CN116837056A - Method for promoting spirulina to accumulate phycocyanin by combining salt stress with betaine - Google Patents
Method for promoting spirulina to accumulate phycocyanin by combining salt stress with betaine Download PDFInfo
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- CN116837056A CN116837056A CN202310971348.4A CN202310971348A CN116837056A CN 116837056 A CN116837056 A CN 116837056A CN 202310971348 A CN202310971348 A CN 202310971348A CN 116837056 A CN116837056 A CN 116837056A
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- 235000016425 Arthrospira platensis Nutrition 0.000 title claims abstract description 87
- 229940082787 spirulina Drugs 0.000 title claims abstract description 85
- KWIUHFFTVRNATP-UHFFFAOYSA-N glycine betaine Chemical compound C[N+](C)(C)CC([O-])=O KWIUHFFTVRNATP-UHFFFAOYSA-N 0.000 title claims abstract description 66
- 229960003237 betaine Drugs 0.000 title claims abstract description 50
- 108010053210 Phycocyanin Proteins 0.000 title claims abstract description 49
- 150000003839 salts Chemical class 0.000 title claims abstract description 28
- 238000000034 method Methods 0.000 title claims abstract description 22
- 230000001737 promoting effect Effects 0.000 title claims abstract description 16
- 239000001963 growth medium Substances 0.000 claims abstract description 58
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 50
- 239000011780 sodium chloride Substances 0.000 claims abstract description 25
- 241000195493 Cryptophyta Species 0.000 claims abstract description 17
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- 238000012258 culturing Methods 0.000 claims abstract description 15
- 238000005286 illumination Methods 0.000 claims abstract description 14
- 238000009825 accumulation Methods 0.000 claims description 13
- 230000001954 sterilising effect Effects 0.000 claims description 10
- 239000012153 distilled water Substances 0.000 claims description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
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- 238000000926 separation method Methods 0.000 claims description 4
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- 238000003199 nucleic acid amplification method Methods 0.000 claims description 3
- 238000004659 sterilization and disinfection Methods 0.000 claims description 3
- 241000620196 Arthrospira maxima Species 0.000 claims description 2
- 230000035882 stress Effects 0.000 description 18
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- 230000012010 growth Effects 0.000 description 6
- 239000002207 metabolite Substances 0.000 description 5
- 238000007710 freezing Methods 0.000 description 4
- 230000008014 freezing Effects 0.000 description 4
- 230000036579 abiotic stress Effects 0.000 description 3
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- 238000002835 absorbance Methods 0.000 description 2
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- 239000003963 antioxidant agent Substances 0.000 description 2
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- 230000009286 beneficial effect Effects 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
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- 239000000411 inducer Substances 0.000 description 2
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- 238000004108 freeze drying Methods 0.000 description 1
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- VZCCETWTMQHEPK-UHFFFAOYSA-N gamma-Linolensaeure Natural products CCCCCC=CCC=CCC=CCCCCC(O)=O VZCCETWTMQHEPK-UHFFFAOYSA-N 0.000 description 1
- VZCCETWTMQHEPK-QNEBEIHSSA-N gamma-linolenic acid Chemical compound CCCCC\C=C/C\C=C/C\C=C/CCCCC(O)=O VZCCETWTMQHEPK-QNEBEIHSSA-N 0.000 description 1
- 235000020664 gamma-linolenic acid Nutrition 0.000 description 1
- 229960002733 gamolenic acid Drugs 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
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- 230000000324 neuroprotective effect Effects 0.000 description 1
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- 239000000049 pigment Substances 0.000 description 1
- 239000005648 plant growth regulator Substances 0.000 description 1
- 150000008442 polyphenolic compounds Chemical class 0.000 description 1
- 235000013824 polyphenols Nutrition 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
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- 238000012545 processing Methods 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 150000003856 quaternary ammonium compounds Chemical class 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/795—Porphyrin- or corrin-ring-containing peptides
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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Abstract
The invention discloses a method for promoting spirulina to accumulate phycocyanin by combining salt stress with betaine, which comprises the following steps: firstly, culturing spirulina to a late logarithmic phase, and collecting algae cells as seeds; adding a certain amount of sodium chloride into spirulina culture medium (such as Zarouk culture medium) to make sodium chloride concentration reach 5-600 mmol/L; preparing glycine betaine mother liquor, adding the mother liquor into spirulina culture medium to reach final concentration of 5-5000 μmol/L, and culturing spirulina under illumination. Separating and enriching the spirulina cells obtained by culture, and extracting phycocyanin. The method is simple and easy to implement, can greatly shorten the culture time of the spirulina, and obviously increases the yield of phycocyanin, thereby greatly improving the efficiency of producing phycocyanin by microalgae.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to a method for promoting spirulina to accumulate phycocyanin by combining salt stress with betaine.
