CN101985645A - Method for biosynthesizing astaxanthin by using exponential intermittent feed supplement mode - Google Patents
Method for biosynthesizing astaxanthin by using exponential intermittent feed supplement mode Download PDFInfo
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Abstract
The invention relates to a method for biosynthesizing astaxanthin by using an exponential intermittent feed supplement mode. The method comprises the following steps: S1, taking subsidiary agricultural products rich in synthetic precursors of the astaxanthin to prepare an adding fluid and sterilizing the adding fluid for 15-30 minutes at the temperature of 115-121 DEG C; S2, taking and cultivating Phaffia rhodozyma in a liquid fermentation medium, feeding 0.5-5% the adding fluid in a batch mode at intervals of 3-12 hours after the exponential phase, and after carrying out fed-batch for 1-4 times, continuing to carry out cultivation 12-48 hours; and S3, extracting the astaxanthin. In the method of the invention, a small amount of astaxanthin synthetic accelerator is added in the exponential phase of the fermentation, thus avoiding premature addition of the astaxanthin synthetic accelerator from inhibiting yeast plant growth and greatly improving the yield of the astaxanthin at the same time, wherein the yield of the astaxanthin is improved by 20-95% compared with a control group. The extracted astaxanthin can be used as feed additives in high-grade aquatic products industry, fish breeding and poultry raising industry and the like. The invention has the advantages that the raw material price is low, the operation is simple and the popularization and application value is high.
Description
Technical field
The present invention relates to the biotechnology correlative technology field, relate in particular to a kind of utilization and be rich in the method that the agricultural byproducts fermenting raw materials of astaxanthin precursor is produced astaxanthin.
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.Because of it has special colouring function, can make epidermis and the yolk of meat, shrimp shell and poultry of fish painted, make its lovely luster, so can be used as the feed nutrition reinforcer of the poultry of high-grade aquaculture; In addition, astaxanthin also 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 requirement 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.
Astaxanthin is the main component of carotenoid, accounts for 40-95% greatly, and red Fife's yeast 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 bacterial classification of astaxanthin scale operation of tool researching value.
Description of drawings
Fig. 1 is the technical solution adopted in the present invention schematic flow sheet.
Summary of the invention
The present invention relates to a kind of utilization and be rich in the method for the agricultural byproducts of astaxanthin precursor by exponential phase fed-batch fermentative production astaxanthin.Used agricultural byproducts, as Semen Maydis powder, Radix Dauci Sativae etc. after treatment, are made the annex solution of fermention medium.Annex solution was added in red Fife's zymic liquid nutrient medium in the different time, ferment, collect yeast cell, extract astaxanthin to certain hour.Output when purpose of the present invention is intended to improve red Fife's yeast microorganism method fermentative production astaxanthin, and reduce production costs.
In order to achieve the above object, the method for a kind of utilization index fed-batch of the present invention mode biosynthesizing astaxanthin comprises the steps:
S1, get the agricultural byproducts that are rich in the synthetic precursor of astaxanthin, to squeeze the juice or breaking method prepares annex solution; And 115-121 ℃ of sterilization 15-30 minute;
S2, get the phaffiafhodozyma bacterium and in liquid fermentation medium, cultivate, after exponential phase,, after 1-4 time stream adds, continue to cultivate 12-48 hour to add the annex solution of 0.5-5% every 3-12 hour stream;
Extract astaxanthin in S3, the product behind S2.
Under the optimal way, the method for above-mentioned utilization index fed-batch mode biosynthesizing astaxanthin is rich in the synthetic precursor of astaxanthin and comprises Lyeopene, β-Hu Luobusu, zeaxanthin.In step S1, the agricultural byproducts that are rich in the synthetic precursor of astaxanthin comprise one or more in tomato, Radix Dauci Sativae, Semen Maydis powder, yellow ginger, the pumpkin.Materials such as tomato, Radix Dauci Sativae add as required during the preparation annex solution, and under the optimal way, the proportioning of solid feed and water is taken as 1: 0.5-2; But it is rare that the concentration that needs the final annex solution of assurance was difficult for, and adds effect in order to avoid influence subsequent flows.
