CN108823276B - Method for producing astaxanthin by using starch wastewater - Google Patents
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- 239000001168 astaxanthin Substances 0.000 title claims abstract description 69
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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
- C12P39/00—Processes involving microorganisms of different genera in the same process, simultaneously
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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 belongs to the technical field of biology, and discloses a method for producing astaxanthin by using starch wastewater, which comprises the following steps: step 1) pretreating starch wastewater, step 2) preparing mixed bacteria liquid, step 3) performing mixed culture of bacteria and algae, and step 4) extracting astaxanthin. The method disclosed by the invention not only treats the starch wastewater, but also prepares the astaxanthin, achieves two purposes, and has a wide application prospect.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for producing astaxanthin by using starch wastewater.
Background
Astaxanthin (astaxanthin), the chemical name of which is 3,3 ' -dihydroxy-4, 4 ' -diketo-beta, beta ' -carotene, is a terpene unsaturated compound with the chemical formula of C40H52O4, and has two beta-ionone rings and 11 conjugated double bonds in the molecular structure, and the chemical structure is similar to that of beta-carotene. Astaxanthin is widely found in the natural world, such as most crustaceans and salmonids, leaves, flowers, fruits of plants, and feathers of flamingos, and is a major carotenoid in marine organisms, especially the feathers of shrimps, crabs, fish, algae, yeasts, and birds.
Astaxanthin is one of the strongest natural antioxidants in the world, effectively eliminates oxygen free radicals in cells, enhances the regeneration capacity of the cells, maintains the balance of the organism, reduces the accumulation of aging cells, and protects the health of the cells and DNA from inside to outside, thereby protecting the health of skin, promoting the growth of hair, resisting aging, relieving sports fatigue and enhancing the vitality. Since 2008, a large number of studies at home and abroad prove that astaxanthin has strong antioxidant activity and has positive promoting effects in the aspects of improving immunity, preventing the occurrence and development of chronic diseases such as tumors, cardiovascular diseases, diabetes and the like, delaying aging and the like. At present, the compound is mainly used as high-grade health food and medicine for human beings, and feed additives for aquaculture animals, poultry and livestock. With the discovery of various physiological functions of astaxanthin, research on astaxanthin is hot at home and abroad. The production of astaxanthin has two modes of artificial synthesis and biological acquisition. The artificially synthesized astaxanthin is expensive, has obvious difference with natural astaxanthin in structure, function, application, safety and other aspects, and has weak absorption capacity of animals on the chemically synthesized astaxanthin and can not be converted into natural configuration in vivo. The biological acquisition is mainly from waste, phaffia rhodozyma and microalgae in the aquatic product processing industry. Among them, the astaxanthin content in the waste is low, and the extraction cost is high, so that the method is not suitable for large-scale production. The average astaxanthin content in the natural Phaffia rhodozyma is only 0.40%. In contrast, Haematococcus pluvialis has an astaxanthin content as high as 1.5% to 3.0%, and is therefore considered a "concentrate" of natural astaxanthin. Haematococcus pluvialis is recognized as the best organism for producing natural astaxanthin in nature, so that the extraction of astaxanthin by using the microalgae has a wide development prospect and becomes a research hotspot for producing natural astaxanthin internationally.
The literature, "influence of nutritional stress on astaxanthin accumulation of haematococcus pluvialis, 2000 of aquatic biology reports" finds that by changing nutritional conditions, haematococcus pluvialis can be induced to accumulate astaxanthin, the rate of pigments is inversely proportional to the primary nitrogen concentration, the relative lack of a nitrogen source is relatively beneficial to cell proliferation and pigment accumulation, and balancing the nitrogen source, cell proliferation and astaxanthin yield is the key point of attention for astaxanthin production. Chinese patent CN103114121A, a method for producing astaxanthin by haematococcus pluvialis, discloses a method for producing astaxanthin by haematococcus pluvialis, which comprises the steps of naturally culturing the haematococcus pluvialis in water of a culture pond for 6-8 days, allowing the haematococcus pluvialis to proliferate, adding potassium dihydrogen phosphate and sodium nitrate into the pond water, covering a red film on the pond, and enabling the content of astaxanthin to reach more than 1% under the irradiation of light waves with the wavelength of 630-760 nm. In the literature, "the influence of temperature on the biomass and the yield of astaxanthin of Haematococcus pluvialis (Haematococcus pluvialis)" in Wuhan botanicals research 2005, "the influence of temperature on the biomass and the yield of astaxanthin of Haematococcus pluvialis was studied in the process of culturing Haematococcus pluvialis using a circular culture pond simulation system, and the results show that the biomass and the yield of astaxanthin of Haematococcus pluvialis are highest at 25 ℃, and are respectively 2.68g/L and 13.53 mg/L.
