阅读说明：本技术 茴香霉素的制备方法、茴香霉素的检测方法及抑菌应用 (Preparation method of anisomycin, detection method of anisomycin and antibacterial application ) 是由 葛蓓孛 张克诚 施李鸣 韦秋合 张维 杨淼泠 于 2020-12-25 设计创作，主要内容包括：本发明公开了一种茴香霉素的制备方法、茴香霉素的检测方法及抑菌应用,该茴香霉素的制备方法,包括：取小白链霉菌CK-15的菌株种子液接种于种子培养基,28℃、220rpm培养24h后,按照5％接种量转接至100ml发酵培养基中,30℃发酵5d,将得到的发酵液经离心处理,收集第一上清液；将第一上清液的pH调至9.0,用与上清液等体积的乙酸乙酯萃取第一有机相；用与第一有机相等体积的pH为2.0的酸水对第一有机相反向萃取,收集水相并将水相的pH调至9.0；用与水相等体积的乙酸乙酯萃取,收集第二有机相并旋干。本发明的茴香霉素的制备方法,通过小白链霉菌CK-15菌株进行发酵制备茴香霉素,相比于采用化学合成方法制备茴香霉素,该制备方法简单,方便操作,成本较低。(The invention discloses a preparation method of anisomycin, a detection method of anisomycin and antibacterial application, wherein the preparation method of anisomycin comprises the following steps: inoculating a strain seed liquid of the streptomyces albidoflavus CK-15 strain to a seed culture medium, culturing at 28 ℃, 220rpm for 24 hours, transferring the strain seed liquid to 100ml of fermentation culture medium according to the inoculation amount of 5%, fermenting at 30 ℃ for 5 days, centrifuging the obtained fermentation liquid, and collecting a first supernatant; adjusting the pH of the first supernatant to 9.0, and extracting the first organic phase with an equal volume of ethyl acetate to the supernatant; counter-extracting the first organic phase with a volume of acid water equal to the first organic phase at a pH of 2.0, collecting the aqueous phase and adjusting the pH of the aqueous phase to 9.0; extracted with an equal volume of ethyl acetate to the aqueous phase, the second organic phase was collected and spin dried. Compared with the preparation method of the anisomycin by adopting a chemical synthesis method, the preparation method of the anisomycin has the advantages of simplicity, convenient operation and lower cost.)

1. A preparation method of anisomycin is characterized by comprising the following steps:

inoculating a strain seed liquid of the streptomyces albidoflavus CK-15 strain to a seed culture medium, culturing at 28 ℃, 220rpm for 24 hours, transferring the strain seed liquid to 100ml of fermentation culture medium according to the inoculation amount of 5%, fermenting at 30 ℃ for 5 days, centrifuging the obtained fermentation liquid, and collecting a first supernatant;

adjusting the pH of the first supernatant to 9.0, and extracting a first organic phase with an equal volume of ethyl acetate to the supernatant;

counter-extracting the first organic phase with a volume of acid water equal to the first organic phase at a pH of 2.0, collecting the aqueous phase and adjusting the pH of the aqueous phase to 9.0;

extracted with an equal volume of ethyl acetate to the aqueous phase, the second organic phase was collected and spin dried.

2. The method of claim 1, further comprising:

pretreating the second organic phase by using macroporous adsorption resin HP-20, sequentially performing ODS and Sephadex LH-20 chromatography, performing gradient elution by adopting the ratio of ethyl acetate to methanol from 20:1 to 1:20, and removing impurities in the second organic phase to obtain an anisomycin crude product;

dissolving the crude product of the anisomycin with methanol, filtering the solution by a filter membrane, collecting a liquid phase when the anisomycin has a peak value by HPLC, concentrating and drying the liquid phase at a temperature of below 60 ℃ under reduced pressure, and freeze-drying to obtain light yellow anisomycin powder.

3. The method according to claim 1, wherein the seed medium is prepared by mixing 2g of glucose, 0.6g of peptone, 0.6g of yeast powder, 1g of NaCl and 100ml of distilled water, and sterilizing at 121 ℃ for 30 min.

4. The method according to claim 1, wherein the fermentation medium comprises 10g of corn flour, 20g of soluble starch, 10g of soybean meal, and 0.2g of KH₂PO₄3g of NaCl, 3g of NH₄Cl, 4g of CaCO₃1L of distilled water, and the pH was 7.0.

5. The method according to claim 1 or 3, wherein the seed solution of the strain is prepared by: cutting 1cm × 1cm of the strain block, inoculating into the seed culture medium, and culturing at 28 deg.C and 220rpm for 24 hr to obtain seed solution.

