CN115627237A - Method for improving haematococcus pluvialis astaxanthin yield by coupling proline with various stresses - Google Patents
Method for improving haematococcus pluvialis astaxanthin yield by coupling proline with various stresses Download PDFInfo
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Abstract
The invention belongs to the technical field of bioengineering, and discloses a method for improving the yield of astaxanthin of haematococcus pluvialis by using proline coupling multiple stresses, wherein the method comprises the steps of adopting abiotic stress coupling proline induction conditions, and firstly culturing haematococcus pluvialis in a growth period to enable algal cells to grow and accumulate biomass; and then culturing the microalgae by coupling abiotic stress with proline in an induction period. According to the invention, under the condition of abiotic stress, the addition of 100 mu mol/L proline can obviously improve the astaxanthin content and the astaxanthin yield of microalgae, and by the culture method, the rapid accumulation of astaxanthin in algal cells is realized, so that a new research direction is provided for efficiently culturing haematococcus pluvialis to produce astaxanthin. The invention obviously improves the astaxanthin content in haematococcus pluvialis, and when the concentration of the added proline is 100 mu mol/L, the content and the yield of the microalga astaxanthin are respectively 42.77mg/g and 3.27mg/L/d, which are improved by 61.40 percent and 55.45 percent compared with a control group.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to a method for improving the yield of astaxanthin of haematococcus pluvialis by coupling proline with various stresses.
Background
Astaxanthin is a carotenoid of red fat-soluble ketone, and can effectively inhibit lipid peroxidation caused by free radicals due to excellent antioxidant performance. Meanwhile, the composition has physiological effects in various aspects such as inhibiting tumorigenesis, enhancing immunity, eliminating free radicals in vivo and the like. Is widely applied to health care products, cosmetics and medicines. Haematococcus pluvialis is a freshwater alga which can accumulate a large amount of astaxanthin under certain conditions, and is a high-quality source for producing natural astaxanthin. However, the industrial production of astaxanthin is limited by low productivity and high cost. Therefore, it is important to find an effective strategy for increasing the yield of natural astaxanthin.
A large number of studies indicate that abiotic stress conditions, including strong light, nitrogen deficiency and high salt stress, can promote the accumulation of astaxanthin in haematococcus pluvialis cells. In addition, part of the phytohormones or chemical inducers can also promote the accumulation of astaxanthin by Haematococcus pluvialis under abiotic stress. However, most of the inducers can only unilaterally increase the astaxanthin content in Haematococcus pluvialis, and have little influence on biomass, thus resulting in no improvement in astaxanthin yield.
Proline is a multifunctional amino acid, widely exists in plants, and can be accumulated in large quantities when the proline is used for coping with environmental stress, so that the plants can better cope with the adverse effects caused by abiotic stress. Reports show that the added exogenous proline increases the chlorophyll content of leaves and slows down the reduction of the photosynthetic activity under the salt stress, thereby maintaining the growth of plants under the salt stress. The addition of proline in the culture process of haematococcus pluvialis can improve the biomass of the haematococcus pluvialis under abiotic stress and the content of astaxanthin, so that the yield of astaxanthin is improved.
Through the above analysis, the problems and defects of the prior art are as follows: the existing strategy for producing astaxanthin by haematococcus pluvialis cannot simultaneously improve the biomass of microalgae and the content of astaxanthin, so that the yield of astaxanthin cannot be further improved.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a method for improving the yield of astaxanthin of haematococcus pluvialis by coupling proline with various stresses; thereby solving the problems that haematococcus pluvialis cannot improve the biomass and further cannot improve the yield due to adverse environmental factors in the prior art.
The invention is realized by a method for improving the astaxanthin yield of haematococcus pluvialis by using proline coupled with various stresses, which comprises the following steps: the method for producing astaxanthin by haematococcus pluvialis comprises two culture stages of a growth phase and an induction phase, wherein microalgae are cultured in the growth phase to enable the haematococcus pluvialis to accumulate biomass; and then the culture is carried out in an induction period by coupling abiotic stress with proline to promote the accumulation of astaxanthin.
Further, the method for improving the yield of astaxanthin from haematococcus pluvialis by coupling proline with various stresses further comprises the following steps: firstly, carrying out algae seed culture in a growth period to ensure that algae cells quickly grow and accumulate certain biomass; and adding proline under the conditions of nitrogen deficiency, high light and high salt to culture haematococcus pluvialis, placing the haematococcus pluvialis in an environment with a certain temperature to culture, and detecting and analyzing the biomass, astaxanthin content and yield of the haematococcus pluvialis.
