CN107189946A - A kind of method for avoiding microalgae Xanthophyll cycle to improve astaxanthin yield - Google Patents
A kind of method for avoiding microalgae Xanthophyll cycle to improve astaxanthin yield Download PDFInfo
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Abstract
The present invention relates to avoid Xanthophyll cycle during a kind of microdisk electrode to improve the new method of astaxanthin yield.Cultivate microalgae production astaxanthin and generally use two-phase method, the first stage realizes the accumulation of the amount reproduction and biomass of cell by heterotrophism, autotrophy or the mode of raising together with, and the accumulation that the methods such as Nutrient Stress realize intracellular astaxanthin is coerced and be aided with to second stage by light.But second stage is turned by the first stage phenomenons such as Xanthophyll cycle often occur, have a strong impact on the yield of astaxanthin.Therefore the present invention is by the control to condition of culture in conversion process, such as：Control culture medium composition, sunshade, dim light transition, progressively raising Initial seeding density, the means such as dilution, the outdoor inoculation time of adjustment, adjust cell state, make the ability of the strong light of cell enhancing resistance, therefore the Xanthophyll cycle problem under the strong light of microalgae is substantially overcomed, the efficiency that microalgae produces astaxanthin can be greatly enhanced, low cost, high efficiency and large-scale culturing micro-algae production astaxanthin is realized.
Description
Technical field
The invention belongs to technical field of microalga biology, and in particular to a kind of adjustable micro-algal cell state improves astaxanthin yield
Method.
Background technology
Astaxanthin (Astaxanthin), chemical name is 3,3 '-dihydroxy -4,4 '-diketo-β, β '-carrotene, point
Minor is C40H52O4, relative molecular mass is 596.86, also known as astaxanthin, ASX or Astaxanthin, is a kind of keto-acid class
Carrotene.Color and luster is pink, has fat-soluble, water insoluble, is soluble in chloroform, acetone, benzene and carbon disulfide etc. organic molten
Agent.The chemical constitution of astaxanthin is to be linked by 4 isoprene units with conjugated double bond pattern, and there are 2 iso-amylene lists at two ends again
Position composition six-membered ring structure.Due in the chemical constitution of astaxanthin containing conjugation unsaturated double-bond system one long, therefore, it
It is easy to the effect of light, heat, oxide etc. and destroys its structure.The chemical constitution of astaxanthin sees below formula：
Astaxanthin is one kind of carotenoid, is also the highest level product of class Hu Luosu synthesis, and beta carotene, leaf are yellow
Element, canthaxanthin, lycopene etc. are all the intermediate products of carotenogenesis.Therefore, in nature, astaxanthin has most
Strong inoxidizability.Natural astaxanthin is that the mankind have found antioxidant most strong in nature, its antioxidation activity so far
Considerably beyond existing antioxidant, it is described as " super oxidant ".Astaxanthin is with a wide range of applications, and can not only use
Make the feed addictive and human food's additive of aquaculture, also have in fields such as medicine, cosmetics and nutrient and healthcare products
Very big application potential.
Comparatively speaking, there is obvious advantage using microalgae production astaxanthin.Wherein, haematococcus pluvialis, which contain, accounts for carefully
Born of the same parents' dry weight 1-5% astaxanthin, is the natural species of content astaxanthin highest in nature.First, astaxanthin is main in microalgae
If existing in the form of monoesters, its structure is transconfiguration, and the cis-structure bioavilability compared with chemical synthesis is high；Secondly,
The growth cycle of microalgae is short, production equipment floor space is small, and product quality and yield are stablized relatively；Finally, (such as rain life is red for microalgae
Ball algae etc.) inherently a kind of product of high value, containing substantial amounts of protein, grease, polysaccharide isoreactivity composition, it can separate
These materials are extracted, the comprehensive utilization of microalgae cell is realized.
At present, the process of astaxanthin is produced according to microalgae, by taking haematococcus pluvialis as an example, is typically divided into microdisk electrode process
Two stages of microdisk electrode and astaxanthin accumulation.First stage (microdisk electrode) is substantially carried out the culture of haematococcus pluvialis, makes it
Fast-growth, the stage can be light autotrophy, heterotrophism and raise together with.Second stage (astaxanthin accumulation) be by such as bloom shine,
A series of coercions such as high temperature, high salt, nutrition salt-hunger, promote haematococcus pluvialis to be changed into thickness under severe living environment
Wall spore, to reach the purpose of accumulation astaxanthin.
In the two stages, the nutrition and environmental condition needed for microalgae are different, and research is concentrated mainly on both at home and abroad at present
In terms of the condition selection and control and the influence of envirment factor in the two stages.Under normal circumstances, the first stage not with
Accumulate for the purpose of astaxanthin, but be to increase cell number and weight, now by using the condition of culture for comparing mitigation,
Such as：Dim light, less salt；Second stage is then transferred to, is now aided with the stress conditions such as strong light, high temperature, high salt and addition nitrogen phosphorus lacks
Culture medium, promote the accumulation of astaxanthin, this phase cell number is not further added by, increase sometimes with the severe degree of stress conditions
Plus, cell number declines, but due to cellular spore and expands, and cell weight is slowly increased, when coercing cultivation terminates with starting
Compare, the cell weight in unit volume nutrient solution increases by 2~4 times, reaches about 2~3g/L.Astaxanthin accumulation stage culture medium
Culture medium with the microdisk electrode stage is incomplete same, the latter N, P it is abundant and require reasonable mixture ratio between each element (required carbon, nitrogen,
Phosphorus, sulphur, the element such as sodium, calcium, potassium, magnesium), the former only needs a small number of salts substances such as addition calcium salt and general lack of nitrogen, phosphorus.
However, going to second stage (astaxanthin accumulation) from previous stage (microdisk electrode) has a subject matter, i.e.,：
Cell is already adapted to the mitigating circumstances of first stage, when going to second stage, and environment changes and harsh stress conditions, Yi Zao
It is difficult in adapt into cell, occurs situations such as death, damage.For example：When cell is cultivated in the first stage, cell is already adapted to dim light
Environment, when going to second stage, photosynthetical system is exposed under strong light, is highly prone to destruction, is caused cell death, Xanthophyll cycle etc. no
Beneficial to the phenomenon of astaxanthin accumulation.
