CN1461807A - Fermentation method of producing antifungus active substance using ocean streptomycete - Google Patents
Fermentation method of producing antifungus active substance using ocean streptomycete Download PDFInfo
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- CN1461807A CN1461807A CN 02109774 CN02109774A CN1461807A CN 1461807 A CN1461807 A CN 1461807A CN 02109774 CN02109774 CN 02109774 CN 02109774 A CN02109774 A CN 02109774A CN 1461807 A CN1461807 A CN 1461807A
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Abstract
A process for preparing the antifungal active substance from S.albus var-fungatus Marinus Machengxing et al by fermenting includes such steps as choosing antifungal marine microbes, and fermenting. Its culture medium is prepared from soybean cake powder, starch, corn flour, potassium dihydrogen phosphate, potassium nitrate, magnesium sulfate, iron sulfate, and water. Its advantage is high purity of product.
Description
Technical field
The present invention relates to a kind of method of marine streptomyces fermentative production antifungus active substance, the method for the antimycotic mutation fermentative production of specifically a kind of marine actinomycete streptomyces albus antifungus active substance.
Background technology
As everyone knows, microbiotic is most important to human health, and people continually develop medicines such as new microbiotic, immunological reagent and tackle various new old stick-in-the-mud diseases such as Resistant strain, fungi infestation, AIDS.Because the excessive exploitation of land resources, the actinomycetes of seeking to produce new natural physiological active material from soil microorganisms are more and more difficult, so people have invested the ocean to sight.Biological phenomena in the ocean is much more ancient than land, live in biology in the ocean and be the land biology ten times, yet up to the present, the marine organisms kind that people can list only accounts for 10% of its total amount, that is studied then has only 1%, and the ocean is the treasure-house of new resources, and the marine microorganism that some are new can both be found every day in the whole world now, along with deepening continuously of marine scientific research, using modern biotechnology exploitation ocean new resources has become the focus that the current whole world is paid close attention to.The new biologically active substance of development research is used and develops into new high-tech industry from this huge treasure-house of marine microorganism, is one of important step that realizes the resource continuous development utilization of marine microorganism.
Relevant marine microorganism produces the research of antibacterial substance, as far back as the forties the research report is just arranged, from the isolating microorganism in ocean, 20～30% have anti-microbial activity, and cephamycin and Micronomicin Sulfate are exactly that marine microorganism produces and is applied to clinical.External as the U.S., Japan, Europe is dropped into very big strength Living marine resources is researched and developed (Fang Jinrui etc., Japan is from the new development of marine microorganism development of new biologically active substance research, China's marine drug, 1995,40,21-25, Zhang Rongqing, marine natural active substance foreign study is dynamic, China's the 5th the academic exploitation of ocean lakes and marhshes medicine Conference Papers collection (first volume), 1998,237-241, Guo Yuewei, the present Research of Europe marine drug and the notice that China's marine drug is studied, marine natural product and biochemical drug paper compilation, 2000,35-40).More external scholars (Abbanat D.et al, Cell wall active antifungal compoundsproduced by the.marine fungus hypoxylon oceanicum LL-15G256.I.Taxonomy and fermentation.J Antibiot (Tokyo) .1998; 51 (3): lucky youth is seen on 296-302., ridge, fermentation と industry, 1979,37 (6): 504-513, Okami, Y.andHotta, D.J.Antibiotics, 1979,32:964～966) from marine microorganism, obtained new or known antibiotics.China to the research of marine microorganism focus mostly in Guangdong, the ALONG COASTAL FUJIAN marine site, Huang Weizhen, Fang Jinrui (yellow dimension is true etc., dwell at the bottom of the ALONG COASTAL FUJIAN antimicrobial substance of actinomycetes and generation thereof, China's marine drug, 1991,3:1～6, Fang Jinrui etc., separation and the evaluation of the microbiotic 2B-B that marine actinomycete produces, China's microbiotic magazine, 1991. (4): 305～306) in the research of ALONG COASTAL FUJIAN marine microorganism, also obtained new and known microbiotic.
