CN101818141A - Composite mutagenesis method of Phaffia rhodozyma strain - Google Patents
Composite mutagenesis method of Phaffia rhodozyma strain Download PDFInfo
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- CN101818141A CN101818141A CN200910188300A CN200910188300A CN101818141A CN 101818141 A CN101818141 A CN 101818141A CN 200910188300 A CN200910188300 A CN 200910188300A CN 200910188300 A CN200910188300 A CN 200910188300A CN 101818141 A CN101818141 A CN 101818141A
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Abstract
The invention relates to a composite mutagenesis method of a Phaffia rhodozyma strain, which comprises the steps of: firstly, carrying out ultraviolet mutagenesis on the Phaffia rhodozyma strain twice to obtain a mutant strain with stable hereditary features in five generations; secondly, carrying out low-temperature treatment on the mutant strain; and thirdly, carrying out singlet oxygen mutagenesis treatment on the mutant stain subjected to the low-temperature treatment to obtain a mutant strain with better genetic stability in ten generations. The mutant strain is fermented and cultured, the biomass liveweight and the pigment content of the mutant strain are measured to be higher than those of the original strain. A Phaffia rhodozyma mutant strain with higher astaxanthin yield is obtained by low-temperature composite mutagenesis, which lays a foundation for the Phaffia rhodozyma industrialization.
Description
Technical field
The present invention relates to a kind of mutafacient system of Phaffia rhodozyma strain, relate in particular to the method that obtains red Fife's yeast mutagenicity high-yield bacterial strain by the low temperature complex mutation.
Background technology
Astaxanthin (Astaxanthin) is 3,3 '-dihydroxyl-4, and 4 '-diketo-β, β ' carotene is a kind of keto-acid carotenoid.Color and luster is gorgeous redness, has fat-soluble.Astaxanthin has many other physiological functions, as the effect that has height quencher antozone and remove free radical; Antioxygenation, the energy force rate β-Hu Luobusu of its lipotropism fat oxidation is high 10 times, and is higher 100 times than VITAMIN, can prevent tissue effectively.The oxidized damage of cell and DNA, raise immunity, antitumous effect, protection cardiovascular health etc.Astaxanthin has broad application prospects in industry such as medicine, food, feed and makeup as functional pigment, is subjected to the concern of domestic and international researchist and every profession and trade in recent years.
The price of world market is 2500 dollars every kilogram at present, and the market capacity more than 1,000,000,000 is arranged global every year.Therefore, step up the exploitation of astaxanthin product, the particularly production of natural astaxanthin product has very high marketable value and ten minutes vast market prospect.Now, the shared market share of natural astaxanthin is very little.With regard to present production and market situation, natural astaxanthin owing to yield poorly, production cost is high is difficult to and the astaxanthin of chemosynthesis is at war with on price.But improvement and the reduction of optimization and production cost and the raising of public's cultural quality along with production technology, approval and the fishery products or the legislation that require purchase to contain natural astaxanthin upward require to use the natural pigment additive, and natural astaxanthin is compared with synthesizing astaxanthin has just possessed a special price advantage.
Red Fife's yeast is the maximum microorganism of research, and it has some essential feature as the astaxanthin biological source: astaxanthin is main carotenoid, accounts for 40-95% greatly, can utilize multiple sugar to carry out the heterotrophism metabolism as carbon source; Incubation time is short; Do not need illumination; Can in fermentor tank, realize high-density culture.Thereby red Fife's yeast is considered to a kind of source of astaxanthin scale operation of tool researching value.
Though in red Fife's yeast the content of astaxanthin be the Crustacean content astaxanthin 5-50 doubly, but the 1%-10% that has only content astaxanthin in the Haematocoocus Pluvialls, want to utilize red Fife's yeast to carry out the suitability for industrialized production of astaxanthin, the breeding high-yield bacterial strain is a key in application.
Description of drawings
Fig. 1 is that the present invention adopts the technical scheme schematic flow sheet.