Although there have been many improvement on this method this area, by the correlation to having applied both at home and abroad specially
Sharp analysis shows, these patents mostly concentrate on the bioreactor and device, Growth Medium For Haematococcus Pluvialis, astaxanthin of culture
New method for extracting etc..However, haematococcus pluvialis (Haematococcus pluvialis), chlorella (Chlorella
) etc. zofingiensis the patent in terms of Transition Technology of the astaxanthin microalgae between culture and stress can be produced not yet to retrieve
Arrive.
Therefore, it is badly in need of the new cultural method of research and development for this case, in this area, to overcome this defect, improves shrimp
Blue or green element yield.
Invention summary
In view of the above-mentioned problems, this application provides a kind of effective solution.To overcome two-stage incubation from thin
Intracellular growth condition is unfavorable for the defect of production of astaxanthin when changing astaxanthin accumulation condition, the present invention devises a kind of regulation rain life
The method that haematococcus cell state improves astaxanthin yield, this method has three kinds of modes：(1) high-density cells obtained will be cultivated
Directly strong photoinduction, then returns again to normal second stage induction；(2) it is the high-density cells dilution for cultivating acquisition is laggard
Row dim light is induced, and then returns again to normal second stage induction；(3) high-density cells that obtain will be cultivated and goes to normal the
Two-stage induces, but by way of shading (including with transparent material or opaque material shading) and optimization inoculation time
(light intensity weaker time point is inoculated with i.e. in one day) makes cell adapted stressful environmental, then cancels shading.
The method of the present invention has following advantage：
(1) astaxanthin yield of frustule is improved；
(2) means are various, can select most convenient mode according to actual conditions；
(3) reactor that Transition Technology can be induced directly using first stage or second stage, it is not necessary to extra increase
Equipment.
According to the one side of the application, this application provides avoid Xanthophyll cycle during a kind of microdisk electrode to improve shrimp
The new method of blue or green element yield, methods described includes by heterotrophism, light autotrophy or raises together with cultural method acquisition microalgae cell；Adjustment is micro-
Frustule state；Photoinduction or the culture of light autotrophy are carried out to accumulate astaxanthin with the microalgae cell after cell state will be adjusted.
According to some embodiments of the application, the adjustment microalgae cell state is by controlling condition of culture come real
It is existing.
According to some embodiments of the application, the adjustment microalgae cell state is will to cultivate the high-density cells obtained
Directly strong photoinduction, then returns again to normal second stage induction；It will be carried out after the high-density cells dilution for cultivating acquisition weak
Photoinduction, then returns again to normal second stage induction；Or the high-density cells for cultivating acquisition are gone to normal second
Stage induces, but by way of shading and time of optimization inoculation makes cell adapted stressful environmental, then cancels shading, wherein
The mode of the shading includes using transparent material or opaque material shading；And the time of the optimization inoculation referred at one day
Interior light intensity weaker time point is inoculated with, and wherein high-density cells refer to that cell density is 0.5-5g/L, and light intensity is weaker to be referred to
Light intensity is less than 10klux.
According to some embodiments of the application, the control condition of culture includes：Adjust the culture medium of microalgae cell, control
The pH of culture medium processed is between 4-10, and carbon source concentration (selected from sodium acetate etc.) is 0-60mM, nitrogen concentration (selected from sodium nitrate etc.)
It is that 0-100mM and/or phosphorus concentration (selected from phosphoglycerol disodium etc.) be 0-10mM, and controls temperature for 5~50 DEG C；It is thin to microalgae
Born of the same parents carry out illumination：Intensity of illumination can be gradually increased with adjustment process, and scope is between 0~200klx, and preferably described illumination is company
Continuous or intermittent illumination, the light source of preferably described illumination can be natural light or artificial light, light quality can for feux rouges, blue light, gold-tinted or
White light；Control the mode of light intensity：During using artificial light source, change the intensity of light of artificial light source；Using the DT, it can pass through
Curtain, puggaree, plastic sheeting block reactor；The density of regulating cell, for example, cause cell density in 0.1g/L-10g/
Between L；Or optimization inoculation time, for example fine day control inoculation time in the afternoon 3 points between at 12 points in evening.
According to some embodiments of the application, the incubation time of the adjustment microalgae cell state is small for 0.1~300
When, at the end of preferably described microalgae state adjustment, the concentration of the nutritional ingredient such as carbon, nitrogen and/or phosphorus in control culture medium is relatively low
Even zero, i.e., less than 10mM.
According to some embodiments of the application, described microalgae, which is selected from, to be given birth to the microalgae of synthesizing astaxanthin, such as rain
Haematococcus (Haematococcus pluvialis), chlorella (Chlorella zofingiensis).
According to some embodiments of the application, the adjustment microalgae cell state includes：Adjust the size of cell：For example
Each cell is reduced between 0.1ng to 50ng between each cell 0.01ng to 5ng；The rise of chlorophyll：For example leaf is green
The weight/mass percentage composition of element is increased between 0.1% to 5% between 0.04% to 2%；Adjust the form of cell：Motor cell
It is as many as possible, 10% can be more than, maximum can reach 100%；Or increase cell quantity and/or increase dry cell weight.
According to some embodiments of the application, it is described by heterotrophism, light autotrophy or raise together with cultural method obtain microalgae it is thin
During born of the same parents, the microalgae cell can be diluted directly or after first being concentrated or first and be used to adjust microalgae cell state afterwards.
According to some embodiments of the application, the micro algae culturing liquid obtained after the adjustment microalgae cell state can be with
Directly carry out photoinduction or culture, added again after can also diluting the culture medium progress photoinduction that is used required for normal induction or
Culture.