In addition, in step S2, liquid fermentation medium comprises: glucose 1-4%, ammonium sulfate 0.3-0.6%, potassium primary phosphate 0.1-0.2%, MgSO
47H
2O 0.05-0.1%, yeast soak powder 0.1-0.3%, CaCl
20.01-0.05%.Optimum liquid fermentation medium comprises: glucose 3%, ammonium sulfate 0.5%, potassium primary phosphate 0.1%, MgSO
47H
2O 0.05%, yeast soak powder 0.1%, CaCl
20.01%.Also can adopt the composition of other substratum as required.Such as, glucose 3.6%, extractum carnis 0.5%, potassium primary phosphate 0.1%, MgSO
47H
2O 0.05%, and yeast soaks powder 0.1%, CaCl
20.01%.
In addition, also comprise the step of actication of culture before the step S2: the phaffiafhodozyma bacterium bacterial classification of slant preservation is carried out activation culture in 20-26 ℃ on the YM slant medium, then in the YM substratum, ventilate and cultivate, obtain the YM seed liquor in 20-26 ℃ of liquid.And in step S2, the cultivation before exponential phase should insert the YM seed liquor with 6%-12% under the optimum way in liquid fermentation medium, cultivates 12-48 hour, arrives exponential phase.Wherein, 6%-12% is the volume ratio that accounts for fermented liquid.And the composition of YM substratum or YM slant medium comprises: glucose 1%, peptone 0.5%, wort 0.3%, yeast extract paste 0.3%.
The present invention is by adding a spot of astaxanthin synthesis accelerant in the exponential phase of fermentation, having avoided these materials to add inhibiting while to yeast cell growth too early, significantly improved the output of astaxanthin, and comparison you can well imagine 20-95% high according to component.Therefrom the astaxanthin of Ti Quing can be used for the fodder additives of high-grade water industry and aquaculture etc.The present invention adopts cheap raw material, and is simple to operate, has highly application value.
Embodiment
As shown in Figure 1, the technical solution used in the present invention: some intermediate products of red Fife's yeast metabolism process are provided or are rich in the material of astaxanthin synthetic precursor, exponential phase fed-batch in fermention medium, obtain the high red Fife's yeast of content astaxanthin.Facts have proved: after fermentation has entered exponential phase, add some cheap materials, not only improved the output of astaxanthin, also reduced cost simultaneously.Concrete implementing process is as follows:
One, raw material choosing and handling
Selection contains some agricultural byproducts of the synthetic precursor of astaxanthin, and wherein the synthetic precursor of astaxanthin has Lyeopene, β-Hu Luobusu etc.According to the feature of raw material, adopt the mode squeeze the juice or to pulverize to obtain raw material, be made into behind the liquid 121 ℃, sterilized 20-30 minute.
Two, the activation of bacterial classification
The bacterial classification of slant preservation is carried out activation culture in 20-26 ℃ on the YM slant medium, in the YM substratum, ventilate and cultivate in 20-26 ℃ of liquid.
Three, liquid culture
Liquid fermentation medium comprises: glucose 1-4%, ammonium sulfate 0.3-0.6%, potassium primary phosphate 0.1-0.2%, MgSO
47H
2O 0.05-.0.1%, yeast soaks powder 0.1-0.3%, CaCl
20.01-0.05%.
In liquid fermentation medium, insert the YM seed liquor with 6%-12%, cultivated 12-48 hour, arrive exponential phase.
Four, the stream that is rich in the annex solution of precursor substance adds
After thalline arrived exponential phase, every 3-12 hour, stream added annex solution 1-4 time of 0.5-5%, finishes until logarithmic phase, after stopping stream and adding, continued cultivation 12-48 hour.Wherein, 0.5%-5% is meant the per-cent that accounts for liquid fermentation medium.
Five, the extraction of astaxanthin
Measure the content astaxanthin in the yeast cell, extract astaxanthin then according to a conventional method.
Embodiment 1.
