CN109295147A - A method of promoting Determination of Astaxanthin in Haematococcus Pluvialis accumulation - Google Patents
A method of promoting Determination of Astaxanthin in Haematococcus Pluvialis accumulation Download PDFInfo
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- CN109295147A CN109295147A CN201811067412.1A CN201811067412A CN109295147A CN 109295147 A CN109295147 A CN 109295147A CN 201811067412 A CN201811067412 A CN 201811067412A CN 109295147 A CN109295147 A CN 109295147A
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- astaxanthin
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Abstract
The invention discloses a kind of methods of promotion Determination of Astaxanthin in Haematococcus Pluvialis accumulation, feature is the haematococcus pluvialis cell cultivated haematococcus pluvialis in low light conditions to logarithmic growth later period and/or plateau, liquor kalii iodide is added in Growth Medium For Haematococcus Pluvialis, the final concentration of 0.5-3.0 mM for making potassium iodide, is subsequently placed in 100-250 μm of ol/m2Determination of Astaxanthin in Haematococcus Pluvialis accumulation is induced under the conditions of/s bloom, advantage is that the content of Determination of Astaxanthin in Haematococcus Pluvialis can be improved 53.4%-82.8% by potassium iodide.
Description
Technical field
The present invention relates to a kind of methods of astaxanthin accumulation, more particularly, to a kind of promotion Determination of Astaxanthin in Haematococcus Pluvialis product
Tired method.
Background technique
Haematococcus pluvialis (Haematococcus pluvialis) it is Chlorophyta, volvocales, haematococcus section, haematococcus
A kind of unicellular microalgae of fresh water.When by environment-stress, it can become because accumulating a large amount of astaxanthins red.Astaxanthin is to certainly
550 times stronger than vitamin E by the Scavenging activity of base and singlet active oxygen, antioxidant activity is approximately higher than other carotenoid
10 times, it is known as " super antioxidant ", there is high potentiality to be exploited and economic valence in industries such as medicine, foods and cosmetics
Value (Borowitzka, et al., 1991).Animal and human experimentation the result shows that, natural astaxanthin without pathogenic effect or
Toxic side effect, it is safe to the human body.Natural astaxanthin has passed through food and drug administration (FDA) and EU Committee
Safety certification also regards as new resource food by ministry of Health of China.The price of astaxanthin at 2500 dollars of per kilogram or so,
Global astaxanthin market is up to 200,000,000 dollars within 2015, to about 670 tonnes of demand of the year two thousand twenty astaxanthin, market valuation about 11
Hundred million dollars (F é lixvalenzuela, et al., 2006).
Have at present and multiple has promoted haematococcus pluvialis Prawn by changing the i.e. artificial creation stressful environmental of condition of culture
The report of green element accumulation, including it is high temperature stress (Tjahjono, et al., 1994), salt stress (Jiang Hongxia etc., 2017), high
Light stress (F á bregas, et al., 2003) lacks Nutrient Stress (Yang Jin etc., 2011).Some methods are difficult to industry
Metaplasia produces, such as high temperature stress needs to increase the temperature of culture solution, increases cost；Sodium chloride is added in culture medium and causes salt stress
When can kill frustule and be extremely difficult to the purpose of high-yield astaxanthin；Scarce Nutrient Stress needs replacing the training for lacking nitrogen or phosphorus or sulphur
Base is supported, will increase process and great amount of cost.Bloom stress is the most popular method of industrialized production astaxanthin, but bloom also induces
Generate the yield that a large amount of active oxygens kill part frustule and reduce astaxanthin.
Summary of the invention
Haematococcus pluvialis shrimp blueness is greatly promoted using potassium iodide technical problem to be solved by the invention is to provide a kind of
The method for promoting Determination of Astaxanthin in Haematococcus Pluvialis accumulation of the accumulation of element.
The technical scheme of the invention to solve the technical problem is: a kind of promotion Determination of Astaxanthin in Haematococcus Pluvialis product
Tired method first cultivates haematococcus pluvialis under low light to logarithmic growth later period and/or plateau, then by suitable iodate
Algae solution is added in potassium, then algae solution is placed under intense light conditions to the accumulation for inducing astaxanthin.
The low light refers to that intensity of illumination is 30-50 μm of ol/m2Every square of metre per second (m/s) of/s(micromole).