Summary of the invention
Purpose of the present invention is intended to obtain by complex mutation the high-yield astaxanthin bacterial strain of a strain stabilization characteristics of genetics, and the industrialization of producing astaxanthin for red Fife's yeast fermentation lays the foundation.
In order to achieve the above object, the composite mutagenesis method of a kind of Phaffia rhodozyma strain of the present invention comprises the steps:
S1, get red Fife's yeast starter nutrient solution, be coated with flat board after the dilution;
S2, a ultraviolet mutagenesis: under dark surrounds, behind the ultra violet lamp flat board, cultivated 4-5 days down at 20-25 ℃;
S3, secondary ultraviolet mutagenesis: the bacterial strain of screening pigment production height and inheritance stability, repeat to be coated with flat board and step S2;
S4, subzero treatment: the bacterial strain of screening pigment production height and inheritance stability, make seed liquor, be stored under the 1-5 ℃ of environment 2-4 days;
S5, singlet oxygen are handled: seed liquor is transferred to be cultured to the logarithmic growth middle and later periods in the fermention medium, extract the unicellular state of bacterial strain, add the H that concentration is 10-15mmol/L respectively
2O
2, the vibration mixing is placed in the 1-5 ℃ of environment to be cultivated 10-18 hour;
Bacterial strain after S6, singlet oxygen are handled is coated with flat board and cultivated 7-10 days down at 20-25 ℃ after washing, dilution.
Under the optimal way, the composite mutagenesis method of Phaffia rhodozyma strain of the present invention, in above-mentioned steps S5, strain culturing to the method for logarithmic phase is: bacterial strain is moved on to from the inclined-plane the fermention medium cultivated 36-60 hour, changed the fresh liquid culture medium culturing again over to 24-48 hour, to the logarithmic growth middle and later periods.In above-mentioned steps S5, the method of extracting the unicellular state of bacterial strain is: get 1 milliliter of bacterium liquid with aseptic centrifuge tube, with 3500 rev/mins of centrifugal 5 minutes collection thalline, use the damping fluid washed twice, revert to original volume with damping fluid again, vibrate on the vortex oscillation device 20-30 second, thalline is scattered becomes unicellular state.
In addition, under the optimal way, the detailed process of step S1 is: behind the screening culture medium heat sterilization, dull and stereotyped while hot, the 15-20ml/ plate solidifies stand-by; Get red Fife's yeast starter nutrient solution, with aqua sterilisa dilution 10
5-10
7Doubly, measure diluent and be coated with flat board.And the dull and stereotyped substratum of system is preferentially selected for use: glucose 10 grams; Peptone 5 grams, yeast extract paste 3 grams, wort 3 grams, agar 20 grams, 1 liter in water; After sterilization was cooled to 45 ℃, adding an amount of was the diphenylamine solution of 50-80 μ mol/L through filtration sterilization concentration.
Under the optimal way, the detailed process of ultraviolet mutagenesis is: the ultraviolet lamp with 15w shines at distance 20cm place, and irradiation time is 10-100 second.In addition, in the above-mentioned steps S1-S6 process of the present invention, select for use sterilized water to dilute; Damping fluid is preferentially selected the 0.2mol/L phosphoric acid of pH5.0 for use.
And the screening method in the above-mentioned steps comprises the steps:
S31, after mutagenesis has been cultivated, detect by an unaided eye, choose the big redder single bacterium colony of color of bacterium colony in the flat board in screening, be coated onto and cultivate preservation in the slant medium with connecing the collarium picking;
S32, multiple sieve: the strain fermentation of primary dcreening operation is cultivated, measured its biomass and pigment content, choose the wherein the highest bacterial classification of pigment production.
A kind of method of screening red Fife's yeast superior strain of the present invention.Twice ultraviolet mutagenesis of Phaffia rhodozyma strain at first obtains the mutant strain of stabilization characteristics of genetics in five generations an of strain, then again with this mutant strain subzero treatment.Through the mutant strain of subzero treatment, pass through the singlet oxygen mutagenic treatment again, obtain a strain has good genetic stability in 10 generations mutant strain.With the mutant strain fermentation culture, measure its biomass and pigment content, all be higher than original strain.The present invention obtains the higher red Fife's yeast mutagenic strain of a strain astaxanthin yield by the low temperature complex mutation, for next step red Fife's yeast industry production lays the foundation.
