CN105779292A - Ultralow-temperature preservation method for haematococcus pluvialis subjected to aseptic treatment - Google Patents
Ultralow-temperature preservation method for haematococcus pluvialis subjected to aseptic treatment Download PDFInfo
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Abstract
The invention discloses an ultralow-temperature preservation method for haematococcus pluvialis subjected to aseptic treatment. The ultralow-temperature preservation method has the advantages that systematic sterilization work is carried out before the haematococcus pluvialis is preserved, the thawed aseptic haematococcus pluvialis is excellent in resuscitation survival rate owing to specific low-temperature preservation conditions, and the resuscitation survival rate of the haematococcus pluvialis can reach 66.13%; sterilized bacterial liquid on LB panels is free of bacterial colonies, and is free of bacterial colonies after the bacterial liquid is continuously cultivated thrice and is subjected to aseptic detection by the aid of the LB panels, sterilized haematococcus pluvialis strains are difficult to age, and accordingly effects of keeping the germplasm genetic stability and bringing convenience for storing the haematococcus pluvialis for a long time can be realized.
Description
Technical field
The present invention relates to the technical field of the microdisk electrode with high added value, be more particularly to the ultralow temperature method for preserving of a kind of Haematocoocus Pluvialls kind matter processed through asepticization
Background technology
Haematocoocus Pluvialls, it is under the jurisdiction of Chlorophyta (Chlorophyta) Chlorophyceae (Chlorophyceae) volvocales (Volvocales) Haematococcus Pluvialis section (Haematococcaceae) haematococcus (haematococcus), when environment is bad, its green swarm cell is converted into red akinete, and accumulates astaxanthin.Astaxanthin is the bioactive product of a kind of high value, has powerful antioxidant activity, has extensive use in the industries such as aquaculture, food, cosmetics, medicine.Owing to the content astaxanthin in Haematocoocus Pluvialls is the highest, the best biogenetic derivation being acknowledged as in nature astaxanthin, therefore become the focus of the application of algae exploitation in recent years.And asepticization of Haematocoocus Pluvialls is the basic and key link of the exploitation application such as high-density breeding and physiological and biochemical research.And how keeping asepticization of algae kind and fine quality is also one of bottleneck problem restricting Haematococcus Pluvialis developmental research.
Asepticization of microalgae processes and includes physical method and chemical method, and physical method is method of dilution butteron on plate, ultrasonic cleaning process, ultraviolet radiation method etc. such as, but physical method is generally difficult to obtain pure strain, and chemical method generally uses antibiotic method.The growth and breeding of antibacterial is had very high inhibition effect by antibiotic.Utilize chlorella for the strong feature of anti-biotic resistance energy force rate antibacterial, adopt antibiotic treatment chlorella, aseptic strain can be obtained.But too high antibiotic concentration will cause the death of algae kind, too low antibiotic concentration is then to the antibacterial unrestraint effect in algae solution.And different class of antibiotic and different orders of addition there is also difference for the rejection ability of antibacterial in algae solution.
The mode of traditional liquid subculture preservation algae kind exist complex operation, workload greatly, easily pollute, the problem such as genetic drift, algae kind merit degeneration.Algae Germplasm cryopreservation technology has maintenance blastogenesis stability, is easy to long-term preservation, can farthest reduce the advantage of the aspects such as pollution, therefore increasingly receives publicity, and is widely used in the long term storage of a large amount of microalgae.And for Haematocoocus Pluvialls ultralow temperature preservation, due to affected by various factors, therefore also do not have correlation technique to report.
Summary of the invention
For the problems referred to above, the present invention provides the ultralow temperature method for preserving of a kind of Haematocoocus Pluvialls processed through asepticization, the method utilizing the present invention, and algae strain can be made to reach asepticization, keep the merit of algae strain, in order to carry out exploitation application and the physiological and biochemical research such as algae kind high-density breeding.
