Method for quickly screening high-yield astaxanthin phaffia rhodozyma strains

A technology of Phaffia rhodochrous and yeast strains is applied in the field of rapid screening of high-yielding astaxanthin strains of Phaffia rhodozyma, which can solve the problems of high cost, complicated chemical synthesis method of astaxanthin, etc. simple craftsmanship

Inactive Publication Date: 2018-03-09
BEIJING FORESTRY UNIVERSITY
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Problems solved by technology

The chemical synthesis of astaxanthin requires multi-step chemical and biocatalytic re

Method used

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  • Method for quickly screening high-yield astaxanthin phaffia rhodozyma strains

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Embodiment 1

[0030] Activation: After activation, the wild-type Phaffia rhodozyma strain was cultured in a constant temperature incubator at 22°C for 125 hours at a speed of 120r / min;

[0031] Mutagenesis: Take the wild-type single colony strain and add it to the seed medium for 24 hours on a shaking table with a rotation speed of 120-130r / min and a temperature of 22-25°C. The time is 160s, the mutagenized bacterial solution is transferred to a centrifuge tube containing sterile saline, after gradient dilution, spread on a plate, and incubate at 22°C for 120h;

[0032] Preliminary screening: the single colony grown on the plate was transferred to a 24-well plate with 2mL of culture solution for shaking culture at a rotation speed of 125r / min, and the dark orange mutant strain was selected as the candidate strain;

[0033] Re-screening: the candidate strains are made into temporary mounts, observed with a laser scanning confocal microscope, and mutant strains with higher fluorescence intens...

Embodiment 2

[0040] Activation: activation of the wild-type Phaffia rhodozyma strain, cultured in a constant temperature incubator at 25°C and 125r / min for 120h;

[0041] Mutagenesis: Take a small amount of cultured strains and add them to the seed medium, culture them on a shaking table for 24 hours, and mutagenize the yeast with plasma beams at room temperature at normal pressure and constant pressure for 180 seconds. Transfer the mutagenized bacteria solution into a centrifuge tube containing sterile saline Spread the plate after serial dilution and incubate at 22°C for 120 hours;

[0042] Preliminary screening: the strains were transferred to a 24-well plate with 2mL of culture medium, cultured on a shaker at 120r / min for 46 hours, and dark orange mutant strains were selected as candidate strains;

[0043] Re-screening: the candidate strains are made into temporary slides, observed with a laser scanning confocal microscope, and mutant strains with higher fluorescence intensity are scre...

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Abstract

The invention relates to a method for quickly screening high-yield astaxanthin phaffia rhodozyma strains (Phaffia rhodozyma CGMCC2.1557). The method includes steps: 1) activating and culturing wild-type phaffia rhodozyma strains in a constant-temperature incubator at 22-25 DEG C, taking a small quantity of single colonies to add into a seed medium, and performing shake cultivation by 120-130r/min;2) performing ARTP (atmospheric and room temperature plasma) mutagenesis of the wild-type phaffia rhodozyma strains for 150-180s; 3) transferring bacteria liquid into a centrifugal tube after mutagenesis, and performing gradient dilution and flat plate coating; 4) transferring the strains to a 24-pore plate, performing shake cultivation for 45-50h, and observing through a confocal laser scanningmicroscope to screen candidate mutant strains; 5) carrying out shake cultivation in the seed medium; 6) performing proportional inoculation of seed liquid in a fermentation medium, and starting shakecultivation; 7) ultrasonically extracting astaxanthin for 1-1.2h, and detecting the astaxanthin content of fermented liquid by high performance liquid chromatography; 8) screening out high-yield astaxanthin mutant strains, wherein yield is increased by 25-55% compared with that of the mild-type strains. The high-yield astaxanthin phaffia rhodozyma strains are screened by two steps including colonycolor primary screening and cell fluorescence intensity re-screening, the method is simple in operation, safe and free of pollution, the mutant strains are stable, and industrial production cost is reduced.

Description

technical field [0001] The invention relates to a method for rapidly screening a high-yield astaxanthin-producing Phaffia rhodozyma strain (Phaffia rhodozymaCGMCC2.1557). Background technique [0002] Astaxanthin, also known as astaxanthin, lobster shell pigment. The chemical name is 3,3'-dihydroxy-4,4'-diketo-β,β'-carotene, which is a keto carotenoid. Pure astaxanthin powder is dark reddish brown, insoluble in water, and easily soluble in organic solvents such as acetone, chloroform and benzene. Astaxanthin widely exists in the biological world, especially in shrimp, crab, fish, algae, yeast and bird feathers. It is one of the main carotenoids in marine organisms. Since the 1990s, astaxanthin has attracted great attention and done a lot of research in scientific circles at home and abroad. Astaxanthin has significant effects on coloring, anti-oxidation, anti-cancer and enhancing immune function. Astaxanthin also participates in the physiological metabolism of vision, th...

Claims

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Application Information

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IPC IPC(8): C12N13/00C12R1/645
CPCC12N13/00
Inventor 任迪峰马珺申灿灿王璟悦
Owner BEIJING FORESTRY UNIVERSITY
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