CN103289924B - Streptomyces sp. with antibacterial function and application thereof - Google Patents
Streptomyces sp. with antibacterial function and application thereof Download PDFInfo
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- 230000000844 anti-bacterial effect Effects 0.000 title claims abstract description 19
- 241000187180 Streptomyces sp. Species 0.000 title claims abstract description 15
- 239000003814 drug Substances 0.000 claims abstract description 28
- 241000894006 Bacteria Species 0.000 claims abstract description 25
- 241000191967 Staphylococcus aureus Species 0.000 claims abstract description 22
- 229940079593 drug Drugs 0.000 claims abstract description 19
- 241001655322 Streptomycetales Species 0.000 claims description 37
- 230000001580 bacterial effect Effects 0.000 claims description 29
- 239000002207 metabolite Substances 0.000 claims description 19
- 238000000855 fermentation Methods 0.000 claims description 14
- 230000004151 fermentation Effects 0.000 claims description 14
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- 241000589771 Ralstonia solanacearum Species 0.000 claims description 12
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Abstract
The invention discloses a streptomyces sp. with an antibacterial function and an application thereof. The metabolic product of the streptomyces sp. GIM4.116 has antibacterial activity, particularly has good activity for escherichia coli and staphylococcus aureus with multiple drug resistances, and can be used for developing drugs for resisting the bacteria. The streptomyces sp. also has a good inhibition function on ralstonia solaanacearum capable of causing plant ralstonia solaanacearum disease, can also be used for developing drugs for inhibiting the ralstonia solaanacearum disease caused by the ralstonia solaanacearum, and has a very good application prospect.
Description
Technical field:
The invention belongs to microorganism field, be specifically related to a strain and have streptomycete and the application thereof of anti-microbial effect.
Background technology:
Antibiotics being widely used clinically, the infectious diseases being caused by microorganism is controlled effectively, but also cause simultaneously many pathogenic bacterium to Tri-Biocin deposits yields serious resistance, be multi-drug resistant bacteria the pathogenic bacteria that can present resistance to three classes or the above antibacterials of three classes clinically.The microbial infection of multidrug resistant presents complicacy, the feature such as intractable, the health that is endangering greatly the mankind.Vancomycin is acknowledged as the most effective microbiotic at present, but the streptococcus aureus of vancomycin resistance also occurs.The Novel super germ NDM-1 finding in South Asia for 2010, it all has resistance to all known microbiotic beyond Tigecycline and colistin.Resistant strain increase gradually and the continuous enhancing of resistance has limited existing antibiotic clinical therapeutic efficacy, find and development of new microbiotic resource imperative.Bacterial wilt is the kind of plant destructive disease being caused by Ralstonia solanacearum (Ralstonia solanacearum), and in the whole world, there is distribution in many places, but it is the most serious with subtropical and tropical zones, harm to occur.It can infect more than 200 kind of raise crop of 40 sections and wild plant, comprising potato, tobacco, tomato, eggplant, hot pepper, green pepper and peanut etc.The control of Plant diseases is mainly taking chemical pesticide as main at present.Chemical pesticide has the property of medicine by force, easily makes phytopathogen produce the features such as resistance and difficult degradation, cause serious pollution to environment around, make that agricultural-food agriculture is residual to exceed standard, threaten people's health by food chain, this has had a strong impact on agriculture Sustainable development.Biological pesticide refers to and directly utilizes biogenic biologically active substance or living organisms as agricultural chemicals, and the agricultural chemicals identical with natural compounds structure of synthetic.It is extensive that biological pesticide has raw material sources, little to non-target organism safety, toxic side effect, to features such as environment compatibility are good, become the new trend of global agricultural chemicals industry development.Food crop 100,000,000 hm plant throughout the year in Shi Ge large agricultural country of China
2above, cotton 4,800,000 hm
2above, oil crops 1,400 ten thousand hm
2above, vegetable melon and fruit 1,800 ten thousand hm
2above, need to prevent and treat area throughout the year exceed 300,000,000 hm
2, but adopt biological medicament prevention and elimination of disease and pests only account for 10% left and right.Existing biological pesticide can not meet the demand of domestic market far away, and has the problems such as imitated external product, shortage originality, poor stability.Therefore, continually develop the novel biological pesticide with independent intellectual property right and have extremely important meaning for the Economic development of China.Actinomycetes metabolism has diversity, and the biologically active substance that can produce is extremely abundant, is the main source of new antibiotic.Marine actinomycete, due to its unique living environment, may contain the meta-bolites with special anti-microbial effect.
