A method for increasing the yield of astaxanthin from Haematococcus pluvialis by using dopamine

Abstract

The invention discloses a method for improving the yield of astaxanthin in Haematococcus pluvialis by using dopamine, and belongs to the field of bioengineering technology. The invention improves the yield and yield of astaxanthin by adding dopamine in the culture medium in the second culture stage of Haematococcus pluvialis, that is, the red stage. The method of the invention can effectively improve the yield and yield of astaxanthin in Haematococcus pluvialis, and has simple procedures, easy operation, low dependence on the environment, and no harm to the environment during the culture process, and can be applied to the large-scale production of astaxanthin in actual production.

Description

Technical Field

[0001] The invention relates to a method for improving the astaxanthin yield of Haematococcus pluvialis by utilizing dopamine, and belongs to the technical field of bioengineering. Background Art

[0002] Astaxanthin is widely used in the medical, food, cosmetics and aquaculture industries due to its strong antioxidant capacity. Haematococcus pluvialis has also attracted much attention as the best source of natural astaxanthin. Haematococcus pluvialis is a freshwater unicellular green algae. Its growth process goes through two stages, namely the green stage and the red stage. The cells in the green stage are highly active and have a high division rate, and the biomass of Haematococcus pluvialis can be accumulated more quickly in this stage. The cells in the red stage have low activity and a low division rate, and astaxanthin begins to accumulate in large quantities in the red stage. It will produce astaxanthin when subjected to abiotic stresses such as high light, nitrogen deficiency, high salt and high temperature.

[0003] Globally, Haematococcus pluvialis is one of the few algae species that can be cultivated on a large scale industrially. However, it has a slow growth rate, a long astaxanthin accumulation period, a high risk of contamination in large-scale cultivation, and high costs for cultivation and astaxanthin extraction. Although the global market demand for natural astaxanthin is increasing rapidly, the above shortcomings have hindered the commercialization of astaxanthin. In order to increase the economic benefits of microalgae-derived astaxanthin, it is urgent to find effective strategies to improve the efficiency of astaxanthin production in Haematococcus pluvialis.

[0004] In order to solve the above problems, the Haematococcus pluvialis industry often adopts a two-step cultivation model, namely the first step is to accumulate biomass in the green stage. In this stage, the Haematococcus pluvialis cells are highly active and a higher nutritional growth rate and biomass can be obtained under suitable conditions. The second step is to promote the synthesis of astaxanthin in the red stage. In this stage, specific conditions are used to promote the synthesis and accumulation of astaxanthin in algal cells. This can effectively alleviate the contradiction between the growth of Haematococcus pluvialis and the synthesis of astaxanthin. However, the current cultivation strategy still cannot meet the high-yield requirements required for production. Therefore, promoting the accumulation of astaxanthin in the second stage of Haematococcus pluvialis will promote the development of the Haematococcus pluvialis industry.

[0005] Through the above analysis, the problems and defects of the existing technology are: the astaxanthin production of Haematococcus pluvialis in the second stage is low and cannot meet the needs of large-scale production. Summary of the invention

[0006] In order to solve the problems of low astaxanthin yield and long accumulation period of existing Haematococcus pluvialis, the present invention provides a method for improving the astaxanthin yield of Haematococcus pluvialis by using dopamine, wherein dopamine is added in the second culture stage of Haematococcus pluvialis to improve the astaxanthin yield of Haematococcus pluvialis, and the specific culture method is as follows:

[0007] (1) The first stage of cultivation is the green stage: the Haematococcus pluvialis is cultivated to the logarithmic growth phase to obtain the seed solution (there is no special requirement for the inoculation amount of Haematococcus pluvialis in this step, which can be determined according to the actual situation. The larger the inoculation amount, the shorter the time to reach the logarithmic growth phase; when the amount of Haematococcus pluvialis in the seed solution is 7.0×10 5 ~8.0×10 5 cells / mL, it reaches the logarithmic growth phase).

[0008] (2) The second stage of cultivation is the red stage: the seed solution cultured in step (1) is inoculated into a culture medium containing NaCl and dopamine and cultured to the logarithmic phase to obtain Haematococcus pluvialis containing astaxanthin.

[0009] (3) Extract astaxanthin from Haematococcus pluvialis.

[0010] Preferably, the culture medium used for culturing Haematococcus pluvialis in step (1) and step (2) is BG-11 culture medium.

[0011] Preferably, the culture conditions in step (1) and step (2) are: the ambient temperature is 25° C., and the sterile air ventilation volume is 1 vvm.