Further, the method for improving the yield of astaxanthin from haematococcus pluvialis by coupling proline with various stresses comprises the following steps:
step one, culturing haematococcus pluvialis seed liquid to accumulate biomass;
step two, culturing microalgae by using proline coupled with various abiotic stresses to promote haematococcus pluvialis to accumulate astaxanthin;
and step three, extracting astaxanthin in the algal cells in the culture solution obtained in the step two by using an organic solvent.
Further, in the first step, a culture medium adopted by haematococcus pluvialis seed liquid culture is a BG-11 culture medium.
Further, the temperature adopted by the haematococcus pluvialis seed liquid culture in the step one is 23-25 ℃ and the illumination is 25-50 mu mol/m 2 /s。
Further, the haematococcus pluvialis seed solution in the first step is cultured in a 3000mL round-bottom bottle, and sterile air is introduced by using an air pump. .
Further, the haematococcus pluvialis is haematococcus pluvialis LUGU.
Further, the culturing haematococcus pluvialis with the proline-coupled abiotic stress in the second step comprises: adding proline with different concentrations into a culture medium, and resuspending the seed solution into a 500mL conical flask to ensure the inoculation amount of microalgae; continuously introducing air, and culturing in an environment with certain temperature and illumination intensity to obtain the final culture solution.
Further, the culture medium is nitrogen-deficient BG-11 culture medium.
Further, the culture temperature is 28 ℃, the salinity is 1g/L, and the illumination intensity is 250 mu mol/m 2 /s。
Furthermore, the concentration of the proline added is 50, 100 and 150 mu mol/L respectively.
Further, the inoculation amount of the microalgae is 0.35g/L.
Further, the extraction of astaxanthin in algal cells in the culture solution obtained in the second step by using an organic solvent in the third step comprises:
centrifuging the final culture solution obtained in the second step for 5min at a speed of 5000r/min, and washing with distilled water twice; treating with 5% potassium hydroxide solution in 30% methanol at 65 deg.C for 15min to destroy chlorophyll; centrifuging algae cells at 5000r/min for 5min, and washing with distilled water for 3 times; carrying out ultrasonic treatment on the treated algae cells by using 5mL of dimethyl sulfoxide under 200W power for 10min, and extracting in a water bath at 45 ℃ for 20min; repeating the extraction process until the particles become colorless; total astaxanthin was determined by absorbance measurement at 490 nm.
By combining the technical scheme and the technical problem to be solved, the technical scheme to be protected by the invention has the advantages and positive effects that:
first, aiming at the technical problems and difficulties in solving the problems in the prior art, the technical problems to be solved by the technical scheme of the present invention are closely combined with results, data and the like in the research and development process, and some creative technical effects are brought after the problems are solved. The specific description is as follows:
according to the method for improving the yield of astaxanthin of haematococcus pluvialis by coupling various stresses with proline, firstly, haematococcus pluvialis is cultured in a growth period, so that algae cells grow rapidly and accumulate certain biomass. The seed solution is then diluted during the induction phase and cultured with proline. Meanwhile, proline is added into a BG-11 culture medium under the conditions of nitrogen deficiency, high light and high salt to culture haematococcus pluvialis, the haematococcus pluvialis is cultured in a certain temperature environment, and the biomass, the astaxanthin content and the yield of the haematococcus pluvialis are detected and analyzed.
According to the invention, by combining an abiotic stress and a proline induction culture mode, when the concentration of proline is 100 mu mol/L, the content of the microalgae astaxanthin and the yield of the astaxanthin are respectively increased by 61.40% and 55.45% compared with a control group; the culture method can obviously improve the astaxanthin content and the astaxanthin yield of haematococcus pluvialis.
By the culture method, the rapid accumulation of astaxanthin in algal cells is realized, and a new research direction is provided for efficiently culturing microalgae to produce astaxanthin. The invention obviously improves the astaxanthin content of the microalgae, when the concentration of the added proline is 100 mu mol/L, the astaxanthin content and the yield of the microalgae are respectively 42.77mg/g and 3.27mg/L/d, which are improved by 61.40 percent and 55.45 percent compared with a control group.