6. A method for detecting anisomycin prepared by the process according to any one of claims 1 to 5, comprising:

dissolving the second organic phase with methanol, centrifuging at high speed for 10min, and filtering the second supernatant with 0.22 μm organic phase filter membrane or water phase filter membrane to remove impurities;

comparing the HPLC chromatogram of the second supernatant after filtration with the standard of anisomycin; wherein, HPLC detection conditions are as follows: agilent 150X 21mm C18-SB reverse phase chromatography column was used with detection wavelengths of 223nm and 280nm and flow rate of 0.5ml/min, mobile phase A was water containing 0.2% formic acid or TFA and phase B was 100% methanol.

7. Use of anisomycin prepared by the process according to any one of claims 1 to 5 for its bacteriostatic application, comprising: respectively placing test pathogenic fungus cakes with a preset diameter on the upper surfaces of a PDA culture medium without adding the anisomycin and a PDA culture medium containing 100 mug/ml of anisomycin, culturing at a constant temperature of 24 ℃ for 3-4 days, measuring the colony diameter by a cross method, and calculating the hypha growth inhibition rate: wherein the content of the first and second substances,

hypha growth inhibition rate (control colony diameter-treated colony diameter)/control colony diameter × 100%; the diameter of the zone of inhibition was determined by the oxford cup method.

8. Bacteriostatic use according to claim 7, wherein the bacteria on the pathogenic fungal cake to be tested are botrytis cinerea, apple tree rot, botrytis cinerea, colletotrichum cucumerinum, alternaria nicotianae, alternaria zeae, botrytis cinerea or fusarium graminearum.

Technical Field

The invention relates to the technical field of microbial fermentation, in particular to a method for preparing anisomycin by fermentation, a detection method of anisomycin and antibacterial application.

Background

Anisomycin is a nitrogen-containing heterocyclic aromatic antibiotic produced by actinomycetes such as Streptomyces griseus, and has good antifungal, antiprotozoal and antitumor effects. The derivative is widely applied to the research in the fields of cell and molecular genetics at present, and has great potential for being used as an immunosuppressive drug.

Currently, the anisomycin is prepared by a chemical synthesis method, and the anisomycin prepared by the chemical synthesis method has the problems of high chiral catalyst cost, harsh reaction conditions, difficult enantiomer separation, serious environmental pollution and the like.

Disclosure of Invention

Aiming at the problems in the prior art, the first purpose of the invention is to provide a preparation method of anisomycin.

In order to realize the purpose, the technical scheme of the preparation method of the anisomycin is as follows:

a preparation method of anisomycin comprises the following steps:

inoculating strain seed liquid of Streptomyces albugineus CK-15(Streptomyces albugineus strain CK-15) into a seed culture medium, culturing at 28 ℃ and 220rpm for 24h, transferring into 100ml of fermentation culture medium according to the inoculum size of 5%, fermenting for 5d at 30 ℃, centrifuging the obtained fermentation liquid, and collecting a first supernatant;

adjusting the pH of the first supernatant to 9.0, and extracting a first organic phase with an equal volume of ethyl acetate to the supernatant;

counter-extracting the first organic phase with a volume of acid water equal to the first organic phase at a pH of 2.0, collecting the aqueous phase and adjusting the pH of the aqueous phase to 9.0;

extracted with an equal volume of ethyl acetate to the aqueous phase, the second organic phase was collected and spin dried.

Further, the method further comprises:

pretreating the second organic phase by using macroporous adsorption resin HP-20, sequentially performing ODS and Sephadex LH-20 chromatography, performing gradient elution by adopting the ratio of ethyl acetate to methanol from 20:1 to 1:20, and removing impurities in the second organic phase to obtain an anisomycin crude product;

dissolving the crude anisomycin product in methanol, filtering the solution by a filter membrane, collecting a Liquid phase when the anisomycin has a peak value by using High Performance Liquid Chromatography (HPLC), concentrating and drying the Liquid phase at a temperature of below 60 ℃ under reduced pressure, and freeze-drying the concentrated Liquid phase to obtain light yellow anisomycin powder.

Further, the seed culture medium is prepared by mixing 2g of glucose, 0.6g of peptone, 0.6g of yeast powder, 1g of NaCl and 100ml of distilled water, and sterilizing at 121 ℃ for 30 min.

Further, theThe fermentation medium comprises 10g of corn flour, 20g of soluble starch, 10g of soybean cake powder and 0.2g of KH₂PO₄3g of NaCl, 3g of NH₄Cl, 4g of CaCO₃1L of distilled water, and the pH was 7.0.

