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Astaxanthin-producing strain, mutagenesis and screening method and application thereof

A technology for the production of bacterial strains and astaxanthin, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve the problems of long cycle, pollution, low astaxanthin content, etc., achieve stable output and large application prospects Effect

Active Publication Date: 2013-09-18
XIAMEN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The price of chemically synthesized astaxanthin is relatively cheap, and it still has certain competitiveness in feed additives. However, its structure is mostly cis, and the utilization rate of animals is low. With people's awareness of environmental protection, it is facing the threat of being eliminated. ; For countries with developed shrimp and crab processing industries, crustacean waste is a good source of astaxanthin, but its astaxanthin content is very low, and problems such as organic solvent pollution in the extraction process and low product purity are limited. Its further development; the content of astaxanthin in Haematococcus pluvialis generally accounts for 0.2% to 2% of dry cell weight, up to 4%. It is an excellent source of astaxanthin, so it has been widely studied and However, due to the extremely harsh growth conditions of Haematococcus pluvialis, high requirements on water quality, environment and light, long cycle, large floor area and other factors, its large-scale production is relatively difficult; Phaffia rhodozyme is a microorganism The strain with the second highest content of astaxanthin in the medium, due to its advantages of simple cultivation method, short cycle and high astaxanthin activity, people have gradually shifted their attention from Haematococcus pluvialis to Phaffia rhodozyme in the past two decades

Method used

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  • Astaxanthin-producing strain, mutagenesis and screening method and application thereof
  • Astaxanthin-producing strain, mutagenesis and screening method and application thereof
  • Astaxanthin-producing strain, mutagenesis and screening method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034](1) The starting strain of Phaffia rhodozyma was inoculated into the slant of YM agar medium, and cultured at 22° C. for 3 days. YM agar medium (g / L) is glucose 10, peptone 5, yeast powder 3, wort juice 3, pH 5.0.

[0035] (2) Pick a loop of bacteria from the activated slant and inoculate it into a shake flask, culture at 22° C. and 200 rpm for 36 hours, wherein the culture medium of the shake flask is YEPD. YEPD medium (g / L) is glucose 20, peptone 20, yeast powder 10; natural pH.

[0036] (3) Protoplast preparation: the above-mentioned cultivated bacterial liquid was collected by centrifugation to collect the bacterial cells, and the bacterial cells were washed twice with 0.6 times volume of sodium citrate buffer solution (1.0 mol / L). Then add 0.6 times the volume of wall-breaking enzyme mixed solution (0.5% wall-breaking enzyme, 0.1% cellulase and 0.01% lysozyme) to the thalline, at 28 ° C, 54 rpm shaker for 60 minutes; take out the treated bacteria solution, and the...

Embodiment 2

[0069] (1) The experimental procedure is the same as (1)-(2) of Example 1.

[0070] (2) Protoplast preparation: the cultured bacterial solution was collected by centrifugation, and the bacterial cells were washed twice with 1.0 times volume of sodium citrate buffer solution (0.8 mol / L). Then add 1.0 times the volume of wall-breaking enzyme mixture (0.6% wall-breaking enzyme, 0.05% cellulase and 0.005% lysozyme) to the thalline, at 30 ° C, place 40min in a 54rpm shaker; take out the treated bacteria solution, and the protoplasts were collected by centrifugation.

[0071] (4) The prepared protoplasts were washed twice with 1.0 times volume of sodium citrate buffer (0.8mol / L), and then adjusted to make its concentration approximately 10 with this sodium citrate buffer. 7 ~10 8 / mL.

[0072] (5) Add 1.0 times the volume of 20 μg / mL NTG to the treated protoplasts, and place in a shaker at 22°C and 54 rpm for 30 minutes;

[0073] (6) The liquid treated with NTG was taken out, th...

Embodiment 3

[0076] The astaxanthin production strain obtained in Example 1 was inserted into YM medium for shake flask culture, the liquid filling volume was 8% to 12%, the inoculum volume was 10%, 22°C constant temperature, 200rpm, cultured for 5 days, the result Such as Figure 9 shown. from Figure 9 It can be seen that the astaxanthin production strain is cultured in shake flasks on the YM medium, and the biomass reaches a maximum value of 7.083g / L at 46h. According to the chromatographic conditions provided in Example 1, it is recorded that astaxanthin reaches The highest is 25.57mg / L, at this time the biomass is 6.233g / L, that is, the astaxanthin content is 4743mg / kg (dry cell weight).

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Abstract

The invention discloses an astaxanthin-producing strain, a mutagenesis and screening method and application thereof, and relates to a microbiological strain and an obtaining method thereof. The astaxanthin-producing strain is a mutagenic strain N1806-04 of phaffia rhodozyma. The method comprises the following steps of: preparing phaffia rhodozyma protoplast by an enzyme method by taking the phaffia rhodozyma as an original strain; and selectively breeding the astaxanthin-producing strain by an NTG mutagenesis method, a beta-ionone screening method and the like to obtain the astaxanthin-producing strain, wherein the yield of the astaxanthin is stable after five times of transfer of culture. By performing scale-up culturing on the astaxanthin-producing strain in a fermentation tank, the yield and the content of the astaxanthin can reach 500 to 600mg / L and 5,000 to 6,300mg / kg (dry weight of cell) respectively and the biomass can reach 80 to 110g / L. The yield and the content of the astaxanthin and the corresponding biomass all meet the requirement of industrial production and have a great application prospect.

Description

technical field [0001] The invention relates to a microbial strain and an acquisition method thereof, in particular to an astaxanthin production strain and a mutagenesis screening method and application thereof. Background technique [0002] Astaxanthin, also known as astaxanthin, astaxanthin, lobster shell pigment, etc., is naturally purple-red. It widely exists in the biological world, especially in crustaceans, salmon, rainbow trout, etc. It is their Main pigments (1, Johnson E A, An G H. Anstaxanthin from microbial sources. Critical Reviews in Biotechnology, 1991, 11: 297-326; 2, Torrissen O J, Christiansen R. Requirements for carotenoids in fish diets. J Appl Ichthyol., 1995, 11: 225-230; 3. Pharmaceuticals M. Aquaxan TM HD algal meal use in aquaculture diets: Enhancing nutritional performance and pigmentation. Inc. Technical Report TR, 1999, 2102.001). Its chemical name is 3,3'-dihydroxy-4,4'-diketone-β,β'-carotene, and its molecular formula is C 40 h 52 o 4 . A...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/16C12N15/01C12P23/00C12R1/645
Inventor 卢英华王宝贝敬科举凌雪萍
Owner XIAMEN UNIV
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