CN108048366A - One plant of marine actinomycete and its application - Google Patents
One plant of marine actinomycete and its application Download PDFInfo
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- CN108048366A CN108048366A CN201810052133.1A CN201810052133A CN108048366A CN 108048366 A CN108048366 A CN 108048366A CN 201810052133 A CN201810052133 A CN 201810052133A CN 108048366 A CN108048366 A CN 108048366A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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Abstract
The invention discloses one plant of marine actinomycete, the marine actinomycete is ash slightly red streptomyces（streptomyces griseorubens）F8 was preserved in China General Microbiological culture presevation administrative center, deposit number on November 17th, 2017：CGMCC No. 14923.The marine actinomycete of the present invention is ash slightly red streptomyces（streptomyces griseorubens）F8 has good effect to inhibiting aerobacteria and proteus growth, has broad application prospects.
Description
Technical field
The invention belongs to marine actinomycete development of resources with using field, and in particular to one plant of marine actinomycete and its should
With.Background technology
Actinomyces are the important microbe resources for generating natural bioactivity substance, and utilization receive various countries' attention always.
More than 20000 found so far are planted in microbe-derived bioactive substance, there are about 50% are generated by actinomyces.Therefore,
The exploitation of actinomycetes population and use new method are always the research of actinomyces metabolite to the deep excavation of actinomycetes population
Important content.Ocean has unique physics, chemistry and ecological environment such as low temperature, low nutrition, with high salt, no light, anoxic, therefore
Marine actinomycete necessarily leads to the metabolic pathway different from land microorganism and body defenses mechanism, this is to finding that newtype drug is first
Compound, the new mechanism of research drug effect, novel targets etc. are led to have great importance.
Aerobacteria is the coarse clostridium of Gram-positive, and pathogenic conditions are similar to clostridium tetani.Aerobacteria can produce
Raw strong exotoxin, but there are many aggressive enzyme, and have pod membrane, its powerful invasiveness is formed, causes Infective.Aerogenesis
Though bacillus generate ectotoxic toxicity not as good as botulinum toxin and tetanus toxin it is strong, species is more, exotoxin have α, β, γ, δ,
ε, η, θ, ι, κ, λ, μ and ν and a variety of enzymes with toxic action, such as lecithinase, plasmase, hyaluronidase, collagen
Enzyme and dna enzymes etc. form powerful invasiveness.
Proteus is Gram-negative bacteria, can cause multi-infection.Common infection has respiratory tract infection, diarrhea, urinary tract
Infection, peritonitis, tympanitis, mastoiditis, endocarditis, meningitis, septicaemia can also cause food poisoning.Poisoning food master
To be secondly bean product and cold vegetable dish in sauce based on animal food, for morbidity season mostly in summer, autumn, poisoning reason is contaminated food
Product not thoroughly heating before consumption, proteus food poisoning are one of China's common meal poisonings.
The content of the invention
The present invention provides a kind of marine actinomycetes.
The present invention also provides the applications of the marine actinomycete.
The purpose of the present invention is what is be achieved through the following technical solutions：
One plant of marine actinomycete, the marine actinomycete are ash slightly red streptomyces（streptomyces griseorubens）F8,
China General Microbiological culture presevation administrative center, deposit number are preserved on November 17th, 2017：CGMCC No.
14923。
The ash of the present invention slightly red streptomyces sampling status in Shandong Rizhao coastline ooze, is screened through humic acid medium,
Then line purifying is carried out using Gause I culture medium to obtain.
The application of the marine actinomycete inhibits to apply in aerobacteria and proteus growth for marine actinomycete.
Detection ash slightly red streptomyces（streptomyces griseorubens）F8 has suppression to aerobacteria and proteus
It makes and uses, bacteriostatic experiment is carried out using Odontothrips loti, specific method is as follows：
（1）Ash is omited into red streptomyces with bamboo stick（streptomyces griseorubens）F8 is inoculated into Gause I culture medium
In triangular flask, 28 DEG C, shaking table culture 7 days under the conditions of 180rpm/min, bacterium solution centrifuges l0min under the conditions of 10000rpm/min,
Supernatant is taken to filter degerming, obtains fermented supernatant fluid；
（2）Proteus, aerobacteria are inoculated in LB culture mediums respectively, 37 DEG C, shaking table culture 8- under the conditions of 150 rpm/min
10h obtains proteus pathogen and aerobacteria pathogen respectively；
（3）Proteus pathogen and each 100ul of aerobacteria pathogen is taken to be respectively coated on respective LB culture medium flat plates,
It is separated by 3cm on tablet again and is equidistantly placed 4 sterile Oxford cups, draws 120ul fermented supernatant fluids and add in Oxford cup, move
To 37 DEG C of incubator culture 18-24h, observe and inhibition circle whether is formed around Oxford cup；
（4）It is measured, ash slightly red streptomyces（streptomyces griseorubens）F8 is to aerobacteria and proteus
Inhibition zone reach 2.5cm.