The haematococcus pluvialis cell in logarithmic growth later period or plateau refers in light conditions culture 15-
20 days until OD680Algae solution equal to 0.3, i.e. frustule concentration reach 2.5 × 105A/ml.
Final concentration of 0.5-3.0 mM(mM every liter of the potassium iodide in algae solution).
The strong light refers to that intensity of illumination is 100-250 μm of ol/m2Every square of metre per second (m/s) of/s(micromole).
Compared with the prior art, the advantages of the present invention are as follows: promote rain using potassium iodide present invention firstly discloses a kind of
The technical method of astaxanthin Rapid Accumulation in raw haematococcus, principle are that will there is the potassium iodide of antioxidation to be added to for utilization
It in the culture medium of haematococcus pluvialis, is subsequently placed under the conditions of bloom and induces, promote Determination of Astaxanthin in Haematococcus Pluvialis content to reach
Purpose.Suitable antioxidant is added in culture medium this method, then with high photoinduction, it is possible to reduce active oxygen is thin to algae
To improve the yield of astaxanthin, significant effect that is easy to operate, promoting astaxanthin accumulation is existed using induction light intensity for the damage of born of the same parents
100-250 µmol/m2Within the scope of/s, the potassium iodide of 0.5-3 mM is mentioned compared to potassium iodide control group astaxanthin yield is not added
High 53.4-77.6%, is greatly promoted the accumulation of Astaxanthin In Haematococcus Pluvialis.
Specific embodiment
Present invention is further described in detail with reference to embodiments.
One, specific embodiment
1, haematococcus pluvialis Multiplying culture: haematococcus pluvialis NMB3# culture medium (see Table 1) culture, in the triangular flask of 250 ml
Add the culture medium of 150 mL to repeat as a biology, uses preceding sterilizing.It is 30-50 μm of ol/m in intensity of illumination2The people of/s
(combination for referring to natural light or artificial light or natural light and artificial light) is cultivated in work illumination box, the photoperiod is 12 h:
12 h, cultivation temperature are 24 DEG C, and culture 15-20 angel concentration of algae reaches logarithmic growth phase (about 2.5 × 105A/ml) it is used for afterwards
Astaxanthin induction culture.
1 NMB3# culture medium prescription of table
2, potassium iodide induction haematococcus pluvialis produces astaxanthin culture: potassium iodide being added in the culture solution of experimental group, makes iodine
Change the final concentration of 0.5-3.0 mM of potassium, control group does not add potassium iodide.Then algae solution being respectively placed in bloom, (strong light refers to certainly
The combination of right light or artificial light or naturally photosynthetic artificial light or complex light or monochromatic light (such as feux rouges, blue-ray light)) it is strong
Degree is 100,150,200 and 270 μm of ol/m2It is cultivated 14 days under the conditions of/s, continuous illumination, collects frustule and contain for astaxanthin
It is fixed to measure.
3, content astaxanthin measures: content astaxanthin measuring method is the Boussiba method of improvement.Take the raw red ball of 10 mL rain
Algae algae solution (Va), 8000 rpm are centrifuged 5 min and collect frond, and 5 mL water-methanols-potassium hydroxide solution (65:30:5), whirlpool is added
Rotation, which mixes, is placed on 5 min of warm bath in 70 DEG C of thermostat water baths, and 8000 rpm are centrifuged 5 min, remove supernatant；5 mL are added to contain
The dimethyl sulfoxide (DMSO) of 1wt% glacial acetic acid, 70 DEG C of 5 min of warm bath, it is astaxanthin solution that supernatant, which is collected by centrifugation,；It repeats
Operation, until algae-residue bleaches.Astaxanthin solution is settled to volume using DMSOVb, it is control with DMSO, in 492 nm
Absorbance is measured under wavelength.Calculate Astaxanthin In Haematococcus Pluvialis content CAstaxanthin:
Wherein: algae solution volume is Va；Extracting liquid volume is Vb；N is cell density.
4, interpretation of result: with the extension of light application time, the algae solution color of control group and processing group is gradually reddened by green.It lures
Culture 14 days is led, the algae solution color of potassium iodide processing group is obviously redder than control group.The measurement result of astaxanthin is as shown in table 2.
