CN107858395A - Prepare the synchronous extractive fermentation method of natural astaxanthin and other carotenoid - Google Patents
Prepare the synchronous extractive fermentation method of natural astaxanthin and other carotenoid Download PDFInfo
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- CN107858395A CN107858395A CN201711389247.7A CN201711389247A CN107858395A CN 107858395 A CN107858395 A CN 107858395A CN 201711389247 A CN201711389247 A CN 201711389247A CN 107858395 A CN107858395 A CN 107858395A
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- astaxanthin
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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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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C403/00—Derivatives of cyclohexane or of a cyclohexene or of cyclohexadiene, having a side-chain containing an acyclic unsaturated part of at least four carbon atoms, this part being directly attached to the cyclohexane or cyclohexene or cyclohexadiene rings, e.g. vitamin A, beta-carotene, beta-ionone
- C07C403/24—Derivatives of cyclohexane or of a cyclohexene or of cyclohexadiene, having a side-chain containing an acyclic unsaturated part of at least four carbon atoms, this part being directly attached to the cyclohexane or cyclohexene or cyclohexadiene rings, e.g. vitamin A, beta-carotene, beta-ionone having side-chains substituted by six-membered non-aromatic rings, e.g. beta-carotene
Abstract
The present invention relates to a kind of synchronous extractive fermentation method for preparing natural astaxanthin and other carotenoid, it is characterised in that：During the fermentation, when cell concentration OD reaches between 0.6 140, extractant is added, 1% 100%, cultivation temperature is 15 40 DEG C for the volume ratio of the extractant and zymotic fluid, and pH is for 5 10.The present invention compared with prior art, has advantages below and effect：Extractant is added during the fermentation, is not only increased the yield and yield of astaxanthin, be also simplify subsequent purification technique, so as to effectively reduce production cost.
Description
Technical field
The present invention relates to a kind of synchronous extractive fermentation method, more particularly to one kind prepares natural astaxanthin and other class Hu trailing plants
Bu Su synchronous extractive fermentation method.
Background technology
Carotenoid may be used for the natural pigment of feed addictive, food additives, medicine and cosmetics etc..Class
Carrotene includes：Such as astaxanthin (astaxanthin), canthaxanthin (canthaxanthin), luteole
(zeaxanthin), β-kryptoxanthin (β-cryptoxanthin), lycopene (lycopene), beta carotene (β-
Carotene), phoenicoxanthin (phoenicoxanthin), ADX (adonixanthin), echinenone
And 3-hydroxyechinenon (3-hydroxyechinenone) etc. (echinenone).In carotenoid, astaxanthin have compared with
High inoxidizability and more extensive bioactivity, can be used as cultivating fish i.e. salmon, trout, sea bream etc. body colour improver or
The feed addictives such as the yolk yellow improver of poultry.In addition, natural additive for foodstuff or health food of the astaxanthin as safety
Product raw material, moreover it is possible to prevention of arterial atherosclerotic cardiovascular disease and diabetes, strengthen muscular endurance, lifter motion performance.Cause
This, the market demand is very big and in rapid growth, and survey data is shown within 2016, global synthesizing astaxanthin and natural astaxanthin
Overall market scale at 5.5 hundred million dollars, and it is expected that be up to 25.7 hundred million dollars in 2025.
At present, the preparation method of astaxanthin mainly has extraction method and micro-organisms side in chemical synthesis, natural materials
Method.For security consideration, astaxanthin prepared by chemical synthesis can only be used as feed addictive to sell.Because astaxanthin is present in
In the shell-fish such as the fish such as sea bream, salmon and shrimp, crab, krill, astaxanthin can by being extracted in above-mentioned fish and shell-fish, but
Efficiency low cost is high.The method of micro-organisms astaxanthin mainly has three kinds at present：Such as utilize green algae haematococcus pluvialis
(Haematococcus pluvialis) cultivation (it is a kind of cultivate Haematococcus pluvialis production astaxanthin method,
CN1181184C), (it is red to produce astaxanthin ocean using red yeast phaffiafhodozyma (Phaffia rhodozyma) fermentation method
Yeast YS-185 and its astaxanthin manufacture method, the B of CN 101705193), using the fermentation method of lactobacillus, (one kind passes through hair
Kefir milk bacillus produces the method for astaxanthin, the B of CN 103436585) and using belonging to paracoccus (Paracoccus) bacterium
Fermentation method (preparing the method for astaxanthin, CN 102933718 B, PCT/JP2011/056033 by fermenting).On however,
State preparation method and the problem of respective all be present, chemical synthesis has safety problem and do not welcome by consumer.Shrimp, crab etc. are natural
The preparation cost extracted in material is high, and the productivity ratio of algae or yeast is low and extraction is more difficult due to firm cell
Wall, and algae produces predominantly astaxanthin ester rather than pure astaxanthin further increases separation costs.Paracoccus bacterium then exists
Substantial amounts of accessory substance such as PHB (Poly-β-hydroxybutyric Acid) and canthaxanthin etc. are accumulated while producing astaxanthin.
The content of the invention
It is an object of the invention to overcome above shortcomings in the prior art, and natural shrimp can be improved by providing one kind
The synchronous extractive fermentation method for preparing natural astaxanthin and other carotenoid of blue or green element and other carotenoid preparation efficiencies.
Technical scheme is used by the present invention solves the above problems：This prepares natural astaxanthin and other carotenoid
Synchronous extractive fermentation method, it is characterised in that：During the fermentation, when cell concentration (OD) reaches between 0.6-140, add
Enter, in 1%-100%, cultivation temperature is 15-40 DEG C for the volume ratio of the extractant and zymotic fluid, and pH is for 5-10.
Preferably, extractant of the present invention is the organic matter that Determination of oil-water partition coefficient is more than 4.
