CN106676025B - Method for co-culturing epicoccum nigrum and aspergillus fungi and obtained compound - Google Patents
Method for co-culturing epicoccum nigrum and aspergillus fungi and obtained compound Download PDFInfo
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Abstract
The invention relates to a method for co-culturing epiphyte nigrococcus and aspergillus. The co-cultivation method can lead aspergillus to produce secondary metabolites which have extremely low yield when being cultured alone and/or are not produced when being cultured alone; the method comprises the following steps: a) respectively inoculating the epicoccum nigrum and the aspergillus fungi to an activation culture medium for strain activation; b) inoculating the activated epicoccum nigrum and the aspergillus fungi into a solid culture medium of the same culture container for standing culture; or inoculating the activated epicoccum nigrum and the aspergillus fungi into a liquid culture medium of the same culture vessel for shaking culture; c) when spores of the Aspergillus fungus in the solid medium become yellow, the Aspergillus fungus is collected and the medium is used to obtain a product. Also relates to secondary metabolites produced by the co-cultivation process.
Description
Technical Field
The invention relates to the field of microorganisms, in particular to a method for co-culturing epicoccum nigrum and aspergillus fungi and an obtained compound.
Background
Aspergillus spp is an important filamentous fungus, many groups of which are closely related to human life and health, and has been widely used in both traditional wine making and sauce making and modern food processing industries, such as: aspergillus together with yeast, bacteria or combined bacteria ferment soybean for producing soybean paste and soy sauce; mixing fermented rice to produce rice wine; the product can be used for ripening cheese, making cheese have special flavor, and smoking ham, sausage, etc. For years, a large number of secondary metabolites have been found in Aspergillus fungi, and have important research and application values. Some of these are mycotoxins with strong carcinogenicity, such as: aflatoxins (Aflatoxins) and variegated aspergillins (sterigmatocystins) produced by fungi of the genus aspergillus are found in food and agricultural crops worldwide; aspergillus fungus is also an important natural medicine source, and various secondary metabolites produced by the Aspergillus fungus are widely applied to clinic, such as Lovastatin (Lovastatin) produced by Aspergillus terreus can be used as a specific blood pressure lowering medicament for resisting cholesterol. However, with the continuous and intensive research, the compounds obtained from the traditional screening mode for finding natural products in fungi are repeatedly found in a large amount, aspergillus can only produce limited metabolites under the conventional culture conditions of a laboratory, new compounds with activity in aspergillus are difficult to find, and the existing culture method greatly limits the discovery of the new compounds.
In recent years, several new research strategies have been used to solve the dilemma encountered in the conventional methods for discovering new metabolites in fungi, and the completion of extensive fungal genome sequencing has provided for the development of new natural product discovery methods for fungi. The model fungal genome bioinformatics analysis found that a large number of the gene clusters encoding the natural products were in a "silent" state, indicating that a large number of potential compounds with novel structures were hidden in the "silent" gene cluster and not found. Based on this, it was found that co-cultivation with other organisms under laboratory conditions can exert an external stress on the fungus, thereby inducing the expression of "silent" gene clusters in the fungus, and thus the production or increased yield of new compounds. The co-cultivation of bacteria and fungi that has been found to date results in the production of new compounds: the co-culture of Rhizopus (Rhizopus microsporus) and burkholderia (Burkholderiagladioli) can induce the synthesis of antibacterial polyketide compounds; the co-cultivation of Fusarium (Fusarium tricinctum) with Bacillus (Bacillus subtilis) can lead to the production of large amounts of secondary metabolites; coculture of Fusarium (F. pallidoroseum) with Saccharopolyspora erythraea induced the production of Tetramic Acid homologues. Co-cultivation of bacteria with aspergillus may induce the production of new compounds, such as: the co-cultivation of Aspergillus marinus fungus (Emericella sp.) with Actinomycetes (Salinippora arenicola) induces the production of the compounds, emericelamides A and B; aspergillus fumigatus (A. fumigatus) produces the compounds N-Formyl Alkaloids and diketopiperazines when co-cultured with actinomycetes (Streptomyces spp.); co-cultivation of aspergillus terreus (a. terreus) with bacillus (b.subtilis and b.cereus) induced a 34-fold increase in production of the relevant compounds. As for the method of producing novel secondary metabolites by co-culture between fungi, there are only few reports, such as that co-culture between fusarium strains (f.tricinctum and f.begoniae) induces production of depsipeptides; whereas co-culture of Trichophyton rubrum (Trichophyton rubrum) and Chitosa pallida (Bionectria ochroleuca) induced the production of 5 compounds. At present, no report exists on the production of new structural compounds by the aspergillus fungi through the mode of co-culture with other fungi.
