CN102838650B - Method of extracting dustanin from Moelleriella ochracea - Google Patents
Method of extracting dustanin from Moelleriella ochracea Download PDFInfo
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- CN102838650B CN102838650B CN201210288827.8A CN201210288827A CN102838650B CN 102838650 B CN102838650 B CN 102838650B CN 201210288827 A CN201210288827 A CN 201210288827A CN 102838650 B CN102838650 B CN 102838650B
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- mycelium
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Abstract
The invention discloses a method of extracting dustamin from moelleriella ochracea. The method includes that a bacterial strain is firstly activated; primary culture and secondary culture are sequentially carried out; fermentation liquor is sucked and filtered; mycelium is dried, ground, immersed by organic solvents, and rotatably steamed to obtain concrete; a little organic solvents are used for dissolving silicagel columns of 200-300 meshes on the concrete; gradient elution with petroleum ether and acetone in a volume ratio of 50:1 is carried out on the concrete; partial eluent with the petroleum ether and acetone in a volume ratio of 20:1 is collected and concentrated through rotary steam; chloroform is precipitated, and dried by natural volatilization; and methyl alcohol is crystallized to form the dustamin in the moelleriella ochracea. The method of extracting the dustmin from the moelleriella ochracea is simple in raw materials drawing, simple in extraction procedures, low in cost, and high in purity of produced compounds.
Description
Technical field
The present invention relates to a kind ofly from reddish brown little Mo, strangle the method for extracting dustanin bacterium.
background technology
Reddish brown little Mo strangle bacterium (
moelleriella ochracea) be Ascomycota, minute Gammaproteobacteria, Hypocreales, Clavicipitaceae, do not strangle Pseudomonas, it is the important pathogen fungi of aleyrodid, made full use of its Natural control action to aleyrodid in the past, aspect biological control, obtained unusual effect, along with going deep into this bacterium research comprehensively, people are separated from its meta-bolites, be purified into some activated materials, as dustanin, and its molecular formula C
30h
50o
2, molecular weight 444, is five yuan of carbocyclic ring materials of triterpenes, has the effect of anti-mycobacterium tuberculosis, IC
50=12.5ug/ml, visible, do not strangle bacterium and medically there is potential using value.
Summary of the invention
The object of the invention is to disclose and a kind ofly from reddish brown little Mo, strangle the method for extracting dustanin bacterium, to promote its pharmacological further research and better development and application clinically.
The technical scheme that the present invention takes is as follows:
From reddish brown little Mo, strangle a method of extracting dustanin bacterium, comprise the following steps:
1) reddish brown little Mo strangles after bacterium activation, get in bacterium piece inoculation PDB substratum, under 120～160r/min, 20～28 ℃ of conditions, cultured continuously 5～10 days is elementary cultivation, by 8%～10% inoculum size, be again transferred in PDB substratum, continuing cultured continuously under 120～160r/min, 20～28 ℃ of conditions 25～30 days is secondary cultivation;
2) bacterium liquid is separated with mycelium, after mycelium is dry at 28-35 ℃, grind to form powdery, the ethyl acetate that adds 1～2 times of volume is soaked 10～14 hours, and ultrasonic 20～40 minutes, soak solution revolved and steams to obtain mycelium medicinal extract, reclaimed ethyl acetate; Bacterium liquid is extracted with ethyl acetate, and extraction liquid concentrated by rotary evaporation obtains bacterium immersion cream, reclaims ethyl acetate;
3) mycelium medicinal extract and bacterium immersion cream are merged, upper 200～300 order silicagel columns, with sherwood oil and acetone gradient elution, collect sherwood oil: acetone volume ratio=20:1 part elutriant, concentrated by rotary evaporation, chloroform dissolves, lysate volatilizes naturally, then obtains dustanin through recrystallizing methanol.
