Method for promoting spirulina to efficiently express phycocyanin

Abstract

The invention discloses a method for promoting spirulina to efficiently express phycocyanin, which is characterized in that after a spirulina seed culture solution is cultured, the spirulina is cultured by using two-stage light regulation of strong red light and weak blue light in cooperation with high nitrogen concentration. The high spirulina biomass can be realized; meanwhile, the spirulina can be effectively promoted to efficiently express phycocyanin. The content of phycocyanin in spirulina cells produced by the method is obviously higher than the conventional level of a photobioreactor and is 25.18%, the yield can reach 98.55 mg / L / d, and preconditions are provided for extraction of high-purity pharmaceutical-grade phycocyanin; and the method defines the optimal matching point of the nitrogen source concentration and light regulation, realizes the optimal distribution of the metabolic flux, provides a reliable technical path for large-scale production, and has great value in industrial application.

Description

TECHNICAL FIELD

[0001] The present application relates to Spirulina, and in particular to a method for promoting high-efficiency expression of phycocyanin by Spirulina. BACKGROUND

[0002] Phycocyanin is a water-soluble pigment protein mainly existing in blue-green algae and red algae. Due to its unique properties, phycocyanin has been widely used in food coloring, cosmetics, natural dyes, pharmacological research, and biological fluorescence labeling, etc. More notably, a large number of studies have continuously confirmed that phycocyanin has significant anti-inflammatory, antioxidant, antiviral, antitumor activity and antibacterial effect, highlighting its important biomedical value. Therefore, phycocyanin, especially A 620 / A 28 High-purity medical-grade and analytical-grade products with an absorbance ratio of 4.0 or more have great application potential and considerable market size in the high-end medical and diagnostic markets. However, such high-purity phycocyanin is very expensive, and the existing production capacity is difficult to meet the growing demand.

[0003] Currently, the main source of high-purity phycocyanin is still natural Spirulina. Spirulina is a photoautotrophic microorganism, which has traditionally been cultivated on a large scale in outdoor open ponds or raceway ponds relying on natural sunlight. Although this open cultivation mode is low in cost, its yield and phycocyanin quality are extremely susceptible to adverse effects of outdoor environments such as fluctuations in light intensity, temperature changes, pollution, etc., leading to unstable production. Cultivating Spirulina under simulated sunlight in a photobioreactor (PBR) controlled conditions, although significantly improves the stability of biomass and total phycocyanin yield compared to outdoor cultivation, still has limited improvement in the intracellular phycocyanin content of Spirulina, which is usually below 20%, greatly increasing the difficulty and cost of subsequent separation and purification to obtain high-purity products.

[0004] In the prior art, the main research direction is to regulate light conditions to improve yield. For example, maintaining a high light intensity at a lower algal liquid density can promote the accumulation of Spirulina biomass, thereby indirectly increasing the total yield of phycocyanin. Using red light irradiation can quickly promote the photosynthesis of Spirulina and accelerate biomass growth. However, the rapid growth induced by red light will greatly accelerate the nitrogen metabolism of the cells, leading to rapid depletion of nitrogen sources in the culture medium. When the nitrogen source is scarce, phycocyanin, as an important intracellular nitrogen reservoir, will be degraded by the cells to provide the necessary nutrients for survival, which greatly limits the further accumulation and maintenance of phycocyanin in the cells. In addition, recent technologies attempt to promote phycocyanin synthesis by adding chemicals during cultivation, such as the published patent CN 118772264 A proposes to add rare earth ion yttrium (Y 3+) is carried out. This method can improve the content and yield of phycocyanin to some extent, but it introduces the risk of rare earth ion pollution. Phycocyanin products containing residual rare earth ions may pose a potential threat to human health, and removing these exogenous ions also significantly increases the complexity and cost of the downstream purification process, posing a double challenge of safety and economy. SUMMARY

[0005] The purpose of the application is to provide a method for promoting high-efficiency expression of phycocyanin in Spirulina based on two-stage light regulation and nitrogen concentration synergy.