Preferably, Batch fermentation adds extractant, fed-batch hair in Exponential growth stage, OD in 0.6-4 in the present invention
Ferment exists, and OD is that 10-140 adds extractant.
The extractant be alkanes, alcohols, esters, the one of which of edible oil or its combination, the alkanes bag
Decane, hendecane or dodecane are included until hexadecane, the alcohols include decyl alcohol or laruyl alcohol and other to thalli growth without bright
The aobvious alcohols resisted, the esters include isopropyl myristate, methyl myristate, methyl laurate, methyl caprate, Pork and beans
Cool acetoacetic ester, ethyl myristate or ethyl laurate and other to thalli growth without the esters substantially resisted, the edible oil
Including Semen setariae oil, peanut oil, olive oil, Canola oil, sunflower oil, soybean oil, sesame oil, rice bran oil, grape-kernel oil, palm
The various animals and plants lard oils such as oil, butter or lard.Preferably, use cheap and safe and non-toxic organic matter.
Preferably, the present invention adds derivant during the fermentation, derivant reaches 0.6-140 in cell concentration (OD)
Between when add, the derivant is lactose or IPTG, if the derivant is lactose, dosage 0.1-100mmol/
L；If the derivant is IPTG, addition 0.001-10mmol/L.The related gene of the carotenogenesis of thalline
Expression is suppressed, it is necessary to be induced using derivant by lacI repressors (lacI repressor).Induction can use lactose
Or isopropylthiogalactoside (IPTG).
Preferably, after present invention fermentation, zymotic fluid is separated into organic phase and aqueous phase after centrifugal treating, and dissociate shrimp
Blue or green element mainly in the organic phase, can pass through separation circuit purification astaxanthin.
Preferably, separation circuit of the present invention be crystallize, evaporate, being concentrated under reduced pressure, filter in one kind or its group
Close.
Preferably, the strain that fermentation process of the present invention adds be to produce the strain of astaxanthin (primary product is that shrimp is blue or green
Element).
Preferably, the present invention is during the fermentation, concentration of glucose control exists in below 20g/L, oxyty control
More than 1mg/L, the generation concentration of acetate of accessory substance is controlled in below 10g/L.
Preferably, in fermentation process of the present invention, inorganic salts can select the one or more in following material, di(2-ethylhexyl)phosphate
The phosphoric acid salts such as hydrogen sodium, potassium dihydrogen phosphate, dipotassium hydrogen phosphate；The magnesium salts such as magnesium sulfate, magnesium chloride；Ferric sulfate, ironic citrate, chlorine
Change the molysite classes such as iron；The calcium salt class such as calcium chloride, calcium carbonate；The sodium salts such as sodium carbonate, sodium chloride；The manganese salts such as manganese chloride, manganese sulfate
Class；The cobalt salt class such as cobalt chloride；The copper salt kinds such as copper sulphate；The zincum salts such as zinc acetate, zinc sulfate；The molybdenum salt such as sodium molybdate；Nickel sulfate
Deng nickel salt class；Boric acid.Inorganic salts addition is adjusted accordingly according to its species difference, is 0.0001- relative to 1L culture mediums
20g.Wherein, phosphoric acid salt, magnesium salts, calcium salt class, the preferred 0.02-20g/L of the concentration of sodium salt and molysite class in the medium,
When adding manganese salt class, cobalt salt class, copper salt kind, zincum salts, molybdenum salt, nickel salt class and/or boric acid, concentration 0.1-20mg/L.
The present invention compared with prior art, has advantages below and effect：Extractant is added during the fermentation, is not only carried
The high yield and yield of astaxanthin, also simplify subsequent purification technique, so as to effectively reduce production cost.
Brief description of the drawings
Fig. 1 is the procedure chart for preparing natural astaxanthin and the synchronous extractive fermentation method of other carotenoid of the present invention
Schematic diagram.
Fig. 2 is impact effect table of the different derivants of the embodiment of the present invention 4 under different conditions.
Fig. 3 is the extractant and zymotic fluid scale effect effect table of the embodiment of the present invention 3.
Fig. 4 is the single-phase fermentation of Batch fermentation in the embodiment of the present invention 1 and synchronous extractive fermentation contrast table.
Fig. 5 is the impact effect table of the different extractants of Batch fermentation in the embodiment of the present invention 2.
Fig. 6 is the effect table that fed-batch fermentation induces in different times in the embodiment of the present invention 5.
Fig. 7 is the single-phase and synchronous extractive fermentation contrast table of fed-batch fermentation in the embodiment of the present invention 6.
Embodiment
Below in conjunction with the accompanying drawings and the present invention is described in further detail by embodiment, and following examples are to this hair
Bright explanation and the invention is not limited in following examples.
Referring to Fig. 1 to Fig. 7.
Embodiment 1：
Step 1) prepares 5L culture mediums, including 10g/L glucose, 2g/L (NH4)2SO4, 4.2g/L KH2PO4And 11.24g/
L K2HPO4, 1.7g/L citric acids, 0.5g/L MgSO4With 10mL/L trace element solutions.Wherein, trace element solution contains
0.25g/L CoCl2·6H2O, 1.5g/L MnSO4·4H2O, 0.15g/L CuSO4·2H2O, 0.3g/L H3BO3, 0.25g/L
Na2MoO4·2H2O, 0.8g/L Zn (CH3COO)2, 5g/L ironic citrates (III) and 0.84g/L EDTA, trace element solution
PH is 8.0.
By medium sterilization, 121 DEG C sterilize 15-20 minutes step 2).(note, glucose solution will individually sterilize, Ran Houzai
Mixed with other compositions).
Step 3) next, take 100mL culture medium to load identical #1 and #2, two 250mL fermentation respectively
In tank, and fermentation tank is all placed at a temperature of 121 DEG C.
