CN103820520A - High-yield natural astaxanthin fermentation method - Google Patents
High-yield natural astaxanthin fermentation method Download PDFInfo
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Abstract
The invention discloses a high-yield natural astaxanthin fermentation method, which comprises the steps as follows: activating phaffia rhodozyma CGMCC6355 bacterial strains and preparing a seed liquid; inoculating the seed liquid to a fermentation culture medium to perform fermentation culture, and extracting astaxanthin after the end of the fermentation culture, wherein before the fermentation culture, adding an oxygen vector with a volume ratio of 0.5-6 percent to the fermentation culture medium; during the fermentation culture, adding a sugar source to enable the concentration of reducing sugar in a fermentation liquid to reach 2-3 percent when the concentration of the reducing sugar in the fermentation liquid is consumed to below 2 percent, and adding an astaxanthin precursor substance to the fermentation liquid every other 8-24 hours. The astaxanthin produced by adopting the method can be applied to high-grade feed additives for the aquaculture industry and the like, and has the effects on improving the egg laying rate of aquatic animals, boosting the growth, and resisting and preventing diseases. The high-yield natural astaxanthin fermentation method is low in cost, high in the utilization ratio of raw materials, simple to operate, and easy to achieve large-scale industrial production.
Description
Technical field
The present invention relates to microorganism fermentative production field, be specifically related to a kind of fermentation process of high yield natural astaxanthin.
Background technology
Astaxanthin (3,3 '-dihydroxyl-4,4 '-diketone-β, β '-carotene) be a kind of keto-acid carotenoid, have extremely strong anti-oxidant activity, its anti-oxidant activity is 500 times of vitamin-E, is improving immunizing power, the generation of the chronic diseases such as prophylaxis of tumours, cardiovascular disorder, diabetes, the aspect such as delay senility has positive promoter action.
At present, every kilogram of synthesizing astaxanthin reaches 2500 yuan, and every kilogram of natural astaxanthin reaches 7000-10000 unit.Due to the difference of price, on market, 97% share is all occupied by the astaxanthin of chemosynthesis.But chemical synthesis complex process, need to just can complete through multistep chemistry and biocatalytic reaction, and cost is very high, and synthetic astaxanthin is completely different compared with natural astaxanthin aspect structure, function, application and security.U.S. FDA expressly forbids that the astaxanthin of chemosynthesis enters health-product market.The pure natural active material that natural astaxanthin has, safe, nuisanceless and noresidue, be used in the industrial circle such as medicine, food and feed by majority state approval.
Astaxanthin is second largest product of sales volume in carotenoid.Astaxanthin adds in breeding bait as fodder additives, can not only promote fish-egg fertilization, reduce the mortality ratio of fetal development, promote individual growth, increase ripe speed and breeding potential, improve immunizing power and the viability of animal, and can make astaxanthin be deposited in the skin and muscle of fish, skin and muscle are taken on a red color, this fish nutrition is high, bright in colour, features good taste, receives favor on market greatly.In addition, astaxanthin also can be served as nutritive substance, promotes the growth of poultry and improves laying rate, and astaxanthin pigment can be deposited in yolk, makes the intensification that reddens of egg yellow, increases economic efficiency.At present, domestic, particularly propagating artificially of fish, shrimp, crab etc. just greatly developed in region following the line of the sea, therefore, also increasing to the demand of the astaxanthin as fodder additives.
The production of China's natural astaxanthin is scarcely out of swaddling-clothes at present, and extensive work mainly concentrates on laboratory study level at present, and the market value of domestic natural astaxanthin powder is 7900 yuan/kilogram, and therefore the production of astaxanthin has very large market potential.Only micro-algae is cultivated the astaxanthin listing supply of producing in the world, shows that after deliberation its various aspects of performance of Production by Microorganism Fermentation astaxanthin is all better than astaxanthin and synthesis method astaxanthin that micro-algae produces.Publication number is that the Chinese patent literature of CN102864087A discloses a kind of high yield Phaffia rhodozyma strain obtaining after mutagenesis is merged, the output that this bacterial strain is produced astaxanthin is higher 14.3 times than parent, but the output of astaxanthin is still lower, for 68mg/L, therefore, be necessary the processing condition of fermentation to be optimized.
Summary of the invention
The invention provides a kind of fermentation process of high yield natural astaxanthin, improved the productive rate of astaxanthin, and reduced the cost of fermentation.