[0006] Technical solution: The method for promoting high-efficiency expression of phycocyanin in Spirulina according to the application is characterized by the following steps:

[0007] (1) Inoculate Spirulina into a culture medium and cultivate at a temperature of 28-32℃ and a light intensity of 5000-7000 lux until the OD 560 of the logarithmic growth phase is between 0.2 and 0.3 to obtain a seed culture solution;

[0008] (2) Inoculate the seed culture solution into a high-nitrogen culture medium and cultivate at a temperature of 28-32℃ and a red light intensity of 11000-14000 lux until the OD 560 of the logarithmic growth phase is between 0.7 and 1.1;

[0009] (3) Continue to cultivate at a temperature of 28-32℃ and a blue light intensity of 5000-8000 lux to obtain Spirulina algal liquid with high-efficiency expression of phycocyanin.

[0010] Preferably, the Spirulina in step 1 is Arthrospira platensis or Arthrospira maxima.

[0011] Preferably, the Spirulina in step 1 is Arthrospira platensis.

[0012] Preferably, the culture medium in step 1 is Zarrouk culture medium added with A5 trace elements.

[0013] Preferably, the high-nitrogen culture medium in step 2 is Zarrouk culture medium added with A5 trace elements and with a final concentration of NaNO3 of 3-4 g / L, the wavelength range of the red light is 650-670 nm, the inoculation amount of the seed culture solution is 10% of the volume of the high-nitrogen culture medium, and the cultivation time after inoculation of the seed culture solution is 3-5 days.

[0014] Preferably, the wavelength range of the blue light in step 3 is 440-460 nm, and the continued cultivation time is 2-4 days.

[0015] Beneficial effects: compared with the prior art, the present application has the following remarkable advantages: 1. The method is based on two-stage light regulation and nitrogen concentration cooperation, which can effectively promote the high efficient expression of phycocyanin in Spirulina while achieving high biomass of Spirulina, and the intracellular phycocyanin content is significantly higher than the conventional level of photobioreactor, which is 25.18%, and the phycocyanin content yield can reach 98.55mg / L / d; 2. The method determines the best matching point of nitrogen source concentration and light regulation, optimizes the distribution of metabolic flow, provides a reliable technical path for large-scale production, and has great value in industrial application. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 Statistical chart of Spirulina and phycocyanin production rate for different culture methods. DETAILED DESCRIPTION

[0017] The technical solutions of the present application are further described below.

[0018] Example 1: Spirulina culture based on two-stage light quality regulation and nitrogen concentration cooperation

[0019] (1) Prepare Zarrouk culture medium with A5 trace elements, sterilize at 121℃ for 15 minutes, and then inoculate the purified single colony of Spirulina platensis on the solid culture medium, and culture at 30±0.5℃ and white light with light intensity of 6000lux to OD 560 0.25 logarithmic phase, obtain seed culture solution,

[0020] The formula of Zarrouk culture medium with A5 trace elements is: 16.8g / L NaHCO3, 2.5g / L NaNO3, 0.5g / L K2HPO4, 1.0g / L K2SO4, 1.0g / L NaCl, 0.08g / L Na2EDTA, 0.2g / L MgSO4·7H2O, 0.01g / L FeSO4·7H2O, 0.04g / L CaCl2·2H2O, 0.222g / L ZnSO4·7H2O, 0.079g / L CuSO4·5H2O, 2.86g / L H3BO3, 1.81g / L MnCl2·4H2O, 0.0177g / L NaMoO4;

[0021] (2) Prepare high-nitrogen Zarrouk culture medium with A5 trace elements, and sterilize the culture medium at 121℃ for 15 minutes. Inoculate the seed culture solution into the high-nitrogen Zarrouk culture medium with inoculation amount of 10% of the volume of the high-nitrogen culture medium, and culture at 30±0.5℃ and wavelength 660nm red light with light intensity of 125000lux for 4d to the middle logarithmic phase,

[0022] The high-nitrogen Zarrouk medium with the addition of A5 trace elements is prepared by adjusting the final concentration of NaNO3 to 3.5 g / L based on the formula described in step 1.

