CN103820520B - High-yield natural astaxanthin fermentation method - Google Patents
High-yield natural astaxanthin fermentation method Download PDFInfo
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- CN103820520B CN103820520B CN201410078929.6A CN201410078929A CN103820520B CN 103820520 B CN103820520 B CN 103820520B CN 201410078929 A CN201410078929 A CN 201410078929A CN 103820520 B CN103820520 B CN 103820520B
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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 fermentable production field is and in particular to a kind of fermentation process of high yield natural astaxanthin.
Background technology
Astaxanthin (3,3 '-dihydroxy -4,4 '-diketone-β, β '-carotene) it is a kind of keto-acid carotenoid, there is pole
Strong antioxidant activity, its antioxidant activity is 500 times of vitamin e, in enhance immunity, prophylaxis of tumours, cardiovascular disease,
The generation of the chronic diseases such as diabetes, the aspect such as slow down aging has positive facilitation.
At present, every kilogram of synthesizing astaxanthin reaches 2500 yuan, and every kilogram of natural astaxanthin reaches 7000-10000 unit.Due to price
Difference, the astaxanthin that on market, 97% share is all synthesized chemically occupies.But chemical synthesiss complex process, need through
Multi-step chemical and biocatalytic reaction just can complete, and cost is very high, and the astaxanthin synthesizing is in structure, function, application and safety
Property aspect is completely different compared with natural astaxanthin.U.S. fda has forbidden that the astaxanthin of chemosynthesis enters health-product market in plain text.
Pure natural active material that natural astaxanthin has, safe, nuisanceless and noresidue, are used in doctor by majority state approval
The industrial circles such as medicine, food and feedstuff.
Astaxanthin is second largest product of sales volume in carotenoid.Astaxanthin is added to cultivation as feed additive
In bait, fish roe can not only be promoted to be fertilized, reduce the mortality rate of fetal development, promote individual growth, increase ripe speed and numerous
Grow rate, improve immunity and the viability of animal, and astaxanthin can be made to be deposited in the skin and muscle of fish, make skin and flesh
Meat takes on a red color, and this fish nutrition is high, bright in colour, features good taste, commercially receives greatly to favor.In addition, astaxanthin also can be used as battalion
Foster material, promotes the growth of poultry and improves laying rate, astaxanthin pigment can be deposited in egg yolk, so that egg yellow is reddened and adds
Deep, increase economic efficiency.At present, domestic, particularly the artificial cultivation of fish, shrimp, Eriocheir sinensiss etc. is just being greatly developed in region following the line of the sea, therefore,
Also increasing to the demand of the astaxanthin as feed additive.
The production of China's natural astaxanthin is scarcely out of swaddling-clothes at present, and extensive work is concentrated mainly on laboratory at present and grinds
Study carefully level, the market price of domestic natural astaxanthin powder is 7900 yuan/kilogram, and the production of therefore astaxanthin has very big market
Potentiality.Through research, the astaxanthin listing supply that only microdisk electrode produces in the world, shows that Production by Microorganism Fermentation shrimp is blue or green
Its various aspects of performance plain is superior to astaxanthin and the synthetic method astaxanthin of microalgae production.The China of Publication No. cn102864087a
The high yield Phaffia rhodozyma strain that patent documentation obtains after disclosing a kind of fusion through mutation, this bacterial strain produces the yield of astaxanthin
Higher 14.3 times than parent, but the yield of astaxanthin is still relatively low, is 68mg/l, therefore, it is necessary to enter to the process conditions of fermentation
Row optimizes.
Content of the invention
The invention provides a kind of fermentation process of high yield natural astaxanthin, improve the yield of astaxanthin, and reduce
The cost of fermentation.
A kind of fermentation process of high yield natural astaxanthin, comprising:
(1) activate red phaffia rhodozyma cgmcc6355 bacterial strain, prepare seed liquor；
(2) described seed liquor is seeded in fermentation medium and carries out fermentation culture, after fermentation culture terminates, extract shrimp
Blue or green element；Wherein,
Before fermentation culture, add the carrier of oxygen, the body of the described carrier of oxygen and fermentation medium in described fermentation medium
Long-pending ratio is 0.5～6%；
During described fermentation culture, when the mass concentration of reducing sugar in fermentation liquid is consumed to below 2%, adding sugar source makes to send out
In zymotic fluid, the mass concentration of reducing sugar reaches 2～3%；
During described fermentation culture, every 8～24 hours to fermentation liquid in add astaxanthin precursor substance.
Red phaffia rhodozyma cgmcc6355 described herein, that is, zl20121032194.9 patent is disclosed merges through mutation
The high yield Phaffia rhodozyma strain obtaining afterwards, is named as red phaffia rhodozyma (phaffia rhodozyma) cz10, and deposit number is
cgmcc no.6355.
Initial concentration of reduced sugar in described fermentation medium is 3～4%.