Disclosure of Invention
The invention provides a method for co-culturing epicoccum nigrum and aspergillus fungi and an obtained compound, and discloses a method for discovering a new natural product by co-culturing the epicoccum nigrum and the aspergillus fungi for the first time, and discovers that the color change of aspergillus spores can be used as an indication reaction of the change of a secondary metabolite and used for inducing or activating the expression of related gene clusters in aspergillus, so that the aspergillus generates a new structural compound with extremely low yield and/or no yield when being cultured independently.
In one aspect of the present invention, there is provided a method for co-culturing epicoccum nigrum with a fungus of the genus aspergillus, said co-culturing method allowing aspergillus to produce secondary metabolites that are produced in very low yields and/or are not produced when cultured alone; the method comprises the following steps:
a) respectively inoculating the epicoccum nigrum and the aspergillus fungi to an activation culture medium for strain activation;
b) inoculating the activated epicoccum nigrum and the aspergillus fungi into a solid culture medium of the same culture container for standing culture; or inoculating the activated epicoccum nigrum and the aspergillus fungi into a liquid culture medium of the same culture vessel for shaking culture;
c) when spores of the Aspergillus fungus in the solid medium become yellow, the Aspergillus fungus is collected and the medium is used to obtain a product.
In the above co-culture method, the Epicoccum nigrum is Epicoccum nigrum YL001, which is deposited in the common microorganism center of the China Committee for culture Collection of microorganisms at the address of: west road No.1, north chen, chaoyang district, beijing, zip code 10010, the accession number: CGMCC No.11397, preservation date of 2015, 10 months and 16 days.
The co-cultivation method as described above, wherein the Aspergillus fungus comprises one of Aspergillus nidulans (Aspergillus nidulans), Aspergillus terreus, and Aspergillus oryzae (Aspergillus oryzae) (all commercially available or commercially available from a strain collection agency such as the general microbiological culture Collection center of China Committee for culture Collection of microorganisms).
In the co-culture method, the solid culture medium in the step b) is a solid fungus culture medium, preferably a PDA culture medium, and the liquid culture medium is a liquid fungus culture medium, preferably a PDB culture medium.
In the co-culture method, the temperature of the standing culture in the step b) is 25-28 ℃; the shaking culture temperature is 25-28 ℃, and the speed is 100-250 rpm/min.
In the co-culture method, the activated culture medium of the epicoccum nigrum is a PDA solid culture medium, and the activated culture medium of the aspergillus fungi is a GMM culture medium.
The co-culture method described above, wherein the PDA solid medium comprises per liter: 37g of PDA culture medium powder finished product, the balance of distilled water and natural pH value; the GMM solid medium comprises per liter: 10g of glucose, 50mL of nitrate mother liquor, 1mL of trace element mother liquor, 1g of yeast extract, 15-20g of agar and the balance of distilled water, wherein the pH value is 6.5.
The co-culture method as described above, wherein the nitrate mother liquor comprises per liter: NaNO3120g，KCl 10.4g，MgSO4·7H2O 10.4g，K2HPO4·3H230.4g of O, and the balance of distilled water.
