CN113684144A - Streptomyces ambaris intestinalis WA5-1-7 and application thereof - Google Patents
Streptomyces ambaris intestinalis WA5-1-7 and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of microorganisms, and particularly relates to a streptomyces entomogenous Wa5-1-7 of cockroach in intestinal tract and application thereof, wherein the strain WA5-1-7 is preserved in the Guangdong province collection center of microbial strains, and the preservation number is GDMCC NO. 61630; the endophyte is obtained by separating from periplaneta americana intestinal tracts, and the species of the endophyte is preliminarily identified through morphological observation, molecular biological identification, phylogenetic tree analysis and the like. The secondary metabolite of the strain WA5-1-7 provided by the invention has a strong inhibiting effect on methicillin-resistant staphylococcus aureus MRSA, staphylococcus aureus and candida albicans, has a certain antibacterial activity on klebsiella pneumoniae and pseudomonas aeruginosa, the content of actinomycin in a fermentation liquid crude extract is up to 80%, and the yields of actinomycin D and actinomycin X2 respectively reach 138.36mg/L and 227.21 mg/L.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a streptomyces entomogenes WA5-1-7 of cockroach in intestinal tract and application thereof.
Background
Actinomycetes are a resource treasure house for finding natural products and various active lead compounds. The number of streptomyces is a major part of actinomycetes, and about 80% or more of various antibiotics such as streptomycin are derived from streptomyces. However, in recent years, the hit rate of the candidate drugs of terrestrial and marine actinomycetes has been decreasing, which has led scientists to shift their attention to endophytic actinomycetes, which are in special niches and may become a natural bioactive material-rich resource. Insect endophytes are special microorganisms inhabiting insects, and no endophyte inhabiting insects are found at present, so huge microbial resources are accumulated in the insects.
American cockroaches (Periplaneta americana) are colloquially called "cockroaches", and belong to the order of the class entomophyes of the phylum arthropoda, the order blattaria of the subclass pteridophyes, and are one of the most tenacious and ancient groups of insects known to be of the earth's greatest vitality. It is recorded in Shen nong Ben Cao Jing, Ben Cao gang mu and Xin Xiu Ben Cao, etc., and has the effects of promoting blood circulation, removing blood stasis, invigorating spleen, eliminating infantile malnutrition, inducing diuresis, relieving swelling, promoting granulation, etc., with cold nature, salty taste, liver meridian tropism, spleen and kidney meridian. Modern researches show that the periplaneta americana has pharmacological actions of resisting bacteria, inflammation and tumors, repairing tissues, enhancing the immunity of organisms and the like. The periplaneta americana still has extremely strong vitality and reproductive capacity after living in a kitchen, a sewer, a garbage pile and other dark and humid environments with a large amount of pathogenic microorganisms, and the pharmacological action, the strong environmental adaptability and the capability of defending infection of external pathogenic bacteria of the periplaneta americana are prompted, and the periplaneta americana possibly has a close relationship with the abundant microbial flora in the intestinal tract.
Disclosure of Invention
The invention aims to provide a streptomyces endophyticus WA5-1-7 separated and screened from periplaneta americana intestinal tracts and application thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
the first purpose of the invention is to provide a streptomyces entomogenes WA5-1-7 in intestinal tract of insect blattaria, wherein the streptomyces WA5-1-7 is an endophyte obtained by separating and screening from the intestinal tract of periplaneta americana, is preserved in the microbial strain preservation center of Guangdong province, and has the address of China, Guangdong and Guangdong institute for microbiology, the preserved strain name is Streptomyces sp.WA5-1-7, the taxonomic name is Streptomyces sp, the preservation number is GDMCC NO.61630, the preservation date is 2021, 4, 26 days, and the preservation life is 30 years.
Another object of the present application is to provide a method for identifying Streptomyces WA5-1-7 as described in the above protocol, comprising the following steps:
(1) 159 actinomycetes are separated from periplaneta americana intestinal tracts, 15 strains with good candida albicans resistant activity are screened out, and a strain WA5-1-7 with good antibacterial property is further screened out;
(2) performing morphological observation, molecular biological identification and phylogenetic tree analysis on the bacterial strain WA5-1-7 with good antibacterial property selected in the step (1) to perform biological classification identification.
Preferably, the morphological observation in the step (2) is to observe the growth morphology of the strain WA5-1-7 on a Gauss No.1 culture medium, observe the morphology of the strain WA5-1-7 by a gram stain method, and observe the morphology of the strain WA5-1-7 by a scanning electron microscope.