Described fermentation medium mainly includes carbon source, nitrogen source and inorganic salt, and wherein, carbon source can adopt agricultural byproducts, agricultural production
Product garbage and national treasury stock etc., concretely at least in cane molasses, distiller grains, Microcrystalline Cellulose, Semen Oryzae and corn cob
Kind, it is used for preparing astaxanthin fermentation culture medium, it is possible to decrease fermentation costs, so that resource is fully used.
Agricultural byproducts, agricultural product castoff etc. generally need to first pass through certain pretreatment when configuring fermentation medium, make
Its degraded generates fermentability saccharide, provides the carbon source of growth for yeast.
As with corn cob as raw material, preprocess method is specifically as follows: is placed in 4% hydroxide after corn cob is pulverized
Process overnight in sodium solution, filter after the completion of process, filtering residue adds the citric acid of the 0.05mol/l of ph4.8 after washing, being dried
Sodium buffer, then carry out sterilizing, enzyme hydrolysiss, wherein enzyme hydrolysiss when the enzyme that adopts can be cellulase and xylanase composition
Mixing enzyme system.
As with Semen Oryzae as raw material, preprocess method is specifically as follows: after Semen Oryzae washing is soaked, pulverizing is sized mixing, serosity
After 85～90 DEG C of liquefaction, sterilizing, filtration, adjust ph4.5, then carry out saccharifying, high temperature destroy the enzyme treatment.
As with cane molasses as raw material, preprocess method is specifically as follows: cane molasses are diluted with water to pol is
45～500Bx, is subsequently adding concentrated sulphuric acid, adjusts ph4.0～4.5, and then at 90～95 DEG C of ventilation process 10min, process completes cold
But adjusting ph with sodium hydroxide afterwards is 5.0～6.0, stands overnight, and separates and obtains molasses clear liquid.
After above-mentioned agricultural byproducts pretreatment, that is, obtain pretreatment fluid, it is 3～4% that pretreatment fluid is diluted to concentration of reduced sugar,
Can mix with nitrogen source (as yeast extract), inorganic salt, obtain described fermentation medium.
The suitable carrier of oxygen can promote oxygen to transmit, and improves dissolved oxygen amount in fermentation liquid, thus promoting chemical activators, improves
Astaxanthin yield, the described carrier of oxygen can be liquid alkane, Oleic acid, toluene, perfluocarbon or Oleum Glycines, more preferably for Oleum Glycines
Or n-dodecane.
During with Oleum Glycines for the carrier of oxygen, the addition of described Oleum Glycines is the 2.0～5.0% of fermentation medium volume, more preferably
5%.
During with n-dodecane for the carrier of oxygen, the addition of described n-dodecane is the 0.5～2.0% of fermentation medium volume,
More preferably 1%.
Preferably, the inoculum concentration of described seed liquor is 6～10%.
Preferably, during described fermentation culture, temperature is 21～23 DEG C, and ventilation is 1.0～1.2vvm, controls fermentation liquid
Ph is 5.0 ± 0.5.
Preferably, during described fermentation culture, controlling mixing speed to maintain the concentration of dissolved oxygen in fermentation liquid is 20～60%.
Red phaffia rhodozyma can carry out growing using multiple reducing sugar, synthesizing astaxanthin, with the carrying out of fermentation, reducing sugar
Gradually it is consumed, be then unfavorable for zymogenic breeding and metabolic activity when reducing sugar is too low, accordingly, it would be desirable to during the fermentation
Need to add certain sugar source (carbon source), described sugar source is glucose, sucrose or cellobiose, more preferably sucrose.Certainly,
Concentration of reduced sugar is also unsuitable too high, and general sugar source of adding makes the concentration of reduced sugar in fermentation liquid reach 2～3%.
Add the synthesis that astaxanthin precursor mass-energy promotes astaxanthin during the fermentation, described astaxanthin precursor substance is
Radix Dauci Sativae juice, Fructus Lycopersici esculenti juice, Rhizoma Curcumae Longae juice, tea juice etc., more preferably for Radix Dauci Sativae juice or Fructus Lycopersici esculenti juice, more preferably Fructus Lycopersici esculenti juice.
The addition of astaxanthin precursor substance, add interval time, add number of times and all can produce shadow to the yield of astaxanthin
Ring.
The interpolation number of times of described astaxanthin precursor substance is 2～4 times, more preferably 4 times.
Adjacent astaxanthin precursor substance twice adds and is preferably spaced 8～24 hours, more preferably 12 hours.
The addition of described astaxanthin precursor substance is the 4～5% of fermentation medium volume every time.
It is further continued for fermentation after last interpolation astaxanthin precursor substance can stop for a period of time fermenting, general continuation is sent out
Conveniently more excellent ferments 36 hours ferment for continuation within 24～48 hours.