The starch processing is a process technology for preparing starch by soaking, grinding and other processes of crops such as corn, sorghum, wheat, potato, sweet potato and the like. The starch processing industry has the characteristics of high water consumption, strong pollution and the like, the COD of the wastewater is high, usually 1000-30000 mg/L, and the wastewater also comprises high-concentration SS, NH3-N, pigment, starch, cellulose and the like. Starch manufacturing enterprises discharge a large amount of high-concentration organic wastewater every year, and the wastewater treatment standard reaching rate of the starch manufacturing enterprises is low, so that the environment is seriously polluted, and how to purify and utilize the organic wastewater is also a technical problem to be solved.
Disclosure of Invention
The invention aims to solve the technical problems of low astaxanthin yield and difficult treatment of starch wastewater, and provides a method for producing astaxanthin by using starch wastewater.
The invention is realized by the following technical scheme:
a method for producing astaxanthin by using starch wastewater comprises the following steps: step 1) pretreating starch wastewater, step 2) preparing mixed bacteria liquid, step 3) performing mixed culture of bacteria and algae, and step 4) extracting astaxanthin.
Further, the method comprises the steps of:
step 1) pretreating starch wastewater;
step 2) preparing mixed bacteria liquid: mixing a bacillus amyloliquefaciens culture solution, a lactobacillus plantarum culture solution and a rhodopseudomonas palustris culture solution according to a volume ratio of 5-7:3-5:3-5 to obtain a composite bacterial solution;
step 3), mixed culture of bacteria and algae: injecting the pretreated starch wastewater into a biological reaction tank, firstly, inoculating the composite bacterial liquid into the starch wastewater according to the volume ratio of 1 (200-; then centrifuging the liquid at the speed of 2000-3000rpm for 10-20min, and collecting the precipitate;
and 4) extracting astaxanthin.
Preferably, the first and second electrodes are formed of a metal,
the step 1) of starch wastewater pretreatment comprises the following steps: the starch wastewater firstly enters a sedimentation tank, the sedimentation time is 12-24h, and then the starch wastewater enters an acid-base adjusting tank, and the pH value is adjusted to 7.0-7.5.
Preferably, the first and second electrodes are formed of a metal,
the step 4) of extracting astaxanthin comprises the following steps: washing the precipitate with water twice, and freeze-drying to obtain powder; then, as 1kg of powder: adding solvent in an amount of 1-2L of solvent, performing ultrasonic wall breaking, performing ultrasonic power of 300W, performing ultrasonic time of 10s each time, performing intermittent time of 10s, performing total ultrasonic time of 60min, standing for 30min, centrifuging at 3000rpm for 5min, collecting supernatant and precipitate, adding acetone with the same volume into the precipitate, performing ultrasonic treatment for 30min, performing ultrasonic power of 300W, performing ultrasonic time of 10s each time, performing intermittent time of 10s, centrifuging at 3000rpm for 3min, and collecting supernatant; and combining the two supernatants, evaporating to dryness at low temperature, dissolving the residue with diethyl ether, adding 0.1mol/L KOH/methanol solution with the same mass, and saponifying at 4 ℃ for 12 hours to obtain an astaxanthin crude extract.
Preferably, the first and second electrodes are formed of a metal,
the preparation method of the bacillus amyloliquefaciens culture solution comprises the following steps: inoculating the bacillus amyloliquefaciens to an LB solid culture medium for culture to obtain a single colony; selecting single colony, inoculating to LB liquid culture medium, performing amplification culture to obtain 5-7 × 108cfu/ml of Bacillus amyloliquefaciens culture solution.
Preferably, the first and second electrodes are formed of a metal,
the preparation method of the lactobacillus plantarum culture solution comprises the following steps: inoculating Lactobacillus plantarum to MRS solid culture medium, culturing to obtain single colony, inoculating to MRS liquid culture medium, and culturing to 3-5 × 108cfu/ml of lactobacillus plantarum culture fluid.