According to some embodiments of the application, the adjustment microalgae cell state is in shaking flask, stirring-type or gas-lifting type
Or bubbling fermentation tank, raceway pond, circle pond, flat plate photobioreactor, duct type bioreactor, pillar photo-biological are anti-
Answer device, film to stand in any device available for microdisk electrode such as bag and Pig to carry out, or microalgae cell is coated in solid
The adherent method of the semisolids such as film surface is cultivated, and the photoinduction carried out after preferably described adjustment microalgae cell state or light
Culture, the culture device that can more renew can also be carried out in same culture device.
According to the one side of the application, this application provides a kind of culture medium for adjusting microalgae cell state, the training
Foster base contains nitrogen source, organic carbon source (selected from sodium acetate etc.), inorganic carbon source (selected from carbon dioxide etc.), auxin, nothing
Machine salt, trace element and water, or be made up of auxin, inorganic salts, trace element and water.
According to some embodiments of the application, the cell state adjustment culture medium is used for can be with the micro- of synthesizing astaxanthin
The state adjustment of frustule.
In summary, supplement and perfect, further raising of the present invention for traditional microalgae production of astaxanthin two-stage method
The efficiency of production of astaxanthin, important technology hand is provided for the extensive Industrialization that solves to come from microalgae astaxanthin
Section.
Brief description of the drawings
Fig. 1 shows the process that haematococcus pluvialis first stage cell is obtained by heterotrophism mode；
Fig. 2 shown after transition incubation of the haematococcus pluvialis in 1L bioreactors, transition 144h, and cell is from thickness
Wall spore is changed into green cell, but single celled dry weight is reduced, and is shown as cell and is diminished；
Fig. 3 shows that haematococcus pluvialis (pass through Transition Technology of the present invention in 1L bioreactor Fiber differentiations process containing cell
Cell induction, the cell induction data without Transition Technology compare afterwards)；After photoinduction culture 10 days, by the cell of Transition Technology
Dry weight reaches 1.4g/L, and astaxanthin rises to 4.6%, and astaxanthin yield reaches 64mg/L, considerably beyond without Transition Technology
And the cell directly induced；With
Fig. 4 shows the concrete condition of cell photosynthetical system after transition under different light intensity and nitrogen source.
Detailed description of the invention
Unless otherwise defined, all technical terms or proprietary vocabulary used herein have the common of technical field
The implication that technical staff is generally understood.
The survival rate of cell when the method for the present invention can improve photoinduction, promotes the Rapid Accumulation of intracellular astaxanthin,
Production efficiency is significantly improved, production cost is reduced, and there is provided the astaxanthin of high-quality.
The method of the present invention includes by heterotrophism, light autotrophy or raises together with the technique that cultural method obtains microalgae cell；Adjustment
The Transition Technology of microalgae cell state；Photoinduction or light autotrophy culture are carried out with product with the microalgae cell after cell state will be adjusted
The technique of tired astaxanthin.
According to some embodiments of the application, the technique for obtaining microalgae cell be using the heterotrophism of microalgae, light autotrophy or
The cultural method such as raise together with.According to some embodiments of the present invention, it is existing to obtain the condition of culture and culture medium of microalgae cell
Known condition of culture and culture medium in technology.
The method that haematococcus pluvialis cell state improves astaxanthin yield is adjusted this application provides a kind of.This method has three
The mode of kind：(1) the directly strong photoinduction of high-density cells obtained will be cultivated, then returns again to normal second stage induction；(2)
Dim light induction will be carried out after the high-density cells dilution for cultivating acquisition, then return again to normal second stage induction；Or (3)
The high-density cells that obtain will be cultivated and go to the induction of normal second stage, but by way of shading (including with transparent material
Or opaque material shading) make cell adapted stressful environmental, then cancel shading.
According to some embodiments of the application, high-density cells refer to that cell density is 0.3-5g/L, such as 0.3-4g/
L、0.3-3g/L、0.3-2g/L、0.3-1g/L、0.3-0.5g/L、0.5-5g/L、0.5-4g/L、0.5-3g/L、0.5-2g/L、
0.5-1g/L, 1-5g/L, 1-4g/L, 1-3g/L, 1-2g/L, 2-5g/L, 2-4g/L, 2-3g/L, 3-5g/L, 3-4g/L or 4-
5g/L。
According to some embodiments of the application, the cell density of normal second stage induction is 0.01-5g/L, for example
0.01-3g/L、0.01-1g/L、0.01-0.5g/L、0.01-0.1g/L、0.1-5g/L、0.1-3g/L、0.1-1g/L、0.1-
0.5g/L, 0.5-5g/L, 0.5-3g/L, 0.5-1g/L, 1-5g/L, 1-3g/L or 3-5g/L.
According to some embodiments of the application, the adjustment microalgae cell state is by controlling condition of culture come real
It is existing.
According to some embodiments of the application, the control condition of culture includes the culture of adjustment microalgae cell
Base；Illumination is carried out to microalgae cell；Control light intensity or the density of regulating cell.
According to some embodiments of the application, the adjustment microalgae cell state is by optimizing the time of inoculation come real
It is existing.According to some embodiments of the application, the time of the optimization inoculation is to carry out at the time point that light intensity is weaker in one day
Inoculation, for example fine day control inoculation time in the afternoon 3 points between at 12 points in evening, such as at 5 points in afternoon at 12 points in evening or under
7 points of noon at 12 points in evening.
According to some embodiments of the application, the light intensity is weaker to refer to that light intensity is less than 10klux, such as less than
8klux, less than 5klux, less than 3klux, less than 1klux, less than 0.5klux or less than 0.1klux.
According to some embodiments of the application, the culture medium of the adjustment microalgae cell includes controlling the pH of culture medium to exist
Between 4-10, carbon source concentration (selected from sodium acetate etc.) be 0-60mM, nitrogen concentration (being selected from sodium nitrate etc.) be 0-100mM and/or
Phosphorus concentration (selected from phosphoglycerol disodium etc.) is 0-10mM, and controls temperature to be 5~50 DEG C, is preferably also existed including magnesium density
In the range of 0.00001-0.001mM.