In the above co-culture method, each 100mL of the microelement mother liquor comprises ZnSO4·7H2O 2.2g，H3BO31.1g，MnCl2·4H2O 0.5g，FeSO4·7H2O 0.16g，CoCl2·5H2O 0.16g，CuSO4·5H2O 0.16g，(NH4)6Mo7O24·4H2O 0.11g，Na2EDTA 5.0g, the balance being distilled water.
In another aspect of the present invention, there is provided a compound obtained by co-culturing the above-mentioned Epicoccum nigrum and Aspergillus nidulans in PDA medium, wherein the compound has a general formula shown in formula 1:
in formula 1, R is CHO or
In still another aspect of the present invention, there is provided a compound obtained by co-culturing the aforementioned Epicoccum nigrum and Aspergillus terreus in PDA medium, wherein the chemical formula of the compound Fallacino is shown in formula 2.
In still another aspect of the present invention, there is provided a compound obtained by co-culturing epicoccum nigrum and aspergillus oryzae in PDA medium as described above, wherein the chemical formula of the compound Parasperone a is shown in formula 3.
In still another aspect of the present invention, there is provided a compound obtained by co-culturing the above-mentioned Epicoccum nigrum and Aspergillus nidulans in a PDB medium, wherein the compound has a formula shown in formula 4:
in the formula 4, the first step is,
or
Or
The invention utilizes the coculture of the epicoccum nigrum and aspergillus nidulans, aspergillus terreus and aspergillus oryzae of aspergillus, takes the spore discoloration of aspergillus as an indication to regulate or activate the expression of related gene clusters of related secondary metabolites in the aspergillus, thereby leading to the increase of the yield of related compounds or the generation of new compounds. This provides new methods for the study of strategies for co-cultivation between filamentous fungi to increase their natural product yield or to activate expression of "silent" gene clusters in fungi and to discover new natural products.
Drawings
FIG. 1 is a graph showing the growth of coculture of Epicoccum nigrum and Aspergillus nidulans on PDA medium in example 3 of the present invention;
FIG. 2 is a graph showing the growth of coculture of Epicoccum nigrum and Aspergillus terreus on PDA medium in example 3 of the present invention;
FIG. 3 is a graph showing the growth of coculture of Epicoccum nigrum and Aspergillus oryzae on PDA medium in example 3 of the present invention;
FIG. 4 shows the HPLC analysis of the compounds of example 5 of the present invention when Epicoccum nigrum and Aspergillus nidulans are co-cultured on PDA medium;
FIG. 5 shows the HPLC analysis of the compounds of example 6 of the present invention when Epicoccum nigrum and Aspergillus terreus were co-cultured in PDA medium;
FIG. 6 shows HPLC assay detection of compounds from Epicoccum nigrum and Aspergillus oryzae cocultured on PDA medium in accordance with example 7 of the present invention;
FIG. 7 shows HPLC analysis detection of co-culture compound of Epicoccum nigrum and Aspergillus nidulans liquid in PDB medium.
In the part marked a in the figure, the aspergillus spores have turned yellow.
Detailed Description
The following detailed description of the present invention, taken in conjunction with the accompanying drawings and examples, is provided to enable the invention and its various aspects and advantages to be better understood. However, the specific embodiments and examples described below are for illustrative purposes only and are not limiting of the invention.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1
The epicoccum nigrum strain activation culture medium used in the invention is a PDA culture medium, and the high-pressure moist heat sterilization is carried out for 20-30 minutes at 121 ℃. Pouring into a culture dish of 90mm, inoculating Epicoccum nigrum mycelium, standing at 25 deg.C for 3-7 days, and storing at 4 deg.C for culture inoculation.
Example 2
The aspergillus activation culture medium used by the invention is a GMM culture medium, and each 1L of the solid culture medium is prepared according to the following method: 10g of glucose, 50mL of nitrate mother liquor, 1mL of trace element mother liquor, 1g of yeast extract and 15-20g of agar, wherein the components are sequentially added, and finally distilled water is added to the mixture until the final volume is 1L, the pH value is adjusted to 6.5, and the mixture is sterilized by moist heat under high pressure at 121 ℃ for 20-30 minutes. Pouring into a 90mm culture dish, streaking, and standing at 37 deg.C for 3-4 days. Spores were collected in sterile water, collected on each plate, made into a spore suspension of 2mL final volume, and stored at 4 ℃ for culture inoculation.