[0023] (3) The culture solution is further cultured under the condition of blue light with a wavelength of 450 nm and a light intensity of 6500 lux for 3 days to obtain a spirulina algal solution with high expression of phycocyanin,

[0024] The algal solution is filtered using a pre-dried and weighed cellulose acetate filter membrane. The filter membrane is washed with distilled water and a 0.9% sodium chloride solution to remove insoluble salts remaining on the filter membrane. Then, the filter membrane is dried at 80°C for 24 hours. After cooling, the total weight of the filter membrane and the spirulina is measured. The biomass is calculated according to the formula M (mg / L / d) = 1000 (b-a) ÷ (x×d), where a is the weight of the cellulose acetate filter membrane (0.7034 g), b is the total weight of the cellulose acetate filter membrane and the spirulina (0.7308 g), x is the volume of the algal solution (0.01 L), and d is the culture time (7 days). The calculated spirulina biomass is 391.43 mg / L / d.

[0025] (4) 0.01 L of the algal solution cultured for 7 days is taken and centrifuged at 4°C and 5000 r / min for 10 min to obtain spirulina cells. The cells are washed twice with deionized water at 4°C and 5000×g for 10 min to remove medium impurities. The obtained precipitate is resuspended with 0.03 L of deionized water to form a spirulina solution. The solution is frozen in a -80°C freezer for 1 h and then thawed at room temperature for 1 h, repeating this process 3 times. The thawed solution is broken using a 200W ultrasonic wave in an ice water bath, with a 2s ultrasonic wave followed by a 3s interval, for a total of 5 min. After centrifugation at 4°C and 5000 r / min for 10 min, the supernatant is obtained to obtain a crude phycocyanin solution with a volume of 32.85 mL.

[0026] (5) The absorbance of the crude solution at 620 nm and 652 nm is measured using a UV-visible spectrophotometer. The concentration of phycocyanin is calculated according to the formula C (mg / mL) = (A 620 -0.474×A 652 ) ÷ 5.34, where A 620 = 1.32 and A 652 = 0.40. The calculated concentration of phycocyanin is 0.21 mg / mL.

[0027] The yield of phycocyanin is calculated according to the formula Y (mg / L / d) = (C×V) ÷ (y×d), where C is the concentration of phycocyanin (mg / mL), V is the volume of the crude phycocyanin solution (mL), y is the volume of the algal solution (0.01 L), and d is the culture time (7 days). The calculated yield of phycocyanin is 98.55 mg / L / d.

[0028] According to the formula F=Y÷M, the phycocyanin content was calculated to be 25.18%.

[0029] Comparative Example 1: Standard Spirulina Cultivation

[0030] The seed culture solution of Spirulina was prepared according to the method described in Reference Example 1.

[0031] The 10% seed culture solution was inoculated into the Zarrouk culture medium added with A5 trace elements, and cultured at 30±0.5°C and under white light with light intensity of 9500 lux for 8 days. The phycocyanin content no longer increased, and the algal liquid was collected.

[0032] The crude extract of phycocyanin was prepared after collecting the Spirulina cells according to the method described in Reference Example 1. According to the formula described in Example 1, the Spirulina biomass was calculated to be 253.53 mg / L / d, the phycocyanin yield was 28.75 mg / L / d, and the phycocyanin content was 11.34%.

[0033] Comparative Example 2: Single Red Light Spirulina Cultivation

[0034] The seed culture solution of Spirulina was prepared according to the method described in Reference Example 1.

[0035] The 10% seed culture solution was inoculated into the Zarrouk culture medium added with A5 trace elements, and cultured at 30±0.5°C and under 660 nm red light with light intensity of 9500 lux for 7 days. The phycocyanin content no longer increased, and the algal liquid was collected.

[0036] The crude extract of phycocyanin was prepared after collecting the Spirulina cells according to the method described in Reference Example 1. According to the formula described in Example 1, the Spirulina biomass was calculated to be 266.86 mg / L / d, the phycocyanin yield was 22.41 mg / L / d, and the phycocyanin content was 8.40%.