In the method for the invention, in the Transition Technology of adjustment microalgae cell state, by controlling pH constant and carbon, nitrogen
The dense of the nutritional ingredients such as carbon, nitrogen and/or phosphorus is controlled and/or the elemental stable such as phosphorus is in the range of finite concentration, and at the end of transition
Degree relatively low even zero, i.e., in below 10mM, be, for example, less than 9mM, 8mM, 7mM, 6mM, 5mM, 4mM, 3mM, 29mM, 1mM,
0.5mM or 0.1mM.
In the Transition Technology for adjusting microalgae cell state, by elemental stables such as control of additive raw material carbon, nitrogen and/or phosphorus certain
In concentration range.For example, can be controlled the content of carbon in algae solution in the range of 0-60mM by feed supplement, the content control of nitrogen exists
In the range of 0-100mM, the content of phosphorus is controlled in the range of 0-10mM.
According to some embodiments of the application, in the Transition Technology for adjusting microalgae cell state, by feed supplement by algae solution
Middle carbon source concentration control is in the range of 0-60mM, such as 0-50mM, 0-40mM, 0-30mM, 0-20mM, 0-10mM, 10-
60mM、10-50mM、10-40mM、10-30mM、10-20mM、20-60mM、20-50mM、20-40mM、20-30mM、30-50mM、
30-40mM or 40-50mM, preferably 0-40mM.According to some embodiments of the application, the transition of microalgae cell state is adjusted
In technique, nitrogen concentration is controlled in the range of 0-100mM by feed supplement, such as 0-8mM, 0-5mM, 0-3mM, 0-1mM, 0-
0.5mM、0.5-10mM、0.5-8mM、0.5-5mM、0.5-3mM、0.5-1mM、1-10mM、1-8mM、1-5mM、1-3mM、3-
10mM, 3-8mM, 3-5mM, 5-10mM, 5-8mM or 8-10mM, preferably 0.5-50mM.According to some embodiments of the application,
In the Transition Technology for adjusting microalgae cell state, the concentration of phosphorus is controlled in the range of 0-10mM by feed supplement, such as 0-
0.5mM、0-0.1mM、0-0.5mM、0-0.01mM、0.01-1mM、0.01-0.5mM、0.01-0.1mM、0.1-1mM、0.1-
0.5mM or 0.5-1mM, preferably 0-5mM.
In an embodiment, by control of additive raw material carbon, three kinds of elemental stables of nitrogen and phosphorus in the range of finite concentration.
For example, can be controlled the content of carbon in algae solution in the range of 0.5-60mM by feed supplement, the content of nitrogen is controlled in 0.5-50mM
In the range of, the content of phosphorus is controlled in the range of 0.01-5mM.
In a detailed embodiment, in addition to by feed supplement the content of magnesium in algae solution is controlled in 0.00001-
In the range of 0.001mM, such as 0.00001-0.0005mM, 0.00001-0.0001mM, 0.00001-0.00005mM,
0.00005-0.001mM、0.00005-0.0005mM、0.00005-0.0001mM、0.0001-0.001mM、0.0001-
0.0005mM or 0.0005-0.001mM.
In a detailed embodiment, described microalgae is selected from haematococcus pluvialis (Haematococcus
Pluvialis), chlorella (Chlorella zofingiensis) etc..
In a detailed embodiment, the process of the adjustment microalgae cell state includes：Connect in bioreactor
Enter microalgae algae solution, and add culture medium controls pH to be 5.0-10.0 while being intermittently passed through carbon dioxide, and cultivation temperature is 10-40
DEG C, control pH is less than 10.0, and control dissolved oxygen is more than 0.1%.
In one embodiment, during adjustment microalgae cell state, by being passed through carbon dioxide by the pH of algae solution
Control a steady state value in the range of 5.0-10.0, such as pH value be 5.0-9.0,5.0-8.0,5.0-7.0,5.0-6.0,
6.0-10.0,6.0-9.0,6.0-8.0,6.0-7.0,7.0-10.0,7.0-9.0,7.0-8.0,8.0-10.0,8.0-9.0 or
9.0-10.0；Such as pH can be 7.0,7.1,7.2,7.3,7.4,7.5,7.6,7.7,7.8,7.9,8.0, preferably 7.5.
It should be understood that a little variation of pH value is allowed.For example, pH can be allowed to have ± Y variation, wherein Y≤1.0, example
Such as Y≤0.2, Y≤0.1.In certain embodiments, Y=0.Therefore, in a specific embodiment, it is X by the pH controls of algae solution
± Y, wherein, the 7.0≤X ± Y≤9.0.For example, in an embodiment of the invention, by being passed through carbon dioxide by algae
The pH of liquid is controlled within the scope of 8.0 ± 0.3.
In one embodiment, in the Transition Technology of adjustment microalgae cell state, cultivation temperature is 5~50 DEG C, such as 5
~40 DEG C, 10~50 DEG C；It is preferred that 10~40 DEG C, such as 10~30 DEG C, 10~20 DEG C, 20~40 DEG C, 20~30 DEG C or 30~40
℃。
In a detailed embodiment, dilution of the microalgae Transition Technology to microalgae high concentration algae solution is to use transition culture
Base by the algae solution that heterotrophism/light autotrophy is obtained be diluted to cell density be 0.1-20 g/l, pH be 5.0-9.0.
In a detailed embodiment, the Transition Technology includes being transferred in photoinduction device by the algae solution after dilution
Row dim light transition, continuous illumination or intermittent illumination, cultivation temperature are 5~50 DEG C, and intensity of illumination is 0.1~50klx, transient period
For 1~168 hour.
In a detailed embodiment, Transition Technology add adjustment microalgae cell state culture medium contain nitrogen source,
Organic carbon source, a small amount of inorganic salts, auxin, trace element and water are made up of these compositions.