Wherein, the preparation method (1L) of the nitrate mother liquor is as follows: sodium nitrate NaNO3120g, potassium chloride KCl 10.4g, magnesium sulfate MgSO4·7H2O10.4 g, dipotassium hydrogen phosphate K2HPO4·3H2O30.4 g, adding the above components in sequence, adding distilled water to a final volume of 1L, sterilizing at 121 deg.C for 20-30 min, and storing at room temperature;
the preparation method of the microelement mother liquor (100mL) comprises the following steps: zinc sulfate ZnSO4·7H2O2.2 g, boric acid H3BO31.1g of manganese chloride MnCl2·4H20.5g of O, ferrous sulfate FeSO4·7H2O0.16 g, CoCl2·5H20.16g of O, copper sulfate CuSO4·5H2O0.16 g, ammonium molybdate (NH)4)6Mo7O24·4H2O0.11 g, EDTA sodium salt Na2EDTA 5.0g, adding above components sequentially, adding distilled water to final volume of 100mL, sterilizing at 121 deg.C under high pressure and humid heat for 20-30 min, and storing at room temperature.
Example 3
The method for co-culturing the epiphyte nigrococcus and the aspergillus comprises the following steps: the epiphytic coccus nigricans, the aspergillus nidulans, the aspergillus terreus and the aspergillus oryzae are respectively inoculated on the same culture dish poured with the PDA culture medium at the same time for co-culture. The culture temperature is 25-28 ℃, and standing culture is carried out; after culturing for a period of time, the growth of aspergillus on one side close to the epicoccum nigrum is obviously inhibited, and aspergillus spores obviously change color; the culture time is 4 to 7 days.
Example 4
The method for co-culturing the epicoccum nigrum and the aspergillus is characterized in that the epicoccum nigrum hyphae and the aspergillus nidulans spores are simultaneously inoculated into the same triangular flask filled with a PDB liquid culture medium for co-culture. The culture temperature is 25-28 ℃, the shaking table is oscillated at 100-250 rpm/min, and the culture time is 4-7 days.
Example 5
HPLC analysis of cocultured compounds of epicoccum nigrum and aspergillus nidulans: the co-cultures were analyzed for changes in compounds by HPLC. HPLC analysis was performed under the following conditions: the device comprises the following steps: waters e2695 system; a detector: waters 2998(200-600 nm); a chromatographic column: 38020-41COSMOSIL5C18-MS-II Packed Column, 4.6mm I.D.x250mm; elution speed: 1mL/min, eluent: acid water (0.1% formic acid) and acetonitrile (0.1% formic acid) increased the acetonitrile content from 20% acetonitrile/acid water to 100% acetonitrile within 20 min.
The compound extraction is carried out by co-culturing the epiphytic and the aspergillus nidulans for 4 to 7 days to obtain hyphae and a culture medium. 1 plate of the culture was divided into small pieces and placed in a triangular flask, and 20-40mL of MEA (ethyl acetate: methanol: glacial acetic acid: 89:10:1) was added for ultrasonic extraction (ultrasonic frequency 80Hz, time 1 hour); collecting extractive solution, and performing rotary evaporation (temperature 30 deg.C, rotation speed 100rpm, vacuum degree less than 3 mmHg); evaporating to dryness, dissolving with 0.30-0.60mL methanol, diluting with appropriate concentration, injecting 20-40ul, washing column with 20-100% methanol, and performing HPLC analysis. Formula 1 was detected at the peaks of retention times 22.697min and 23.389 min; formula 4 was detected at the peak at retention times from 20min to 25 min. This compound was not detected in the extracts from epicoccum nigrum cultured alone for 4 to 7 days, whereas a trace amount or no compound was detected in the extracts from aspergillus nidulans cultured alone for 4 to 7 days.