[0037] Comparative Example 3: Single Blue Light Spirulina Cultivation

[0038] The 10% seed culture solution was inoculated into the Zarrouk culture medium added with A5 trace elements, and cultured at 30±0.5°C and under 450 nm blue light with light intensity of 9500 lux for 8 days. The phycocyanin content no longer increased, and the algal liquid was collected.

[0039] The crude extract of phycocyanin was prepared after collecting the Spirulina cells according to the method described in Reference Example 1. According to the formula described in Example 1, the Spirulina biomass was calculated to be 125.35 mg / L / d, the phycocyanin yield was 28.25 mg / L / d, and the phycocyanin content was 22.54%.

[0040] Comparative Example 4: White Light Plus Blue Light Two-Stage Light Quality Regulation Spirulina Cultivation

[0041] The seed culture solution of Spirulina was prepared according to the method described in Reference Example 1.

[0042] The seed culture solution was inoculated into the Zarrouk culture medium added with A5 trace elements, and cultured at 30±0.5°C under 660 nm red light with an intensity of 9500 lux for 4 days to the middle logarithmic phase. Then, the culture was further cultured under 450 nm blue light with an intensity of 9500 lux for 3 days until the phycocyanin content no longer increased, and the algal liquid was collected.

[0043] The crude extract of phycocyanin was prepared after collecting the Spirulina cells according to the method described in Reference Example 1. The biomass of Spirulina was calculated to be 318.84 mg / L / d, the yield of phycocyanin was 58.57 mg / L / d, and the phycocyanin content was 18.37% according to the formula described in Example 1.

[0044] Comparative Example 5: Two-stage light quality regulation of Spirulina culture under red light and blue light

[0045] The seed culture solution of Spirulina was prepared according to the method described in Reference Example 1.

[0046] The seed culture solution was inoculated into the Zarrouk culture medium added with A5 trace elements, and cultured at 30±0.5°C under 660 nm red light with an intensity of 9500 lux for 4 days to the middle logarithmic phase. Then, the culture was further cultured under 450 nm blue light with an intensity of 9500 lux for 3 days until the phycocyanin content no longer increased, and the algal liquid was collected.

[0047] The crude extract of phycocyanin was prepared after collecting the Spirulina cells according to the method described in Reference Example 1. The biomass of Spirulina was calculated to be 318.84 mg / L / d, the yield of phycocyanin was 58.57 mg / L / d, and the phycocyanin content was 18.37% according to the formula described in Example 1.

[0048] Comparative Example 6: Two-stage light quality regulation of Spirulina culture under optimized light intensity

[0049] The seed culture solution of Spirulina was prepared according to the method described in Reference Example 1.

[0050] The seed culture solution was inoculated into the Zarrouk culture medium added with A5 trace elements, and cultured at 30±0.5°C under 660 nm red light with an intensity of 9500 lux for 4 days to the middle logarithmic phase. Then, the culture was further cultured under 450 nm blue light with an intensity of 9500 lux for 3 days until the phycocyanin content no longer increased, and the algal liquid was collected.

[0051] The crude phycocyanin extract was prepared according to the method described in Reference Example 1 after collecting the Spirulina cells. The Spirulina biomass was calculated to be 368.01 mg / L / d, the phycocyanin yield was 78.57 mg / L / d, and the phycocyanin content was 21.35% according to the formula described in Example 1.

[0052] Comparative Example 7: Spirulina culture with low nitrogen concentration

[0053] The seed culture solution of Spirulina was prepared according to the method described in Reference Example 1.

[0054] The 10% seed culture solution was inoculated into the low-nitrogen Zarrouk medium with a final concentration of 1.5 g / L NaNO3 and A5 trace elements, and cultured at 30±0.5°C under 660 nm red light with an intensity of 12500 lux for 4 days to the middle logarithmic phase. Then the culture was further cultured under 450 nm blue light with an intensity of 6500 lux for 3 days until the phycocyanin content no longer increased, and the algal solution was collected.