The coli strain E for the high-yield astaxanthin that step 4) transforms genetic engineering is inoculated into the (culture of 20mL culture mediums
Base is derived from step 1)) in, and cultivated 14 hours or so at 37 DEG C, bacterium solution is made.
5mL fermentation steps 4 are taken respectively after step 5)) in bacterium solution be inoculated into #1 the and #2 fermentation tanks in step 3), #
1 and #2 fermentation tanks are using 15% ammonia spirit control pH 7.1, and start temperature is controlled at 37 DEG C, and temperature control is 30 after induction
DEG C, using air as source of oxygen, throughput control in 1vvm or so and as far as possible guarantee oxygen concentration is lured in more than 1mg/L
It is 0.1mmol/L IPTG to lead agent.
After step 6) induction, when cell concentration (OD) reaches 2 in #2 fermentation tanks, extractant (myristic acid isopropyl is added
Ester), extractant and #2 ferment tank liquid volume ratio are 20 to 100.#1 fermentation tanks are not added with extracting liquid, other conditions and #2 fermentation tanks
It is identical.Whole fermentation process is 24 hours or so.After fermentation ends, the separately sampled measurement intracellular astaxanthin of #1 and #2 fermentation tanks
Content, see Fig. 4.
Step 7) takes that zymotic fluid in #2 fermentation tanks carries out centrifugal treating or membrane filtration handles to obtain wet thallus, after drying
Obtained drying thalline can be directly used as feed addictive.Zymotic fluid in #2 fermentation tanks separates organic after centrifugal treating
Phase and aqueous phase, it is mainly astaxanthin wherein in organic phase, other carotenoid contents are very low, therefore can pass through crystallization, steam
Send out, be concentrated under reduced pressure, filtering etc. and obtaining high-purity astaxanthin, serving as medicine or cosmetics, preferably crystallize.Organic phase can pass through
Evaporation or rectifying etc. recycle.Zymotic fluid in #1 fermentation tanks carries out centrifugal treating or membrane filtration handles to obtain wet thallus, passes through
The drying thalline obtained after drying can be directly used as feed addictive.Zymotic fluid in #1 fermentation tanks is after centrifugal treating, water
The ethyl acetate that case aqueous phase adds 10% (volume content) is obtained by extraction.The shrimp in #2 fermentation tanks organic phase and aqueous phase is measured respectively
Aqueous phase Prawn green grass or young crops cellulose content in blue or green cellulose content, and #1 fermentation tanks.
As a result as shown in figure 4, from figure it is upper can clearly find out used synchronous extractive fermentation method of the invention it
Afterwards, the yield of astaxanthin is obviously improved.
Embodiment 2：
Step 1) prepares 5L culture mediums, including 10g/L glucose, 2g/L (NH4)2SO4, 4.2g/L KH2PO4And 11.24g/
L K2HPO4, 1.7g/L citric acids, 0.5g/L MgSO4With 10mL/L trace element solutions.Wherein, trace element solution contains
0.25g/L CoCl2·6H2O, 1.5g/L MnSO4·4H2O, 0.15g/L CuSO4·2H2O, 0.3g/L H3BO3, 0.25g/L
Na2MoO4·2H2O, 0.8g/L Zn (CH3COO)2, 5g/L ironic citrates (III) and 0.84g/L EDTA, trace element solution
PH is 8.0.
By medium sterilization, 121 DEG C sterilize 15-20 minutes step 2).(note, glucose solution will individually sterilize, Ran Houzai
Mixed with other compositions).
Step 3) next, take 100mL culture medium to load identical #3 fermentation tanks, #4 fermentation tanks, #5 hairs respectively
Fermentation tank, #6 fermentation tanks, be all 250mL fermentation tank in, and fermentation tank is all placed at a temperature of 121 DEG C.
The coli strain E for the high-yield astaxanthin that step 4) transforms genetic engineering is inoculated into the (culture of 20mL culture mediums
Base is derived from step 1)) in, and cultivated 14 hours or so at 37 DEG C, bacterium solution is made.
5mL fermentation steps 4 are taken respectively after step 5)) in bacterium solution be inoculated into #3 fermentation tanks in step 3), #4 fermentations
In tank, #5 fermentation tanks, #6 fermentation tanks, all fermentation tanks control pH 7.1 using 15% ammonia spirit.Fermentation start temperature control
System is at 37 DEG C, and temperature control is at 30 DEG C after induction, and using air as source of oxygen, throughput control is in 1vvm or so and as far as possible
Ensure oxygen concentration in more than 1mg/L, derivant is 0.1mmol/L IPTG.
After step 6) induction, when cell concentration (OD) reaches 2 in fermentation tank, adding extractant, (#3 fermentation tanks add 12
Alkane adds laruyl alcohol as extractant, #4 fermentation tanks and adds IPM as extraction as extractant, #5 fermentation tanks
Agent, #6 fermentation tanks add methyl laurate as extractant), extractant and ferment tank liquid volume ratio are 20 to 100.Entirely
Fermentation process is 24 hours or so.After fermentation ends, the separately sampled measurement intracellular content astaxanthin of fermentation tank, Fig. 5 is seen.
Step 7) takes that zymotic fluid in fermentation tank carries out centrifugal treating or membrane filtration handles to obtain wet thallus, after drying
To drying thalline can be directly used as feed addictive.Zymotic fluid in fermentation tank separated after centrifugal treating organic phase and
It is mainly astaxanthin in aqueous phase, wherein organic phase, other carotenoid contents are very low, therefore can pass through and crystallize, evaporate, subtracting
Pressure concentration, filtering etc. obtain high-purity astaxanthin, serve as medicine or cosmetics, preferably crystallize.Organic phase can pass through evaporation
Or rectifying etc. recycles.After separating organic phase and aqueous phase, #3 fermentation tanks, #4 fermentation tanks, #5 fermentation tanks, #6 are fermented respectively
The content astaxanthin of aqueous phase and organic phase in tank is sampled measurement, and end product is shown in Fig. 5.
