CN104745479A - Method for culturing haematococcus pluvialis - Google Patents
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Abstract
The invention relates to a method for culturing haematococcus pluvialis, which comprises the following steps: 1)under illumination open type culture condition, a first phase culture is carried out on haematococcus pluvialis in a nutrient solution to obtain haematococcus pluvialis containing swarm cells; 2)under illumination open type culture condition, a second phase culture is carried out on haematococcus pluvialis containing the swarm cells in a nutrient solution, at least parts of swarm cells can be converted to non motile cells; wherein antibiotic A and antibiotic B are added in the step 1) and step 2) in order; or antibiotic A is added in the step 1), antibiotic B is added in the step 2), and the antibiotic A and the antibiotic B are different. The method of the invention can obviously increase the content of astaxanthin in haematococcus pluvialis and output of haematococcus pluvialis.
Description
Technical field
The present invention relates to a kind of method of cultivating Haematocoocus Pluvialls.
Background technology
Astaxanthin (being also called astacin) is a kind of haematochrome, and its chemical structure is similar to β-carotene.Astaxanthin is the one of carotenoid.Research finds, astaxanthin has important physiological function, and its resistance of oxidation is comparatively strong, can the peroxidation of more effective prevention unsaturated fatty acids.In addition, astaxanthin also has very strong Tumor suppression growth, strengthens immunologic function, resists the multiple physiological functions such as uv damage.Thus, the fields such as food, medicine, healthcare products and makeup are widely used in.
At present, the astaxanthin on market mainly contains synthetic and natural astaxanthin two kinds.The astaxanthin of synthetic not only its tinting strength and biological value much lower (Bowen, etal., 1999) compared with natural astaxanthin, and be not allowed to be applied to the industries such as food.Natural astaxanthin is then because be more prone to be absorbed and do not relate to chemical technology and more welcome.The natural astaxanthin of present discovery mainly contains four kinds of sources: the waste of (1) Crustacean processing; (2) fungi, some fungi can synthesizing astaxanthin, as red phaffia rhodozyma, rhodothece rubra etc.(3) bacterium, the bacteriums such as lactic acid mycobacterium, tyrothricin (Yokoyama & Miki, 1995) can synthesizing astaxanthin.(4) micro-algae, Haematocoocus Pluvialls is the species that known natural astaxanthin content is the highest at present, and its accumulation volume about can reach 2% ~ 4% of dry cell weight, even higher, is described as the concentrate of natural astaxanthin and best biogenetic derivation.In addition, chlamydomonas, the green algas such as Chlorococcum, chlorella, grid algae also can accumulate a small amount of astaxanthin under hostile environment condition, but its content astaxanthin is far below the content astaxanthin of Haematocoocus Pluvialls, generally all below 0.5%.Therefore, Haematococcus pluvialis production natural astaxanthin is utilized to be up to the present feasible way.
At present, utilize the method for haematococcus pluvialis to produce astaxanthin varied, utilize complicated nutrient solution such as, in patent application CN1181184 openly, in bio-reactor, expand growth, be converted into astaxanthin, reach the object of quick, efficient Accumulation of Astaxanthin; Open in patent application CN1004536325, utilize suite of equipment, in the nutrient solution that the content of nitrogen and phosphorous is suitable, control Haematocoocus Pluvialls with LED light source and expand growth and accumulation astaxanthin.At present, the principal element hindering Haematocoocus Pluvialls scale operation is biological pollution, and in Haematocoocus Pluvialls culturing process, population density is low and yield per unit that is that cause is low, thus makes manufacturing cost higher.When carrying out small-scale cultivation Haematocoocus Pluvialls in the bio-reactor that duct type bioreactor or small-sized immobilization system form or its airlift bioreactor, the defect existed is that duct wall is easily covered by frustule and to cause between incubation period light transmission to decline and cleaning difficulty, although adopted dissimilar paddle wheel to be uniformly mixed, be only suitable for the bench-scale testing in laboratory.But Haematocoocus Pluvialls still exists specificity miscellaneous bacteria and easily pollutes when open large scale culturing, have a strong impact on product production and quality, restrict the defect of its application scale.If frustules a large amount of in the haematococcus pluvialis cell of results is by living contaminants, the content of the natural astaxanthin of frustule will sharply decline.Particularly, when adopting extensive open pond large-scale production, Haematocoocus Pluvialls is even caused to have no harvest once eruption and prevalence.
Therefore, be badly in need of now a kind of extensive open cultivation Haematocoocus Pluvialls of exploitation, improve the method for the output of natural astaxanthin, solve a haematococcus pluvialis growing difficult problem slowly.
Summary of the invention
The object of the invention is yielding poorly of the natural astaxanthin overcoming prior art, haematococcus pluvialis growing is defect slowly, provides a kind of cultural method that can significantly improve the Haematocoocus Pluvialls of the output of Determination of Astaxanthin in Haematococcus Pluvialis content and Haematocoocus Pluvialls under open culture condition.