Preferably, the molecular biological identification and phylogenetic tree analysis process in step (2) is as follows:
s1, extracting DNA of the strain WA5-1-7, selecting a bacterial 16S rRNA universal primer 27F and a primer 1492R to perform PCR amplification on the extracted DNA to obtain a PCR amplification product;
s2, carrying out agarose gel electrophoresis detection on the PCR amplification product obtained in the step S1, sequencing, submitting a sequence obtained by sequencing to NCBI for BLAST homology comparison, and simultaneously submitting sequence information to a GenBank database to obtain a login number of a strain WA 5-1-716S rRNA;
s3, performing BLAST homology comparison on the 16S rRNA sequence of the strain WA5-1-7 and a database sequence in NCBI, selecting representative strains in different published species, performing phylogenetic tree analysis on the multi-sequence comparison result by using an adjacency method in MEGA5.0 software, and judging the species to which the strain WA5-1-7 belongs.
Preferably, the sequence information of the primer 27F in step S1 is shown in SEQ ID NO.1, and the sequence information of the primer 1492R is shown in SEQ ID NO. 2.
27F：5′-AGAGTTTGATCCTGGCTCAG-3′(SEQ ID NO.1)；
1492R：5′-TACGGCTACCTTGTTACGACTT-3′(SEQ ID NO.2)。
Preferably, the sequence information of 16S rRNA obtained by sequencing WA 5-1-716S rRNA of the strain in the step S2 is shown as SEQ ID NO. 3.
GCTTGTCCGACGTTGCTTACCATGCGAGTCGTAACATGGTAGCCGTAA GGTGGTGGATTAGTGGCGAACGGGTGAGTAACACGTGGGCAATCTGCCCT TCACTCTGGGACAAGCCCTGGAAACGGGGTCTAATACCGGATAACACTCT GTCCCGCATGGGACGGGGTTGAAAGCTCCGGCGGTGAAGGATGAGCCCGC GGCCTATCAGCTTGTTGGTGGGGTAATGGCCTACCAAGGCGACGACGGGT AGCCGGCCTGAGAGGGCGACCGGCCACACTGGGACTGAGACACGGCCCA GACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGAAAGCC TGATGCAGCGACGCCGCGTGAGGGATGACGGCCTTCGGGTTGTAAACCTC TTTCAGCAGGGAAGAAGCGAAAGTGACGGTACCTGCAGAAGAAGCGCCG GCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGCGCAAGCGTTGTC CGGAATTATTGGGCGTAAAGAGCTCGTAGGCGGCTTGTCACGTCGGATGTG AAAGCCCGGGGCTTAACCCCGGGTCTGCATTCGATACGGGCTAGCTAGAGT GTGGTAGGGGAGATCGGAATTCCTGGTGTAGCGGTGAAATGCGCAGATATC AGGAGGAACACCGGTGGCGAAGGCGGATCTCTGGGCCATTACTGACGCTG AGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCAC GCCGTAAACGTTGGGAACTAGGTGTTGGCGACATTCCACGTCGTCGGTGC CGCAGCTAACGCATTAAGTTCCCCGCCTGGGGAGTACGGCCGCAAGGCTA AAACTCAAAGGAATTGACGGGGGCCCGCACAAGCAGCGGAGCATGTGGC TTAATTCGACGCAACGCGAAGAACCTTACCAAGGCTTGACATATACCGGAA AGCATCAGAGATGGTGCCCCCCTTGTGGTCGGTATACAGGTGGTGCATGGC TGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGC AACCCTTGTTCTGTGTTGCCAGCATGCCCTTCGGGGTGATGGGGACTCACA GGAGACTGCCGGGGTCAACTCGGAGGAAGGTGGGGACGACGTCAAGTCA TCATGCCCCTTATGTCTTGGGCTGCACACGTGCTACAATGGCCGGTACAAT GAGCTGCGATGCCGCGAGGCGGAGCGAATCTCAAAAAGCCGGTCTCAGTT CGGATTGGGGTCTGCAACTCGACCCCATGAAGTCGGAGTTGCTAGTAATCG CAGATCAGCATTGCTGCGGTGAATACGTTCCCGGGCCTTGTCACACCGCCC GTCTCGTCTACGTAATAG(SEQ ID NO.3)。
Preferably, the strain WA5-1-7 is analyzed in step S3 to belong to the genus Streptomyces, and the homology reaches 99%.
The third purpose of the invention is to provide the application of the secondary metabolite of the streptomyces WA5-1-7 in preparing a bacteriostatic composition.
Preferably, the antibacterial composition can strongly inhibit the growth of methicillin-resistant staphylococcus aureus MRSA, staphylococcus aureus, candida albicans, klebsiella pneumoniae, pseudomonas aeruginosa.