In order to achieve the above object, the present invention takes techniques below measure:
A kind of ultralow temperature method for preserving of the Haematocoocus Pluvialls processed through asepticization, including:
1) in the Haematocoocus Pluvialls algae solution be in the exponential growth later stage, add the ampicillin of final concentration of 100～500ug/ml, cultivate 1～3 day, continuously add the gentamycin of final concentration of 1～10ug/ml, cultivate 1～3 day, continuously add the kanamycin of final concentration of 1～10ug/ml, be further cultured for 2 days；Algae solution after asepticization of learning from else's experience process is coated on LB flat board, after preliminary identification is aseptic, then through 3 Secondary Culture, thoroughly eliminates the impact of residual antibiotic, again carries out Sterility testing with LB flat board, and Haematocoocus Pluvialls aseptic after testing is for ultralow temperature preservation.
Above condition of culture is room temperature (20～25 degrees Celsius) quiescent culture.
In approach described above, it is preferred that the final concentration of 100～400ug/ml of ampicillin, cultivate 1～2 day, continuously add the gentamycin of final concentration of 1～8ug/ml, cultivate 1～2 day, continuously add the kanamycin of final concentration of 1～8ug/ml, be further cultured for 2 days.
In approach described above, it is preferred that the final concentration of 100～300ug/ml of ampicillin, cultivate 1～2 day, continuously add the gentamycin of final concentration of 1～5ug/ml, cultivate 1～2 day, continuously add the kanamycin of final concentration of 1～5ug/ml, be further cultured for 2 days；
In approach described above, it is preferred that the final concentration of 300ug/ml of ampicillin, after cultivating 2 days, continuously add the gentamycin of final concentration of 5ug/ml, cultivate 2 days, continuously add the kanamycin of final concentration of 5ug/ml, be further cultured for 2 days.
2). Haematocoocus Pluvialls akinete liquid and glycerol are mixed for 1:1 by volume so that in mixed liquor final concentration of the 1.5 × 10 of cell density5～5 × 105Individual/ml, final concentration of 5～25% (volume ratio) room temperature of glycerol stands 5～20min, is then placed in programmed cooling instrument, 0.5 DEG C/min～5 DEG C/min, pre-freeze, to-30 DEG C～-80 DEG C, keeps 30min～120min in this temperature, is then placed in liquid nitrogen container and preserves.
In approach described above, it is preferred that:
Final concentration of the 5%～15% of glycerol；Rate of temperature fall is 0.5 DEG C/min～2 DEG C/min, and pre-freeze, to-30 DEG C～-60 DEG C, keeps 30min～60min in this temperature.
In approach described above, it is preferred that:
Final concentration of the 15% of glycerol；In mixed liquor final concentration of the 5 × 10 of cell density5Individual/ml；Rate of temperature fall is 1 DEG C/min, and pre-freeze, to-40 DEG C, keeps 30min.
Approach described above, the Haematocoocus Pluvialls of low-temperature preservation is recovered in the following manner:
In liquid nitrogen container after preservation three months, sample is taken out, put into rapidly in 20 DEG C～60 DEG C water-baths and thaw, until last ice crystal disappears, then as carrying out the centrifugal 5min of 5000rpm in centrifuge, it is resuspended to add sterilized BG11 culture medium, recentrifuge, rotating speed is 5000rpm, and centrifugation time is 5min.Frustule after centrifugal is inoculated in fresh BG11 culture medium, after cultivating 7d, measures their cell density with cell counting count board.
In approach described above, it is preferred that: bath temperature is 40 DEG C.
Compared with prior art, the invention have the advantages that
1. utilizing method provided by the invention, the aseptic Haematocoocus Pluvialls after defrosting has good recovery survival rate, up to 66.13%.
2. utilizing method provided by the invention, the algae solution after degerming occurs without bacterial clump on LB flat board, and after 3 Secondary Culture, again carries out Sterility testing with LB flat board, also occurs without bacterial clump, and the algae strain after degerming is not easily aging.
Detailed description of the invention
Technical scheme of the present invention, if not otherwise specified, is the conventional scheme of this area.Described reagent or material, if not otherwise specified, derive from commercial channel.
