CN1986773A - Medium temperature type astaxanthin producing bacterial strain and its culture process - Google Patents
Medium temperature type astaxanthin producing bacterial strain and its culture process Download PDFInfo
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Abstract
The present invention provides Phaffia rhodozyma A-1 CGMCC No.1837 as one kind of medium temperature type astaxanthin producing bacterium. The present invention provides also the culture process of the medium temperature type astaxanthin producing bacterium. The medium temperature type astaxanthin producing bacterium has simple nutrient requirement for production and industrial production power consumption lower than that for low temperature type astaxanthin producing bacterium. The culture process of the medium temperature type astaxanthin producing bacterium is simple, short in culture period, high in astaxanthin yield and favorable to industrial production.
Description
Technical field
The present invention relates to the production of astaxanthin field, relate to the blue or green rope of a kind of middle warm type shrimp particularly and produce bacterium and cultural method thereof.
Background technology
Fife's yeast is separated at the beginning of 1967, is named as Phaffia rhodozyma in 1976.Nineteen ninety-five, the part bacterial strain was found its sexual circulation, re-positioned at Basidiomycota (Xanthophyllomycesdendrorhous).Its biological characteristics mainly contains: withered red bacterium colony, sprout repeatedly in the single site of cell, and cell walls contains wood sugar, diazonium B stained positive, distinctive 18S rRNA sequence, the astaxanthin of generation 3R, 3 ' R ' configuration.
Astaxanthin is a kind of carotenoid, and chemical name is 3,3 '-dihydroxyl-B, B-carotene-4,4 ' diketone.Astaxanthin is a kind of fat-soluble pigment, and is water insoluble, is soluble in organic solvents such as tetrahydrofuran (THF), methylene dichloride, chloroform, methyl alcohol, ethanol.Natural astaxanthin has the specific molecule structure, and experiment in vitro finds that it is the powerful quencher of singlet oxygen, has the great ability of removing oxyradical, is a kind of utmost point good antioxidant.That studies show that in recent years, astaxanthin have is anticancer, enhancing immunity system function, anti-infective isoreactivity.Astaxanthin not only can be used as human healthcare products, also can be used as the fodder additives of egg fowl industry, is widely used in culture fishery mainly as tinting material at present.
The production of astaxanthin has synthetic and biology obtains dual mode.FDA (FDA) has forbidden that the astaxanthin of chemosynthesis enters health food market.The rice source of the blue or green rope of natural shrimp mainly is Haematocoocus Pluvialls (Haematococcus pluvialis) and Fife's yeast (Phaffia rhodozyma).Utilize Fife's yeast (P.rhodozyma) fermentative production astaxanthin, have high cell growth speed, fermentation period short, be easy to advantage such as industrialization.And contain rich in protein, lipid and VITAMIN in the yeast, can directly use as fodder additives.But because the astaxanthin cell content of Fife's yeast original strain is very low, and requires culture temperature on the low side, cause complex manufacturing, have only the external major company of minority to produce at present.Fife's yeast synthesizing astaxanthin generally needs low temperature to cultivate (20-22 ℃), and the fermentation culture process must cool, and consumes energy is more.These drawbacks limit the production of astaxanthin industrial expansion, middle warm type astaxanthin produces the acquisition of bacterium and the foundation of cultural method will bring new opportunity for the development in this field.
Summary of the invention
(1) technical problem that will solve
The purpose of this invention is to provide a kind of middle warm type astaxanthin and produce bacterium, another object of the present invention provides the cultural method of warm type astaxanthin generation bacterium in this.
(2) technical scheme
Definition: on meaning of the present invention, middle warm type astaxanthin produces bacterium, and to refer in particular to optimum growth temperature be that 23-26 ℃ astaxanthin produces bacterium, and the low temperature modification astaxanthin produces bacterium, and to refer in particular to optimum growth temperature be that 20-22 ℃ astaxanthin produces bacterium.
The present inventor is by chemistry, physical mutagenesis technology, cooperate the screening strategy that adds inhibitor, select a kind of middle warm type astaxanthin and produce bacterium Fife's yeast (Phaffia rhodozyma) A-1, this bacterial strain has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on October 13rd, 2006, be called for short CGMCC, deposit number is CGMCC No.1837.