Compared with prior art, the invention has the benefit that
(1) present invention utilizes the Phaffia rhodozyma strain of high yield, and astaxanthin yield is high, and can be with agricultural byproducts, agricultural production
Product garbage and national treasury stock fermenting and producing astaxanthin, substantially reduce production cost, have reached resource synthetic development utilization.
(2) present invention uses carrier of oxygen strengthening oxygen transmission Two Liquid Phases fermentation technique, promotes oxygen transmission, improves molten in fermentation liquid
Oxygen amount, and add astaxanthin precursor substance and carbohydrate carbon source during the fermentation, promote in growth and the thalline of red phaffia rhodozyma
The synthesis of astaxanthin, improves the yield of astaxanthin.
(3) shrimp adding the inventive method preparation that weight content is 1.0～2.0 ‰ in aquatic animal feedstuff is blue or green
Element, can substantially put forward aquatic animal spawning rate, rate of fertilization and immunity and resistances against diseases.The present invention containing 1.0～2.0 ‰ astaxanthins
Aquatic animal feedstuff, take the lead at home establishing astaxanthin to aquatic animal defending and fighting against diseases application technology parameter,
Provide scientific basis for aquaculture Harmless defending and fighting against diseases technology.
Brief description
Fig. 1 is the ultraviolet spectrogram of astaxanthin.
Fig. 2 is the infrared spectrogram of astaxanthin.
Fig. 3 is astaxanthin1H-nmr spectrogram.
Fig. 4 is the mass spectrum of astaxanthin.
Specific embodiment
Explain the present invention with reference to specific embodiment further.
Strain: red phaffia rhodozyma (phaffia rhodozyma) cz10, deposit number is cgmcc no.6355.
Ym slant medium: 100The beerwort of brix；If solid medium need to separately add 2% agar powder.Ph value is certainly
So, 115 DEG C of sterilizing 20min.
Shake flask medium (g/l): glucose 10.0, peptone 5.0, concentrate yeast extract powder 5.0, cacl2·2h2O0.2,
mgso4·7h2O2.75, kh2po41.5, (nh4)2so43.0, ph5.0.
Fermentation medium (g/l): the pretreatment fluid of agricultural byproducts is diluted with water to and reduces Sugar concentration 3%, yeast extract 5,
(nh4)2so46.0, kh2po43.0, mgso4·7h2O3.0, ph5.0 ± 0.5；
Wherein, agricultural byproducts can be corn cob, Semen Oryzae, cane molasses etc., the preparation side of the pretreatment fluid of agricultural byproducts
Method is as follows:
1st, corn cob pretreatment fluid
After corn cob is pulverized, the sodium hydroxide solution with 4% processes overnight, after filtration, washes with water to neutrality, drying is standby
With.It is subsequently adding the sodium citrate buffer solution of the 0.05mol/l of ph4.8,115 DEG C of sterilizing 20min, after cooling, add cellulose
Enzyme, xylanase mixing enzyme system, carry out enzyme hydrolysiss under 45 DEG C of stirrings.
2nd, Semen Oryzae pretreatment fluid
After Semen Oryzae washing is soaked, pulverizing is sized mixing, and liquefies at 85～90 DEG C, is heated to 100 DEG C of sterilizings, filters, be cooled to
45 DEG C, adjust ph4.5, carry out saccharifying, high temperature enzyme denaturing obtains sugar liquid.
3rd, cane molasses pretreatment fluid
After cane molasses dilute with water, measure pol with saccharometer, until pol is 45～500Bx, then stirring adds
Enter concentrated sulphuric acid, adjust ph4.0～4.5, put into defecator and be heated to 90～95 DEG C, divulge information 30min, after ventilation, be incubated 10min, cold
But adjusting ph with sodium hydroxide afterwards is 5.0～6.0, stands overnight, and obtains molasses clear liquid after separating.
It is raw material using agricultural byproducts, configure fermentation medium after pretreatment, can be used for the fermenting and producing of astaxanthin,
Wherein, corn cob, Semen Oryzae and three kinds of raw materials of cane molasses, no matter using configuring fermentation medium after which kind of pretreatment, prawn is blue or green
The yield impact of element less, if no special instructions, in the optimization process to astaxanthin fermentation condition, send out by the embodiment of the present application 1
The agricultural byproducts being adopted during the configuration of ferment culture medium are cane molasses.
Content of reducing sugar measures: is measured using 3,5- dinitrosalicylic acid system (dns).
The optimization of embodiment 1 astaxanthin fermentation condition
1st, the optimization of the carrier of oxygen
(1) slant strains having activated are inoculated in the 250ml conical flask of the ym culture medium of liquid containing 30ml, 22 ± 1 DEG C,
180r/min cultivates 20h；Then it is inoculated in the 1000ml conical flask containing 100ml Medium of shaking flask fermentation by inoculum concentration 10%, 22
± 1 DEG C, 180r/min cultivates 20h.
