CN106811417A - A kind of culture medium and its cultural method for Alexandrium mimutum Halim - Google Patents
A kind of culture medium and its cultural method for Alexandrium mimutum Halim Download PDFInfo
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Abstract
The present invention relates to microbial technology field, and in particular to a kind of culture medium and its cultural method for Alexandrium mimutum Halim.Culture medium specifically includes each component of following molar concentration：Sodium nitrate is 1766 μm of ol/L, sodium dihydrogen phosphate is 18.15 μm of ol/L, iron chloride is 5 μm of ol/L, Na2EDTA is 5 μm of ol/L, and copper sulphate is 0.02 μm of ol/L, and sodium molybdate is 0.015 μm of ol/L, zinc sulfate is 0.04 μm of ol/L, cobalt chloride is 0.025 μm of ol/L, and manganese chloride is 0.45 μm of ol/L, natural sea-water.Using the Alexandrium mimutum Halim of the medium culture, poison amount of producing and can be greatly improved, and cultural method therein is continuous cultural method, it is time saving and energy saving.
Description
Technical field
The present invention relates to microbial technology field, and in particular to a kind of culture medium and its training for Alexandrium mimutum Halim
The method of supporting.
Background technology
Alexandrium mimutum Halim（Alexandrium minutum）It is subordinate to Protista（Protoctista）, Pyrrhophyta
（Dinoflagellata）, Alexandria category（Alexandrium）, it is a kind of Marine Planktonic dinoflagellate for having a first piece, cell size
At 13-30 μm, it is widely distributed and with produce paralytic shellfish poisoning (PSP)（paralytic shellfish toxins PSTs）Red tide
It is relevant.Its gonyatoxin (gonyautoxin) GTX1, GTX2, GTX3, GTX4 for producing in vivo, is enriched with, most through mollusk
Shellfish, fish and the mankind are passed to by food chain eventually, the serious paralytic shellfish poisoning (PSP) of people can be caused to be poisoned（paralytic
shellfish poisoning PSP）, the country occurred many causes eat cause containing the basket whelk of paralytic shellfish poisoning (PSP) it is dead
The accident died.
Paralytic shellfish poisoning (PSP) is the alkaloid that there is a class neuron excitotoxicity to act on, and its mechanism of poisoning is that toxin combines sodium
Ion channel structure, hinders the flowing of sodium ion.There is no antidote after poisoning, unique rescue method is artificial respiration and defeated
Liquid.PSTs is because its toxicity is strong, colorless and odorless and with paralysis effect, militarily there is the possibility for being applied to chemical and biological weapons,
Medically then as the precursor substance for exploring Novel anesthetic, and in terms of environment and food inspection, the existing part of foreign countries
PSTs toxin standard items are sold, but price is expensive.
Preparing gonyatoxin needs substantial amounts of raw material, is generally obtained by cultivating the malicious microalgae of product.Small sub- Lishan Mountain
Big algae produces poison amount and is not only subject to the such environmental effects such as light intensity, temperature, salinity, also by the nutrient formulations such as nitrogen phosphorus, training method
Influence.Know that Alexandrium mimutum Halim is generally incubated in f/2 sea water mediums and K sea water mediums, but they are simultaneously
It is non-to produce the optimal nutrient formulation of poison, if with its large-scale culture marine alga, the waste and cost raising of resource can be caused.
The content of the invention
The invention aims to solve the problems, such as above-mentioned Alexandrium mimutum Halim institute, there is provided one kind is for small
The culture medium and its cultural method of Alexandrium.
Further, since the growth of Alexandrium is more sensitive to disturbance, this case uses semi-continuous training method, culture
Cycle is shorter, time saving and energy saving.It is that a kind of Alexandrium mimutum Halim that is especially suitable for produces malicious culture medium, its method is a kind of efficient
Cultural method.
Here, a kind of culture medium for cultivating Alexandrium mimutum Halim and Semi-continuous cultivation method, Neng Gouxian are disclosed
Write to improve and produce malicious content.
