CN107686813A - A kind of Euglena high-density cultivation method - Google Patents
A kind of Euglena high-density cultivation method Download PDFInfo
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- CN107686813A CN107686813A CN201710886276.8A CN201710886276A CN107686813A CN 107686813 A CN107686813 A CN 107686813A CN 201710886276 A CN201710886276 A CN 201710886276A CN 107686813 A CN107686813 A CN 107686813A
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Abstract
The invention discloses a kind of Euglena high-density cultivation method, belong to Euglena preparing technical field.The preparation method includes：Spreading cultivation for Euglena algae kind, Euglena mixotrophic cultivation base of the culture medium that spreads cultivation needed for using production are carried out first；Then the Euglena algae kind to spread cultivation is inoculated into plastic sheeting bioreactor, the addition of Euglena mixotrophic cultivation base is carried out stage by stage, and the inoculum concentration per the stage is about 30% or so, using artificial LED light source culture.On the one hand the present invention can utilize the organic matter in culture medium, obtain the fast breeding of Euglena by using mixotrophic cultivation pattern；On the other hand, under illumination, the condition of culture ventilated, CO is fixed to greatest extent2And improve the chlorophyll content of Euglena individual.The Euglena cell concentration obtained using the inventive method can reach 3,000,000/mL, and Euglena dry cell weight is significantly improved.
Description
Technical field
The present invention relates to Euglena preparing technical field, and in particular to a kind of Euglena high-density cultivation method.
Background technology
Euglena is a kind of osculant biology between animal and plant between the two, and the protein with animal and plant forms, and is
Good food organisms；50% is protein in Euglena dry weight, and its quality is better than protein caused by plant, includes 20 kinds
Amino acid, wherein there are 8 kinds of necessary amino acid of human body such as isoleucine, food is more suitable for than chlorella, 2013, China
Approval Euglena is new raw-food material, and this causes Euglena to have good market prospects.In the last few years, the state such as Japan developed in succession
Patented product of the Euglena as fish food, China start late in this field, and correlation technique is also and immature, explore Euglena
High Density Cultivation, be advantageously implemented the production industrialization of China Euglena, promote fast development of the China in the field.
At present, the main autotrophy of Euglena, Heterotrophic culture method.Wherein the Euglena speed of growth is slow under autotrophy state, production week
Phase is long, production cost is higher, is unfavorable for large-scale production；And during Heterotrophic culture, the growth rate of Euglena is slower than bacterium, Wu Facheng
For sociales.
The content of the invention
It is an object of the invention to provide a kind of Euglena high-density cultivation method, by plastic sheeting bioreactor
Middle addition Heterotrophic culture base, the photo-irradiation treatment of certain time is given, making Euglena, growth rate improves, numerous under simultaneous foster pattern
Speed quickening is grown, growth cycle is short so as to reaching, the high purpose of culture density.
Its technical solution includes：
A kind of Euglena high-density cultivation method, comprises the following steps successively：
A, algae kind is spread cultivation, and Euglena mixotrophic cultivation base is configured in triangular flask, it is sterilized, then by seed bottle
Algae kind is inoculated into the Euglena mixotrophic cultivation base of sterilizing cooling, LED light source irradiation culture；
B, it is inoculated with, Euglena mixotrophic cultivation base is configured in moveable culture medium batch tank, it is sterilized, rear cooling
To room temperature；Euglena mixotrophic cultivation base after cooling is transferred to multiple plastic sheeting bioreactors by the closed conduct of sterilizing
In, the Euglena algae kind for growing to exponential phase of growth is inoculated into each plastic sheeting bioreactor, is irradiated and trained using LED light source
Support；
C, expand culture, then continue to the Euglena added into each plastic sheeting bioreactor after being cooled down obtained by step b
Mixotrophic cultivation base is irradiated using LED light source and cultivated, cultivated 2~4 days to certain volume.
As the preferred scheme of the present invention, step a, the Euglena mixotrophic cultivation base described in b, c, in every liter of culture medium
Contain 0.5~3g of peptone, 0.8~3g of dusty yeast, 0.02~0.1g of potassium dihydrogen phosphate, 0.05~0.2g of magnesium sulfate, calcium chloride
0.03~0.1g, Fe-EDTA mother liquors 1mL, micro- mother liquor 1mL and vitamin stock solution 1mL, the Euglena mixotrophic cultivation base
PH value is 3~7.