Described bacterium liquid is extracted with ethyl acetate concrete operations: by bacterium liquid and ethyl acetate by volume 1:1 mix, pour in separating funnel, shaking flask extraction 20～40min, static 12～24 hours, extracts 1-3 time.
Described ultrasonic power is 300～500W, and frequency is 30～40KHz.
Being prepared as of described PDB substratum: 200g potato, 20g glucose, boiling water boiling 30min adds single water that steams and is settled to 1L after 4 layers of filtered through gauze.
Remarkable advantage of the present invention:
1) raw material is drawn materials simply;
2) extraction step is simple, and cost is low;
3) compound purity making is high.
Accompanying drawing explanation
Fig. 1 is the purity proof diagram of the dustanin of extraction.
Embodiment
embodiment 1
1) get reddish brown little Mo and strangle bacterium (Qiu Junzhi etc., reddish brown little Mo strangles bacterium in Chinese discovery. fungus journal, 2009.28 (1): 148-150) be material, the bacterium piece that under aseptic condition, picking length and width are about 1cm is to PDB substratum (200g potato, 20g glucose, boiling water boiling 30min, after 4 layers of filtered through gauze, add single water that steams and be settled to 1L, divide and install in 250ml triangular flask, every bottled 100ml, sterilizing 25min under 121 ℃ of high pressure) in, at 160r/min, under 25 ℃ of conditions, cultured continuously 8 days is elementary cultivation, by 9% inoculum size, be again transferred in PDB substratum, continuation is at 160r/min, under 25 ℃ of conditions, cultured continuously 28 days is secondary cultivation,
2) bacterium liquid and mycelium are separated, mycelium is at room temperature dry grinds to form powdery afterwards, and the ethyl acetate that adds 2 times of volumes (w/v) is soaked 12 hours, and ultrasonic 30 minutes, soak solution revolved and steams to obtain medicinal extract, reclaimed ethyl acetate; By bacterium liquid and ethyl acetate by volume 1:1 mix, pour in separating funnel, shaking flask extraction 30min, static 16 hours, pour out ethyl acetate, its concentrated by rotary evaporation is obtained to bacterium immersion cream, reclaim ethyl acetate.
3) merge bacterium immersion cream and mycelium medicinal extract, chloroform dissolves medicinal extract, and upper 200 order silicagel columns, with sherwood oil: acetone volume ratio=50-10:1 gradient elution, collection sherwood oil: acetone volume ratio=20:1 part elutriant.Concentrated by rotary evaporation, chloroform dissolves and washes out, and naturally volatilizes, and recrystallizing methanol obtains reddish brown little Mo and strangles the dustanin in bacterium.
4) purity of dustanin is verified by a silica-gel plate, as shown in Figure 1.Developping agent is methyl alcohol: methylene chloride volume ratio=1:20, R
f=0.43, according to document (Isaka M, Hywel-Jones NL, Sappan M, Mongkolsamrit S, Saidaengkham S. Hopane triterpenes as chemotaxonomic markers for the scale insect pathogens
hypocrellas. lat. and
aschersonia. Mycological Research, 2009,113 (4): 491-497) can determine the dustanin that the purity that obtains is 99%.
Extraction yield: 3.23%(extraction yield %=pure dustanin weight/bacterium immersion cream and mycelium medicinal extract total amount * 100%).