In order to reach foregoing invention purpose, the present invention uses following technical scheme：
A kind of culture medium for Alexandrium mimutum Halim, specifically includes each component of following molar concentration：Sodium nitrate is 1766 μ
Mol/L, sodium dihydrogen phosphate is 18.15 μm of ol/L, and iron chloride is 5 μm of ol/L, Na2EDTA is 5 μm of ol/L, and copper sulphate is 0.02 μ
Mol/L, sodium molybdate is 0.015 μm of ol/L, and zinc sulfate is 0.04 μm of ol/L, and cobalt chloride is 0.025 μm of ol/L, and manganese chloride is 0.45
μm ol/L, natural sea-water.
Used as further preferred, the culture medium also includes 0.01 μm of ol/L of samaric nitrate.
Used as further preferred, the collocation method of the culture medium is specific as follows：
（1）Natural sea-water is stood, and takes supernatant seawater through 0.22 μm of membrane filtration, and in transparent culture vessel, 121 DEG C of high pressures are steamed
Vapour sterilizes 20 minutes；
（2）Each component is configured to storing solution first, sodium nitrate Stock concentrations are 1766mmol/L, sodium dihydrogen phosphate storing solution
Concentration is 18.15mmol/L, and minor metallic element storing solution is configured to combine storing solution, chloride containing iron 5mmol/L,
Na2EDTA5mmol/L, copper sulphate 0.02mmol/L, sodium molybdate 0.015mmol/L, zinc sulfate 0.04mmol/L, cobalt chloride
0.025mmol/L, manganese chloride 0.45mmol/L, by storing solution also in 121 DEG C of high pressure steam sterilizations 20 minutes；
（3）In superclean bench or culturing room after ultraviolet sterilization 30 minutes, by 1 milliliter of nitrogen storing solution, 1 milliliter of phosphorus deposit
Liquid, 1 milliliter of minor metallic element storing solution are added in sterilizing seawater, are settled to 1L, are configured to it dense specified in formula
Degree, culture medium is to complete to prepare.
Used as further preferred, natural sea-water salinity is adjusted to 24 ‰ salinity by adding sodium chloride or pure water.
A kind of cultural method using above-mentioned medium culture Alexandrium mimutum Halim, specifically includes following steps：
（1）To be inoculated in above-mentioned culture medium in exponential phase algae kind, regulation initial density is 5000 ± 200cell/ml,
Temperature is 25 DEG C, and illumination is 125 ± 12.5 μm of ol/ (m2S), light application time ratio is quiescent culture under the conditions of 12h: 12h round the clock
8-9 days；
（2）The culture medium of isometric above-mentioned brand-new is added wherein, culture is enlarged, and is cultivated 4-5 days；
（3）Repeat step（2）Incubation, be continuously increased the volume of nutrient solution；
（4）In a step（2）Repetitive process after the completion of, by incubator algae solution take out part, be placed in 20 DEG C of temperature, light
According to 125 ± 12.5 μm of ol/ (m2S), light application time ratio is quiescent culture under the conditions of 12h: 12h round the clock, can be received after 3 ~ 5 days
Frustule is obtained, meanwhile, to the culture medium of isometric brand-new that remaining algae solution is supplemented in incubator, with remaining algae solution in incubator
Mixing, repeat step（2）Incubation；
（5）Continuous repeat step（2）And step（4）, get the Alexandrium mimutum Halim of content of toxins high.
As further preferred, step（4）In, to sodium nitrate is added in the algae solution taken out, addition is every liter of algae solution
1766umol。
Compared with prior art, beneficial effect is the present invention：
1st, product poison amount is improve, poison amount is produced than existing（5.31μmol/L）Improve 48.6%, up to 7.89 μm ol/L knee ditches
Algae toxins；
2nd, it is Semi-continuous cultivation mode, can constantly obtains frustule, shortens cultivation cycle, improve production efficiency.Incubation time
It was shorten to 7 ~ 10 days from existing 22 ~ 30 days.
3rd, nutrient categories are reduced, operation is simplified, spending has been saved, for example, of the invention compared with existing culture medium
Omit the addition of vitamin.
Specific embodiment
Explanation is further described to technical scheme below by specific embodiment.
