CN110157620B - Culture method for improving content of phycoerythrin synthesized by porphyridium - Google Patents
Culture method for improving content of phycoerythrin synthesized by porphyridium Download PDFInfo
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- CN110157620B CN110157620B CN201910313268.3A CN201910313268A CN110157620B CN 110157620 B CN110157620 B CN 110157620B CN 201910313268 A CN201910313268 A CN 201910313268A CN 110157620 B CN110157620 B CN 110157620B
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Abstract
The invention discloses a culture method for improving the content of phycoerythrin synthesized by porphyridium, which aims to induce more phycoerythrin synthesized by porphyridium cells by changing environmental conditions, reducing temperature and illumination intensity. The phycoerythrin content of the porphyridium cultured by the method is 109.2-205.3 mg/L, the biomass is 5.3-7.7 g/L, and the porphyridium has important value in the fields of medicines, foods and the like.
Description
Technical Field
The invention belongs to the technical field of marine microorganisms, and particularly relates to a culture method for improving the content of phycoerythrin synthesized by porphyridium.
Background
Phycoerythrin is a food-grade pigment with excellent water solubility, is mainly present at the outermost end of phycobilisomes, participates in photosynthesis as a main light supplement antenna of porphyridium algae cells, is mainly formed by covalently combining chromophore and apoprotein with an open-chain tetrapyrrole structure through thioether bonds, has wide application in the industries of food, cosmetics, medicines and the like due to the good fluorescent intensity, antioxidation, free radical elimination, high chroma and the like of the phycoerythrin, can also be used as a fluorescent marker in the fields of molecular biology, clinical medicine and the like, for example, the phycoerythrin can be used in photodynamic tumor treatment, and has great potential as a good photosensitizer due to the safe and natural characteristics.
Porphyridium purpureum is the only single original unicellular red alga first discovered by Naegeli in 1849. It belongs to the phylum Rhodophyta (Rhodophyta), Protoferridophyceae (Porphyridiales), Porphyridiales (Porphyridiales), Porphyridiaceae (Porphyridiales) and Porphyridium (Porphyridium). Porphyridium has the characteristics of high biomass yield and rich products with high added value in a plurality of microalgae strains. And the porphyridium is distributed in seawater, fresh water, salt water and moist land, has stronger salt resistance, and the porphyridium cell can synthesize various bioactive substances in the growth process, thereby having wide application prospect in the industries of medicine, cosmetics, food, health products, textile, printing and dyeing, metallurgical petroleum and the like. The porphyridium cell can synthesize various bioactive substances in the growth process, including: phycobiliproteins (phycobiliproteins are classified into phycoerythrin PE, phycocyanin PC and allophycocyanin A-PC, wherein the content of phycoerythrin is at most, about 84%), porphyridium polysaccharide (mainly extracellular sulfated lipopolysaccharide), and polyunsaturated fatty acids (mainly arachidonic acid ARA and eicosapentaenoic acid EPA). The porphyridium polysaccharide is successfully applied to the aspects of food, medicine, clinical medicine and the like due to the antioxidant capacity of the porphyridium polysaccharide, unsaturated fatty acids ARA and EPA are applied to energy, health care products and medicine in different degrees, the market is relatively mature, the market of phycoerythrin and the like is relatively thin, at present, the domestic merchant for independently selling products of phycoerythrin and the like only belongs to Shanghai Langya biological technology limited company, and other agents completely depend on import, so the price of the porphyridium polysaccharide is very expensive.
The porphyridium is one of the main sources of phycoerythrin, and the phycoerythrin content in vivo and the biomass of the porphyridium have crucial determinants for the extraction of the phycoerythrin. In the existing research, how to increase the raw material source is more and more important except for extracting and purifying phycoerythrin, however, due to the limitation of production capacity, the method for culturing porphyridium usually achieves the purpose of improving the content of phycoerythrin by adding nutrient elements and maintaining single culture means such as low temperature and low light, and the like, and the method can improve the content of phycoerythrin to a certain extent, but generally has the problem that the content of phycoerythrin is not high enough when the biomass content is low.
Disclosure of Invention
The invention aims to overcome the defects of the prior art, provides a culture method for improving the content of phycoerythrin synthesized by porphyridium, and solves the problems in the background technology.
The technical scheme adopted by the invention for solving the technical problems is as follows: the culture method for improving the content of phycoerythrin synthesized by porphyridium is provided, the porphyridium seed solution is cultured by controlling the temperature and the illumination intensity in a segmented manner, and the culture method comprises the following steps:
(1) controlling the temperature to be 23-27 ℃ and the illumination intensity to be 145-185 mu mol/m < 2 > 2s for culturing for 1-3 days; the porphyridium cell is adapted to the growth environment and rapidly enters the logarithmic phase of growth.
