CN108048366B - A marine actinomycete and its application - Google Patents
A marine actinomycete and its application Download PDFInfo
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- CN108048366B CN108048366B CN201810052133.1A CN201810052133A CN108048366B CN 108048366 B CN108048366 B CN 108048366B CN 201810052133 A CN201810052133 A CN 201810052133A CN 108048366 B CN108048366 B CN 108048366B
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Abstract
The invention discloses a marine actinomycete, which is streptomyces griseofulensis（streptomyces griseorubens）f8, deposited in the China general microbiological culture Collection center in 2017 at 11 and 17 months, with the deposit number: CGMCC No. 14923. The marine actinomycete is streptomyces griseofulensis（streptomyces griseorubens）f8 has good effect on inhibiting the growth of aerobacter and proteus and has wide application prospect.
Description
Technical Field
The invention belongs to the field of marine actinomycete resource development and utilization, and particularly relates to a marine actinomycete and application thereof. Background
Actinomycetes are important microbial resources for producing natural bioactive substances, and development and utilization of actinomycetes are always regarded by various countries. About 50% of the over 20000 species of biologically active substances derived from microorganisms found to date are produced by actinomycetes. Therefore, the development of actinomycete resources and the deep excavation of actinomycete resources by adopting the new method are always important contents for research of actinomycete metabolites. The ocean has unique physical, chemical and ecological environments of low temperature, low nutrition, high salt content, no illumination, oxygen deficiency and the like, so that the marine actinomycetes inevitably generate different metabolic pathways and body defense mechanisms compared with terrestrial microorganisms, and the marine actinomycetes have important significance for finding novel medicine lead compounds, researching new mechanisms and new targets of medicine action and the like.
The aerobacter is a gram-positive large clostridium, and the pathogenic condition is similar to that of clostridium tetani. The aerobacter can generate strong exotoxin, has various invasive enzymes and capsules, forms strong invasiveness and causes infection and pathogenesis. Although the toxin produced by aerobacter is not as strong as botulinum toxin and tetanus toxin, the toxin is various, and the toxin has alpha, beta, gamma, delta, epsilon, eta, theta, iota, kappa, lambda, mu and nu, and various enzymes with toxic action, such as lecithinase, fibrinolytic enzyme, hyaluronidase, collagenase and dnase, and the like, and constitutes strong invasiveness.
Proteus is a gram-negative bacterium that can cause a variety of infections. Common infections include respiratory tract infection, diarrhea, urinary tract infection, peritonitis, otitis media, mastoiditis, endocarditis, meningitis, septicemia, and food poisoning. The poisoning food mainly comprises animal food, bean products and cold vegetable, the disease season is more in summer and autumn, the poisoning reason is that the polluted food is not heated thoroughly before eating, and the proteus vulgaris food poisoning is one of common food poisoning in China.
Disclosure of Invention
The invention provides a marine actinomycete.
The invention also provides application of the marine actinomycete.
The purpose of the invention is realized by the following technical scheme:
a strain of marine actinomycete, wherein the marine actinomycete is streptomyces griseofulensis（streptomyces griseorubens）f8, deposited in the China general microbiological culture Collection center in 2017 at 11 and 17 months, with the deposit number: CGMCC number 14923.
The streptomyces griseofulvin is obtained by sampling sea mud on a sunshine coastline in Shandong, screening by a humic acid culture medium and then carrying out streak purification by using a Gao's first culture medium.
The application of the marine actinomycetes is the application of the marine actinomycetes in inhibiting the growth of aerobacter aerogenes and proteus.