Embodiment
Technical scheme that the present invention as shown in Figure 1 adopts: adopt repetition uv irradiating and singlet oxygen mutagenesis bonded mode that this yeast is carried out mutagenic and breeding, each sudden change is screened, choose optimum forward mutation by the screening culture medium that has pentanoic.By continuous passage, investigate the stability of mutant strain.And pigment is carried out purifying with the method for thin-layer chromatography.Facts have proved: ultraviolet mutagenesis, through after the subzero treatment and after singlet oxygen (hydrogen peroxide) complex mutation handles, obtain mutant strain, stabilization characteristics of genetics not only, pigment content and biomass all are higher than original strain.Concrete implementing process of the present invention is as follows:
One, ultraviolet mutagenesis
Behind the screening culture medium heat sterilization, dull and stereotyped while hot, about the 15-20ml/ plate, solidify stand-by.Get seed culture fluid, with aqua sterilisa dilution 10
5-10
7Doubly, accurately measure diluent 0.2ml and be coated with flat board.Ultraviolet lamp with 15w shines at distance 20cm place then, and irradiation time is 10-100S.Flat board after handling was cultivated 4-5 days down at 20-25 ℃, and these operations are carried out in the dark.
Wherein, the configuration preferred embodiment of screening culture medium is: glucose 10g; Peptone 5g, yeast extract paste 3g, wort 3g, agar 20g, water 1L.After sterilization is cooled to 45 ℃, adding an amount of diphenylamine solution through filtration sterilization, is the final concentration 50-80 μ mol/L of pentanoic.
Twice of above-mentioned ultraviolet mutagenesis process repetitive operation.
In addition, wattage, irradiation distance and the time of lamp is set in the selection in above-mentioned ultraviolet mutagenesis lamp source as required.The present invention only provides preferred version in, and other schemes through twice ultraviolet mutagenesis are equivalent of the present invention.In like manner, the selection of screening culture medium also selects conventional screening culture medium or special screening culture medium all can as required.
Two, subzero treatment
The seed liquor of selected mutant strain is stored under the 1-5 ℃ of environment 2-4 days.Above-mentioned seed liquor transferred in the fermention medium cultivated plain output of colour examining and biomass 96 hours.
Three, singlet oxygen mutagenesis
Choosing gain mutant bacterial strain through the ultraviolet mutagenesis of subzero treatment carries out singlet oxygen and handles.Concrete grammar is: bacterial strain is moved on to from the inclined-plane the liquid nutrient medium cultivated 36-60 hour, change the fresh liquid culture medium culturing again over to and arrived the logarithmic growth middle and later periods in 24-48 hour, get the centrifugal 5min of 1ml bacterium liquid 3500r/min with aseptic centrifuge tube and collect thalline, 0.2mol/L phosphoric acid buffer washed twice with pH5.0, revert to original volume with damping fluid again, the 20-30s that vibrates on the vortex oscillation device scatters thalline and becomes unicellular state.Add 3%H more respectively
2O
2(ultimate density is 10-15mmol/L) vibration mixing is placed in 4 ℃ of refrigerators to be preserved 10-18 hour, and centrifugal abandoning supernatant reverts to original volume 1-2 time with the damping fluid washing again, is coated with flat board after the dilution.Cultivate 7-10 days countings for 20-25 ℃.
Four, mutagenic strain pigment analysis
1, after mutagenesis has been cultivated, detect by an unaided eye, in the screening flat board, choose the big redder single bacterium colony of color of bacterium colony, be coated onto cultivation preservation in the slant medium with connecing the collarium picking.
2, multiple sieve: the strain fermentation of primary dcreening operation is cultivated, measured its biomass and pigment content, choose the wherein the highest bacterial classification of pigment production.
