CN109609385A - A kind of cultural method of haematococcus pluvialis - Google Patents

A kind of cultural method of haematococcus pluvialis Download PDF

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CN109609385A
CN109609385A CN201910149444.4A CN201910149444A CN109609385A CN 109609385 A CN109609385 A CN 109609385A CN 201910149444 A CN201910149444 A CN 201910149444A CN 109609385 A CN109609385 A CN 109609385A
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haematococcus pluvialis
culture solution
culture
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cultural method
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陈国荣
黄轲
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Fujian Kang Shi Mei Biotechnology Co Ltd
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/12—Unicellular algae; Culture media therefor

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Abstract

It is the culture feature according to haematococcus pluvialis growing stage and induction period, on the basis of basic culture solution, by the way that NaHCO is added in growth phase culture solution the invention discloses a kind of cultural method of haematococcus pluvialis3, ampicillin, vitamin B12, to inhibit living contaminants, NaAc, gibberellin are added in induction period culture solution, to promote astaxanthin accumulation, to significantly improve the yield and Determination of Astaxanthin in Haematococcus Pluvialis content of haematococcus pluvialis.

Description

A kind of cultural method of haematococcus pluvialis
Technical field
Present invention relates particularly to a kind of cultural methods of haematococcus pluvialis.
Background technique
Astaxanthin has significant colorability, and there is anticancer, anti-oxidant, ultra-violet radiation resisting and strengthen immunity etc. Multiple efficacies have vast potential for future development in fields such as health care product, cosmetics, medicine, aquacultures.Foundation production technology, Astaxanthin can be divided into artificial synthesized astaxanthin and natural astaxanthin.Artificial synthesized astaxanthin is not only expensive, and its Stability, oxidation activity, bio-absorbable effect, colorability etc. are lower than natural astaxanthin.Therefore, natural astaxanthin The current main research direction of exploitation.
Currently, the source of natural astaxanthin generally has following three kinds: waste, fungi (the red hair of processing of aquatic products industry Husband's yeast) and microalgae (haematococcus pluvialis).Wherein, the content of astaxanthin is lower in waste and fungi, is unsuitable for extensive life It produces.And haematococcus pluvialis can largely accumulate astaxanthin under stress conditions, content astaxanthin is up to 1.5%-3.0%.Therefore, The pilot scale culture of haematococcus pluvialis can be developed, with the natural astaxanthin of mass production high-quality.
Summary of the invention
The purpose of the present invention is to provide a kind of cultural methods of haematococcus pluvialis, and the yield of haematococcus pluvialis can be improved And Determination of Astaxanthin in Haematococcus Pluvialis content, and can realize the continuous grown cultures of haematococcus pluvialis.
To achieve the above object, the present invention adopts the following technical scheme:
A kind of cultural method of haematococcus pluvialis, including growth phase and induction period.
Wherein, growth phase is cultivated using Semi-continuous cultivation mode, condition of culture are as follows: and 20 ~ 25 DEG C of temperature, illumination Intensity 1000-1200 lx, light application time 12 ~ 15h/ days, culture solution pH 8.0；It cultivates formula of liquid are as follows: NaNO3 0.20~ 0.35 g/L、MgSO4·7H2O 0.06~0.20 g/L、KH2PO4 0.01~0.03 g/L、CaCl2 0.01~0.04 g/L、 EDTA 0.0003~0.0005 g/L、FeSO4·7H2O 0.001~0.004 g/L、ZnSO4·7H2O 0.001~0.004 g/ L、MnCl2·4H2O 0.00003~0.00005 g/L、CoNO3·6H2O 0.00005~0.00008 g/L、KOH 0.05~ 0.12 g/L、H3BO3 0.0001~0.0002 g/L、CuSO4·5H2O 0.00002~0.00005 g/L、MoO3 0.00001~ 0.00002 g/L, vitamin B12 0.00002~0.00005 g/L、NaHCO30.02 ~ 0.06 g/L, ampicillin 0.01 ~ 0.02 g/L。
