7-SW9 preferably comprises the following steps: diluting a seawater sample with sterile distilled water, smearing the diluted seawater sample on a PDA culture medium, placing the PDA culture medium in a fungus incubator for static culture, transferring each fungus colony to the PDA culture medium for continuous culture after fungus colonies grow out on the surface of the PDA culture medium, repeatedly transferring until the colony on the PDA culture medium is a colony of a single strain, and then carrying out strain identification. In the present invention, the PDA medium is preferably sterilized before use, and the present invention is not particularly limited in the sterilization method, and a sterilization method of a medium known to those skilled in the art may be used. In the present invention, the dilution factor is preferably 5 to 20 times, more preferably 10 times. In the present invention, the temperature of the stationary culture is preferably 25 to 30℃and more preferably 28 ℃. In the invention, the bacterial colony formed by the marine fungus aspergillus versicolor M-7-SW9 on the PDA culture medium is white aerial hypha, and the growing spores are green spores. In the invention, the strain identification is preferably carried out by using a DP305 kit (TIANGEN biotechnology Co.) and the sequence of the strain is obtained by extracting and cloning the transcription spacer sequence (ITS 1-5.8S-ITS2 full-length sequence) of the ribosomal rDNA gene of the strain, then carrying out gene sequencing, comparing the obtained strain sequence with the gene sequence of a known strain in Genbank, wherein the gene sequence of the strain is shown as SEQ ID NO.1, and the DNA sequence of the strain has 99% homology with Aspergillus versicolor Aspergillus versicolor (accessionno. MH911415.1), so the strain is named as marine fungus Aspergillus versicolor (Aspergillus versicolor) M-7-SW9.
The invention provides a mixed source terpenoid, which has a structure shown in a formula I:
the invention provides an extraction method of the mixed source terpenoid, which comprises the following steps:
fermenting and culturing the marine fungus aspergillus versicolor M-7-SW9 on a PDB liquid culture medium, and extracting the obtained fermentation product by adopting a polar organic solvent to obtain a fermentation crude extract;
separating the fermented crude extract by normal phase silica gel column chromatography to obtain an eluting component; the elution mode of the normal phase silica gel column chromatographic separation is gradient elution, and the eluent adopted by the gradient elution is low-polarity solvent-high-polarity solvent; the volume ratio of the low-polarity solvent to the high-polarity solvent is 50-0: 1, a step of;
separating the eluted component by reverse phase silica gel column chromatography to obtain a crude product; the elution mode of reversed-phase silica gel column chromatographic separation is gradient elution, and the eluent adopted by the gradient elution is water-alcohol solvent; the volume ratio of water to alcohol in the water-alcohol solvent is 4-0: 1, a step of;
purifying the crude product to obtain a mixed source terpenoid with a structure shown in a formula I; the purification comprises gel column chromatography and thin layer chromatography which are sequentially carried out, or high performance liquid chromatography purification;
the eluent adopted by the gel column chromatography is methylene dichloride-alcohol solvent; the volume ratio of the dichloromethane to the alcohol in the dichloromethane-alcohol solvent is 2-0: 1, a step of;
the developing agent adopted by the thin layer chromatography is a petroleum ether-ethyl acetate mixed solvent, and the volume ratio of petroleum ether to ethyl acetate in the petroleum ether-ethyl acetate mixed solvent is 2:1;
the mobile phase of the high performance liquid chromatography purification is a water-methanol mixed solvent, and the volume ratio of methanol to water in the water-methanol mixed solvent is 1:1.
The marine fungus aspergillus omutatus M-7-SW9 is fermented and cultured on a PDB liquid culture medium, and then the obtained fermentation product is extracted by adopting a polar organic solvent to obtain a fermentation crude extract.
