CN108220198A - It is a kind of that there is the marine actinomycete for inhibiting fungi activity - Google Patents
It is a kind of that there is the marine actinomycete for inhibiting fungi activity Download PDFInfo
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- CN108220198A CN108220198A CN201810084193.1A CN201810084193A CN108220198A CN 108220198 A CN108220198 A CN 108220198A CN 201810084193 A CN201810084193 A CN 201810084193A CN 108220198 A CN108220198 A CN 108220198A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N1/20—Bacteria; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N1/02—Separating microorganisms from their culture media
Abstract
A kind of to have the marine actinomycete for inhibiting fungi activity, it uses marine actinomycete ZOUSKL 019, is identified and is analyzed by following steps：a）It takes strain appropriate, cultivates in the incubator；It with the appropriate robe of transfer needle picking and thalline, is inoculated into seed culture fluid, obtains seed culture fluid；The seed culture fluid is taken to be inoculated into culture solution to ferment, obtains strain fermentation object；b）The resin for being enriched strain cultured solution addition acetone is stirred overnight, clear liquid is taken after filtering, is concentrated to dryness, obtains total medicinal extract；c）Medicinal extract mixes silica gel and crosses column, with petroleum ether：Chloroform：Methanol elution gradient obtains 10 component Fr1-Fr10；Taking active constituent 5, elution, purification on normal-phase silica gel pressured column, most afterwards through half preparation reversed-phase high performance liquid chromatography, Methanol-water elution respectively obtains compound 1, compound 2, compound 3 through SephadexLH -20；d）The compound obtained using srb assay.
Description
Technical field
The present invention relates to a kind of ocean unwrapping wire for having and inhibiting excrement bacteroid, Escherichia coli and Salmonella typhi activity
Bacterium belongs to microbial medicine technical field.
Background technology
Marine microorganism is important marine pharmaceutical resource, through FDA approvals listing and 20 seas in clinical research
In foreign drug, the real source of 17 marine drugs is marine microorganism, wherein 15 marine drugs be by sponge, coral,
The metabolism of the marine organisms such as fish symbiotic and epiphyte microorganism generates.Wherein marine actinomycete accounts in marine microorganism drugs discovery again
According to critical role.
Invention content
It is an object of the invention to using marine microorganism as research object, from existing known marine actinomycete ZOUSKL-
Find and find wherein to have in 019 its metabolite inhibit excrement bacteroid, Escherichia coli and Salmonella typhi activity, for food
What product safety, healthy diet provided safeguard has the marine actinomycete for inhibiting fungi activity.
The purpose of the present invention is by following technical solution to complete, a kind of to have the ocean unwrapping wire for inhibiting fungi activity
Bacterium, it uses marine actinomycete ZOUSKL-019, is identified and analyzed by following steps：
a）Strain fermentation：By the conventional method of culture microorganism, take strain appropriate, be inoculated on slant medium, 25-30
It is cultivated 3-5 days in DEG C incubator；With the appropriate robe of transfer needle picking and thalline, it is inoculated into the seed culture fluid containing 100ml
It in 500mL conical flasks, is placed in shaking table, 46-50h is cultivated under the conditions of 25-30 DEG C, 100-150r/min, obtain seed
Culture solution；The seed culture fluid is taken to be inoculated by 5% inoculum concentration in the 500mL conical flasks of built-in 150mL culture solutions, is placed in
25-30 DEG C, the shaking table top fermentation of 100-150r/min 5-10 days, before culture terminates, the trees of XAD-16 are added according to 30g/L
Fat, filtering resin is to get strain fermentation object；
b）The preparation of extract：The resin for being enriched strain cultured solution addition acetone is stirred overnight, clear liquid is taken after filtering, is concentrated
To doing, after acetone extraction resin 2 times, combining extraction liquid is concentrated to dryness, and obtains total medicinal extract；
c）The tracking separation of active constituent：Medicinal extract mixes silica gel and crosses column, with petroleum ether：Chloroform：Methanol elution gradient obtains 10 groups
Divide Fr1-Fr10；Each component carries out active testing through above-mentioned activity test method, take active constituent 5 through SephadexLH-
20, chloroform-methanol（1：1）Elution, purification on normal-phase silica gel pressured column most prepare reversed-phase high performance liquid chromatography through half afterwards, and Methanol-water is washed
It is de-, respectively obtain compound 1, compound 2, compound 3；
d）Active testing：Using srb assay with reference to the variation of micro- sem observation cellular morphology, obtained compound 1, compound 2, chemical combination
Object 3, which is respectively provided with, inhibits excrement bacteroid, Escherichia coli and Salmonella typhi activity, inhibits loop diameter point under 10 mg/mL concentration
It is not 10 mm, 8.5 mm, 12 mm.