The morphological feature that astaxanthin of the present invention produces bacterium is:
The individual morphology feature: unicellular based on oval, size 3～6 μ m * 5～12 μ m.Sometimes several cells connect together, and cell is the pseudohypha shape sometimes, long * wide be 3～5 μ m * 10～17 μ m.
Dull and stereotyped cultural characteristic: the colony diameter of this bacterium on YM (yeast powder and maltose) substratum is 1.0～5.0mm, and orange is to scarlet, and the bacterium colony red color is dark more glossy more, and the amount of its astaxanthin-containing is many more.
The physiological and biochemical property that astaxanthin of the present invention produces bacterium is: aerobic bacteria needs could grow in aerobic environment.The suitableeest growth pH is 6.0, and the optimal pH of chromogenesis is 5.0.Belong to the facultative cold low temperature modification microorganism of having a liking for.Optimum growth temperature is 23～26 ℃.When glucose or sucrose concentration were above greater than 8%, biomass, amount of pigment all obviously descended.The growth that yeast extract paste both had been beneficial to thalline also is beneficial to chromogenesis.With (NH
4)
2SO
4Be only nitrogen source, pigment content is higher, but biomass is not high.Urea had not only helped this bacteria growing as only nitrogen source but also had helped the accumulation of pigment.
The trophology that astaxanthin of the present invention produces bacterium is characterized as: glucose, yeast powder, urea add the needs that an amount of phosphoric acid salt and magnesium salts can satisfy this bacterial strain pigment accumulation and cell growth again.
The breeding technique route that astaxanthin of the present invention produces bacterium is: the cell of fresh activation 30h suspends with physiological saline through centrifugal supernatant discarded, adds nitrosoguanidine (NTG) physiological saline saturated solution, and thorough mixing acts on 0-20 minute.After mutagenesis finished, the fully centrifugal and washing of reaction solution was suitably coated the PDA culture medium flat plate after the dilution, and flat board contains the inhibition pigment accumulation of proper concn or the selective agent of cell growth (Simvastatin or yak base lemonol).The dull and stereotyped inversion 23～26 ℃ of lucifuges cultivated 7 days.Select the scarlet cell to continue to cultivate dilution and carry out physical mutagenesis, take
60The Co irradiation, irradiation dose is 4kGy.Bacterium liquid after the mutagenesis is coated the above-mentioned flat board that contains inhibitor, and 23～26 ℃ of lucifuges are inverted and were cultivated 7 days, and it is obviously red in single bacterium colony of starting strain to select visual inspection, enter to shake the multiple sieve of bottle.
The bacterium colony that the primary dcreening operation flat board grows is inoculated in 5ml liquid nutrient medium (glucose 40g/L, yeast powder 4g/L, urea 3.6g/L, potassium primary phosphate 2g/L, sal epsom 0.5g/L, pH6.0) test tube is housed.Directly change over to behind the activation culture 30h in the triangular flask of the 500ml that the 50ml liquid nutrient medium is housed and cultivate.After cultivating 120h, collecting cell, broken wall extracts pigment, and the liquid spectrum is measured the content of pure astaxanthin.Choose the optimum muton 3-5 strain of multiple sieve back performance, enter next round mutagenesis.
Astaxanthin of the present invention produces the cultural method of bacterium, may further comprise the steps:
(1) fermentor tank seed culture: this astaxanthin of picking produces single bacterium colony of bacterium, is linked in the 50mL liquid seed culture medium, and at 23-26 ℃, 200-260r/min cultivates 36-40h, nutrient solution OD down
600=7.5-8.5; According to 5% inoculum size above-mentioned seed culture fluid is linked in the described liquid seed culture medium, at 23-26 ℃, 200-260r/min cultivates 36-60h, nutrient solution OD down again
600=7.5-8.5; According to 5% inoculum size the seed culture fluid that obtains is transferred in the described liquid seed culture medium then, at 23-26 ℃, 200-260r/min cultivates 36-60h, nutrient solution OD down
600=7.5-8.5;
(2) fed-batch fermentation: the seed liquor that described step (1) is obtained according to 5% inoculum size is linked into carries out batch culture in the fermentor tank that liquid batch basis substratum is housed; The NaOH that with volume ratio is 10% HCl and 10mol/L is controlled at 4.8-5.2 with the pH of described basic medium, and air flow is 0.7-0.9L/L/min; By regulating rotating speed dissolved oxygen is controlled at 20-70%; Feed supplement for the first time makes glucose concn return to 40g/L when the glucose concn of described basic medium is reduced to 20g/L, and the glucose concn of controlling described basic medium all the time is at 20-40g/L, and total glucose consumption reaches 100g/L; Incubation time is 60-80h.