(2) fermenter volume is 10l, and liquid amount is 6l, adds the carrier of oxygen, 121 DEG C of sterilizings in fermentation medium simultaneously
20min.After cooling, seed culture fluid is seeded to fermentation medium by 10% inoculum concentration and carries out fermentation culture, cultivation temperature 22 ± 1
DEG C, controlling fermentation ph by stream plus 2mol/l naoh and 2mol/l hcl is 5.0 ± 0.5, ventilation 1.2vvm, by adjusting
Mixing speed is come to control dissolved oxygen concentration be 30%.
(3) during fermentation culture, every 12h stream plus astaxanthin precursor substance Radix Dauci Sativae juice, stream plus 240ml(send out every time
The 4% of ferment culture volume), stream adds 4 times altogether.During this 48h, detect fermentation liquid concentration of reduced sugar every 4h, when being reduced to
When less than 2%, adding the glucose concentrated solution that concentration is 50% makes concentration of reduced sugar in fermentation liquid (mass concentration) reach 2%～3%,
Until 48h stops.Stop fermentation after continuing fermentation 24h afterwards.
(4) after fermentation ends, collects thalline, extract astaxanthin, calculate astaxanthin yield.
The method extracting astaxanthin is: fermentation liquid centrifuge washing 3 times, 4000r/min are centrifuged 10min, collects thalline.
Under the conditions of lucifuge, thalline is suspended in 55 DEG C of dimethyl sulfoxide, vibration, acetone extract, centrifugation, supernatant is concentrated in vacuo, use
Normal hexane dissolves again, concentrated in vacuo, is more again dissolved with ethyl acetate, concentrated in vacuo, finally uses anhydrous alcohol solution, vacuum
It is concentrated to give astaxanthin dry powder.
Through structural confirmation and test analysis (Fig. 1～Fig. 4), the microbial source natural astaxanthin dry powder obtaining concentrated in vacuo
Collection of illustrative plates is consistent with the collection of illustrative plates of document report.Astaxanthin molecular weight is 644, and molecular formula is c43h64o4, structural formula is:
The difference impact to astaxanthin fermentation for the addition of table 1-1 n-dodecane
The difference impact to astaxanthin fermentation for the addition of table 1-2 Oleum Glycines
Carrier of oxygen Two Liquid Phases ferment, the oxygen transmission during forced fermentation, essentially from two aspects: one is that oxygen carries in oxygen
Dissolubility in water for dissolubility in the body hyperoxia significantly；On the other hand it is carrier of oxygen dispersion in the medium, emulsifying, increase
The phase area of oxygen transmission, thus improve the volume carry-over factor of oxygen.Can be seen that positive the 12 of 1% by table 1-1, table 1-2
Alkane and 5% Oleum Glycines have the facilitation of maximum to the synthesis of astaxanthin, the yield of astaxanthin up to 88.2%mg/l,
87.3mg/l.The carrier of oxygen of low concentration makes oxygen concentration in fermentation tank too low, does not promote the synthesis of astaxanthin to greatest extent.High
The concentration carrier of oxygen can form big drop in water, bubbles through the water column, and reduces oxygen transmission, thus cell growth inhibiting is it is suppressed that shrimp
The synthesis of blue or green element.Compared with 5% Oleum Glycines, oxygen has higher dissolubility in n-dodecane.
2nd, the optimization of precursor substance
(1) slant strains having activated are inoculated in the 250ml conical flask of the ym culture medium of liquid containing 30ml, 22 ± 1 DEG C,
180r/min cultivates 20h；Then it is inoculated in the 1000ml conical flask containing 100ml Medium of shaking flask fermentation by inoculum concentration 10%, 22
± 1 DEG C, 180r/min cultivates 20h.
(2) fermenter volume 10l, liquid amount is 6l, adds the carrier of oxygen (1% fermentation medium in fermentation medium simultaneously
The n-dodecane of volume), 121 DEG C of sterilizing 20min.After cooling, seed culture fluid is seeded to fermentation medium by 10% inoculum concentration
Carry out fermentation culture, 22 ± 1 DEG C of cultivation temperature, by stream plus 2mol/l naoh and 2mol/l hcl control fermentation ph be 5.0 ±
0.5, ventilation 1.2vvm, are 30% by adjust mixing speed controlling dissolved oxygen concentration.
(3) during fermentation culture, every 12h stream plus astaxanthin precursor substance Radix Dauci Sativae juice, stream plus 240ml(send out every time
The 4% of ferment culture medium), stream adds 4 times altogether.During this 48h, every 4h detect fermentation liquid concentration of reduced sugar, when be reduced to 2% with
When lower, adding the glucose concentrated solution that concentration is 50% makes reducing sugar sugar concentration (mass concentration) in fermentation liquid reach 2%～3%, directly
Stop to 48h.Stop fermentation after continuing fermentation 24h afterwards.
(4) after fermentation ends, collects thalline, extract astaxanthin, calculate astaxanthin yield.