As preferred：The step a）In, the composition of the seed culture fluid and culture solution is：1% glucose,
1% soy meal, 1% yeast extract, 0.5% soluble starch, 0.025% are dipotassium hydrogen phosphates, remaining is natural Chen Hai
Water is formulated, and pH value is adjusted to 7.2-7.4.
The present invention is using marine microorganism as research object, from its metabolism of existing known marine actinomycete ZOUSKL-019
Find and find wherein to have in product inhibit excrement bacteroid, Escherichia coli and Salmonella typhi activity, for food security, drink
Food health provides safeguard.
Specific embodiment
Below in conjunction with specific embodiment, the present invention will be described in detail：It is of the present invention a kind of with inhibition fungi
The marine actinomycete of activity, it uses marine actinomycete ZOUSKL-019, is identified and analyzed by following steps：
a）Strain fermentation：By the conventional method of culture microorganism, take strain appropriate, be inoculated on slant medium, 25-30
It is cultivated 3-5 days in DEG C incubator；With the appropriate robe of transfer needle picking and thalline, it is inoculated into the seed culture fluid containing 100ml
It in 500mL conical flasks, is placed in shaking table, 46-50h is cultivated under the conditions of 25-30 DEG C, 100-150r/min, obtain seed
Culture solution；The seed culture fluid is taken to be inoculated by 5% inoculum concentration in the 500mL conical flasks of built-in 150mL culture solutions, is placed in
25-30 DEG C, the shaking table top fermentation of 100-150r/min 5-10 days, before culture terminates, the trees of XAD-16 are added according to 30g/L
Fat, filtering resin is to get strain fermentation object；
b）The preparation of extract：The resin for being enriched strain cultured solution addition acetone is stirred overnight, clear liquid is taken after filtering, is concentrated
To doing, after acetone extraction resin 2 times, combining extraction liquid is concentrated to dryness, and obtains total medicinal extract；
c）The tracking separation of active constituent：Medicinal extract mixes silica gel and crosses column, with petroleum ether：Chloroform：Methanol elution gradient obtains 10 groups
Divide Fr1-Fr10；Each component carries out active testing through above-mentioned activity test method, take active constituent 5 through SephadexLH-
20, chloroform-methanol（1：1）Elution, purification on normal-phase silica gel pressured column most prepare reversed-phase high performance liquid chromatography through half afterwards, and Methanol-water is washed
It is de-, respectively obtain compound 1, compound 2, compound 3；
d）Active testing：Using srb assay with reference to the variation of micro- sem observation cellular morphology, obtained compound 1, compound 2, chemical combination
Object 3, which is respectively provided with, inhibits excrement bacteroid, Escherichia coli and Salmonella typhi activity, inhibits loop diameter point under 10 mg/mL concentration
It is not 10 mm, 8.5 mm, 12 mm.
Step a of the present invention）In, the composition of the seed culture fluid and culture solution is：1% glucose, 1%
Soy meal, 1% yeast extract, 0.5% soluble starch, 0.025% are dipotassium hydrogen phosphates, remaining is prepared for natural Chen Haishui
It forms, pH value is adjusted to 7.2-7.4.
Other embodiments of the invention, can be on the basis of disclosed above, can by numerical value and the simple replacement of feature
To obtain countless embodiments, and those skilled in the art can be advantageously carried out this hair on the basis of the present invention is understood
It is bright.
