CN103695314B - A kind of store method of Vegetative Cell of Haematococcus Pluvialis - Google Patents
A kind of store method of Vegetative Cell of Haematococcus Pluvialis Download PDFInfo
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- CN103695314B CN103695314B CN201310734011.8A CN201310734011A CN103695314B CN 103695314 B CN103695314 B CN 103695314B CN 201310734011 A CN201310734011 A CN 201310734011A CN 103695314 B CN103695314 B CN 103695314B
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Abstract
The object of this invention is to provide a kind of store method of haematococcus pluvialis cell, is be row amplification cultivation in the nutrient solution of 20-30:1 by Haematocoocus Pluvialls at the concentration ratio of nitrogen, phosphoric; The temperature 12-20 DEG C cultivated, and the light that light source is sent is the red film of 0.05-0.12mm by thickness, intensity of illumination is 500-1500lx.The present invention compared with prior art, shelf time is long, is not easy aging, and the vegetative cell in nutrient solution can keep nourishing and growing, 20-35 days when being cultured to plateau maximum density, after plateau, also can keep 20-35 days, carry out next step nutrition cultivation or conversion, and continue to keep growth conditions fast, the method is simple and easy to do, and the facility reducing conservation drops into, and reduces cost of labor.
Description
Technical field
The invention belongs to alga cells and cultivate Techniques of preserving field, be specifically related to a kind of store method of Vegetative Cell of Haematococcus Pluvialis.
Background technology
Natural astaxanthin (Astaxanthin) is the one of carotenoid, is the strongest material of occurring in nature resistance of oxidation found up to now; It has protection skin, improves immunizing power, suppresses cancer, alleviating fatigue, delays senility and the function of prevention and therapy cardiovascular and cerebrovascular diseases aspect, mainly as the natural colorant of fish, be also widely used in fishery products, makeup, the industries such as medicine.And Haematocoocus Pluvialls Haematococcus pluvialis is a kind of natural astaxanthin unicell green alga of production, its astaxanthin substances content is high, is regarded as " concentrate " of natural astaxanthin, and the stability of natural astaxanthin structure and security high; Therefore, cultivating Haematocoocus Pluvialls has become the following optimal path obtaining natural astaxanthin, thus obtains carrying forward vigorously of various countries.
Current cultivation haematococcus pluvialis to produce astaxanthin adopts the technological approaches of single stage method or two step method, single stage method such as notification number is CN101586140, the patent of invention that patent name is " a kind of simple and easy method cultivating haematococcus pluvialis to produce astaxanthin ", discloses the accumulation that substratum (nutrient solution) more complicated with composition in a bioreactor completes nourishing and growing of Haematocoocus Pluvialls and Haematocoocus Pluvialls conversion (spore conversion) and astaxanthin.Two step method such as publication number is CN1966660, patent name is the patent of invention of " devices and methods therefor of large scale culturing Haematocoocus Pluvialls and conversion astaxanthin ", disclose a kind of culture apparatus of the photo-bioreactor system be arranged on anchor, two the unit light bio-reactors connected carry out nourishing and growing with nutrient solution respectively and Haematocoocus Pluvialls transforms and the accumulation of astaxanthin.
No matter but be single stage method or two step method, all need accumulation and the preservation of a large amount of Vegetative Cell of Haematococcus Pluvialis.And the preservation of vegetative cell and growth are conflicts: grow rapidly, easily enter plateau ahead of time, the shelf time is just short; Poor growth, the shelf time is relatively just long, will affect the semi-invariant of vegetative cell.The preservation of vegetative cell and aging be also the condition of conflict: the shelf time is long, easily occurs aging, and impact continues to cultivate, transform.The semi-invariant of Vegetative Cell of Haematococcus Pluvialis is not enough, will the efficiency of conversion be caused not high.Therefore, the efficient preservation of Vegetative Cell of Haematococcus Pluvialis is the prerequisite continuing to nourish and grow and transform, and is directly connected to the production benefit of astaxanthin and the development of Haematocoocus Pluvialls industry.