Goal of the invention:
The object of this invention is to provide a kind of streptomycete and application thereof with anti-microbial effect.
Streptomycete of the present invention (Streptomyces sp.) GIM4.116, belong to actinomycetales (Actinomycete) streptomyces (Streptomyces), be preserved in Chinese Typical Representative culture collection center (CCTCC) on March 4th, 2013, address: Wuhan University of Wuhan, China city, deposit number is CCTCC NO:M2013064.
The present invention found through experiments, the M of streptomycete (Streptomyces sp.) GIM4.116
2 +the crude extract of the meta-bolites of substratum fermentation all has stronger bacteriostatic activity to common intestinal bacteria, streptococcus aureus and Candida albicans, and especially to Candida albicans, antibacterial circle diameter has reached 29.33mm.The M of streptomycete GIM4.116
2 +the crude extract of the meta-bolites of substratum fermentation all has stronger inhibition to resistance to 11 kinds of antibiotic coli strains 460 and bacterial strain 480, resistance to 13 kinds of antibiotic staphylococcus aureus strains 206 are also had to stronger inhibition, along with resistant organism Enrichment antibacterial circle diameter decreases, but still have obvious inhibition.The M of streptomycete GIM4.116
2 +the crude extract of the tunning of substratum all has stronger inhibition to the pathogenic bacteria bacterial strain that causes three kind of plant bacterial wilts, and the about 24mm of antibacterial circle diameter illustrates that streptomycete GIM4.116 is also having great application potential aspect control plant-bacterial-wilt.
As can be seen here, streptomycete of the present invention (Streptomyces sp.) GIM4.116 can be in the application of preparing in antibacterials.
Described application, is using streptomycete (Streptomyces sp.) GIM4.116 as fermentation strain, M
2 +substratum is as fermention medium, carries out the application in preparation antibacterials of meta-bolites (extract after more preferably meta-bolites being extracted with ethyl acetate) after fermentation culture, described M
2 +every liter of substratum contains Fructus Hordei Vulgaris extract 10g, glucose 4g, and yeast extract 4g, surplus is water, pH7.8.
Described antibacterials are preferably anti-common intestinal bacteria, anti-ly have multi-drug resistant intestinal bacteria (Eshcerichia coli), anti-common streptococcus aureus, an anti-medicine with multi-drug resistant streptococcus aureus (Staphylococcus aureus) or anti-candida albicans.
The present invention is using streptomycete (Streptomyces sp.) GIM4.116 as fermentation strain, M
2 +substratum is as fermention medium, the intestinal bacteria of meta-bolites after fermenting to multi-drug resistant and the streptococcus aureus of multi-drug resistant all have good restraining effect, can be for the preparation of the medicine of anti-multi-drug resistant intestinal bacteria and multi-drug resistant streptococcus aureus.
Described antibacterials are preferably the medicine that suppresses the withered Raul Salmonella of green grass or young crops that causes plant-bacterial-wilt.
The withered Raul Salmonella of described green grass or young crops is bacterial wilt of tomato pathogenic bacteria (R.Solanacearum) GIM1.356, potato bacterial wilt pathogenic bacteria (R.Solanacearum) GIM1.74 or cotton Stalk Rot (R.Solanacearum) GIM1.68.
The invention provides a kind of streptomycete (Streptomyces sp.) GIM4.116, its meta-bolites has anti-microbial activity, particularly multi-drug resistant intestinal bacteria and multi-drug resistant streptococcus aureus are had to good activity, therefore can be for exploitation for resisting the medicine of this bacterioid.It also has good restraining effect to the withered Raul Salmonella of green grass or young crops that causes plant-bacterial-wilt, therefore, can suppress for exploitation the medicine of the withered Raul Salmonella of green grass or young crops that causes plant-bacterial-wilt, has extraordinary application prospect.
