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Lovastatin high-yield monascus and high-throughput screening method thereof

A screening method, lovastatin technology, applied in the field of microbiology, can solve the problems of low lovastatin yield, low screening efficiency, and complicated screening process of Monascus, achieving high accuracy, high throughput, and simple operation steps Effect

Active Publication Date: 2018-04-24
TIANSHUI NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The technical problem to be solved by the present invention is to provide a high-throughput screening method for lovastatin high-yielding Monascus bacterium and its high-yield lovastatin technology, and solve the screening process after mutagenesis of lovastatin-producing Monascus bacterium in the prior art Problems such as complexity, low screening efficiency, and low production of lovastatin from the existing Monascus

Method used

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  • Lovastatin high-yield monascus and high-throughput screening method thereof
  • Lovastatin high-yield monascus and high-throughput screening method thereof
  • Lovastatin high-yield monascus and high-throughput screening method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0032] Example 1. High-throughput screening of high-yield lovastatin strains of Monascus mutated by space

[0033] Weigh an appropriate amount of Monascus lyophilized powder, dilute it with peptone sodium chloride activation solution 50 times, spread it on a malt extract agar medium, and cultivate it at 30°C for 5-7 days. After the activation of the strain, the colonies of Monascus in the plate were scraped with an inoculating spatula, and inserted into a 96-well deep-well plate containing 1 ml of the primary fermentation medium, and cultured on a shaker at 27° C. and 180 rpm for 7 days. The initial screening fermentation medium components are: 7% rice flour, 1.5% ammonium chloride, 0.15% dipotassium hydrogen phosphate, 0.05% MgSO 4 ·7H 2 O, pH is natural.

[0034] Centrifuge the 96-well plate with fermentation broth at 4000 rpm for 5 minutes, use a multi-channel pipette to take 0.2 ml of the supernatant, adjust the pH to about 3.0 with phosphoric acid, add an equal volume of ethyl...

Embodiment 2

[0039] Example 2. Re-screening of high-yield lovastatin strain of Monascus 30192 carried by space

[0040] Six strains, including 37-2, B11-1, 20-3, 20-2, A25, and B40, were selected for shake flask re-screening. The seed solution of the above strain was inoculated into a 250 mL Erlenmeyer flask containing 40 mL of fermentation medium according to an inoculum amount of 7%, and cultured on a shaker at 27°C and 180 rpm. Fermentation medium components are 7% rice flour, 1.5% ammonium chloride, 0.15% dipotassium hydrogen phosphate, 0.05% MgSO 4 ·7H 2 O, pH is natural. After 7 days of fermentation, the production of lovastatin was measured.

[0041] The content of lovastatin was detected by liquid chromatography. The specific steps are as follows: centrifuge the fermentation broth at 4000 rpm for 5 minutes, take 4 mL of the supernatant, adjust the pH to 3 with phosphoric acid, add an equal volume of ethyl acetate, 50 ℃, 150 rpm water bath shaker Extraction for 2 hours, 3000rpm, centri...

Embodiment 3

[0043] Example 3. The influence of carbon source and carbon source content on the production of lovastatin

[0044] 7% carbon source, 1.5% peptone, 0.2% ammonium chloride, MgSO 4 ·7H 2 O 0.05% and potassium dihydrogen phosphate 0.15% are fermentation medium to detect the effects of glycerol, rice flour, lactose, glucose, and maltose as carbon sources on the production of lovastatin. The inoculum volume is 7%, and the liquid volume in a 250mL flask is 40mL After 7 days of fermentation in a shaker at 27°C and 180 rpm, the content of lovastatin in the fermentation broth was detected. The detection method is the same as in Example 2.

[0045] The result is image 3 As shown, when glycerol was used as the carbon source, the yield of lovastatin was the highest, followed by rice flour, with yields of 590 mg / L and 567.5 mg / L, respectively. Glycerol is not only used as a nutrient for monascus to absorb, but because of its molecular structure, it may be used as a precursor to promote the sy...

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Abstract

The invention relates to the technical field of microorganisms, and in particular to lovastatin high-yield monascus and a high-throughput screening method thereof. The high-throughput screening methodprovided by the invention has the characteristics of being simple in operating steps, high in throughput, high in accuracy, low in equipment input and the like; and the method, as being slightly changed, can be applied to high-throughput screening of production strains of other secondary metabolites. The monascus Monascus anka Mon20-2 (CCTCC M2015356) provided by the invention is high in lovastatin content; and with the application of a fermentation process provided by the invention, lovastatin yield in mass production can reach 2952.0mg / L, much better than that reported by the prior art.

Description

Technical field [0001] The invention relates to the technical field of microorganisms, in particular to a lovastatin high-producing monascus and a high-throughput screening method thereof. Background technique [0002] Monascus spp. is a genus of small filamentous saprophytic fungi, belonging to Ascomycotina (Ascomycotina), Plectomycetes (Plectomycetes), Eurotiales, and Monascaceae (Monascaceae). There is only one genus of Monascus, established in 1884 by French scholar Van Tieghem. The main species are M. anka, M. fuliginosus, M. purpureus and M. ruber. [0003] Monascus can produce primary metabolites of various aromatic substances such as alcohol, vinegar and acid, and various hydrolytic enzymes, which can produce high-quality aroma and sweet taste in fermented food. It is also an excellent brewing material. Especially the secondary metabolites produced by it, such as pigments, antibiotics, cholesterol inhibitors, Chinese medicine ingredients, etc., have become a hot spot of r...

Claims

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Application Information

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IPC IPC(8): C12N1/14C12N1/02C12P17/06C12R1/645
CPCC12N1/02C12N1/14C12P17/06C12N1/145C12R2001/645
Inventor 李一婧马伟超王廷璞包文生罗爱玉
Owner TIANSHUI NORMAL UNIV
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