159 actinomycetes which are separated from periplaneta americana intestinal tracts in the previous period are extracted by the inventor, 15 actinomycetes in the actinomycetes have good candida albicans activity, and a bacterial strain WA5-1-7 with good candida albicans activity is selected. The strain WA5-1-7 is biologically classified by methods such as morphological observation, molecular biological identification, phylogenetic tree analysis and the like, and the strain is preliminarily determined to be Streptomyces (Streptomyces sp) with the preservation number of GDMCC NO. 61630. This suspension was inoculated at 5% inoculum size into a 500mL Erlenmeyer flask containing 300mL of pH7.2 ISP2 fermentation medium and fermented in a liquid shake flask at 28 ℃ at 160 rpm. Bacteriostatic experiments carried out by an Oxford cup method show that the fermentation supernatant and the crude extract of the fermentation liquid ethyl acetate have strong inhibitory action on methicillin-resistant Staphylococcus aureus MRSA, Staphylococcus aureus and Candida albicans, and have certain bacteriostatic activity on Klebsiella pneumoniae and Pseudomonas aeruginosa; and finally, detecting the actinomycin content in the crude fermentation extract of the strain by a high performance liquid external standard method, wherein the actinomycin content in the crude fermentation extract is up to 80%, and the yields of actinomycin D and actinomycin X2 respectively reach 138.36mg/L and 227.21 mg/L.
Compared with the prior art, the invention has the following beneficial effects:
the intestinal endogenous streptomyces blattaria provided by the invention has strong antibacterial activity of secondary metabolites, has strong inhibiting effect on methicillin-resistant staphylococcus aureus MRSA, staphylococcus aureus and candida albicans, and has certain antibacterial activity on klebsiella pneumoniae and pseudomonas aeruginosa; the actinomycin content and the yield are high, the actinomycin content in the fermentation crude extract is up to 80 percent, the actinomycin D and actinomycin X2 yields respectively reach 138.36mg/L and 227.21mg/L, and the method has wide development space and good development and application prospects.
Drawings
FIG. 1 is a diagram showing the growth of the strain WA5-1-7 on a Gao's number 1 plate;
FIG. 2 is a morphological diagram of strain WA5-1-7 under gram stain under oil mirror 100 x;
FIG. 3 is a morphology diagram of strain WA5-1-7 under a scanning electron microscope of 2 kx;
FIG. 4 is a morphology diagram of strain WA5-1-7 under a scanning electron microscope of 10 kx;
FIG. 5 is an agarose gel electrophoresis of the 16S rRNA amplification product of strain WA 5-1-7;
FIG. 6 is a drawing showing the construction of a phylogenetic tree of 16S rRNA amplification products of the strain WA 5-1-7;
FIG. 7 is a graph of anti-MRSA activity of fermentation broths on different days of fermentation;
FIG. 8 is a graph showing statistics of MRSA activity of fermentation broths on different days of fermentation;
FIG. 9 is a graph showing the analysis of the antibiogram of a crude extract of a fermentation broth;
FIG. 10 is a graph showing the results of chromatographic analysis of crude extract of the supernatant of WA5-1-7 fermentation broth.
Detailed Description
The present invention will be described in more detail with reference to the following examples. It should not be understood that the scope of the above-described subject matter of the present invention is limited to the following examples.
The gram staining kit is purchased from Guangdong Huanji microbial technology GmbH, and has the model of 029010; the Ezup column type bacterial genome DNA extraction kit is purchased from Shanghai Biotechnology limited company with the model number of NO. B518255; the actinomycin D and the actinomycin X2 are separated, purified and identified from the subject group; the ampicillin is purchased from Guangzhou Qiyun biotechnology limited, and the model is K0020; the amphotericin B is purchased from Shanghai Allantin Biotechnology Co., Ltd, and has a model number of A105482-250 mg; the ciprofloxacin is purchased from Shanghai Merlin Biotechnology Co., Ltd, and has the model of C824343-25 g; the YMC-Pack ODS-AQ (4.6X 250mm) column was purchased from the Japan YMC corporation, and has a model number of AQ12S05-2546 WT; the methanol, the ethyl acetate, the n-butanol, the dichloromethane and the absolute ethanol are purchased from Guangdong Guanghua science and technology GmbH and are analytically pure; the reagents or biological materials are commercially available, unless otherwise specified.
