CN107841465A - A kind of haematococcus pluvialis highly effective sea-water cultural method - Google Patents
A kind of haematococcus pluvialis highly effective sea-water cultural method Download PDFInfo
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Abstract
The present invention provides a kind of haematococcus pluvialis highly effective sea-water cultural method, its condition of culture of reasonable adjusting on the basis of traditional " two-step method ", and it is that algae supplements natural nutrient component to add seawater, takes full advantage of the abundant seawater resources in the coastal areas such as Hainan；Pumpkin seed powder, sweet potato powder and Brainea insignis ethanol extract are added in the medium, not only supplement enough nutritional ingredients, and play a part of preventing biological pollution；Simultaneously by mixing the use of rotating of light source, efficiently promote the accumulation of algae reproduction and astaxanthin.Using the inventive method, cell density is up to 10.56 × 105Individual/ml, dry cell weight are up to 3.15mg/ml, and content astaxanthin is up to 104.12mg/g, and unicellular content astaxanthin is up to 3.51 × 10‑2Ug/, the inventive method can not only effectively improve the fertility of haematococcus pluvialis, and single celled Accumulation of Astaxanthin amount significantly improves.
Description
Technical field
The present invention relates to technical field of microalga biology, and in particular to a kind of haematococcus pluvialis highly effective sea-water cultural method.
Background technology
Natural astaxanthin is a kind of efficient bioactive substance, and it has extremely strong inoxidizability, widely should be had
Use prospect.Compared to other biological, haematococcus pluvialis are acknowledged as the optimal biological source of natural astaxanthin.
Newest result of study shows, under splendid condition of culture, the content of Determination of Astaxanthin in Haematococcus Pluvialis is up to
To 4-5%.At present, the method for large-scale cultivation haematococcus pluvialis, mainly there is " two-step method " and " one-step method ", " two-step method " i.e.：
The first step is vegetative growth phase, i.e., it is thin to obtain " green " of high-density growth to cultivate haematococcus pluvialis under appropriate conditions
Born of the same parents, this stage haematococcus pluvialis growing is vigorous, but content astaxanthin is few in vivo, and this stage is generally carried out indoors；Second step
To induce the accumulation stage of astaxanthin, i.e., green swarm cell is promoted to be changed into the motionless of " red " under conditions of various abiotic stress
Spore, induction accumulation astaxanthin, this stage mainly open and carried out in pond or closed photo bioreactor out of doors；" step
Method " is to carry out two stages in " two-step method " in same bioreactor, by nutritional ingredient and external rings
The regulation and control in border, both completed the substantial increase of " green " cellular biomass, realized the accumulation of these cell degree astaxanthins again.
Although " one-step method " has operation and prevents the advantage in pollution, rain in the actual production of current business be present and give birth to red ball
The problem of specific yield of astaxanthin is relatively low in algae, there are some researches show its astaxanthin yield be only " two-step method " half, therefore,
This area is still used as main cultural method using " two-step method " at present.
In recent years, on improving the biomass of haematococcus pluvialis and the accumulation of intracellular astaxanthin, rain is progressively expanded
The industrialization production of the cultivation scale of raw haematococcus to realize astaxanthin etc. has become the focus of research.
The content of the invention
For in place of above the deficiencies in the prior art, the present invention provides a kind of haematococcus pluvialis highly effective sea-water cultural method.