The present inventor finds Haematocoocus Pluvialls to add in nutrient solution to carry out first stage cultivation under study for action, obtains the Haematocoocus Pluvialls containing swarm cell; Again the Haematocoocus Pluvialls containing swarm cell obtained is joined in nutrient solution and carry out subordinate phase cultivation, at least part of swarm cell is made to change non motile cell into, wherein, microbiotic A and microbiotic B is added successively in step (1) and/or step (2), or first add microbiotic A in step (1), in step (2), add microbiotic B again, microbiotic A is different with microbiotic B.Adopt the method cultivation Haematocoocus Pluvialls can improve the output of natural astaxanthin.
Therefore, to achieve these goals, the invention provides a kind of method of cultivating Haematocoocus Pluvialls, the method comprises the following steps:
(1) under the condition of the open cultivation of illumination, Haematocoocus Pluvialls is carried out first stage cultivation in nutrient solution, obtain the Haematocoocus Pluvialls containing swarm cell;
(2) under the condition of the open cultivation of illumination, the Haematocoocus Pluvialls containing swarm cell step (1) obtained carries out subordinate phase cultivation in nutrient solution, makes at least part of swarm cell be converted into non motile cell;
Wherein, in step (1) and/or step (2), microbiotic A and microbiotic B is added successively; Or first add microbiotic A in step (1), then add microbiotic B in step (2), microbiotic A is different with microbiotic B.
The inventive method is adopted to cultivate Haematocoocus Pluvialls, greatly can improve the ratio that the first stage cultivates swarm cell in the Haematocoocus Pluvialls obtained, swarm cell can reach 50%-90%, greatly can improve the ratio that subordinate phase cultivates non motile cell in the Haematocoocus Pluvialls obtained, non motile cell can reach 40%-80%, thus greatly improve the content of Determination of Astaxanthin in Haematococcus Pluvialis and the output of Haematocoocus Pluvialls, the content of astaxanthin in the Haematocoocus Pluvialls after cultivation (with dry weight basis) can be made to reach 1.2-2.5 % by weight, the dry weight of haematococcus pluvialis cell reaches 0.5-1.5mg/mL.
Other features and advantages of the present invention are described in detail in embodiment part subsequently.
Embodiment
Below the specific embodiment of the present invention is described in detail.Should be understood that, embodiment described herein, only for instruction and explanation of the present invention, is not limited to the present invention.
The invention provides a kind of method of cultivating Haematocoocus Pluvialls, the method comprises the following steps: Haematocoocus Pluvialls, under the condition of the open cultivation of illumination, is carried out first stage cultivation by (1) in nutrient solution, obtains the Haematocoocus Pluvialls containing swarm cell; (2) under the condition of the open cultivation of illumination, the Haematocoocus Pluvialls containing swarm cell step (1) obtained carries out subordinate phase cultivation in nutrient solution, makes at least part of swarm cell be converted into non motile cell; Wherein, in step (1) and/or step (2), microbiotic A and microbiotic B is added successively; Or first add microbiotic A in step (1), then add microbiotic B in step (2), microbiotic A is different with microbiotic B.
What it should be appreciated by those skilled in the art is, microbiotic A and microbiotic B is added successively in step (1) and/or step (2), or first add microbiotic A in step (1), in step (2), adding microbiotic B again, is four kinds of technical schemes in fact: only in the first stage cultivates, add microbiotic A and microbiotic B successively; Only in subordinate phase is cultivated, add microbiotic A and microbiotic B successively; Microbiotic A and microbiotic B is added all successively in the first stage cultivates and subordinate phase is cultivated; In the first stage cultivates, add microbiotic A, in subordinate phase is cultivated, add microbiotic B, wherein, microbiotic A always added before microbiotic B, and microbiotic A is different with microbiotic B.
According to method of the present invention, as long as add different microbiotic A and microbiotic B successively can realize object of the present invention, but in order to more be conducive to the output improving astaxanthin, preferably, first add microbiotic A in step (1), then add microbiotic B in step (2).
Preferably, in the cultivation in per stage, first in nutritive medium, add microbiotic, then add Haematocoocus Pluvialls.
According to method of the present invention, the kind of microbiotic A can be conventional microbiotic, in order to improve the ratio of swarm cell in Haematocoocus Pluvialls further, and be conducive to follow-up subordinate phase cultivation, and then be more conducive to the output improving astaxanthin, preferably, the kind of microbiotic A is selected from least one in penbritin, Streptomycin sulphate, kantlex, paraxin, penicillin, grisovin, gentamicin sulphate and cephamycin.
More preferably, when microbiotic A is the combination of penbritin, Streptomycin sulphate, kantlex, paraxin, penicillin, grisovin, gentamicin sulphate and cephamycin, more be conducive to the ratio improving swarm cell in Haematocoocus Pluvialls, thus be more conducive to the output improving astaxanthin.