EXAMPLE 1 morphological identification of Strain WA5-1-7
1.1 growth morphology of Strain WA5-1-7 on Gao's No.1 Medium
Finding out a seed-preserving tube of the strain WA5-1-7 from a refrigerator fungus library stored at the temperature of-20 ℃, sucking 100 mu L of bacterial liquid to a plate of Gaoshi No.1 agar, uniformly coating a glass rod, performing the operation in an ultra-clean bench, placing the plate in a constant-temperature incubator, performing inverted culture at the temperature of 28 ℃ for 5d, after the colony state of the strain WA5-1-7 in the plate is stable, selecting spores of a single colony by using an inoculating loop which is sterilized by flame, performing partition streaking and inoculating to the plate of the Gaoshi No.1 agar, performing inverted culture in the incubator at the temperature of 28 ℃ for 5d, sealing by a sealing film, and placing the plate in a refrigerator at the temperature of 4 ℃ for later use. Inoculating strain WA5-1-7 in Gao's No.1 plate by smear method, culturing in 28 deg.C incubator for 5d, and observing growth form of colony and generation of aerial hypha, substrate hypha and soluble pigment after spore of strain is mature;
the preparation method of the Gauss No.1 agar comprises the following steps: dissolving 20.0g of soluble starch, 0.5g of sodium chloride, 0.05g of dipotassium phosphate, 0.01g of ferrous sulfate, 0.05g of magnesium sulfate, 1.0g of potassium nitrate and 15.0g of agar in distilled water, fixing the volume to 1000mL, adjusting the pH value to 7.3, subpackaging, and sterilizing for 20min at 121 ℃ by high-pressure steam.
The experimental results are as follows: the bacterial strain WA5-1-7 is inoculated on a Gao's No.1 plate, cultured in a constant temperature incubator at 28 ℃ for 5d and observed colony morphology characteristics, as shown in figure 1, the bacterial strain grows well on the Gao's No.1 plate, the colony is circular, is dry and non-transparent, presents an inner circular ring and an outer circular ring, hyphae extend to the periphery, hyphae in the base are light yellow, and aerial hyphae are white.
1.2 gram staining method for observing the form of the Strain WA5-1-7
Inoculating loop sterilized by alcohol lamp flame selecting monoclonal thallus from Gao's No.1 plate and placing in sterile dH containing 0.5mL2And (3) resuspending in a centrifugal tube of O, sucking 1 drop of bacteria liquid, dripping the bacteria liquid onto a clean glass slide, baking the bacteria liquid on an alcohol lamp, performing gram staining according to the instruction of a gram staining kit, and then placing the bacteria liquid under an optical microscope for observation. Firstly, finding a visual field area of a sample under a low-power lens, using a coarse focusing screw to heighten an objective lens, dripping 1-2 drops of cedar oil on the sample area, switching the objective lens to 100 times (oil lens), raising a condenser to the highest position and opening an aperture to the maximum, slowly lowering a lens barrel under horizontal watching on the side, immersing the oil lens in oil but not crushing a glass slide, slowly adjusting a fine adjuster until an object image appears in the visual field and the focus of bacteria is clear, and observing and recording the color and morphological characteristics of the strains.
As shown in FIG. 2, the color of the test sample WAs purple when subjected to gram staining, and the strain WA5-1-7 WAs determined to be a gram-positive bacterium.
1.3 scanning Electron microscope Observation of the morphology of Strain WA5-1-7
Preparation of 2.5% glutaraldehyde solution: weighing Na2HPO4·12H21.64g of O7 crystal and deionized water are dissolved and the volume is constant to 1L, and the solution A is obtained. 31.21g NaH was weighed2PO42H2O, dissolving in deionized water and making the volume to 1L to obtain solution B. 50mL of 50% glutaraldehyde is added into 0.2moL of phosphate buffer solution (prepared by 360mL of solution A and 140mL of solution B, and the pH value is 7.2), deionized water is added to the solution to reach 1L, and 20.0g of activated carbon is added. Sealing and protecting from light, and placing in a refrigerator at 4 deg.C for use.
Placing 1.0mL of the prepared 2.5% glutaraldehyde solution in 1.5mL of an autoclaved EP tube, adding an inoculating loop into a strain WA5-1-7 thallus growing on a Gaoshi No.1 flat plate, placing the mixture in a refrigerator at 4 ℃, fixing for about 12h, centrifuging at 2000rpm for 5min, and removing the supernatant; then adding 1.0mL of Phosphate Buffer Solution (PBS) for repeated blowing, centrifuging at 2000rpm for 5min, discarding the supernatant, repeating for 3 times, each time for 10 min; performing gradient dehydration with ethanol (30%, 50%, 70%, 80%, 90%, 100%) of different concentrations, centrifuging under the same conditions, and discarding supernatant, wherein each dehydration is performed for 6 min; then, dripping the sample on a glass slide, and naturally drying at room temperature; finally, the processed sample is placed in an ion sputtering instrument (Hitachi MC1000) to spray a gold bond film, and then a scanning electron microscope (Hitachi S-3400N) is used for observing the characteristics of hyphae and the shape of spores. The experimental results are shown in fig. 3 and 4, and the scanning electron microscope results show that the intrabasal hyphae and the aerial hyphae of the strain are rich, and the spores fall off in a columnar shape after being mature, so that the morphological characteristics of the strain are basically consistent with those of streptomyces.