Preferably, the first and second electrodes are formed of a metal,
the above-mentionedThe preparation method of the rhodopseudomonas palustris culture solution comprises the following steps: performing streak culture on rhodopseudomonas palustris on an LB (Langerhans) plate to obtain a single colony; selecting single colony, inoculating to amplification culture medium, and culturing to 3-5 × 108cfu/ml of Rhodopseudomonas palustris culture solution.
Preferably, the first and second electrodes are formed of a metal,
the components of the expanding culture medium are as follows: 10g/L glucose, 8g/L corn steep liquor, 1g/L ammonium chloride, 0.1g/L potassium dihydrogen phosphate, 0.1g/L dipotassium hydrogen phosphate, 0.01g/L ferrous sulfate heptahydrate and 0.01g/L magnesium sulfate heptahydrate.
Preferably, the first and second electrodes are formed of a metal,
the solvent is prepared by uniformly mixing ethyl acetate and ethanol according to the volume ratio of 1: 2.
The strains specifically used in the embodiment of the invention are Bacillus amyloliquefaciens ATCC23843, Lactobacillus plantarum ATCC8014 and Rhodopseudomonas palustris ATCC 17001.
In other technical solutions of the present invention, the concentration of the bacterial strain in the composite bacterial liquid is not particularly limited, and may be specifically selected according to specific situations, which is not described in detail herein.
The strains of the present invention belong to known strains and can be purchased from ATCC and other commercial sources. The culture procedure of each strain of the present invention is a routine technique in the art, and is not limited to the embodiment described in the present invention. The starting materials or reagents used in the present invention are commercially available unless otherwise specified.
Compared with the prior art, the invention has the advantages that the following aspects are mainly included but not limited:
the content of ammonia nitrogen in the starch wastewater is too high, and the ammonia nitrogen of a polluted water body in the form of free ammonia and ammonium ions has great toxicity to haematococcus pluvialis, so that the haematococcus pluvialis can die; after the starch wastewater is pretreated, the toxicity is reduced, the composite bacterial liquid adopted by the invention can utilize ammonium ions and degrade starch and other wastes to generate reducing carbohydrate substances, and after the starch wastewater is treated, the ammonia nitrogen content in the wastewater is reduced, and the wastewater contains rich carbon sources and pigments and is suitable for the production of haematococcus pluvialis; the carbon dioxide generated by the strain can meet the requirement of the rapid growth of algae cells, so that the accumulation rate of astaxanthin is increased; haematococcus pluvialis can generate oxygen and small molecular compounds for use in common bacteria to form symbiotic relationship; the haematococcus pluvialis can utilize nitrate ions in wastewater as a nitrogen source, the growth rate is improved, and ammonium ions generated by reduction reaction are utilized by bacterial strains in turn; sufficient nitrogen source is beneficial to the increase of algae biomass but not beneficial to the accumulation of astaxanthin, and as the nitrogen source in the wastewater is reduced, the haematococcus pluvialis growth rate is slowed down, but the astaxanthin accumulation rate is obviously increased. The composite bacterial liquid provided by the invention adopts three strains which are symbiotic with each other, has good synergistic performance, and can greatly reduce pollutants in wastewater, thereby conforming to the living environment of haematococcus pluvialis. The method disclosed by the invention not only treats the starch wastewater, but also prepares the astaxanthin, achieves two purposes, and has a wide application prospect.
Drawings
FIG. 1: the effect of incubation time on biomass;
FIG. 2: effect of incubation time on astaxanthin content.
Detailed Description
Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the products and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations and modifications, or appropriate alterations and combinations, of the products and methods described herein may be made and utilized without departing from the spirit, scope, and spirit of the invention. For a further understanding of the present invention, reference will now be made in detail to the following examples.