In the present invention, the PDB liquid medium preferably uses water as a solvent, and preferably includes: 200-300 g/L of potato flour and/or wheat flour, preferably 250g/L; glucose 20-30 g/L, preferably 25g/L; 5-8 g/L peptone, preferably 6-7 g/L peptone; 3-5 g/L yeast extract powder, preferably 4-4.5 g/L yeast extract powder; 35 to 40g/L of sodium chloride, preferably 36 to 38g/L. In the present invention, the temperature of the fermentation culture is preferably 25 to 30 ℃, more preferably 28 ℃; the fermentation culture time is preferably 7 to 14 days, more preferably 14 days.
In the present invention, the step of fermentation culture preferably includes: inoculating marine fungus Aspergillus versicolor M-7-SW9 growing in a flat plate culture medium into a first PDB liquid culture medium for first fermentation culture to obtain bacterial liquid; and inoculating the bacterial liquid into a second PDB liquid culture medium for second fermentation culture. In the present invention, the first PDB liquid medium and the second PDB liquid medium are preferably sterilized before use, and the sterilization method is not particularly limited, and a method for sterilizing a plate medium, which is well known to those skilled in the art, may be used. In the present invention, the size of the plate medium is preferably 1.5cm×1.5cm; the volume of the first PDB liquid culture medium is preferably 200-300 mL, more preferably 250mL; the temperature of the first fermentation culture is preferably 25-30 ℃, more preferably 28 ℃; the first fermentation culture time is preferably 7 to 14 days, more preferably 7 days; the first fermentation culture is preferably carried out on a shaker, preferably at a rotational speed of 150 to 250r/min, more preferably 200r/min. In the present invention, the volume of the second PDB liquid medium is preferably 250-350L, more preferably 300L; the temperature of the second fermentation culture is preferably 25-30 ℃, more preferably 28 ℃; the second fermentation culture time is preferably 7 to 14 days, more preferably 7 days; the second fermentation culture is preferably carried out in a fermenter; the filling volume fraction of the second PDB liquid medium in the fermenter is preferably 50 to 70%, more preferably 60%.
In the present invention, the polar organic solvent preferably includes one or more of methylene chloride, ethyl acetate and lower alcohols, and the lower alcohols preferably include one or more of methanol, ethanol, propanol and isopropanol. In the present invention, the temperature of the extraction is preferably 20 to 40 ℃, more preferably 25 to 30 ℃, and the time of the extraction is preferably 3 to 7 days, more preferably 4 to 5 days.
After the extraction, the invention preferably further comprises extracting the extracted system, and concentrating the obtained organic phase to obtain a fermentation crude extract. In the present invention, the extractant used for the extraction preferably includes ethyl acetate, petroleum ether or n-butanol; the number of the extractions is preferably 3 to 5, more preferably 4. The concentration is not particularly limited, and a concentration method well known to those skilled in the art may be adopted, and in particular, the concentration may be performed by vacuum heating concentration using a rotary evaporator until no extractant remains.
After the fermentation crude extract is obtained, the invention carries out normal phase silica gel column chromatography separation on the fermentation crude extract to obtain an elution component. In the invention, the elution mode of the normal phase silica gel column chromatographic separation is gradient elution, and the eluent adopted by the gradient elution is low-polarity solvent-high-polarity solvent; the low polarity solvent preferably comprises petroleum ether and/or dichloromethane; the high-polarity solvent preferably comprises one or more of ethyl acetate, ethanol, propanol and isopropanol; the volume ratio of the low-polarity solvent to the high-polarity solvent in the low-polarity solvent-high-polarity solvent is 50-0: 1, a step of; the volume ratio of the low-polarity solvent to the high-polarity solvent in the gradient elution process is respectively 50: 1. 40: 1. 30: 1. 20: 1. 10:1 and 0:1, wherein the eluting component is the component with the volume ratio of the low polar solvent to the high polar solvent being 10:1.
After the elution component is obtained, the elution component is subjected to reversed-phase silica gel column chromatography to obtain a crude product. In the invention, the elution mode of reversed-phase silica gel column chromatographic separation is gradient elution, and the eluent adopted by the gradient elution is water-alcohol solvent; the volume ratio of water to alcohol in the water-alcohol solvent is 4-0: 1. in the present invention, the alcohol preferably includes methanol and/or ethanol; in the gradient elution process, the volume ratio of water to alcohol is respectively 4: 1. 3: 1. 2: 1. 1:1 and 0:1, wherein the components with the volume ratio of water to alcohol being 1:1 are the crude products.