Astaxanthin of the present invention produces the cultural method of bacterium, the consisting of of used substratum:
Described seed culture medium: glucose 40g/L, yeast powder 4g/L, urea 3.6g/L, potassium primary phosphate 2g/L, sal epsom 0.5g/L, pH6.0;
Described basic medium: glucose 40g/L, yeast powder 10g/L, urea 3.6g/L, ammonium sulfate 5.0g/L, potassium primary phosphate 2g/L, sal epsom 0.5g/L, pH6.0;
Described supplemented medium: 40% glucose (W/V) feed supplement liquid.
Adopt method of the present invention to cultivate 60-80h hour, the output of astaxanthin reaches 30-35mg/L.And adopt 20-22 ℃ of cultivation with aforesaid method, then cell growth and pigment are synthetic all than cultivating slowly at 23-26 ℃, and the pigment resultant quantity is not seen and increased.
(3) beneficial effect
Warm type production of astaxanthin bacterium Fife yeast (Phaffia rhodozyma) A-1 nutritional requirement is simple in provided by the invention, 23-26 ℃ of following culturing cell growth rapidly, and astaxanthin accumulates in a large number, for low temperature modification (20-22 ℃) the production of astaxanthin bacterium of present industrial widespread usage, effectively reduce production energy consumption, thereby reach the purpose that reduces production costs.
The cultural method of warm type production of astaxanthin bacterium is compared with present industrial process in provided by the invention, technology is easy, and culture cycle is short, the astaxanthin yield height, the output of cultivating 60-80h hour astaxanthin reaches 30-35mg/L, and is very favourable to suitability for industrialized production.
The warm type astaxanthin produces bacterium Fife's yeast (Phaffia rhodozyma) A-1 in of the present invention, this bacterial strain has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on October 13rd, 2006, be called for short CGMCC, deposit number is CGMCC No1837.
Embodiment
Following examples are used to illustrate the present invention, but are not used for limiting the scope of the invention.
The warm type astaxanthin produces the mutagenesis screening of bacterium among the embodiment 1
One, chemomorphosis
(1) get the new fresh thalli (starting strain is Phaffia rhodozyma JMC9042, purchases in Japanese physics and chemistry institute) of activation 30h, centrifugal, supernatant discarded suspends with 0.5ml physiological saline, adds the NTG physiological saline saturated solution of 0.1ml, acts on 10 minutes;
(2) reaction solution is through centrifugal, and physiological saline washs three times;
(3) thalline after the detoxification suspends with the physiological saline of 1ml, and (extent of dilution is 10 to the dilution proper concn
6-10
8) after coat PDA flat board (part PDA substratum contains the selective agent Simvastatin 4-8mg/L of proper concn), pH=6.0.Dull and stereotyped lucifuge, is inverted and was cultivated 7-10 days by 24 ℃;
(4) cell after the mutagenesis is inverted cultivation after 7-10 days through flat board, selects the obvious red single bacterium colony in starting strain of visual inspection and is re-seeded into new flat board (not adding selective agent).Cultivate in inversion,, then enter and shake the multiple sieve of bottle if the bacterium colony that newly grows is still red in starting strain through 5-7 days;
(5) the outstanding bacterium colony that grows of primary dcreening operation flat board is inoculated in 5ml liquid nutrient medium test tube is housed.Directly change over to behind the activation culture 48h in the triangular flask of the 500ml that the 50ml liquid nutrient medium is housed and cultivate (substratum consists of: glucose 40g/L, and yeast powder 4g/L, urea 3.6g/L, potassium primary phosphate 2g/L, sal epsom 0.5g/L, pH6.0).After cultivating 120h, collecting cell, broken wall extracts pigment, and the liquid spectrum is measured the content of pure astaxanthin.Choose the optimum muton 3-5 strain of multiple sieve back performance, enter next round mutagenesis.