Brief description of the drawings:
Fig. 1 is the fibrillae of spores structure iron of streptomycete GIM4.116
Fig. 2 is the phylogenetic tree that the relevant bacterial strain of streptomycete GIM4.116 and part builds according to 16S rRNA gene order.
Embodiment:
Following examples are to further illustrate of the present invention, instead of limitation of the present invention.
Embodiment 1: the Isolation and Identification of streptomycete GIM4.116
1, the separation of streptomycete GIM4.116
Adopt Gause I substratum from the oceanic sediment of the South Sea, to isolate a strain bacterial strain GIM4.116.Be inoculated in Gao Shi No. 1 slant medium (Zulkovsky starch 20g, KNO
31g, K
2hPO
40.5g, MgSO
47H
2o0.5g, NaCl0.5g, FeSO
47H
2o0.01g, distilled water 1000mL, pH7.2) upper, cultivate 7 days for 28 DEG C, take out and be placed on 4 DEG C of Refrigerator stores.
2, identification of strains
Bacterial strain GIM4.116 is carried out to morphological specificity, cultural characteristic and the qualification of 16S rRNA gene sequencing, specific as follows:
1) morphological specificity of bacterial strain
Bacterial strain GIM4.116 is inoculated in to No. 1 substratum of Gao Shi, and 28 DEG C of inserted sheets were cultivated after 7 days, got sheet, observed the morphological specificity of thalline by ordinary method.
Result is as follows: substrate mycelium and aerial hyphae are abundant.Substrate mycelium is not without tabula, rupture; Aerial hyphae is flexible, multi-branched; The straight shape of fibrillae of spores, middle length; Spore is subsphaeroidal, smooth surface (Fig. 1).These features are consistent with streptomyces.
2) cultural characteristic of bacterial strain
Bacterial strain GIM4.116 is inoculated in to No. 1 substratum of Gao Shi, and 28 DEG C of inserted sheets were cultivated after 7 days, got sheet, observed the cultural characteristic of bacterium colony by ordinary method.
Result is as follows: diameter 3.0-4.0mm; Bacterium colony is rounded, surface irregularity, edge white, middle light grey, can be lilac by lysochrome; Substrate mycelium is ecru, and it is light grey that aerial hyphae is.
3) 16S rRNA gene sequencing
Adopt CTAB method to extract the genomic dna of bacterial strain GIM4.116, by DNA concentration dilution to 50-100ng/ μ L, as the template of pcr amplification.Adopt bacterium universal primer F27(5 '-AGAGTTTGATCCTGGCTCAG-3 ') and 1492R(5 '-TACGACTTA ACCCCAATCGC-3 ') carry out the pcr amplification of 16S rRNA gene.
25 μ L PCR reaction systems: 10 × Buffer2.5 μ L, dNTP (10mmol/L) 0.5 μ L, primers F 27(10 μ mol/L) 0.5 μ L, primer 1492R (10 μ mol/L) 0.5 μ L, Taq enzyme (5U/ μ L) 0.25 μ L, DNA profiling 1 μ L, sterilizing distilled water is supplemented to 25 μ L.
PCR reaction conditions: 94 DEG C of 5min; 30 circulations (94 DEG C of 30s, 56 DEG C of 30s, 72 DEG C of 1.5min); 72 DEG C of 10min.
PCR product after testing with clone after, by Shanghai, Ying Jun biotech company completes order-checking.Its sequence is as shown in SEQ ID NO:1.In this sequence and GenBank database, known array carries out BLAST comparative analysis, from database, obtain the 16S rRNA gene order of the bacterial strain that similarity is larger, adopt software MEGA5.0 to build the phylogenetic tree of bacterial strain with Neighbor-Joining method, Bootstrap is made as 1000 times, and phylogenetic tree is shown in Fig. 2.Find bacterial strain GIM4.116 of the present invention and Streptomyces griseoplanus AS4.1868 through comparative analysis
t16S rRNA gene order similarity is the highest, is 99.79%, but on phylogenetic tree with Streptomyces globisporus NBRC12867
t, Streptomyces badius NRRL B-2567
t, Streptomyces parvus NBRC3388
t, Streptomyces sindenensis NBRC3399
t, Streptomyces pluricolorescens NBRC12808
twith Streptomyces rubiginosohelvolus NBRC12912
tgather in a branch Deng 6 strain bacterium, there is nearer sibship, in conjunction with its form and cultural characteristic, be accredited as streptomycete (Streptomyces sp.), called after: streptomycete (Streptomyces sp.) GIM4.116, be preserved in Chinese Typical Representative culture collection center (CCTCC), address on March 4th, 2013: Wuhan University of Wuhan, China city, deposit number is CCTCC NO:M2013064.