Claims (2)
1. a kind of have the marine actinomycete for inhibiting fungi activity, it is characterised in that it uses marine actinomycete ZOUSKL-019,
It is identified and is analyzed by following steps：
a）Strain fermentation：By the conventional method of culture microorganism, take strain appropriate, be inoculated on slant medium, 25-30
It is cultivated 3-5 days in DEG C incubator；With the appropriate robe of transfer needle picking and thalline, it is inoculated into the seed culture fluid containing 100ml
It in 500mL conical flasks, is placed in shaking table, 46-50h is cultivated under the conditions of 25-30 DEG C, 100-150r/min, obtain seed
Culture solution；The seed culture fluid is taken to be inoculated by 5% inoculum concentration in the 500mL conical flasks of built-in 150mL culture solutions, is placed in
25-30 DEG C, the shaking table top fermentation of 100-150r/min 5-10 days, before culture terminates, the trees of XAD-16 are added according to 30g/L
Fat, filtering resin is to get strain fermentation object；
b）The preparation of extract：The resin for being enriched strain cultured solution addition acetone is stirred overnight, clear liquid is taken after filtering, is concentrated
To doing, after acetone extraction resin 2 times, combining extraction liquid is concentrated to dryness, and obtains total medicinal extract；
c）The tracking separation of active constituent：Medicinal extract mixes silica gel and crosses column, with petroleum ether：Chloroform：Methanol elution gradient obtains 10 groups
Divide Fr1-Fr10；Each component carries out active testing through above-mentioned activity test method, take active constituent 5 through SephadexLH-
20, chloroform-methanol（1：1）Elution, purification on normal-phase silica gel pressured column most prepare reversed-phase high performance liquid chromatography through half afterwards, and Methanol-water is washed
It is de-, respectively obtain compound 1, compound 2, compound 3；
d）Active testing：Using srb assay with reference to the variation of micro- sem observation cellular morphology, obtained compound 1, compound 2, chemical combination
Object 3, which is respectively provided with, inhibits excrement bacteroid, Escherichia coli and Salmonella typhi activity, inhibits loop diameter point under 10 mg/mL concentration
It is not 10 mm, 8.5 mm, 12 mm.
2. according to claim 1 have the marine actinomycete for inhibiting fungi activity, it is characterised in that the step a）
In, the composition of the seed culture fluid and culture solution is：1% glucose, 1% soy meal, 1% yeast extract, 0.5%
Soluble starch, 0.025% are dipotassium hydrogen phosphates, remaining is formulated for natural Chen Haishui, and pH value is adjusted to 7.2-7.4.
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| CN201810084193.1A CN108220198A (en) | 2018-01-29 | 2018-01-29 | It is a kind of that there is the marine actinomycete for inhibiting fungi activity |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108410750A (en) * | 2018-01-29 | 2018-08-17 | 浙江海洋大学 | A kind of marine actinomycete with anti-tumor activity |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101691557A (en) * | 2009-09-30 | 2010-04-07 | 广东省农业科学院植物保护研究所 | Method for preparing marine actinomyces and metabolite thereof and application |
| CN102352327A (en) * | 2011-09-06 | 2012-02-15 | 大连理工大学 | Marine actinomycete L131 and metabolin, preparation method and application of metabolin |
| CN103114064A (en) * | 2013-03-08 | 2013-05-22 | 浙江省柑桔研究所 | Marine actinomycete with antibacterial activity to multiple plant pathogens |
| CN104744533A (en) * | 2015-01-30 | 2015-07-01 | 中国科学院南海海洋研究所 | Angucycline compounds and application of angucycline compounds in preparation of anti-tumour or antibacterial medicine |
| CN107299064A (en) * | 2016-09-29 | 2017-10-27 | 天津大学 | One plant of marine actinomycete S 19 3 with broad spectrum antibiotic activity |
-
2018
- 2018-01-29 CN CN201810084193.1A patent/CN108220198A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101691557A (en) * | 2009-09-30 | 2010-04-07 | 广东省农业科学院植物保护研究所 | Method for preparing marine actinomyces and metabolite thereof and application |
| CN102352327A (en) * | 2011-09-06 | 2012-02-15 | 大连理工大学 | Marine actinomycete L131 and metabolin, preparation method and application of metabolin |
| CN103114064A (en) * | 2013-03-08 | 2013-05-22 | 浙江省柑桔研究所 | Marine actinomycete with antibacterial activity to multiple plant pathogens |
| CN104744533A (en) * | 2015-01-30 | 2015-07-01 | 中国科学院南海海洋研究所 | Angucycline compounds and application of angucycline compounds in preparation of anti-tumour or antibacterial medicine |
| CN107299064A (en) * | 2016-09-29 | 2017-10-27 | 天津大学 | One plant of marine actinomycete S 19 3 with broad spectrum antibiotic activity |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108410750A (en) * | 2018-01-29 | 2018-08-17 | 浙江海洋大学 | A kind of marine actinomycete with anti-tumor activity |
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Application publication date: 20180629 |