Further, the preparation method of the strain seed solution comprises the following steps: cutting 1cm × 1cm of the strain block, inoculating into the seed culture medium, and culturing at 28 deg.C and 220rpm for 24 hr to obtain seed solution.

Compared with the preparation method of the anisomycin by adopting a chemical synthesis method, the preparation method of the anisomycin has the advantages of simplicity, convenient operation and lower cost.

The second purpose of the invention is to provide a method for detecting the anisomycin prepared by the method.

In order to realize the purpose, the technical scheme of the method for detecting the anisomycin is as follows:

a method for detecting anisomycin prepared by adopting the preparation method comprises the following steps:

dissolving the second organic phase with methanol, centrifuging at high speed for 10min, and filtering the second supernatant with 0.22 μm organic phase filter membrane or water phase filter membrane to remove impurities;

comparing the HPLC chromatogram of the second supernatant after filtration with the standard of anisomycin; wherein, HPLC detection conditions are as follows: agilent 150X 21mm C18-SB reverse phase chromatography column was used with detection wavelengths of 223nm and 280nm and flow rate of 0.5ml/min, mobile phase A was water containing 0.2% formic acid or TFA and phase B was 100% methanol.

The method for detecting anisomycin of the present invention can be used for detecting and verifying the method for preparing anisomycin.

The third purpose of the invention is to provide the bacteriostatic application of the anisomycin prepared by the method.

In order to achieve the purpose, the antibacterial application technical scheme of the anisomycin is as follows:

the anisomycin prepared by the preparation method has bacteriostatic application, and comprises the following steps: respectively placing test pathogenic fungus cakes with a preset diameter on the upper surfaces of a PDA culture medium without adding the anisomycin and a PDA culture medium containing 100 mug/ml of anisomycin, culturing at a constant temperature of 24 ℃ for 3-4 days, measuring the colony diameter by a cross method, and calculating the hypha growth inhibition rate: wherein the content of the first and second substances,

hypha growth inhibition rate (control colony diameter-treated colony diameter)/control colony diameter × 100%; the diameter of the zone of inhibition was determined by the oxford cup method.

Further, the bacteria on the test pathogenic fungal cake are Botrytis cinerea (Phyllosticta ampeliica), apple tree rot (Valsa ceratosperma), grape canker (Botryosphaeria dothidea), cucumber anthracnose (Colletotrichum lagenarium), tobacco brown spot (Alternaria alternata), maize plaque (Bipolaris maydis), tomato leaf mold (Fusariua fulva), gray mold (Botrytis cinerea) or Fusarium graminearum (Fusarium graminearum).

The anisomycin prepared by the preparation method has better antagonistic action.

Drawings

FIG. 1 is a graph of a prediction of the secondary metabolic biosynthesis gene cluster for the whole genome antifungal activity of Streptomyces albus CK-15 in an example of the present invention;

FIG. 2 is a chromatogram of an anisomycin standard according to an example of the present invention;

FIG. 3 is a chromatogram of the product obtained using the process for the preparation of anisomycin of this example.

Detailed Description

In order to make those skilled in the art better understand the technical solution of the present invention, the technical solution of the present invention is described clearly and completely below with reference to the drawings in the examples of the present invention, and it is obvious that the described examples are only a part of examples of the present invention, and not all examples. All other embodiments obtained by a person skilled in the art based on examples in the present invention shall fall within the scope of protection of the present invention without making creative efforts.

In this example, the inventors analyzed and identified the antifungal activity of the secondary metabolites of Streptomyces albus. Sequencing the whole genome of Streptomyces albulus strain CK-15 and bioinformatics analysis of secondary metabolites: first, the resulting bacteria are cultured in a liquid medium, CK-15 whole genome DNA is extracted, and whole genome sequencing is performed after RNA enzyme degradation and other treatments. Secondly, the CK-15 complete genome DNA group extraction is carried out, and because the third-generation sequencing of the complete genome needs DNA samples with sufficient concentration and high purity, the complete genome DNA is extracted by adopting a hand-held method.

The sequencing assembly result shows that the total length of the whole genome is 9426086bp, and the GC content is 72.33%. The gene function annotation is carried out on the whole genome by combining NR, COG, KEGG and GO databases, and the result shows that the whole genome predicts 9077 open reading frames which account for 83.16% of the whole genome length. The composition of the CK-15 genome to be sequenced is obtained by predicting coding genes, repetitive sequences, non-coding RNAs and the like, wherein the genome codes 68 tRNAs (total length 5156bp), 7 5s rRNAs (total length 803bp), 7 16s rRNAs (total length 10619bp), 7 23s rRNAs (total length 21819bp) and 20 sRNAs (total length 1487 bp).