Background
Spirulina contains essential fatty acids (gamma-linolenic acid), polyphenols, pigments, vitamins (B1, B3, etc.), minerals, and carbohydrates and high protein content. Notably, bioactive compounds isolated from spirulina have therapeutic properties including anti-inflammatory, antioxidant and antibacterial activity. Thus, spirulina biomass finds a great number of applications in foods, beverages, baked products, dairy products, candies, and the like. Phycocyanin is a very important class of spirulina products, a blue pigment-protein complex, which has been approved by the U.S. Food and Drug Administration (FDA) as a natural blue food colorant. Phycocyanin is used in pharmaceutical industry and functional food due to its antioxidant, neuroprotective, anti-inflammatory and liver protecting properties. Compared with artificially synthesized amino acids and blue pigment, the spirulina-based protein and phycocyanin have higher quality and bioavailability. Although the market demand for spirulina PCs is increasing, challenges remain in the cultivation and processing of spirulina biomass. Most notably, in large-scale planting processes, cost-intensive and low yields of target products are major constraints for economic viability.
Biosynthesis of microalgae metabolites can be triggered by some abiotic stresses, while altering the microalgae metabolite spectrum, thereby enabling cost-effective and sustainable microalgae derivative product production. Salinity is a very important influencing factor for microbial growth and productivity. While abiotic stress factors may increase the accumulation of microalgae metabolites, it may inhibit the growth of algal cells, even reducing biomass concentration. At present, the technology of using plant growth regulator, chemical inducer and the like is an effective means for reducing production cost, improving microalgae biomass and enhancing the synthesis of high-added-value metabolites by microalgae. Glycine betaine is a quaternary ammonium compound present in microorganisms, plants and animals. A number of studies have shown that glycine betaine can resist various forms of stress, including salinity, nutrient deficiency, temperature changes, and drought. Among compatible solutes, glycine betaine increases the tolerance of cells to various abiotic stresses in various ways, effectively protecting cell growth and accumulation of metabolites. Thus, exogenous betaine addition may have a positive effect on spirulina growth and phycocyanin production under salt stress. More importantly, the glycine betaine is cheap and easy to obtain, and the use of the glycine betaine as an inducer has economic benefit.
Disclosure of Invention
The invention aims to solve the problems in the prior art, and provides a method for improving the biomass of spirulina and the yield of phycocyanin by combining salt stress with betaine, so as to solve the problems that the biomass of spirulina is low and the yield of phycocyanin cannot be improved due to adverse environmental factors in the prior art. The method is two-stage culture, firstly, utilizing light to autotrophy culture spirulina to collect algae cells in the late stage of logarithmic growth phase; adding sodium chloride and glycine betaine into Zarouk culture medium, continuously introducing air to culture spirulina under illumination, and concentrating the cultured spirulina cells in the stationary phase by filtration separation method to extract phycocyanin.
In order to achieve the above purpose, the present invention adopts the following technical scheme.
A method for promoting spirulina to accumulate phycocyanin by combining salt stress with betaine, comprising the following steps:
step 1, selecting a proper culture medium as a basic culture medium to culture spirulina, and collecting algae cells as seeds required by amplification culture when the spirulina grows to the late stage of the logarithmic growth phase;
step 2, preparing high-concentration sodium chloride mother liquor and adding the high-concentration sodium chloride mother liquor into a spirulina basic culture medium to keep the final concentration of sodium chloride between 5 and 600 mmol/L;
and 3, sterilizing the culture medium obtained in the step 2 at the temperature of 115 ℃ for 30min under high temperature and high pressure, inoculating the spirulina seed liquid obtained in the step 1, adding glycine betaine mother liquor prepared by distilled water, keeping the final concentration of glycine betaine in the culture medium between 5 mu mol/L and 150mmol/L, providing illumination for the culture medium for culturing, enriching the spirulina cells cultured to a stationary phase by a filtration separation method, and extracting phycocyanin.