Embodiment 1:
The method of the present invention, for Haematocoocus Pluvialls FACHB-712, is illustrated by the present embodiment.The method detection of the coated LB flat board of Haematocoocus Pluvialls used by the present embodiment is the Haematocoocus Pluvialls containing bacterium.
A kind of ultralow temperature method for preserving of the Haematocoocus Pluvialls processed through asepticization, including:
1). process group (totally three groups of repetitions): add the ampicillin of final concentration of 300ug/ml in the Haematocoocus Pluvialls FACHB-712 algae solution 30ml be in the exponential growth later stage, cultivate 2 days, continuously add the gentamycin of final concentration of 5ug/ml, cultivate 2 days, continuously add the kanamycin of final concentration of 5ug/ml, be further cultured for 2 days.
Matched group: except without antibiotic, all the other condition of culture are all identical with process group.
Above condition of culture: intensity of illumination is 10umol/m2/ s, Light To Dark Ratio is 12h:12h, and cultivation temperature is 22 ± 1 DEG C, quiescent culture.
Utilize said method, detect after utilizing said method to process Haematocoocus Pluvialls, the specific growth rate of process group is compared with matched group, process group is more slightly higher than matched group at the middle and late stage of exponential growth, the final Biomass of process group simultaneously is also high than matched group, illustrate to utilize the Haematocoocus Pluvialls that above-mentioned sterilisation process processes to have no effect on its proliferation rates, the growth of Haematocoocus Pluvialls can be promoted on the contrary.
Wherein: specific growth rate u=(lnNt-lnN0)/T, cell number when wherein Nt is cultivate t days, cell number when N0 is initial, T is incubation time.
Then taking 100ul algae solution after asepticization processes to coat on LB flat board, assert aseptic, then through 3 Secondary Culture, thoroughly eliminate the impact of residual antibiotic, again detect with LB flat board, Haematocoocus Pluvialls aseptic after testing is used for ultralow temperature preservation.
Step 1) in three process groups after asepticization processes, through LB flat board primary detection, aseptic；Go down to posterity after 3 these elimination antibiotic remains impacts, again detect all aseptic with LB flat board.
2) Haematocoocus Pluvialls akinete liquid and glycerol are mixed for 1:1 by volume so that in mixed liquor final concentration of the 3 × 10 of cell density5Individual/ml, final concentration of 15% (volume ratio) room temperature of glycerol stands 10min, is then placed in programmed cooling instrument, and rate of temperature fall is 1 DEG C/min, and pre-freeze, to-40 DEG C, keeps 30min, is then placed in liquid nitrogen container and preserves.
In liquid nitrogen container after preservation three months, sample is taken out, put into rapidly in 40 DEG C of water-baths and thaw, until last ice crystal disappears, it is subsequently placed in eppendorf5810R centrifuge and carries out the centrifugal 5min of 5000rpm, add sterilized BG11 culture medium and carry out resuspended, recentrifuge, rotating speed is 5000rpm, and centrifugation time is 5min.Frustule after eccentric cleaning is inoculated in fresh BG11 culture medium, after cultivating 7d, measures their cell density with cell counting count board.The density of the living cells when growth curve of the not freezing frustule according to different vaccination density can extrapolate freezing sample and control sample inoculation.The percent value of the two is the survival rate of freezen protective, and the survival rate utilizing the calculated Haematocoocus Pluvialls of said method is 66.13%, and the Haematocoocus Pluvialls after recovery is aseptic through the detection of LB flat board, and no significant difference before specific growth rate and low-temperature preservation.