A fermentation process for high yield natural astaxanthin, comprising:
(1) activate red phaffia rhodozyma CGMCC6355 bacterial strain, preparation seed liquor;
(2) described seed liquor is seeded in fermention medium and carries out fermentation culture, after fermentation culture finishes, extract astaxanthin; Wherein,
Before fermentation culture, in described fermention medium, add oxygen carrier, the volume ratio of described oxygen carrier and fermention medium is 0.5～6%;
When described fermentation culture, in the time that the mass concentration of reducing sugar in fermented liquid is consumed to below 2%, adds sugared source and make the mass concentration of reducing sugar in fermented liquid reach 2～3%;
When described fermentation culture, in fermented liquid, added astaxanthin precursor substance every 8～24 hours.
Red phaffia rhodozyma CGMCC6355 described in the application, it is the disclosed high yield Phaffia rhodozyma strain obtaining after mutagenesis is merged of ZL20121032194.9 patent, the red phaffia rhodozyma of called after (Phaffia rhodozyma) CZ10, deposit number is CGMCC NO.6355.
Initial concentration of reduced sugar in described fermention medium is 3～4%.
Described fermention medium mainly comprises carbon source, nitrogenous source and inorganic salt, wherein, carbon source can adopt agricultural byproducts, agricultural product castoff and national treasury stock etc., specifically can be at least one in cane molasses, vinasse, Microcrystalline Cellulose, long-grained nonglutinous rice and corn cob, it is for the preparation of astaxanthin fermentation substratum, can reduce fermentation costs, resource is fully used.
Agricultural byproducts, agricultural product castoff etc. need first pass through certain pre-treatment in configuration when fermention medium conventionally, make its degraded generation fermentability carbohydrate, for yeast provides the carbon source of growth.
As take corn cob as starting material, pretreatment process is specifically as follows: corn cob is pulverized in the sodium hydroxide solution that is placed on 4% and processed and spend the night, after finishing dealing with, filter, filter residue adds the sodium citrate buffer solution of the 0.05mol/L of pH4.8 after washing, being dried, then carry out sterilizing, enzymic hydrolysis, the enzyme wherein adopting when enzymic hydrolysis can be the mixed enzyme system of cellulase and zytase composition.
As take long-grained nonglutinous rice as starting material, pretreatment process is specifically as follows: after long-grained nonglutinous rice washing is soaked, pulverizing is sized mixing, and slurries sterilizing after 85～90 ℃ of liquefaction, filtration regulate pH4.5, then carry out saccharification, the high temperature enzyme processing of going out.
As take cane molasses as starting material, pretreatment process is specifically as follows: it is 45～50 that cane molasses is diluted with water to pol
0bx, then adds the vitriol oil, regulates pH4.0～4.5, then ventilates and process 10min in 90～95 ℃, finishes dealing with that cooling rear to regulate pH with sodium hydroxide be 5.0～6.0, and hold over night, separates and obtain molasses clear liquid.
After above-mentioned agricultural byproducts pre-treatment, obtain pretreatment fluid, it is 3～4% that pretreatment fluid is diluted to concentration of reduced sugar, can mix with nitrogenous source (as yeast extract paste), inorganic salt, obtains described fermention medium.
Suitable oxygen carrier can promote oxygen transmission, improve dissolved oxygen amount in fermented liquid, thereby promote chemical activators, improve astaxanthin yield, described oxygen carrier can be liquid alkane, oleic acid, toluene, perfluoro-carbon or soya-bean oil, is more preferably soya-bean oil or n-dodecane.
During take soya-bean oil as oxygen carrier, the addition of described soya-bean oil is 2.0～5.0% of fermention medium volume, more preferably 5%.
During take n-dodecane as oxygen carrier, the addition of described n-dodecane is 0.5～2.0% of fermention medium volume, more preferably 1%.
Preferably, the inoculum size of described seed liquor is 6～10%.
Preferably, when described fermentation culture, temperature is 21～23 ℃, and air flow is 1.0～1.2vvm, and the pH of controlled fermentation liquid is 5.0 ± 0.5.
Preferably, when described fermentation culture, the concentration that control stirring velocity maintains dissolved oxygen in fermented liquid is 20～60%.
Red phaffia rhodozyma can utilize that multiple reducing sugar is grown, synthesizing astaxanthin, along with the carrying out of fermentation, reducing sugar is consumed gradually, in the time that reducing sugar is too low, be unfavorable for zymogenic breeding and Metabolic activity, therefore, need to need during the fermentation to add certain sugared source (carbon source), described sugared source is glucose, sucrose or cellobiose, more preferably sucrose.Certainly, concentration of reduced sugar is also unsuitable too high, generally adds sugared source and makes the concentration of reduced sugar in fermented liquid reach 2～3%.