Marine site, Liaoning Province is vast, the marine actinomycete aboundresources, and the contriver is in Liaoning marine microorganism resource is investigated, separate and obtain 1116 strain marine actinomycetes, streptomyces accounts for 94.74%, contains white spore, pink spore, the ball spore, the cinder ash, blueness, grey purple and brown 7 monoids of ash, micromonospora accounts for 2.86%, and Nocardia accounts for 2.40%.The bacteriostatic activity experiment shows a wherein strain marine actinomycete, the secondary metabolite that is the antimycotic mutation of marine actinomycete streptomyces albus (S.albus var.fungatus Marinus Machengxing et al) is to human body skin pathomycete trichophyton, trichophyton gypseum, Candida albicans and subtilis, streptococcus aureus, intestinal bacteria all have very strong restraining effect, laboratory initial analysis research thinks that it may be the biologically active substance that suppresses the synthetic class of fungal cell wall, with KETOKONAZOL, aerosporin, the nystatin chromatography finds that relatively a few class microbiotic of its character and this are all not too identical, may be the novel anti biomass.The antifungus active substance that the antimycotic mutation of marine actinomycete streptomyces albus produces has good development prospect.
Summary of the invention
The object of the present invention is to provide the method for the antimycotic mutation fermentative production of a kind of marine actinomycete streptomyces albus antifungus active substance.
To achieve these goals, the technical solution used in the present invention is, on experimental basis in the past, marine streptomyces fermentative production antifungus active substance is optimized test with the homogeneous design method, select the optimal medium prescription of fermentative production antifungus active substance at last, specific operation process is as follows:
(1) antimycotic marine microorganism bacterial classification chooses
Choose seawater and ooze sample, separation and purification streptomycete therefrom, do indicator with Candida albicans, by digging piece method picking Candida albicans there is inhibiting marine streptomyces, marine streptomyces is identified, obtained the antimycotic mutation of marine actinomycete streptomyces albus (S.albus var.fungatus Marinus Machengxing et al);
(2) fermentative production active substance
Adopt shake flask culture to produce antifungus active substance, the composition of fermention medium is by weight percentage: soybean cake powder 1.5--2.5%, starch 0～3%, Semen Maydis powder 0.5～3.0%, potassium primary phosphate 0～0.25%, saltpetre 0～0.15%, sal epsom 0～0.25, ferric sulfate 0～0.005%, surplus are water;
The preferred version of above-mentioned substratum is: soybean cake powder 1.5--2.5%, starch 2--3%, Semen Maydis powder 1.0--3.0%, dipotassium hydrogen phosphate 0--0.2, saltpetre 0～0.1%, sal epsom 0～0.2%, ferric sulfate 0～0.003%, surplus are water.
Above-mentioned fermentation time is 24～168 hours, preferred 96 hours.
The present invention has following advantage
1. the method for fermentative Production antifungus active substance provided by the invention, the antifungus active substance content height of final product, the further development and use of active substance of being produced for the further antimycotic mutation of ocean streptomyces albus lay the first stone.
2. the used fermented material of substratum of the present invention all is common, cheap agricultural-food, can enlarge production on a large scale.
Description of drawings
Fig. 1 be in the active substance antibiotic concentration with the fermentation time change curve.
Embodiment
(1) antimycotic marine microorganism bacterial classification chooses
Coastal from Liaoning (ground such as Dalian black stone reef, Jinzhou Wang Jia shack, Panjin three road ditches) choose seawater and ooze sample, seawater and ooze sample add the 100ppm potassium bichromate with the synthetic agar of 3% sodium-chlor Gao Shi and make the high substratum of selecting, dilution coating plate culture, through 28 ℃ of cultivations 14 days, the picking streptomycete carried out separation and purification and cultivates.Do indicator with Candida albicans, select Candida albicans is had inhibiting marine streptomyces by digging the piece method, " classification of actinomycetes basis " with reference to Ruan Jisheng identified marine streptomyces, obtained the antimycotic mutation of marine actinomycete streptomyces albus (S.albus var.fungatus Marinus Machengxing et al).