Embodiment 2: the screening of bacteriostatic activity substratum
Streptomycete GIM4.116 is inoculated in respectively to Gause I substratum, GBP substratum (glucose 10g, extractum carnis 3g, peptone 5g, distilled water 1000mL, pH7.2), the calcium chloride substratum (CaCl of 200mL
245g, yeast extract 40g, glucose 5g, distilled water 1000mL, pH7.8) and M
2 +in substratum (Fructus Hordei Vulgaris extract 10g, glucose 4g, yeast extract 4g, distilled water 1000mL, pH7.8).28 DEG C of shaking culture 7 days, fermented liquid is at the centrifugal 20min of 8000r/min, and supernatant liquor adopts 8 layers of filtered through gauze, and filtrate is with the extraction of equal-volume ethyl acetate, and extract rotary evaporation obtains the crude extract of meta-bolites, meta-bolites is dissolved and is settled to l mL with methyl alcohol.Taking intestinal bacteria Escherichia coli ATCC8739, streptococcus aureus Staphylococcus aureus ATCC6538, Candida albicans Candida albicans ATCC10231 as antagonism object, the bacteriostatic activity of the tunning crude extract of employing Oxford cup mensuration different culture media to aimed strain.
Result is as shown in table 1.M
2 +the tunning crude extract of substratum all has stronger bacteriostatic activity to intestinal bacteria, streptococcus aureus, Candida albicans, and especially to Candida albicans, antibacterial circle diameter has reached 29.33mm.The crude extract of the meta-bolites of calcium chloride substratum fermentation does not all have bacteriostatic activity to intestinal bacteria, streptococcus aureus, Candida albicans.The crude extract of the meta-bolites of Gause I and GBP substratum fermentation only has bacteriostatic activity to streptococcus aureus, and to intestinal bacteria, Candida albicans all without any inhibition.As can be seen here, there is larger difference in the meta-bolites bacteriostatic activity that streptomycete GIM4.116 adopts different culture media fermentation to obtain, and the fermention medium of the best bacteriostatic activity of streptomycete GIM4.116 is M
2 +substratum.
Table 1: the tunning bacteriostatic activity of different culture media
Note: "-" indicates to produce without pressing down circle
Embodiment 3: the inhibition of streptomycete GIM4.116 tunning to multi-drug resistant bacteria
According to the method for embodiment 2, adopt M
2 +substratum fermentation streptomycete GIM4.116, and obtain the crude extract of the meta-bolites of streptomycete GIM4.116.With coli strain 460 and the resistance to 11 kinds of microbiotic of bacterial strain 480(, in table 2), the resistance to 13 kinds of microbiotic of staphylococcus aureus strains 206(, in table 2), Salmonellas bacterial strain 31 and the resistance to 9 kinds of microbiotic of bacterial strain 47(, in table 2) etc. 5 strain multi-drug resistant bacterias be antibacterial object, they are so kind as to give by professor Jiang Hongxia of College of Veterinary Medicine, South China Agricultural University.Adopt the dilution of gradient dilution method, and carry out blood counting chamber counting, make different concns (10
5, 10
6, 10
7individual/mL) resistant organism bacteria suspension, get respectively 100 μ L and coat on nutrient agar (peptone 10g, extractum carnis 3g, NaCl5g, distilled water 1000mL, pH7.2～7.6).Adopt Oxford cup to measure M
2 +the bacteriostatic activity of the crude extract of the meta-bolites of substratum to 5 strain multi-drug resistant bacterias.