Add during the fermentation astaxanthin precursor mass-energy and promote the synthetic of astaxanthin, described astaxanthin precursor substance is Radix Dauci Sativae juice, tomato juice, turmeric juice, tea juice etc., is more preferably Radix Dauci Sativae juice or tomato juice, more preferably tomato juice.
The addition of astaxanthin precursor substance, add interval time, add number of times and all can exert an influence to the productive rate of astaxanthin.
The interpolation number of times of described astaxanthin precursor substance is 2～4 times, more preferably 4 times.
The astaxanthin precursor substance of adjacent twice adds interval and is preferably 8～24 hours, more preferably 12 hours.
The addition of each described astaxanthin precursor substance is 4～5% of fermention medium volume.
Last interpolation continues fermentation for some time after astaxanthin precursor substance again and can stop fermentation, and general to continue fermentation 24～48 hours more suitable, more excellent in continuing fermentation 36 hours.
Compared with prior art, beneficial effect of the present invention is:
(1) the present invention utilizes the Phaffia rhodozyma strain of high yield, and astaxanthin productive rate is high, and can, with agricultural byproducts, agricultural product castoff and national treasury stock fermentative production astaxanthin, greatly reduce production costs, and has reached resource synthetic development utilization.
(2) the present invention uses oxygen carrier strengthening oxygen to transmit Two Liquid Phases fermentation technique, promote oxygen transmission, improve dissolved oxygen amount in fermented liquid, and add during the fermentation astaxanthin precursor substance and carbohydrate carbon source, promote the growth of red phaffia rhodozyma and synthesizing of the interior astaxanthin of thalline, improved the output of astaxanthin.
(3) in aquatic animal feed, adding weight content is astaxanthin prepared by the inventive method of 1.0～2.0 ‰, can obviously put forward aquatic animal spawning rate, rate of fertilization and immunizing power and resistance against diseases.The aquatic animal feed that contains 1.0～2.0 ‰ astaxanthins of the present invention, takes the lead in having set up astaxanthin at home to aquatic animal defending and fighting against diseases utilisation technology parameter, for aquaculture Harmless defending and fighting against diseases technology provides scientific basis.
Accompanying drawing explanation
Fig. 1 is the ultraviolet spectrogram of astaxanthin.
Fig. 2 is the infrared spectrogram of astaxanthin.
Fig. 3 is astaxanthin
1h-NMR spectrogram.
Fig. 4 is the mass spectrum of astaxanthin.
Embodiment
Further explain the present invention below in conjunction with embodiment.
Bacterial classification: red phaffia rhodozyma (Phaffia rhodozyma) CZ10, deposit number is CGMCC No.6355.
YM slant medium: 10
0the wort of Brix; If solid medium need separately add 2% agar powder.PH value nature, 115 ℃ of sterilizing 20min.
Shake-flask culture base (g/L): glucose 10.0, peptone 5.0, concentrated yeast extract powder 5.0, CaCl
22H
2o0.2, MgSO
47H
2o2.75, KH
2pO
41.5, (NH4)
2sO
43.0, pH5.0.
Fermention medium (g/L): the pretreatment fluid of agricultural byproducts is diluted with water to reducing sugar mass concentration 3%, yeast extract paste 5, (NH
4)
2sO
46.0, KH
2pO
43.0, MgSO
47H
2o3.0, pH5.0 ± 0.5;
Wherein, agricultural byproducts can be corn cob, long-grained nonglutinous rice, cane molasses etc., and the preparation method of the pretreatment fluid of agricultural byproducts is as follows:
1, corn cob pretreatment fluid
After corn cob is pulverized, spend the night with 4% sodium hydroxide solution processing, after filtration, wash to neutrality drying for standby with water.Then add the sodium citrate buffer solution of the 0.05mol/L of pH4.8,115 ℃ of sterilizing 20min, add cellulase, zytase mixed enzyme system after cooling, under 45 ℃ of stirrings, carry out enzymic hydrolysis.
2, long-grained nonglutinous rice pretreatment fluid
After long-grained nonglutinous rice washing is soaked, pulverizing is sized mixing, and 85～90 ℃ of liquefaction, is heated to 100 ℃ of sterilizings, filters, and is cooled to 45 ℃, regulates pH4.5, carries out saccharification, and the high temperature enzyme that goes out obtains liquid glucose.
3, cane molasses pretreatment fluid
By after cane molasses dilute with water, measure pol with saccharometer, until pol is 45～50
0bx, then stirs and adds the vitriol oil, regulates pH4.0～4.5, and put into clarifying tank and be heated to 90～95 ℃, ventilation 30min, insulation 10min after ventilating, it is cooling that rear to regulate pH with sodium hydroxide be 5.0～6.0, hold over night, obtains molasses clear liquid after separation.