And after obtaining a crude product, purifying the crude product to obtain the mixed source terpenoid with the structure shown in the formula I. In the present invention, the purification includes gel column chromatography and thin layer chromatography which are sequentially performed, or high performance liquid chromatography purification. In the invention, the eluent adopted by the gel column chromatography is methylene dichloride-alcohol solvent; the volume ratio of the dichloromethane to the alcohol in the dichloromethane-alcohol solvent is 2-0: 1, a step of; the alcohol preferably comprises methanol and/or ethanol. In the invention, the developing agent adopted by the thin layer chromatography is a petroleum ether-ethyl acetate mixed solvent, the volume ratio of petroleum ether to ethyl acetate in the petroleum ether-ethyl acetate mixed solvent is 2:1, and R separated by the thin layer chromatography is as follows f The component with the value of 0.6-0.7 is the mixed source terpenoid with the structure shown in the formula I. In the invention, the mobile phase of the high performance liquid chromatography purification is a water-methanol mixed solvent, the volume ratio of methanol to water in the water-methanol mixed solvent is 1:1, and the component with the retention time of 18.5min of the high performance liquid chromatography purification is a mixed source terpenoid with the structure shown in the formula I。
The invention also provides the application of the mixed source terpenoid in the preparation of novel aquatic disease resistant bacterial medicines. In the present invention, the bacteria resistant to water-borne diseases preferably include Edwardsiella tarda and/or Vibrio harveyi.
The technical solutions of the present invention will be clearly and completely described in the following in connection with the embodiments of the present invention. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
Diluting a seawater sample with sterile distilled water for 10 times, smearing the diluted seawater sample on a PDA culture medium, then placing the PDA culture medium in a fungus incubator, standing and culturing at 28 ℃, transferring each fungus colony to the sterilized PDA culture medium for continuous culturing after fungus colonies grow out on the surface of the sterilized PDA culture medium, and repeatedly transferring until the colony on the PDA culture medium is a colony of a single strain; the bacterial colony formed by the marine fungus aspergillus omutatus M-7-SW9 on the PDA culture medium is white aerial hypha, and the growing spores are gray green spores; the strain identification is carried out by adopting a DP305 kit (TIANGEN biotechnology company), the sequence of the strain is obtained by extracting and cloning a ribosomal rDNA gene transcription spacer sequence (ITS 1-5.8S-ITS2 full-length sequence), then gene sequencing is carried out, the obtained strain sequence is compared with the gene sequence of a known strain in Genbank, the gene sequence of the strain is shown as SEQ ID NO.1, and the DNA sequence of the strain has 99% homology with Aspergillus versicolor Aspergillus versicolor (accessionno. MH911415.1), so the strain is named as marine fungus Aspergillus versicolor (Aspergillus versicolor) M-7-SW9.
Example 2
Inoculating marine fungus Aspergillus versicolor (Aspergillus versicolor) M-7-SW9 (1.5 cm×1.5 cm) grown in a plate culture medium into 250mL sterilized PDB liquid culture medium, fermenting at 28deg.C on a shaker (200 r/min) for 7 days, inoculating the obtained bacterial liquid into 300L sterilized PDB liquid culture medium, fermenting for 7 days with a 500L fermenter, extracting with ethanol soaking fermentation system, extracting with ethyl acetate for 4 times, mixing organic phases, and concentrating to obtain fermented crude extract; the PDB liquid culture medium takes water as a solvent, and comprises the following components: 200g of potato powder, 20g/L of glucose, 5g/L of peptone, 3g/L of yeast extract powder and 35g/L of sodium chloride.