The formula of the LB culture mediums is：Dusty yeast 5g, peptone 10g, NaCl 10g, distilled water 1L.
The formula of humic acid medium of the present invention is：Humic acid 1g, Na2HPO4 0.5g, KCl 1.7g MgSO40.5g,
FeSO4·7H2O0.01g, CaCO30.02g, multi-vitamins 1mL, seawater are supplemented to 1L, pH 7.5.
The formula of Gause I culture medium of the present invention is：Soluble starch 20g, KNO31g, K2HPO40.5g, FeSO4·
7H2O 0.01g, MgSO4·7H2O 0.5g, NaCl 0.5g, seawater are supplemented to 1L, pH 7.5.
Beneficial effects of the present invention：The marine actinomycete of the present invention is ash slightly red streptomyces（streptomyces griseorubens）F8 has good effect to inhibiting aerobacteria and proteus growth, has broad application prospects.
Culture presevation information
The preservation time：On November 17th, 2017,
Depositary institution：China Committee for Culture Collection of Microorganisms's common micro-organisms center,
Deposit number：CGMCC No.14923,
Depositary institution address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica, postcode：
100101
Classification And Nomenclature：Ash slightly red streptomycesstreptomyces griseorubens。
Description of the drawings
Fig. 1 is present invention ash slightly red streptomyces（streptomyces griseorubens）F8 is to the antibacterial of proteus
Design sketch.
Fig. 2 is present invention ash slightly red streptomyces（streptomyces griseorubens）F8 is to the antibacterial of aerobacteria
Design sketch.
Specific embodiment
For the specific embodiment enumerated, the present invention is described further below, but not thereby limiting the invention
Content.
The formula of humic acid medium of the present invention is：Humic acid 1g, Na2HPO4 0.5g, KCl 1.7g MgSO40.5g,
FeSO4·7H2O0.01g, CaCO30.02g, multi-vitamins 1mL, seawater are supplemented to 1L, pH 7.5.
The formula of Gause I culture medium of the present invention is：Soluble starch 20g, KNO31g, K2HPO40.5g, FeSO4·
7H2O 0.01g, MgSO4·7H2O 0.5g, NaCl 0.5g, seawater are supplemented to 1L, pH 7.5.
Embodiment 1
One plant of marine actinomycete, the marine actinomycete are ash slightly red streptomyces（streptomyces griseorubens）F8,
China General Microbiological culture presevation administrative center, deposit number are preserved on November 17th, 2017：CGMCC No.
14923。
The ash of the present invention slightly red streptomyces sampling status in Shandong Rizhao coastline ooze, is screened through humic acid medium,
Then line purifying is carried out using Gause I culture medium to obtain.
The application of the marine actinomycete inhibits to apply in aerobacteria and proteus growth for marine actinomycete.
Detection ash slightly red streptomyces（streptomyces griseorubens）F8 has suppression to aerobacteria and proteus
It makes and uses, bacteriostatic experiment is carried out using Odontothrips loti, specific method is as follows：
（1）Ash is omited into red streptomyces with bamboo stick（streptomyces griseorubens）F8 is inoculated into Gause I culture medium
In triangular flask, 28 DEG C, shaking table culture 7 days under the conditions of 180rpm/min, bacterium solution centrifuges l0min under the conditions of 10000rpm/min,
Supernatant is taken to filter degerming, obtains fermented supernatant fluid；
（2）Proteus, aerobacteria are inoculated in LB culture mediums respectively, 37 DEG C, shaking table culture 8- under the conditions of 150 rpm/min
10h obtains proteus pathogen and aerobacteria pathogen respectively；
（3）Proteus pathogen and each 100ul of aerobacteria pathogen is taken to be respectively coated on respective LB culture medium flat plates,
It is separated by 3cm on tablet again and is equidistantly placed 4 sterile Oxford cups, draws 120ul fermented supernatant fluids and add in Oxford cup, move
To 37 DEG C of incubator culture 18-24h, observe and inhibition circle whether is formed around Oxford cup；
（4）It is measured, ash slightly red streptomyces（streptomyces griseorubens）F8 is to aerobacteria and proteus
Inhibition zone reach 2.5cm.
The formula of the LB culture mediums is：Dusty yeast 5g, peptone 10g, NaCl 10g, distilled water 1L.