Example 2 molecular biological identification of Strain WA5-1-7
2.116S rRNA gene sequence extraction and alignment
Extracting DNA of the strain WA5-1-7 by using an Ezup column type bacterial genome DNA extraction kit, selecting a bacterial universal primer 27F and a primer 1492R to perform PCR amplification on the extracted gene sequence, wherein the primer information is shown as SEQ ID NO.1 and SEQ ID NO. 2;
the PCR reaction system is as follows:
TABLE 1 PCR reaction System
| Reagent | Volume (μ L) |
| Primer STAR Max Premix(2×) | 12.5 |
| 100 μ M Forward primer 27F | 1.0 |
| 100 mu M downstream primer 1492R | 1.0 |
| DNA template | 1.5 |
| Sterilized water | 9.0 |
Amplification conditions: preheating at 94 deg.C for 3min, denaturing at 95 deg.C for 30s, annealing at 55 deg.C for 30s, extending at 72 deg.C for 1min for 35 cycles, extending at 72 deg.C for 10min, and storing at 4 deg.C).
Detecting the PCR amplification product by 1% agarose gel electrophoresis (constant voltage 120V, 15min), wherein a bright band appears at 1000-2000 bp, which accords with the characteristics of actinomycetes, sending the product to Huada gene company for sequencing as shown in figure 5, submitting the sequence obtained by sequencing to NCBI for BLAST homology comparison, and simultaneously submitting sequence information to GenBank database to obtain the accession number MN759381 of WA 5-1-716S rRNA.
2.216S rRNA phylogenetic tree analysis
BLAST homologous comparison is carried out on the 16S rRNA sequence of the strain WA5-1-7 and a database sequence in NCBI, representative strains in different published species are selected, a phylogenetic tree analysis is carried out on the multi-sequence comparison result by using a Neighbor-Joining method (Neighbor-Joining) in MEGA5.0 software, and the species to which the strain WA5-1-7 belongs is judged.
Homology comparison and phylogenetic tree analysis of the strain WA5-1-7 resulted:
sequencing a PCR amplification product of the strain WA5-1-7 into a 1371bp 16S rRNA sequence, uploading the sequence to a GenBank database for BLAST homology comparison, selecting a representative strain of different species, performing phylogenetic tree analysis by multi-sequence comparison as shown in FIG. 6, wherein the result shows that the strain is in the same branch with Streptomyces sp.CP010833.1, the homology reaches 99%, and the strain WA5-1-7 is identified as Streptomyces sp. The strain sequence is submitted to a GenBank database of NCBI to obtain a strain WA5-1-7 sequence registration number MN759381, and the 16S rRNA gene sequence of the strain WA5-1-7 is shown as SEQ ID NO.3 after sequencing.
EXAMPLE 3 fermentation of strains and determination of anti-MRSA Activity of fermentation broths on different days
Inoculating a single colony on a Gaoshi No.1 plate into an ISP-1 seed liquid culture medium containing 50mL, placing the mixture in a constant-temperature shaking table at 28 ℃ and 160rpm for culturing for 2-3 days until the seed liquid is turbid, inoculating 5% of the inoculation amount into a 500mL conical flask filled with 300mL of the ISP-2 culture medium, placing the conical flask in the constant-temperature shaking table at 28 ℃ and 160rpm for fermenting, taking 1mL of fermentation liquid every other day from the 4 th day of fermentation, and storing the fermentation liquid in a refrigerator at 4 ℃ for later use. The anti-MRSA activity of the fermentation broth was measured using the oxford cup method: the obtained fermentation broth sample was centrifuged at 12000rpm for 5min for use. Thawing the TSA culture medium sterilized by high pressure steam in a microwave oven, adding appropriate amount of fresh culture medium with final concentration of 4 × 10 when the temperature of the culture medium is reduced to 43 deg.C6And (3) uniformly mixing the CFU/mL MRSA bacterial solution, pouring a proper amount of uniformly mixed culture medium into a culture dish by adopting a double-layer plate-pouring method, placing an oxford cup on the culture medium until the culture medium is solidified, adding 200 mu L of fermentation supernatant into the oxford cup, placing the well-placed flat plate into a constant-temperature incubator at 37 ℃ for culturing for 12 hours, and observing and recording the result. In which each strain was replicated 3 times, 128. mu.g/mL actinomycin D dissolved in methanol, 128. mu.g/mL actinomycin X2 were used as positive controls, and methanol was used as negative control.