Two, physical mutagenesis (
60Co)
The process of physical mutagenesis is similar to chemomorphosis, and difference is that mutagenic compound are changed into by NTG
60The Co irradiation, irradiation dose is 4kGy.
Through repeatedly screening, the warm type astaxanthin produces bacterium Fife's yeast (Phaffia rhodozyma) A-1 in obtaining.
The warm type astaxanthin produces cultivation and the content astaxanthin mensuration of bacterium among the embodiment 2
1. middle warm type astaxanthin produces the cultivation of bacterium:
(1) fermentor tank seed culture: this astaxanthin of picking produces single bacterium colony of bacterium, is linked in the 50mL liquid seed culture medium, and at 23 ℃, 200r/min cultivates 36h, nutrient solution OD down
600=7.5; According to 5% inoculum size above-mentioned seed culture fluid is linked in the described liquid seed culture medium of 50mL, at 23 ℃, 200r/min cultivates 36h, nutrient solution OD down again
600=7.5; According to 5% inoculum size the seed culture fluid that obtains is transferred in the described liquid seed culture medium of 100mL then, at 23 ℃, 200r/min cultivates 36h, nutrient solution OD down
600=7.5;
(2) fed-batch fermentation: the seed liquor that described step (1) is obtained according to 5% inoculum size is linked in the 7.5L fermentor tank that 4.0L liquid batch basis substratum is housed carries out batch culture; The NaOH that with volume ratio is 10% HCl and 10mol/L is controlled at 4.8 with the pH of described basic medium, and air flow is 0.7L/L/min; By regulating rotating speed dissolved oxygen is controlled at 20%; Feed supplement for the first time makes glucose concn return to 40g/L when the glucose concn of described basic medium is reduced to 20g/L, and the glucose concn of controlling described basic medium all the time is at 20-40g/L, and total glucose consumption reaches 100g/L; Incubation time is 60h.
2. content astaxanthin is measured:
(1) extraction of astaxanthin:
Get the bacterium liquid of the above-mentioned cultivation 60h of 1.0ml, centrifugal, supernatant discarded adds 70 ℃ methyl-sulphoxide 0.2ml, and vortex vibration 30 seconds adds 1ml methyl alcohol-methylene dichloride (3: 1) mixed organic solvents and extracts pigment.Centrifugal, collect supernatant (containing pigment).If precipitation also has color, repeat said process, colourless up to precipitation, merge supernatant, centrifugal (120,000r/min, 10 minutes) ,-20 ℃ keep in Dark Place, (sample can directly detect) to be measured.
(2) high-pressure liquid phase chromatogram therapy determining astaxanthin
Waters 600E high pressure liquid chromatograph; ZY1104 type liquid-phase chromatographic column (filler C-18, dp10 μ m), column length 25cm, internal diameter 4mm; 2487 twin-beam UV-detector.Moving phase is methyl alcohol-methylene dichloride (9: 1); Flow velocity 1.0mL/min; Detect wavelength 476nm.
Measurement result is: the output of astaxanthin reaches 30mg/L.Measurement result shows: adopt the cultural method of middle warm type production of astaxanthin bacterium of the present invention, culture cycle is short, the astaxanthin yield height.
The warm type astaxanthin produces cultivation and the content astaxanthin mensuration of bacterium among the embodiment 3
1. middle warm type astaxanthin produces the cultivation of bacterium:
(1) fermentor tank seed culture: this astaxanthin of picking produces single bacterium colony of bacterium, is linked in the 50mL liquid seed culture medium, and at 24 ℃, 220r/min cultivates 40h, nutrient solution OD down
600=8.0; According to 5% inoculum size above-mentioned seed culture fluid is linked in the described liquid seed culture medium of 50mL, at 24 ℃, 220r/min cultivates 36h, nutrient solution OD down again
600=8.0; According to 5% inoculum size the seed culture fluid that obtains is transferred in the described liquid seed culture medium of 100mL then, at 24 ℃, 220r/min cultivates 36h, nutrient solution OD down
600=8.0;
(2) fed-batch fermentation: the seed liquor that described step (1) is obtained according to 5% inoculum size is linked in the 7.5L fermentor tank that 4.0L liquid batch basis substratum is housed carries out batch culture; The NaOH that with volume ratio is 10% HCl and 10mol/L is controlled at 5.0 with the pH of described basic medium, and air flow is 0.8L/L/min; By regulating rotating speed dissolved oxygen is controlled at 40%; Feed supplement for the first time makes glucose concn return to 40g/L when the glucose concn of described basic medium is reduced to 20g/L, and the glucose concn of controlling described basic medium all the time is at 20-40g/L, and total glucose consumption reaches 100g/L; Incubation time is 70h.