Specifically, in step 1, a suitable culture medium is selected as a basal medium for culturing spirulina, wherein the suitable culture medium is a basal medium which does not induce salt stress.
Further, the temperature of the spirulina cultured in the step 1 is 20-35 ℃, and the illumination is 3000-9000 lux.
Specifically, in the step S2, the high-concentration sodium chloride mother solution is prepared and added into the spirulina basic culture medium, or a certain amount of sodium chloride is directly weighed and added into the spirulina basic culture medium, so that the final concentration of the sodium chloride is kept between 5 and 600 mmol/L.
Specifically, in the step 3, the culture medium obtained in the step 2 is sterilized at the temperature of 115 ℃ for 30min under high temperature and high pressure, or is directly used without sterilization, and is inoculated into the spirulina seed liquid obtained in the step 1.
Further, adding glycine betaine mother liquor prepared by distilled water in the step 3, or directly weighing a certain amount of glycine betaine and adding the glycine betaine mother liquor into the spirulina basic culture medium, so that the final concentration of the glycine betaine in the culture medium is kept between 5 mu mol/L and 150 mmol/L.
Further, the culture medium is provided with illumination for culture in the step 3, and the illumination culture temperature is 10-40 ℃.
Preferably, the spirulina species is spirulina platensis or spirulina maxima, including but not limited to arthrospira and spirulina species.
The invention also provides a method for detecting the phycocyanin content in the algae cells, which comprises the following steps:
preparation of phycocyanin: filtering the stationary-phase algae cell culture solution obtained according to the technical scheme through a screen, repeatedly washing for 2-3 times with distilled water, freezing at-80 ℃ for more than 2 h, and freeze-drying; weighing the freeze-dried dry algae powder, adding 0.1mol/L phosphate buffer (pH=7), repeatedly freezing and thawing for 3 times, and centrifuging to obtain phycocyanin; the phycocyanin content in the algal cells was determined by spectrophotometric measurement of absorbance values at 615nm, 652nm and 720 nm.
Further, the screen mesh is 300 mesh in pore diameter; repeatedly freezing and thawing, and cycling at-20deg.C and 4deg.C.
The beneficial effects of the invention are as follows:
the method is simple and easy to operate, and spirulina strains screened by the method can be cultured according to a conventional method. According to the invention, photoautotrophy is utilized, and then the induction factor is added to induce the algae cells under salt stress to accumulate phycocyanin, so that the rapid high-density growth of microalgae is realized, and the high-quality culture of the microalgae is realized; compared with the culture under the condition of only salt stress, the culture method has the advantages that the accumulation of phycocyanin in microalgae cells can be effectively promoted by exogenously adding glycine betaine under the condition of salt stress, the phycocyanin content is greatly improved, the maximum phycocyanin content in the microalgae cells reaches 22.31%, and the maximum phycocyanin content is improved by 106.38% compared with a control group, so that the salt stress combined glycine betaine can be proved to be capable of remarkably promoting the accumulation of phycocyanin in spirulina. Therefore, under the condition of salt stress, the combination of the action of exogenous chemical regulators is more beneficial to the growth of spirulina and the accumulation of phycocyanin content.
Drawings
FIG. 1 is a flow chart of a method for promoting spirulina to accumulate phycocyanin by combining salt stress and exogenous addition of glycine betaine;
fig. 2 is a graph of analysis of the results of comparative example 1 and examples 1 to 3.
Detailed Description
The present invention will be described in further detail with reference to the specific examples and fig. 1, but the scope of the present invention is not limited to the above.