Secondly, considering the technical scheme as a whole or from the perspective of products, the technical effect and advantages of the technical scheme to be protected by the invention are specifically described as follows:
the method has simple process, easy operation and obvious effect, can obviously improve the astaxanthin content and the yield of the haematococcus pluvialis, simultaneously provides a new technical approach for the industrial production of the astaxanthin by the haematococcus pluvialis, and provides a new idea for improving the yield of the astaxanthin.
Third, as an inventive supplementary proof of the claims of the present invention, there are also presented several important aspects:
the expected income and commercial value after the technical scheme of the invention is converted are as follows: provides a new technical approach for the industrial production of astaxanthin by haematococcus pluvialis and provides a new idea for improving the yield of astaxanthin.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required to be used in the embodiments of the present invention will be briefly described below, and it is obvious that the drawings described below are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a flow chart of a method for increasing astaxanthin production from Haematococcus pluvialis using proline coupled to various stresses according to an embodiment of the present invention;
FIG. 2A is a schematic diagram showing the effect of different concentrations of proline on Haematococcus pluvialis biomass according to an embodiment of the present invention;
FIG. 2B is a graph showing the effect of different concentrations of proline on astaxanthin content provided by an example of the present invention;
fig. 2C is a schematic diagram showing the effect of varying concentrations of proline on astaxanthin yield at day 13 as provided by the examples of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and do not limit the invention.
In view of the problems of the prior art, the present invention provides a method for increasing astaxanthin production by Haematococcus pluvialis by using proline coupled with various stresses, and the present invention is described in detail below with reference to the accompanying drawings.
1. The embodiments are explained. This section is an explanatory embodiment expanding on the claims so as to fully understand how the present invention is embodied by those skilled in the art.
As shown in fig. 1, the method for improving astaxanthin production by haematococcus pluvialis by coupling proline with various stresses provided by the embodiment of the invention comprises the following steps:
s101, culturing haematococcus pluvialis seed liquid to accumulate biomass;
s102, culturing microalgae by coupling proline with various abiotic stresses to promote haematococcus pluvialis to accumulate astaxanthin;
s103, extracting astaxanthin in algal cells in the culture solution obtained in the step S102 by using an organic solvent.
As a preferred embodiment, the method for improving microalgae astaxanthin by using proline-coupled multiple stress provided by the embodiment of the invention specifically comprises the following steps:
(1) Culturing seed liquid in the growth period of haematococcus pluvialis: at 23-25 deg.C, illuminating 25-50 μmol/m 2 Culturing Haematococcus pluvialis in a 3000mL round-bottomed bottle by using a BG-11 culture medium in a/s environment, and introducing sterile air by using an air pump; the Haematococcus pluvialis LUGU is Haematococcus pluvialis.
(2) Culturing Haematococcus pluvialis by coupling abiotic stress with proline in an induction period: use of a composition containing 1g/L NaCl and lacking NaNO 3 The BG-11 culture medium is used as a basic culture medium, proline with different concentrations is added, the seed solution is resuspended in a 500mL conical flask, the volume of the culture medium is 300mL, and the inoculation amount of microalgae is ensured to be 0.35g/L; continuously introducing sterile air, and placing at 28 deg.C under illumination intensity of 250 μmol/m 2 Culturing at/s to obtain final culture solution.
(3) Extracting astaxanthin in algal cells in the culture solution obtained in the step (2) by using an organic solvent: centrifuging the final culture solution obtained in step (2) at 5000r/min for 5min, washing with distilled water twice, and treating with 5% (w/v) potassium hydroxide solution in 30% (v/v) methanol at 65 deg.C for 15min to destroy chlorophyll. The algal cells were centrifuged at 5000r/min for 5min and washed 3 times with distilled water. The treated algal cells were sonicated with 5mL of dimethyl sulfoxide at 200W power for 10min and extracted in a water bath at 45 ℃ for 20min. The extraction process was repeated until the particles became colorless. Total astaxanthin was determined by absorbance measurement at 490 nm.
2. Application examples. In order to prove the creativity and the technical value of the technical scheme of the invention, the part is the application example of the technical scheme of the claims on specific products or related technologies.