2 various concentration potassium iodide of table induces the yield of 14 days Determination of Astaxanthin in Haematococcus Pluvialis under different light-intensity conditions
(mg/L)
By above-mentioned table 2 it is found that 100 μm of ol/m of intensity of illumination2When/s, the astaxanthin yield of control group is 5.80 ± 0.18
Mg/L, i.e. every liter of culture solution can extract 5.80 ± 0.18 milligrams of astaxanthins, and 0.5,1.0 and 3.0 mM potassium iodide processing groups
Astaxanthin yield be respectively 9.62 ± 0.22,10.65 ± 0.31 and 10.34 ± 0.26 mg/L, than control group distinguish
Improve 65.9%, 83.6% and 78.3%；
Intensity of illumination is 150 μm of ol/m2When/s, the astaxanthin yield of control group is 6.52 ± 0.20 mg/L, and 0.5,1.0
Astaxanthin yield with 3.0 mM potassium iodide processing groups is respectively 10.03 ± 0.23,11.08 ± 0.30 and 10.86 ±
0.18 mg/L has been respectively increased 53.8%, 69.9% and 66.6% than control group；
Intensity of illumination is 200 μm of ol/m2When/s, the astaxanthin yield of control group is 6.83 ± 0.23 mg/L, and 0.5,1.0
Astaxanthin yield with 3.0 mM potassium iodide processing groups is respectively 10.61 ± 0.26,11.74 ± 0.32 and 11.42 ±
0.18 mg/L has been respectively increased 55.3%, 71.9% and 67.2% than control group；
Intensity of illumination is 250 μm of ol/m2When/s, the astaxanthin yield of control group is 7.10 ± 0.25 mg/L, and 0.5,1.0
Astaxanthin yield with 3.0 mM potassium iodide processing groups is respectively 11.03 ± 0.30,12.27 ± 0.29 and 11.80 ±
0.18 mg/L has been respectively increased 55.4%, 72.8% and 66.2% than control group.
In conclusion induction light intensity is in 100-250 μm of ol/m2Within the scope of/s, the potassium iodide within the scope of 0.5-3 mM can
It is greatly promoted the accumulation of Astaxanthin In Haematococcus Pluvialis.
This result illustrates the feasibility and reliability of the method for the present invention, is to improve Determination of Astaxanthin in Haematococcus Pluvialis content
Effective ways.
The history of life and astaxanthin accumulation characteristic based on haematococcus pluvialis, cultivation haematococcus pluvialis obtain general when astaxanthin
Using two-phase method.First stage is the frustule of low illumination mass propgation green, to obtain maximum biomass as target；The
Two-stage is so that algae is in adverse circumstances state and largely accumulate astaxanthin.Because haematococcus pluvialis is single celled algae, right
Environmental change is very sensitive.Bloom is one of induction Determination of Astaxanthin in Haematococcus Pluvialis accumulation most efficient method.Under the conditions of bloom
Frustule can generate active oxygen, they are the haematococcus pluvialis accumulation essential factors of astaxanthin, but excessively high active oxygen
To the toxic effect of frustule, including inhibit photosynthesis, frustule can transfer antioxidant system again to reduce intracellular work
Property oxygen content.It is increased with active oxygen and what Determination of Astaxanthin in Haematococcus Pluvialis accumulated is that the significant of Antioxidant Indexes in frustule mentions
Height, activity, total antioxidant capacity and freedom including superoxide dismutase, catalase and glutathione peroxidase
Base Scavenging activity.Active o content, frustule will reduce anti-oxidant required substance in frustule when by adjusting high photoinduction
With energy, bloom inhibition photosynthetic to frustule will also be alleviated, it is meant that have more substances and energy can by with
In the synthesis of astaxanthin.Therefore, the present invention adds suitable antioxidant potassium iodide to haematococcus pluvialis culture solution by external source
In, it is subsequently placed under the conditions of bloom and induces, achieved the purpose that improve the content astaxanthin in haematococcus pluvialis.
Two, comparative test
1, experimental design
By haematococcus pluvialis intensity of illumination be 30-50 μm of ol/m216 days are cultivated in the artificial lighting incubator of/s to logarithm life
For a long time.The potassium iodide of 1.0 mM is added in experimental group 1, and the sodium chloride (Jiang Hongxia etc., 2017) of 80 mM is added in experimental group 2,
Control group does not add promotor, is 150 μm of ol/m in intensity of illumination2Fiber differentiation 14 days in the artificial lighting incubator of/s is surveyed
Determine content astaxanthin.