As shown in fig. 1, the invention provides a method for promoting spirulina to accumulate phycocyanin by combining salt stress with betaine, which comprises the following steps:
step 1, selecting a proper culture medium as a basic culture medium to culture spirulina, and collecting algae cells as seeds required by amplification culture when the spirulina grows to the late stage of the logarithmic growth phase;
step 2, preparing high-concentration sodium chloride mother liquor and adding the high-concentration sodium chloride mother liquor into a spirulina basic culture medium to keep the final concentration of sodium chloride between 5 and 600 mmol/L;
and 3, sterilizing the culture medium obtained in the step 2 at the temperature of 115 ℃ for 30min under high temperature and high pressure, inoculating the spirulina seed liquid obtained in the step 1, adding glycine betaine mother liquor prepared by distilled water, keeping the final concentration of glycine betaine in the culture medium between 5 mu mol/L and 150mmol/L, providing illumination for the culture medium for culturing, enriching the spirulina cells cultured to a stationary phase by a filtration separation method, and extracting phycocyanin.
Specifically, in step 1, a suitable culture medium is selected as a basal medium for culturing spirulina, wherein the suitable culture medium is a basal medium which does not induce salt stress.
Further, the temperature of the spirulina cultured in the step 1 is 20-35 ℃, and the illumination is 3000-9000 lux.
Specifically, in the step S2, the high-concentration sodium chloride mother solution is prepared and added into the spirulina basic culture medium, or a certain amount of sodium chloride is directly weighed and added into the spirulina basic culture medium, so that the final concentration of the sodium chloride is kept between 5 and 600 mmol/L.
Specifically, in the step 3, the culture medium obtained in the step 2 is sterilized at the temperature of 115 ℃ for 30min under high temperature and high pressure, or is directly used without sterilization, and is inoculated into the spirulina seed liquid obtained in the step 1.
Further, adding glycine betaine mother liquor prepared by distilled water in the step 3, or directly weighing a certain amount of glycine betaine and adding the glycine betaine mother liquor into the spirulina basic culture medium, so that the final concentration of the glycine betaine in the culture medium is kept between 5 mu mol/L and 150 mmol/L.
Further, the culture medium is provided with illumination for culture in the step 3, and the illumination culture temperature is 10-40 ℃.
Comparative example 1
Preparing a Zarouk culture medium as a basic culture medium of spirulina; sterilizing at high pressure and high temperature for 30min, inoculating spirulina with algae cell concentration controlled at 0.45 g/L, adding glycine betaine into the culture medium to final concentration of 0 μm, culturing at 28deg.C under 5500 lux, and culturing under shaking. The maximum phycocyanin content of the spirulina cultivated in the control example reaches 10.81%; the maximum biomass reached 2.04 g/L.
Example 1
Preparing a Zarouk culture medium as a basic culture medium of spirulina; sterilizing at high pressure and high temperature for 30min, inoculating spirulina with algae cell concentration controlled at 0.45 g/L, adding glycine betaine to the culture medium to final concentration of 150 μm, culturing at 28deg.C under 5500 lux, and culturing under shaking. The maximum phycocyanin content of the spirulina cultivated in the embodiment reaches 11.45%; the maximum biomass reached 2.18 g/L.
Example 2
Preparing a Zarouk culture medium containing 100 mM sodium chloride as a basic culture medium of spirulina; sterilizing at high pressure and high temperature for 30min, inoculating spirulina with algae cell concentration controlled at 0.45 g/L, adding glycine betaine into the culture medium to final concentration of 0 μm, culturing at 28deg.C under 5500 lux, and culturing under shaking. The maximum phycocyanin content of the spirulina cultivated in the control example reaches 16.24%; the maximum biomass reached 1.88 g/L.
Example 3
Preparing a Zarouk culture medium containing 100 mM sodium chloride as a basic culture medium of spirulina; sterilizing at high pressure and high temperature for 30min, inoculating spirulina with algae cell concentration controlled at 0.45 g/L, adding glycine betaine to the culture medium to final concentration of 150 μm, culturing at 28deg.C under 5500 lux, and culturing under shaking. The maximum phycocyanin content of the spirulina cultivated in the embodiment reaches 22.31%; the maximum biomass reached 2.58 g/L.
The culture solutions obtained in comparative example 1 and examples 1 to 3 were filtered and enriched, and were freeze-dried and weighed after repeated washing with distilled water for 3 times; adding 0.1mol/L phosphate buffer (pH=7) into the freeze-dried algae powder, repeatedly freezing and thawing for 3 times, and centrifuging to obtain phycocyanin. Absorbance values at 615nm, 652nm and 720nm were determined with a spectrophotometer. Phycocyanin content (mg/mL) = (a 615-a 720) -0.474 x (a 652-a 720))/5.34.