embodiment 2
1) get reddish brown little Mo and strangle bacterium (Qiu Junzhi etc., reddish brown little Mo strangles bacterium in Chinese discovery. fungus journal, 2009.28 (1): 148-150) be material, the bacterium piece that under aseptic condition, picking length and width are about 1cm is to PDB substratum (200g potato, 20g glucose, boiling water boiling 30min, after 4 layers of filtered through gauze, add single water that steams and be settled to 1L, divide and install in 250ml triangular flask, every bottled 100ml, sterilizing 25min under 121 ℃ of high pressure) in, at 120r/min, under 20 ℃ of conditions, cultured continuously 5 days is elementary cultivation, by 8%% inoculum size, be again transferred in PDB substratum, continuation is at 120r/min, under 20 ℃ of conditions, cultured continuously 25 days is secondary cultivation,
5) bacterium liquid and mycelium are separated, mycelium is at room temperature dry grinds to form powdery afterwards, and the ethyl acetate that adds 1 times of volume (w/v) is soaked 10 hours, and ultrasonic 20 minutes, soak solution revolved and steams to obtain medicinal extract, reclaimed ethyl acetate; By bacterium liquid and ethyl acetate by volume 1:1 mix, pour in separating funnel, shaking flask extraction 20min, static 12 hours, pour out ethyl acetate, its concentrated by rotary evaporation is obtained to bacterium immersion cream, reclaim ethyl acetate.
6) merge bacterium immersion cream and mycelium medicinal extract, chloroform dissolves medicinal extract, and upper 200 order silicagel columns, with sherwood oil: acetone volume ratio=50-10:1 gradient elution, collection sherwood oil: acetone volume ratio=20:1 part elutriant.Concentrated by rotary evaporation, chloroform dissolves and washes out, and naturally volatilizes, and recrystallizing methanol obtains reddish brown little Mo and strangles the dustanin in bacterium.
7) obtaining purity is 99% dustanin.
Extraction yield: 3. 01%(extraction yield %=pure dustanin weight/bacterium immersion cream and mycelium medicinal extract total amount * 100%).
embodiment 3
1) get reddish brown little Mo and strangle bacterium (Qiu Junzhi etc., reddish brown little Mo strangles bacterium in Chinese discovery. fungus journal, 2009.28 (1): 148-150) be material, the bacterium piece that under aseptic condition, picking length and width are about 1cm is to PDB substratum (200g potato, 20g glucose, boiling water boiling 30min, after 4 layers of filtered through gauze, add single water that steams and be settled to 1L, divide and install in 250ml triangular flask, every bottled 100ml, sterilizing 25min under 121 ℃ of high pressure) in, at 160r/min, under 28 ℃ of conditions, cultured continuously 10 days is elementary cultivation, by 10% inoculum size, be again transferred in PDB substratum, continuation is at 160r/min, under 28 ℃ of conditions, cultured continuously 30 days is secondary cultivation,
8) bacterium liquid and mycelium are separated, mycelium is at room temperature dry grinds to form powdery afterwards, and the ethyl acetate that adds 2 times of volumes (w/v) is soaked 14 hours, and ultrasonic 40 minutes, soak solution revolved and steams to obtain medicinal extract, reclaimed ethyl acetate; By bacterium liquid and ethyl acetate by volume 1:1 mix, pour in separating funnel, shaking flask extraction 40min, static 24 hours, pour out ethyl acetate, its concentrated by rotary evaporation is obtained to bacterium immersion cream, reclaim ethyl acetate.
9) merge bacterium immersion cream and mycelium medicinal extract, chloroform dissolves medicinal extract, and upper 300 order silicagel columns, with sherwood oil: acetone volume ratio=50-1:1 gradient elution, collection sherwood oil: acetone volume ratio=20:1 part elutriant.Concentrated by rotary evaporation, chloroform dissolves and washes out, and naturally volatilizes, and recrystallizing methanol obtains reddish brown little Mo and strangles the dustanin in bacterium.
10) obtaining purity is 99% dustanin.
Extraction yield: 3.14%(extraction yield %=pure dustanin weight/bacterium immersion cream and mycelium medicinal extract total amount * 100%).
comparative example 1
Substratum sabouraud culture medium, its formula: peptone 10.0 g, glucose 40.0 g, add single water that steams and are settled to 1L after mixing, and divide and install in 250ml triangular flask, every bottled 100 ml, sterilizing 20 min under 121 ℃ of high pressure, other step is with embodiment 1.