The technical scheme that the present invention takes is as follows：
A kind of haematococcus pluvialis highly effective sea-water cultural method, comprises the following steps：
S1：Culture medium configures
The culture medium includes following component：Glucose 3-5g/L, KNO30.6-1g/L、NaH2PO40.1-0.3g/L、
MgSO4·7H2O0.1-0.2g/L, Fe-EDTA1-2mg/L, citric acid 0.5-1.0mg/L, cushaw seed 0.3-0.7g/L, kind
Potato powder 0.2-0.4g/L, seawater 50-100mL/L, the pH value of the culture medium is 5.0-6.0；
S2：The haematococcus pluvialis growing stage
Algae kind is inoculated into culture medium with 17-20% inoculum concentration, combined using feux rouges, green glow with blue light to be formed it is mixed
Closing light source is irradiated, intensity of illumination 2500-3000Lx, the photoperiod 12:12, it is 8-15 DEG C to control environment temperature, is passed through dioxy
Change carbon, persistently stirred with 150-200rpm rotating speeds, cultivated 2-3 days, it is 5-15% then to add percent by volume in the medium
Seawater, combined using white light and feux rouges to be formed mixing light source irradiation, adjustment intensity of illumination is 4200-4500Lx, continues to train
Support 2-3 days；
S3：The Accumulation of Astaxanthin stage
Algae solution after step S2 is cultivated is inoculated into new culture medium with 10-15% inoculum concentration, then in culture medium
It is middle to add the seawater of Brainea insignis ethanol extract and percent by volume for 0.2-0.4%, using blue light, purple light and white light combination shape
Into mixing light source irradiation, intensity of illumination 5000-7000Lx, whole day illumination, control 20-25 DEG C of environment temperature, be passed through oxygen, with
200-300rpm rotating speeds persistently stir, and cultivate 8-10 days.
Preferably, the intake of carbon dioxide is to be passed through 0.5-0.7L/min in every liter of culture medium in the step S2；
Preferably, the intensity for mixing feux rouges, green glow and blue light in light source of feux rouges, green glow and blue light composition described in step S2
Ratio is 3:1:2；It is 7 that white light and feux rouges, which combine the intensity of white light and feux rouges in the mixing light source to be formed,：3.
Preferably, the intake of oxygen is to be passed through 0.2-0.4L/mim in every liter of culture medium in the step S3.
Preferably, blue light in the mixing light source that blue light in the step S3, purple light and white light are formed, purple light and white light it is strong
Degree ratio is 2:5:3.
Preferably, the addition of Brainea insignis ethanol extract is to add 16-27mg in every liter of culture medium in the step S3.
Compared with prior art, the beneficial effects of the invention are as follows：
1st, the present invention provides a kind of haematococcus pluvialis highly effective sea-water cultural method, adds in right amount during haematococcus is cultivated
Enter seawater and supplement natural nutrient component for algae, take full advantage of the abundant seawater resources in the coastal areas such as Hainan；
2nd, pumpkin seed powder, sweet potato powder and Brainea insignis ethanol extract are added in the medium, not only supplement enough nutrition
Composition, and play a part of preventing biological pollution；
3rd, in the different phase of algal culture, also carry out mixing the use of rotating of light source, efficiently promote algae reproduction and shrimp
The accumulation of blue or green element.
4th, using the inventive method, its condition of culture of reasonable adjusting on the basis of traditional " two-step method " is thin after culture
Born of the same parents' density is up to 10.56 × 105Individual/ml, dry cell weight are up to 3.15mg/ml, and content astaxanthin is up to 104.12mg/g, slender
Born of the same parents' content astaxanthin is up to 3.51 × 10-2Ug/, compared with comparative example 1-4, algae cell density improves nearly 3 times, and unicellular shrimp is blue or green
Element improves nearly 10 times, and compared with comparative example 5, unicellular content astaxanthin improves nearly 30 times, illustrates that the inventive method can not only have
Effect improves the fertility of haematococcus pluvialis, and its single celled Accumulation of Astaxanthin amount significantly improves.
Embodiment
The invention will now be further described with reference to specific embodiments, advantages of the present invention and feature will be with description and
It is apparent, but protection scope of the present invention is not limited to following examples.