(2) fermentative production of active substance
Adopt shake flask culture to produce the active substance of the above-mentioned antimycotic mutation of marine actinomycete streptomyces albus of obtaining.
A. material 1.1 bacterial strains
(1) the antimycotic mutation of marine actinomycete streptomyces albus, the coastal sampling separation in Liaoning obtains.
(2) detect bacterium: yeast Y761.2 substratum
(1) liquid Y76 substratum
Solid Y76 substratum had both added 2% agar in liquid Y76 substratum.
(2) fermention medium
Adopt shake flask culture to produce antifungus active substance, the composition of fermention medium is by weight percentage: A starch 0～3%, B Semen Maydis powder 0.5～3.0%, C potassium primary phosphate 0～0.25%, D saltpetre 0～0.15%, E sal epsom 0～0.25, F ferric sulfate 0～0.005%, G soybean cake powder 1.5-2.5%,, surplus is a water.
B. the preparation of method 2.1 fermented bacteriums
Get and go down to posterity at antimycotic mutation spore one ring of No. 1 cultured streptomyces albus of Gao Shi, streak inoculation in the plate of No. 1 substratum of Gao Shi, 28 ℃ of constant temperature culture 96 hours.2.2 active substance preparation of fermentation liquid
4 days bacterial classification of above-mentioned cultivation is scraped in the triangular flask that sterilized water and granulated glass sphere are housed, shaking up the back is linked in the given fermention medium of table 1 by 5%, transfer 121 ℃ of sterilizations of PH6.7-7.0 20 minutes: 28 ℃ of shaking tables, 180rpm constant temperature culture on the fermention medium, fermentation time is 24～168 hours.2.3 the preparation of the antimycotic mutation fermented supernatant fluid of streptomyces albus
The antimycotic mutation fermented liquid of streptomyces albus at high speed tabletop centrifuge centrifugal 1 minute of 13000rpm up and down, is got supernatant promptly.2.4 the measuring method 2.4.1 Oxford agar diffusion method of antibiotic concentration: a. is according to the height of the big or small guestimate active material concentration of inhibition zone, and b. is diluted to different multiples with fermented liquid, and the extension rate that can see inhibition zone is as bacteriostatic activity unit.2.4.2 two dosimetries: because streptomyces albus antibiotics that antimycotic mutation is produced is not also determined its structure, belong to unknown antibiotics, so make standard substance with nystatin, two dosimetries are measured the concentration of the antimycotic mutation antibiotics of streptomyces albus.2.5 the preparation of test organisms
Get a ring Y76 inclined-plane seed and be inoculated in the test tube that 10ml liquid Y76 substratum is housed, cultivated 24 hours for 28 ℃.2.6 the preparation of double-layer plate
Get 28 ℃ of Y76 bacterium liquid 0.2ml that cultivate 24 hours with transfer pipet, add and be equipped with in the triangular flask of the Y76 liquid nutrient medium of 50ml temperature between 50-55 ℃, getting 5ml after shaking up adds and to contain the 15ml that has an appointment and solidify the Y76 substratum and do promptly to get double-layer plate in the plate of lower floor.
C. result
Survey the fermented liquid active material concentration with two dosimetries.
The concrete composition and the fermented liquid antibiotic concentration result of fermention medium are as shown in table 1.