According to method of the present invention, in microbiotic A, often kind of antibiotic add-on can be the consumption of this area routine, can be such as: relative to the nutrient solution of 1mL that in microbiotic A, often kind of antibiotic consumption can be 0.025-0.200mg, be preferably 0.050-0.100mg.In step (1), relative to the normal nutrition liquid of 1mL, the haematococcus pluvialis cell initially added can be that 0.05-0.2mg(is with dry weight basis), the swarm cell of the haematococcus pluvialis cell initially added generally containing 15%-40%.
In the present invention, swarm cell refers to the frustule with obvious flagellum.
The content of each component in microbiotic A can be the same or different, and in order to easy to operate, the content of each component is preferably identical.
Under the above-mentioned preferable case that the first stage cultivates, quantity and the vigor of Haematocoocus Pluvialls in first stage cultivation can be improved further, and the ratio of swarm cell in Haematocoocus Pluvialls can be improved further, make the ratio of swarm cell to reach 50%-90%.
The quantity of the quantity of frustule, the quantity of swarm cell and non motile cell is measured according to blood counting chamber in the present invention.
According to method of the present invention, the kind of microbiotic B can be conventional microbiotic, in order to more be conducive to the accumulation volume improving astaxanthin, preferably, the kind of microbiotic B is selected from least one in paraxin, penicillin, grisovin, penicillin and cephamycin.
More preferably, when microbiotic B is the combination of paraxin, penicillin, grisovin, penicillin and cephamycin, be more conducive to the accumulation volume improving astaxanthin.
According to method of the present invention, in microbiotic B, often kind of antibiotic add-on can be the consumption of this area routine, such as, relative to the nutrient solution of 1mL, in microbiotic B, often kind of antibiotic consumption can be 0.010-0.200mg, is preferably 0.020-0.100mg.In step (2), relative to the nutrient solution of 1mL, the haematococcus pluvialis cell containing swarm cell that the step (1) added obtains can be that 0.5-2mg(is with dry weight basis).
The content of each component in microbiotic B can be the same or different, and in order to easy to operate, the content of each component is preferably identical.
Under the above-mentioned preferable case that subordinate phase is cultivated, the ratio of non motile cell in subordinate phase cultivation can be improved further, improve the accumulation volume of astaxanthin further.The ratio of the non motile cell cultivated through subordinate phase is made to reach 60%-80%.
In the present invention, the frustule after non motile cell refers to and loses flagellum, i.e. chlamydospore cell.
According to method of the present invention, wherein, in step (1), the condition of the open cultivation of described illumination can be the conventional condition of cultivating for Haematocoocus Pluvialls, such as, the condition of the open cultivation of this illumination can comprise: intensity of illumination is 1000-2500lux, is more preferably 1300-2000lux; Culture temperature is 18-28 DEG C, is more preferably 20-25 DEG C; Incubation time is 7-20 days, is more preferably 10-15 days.
According to method of the present invention, wherein, in step (2), the condition of the open cultivation of described illumination can be the conventional condition of cultivating for Haematocoocus Pluvialls, such as, the condition of the open cultivation of this illumination can comprise: intensity of illumination is 20000-35000lux, is more preferably 25000-30000lux; Culture temperature is 30-38 DEG C, is more preferably 33-35 DEG C; Incubation time is 10-20 days, is more preferably 12-18 days.
According to method of the present invention, the first stage cultivates and subordinate phase is cultivated and can be carried out in bioreactor, and the cultivation in two stages can be carried out in same bioreactor, also can carry out in two bioreactors.
In the present invention, do not have particular requirement to the nutrient solution that the first stage cultivates, such as, in the first stage cultivates, nutrient solution can be BBM substratum, and BBM substratum can be the one in following two kinds.
The formula of the first BBM substratum is: in 1000ml water, and the content of each component is: NaNO
350-250mg, KH
2pO
4100-300mg, K
2hPO
450-250mg, MgSO
47H
2o50-100mg, CaCl
22H
2o10-100mg, NaCl25-100mg, EDTA10-100mg, KOH50-100mg, FeSO
47H
2o1-50mg, H
3bO
310-50mg, ZnSO
47H
2o1-50mg, MnCl
21-50mg, MoO
31-50mg, CuSO
45H
2o1-50mg, Co(NO
3)
26H
2o0.5-50mg.
The formula of the second BBM substratum is: in 1000ml water, and the content of each component is: NaNO
3100-300mg, MgSO47H2O50-200mg, KH
2pO
4100-300mg, CaCl
22H
2o10-100mg, NaCl25-100mg, EDTA10-100mg, VB
10.1-10mg, MnCl
20.1-10mg, ZnCl
27H
2o1-50mg, Na
2moO
41-50mg, FeCl
36H
2o1-150mg, Co(Cl)
26H
2o0.1-5mg.
Preferably, BBM substratum is: in 1000ml water, and the content of each component is: NaNO
3250mg, MgSO
47H
2o75mg, KH
2pO
4175mg, CaCl
22H
2o25mg, NaCl25mg, EDTA50mg, VB
11mg, MnCl
21.44mg, ZnCl
27H
2o5mg, Na
2moO
44mg, FeCl
36H
2o97mg, Co (Cl)
26H
2o2mg.