Employing agricultural byproducts are starting material, configure after pretreatment fermention medium, can be used for the fermentative production of astaxanthin, wherein, corn cob, long-grained nonglutinous rice and three kinds of raw materials of cane molasses, configure fermention medium after no matter adopting which kind of pre-treatment, productive rate impact on astaxanthin is little, if no special instructions, the embodiment of the present application 1 is in to the optimizing process of astaxanthin fermentation condition, and the agricultural byproducts that adopt when fermention medium configuration are cane molasses.
Reducing sugar content is measured: adopt 3,5-dinitrosalicylic acid method (DNS) to measure.
The optimization of embodiment 1 astaxanthin fermentation condition
1, the optimization of oxygen carrier
(1) slant strains having activated is inoculated in the 250mL Erlenmeyer flask containing 30mL liquid YM substratum, 22 ± 1 ℃, 180r/min cultivates 20h; Then be inoculated in the 1000mL Erlenmeyer flask containing 100mL Medium of shaking flask fermentation by inoculum size 10%, 22 ± 1 ℃, 180r/min cultivates 20h.
(2) fermentor tank volume is 10L, and liquid amount is 6L, adds oxygen carrier, 121 ℃ of sterilizing 20min in fermention medium simultaneously.After cooling, seed culture fluid is seeded to fermention medium by 10% inoculum size and carries out fermentation culture, 22 ± 1 ℃ of culture temperature, add 2mol/L NaOH and 2mol/L HCl controlled fermentation pH is 5.0 ± 0.5 by stream, air flow 1.2vvm, is 30% by regulating stirring velocity to control dissolved oxygen concentration.
(3) in fermentation culture process, add astaxanthin precursor substance Radix Dauci Sativae juice every 12h stream, each stream add 240mL(fermention medium volume 4%), stream adds 4 times altogether.In this 48h process, detect fermented liquid concentration of reduced sugar every 4h, in the time being reduced to below 2%, adding concentration and be 50% glucose concentrated solution and make concentration of reduced sugar in fermented liquid (mass concentration) reach 2%～3%, until 48h stops.Continue afterwards to stop fermentation after fermentation 24h.
(4) after fermentation ends, collect thalline, extract astaxanthin, calculate astaxanthin productive rate.
Extracting the method for astaxanthin is: by fermented liquid centrifuge washing 3 times, and 4000r/min, centrifugal 10min, collects thalline.Under lucifuge condition, thalline is suspended in to 55 ℃ of dimethyl sulfoxide (DMSO), vibration, acetone extract, centrifugal, by supernatant liquor vacuum concentration, again dissolve vacuum concentration with normal hexane, again dissolve by ethyl acetate, vacuum concentration, finally uses anhydrous alcohol solution again, and vacuum concentration obtains astaxanthin dry powder.
Through structural confirmation and test analysis (Fig. 1～Fig. 4), the collection of illustrative plates of the microbial source natural astaxanthin dry powder that vacuum concentration obtains is consistent with the collection of illustrative plates of bibliographical information.Astaxanthin molecular weight is 644, and molecular formula is C
43h
64o
4, structural formula is:
The impact of the different add-ons of table 1-1 n-dodecane on astaxanthin fermentation
The impact of the different add-ons of table 1-2 soya-bean oil on astaxanthin fermentation
The fermentation of oxygen carrier Two Liquid Phases, the oxygen transmission in forced fermentation process, mainly from two aspects: the one, the solubleness of oxygen in oxygen carrier is the solubleness of hyperoxia in water greatly; Be dispersion, the emulsification of oxygen carrier in substratum on the other hand, increased the phase area that oxygen transmits, thereby improved the volume transmission coefficient of oxygen.Can be found out by table 1-1, table 1-2,1% n-dodecane and 5% soya-bean oil have maximum promoter action to astaxanthin synthetic, and the productive rate of astaxanthin can reach 88.2%mg/L, 87.3mg/L.The oxygen carrier of lower concentration makes in fermentor tank oxygen concn too low, does not promote to greatest extent the synthetic of astaxanthin.High oxygen concentration carrier can form large drop in water, bubbles through the water column, and reduces oxygen transmission, thereby cell growth inhibiting has suppressed the synthetic of astaxanthin.Compared with 5% soya-bean oil, oxygen has higher solubleness in n-dodecane.
2, the optimization of precursor substance
(1) slant strains having activated is inoculated in the 250mL Erlenmeyer flask containing 30mL liquid YM substratum, 22 ± 1 ℃, 180r/min cultivates 20h; Then be inoculated in the 1000mL Erlenmeyer flask containing 100mL Medium of shaking flask fermentation by inoculum size 10%, 22 ± 1 ℃, 180r/min cultivates 20h.