Table 1
| Embodiment number | Factor % | Fermented liquid antibiotic concentration (ppm) |
| A?????B?????C??????D??????E??????F?????????G | ||
| 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 | 0?????2.5???0.25???0.03???0.15???0.003?????2 0.5???1.5???0.25???0.06???0.05???0?????????2 1.0???0.5???0.20???0.12???0.25???0.003?????2 1.5???2.5???0.20???0.15???0.10???0?????????2 2.0???1.5???0.15???0??????0??????0.004?????2 2.5???0.5???0.15???0.06???0.20???0.001?????2 0?????3.0???0.10???0.09???0.05???0.004?????2 0.5???2.0???0.10???0.15???0.25???0.001?????2 1.0???1.0???0.05???0??????0.15???0.005?????2 1.5???3.0???0.05???0.03???0??????0.002?????2 2.0???2.0???0??????0.09???0.20???0.005?????2 2.5???1.0???0??????0.12???0.10???0.002?????2 2.0???3.0???0.2????0.05???0.1????0.003?????1.5 2.5???3.0???0.1????0??????0??????0?????????1.8 3.0???1.0???0.15???0??????0.05???0.001?????2.5 | ?47.98 ?24.84 ?52.39 ?10 ?15 ?88.69 ?67.36 ?29.93 ?62.22 ?99.18 ?94.85 ?105.31 ?110.25 ?99.23 ?148.79 |
The antimycotic mutation product of its streptomyces albus active substance change curve is in time seen Fig. 1.
As can be seen from Figure 1: active material concentration increases along with fermentation time and raises, and reaches maximum at 96 hours, then slowly descends.
Antimycotic mutation thalline of the marine actinomycete streptomyces albus of gained and fermented liquid detect its acute toxicology and skin irritation test through Sanitation and Anti Epidemic Station, Liaoning Prov. in the foregoing description, show that antimycotic mutation thalline of marine actinomycete streptomyces albus and fermented liquid are nontoxic, LD
50＞15g/kg body weight has no stimulation to skin.Active substance in its fermented liquid is to human body skin pathomycete trichophyton, trichophyton gypseum, and Candida albicans and subtilis, streptococcus aureus, intestinal bacteria all have the strong restraining effect of root.
Claims (5)
1. the fermentation process of a producing antifungus active substance using ocean streptomycete is characterized in that:
(1) chooses the antimycotic mutation of marine actinomycete streptomyces albus of marine streptomyces bacterial classification;
(2) adopt shake flask culture to produce antifungus active substance, the composition of fermention medium is by weight percentage: soybean cake powder 1.5-2.5%, starch 0～3%, Semen Maydis powder 0.5～3.0%, potassium primary phosphate 0～0.25%, saltpetre 0～0.15%, sal epsom 0～0.25, ferric sulfate 0～0.005%, surplus are water.
2. the fermentation process of antifungus active substance as claimed in claim 1, it is characterized in that consisting of of substratum: soybean cake powder 1.5--2.5%, starch 2--3%, Semen Maydis powder 1.0--3.0%, dipotassium hydrogen phosphate 0-0.2, saltpetre 0～0.1%, sal epsom 0～0.2%, ferric sulfate 0～0.003%, surplus are water.
3. the fermentation process of antifungus active substance as claimed in claim 1 is characterized in that fermentation time is 24～168 hours.
4. the fermentation process of antifungus active substance as claimed in claim 1 is characterized in that fermentation time is % hour.
5. the fermentation process of antifungus active substance as claimed in claim 1, it is characterized in that the antimycotic mutation of marine actinomycete streptomyces albus that step (1) obtains is to choose seawater and ooze sample, separation and purification streptomycete therefrom, do indicator with Candida albicans, by digging piece method picking Candida albicans there is inhibiting marine streptomyces, marine streptomyces is identified.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103088068A (en) * | 2011-11-04 | 2013-05-08 | 天津绿动植物营养技术开发有限公司 | Preparation method and application of soil rare actinomyces broth |
| CN106591184A (en) * | 2016-12-09 | 2017-04-26 | 河北路克希德生物科技有限公司 | Antifungal activity substance, and preparation method and application thereof |
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2002
- 2002-05-31 CN CN 02109774 patent/CN1461807A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103088068A (en) * | 2011-11-04 | 2013-05-08 | 天津绿动植物营养技术开发有限公司 | Preparation method and application of soil rare actinomyces broth |
| CN106591184A (en) * | 2016-12-09 | 2017-04-26 | 河北路克希德生物科技有限公司 | Antifungal activity substance, and preparation method and application thereof |
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