As another preferred scheme of the present invention, in step a, algae kind inoculum concentration is 10%, LED light source irradiation culture 2
~4 days, LED illumination intensity was 1500~3000lx, and temperature is 22~28 DEG C.
Preferably, in step b, the inoculum concentration for growing to the Euglena algae kind of exponential phase of growth is 30%, LED light source irradiation training
Support 2~3 days.
Further, interval shaking flask mode, interval time 4h are used in step a.
Further, step b, the plastic sheeting bioreactor bottom ventilation described in c, ventilation ratio are 0.2~1：1v/
vm。
Further, step b, in c, LED light source irradiation cultivation temperature is 22~28 DEG C, intensity of illumination be 1500~
3000lx, carbon dioxide content account for 0.02~1%.
Further, in step c, added first into each plastic sheeting bioreactor after being cooled down obtained by 6L steps b
Euglena mixotrophic cultivation base is cultivated, and condition of culture is identical with step b, after the completion of culture, then to each plastic sheeting biological respinse
Euglena mixotrophic cultivation base to volume after being cooled down in device obtained by addition step b is that 30L is cultivated.
Advantageous effects are caused by the present invention：
(1) Euglena culture medium uses mixotrophic cultivation base, and for the autotrophy culture medium of prior art, training is supported using simultaneous
Base is supported, the growth rate of Euglena is substantially accelerated, and is advantageous to shorten the production cycle, reduces production cost.
(2) present invention use plastic sheeting bioreactor, compared to traditional plastic-steel, glass Photoreactor, with into
The features such as this is low, easy for installation, operation is flexible, and each reactor is relatively independent, can effectively avoid the problem of cross pollution.
(3) pattern that the present invention is cultivated using multiple supplemented medium, can be with compared to disposable addition culture medium
Cultivation cycle is substantially reduced, and early stage once pollutes, and can carry out respective handling, avoids the waste of culture medium, drop
Low production cost.
(4) Euglena fixes CO2Ability be current agricultural productivity it is higher US corn production 50 times.Using simultaneous
The pattern of supporting culture Euglena, the organic matter in culture medium on the one hand can be utilized, obtain the fast breeding of Euglena；On the other hand, exist
Under illumination, the condition of culture ventilated, CO is fixed to greatest extent2, improve the individual chlorophyll content of Euglena.So improve and turn
Rate, production cost is reduced, be advantageous to the industrialization culture of Euglena.
Embodiment
The present invention proposes a kind of Euglena high-density cultivation method, in order that advantages of the present invention, technical scheme are more clear
Chu, clearly, the present invention is elaborated with reference to specific embodiment.
Signified " high density " refers in Euglena High Density Cultivation in the present invention：The density of Euglena reaches in every milliliter of nutrient solution
More than 1000000.
Embodiment 1：
Euglena high-density cultivation method of the present invention, comprises the following steps：
The first step, algae kind spread cultivation, and Euglena mixotrophic cultivation base are configured in 5.0L triangular flask, 121 in high-pressure sterilizing pot
DEG C, 20min sterilizings.Then the algae kind in seed bottle is inoculated into the culture medium of above-mentioned sterilizing cooling, the Euglena algae kind expands
It is 10% to train inoculum concentration.Algae kind spreads cultivation every 4 hours shaking flasks, and artificial LED light source irradiation intensity of illumination be 2500lx, culture 3 days.It is naked
Algae mixotrophic cultivation based formulas is to contain in every liter of culture medium：There are peptone 1.0g, dusty yeast 0.9g, potassium dihydrogen phosphate 0.05g, sulphur
Sour magnesium 0.1g, calcium chloride 0.06g, Fe-EDTA mother liquor 1mL, micro- mother liquor 1mL and vitamin stock solution 1mL, it is 6 to control pH；
Second step, inoculation, Euglena mixotrophic cultivation base is configured in moveable culture medium batch tank, is entered using high-temperature steam
Row sterilizing, after be cooled to room temperature.Above-mentioned culture medium is transferred to each plastic sheeting bioreactor by the closed conduct of sterilizing
In, each transfer amount about 2.0L.Then the Euglena algae kind for growing to exponential phase of growth is inoculated into each plastic sheeting bioreactor
In, the inoculum concentration of Euglena algae kind is 30%.Plastic sheeting bioreactor bottom is ventilated, and is shone using artificial LED light source irradiation light
Intensity is 2500lx；Ventilation ratio 0.5：1v/vm, carbon dioxide content account for 0.05%, cultivate 2 days.