(2) fermentor tank volume 10L, liquid amount is 6L, adds oxygen carrier (n-dodecane of 1% fermention medium volume), 121 ℃ of sterilizing 20min in fermention medium simultaneously.After cooling, seed culture fluid is seeded to fermention medium by 10% inoculum size and carries out fermentation culture, 22 ± 1 ℃ of culture temperature, add 2mol/L NaOH and 2mol/L HCl controlled fermentation pH is 5.0 ± 0.5 by stream, air flow 1.2vvm, is 30% by regulating stirring velocity to control dissolved oxygen concentration.
(3) in fermentation culture process, add astaxanthin precursor substance Radix Dauci Sativae juice every 12h stream, each stream add 240mL(fermention medium 4%), stream adds 4 times altogether.In this 48h process, detect fermented liquid concentration of reduced sugar every 4h, in the time being reduced to 2% below, adding concentration and be 50% glucose concentrated solution and make the sugared concentration of reducing sugar (mass concentration) in fermented liquid reach 2%～3%, until 48h stops.Continue afterwards to stop fermentation after fermentation 24h.
(4) after fermentation ends, collect thalline, extract astaxanthin, calculate astaxanthin productive rate.
Table 1-3 adds the impact of different precursor substances on astaxanthin fermentation
| Precursor substance | Dry cell weight (g/L) | Astaxanthin productive rate (mg/L) |
| Nothing | 18.3 | 83.4 |
| Radix Dauci Sativae juice | 18.5 | 88.2 |
| Tomato juice | 19.2 | 94.3 |
| Turmeric juice | 17.8 | 84.3 |
| Tea juice | 18.1 | 83.8 |
The impact of different joining days of table 1-4 tomato juice on astaxanthin fermentation
The interpolation of astaxanthin precursor substance, can make the concentration of reaction substrate increase, thereby reaction is carried out to positive dirction, promotes the synthetic of astaxanthin.By finding out in table 1-3,1-4, add tomato juice precursor substance best to the synthetic promoter action effect of astaxanthin, and the timed interval of adding tomato juice precursor substance can cause the productive rate difference of astaxanthin, to add a tomato juice every 12h, add altogether 4 times for top condition, the productive rate of astaxanthin is 94.3mg/L.
3, add the optimization in sugared source
(1) slant strains having activated is inoculated in the 250mL Erlenmeyer flask containing 30mL liquid YM substratum, 22 ± 1 ℃, 180r/min cultivates 20h; Then be inoculated in the 1000mL Erlenmeyer flask containing 100mL Medium of shaking flask fermentation by inoculum size 10%, 21 ℃, 180r/min cultivates 20h.
(2) fermentor tank volume 10L, liquid amount is 6L, adds oxygen carrier (n-dodecane of 1% fermention medium volume), 121 ℃ of sterilizing 20min in fermention medium simultaneously.After cooling, seed culture fluid is seeded to fermention medium by 10% inoculum size and carries out fermentation culture, 22 ± 1 ℃ of culture temperature, add 2mol/L NaOH and 2mol/L HCl controlled fermentation pH is 5.0 ± 0.5 by stream, air flow 1.2vvm, is 30% by regulating stirring velocity to control dissolved oxygen concentration.
(3) in fermentation culture process, add astaxanthin precursor substance tomato juice every 12h stream, each stream add 240mL(fermention medium 4%), stream adds 4 times altogether.In this 48h process, detect fermented liquid concentration of reduced sugar every 4h, in the time being reduced to 2% below, adding concentration and be 50% carbohydrate concentrated solution and make the sugared concentration of reducing sugar (mass concentration) in fermented liquid reach 2%～3%, until 48h stops.Continue afterwards to stop fermentation after fermentation 24h.
(4) after fermentation ends, collect thalline, extract astaxanthin, calculate astaxanthin productive rate.
Because growth needs carbon source, during the fermentation, carbon source can along with fermentation carrying out and slow consumption, therefore phaffia rhodozyma high-density culture to be realized and carbon source must be added during the fermentation, maintain growth and the fermentation of cell, simultaneously because high C/N ratio is conducive to chemical activators, so accumulate astaxanthin in the fermentation later stage supplemented with being beneficial to phaffia rhodozyma.