Embodiment 1
A, ultraviolet mutagenesis for the first time: behind the screening culture medium heat sterilization, fall dull and stereotypedly while hot, about the 15-20ml/ plate, solidify stand-by.Get seed culture fluid, with aqua sterilisa dilution 10
5Doubly, accurately measure diluent 0.2ml and be coated with flat board.Ultraviolet lamp with 15w shines at distance 20cm place then, and irradiation time is 70S.Flat board after handling was cultivated 4-5 days down at 20-25 ℃, and these operations are carried out in the dark.
B, select the scarlet bacterial strain of gained during this,, cultivate, survey its biomass, look output and genetic stability as the purpose bacterial strain.Select the bacterial strain of pigment production height and inheritance stability to carry out the ultraviolet mutagenesis second time.
C, the seed liquor of selected mutant strain is stored under 4 ℃ of environment 3 days.
D, choose gain mutant bacterial strain through the ultraviolet mutagenesis of subzero treatment and carry out singlet oxygen and handle.Concrete grammar is: inferior bacterial strain is moved on to from the inclined-plane the liquid nutrient medium cultivated 48 hours, change the fresh liquid culture medium culturing again over to and arrived the logarithmic growth middle and later periods in 24 hours, get the centrifugal 5min of 1ml bacterium liquid 3500r/min with aseptic centrifuge tube and collect thalline, 0.2mol/L phosphoric acid buffer washed twice with pH5.0, revert to original volume with damping fluid again, the 20-30s that vibrates on the vortex oscillation device scatters thalline and becomes unicellular state.Add 3%H more respectively
2O
2(ultimate density is 10mmol/L) vibration mixing is placed on to preserve in 4 ℃ of refrigerators and spends the night, and centrifugal abandoning supernatant reverts to original volume 1-2 time with the damping fluid washing again, is coated with flat board after the dilution, cultivates 7-10 days countings for 20-25 ℃.
E, primary dcreening operation: after mutagenesis has been cultivated, detect by an unaided eye, in the screening flat board, choose the big redder single bacterium colony of color of bacterium colony, be coated onto cultivation preservation in the slant medium with connecing the collarium picking.Multiple sieve: the strain fermentation of primary dcreening operation is cultivated, measured its biomass and pigment content.
Through twice ultraviolet mutagenesis, subzero treatment and H
2O
2Handle, obtaining a strain has the mutant strain of good genetic stability in 10 generations, the pigment production of this mutant strain is 5.11mg/L, and biomass is 10.7g/L, (2.63mg/L 9.4g/L) has improved 94.2% and 13.8% than the red Fife's yeast of starting strain AS2.1557 respectively.
Embodiment 2
Be with the difference of example 1
The time of A, twice ultraviolet mutagenesis of B is 80 seconds;
C, the seed liquor of selected mutant strain is stored under 3 ℃ of environment 3 days.
When D, singlet oxygen mutagenic treatment, H
2O
2Ultimate density is 15mmol/L;
Other are all identical with example 1.
Through twice ultraviolet mutagenesis, subzero treatment and H
2O
2Handle, obtaining a strain has the mutant strain of good genetic stability in 10 generations, the pigment production of this mutant strain is 5.56mg/L, and biomass is 12g/L, (2.63mg/L 9.4g/L) has improved 111% and 28% than the red Fife's yeast of starting strain AS2.1557 respectively.
Embodiment 3.
The time of A, twice ultraviolet mutagenesis of B is 75 seconds;
C, the seed liquor of selected mutant strain is stored under 5 ℃ of environment 4 days.
When D, singlet oxygen mutagenic treatment, H
2O
2Ultimate density is 10mmol/L;
Other are all identical with example 1.
Through twice ultraviolet mutagenesis, subzero treatment and H
2O
2Handle, obtaining a strain has the mutant strain of good genetic stability in 10 generations, the pigment production of this mutant strain is 5.47mg/L, and biomass is 10.2g/L, (2.63mg/L 9.4g/L) has improved 108% and 8.5% than the red Fife's yeast of starting strain AS2.1557 respectively.