Summary of the invention
The object of this invention is to provide a kind of store method of Vegetative Cell of Haematococcus Pluvialis, the vigor of quick growth conditions can also be kept after making the Haematocoocus Pluvialls of preservation have continuation cultivation, thus make up the deficiencies in the prior art.
Store method of the present invention, be by haematococcus pluvialis cell in nutrient solution after row amplification cultivation; Again the haematococcus pluvialis cell after cultivation is carried out preservation at the conserving liquid that the concentration ratio of nitrogen, phosphoric is 20-30:1 to cultivate, the temperature 12-20 DEG C of preservation, the wavelength of light is 660-740nm, and intensity of illumination is 500-1500lx.
In described conserving liquid, its a kind of proportioning includes following component: 1000mg/L NaNO
3, 5mg/L KH
2pO
4, 2.5mg/L FeSO
47H
2o, 0.25mg/L MnSO
4and 5.0mg/LEDTA-Na
2.
Preserving the light cultivated is the light that light source is sent, and is the red film of 0.08-0.12mm by thickness, and obtaining wavelength is the light of 660-740nm.
Also be added with Ampicillin Trihydrate in above-mentioned conserving liquid, adding concentration is 0.10-1.0 ten thousand u/L.Ampicillin Trihydrate can effectively prevent haematococcus pluvialis cell from forming cohesion, thus prevents Haematocoocus Pluvialls condensation in nutrient solution, when continued growth, shortens lag phase.
Density when wherein Haematocoocus Pluvialls is preserved is 9-15 ten thousand/ml.
The present invention compared with prior art, shelf time is long, be not easy aging, vegetative cell in nutrient solution can keep nourishing and growing, being cultured to plateau reaches maximum density, after plateau, also can keep 20-35 days, and carry out next step nutrition cultivation or conversion, can continue to keep growth conditions fast.The method is simple and easy to do, and the facility reducing conservation drops into, and reduces cost of labor.
Embodiment
Applicant finds in long-term research, and Vegetative Cell of Haematococcus Pluvialis is being carried out to amplification cultivation simultaneously, adjustment nutrient composition, make the concentration ratio of nitrogen in conserving liquid, phosphoric be 20-30:1, namely the concentration ratio of nitrogen, phosphoric substantially exceeds common ratio; And make temperature be 12-20 DEG C; Adding through optical wavelength between the light source irradiated and nutrient solution is the red film of 660-740nm; And the surperficial illumination condition of nutrient solution is 500-1500lx.Above-mentioned condition can effectively make the shelf time of Haematocoocus Pluvialls elongated, is not easy aging.
Below in conjunction with embodiment, store method of the present invention is described in detail.
Embodiment 1:
The NaNO of the preparation of nutrient solution: 120mg/L
3, 15mg/L KH
2pO
4, 2.5mg/L FeSO
47H
2the MnSO of O, 0.25mg/L
4, 2.5mg/L EDTA-Na
2.
The Vegetative Cell of Haematococcus Pluvialis density of inoculation is 3-5 ten thousand/ml, and at 20 DEG C, natural light irradiation, cultivation light intensity is 2000lx, and cultivate after 5 days, density is 9-15 ten thousand/ml, enters preservation state.
The preparation of conserving liquid, in conserving liquid, the content of main component is the NaNO of 1000mg/L
3, and the KH of 50mg/L
2pO
4, the FeSO of 2.5mg/L
47H
2the MnSO of O, 0.25mg/L
4, 5.0mg/L EDTA-Na
2.Conserving liquid directly can add NaNO in nutrient solution
3and KH
2pO
4make.
The control of preservation condition: adding through optical wavelength between the light source irradiated and nutrient solution is the red film of 660-740nm; And the surperficial illumination condition of conserving liquid is 500-1500lx.
Control temperature is 12-20 DEG C, and the additive capacity of Ampicillin Trihydrate is 0.10-1.0 ten thousand u/L.