Table 1-3 adds the impact to astaxanthin fermentation for the different precursor substances
| Precursor substance | Dry cell weight (g/l) | Astaxanthin yield (mg/l) |
| No | 18.3 | 83.4 |
| Radix Dauci Sativae juice | 18.5 | 88.2 |
| Fructus Lycopersici esculenti juice | 19.2 | 94.3 |
| Rhizoma Curcumae Longae juice | 17.8 | 84.3 |
| Tea juice | 18.1 | 83.8 |
The difference impact to astaxanthin fermentation for the joining day of table 1-4 Fructus Lycopersici esculenti juice
The interpolation of astaxanthin precursor substance, can make the concentration of reaction substrate increase, so that react carrying out to positive direction, promote
Enter the synthesis of astaxanthin.Add the synthesis facilitation to astaxanthin for the Fructus Lycopersici esculenti juice precursor substance by can be seen that in table 1-3,1-4
Effect preferably, and adds the yield difference that the time interval of Fructus Lycopersici esculenti juice precursor substance can lead to astaxanthin, to add one every 12h
Secondary Fructus Lycopersici esculenti juice, adding altogether 4 times is optimum condition, and the yield of astaxanthin is 94.3mg/l.
3rd, add the optimization of sugar source
(1) slant strains having activated are inoculated in the 250ml conical flask of the ym culture medium of liquid containing 30ml, 22 ± 1 DEG C,
180r/min cultivates 20h；Then it is inoculated in the 1000ml conical flask containing 100ml Medium of shaking flask fermentation by inoculum concentration 10%, 21
DEG C, 180r/min cultivates 20h.
(2) fermenter volume 10l, liquid amount is 6l, adds the carrier of oxygen (1% fermentation medium in fermentation medium simultaneously
The n-dodecane of volume), 121 DEG C of sterilizing 20min.After cooling, seed culture fluid is seeded to fermentation medium by 10% inoculum concentration
Carry out fermentation culture, 22 ± 1 DEG C of cultivation temperature, by stream plus 2mol/l naoh and 2mol/l hcl control fermentation ph be 5.0 ±
0.5, ventilation 1.2vvm, are 30% by adjust mixing speed controlling dissolved oxygen concentration.
(3) during fermentation culture, every 12h stream plus astaxanthin precursor substance Fructus Lycopersici esculenti juice, stream plus 240ml(send out every time
The 4% of ferment culture medium), stream adds 4 times altogether.During this 48h, every 4h detect fermentation liquid concentration of reduced sugar, when be reduced to 2% with
When lower, adding the saccharide concentrated solution that concentration is 50% makes reducing sugar sugar concentration (mass concentration) in fermentation liquid reach 2%～3%, until
48h stops.Stop fermentation after continuing fermentation 24h afterwards.
(4) after fermentation ends, collects thalline, extract astaxanthin, calculate astaxanthin yield.
Because growth needs carbon source, during the fermentation, carbon source can with fermentation carrying out and slow consumption, therefore
Realize phaffia rhodozyma High Density Cultivation and must add carbon source during the fermentation, maintain growth and the fermentation of cell, simultaneously by
Be conducive to chemical activators in high c/n ratio, thus after fermentation the phase supplemented with beneficial to phaffia rhodozyma accumulate astaxanthin.
Table 1-5 adds the impact to astaxanthin fermentation for the different carbon source
| Carbon source (concentration is 50%) | Dry cell weight (g/l) | Astaxanthin yield (mg/l) |
| No | 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 table 1-5, the yield of astaxanthin can be improved by adding carbon source, but the difference on effect of different carbon source is larger,
Glucose and Fructose are monosaccharide, and sucrose and cellobiose are disaccharide, and the degradation product of sucrose is glucose sugar and Fructose, cellobiose
Degradation product be glucose.Compare glucose as can be seen from the table, Fructose is more suitable for red phaffia rhodozyma as carbon source and sends out
Ferment produces astaxanthin, and sucrose is better than cellobiose for the accumulative effect of astaxanthin.Because directly adding glucose can produce " Portugal
Grape sugar effect ", in fermentation tank, concentration of reduced sugar is too high can suppress thalli growth, and sucrose hydrolysis are slower, be not susceptible to " glucose effect
Should ", and degradation product is glucose and Fructose, is suitable for red phaffia rhodozyma growth, is conducive to astaxanthin accumulation.So selecting sucrose to make
For preferably adding carbon source.The yield of final astaxanthin reaches 103.7mg/l.