Wherein, isopropyl myristate is best as the resultant effect of extractant.Comparison diagram 4 is it can be seen that all use
The fermentation process of the present invention, astaxanthin yield is not all than using the high of fermentation process of the present invention.
Embodiment 3：
Step 1) prepares 5L culture mediums, including 10g/L glucose, 2g/L (NH4)2SO4, 4.2g/L KH2PO4And 11.24g/
L K2HPO4, 1.7g/L citric acids, 0.5g/L MgSO4With 10mL/L trace element solutions.Wherein, trace element solution contains
0.25g/L CoCl2·6H2O, 1.5g/L MnSO4·4H2O, 0.15g/L CuSO4·2H2O, 0.3g/L H3BO3, 0.25g/L
Na2MoO4·2H2O, 0.8g/L Zn (CH3COO)2, 5g/L ironic citrates (III) and 0.84g/L EDTA, trace element solution
PH is 8.0.
By medium sterilization, 121 DEG C sterilize 15-20 minutes step 2).(note, glucose solution will individually sterilize, Ran Houzai
Mixed with other compositions).
Step 3) next, take 100mL culture medium to load identical #7 fermentation tanks, #8 fermentation tanks, #9 hairs respectively
Fermentation tank, #10 fermentation tanks, #11 fermentation tanks, #12 fermentation tanks, #13 fermentation tanks, #14 fermentation tanks, all fermentation tanks are all to be
250mL, and fermentation tank is all placed at a temperature of 121 DEG C.
The coli strain E for the high-yield astaxanthin that step 4) transforms genetic engineering is inoculated into the (culture of 20mL culture mediums
Base is derived from step 1)) in, and cultivated 14 hours or so at 37 DEG C, bacterium solution is made.
5mL fermentation steps 4 are taken respectively after step 5)) in bacterium solution be inoculated into #7 fermentation tanks in step 3), #8 fermentations
In tank, #9 fermentation tanks, #10 fermentation tanks, #11 fermentation tanks, #12 fermentation tanks, #13 fermentation tanks, #14 fermentation tanks, all fermentation tanks are equal
PH is controlled 7.1 using 15% ammonia spirit.Start temperature of fermenting control is at 37 DEG C, and temperature control uses at 30 DEG C after induction
For air as source of oxygen, throughput control is in 1vvm or so and ensures that oxygen concentration is in more than 1mg/L, derivant as far as possible
0.1mmol/L IPTG。
After step 6) induction, when cell concentration (OD) reaches 2 in fermentation tank, extractant (IPM conduct is added
Extractant), extractant and ferment tank liquid volume ratio are #7 fermentation tanks 0, #8 fermentation tanks 1 to 100, #9 fermentation tanks 1 to 50, #
10 fermentation tanks 1 to 20, #11 fermentation tanks 1 to 10, #12 fermentation tanks 1 to 5, #13 fermentation tanks 1 to 2, #14 fermentation tanks 1 to 1.Whole hair
Ferment process is 24 hours or so.After fermentation ends, each separately sampled measurement intracellular content astaxanthin of fermentation tank, Fig. 3 is seen.
Step 7) takes that zymotic fluid in fermentation tank carries out centrifugal treating or membrane filtration handles to obtain wet thallus, after drying
To drying thalline can be directly used as feed addictive.Zymotic fluid in fermentation tank separated after centrifugal treating organic phase and
It is mainly astaxanthin in aqueous phase, wherein organic phase, other carotenoid contents are very low, therefore can pass through and crystallize, evaporate, subtracting
Pressure concentration, filtering etc. obtain high-purity astaxanthin, serve as medicine or cosmetics, preferably crystallize.Organic phase can pass through evaporation
Or rectifying etc. recycles.After separating organic phase and aqueous phase, #7 fermentation tanks, #8 fermentation tanks, #9 fermentation tanks, #10 are sent out respectively
Fermentation tank, #11 fermentation tanks, #12 fermentation tanks, #13 fermentation tanks, the content astaxanthin of the aqueous phase of #14 fermentation tanks and organic phase are taken
Sample measures, and end product is shown in Fig. 3.
It can be seen that when ratio is 1 to 5 in Fig. 3, resultant effect is best, astaxanthin yield highest.
Embodiment 4：
Step 1) prepares 5L culture mediums, including 10g/L glucose, 2g/L (NH4)2SO4, 4.2g/L KH2PO4And 11.24g/
L K2HPO4, 1.7g/L citric acids, 0.5g/L MgSO4With 10mL/L trace element solutions.Wherein, trace element solution contains
0.25g/L CoCl2·6H2O, 1.5g/L MnSO4·4H2O, 0.15g/L CuSO4·2H2O, 0.3g/L H3BO3, 0.25g/L
Na2MoO4·2H2O, 0.8g/L Zn (CH3COO)2, 5g/L ironic citrates (III) and 0.84g/L EDTA, trace element solution
PH is 8.0.
By medium sterilization, 121 DEG C sterilize 15-20 minutes step 2).(note, glucose solution will individually sterilize, Ran Houzai
Mixed with other compositions).
Step 3) is all 250mL hair next, take 100mL culture medium to load identical 9 fermentation tanks respectively
In fermentation tank, and fermentation tank is all placed at a temperature of 121 DEG C.
The coli strain E for the high-yield astaxanthin that step 4) transforms genetic engineering is inoculated into the (culture of 20mL culture mediums
Base is derived from step 1)) in, and cultivated 14 hours or so at 37 DEG C, bacterium solution is made.