Example 1
A method for producing astaxanthin by using starch wastewater comprises the following steps:
step 1) pretreatment of starch wastewater: the starch wastewater firstly enters a sedimentation tank, the sedimentation time is 24 hours, and then the starch wastewater enters an acid-base adjusting tank, and the pH value is adjusted to 7.0;
step 2) preparing mixed bacteria liquid: will solveInoculating the bacillus amyloliquefaciens to an LB solid culture medium for culture to obtain a single colony; selecting single colony, inoculating to LB liquid culture medium, and performing amplification culture to obtain 5 × 108cfu/ml of a bacillus amyloliquefaciens culture solution;
inoculating Lactobacillus plantarum to MRS solid culture medium, culturing to obtain single colony, inoculating to MRS liquid culture medium, and culturing to 5 × 108cfu/ml of lactobacillus plantarum culture fluid;
performing streak culture on rhodopseudomonas palustris on an LB (Langerhans) plate to obtain a single colony; picking single colony, inoculating to amplification culture medium, and culturing to 3 × 108cfu/ml of Rhodopseudomonas palustris culture solution; the components of the expanding culture medium are as follows: 10g/L glucose, 8g/L corn steep liquor, 1g/L ammonium chloride, 0.1g/L potassium dihydrogen phosphate, 0.1g/L dipotassium hydrogen phosphate, 0.01g/L ferrous sulfate heptahydrate and 0.01g/L magnesium sulfate heptahydrate;
mixing a bacillus amyloliquefaciens culture solution, a lactobacillus plantarum culture solution and a rhodopseudomonas palustris culture solution according to a volume ratio of 5:3:3 to obtain a composite bacterial solution;
step 3), mixed culture of bacteria and algae: injecting the pretreated starch wastewater into a biological reaction tank, firstly, inoculating a composite bacterial liquid into the starch wastewater according to the volume ratio of 1:400, carrying out biological treatment for 48h, then, inoculating haematococcus pluvialis in logarithmic phase into the starch wastewater according to the volume ratio of 1:200, wherein the cell density of the haematococcus pluvialis is 2 multiplied by 104The culture medium is cultured for 8 days, the light intensity is 5000lux, the light-dark ratio is 12: 12; centrifuging the liquid at 2000rpm for 20min, and collecting precipitate;
step 4) astaxanthin extraction: washing the precipitate with water twice, and freeze-drying to obtain powder; then, as 1kg of powder: adding solvent into 1L of solvent (prepared by mixing ethyl acetate and ethanol at a volume ratio of 1: 2), breaking cell wall by ultrasonic treatment, performing ultrasonic treatment at an ultrasonic power of 300W, performing ultrasonic treatment at an ultrasonic time of 10s each time, performing intermittent treatment at an ultrasonic time of 10s for a total time of 60min, standing for 30min, centrifuging at 3000rpm for 5min, collecting supernatant and precipitate, adding acetone with the same volume into the precipitate, performing ultrasonic treatment at an ultrasonic power of 300W for 10s each time, performing centrifugal treatment at 3000rpm for 3min, and collecting supernatant; combining the two supernatants, evaporating to dryness at low temperature, dissolving the residue with diethyl ether of twice weight, adding KOH/methanol solution of 0.1mol/L with the same mass as the dissolved substance, and saponifying at 4 deg.C for 12 hr to obtain astaxanthin crude extract.
Example 2
A method for producing astaxanthin by using starch wastewater comprises the following steps:
step 1) pretreatment of starch wastewater: the starch wastewater firstly enters a sedimentation tank, the sedimentation time is 12 hours, and then the starch wastewater enters an acid-base adjusting tank, and the pH value is adjusted to 7.5;
step 2) preparing mixed bacteria liquid: inoculating the bacillus amyloliquefaciens to an LB solid culture medium for culture to obtain a single colony; selecting single colony, inoculating to LB liquid culture medium, and performing amplification culture to obtain 7 × 108cfu/ml of a bacillus amyloliquefaciens culture solution;
inoculating Lactobacillus plantarum to MRS solid culture medium, culturing to obtain single colony, inoculating to MRS liquid culture medium, and culturing to obtain 3 × 10 lactobacillus plantarum strain8cfu/ml of lactobacillus plantarum culture fluid;