If without specified otherwise, the raw material employed in embodiments of the invention is raw material commonly used in the art, implements
Method employed in example, is the conventional method of this area.
Embodiment 1：
A kind of culture medium for Alexandrium mimutum Halim, specifically includes each component of following molar concentration：Sodium nitrate is 1766 μ
Mol/L, sodium dihydrogen phosphate is 18.15 μm of ol/L, and iron chloride is 5 μm of ol/L, Na2EDTA is 5 μm of ol/L, and copper sulphate is 0.02 μ
Mol/L, sodium molybdate is 0.015 μm of ol/L, and zinc sulfate is 0.04 μm of ol/L, and cobalt chloride is 0.025 μm of ol/L, and manganese chloride is 0.45
μm ol/L, natural sea-water.
The collocation method of the culture medium is specific as follows：
（1）Natural sea-water is stood, and takes supernatant seawater through 0.22 μm of membrane filtration, and in transparent culture vessel, 121 DEG C of high pressures are steamed
Vapour sterilizes 20 minutes；Natural sea-water salinity is adjusted to 24 ‰ salinity by adding sodium chloride or pure water；
（2）Each component is configured to storing solution first, sodium nitrate Stock concentrations are 1766mmol/L, sodium dihydrogen phosphate storing solution
Concentration is 18.15mmol/L, and minor metallic element storing solution is configured to combine storing solution, chloride containing iron 5mmol/L,
Na2EDTA5mmol/L, copper sulphate 0.02mmol/L, sodium molybdate 0.015mmol/L, zinc sulfate 0.04mmol/L, cobalt chloride
0.025mmol/L, manganese chloride 0.45mmol/L, by storing solution also in 121 DEG C of high pressure steam sterilizations 20 minutes；
（3）In superclean bench or culturing room after ultraviolet sterilization 30 minutes, by 1 milliliter of nitrogen storing solution, 1 milliliter of phosphorus deposit
Liquid, 1 milliliter of minor metallic element storing solution are added in sterilizing seawater, are settled to 1L, are configured to it dense specified in formula
Degree, culture medium is to complete to prepare.
A kind of cultural method using above-mentioned medium culture Alexandrium mimutum Halim, specifically includes following steps：
（1）To be inoculated in above-mentioned culture medium in exponential phase algae kind, regulation initial density is 5000 ± 200cell/ml,
Temperature is 25 DEG C, and illumination is 125 ± 12.5 μm of ol/ (m2S), light application time ratio is quiescent culture under the conditions of 12h: 12h round the clock
8-9 days；
（2）The culture medium of isometric above-mentioned brand-new is added wherein, culture is enlarged, and is cultivated 4-5 days；
（3）Repeat step（2）Incubation, be continuously increased the volume of nutrient solution；
（4）In a step（2）Repetitive process after the completion of, the algae solution in incubator is taken out into part, and be added to nitre
Sour sodium, addition is every liter of algae solution 1766umol；It is subsequently placed in 20 DEG C of temperature, 125 ± 12.5 μm of ol/ (m of illumination2S), round the clock
Light application time ratio is quiescent culture under the conditions of 12h: 12h, and frustule can be harvested after 3 ~ 5 days, meanwhile, supplemented in incubator
The culture medium of isometric brand-new of remaining algae solution, mixes, repeat step with remaining algae solution in incubator（2）Incubation；
（5）Continuous repeat step（2）And step（4）, get the Alexandrium mimutum Halim of content of toxins high.
To sodium nitrate is added in the algae solution taken out, addition is every liter of algae solution 1766umol.
Embodiment 2：
A kind of culture medium for Alexandrium mimutum Halim, specifically includes each component of following molar concentration：Sodium nitrate is 1766 μ
Mol/L, sodium dihydrogen phosphate is 18.15 μm of ol/L, and iron chloride is 5 μm of ol/L, Na2EDTA is 5 μm of ol/L, and copper sulphate is 0.02 μ
Mol/L, sodium molybdate is 0.015 μm of ol/L, and zinc sulfate is 0.04 μm of ol/L, and cobalt chloride is 0.025 μm of ol/L, and manganese chloride is 0.45
μm ol/L, 0.01 μm of ol/L of samaric nitrate, natural sea-water.