Choosing of A, raw material: select to contain the synthetic precursor of astaxanthin fresh sophisticated tomato cleaning, stripping and slicing, squeeze the juice, filter.121 ℃ of sterilizations of filtrate 20 minutes, standby in 4 ℃ of preservations.
The activation of B, bacterial classification: the bacterial classification of slant preservation is carried out activation culture in 23 ℃ on the YM slant medium, in the YM substratum, ventilate and cultivate in 23 ℃ of liquid.
C, liquid culture: the YM seed liquor with in 8% the inoculum size liquid medium within, was cultivated 36 hours down in 23 ℃.
D, the stream that is rich in the annex solution of precursor add: ferment after 36 hours, added 1% tomato juice every 12 hours, add twice continuously after, stop stream and add, continue to cultivate 36 hours.
The extraction of E, astaxanthin: measure the content astaxanthin in red Fife's yeast.
The blue or green cellulose content of gained has improved 91% than control group.
Embodiment 2.
Getting Radix Dauci Sativae implements by A, B step in the example 1.
C, liquid culture: the YM seed liquor with in 10% the inoculum size liquid medium within, was cultivated 24 hours down in 22 ℃.
D, the stream that is rich in the annex solution of precursor add: ferment after 24 hours, added 0.5% Radix Dauci Sativae juice in each 6 hours, add three times continuously after, stop stream and add, continue to cultivate 24 hours.
The extraction of E, astaxanthin: measure the content astaxanthin in red Fife's yeast.
The blue or green cellulose content of gained has improved 27% than control group.
Embodiment 3.
A, get Semen Maydis powder and carry out gelatinization, 121 ℃ of sterilizations are 20 minutes then, and are standby in 4 ℃ of preservations.
The activation of B, bacterial classification: the bacterial classification of slant preservation is carried out activation culture in 24 ℃ on the YM slant medium, in the YM substratum, ventilate and cultivate in 24 ℃ of liquid.
C, liquid culture: the YM seed liquor with in 12% the inoculum size liquid medium within, was cultivated 48 hours down in 24 ℃.
D, the stream that is rich in the annex solution of precursor add: ferment after 48 hours, added 2% corn steep liquor in each 3 hours, add 4 times continuously after, stop stream and add, continue to cultivate 48 hours.
The extraction of E, astaxanthin: measure the content astaxanthin in red Fife's yeast.
The blue or green cellulose content of gained has improved 47% than control group.
Embodiment 4
A, get pumpkin cleaning, stripping and slicing, squeeze the juice, filter.About 115 ℃ of sterilizations of filtrate 25min, standby in 4 ℃ of preservations.
The activation of B, bacterial classification: the bacterial classification of slant preservation is carried out activation culture in 22 ℃ on the YM slant medium, in the YM substratum, ventilate and cultivate in 22 ℃ of liquid.
C, liquid culture: the YM seed liquor with in 7% the inoculum size liquid medium within, was cultivated 30 hours down in 22 ℃.
D, the stream that is rich in the annex solution of precursor add: ferment after 30 hours, added 2% Sucus Cucurbitae moschatae every 15 hours, add 2-3 time continuously after, stop stream and add, continue cultivation 30 hours.
The extraction of E, astaxanthin: measure the content astaxanthin in red Fife's yeast.
The blue or green cellulose content of gained has improved 60-85% than control group, is more excellent preparation scheme.
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 (6)
1. the method for a utilization index fed-batch mode biosynthesizing astaxanthin is characterized in that, comprises the steps:
S1, get the agricultural byproducts that are rich in the synthetic precursor of astaxanthin, to squeeze the juice or breaking method prepares annex solution; And 115-121 ℃ of sterilization down;
S2, get the phaffiafhodozyma bacterium and in liquid fermentation medium, cultivate, after exponential phase,, after 1-4 time stream adds, continue to cultivate 12-48 hour to add the described annex solution of 0.5-5% every 3-12 hour stream;
Extract astaxanthin in S3, the product behind S2.
2. according to the method for the described utilization index fed-batch of claim 1 mode biosynthesizing astaxanthin, it is characterized in that the agricultural byproducts that are rich in the synthetic precursor of astaxanthin among the step S1 comprise one or more in tomato, Radix Dauci Sativae, Semen Maydis powder, yellow ginger, the pumpkin.