The condition of culture of induction period are as follows: 30 ~ 35 DEG C of temperature, intensity of illumination 10000-15000 lx, light application time 12 ~ 15h/ days, culture solution pH 7.0；It cultivates formula of liquid are as follows: NaNO3 0.20~0.35 g/L、MgSO4·7H2O 0.06~0.20 g/L、KH2PO4 0.01~0.03 g/L、CaCl2 0.01~0.04 g/L、EDTA 0.0003~0.0005 g/L、FeSO4·7H2O 0.001~0.004 g/L、ZnSO4·7H2O 0.001~0.004 g/L、MnCl2·4H2O 0.00003~0.00005 g/L、 CoNO3·6H2O 0.00005~0.00008 g/L、KOH 0.05~0.12 g/L、H3BO3 0.0001~0.0002 g/L、 CuSO4·5H2O 0.00002~0.00005 g/L、MoO30.00001 ~ 0.00002 g/L, vitamin B12 0.00002~ 0.00005 1.0 ~ 1.2 g/L of g/L, NaAc, 0.002 ~ 0.003 g/L of gibberellin.
Remarkable advantage of the invention is:
(1) present invention in growth phase by the addition of a small amount of antibiotic, to avoid living contaminants, to realize haematococcus pluvialis High-density growth.
(2) present invention forms the condition of salt stress in induction period by the addition of NaAc, to promote the accumulation of astaxanthin； And the addition of gibberellin is also beneficial to the accumulation of astaxanthin.
(3) it carries out cultivating the yield that haematococcus pluvialis can be improved using the method for the present invention and Determination of Astaxanthin in Haematococcus Pluvialis contains Amount, and it can realize the continuous growth of haematococcus pluvialis, help to improve growth efficiency.
Specific embodiment
In order to make content of the present invention easily facilitate understanding, With reference to embodiment to of the present invention Technical solution is described further, but the present invention is not limited only to this.
Embodiment 1
A kind of cultural method of haematococcus pluvialis, including growth phase and induction period.
Wherein, growth phase is cultivated using Semi-continuous cultivation mode, condition of culture are as follows: 23 DEG C of temperature, illumination is strong Spend 1100 lx, light application time 12h/ days, culture solution pH 8.0；It cultivates formula of liquid are as follows: NaNO3 0.20 g/L、MgSO4· 7H2O 0.06 g/L、KH2PO4 0.01 g/L、CaCl2 0.01 g/L、EDTA 0.0003 g/L、FeSO4·7H2O 0.001 g/L、ZnSO4·7H2O 0.001 g/L、MnCl2·4H2O 0.00003 g/L、CoNO3·6H2O 0.00005 g/L、KOH 0.05 g/L、H3BO3 0.0001 g/L、CuSO4·5H2O 0.00002 g/L、MoO30.00001 g/L, vitamin B12 0.00002 g/L、NaHCO30.02 g/L, 0.01 g/L of ampicillin.
The condition of culture of induction period are as follows: 32 DEG C of temperature, 15000 lx of intensity of illumination, light application time 12 ~ for 24 hours/day, culture Liquid pH 7.0；It cultivates formula of liquid are as follows: NaNO3 0.20 g/L、MgSO4·7H2O 0.06 g/L、KH2PO4 0.01 g/L、 CaCl2 0.01 g/L、EDTA 0.0003 g/L、FeSO4·7H2O 0.001 g/L、ZnSO4·7H2O 0.001 g/L、 MnCl2·4H2O 0.00003 g/L、CoNO3·6H2O 0.00005 g/L、KOH 0.05 g/L、H3BO3 0.0001 g/L、 CuSO4·5H2O 0.00002 g/L、MoO30.00001 g/L, vitamin B12It is 0.00002 1.2 g/L of g/L, NaAc, red 0.002 g/L of mycin.
After culture the yield of haematococcus pluvialis cell be 2.2mg/mL(by dry weight), content astaxanthin reach 36.9mg/g (by dry weight).
Embodiment 2
A kind of cultural method of haematococcus pluvialis, including growth phase and induction period.
Wherein, growth phase is cultivated using Semi-continuous cultivation mode, condition of culture are as follows: 25 DEG C of temperature, illumination is strong Spend 1000 lx, light application time 15h/ days, culture solution pH 8.0；It cultivates formula of liquid are as follows: NaNO3 0.25 g/L、MgSO4· 7H2O 0.14 g/L、KH2PO4 0.02 g/L、CaCl2 0.02 g/L、EDTA 0.0004 g/L、FeSO4·7H2O 0.002 g/L、ZnSO4·7H2O 0.002 g/L、MnCl2·4H2O 0.00004 g/L、CoNO3·6H2O 0.00006 g/L、KOH 0.08 g/L、H3BO3 0.0001 g/L、CuSO4·5H2O 0.00003 g/L、MoO30.00001 g/L, vitamin B12 0.00003 g/L、NaHCO30.06 g/L, 0.012 g/L of ampicillin.