Embodiment 1：
A kind of haematococcus pluvialis highly effective sea-water cultural method, comprises the following steps：
S1：Culture medium configures
The culture medium includes following component：Glucose 3g/L, KNO30.6g/L、NaH2PO40.1g/L、MgSO4·
7H2O0.1g/L, Fe-EDTA1mg/L, citric acid 0.5mg/L, cushaw seed 0.3g/L, sweet potato powder 0.2g/L, seawater 50mL/
L, the pH value of the culture medium is 5.0；
S2：The haematococcus pluvialis growing stage
Algae kind is inoculated into culture medium with 17% inoculum concentration, the mixing to be formed is combined with blue light using feux rouges, green glow
(intensity of feux rouges, green glow and blue light is 3 for light source irradiation:1:2).Intensity of illumination is 2500Lx, the photoperiod 12:12, control
Environment temperature processed is 8 DEG C, is passed through carbon dioxide, and the intake of carbon dioxide is to be passed through 0.5L/min in every liter of culture medium, with
150rpm rotating speeds persistently stir, and cultivate 2 days, then add the seawater that percent by volume is 5% in the medium, using white light and
Feux rouges combines the mixing light source to be formed irradiation, and (intensity of white light and feux rouges is 7：3) it is 4200Lx, to adjust intensity of illumination, after
Continuous culture 2 days；
S3：The Accumulation of Astaxanthin stage
Algae solution after step S2 is cultivated is inoculated into new culture medium with 10% inoculum concentration, is then added in the medium
Enter Brainea insignis ethanol extract (adding 16mg in every liter of culture medium) and percent by volume for 0.2% seawater, using blue light, purple
(intensity of blue light, purple light and white light is 2 for the mixing light source irradiation that light and white light combination are formed:5:3), intensity of illumination
5000Lx, whole day illumination, 20 DEG C of environment temperature is controlled, be passed through oxygen (being passed through 0.2L/mim in every liter of culture medium) and turned with 200rpm
Fast lasting stirring, is cultivated 8 days.
Embodiment 2：
A kind of haematococcus pluvialis highly effective sea-water cultural method, comprises the following steps：
S1：Culture medium configures
The culture medium includes following component：Glucose 5g/L, KNO31g/L、NaH2PO40.3g/L、MgSO4·
7H2O0.2g/L, Fe-EDTA2mg/L, citric acid 1.0mg/L, cushaw seed 0.7g/L, sweet potato powder 0.4g/L, seawater
100mL/L, the pH value of the culture medium is 6.0；
S2：The haematococcus pluvialis growing stage
Algae kind is inoculated into culture medium with 20% inoculum concentration, the mixing to be formed is combined with blue light using feux rouges, green glow
(intensity of feux rouges, green glow and blue light is 3 for light source irradiation:1:2).Intensity of illumination is 3000Lx, the photoperiod 12:12, control
Environment temperature processed is 15 DEG C, is passed through carbon dioxide, and the intake of carbon dioxide is to be passed through 0.7L/min in every liter of culture medium, with
200rpm rotating speeds persistently stir, and cultivate 3 days, the seawater that percent by volume is 15% are then added in the medium, using white light
The mixing light source to be formed irradiation is combined with feux rouges, and (intensity of white light and feux rouges is 7：3) it is 4500Lx, to adjust intensity of illumination,
Continue culture 3 days；
S3：The Accumulation of Astaxanthin stage
Algae solution after step S2 is cultivated is inoculated into new culture medium with 15% inoculum concentration, is then added in the medium
Enter Brainea insignis ethanol extract (adding 27mg in every liter of culture medium) and percent by volume for 0.4% seawater, using blue light, purple
(intensity of blue light, purple light and white light is 2 for the mixing light source irradiation that light and white light combination are formed:5:3), intensity of illumination
7000Lx, whole day illumination, 25 DEG C of environment temperature is controlled, be passed through oxygen (being passed through 0.4L/mim in every liter of culture medium) and turned with 300rpm
Fast lasting stirring, is cultivated 10 days.