4th, the optimization of inoculum concentration
With reference to above-mentioned " 1～3 " method and step and optimal conditions, different vaccination amount 5%, 10%, 15%, 20% is inoculated by the present invention
In fermentation medium, carry out single factor exploration.Fermentation temperature is 22 ± 1 DEG C, and fermentation ph controls 5.0 ± 0.5, ventilation
1.2vvm, adjusts rotating speed to control dissolved oxygen concentration to be 30%, adds the carrier of oxygen 1% n-dodecane, during fermentation culture, often
Every 12h stream plus astaxanthin precursor substance Fructus Lycopersici esculenti juice, each stream adds the 4% of 240ml(fermentation medium), stream adds 4 times altogether.In this 48h
During, detect fermentation liquid concentration of reduced sugar every 4h, when being reduced to below 2%, add the sucrose concentrated solution that concentration is 50%
Reducing sugar sugar concentration (mass concentration) in fermentation liquid is made to reach 2%～3%, until 48h stops.Continue fermentation 24h afterwards to stop sending out
Ferment.
The impact to astaxanthin yield for the table 1-6 inoculum concentration
| Inoculum concentration (v/v) % | 5 | 10 | 15 | 20 |
| Dry cell weight (g/l) | 20.4 | 20.9 | 20.7 | 18.2 |
| Astaxanthin yield (mg/l) | 94.5 | 103.7 | 98.4 | 85.6 |
From table 1-6, optimum inoculation amount is 10%, and its astaxanthin yield is up to 103.7mg/l.
5th, the optimization of fermentation liquid dissolved oxygen concentration
The present invention controls different dissolved oxygen concentration 20%, 30%, 40%, 50%, 60% by adjusting mixing speed, for method
Husband's yeast growth and astaxanthin yield carry out single factor exploration, and other Step By Conditions are with reference to " 4, the optimization of inoculum concentration ", fermentation
Temperature is 22 ± 1 DEG C, and fermentation ph is 5.0 ± 0.5, ventilation 1.2vvm, and inoculum concentration 10% adds the carrier of oxygen 1% n-dodecane,
During fermentation culture, every 12h stream plus astaxanthin precursor substance Fructus Lycopersici esculenti juice, flow every time and add the 4% of 240ml(fermentation medium),
Stream adds 4 times altogether.During this 48h, detect fermentation liquid concentration of reduced sugar every 4h, when being reduced to below 2%, add concentration
Sucrose concentrated solution for 50% makes reducing sugar sugar concentration (mass concentration) in fermentation liquid reach 2%～3%, until 48h stops.Afterwards
Continue fermentation 24h and stop fermentation.
Table 1-7 fermentation liquid dissolved oxygen concentration is to astaxanthin yield impact
| Dissolved oxygen concentration (%) | 20 | 30 | 40 | 50 | 60 |
| Dry cell weight (g/l) | 18.4 | 20.9 | 21.5 | 21.9 | 21.3 |
| Astaxanthin yield (mg/l) | 83.2 | 103.7 | 110.2 | 104.8 | 89.2 |
When can be seen that control dissolved oxygen least concentration for 40% by table 1-7, the yield of astaxanthin is up to 110.2mg/
l.When dissolved oxygen concentration is less than 40%, because phaffia rhodozyma is aerobic funguses, so thalli growth is bad, astaxanthin yield
Low；When oxyty is higher than 40%, the impact to maximum biomass for the dissolved oxygen less, but raises with dissolved oxygen concentration, and shrimp is blue or green
Element synthesis is suppressed.So selecting to control dissolved oxygen optimum concentration to be 40%.
6th, add the optimization of precursor after fermentation time
Change and add the yield that the fermentation time after precursor substance investigates astaxanthin, other Step By Conditions with reference to " 5, send out
The optimization of zymotic fluid dissolved oxygen concentration ".Fermentation temperature is 22 ± 1 DEG C, and fermentation ph is 5.0 ± 0.5, ventilation 1.2vvm, and regulation is stirred
Mix rotating speed controlling dissolved oxygen to be 40%, inoculum concentration 10%, add the carrier of oxygen 1% n-dodecane, during fermentation culture, every 12h
The 4% of stream plus astaxanthin precursor substance Fructus Lycopersici esculenti juice, each stream plus 240ml(fermentation medium), stream adds 4 times altogether.In this 48h process
In, detect fermentation liquid concentration of reduced sugar every 4h, when being reduced to below 2%, adding the sucrose concentrated solution that concentration is 50% makes to send out
In zymotic fluid, reducing sugar sugar concentration (mass concentration) reaches 2%～3%, until 48h stops.Then investigate respectively again fermentation 24,36,
48th, after 72h astaxanthin yield.
Table 1-8 adds the impact to astaxanthin yield for the precursor substance after fermentation time
| Fermentation time (h) | 24 | 36 | 48 | 72 |
| Dry cell weight (g/l) | 21.5 | 21.7 | 21.3 | 20.4 |
| Astaxanthin yield (mg/l) | 110.2 | 114.5 | 111.3 | 98.6 |
Be can be seen that after addition Fructus Lycopersici esculenti juice precursor substance 4 times by table 1-8, then the 36h that ferments, the yield of astaxanthin is up to
To 114.5mg/l, fermentation time is long, and thalline yield declines, and the yield of astaxanthin is declined slightly, and expands fermentation costs,
Therefore select 36h as optimal fermentation time.