Example 6
HPLC analysis of epicoccum nigricans and aspergillus terreus co-cultured compounds: changes in the compounds in the co-cultures were investigated by HPLC. HPLC analysis was performed under the following conditions: the device comprises the following steps: waters e2695 system detector: waters 2998(200-600 nm); a chromatographic column: 38020-41COSMOSIL5C18-MS-II Packed Column, 4.6mm I.D.x250 mm; elution speed: 1mL/min, eluent: acid water (0.1% formic acid) and methanol (0.1% formic acid) increased the methanol content from 20% methanol/acid water to 100% methanol within 20 min.
The compound extraction is carried out on hyphae and a culture medium obtained by co-culturing the epicoccum nigrum and the aspergillus terreus for 4 to 7 days. 1 plate of the culture was divided into small pieces and placed in a triangular flask, and 20-40mL of MEA (ethyl acetate: methanol: glacial acetic acid: 89:10:1) was added for ultrasonic extraction (ultrasonic frequency 80Hz, time 1 hour); collecting extractive solution, and performing rotary evaporation (temperature 30 deg.C, rotation speed 100rpm, vacuum degree less than 3 mmHg); evaporating to dryness, dissolving with 0.30-0.60mL methanol, diluting with appropriate concentration, injecting 20-40ul, washing column with 20-100% methanol, and performing HPLC analysis. Equation 2 is detected at the peak of retention time 18.072. Meanwhile, such compounds were not detected in the extracts obtained by culturing Epicoccum nigrum or Aspergillus terreus alone for 4 to 7 days.
Example 7
HPLC analysis of cocultured compounds of epicoccum nigrum with aspergillus oryzae: changes in the compounds in the co-cultures were investigated by HPLC. HPLC was performed under the following conditions: the device comprises the following steps: waters e2695 system detector: waters 2998(200-600 nm); a chromatographic column: 38020-41COSMOSIL5C18-MS-II Packed Column, 4.6mm I.D.x250 mm; elution speed: 1mL/min, eluent: acid water (0.1% formic acid) and methanol (0.1% formic acid) increased the methanol content from 20% methanol/acid water to 100% methanol within 20 min; .
The compound extraction was performed on the mycelia and the medium obtained by co-culturing Epicoccum nigrum and Aspergillus oryzae for 4 to 7 days. 1 plate of the culture was divided into small pieces and placed in a triangular flask, and 20-40mL of MEA (ethyl acetate: methanol: glacial acetic acid: 89:10:1) was added for ultrasonic extraction (ultrasonic frequency 80Hz, time 1 hour); collecting extractive solution, and performing rotary evaporation (temperature 30 deg.C, rotation speed 100rpm, vacuum degree less than 3 mmHg); evaporating to dryness, dissolving with 0.30-0.60mL methanol, diluting with appropriate concentration, injecting 20-40ul, washing column with 20-100% methanol, and performing HPLC analysis. Equation 3 is detected at the peak of retention time 15.226. Meanwhile, no such compound was detected in the culture broth obtained by culturing epicoccum nigrum or aspergillus oryzae alone for 4 to 7 days.
Example 8
Separation and purification of coculture compound of epicoccum nigrum and aspergillus nidulans: organic compounds were extracted from the culture media obtained in examples 3 and 4, the co-cultured strain culture media were separated 3 times using ethyl acetate, and the extract was dried under reduced pressure at 30 ℃ using a rotary vacuum evaporator, and then the resulting extract was subjected to silica gel column gradient elution, resulting in 7 fractions. The resulting fractions were analyzed by HPLC, and as a result, it was confirmed that the compounds of formula 1 and formula 4 were contained in fractions eluted with 100% dichloromethane. The fractions were prepared by semi-preparative liquid chromatography. The compound of formula 1 is obtained at retention times of 22.697min and 23.389min, respectively, and the compound of formula 4 is obtained at retention times of 20min to 25 min.