5mL fermentation steps 4 are taken respectively after step 5)) in bacterium solution be inoculated into all fermentation tanks in step 3) and use
15% ammonia spirit controls pH, and start temperature is controlled at 37 DEG C, and 9 fermentation tanks are separately added into the derivant species in Fig. 2 7.1
Concentration, temperature control is at 30 DEG C after induction, and using air as source of oxygen, throughput control is protected in 1vvm or so and as far as possible
Oxygen concentration is demonstrate,proved in more than 1mg/L, derivant is 0.1mmol/L IPTG.
After step 6) induction, when cell concentration (OD) reaches 2 in fermentation tank, extractant (IPM conduct is added
Extractant), extractant and ferment tank liquid volume ratio are 1 to 100.Whole fermentation process is 24 hours or so.Fermentation ends
Afterwards.
Step 7) takes that zymotic fluid in fermentation tank carries out centrifugal treating or membrane filtration handles to obtain wet thallus, after drying
To drying thalline can be directly used as feed addictive.Zymotic fluid in fermentation tank separated after centrifugal treating organic phase and
It is mainly astaxanthin in aqueous phase, wherein organic phase, other carotenoid contents are very low, therefore can pass through and crystallize, evaporate, subtracting
Pressure concentration, filtering etc. obtain high-purity astaxanthin, serve as medicine or cosmetics, preferably crystallize.Organic phase can pass through evaporation
Or rectifying etc. recycles.It is blue or green to the shrimp of the aqueous phase in each fermentation tank and organic phase respectively after separating organic phase and aqueous phase
Cellulose content is sampled measurement, and end product is shown in Fig. 2.
Embodiment 5：
In fed batch fermentation, it is respectively mid-term (OD 25-54) in early days (OD 5-24) to define induction time, in
Later stage (OD 55-84) and later stage (OD 85-140).
Step 1) prepares 5L culture mediums, including 10g/L glucose, 2g/L (NH4)2SO4, 4.2g/L KH2PO4And 11.24g/
L K2HPO4, 1.7g/L citric acids, 0.5g/L MgSO4With 10mL/L trace element solutions.Wherein, trace element solution contains
0.25g/L CoCl2·6H2O, 1.5g/L MnSO4·4H2O, 0.15g/L CuSO4·2H2O, 0.3g/L H3BO3, 0.25g/L
Na2MoO4·2H2O, 0.8g/L Zn (CH3COO)2, 5g/L ironic citrates (III) and 0.84g/L EDTA, trace element solution
PH is 8.0.
By medium sterilization, 121 DEG C sterilize 15-20 minutes step 2).(note, glucose solution will individually sterilize, Ran Houzai
Mixed with other compositions).
Step 3) is all 250mL hair next, take 100mL culture medium to load identical 9 fermentation tanks respectively
In fermentation tank, and fermentation tank is all placed at a temperature of 121 DEG C.
The coli strain E for the high-yield astaxanthin that step 4) transforms genetic engineering is inoculated into the (culture of 20mL culture mediums
Base is derived from step 1)) in, and cultivated 14 hours or so at 37 DEG C, bacterium solution is made.
5mL fermentation steps 4 are taken respectively after step 5)) in bacterium solution be inoculated into the fermentation tank in step 3) and use 15%
Ammonia spirit controls pH, and start temperature is controlled at 37 DEG C 7.1.Fermentation tank is separately added into the concentration of the derivant species in Fig. 2,
Temperature control is at 30 DEG C after induction, and using air as source of oxygen, throughput controls in 1vvm or so and ensures oxygen as far as possible
Concentration is in more than 1mg/L.It is different from Batch fermentation, once OD reaches about 6-8, a feed supplement is proceeded by, feed supplement is at intervals of 4-6
Hour, and determine whether to continue feed supplement (specifically, if oxygen dissolving value continues greatly by oxygen dissolving value or detection concentration of glucose
Exceed half an hour more than 20g/L in 5mg/L or concentration of glucose, then stop feed supplement).Feed medium contains 500g/L glucose
And 5g/LMgSO4.Induction time be respectively early stage (OD 5-24), mid-term (OD 25-54), the middle and later periods (OD 55-84) and
Later stage (OD 85-140).Derivant uses 0.03mmol/LIPTG or 10mmol/L lactose.Fed-batch fermentation can obtain
Cell concentration more, so as to greatly improve astaxanthin yield.Fig. 6 results illustrate that different time induces to be had to the yield of astaxanthin
Very big influence.
Embodiment 6：
In fed batch fermentation, it is respectively mid-term (OD 25-54) in early days (OD 5-24) to define induction time, in
Later stage (OD 55-84) and later stage (OD 85-140).It is that single-phase and synchronous extractive fermentation uses the middle and later periods in the present embodiment
Induction.
Step 1) prepares 5L culture mediums, including 10g/L glucose, 2g/L (NH4)2SO4, 4.2g/L KH2PO4And 11.24g/
L K2HPO4, 1.7g/L citric acids, 0.5g/L MgSO4With 10mL/L trace element solutions.Wherein, trace element solution contains
0.25g/L CoCl2·6H2O, 1.5g/L MnSO4·4H2O, 0.15g/L CuSO4·2H2O, 0.3g/L H3BO3, 0.25g/L
Na2MoO4·2H2O, 0.8g/L Zn (CH3COO)2, 5g/L ironic citrates (III) and 0.84g/L EDTA, trace element solution
PH is 8.0.
By medium sterilization, 121 DEG C sterilize 15-20 minutes step 2).(note, glucose solution will individually sterilize, Ran Houzai
Mixed with other compositions).
Step 3) is all 250mL hair next, take 100mL culture medium to load identical 2 fermentation tanks respectively
In fermentation tank, and fermentation tank is all placed at a temperature of 121 DEG C.