Ash slightly red streptomyces（streptomyces griseorubens）The acquisition of f8
（1）1g oozes are weighed, 10ml sodium taurocholates (0.1 % w/v) and bead 10 is added in, is vibrated under the conditions of 200r/min
1 min is centrifuged under the conditions of 30min, 500g centrifugal force, upper strata ooze suspension is taken to be transferred to clean pipe（1）In；Lower floor's ooze sinks
It forms sediment and adds in 4 DEG C of Tris buffer solutions (pH7.4) of l0ml, 0.05mol/L, 200r/min vibration 30min, 500g centrifugal force items
1 min is centrifuged under part, upper strata suspension is taken to be merged into pipe（1）In, obtain first step ooze treatment fluid.
（2）Take step（1）In lower floor ooze precipitation, add in l0ml sodium taurocholates (0.1 % w/v), 30w ultrasound baths
1 min is handled, adds 10ml sodium taurocholates (0.1 % w/v), 200r/min vibrates 30min, centrifuged under the conditions of 500g centrifugal force
1 min takes upper strata suspension to be transferred to clean pipe（2）In；Lower sediment adds in 4 DEG C of Tris buffer solutions of l0ml, 0.05mol/L
(pH7.4), 200r/min vibrates 30min, centrifuges 1 min under the conditions of 500g centrifugal force, upper strata suspension is taken to be merged into pipe（2）In,
Obtain second step ooze treatment fluid.
（3）By step（2）Last remaining precipitation is suspended into again in 4 DEG C of 40m1 water, 200r/min vibration 30min,
Upper strata suspension is taken, obtains the 3rd step ooze treatment fluid.
（4）20min is centrifuged under the conditions of the first step, second step and the 3rd step ooze treatment fluid are distinguished 5000g centrifugal force, is abandoned
Supernatant, sediment are resuspended in 10ml physiological saline, do 10 respectively-2To 10-4It is serially diluted, each dilution factor respectively takes 0.2m1
It is coated on above the culture dish of humic acid medium.（The naphthalene that humic acid medium addition concentration is 50ug/ml before culture dish
Batanone acid is to inhibit the bacterium of fast-growth, especially gramnegative bacterium；The dichromic acid that concentration is 50ug/ml is added simultaneously
Potassium solution is to inhibit bacteria the growth with fungi.）
Picking single bacterium colony is further to be purified, and is separated the culture medium used in single bacterium colony and is carried out continuously 3 for Gause I culture medium
It is secondary to rule to be purified into single bacterium colony.
The single bacterium colony being purified is fermented on Gause I culture medium, centrifuging and taking supernatant after the completion of fermentation,
Bacteriostatic test is carried out after 0.22 μm of membrane filtration degerming.
Ash slightly red streptomyces（streptomyces griseorubens）The 16 SrDNA identifications of f8
It is identified by 16SrDNA, determines that the marine actinomycete f8 that the present invention obtains belongs to ash slightly red streptomyces（streptomyces griseorubens）, it is as follows：
First, genomic DNA is extracted
（1）The activation of bacterial strain：In an aseptic environment, pure one rings of bacterium colony of bacterial strain f8 are taken, are inoculated on 5ml Gause I culture mediums,
180r/min, 28 DEG C of activation 36h.
（2）Extract genomic DNA：Use the gram-positive bacterium genomic DNA of Beijing Suo Laibao Science and Technology Ltd
Extracts kit, the genomic DNA of the f8 bacterial strains after extraction activation.
（3）Examine genomic DNA fragment：By step（2）The genomic DNA of acquisition is into row agarose gel electrophoresis, with inspection
It tests and whether extracts target gene group DNA.Ago-Gel formula is：0.25 mg of agarose, 1 × TAE 25ml, Gelred
2.5μl.Deposition condition is：120 V、20 min.
2nd, PCR product recycles
Use healthy and free from worry life science（Wujiang）The PCR plastic recovery kits recycling PCR product of Co., Ltd.
Target gene fragment is expanded using actinomyces universal primer.50 μ l PCR systems and PCR reaction conditions point
Not as shown in Table 1 and Table 2.
Primer sequence is as follows
Primer 1：5'-AGAGTTTGATCCTGGCTCAG-3'
Primer 2：5'-AAGGAGGTGATCCAGCCGCA-3'；
1 50 μ l PCR systems of table
2 PCR reaction conditions of table
3rd, sequencing compares
（1）Sequencing：PCR purified products are sent to Jinan City, Shandong Province Li Ge Science and Technology Ltd.s and are sequenced.