Analysis and prediction of secondary metabolism gene cluster of CK-15 antifungal activity: analyzing the whole genome sequence by utilizing databases such as anti-SMASH, ThioFinder and the like, and analyzing to obtain that the whole genome totally contains 39 secondary metabolic gene clusters, wherein the types of the gene clusters comprise: nucleosides, polyketides, nonribosomal peptides, butyrolactone, lactobacillus peptides, bacteriocins, siderophiles, tetrahydropyrimidines, terpenes, etc., wherein the antifungal substances include gougerotin, natamycin, oxazoleoxidomycin, Warkmycin, tomycin, anisomycin, nystatin.

Referring to FIG. 1, only 5 of the 7 antifungal metabolites analyzed above were expressed by transcriptome analysis, thus demonstrating that gougerotin, natamycin, toyocamycin, anisomycin and nystatin were produced, and that the biosynthesis amounts of Oxazolepoxydomycin and Warkmycin were very small and negligible. In summary, Streptomyces albulus CK-15 antifungal activity secondary metabolites are considered to contain gougerotin, natamycin, toyocamycin, anisomycin and nystatin.

In summary, the present example provides a method for preparing anisomycin, comprising: inoculating a strain seed liquid of the streptomyces albidoflavus CK-15 strain to a seed culture medium, culturing at 28 ℃, 220rpm for 24 hours, transferring the strain seed liquid to 100ml of fermentation culture medium according to the inoculation amount of 5%, fermenting at 30 ℃ for 5 days, centrifuging the obtained fermentation liquid, and collecting a first supernatant;

adjusting the pH of the first supernatant to 9.0, and extracting the first organic phase with an equal volume of ethyl acetate to the supernatant;

counter-extracting the first organic phase with a volume of acid water equal to the first organic phase at a pH of 2.0, collecting the aqueous phase and adjusting the pH of the aqueous phase to 9.0;

extracted with an equal volume of ethyl acetate to the aqueous phase, the second organic phase was collected and spin dried.

Compared with the preparation method of the anisomycin by adopting a chemical synthesis method, the preparation method of the anisomycin by fermenting the streptomyces albidoflavus CK-15 strain is simple, convenient to operate and low in cost.

In this example, the seed medium was mixed with 2g of glucose, 0.6g of peptone, 0.6g of yeast powder, 1g of NaCl and 100ml of distilled water, and sterilized at 121 ℃ for 30 min.

In this example, 10g corn flour, 20g soluble starch, 10g soybean meal, 0.2g KH was used as the fermentation medium₂PO₄3g of NaCl, 3g of NH₄Cl, 4g of CaCO₃1L of distilled water, and the pH was 7.0.

In this example, the strain seed solution was prepared by: cutting 1cm × 1cm of the strain block, inoculating into seed culture medium, and culturing at 28 deg.C and 220rpm for 24 hr to obtain seed solution.

It should be noted that after the second organic phase is collected, it needs to be stored at-30 ℃ to prevent degradation. After collecting the second organic phase, to detect and verify the presence of anisomycin in the second organic phase, the method and difficulty of preparation of anisomycin were determined. Therefore, the present example provides a method for detecting anisomycin prepared by the above preparation method, comprising:

dissolving the second organic phase with methanol, centrifuging at high speed for 10min, and filtering the second supernatant with 0.22 μm organic phase filter membrane or water phase filter membrane to remove impurities;

referring to fig. 2 and 3, HPLC chromatograms of the standard of anisomycin and the filtered second supernatant were compared; wherein, HPLC detection conditions are as follows: agilent 150X 21mm C18-SB reverse phase chromatography column was used with detection wavelengths of 223nm and 280nm and flow rate of 0.5ml/min, mobile phase A was water containing 0.2% formic acid or TFA and phase B was 100% methanol. The detection program setting method is as follows:

HPLC detection conditions of anisomycin standard substance and CK-15 intermediate product

Setting mass spectrum detection parameters: in the positive ion mode, the high attracting gas flow rate (drying gas flow) is set to 10ml/min, the pressure of a sprayer (nebulizer pressure) is set to 30psi, the temperature of drying gas (drying gas temperature) is set to 350 ℃, the molecular weight scanning range is 50-800(m/z), and the bombardment voltage setting range of the multistage mass spectrum fragmentation analysis is 0.8-1.1V.

As is clear from a comparison of FIGS. 2 and 3, it can be fully explained that the intermediate product of CK-15 fermentation contains anisomycin.