Embodiment 3：
A kind of haematococcus pluvialis highly effective sea-water cultural method, comprises the following steps：
S1：Culture medium configures
The culture medium includes following component：Glucose 4g/L, KNO30.7g/L、NaH2PO40.2g/L、MgSO4·
7H2O0.2g/L, Fe-EDTA2mg/L, citric acid 0.7mg/L, cushaw seed 0.5g/L, sweet potato powder 0.3g/L, seawater 70mL/
L, the pH value of the culture medium is 5.5；
S2：The haematococcus pluvialis growing stage
Algae kind is inoculated into culture medium with 20% inoculum concentration, the mixing to be formed is combined with blue light using feux rouges, green glow
(intensity of feux rouges, green glow and blue light is 3 for light source irradiation:1:2).Intensity of illumination is 3000Lx, the photoperiod 12:12, control
Environment temperature processed is 10 DEG C, is passed through carbon dioxide, and the intake of carbon dioxide is to be passed through 0.6L/min in every liter of culture medium, with
170rpm rotating speeds persistently stir, and cultivate 2 days, the seawater that percent by volume is 10% are then added in the medium, using white light
The mixing light source to be formed irradiation is combined with feux rouges, and (intensity of white light and feux rouges is 7：3) it is 4500Lx, to adjust intensity of illumination,
Continue culture 2 days；
S3：The Accumulation of Astaxanthin stage
Algae solution after step S2 is cultivated is inoculated into new culture medium with 15% inoculum concentration, is then added in the medium
Enter Brainea insignis ethanol extract (adding 20mg in every liter of culture medium) and percent by volume for 0.3% seawater, using blue light, purple
(intensity of blue light, purple light and white light is 2 for the mixing light source irradiation that light and white light combination are formed:5:3), intensity of illumination
5000-7000Lx, whole day illumination, control 22 DEG C of environment temperature, be passed through oxygen (being passed through 0.3L/mim in every liter of culture medium) with
250rpm rotating speeds persistently stir, and cultivate 9 days.
Comparative example 1：
A kind of haematococcus pluvialis highly effective sea-water cultural method, comprises the following steps：
S1：Culture medium configures
The culture medium includes following component：Glucose 2g/L, KNO30.5g/L、NaH2PO40.1g/L、MgSO4·
7H2O0.05g/L, Fe-EDTA0.5mg/L, citric acid 0.2mg/L, cushaw seed 0.2g/L, sweet potato powder 0.1g/L, seawater
45mL/L, the pH value of the culture medium is 4.5；
Step S2 and step 3 are same as Example 1, do not add Brainea insignis extract in step S3.
Comparative example 2：
A kind of haematococcus pluvialis highly effective sea-water cultural method, comprises the following steps：
S1：Culture medium configures
The culture medium includes following component：Glucose 5g/L, KNO31g/L、NaH2PO40.3g/L、MgSO4·
7H2O0.2g/L, Fe-EDTA2mg/L, citric acid 1.0mg/L, cushaw seed 0.7g/L, sweet potato powder 0.4g/L, seawater
100mL/L, the pH value of the culture medium is 6.0；
Step S2, step S3 is same as Example 1, does not add Brainea insignis extract in step S3.
Comparative example 3：
A kind of haematococcus pluvialis highly effective sea-water cultural method, comprises the following steps：
S1：Culture medium configures
The culture medium includes following component：It is same as Example 1；
S2：The haematococcus pluvialis growing stage
Algae kind is inoculated into culture medium with 10% inoculum concentration, the mixing light source to be formed is combined with blue light using feux rouges and shines
Penetrate (intensity of feux rouges and blue light be 3:2：3), intensity of illumination 2000Lx, photoperiod 12:12, control the environment temperature to be
5 DEG C, carbon dioxide is passed through, the intake of carbon dioxide is to be passed through 0.2L/min in every liter of culture medium, is continued with 100rpm rotating speeds
Stirring, cultivate 2 days, then add the seawater that percent by volume is 2% in the medium, combine what is formed using white light and feux rouges
Mixing light source irradiation, (intensity of white light and feux rouges is 3：7) it is 3500Lx, to adjust intensity of illumination, continues culture 2 days；
S3：The Accumulation of Astaxanthin stage
Algae solution after step S2 is cultivated is inoculated into new culture medium with 5% inoculum concentration, is then added in the medium
Enter Brainea insignis ethanol extract (adding 10mg in every liter of culture medium) and percent by volume for 0.1% seawater, using blue light, purple
(intensity of blue light, purple light and white light is 2 for the mixing light source irradiation that light and white light combination are formed:3:5), intensity of illumination
4000Lx, whole day illumination, 15 DEG C of environment temperature is controlled, be passed through oxygen (being passed through 0.1L/mim in every liter of culture medium) and turned with 150rpm
Fast lasting stirring, is cultivated 8 days.
Comparative example 4：
A kind of haematococcus pluvialis highly effective sea-water cultural method, comprises the following steps：
S1：Culture medium configures
The culture medium includes following component：It is same as Example 1.