3. according to the method for claim 1 or 2 described utilization index fed-batch mode biosynthesizing astaxanthins, it is characterized in that,
In step S2, described liquid fermentation medium comprises: glucose 3%, ammonium sulfate 0.5%, potassium primary phosphate 0.1%, MgSO
47H
2O 0.05%, yeast soak powder 0.1%, CaCl
20.01%.
4. according to the method for the described utilization index fed-batch of claim 3 mode biosynthesizing astaxanthin, it is characterized in that,
The step that also comprises actication of culture before the step S2: the phaffiafhodozyma bacterium bacterial classification of slant preservation is carried out activation culture in 20-26 ℃ on the YM slant medium, then in the YM substratum, ventilate and cultivate, obtain the YM seed liquor in 20-26 ℃ of liquid.
5. according to the method for the described utilization index fed-batch of claim 4 mode biosynthesizing astaxanthin, it is characterized in that,
In step S2, in described liquid fermentation medium, insert described YM seed liquor with 6%-12%, cultivated 12-48 hour, arrive exponential phase.
6. according to the method for the described utilization index fed-batch of claim 5 mode biosynthesizing astaxanthin, it is characterized in that in step S1, the solid feed of described annex solution and the proportioning of water are taken as 1: 0.5-2.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103436585A (en) * | 2013-09-03 | 2013-12-11 | 广州元大生物科技发展有限公司 | Method for producing astaxanthin through lactobacillus fermentum |
| CN103820520A (en) * | 2014-03-06 | 2014-05-28 | 浙江皇冠科技有限公司 | High-yield natural astaxanthin fermentation method |
| CN104762327A (en) * | 2014-08-11 | 2015-07-08 | 四川安益生物科技有限公司 | Pumpkin yellow pigment yeast product and preparation method thereof |
| CN106834405A (en) * | 2017-01-09 | 2017-06-13 | 山东理工大学 | It is a kind of to add the method that carrot juice induces haematococcus pluvialis efficient accumulation astaxanthin |
| CN108893517A (en) * | 2018-07-19 | 2018-11-27 | 威海利达生物科技有限公司 | A kind of fermentation medium and method of red phaffia rhodozyma fermenting and producing astaxanthin |
| CN112266945A (en) * | 2020-09-29 | 2021-01-26 | 自然资源部第三海洋研究所 | Method for extracting astaxanthin from phaffia rhodozyma |
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2009
- 2009-10-30 CN CN2009101882997A patent/CN101985645A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103436585A (en) * | 2013-09-03 | 2013-12-11 | 广州元大生物科技发展有限公司 | Method for producing astaxanthin through lactobacillus fermentum |
| CN103436585B (en) * | 2013-09-03 | 2015-03-04 | 广州元大生物科技发展有限公司 | Method for producing astaxanthin through lactobacillus fermentum |
| CN103820520A (en) * | 2014-03-06 | 2014-05-28 | 浙江皇冠科技有限公司 | High-yield natural astaxanthin fermentation method |
| CN104762327A (en) * | 2014-08-11 | 2015-07-08 | 四川安益生物科技有限公司 | Pumpkin yellow pigment yeast product and preparation method thereof |
| CN106834405A (en) * | 2017-01-09 | 2017-06-13 | 山东理工大学 | It is a kind of to add the method that carrot juice induces haematococcus pluvialis efficient accumulation astaxanthin |
| CN106834405B (en) * | 2017-01-09 | 2020-09-08 | 山东理工大学 | Method for inducing haematococcus pluvialis to efficiently accumulate astaxanthin by adding carrot juice |
| CN108893517A (en) * | 2018-07-19 | 2018-11-27 | 威海利达生物科技有限公司 | A kind of fermentation medium and method of red phaffia rhodozyma fermenting and producing astaxanthin |
| CN112266945A (en) * | 2020-09-29 | 2021-01-26 | 自然资源部第三海洋研究所 | Method for extracting astaxanthin from phaffia rhodozyma |
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