The coli strain E for the high-yield astaxanthin that step 4) transforms genetic engineering is inoculated into the (culture of 20mL culture mediums
Base is derived from step 1)) in, and cultivated 14 hours or so at 37 DEG C, bacterium solution is made.
5mL fermentation steps 4 are taken respectively after step 5)) in bacterium solution be inoculated into the fermentation tank in step 3) and use 15%
Ammonia spirit controls pH, and start temperature is controlled at 37 DEG C 7.1.Fermentation tank is separately added into the concentration of the derivant species in Fig. 2,
Temperature control is at 30 DEG C after induction, and using air as source of oxygen, throughput controls in 1vvm or so and ensures oxygen as far as possible
Concentration is in more than 1mg/L.It is different from Batch fermentation, once OD reaches about 6-8, a feed supplement is proceeded by, feed supplement is at intervals of 4-6
Hour, and determine whether to continue feed supplement (specifically, if oxygen dissolving value continues greatly by oxygen dissolving value or detection concentration of glucose
Exceed half an hour more than 20g/L in 5mg/L or concentration of glucose, then stop feed supplement).Feed medium contains 500g/L glucose
And 5g/LMgSO4.
Step 6) induction time is respectively middle and later periods (OD 55-84).Derivant using 0.03-0.1mmol/LIPTG or
10-20mmol/L lactose.In synchronous extractive fermentation tank, also it is possible to additionally incorporate IPM as extractant, extractant and
Ferment tank liquid volume ratio is 20 to 100.
Fed-batch fermentation can obtain cell concentration more, so as to greatly improve astaxanthin yield.Meanwhile Fig. 7 results
Illustrate, after synchronous extractive fermentation method, the yield and yield of astaxanthin are obtained for further lifting.
The engineering bacteria used in the present invention can be engineered strain, such as strain (Z1 strains, E strains and G strains).
The nitrogen source of culture medium in the present invention can select inorganic nitrogen-sourced or organic nitrogen source.As inorganic nitrogen-sourced, such as can
To enumerate：The ammonium salt class such as ammonium sulfate, ammonium chloride, ammonium nitrate, ammonium phosphate；The Nitrateses such as potassium nitrate；Also include ammonia and urea etc.,
One or more can be wherein used, wherein preferentially using ammonium sulfate and potassium nitrate.Addition according to the species of nitrogen source and
Difference, as long as appropriate adjustment, inorganic nitrogen-sourced addition is 0.1-15g, is preferably 0.2-5g in generally every 1L culture mediums.
As organic nitrogen source, such as can enumerate：Corn steep liquor (including filtration treatment thing), dry yeast, medicinal culture medium, Soybean Meal,
Soy meal, peanut powder and solvable vinasse etc., one or more can be used in above-mentioned organic nitrogen source.Concentration is added according to nitrogen
The species in source and it is different, as long as appropriate adjustment, the addition concentration for being generally incubated organic nitrogen source in base is 0-60g/L, preferably
For 0-30g/L.It is preferential to use relatively cheap inorganic nitrogen-sourced of price, or the organic nitrogen source of steady quality.Nitrogen source generally addition exists
Originate in culture medium, but it is also preferred that successively or continuously additional services.
As inorganic salts, such as can enumerate：The phosphoric acid salts such as sodium dihydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate；
The magnesium salts such as magnesium sulfate, magnesium chloride；The molysite class such as ferric sulfate, ironic citrate, iron chloride；The calcium salt class such as calcium chloride, calcium carbonate；Carbon
The sodium salts such as sour sodium, sodium chloride；The manganese salt class such as manganese chloride, manganese sulfate；The cobalt salt class such as cobalt chloride；The copper salt kinds such as copper sulphate；Acetic acid
The zincum salts such as zinc, zinc sulfate；The molybdenum salt such as sodium molybdate；The nickel salt class such as nickel sulfate；Boric acid etc., it can use in above-mentioned inorganic salts
It is one or more kinds of.The addition of inorganic salts adjusts according to its species, as long as appropriate adjustment, is commonly angled relative to 1L
Culture medium is 0.0001-20g.Wherein, phosphoric acid salt, magnesium salts, calcium salt class, the concentration of sodium salt and molysite class in the medium
It is preferred that 0.02-20g/L, when adding manganese salt class, cobalt salt class, copper salt kind, zincum salts, molybdenum salt, nickel salt class, boric acid etc., 0.1-
20mg/L is preferable concentration.Inorganic salts generally addition can also successively or continuously add confession in culture medium is originated
Give.
The related gene expression of the carotenogenesis of thalline is suppressed by lacI repressors (lacI repressor),
Derivant is needed to use to induce.Induction can use lactose or isopropylthiogalactoside (IPTG).Induction time is with thin
Born of the same parents' concentration is as standard, i.e., cell concentration (OD) reaches between 0.6-140.Wherein, Batch fermentation preferably has 0.6-4, feed supplement point
Wholesale ferment is preferably 10-140.Dosage as derivant lactose is in 0.1-100mmol/L, preferably 5-30mmol/L；As
Derivant IPTG dosage is in 0.001-10mmol/L, preferably 0.03-1mmol/L.In addition, lactose can be used as derivant
May be used as carbon source again, used as single carbon source, 80mmol/L can be brought up to by adding concentration, preferably 60mmol/L with
Under.
In the present invention, synchronous extractive fermentation refers to the fermentation for adding extractant, including two-phase, aqueous phase and organic phase.