（2）Sequence alignment：The strain sequence that PCR product recycling obtains is subjected to nucleotide sequence BLAST ratios on NCBI
To rear, discovery and bacterial strain ash slightly red streptomyces（streptomyces griseorubens）Similitude reaches 99%, therefore the suppression
Aerogenesis bar processed and the bacterial strain of proteus are ash slightly red streptomyces.
Claims (4)
1. one plant of marine actinomycete, which is characterized in that the marine actinomycete is ash slightly red streptomyces（streptomyces griseorubens）F8, China General Microbiological culture presevation administrative center is preserved on November 17th, 2017, and preservation is compiled
Number：CGMCC No. 14923.
2. the application of marine actinomycete described in a kind of claim 1, which is characterized in that the application of the marine actinomycete is ocean
Actinomyces inhibit to apply in aerobacteria and proteus growth.
3. the application of marine actinomycete according to claim 2, which is characterized in that the marine actinomycete inhibits aerobacteria
The application grown with proteus, specific method are as follows：
（1）Ash slightly red streptomyces f8 is inoculated into Gause I culture medium triangular flask with bamboo stick, 28 DEG C, 180rpm/min conditions
Lower shaking table culture 7 days, bacterium solution centrifuges 10min under the conditions of 10000rpm/min, and supernatant is taken to filter degerming, obtains fermentation supernatant
Liquid；
（2）Proteus, aerobacteria are inoculated in LB culture mediums respectively, 37 DEG C, shaking table culture 8- under the conditions of 150 rpm/min
10h obtains proteus pathogen and aerobacteria pathogen respectively；
（3）Proteus pathogen and each 100ul of aerobacteria pathogen is taken to be respectively coated on respective LB culture medium flat plates,
It is separated by 3cm on tablet again and is equidistantly placed 4 sterile Oxford cups, draws 120ul fermented supernatant fluids and add in Oxford cup, move
To 37 DEG C of incubator culture 18-24h, observe and inhibition circle whether is formed around Oxford cup；
（4）Measured, ash slightly red streptomyces f8 reaches 2.5cm to the inhibition zone of aerobacteria and proteus.
4. the application of marine actinomycete according to claim 3, which is characterized in that the formula of the LB culture mediums is：Yeast
Powder 5g, peptone 10g, NaCl 10g, distilled water 1L.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109280692A (en) * | 2018-10-31 | 2019-01-29 | 广东药科大学 | A kind of the antifungal activity screening technique and its fermentation process of blattaria endogeny rayungus |
| CN109652483A (en) * | 2018-12-03 | 2019-04-19 | 曲阜师范大学 | A kind of marine actinomycete liquid fermentation production extracts compound and its preparation method and application |
| CN109666029A (en) * | 2018-12-03 | 2019-04-23 | 曲阜师范大学 | Cyclic dipeptide compound and its preparation method and application |
| CN111893067A (en) * | 2020-08-05 | 2020-11-06 | 曲阜师范大学 | Microbacterium siberia for producing low-temperature protease and application thereof |
| CN112708638A (en) * | 2020-12-25 | 2021-04-27 | 中国科学院微生物研究所 | Extract with nematode inhibiting effect, streptomycete and application of extract and streptomycete |
| CN113493750A (en) * | 2021-06-08 | 2021-10-12 | 曲阜师范大学 | Marine actinomycete and application thereof |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109280692A (en) * | 2018-10-31 | 2019-01-29 | 广东药科大学 | A kind of the antifungal activity screening technique and its fermentation process of blattaria endogeny rayungus |
| CN109652483A (en) * | 2018-12-03 | 2019-04-19 | 曲阜师范大学 | A kind of marine actinomycete liquid fermentation production extracts compound and its preparation method and application |
| CN109666029A (en) * | 2018-12-03 | 2019-04-23 | 曲阜师范大学 | Cyclic dipeptide compound and its preparation method and application |
| CN109652483B (en) * | 2018-12-03 | 2022-07-29 | 曲阜师范大学 | Marine actinomycete liquid fermentation product extraction compound and preparation method and application thereof |
| CN111893067A (en) * | 2020-08-05 | 2020-11-06 | 曲阜师范大学 | Microbacterium siberia for producing low-temperature protease and application thereof |
| CN112708638A (en) * | 2020-12-25 | 2021-04-27 | 中国科学院微生物研究所 | Extract with nematode inhibiting effect, streptomycete and application of extract and streptomycete |
| CN112708638B (en) * | 2020-12-25 | 2022-04-26 | 中国科学院微生物研究所 | Extract with nematode inhibiting effect, streptomycete and application of extract and streptomycete |
| CN113493750A (en) * | 2021-06-08 | 2021-10-12 | 曲阜师范大学 | Marine actinomycete and application thereof |
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|---|---|
| CN108048366B (en) | 2021-03-30 |
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