[0055] The crude phycocyanin extract was prepared according to the method described in Reference Example 1 after collecting the Spirulina cells. The Spirulina biomass was calculated to be 325.75 mg / L / d, the phycocyanin yield was 49.23 mg / L / d, and the phycocyanin content was 15.11% according to the formula described in Example 1.

[0056] Comparative Example 8: Spirulina culture with high nitrogen concentration

[0057] The seed culture solution of Spirulina was prepared according to the method described in Reference Example 1.

[0058] The 10% seed culture solution was inoculated into the high-nitrogen Zarrouk medium with a final concentration of 4.5 g / L NaNO3 and A5 trace elements, and cultured at 30±0.5°C under 660 nm red light with an intensity of 12500 lux for 4 days to the middle logarithmic phase. Then the culture was further cultured under 450 nm blue light with an intensity of 6500 lux for 3 days until the phycocyanin content no longer increased, and the algal solution was collected.

[0059] The crude phycocyanin extract was prepared according to the method described in Reference Example 1 after collecting the Spirulina cells. The Spirulina biomass was calculated to be 368.04 mg / L / d, the phycocyanin yield was 45.71 mg / L / d, and the phycocyanin content was 12.42% according to the formula described in Example 1.

[0060] The results of the Spirulina and phycocyanin production rates of Example 1 and Comparative Examples 1-8 are shown in Table 1. Figure 1 The method described in Example 1 is based on the two-stage light quality regulation and nitrogen concentration synergy, which can effectively promote the high-efficiency expression of phycocyanin by Spirulina while achieving high Spirulina biomass.

Claims

1. A method for promoting efficient expression of phycocyanin in Spirulina, characterized by the following steps: include: (1) Spirulina was inoculated into the culture medium and cultured at a temperature of 28-32℃ and a light intensity of 5000-7000 lux until the logarithmic growth phase to obtain seed culture solution; (2) Inoculate the seed culture solution into a high-nitrogen medium and culture it under red light at a temperature of 28-32℃ and a light intensity of 11000-14000 lux until the middle of the logarithmic growth phase; (3) Continue to cultivate under blue light at a temperature of 28-32℃ and a light intensity of 5000-8000 lux to obtain Spirulina algal solution that expresses phycocyanin efficiently.

2. The method for promoting efficient expression of phycocyanin in Spirulina according to claim 1, characterized in that, The spirulina mentioned in step 1 is either Spirulina platensis or Spirulina macrophylla.

3. The method for promoting efficient expression of phycocyanin in Spirulina according to claim 2, characterized in that, The spirulina mentioned in step 1 is Spirulina platensis.

4. The method for promoting efficient expression of phycocyanin in Spirulina according to claim 1, characterized in that, The culture medium described in step 1 is Zarrouk medium supplemented with A5 trace elements.

5. The method for promoting efficient expression of phycocyanin in Spirulina according to claim 1, characterized in that, The high-nitrogen culture medium mentioned in step 2 is Zarrouk medium with a final NaNO3 concentration of 3-4 g / L supplemented with A5 trace element.

6. The method for promoting efficient expression of phycocyanin in Spirulina according to claim 1, characterized in that, The wavelength range of the red light mentioned in step 2 is 650-670nm.

7. The method for promoting efficient expression of phycocyanin in Spirulina according to claim 1, characterized in that, The inoculation volume of the seed culture solution in step 2 is 10% of the volume of high-nitrogen culture medium.

8. The method for promoting efficient expression of phycocyanin in Spirulina according to claim 7, characterized in that, Culture for 3-5 days after inoculation with seed culture solution.

9. The method for promoting efficient expression of phycocyanin in Spirulina according to claim 1, characterized in that, The wavelength range of the blue light mentioned in step 3 is 440-460nm.

10. The method for promoting efficient expression of phycocyanin in Spirulina according to claim 9, characterized in that, The culturing time described in step 3 is 2-4 days.

Images