3rd step, expand culture, 6.0L sterilized Euglena is added into each plastic sheeting bioreactor and supports training
Support base, same culture conditions culture 2~3 days.Continue the sterilized Euglena added into each plastic sheeting bioreactor afterwards
Mixotrophic cultivation base was to cumulative volume 30.0L, with second step the same terms culture 2 days.
Through biochemistry detection, the Euglena cell concentration that the present embodiment is prepared is 1,500,000/mL.
Embodiment 2：
Difference from Example 1 is：Artificial LED light source is used to irradiate intensity of illumination for 3000lx in second step；It is logical
Gas is than 1：1v/vm, carbon dioxide content account for 1%, cultivate 3 days.
Through biochemistry detection, the Euglena cell concentration that the present embodiment is prepared is 2,400,000/mL.
Comparative example 1：
Common autotrophy cultural method, comprises the following steps：
The first step, algae kind spread cultivation, and Euglena autotrophy culture medium are configured in 5.0L triangular flask, 121 in high-pressure sterilizing pot
DEG C, 20min sterilizings.Then the algae kind in seed bottle is inoculated into the culture medium of above-mentioned sterilizing cooling, the Euglena algae kind expands
It is 10% to train inoculum concentration.Algae kind spreads cultivation every 4 hours shaking flasks, and artificial LED light source irradiation intensity of illumination be 3000lx, culture 5 days.It is naked
Algae autotrophy culture medium prescription is to contain in every liter of culture medium：Magnesium sulfate 0.05g, sodium nitrate 0.08g, potassium dihydrogen phosphate 0.08g, chlorine
Change calcium 0.05g, Fe-EDTA mother liquor 1mL, micro- mother liquor 1mL, vitamin stock solution 1mL, it is 6 to control pH；
Second step, inoculation, Euglena autotrophy culture medium is configured in moveable culture medium batch tank, is entered using high-temperature steam
Row sterilizing, after be cooled to room temperature.Above-mentioned culture medium is transferred to each plastic sheeting bioreactor by the closed conduct of sterilizing
In, each transfer amount about 2.0L.Then the Euglena algae kind for growing to exponential phase of growth is inoculated into each plastic sheeting bioreactor
In, the inoculum concentration of Euglena algae kind is 30%.Plastic sheeting bioreactor bottom is ventilated, and is shone using artificial LED light source irradiation light
Intensity is 3000lx；Ventilation ratio 1：1v/vm, carbon dioxide content account for 0.1%, cultivate 6 days.
3rd step, expand culture, 6.0L sterilized Euglena autotrophy training is added into each plastic sheeting bioreactor
Support base, same culture conditions culture 4~7 days.Continue the sterilized Euglena added into each plastic sheeting bioreactor afterwards
Autotrophy culture medium was to cumulative volume 30.0L, with second step the same terms culture 6 days.
Through biochemistry detection, the Euglena cell concentration that the comparative example is prepared is 900,000/mL.
The relevant technology contents do not addressed in aforesaid way are taken or used for reference prior art and can be achieved.
It should be noted that those skilled in the art can also make such or such appearance under the teaching of this specification
Easy variation pattern, such as equivalent way, or obvious mode of texturing.Above-mentioned variation pattern all should protection scope of the present invention it
It is interior.
Claims (8)
1. a kind of Euglena high-density cultivation method, it is characterised in that comprise the following steps successively：
A, algae kind is spread cultivation, and Euglena mixotrophic cultivation base is configured in triangular flask, it is sterilized, then by the algae kind in seed bottle
It is inoculated into the Euglena mixotrophic cultivation base of sterilizing cooling, LED light source irradiation culture；
B, be inoculated with, in moveable culture medium batch tank configure Euglena mixotrophic cultivation base, it is sterilized, after be cooled to room
Temperature；Euglena mixotrophic cultivation base after cooling is transferred in multiple plastic sheeting bioreactors by the closed conduct of sterilizing,
The Euglena algae kind for growing to exponential phase of growth is inoculated into each plastic sheeting bioreactor, is irradiated and cultivated using LED light source；
C, expand culture, then continue to add the Euglena after step b gained cools down into each plastic sheeting bioreactor and support
Culture medium is irradiated using LED light source and cultivated, cultivated 2~4 days to certain volume.