Subjecting the crude extract to normal phase silica gel column chromatography, wherein the volume ratio is sequentially 50-0: 1, the petroleum ether-ethyl acetate solvent and the volume ratio are sequentially 50: 1. 40: 1. 30: 1. 20: 1. 10:1 and 0:1, sequentially carrying out gradient elution on dichloromethane-methanol solvent, and collecting components with the volume ratio of dichloromethane to methanol being 10:1 to obtain eluted components;
the volume ratio of water to methanol is 4: 1. 3: 1. 2: 1. 1:1 and 0: and (3) carrying out reverse phase silica gel column chromatography on the eluted component by using a water-methanol solvent, collecting the component with the volume ratio of water to methanol of 1:1, then taking the water-methanol mixed solvent with the volume ratio of 1:1 as a mobile phase, and collecting the component with the retention time of 18.5min at the wavelength of 210nm to obtain the mixed source terpenoid with the structure shown in the formula I.
Physicochemical and spectral characteristics of the mixed source terpenoid: colorless crystals; the melting point is 178-181 ℃; specific optical rotation [ alpha ]] 20 D -41 (c 0.10, meoh); nuclear magnetic resonance hydrogen spectrum (deuterated dimethyl sulfoxide as solvent) delta H 6.14 (s), 5.38(s), 5.08(s), 4.51(s), 3.73(s), 2.01(s), 1.96 (d, 13.9), 1.84(s), 1.77 (d, 10.9), 1.68 (t, 13.6), 1.62 (m), 1.53 (m), 1.50 (m), 1.46 (m), 1.40 (m), 7.53 (d, 7.6), 1.21 (br s), 1.17(s), 0.98(s), 0.87(s), 0.80(s); nuclear magnetic resonance carbon spectrum (deuterated dimethyl sulfoxide as solvent) delta C 170.6(C),170.3(C),166.8(C),137.7(C),136.3(C),127.2(CH),116.3(CH 2 ),89.2(C),77.0(CH),69.8(C),52.4(CH 3 ),48.7(C),38.4(C),36.5(C),32.8(CH 2 ),30.4(C),28.0(CH 3 ),25.5(CH 2 ),22.0(CH 2 ),21.8(CH 3 ),21.5(CH 3 ),18.4(CH 3 ),17.4(CH 3 ),17.2(CH 2 ),16.0(CH 3 ) The method comprises the steps of carrying out a first treatment on the surface of the High resolution mass spectrum m/z 431.2423[ M+H ]] + ，C 25 H 35 O 6 + Calculated as 435.2428.
Comparative example 1
Compound 1 and compound 2, having the structural formula:
test example 1
Bacterial inhibitor activity against aquatic disease
The activity of the compound shown in the formula I against water-borne disease bacteria is detected by a minimum inhibitory concentration method. The following 4 aquatic pathogenic strains were selected: edwardsiella tarda, vibrio harveyi, micrococcus luteus and Vibrio parahaemolyticus were tested for antibacterial activity.
(1) Antibacterial activity test (MIC method): the minimum inhibitory concentration is the minimum drug concentration that can inhibit bacterial growth in vitro. In a 96-micro-well plate, medicines with different concentrations are added into bacterial suspension of bacteria to be detected, after culturing, observation is carried out, if the indicator bacteria grow in a certain hole, the medicine concentration in the hole cannot inhibit the growth of the bacteria, the liquid in the hole is turbid, and the transmittance is obviously reduced. Otherwise, the liquid in the hole is clear, and the transmittance is not obviously reduced. The minimum sample concentration at which complete inhibition of growth of indicator bacteria in the wells was the MIC value of the compound.
(2) Preparation of bacterial suspension
Edwardsiella tarda and Vibrio harveyi are inoculated onto a medium (TSB medium for Edwardsiella tarda; LB medium for Vibrio harveyi) and cultured at 28℃for 24 hours, and after 4mL of a sterile 0.85% NaCl solution is aspirated to wash the culture, and the bacteria are gently scraped off with a glass spatula. The appropriate amount of the bacterial suspension was pipetted into a sterile test tube and then the bacterial suspension was brought to 0.5 M.RTM.with 0.85% NaCl solution (i.e.1.5X10) 8 CFU/mL) and further diluted to 5 x 10 with 0.85% nacl solution 5 CFU/mL；
Wherein, the preparation method of the 0.85% sodium chloride solution comprises the following steps: after 8.5g of sodium chloride was dissolved in water, the volume was set to 1000mL.
