CN105567776A - Method for preparing astaxanthin through yeast fermentation - Google Patents
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- CN105567776A CN105567776A CN201610078105.8A CN201610078105A CN105567776A CN 105567776 A CN105567776 A CN 105567776A CN 201610078105 A CN201610078105 A CN 201610078105A CN 105567776 A CN105567776 A CN 105567776A
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P23/00—Preparation of compounds containing a cyclohexene ring having an unsaturated side chain containing at least ten carbon atoms bound by conjugated double bonds, e.g. carotenes
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Abstract
The invention discloses a method for preparing astaxanthin through yeast fermentation. Fermental cultivation is conducted on yeast in a fermental cultivation base, the ph of fermentation liquor is regulated to 9-10 after fermentation is finished, the fermentation liquor is processed through the optical pulse technology for 3-8 min, high-voltage pulse processing is conducted, centrifugal washing is conducted on the yeast fermentation liquor to obtain yeast cells, the yeast cells are frozen at a temperature of -50 DEG C for 2 hours and freeze-dried into yeast powder, the yeast powder is extracted through alcohol, and extract liquor is concentrated to obtain astaxanthin. According to the fermentation cultivation method, a fermentation medium is prepared from 1% of glucose, 1-3% of corn steep liquor, 2.5% of peptone, 0.02% of magnesium sulfate, 0.15% of monopotassium phosphate, 20% of sodium chloride and the balance water; the initial pH is 8, the inoculation amount is 3-8%, the shaking table rotating speed is 50-80 r/min, cultivation is conducted at a temperature of 24-28 DEG C for 7-10 hours, the temperature is regulated to 12-17 DEG C, static cultivation is conducted for 0.1-0.5 hour, the temperature rises to 24-30 DEG C in a stepped mode, pH is regulated to 4.6-6.5, 2.5% of egg peptone and 2% of yeast powder are added, the shaking table rotating speed is 50-100 r/min, and fermentation continues to be conducted for 35-65 hours. The astaxanthin extraction rate can reach up to 95% or above, the astaxanthin yield is effectively increased, and the astaxanthin content of yeast cells relative to stem cells reaches 9700 ug/g.
Description
Technical field:
The invention belongs to field of microbial fermentation, particularly metabolism of yeasts product extracts.
Background technology:
Red phaffia rhodozyma (Phaffiarhodozyma) can synthesizing astaxanthin, the blue or green plain accumulation volume of red phaffia rhodozyma wild strain Prawn reaches about 0.05% of dry cell weight, can 0.3% be reached at most in some mutating strain series, and astaxanthin is main component in synthesized carotenoid, because of but the current bacterial strain that generally adopts of Production of Astaxanthin from Fermentation by Microorganisms.
The extracting method of the patent application astaxanthin in phaffiarhodozyma of Zhejiang Polytechnical University, publication number is CN1548420, and on November 24th, 2004 is open.The organic acid containing antioxidant or acidic organic solvent extraction astaxanthin is adopted in this patent.
The name of Shandong Agricultural University's application is called in technology for preparing astaxanthin using microwave-assisted dimethyl sulfoxide method, number of patent application is CN200910017325.X, the invention provides a kind of technology for preparing astaxanthin using microwave-assisted dimethyl sulfoxide method, dimethyl sulfoxide (DMSO) is added after red phaffia rhodozyma fermented liquid is centrifugal, auxiliary broken wall under being placed in microwave, add organic solvent again, insulation lixiviate, make astaxanthin forward extracellular in cell and be dissolved in organic solvent, after mixing solutions is centrifugal, obtaining astaxanthin concentrated solution through low temperature vacuum concentration.Microwave frequency measurment technology is successfully combined with dimethyl sulfoxide (DMSO) broken wall by this invention, reduces cost, improves efficiency, has saved extraction time, astaxanthin structure effectively can be prevented not to be destroyed, improve the extraction effect of red Astaxanthin from Xanthophyllomyces dendrorhous during Storage.
Summary of the invention:
The technical problem that the present invention solves is to provide a kind of method that saccharomycetes to make fermentation prepares astaxanthin.
Technical solution of the present invention is as follows:
Yeast carries out fermentation culture in the fermentation medium, and be adjusted to 9-10 at fermentation ends secondary fermentation liquid ph, pulsed technique process 3-8 minute, burst process parameter is: strength of electric field 42-52KV/cm, burst length 500-600 μ S, pulse-repetition 200-300Hz.Obtain yeast cell by after yeast fermentation broth centrifuge washing, yeast cell obtains yeast powder through freeze-drying method, alcoholic extract yeast powder, and concentrated extract obtains astaxanthin.