As the organic phase of synchronous extractive fermentation, such as can enumerate：The alkanes such as decane, hendecane, dodecane；Decyl alcohol (1-
Decanol), the alcohols such as laruyl alcohol (Dodecanol)；Isopropyl myristate (isopropyl myristate), myristic acid
Methyl esters (Methyl tetradecanoate), methyl laurate (Methyl laurate), methyl caprate (Methyl
Caprate), ethyl myristate (Ethyl laurate), ethyl myristate (Ethyl myristate), ethyl laurate
Esters such as (Ethyl laurate)；And other grow what nothing significantly inhibited, in general, Determination of oil-water partition coefficient to microorganism
(lopPOW) can be used more than 4 organic matter, as preferential, use cheap and safe and non-toxic organic matter such as bay
Sour methyl esters, laruyl alcohol etc..Extractant carries out situ extracting (In situ extraction) to astaxanthin for of dissociating etc..As
The extractant of synchronous extractive fermentation can also can add in culture starting addition in ferment middle or later stage.Prioritizing selection is being sent out
Ferment mid-term adds, i.e., between cell concentration (OD) reaches 0.6-140, preferably 30-80.Can also with reference to IPTG induction times,
It can add in induction or be added after induction in 6 hours.Extractant addition, using liquid phase of fermenting to participate in, that is, extract
Agent is taken to account for the ratio of total zymotic fluid in 1%-100%, preferably 4-30%, more preferably 5-20%.
Culture is carried out in appropriate culture vessel.Culture vessel can suitably select according to culture capacity, such as have examination
Pipe, flask, fermentation tank etc..
Cultivation temperature is, for example, 15-40 DEG C, preferably 20-37 DEG C, more preferably 25-35 DEG C.It is usually 1-14 during culture
My god, preferably 1-7 days, more preferably 2-4 days, cultivated under aerobic conditions.
As aerobic condition, such as can enumerate：Air agitation culture or shaken cultivation etc., preferably using dissolved oxygen electrode
Constantly simultaneously feedback control enters oxygen to monitoring oxyty.Culture originates oxyty and shown close to saturated concentration, but with micro- life
Thing starts to grow, and zmount of oxygen consumption gradually increases, and oxyty also gradually reduces therewith.It is that microorganism oxygen consumption is most active to cultivate mid-term
Period.Wherein, during air agitation deficiency, oxyty reaches zero, i.e. oxygen depletion, growth and carotenoid to microorganism
Production bring harmful effect.Because production of the culture mid-term to astaxanthin is most important, thus preferred control oxyty with
Oxygen is set not exhaust.The control of oxyty such as can by changing throughput to mix oxygen concentration in revolution and ventilation gas come
Carry out.Culture mid-term culture in oxyty be preferably controlled in more than 1mg/L, more preferably more than 1.5mg/L, further it is excellent
Select more than 2mg/L.The upper limit for cultivating the control range of oxyty in the culture of mid-term is not particularly limited, but preferred 8mg/
Below L, further preferred below 6mg/L.Oxyty can be controlled in certain constant range, can also rank such as 1-3mg/L
Section property or continuously improve culture in oxyty reach highest oxyty, such as from 1mg/L to 1.5mg/L to
2mg/L to 3mg/L.Control to oxyty can reduce the generation of the accessory substances such as canthaxanthin, so as to improve the production of astaxanthin
Amount and purity.
The pH of culture medium is adjusted to 5-10, preferably 6-9, more preferably 6.5-7.5.As the method for maintaining pH, preferably use
The method of the negative-feedback PID control of pH electrodes.As pH alkaline conditioners, such as can enumerate：Ammoniacal liquor, sodium hydroxide are water-soluble
Liquid, potassium hydroxide aqueous solution, aqueous sodium carbonate, ammonia or their mixture.As pH acid regulators, such as can arrange
Lift：Aqueous sulfuric acid, hydrochloric acid or their mixture.
It is preferred that using defoamer to suppress the foaming of fermentation process.Such as it can enumerate：Polyethers system defoamer, the defoaming of alcohol system
Agent, fatty acid series defoamer, ester system defoamer, silicon-type defoamer, sulfonic acid system defoamer etc..The addition of defoamer according to
The species of defoamer and it is different, as long as appropriate adjustment, but it is 0.01-10g to be commonly angled relative to 1L culture mediums.Defoamer can be with
Addition originating in culture medium before sterilization, can also be in culture midway continuously or intermittently additional addition defoamer.Typically
The method added automatically after being steeped by using sensor senses, it is added at a certain time interval using program timer
Method that method, change and raising carbon source, nitrogen source or the pH adjusting agent of Adaptable growth speed etc. are added after mixing etc..Preferentially make
With the few defoamer of the inhibitory action to producing bacterium, can select any.
Zymotic fluid carries out centrifugal treating or membrane filtration handles to obtain wet thallus, and the drying thalline obtained after drying can be straight
Connect and be used as feed addictive.The zymotic fluid of synchronous extractive fermentation separates organic phase and aqueous phase after centrifugal treating, wherein organic
It is mainly astaxanthin in phase, other carotenoid contents are very low, therefore can pass through and crystallize, evaporate, being concentrated under reduced pressure, filtering
High-purity astaxanthin is obtained, is preferably crystallized, serves as medicine or cosmetics.Organic phase can pass through the recovery profit such as evaporation or rectifying
With.
The strain used in the embodiment of the present invention, elaborated inside the patent that application is 2017110721556.
Above content described in this specification is only illustration made for the present invention.Technology belonging to the present invention
The technical staff in field can make various modifications or supplement to described specific embodiment or using similar mode
Substitute, content without departing from description of the invention or surmount scope defined in the claims, this all should be belonged to
The protection domain of invention.
Claims (10)
- A kind of 1. synchronous extractive fermentation method for preparing natural astaxanthin and other carotenoid, it is characterised in that：Fermenting Cheng Zhong, when cell concentration OD reaches between 0.6-140, extractant is added, the volume ratio of the extractant and zymotic fluid exists 1%-100%, cultivation temperature are 15-40 DEG C, and pH is for 5-10.