In a detailed embodiment, when the algae kind for producing astaxanthin is haematococcus pluvialis, used in Transition Technology
The culture medium of adjustment microalgae cell state is substantially consisted of the following composition：0.1~5.0 g/l of sodium acetate, NaNO30.05~
1.5 g/l of CaCl2·7H20.05~1.5 g/l of O, KH2PO40.01~1.5 g/l, MgSO4·7H2O 0.01~1.0
G/l, FeSO4·7H20.01~0.05 g/l of O, auxin 0.001-35 mg/litres, the milli of trace element 0.5~4
Rise and water.
In a specific embodiment, the Transition Technology can be in shaking flask, mechanical agitation type, gas-lifting type or bubble type
Carried out in bioreactor, can also open type raceway pond or circle pond, enclosed flat plate photobioreactor or pipe
It is any available for micro- with Pig bioreactor etc. that road formula bioreactor or pillar bioreactor or film found bag
Carried out in the device of algae light autotrophy culture, light source is natural light or various artificial lights.
According to some embodiments of the application, the adjustment microalgae cell state includes the size of adjustment cell, Ye Lv
The rise of element, adjusts the form or increase cell quantity and/or increase dry cell weight of cell.According to some implementations of the application
Mode, the size of the adjustment cell is each cell is reduced to each cell 0.01ng to 5ng between 0.1ng to 50ng
Between, such as each cell is reduced between 1ng to 25ng between each cell 0.05ng to 3ng, each cell from 5ng to
It is reduced between 10ng between each cell 0.1ng to 1ng, each cell is reduced to each cell between 5ng to 10ng
Between 0.5ng to 1ng.
According to some embodiments of the application, the rise of the chlorophyll be make the weight/mass percentage composition of chlorophyll from
It is increased between 0.04% to 2% between 0.1% to 5%, for example weight/mass percentage composition is increased between 0.06% to 1.0%
Between 0.6% to 2% or weight/mass percentage composition is increased between 0.8% to 2% between 0.08% to 1.2%.
According to some embodiments of the application, methods known in the art can be used to detect the content of chlorophyll.
According to some embodiments of the application, the form of the adjustment cell is to make motor cell as many as possible, can be more than 10%, example
Such as larger than 10%, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80% or
More than 90%, maximum can reach 100%.
According to some embodiments of the application, the micro algae culturing liquid obtained after the adjustment microalgae cell state can be with
Directly carry out photoinduction or culture.According to some embodiments of the application, what is obtained after the adjustment microalgae cell state is micro-
Algae culturing liquid adds the culture medium used required for normal induction again after can diluting, carry out photoinduction or culture.
According to some embodiments of the application, the adjustment microalgae cell state is in shaking flask, stirring-type or gas-lifting type
Or bubbling fermentation tank, raceway pond, circle pond, flat plate photobioreactor, duct type bioreactor, pillar photo-biological are anti-
Answer device, film to stand in any device available for microdisk electrode such as bag and Pig to carry out, or microalgae cell is coated in solid
The adherent method of the semisolids such as film surface is cultivated.According to some embodiments of the application, the adjustment microalgae cell state
The photoinduction carried out afterwards or optical culture, the culture device that can more renew.According to some embodiments of the application, the tune
The photoinduction carried out after whole microalgae cell state or optical culture, can be carried out in same culture device.
Microalgae suitable for the application can be with the microalgae of synthesizing astaxanthin, including but not limited to haematococcus pluvialis including those
(Haematococcus pluvialis), chlorella (Chlorella zofingiensis) etc..Preferred embodiment
In, the present invention produces astaxanthin using haematococcus pluvialis (Haematococcus pluvialis).
Nitrogen source, organic/inorganic carbon source, plant can generally be contained by adjusting the culture medium of the Transition Technology of microalgae cell state
Growth hormone, a small amount of inorganic salts, trace element and water.
This kind of culture medium includes C culture mediums (Ichimura, T.1971Sexual cell division and
conjugation-papilla formation in sexual reproduction of Closterium
Strigosum.In Proceedings of the Seventh International Seaweed Symposium,
University of Tokyo Press, Tokyo, p.208-214.), MCM culture mediums (Borowitzka et al.,
1991), BG-11 culture mediums (Boussiba and Vonshak, 1991), BBM culture mediums (Nichols and Bold,
1969), BAR culture mediums (Barbera et al., 1993) .KM culture mediums (Kobayashi et al., 1991), Z8 culture mediums
(Renstrom et al., 1981), A9 culture mediums (Lee and Pirt, 1981), OHM culture mediums (Fa ' bregas et
Al., 2000), KM1 culture mediums (Usha et al.1999) (Garc ' 1a-Malea et al., 2005), HK2 culture mediums
(Chen et al., 1997), HK3 culture mediums (Gong and Chen, 1998) etc..
C culture mediums used in the present invention are substantially by nitrate, sodium acetate and a small amount of inorganic salts, trace element and water group
Into with the addition of some auxins on this basis.
Term used herein " substantially by ... constitute " is represented in the composition of the present invention except containing key component
Outside nitrate, sodium acetate and a small amount of inorganic salts, trace element and water, some fundamental characteristics for composition can be also included
Or new characteristic (can maintain microalgae to reach higher level in shorter cultivation cycle inner cell density, while active material
Content has a more substantial increase compared with cellar culture) component that does not influence substantially.Term used herein " by ... group
Into " represent that the composition of the present invention is made up of pointed concrete component, without other components, but can be with content logical
Impurity in normal scope.
In the culture medium, each component of culture medium can change without to microalgae cell density and product within the specific limits
Matter has very big materially affect.Therefore, the consumption of these components should not by embodiment strict limitation.Such as those skilled in the art
Known, a small amount of inorganic salts can be also added in culture medium, such as magnesium sulfate, calcium chloride, ferrous sulfate and phosphate, and
A small amount of trace element such as Mn, Zn, B, I, M, Cu, Co, and auxin addition, including single hormone or a variety of
The combination of hormone.The consumption of inorganic salts and trace element can be determined according to Conventional wisdom.