In this example, the method of preparing anisomycin further comprises:

the second organic phase is pretreated by macroporous adsorption resin HP-20, specifically, the second organic phase is subjected to gradient elution by using water and ethanol in a concentration ratio of 0:100 to 100:0, for example, the second organic phase can be subjected to gradient washing by using ethanol with the content of 100%, 80%, 60%, 40%, 20% and 0% (namely water) in sequence.

Sequentially performing ODS and Sephadex LH-20 chromatography, performing gradient elution by adopting the ratio of ethyl acetate to methanol from 20:1 to 1:20, and removing impurities in a second organic phase to obtain an anisomycin crude product;

dissolving the crude product of anisomycin with methanol, filtering with a filter membrane, collecting the liquid phase with peak value of anisomycin by HPLC, concentrating the liquid phase at below 60 deg.C under reduced pressure, drying, and freeze drying to obtain light yellow anisomycin powder. Specifically, in this example, the crude product was dissolved in methanol to prepare a 10mg/mL sample, which was filtered through a filter membrane and used. The analytical chromatography shows that the anisomycin standard produces a peak in about 10min, the mobile phase is methanol-water (volume ratio 40:60), and the flow rate: 0.7ml/min, and the detection wavelength is 225 nm; column temperature: and (2) at 25 ℃, carrying out sample introduction of 10 mu L, carrying out separation preparation by semi-preparative HPLC, collecting and retaining time of about 10 minutes, concentrating and drying the mixture at the temperature of below 60 ℃ under reduced pressure, and finally carrying out freeze drying to obtain light yellow powder 170mg, namely the anisomycin, wherein the mass fraction of the fennel obtained by the preparation is 98.1% by peak area normalization method.

The present example further provides a bacteriostatic application of anisomycin prepared by the preparation method, comprising: respectively placing test pathogenic fungus cakes with a preset diameter on the upper surfaces of a PDA culture medium without adding the anisomycin and a PDA culture medium containing 100 mug/ml of anisomycin, culturing at a constant temperature of 24 ℃ for 3-4 days, measuring the colony diameter by a cross method, and calculating the hypha growth inhibition rate: wherein the content of the first and second substances,

hypha growth inhibition rate (control colony diameter-treated colony diameter)/control colony diameter × 100%; the diameter of the zone of inhibition was determined by the oxford cup method.

Anisomycin prepared by the preparation method of the example has a good antagonistic effect.

It is to be noted that, in this example, the diameter of the cake of pathogenic fungi to be tested is 5 mm. A plurality of test pathogenic fungus cakes can be respectively put on a plurality of different PDA culture media without the addition of the anisomycin and PDA culture media containing 100 mug/ml of the anisomycin so as to carry out a plurality of groups of comparison tests, for example, 3 groups of comparison tests, and more accurately obtain the data of the anisomycin antibacterial action; in calculating the hyphal growth inhibition ratio, the control colony diameter and the treated colony diameter may be calculated by averaging the data of the respective groups.

In this example, the bacteria on the test pathogenic fungal cake may be one of Botrytis cinerea (Phyllosticta ampeliica), apple tree rot (Valsa ceratosperma), grape canker (Botrytis dothiea), cucumber anthracnose (Colletotrichum lagenarium), tobacco brown spot (Alternaria alternata), maize macula (Bipolaris maydis), tomato leaf mold (Fulvia fulva), gray mold (Botrytis cinerea) or Fusarium graminearum (Fusarium graminearum), and the inhibition effect of the growth of the 9 plant hyphae by the anisomycin prepared by the preparation method of this example is shown in the table below:

as can be seen from the table above, the anisomycin has good antibacterial activity to 9 different pathogenic fungi, and the results show that the growth of 9 plant pathogenic fungi is inhibited by the anisomycin, the inhibition efficiency of the anisomycin to the botrytis cinerea and the apple tree rot pathogen is over 90 percent, the inhibition efficiency to the botrytis cinerea, the cucumber colletotrichum, the tobacco brown spot pathogen, the maize microsporum, the tomato leaf mold pathogen and the gray mold is 89 to 78 percent, and the inhibition effect to the growth of the mycelium of the fusarium graminearum is only 58 percent. These results demonstrate the bacteriostatic effect of anisomycin on a variety of phytopathogens.

Finally, it is to be understood that the above embodiments are merely exemplary embodiments taken to illustrate the principles of the present invention, which is not intended to be limiting. It will be apparent to those skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope of the invention, and these changes and modifications are to be considered as within the scope of the invention.