Extraction yield: 1.52%(extraction yield %=pure dustanin weight/bacterium immersion cream and mycelium medicinal extract total amount * 100%).
Contrast
embodiment 2
Substratum M102 substratum, its formula: sucrose 30g, malt extract 20 g, peptone 2.0 g, yeast extract 1.0 g, KCl 0.5 g, MgSO
40.5 g, KH
2pO
40.5 g, adds single water that steams and is settled to 1L after mixing, and divide and install in 250ml triangular flask, every bottled 100ml, sterilizing 20min under 121 ℃ of high pressure, other step is with embodiment 1.
Extraction yield: 1.78%(extraction yield %=pure dustanin weight/bacterium immersion cream and mycelium medicinal extract total amount * 100%).
Claims (1)
1. from reddish brown little Mo, strangle a method of extracting dustanin bacterium, it is characterized in that the method comprises the following steps:
1) reddish brown little Mo strangles after bacterium activation, get in bacterium piece inoculation PDB substratum, under 120～160r/min, 20～28 ℃ of conditions, cultured continuously 5～10 days is elementary cultivation, by 8%～10% inoculum size, be again transferred in PDB substratum, continuing cultured continuously under 120～160r/min, 20～28 ℃ of conditions 25～30 days is secondary cultivation;
2) bacterium liquid is separated with mycelium, after mycelium is dry at 28-35 ℃, grind to form powdery, the ethyl acetate that adds 1～2 times of volume is soaked 10～14 hours, and ultrasonic 20～40 minutes, soak solution revolved and steams to obtain mycelium medicinal extract, reclaimed ethyl acetate; Bacterium liquid is extracted with ethyl acetate, and extraction liquid concentrated by rotary evaporation obtains bacterium immersion cream, reclaims ethyl acetate;
3) mycelium medicinal extract and bacterium immersion cream are merged, upper 200～300 order silicagel columns, with sherwood oil and acetone gradient elution, collect sherwood oil: acetone volume ratio=20:1 part elutriant, concentrated by rotary evaporation, chloroform dissolves, lysate volatilizes naturally, then obtains dustanin through recrystallizing methanol;
Described bacterium liquid is extracted with ethyl acetate concrete operations: by bacterium liquid and ethyl acetate by volume 1:1 mix, pour in separating funnel, shaking flask extraction 20～40min, static 12～24 hours, extracts 1-3 time;
Described ultrasonic power is 300～500W, and frequency is 30～40KHz.
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| CN103276037B (en) * | 2013-06-09 | 2014-10-29 | 福建农林大学 | Method for extracting beauveria bassiana lactide I from moelleriellaochracea |
| CN103276041B (en) * | 2013-06-09 | 2014-07-30 | 福建农林大学 | Method for extracting hopene from moelleriellaochracea |
| CN103265599B (en) * | 2013-06-09 | 2015-04-01 | 福建农林大学 | Method for extracting ergosterol from Moelleriella ochracea |
| CN103290089B (en) * | 2013-06-15 | 2014-07-30 | 福建农林大学 | Method for high-efficient extraction of 3 beta-acetyl-15 alpha, 22-dihydroxyhopane/hopanone from moelleriella ochracea |
| CN103290088B (en) * | 2013-06-15 | 2014-10-08 | 福建农林大学 | Method for extracting triterpenoid type substance, namely zeorin from moelleriella ochracea |
| CN103540577B (en) * | 2013-09-25 | 2015-04-29 | 福建农林大学 | Culture medium and method for producing lipase from moelleriella ochracea through fertilization |
| CN107955819A (en) * | 2017-12-23 | 2018-04-24 | 福建农林大学 | A kind of culture medium and method for not strangling bacterium fermenting and producing triterpene |
| CN115385979B (en) * | 2022-09-07 | 2024-01-30 | 福建农林大学 | Method for extracting hopane type triterpene compound from Raffaelea lauricola |
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