Embodiment 4.
Behind S1, the screening culture medium heat sterilization, dull and stereotyped while hot, the 18ml/ plate solidifies stand-by; Get red Fife's yeast starter nutrient solution, with aqua sterilisa dilution 10
6Doubly, measure diluent and be coated with flat board.Under dark surrounds, behind the ultra violet lamp flat board, cultivated 4 days down at 23 ℃;
The bacterial strain of S2, screening pigment production height and inheritance stability repeats to be coated with flat board and above-mentioned steps;
The bacterial strain of S3, screening pigment production height and inheritance stability makes seed liquor, is stored in 4 ℃ of environment following 4 days;
S4, seed liquor transferred to be cultured to the logarithmic growth middle and later periods in the fermention medium, extract the unicellular state of bacterial strain, add the H that concentration is 12mmol/L respectively
2O
2, the vibration mixing is placed in 4 ℃ of environment to be cultivated 12 hours;
S5, choose gain mutant bacterial strain through the ultraviolet mutagenesis of subzero treatment and carry out singlet oxygen and handle.Concrete grammar is: bacterial strain is moved on to from the inclined-plane the liquid nutrient medium cultivated 55 hours, change the fresh liquid culture medium culturing again over to and arrived the logarithmic growth middle and later periods in 45 hours, get 3000 rev/mins of centrifugal 7 minutes collection thalline of 2ml bacterium liquid with aseptic centrifuge tube, 0.2mol/L phosphoric acid buffer washed twice with pH5.0, revert to original volume with damping fluid again, vibration 20-30 second on the vortex oscillation device, thalline is scattered and become unicellular state.Add 3%H more respectively
2O
2The vibration mixing is placed in 1 ℃ of refrigerator to be preserved 11 hours, and centrifugal abandoning supernatant reverts to original volume 2 times with the damping fluid washing again, is coated with flat board after the dilution, cultivated 9 days countings for 20 ℃.
Through twice ultraviolet mutagenesis, subzero treatment and H
2O
2Handle, obtaining a strain has the mutant strain of good genetic stability in 10 generations, the pigment production of this mutant strain is 5.1mg/L, and biomass is 10.9g/L, (2.63mg/L 9.4g/L) has improved 93.9% and 20% than the red Fife's yeast of starting strain AS2.1557 respectively.
The above; only be the preferable embodiment of the present invention; but protection scope of the present invention is not limited thereto; anyly be familiar with those skilled in the art in the technical scope that the present invention discloses; be equal to replacement or change according to technical scheme of the present invention and inventive concept thereof, all should be encompassed within protection scope of the present invention.
Claims (7)
1. the composite mutagenesis method of a Phaffia rhodozyma strain is characterized in that, comprises the steps:
S1, get red Fife's yeast starter nutrient solution, be coated with flat board after the dilution;
S2, a ultraviolet mutagenesis: under dark surrounds, behind the ultra violet lamp flat board, cultivated 4-5 days down at 20-25 ℃;
S3, secondary ultraviolet mutagenesis: the bacterial strain of screening pigment production height and inheritance stability, repeat to be coated with flat board and step S2;
S4, subzero treatment: the bacterial strain of screening pigment production height and inheritance stability, make seed liquor, be stored under the 1-5 ℃ of environment 2-4 days;
S5, singlet oxygen are handled: described seed liquor is transferred to be cultured to the logarithmic growth middle and later periods in the fermention medium, extract the unicellular state of bacterial strain, add the H that concentration is 10-15mmol/L respectively
2O
2, the vibration mixing is placed in the 1-5 ℃ of environment to be cultivated 10-18 hour;
Bacterial strain after S6, singlet oxygen are handled is coated with flat board and cultivated 7-10 days down at 20-25 ℃ after washing, dilution.