0.5 mahalanobis turbidity standard: 0.5mL of BaCl with the concentration of 0.048mol/L is added 2 Added to 99.5mL of H with the concentration of 0.18mol/L 2 SO 4 Is constantly agitated to maintain the suspension.
(3) Preparation of drug solution to be tested
The medicine to be tested: a mixed source terpenoid compound with a structure shown in a formula I, a positive control drug (chloramphenicol), a compound 1 and a compound 2.
Respectively dissolving the mixed source terpenoid and chloramphenicol in 100 mu LDMSO, and fully and uniformly mixing to obtain a drug solution to be detected, wherein the final concentration of the drug solution to be detected is 2560 mu g/mL; the 50. Mu.L of the drug solution to be tested was sucked into a centrifuge tube, 50. Mu.L of DMSO was added to obtain a drug solution to be tested having a halved concentration, and the above-mentioned operations were repeated to obtain 11 sets of drug solutions to be tested having a halved concentration in order (the concentrations were 2560. Mu.g/mL, 1280. Mu.g/mL, 640. Mu.g/mL, 320. Mu.g/mL, 160. Mu.g/mL, 80. Mu.g/mL, 40. Mu.g/mL, 20. Mu.g/mL, 10. Mu.g/mL, 5. Mu.g/mL and 2.5. Mu.g/mL in order).
(4) Blank control: DMSO was used as a blank.
(5) MIC value determination procedure:
and (5.1) adopting aseptic operation, adding the to-be-detected medicine solutions with different concentrations into aseptic 96-well plates respectively, adding the to-be-detected medicine solution into the 1 st to 11 th wells, wherein each well is 5 mu L, and the 12 th well is not added with the to-be-detected medicine solution as a growth control.
(5.2) the suspension of the indicator bacteria corresponding to the turbidity of 0.5 McAb was diluted 1000-fold with a liquid medium (Edwardsiella tarda TSB medium; vibrio harveyi LB medium), and then 95. Mu.L was sequentially added to a 96-well plate so that the final concentrations of the drug solutions to be measured in the 1 st to 11 th wells were 128. Mu.g/mL, 64. Mu.g/mL, 32. Mu.g/mL, 16. Mu.g/mL, 8. Mu.g/mL, 4. Mu.g/mL, 2. Mu.g/mL, 1. Mu.g/mL, 0.5. Mu.g/mL, 0.25. Mu.g/mL and 0.125. Mu.g/mL in this order. After gentle shaking and mixing, the 96-well plate is sealed and placed in a 28 ℃ incubator for bacterial culture for 24 hours.
(5.3) absorbance of each well was measured at a wavelength of 600nm using an enzyme-labeled instrument to completely inhibit growth of indicator bacteria in the wells at the lowest sample concentration as MIC value of the drug to be tested. The method is significant only when the indicator bacteria in the negative control hole obviously grow; when single jump hole appears in the experiment, the highest drug concentration for inhibiting the growth of the strain is recorded; if a plurality of jump holes appear, the result is not reported, and the experiment is required to be repeated.