In a specific embodiment, the high density algae solution obtained was cultivated the two-step method first stage (in open type
When reactor is induced, preferably without organic carbon source, the photoinduction stage can be so avoided to grow excessive miscellaneous bacteria；But in closed photo
When thing reactor is induced, organic carbon source can be contained, promote cell concentration increase) operation should be diluted, with transitional culture medium to highly dense
The algae solution of degree is diluted, and cell density is maintained 0.1~20 g/l, pH is 4.0~10.0.In some embodiments,
Highdensity algae solution is diluted with the culture medium without organic carbon source with water, make cell density maintain 0.1~10 gram/
Rise, adjust pH to 5.0~8.0.In other embodiments, algae solution is diluted, cell density is maintained 1~8 g/l, regulation
PH to 5.0~8.0.In a preferred embodiment, cell density is maintained 1.0~5.0 g/l, be passed through CO2And adjust pH
To 5.0~8.0, incubation can also continue to be passed through CO2To control pH 5.0~8.0.
In an embodiment, the transitional culture medium of adjustment microalgae cell state contains：MgCl2·7H2O0.01~
0.1 g/l, 0.1~1 g/l of KCl, CaCl20.01~0.2 g/l, FeSO4·7H20.01~0.06 g/l of O, EDTA
0.020~0.052 g/l and auxin 0.001-35 mg/litres.
In an embodiment, the auxin in transitional culture medium contains：2,4 dichlorophenoxyacetic acid
0.001-5 mg/litres, benayl aminopurine 0.001-5 mg/litres, Exogenous gibberellic acid 0.001-5 mg/litres, 3- indolebutyric acids
0.001-5 mg/litres, methyl α-naphthyl acetate 0.001-5 mg/litres and/or brassin 0.001-5 mg/litres.
In an embodiment, the auxin in transitional culture medium contains：Benayl aminopurine 0.001-5 millis
G/l and 3- indolebutyric acid 0.001-5 mg/litres.
The culture medium used is diluted without autoclaving, and pH to 5.0~9.0 is adjusted after preparing and be can be used.
It should be understood that in some embodiments, it is not necessary to which the first stage culture gained frustule progress to two-step method is dilute
Release, and directly implement strong light transition to it, this depends on cell density, nutrient solution composition and actual transition condition (such as light intensity, temperature
Degree, whether using shading measure etc.) between Proper Match.
The purpose of the Transition Technology of adjustment microalgae cell state described herein is to allow production astaxanthin microalgae entering the side of body
Appropriate adjustment is carried out before the condition of compeling to its cell more to adapt to the stress conditions of photoinduction, it is carried out after photoinduction,
Frustule rapid, high volume dynamic accumulation astaxanthin, while properly increasing the frustule concentration in nutrient solution.
According to some embodiments of the present invention, the control condition of culture includes carrying out illumination to microalgae cell.According to
Some embodiments of the present invention, intensity of illumination can be gradually increased with adjustment process, scope between 0~200klx, such as 0~
150klx, 0~100klx, 0~50klx, 0.1~10klx, 0.1~5klx, 0.1~1klx, 1~200klx, 1~150klx,
1~100klx, 1~50klx, 1~10klx, 1~5klx, 5~200klx, 5~150klx, 5~100klx, 5~50klx, 5
~10klx, 10~200klx, 10~150klx, 10~100klx, 10~50klx, 50~200klx, 50~150klx, 50~
100klx, 100~200klx, 100~150klx or 150~200klx.
According to some embodiments of the present invention, the illumination is accomplished continuously or intermittently illumination.According to some realities of the present invention
Mode is applied, the light source of the illumination can be natural light or artificial light.According to some embodiments of the present invention, the illumination
Light quality can be feux rouges, blue light, gold-tinted or white light.
According to some embodiments of the present invention, the mode for controlling light intensity is when using artificial light source, to change people
The intensity of light of work light source；Using the DT, reactor can be blocked by curtain, puggaree, plastic sheeting.
According to some embodiments of the present invention, the control condition of culture includes the density of regulating cell, for example, cause
Cell density is between 0.1g/L-10g/L, such as 0.1g/L-8g/L, 0.1g/L-5g/L, 0.1g/L-3g/L, 0.1g/L-1g/
L、1g/L-10g/L、1g/L-8g/L、1g/L-5g/L、1g/L-3g/L、3g/L-10g/L、3g/L-8g/L、3g/L-5g/L、5g/
L-10g/L, 5g/L-8g/L or 8g/L-10g/L.
When generally carrying out Transition Technology, temperature control is at 5~50 DEG C, and intensity of illumination is 0.1~50klx (low cell densities
During dim light transition) or 50klx-200klx when shading transition (during the strong light transition of high-cell density and), continuous illumination or interval
Illumination, cultivation cycle is 0.1~300 hour, and throughput is 0.1~10.0vvm.Wherein described reactor includes all envelopes
Enclosed bioreactor (shaking flask, duct type, flat, pillar, film found bag and Pig etc.) and all open biologies are anti-
Answer device (raceway pond, circle pond and bubble type big basin etc.).
Generally, cultivation temperature is can be controlled in the range of 15~35 DEG C, such as 18~35 DEG C, 20~35 DEG C, 20~30 DEG C
Deng.Generally, intensity of illumination is 0~200klx, for example, 0~30,30~40,0~40,1~30,1~20,1~10klx etc., can
Depending on the specific condition of production.Generally, such as cause algae solution to be sufficiently mixed by gas, then throughput be controllable to 0.1~
2.0vvm, for example, 0.2~1.8,0.5~1.5,0.8~1.5,1.0~1.5vvm etc..Meanwhile, it is passed through certain density CO2With
Inorganic carbon source and control pH are provided, for example, 0.5%-10% CO2.In other embodiments, cultivation temperature control 10~
50 DEG C, intensity of illumination is 1~10klx, and throughput is 0.05~2.0vvm.
In other embodiments, the transient period is 0.1~300 hour, for example, according to actual weather condition, culture
Cycle can for 0.1~250 hour, 0.1~200 hour, 0.1~100 hour, 0.5~50 hour, 50~150 hours, 150
~300 hours, 100~300 hours, any duration in the range of 0.5~8 hour, and 0.5~300 hour.