The condition of culture of induction period are as follows: 35 DEG C of temperature, intensity of illumination 12000 lx, light application time 15h/ days, culture solution pH 7.0；It cultivates formula of liquid are as follows: NaNO3 0.25 g/L、MgSO4·7H2O 0.14 g/L、KH2PO4 0.02 g/L、CaCl2 0.02 g/L、EDTA 0.0004 g/L、FeSO4·7H2O 0.008 g/L、ZnSO4·7H2O 0.002 g/L、MnCl2· 4H2O 0.00004 g/L、CoNO3·6H2O 0.00006 g/L、KOH 0.08 g/L、H3BO3 0.0001 g/L、CuSO4· 5H2O 0.00003 g/L、MoO30.00001 g/L, vitamin B120.00003 1.1 g/L of g/L, NaAc, gibberellin 0.003 g/L。
After culture the yield of haematococcus pluvialis cell be 1.9mg/mL(by dry weight), content astaxanthin reach 34.7mg/g (by dry weight).
Embodiment 3
A kind of cultural method of haematococcus pluvialis, including growth phase and induction period.
Wherein, growth phase is cultivated using Semi-continuous cultivation mode, condition of culture are as follows: 20 DEG C of temperature, illumination is strong Spend 1200 lx, light application time 14h/ days, culture solution pH 8.0；It cultivates formula of liquid are as follows: NaNO3 0.35 g/L、MgSO4· 7H2O 0.20 g/L、KH2PO4 0.03 g/L、CaCl2 0.04 g/L、EDTA 0.0005 g/L、FeSO4·7H2O 0.004 g/L、ZnSO4·7H2O 0.004 g/L、MnCl2·4H2O 0.00005 g/L、CoNO3·6H2O 0.00008 g/L、KOH 0.12 g/L、H3BO3 0.0002 g/L、CuSO4·5H2O 0.00005 g/L、MoO30.00002 g/L, vitamin B12 0.00005 g/L、NaHCO30.04 g/L, 0.02 g/L of ampicillin.
The condition of culture of induction period are as follows: 30 DEG C of temperature, intensity of illumination 10000 lx, light application time 12h/ days, culture solution pH 7.0；It cultivates formula of liquid are as follows: NaNO3 0.35 g/L、MgSO4·7H2O 0.20 g/L、KH2PO4 0.03 g/L、CaCl2 0.04 g/L、EDTA 0.0005 g/L、FeSO4·7H2O 0.004 g/L、ZnSO4·7H2O 0.004 g/L、MnCl2· 4H2O 0.00005 g/L、CoNO3·6H2O 0.00008 g/L、KOH 0.12 g/L、H3BO3 0.0002 g/L、CuSO4· 5H2O 0.00005 g/L、MoO30.00002 g/L, vitamin B120.00005 1.0 g/L of g/L, NaAc, gibberellin 0.003 g/L。
After culture the yield of haematococcus pluvialis cell be 1.8mg/mL(by dry weight), content astaxanthin reach 33.6mg/g (by dry weight).
Comparative example
A kind of cultural method of haematococcus pluvialis, including growth phase and induction period.
Wherein, growth phase is cultivated using Semi-continuous cultivation mode, condition of culture are as follows: 23 DEG C of temperature, illumination is strong Spend 1200 lx, light application time 14h/ days, culture solution pH 8.0；It cultivates formula of liquid are as follows: NaNO3 0.20 g/L、MgSO4· 7H2O 0.06 g/L、KH2PO4 0.01 g/L、CaCl2 0.01 g/L、EDTA 0.0003 g/L、FeSO4·7H2O 0.001 g/L、ZnSO4·7H2O 0.001 g/L、MnCl2·4H2O 0.00003 g/L、CoNO3·6H2O 0.00005 g/L、KOH 0.05 g/L、H3BO3 0.0001 g/L、CuSO4·5H2O 0.00002 g/L、MoO30.00001 g/L, vitamin B12 0.00002 g/L、NaHCO3 0.02g/L。
The condition of culture of induction period are as follows: 32 DEG C of temperature, intensity of illumination 15000 lx, light application time 12h/ days, culture solution pH 7.0；It cultivates formula of liquid are as follows: NaNO3 0.20 g/L、MgSO4·7H2O 0.06 g/L、KH2PO4 0.01 g/L、CaCl2 0.01 g/L、EDTA 0.0003 g/L、FeSO4·7H2O 0.001 g/L、ZnSO4·7H2O 0.001 g/L、MnCl2· 4H2O 0.00003 g/L、CoNO3·6H2O 0.00005 g/L、KOH 0.05 g/L、H3BO3 0.0001 g/L、CuSO4· 5H2O 0.00002 g/L、MoO30.00001 g/L, vitamin B12 0.00002 g/L、NaAc 1.2 g/L、3-IBA 0.003 g/L。
After culture the yield of haematococcus pluvialis cell be 1.2mg/mL(by dry weight), content astaxanthin reach 25.3mg/g (by dry weight).
The foregoing is merely presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with Modification, is all covered by the present invention.