(2) The temperature is controlled to be 18-22 ℃, and the illumination intensity is controlled to be 90-130 mu mol/m2s culturing for 7-9 days; by changing the environmental conditions, reducing the temperature and the illumination intensity, the porphyridium cells are induced to synthesize more phycoerythrin. The low temperature is favorable for keeping the activity of the protein, and the reduction of the illumination is favorable for the synthesis of the phycoerythrin.
(3) Controlling the temperature at 12～At 18 ℃ and the illumination intensity of 35-75 mu mol/m2And (5) culturing for 1-3 days under s, and finishing the culture. By further changing the environmental conditions, the temperature and the illumination intensity are further reduced, and more phycoerythrin is induced to be accumulated in the porphyridium cells. The low temperature is favorable for keeping the activity of the protein, and the reduction of illumination is favorable for the accumulation of phycoerythrin.
In a preferred embodiment of the present invention, the Porphyridium includes Porphyridium purpureum CoE 1.
In a preferred embodiment of the present invention, the raw algae liquid is cultured in an artificial seawater culture medium.
In a preferred embodiment of the present invention, the preparation steps of the raw algae solution are: inoculating the porphyridium liquid cultured normally to late logarithmic phase of growthPlanting in artificial seawater culture medium, and controlling initial OD680The value is 0.4 to 0.6.
In a preferred embodiment of the present invention, the normal culture conditions are a temperature of 25 ℃ and a light intensity of 165. mu. mol/m2s, the ventilation volume of sterile air is 1L/min.
In a preferred embodiment of the invention, the end of the logarithmic phase of growth refers to day 8. The biomass of the porphyridium is autotrophically cultured until the biomass reaches a higher value of about 8 g/L-12 g/L after 18 days, and the biomass yield reaches a higher value of about 700 mg/L/d-900 mg/L/d after 6-10 days of culture, namely 6-10 days are the final stage of the logarithmic phase of growth. According to other experiments, the OD of the initial biomass inoculated into the artificial seawater culture medium was indicated680The value is 0.4-0.6, which is the optimal inoculation amount.
In a preferred embodiment of the present invention, the raw algae liquid is cultured under continuous illumination.
In a preferred embodiment of the present invention, a white fluorescent lamp is used as the illumination source.
In a preferred embodiment of the present invention, the porphyridium seed solution is cultured by controlling the temperature and the illumination intensity in sections in an illumination incubator, a column type photobioreactor or an open pond.
In a preferred embodiment of the present invention, the phycoerythrin content of the cultured porphyridium is 109.2-205.3 mg/L, and the biomass is 5.3-7.7 g/L.
Compared with the background technology, the technical scheme has the following advantages:
the method has the advantages of simple conditions, mild environment, easy control, no need of additives, and capability of inducing more phycoerythrin from porphyridium cells by changing the environmental conditions, reducing the temperature and the illumination intensity. The phycoerythrin content of the porphyridium cultured by the method is 109.2-205.3 mg/L, the biomass is 5.3-7.7 g/L, and the porphyridium has important value in the fields of medicines, foods and the like.
Drawings
FIG. 1 is a graph showing the trend of biomass change during the cultivation of Porphyridium;
FIG. 2 is a graph showing the variation of phycoerythrin content during the cultivation of Porphyridium;
wherein the histogram for each column is comparative example 1 on the left and example 2 on the right.
Detailed Description
Example 1
The cultivation method for increasing the content of phycoerythrin synthesized by Porphyridium by controlling temperature and illumination intensity in sections in the embodiment adopts Porphyridium purpureum CoE1 for cultivation, and comprises the following steps:
(0) and (4) normal culture: under the conditions of 25 ℃ and 165 mu mol/m of illumination intensity2s, sterile air ventilation volume of 1L/min, inoculating the porphyridium liquid cultured normally to late logarithmic phase of growth into artificial seawater culture medium, and controlling initial OD680The value is 0.4 to 0.6.
The artificial seawater culture medium comprises the following components:
TABLE 1 Artificial seawater culture Medium (ASW)
Table 1 ASW medium
Mother liquor of trace metal salt solution (mg/L): ZnCl2 4.0，H3BO3 60.0，CoCl2·2H2O 4.0，CuCl2·2H2O 4.0，MnCl2·4H2O 40.0，(NH4)6Mo7O24·4H2O 37.0。
② chelating iron solution: FeCl3·4H2O 0.24g/100mL，Na2EDTA(pH7.6)0.05mol/L。
(1) The temperature is controlled at 23 ℃, and the illumination intensity is 145 mu mol/m2s culturing for 1 day;
(2) the temperature is controlled at 18 ℃ and the illumination intensity is controlled at 90 mu mol/m2s culturing for 7 days;
(3) the temperature is controlled at 12 ℃, and the illumination intensity is controlled at 35 mu mol/m2And s culturing for 1 day to finish the culture. The content of phycoerythrin synthesized by porphyridium is improved by controlling the temperature and the illumination intensity in sections and changing the culture conditions.