2. according to the composite mutagenesis method of the described Phaffia rhodozyma strain of claim 1, it is characterized in that,
Among the step S5, strain culturing to the method for logarithmic phase is: bacterial strain is moved on to from the inclined-plane the fermention medium cultivated 36-60 hour, changed the fresh liquid culture medium culturing again over to 24-48 hour, to the logarithmic growth middle and later periods;
Among the step S5, the method of extracting the unicellular state of bacterial strain is: get 1 milliliter of bacterium liquid with aseptic centrifuge tube, with 3500 rev/mins of centrifugal 5 minutes collection thalline, use the damping fluid washed twice, revert to original volume with damping fluid again, vibrate on the vortex oscillation device 20-30 second, thalline is scattered becomes unicellular state.
3. according to the composite mutagenesis method of the described Phaffia rhodozyma strain of claim 2, it is characterized in that,
The detailed process of step S1 is: behind the screening culture medium heat sterilization, dull and stereotyped while hot, the 15-20ml/ plate solidifies stand-by; Get red Fife's yeast starter nutrient solution, with aqua sterilisa dilution 10
5-10
7Doubly, measure diluent and be coated with flat board.
4. according to the composite mutagenesis method of the described Phaffia rhodozyma strain of claim 3, it is characterized in that,
The detailed process of step S2 is: the ultraviolet lamp with 15w shines at distance 20cm place, and irradiation time is 10-100 second.
5. according to the composite mutagenesis method of the arbitrary described Phaffia rhodozyma strain of claim 1-4, it is characterized in that, in the step S1-S6 process, select the sterilized water dilution for use; Damping fluid is selected the 0.2mol/L phosphoric acid of pH5.0 for use.
6. according to the composite mutagenesis method of the arbitrary described Phaffia rhodozyma strain of claim 1-4, it is characterized in that screening method comprises among the step S1-S6:
S31, after mutagenesis has been cultivated, detect by an unaided eye, choose the big redder single bacterium colony of color of bacterium colony in the flat board in screening, be coated onto and cultivate preservation in the slant medium with connecing the collarium picking;
S32, multiple sieve: the strain fermentation of primary dcreening operation is cultivated, measured its biomass and pigment content, choose the wherein the highest bacterial classification of pigment production.
7. according to the composite mutagenesis method of the described Phaffia rhodozyma strain of claim 6, it is characterized in that,
The dull and stereotyped used substratum of step S1-S3 is: glucose 10 grams; Peptone 5 grams, yeast extract paste 3 grams, wort 3 grams, agar 20 grams, 1 liter in water; After sterilization was cooled to 45 ℃, adding an amount of was the diphenylamine solution of 50-80 μ mol/L through filtration sterilization concentration.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102864087A (en) * | 2012-09-03 | 2013-01-09 | 浙江皇冠科技有限公司 | Phaffia rhodozyma strain with high yield of natural astaxanthin as well as breeding method and application thereof |
| CN108070632A (en) * | 2017-12-19 | 2018-05-25 | 博益德（北京）生物科技有限公司 | A kind of blutene with screening rhodotorula effect and its application |
| CN116590160A (en) * | 2023-07-11 | 2023-08-15 | 山东合成远景生物科技有限公司 | Phaffia rhodozyma mutant strain HCYJ-07 and application thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102864087A (en) * | 2012-09-03 | 2013-01-09 | 浙江皇冠科技有限公司 | Phaffia rhodozyma strain with high yield of natural astaxanthin as well as breeding method and application thereof |
| CN108070632A (en) * | 2017-12-19 | 2018-05-25 | 博益德（北京）生物科技有限公司 | A kind of blutene with screening rhodotorula effect and its application |
| CN116590160A (en) * | 2023-07-11 | 2023-08-15 | 山东合成远景生物科技有限公司 | Phaffia rhodozyma mutant strain HCYJ-07 and application thereof |
| CN116590160B (en) * | 2023-07-11 | 2023-09-05 | 山东合成远景生物科技有限公司 | Phaffia rhodozyma mutant strain HCYJ-07 and application thereof |
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