A method for promoting growth of a green algae stage of haematococcus pluvialis using l-dopamine

Abstract

This invention relates to the field of bioengineering technology, specifically to a method for promoting the growth of Haematococcus pluvialis in its green algal stage using L-Abrine. The method significantly promotes cell proliferation and biomass accumulation in the green algal stage of Haematococcus pluvialis by adding an appropriate concentration of L-Abrine to the culture medium. Experimental results show that L-Abrine significantly promotes biomass accumulation in Haematococcus pluvialis, increasing biomass by 36.36%–54.54% compared to the control group. This method is simple to operate, cost-effective, and has stable results. It requires no changes to existing culture equipment and conditions; efficient growth promotion can be achieved simply by adding L-Abrine. This invention provides a new technical approach for the large-scale cultivation of Haematococcus pluvialis and the efficient production of astaxanthin, with promising industrial application prospects, and provides sufficient material conditions for the subsequent synthesis of astaxanthin from Haematococcus pluvialis.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, specifically to a method for promoting the green algae growth of Haematococcus pluvialis using L-caryophyllene. Background Technology

[0002] Astaxanthin is a natural and potent antioxidant with the ability to efficiently capture free radicals and block oxidation chain reactions, making it irreplaceable in food, medicine, cosmetics, and feed. The main source of natural astaxanthin is Haematococcus pluvialis, which is one of the main sources of natural astaxanthin production worldwide. The antioxidant activity of natural astaxanthin from Haematococcus pluvialis is significantly better than that of synthetic products, and its astaxanthin content is much higher than that of other biological sources. It is currently recognized as a high-quality biological resource for natural astaxanthin and is also the core carrier for the industrial production of natural astaxanthin.

[0003] Although Haematococcus pluvialis is currently the best source of natural astaxanthin, large-scale production still faces many limitations and difficulties: long growth cycle, high production environment requirements, high production costs, low actual yield and growth stability, and susceptibility to fluctuations in environmental factors are still pain points that restrict the development of the Haematococcus pluvialis industry.

[0004] The growth process of Haematococcus pluvialis can be divided into two stages: the green algae stage, which is suitable for cell growth and division, and involves vigorous biomass accumulation and reproduction; and the red algae stage, in which growth and division cease and astaxanthin begins to accumulate in large quantities. Sufficient biomass accumulation in the green algae stage is the key to astaxanthin production, determining the material conditions for subsequent astaxanthin synthesis in cells. The current problem with large-scale production is that the biomass accumulation in the green algae stage of Haematococcus pluvialis is insufficient, making it difficult to produce astaxanthin efficiently and in large quantities. Summary of the Invention

[0005] To address the problems existing in related technologies, this invention provides a method for promoting the green algae stage of Haematococcus pluvialis using L-carboxylic acid, the specific steps of which are as follows: (1) Use a culture medium to culture Haematococcus pluvialis seed liquid.

[0006] (2) Add L-red alkaloid to the culture medium to culture Haematococcus pluvialis until the green algae stage of Haematococcus pluvialis is completed.

[0007] Preferably, the culture medium in step (1) of the present invention is BG-11 culture medium, the amount of which is 2.5L, and the Haematococcus pluvialis in the seed liquid is in the logarithmic growth phase.

[0008] Preferably, the culture conditions for the Haematococcus pluvialis seed solution in step (1) of the present invention are: ambient temperature 25±1℃, light intensity 30μmol·m-2 ·s -1 The sterile air contains 0.5% CO2, and the sterile air ventilation rate is 1 vvm.

[0009] Preferably, the concentration of L-caryophylline in the culture medium in step (2) of the present invention is 0.5~1μmol / L, and after adding L-caryophylline, Haematococcus pluvialis is transferred to a 1L column photobioreactor for culture.

[0010] Preferably, the culture conditions for Haematococcus pluvialis in step (2) of the present invention are: ambient temperature 25±1℃, light intensity 30μmol·m -2 ·s -1 The sterile air contains 0.5% CO2, and the sterile air ventilation rate is 1 vvm.

[0011] The beneficial effects of this invention are: (1) The present invention can significantly promote cell proliferation and biomass accumulation in the green algae stage of Haematococcus pluvialis by adding L-red alkaloids, and the biomass is increased by 36.36%-54.54% compared with the control group.