In this application, " transient period " includes whole transition incubation to light, for example, transient period during outdoor culture
There is no the time of illumination including night.
In this application, " incubation time " refer to using microalgae is implemented under herein described intensity of illumination transition culture when
Between.Due to the micro- 0-200Lux of herein described intensity of illumination, the i.e. time include night there is no illumination (i.e.：Intensity of illumination is
Time 0Lux).The time is any duration in the range of 0.1~300 hour.
The illumination that the transition incubation step of the application needs can carry out photoinduction culture by the way of artificial lighting, also may be used
Out of doors transition is carried out using the mode of natural lighting.
In a specific embodiment, for the microalgae cell of first stage, treat that cell photosynthetical system is repaiied in nutrient solution
After multiple, Transition Technology can be terminated, into photoinduction culture, the cell photosynthetical system reparation include occurring one of situations below or
Combination：The rise of cell chlorophyll, cellular colours greening, photosynthetical system II efficiency are improved.
In a specific embodiment, for the microalgae cell of first stage, treat that the appearance of cell intracellular is bright in nutrient solution
Aobvious astaxanthin particle, can also terminate Transition Technology, into photoinduction culture.It is individual in a specific embodiment, by microscopy or
There is astaxanthin particle to observe cell intracellular in this area other method.
In this application when " about " is used to modify numerical value, refer to the numerical value can fluctuate ± 10%, ± 9%,
± 8%, in the range of ± 7%, ± 6%, ± 5%, ± 4%, ± 3%, ± 2% or ± 1%.
Unless be otherwise noted in this application or otherwise clearly contradicted, in the context of description the application (including
In the context of claim) term " one kind " that uses, " one ", " described ", "the" and " at least one " and similar refer to
In generation, is interpreted covering odd number and plural number.Unless be otherwise noted in this application or otherwise clearly contradicted, it is herein described
All methods can in any suitable order be carried out according to the understanding of those skilled in the art.
The relevant content of the present invention will be further described by embodiment below.It should be understood that " containing in the application
By ", "comprising" also include " by ... constitute ", " by ... constitute " implication.
All patents, patent application and the bibliography quoted in the application are incorporated by this Shen by reference
Please, its incorporated extent is individually recited as reference just as each document.If the application and provided herein is document between
In the presence of conflict, content that should be in the application is defined.
This application describes preferred embodiment and embodiment, those skilled in the art are reading the basis of the application
On, appropriate change can be carried out to embodiment described herein and embodiment.Therefore, the application, which includes law, allows model
Enclose all equivalent modifications and variations of theme in interior claims to the application.
Embodiment
The present invention is described in detail below with reference to embodiment and accompanying drawing.It will be understood by those skilled in the art that,
Following embodiments are for illustrative purposes, to should not be construed as in any way limitation of the present invention.The guarantor of the present invention
Shield scope is limited by appended claims.
The Transition Technology of embodiment 1. causes the state of microalgae cell to change
As shown in figure 1, when cultivating in the first stage, obtaining highdensity haematococcus pluvialis by the method for Heterotrophic culture thin
Born of the same parents.Transitional culture medium and water are added in 1L bioreactors to steam sterilizing is carried out after 1L, then when temperature drops to 25 DEG C
The cell that the haematococcus pluvialis heterotrophism of 12ml high density (26g/L) is obtained is accessed, starts transition culture, temperature maintains 28~38
DEG C, air mass flow is 1vvm, artificial illumination in 24 hours, is about 50klx by force per sidelight.
In culture, that is, start batch (-type) and be passed through 1% carbon dioxide, pH is constant in 8-9.Microalgae is promoted to start to adapt to the side of body
Compel environment.After 144h, cell is changed into green cell from akinete, and Transition Technology terminates.
Fig. 2 shows transition incubation of the haematococcus pluvialis in 1L bioreactors, and (process used is preferably
Transition Technology), after transition 144h, cell is changed into green cell from akinete, but single celled dry weight is reduced, and shows as thin
Born of the same parents diminish.As seen from Figure 1, in Transition Technology culture, the state of microalgae cell is changed.
Embodiment 2. causes dry cell weight increase, astaxanthin yield increase via the cultural method of Transition Technology
After the present embodiment cultivates microalgae cell in the first stage, by the Transition Technology of the present invention, then carry out cell and lure
Lead, directly carrying out cell induction with the Transition Technology without the present invention compares.
Fig. 3 is shown via transition and without the haematococcus pluvialis cell of Transition Technology, is induced and is trained in 1L bioreactors
The contrast of 10 days culture situations in supporting.As seen from Figure 3 after photoinduction culture 10 days, reached by the dry cell weight of Transition Technology
To 1.4g/L, astaxanthin rises to 4.6%, and astaxanthin yield reaches 64mg/L.And without the haematococcus pluvialis of Transition Technology
After cell photoinduction culture 10 days, 0.3g/L is only reached by the dry cell weight of Transition Technology, astaxanthin rises to 0.8%, shrimp
Blue or green element yield reaches 2.4mg/L.
Therefore as seen from Figure 3, the Transition Technology of patent of the present invention so that dry cell weight increase, astaxanthin yield increase
Plus, considerably beyond the mode directly induced without Transition Technology.
Change of the cell that embodiment 3. is handled through Transition Technology in intracellular photosynthetical system
Fig. 4 show the cell handled through Transition Technology in terms of intracellular photosynthetical system with the difference compareed, be respectively everywhere
The difference (figure A) of the intracellular photosynthetical system II of reason electron transmission efficiency (ETR), the difference (figure of non-photochemical quenching (NPQ)
B), photosynthetical system II photosynthetic efficiency Y (II) (figure C) and other non-photochemistry energy (Y (NO) schemes D) differences.Result above table
Bright, by Transition Technology, the photosynthetical system II of intracellular efficiency is improved, especially transition when pass through the increasing of nitrogen
Plus, photosynthetical system is significantly recovered.Non- photochemical quenching part is relatively small, show the ability of the strong light of cell tolerance it is relatively strong and
Because photooxidation is preferably minimized to the damage that cell is caused.