Detection of Streptomyces griseofulensis（streptomyces griseorubens）f8 treatment of Aerobacter aerogenesThe bacillus shapers have an inhibiting effect, and an oxford cup method is adopted for carrying out an antibacterial experiment, and the specific method comprises the following steps:
(1) subjecting Streptomyces griseofulensis to bamboo stick treatment（streptomyces griseorubens）f8 inoculating into a Gao's first culture medium triangular flask, shake culturing at 28 deg.C and 180rpm/min for 7 days, centrifuging the bacterial liquid at 10000rpm/min for l0min, and suction filtering the supernatant to remove bacteria to obtain fermented supernatant;
(2) respectively inoculating proteus and aerobacter in LB culture medium, and shake culturing at 37 deg.C and 150 rpm/min for 8-10h to respectively obtain proteus pathogenic bacteria and aerobacter pathogenic bacteria;
(3) respectively coating 100ul of bacillus proteus pathogenic bacteria and 100ul of bacillus aerogenes pathogenic bacteria on respective LB culture medium flat plates, placing 4 aseptic oxford cups on the flat plates at equal intervals of 3cm, sucking 120ul of fermentation supernatant, adding the fermentation supernatant into the oxford cups, moving the oxford cups to a 37 ℃ incubator for culturing for 18-24 hours, and observing whether inhibition rings are formed around the oxford cups or not;
(4) through measurement, the streptomyces griseofulensis（streptomyces griseorubens）f8 the inhibition zone for aerogenic bacillus and proteus reaches 2.5 cm.
The LB culture medium comprises the following components in percentage by weight: 5g of yeast powder, 10g of peptone, 10g of NaCl and 1L of distilled water.
The formula of the humic acid culture medium is as follows: humic acid 1g, Na2HPO4 0.5g，KCl 1.7g MgSO40.5g，FeSO4·7H2O0.01g，CaCO30.02g, 1mL of vitamin complex, and 1L of seawater supplemented, wherein the pH is 7.5.
The formula of the Gao's No. one culture medium is as follows: soluble starch 20g, KNO31g，K2HPO40.5g，FeSO4·7H2O 0.01g，MgSO4·7H20.5g of O, 0.5g of NaCl and 1L of seawater are supplemented, and the pH value is 7.5.
The invention has the beneficial effects that: the marine actinomycete is streptomyces griseofulensis（streptomyces griseorubens）f8 has good effect on inhibiting the growth of aerobacter and proteus and has wide application prospect.
Information on strain preservation
Preservation time: 11/month/17/2017,
the preservation unit: china general microbiological culture Collection center,
the preservation number is: the CGMCC No.14923 is used as a raw material,
the address of the depository: the microbiological research institute of western road 1, 3, national academy of sciences, north-kyo, chaoyang, the postal code: 100101
And (3) classification and naming: streptomyces griseofulensisstreptomyces griseorubens。
Drawings
FIG. 1 shows Streptomyces griseofulensis of the present invention（streptomyces griseorubens）f8 bacteriostatic effect graph on proteus.
FIG. 2 shows Streptomyces griseofulensis of the present invention（streptomyces griseorubens）f8 bacteriostatic effect graph for aerobacter.
Detailed Description
The present invention will be further described below with reference to specific embodiments, but the present invention is not limited thereto.
The formula of the humic acid culture medium is as follows: humic acid 1g, Na2HPO4 0.5g，KCl 1.7g MgSO40.5g，FeSO4·7H2O0.01g，CaCO30.02g, 1mL of vitamin complex, and 1L of seawater supplemented, wherein the pH is 7.5.
The formula of the Gao's No. one culture medium is as follows: soluble starch 20g, KNO31g，K2HPO40.5g，FeSO4·7H2O 0.01g，MgSO4·7H20.5g of O, 0.5g of NaCl and 1L of seawater are supplemented, and the pH value is 7.5.
Example 1
A strain of marine actinomycete, wherein the marine actinomycete is streptomyces griseofulensis（streptomyces griseorubens）f8, deposited in the China general microbiological culture Collection center in 2017 at 11 and 17 months, with the deposit number: CGMCC number 14923.
The streptomyces griseofulvin is obtained by sampling sea mud on a sunshine coastline in Shandong, screening by a humic acid culture medium and then carrying out streak purification by using a Gao's first culture medium.