[0012] Preferably, the light intensity in step (1) is 30 μmol·m -2 ·s -1 .

[0013] Preferably, the light intensity in step (2) is 250 μmol·m -2 ·s -1

[0014] Preferably, the content of NaCl in the culture medium in step (2) is 1 g·L -1 .

[0015] Preferably, the final concentration of dopamine in the culture medium in step (2) is 12.5-25 μmol / L.

[0016] Preferably, the inoculation amount of Haematococcus pluvialis in step (2) is 2.6×10 5 cells / mL.

[0017] Preferably, the culture days in step (2) are 13 days.

[0018] Preferably, the astaxanthin extraction method in step (3) is as follows: 5 mL of the Haematococcus pluvialis culture solution obtained in step (2) is taken in a sterile clean bench and transferred into a centrifuge tube, centrifuged at 3500 r / min for 5 minutes, the supernatant is discarded, the algal cells are collected, 5 ml of a mixture of dimethyl sulfoxide and glacial acetic acid (the volume ratio of dimethyl sulfoxide: glacial acetic acid is 100:1) is added, the mixture is water bathed at 65°C for 15 minutes, and then centrifuged at 3000 rpm for 5 minutes, the supernatant is collected into a new centrifuge tube, 5 ml of a mixture of dimethyl sulfoxide and glacial acetic acid is added to the precipitate, the water bath and centrifugation operations are repeated until the algae turn white, and the supernatants are combined to obtain astaxanthin.

[0019] Preferably, the astaxanthin concentration is measured by using a spectrophotometer to measure the absorbance value at 530 nm, substituting it into a standard curve for calculation, and finally measuring the astaxanthin concentration of Haematococcus pluvialis.

[0020] VVM is a unit used to describe the ventilation ratio during the fermentation process. Its full name is air volume / culture volume / min. This unit represents the ratio of the ventilation volume per minute to the actual liquid volume in the tank.

[0021] Beneficial effects of the present invention

[0022] (1) The results of the present invention show that the addition of dopamine can significantly increase the production and yield of astaxanthin in Haematococcus pluvialis. Through this cultivation method, the astaxanthin production potential of Haematococcus pluvialis is significantly improved, and the astaxanthin accumulation of Haematococcus pluvialis is improved. The maximum astaxanthin content of Haematococcus pluvialis reaches 32.37 mg·g -1 , compared with the case without dopamine, the astaxanthin yield was increased by 41.25% and reached 3.51 mg·L -1 ·d -1 , an increase of 52.04% compared to without dopamine.

[0023] (2) The present invention creates an optimal environment for astaxanthin accumulation in Haematococcus pluvialis by controlling culture conditions (such as temperature, light intensity, ventilation, etc.). This optimization helps to increase the astaxanthin content of the algae.

[0024] (3) The present invention helps to improve the production efficiency of astaxanthin from Haematococcus pluvialis, and is expected to reduce dependence on natural resources and impact on the environment, thereby promoting environmental sustainability.

[0025] (4) The present invention provides an efficient culture strategy for the large-scale production of Haematococcus pluvialis astaxanthin, and has high commercial application value.

[0026] (5) The process of the present invention is simple, easy to operate and highly efficient. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The present invention provides a flowchart of a method for promoting the growth of Haematococcus pluvialis and the accumulation of astaxanthin by using dopamine.

[0028] Figure 2 This is a graph showing the effects of different concentrations of dopamine on the biomass of Haematococcus pluvialis provided in Examples 1 and 2 of the present invention and Comparative Examples 1 and 2.

[0029] Figure 3 This is a graph showing the effect of different concentrations of dopamine on the astaxanthin content of Haematococcus pluvialis provided by Examples 1 to 2 of the present invention and Comparative Examples 1 to 2.

[0030] Figure 4 This is a graph showing the effect of different concentrations of dopamine on the astaxanthin yield of Haematococcus pluvialis provided in Examples 1 to 2 of the present invention and Comparative Examples 1 to 2. DETAILED DESCRIPTION

[0031] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and through specific implementation methods. However, the following embodiments are merely simple examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0032] Example 1

[0033] A method for improving the astaxanthin yield of Haematococcus pluvialis by using dopamine. In this embodiment, the final concentration of dopamine in the culture medium is 12.5 μmol / L. The specific culture method is as follows:

[0034] (1) First stage culture: 2.5 L of BG-11 medium was used to culture Haematococcus pluvialis in a 3 L round-bottom flask. The culture environment temperature was 25 °C and the light intensity was 30 μmol·m -2 ·s -1 The ventilation volume of sterile air is 1vvm. When Haematococcus pluvialis grows to the logarithmic growth period, the seed liquid is obtained and set aside.