Table 2: the resistance of multidrug resistant bacteria strain
Note: "+" represents this antibiosis to have resistance, "-" represents to have no drug resistance
Result is as shown in table 3, and streptomycete GIM4.116 adopts M
2 +the crude extract of the meta-bolites of substratum fermentation has stronger inhibition to resistance to 11 kinds of microbiotic coli strains 460 and bacterial strain 480, resistance to 13 kinds of antibiotic staphylococcus aureus strains 206 are also had to stronger inhibition, along with resistant organism Enrichment antibacterial circle diameter decreases, but still have obvious inhibition.The crude extract of the meta-bolites of streptomycete GIM4.116 is to resistance to 9 kinds of antibiotic Salmonellas bacterial strains 31 and the equal unrestraint effect of bacterial strain 47.Streptomycete GIM4.116 has great application potential aspect the control intestinal bacteria of multi-drug resistant and streptococcus aureus.
Table 3: the inhibition of streptomycete GIM4.116 tunning to multi-drug resistant bacteria
Note: "-" indicates to produce without pressing down circle
Embodiment 4: the inhibition of streptomycete GIM4.116 tunning to plant-bacterial-wilt pathogenic bacteria
Ralstonia solanacearum (Ralstonia solanacearum) GIM1.356(bacterial wilt of tomato pathogenic bacteria), R.solanacearum GIM1.74(potato bacterial wilt pathogenic bacteria), R.solanacearum GIM1.68(cotton Stalk Rot) provide by microbial strains preservation center, Guangdong Province, can buy from this preservation center.After adopting TM liquid nutrient medium to cultivate, be diluted to 10
7individual/mL, gets 100 μ L and coats (peptone 10g, glucose 5g on TM solid medium, hydrolysis casein food grade 1g, agar 15g, distilled water 1L, pH7.0), the inhibition of the crude extract of the meta-bolites of employing Oxford agar diffusion method mensuration streptomycete GIM4.116 fermentation to three strain plant-bacterial-wilt pathogenic bacterias.
Result is as shown in table 4, adopts M according to the method for embodiment 2
2 +substratum fermentation streptomycete GIM4.116, and obtain the crude extract of the meta-bolites of streptomycete GIM4.116.Crude extract all has stronger inhibition to the pathogenic bacteria bacterial strain that causes three kind of plant bacterial wilts, and the about 24mm of antibacterial circle diameter illustrates that streptomycete GIM4.116 is having great application potential aspect control plant-bacterial-wilt.
Table 4: the inhibition of streptomycete GIM4.116 tunning to Stalk Rot
Claims (7)
1. streptomycete (Streptomyces sp.) GIM4.116, its deposit number is CCTCC NO:M2013064.
2. the application of streptomycete claimed in claim 1 (Streptomyces sp.) GIM4.116 in preparation antibacterials.
3. application according to claim 2, is characterized in that, it is using streptomycete (Streptomyces sp.) GIM4.116 as fermentation strain, M
2 +substratum is as fermention medium, carries out the application in preparation antibacterials of meta-bolites after fermentation culture, described M
2 +every liter of substratum contains Fructus Hordei Vulgaris extract 10g, glucose 4g, and yeast extract 4g, surplus is water, pH7.8.
4. according to the application described in claim 2 or 3, it is characterized in that, described antibacterials are the medicine of Chinese People's Anti-Japanese Military and Political College enterobacteria (Eshcerichia coli) or streptococcus aureus (Staphylococcus aureus).
5. application according to claim 4, it is characterized in that, described intestinal bacteria are common intestinal bacteria or the intestinal bacteria with multi-drug resistant, and described streptococcus aureus is common streptococcus aureus or the streptococcus aureus with multi-drug resistant.
6. according to the application described in claim 2 or 3, it is characterized in that, described antibacterials are to suppress the medicine of the withered Raul Salmonella of green grass or young crops that causes plant-bacterial-wilt.
7. application according to claim 6, it is characterized in that, the withered Raul Salmonella of described green grass or young crops is bacterial wilt of tomato pathogenic bacteria (R.Solanacearum) GIM1.356, potato bacterial wilt pathogenic bacteria (R.Solanacearum) GIM1.74 or cotton Stalk Rot (R.Solanacearum) GIM1.68.
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