In formula 1, R is CHO or
In the formula 4, the first step is,
or
Or
Example 9
Separation and purification of the coculture compound of epicoccum nigrum and aspergillus terreus: organic compounds were extracted from the culture medium obtained in example 3, the co-cultured strain culture medium was separated 3 times using ethyl acetate, and the extract was dried under reduced pressure at 30 ℃ using a rotary vacuum evaporator, and then the resulting extract was subjected to silica gel column gradient elution, resulting in 7 fractions. The obtained fractions were analyzed by HPLC, and as a result, it was confirmed that in dichloromethane: methanol 100: 3 the eluted fraction contains formula 2. The components are prepared by glucose gel column chromatography and semi-preparative liquid chromatography respectively. The compound of formula 2 is obtained at a retention time of 15.397 min.
Example 10
Separation and purification of coculture compounds of epicoccum nigrum and aspergillus oryzae: the organic compound was extracted from the medium obtained in example 3, and the co-cultured strain medium was separated 3 times using ethyl acetate. The extract was dried under reduced pressure at 30 ℃ using a rotary vacuum evaporator, and the resulting extract was subjected to chromatography on glucose gel column, eluting with methanol, to give a fraction containing the compound of formula 3. The fractions obtained were analyzed by semi-preparative liquid chromatography and prepared under isocratic conditions of 70% methanol/acid water to obtain the compound represented by formula 3.
Finally, it should be noted that: it should be understood that the above examples are only for clearly illustrating the present invention and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications of the invention may be made without departing from the scope of the invention.
Claims (6)
1. A method for co-culturing Epicoccum nigrum (Epicoccum nigrum) with Aspergillus sp, wherein the co-culturing method allows Aspergillus to produce secondary metabolites that are produced in very low yields and/or not produced when cultured alone; the method comprises the following steps:
a) respectively inoculating the epicoccum nigrum and the aspergillus fungi to an activation culture medium for strain activation;
b) inoculating the activated epicoccum nigrum and the aspergillus fungi into a solid culture medium of the same culture container for standing culture; or inoculating the activated epicoccum nigrum and the aspergillus fungi into a liquid culture medium of the same culture vessel for shaking culture;
c) collecting the Aspergillus fungus and the culture medium to obtain a product when spores of the Aspergillus fungus in the solid culture medium turn yellow,
the general formula of the product is shown as formula 1 or formula 4:
in the formula 4, the first step is,
the Epicoccum nigrum is Epicoccum nigrum (Epicoccum nigrum) YL001, is preserved in China general microbiological culture collection center, and has the preservation number: CGMCC No.11397, wherein the Aspergillus fungus is selected from Aspergillus nidulans (Aspergillus nidulans), Aspergillus terreus (Aspergillus terreus) or Aspergillus oryzae (Aspergillus oryzae)One of (1) and (b).
2. The co-cultivation process according to claim 1, wherein the solid medium in step b) is a solid fungal medium; the liquid culture medium is a liquid fungus culture medium.
3. The co-culture method according to claim 2, wherein the solid medium in step b) is PDA medium; the liquid culture medium is a PDB culture medium.
4. The co-culture method according to claim 1, wherein the temperature of the static culture in the step b) is 25 to 28 ℃; the shaking culture temperature is 25-28 ℃, and the speed is 100-250 rpm/min.
5. The co-culture method according to claim 1, wherein the activated medium of epicoccum nigrum is PDA solid medium and the activated medium of aspergillus is GMM medium.
6. The co-culture method as claimed in claim 5, wherein said PDA solid medium comprises per liter: 37g of PDA culture medium powder finished product, the balance of distilled water and natural pH value; the GMM solid medium comprises per liter: 10g of glucose, 50mL of nitrate mother liquor, 1mL of trace element mother liquor, 1g of yeast extract, 15-20g of agar and the balance of distilled water, wherein the pH value is 6.5.
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