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Rapid screening method of high-yielding coenzyme Q10 strain

A technology of high-yielding strains and coenzymes, applied in the preparation of test samples, measuring devices, instruments, etc., can solve the problems of high measurement results, large errors, and affecting screening efficiency, so as to improve efficiency, shorten the breeding cycle, and ensure reliability sexual effect

Active Publication Date: 2013-07-10
XIAMEN KINGDOMWAY GROUP +1
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AI Technical Summary

Problems solved by technology

Since the strain selection has to go through two stages of primary screening and re-screening, there are many single colonies in the primary screening, generally 500-1000, if a more tedious determination method is used, it will definitely affect the efficiency of strain screening
coenzyme Q 10 Conventional UV spectrophotometry and high performance liquid chromatography are usually used for detection. The disadvantages of these two detection methods are: (1) The conventional UV spectrophotometry results are high due to the influence of impurities such as nucleic acid and protein, and the error is large ; (2) High-performance liquid chromatography is complex and time-consuming, unable to measure a large number of samples, and the efficiency is low, which affects the overall breeding cycle
This method can quickly screen the mutagenized cells by color and cell size, but the visual resolution is limited, and some high-yielding strains are often missed. The extraction and detection methods are relatively time-consuming, which affects the overall screening efficiency.

Method used

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  • Rapid screening method of high-yielding coenzyme Q10 strain
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  • Rapid screening method of high-yielding coenzyme Q10 strain

Examples

Experimental program
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Effect test

Embodiment 1

[0021] Embodiment 1: Determination of the most suitable amount of reducing agent

[0022] Taking the reducing agent as sodium borohydride as an example:

[0023] The preparation concentration is 80mg / L oxidized coenzyme Q 10 For the standard product, accurately draw 3mL into the quartz cuvette, then add 2μL, 6μL, 12μL, 16μL, 20μL, 32μL, 40μL, 50μL of sodium borohydride solution with a concentration of 9mg / mL, shake evenly, and use absolute ethanol as For comparison, measure the absorbance at 275 nm after the bubbles disappear, and record the final stable value. The results are shown in Table 1.

[0024] Table 1 The most suitable amount of reducing agent

[0025]

[0026] It can be seen from Table 1 that after adding the reducing agent, the absorbance value at 275nm wavelength will gradually decrease with the increase of the dosage of sodium borohydride solution. It was determined that when 20 μL of sodium borohydride was added, coenzyme Q 10 has been fully restored.

[0...

Embodiment 2

[0032] Example 2: Preparation of Coenzyme Q10 Concentration-Absorbance Difference Standard Curve

[0033] Determination of oxidized coenzyme Q at concentrations of 8mg / L, 16mg / L, 32mg / L, 48mg / L, 64mg / L, and 80mg / L 10 Standards and Reduced Coenzyme Q 10 △A (oxidized OD275nm-reduced OD275nm) can be obtained from the absorbance value of the standard at 275nm, and then for different concentrations of coenzyme Q 10 Standard products do linear regression, that is, coenzyme Q 10 Concentration-absorbance difference standard curve, such as Figure 4 shown.

Embodiment 3

[0035] 1. Cell mutagenesis fermentation

[0036] The starting strain Rhodobacter sphaeroides (Rhodobacter sphaeroides) JDW-610 species conservation slant was separated by drawing a line, and then the mature single colony was inserted into a fresh slant, cultured at 32°C for 2 days, and then washed with 5 mL of sterile water. Transfer the suspension into a conical flask containing 25mL pH6.0 PBS buffer solution and several glass beads, mix well, add NTG to make the NTG concentration in the final bacterial solution 0.2mg / mL, and treat the bacterial solution in a water bath at 32°C for 15min. The reaction was terminated by diluting with pH 6.0 PBS buffer solution, and the bacterium solution after mutagenesis was applied to a plate medium containing 0.002 g / L sodium azide, and cultured at 32° C. for 5 days.

[0037] The Rhodobacter sphaeroides JDW-610 was deposited in the General Microbiology Center of the China Committee for the Collection of Microorganisms on December 21, 2010, ...

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Abstract

The invention provides a rapid screening method of a coenzyme high-yielding Q10 strain, and relates to the coenzyme Q10. The method comprise: (1) extracting coenzyme Q10 samples from a broth by using supersonic wave; (2) primarily screening high-content coenzyme Q10 strains by using a microplate reader; and (3) secondarily screening by using HPLC. The method is improved on the basis of a conventional UV spectrophotometry, the coenzyme Q10 samples can be obtained through an ultrasonic crushing method for the strain broth, the coenzyme Q10 samples are converted into reduced-form coenzyme Q10 by using a reducing agent, the reduced-form coenzyme Q10 is used as a contrast to eliminate errors caused by proteins, nucleic acids and other impurities in the extracted liquid, and meanwhile the microplate reader is used for rapid detection of the coenzyme Q10 samples in batches, thereby both improving determination efficiency and guaranteeing reliability of the method. Finally, the HPLC method is used for secondary screening of few primarily screened high-yield strains to obtain the high-yielding coenzyme Q10 strain, thereby greatly shortening a breeding period and increasing efficiency of screening the high-yielding coenzyme Q10 strain.

Description

technical field [0001] The present invention relates to coenzyme Q 10 , especially involving a coenzyme Q 10 A rapid screening method for high-yield strain breeding. Background technique [0002] coenzyme Q 10 It is one of the indispensable important elements in human life. It is an important physiologically active substance that can activate the energy of human cells, improve human immunity, enhance cell antioxidant capacity and delay aging. It has become a research hotspot in the field of biomedicine in the world. In recent years, it has been widely used in health care products, cosmetics and food industries. With coenzyme Q 10 With the increasing demand, scholars from all over the world are focusing on coenzyme Q 10 production research. The current production methods mainly include direct extraction method, chemical synthesis method and microbial fermentation method, among which microbial fermentation method has the most development prospect and is the production m...

Claims

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Application Information

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IPC IPC(8): G01N21/33G01N30/02G01N1/28
Inventor 郑毅朱志春陈金卿陈俊煌吴美琼
Owner XIAMEN KINGDOMWAY GROUP
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