In the present invention, particular requirement is not had, in order to more be conducive to the accumulation of astaxanthin to the nutrient solution that subordinate phase is cultivated, preferably, in subordinate phase is cultivated, nutritive medium is for coercing substratum, the formula of coercing substratum can be: in 1000ml water, and the content of each component is: NaNO
3750-850mg, KH
2pO
4175mg, K
2hPO
475mg, MgSO
47H
2o75mg, CaCl
22H
2o25mg, NaCl400-1000mg, EDTA50mg, KOH31mg, FeSO
47H
2o100-200mg, H
3bO
311.42mg, ZnSO
47H
2o8.82mg, MnCl
21.44mg, MoO
30.71mg, CuSO
45H
2o1.57mg, Co(NO
3)
26H
2o0.49mg, sodium acetate 200-400mg.
In the present invention, adopt method of the present invention to cultivate Haematocoocus Pluvialls, can significantly improve the output of astaxanthin, the content of Determination of Astaxanthin in Haematococcus Pluvialis (with dry weight basis) can reach 1.2-2.5 % by weight.
One of the present invention preferred embodiment in, cultivate the method for Haematocoocus Pluvialls and comprise the following steps:
(1) first in the nutrient solution in first stage bioreactor, microbiotic A is added, relative to the nutrient solution of 1mL, in microbiotic A, often kind of antibiotic add-on is 0.050-0.100mg, again the Haematocoocus Pluvialls of the swarm cell containing 15%-40% is joined in the nutrient solution containing microbiotic A, relative to the nutrient solution of 1mL, the add-on of Haematocoocus Pluvialls is that 0.05-0.2mg(is with dry weight basis).At intensity of illumination 1300-2000lux, carry out the first stage under temperature 20-25 DEG C of condition and cultivate 10-15 days, obtain the Haematocoocus Pluvialls containing swarm cell; Wherein, microbiotic A is the combination of penbritin, Streptomycin sulphate, kantlex, paraxin, penicillin, grisovin, gentamicin sulphate and cephamycin, and the consumption of each component is identical; Nutrient solution is BBM substratum, fills a prescription to be: in 1000ml water, and the content of each component is: NaNO
3250mg, MgSO
47H
2o75mg, KH
2pO
4175mg, CaCl
22H
2o25mg, NaCl25mg, EDTA50mg, VB
11mg, MnCl
21.44mg, ZnCl
27H
2o5mg, Na
2moO
44mg, FeCl
36H
2o97mg, Co (Cl)
26H
2o2mg.
(2) in the nutrient solution in subordinate phase bioreactor, microbiotic B is added, relative to the nutritive medium of 1mL, in microbiotic B, often kind of antibiotic add-on is 0.020-0.100mg, the Haematocoocus Pluvialls containing swarm cell step (1) obtained again joins in the nutrient solution containing microbiotic B, relative to 1mL nutrient solution, the add-on of the Haematocoocus Pluvialls (wherein the ratio of non motile cell is 10%-50%) containing swarm cell that step (1) obtains is 0.5-2mg.At intensity of illumination 25000-30000lux, carry out subordinate phase under temperature 33-35 DEG C of condition and cultivate 12-18 days; Wherein, microbiotic B is the combination of paraxin, grisovin, penicillin and cephamycin, and the consumption of each component is identical, and nutrient solution is for coercing substratum, and filling a prescription is: in 1000ml water, and the content of each component is: NaNO
3750-850mg, KH
2pO
4175mg, K
2hPO
475mg, MgSO
47H
2o75mg, CaCl
22H
2o25mg, NaCl400-1000mg, EDTA50mg, KOH31mg, FeSO
47H
2o100-200mg, H
3bO
311.42mg, ZnSO
47H
2o8.82mg, MnCl
21.44mg, MoO
30.71mg, CuSO
45H
2o1.57mg, Co(NO
3)
26H
2o0.49mg, sodium acetate 200-400mg.
Embodiment
In following examples and comparative example:
Haematocoocus Pluvialls is purchased from Inst. of Hydrobiology, Chinese Academy of Sciences's algae kind storehouse, and algae kind is numbered FACHB-875, FACHB-797, FACHB-871; Penbritin, Streptomycin sulphate, kantlex, paraxin, penicillin, grisovin, gentamicin sulphate and cephamycin are purchased from traditional Chinese medicines; First stage bioreactor and subordinate phase bioreactor are board-like photoreactor.
The formula of BBM substratum is, in 1000ml water, the content of each component is: NaNO
3250mg, MgSO
47H
2o75mg, KH
2pO
4175mg, CaCl
22H
2o25mg, NaCl25mg, EDTA50mg, VB
11mg, MnCl
21.44mg, ZnCl
27H
2o5mg, Na
2moO
44mg, FeCl
36H
2o97mg, Co (Cl)
26H
2o2mg.