Table 1-5 adds the impact of different carbon sources on astaxanthin fermentation
| Carbon source (concentration is 50%) | Dry cell weight (g/L) | Astaxanthin productive rate (mg/L) |
| Nothing | 18.8 | 88.6 |
| Glucose | 19.2 | 94.3 |
| Fructose | 19.8 | 98.4 |
| Sucrose | 20.9 | 103.7 |
| Cellobiose | 20.5 | 98.5 |
As show 1-5, can improve the productive rate of astaxanthin by adding carbon source, but the difference on effect of different carbon sources is larger, glucose and fructose are monose, and sucrose and cellobiose are disaccharides, the degradation product of sucrose is glucose sugar and fructose, and the degradation product of cellobiose is glucose.Compare as can be seen from the table glucose, fructose is more suitable for red phaffia rhodozyma fermentative production astaxanthin as carbon source, and sucrose is better than cellobiose for the accumulative effect of astaxanthin.Can produce " glucose effect " because directly add glucose, in fermentor tank, concentration of reduced sugar is too high can suppress thalli growth, and sucrose hydrolysis is slower, be difficult for occurring " glucose effect ", and degradation product is glucose and fructose, be applicable to red phaffia rhodozyma growth, be conducive to astaxanthin accumulation.So select sucrose as preferably adding carbon source.The productive rate of final astaxanthin reaches 103.7mg/L.
4, the optimization of inoculum size
With reference to above-mentioned " 1～3 " method steps and optimal conditions, different vaccination amount 5%, 10%, 15%, 20% is inoculated in fermention medium by the present invention, carries out single factor investigation.Leavening temperature is 22 ± 1 ℃, fermentation pH is controlled at 5.0 ± 0.5, air flow 1.2vvm, it is 30% that adjusting rotary speed is controlled dissolved oxygen concentration, add oxygen carrier 1% n-dodecane, in fermentation culture process, add astaxanthin precursor substance tomato juice every 12h stream, each stream add 240mL(fermention medium 4%), stream adds 4 times altogether.In this 48h process, detect fermented liquid concentration of reduced sugar every 4h, in the time being reduced to 2% below, adding concentration and be 50% sucrose concentrated solution and make the sugared concentration of reducing sugar (mass concentration) in fermented liquid reach 2%～3%, until 48h stops.Continue afterwards fermentation 24h and stop fermentation.
The impact of table 1-6 inoculum size on astaxanthin productive rate
| Inoculum size (v/v) % | 5 | 10 | 15 | 20 |
| Dry cell weight (g/L) | 20.4 | 20.9 | 20.7 | 18.2 |
| Astaxanthin productive rate (mg/L) | 94.5 | 103.7 | 98.4 | 85.6 |
From table 1-6, optimum inoculation amount is 10%, and its astaxanthin productive rate is up to 103.7mg/L.
5, the optimization of fermented liquid dissolved oxygen concentration
The present invention is by regulating stirring velocity to control different dissolved oxygen concentration 20%, 30%, 40%, 50%, 60%, carry out single factor investigation for phaffia rhodozyma growth and astaxanthin yield, other Step By Conditions are with reference to the optimization of inoculum size " 4, ", leavening temperature is 22 ± 1 ℃, fermentation pH is 5.0 ± 0.5, air flow 1.2vvm, inoculum size 10%, add oxygen carrier 1% n-dodecane, in fermentation culture process, add astaxanthin precursor substance tomato juice every 12h stream, each stream add 240mL(fermention medium 4%), stream adds 4 times altogether.In this 48h process, detect fermented liquid concentration of reduced sugar every 4h, in the time being reduced to 2% below, adding concentration and be 50% sucrose concentrated solution and make the sugared concentration of reducing sugar (mass concentration) in fermented liquid reach 2%～3%, until 48h stops.Continue afterwards fermentation 24h and stop fermentation.
Table 1-7 fermented liquid dissolved oxygen concentration affects astaxanthin productive rate
| Dissolved oxygen concentration (%) | 20 | 30 | 40 | 50 | 60 |
| Dry cell weight (g/L) | 18.4 | 20.9 | 21.5 | 21.9 | 21.3 |
| Astaxanthin productive rate (mg/L) | 83.2 | 103.7 | 110.2 | 104.8 | 89.2 |
By table, 1-7 can find out, when control dissolved oxygen minimum concentration is 40%, the productive rate of astaxanthin is up to 110.2mg/L.When dissolved oxygen concentration is lower than 40% time, because phaffia rhodozyma is aerobic type fungi, so thalli growth is bad, astaxanthin productive rate is on the low side; When oxyty is higher than 40% time, dissolved oxygen is little on the impact of maximum biomass, but along with dissolved oxygen concentration raises, chemical activators is suppressed.So selecting to control dissolved oxygen optimal concentration is 40%.