The collocation method of the culture medium is specific as follows：
（1）Natural sea-water is stood, and takes supernatant seawater through 0.22 μm of membrane filtration, and in transparent culture vessel, 121 DEG C of high pressures are steamed
Vapour sterilizes 20 minutes；Natural sea-water salinity is adjusted to 24 ‰ salinity by adding sodium chloride or pure water；
（2）Each component is configured to storing solution first, sodium nitrate Stock concentrations are 1766mmol/L, sodium dihydrogen phosphate storing solution
Concentration is 18.15mmol/L, and minor metallic element storing solution is configured to combine storing solution, chloride containing iron 5mmol/L,
Na2EDTA5mmol/L, copper sulphate 0.02mmol/L, sodium molybdate 0.015mmol/L, zinc sulfate 0.04mmol/L, cobalt chloride
0.025mmol/L, manganese chloride 0.45mmol/L, samaric nitrate 0.01mmol/L, by storing solution also in 121 DEG C of high pressure steam sterilizations 20
Minute；
（3）In superclean bench or culturing room after ultraviolet sterilization 30 minutes, by 1 milliliter of nitrogen storing solution, 1 milliliter of phosphorus deposit
Liquid, 1 milliliter of minor metallic element storing solution are added in sterilizing seawater, are settled to 1L, are configured to it dense specified in formula
Degree, culture medium is to complete to prepare.
A kind of cultural method using above-mentioned medium culture Alexandrium mimutum Halim, specifically includes following steps：
（1）To be inoculated in above-mentioned culture medium in exponential phase algae kind, regulation initial density is 5000 ± 200cell/ml,
Temperature is 25 DEG C, and illumination is 125 ± 12.5 μm of ol/ (m2S), light application time ratio is quiescent culture under the conditions of 12h: 12h round the clock
8-9 days；
（2）The culture medium of isometric above-mentioned brand-new is added wherein, culture is enlarged, and is cultivated 4-5 days；
（3）Repeat step（2）Incubation, be continuously increased the volume of nutrient solution；
（4）In a step（2）Repetitive process after the completion of, the algae solution in incubator is taken out into part, and be added to nitre
Sour sodium, addition is every liter of algae solution 1766umol；It is subsequently placed in 20 DEG C of temperature, 125 ± 12.5 μm of ol/ (m of illumination2S), round the clock
Light application time ratio is quiescent culture under the conditions of 12h: 12h, and frustule can be harvested after 3 ~ 5 days, meanwhile, supplemented in incubator
The culture medium of isometric brand-new of remaining algae solution, mixes, repeat step with remaining algae solution in incubator（2）Incubation；
（5）Continuous repeat step（2）And step（4）, get the Alexandrium mimutum Halim of content of toxins high.
The Alexandrium mimutum Halim that embodiment 1 and embodiment 2 are cultivated is measured, concrete outcome is：
It is every liter of algae solution of 6.63umol gonyatoxins that the frustule harvested in embodiment 1 produces poison amount.The algae harvested in embodiment 2
It is every liter of algae solution of 7.89umol gonyatoxins that cell produces poison amount.The addition of samaric nitrate, is positive, phase for follow-up influence
Than being not added with, it is significantly improved.
Claims (6)
1. a kind of culture medium for Alexandrium mimutum Halim, it is characterised in that specifically include each component of following molar concentration：
Sodium nitrate is 1766 μm of ol/L, and sodium dihydrogen phosphate is 18.15 μm of ol/L, and iron chloride is 5 μm of ol/L, Na2EDTA is 5 μm of ol/L,
Copper sulphate is 0.02 μm of ol/L, and sodium molybdate is 0.015 μm of ol/L, and zinc sulfate is 0.04 μm of ol/L, and cobalt chloride is 0.025 μm of ol/
L, manganese chloride is 0.45 μm of ol/L, natural sea-water.
2. a kind of culture medium for Alexandrium mimutum Halim according to claim 1, it is characterised in that the culture medium
Also include 0.01 μm of ol/L of samaric nitrate.