2. Euglena high-density cultivation method according to claim 1, it is characterised in that：Step a, the Euglena described in b, c
Mixotrophic cultivation base, in every liter of culture medium containing 0.5~3g of peptone, 0.8~3g of dusty yeast, 0.02~0.1g of potassium dihydrogen phosphate,
0.05~0.2g of magnesium sulfate, 0.03~0.1g of calcium chloride, Fe-EDTA mother liquors 1mL, micro- mother liquor 1mL and vitamin stock solution
1mL, the pH value of the Euglena mixotrophic cultivation base is 3~7.
3. Euglena high-density cultivation method according to claim 1, it is characterised in that：In step a, algae kind inoculum concentration is
10%, LED light source irradiation culture 3~4 days, LED illumination intensity is 1500~3000lx, and temperature is 22~28 DEG C.
4. Euglena high-density cultivation method according to claim 1, it is characterised in that：In step b, exponential growth is grown to
The inoculum concentration of the Euglena algae kind of phase is 30%, LED light source irradiation culture 2~4 days.
5. Euglena high-density cultivation method according to claim 1, it is characterised in that：Interval shaking flask side is used in step a
Formula, interval time 4h.
6. Euglena high-density cultivation method according to claim 1, it is characterised in that：Step b, the plastic sheeting described in c
Bioreactor bottom is ventilated, and ventilation ratio is 0.2~1：1v/vm.
7. Euglena high-density cultivation method according to claim 1, it is characterised in that：Step b, in c, LED light source irradiation,
Cultivation temperature is 22~28 DEG C, intensity of illumination is 1500~3000lx, and carbon dioxide content accounts for 0.02~1%.
8. Euglena high-density cultivation method according to claim 1, it is characterised in that：It is thin to each plastics first in step c
The Euglena mixotrophic cultivation base added in membrane bioreactor after being cooled down obtained by 6L steps b is cultivated, condition of culture and step b phases
Together, after the completion of culture, then the Euglena mixotrophic cultivation base after being cooled down obtained by step b is added extremely into each plastic sheeting bioreactor
Volume is that 30L is cultivated.
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| CN201710886276.8A CN107686813A (en) | 2017-09-27 | 2017-09-27 | A kind of Euglena high-density cultivation method |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108277163A (en) * | 2018-04-17 | 2018-07-13 | 宁波浮田生物技术有限公司 | A method of isolating and purifying Euglena algae |
| CN111334434A (en) * | 2020-03-12 | 2020-06-26 | 优格天成生物技术(义乌)有限公司 | Gymnodinium culture medium and application thereof |
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| TW200512289A (en) * | 2003-09-26 | 2005-04-01 | Wan-Ju Liao | Method for culturing chlorella species |
| CN101633894A (en) * | 2009-08-20 | 2010-01-27 | 北京芳能科技有限公司 | Culture medium of euglena gracilis and open type high-density culture method |
| CN104357330A (en) * | 2014-11-11 | 2015-02-18 | 甘肃德福生物科技有限公司 | Chlorella autotrophic-heterotrophic mixed culture method |
| CN106867909A (en) * | 2017-04-13 | 2017-06-20 | 河南师范大学 | A kind of cultural method of very thin Euglena |
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2017
- 2017-09-27 CN CN201710886276.8A patent/CN107686813A/en active Pending
Patent Citations (4)
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| TW200512289A (en) * | 2003-09-26 | 2005-04-01 | Wan-Ju Liao | Method for culturing chlorella species |
| CN101633894A (en) * | 2009-08-20 | 2010-01-27 | 北京芳能科技有限公司 | Culture medium of euglena gracilis and open type high-density culture method |
| CN104357330A (en) * | 2014-11-11 | 2015-02-18 | 甘肃德福生物科技有限公司 | Chlorella autotrophic-heterotrophic mixed culture method |
| CN106867909A (en) * | 2017-04-13 | 2017-06-20 | 河南师范大学 | A kind of cultural method of very thin Euglena |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108277163A (en) * | 2018-04-17 | 2018-07-13 | 宁波浮田生物技术有限公司 | A method of isolating and purifying Euglena algae |
| CN108277163B (en) * | 2018-04-17 | 2022-02-01 | 宁波浮田生物技术有限公司 | Method for separating and purifying euglena species |
| CN111334434A (en) * | 2020-03-12 | 2020-06-26 | 优格天成生物技术(义乌)有限公司 | Gymnodinium culture medium and application thereof |
| CN111334434B (en) * | 2020-03-12 | 2021-05-04 | 优格天成生物技术(义乌)有限公司 | Gymnodinium culture medium and application thereof |
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