[0012] (2) It is easy to operate, cost controllable, and has stable effect. It does not require changes to existing culture equipment and conditions. It can achieve efficient growth promotion by simply adding L-red bean alkaloid.

[0013] (3) This invention provides a new technical approach for the large-scale cultivation of Haematococcus pluvialis and the efficient production of astaxanthin, and has good prospects for industrial application. It provides sufficient material conditions for the subsequent synthesis of astaxanthin from Haematococcus pluvialis. Attached Figure Description

[0014] Figure 1 This is a flowchart of the method for increasing the biomass accumulation of Haematococcus pluvialis using L-redoxamine according to the present invention.

[0015] Figure 2 This is a graph showing the effect of different concentrations of L-redoxaline on the biomass of Haematococcus pluvialis in Examples 1-3 of this invention. Detailed Implementation

[0016] The present invention will be further described in detail below with reference to specific embodiments and accompanying drawings, but the scope of protection of the present invention is not limited to the contents described therein; unless otherwise specified, all reagents used in the present invention are commercially available analytical grade reagents, and both the reagents and Haematococcus pluvialis can be purchased through conventional commercial channels.

[0017] Example 1 The steps for culturing Haematococcus pluvialis using L-erythrine are as follows: Figure 1 As shown, the specific steps are as follows: (1) Cultivation of Haematococcus pluvialis seed culture using BG-11 medium (blue-green algae medium): Add 2.5 L of BG-11 medium to an Erlenmeyer flask, inoculate with Haematococcus pluvialis, and culture at 25±1℃ with a light intensity of 30 μmol·m -2 ·s -1 The Haematococcus pluvialis was cultured in a sterile environment containing 0.5% CO2 at a ventilation rate of 1 vvm until it reached the logarithmic growth phase, thus obtaining the seed culture.

[0018] (2) Dissolve L-caryophylline in methanol (methanol is only used as a co-solvent, and the concentration is not critical) to obtain an L-caryophylline solution. Add 0.675 mL of the L-caryophylline solution to BG-11 medium to make the concentration of L-caryophylline in BG-11 medium 0.5 μmol / L. Transfer the solution to a 1L column photobioreactor and inoculate with Haematococcus pluvialis seed culture at an inoculation amount of 1.5 × 10⁻⁶. 5 cells / mL, and placed at 25±1℃ with a light intensity of 30 μmol·m -2 ·s -1 The culture medium was cultured in a controlled environment with sterile air containing 0.5% CO2 continuously introduced at a flow rate of 1 vvm. After 11 days of culture, the final culture medium was obtained.

[0019] (3) Determination of Haematococcus pluvialis biomass in the culture medium obtained in step (2) using the dry weight method: Take 10 mL of culture medium in a sterile laminar flow hood, transfer it to a centrifuge tube, centrifuge at 4000 rpm for 3 min, discard the supernatant, collect Haematococcus pluvialis cells, freeze them overnight in a -80℃ freezer, freeze-dry them for 24 h, and weigh them. The final dry weight of Haematococcus pluvialis biomass was measured, and the results are as follows: Figure 2 As shown, after 11 days of cultivation, the biomass of Haematococcus pluvialis was 0.75 g / L, which was 36.36% higher than that of the control group.

[0020] Example 2 The steps for culturing Haematococcus pluvialis using L-erythrine are as follows: Figure 1 As shown, the specific steps are as follows: (1) Cultivation of seed culture using BG-11 medium: Add 2.5 L of BG-11 medium to an Erlenmeyer flask, inoculate with Haematococcus pluvialis, and incubate at 25 ± 1 °C with a light intensity of 30 μmol·m⁻¹. -2 ·s -1 The Haematococcus pluvialis was cultured in a sterile environment containing 0.5% CO2 at a ventilation rate of 1 vvm until it reached the logarithmic growth phase, thus obtaining the seed culture.