- 2. the synchronous extractive fermentation method according to claim 1 for preparing natural astaxanthin and other carotenoid, it is special Sign is：The extractant is the organic matter that Determination of oil-water partition coefficient is more than 4.
- 3. the synchronous extractive fermentation method according to claim 1 for preparing natural astaxanthin and other carotenoid, it is special Sign is：Batch fermentation is that cell concentration OD adds extractant in the range of 0.6-4 in Exponential growth stage, and fed-batch fermentation exists Cell concentration OD is that 10-140 adds extractant.
- 4. the synchronous extractive fermentation method according to claim 2 for preparing natural astaxanthin and other carotenoid, it is special Sign is：The extractant is that alkanes, alcohols, esters, the one of which of edible oil or its combination, the alkanes include Decane, hendecane or dodecane until hexadecane, the alcohols include decyl alcohol or laruyl alcohol and other to thalli growth without obvious The alcohols of resistance, the esters include isopropyl myristate, methyl myristate, methyl laurate, methyl caprate, nutmeg Acetoacetic ester, ethyl myristate or ethyl laurate and other to thalli growth without the esters substantially resisted, the edible oil bag Include Semen setariae oil, peanut oil, olive oil, Canola oil, sunflower oil, soybean oil, sesame oil, rice bran oil, grape-kernel oil, palm The various animals and plants lard oils such as oil, butter or lard.
- 5. the synchronous extractive fermentation method according to claim 1 for preparing natural astaxanthin and other carotenoid, it is special Sign is：Derivant is added during the fermentation, and derivant adds when cell concentration OD reaches between 0.6-140, described to lure It is lactose or IPTG to lead agent, if the derivant is lactose, dosage 0.1-100mmol/L；If the derivant For IPTG, then addition is 0.001-10mmol/L.
- 6. the synchronous extractive fermentation method according to claim 1 for preparing natural astaxanthin and other carotenoid, it is special Sign is：After fermentation, zymotic fluid is separated into organic phase and aqueous phase after centrifugal treating, wherein free astaxanthin and/or Other carotenoid mainly in the organic phase, can further purify astaxanthin and/or other class Hu trailing plants by separation circuit Bu Su.
- 7. the synchronous extractive fermentation method according to claim 5 for preparing natural astaxanthin and other carotenoid, it is special Sign is：The separation circuit be evaporate, be concentrated under reduced pressure, crystallizing, filter in one kind or its combine.
- 8. the synchronous extractive fermentation method according to claim 1 for preparing natural astaxanthin and other carotenoid, it is special Sign is：The strain that fermentation process adds is the strain of production astaxanthin.
- 9. the synchronous extractive fermentation method according to claim 1 for preparing natural astaxanthin and other carotenoid, it is special Sign is：During the fermentation, concentration of glucose control is secondary in more than 1mg/L, control in below 20g/L, oxyty control The generation concentration of the acetate of product is in below 10g/L.
- 10. the synchronous extractive fermentation method according to claim 1 for preparing natural astaxanthin and other carotenoid, its It is characterised by：In fermentation process, inorganic salts can select the one or more in following material, sodium dihydrogen phosphate, biphosphate The phosphoric acid salts such as potassium, dipotassium hydrogen phosphate；The magnesium salts such as magnesium sulfate, magnesium chloride；The molysite class such as ferric sulfate, ironic citrate, iron chloride； The calcium salt class such as calcium chloride, calcium carbonate；The sodium salts such as sodium carbonate, sodium chloride；The manganese salt class such as manganese chloride, manganese sulfate；The cobalts such as cobalt chloride Salt；The copper salt kinds such as copper sulphate；The zincum salts such as zinc acetate, zinc sulfate；The molybdenum salt such as sodium molybdate；The nickel salt class such as nickel sulfate；Boric acid, Addition is different according to the species of inorganic salts, is 0.0001-20g relative to 1L culture mediums, phosphoric acid salt, magnesium salts, calcium salt Class, the preferred 0.02-20g/L of the concentration of sodium salt and molysite class in the medium, add manganese salt class, cobalt salt class, copper salt kind, zinc salt When class, molybdenum salt, nickel salt class and/or boric acid, concentration 0.1-20mg/L.
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| CN114751848A (en) * | 2022-04-14 | 2022-07-15 | 厦门昶科生物工程有限公司 | Method for preparing astaxanthin essential oil based on phaffia rhodozyma |
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| CN103627650A (en) * | 2013-08-15 | 2014-03-12 | 上海理工大学 | Serratia marcescens strain and synchronous extraction and fermentation method thereof |
| CN103820520A (en) * | 2014-03-06 | 2014-05-28 | 浙江皇冠科技有限公司 | High-yield natural astaxanthin fermentation method |
| CN106565576A (en) * | 2016-11-04 | 2017-04-19 | 嘉必优生物技术（武汉）股份有限公司 | Method for extracting carotenoid |
| CN107129995A (en) * | 2014-09-03 | 2017-09-05 | 武汉生物技术研究院 | A kind of construction method for producing astaxanthin genetic engineering bacterium |
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| CN103627650A (en) * | 2013-08-15 | 2014-03-12 | 上海理工大学 | Serratia marcescens strain and synchronous extraction and fermentation method thereof |
| CN103820520A (en) * | 2014-03-06 | 2014-05-28 | 浙江皇冠科技有限公司 | High-yield natural astaxanthin fermentation method |
| CN107129995A (en) * | 2014-09-03 | 2017-09-05 | 武汉生物技术研究院 | A kind of construction method for producing astaxanthin genetic engineering bacterium |
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| CN114751848A (en) * | 2022-04-14 | 2022-07-15 | 厦门昶科生物工程有限公司 | Method for preparing astaxanthin essential oil based on phaffia rhodozyma |
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