The formula of coercing substratum is, in 1000ml water, the content of each component is: NaNO
3750mg, KH
2pO
4175mg, K
2hPO
475mg, MgSO
47H
2o75mg, CaCl
22H
2o25mg, NaCl800mg, EDTA50mg, KOH31mg, FeSO
47H
2o150mg, H
3bO
311.42mg, ZnSO
47H
2o8.82mg, MnCl
21.44mg, MoO
30.71mg, CuSO
45H
2o1.57mg, Co(NO
3)
26H
2o0.49mg, sodium acetate 300mg.
The content of astaxanthin measures according to HPLC method.
Swarm cell and non motile cell count and adopt counting method of blood cell to measure.
Embodiment 1
The present embodiment is for illustration of the cultural method of Haematocoocus Pluvialls of the present invention
(1) first in the BBM substratum of the 20L in first stage bioreactor, microbiotic A is added, microbiotic A is the combination of penbritin, Streptomycin sulphate, kantlex, paraxin, penicillin, grisovin, gentamicin sulphate and cephamycin, wherein, the add-on of each component in microbiotic A is 1g.Again 1g Haematocoocus Pluvialls (is numbered FACHB-875, with dry weight basis, wherein, the ratio of swarm cell is 15%) join containing microbiotic A BBM substratum, at intensity of illumination 1300lux, carry out the first stage under temperature 20 DEG C of conditions and cultivate 10 days, in mensuration Haematocoocus Pluvialls, the quantity of swarm cell accounts for the ratio of total Haematocoocus Pluvialls total amount is 50%.
(2) add microbiotic B to coercing in substratum of the 20L in subordinate phase bioreactor, microbiotic B is the combination of paraxin, penicillin, grisovin and cephamycin, and wherein, the add-on of each component in microbiotic B is 0.4g.The ratio of the Haematocoocus Pluvialls 10g(wherein non motile cell containing swarm cell step (1) obtained again is 50%) join and coerce in substratum containing microbiotic B, at intensity of illumination 25000lux, carry out subordinate phase under temperature 33 DEG C of conditions and cultivate 12 days, measuring the ratio that the quantity of non motile cell accounts for Haematocoocus Pluvialls total amount is 70%.
(3), after cultivation terminates, measure content and the haematococcus pluvialis cell dry weight of Determination of Astaxanthin in Haematococcus Pluvialis, the results are shown in Table 1.
Embodiment 2
The present embodiment is for illustration of the cultural method of Haematocoocus Pluvialls of the present invention
(1) first in the BBM substratum of the 20L in first stage bioreactor, microbiotic A is added, microbiotic A is the combination of penbritin, Streptomycin sulphate, kantlex, paraxin, penicillin, grisovin, gentamicin sulphate and cephamycin, wherein, the add-on of each component in microbiotic A is 2g.Again 4g Haematocoocus Pluvialls (is numbered FACHB-797, with dry weight basis, wherein, the ratio of swarm cell is 40%) join containing microbiotic A BBM substratum, at intensity of illumination 2000lux, carry out the first stage under temperature 25 DEG C of conditions and cultivate 15 days, in mensuration Haematocoocus Pluvialls, the quantity of swarm cell accounts for the ratio of total Haematocoocus Pluvialls total amount is 80%.
(2) add microbiotic B to coercing in substratum of the 20L in subordinate phase bioreactor, microbiotic B is the combination of paraxin, penicillin, grisovin and cephamycin, and wherein, the add-on of each component in microbiotic B is 2g.The ratio of the Haematocoocus Pluvialls 40g(wherein non motile cell containing swarm cell step (1) obtained again is 20%) join and coerce in substratum containing microbiotic B, at intensity of illumination 30000lux, carry out subordinate phase under temperature 35 DEG C of conditions and cultivate 18 days, measuring the ratio that the quantity of non motile cell accounts for Haematocoocus Pluvialls total amount is 80%.
(3), after cultivation terminates, measure content and the haematococcus pluvialis cell dry weight of Determination of Astaxanthin in Haematococcus Pluvialis, the results are shown in Table 1.
Embodiment 3
The present embodiment is for illustration of the cultural method of Haematocoocus Pluvialls of the present invention
(1) first in the BBM substratum of the 20L in first stage bioreactor, microbiotic A is added, microbiotic A is the combination of penbritin, Streptomycin sulphate, kantlex, paraxin, penicillin, grisovin, gentamicin sulphate and cephamycin, wherein, the add-on of each component in microbiotic A is 1.6g.Again 3g Haematocoocus Pluvialls (is numbered FACHB-871, with dry weight basis, wherein, the ratio of swarm cell is 30%) join containing microbiotic A BBM substratum, at intensity of illumination 1600lux, carry out the first stage under temperature 22 DEG C of conditions and cultivate 13 days, in mensuration Haematocoocus Pluvialls, the quantity of swarm cell accounts for the ratio of total Haematocoocus Pluvialls total amount is 90%.