S2：The haematococcus pluvialis growing stage
Algae kind is inoculated into culture medium with 25% inoculum concentration, the mixing to be formed is combined with blue light using feux rouges, green glow
(intensity of feux rouges, green glow and blue light is 5 for light source irradiation:1:1), intensity of illumination 3500Lx, photoperiod 12:12, control
Environment temperature processed is 20 DEG C, is passed through carbon dioxide, and the intake of carbon dioxide is to be passed through 1.0L/min in every liter of culture medium, with
300rpm rotating speeds persistently stir, and cultivate 3 days, the seawater that percent by volume is 20% are then added in the medium, using white light
The mixing light source to be formed irradiation is combined with feux rouges, and (intensity of white light and feux rouges is 5:5) it is 5000Lx, to adjust intensity of illumination,
Continue culture 3 days；
S3：The Accumulation of Astaxanthin stage
Algae solution after step S2 is cultivated is inoculated into new culture medium with 20% inoculum concentration, is then added in the medium
Enter Brainea insignis ethanol extract (adding 35mg in every liter of culture medium) and percent by volume for 0.5% seawater, using blue light, purple
(intensity of blue light, purple light and white light is 2 for the mixing light source irradiation that light and white light combination are formed:5:3), intensity of illumination
10000Lx, whole day illumination, 35 DEG C of environment temperature is controlled, is passed through oxygen (being passed through 0.6L/mim in every liter of culture medium) with 300rpm
Rotating speed persistently stirs, and cultivates 10 days.
Comparative example 5：
A kind of haematococcus pluvialis cultural method, comprises the following steps：
By algae kind using appropriate concentration transfer into the 250ml containing MAV culture mediums conical flask (nutrient solution volume as
In 150ml), the formula of MAV mother liquors is：KNO3100g、KH2PO310g、FeSO4·7H2O2.5g、MnSO40.25g、EDTA-
Na210g、VB16mg、VB1250ug, sterilization fresh water 1000mL.Nutrient solution is with sterilizing the volume ratio of fresh water as 1 by mother liquor:1000
Configuration forms, medium pH 7.2.The conical flask of inoculation haematococcus pluvialis is placed in quiescent culture in illumination box, cultivates light
Illumination is 5000lx, and cultivation temperature is (22 ± 1) DEG C, and the photoperiod is arranged to 12：12, the cultivation cycle of haematococcus pluvialis is 15d,
Daily shaking flask 3-4 times during culture.
Experimental example 1：The measure of biomass
Cell density carries out microscopic counting with blood counting chamber, 10mL is taken after algae solution is shaken up, after being fixed with glutaraldehyde
Shake scattered, count under the microscope.It is another to take 20mL algae solutions, centrifuge after abandoning supernatant, weight is dried in 80 DEG C of baking ovens, take out, claim
It is heavy, the unit volume dry mass of frustule in unit of account volume algae solution.Experimental result is shown in Table 1.
Experimental example 2：Content astaxanthin determines
Take 20mL algae solutions to centrifuge, add 5%KOH and 30% methanol after abandoning supernatant, centrifugation, abandon supernatant, add 3mL and contain less
Measure the dimethyl sulfoxide (DMSO) of acetic acid.Shake up, 70 DEG C of insulation 5min, insulating process jiggles centrifuge tube, centrifuges, take supernatant to be placed in
In 10mL volumetric flasks.Extract to frond and turn white repeatedly, high speed centrifugation, collect supernatant, be settled to 10mL with dimethyl sulfoxide (DMSO), take
1mL uses 10 times of dimethyl sulfoxide (DMSO) again, takes and determines absorbance at 492nm in right amount, using dimethyl sulfoxide (DMSO) as blank control.Astaxanthin
Cubage formula be：
In formula, C represents the content astaxanthin (mg/mL) in unit volume；Extinction coefficientRefer to the sample that concentration is 1%
=2220, A492For the light absorption value of 1cm liquid layers mixture at 490nm；Then the content astaxanthin in unit volume is scaled list
The content of position dry mass content astaxanthin (mg/g) and individual cells astaxanthin (ug/).Experimental result is shown in Table 1.