Claims (4)

1. a kind of cultural method of haematococcus pluvialis, including growth phase and induction period, it is characterised in that: basic training used Nutrient solution formula are as follows: NaNO3 0.20~0.35 g/L、MgSO4·7H2O 0.06~0.20 g/L、KH2PO4 0.01~0.03 g/L、 CaCl2 0.01~0.04 g/L、EDTA 0.0003~0.0005 g/L、FeSO4·7H2O 0.001~0.004 g/L、ZnSO4· 7H2O 0.001~0.004 g/L、MnCl2·4H2O 0.00003~0.00005 g/L、CoNO3·6H2O 0.00005~ 0.00008 g/L、KOH 0.05~0.12 g/L、H3BO3 0.0001~0.0002 g/L、CuSO4·5H2O 0.00002~ 0.00005 g/L、MoO30.00001 ~ 0.00002 g/L, vitamin B120.00002~0.00005 g/L；
Culture solution used in growth phase is that NaHCO is added in basic culture solution30.02 ~ 0.06 g/L, ampicillin 0.01 ~ 0.02 g/L；
Culture solution used in induction period is addition 1.0 ~ 1.2 g/L of NaAc, gibberellin 0.002 ~ 0.003 in basic culture solution g/L。
2. the cultural method of haematococcus pluvialis according to claim 1, it is characterised in that: growth phase uses Semi-continuous cultivation Mode is cultivated.
3. the cultural method of haematococcus pluvialis according to claim 1, it is characterised in that: the condition of culture of growth phase are as follows: 20 ~ 25 DEG C of temperature, intensity of illumination 1000-1200 lx, light application time 12 ~ 15h/ days, culture solution pH 8.0.
4. the cultural method of haematococcus pluvialis according to claim 1, it is characterised in that: the condition of culture of induction period are as follows: 30 ~ 35 DEG C of temperature, intensity of illumination 10000-15000 lx, light application time 12 ~ 15h/ days, culture solution pH 7.0.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110157620A (en) * 2019-04-18 2019-08-23 厦门大学 A kind of cultural method improving purple ball algae synthesis phycoerythrin content
CN110408671A (en) * 2019-07-24 2019-11-05 嘉必优生物技术（武汉）股份有限公司 The method for being mixed schizochytrium and haematococcus pluvialis production DHA and astaxanthin

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104662162A (en) * 2013-09-26 2015-05-27 华东理工大学 Method using micro-algae for high-efficiency production of astaxanthin
CN104745479A (en) * 2013-12-26 2015-07-01 中粮营养健康研究院有限公司 Method for culturing haematococcus pluvialis
CN106906142A (en) * 2017-03-10 2017-06-30 烟台布鲁拜尔生物制药有限公司 A kind of large-scale method for producing of high content astaxanthin blood cell algae
CN108410939A (en) * 2018-07-12 2018-08-17 烟台大学 A method of improving Determination of Astaxanthin in Haematococcus Pluvialis content

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104662162A (en) * 2013-09-26 2015-05-27 华东理工大学 Method using micro-algae for high-efficiency production of astaxanthin
CN104745479A (en) * 2013-12-26 2015-07-01 中粮营养健康研究院有限公司 Method for culturing haematococcus pluvialis
CN106906142A (en) * 2017-03-10 2017-06-30 烟台布鲁拜尔生物制药有限公司 A kind of large-scale method for producing of high content astaxanthin blood cell algae
CN108410939A (en) * 2018-07-12 2018-08-17 烟台大学 A method of improving Determination of Astaxanthin in Haematococcus Pluvialis content

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110157620A (en) * 2019-04-18 2019-08-23 厦门大学 A kind of cultural method improving purple ball algae synthesis phycoerythrin content
CN110157620B (en) * 2019-04-18 2021-03-30 厦门大学 Culture method for improving content of phycoerythrin synthesized by porphyridium
CN110408671A (en) * 2019-07-24 2019-11-05 嘉必优生物技术（武汉）股份有限公司 The method for being mixed schizochytrium and haematococcus pluvialis production DHA and astaxanthin

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