Fermentation culture method is as follows: fermention medium consists of glucose 1%, corn steep liquor 1-3%, peptone 2.5%, magnesium sulfate 0.02%, potassium primary phosphate 0.15%, sodium-chlor 20%, and all the other are water; Initial pH is 8, inoculum size 3-8%, shaking speed is 50-80r/min, cultivate for 24-28 DEG C and to adjust temperature after 7-10 hour and be 12-17 DEG C and carry out low temperature static gas wave refrigerator 0.1-0.5 hour, stepped appearance is warmed up to 24-30 DEG C afterwards, pH is adjusted to 4.6-6.5, and the addition according to 2.5% adds egg peptone, and the amount according to 2% adds yeast powder; Shaking speed is 50-100r/min, continues fermentation 35-65 hour.
In above-mentioned alcoholic extract technique, the solid-liquid ratio of yeast powder and alcohol is 1:8-10; Described alcohol is the ethanolic soln of 85-100%.
In Astaxanthin extraction process, concentrated employing distillation and concentration method.
Yeast of the present invention is phaffiafhodozyma CICC33064.
Beneficial effect:
The present invention utilizes the process of complex art means to realize the operational path of production of astaxanthin, extraction process of the present invention makes the extraction yield of astaxanthin reach more than 95%, and the present invention adopts stimulates cultural method and light pulse treatment process effectively to improve the output of astaxanthin in the temperature adjustment of Fermentative growth phase.The content astaxanthin of yeast cell reaches more than 9300 micrograms/gram stem cell.
Embodiment: describe yeast strain ferments in the present invention below by specific embodiment and produce the extraction process of astaxanthin and gsh.Unless stated otherwise, technique means used in the present invention is method known in those skilled in the art.In addition, embodiment is interpreted as illustrative, but not limits the scope of the invention, and the spirit and scope of the invention only limited by claims.To those skilled in the art, under the prerequisite not deviating from essence of the present invention and scope, the various change carry out separation and Extraction condition in these embodiments or change also belong to protection scope of the present invention.
In the present invention, the measuring method of astaxanthin is as follows:
1.10ml fermented liquid 3000r/min is centrifugal, and 15min washes twice, and removes supernatant liquor.
2., with the dimethyl sulfoxide (DMSO) broken wall of 2.5ml, stir and then use the centrifugal 15min of 3000r/min twice to throw out colourless, supernatant liquor acetone extract.
3. adding distil water washing acetone extract liquid is to clear, is settled to 25ml, measures the absorbancy of wavelength when 474nm.
The total astaxanthin mass concentration of cell (μ g/ml)=(A*Va)/(E*Vf)
In formula: A-optical density(OD)
Va-extraction liquid volume, ml
E-optical extinction coefficient is 0.21
Vf-samples fermentating liquid volume, ml
Embodiment 1
Technical solution of the present invention is as follows:
Yeast carries out fermentation culture in the fermentation medium, and after fermentation ends, obtain yeast cell by after yeast fermentation broth centrifuge washing, yeast cell obtains yeast powder through freeze-drying method, alcoholic extract yeast powder, and concentrated extract obtains astaxanthin.
Fermentation culture method is as follows: fermention medium consists of glucose 1%, corn steep liquor 1-3%, peptone 2.5%, magnesium sulfate 0.02%, potassium primary phosphate 0.15%, sodium-chlor 20%, and all the other are water; Initial pH is 8, inoculum size 3-8%, and shaking speed is 50-80r/min, 24-28 DEG C and cultivates 65 hours.20 liters of fermentor tanks.
In alcoholic extract technique, the solid-liquid ratio of yeast powder and alcohol is 1:10; Described alcohol is the ethanolic soln of 85-100%.
In Astaxanthin extraction process, concentrated employing distillation and concentration method.
Cell concn reaches 8.5 grams per liters, and content astaxanthin is 2540 μ g/g stem cells.Described yeast is phaffiafhodozyma CICC33064.
Embodiment 2
Yeast carries out fermentation culture in the fermentation medium, and after fermentation ends, obtain yeast cell by after yeast fermentation broth centrifuge washing, yeast cell obtains yeast powder through freeze-drying method, alcoholic extract yeast powder, and concentrated extract obtains astaxanthin.
Fermentation culture method is as follows: fermention medium consists of glucose 1%, corn steep liquor 1-3%, peptone 2.5%, magnesium sulfate 0.02%, potassium primary phosphate 0.15%, sodium-chlor 20%, and all the other are water; Initial pH is 8, inoculum size 3-8%, and shaking speed is 50-80r/min, 24-28 DEG C and cultivates 75 hours.20 liters of fermentor tanks.
In alcoholic extract technique, the solid-liquid ratio of yeast powder and alcohol is 1:10; Described alcohol is the ethanolic soln of 85-100%.In Astaxanthin extraction process, concentrated employing distillation and concentration method.