Claims (7)
1. a ultralow temperature method for preserving for the Haematocoocus Pluvialls processed through asepticization, including:
1) in the Haematocoocus Pluvialls algae solution be in the exponential growth later stage, add the ampicillin of final concentration of 100～500ug/ml, cultivate 1～3 day, continuously add the gentamycin of final concentration of 1～10ug/ml, cultivate 1～3 day, continuously add the kanamycin of final concentration of 1～10ug/ml, be further cultured for 2 days；Algae solution after asepticization of learning from else's experience process is coated on LB flat board, after preliminary identification is aseptic, then through 3 Secondary Culture, thoroughly eliminates the impact of residual antibiotic, again carries out Sterility testing with LB flat board, and Haematocoocus Pluvialls aseptic after testing is for ultralow temperature preservation；
2) Haematocoocus Pluvialls akinete liquid and glycerol are mixed for 1:1 by volume so that in mixed liquor final concentration of the 1.5 × 10 of cell density5~5×105Individual/ml, final concentration of the 5 ~ 25% of glycerol, room temperature stands 5 ~ 20min, is then placed in programmed cooling instrument, 0.5 DEG C/min～5 DEG C/min, and pre-freeze, to-30 DEG C～-80 DEG C, keeps 30min～120min in this temperature, is then placed in liquid nitrogen container and preserves.
2. method according to claim 1, it is characterised in that: the final concentration of 100～400ug/ml of ampicillin, cultivates 1～2 day, continuously add the gentamycin of final concentration of 1～8ug/ml, cultivate 1～2 day, continuously add the kanamycin of final concentration of 1～8ug/ml, be further cultured for 2 days.
3. method according to claim 1, it is characterised in that: the final concentration of 100～300ug/ml of ampicillin, cultivates 1～2 day, continuously add the gentamycin of final concentration of 1～5ug/ml, cultivate 1～2 day, continuously add the kanamycin of final concentration of 1～5ug/ml, be further cultured for 2 days.
4. method according to claim 1, it is characterised in that: the final concentration of 300ug/ml of ampicillin, after cultivating 2 days, continuously add the gentamycin of final concentration of 5ug/ml, cultivate 2 days, continuously add the kanamycin of final concentration of 5ug/ml, be further cultured for 2 days.
5. method according to claim 1, it is characterised in that: final concentration of the 5%～15% of glycerol；Rate of temperature fall is 0.5 DEG C/min～2 DEG C/min, and pre-freeze, to-30 DEG C～-60 DEG C, keeps 30min～60min in this temperature.
6. method according to claim 1, it is characterised in that: final concentration of the 15% of glycerol；In mixed liquor final concentration of the 5 × 10 of cell density5Individual/ml；Rate of temperature fall is 1 DEG C/min, and pre-freeze, to-40 DEG C, keeps 30min.
7. method according to claim 1, after Haematocoocus Pluvialls low-temperature preservation, the method of recovery includes: in liquid nitrogen container after preservation three months, is taken out by sample, puts into rapidly in 20 DEG C～60 DEG C water-baths and thaw, until last ice crystal disappears, then as carrying out the centrifugal 5min of 5000rpm in centrifuge, it is resuspended to add sterilized BG11 culture medium, recentrifuge, rotating speed is 5000rpm, and centrifugation time is 5min；Frustule after centrifugal is inoculated in fresh BG11 culture medium, after cultivating 7d, measures their cell density with cell counting count board.