performing streak culture on rhodopseudomonas palustris on an LB (Langerhans) plate to obtain a single colony; selecting single colony, inoculating to amplification culture medium, and culturing to 5 × 108cfu/ml of Rhodopseudomonas palustris culture solution; the components of the expanding culture medium are as follows: 10g/L glucose, 8g/L corn steep liquor, 1g/L ammonium chloride, 0.1g/L potassium dihydrogen phosphate, 0.1g/L dipotassium hydrogen phosphate, 0.01g/L ferrous sulfate heptahydrate and 0.01g/L magnesium sulfate heptahydrate;
mixing a bacillus amyloliquefaciens culture solution, a lactobacillus plantarum culture solution and a rhodopseudomonas palustris culture solution according to a volume ratio of 7:5:5 to obtain a composite bacterial solution;
step 3), mixed culture of bacteria and algae: injecting the pretreated starch wastewater into a biological reaction tank, firstly, inoculating a compound bacterial liquid into the starch wastewater according to the volume ratio of 1:200, carrying out biological treatment for 24h, then, taking haematococcus pluvialis in logarithmic phase, and carrying out biological treatment on haematococcus pluvialis in logarithmic phase according to the volume ratioInoculating starch wastewater according to the volume ratio of 1:200, wherein the cell density of algae is 1 × 104The culture medium is cultured for 10 days, the light intensity is 6000lux, the light-dark ratio is 12: 12; centrifuging the liquid at 3000rpm for 10min, and collecting precipitate;
step 4) astaxanthin extraction: washing the precipitate with water twice, and freeze-drying to obtain powder; then, as 1kg of powder: adding solvent into 1.5L solvent (prepared by mixing ethyl acetate and ethanol at a volume ratio of 1: 2), breaking cell wall by ultrasonic treatment, with ultrasonic power of 300W, ultrasonic time of 10s each time, intermittent time of 10s, total ultrasonic time of 60min, standing for 30min, centrifuging at 3000rpm for 5min, collecting supernatant and precipitate, adding acetone with the same volume into the precipitate, treating with ultrasonic treatment for 30min, ultrasonic power of 300W, ultrasonic time of 10s each time, intermittent time of 10s, centrifuging at 3000rpm for 3min, and collecting supernatant; and combining the two supernatants, evaporating to dryness at low temperature, dissolving the residue with diethyl ether, adding 0.1mol/L KOH/methanol solution with the same mass, and saponifying at 4 ℃ for 12 hours to obtain an astaxanthin crude extract.
Example 3
The treatment effect of the composite bacterial liquid on starch wastewater is as follows:
taking the composite bacterial liquid of the example 1 as an example, the control group 1 is two strains of bacillus amyloliquefaciens and lactobacillus plantarum, and the rest is the same as the example 1; control 2 was lactobacillus plantarum and rhodopseudomonas palustris, the remainder being as in example 1; control 3 was Bacillus amyloliquefaciens and Rhodopseudomonas palustris, the remainder of the example 1. Specific results are shown in table 1:
TABLE 1
| After pretreatment | Experimental | Control group | 1 | |
|
|
| COD （mg/L） | 1389 | 203 | 451 | 612 | 574 | |
| NH3-N （mg/L） | 377 | 86 | 155 | 169 | 203 | |
| Sulfide (mg/L) | 203 | 38 | 67 | 54 | 81 | |
| Starch (mg/L) | 589 | 73 | 115 | 141 | 93 | |
| Protein (mg/L) | 325 | 71 | 82 | 91 | 74 |
As shown in Table 1, the wastewater treated by the microorganisms has greatly reduced pollutants, and is suitable for culturing Haematococcus pluvialis.
Example 4
Determination of biomass and astaxanthin content: the astaxanthin content was determined by HPLC. Filtering the crude astaxanthin extract after astaxanthin saponification through a 0.45 mu m membrane before loading, wherein the mobile phase comprises dichloromethane, methanol, acetonitrile and water, and the ratio is 5:85:5:5, and performing ultrasonic degassing for 30min by using the mobile phase before using. The flow rate is 1ml/min, the detection wavelength is 480nm, the column temperature is 25 ℃, the sample injection amount is 20 mu l, and the astaxanthin content is calculated. And simultaneously weighing the dry powder and calculating the biomass.
The experimental group is example 1, while the control group is set up: haematococcus pluvialis was cultured in a conventional culture medium (BG-11), and the procedure was as in example 1. Specific results are shown in table 2:
TABLE 2
| Group of | Biomass g/L | Astaxanthin content mg/g |
| Experimental group | 5.79 | 8.19 |
| Control group | 5.33 | 6.27 |
As shown in Table 2, compared with the conventional culture mode, the biomass and astaxanthin contents of the invention are improved and respectively reach 108.6 percent and 130.6 percent; wherein, the content of the astaxanthin is greatly improved.