2. content astaxanthin is measured:
(1) extraction of astaxanthin:
Get the bacterium liquid of the above-mentioned cultivation 70h of 1.0ml, centrifugal, supernatant discarded adds 70 ℃ methyl-sulphoxide 0.2ml, and vortex vibration 30 seconds adds 1ml methyl alcohol-methylene dichloride (3: 1) mixed organic solvents and extracts pigment.Centrifugal, collect supernatant (containing pigment).If precipitation also has color, repeat said process, colourless up to precipitation, merge supernatant, centrifugal (120,000r/min, 10 minutes) ,-20 ℃ keep in Dark Place, (sample can directly detect) to be measured.
(2) high-pressure liquid phase chromatogram therapy determining astaxanthin
Waters 600E high pressure liquid chromatograph; ZY1104 type liquid-phase chromatographic column (filler C-18, dp10 μ m), column length 25cm, internal diameter 4mm; 2487 twin-beam UV-detector.Moving phase is methyl alcohol-methylene dichloride (9: 1); Flow velocity 1.0mL/min; Detect wavelength 476nm.
Measurement result is: the output of astaxanthin reaches 32mg/L.Measurement result shows: adopt the cultural method of middle warm type production of astaxanthin bacterium of the present invention, culture cycle is short, the astaxanthin yield height.
The warm type astaxanthin produces cultivation and the content astaxanthin mensuration of bacterium among the embodiment 4
1. middle warm type astaxanthin produces the cultivation of bacterium:
(1) fermentor tank seed culture: this astaxanthin of picking produces single bacterium colony of bacterium, is linked in the 50mL liquid seed culture medium, and at 25 ℃, 200r/min cultivates 40h, nutrient solution OD down
600=8.0; According to 5% inoculum size above-mentioned seed culture fluid is linked in the described liquid seed culture medium of 50mL, at 25 ℃, 200r/min cultivates 48h, nutrient solution OD down again
600=8.0; According to 5% inoculum size the seed culture fluid that obtains is transferred in the described liquid seed culture medium of 100mL then, at 25 ℃, 200r/min cultivates 48h, nutrient solution OD down
600=8.0;
(2) fed-batch fermentation: the seed liquor that described step (1) is obtained according to 5% inoculum size is linked in the 7.5L fermentor tank that 4.0L liquid batch basis substratum is housed carries out batch culture; The NaOH that with volume ratio is 10% HCl and 10mol/L is controlled at 5.2 with the pH of described basic medium, and air flow is 0.9L/L/min; By regulating rotating speed dissolved oxygen is controlled at 50%; Feed supplement for the first time makes glucose concn return to 40g/L when the glucose concn of described basic medium is reduced to 20g/L, and the glucose concn of controlling described basic medium all the time is at 20-40g/L, and total glucose consumption reaches 100g/L; Incubation time is 72h.
2. content astaxanthin is measured:
(1) extraction of astaxanthin:
Get the bacterium liquid of the above-mentioned cultivation 72h of 1.0ml, centrifugal, supernatant discarded adds 70 ℃ methyl-sulphoxide 0.2ml, and vortex vibration 30 seconds adds 1ml methyl alcohol-methylene dichloride (3: 1) mixed organic solvents and extracts pigment.Centrifugal, collect supernatant (containing pigment).If precipitation also has color, repeat said process, colourless up to precipitation, merge supernatant, centrifugal (120,000r/min, 10 minutes) ,-20 ℃ keep in Dark Place, (sample can directly detect) to be measured.
(2) high-pressure liquid phase chromatogram therapy determining astaxanthin
Waters 600E high pressure liquid chromatograph; ZY1104 type liquid-phase chromatographic column (filler C-18, dp10 μ m), column length 25cm, internal diameter 4mm; 2487 twin-beam UV-detector.Moving phase is methyl alcohol-methylene dichloride (9: 1); Flow velocity 1.0mL/min; Detect wavelength 476nm.