The application of the marine actinomycetes is the application of the marine actinomycetes in inhibiting the growth of aerobacter aerogenes and proteus.
Detection of Streptomyces griseofulensis（streptomyces griseorubens）f8 has an inhibiting effect on aerogenic bacillus and proteus, and an oxford cup method is adopted for carrying out an antibacterial experiment, and the specific method is as follows:
(1) subjecting Streptomyces griseofulensis to bamboo stick treatment（streptomyces griseorubens）f8 inoculating into a Gao's first culture medium triangular flask, shake culturing at 28 deg.C and 180rpm/min for 7 days, centrifuging the bacterial liquid at 10000rpm/min for l0min, and suction filtering the supernatant to remove bacteria to obtain fermented supernatant;
(2) respectively inoculating proteus and aerobacter in LB culture medium, and shake culturing at 37 deg.C and 150 rpm/min for 8-10h to respectively obtain proteus pathogenic bacteria and aerobacter pathogenic bacteria;
(3) respectively coating 100ul of bacillus proteus pathogenic bacteria and 100ul of bacillus aerogenes pathogenic bacteria on respective LB culture medium flat plates, placing 4 aseptic oxford cups on the flat plates at equal intervals of 3cm, sucking 120ul of fermentation supernatant, adding the fermentation supernatant into the oxford cups, moving the oxford cups to a 37 ℃ incubator for culturing for 18-24 hours, and observing whether inhibition rings are formed around the oxford cups or not;
(4) through measurement, the streptomyces griseofulensis（streptomyces griseorubens）f8 the inhibition zone for aerogenic bacillus and proteus reaches 2.5 cm.
The LB culture medium comprises the following components in percentage by weight: 5g of yeast powder, 10g of peptone, 10g of NaCl and 1L of distilled water.
Streptomyces griseofulensis（streptomyces griseorubens）Acquisition of f8
(1) Weighing 1g of sea mud, adding 10ml of sodium cholate (0.1% w/v) and 10 glass beads, oscillating for 30min under the condition of 200r/min, centrifuging for 1 min under the condition of 500g of centrifugal force, and taking the upper-layer sea mud suspension to transfer into a clean pipe (1); adding the lower layer sea mud precipitate into L0ml, 0.05 mol/L4 deg.C Tris buffer solution (pH7.4), oscillating at 200r/min for 30min, centrifuging at 500g centrifugal force for 1 min, collecting the upper layer suspension, and mixing in tube (1) to obtain the first step sea mud treatment solution.
(2) Taking the lower layer sea mud precipitate in the step (1), adding l0ml sodium cholate (0.1% w/v), treating for 1 min by 30w ultrasonic water bath, adding 10ml sodium cholate (0.1% w/v), oscillating for 30min at 200r/min, centrifuging for 1 min under the centrifugal force condition of 500g, taking the upper layer suspension, and transferring to a clean tube (2); adding L0ml and 0.05 mol/L4 ℃ Tris buffer solution (pH7.4) into the lower layer sediment, oscillating for 30min at 200r/min, centrifuging for 1 min under the centrifugal force condition of 500g, taking the upper layer suspension, and merging the upper layer suspension into a tube (2) to obtain the second step sea mud treatment solution.
(3) And (3) re-suspending the precipitate remained at the end of the step (2) into 40m1 water at the temperature of 4 ℃, oscillating for 30min at the speed of 200r/min, and taking the upper suspension to obtain the sea mud treatment liquid in the third step.
(4) Centrifuging the sea mud treatment solution of the first step, the second step and the third step for 20min under the centrifugal force condition of 5000g respectively, discarding the supernatant, and resuspending the precipitate in 10ml of normal saline respectively to obtain 10-2To 10-4Serial dilutions were made, each dilution taking 0.2m1 coated on a humic acid medium petri dish. (humic acid medium added with nabumetone acid concentration of 50ug/ml before being poured into a culture dish to inhibit fast-growing bacteria, especially gram-negative bacteria; and potassium dichromate solution added with concentration of 50ug/ml to inhibit growth of bacteria and fungi.)