The preparation process of the seed liquid culture medium comprises the following steps: dissolving glucose 10.0g, yeast extract 2.0g, and tryptone 12.0g in distilled water, diluting to 1000mL, adjusting pH to 7.3, packaging, and sterilizing with high pressure steam at 121 deg.C for 20 min;
the ISP-2 culture medium is prepared by dissolving glucose 4.0g, yeast extract 4.0g, and maltose extract 10.0g in distilled water, diluting to 1000mL, adjusting pH to 7.3, packaging, and sterilizing with high pressure steam at 121 deg.C for 20 min;
the preparation process of the soybean casein agar (TSA) culture medium comprises the following steps: dissolving pancreatin digest of casein 15.0g, papain digest of soybean powder 5.0g, sodium chloride 5.0g, and agar 15.0g in distilled water, diluting to 1000mL, adjusting pH to 7.3, packaging, and sterilizing with high pressure steam at 121 deg.C for 20 min.
Results of anti-MRSA activity of fermentation broths on different days:
as shown in FIG. 7 and FIG. 8, wherein 1 in FIG. 7 is actinomycin D, 2 is actinomycin X2, 3 is a negative control methanol solvent, and 4 is WA 5-1-7; the strain WA5-1-7 has strong anti-MRSA activity in supernatant after fermenting for 4 days, the anti-MRSA activity of the supernatant of fermentation liquor reaches a peak at the 10 th day of fermentation, and the anti-MRSA activity falls back after the fermentation time is prolonged.
EXAMPLE 4 preparation of fermentation broth by crude extraction and analysis of antibiogram
Fermenting the strain for 15 days, centrifuging the fermentation liquor in a high-speed refrigerated centrifuge at the rotating speed of 8000rpm for 15min, collecting 250mL of fermentation liquor supernatant, and mixing the fermentation liquor supernatant with ethyl acetate according to the volume ratio of 1: 1, repeatedly extracting for 3 times, evaporating and concentrating an extract phase to dryness at 40 ℃ by using a rotary evaporator, adding a small amount of methanol into a rotary evaporation bottle, transferring into a penicillin bottle with gram weight, placing into a constant-temperature drying oven at 40 ℃ to volatilize excessive methanol, finally obtaining a solid crude extract of a fermentation liquid, and weighing for later use.
Dissolving a certain amount of crude extract in methanol to prepare 1mg/mL methanol solution of the crude extract. The antibacterial activity of the crude extract was tested using the oxford cup method: melting TSA culture medium and PDA culture medium sterilized by high pressure steam in microwave oven, adding fresh culture medium at 5% volume ratio until the culture medium temperature is reduced to 43 deg.C to obtain final concentration of 4 × 106CFU/mL, wherein the culture medium PDA is used for Candida albicans (Candida albicans ATCC 10231), methicillin-resistant Staphylococcus aureus (MRSA, ATCC 25213), Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), Klebsiella pneumoniae (ATCC 13883), etcAnd (3) using a TSA culture medium, uniformly mixing the culture medium and the bacterial liquid, pouring a proper amount of uniformly mixed culture medium into a culture dish by adopting a double-layer plate inversion method, placing an oxford cup on the culture medium after the culture medium is solidified, adding 100 mu L of crude extract methanol solution into the culture medium, placing a sample-added TSA culture medium flat plate into a constant-temperature incubator at 37 ℃ for culturing for 12 hours, placing a PDA culture medium flat plate into a constant-temperature incubator at 28 ℃ for culturing for 36 hours, and observing and recording results. Wherein each strain was subjected to 3 replicates, MRSA resistance was performed using 128. mu.g/mL actinomycin D dissolved in methanol as a positive control, Staphylococcus aureus resistance and Escherichia coli resistance were performed using 64. mu.g/mL ampicillin as a positive control, Candida albicans resistance was performed using 64. mu.g/mL amphotericin B as a positive control, Pseudomonas aeruginosa resistance and Klebsiella pneumoniae resistance were performed using 64. mu.g/mL ciprofloxacin as a positive control, and methanol as a negative control.
The preparation process of the PDA culture medium comprises the following steps: potato Dextrose Agar (PDA) medium: dissolving potato (powder extracted from potato) 300.0g, glucose 20.0g, agar 15.0g, and chloramphenicol 0.1g in distilled water, diluting to 1000mL, adjusting pH to 7.3, packaging, and sterilizing with high pressure steam at 121 deg.C for 20 min.