[0035] (2) Second stage culture: 350 mL containing 1 g L -1 NaCl-free BG-11 medium was used as the basic medium, dopamine was added to the medium so that its final concentration in the medium was 12.5 μmol / L, and the seeds obtained in step (1) were inoculated into the medium to ensure that the inoculation amount of Haematococcus pluvialis was 2.6×10 5 cells / mL, the culture environment temperature was 25°C, and the light intensity was 250 μmol·m -2 ·s -1 , the sterile air ventilation volume was 1 vvm, and the culture was continued for 13 days to obtain Haematococcus pluvialis containing astaxanthin.

[0036] Example 2

[0037] A method for improving the astaxanthin yield of Haematococcus pluvialis by using dopamine. In this embodiment, the final concentration of dopamine in the culture medium is 25 μmol / L. The specific culture method is as follows:

[0038] (1) First stage culture: 2.5 L of BG-11 medium was used to culture Haematococcus pluvialis in a 3 L round-bottom flask. The culture environment temperature was 25 °C and the light intensity was 30 μmol·m -2 ·s -1 The ventilation volume of sterile air is 1vvm. When Haematococcus pluvialis grows to the logarithmic growth period, the seed liquid is obtained and set aside.

[0039] (2) Second stage culture: 350 mL containing 1 g L -1 NaCl-free BG-11 medium was used as the basic medium, dopamine was added to the medium so that its final concentration in the medium was 25 μmol / L, and the seeds obtained in step (1) were inoculated into the medium to ensure that the inoculation amount of Haematococcus pluvialis was 2.6×10 5 cells / mL, the culture environment temperature was 25°C, and the light intensity was 250 μmol·m -2 ·s -1 , the sterile air ventilation volume was 1 vvm, and the culture was continued for 13 days to obtain Haematococcus pluvialis containing astaxanthin.

[0040] Comparative Example 1

[0041] For comparison, the difference between this comparative example and Example 2 is that no exogenous dopamine is added, and the other steps are the same as those in Example 2, specifically, the steps are:

[0042] (1) First stage culture: 2.5 L of BG-11 medium was used to culture Haematococcus pluvialis in a 3 L round-bottom flask. The culture environment temperature was 25 °C and the light intensity was 30 μmol·m -2 ·s -1 The ventilation volume of sterile air is 1vvm. When Haematococcus pluvialis grows to the logarithmic growth period, the seed liquid is obtained and set aside.

[0043] (2) Second stage culture: 350 mL containing 1 g L -1 NaCl-containing BG-11 medium was used as the basic medium. The seeds obtained in step (1) were inoculated into the medium to ensure that the inoculation amount of Haematococcus pluvialis was 2.6×10 5 cells / mL, the culture environment temperature was 25°C, and the light intensity was 250 μmol·m -2 ·s -1, the sterile air ventilation volume was 1 vvm, and the culture was continued for 13 days to obtain Haematococcus pluvialis containing astaxanthin.

[0044] Comparative Example 2

[0045] A method for improving the astaxanthin yield of Haematococcus pluvialis by using dopamine. In this embodiment, the final concentration of dopamine in the culture medium is 50 μmol / L, and the specific culture method is as follows:

[0046] (1) First stage culture: 2.5 L of BG-11 medium was used to culture Haematococcus pluvialis in a 3 L round-bottom flask. The culture environment temperature was 25 °C and the light intensity was 30 μmol·m -2 ·s -1 The ventilation volume of sterile air is 1vvm. When Haematococcus pluvialis grows to the logarithmic growth period, the seed liquid is obtained and set aside.

[0047] (2) Second stage culture: 350 mL containing 1 g L -1 NaCl-free BG-11 medium was used as the basic medium, dopamine was added to the medium to make the final concentration in the medium 50 μmol / L, and the seeds obtained in step (1) were inoculated into the medium to ensure that the inoculation amount of Haematococcus pluvialis was 2.6×10 5 cells / mL, the culture environment temperature was 25°C, and the light intensity was 250 μmol·m -2 ·s -1 , the sterile air ventilation volume was 1 vvm, and the culture was continued for 13 days to obtain Haematococcus pluvialis containing astaxanthin.