The single colony was picked for further purification and the medium used to isolate the single colony was Gao's-I medium and streaked 3 times in succession to purify the single colony.
Fermenting the purified single colony on a Gao's first culture medium, centrifuging after fermentation, taking supernatant, filtering and sterilizing by a 0.22 mu m filter membrane, and then carrying out an antibacterial test.
Streptomyces griseofulensis（streptomyces griseorubens）Identification of 16SrDNA of f8
Through 16SrDNA identification, the marine actinomycete f8 obtained by the invention is determined to belong to Streptomyces griseofulvin（streptomyces griseorubens）The method comprises the following specific steps:
firstly, extracting genome DNA
(1) Activation of the strain: taking a pure colony of the strain f8 in a sterile environment, inoculating the colony on 5ml of a Gao's first culture medium, activating at 180r/min for 36h at 28 ℃.
(2) Extracting genome DNA: the genomic DNA of the activated f8 strain was extracted using a gram-positive bacterium genomic DNA extraction kit from Beijing Solebao science and technology Co.
(3) Examination of genomic DNA fragments: and (3) carrying out agarose gel electrophoresis on the genomic DNA obtained in the step (2) to check whether the target genomic DNA is extracted. The agarose gel formula is as follows: agarose 0.25 mg, 1 XTAE 25ml, Gelred 2.5. mu.l. The electrophoresis conditions are as follows: 120V and 20 min.
Secondly, PCR product recovery
PCR products were recovered using a PCR gel recovery kit from Kangning Life sciences (Wu Jiang) Co., Ltd.
And (3) amplifying the target gene fragment by using an actinomycete universal primer. The 50. mu.l PCR system and PCR reaction conditions are shown in Table 1 and Table 2, respectively.
The primer sequences are as follows
Primer 1: 5'-AGAGTTTGATCCTGGCTCAG-3'
Primer 2: 5'-AAGGAGGTGATCCAGCCGCA-3', respectively;
TABLE 150. mu.l PCR System
TABLE 2 PCR reaction conditions
Third, sequencing comparison
(1) Sequencing: the PCR-purified product was sent to Leog technologies, Inc., of Jinan, Shandong, for sequencing.
(2) And (3) sequence alignment: the strain sequence obtained by recovering the PCR product is subjected to nucleotide sequence B on NCBIAfter LAST comparison, the strain Streptomyces griseoflavus is foundstreptomyces griseorubens) The similarity reaches 99%, so the strain inhibiting the aerogenic rods and the proteus is streptomyces griseofulensis.
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| CN109280692A (en) * | 2018-10-31 | 2019-01-29 | 广东药科大学 | A kind of antifungal activity screening method of endophytic actinomycetes of cockroaches and fermentation method thereof |
| CN109652483B (en) * | 2018-12-03 | 2022-07-29 | 曲阜师范大学 | A kind of marine actinomycetes liquid fermentation product extraction complex and preparation method and application thereof |
| CN109666029A (en) * | 2018-12-03 | 2019-04-23 | 曲阜师范大学 | Cyclic dipeptide compound and its preparation method and application |
| CN111893067B (en) * | 2020-08-05 | 2022-07-05 | 曲阜师范大学 | A low-temperature protease-producing Exiguobacterium siberia and its application |
| CN112708638B (en) * | 2020-12-25 | 2022-04-26 | 中国科学院微生物研究所 | Nematode-inhibiting extract and Streptomyces and their use |
| CN113493750B (en) * | 2021-06-08 | 2023-07-18 | 曲阜师范大学 | A marine actinomycete and its application |
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| US4162304A (en) * | 1975-03-15 | 1979-07-24 | Beecham Group Limited | Streptomycetal antibiotic |
| CN102191204A (en) * | 2011-04-15 | 2011-09-21 | 暨南大学 | Streptomyces microflavus with antibacterial function and applications thereof |
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