(2) add microbiotic B to coercing in substratum of the 20L in subordinate phase bioreactor, microbiotic B is the combination of paraxin, penicillin, grisovin and cephamycin, and wherein, the add-on of each component in microbiotic A is 1.4g.The ratio of the Haematocoocus Pluvialls 30g(wherein non motile cell containing swarm cell step (1) obtained again is 10%) join and coerce in substratum containing microbiotic B, at intensity of illumination 28000lux, carry out subordinate phase under temperature 34 DEG C of conditions and cultivate 15 days, measuring the ratio that the quantity of non motile cell accounts for Haematocoocus Pluvialls total amount is 60%.
(3), after cultivation terminates, measure content and the haematococcus pluvialis cell dry weight of Determination of Astaxanthin in Haematococcus Pluvialis, the results are shown in Table 1.
Embodiment 4
The present embodiment is for illustration of the cultural method of Haematocoocus Pluvialls of the present invention
Haematocoocus Pluvialls is cultivated according to the method for embodiment 1, unlike, microbiotic A is the combination of kantlex and paraxin, and the add-on of kantlex and paraxin is 4g, altogether 8g.After cultivation terminates, measure content and the haematococcus pluvialis cell dry weight of Determination of Astaxanthin in Haematococcus Pluvialis, the results are shown in Table 1.
Embodiment 5
The present embodiment is for illustration of the cultural method of Haematocoocus Pluvialls of the present invention
Haematocoocus Pluvialls is cultivated according to the method for embodiment 1, unlike, microbiotic A is only penbritin, and the add-on of penbritin is 8g.After cultivation terminates, measure content and the haematococcus pluvialis cell dry weight of Determination of Astaxanthin in Haematococcus Pluvialis, the results are shown in Table 1.
Embodiment 6
The present embodiment is for illustration of the cultural method of Haematocoocus Pluvialls of the present invention
Haematocoocus Pluvialls is cultivated according to the method for embodiment 1, unlike, microbiotic A is only cephamycin, and the add-on of cephamycin is 8g.After cultivation terminates, measure content and the haematococcus pluvialis cell dry weight of Determination of Astaxanthin in Haematococcus Pluvialis, the results are shown in Table 1.
Embodiment 7
The present embodiment is for illustration of the cultural method of Haematocoocus Pluvialls of the present invention
Haematocoocus Pluvialls is cultivated according to the method for embodiment 1, unlike, in microbiotic A, often kind of antibiotic add-on is 4g.After cultivation terminates, measure content and the haematococcus pluvialis cell dry weight of Determination of Astaxanthin in Haematococcus Pluvialis, the results are shown in Table 1.
Embodiment 8
The present embodiment is for illustration of the cultural method of Haematocoocus Pluvialls of the present invention
Haematocoocus Pluvialls is cultivated according to the method for embodiment 1, unlike, microbiotic B is the combination of grisovin and cephamycin, and the add-on of grisovin and cephamycin is 0.8g.After cultivation terminates, measure content and the haematococcus pluvialis cell dry weight of Determination of Astaxanthin in Haematococcus Pluvialis, the results are shown in Table 1.
Embodiment 9
The present embodiment is for illustration of the cultural method of Haematocoocus Pluvialls of the present invention
Haematocoocus Pluvialls is cultivated according to the method for embodiment 1, unlike, microbiotic B is only paraxin, and the add-on of paraxin is 1.6g.After cultivation terminates, measure content and the haematococcus pluvialis cell dry weight of Determination of Astaxanthin in Haematococcus Pluvialis, the results are shown in Table 1.
Embodiment 10
The present embodiment is for illustration of the cultural method of Haematocoocus Pluvialls of the present invention
Haematocoocus Pluvialls is cultivated according to the method for embodiment 1, unlike, in microbiotic B, often kind of antibiotic add-on is 4g.After cultivation terminates, measure content and the haematococcus pluvialis cell dry weight of Determination of Astaxanthin in Haematococcus Pluvialis, the results are shown in Table 1.
Embodiment 11
The present embodiment is for illustration of the cultural method of Haematocoocus Pluvialls of the present invention
Haematocoocus Pluvialls is cultivated according to the method for embodiment 1, unlike, only in cultivating in the first stage, add microbiotic A and microbiotic B successively.After cultivation terminates, measure content and the haematococcus pluvialis cell dry weight of Determination of Astaxanthin in Haematococcus Pluvialis, the results are shown in Table 1.
Embodiment 12
The present embodiment is for illustration of the cultural method of Haematocoocus Pluvialls of the present invention
Haematocoocus Pluvialls is cultivated according to the method for embodiment 1, unlike, only in cultivating in subordinate phase, add microbiotic A and microbiotic B successively.After cultivation terminates, measure content and the haematococcus pluvialis cell dry weight of Determination of Astaxanthin in Haematococcus Pluvialis, the results are shown in Table 1.