The application of embodiment 2 natural astaxanthin
1st, the feed additive of astaxanthin-containing is to the egg laying amount of snapper turtle and incubation rate and surviving and grow reality to young Testudiniss
Test
(1)
The close Testudiniss number of test tank is 552, and the close Testudiniss number in comparison pond is 20, and the female Testudiniss feeding interpolation of test tank contains 1 ‰
The Chelydra sertentina special feed of astaxanthin additive, comparison pond feeds normal Chelydra sertentina special feed.28.6 DEG C of average temperature, through 75 days
Feeding comparative trial, the respectively laying in statistical test pond and comparison pond, fertility rate and hatchability.
Table 2-1 contains the impact experiment to snapper turtle egg laying amount, incubation rate for the 1 ‰ astaxanthin additives
(2)
The young Testudiniss of hatching are carried out feeding the experiment of the Chelydra sertentina special feed of 1 ‰ astaxanthin additives.Matched group 100
Young Testudiniss, the young Testudiniss of test group 541, the Chelydra sertentina special feed containing 1 ‰ astaxanthin additives for the test group feeding, control group fed is normal
Chelydra sertentina special feed.28.6 DEG C of average temperature, through the feeding comparative trial of 162 days, statistical test pond and comparison pond respectively
Young Testudiniss survival rate and the speed of growth.
Table 2-2 contains the impact experiment to snapper turtle children's Testudiniss survival rate and the speed of growth for the 1 ‰ astaxanthin additives
From table 2-1, compared with matched group, the astaxanthin of the present invention is added to snapper turtle special feed by 1 ‰ and raises
After feeding female Testudiniss, the relatively matched group without astaxanthin carries respectively for the yield ovum (piece /) of female Testudiniss, rate of fertilization (%) and incubation rate (%)
High 17.0,18.7 and 9.5 percentage points.After table 2-2 can be seen that and adds astaxanthin young turtle feed, its survival rate
(%), rate of body weight gain (%) is respectively increased 7.2 and 5.1 percentage points.Illustrate that astaxanthin can substantially carry aquatic animal spawning rate, rate of fertilization
With immunity and resistances against diseases.
2nd, the disease-resistant experiment to Penaeus vannamei containing 1.5 ‰ astaxanthin feed additives
Matched group and each three groups of test group.Compare 1 group and 2 groups of comparison respectively puts into Penaeus vannamei 10000 tail, compare 3 groups
Put into 15000 tails.Test 2 groups of 1 composite test and respectively put into Penaeus vannamei 10000 tail, 3 groups of input 8000 tails of test.Matched group
Feeding Penaeus vannamei special feed, the Penaeus vannamei special feed of 1.5 ‰ astaxanthin additives is added in test group feeding.
During test, feed 4 times daily, Feeding time is fixed on daily 7:00,12:00,18:00,23:00, continuously
Feed 90 days.The other medicines such as antibiotic are not used during test.Experimental period, counts the survival rate of each group shrimp and death after terminating
Rate.
Table 2-3 disease-resistant experiment to Penaeus vannamei containing 1.5 ‰ astaxanthin feed additives
From table 2-3, compared with matched group, astaxanthin of the present invention is added to Penaeus vannamei as additive special
The survival rate of Penaeus vannamei in feedstuff, can be significantly improved, it improves 20.12% compared with matched group survival rate.
3rd, the disease-resistant experiment to Lateolabrax japonicus (Cuvier et Va-lenciennes) (Lateolabracis) containing 2 ‰ astaxanthin feed additives
Lateolabrax japonicus (Cuvier et Va-lenciennes) (Lateolabracis) (sea water Lateolabrax japonicus) net cage will be cultivated and be divided into 8 groups, each input Lateolabrax japonicus (Cuvier et Va-lenciennes) (Lateolabracis) 1000 tail, 5 groups of net cages are test group, 3 groups
For matched group, feed test group using the mixed fodder containing 2 ‰ astaxanthin additives, the normal Lateolabrax japonicus (Cuvier et Va-lenciennes) (Lateolabracis) of control group fed is special to be raised
Material.Continuous Observation 80 days, counts natural occurrence mortality rate.
Table 2-4 disease-resistant experiment to Lateolabrax japonicus (Cuvier et Va-lenciennes) (Lateolabracis) containing 2 ‰ astaxanthin feed additives
Table 2-4 shows, the disease such as feeding its hemorrhagic skin Peptic Ulcers of Lateolabrax japonicus (Cuvier et Va-lenciennes) (Lateolabracis) containing 2 ‰ astaxanthin additive premixes
The sickness rate of evil is remarkably decreased, and average survival is 88.9%, and feeds raising without 2 ‰ astaxanthin additive premixes
Material comparison its average survival of Lateolabrax japonicus (Cuvier et Va-lenciennes) (Lateolabracis) is 66.7%.Show to strengthen Lateolabrax japonicus (Cuvier et Va-lenciennes) (Lateolabracis) to Kazakhstan Vickers arc to feed 2 ‰ astaxanthin additives
The resistance of the pathogenic bacterial infections such as bacterium, Aeromonas hydrophila, average survival can improve 22.22%.