6, add the optimization of precursor secondary fermentation time
Change the output of adding fermentation time after precursor substance and investigate astaxanthin, other Step By Conditions are with reference to the optimization of fermented liquid dissolved oxygen concentration " 5, ".Leavening temperature is 22 ± 1 ℃, fermentation pH is 5.0 ± 0.5, air flow 1.2vvm, regulating mixing speed to control dissolved oxygen is 40%, and inoculum size 10% is added oxygen carrier 1% n-dodecane, in fermentation culture process, add astaxanthin precursor substance tomato juice every 12h stream, each stream add 240mL(fermention medium 4%), stream adds 4 times altogether.In this 48h process, detect fermented liquid concentration of reduced sugar every 4h, in the time being reduced to 2% below, adding concentration and be 50% sucrose concentrated solution and make the sugared concentration of reducing sugar (mass concentration) in fermented liquid reach 2%～3%, until 48h stops.And then investigate respectively fermentation 24,36,48, the productive rate of astaxanthin after 72h.
Table 1-8 adds the impact of precursor substance secondary fermentation time on astaxanthin productive rate
| Fermentation time (h) | 24 | 36 | 48 | 72 |
| Dry cell weight (g/L) | 21.5 | 21.7 | 21.3 | 20.4 |
| Astaxanthin productive rate (mg/L) | 110.2 | 114.5 | 111.3 | 98.6 |
By table, 1-8 can find out, add after tomato juice precursor substance 4 times, then the 36h that ferments, the productive rate of astaxanthin is up to 114.5mg/L, and fermentation time is long, thalline production declining, the productive rate of astaxanthin slightly declines, and has expanded fermentation costs, therefore selects 36h as best fermentation time.
The application of embodiment 2 natural astaxanthins
1, egg laying amount and hatching rate and the surviving and growth experiment young tortoise of the fodder additives of astaxanthin-containing to snapper turtle
（1）
The close tortoise number of test tank is 552, and the close tortoise number in contrast pond is 20, and the female tortoise of test tank is fed and adds containing the crocodile tortoise special feed of 1 ‰ astaxanthin additives, the contrast pond normal crocodile tortoise special feed of feeding.28.6 ℃ of average water temperatures, through the feeding comparative trial of 75 days, laying, the fertility rate and hatchability in statistical test pond and contrast pond respectively.
Table 2-1 is the impact experiment on snapper turtle egg laying amount, hatching rate containing 1 ‰ astaxanthin additives
（2）
To the feed experiment of crocodile tortoise special feed of 1 ‰ astaxanthin additives of the young tortoise having hatched.100 young tortoises of control group, 541 young tortoises of test group, test group is fed containing the crocodile tortoise special feed of 1 ‰ astaxanthin additives, the normal crocodile tortoise of control group fed special feed.28.6 ℃ of average water temperatures, through the feeding comparative trial of 162 days, young tortoise surviving rate and the speed of growth in statistical test pond and contrast pond respectively.
Table 2-2 is the impact experiment on snapper turtle children's tortoise surviving rate and the speed of growth containing 1 ‰ astaxanthin additives
From table 2-1, compared with control group, add astaxanthin of the present invention to snapper turtle special feed by 1 ‰ and feed after female tortoise, the control group that output ovum (piece /), rate of fertilization (%) and the hatching rate (%) of female tortoise more do not add respectively astaxanthin has improved 17.0,18.7 and 9.5 percentage points.From table, 2-2 can find out, after adding astaxanthin in young turtle feed, its surviving rate (%), rate of body weight gain (%) improve respectively 7.2 and 5.1 percentage points.Illustrate that astaxanthin can obviously put forward aquatic animal spawning rate, rate of fertilization and immunizing power and resistance against diseases.
2, containing the disease-resistant experiment of 1.5 ‰ astaxanthin feed additives to Penaeus vannamei
Each three groups of control group and test group.Contrast 1 group and respectively drop into Penaeus vannamei 10000 tails with 2 groups of contrasts, contrast 3 groups and drop into 15000 tails.Test 2 groups of 1 composite tests and respectively drop into Penaeus vannamei 10000 tails, test 3 groups and drop into 8000 tails.Control group fed Penaeus vannamei special feed, test group is fed and is added the Penaeus vannamei special feed of 1.5 ‰ astaxanthin additives.
Duration of test, throws something and feeds 4 every day, and Feeding time is fixed on 7:00,12:00,18:00, the 23:00 of every day, throws something and feeds continuously 90 days.Duration of test does not use the other medicines such as microbiotic.Trial period, finishes surviving rate and the mortality ratio of each group of shrimp of rear statistics.
Table 2-3 is containing the disease-resistant experiment of 1.5 ‰ astaxanthin feed additives to Penaeus vannamei
From table 2-3, compared with control group, add to astaxanthin of the present invention as additive in Penaeus vannamei special feed, can obviously improve the surviving rate of Penaeus vannamei, it improves 20.12% compared with control group surviving rate.