Measurement result is: the output of astaxanthin reaches 33mg/L.Measurement result shows: adopt the cultural method of middle warm type production of astaxanthin bacterium of the present invention, culture cycle is short, the astaxanthin yield height.
The warm type astaxanthin produces cultivation and the content astaxanthin mensuration of bacterium among the embodiment 5
1. middle warm type astaxanthin produces the cultivation of bacterium:
(1) fermentor tank seed culture: this astaxanthin of picking produces single bacterium colony of bacterium, is linked in the 50mL liquid seed culture medium, and at 26 ℃, 260r/min cultivates 40h, nutrient solution OD down
600=8.5; According to 5% inoculum size above-mentioned seed culture fluid is linked in the described liquid seed culture medium of 50mL, at 26 ℃, 260r/min cultivates 60h, nutrient solution OD down again
600=8.5; According to 5% inoculum size the seed culture fluid that obtains is transferred in the described liquid seed culture medium of 100mL then, at 26 ℃, 260r/min cultivates 60h, nutrient solution OD down
600=8.5;
(2) fed-batch fermentation: the seed liquor that described step (1) is obtained according to 5% inoculum size is linked in the 7.5L fermentor tank that 4.0L liquid batch basis substratum is housed carries out batch culture; The NaOH that with volume ratio is 10% HCl and 10mol/L is controlled at 5.2 with the pH of described basic medium, and air flow is 0.9L/L/min; By regulating rotating speed dissolved oxygen is controlled at 70%; Feed supplement for the first time makes glucose concn return to 40g/L when the glucose concn of described basic medium is reduced to 20g/L, and the glucose concn of controlling described basic medium all the time is at 20-40g/L, and total glucose consumption reaches 100g/L; Incubation time is 80h.
2. content astaxanthin is measured:
(1) extraction of astaxanthin:
Get the bacterium liquid of the above-mentioned cultivation 80h of 1.0ml, centrifugal, supernatant discarded adds 70 ℃ methyl-sulphoxide 0.2ml, and vortex vibration 30 seconds adds 1ml methyl alcohol-methylene dichloride (3: 1) mixed organic solvents and extracts pigment.Centrifugal, collect supernatant (containing pigment).If precipitation also has color, repeat said process, colourless up to precipitation, merge supernatant, centrifugal (120,000r/min, 10 minutes) ,-20 ℃ keep in Dark Place, (sample can directly detect) to be measured.
(2) high-pressure liquid phase chromatogram therapy determining astaxanthin
Waters 600E high pressure liquid chromatograph; ZY1104 type liquid-phase chromatographic column (filler C-18, dp10 μ m), column length 25cm, internal diameter 4mm; 2487 twin-beam UV-detector.Moving phase is methyl alcohol-methylene dichloride (9: 1); Flow velocity 1.0mL/min; Detect wavelength 476nm.
Measurement result is: the output of astaxanthin reaches 35mg/L.Measurement result shows: adopt the cultural method of middle warm type production of astaxanthin bacterium of the present invention, culture cycle is short, the astaxanthin yield height.
Claims (4)
1, a kind of middle warm type astaxanthin produces bacterium Fife's yeast (Phaffia rhodozyma) A-1 CGMCCNo.1837.
2, a kind of astaxanthin according to claim 1 produces the cultural method of bacterium, it is characterized in that it comprises the steps:
(1) fermentor tank seed culture: this astaxanthin of picking produces single bacterium colony of bacterium, is linked in the 50mL liquid seed culture medium, and at 23-26 ℃, 200-260r/min cultivates 36-40h, nutrient solution OD down
600=7.5-8.5; According to 5% inoculum size above-mentioned seed culture fluid is linked in the described liquid seed culture medium, at 23-26 ℃, 200-260r/min cultivates 36-60h, nutrient solution OD down again
600=7.5-8.5; According to 5% inoculum size the seed culture fluid that obtains is transferred in the described liquid seed culture medium then, at 23-26 ℃, 200-260r/min cultivates 36-60h, nutrient solution OD down
600=7.5-8.5;
(2) fed-batch fermentation: the seed liquor that described step (1) is obtained according to 5% inoculum size is linked into carries out batch culture in the fermentor tank that liquid batch basis substratum is housed; The NaOH that with volume ratio is 10% HCl and 10mol/L is controlled at 4.8-5.2 with the pH of described basic medium, and air flow is 0.7-0.9L/L/min; By regulating rotating speed dissolved oxygen is controlled at 20-70%; Feed supplement for the first time makes glucose concn return to 40g/L when the glucose concn of described basic medium is reduced to 20g/L, and the glucose concn of controlling described basic medium all the time is at 20-40g/L; Incubation time is 60-80h.
