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High-throughput screening method for high-yield monascus pigment strain

A technology of monascus pigment and high-yielding strain, applied in the field of microbiology, can solve the problem of high price of large-scale equipment

Inactive Publication Date: 2014-04-16
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In particular, developed countries such as Europe and the United States have developed fully automatic high-throughput screening workstations, which can achieve efficient and high-throughput screening by applying the most advanced computer technology and robot technology. However, such large-scale equipment is quite expensive. Even in developed countries, only Large laboratories with strong financial resources have the purchasing power

Method used

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  • High-throughput screening method for high-yield monascus pigment strain
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  • High-throughput screening method for high-yield monascus pigment strain

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0034] (1) Preparation of Mycelia Suspension

[0035] At room temperature, scrape and wash the bacteria on the fresh slant together with the surface medium into a 10ml homogenizer, add an appropriate amount of sterile water and gently grind it into a uniform suspension, dilute to 10ml, shake evenly and set aside.

[0036] (2) Mutagenesis treatment

[0037] Place 10ml of mycelia suspension in a 9cm-diameter petri dish with a stirring bar, turn on the magnetic stirrer, and irradiate it for 3 minutes at a distance of 20cm from a 15W ultraviolet lamp, and then mix it with an equal volume of 1.0% lithium chloride solution (10ml) Mix evenly and let it stand for 20 minutes, the lethal rate is more than 90%.

[0038] (3) Primary screening

[0039]All mycelial suspensions after mutagenesis were sequentially inserted into 24 deep-well plates equipped with 1.2ml / well seed medium with an 8-hole row gun, 60 μl per well, and distributed as much as possible, and a total of 324 wells of mix...

Embodiment 1

[0053] Example 1. Feasibility demonstration of high-throughput detection of red yeast pigment output

[0054] Shake flask culture: dig a shovel of Monascus purpureus CGMCC3.4651 and inoculate it into a 500ml Erlenmeyer flask containing 50ml of seed medium, and culture it with shaking at 33°C and 220r / min for 42h. According to the inoculation amount of 5% (v / v), they were respectively transferred to 500ml Erlenmeyer flasks equipped with 50ml fermentation medium, and fermented at 33°C and 220r / min for 48h. Samples were taken every 6 hours to obtain fermentation broth samples.

[0055] Select 30 samples of Monascus fermented broth obtained by sampling at each stage, and use a microplate reader and an ultraviolet spectrophotometer to measure the color value of Monascus pigment.

[0056] According to the detection method of prior art, adopt ultraviolet spectrophotometer method: for each shake flask fermented liquid sample of Monascus, draw 1ml respectively and add and be equipped ...

Embodiment 2、24

[0063] Example 2, 24 deep well plates are used for the feasibility verification of fermenting Monascus

[0064] Shake flask culture: dig a shovel of Monascus and inoculate it into a 500ml Erlenmeyer flask filled with 50ml of seed medium, shake and culture at 33°C and 220r / min for 42h, and the formulation of the seed medium is the same as before. According to the inoculation amount of 5% (v / v), transfer to the 500ml Erlenmeyer flask that 50ml fermentation medium (formula is the same as before) is housed respectively, 33 ℃, 220r / min fermentation 48h. Samples were taken every 6 hours, and at least 3 parallels were performed for rapid determination with a microplate reader.

[0065] 24-well deep-well plate culture: dig 2cm from the slope 2 1. Put the 3mm thick bacterial block into a 10ml homogenizer, add an appropriate amount of sterile water and gently grind it into a uniform suspension, then dilute the suspension to 10ml, and shake evenly. Insert into a 24-deep-well plate cont...

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Abstract

The invention relates to a high-throughput screening method for a high-yield monascus pigment strain. The method comprises the following steps of all mutated monascus suspensions to a deep-well plate for mixed culturing, performing high-throughput detection to obtain a mixture with the highest yield, performing plate separation and purification, and performing shake flask fermentation and detection to obtain a pure high-yield strain. The complete high-throughput screening method integrating a deep-well plate culturing technology and microplate reader detection is convenient, rapid, accurate and efficient.

Description

technical field [0001] The invention belongs to the field of microbiology, and relates to a method used in the fields of microbial mutation breeding and fermentation engineering to realize high-throughput screening of strains, in particular to a high-throughput screening method for high-oxygen-consuming pigment-producing fungi. Background technique [0002] Red yeast rice, called Danqu in ancient times, originated in China. It is a natural pigment produced by the Monascus purpureus strain (Monascus purpureus) during the growth and metabolism process. It is also the only edible natural pigment produced by microorganisms in the world. It has a production and application history of more than 1,000 years in China, and has been widely used as a food coloring agent and flavoring agent for a long time. At present, it is mainly obtained through traditional solid-state fermentation product red yeast rice extract and submerged liquid fermentation of Monascus red yeast extract. Compar...

Claims

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Application Information

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IPC IPC(8): C12N15/01C12N13/00C12R1/645
Inventor 储炬谭俊王永红李友元庄英萍张嗣良郭元昕
Owner EAST CHINA UNIV OF SCI & TECH
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