The analysis result of the antibiogram of the crude extract of the fermentation liquor is as follows:
the supernatant of the fermentation broth was dried and weighed to obtain 0.1145g of crude extract of the fermentation broth. The crude extract of the fermentation liquor is dissolved in methanol to prepare a methanol solution of the crude extract of 1mg/mL, and the methanol solution is further subjected to antibacterial spectrum analysis by an Oxford cup method, wherein the results of the antibacterial zone are shown in Table 2 and FIG. 9:
TABLE 2 statistical table of antibacterial spectrum of crude extract of fermentation broth (diameter of zone of inhibition mm, n ═ 3)
In FIG. 9, A is MRSA, B is Staphylococcus aureus, C is Escherichia coli, D is Candida albicans, E is Klebsiella pneumoniae, F is Pseudomonas aeruginosa, 1 is positive control, 2 is negative control methanol solvent, and 3 is WA 5-1-7; as shown in figure 9, the crude extract has strong inhibiting effect on methicillin-resistant staphylococcus aureus MRSA, staphylococcus aureus, escherichia coli and candida albicans, and has certain bacteriostatic activity on klebsiella pneumoniae and pseudomonas aeruginosa.
EXAMPLE 5 determination of the content of actinomycin in the crude extract
Determining the content of actinomycin in the crude extract by using an external standard method, wherein the chromatographic conditions are as follows: a YMC-Pack ODS-AQ (4.6X 250mm) column was used, the sample size was 10. mu.L, and the mobile phase was methanol: water (80: 20) and a detection wavelength of 254 nm. Accurately weighing control actinomycin D and actinomycin X2 to prepare standard methanol solutions with concentrations of 1.0, 0.5, 0.25, 0.1, 0.05, 0.025 and 0.01mg/mL, performing sample injection analysis according to the chromatographic conditions, repeating the sample injection for three times, recording peak areas, fitting a linear equation by taking the concentrations as horizontal coordinates and the peak areas as vertical coordinates, and obtaining an external standard working curve.
And (3) preparing the crude extract into 0.5mg/mL methanol solution, carrying out sample injection analysis according to the same chromatographic condition, repeatedly carrying out sample injection for three times, recording the peak area of actinomycin, and obtaining the content of actinomycin in the crude extract according to the obtained reference product external standard working curve.
Determination results of crude extract actinomycin content:
and (3) carrying out chromatographic separation on control actinomycin D, actinomycin X2 and crude extract methanol solutions with different concentrations to obtain peak areas of different solutions. The regression equation was obtained by fitting a straight line with the concentration of the control as the abscissa and the peak area as the ordinate, and the working curve of actinomycin D (y: 6766334.53X-424127.78, R2: 0.9980) and the working curve of actinomycin X2 (y: 1761112.43X-5741.18, R2: 0.9999) were obtained. Substituting the peak area of the crude extract into the working curve to obtain the content and percentage of actinomycin D and actinomycin X2 in the crude extract, and calculating the actinomycin yield according to the fermentation volume, wherein the results are shown in FIG. 10 and Table 2.
TABLE 2 determination of actinomycin content
The crude extract of the WA5-1-7 strain has the highest content of actinomycin X2, about 50 percent and the yield is 227.21 mg/L; the content of actinomycin D can reach 30 percent, and the yield is 138.36 mg/L.
The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.
Sequence listing
<110> university of Guangdong department of pharmacy
<120> Streptomyces blattaria intestinal endogenesis WA5-1-7 and application thereof
<130> 2021.7.19
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> Sequence information of primer 27F (Sequence information of primer 27F)
<400> 1
<210> 2
<211> 22
<212> DNA
<213> Sequence information of primer 1492R (Sequence information of primer 1492R)
<400> 2
tacggctacc ttgttacgac tt 22
<210> 3
<211> 1371
<212> DNA
<213> sequence information of 16S rRNA obtained by sequencing WA 5-1-716S rRNA of Strain WA5 (16S rRNA sequence information extracted from strain WA 5-1-716S rRNA sequence)
<400> 3
gcttgtccga cgttgcttac catgcgagtc gtaacatggt agccgtaagg tggtggatta 60
gtggcgaacg ggtgagtaac acgtgggcaa tctgcccttc actctgggac aagccctgga 120
aacggggtct aataccggat aacactctgt cccgcatggg acggggttga aagctccggc 180
ggtgaaggat gagcccgcgg cctatcagct tgttggtggg gtaatggcct accaaggcga 240
cgacgggtag ccggcctgag agggcgaccg gccacactgg gactgagaca cggcccagac 300