Vegetative Cell of Haematococcus Pluvialis preserved according to the method described above, in preservation cultivation after 35 days, density is 15-25 ten thousand/ml; Measuring Motility Index by chlorophyll fluorescence instrument is 0.612.After restoration ecosystem, Motility Index is best 0.752.That is, above-mentioned method not only achieves the long-time preservation of Vegetative Cell of Haematococcus Pluvialis, and the cell preserved does not occur aging, easily enters follow-up conversion and the accumulation of astaxanthin.
Embodiment 2
The NaNO of the preparation of nutrient solution: 130mg/L
3, 18mg/L KH
2pO
4, 2.5mg/L FeSO
47H
2the MnSO of O, 0.25mg/L
4, 2.5mg/L EDTA-Na
2.
The Vegetative Cell of Haematococcus Pluvialis inoculated in the nutrient solution prepared, after making inoculation, in nutrient solution, the density of Vegetative Cell of Haematococcus Pluvialis is 3.2 ten thousand/ml, at 23 DEG C, and natural light irradiation, cultivating light intensity is after 2000lx cultivates 6 days, and density is 13.5 ten thousand cell/ml.Now in nutrient solution, add NaNO
3and KH
2pO
4as conserving liquid, make NaNO in conserving liquid
3content be 1200mg/L, KH
2pO
4for 40mg/L, and add Ampicillin Trihydrate, make the final concentration of Ampicillin Trihydrate in conserving liquid be 0.18 ten thousand u/L.Covered by the red film of nutrient solution, making by the wavelength of light is 660-740nm; And the surperficial illumination condition of conserving liquid is 1300lx, culture temperature is 15 DEG C, enters preservation cultivation conditions.
In preservation cultivation after 30 days, the density that microscopy measures Vegetative Cell of Haematococcus Pluvialis is 19.5 ten thousand cell/ml; Measuring Motility Index by chlorophyll fluorescence instrument is 0.654.After proceeding to normal culture condition again, Motility Index is 0.793 two days later.
To sum up, method of the present invention can effectively solve Vegetative Cell of Haematococcus Pluvialis preserve and aging between contradiction, there is good application value.
Claims (3)
1. a store method for Vegetative Cell of Haematococcus Pluvialis, is characterized in that, described store method is by haematococcus pluvialis cell row amplification cultivation in nutrient solution; Then haematococcus pluvialis cell is carried out preservation at the conserving liquid that the concentration ratio of nitrogen, phosphoric is 20-30:1 to cultivate, the temperature 12-20 DEG C of preservation, the wavelength of light is 660-740nm, and intensity of illumination is 500-1500lx;
Described conserving liquid composed as follows: 1000mg/L NaNO
3, 50mg/L KH
2pO
4, 2.5mg/L FeSO
47H
2o, 0.25mg/L MnSO
4with 5.0mg/L EDTA-Na
2;
Described light is that the light that light source is sent is obtained by the red film that thickness is 0.08-0.12mm;
The density of described Haematocoocus Pluvialls in conserving liquid is 9-15 ten thousand cell/ml.
2. store method as claimed in claim 1, is characterized in that, be added with Ampicillin Trihydrate in described conserving liquid.
3. store method as claimed in claim 2, it is characterized in that, the interpolation concentration of described Ampicillin Trihydrate is 0.10-1.0 ten thousand u/L.
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| KR101725976B1 (en) * | 2014-04-17 | 2017-04-26 | 고려대학교 산학협력단 | Method for Increasing a Productivity of Astaxanthin in Haematococcus Pluvialis by Mature Cyst Inoculated and Iron Ions Mediated Haber-Weiss Reaction at High Temperature |
| CN110199711B (en) * | 2019-06-20 | 2021-11-05 | 黑龙江省农业科学院耕作栽培研究所 | Accurate identification method for soybean pythium root rot resistance resources |
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Non-Patent Citations (2)
| Title |
|---|
| 抗菌素对海洋微藻静置培养生长和保存的影响;骆其君 等;《浙江海洋学院学报（自然科学版）》;20000930;第19卷(第3期);244-249 * |
| 雨生红球藻Haematococcus pluvialis绿色营养细胞的培养条件研究;刘健晖;《中国优秀博硕士学位论文全文数据库基础科学辑》;20070615;A006-76 * |
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