3, cultural method according to claim 2 is characterized in that used substratum consists of in the culturing process:
Described seed culture medium: glucose 40g/L, yeast powder 4g/L, urea 3.6g/L, potassium primary phosphate 2g/L, sal epsom 0.5g/L, pH6.0;
Described basic medium: glucose 40g/L, yeast powder 10g/L, urea 3.6g/L, ammonium sulfate 5.0g/L, potassium primary phosphate 2g/L, sal epsom 0.5g/L, pH6.0;
Described supplemented medium: mass volume ratio is 40% glucose feed supplement liquid.
4, according to the cultural method of claim 2 or 3 described astaxanthins generation bacterium, it is characterized in that cultivating 60-80h hour, the output of astaxanthin reaches 30-35mg/L.
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| CN101717731B (en) * | 2009-12-07 | 2012-08-01 | 中国农业大学 | Phaffiarhodozyma mutant strain MK19 of glucose metabolism de-repression high-yield astaxanthin and culturing method thereof |
| CN104178430A (en) * | 2014-05-29 | 2014-12-03 | 南京工业大学 | Astaxanthin high-yield strain and application thereof |
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| CN105567776A (en) * | 2016-02-04 | 2016-05-11 | 邵素英 | Method for preparing astaxanthin through yeast fermentation |
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| CA2047000A1 (en) * | 1990-07-20 | 1992-01-21 | John W. Chapman | Astaxanthin-generating yeast cells |
| US5212088A (en) * | 1990-10-23 | 1993-05-18 | Phillips Petroleum Company | Spheroplast fusions of phaffia rhodozyma cells |
| US5466599A (en) * | 1993-04-19 | 1995-11-14 | Universal Foods Corporation | Astaxanthin over-producing strains of phaffia rhodozyma |
| KR20050053701A (en) * | 2002-09-27 | 2005-06-08 | 디에스엠 아이피 어셋츠 비.브이. | Astaxanthin production using fed-batch fermentation process by phaffia rhodozyma |
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2006
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101717731B (en) * | 2009-12-07 | 2012-08-01 | 中国农业大学 | Phaffiarhodozyma mutant strain MK19 of glucose metabolism de-repression high-yield astaxanthin and culturing method thereof |
| CN102586349A (en) * | 2012-02-24 | 2012-07-18 | 重庆邮电大学 | Preparation method of ethyl (R)-2-hydroxyl-4-phenylbutyrate by combining microbe reduction and chemical catalytic hydrogenation |
| CN102586349B (en) * | 2012-02-24 | 2014-12-03 | 重庆邮电大学 | Preparation method of ethyl (R)-2-hydroxyl-4-phenylbutyrate by combining microbe reduction and chemical catalytic hydrogenation |
| CN104178430A (en) * | 2014-05-29 | 2014-12-03 | 南京工业大学 | Astaxanthin high-yield strain and application thereof |
| CN104178430B (en) * | 2014-05-29 | 2017-02-15 | 南京工业大学 | Astaxanthin high-yield strain and application thereof |
| CN104830938A (en) * | 2015-05-13 | 2015-08-12 | 威海利达生物科技有限公司 | Method for enhancing mussel astaxanthin fermentation production yield |
| CN104830938B (en) * | 2015-05-13 | 2018-02-13 | 威海利达生物科技有限公司 | A kind of method for improving the red astaxanthin yield in fermenting and producing sea |
| CN105567776A (en) * | 2016-02-04 | 2016-05-11 | 邵素英 | Method for preparing astaxanthin through yeast fermentation |
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| CN100447233C (en) | 2008-12-31 |
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