Example 1
The method provided by the embodiment of the invention is used for detecting and analyzing the microalgae biomass and the astaxanthin content under the condition that the proline concentration is 50 mu mol/L, and comprises the following specific steps:
(1) Culturing seed liquid in the growth period of haematococcus pluvialis: at the temperature of 23-25 ℃ and the illumination of 25-50 mu mol/m 2 Culturing Haematococcus pluvialis in a 3000mL round-bottomed bottle by using a BG-11 culture medium in a/s environment, and introducing sterile air by using an air pump; the Haematococcus pluvialis LUGU is Haematococcus pluvialis;
(2) Coupling 50 mu mol/L proline to the abiotic stress in the induction period to culture haematococcus pluvialis: use of a composition containing 1g/L NaCl and lacking NaNO 3 The BG-11 culture medium is used as a basic culture medium, 50 mu mol/L proline is added, the seed solution is resuspended into a 500mL conical flask, the volume of the culture medium is 300mL, and the inoculation amount of microalgae is ensured to be 0.35g/L; continuously introducing sterile air, and placing at 28 deg.C under illumination intensity of 250 μmol/m 2 Culturing at/s to obtain final culture solution.
(3) Extracting astaxanthin in algal cells in the culture solution obtained in the step (2) by using an organic solvent: centrifuging the final culture solution obtained in step (2) at 5000r/min for 5min, washing with distilled water twice, and treating with 5% (w/v) potassium hydroxide solution in 30% (v/v) methanol at 65 deg.C for 15min to destroy chlorophyll. The algal cells were centrifuged at 5000r/min for 5min and washed 3 times with distilled water. The treated algal cells were sonicated with 5mL of dimethyl sulfoxide at 200W power for 10min and extracted in a water bath at 45 ℃ for 20min. The extraction process was repeated until the particles became colorless. Total astaxanthin was determined by absorbance measurement at 490 nm. The biomass is 1.36g/L, and is slightly improved compared with a control group, but has no significant difference; the astaxanthin content and the astaxanthin yield were 33.95mg/L and 2.61mg/L/d, respectively (see FIGS. 2A to 2C), which were increased by 24.18% and 19.54%, respectively, compared to the control group.
Example 2
The method for detecting and analyzing the microalgae biomass and the astaxanthin content under the condition that the proline concentration is 100 mu mol/L comprises the following specific steps:
(1) Culturing seed liquid in the growth period of haematococcus pluvialis: at the temperature of 23-25 ℃ and the illumination of 25-50 mu mol/m 2 Culturing Haematococcus pluvialis in a 3000mL round-bottomed bottle by using a BG-11 culture medium in a/s environment, and introducing sterile air by using an air pump; the Haematococcus pluvialis LUGU is Haematococcus pluvialis;
(2) Coupling abiotic stress to proline of 100 mu mol/L to culture haematococcus pluvialis: use of a composition containing 1g/LNaCl and lacking NaNO 3 The BG-11 culture medium is used as a basic culture medium, 100 mu mol/L proline is added, the seed solution is resuspended into a 500mL conical flask, the volume of the culture medium is 300mL, and the inoculation amount of microalgae is ensured to be 0.35g/L; continuously introducing sterile air, and placing at 28 deg.C under illumination intensity of 250 μmol/m 2 Culturing at/s to obtain final culture solution.
(3) Extracting astaxanthin in algal cells in the culture solution obtained in the step (2) by using an organic solvent: centrifuging the final culture solution obtained in step (2) at 5000r/min for 5min, washing with distilled water twice, and treating with 5% (w/v) potassium hydroxide solution in 30% (v/v) methanol at 65 deg.C for 15min to destroy chlorophyll. The algal cells were centrifuged at 5000r/min for 5min and washed 3 times with distilled water. The treated algae cells were sonicated with 5mL of dimethyl sulfoxide at 200W power for 10min and extracted in a water bath at 45 ℃ for 20min. The extraction process was repeated until the particles became colorless. Total astaxanthin was determined by absorbance measurement at 490 nm. The biomass is 1.42g/L, which is obviously higher than that of a control group; the astaxanthin content and the astaxanthin yield were 42.77mg/L and 3.27mg/L/d, respectively (see FIGS. 2A to 2C), which were increased by 61.40% and 55.45% over the control group.