After the culture of the embodiment, the phycoerythrin content can reach 109.2mg/L, and the biomass content can reach 5.3 g/L.
Example 2
Example 2 differs from example 1 in that:
(1) the temperature is controlled at 25 ℃ and the illumination intensity is 165 mu mol/m2s culturing for 2 days;
(2) the temperature is controlled at 20 ℃, and the illumination intensity is controlled at 110 mu mol/m2s culturing for 8 days;
(3) the temperature is controlled at 15 ℃, and the illumination intensity is controlled at 55 mu mol/m2And (5) finishing the culture after culturing for 2 days under s. The content of phycoerythrin synthesized by porphyridium is improved by controlling the temperature and the illumination intensity in sections and changing the culture conditions.
After the culture of the embodiment, the phycoerythrin content can reach 205.3mg/L, and the biomass content can reach 8.4 g/L.
Example 3
Example 3 differs from example 1 in that:
(1) the temperature is controlled at 27 ℃, and the illumination intensity is 185 mu mol/m2s culturing for 3 days;
(2) the temperature is controlled at 22 ℃, and the illumination intensity is controlled at 130 mu mol/m2s culturing for 9 days;
(3) the temperature is controlled at 18 ℃ and the illumination intensity is controlled at 75 mu mol/m2And s culturing for 3 days to finish the culture. The content of phycoerythrin synthesized by porphyridium is improved by controlling the temperature and the illumination intensity in sections and changing the culture conditions.
After the culture of the embodiment, the phycoerythrin content can reach 189.2mg/L and the biomass content can reach 7.7 g/L.
Comparative example 1
In this example, Porphyridium purpureum CoE1 was used as a control for 12-day normal culture at 25 deg.C under illumination intensity of 165. mu. mol/m2s, the ventilation volume of sterile air is 1L/min.
The experimental results please refer to the attached figures 1-2, which show that the method can induce more phycoerythrin synthesis of the porphyridium cells compared with the normal culture method.
The above description is only a preferred embodiment of the present invention, and therefore should not be taken as limiting the scope of the invention, which is defined by the appended claims and their equivalents.
Claims (10)
1. A culture method for improving the content of phycoerythrin synthesized by porphyridium is characterized in that porphyridium seed liquid is cultured by controlling temperature and illumination intensity in a segmented mode, and the culture method comprises the following steps:
(1) the temperature is controlled to be 25-27 ℃, and the illumination intensity is 165-185 mu mol/m2Culturing for 2-3 days under s;
(2) the temperature is controlled to be 20-22 ℃, and the illumination intensity is 110-130 mu mol/m2Culturing for 8-9 days under s;
(3) the temperature is controlled to be 15-18 ℃ and the illumination intensity is controlled to be 55-75 mu mol/m2And (5) culturing for 2-3 days under s, and finishing the culture.
2. The culture method of claim 1, wherein the culture method comprises the following steps: the porphyridium comprises porphyridium (A) (B)Porphyridium purpureum)CoE1。
3. The culture method of claim 1, wherein the culture method comprises the following steps: the porphyridium seed liquid is cultured in an artificial seawater culture medium.
4. The culture method for increasing the content of phycoerythrin synthesized by porphyridium as claimed in claim 1, wherein the preparation of the porphyridium seed solution comprises the steps of: inoculating the porphyridium solution cultured normally to late logarithmic phase of growth into artificial seawater culture medium, and controlling initial OD680The value is 0.4 to 0.6.
5. The culture method of claim 4, wherein the culture method comprises the following steps: the normal culture conditions are that the temperature is 25 ℃ and the illumination intensity is 165 mu mol/m2s、The ventilation rate of sterile air is 1L/min.
6. The culture method of claim 4, wherein the culture method comprises the following steps: the end of the log phase of growth refers to day 8.
7. The culture method of claim 1, wherein the culture method comprises the following steps: the porphyridium seed solution is cultured under the condition of continuous illumination.
8. The culture method of claim 1, wherein the culture method comprises the following steps: a white light fluorescent lamp is used as the illumination source.
9. The culture method of claim 1, wherein the culture method comprises the following steps: and culturing the porphyridium seed solution in a light culture box, a column type photobioreactor or an open pond by controlling the temperature and the light intensity in sections.
10. The culture method of claim 1, wherein the culture method comprises the following steps: the phycoerythrin content of the cultured porphyridium is 189.2-205.3 mg/L, and the biomass is 7.7-8.4 g/L.
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| JP5588069B2 (en) * | 2011-07-05 | 2014-09-10 | 株式会社 エフジーケー | Medium for microalgae |
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