Above-mentioned specific embodiment is only the specific embodiment of the present invention, rather than limits the invention, at this
In bright scope of the claims, any modifications and changes that the present invention is made, both fall within protection scope of the present invention.
Claims (1)
1. a kind of fermentation process of high yield natural astaxanthin, comprising:
(1) activate red phaffia rhodozyma cgmcc 6355 bacterial strain, prepare seed liquor；
(2) inoculum concentration is that seed liquor described in 10% is seeded in fermentation medium and carries out fermentation culture, fermentation culture terminates
Afterwards, extract astaxanthin；Wherein,
Before fermentation culture, add the carrier of oxygen in described fermentation medium, the described carrier of oxygen is n-dodecane, described positive 12
The addition of alkane is the 1% of fermentation medium volume；
It is calculated in mass percent, described fermentation medium is that the pretreatment fluid of agricultural byproducts is diluted with water to reduction Sugar concentration
3%, yeast extract 5, (nh4)2so46.0, kh2po43.0, mgso4·7h2O3.0, ph 5.0 ± 0.5；Described agricultural byproducts are
Cane molasses；
During described fermentation culture, when the mass concentration of reducing sugar in fermentation liquid is consumed to below 2%, adding sugar source makes fermentation liquid
The mass concentration of middle reducing sugar reaches 2～3%；Described sugar source is sucrose；Fermentation temperature be 21～23 DEG C, ventilation be 1.0～
1.2vvm, the ph controlling fermentation liquid is 5.0 ± 0.5；
During described fermentation culture, every 12 hours to fermentation liquid in add astaxanthin precursor substance；Described astaxanthin precursor substance
For Fructus Lycopersici esculenti juice；The interpolation number of times of described astaxanthin precursor substance is 4 times, each addition be fermentation medium volume 4～
5%；Complete to ferment after being further continued for fermenting 36 hours after last interpolation astaxanthin precursor substance；
During described fermentation culture, temperature be 21～23 DEG C, ventilation be 1.0～1.2vvm, control fermentation liquid ph be 5.0 ±
0.5, controlling mixing speed to maintain the concentration of dissolved oxygen in fermentation liquid is 40%.
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| RU2785792C1 (en) * | 2022-09-05 | 2022-12-13 | Федеральное государственное автономное образовательное учреждение высшего образования "Уральский федеральный университет имени первого Президента России Б.Н. Ельцина" | Method for preparing nutrient medium for cultivation of yeast xanthophyllomyces dendrorhous (phaffia rhodozyma) based on soybean melace and yeast extract |
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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 |
| CN105614022B (en) * | 2015-12-26 | 2020-02-28 | 山东百龙创园生物科技股份有限公司 | Method for preparing astaxanthin-rich feed additive through symbiotic culture |
| CN105695550B (en) * | 2016-03-31 | 2019-12-17 | 广州元大生物科技发展有限公司 | Method for producing astaxanthin by high-density culture of lactobacillus plantarum |
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| CN106701880A (en) * | 2017-01-17 | 2017-05-24 | 浙江皇冠科技有限公司 | Method for improving Phaffia rhodozyma strain high-yield astaxanthin |
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| CN107858395A (en) * | 2017-12-21 | 2018-03-30 | 杭州爱蔻思生物科技有限公司 | Prepare the synchronous extractive fermentation method of natural astaxanthin and other carotenoid |
| CN108998493B (en) * | 2018-06-25 | 2020-11-06 | 浙江皇冠科技有限公司 | Formula technology and application of fermentation medium for high-yield astaxanthin |
| CN109430536B (en) * | 2018-06-25 | 2022-05-17 | 浙江皇冠科技有限公司 | Natural immunopotentiator for aquatic products and application thereof |
| CN108866139A (en) * | 2018-07-19 | 2018-11-23 | 威海利达生物科技有限公司 | A kind of red phaffia rhodozyma cultural method that astaxanthin yield can be improved |
| 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 |
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| CN101985645A (en) * | 2009-10-30 | 2011-03-16 | 大连工业大学 | Method for biosynthesizing astaxanthin by using exponential intermittent feed supplement mode |
| CN102864087B (en) * | 2012-09-03 | 2013-08-07 | 浙江皇冠科技有限公司 | Phaffia rhodozyma strain with high yield of natural astaxanthin as well as breeding method and application thereof |
| CN103333921A (en) * | 2013-06-28 | 2013-10-02 | 华南农业大学 | Method for promoting high-density fermentation of Pichia pastoris by using oxygen carriers |
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