tcctacggga ggcagcagtg gggaatattg cacaatgggc gaaagcctga tgcagcgacg 360
ccgcgtgagg gatgacggcc ttcgggttgt aaacctcttt cagcagggaa gaagcgaaag 420
tgacggtacc tgcagaagaa gcgccggcta actacgtgcc agcagccgcg gtaatacgta 480
gggcgcaagc gttgtccgga attattgggc gtaaagagct cgtaggcggc ttgtcacgtc 540
ggatgtgaaa gcccggggct taaccccggg tctgcattcg atacgggcta gctagagtgt 600
ggtaggggag atcggaattc ctggtgtagc ggtgaaatgc gcagatatca ggaggaacac 660
cggtggcgaa ggcggatctc tgggccatta ctgacgctga ggagcgaaag cgtggggagc 720
gaacaggatt agataccctg gtagtccacg ccgtaaacgt tgggaactag gtgttggcga 780
cattccacgt cgtcggtgcc gcagctaacg cattaagttc cccgcctggg gagtacggcc 840
gcaaggctaa aactcaaagg aattgacggg ggcccgcaca agcagcggag catgtggctt 900
aattcgacgc aacgcgaaga accttaccaa ggcttgacat ataccggaaa gcatcagaga 960
tggtgccccc cttgtggtcg gtatacaggt ggtgcatggc tgtcgtcagc tcgtgtcgtg 1020
agatgttggg ttaagtcccg caacgagcgc aacccttgtt ctgtgttgcc agcatgccct 1080
tcggggtgat ggggactcac aggagactgc cggggtcaac tcggaggaag gtggggacga 1140
cgtcaagtca tcatgcccct tatgtcttgg gctgcacacg tgctacaatg gccggtacaa 1200
tgagctgcga tgccgcgagg cggagcgaat ctcaaaaagc cggtctcagt tcggattggg 1260
gtctgcaact cgaccccatg aagtcggagt tgctagtaat cgcagatcag cattgctgcg 1320
gtgaatacgt tcccgggcct tgtcacaccg cccgtctcgt ctacgtaata g 1371
Claims (9)
1. An insect cockroach intestinal tract endophytic streptomyces WA5-1-7 is characterized in that the streptomyces WA5-1-7 is endophytic bacteria obtained by separating and screening from the intestinal tract of American cockroach, is preserved in Guangdong province microorganism culture collection center, and has the preservation number of GDMCC NO. 61630.
2. A method for identifying the Streptomyces WA5-1-7 according to claim 1, comprising the steps of:
(1) 159 strains of actinomycetes are separated from periplaneta americana intestinal tracts, 15 strains of the strains with good candida albicans resistance activity are screened out, and a strain WA5-1-7 with good antibacterial property is further screened out;
(2) performing morphological observation, molecular biological identification and phylogenetic tree analysis on the bacterial strain WA5-1-7 with good antibacterial property selected in the step (1) to perform biological classification identification.
3. The method for identifying Streptomyces WA5-1-7 as claimed in claim 2, wherein the morphological observation in step (2) is the observation of the growth morphology of strain WA5-1-7 on Gao's 1 medium, the observation of the morphology of strain WA5-1-7 by gram stain method and the observation of the morphology of strain WA5-1-7 by scanning electron microscope.
4. The method for identifying Streptomyces WA5-1-7 of claim 2, wherein the molecular biological identification and phylogenetic tree analysis process in step (2) comprises:
s1, extracting DNA of the strain WA5-1-7, selecting a bacterial 16S rRNA universal primer 27F and a primer 1492R to perform PCR amplification on the extracted DNA to obtain a PCR amplification product;
s2, carrying out agarose gel electrophoresis detection on the PCR amplification product obtained in the step S1, sequencing, submitting a sequence obtained by sequencing to NCBI for BLAST homology comparison, and simultaneously submitting sequence information to a GenBank database to obtain a login number of a strain WA 5-1-716S rRNA;
s3, performing BLAST homology comparison on the 16S rRNA sequence of the strain WA5-1-7 and a database sequence in NCBI, selecting representative strains in different published species, performing phylogenetic tree analysis on the multi-sequence comparison result by using an adjacency method in MEGA5.0 software, and judging the species to which the strain WA5-1-7 belongs.
5. The method for identifying Streptomyces WA5-1-7 of claim 4, wherein in step S1, the sequence information of primer 27F is shown as SEQ ID No.1, and the sequence information of primer 1492R is shown as SEQ ID No. 2.
6. The method for identifying Streptomyces WA5-1-7 of claim 4, wherein the sequence information obtained by sequencing the strain WA 5-1-716S rRNA in step S2 is shown in SEQ ID NO. 3.
7. The method for identifying Streptomyces WA5-1-7 of claim 4, wherein step S3 is performed to determine that said strain WA5-1-7 belongs to Streptomyces and has homology of 99%.
8. Use of the secondary metabolite of Streptomyces WA5-1-7 of claim 1 in the preparation of a bacteriostatic composition.
9. The use of claim 8, wherein said antimicrobial composition is capable of potently inhibiting the growth of methicillin-resistant staphylococcus aureus MRSA, staphylococcus aureus, candida albicans, klebsiella pneumoniae, pseudomonas aeruginosa.
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