2, experimental result
The astaxanthin yield of control group is 6.41 ± 0.18 mg/L, and the astaxanthin yield of experimental group 1 is 10.92 ± 0.28
Mg/L improves 70.3% than control group；The astaxanthin yield of experimental group 2 is 6.32 ± 0.20 mg/L, with control group without aobvious
Work difference (p- value > 0.05).
Above description is not limitation of the present invention, and the present invention is also not limited to the example above.The art it is common
Within the essential scope of the present invention, the variations, modifications, additions or substitutions made also should belong to protection of the invention to technical staff
Range.
Claims (5)
1. a kind of method for promoting Determination of Astaxanthin in Haematococcus Pluvialis accumulation, it is characterised in that: first by haematococcus pluvialis under low light
Logarithmic growth later period and/or plateau are cultivated, algae solution then is added in suitable potassium iodide, then algae solution is placed in intense light conditions
The accumulation of lower induction astaxanthin.
2. a kind of method for promoting Determination of Astaxanthin in Haematococcus Pluvialis accumulation according to claim 1, it is characterised in that: described
Low light refer to intensity of illumination be 30-50 μm of ol/m2/s。
3. a kind of method for promoting Determination of Astaxanthin in Haematococcus Pluvialis accumulation according to claim 1, it is characterised in that: described
The haematococcus pluvialis cell in logarithmic growth later period or plateau refer at light conditions culture 15-20 days up to OD680
Algae solution equal to 0.3, i.e. algae solution concentration are 2.5 × 105A/ml.
4. a kind of method for promoting Determination of Astaxanthin in Haematococcus Pluvialis accumulation according to claim 1, it is characterised in that: described
Final concentration of 0.5-3.0 mM of the potassium iodide in algae solution.
5. a kind of method for promoting Determination of Astaxanthin in Haematococcus Pluvialis accumulation according to claim 1, it is characterised in that: described
Strong light refer to intensity of illumination be 100-250 μm of ol/m2/s。
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111793667A (en) * | 2020-07-20 | 2020-10-20 | 中国科学院合肥物质科学研究院 | Method for increasing yield of haematococcus pluvialis astaxanthin by adding additive |
| CN112998111A (en) * | 2021-04-16 | 2021-06-22 | 馨辰生物(广东)有限公司 | Haematococcus pluvialis gel candy and preparation method thereof |
| CN115044527A (en) * | 2022-02-15 | 2022-09-13 | 昆明理工大学 | Application of inositol in promoting haematococcus pluvialis to produce astaxanthin |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106868085A (en) * | 2017-01-23 | 2017-06-20 | 宁波大学 | A kind of method for promoting haematococcus pluvialis rapid conversion to accumulate astaxanthin |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106868085A (en) * | 2017-01-23 | 2017-06-20 | 宁波大学 | A kind of method for promoting haematococcus pluvialis rapid conversion to accumulate astaxanthin |
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| KOBAYASHI M: "Astaxanthin biosynthesis enhanced by reactive oxygen species in the green alga Haematococcus pluvialis", 《BIOTECHNOLOGY AND BIOPROCESS ENGINEERING》 * |
| MAKIO KOBAYASHI等: "Enhanced Carotenoid Biosynthesis by Oxidative Stress in Acetate-Induced Cyst Cells of a Green Unicellular Alga,Haematococcus pluvialis", 《APPLIED AND ENVIRONMENTAL MICROBIOLOGY》 * |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111793667A (en) * | 2020-07-20 | 2020-10-20 | 中国科学院合肥物质科学研究院 | Method for increasing yield of haematococcus pluvialis astaxanthin by adding additive |
| CN111793667B (en) * | 2020-07-20 | 2022-03-11 | 中国科学院合肥物质科学研究院 | Method for increasing yield of haematococcus pluvialis astaxanthin by adding additive |
| CN112998111A (en) * | 2021-04-16 | 2021-06-22 | 馨辰生物(广东)有限公司 | Haematococcus pluvialis gel candy and preparation method thereof |
| CN115044527A (en) * | 2022-02-15 | 2022-09-13 | 昆明理工大学 | Application of inositol in promoting haematococcus pluvialis to produce astaxanthin |
| CN115044527B (en) * | 2022-02-15 | 2023-12-15 | 昆明理工大学 | Application of inositol in promoting haematococcus pluvialis to produce astaxanthin |
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