Table 1：Measurement result (means standard deviation)
Using the inventive method, cell density is up to 10.56 × 105Individual/ml, dry cell weight are up to 3.15mg/ml, and shrimp is blue or green
Cellulose content is up to 104.12mg/g, and unicellular content astaxanthin is up to 3.51 × 10-2Ug/, compared with comparative example 1-4, algae is thin
Born of the same parents' density improves nearly 3 times, and unicellular astaxanthin improves nearly 10 times, and compared with comparative example 5, unicellular content astaxanthin improves nearly 30
Times, illustrate that the inventive method can not only effectively improve the fertility of haematococcus pluvialis, and single celled Accumulation of Astaxanthin amount
Significantly improve.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all essences in the present invention
God any modification, equivalent substitution and improvements done etc., should be included within the scope of protection of the invention with principle.
Claims (6)
1. a kind of haematococcus pluvialis highly effective sea-water cultural method, it is characterised in that comprise the following steps：
S1：Culture medium configures
The culture medium includes following component：Glucose 3-5g/L, KNO3 0.6-1g/L、NaH2PO4 0.1-0.3g/L、
MgSO4·7H2O 0.1-0.2g/L, Fe-EDTA 1-2mg/L, citric acid 0.5-1.0mg/L, cushaw seed 0.3-0.7g/L,
Sweet potato powder 0.2-0.4g/L, seawater 50-100mL/L, the pH value of the culture medium is 5.0-6.0；
S2：The haematococcus pluvialis growing stage
Algae kind is inoculated into culture medium with 17-20% inoculum concentration, the mixed light to be formed is combined with blue light using feux rouges, green glow
Source is irradiated, intensity of illumination 2500-3000Lx, the photoperiod 12:12, it is 8-15 DEG C to control environment temperature, is passed through carbon dioxide,
Persistently stirred with 150-200rpm rotating speeds, cultivate 2-3 days, then add the sea that percent by volume is 5-15% in the medium
Water, the mixing light source to be formed irradiation is combined using white light and feux rouges, adjustment intensity of illumination is 4200-4500Lx, continues to cultivate 2-3
My god；
S3：The Accumulation of Astaxanthin stage
Algae solution after step S2 is cultivated is inoculated into new culture medium with 10-15% inoculum concentration, is then added in the medium
Enter the seawater of Brainea insignis ethanol extract and percent by volume for 0.2-0.4%, formed using blue light, purple light and white light combination
Light source irradiation is mixed, intensity of illumination 5000-7000Lx, whole day illumination, 20-25 DEG C of environment temperature is controlled, oxygen is passed through, with 200-
300rpm rotating speeds persistently stir, and cultivate 8-10 days.
A kind of 2. haematococcus pluvialis highly effective sea-water cultural method according to claim 1, it is characterised in that the step S2
The intake of middle carbon dioxide is to be passed through 0.5-0.7L/min in every liter of culture medium.
3. a kind of haematococcus pluvialis highly effective sea-water cultural method according to claim 1, it is characterised in that described in step S2
The intensity of feux rouges, green glow and blue light is 3 in the mixing light source of feux rouges, green glow and blue light composition:1:2；White light and feux rouges group
It is 7 to close the intensity of white light and feux rouges in the mixing light source formed：3.
A kind of 4. haematococcus pluvialis highly effective sea-water cultural method according to claim 1, it is characterised in that the step S3
The intake of middle oxygen is to be passed through 0.2-0.4L/mim in every liter of culture medium.
A kind of 5. haematococcus pluvialis highly effective sea-water cultural method according to claim 1, it is characterised in that the step S3
The intensity of blue light, purple light and white light is 2 in the mixing light source that middle blue light, purple light and white light are formed:5:3.
A kind of 6. haematococcus pluvialis highly effective sea-water cultural method according to claim 1, it is characterised in that the step S3
The addition of middle Brainea insignis ethanol extract is to add 16-27mg in every liter of culture medium.
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|---|---|---|---|---|
| CN113249291A (en) * | 2021-07-01 | 2021-08-13 | 海南三元星生物科技股份有限公司 | Method for inducing haematococcus pluvialis cell type conversion and accumulating astaxanthin by using seawater |
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| CN113278577A (en) * | 2021-07-01 | 2021-08-20 | 海南三元星生物科技股份有限公司 | Culture medium for promoting haematococcus pluvialis cell growth and propagation by using seawater and preparation method |
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