Example 5
Effect of algal cell culture time on biomass and astaxanthin content:
the biomass and the astaxanthin content are detected respectively in 2d, 4d, 6d, 8d, 10d and 12d, as shown in figure 1, the biomass is rapidly increased in the first six days, the biomass is not obviously increased after the sixth day, the biomass is reduced after the eighth day, probably because the nitrogen source in the wastewater is rich in the initial culture period, haematococcus pluvialis can rapidly proliferate, the nitrogen source content in the later period is reduced and is not enough to maintain the proliferation, the reduction of the nitrogen source is beneficial to the accumulation of astaxanthin, when the biomass is increased and released, the yield of astaxanthin is greatly increased (as shown in figure 2), the relationship between the biomass and the yield of astaxanthin needs to be balanced, and the selection of 8-10 days is suitable in combination with figure 1-2.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (4)
1. A method for producing astaxanthin by using starch wastewater is characterized by comprising the following steps:
step 1) pretreating starch wastewater;
step 2) preparing mixed bacteria liquid: mixing a bacillus amyloliquefaciens culture solution, a lactobacillus plantarum culture solution and a rhodopseudomonas palustris culture solution according to a volume ratio of 5-7:3-5:3-5 to obtain a composite bacterial solution;
step 3), mixed culture of bacteria and algae: injecting the pretreated starch wastewater into a biological reaction tank, firstly, inoculating the composite bacterial liquid into the starch wastewater according to the volume ratio of 1 (200-; then centrifuging the liquid at the speed of 2000-3000rpm for 10-20min, and collecting the precipitate;
step 4), extracting astaxanthin;
the step 1) of starch wastewater pretreatment comprises the following steps: the starch wastewater firstly enters a sedimentation tank, the sedimentation time is 12-24h, and then the starch wastewater enters an acid-base adjusting tank, and the pH is adjusted to 7.0-7.5;
the step 4) of extracting astaxanthin comprises the following steps: washing the precipitate with water twice, and freeze-drying to obtain powder; then, as 1kg of powder: adding solvent in an amount of 1-2L of solvent, performing ultrasonic wall breaking, performing ultrasonic power of 300W, performing ultrasonic time of 10s each time, performing intermittent time of 10s, performing total ultrasonic time of 60min, standing for 30min, centrifuging at 3000rpm for 5min, collecting supernatant and precipitate, adding acetone with the same volume into the precipitate, performing ultrasonic treatment for 30min, performing ultrasonic power of 300W, performing ultrasonic time of 10s each time, performing intermittent time of 10s, centrifuging at 3000rpm for 3min, and collecting supernatant; mixing the two supernatants, evaporating to dryness at low temperature, dissolving the residue with diethyl ether, adding KOH/methanol solution with the same mass and concentration of 0.1mol/L, and saponifying at 4 deg.C for 12 hr to obtain astaxanthin crude extract; the solvent is prepared by uniformly mixing ethyl acetate and ethanol according to the volume ratio of 1: 2.
2. The method of claim 1, wherein said de-starching is performedThe preparation method of the bacillus culture solution comprises the following steps: inoculating the bacillus amyloliquefaciens to an LB solid culture medium for culture to obtain a single colony; selecting single colony, inoculating to LB liquid culture medium, performing amplification culture to obtain 5-7 × 108cfu/ml of Bacillus amyloliquefaciens culture solution.
3. The method according to claim 1, wherein the lactobacillus plantarum culture solution is prepared by a method comprising the steps of: inoculating Lactobacillus plantarum to MRS solid culture medium, culturing to obtain single colony, inoculating to MRS liquid culture medium, and culturing to 3-5 × 108cfu/ml of lactobacillus plantarum culture fluid.
4. The method according to claim 1, wherein the preparation method of the rhodopseudomonas palustris culture solution comprises the following steps: performing streak culture on rhodopseudomonas palustris on an LB (Langerhans) plate to obtain a single colony; selecting single colony, inoculating to amplification culture medium, and culturing to 3-5 × 108cfu/ml of Rhodopseudomonas palustris culture solution; the components of the expanding culture medium are as follows: 10g/L glucose, 8g/L corn steep liquor, 1g/L ammonium chloride, 0.1g/L potassium dihydrogen phosphate, 0.1g/L dipotassium hydrogen phosphate, 0.01g/L ferrous sulfate heptahydrate and 0.01g/L magnesium sulfate heptahydrate.
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