Present embodiments are exemplarily illustrated above in association with accompanying drawing.Those skilled in the art are according to this specification
Disclosure is readily apparent that, each embodiment suitably can be adjusted and reconfigured according to actual needs,
Without departing from spirit herein.The protection domain of the application is defined by following claims.
Claims (12)
1. avoid Xanthophyll cycle during a kind of microdisk electrode to improve the new method of astaxanthin yield, it is characterised in that the side
Method includes：
By heterotrophism, light autotrophy or raise together with cultural method obtain microalgae cell；
Adjust microalgae cell state；With
The microalgae cell after cell state will be adjusted to carry out photoinduction or the culture of light autotrophy to accumulate astaxanthin.
2. the method as described in claim 1, it is characterised in that the adjustment microalgae cell state is by controlling condition of culture
To realize.
3. method as claimed in claim 2, it is characterised in that the adjustment microalgae cell state is will to cultivate the highly dense of acquisition
The directly strong photoinduction of cell is spent, normal second stage induction is then returned again to；By after the high-density cells dilution for cultivating acquisition
Dim light induction is carried out, normal second stage induction is then returned again to；Or go to the high-density cells for cultivating acquisition normally
Second stage induction, but by way of shading and time of optimization inoculation makes cell adapted stressful environmental, then cancel screening
Light, wherein the mode of the shading includes using transparent material or opaque material shading；The time of the optimization inoculation refers to
Light intensity weaker time point is inoculated with one day, and wherein high-density cells refer to that cell density is 0.3-5g/L, and light intensity is weaker
Refer to that light intensity is less than 10klux.
4. method as claimed in claim 2, it is characterised in that the control condition of culture includes：
The culture medium of microalgae cell is adjusted, the pH of culture medium is controlled between 4-10, carbon source concentration (selected from sodium acetate etc.) is 0-
60mM, nitrogen concentration (selected from sodium nitrate etc.) are 0-100mM and/or phosphorus concentration (selected from phosphoglycerol disodium etc.) is 0-10mM,
And control temperature to be 5~50 DEG C；
Illumination is carried out to microalgae cell：Intensity of illumination can be gradually increased with adjustment process, and scope is between 0~200klx, preferably
The illumination is accomplished continuously or intermittently illumination, and the light source of preferably described illumination can be natural light or artificial light, light quality can for feux rouges,
Blue light, gold-tinted or white light；
Control the mode of light intensity：During using artificial light source, change the intensity of light of artificial light source；Using the DT, it can pass through
Curtain, puggaree, plastic sheeting block reactor；
The density of regulating cell, for example, cause cell density between 0.1g/L-10g/L；Or
Optimize inoculation time, for example fine day control inoculation time in the afternoon 3 points between at 12 points in evening.
5. method as claimed in claim 2, it is characterised in that the incubation time of the adjustment microalgae cell state is 0.1~
300 hours, at the end of preferably described microalgae state adjustment, the concentration of the nutritional ingredient such as carbon, nitrogen and/or phosphorus in control culture medium
Relatively low even zero, i.e., less than 10mM.
6. method as described in claim 1, it is characterised in that described microalgae be selected from can with the microalgae of synthesizing astaxanthin,
Such as haematococcus pluvialis (Haematococcus pluvialis), chlorella (Chlorella zofingiensis).
7. method as described in claim 1, it is characterised in that the adjustment microalgae cell state includes：
Adjust the size of cell：Such as each cell be reduced between 0.1ng to 50ng each cell 0.01ng to 5ng it
Between；
The rise of chlorophyll：The weight/mass percentage composition of such as chlorophyll be increased between 0.04% to 2% 0.1% to 5% it
Between；
Adjust the form of cell：Motor cell is as more as possible, can be more than 10%, maximum can reach 100%；Or
Increase cell quantity and/or increase dry cell weight.
8. method as described in claim 1, it is characterised in that described by heterotrophism, light autotrophy or to raise together with cultural method and obtain
Microalgae cell when, the microalgae cell can be diluted directly or after first being concentrated or first and be used to adjusting microalgae thin afterwards
Born of the same parents' state.
9. the method as described in claim 1, it is characterised in that the microdisk electrode obtained after the adjustment microalgae cell state
Liquid, can directly carry out photoinduction or culture, add the culture medium used required for normal induction after can also diluting again and carry out
Photoinduction or culture.
10. the method as any one of claim 1-2, it is characterised in that the adjustment microalgae cell state is to shake
Bottle, stirring-type or gas-lifting type or bubbling fermentation tank, raceway pond, circle pond, flat plate photobioreactor, duct type photo-biological are anti-
Answer device, pillar bioreactor, film to stand in any device available for microdisk electrode such as bag and Pig to carry out, or will be micro-
The adherent method that frustule is coated in the semisolids such as solid film surface is cultivated, and after preferably described adjustment microalgae cell state
The photoinduction of progress or optical culture, the culture device that can more renew can also be carried out in same culture device.
11. a kind of culture medium for adjusting microalgae cell state, it is characterised in that the culture medium contains nitrogen source, organic carbon source (choosing
From sodium acetate etc.), inorganic carbon source (selected from carbon dioxide etc.), auxin, inorganic salts, trace element and water, or by planting
Thing growth hormone, inorganic salts, trace element and water composition.
12. the culture medium of microalgae cell state is adjusted as described in claim 3, it is characterised in that the cell state is adjusted
Whole culture medium is used to adjust with the state of the microalgae cell of synthesizing astaxanthin.
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| CN109679853A (en) * | 2019-01-18 | 2019-04-26 | 昆明理工大学 | The method for improving haematococcus pluvialis biomass and astaxanthin yield using fulvic acid |
| CN114456941A (en) * | 2021-12-29 | 2022-05-10 | 扬州大学 | Method for reducing inhibition of microalgae on utilization of organic carbon source by light through light regulation and improving rate of microalgae on both long-term maintenance |
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