Analysis of comparison results: as shown in FIG. 2, the combination of salt stress and glycine betaine induction promotes the accumulation of phycocyanin in spirulina, thus realizing both rapid high-density growth and high-quality cultivation of microalgae. And under the condition of salt stress, the phycocyanin content in the algae cells is improved by 50.23 percent compared with that of a control group, and reaches 16.24 percent. Compared with a control group, the content of phycocyanin in algae cells under the condition of exogenous glycine betaine and salt stress is improved by 106.38 percent, which reaches 22.31 percent, and the content of phycocyanin in algae cells is greatly improved compared with that of normal spirulina.
The foregoing is only a preferred embodiment of the present invention, and the present invention is not limited thereto, and any simple modification, variation and imitation of the foregoing embodiments for the technical content of the present invention falls within the scope of the technical solution of the present invention.
Claims (8)
1. A method for promoting spirulina to accumulate phycocyanin by combining salt stress with betaine, which is characterized by comprising the following steps:
step 1, selecting a proper culture medium as a basic culture medium to culture spirulina, and collecting algae cells as seeds required by amplification culture when the spirulina grows to the late stage of the logarithmic growth phase;
step 2, preparing high-concentration sodium chloride mother liquor and adding the high-concentration sodium chloride mother liquor into a spirulina basic culture medium to keep the final concentration of sodium chloride between 5 and 600 mmol/L;
and 3, sterilizing the culture medium obtained in the step 2 at the temperature of 115 ℃ for 30min under high temperature and high pressure, inoculating the spirulina seed liquid obtained in the step 1, adding glycine betaine mother liquor prepared by distilled water, keeping the final concentration of glycine betaine in the culture medium between 5 mu mol/L and 150mmol/L, providing illumination for the culture medium for culturing, enriching the spirulina cells cultured to a stationary phase by a filtration separation method, and extracting phycocyanin.
2. The method for promoting accumulation of phycocyanin by spirulina by combining salt stress with betaine according to claim 1, characterized in that: the spirulina is cultured by selecting a proper culture medium as a basal medium in the step 1, wherein the proper culture medium is the basal medium which does not induce salt stress.
3. The method for promoting accumulation of phycocyanin by spirulina by combining salt stress with betaine according to claim 1, characterized in that: the temperature of the spirulina cultured in the step 1 is 20-35 ℃, and the illumination is 3000-9000 lux.
4. The method for promoting accumulation of phycocyanin by spirulina by combining salt stress with betaine according to claim 1, characterized in that: and (2) preparing a high-concentration sodium chloride mother solution in the step (S2) and adding the high-concentration sodium chloride mother solution into the spirulina basic culture medium, or directly weighing a certain amount of sodium chloride and adding the sodium chloride into the spirulina basic culture medium, so that the final concentration of the sodium chloride is kept between 5 and 600 mmol/L.
5. The method for promoting accumulation of phycocyanin by spirulina by combining salt stress with betaine according to claim 1, characterized in that: in the step 3, the culture medium obtained in the step 2 is sterilized at the temperature of 115 ℃ for 30min under high temperature and high pressure, or is directly used without sterilization, and is connected with the spirulina seed liquid obtained in the step 1.
6. The method for promoting accumulation of phycocyanin by spirulina by combining salt stress with betaine according to claim 1, characterized in that: and step 3, adding glycine betaine mother liquor prepared by distilled water, or directly weighing a certain amount of glycine betaine and adding the glycine betaine mother liquor into the spirulina basic culture medium, so that the final concentration of the glycine betaine in the culture medium is kept between 5 mu mol/L and 150 mmol/L.
7. The method for promoting accumulation of phycocyanin by spirulina by combining salt stress with betaine according to claim 1, characterized in that: and 3, providing illumination for the culture medium for culture, wherein the illumination culture temperature is 10-40 ℃.
8. The method for promoting accumulation of phycocyanin by spirulina by combining salt stress with betaine according to claim 1, characterized in that: the spirulina is spirulina platensis or spirulina maxima.
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