Cell concn reaches 9 grams per liters, and content astaxanthin is 2480 μ g/g stem cells.The extraction rate reached of astaxanthin is to 85%.
Described yeast is phaffiafhodozyma CICC33064.
Embodiment 3
The technical problem that the present invention solves is to provide a kind of method that saccharomycetes to make fermentation prepares astaxanthin.
Technical solution of the present invention is as follows:
Yeast carries out fermentation culture in the fermentation medium, and be adjusted to 9.5 at fermentation ends secondary fermentation liquid ph, pulsed technique process 5 minutes, burst process parameter is: strength of electric field 48KV/cm, burst length 550 μ S, pulse-repetition 250Hz.Obtain yeast cell by after yeast fermentation broth centrifuge washing, yeast cell obtains yeast powder through freeze-drying method, alcoholic extract yeast powder, and concentrated extract obtains astaxanthin.
Fermentation culture method is as follows: fermention medium consists of glucose 1%, corn steep liquor 2%, peptone 2.5%, magnesium sulfate 0.02%, potassium primary phosphate 0.15%, sodium-chlor 20%, and all the other are water; Initial pH is 8, inoculum size 5%, and shaking speed is 60r/min, cultivate for 25-27 DEG C and to adjust temperature after 8 hours and be 14 DEG C and carry out low temperature static gas wave refrigerator 0.3 hour, stepped appearance is warmed up to 26-28 DEG C afterwards, and pH is adjusted to 5.6, addition according to 2.5% adds egg peptone, and the amount according to 2% adds yeast powder; Shaking speed is 50r/min, continues fermentation 65 hours.
In above-mentioned alcoholic extract technique, the solid-liquid ratio of yeast powder and alcohol is 1:8; Described alcohol is the ethanolic soln of 95%.
In Astaxanthin extraction process, concentrated employing distillation and concentration method.
In above-mentioned hot water extraction's operation, hot water temperature is 95 DEG C, lixiviate 3 times, each lixiviate 10 minutes;
Cell concn reaches more than 13 grams per liters, and content astaxanthin is 9480 μ g/g stem cells.The extraction rate reached of astaxanthin is to 95%.
Described yeast is phaffiafhodozyma CICC33064.
Embodiment 4
The technical problem that the present invention solves is to provide a kind of method that saccharomycetes to make fermentation prepares astaxanthin.
Technical solution of the present invention is as follows:
Yeast carries out fermentation culture in the fermentation medium, and be adjusted to 10 at fermentation ends secondary fermentation liquid ph, pulsed technique process 8 minutes, burst process parameter is: strength of electric field 42KV/cm, burst length 500 μ S, pulse-repetition 200Hz.Obtain yeast cell by after yeast fermentation broth centrifuge washing, yeast cell obtains yeast powder through freeze-drying method, alcoholic extract yeast powder, and concentrated extract obtains astaxanthin.
Fermentation culture method is as follows: fermention medium consists of glucose 1%, corn steep liquor 3%, peptone 2.5%, magnesium sulfate 0.02%, potassium primary phosphate 0.15%, sodium-chlor 20%, and all the other are water; Initial pH is 8, inoculum size 3-8%, shaking speed is 50-80r/min, cultivate for 26-28 DEG C and to adjust temperature after 7 hours and be 17 DEG C and carry out low temperature static gas wave refrigerator 0.1 hour, stepped appearance is warmed up to 24-27 DEG C afterwards, pH is adjusted to 5.5, and the addition according to 2.5% adds egg peptone, and the amount according to 2% adds yeast powder; Shaking speed is 50-100r/min, continues fermentation 60 hours.
In above-mentioned alcoholic extract technique, the solid-liquid ratio of yeast powder and alcohol is 1:8; Described alcohol is the ethanolic soln of 100%.
In Astaxanthin extraction process, concentrated employing distillation and concentration method.
The extraction rate reached of astaxanthin is to 96%.Described yeast is phaffiafhodozyma CICC33064.
In above-mentioned hot water extraction's operation, hot water temperature is 95 DEG C, lixiviate 3 times, each lixiviate 10 minutes;
Cell concn reaches more than 12 grams per liters, and content astaxanthin is 9360 μ g/g stem cells.
Claims (7)
1. a saccharomycetes to make fermentation prepares the method for astaxanthin, yeast carries out fermentation culture in the fermentation medium, 9-10 is adjusted at fermentation ends secondary fermentation liquid ph, burst process 3-8 minute, yeast cell is obtained by after yeast fermentation broth centrifuge washing, yeast cell obtains yeast powder through freeze-drying method, alcoholic extract yeast powder, and concentrated extract obtains astaxanthin.