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Cited By (10)
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| CN106520557A (en) * | 2016-11-24 | 2017-03-22 | 王江新 | Sterilization method of euglenophyta culture solution |
| CN106755250A (en) * | 2016-12-27 | 2017-05-31 | 山东金晶生物技术有限公司 | A kind of preservation of haematococcus pluvialis green cell and the large-scale method for producing of astaxanthin induction |
| CN107058110A (en) * | 2017-07-04 | 2017-08-18 | 中国科学院青岛生物能源与过程研究所 | A kind of preservation of haematococcus pluvialis cell and method for resuscitation |
| CN107593416A (en) * | 2017-08-10 | 2018-01-19 | 内蒙古农业大学 | A kind of cryopreservation method of spirulina algae kind |
| CN107815417A (en) * | 2017-11-23 | 2018-03-20 | 武汉大学深圳研究院 | The Cryopreservation of the hidden dinoflagellates of Kou Shi |
| CN108865894A (en) * | 2018-09-03 | 2018-11-23 | 中国科学院青岛生物能源与过程研究所 | A kind of preparation method of sterile micro- quasi- ball algae |
| CN110577898A (en) * | 2019-10-25 | 2019-12-17 | 四川轻化工大学 | Method for producing astaxanthin by culturing haematococcus pluvialis through discontinuous two-step method |
| CN110982698A (en) * | 2019-11-08 | 2020-04-10 | 厦门大学 | Method for freezing and preserving haematococcus pluvialis by using dimethyl sulfoxide |
| CN114214201A (en) * | 2021-12-23 | 2022-03-22 | 日照职业技术学院 | Method for long-term stable storage of marine nannochloropsis oceanica |
| CN117136833A (en) * | 2023-08-18 | 2023-12-01 | 生态环境部华南环境科学研究所(生态环境部生态环境应急研究所) | Sargassum periwinkle germplasm preservation method |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106520557A (en) * | 2016-11-24 | 2017-03-22 | 王江新 | Sterilization method of euglenophyta culture solution |
| CN106755250A (en) * | 2016-12-27 | 2017-05-31 | 山东金晶生物技术有限公司 | A kind of preservation of haematococcus pluvialis green cell and the large-scale method for producing of astaxanthin induction |
| CN106755250B (en) * | 2016-12-27 | 2020-06-02 | 山东金晶生物技术有限公司 | Haematococcus pluvialis green cell preservation and astaxanthin-induced large-scale production method |
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| CN107058110B (en) * | 2017-07-04 | 2021-03-16 | 中国科学院青岛生物能源与过程研究所 | Preservation and recovery method of haematococcus pluvialis cells |
| CN107593416A (en) * | 2017-08-10 | 2018-01-19 | 内蒙古农业大学 | A kind of cryopreservation method of spirulina algae kind |
| CN107593416B (en) * | 2017-08-10 | 2020-04-10 | 内蒙古农业大学 | Ultralow-temperature preservation method for spirulina seeds |
| CN107815417A (en) * | 2017-11-23 | 2018-03-20 | 武汉大学深圳研究院 | The Cryopreservation of the hidden dinoflagellates of Kou Shi |
| CN107815417B (en) * | 2017-11-23 | 2022-04-29 | 武汉大学深圳研究院 | Low-temperature preservation method of Crypthecodinium cohnii |
| CN108865894A (en) * | 2018-09-03 | 2018-11-23 | 中国科学院青岛生物能源与过程研究所 | A kind of preparation method of sterile micro- quasi- ball algae |
| CN108865894B (en) * | 2018-09-03 | 2021-05-28 | 中国科学院青岛生物能源与过程研究所 | Preparation method of sterile nannochloropsis oculata |
| CN110577898A (en) * | 2019-10-25 | 2019-12-17 | 四川轻化工大学 | Method for producing astaxanthin by culturing haematococcus pluvialis through discontinuous two-step method |
| CN110577898B (en) * | 2019-10-25 | 2023-01-31 | 四川轻化工大学 | Method for producing astaxanthin by culturing haematococcus pluvialis through discontinuous two-step method |
| CN110982698A (en) * | 2019-11-08 | 2020-04-10 | 厦门大学 | Method for freezing and preserving haematococcus pluvialis by using dimethyl sulfoxide |
| CN114214201A (en) * | 2021-12-23 | 2022-03-22 | 日照职业技术学院 | Method for long-term stable storage of marine nannochloropsis oceanica |
| CN114214201B (en) * | 2021-12-23 | 2024-02-27 | 日照职业技术学院 | Method for long-term stable preservation of marine nannochloropsis |
| CN117136833A (en) * | 2023-08-18 | 2023-12-01 | 生态环境部华南环境科学研究所(生态环境部生态环境应急研究所) | Sargassum periwinkle germplasm preservation method |
| CN117136833B (en) * | 2023-08-18 | 2024-05-24 | 生态环境部华南环境科学研究所(生态环境部生态环境应急研究所) | Sargassum periwinkle germplasm preservation method |
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