Embodiment 13
The present embodiment is for illustration of the cultural method of Haematocoocus Pluvialls of the present invention
Haematocoocus Pluvialls is cultivated according to the method for embodiment 1, unlike, in the first stage cultivates and subordinate phase is cultivated, add microbiotic A and microbiotic B all successively, the add-on of each component in each stage in microbiotic A is 0.5g, and the add-on of each component in microbiotic B is 0.2g.After cultivation terminates, measure content and the haematococcus pluvialis cell dry weight of Determination of Astaxanthin in Haematococcus Pluvialis, the results are shown in Table 1.
Comparative example 1
Haematocoocus Pluvialls is cultivated according to the method for embodiment 1, unlike, microbiotic A is identical with the kind of microbiotic B, is all the combination of penbritin, Streptomycin sulphate, kantlex, paraxin, penicillin, grisovin, gentamicin sulphate and cephamycin.In step (2), often kind of antibiotic add-on is 0.2g.After cultivation terminates, measure content and the haematococcus pluvialis cell dry weight of Determination of Astaxanthin in Haematococcus Pluvialis, the results are shown in Table 1.
Comparative example 2
Haematocoocus Pluvialls is cultivated according to the method for embodiment 1, unlike, microbiotic A is identical with the kind of microbiotic B, is all the combination of paraxin, penicillin, grisovin and cephamycin.In step (1), often kind of antibiotic add-on is 2g.Measure content and the haematococcus pluvialis cell dry weight of Determination of Astaxanthin in Haematococcus Pluvialis, the results are shown in Table 1.
Comparative example 3
Haematocoocus Pluvialls is cultivated according to the method for embodiment 1, unlike, microbiotic A is not identical with microbiotic B kind, and microbiotic A is the combination of paraxin, penicillin, grisovin and cephamycin, and often kind of antibiotic add-on is 2g; Microbiotic B is the combination of penbritin, Streptomycin sulphate, kantlex, paraxin, penicillin, grisovin, gentamicin sulphate and cephamycin, and often kind of antibiotic add-on is 0.2g.After cultivation terminates, measure content and the haematococcus pluvialis cell dry weight of Determination of Astaxanthin in Haematococcus Pluvialis, the results are shown in Table 1.
Comparative example 4
Haematocoocus Pluvialls is cultivated according to the method for embodiment 1, unlike, in Haematocoocus Pluvialls culturing process, all do not add the microbiotic of any kind.After cultivation terminates, measure content and the haematococcus pluvialis cell dry weight of Determination of Astaxanthin in Haematococcus Pluvialis, the results are shown in Table 1.
Table 1
Embodiment 1-13 and comparative example 1-4 is compared and can find out, adopts the inventive method can significantly improve the content of Determination of Astaxanthin in Haematococcus Pluvialis and significantly improve the output of Haematocoocus Pluvialls.
Embodiment 1 is compared with embodiment 4-6 respectively and can find out, when microbiotic A is the combination of penbritin, Streptomycin sulphate, kantlex, paraxin, penicillin, grisovin, gentamicin sulphate and cephamycin, the content of Determination of Astaxanthin in Haematococcus Pluvialis and the output of Haematocoocus Pluvialls can be improved further;
Embodiment 1 and embodiment 7 are compared and can find out, relative to the nutritive medium of 1mL, when often kind of antibiotic consumption is within the scope of 0.050-0.100mg in microbiotic A, the content of Determination of Astaxanthin in Haematococcus Pluvialis and the output of Haematocoocus Pluvialls can be improved further;
Embodiment 1 is compared with embodiment 8-9 respectively and can find out, when microbiotic B is the combination of paraxin, penicillin, grisovin and cephamycin, the content of Determination of Astaxanthin in Haematococcus Pluvialis and the output of Haematocoocus Pluvialls can be improved further;
Embodiment 1 and embodiment 10 are compared and can find out, relative to the stress nutrient solution of 1mL, when often kind of antibiotic consumption is within the scope of 0.020-0.100mg in microbiotic B, the content of Determination of Astaxanthin in Haematococcus Pluvialis and the output of Haematocoocus Pluvialls can be improved further.
Embodiment 1 is compared with embodiment 11-13 respectively and can find out, first add microbiotic A in step (1), then add microbiotic B in step (2), the content of Determination of Astaxanthin in Haematococcus Pluvialis and the output of Haematocoocus Pluvialls can be improved further.
In the present invention, method of the present invention is adopted to cultivate Haematocoocus Pluvialls, greatly can improve the content of Determination of Astaxanthin in Haematococcus Pluvialis and the output of Haematocoocus Pluvialls, the content of astaxanthin in the Haematocoocus Pluvialls after cultivation (with dry weight basis) can be made to reach 1.2-2.5 % by weight, and the dry weight of haematococcus pluvialis cell reaches 0.5-1.5mg/mL.
More than describe the preferred embodiment of the present invention in detail; but the present invention is not limited to the detail in above-mentioned embodiment, within the scope of technical conceive of the present invention; can carry out multiple simple variant to technical scheme of the present invention, these simple variant all belong to protection scope of the present invention.