2. according to right 1, saccharomycetes to make fermentation prepares the method for astaxanthin, and burst process parameter is: strength of electric field 42-52KV/cm, burst length 500-600 μ S, pulse-repetition 200-300Hz.
3. according to right 1-2, saccharomycetes to make fermentation prepares the method for astaxanthin, and fermentation culture method is as follows: fermention medium consists of glucose 1%, corn steep liquor 1-3%, peptone 2.5%, magnesium sulfate 0.02%, potassium primary phosphate 0.15%, sodium-chlor 20%, all the other are water; Initial pH is 8, inoculum size 3-8%, shaking speed is 50-80r/min, cultivate for 24-28 DEG C and to adjust temperature after 7-10 hour and be 12-17 DEG C and carry out low temperature static gas wave refrigerator 0.1-0.5 hour, stepped appearance is warmed up to 24-30 DEG C afterwards, pH is adjusted to 4.6-6.5, and the addition according to 2.5% adds egg peptone, and the amount according to 2% adds yeast powder; Shaking speed is 50-100r/min, continues fermentation 35-65 hour.
4. according to right 1, saccharomycetes to make fermentation prepares the method for astaxanthin, and in alcoholic extract technique, the solid-liquid ratio of yeast powder and alcohol is 1:8-10; Described alcohol is the ethanolic soln of 85-100%.
5. according to right 1, saccharomycetes to make fermentation prepares the method for astaxanthin, yeast carries out fermentation culture in the fermentation medium, 9.5 are adjusted at fermentation ends secondary fermentation liquid ph, pulsed technique process 5 minutes, burst process parameter is: strength of electric field 48KV/cm, burst length 550 μ S, pulse-repetition 250Hz.Obtain yeast cell by after yeast fermentation broth centrifuge washing, yeast cell obtains yeast powder through freeze-drying method, alcoholic extract yeast powder, and concentrated extract obtains astaxanthin.
6. according to right 5, saccharomycetes to make fermentation prepares the method for astaxanthin, and fermentation culture method is as follows: fermention medium consists of glucose 1%, corn steep liquor 2%, peptone 2.5%, magnesium sulfate 0.02%, potassium primary phosphate 0.15%, sodium-chlor 20%, and all the other are water; Initial pH is 8, inoculum size 5%, and shaking speed is 60r/min, cultivate for 25-27 DEG C and to adjust temperature after 8 hours and be 14 DEG C and carry out low temperature static gas wave refrigerator 0.3 hour, stepped appearance is warmed up to 26-28 DEG C afterwards, and pH is adjusted to 5.6, addition according to 2.5% adds egg peptone, and the amount according to 2% adds yeast powder; Shaking speed is 50r/min, continues fermentation 65 hours.
7. according to right 1-4, saccharomycetes to make fermentation prepares the method for astaxanthin, yeast carries out fermentation culture in the fermentation medium, 10 are adjusted at fermentation ends secondary fermentation liquid ph, pulsed technique process 8 minutes, burst process parameter is: strength of electric field 42KV/cm, burst length 500 μ S, pulse-repetition 200Hz.Obtain yeast cell by after yeast fermentation broth centrifuge washing, yeast cell obtains yeast powder through freeze-drying method, alcoholic extract yeast powder, and concentrated extract obtains astaxanthin; Fermentation culture method is as follows: fermention medium consists of glucose 1%, corn steep liquor 3%, peptone 2.5% magnesium sulfate 0.02%, potassium primary phosphate 0.15%, sodium-chlor 20%, and all the other are water; Initial pH is 8, inoculum size 3-8%, shaking speed is 50-80r/min, cultivate for 26-28 DEG C and to adjust temperature after 7 hours and be 17 DEG C and carry out low temperature static gas wave refrigerator 0.1 hour, stepped appearance is warmed up to 24-27 DEG C afterwards, pH is adjusted to 5.5, and the addition according to 2.5% adds egg peptone, and the amount according to 2% adds yeast powder; Shaking speed is 50-100r/min, continues fermentation 60 hours.In above-mentioned alcoholic extract technique, the solid-liquid ratio of yeast powder and alcohol is 1:8; Described alcohol is the ethanolic soln of 100%.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107164449A (en) * | 2017-08-03 | 2017-09-15 | 广州立达尔生物科技股份有限公司 | A kind of utilization marigold flower dregs of rice extract solution is method and the application that fermenting raw materials produce astaxanthin |
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Cited By (2)
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| CN107164449A (en) * | 2017-08-03 | 2017-09-15 | 广州立达尔生物科技股份有限公司 | A kind of utilization marigold flower dregs of rice extract solution is method and the application that fermenting raw materials produce astaxanthin |
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Application publication date: 20160511 |