Example 3
The method provided by the embodiment of the invention is used for detecting and analyzing the microalgae biomass and the astaxanthin content under the condition that the proline concentration is 150 mu mol/L, and comprises the following specific steps:
(1) Culturing seed liquid in the growth period of haematococcus pluvialis: at the temperature of 23-25 ℃ and the illumination of 25-50 mu mol/m 2 Culturing Haematococcus pluvialis in a 3000mL round-bottomed bottle by using a BG-11 culture medium in a/s environment, and introducing sterile air by using an air pump; the Haematococcus pluvialis LUGU is Haematococcus pluvialis;
(2) Abiotic stress coupling 150. Mu. Mol/L proline to culture H.pluvialis: use of a composition containing 1g/LNaCl and lacking NaNO 3 The BG-11 culture medium is used as a basic culture medium, 100 mu mol/L proline is added, the seed solution is resuspended in a 500mL conical flask, the volume of the culture medium is 300mL, and the inoculation amount of microalgae is ensured to be 0.35g/L; continuously introducing sterile air, and placing at 28 deg.C with illumination intensity of 250 μmol/m 2 Culturing at/s to obtain final culture solution.
(3) Extracting astaxanthin in algal cells in the culture solution obtained in the step (2) by using an organic solvent: centrifuging the final culture solution obtained in step (2) at 5000r/min for 5min, washing with distilled water twice, and treating with 5% (w/v) potassium hydroxide solution in 30% (v/v) methanol at 65 deg.C for 15min to destroy chlorophyll. The algal cells were centrifuged at 5000r/min for 5min and washed 3 times with distilled water. The treated algae cells were sonicated with 5mL of dimethyl sulfoxide at 200W power for 10min and extracted in a water bath at 45 ℃ for 20min. The extraction process was repeated until the particles became colorless. Total astaxanthin was determined by absorbance measurement at 490 nm. The biomass is 1.41g/L, which is obviously higher than that of a control group; the astaxanthin content and the astaxanthin yield are respectively 36.87mg/L and 2.84mg/L/d (see fig. 2A-2C), which are respectively increased by 34.86 percent and 35.24 percent compared with the control group.
3. Evidence of the relevant effects of the examples. The embodiment of the invention achieves some positive effects in the process of research and development or use, and has great advantages compared with the prior art, and the following contents are described by combining data, diagrams and the like in the test process.
Comparative example:
the comparative example of the present invention provides a composition containing 1g/L NaCl and lacking NaNO 3 The BG-11 culture medium is used as a culture condition of a basal culture medium to detect and analyze microalgae biomass and astaxanthin content, and the method comprises the following specific steps:
(1) Culturing seed liquid in the growth period of haematococcus pluvialis: at the temperature of 23-25 ℃ and the illumination of 25-50 mu mol/m 2 Culturing Haematococcus pluvialis in a 3000mL round-bottomed bottle by using a BG-11 culture medium in a/s environment, and introducing sterile air by using an air pump; the Haematococcus pluvialis LUGU is Haematococcus pluvialis;
(2) Culturing haematococcus pluvialis under abiotic stress: use of a composition containing 1g/L NaCl and lacking NaNO 3 The BG-11 culture medium is used as a basic culture medium, the seed solution is resuspended in a 500mL conical flask, the volume of the culture medium is 300mL, and the inoculation amount of microalgae is ensured to be 0.35g/L; continuously introducing sterile air, and placing at 28 deg.C with illumination intensity of 250 μmol/m 2 Culturing at/s to obtain final culture solution.
(3) Extracting astaxanthin in algal cells in the culture solution obtained in the step (2) by using an organic solvent: centrifuging the final culture solution obtained in step (2) at 5000r/min for 5min, washing with distilled water twice, and treating with 5% (w/v) potassium hydroxide solution in 30% (v/v) methanol at 65 deg.C for 15min to destroy chlorophyll. The algal cells were centrifuged at 5000r/min for 5min and washed 3 times with distilled water. The treated algal cells were sonicated with 5mL of dimethyl sulfoxide at 200W power for 10min and extracted in a water bath at 45 ℃ for 20min. The extraction process was repeated until the particles became colorless. Total astaxanthin was determined by absorbance assay at 490 nm. The biomass is 1.24g/L; the astaxanthin content and the astaxanthin yield were 27.34mg/L and 2.10mg/L/d, respectively (see FIGS. 2A to 2C).
The invention combines the abiotic stress and the proline induction to promote the accumulation of the astaxanthin in the haematococcus pluvialis, and the proline of 100 mu mol/L can obviously improve the astaxanthin content and the astaxanthin yield of the haematococcus pluvialis under the abiotic stress condition.
The above description is only for the purpose of illustrating the embodiments of the present invention, and the scope of the present invention should not be limited thereto, and any modifications, equivalents and improvements made by those skilled in the art within the technical scope of the present invention as disclosed in the present invention should be covered by the scope of the present invention.