It should be noted that in addition, each the concrete technical characteristic described in above-mentioned embodiment, in reconcilable situation, can be combined by any suitable mode.
In addition, also can carry out arbitrary combination between various different embodiment of the present invention, as long as it is without prejudice to thought of the present invention, it should be considered as content disclosed in this invention equally.
Claims (11)
1. cultivate a method for Haematocoocus Pluvialls, it is characterized in that, the method comprises the following steps:
(1) under the condition of the open cultivation of illumination, Haematocoocus Pluvialls is carried out first stage cultivation in nutrient solution, obtain the Haematocoocus Pluvialls containing swarm cell;
(2) under the condition of the open cultivation of illumination, the Haematocoocus Pluvialls containing swarm cell step (1) obtained carries out subordinate phase cultivation in nutrient solution, makes at least part of swarm cell be converted into non motile cell;
Wherein, in step (1) and/or step (2), microbiotic A and microbiotic B is added successively; Or first add microbiotic A in step (1), then add microbiotic B in step (2); Microbiotic A is different with microbiotic B.
2. method according to claim 1, wherein, first adds microbiotic A in step (1), then add microbiotic B in step (2).
3. method according to claim 1 and 2, wherein, described microbiotic A is selected from least one in penbritin, Streptomycin sulphate, kantlex, paraxin, penicillin, grisovin, gentamicin sulphate and cephamycin.
4. method according to claim 3, wherein, described microbiotic A is penbritin, Streptomycin sulphate, kantlex, paraxin, penicillin, grisovin, gentamicin sulphate and cephamycin.
5. method according to claim 1 and 2, wherein, described microbiotic B is selected from least one in paraxin, grisovin, penicillin and cephamycin.
6. method according to claim 5, wherein, described microbiotic B is paraxin, grisovin, penicillin and cephamycin.
7. method according to claim 1 and 2, wherein, relative to the nutritive medium of 1mL, in described microbiotic A, often kind of antibiotic consumption is 0.025-0.200mg, is preferably 0.050-0.100mg.
8. method according to claim 1 and 2, wherein, relative to the nutritive medium of 1mL, in described microbiotic B, often kind of antibiotic consumption is 0.010-0.200mg, is preferably 0.020-0.100mg.
9. method according to claim 1 and 2, wherein, in step (1), the condition of the open cultivation of described illumination comprises: intensity of illumination is 1000-2500lux, is preferably 1300-2000lux; Culture temperature is 18-28 DEG C, is preferably 20-25 DEG C; Incubation time is 7-20 days, is preferably 10-15 days.
10. method according to claim 1 and 2, wherein, in step (2), the condition of the open cultivation of described illumination comprises: intensity of illumination is 20000-35000lux, is preferably 25000-30000lux; Culture temperature is 30-38 DEG C, is preferably 33-35 DEG C; Incubation time is 10-20 days, is preferably 12-18 days.
11. methods according to claim 1 and 2, wherein, in the first stage cultivates, described nutrient solution is BBM substratum; In subordinate phase is cultivated, described nutrient solution is for coercing substratum.
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| CN105420332A (en) * | 2015-12-10 | 2016-03-23 | 天津科技大学 | Method for achieving high yield of astaxanthin through haematococcus pluvialis |
| CN105779292A (en) * | 2016-03-23 | 2016-07-20 | 国家开发投资公司 | Ultralow-temperature preservation method for haematococcus pluvialis subjected to aseptic treatment |
| CN107354122A (en) * | 2017-09-18 | 2017-11-17 | 深圳市德和生物科技有限公司 | A kind of method for promoting haematococcus pluvialis growing multiplication and redden |
| CN109609385A (en) * | 2019-02-28 | 2019-04-12 | 福建康是美生物科技有限公司 | A kind of cultural method of haematococcus pluvialis |
| CN113717917A (en) * | 2021-06-30 | 2021-11-30 | 中国海洋大学 | Combined antibiotic application method for pure culture of haematococcus pluvialis |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105420332A (en) * | 2015-12-10 | 2016-03-23 | 天津科技大学 | Method for achieving high yield of astaxanthin through haematococcus pluvialis |
| CN105779292A (en) * | 2016-03-23 | 2016-07-20 | 国家开发投资公司 | Ultralow-temperature preservation method for haematococcus pluvialis subjected to aseptic treatment |
| CN107354122A (en) * | 2017-09-18 | 2017-11-17 | 深圳市德和生物科技有限公司 | A kind of method for promoting haematococcus pluvialis growing multiplication and redden |
| CN109609385A (en) * | 2019-02-28 | 2019-04-12 | 福建康是美生物科技有限公司 | A kind of cultural method of haematococcus pluvialis |
| CN113717917A (en) * | 2021-06-30 | 2021-11-30 | 中国海洋大学 | Combined antibiotic application method for pure culture of haematococcus pluvialis |
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