3. a kind of culture medium for Alexandrium mimutum Halim according to claim 1, it is characterised in that the culture medium
Collocation method is specific as follows：
（1）Natural sea-water is stood, and takes supernatant seawater through 0.22 μm of membrane filtration, and in transparent culture vessel, 121 DEG C of high pressures are steamed
Vapour sterilizes 20 minutes；
（2）Each component is configured to storing solution first, sodium nitrate Stock concentrations are 1766mmol/L, sodium dihydrogen phosphate storing solution
Concentration is 18.15mmol/L, and minor metallic element storing solution is configured to combine storing solution, chloride containing iron 5mmol/L,
Na2EDTA5mmol/L, copper sulphate 0.02mmol/L, sodium molybdate 0.015mmol/L, zinc sulfate 0.04mmol/L, cobalt chloride
0.025mmol/L, manganese chloride 0.45mmol/L, by storing solution also in 121 DEG C of high pressure steam sterilizations 20 minutes；
（3）In superclean bench or culturing room after ultraviolet sterilization 30 minutes, by 1 milliliter of nitrogen storing solution, 1 milliliter of phosphorus deposit
Liquid, 1 milliliter of minor metallic element storing solution are added in sterilizing seawater, are settled to 1L, are configured to it dense specified in formula
Degree, culture medium is to complete to prepare.
4. a kind of culture medium for Alexandrium mimutum Halim according to claim 1 or 3, it is characterised in that natural sea-water
Salinity is adjusted to 24 ‰ salinity by adding sodium chloride or pure water.
5. a kind of cultural method using above-mentioned medium culture Alexandrium mimutum Halim, it is characterised in that specifically include following
Step：
（1）To be inoculated in above-mentioned culture medium in exponential phase algae kind, regulation initial density is 5000 ± 200cell/ml,
Temperature is 25 DEG C, and illumination is 125 ± 12.5 μm of ol/ (m2S), light application time ratio is quiescent culture under the conditions of 12h: 12h round the clock
8-9 days；
（2）The culture medium of isometric above-mentioned brand-new is added wherein, culture is enlarged, and is cultivated 4-5 days；
（3）Repeat step（2）Incubation, be continuously increased the volume of nutrient solution；
（4）In a step（2）Repetitive process after the completion of, by incubator algae solution take out part, be placed in 20 DEG C of temperature, light
According to 125 ± 12.5 μm of ol/ (m2S), light application time ratio is quiescent culture under the conditions of 12h: 12h round the clock, can be received after 3 ~ 5 days
Frustule is obtained, meanwhile, to the culture medium of isometric brand-new that remaining algae solution is supplemented in incubator, with remaining algae solution in incubator
Mixing, repeat step（2）Incubation；
（5）Continuous repeat step（2）And step（4）, get the Alexandrium mimutum Halim of content of toxins high.
6. cultural method according to claim 5, it is characterised in that step（4）In, to adding nitre in the algae solution taken out
Sour sodium, addition is every liter of algae solution 1766umol.
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| CN109182132A (en) * | 2018-08-16 | 2019-01-11 | 浙江海洋大学 | Produce the cultural method of the Alexandrium mimutum Halim of gonyatoxin |
| CN111122774A (en) * | 2019-12-06 | 2020-05-08 | 国家海洋环境监测中心 | Method for evaluating ecological risk safety threshold of toxic microalgae |
| CN116396863A (en) * | 2023-04-13 | 2023-07-07 | 自然资源部第一海洋研究所 | Alexander algae cultivation method |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109182132A (en) * | 2018-08-16 | 2019-01-11 | 浙江海洋大学 | Produce the cultural method of the Alexandrium mimutum Halim of gonyatoxin |
| CN109182132B (en) * | 2018-08-16 | 2021-09-14 | 浙江海洋大学 | Method for culturing Alexandrium micranthum producing gonyautoxin |
| CN111122774A (en) * | 2019-12-06 | 2020-05-08 | 国家海洋环境监测中心 | Method for evaluating ecological risk safety threshold of toxic microalgae |
| CN116396863A (en) * | 2023-04-13 | 2023-07-07 | 自然资源部第一海洋研究所 | Alexander algae cultivation method |
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