[0021] (2) Dissolve L-caryophylline in methanol (methanol is only used as a co-solvent, and the concentration is not critical) to obtain an L-caryophylline solution. Add 0.675 mL of the L-caryophylline solution to BG-11 medium to make the concentration of L-caryophylline in BG-11 medium 0.75 μmol / L. Transfer the solution to a 1L column photobioreactor and inoculate with Haematococcus pluvialis seed culture at an inoculation amount of 1.5 × 10⁻⁶. 5 cells / mL, and placed at 25±1℃ with a light intensity of 30 μmol·m -2 ·s -1 The culture medium was cultured in a controlled environment with sterile air containing 0.5% CO2 continuously introduced at a flow rate of 1 vvm. After 11 days of culture, the final culture medium was obtained.

[0022] (3) Determination of Haematococcus pluvialis biomass in the culture medium obtained in step (2) using the dry weight method: 10 mL of culture medium was taken out in a sterile laminar flow hood, transferred to a centrifuge tube, centrifuged at 4000 rpm for 3 min, the supernatant was discarded, algal cells were collected, and the cells were frozen overnight at -80℃. After freeze-drying for 24 h, the cells were weighed, and the final dry weight of Haematococcus pluvialis biomass was determined. The results are as follows: Figure 2 As shown, after 11 days of cultivation, the biomass of Haematococcus pluvialis was 0.85 g / L, which was 54.54% higher than that of the control group.

[0023] Example 3 The steps for culturing Haematococcus pluvialis using L-erythrine are as follows: Figure 1 As shown, the specific steps are as follows: (1) Cultivation of seed culture using BG-11 medium: Add 2.5 L of BG-11 medium to an Erlenmeyer flask, inoculate with Haematococcus pluvialis, and incubate at 25 ± 1 °C with a light intensity of 30 μmol·m⁻¹. -2 ·s -1 The Haematococcus pluvialis was cultured in a sterile environment containing 0.5% CO2 at a ventilation rate of 1 vvm until it reached the logarithmic growth phase, thus obtaining the seed culture.

[0024] (2) Dissolve L-caryophylline in methanol (methanol is only used as a co-solvent, and the concentration is not critical) to obtain an L-caryophylline solution. Add 0.675 mL of the L-caryophylline solution to BG-11 medium to make the concentration of L-caryophylline in BG-11 medium 1 μmol / L. Transfer the solution to a 1L column photobioreactor and inoculate with Haematococcus pluvialis seed culture at an inoculation amount of 1.5 × 10⁻⁶. 5 cells / mL, and placed at 25±1℃ with a light intensity of 30 μmol·m -2 ·s -1The culture was cultured in a controlled environment with a continuous flow of sterile air containing 0.5% CO2 at a flow rate of 1 vvm. After 11 days of culture, the final culture medium was obtained.

[0025] (3) Determination of Haematococcus pluvialis biomass in the culture medium obtained in step (2) using the dry weight method: 10 mL of culture medium was taken out in a sterile laminar flow hood, transferred to a centrifuge tube, centrifuged at 4000 rpm for 3 min, the supernatant was discarded, algal cells were collected, and the cells were frozen overnight at -80℃. After freeze-drying for 24 h, the cells were weighed, and the final dry weight of Haematococcus pluvialis biomass was determined. The results are as follows: Figure 2 As shown, after 11 days of cultivation, the biomass of Haematococcus pluvialis was 0.76 g / L, which was 38.18% higher than that of the control group.

[0026] Comparative Example Haematococcus pluvialis was cultured in fresh BG-11 medium, and the biomass of Haematococcus pluvialis was detected and analyzed. The difference between this method and Example 1 is that L-caryophylline was not added to the BG-11 medium; the remaining steps were the same as in Example 1. The results are as follows: Figure 2 As shown, the biomass of Haematococcus pluvialis was 0.55 g / L after 11 days of cultivation.

[0027] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for promoting growth of a green algae stage of Haematococcus pluvialis using L- daunomycin, characterized in that, The specific method is: (1) using a culture medium to culture a Haematococcus pluvialis seed solution; (2) adding L-djenkolic acid to the culture medium to culture the Haematococcus pluvialis until the growth of the Haematococcus pluvialis in the green alga stage is completed.

2. The method for promoting the growth of green algae stage of Haematococcus pluvialis by L-ophiopogonanone according to claim 1, characterized in that, The concentration of the L-djenkolic acid in the culture medium in step (2) is 0.5-1 μmol / L.

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