Experimental results: the mixed source terpene compound has stronger inhibitory activity on Edwardsiella tarda and Vibrio harveyi respectively, the MIC value is 1.0 mug/mL, the MIC value of chloramphenicol to Edwardsiella tarda is 2.3 mug/mL, the MIC value to Vibrio harveyi is 2.2 mug/mL, and the MIC value of compound 1 and compound 2 to Edwardsiella tarda and Vibrio harveyi is 8.0 mug/mL. The mixed source terpene compound provided by the invention has better inhibition effect on Edwardsiella tarda and Vibrio harveyi than chloramphenicol, compound 1 and compound 2.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Sequence listing
<110> university of Rudong
<120> A marine fungus Aspergillus versicolor M-7-SW9, and mixed source terpenoid, and its extraction method and application
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 571
<212> DNA
<213> Aspergillus versicolor
<400> 1
ttccgtaggg gtgacctgcg gaaggatcat tactgagtgc gggctgcctc cgggcgccca 60
acctcccacc cgtgaatacc taacactgtt gcttcggcgg ggaaccccct cgggggcgag 120
ccgccgggga ctactgaact tcatgcctga gagtgatgca gtctgagtct gaatataaaa 180
tcagtcaaaa ctttcaacaa tggatctctt ggttccggca tcgatgaaga acgcagcgaa 240
ctgcgataag taatgtgaat tgcagaattc agtgaatcat cgagtctttg aacgcacatt 300
gcgccccctg gcattccggg gggcatgcct gtccgagcgt cattgctgcc catcaagccc 360
ggcttgtgtg ttgggtcgtc gtcccccccg ggggacgggc ccgaaaggca gcggcggcac 420
cgtgtccggt cctcgagcgt atggggcttt gtcacccgct cgactagggc cggccgggcg 480
ccagccgacg tctccaacca tttttcttca ggttgacctc ggatcaggta gggatacccg 540
ctgaacttaa gcatatcaat aagcggagga a 571
Claims (7)
1. Marine fungus aspergillus versicolorAspergillus versicolor) M-7-SW9, wherein the preservation number of the marine fungus Aspergillus versicolor M-7-SW9 is CCTCC NO: m2021454.
2. A mixed source terpenoid is characterized in that the structure is shown as a formula I:
3. The method for extracting the mixed source terpenoid according to claim 2, comprising the steps of:
(1) Fermenting and culturing the marine fungus aspergillus versicolor M-7-SW9 in a PDB liquid culture medium, extracting the obtained fermentation product by adopting a polar organic solvent, and obtaining a fermentation crude extract; the polar organic solvent is ethanol; the extractant adopted in the extraction is ethyl acetate;
(2) Carrying out normal phase silica gel column chromatography on the fermented crude extract, wherein the volume ratio is sequentially 50-0: 1, the petroleum ether-ethyl acetate solvent and the volume ratio are sequentially 50: 1. 40: 1. 30: 1. 20: 1. 10:1 and 0:1, sequentially carrying out gradient elution on dichloromethane-methanol solvent, and collecting components with the volume ratio of dichloromethane to methanol being 10:1 to obtain eluted components;
(3) The volume ratio of water to methanol is 4: 1. 3: 1. 2: 1. 1:1 and 0: and (3) carrying out reverse phase silica gel column chromatography on the eluted component by using a water-methanol solvent, collecting the component with the volume ratio of water to methanol of 1:1, then carrying out high performance liquid chromatography purification by using a water-methanol mixed solvent with the volume ratio of 1:1 as a mobile phase, and collecting the component with the retention time of 18.5min at the wavelength of 210nm to obtain the mixed source terpenoid.
4. The extraction method according to claim 3, wherein the PDB liquid medium is prepared from 200-300 g/L of potato powder and/or wheat powder, 20-30 g/L of glucose, 5-8 g/L of peptone, 3-5 g/L of yeast extract powder and 35-40 g/L of sodium chloride by using an aqueous solvent.
5. The extraction method according to claim 3 or 4, wherein the fermentation culture is carried out at a temperature of 25-30 ℃ for 7-14 days.
6. The extraction method according to claim 3, wherein the extraction temperature is 20-40 ℃ and the extraction time is 3-7 days.
7. The application of the mixed source terpenoid of claim 2 or the mixed source terpenoid obtained by the extraction method of any one of claims 3-6 in preparing an anti-aquatic disease bacterial drug; the bacteria resistant to the water-borne diseases are Edwardsiella tarda and/or Vibrio harveyi.
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