3, containing the disease-resistant experiment of 2 ‰ astaxanthin feed additives to perch
To cultivate perch (seawater flower perch) net cage and be divided into 8 groups, and respectively drop into perch 1000 tails, 5 networking casees are test group, and 3 groups is control group, use containing the mixed fodder of the 2 ‰ astaxanthin additives test group of throwing something and feeding, the normal perch special feed of control group fed.Continuous Observation 80 days, statistics natural occurrence mortality ratio.
Table 2-4 is containing the disease-resistant experiment of 2 ‰ astaxanthin feed additives to perch
Table 2-4 shows, feed and significantly decline containing the sickness rate of the diseases such as its hemorrhagic skin ulcers of perch of 2 ‰ astaxanthin additive premixes, average survival is 88.9%, is not 66.7% containing its average survival of feed contrast perch of 2 ‰ astaxanthin additive premixes and feed.The 2 ‰ astaxanthin additives that show to feed can strengthen the resistibility of perch to the pathogenic bacterial infection such as Vibrio harveyi, Aeromonas hydrophila, and average survival can improve 22.22%.
Above-mentioned embodiment is only specific embodiments of the invention, rather than limits the invention, and in the protection domain of the claims in the present invention, any modification and change that the present invention is made, all fall into protection scope of the present invention.
Claims (10)
1. a fermentation process for high yield natural astaxanthin, comprising:
(1) activate red phaffia rhodozyma CGMCC6355 bacterial strain, preparation seed liquor;
(2) described seed liquor is seeded in fermention medium and carries out fermentation culture, after fermentation culture finishes, extract astaxanthin; Wherein,
Before fermentation culture, in described fermention medium, add oxygen carrier, the volume ratio of described oxygen carrier and fermention medium is 0.5～6%;
When described fermentation culture, in the time that the mass concentration of reducing sugar in fermented liquid is consumed to below 2%, adds sugared source and make the mass concentration of reducing sugar in fermented liquid reach 2～3%;
When described fermentation culture, in fermented liquid, added astaxanthin precursor substance every 8～24 hours.
2. fermentation process as claimed in claim 1, is characterized in that, described oxygen carrier is liquid alkane, oleic acid, perfluoro-carbon or soya-bean oil etc.
3. fermentation process as claimed in claim 2, is characterized in that, described oxygen carrier is soya-bean oil, and the addition of described soya-bean oil is 2.0～5.0% of fermention medium volume.
4. fermentation process as claimed in claim 2, is characterized in that, described oxygen carrier is n-dodecane, and the addition of described n-dodecane is 0.5～2.0% of fermention medium volume.
5. fermentation process as claimed in claim 1, is characterized in that, described sugared source is glucose, fructose, sucrose or cellobiose etc.
6. fermentation process as claimed in claim 1, is characterized in that, described astaxanthin precursor substance is Radix Dauci Sativae juice, tomato juice, turmeric juice or tea juice etc.
7. fermentation process as claimed in claim 1, is characterized in that, the interpolation number of times of described astaxanthin precursor substance is 2～4 times, and each addition is 4～5% of fermention medium volume.
8. fermentation process as claimed in claim 7, is characterized in that, adds for the last time to continue fermentation after astaxanthin precursor substance again and complete fermentation after 24～48 hours.
9. fermentation process as claimed in claim 1, is characterized in that, when described fermentation culture, temperature is 21～23 ℃, and air flow is 1.0～1.2vvm, and the pH of controlled fermentation liquid is 5.0 ± 0.5.
10. fermentation process as claimed in claim 9, is characterized in that, when described fermentation culture, the concentration that control stirring velocity maintains dissolved oxygen in fermented liquid is 20～60%.
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| CN104673871A (en) * | 2015-02-13 | 2015-06-03 | 集美大学 | Culture medium and method for fermenting astaxanthin through clear liquid reflux |
| CN105614022A (en) * | 2015-12-26 | 2016-06-01 | 山东百龙创园生物科技有限公司 | Method for preparing feed additive rich in astaxanthin through co-culture |
| CN105695550A (en) * | 2016-03-31 | 2016-06-22 | 广州元大生物科技发展有限公司 | Method for producing astaxanthin by Lactobacillus plantarum high-density culture |
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| CN108998493A (en) * | 2018-06-25 | 2018-12-14 | 浙江皇冠科技有限公司 | A kind of formula technique of high-yield astaxanthin fermentation medium and application |
| CN109430536A (en) * | 2018-06-25 | 2019-03-08 | 浙江皇冠科技有限公司 | A kind of innate immunity reinforcing agent and purposes of aquatic products |
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