Claims (10)
1. A method for increasing astaxanthin production in Haematococcus pluvialis by coupling proline to various stresses, wherein the method for increasing astaxanthin production in Haematococcus pluvialis by coupling proline to various stresses comprises: two culture stages of a growth period and an induction period, firstly, culturing microalgae in the growth period to ensure that haematococcus pluvialis accumulates biomass; and then the culture is carried out in an induction period by coupling abiotic stress with proline to promote the accumulation of astaxanthin.
2. The method of increasing astaxanthin production from Haematococcus pluvialis using proline coupled to multiple stresses according to claim 1, wherein the method of increasing astaxanthin production from Haematococcus pluvialis using proline coupled to multiple stresses further comprises: firstly, carrying out algae seed culture in a growth period to enable algae cells to grow rapidly and accumulate certain biomass; and adding proline under the conditions of nitrogen deficiency, high light and high salt to culture haematococcus pluvialis, placing the haematococcus pluvialis in an environment with a certain temperature to culture, and detecting and analyzing the biomass, astaxanthin content and yield of the haematococcus pluvialis.
3. The method of increasing astaxanthin production from Haematococcus pluvialis using proline coupled to multiple stresses according to claim 1, wherein the method of increasing astaxanthin production from Haematococcus pluvialis using proline coupled to multiple stresses comprises the steps of:
step one, culturing haematococcus pluvialis seed liquid, and accumulating biomass;
step two, culturing microalgae by using proline coupled with various abiotic stresses to promote haematococcus pluvialis to accumulate astaxanthin;
and step three, extracting astaxanthin in the algal cells in the culture solution obtained in the step two by using an organic solvent.
4. The method for increasing the yield of astaxanthin in Haematococcus pluvialis under various stress coupled with proline according to claim 3, wherein the culture medium used for the seed liquid culture of Haematococcus pluvialis in the first step is BG-11 medium.
5. The method for increasing astaxanthin production in Haematococcus pluvialis under multiple stress coupled with proline in claim 3, wherein the temperature for the seed culture of Haematococcus pluvialis in the first step is 23-25 ℃ and the illumination is 25-50 μmol/m 2 /s。
6. The method for increasing the yield of astaxanthin in Haematococcus pluvialis under various stress coupled with proline according to claim 3, wherein the Haematococcus pluvialis seed solution in the first step is cultured in a 3000mL round-bottomed flask, and sterile air is introduced with an air pump.
7. The method of claim 3, wherein the Haematococcus pluvialis LuGU is Haematococcus pluvialis, wherein the Haematococcus pluvialis is produced by coupling proline with various stresses.
8. The method of claim 3, wherein the culturing Haematococcus pluvialis with the proline-coupled abiotic stress in step two comprises: adding proline with different concentrations into a culture medium, and resuspending the seed solution into a 500mL conical flask to ensure the inoculation amount of microalgae; continuously introducing air, and culturing in an environment with certain temperature and illumination intensity to obtain the final culture solution.
9.The method for increasing astaxanthin production in Haematococcus pluvialis utilizing proline coupled with multiple stresses according to claim 8, wherein the medium is nitrogen deficient BG-11 medium; the culture temperature is 28 ℃, the salinity is 1g/L, and the illumination intensity is 250 mu mol/m 2 S; the concentration of the added proline is 50, 100 and 150 mu mol/L respectively; the inoculation amount of the microalgae is 0.35g/L.
10. The method for increasing the yield of astaxanthin from Haematococcus pluvialis under stress of proline coupled with multiple stresses according to claim 3, wherein the extraction of astaxanthin from algal cells in the culture broth obtained in the second step of step three with an organic solvent comprises:
centrifuging the final culture solution obtained in the second step for 5min at a speed of 5000r/min, and washing with distilled water twice; treating with 5% potassium hydroxide solution in 30% methanol at 65 deg.C for 15min to destroy chlorophyll; centrifuging the algae cells at 5000r/min for 5min, and washing with distilled water for 3 times; carrying out ultrasonic treatment on the treated algae cells by using 5mL of dimethyl sulfoxide under 200W power for 10min, and extracting in a water bath at 45 ℃ for 20min; repeating the extraction process until the particles become colorless; total astaxanthin was determined by absorbance measurement at 490 nm.
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