[0048] The extraction method and determination method of astaxanthin in all embodiments and comparative examples are as follows: 5 mL of the culture solution obtained from the second stage of cultivation is taken in a sterile clean bench and transferred into a centrifuge tube. After centrifugation at 3500 r / min for 5 minutes, the supernatant is discarded, the algal cells are collected, 5 ml of a mixture of dimethyl sulfoxide and glacial acetic acid (the volume ratio of dimethyl sulfoxide: glacial acetic acid is 100:1) is added, the mixture is water bathed at 65°C for 15 minutes, and then centrifuged at 3000 rpm for 5 minutes, the supernatant is collected into a new centrifuge tube, and 5 ml of a mixture of dimethyl sulfoxide and glacial acetic acid is added to the precipitate. The above water bath and centrifugation operations are repeated until the algae turn white, the supernatants are combined, and the absorbance value is measured at 530 nm using a spectrophotometer. The value is substituted into the standard curve for calculation, and the astaxanthin concentration of Haematococcus pluvialis is finally measured.

[0049] On the 1st, 5th, 9th and 13th days of the second stage of cultivation, the biomass, astaxanthin production and astaxanthin yield of the Haematococcus pluvialis in each embodiment and comparative example were measured. The results are as follows: Figure 2-Figure 4 shown.

[0050] Biomass of Haematococcus pluvialis Figure 2 As shown in the figure, it can be seen that when the culture is 13 days, the biomass of Haematococcus pluvialis in Example 1 is 1.55 g·L -1 , which is 18.32% higher than that of Comparative Example 1; the biomass of Haematococcus pluvialis in Example 2 is 1.41 g·L -1 , which is 7.63% higher than that of comparative example 1; the biomass of Haematococcus pluvialis in comparative example 2 is 1.04 g·L -1 , which is 20.61% lower than that of Comparative Example 1.

[0051] Astaxanthin content Figure 3 As shown in the figure, it can be seen that when cultured for 13 days, the astaxanthin content in Example 1 is 27.49 mg·g -1 , which is 19.95% higher than that of comparative example 1; the astaxanthin content of example 2 is 32.37 mg·g -1 , which is 41.25% higher than that of comparative example 1; the astaxanthin content in comparative example 2 is 12.99 mg·g -1 , which is reduced by 43.31% compared with Comparative Example 1.

[0052] Astaxanthin yield Figure 4 As shown in the figure, it can be seen that the astaxanthin yield in Example 1 is 3.27 mg·L -1 ·d -1 , which was 41.78% higher than that of comparative example 1; the astaxanthin yield in example 2 was 3.51 mg·L -1 ·d -1 , which is 52.04% higher than that of comparative example 1; the yield of cyanocobalamin in comparative example 2 is 1.04 mg·L -1 ·d -1 , which is reduced by 55.00% compared with Comparative Example 1.

[0053] In summary, when dopamine at a concentration of 12.5-25 μmol / L is added to the culture medium of Haematococcus pluvialis in the second stage, the production and yield of astaxanthin can be effectively improved. When the concentration of dopamine is greater than or equal to 50 μmol / L, the production and yield of astaxanthin will be inhibited. Therefore, although dopamine can increase the production and yield of astaxanthin in Haematococcus pluvialis, the higher the concentration, the better. On the contrary, high concentration of dopamine will inhibit the production of astaxanthin.

Claims

1. A method for increasing the yield of astaxanthin from Haematococcus pluvialis using dopamine, characterized in that: Dopamine is added in the second culture stage of Haematococcus pluvialis to increase the astaxanthin yield of Haematococcus pluvialis. The specific culture method is as follows: (1) First stage of cultivation: culturing Haematococcus pluvialis to the logarithmic growth phase to obtain seed solution; (2) Second stage of cultivation: inoculating the seed solution cultured in step (1) into a culture medium containing NaCl and dopamine to continue culturing to obtain Haematococcus pluvialis containing astaxanthin; The inoculum size of Haematococcus pluvialis was 2.6×10 5 cells / mL; The final concentration of dopamine in the culture medium was 12.5 to 25 μmol / L; (3) Extract astaxanthin from Haematococcus pluvialis.

2. The method for increasing the astaxanthin yield of Haematococcus pluvialis by utilizing dopamine according to claim 1, characterized in that: The culture medium used for culturing Haematococcus pluvialis in step (1) and step (2) is BG-11 culture medium.

3. The method for improving the astaxanthin yield of Haematococcus pluvialis by utilizing dopamine according to claim 1, characterized in that: The culture conditions in step (1) and step (2) are: the ambient temperature is 25°C and the sterile air ventilation volume is 1 vvm.

4. The method for increasing the astaxanthin yield of Haematococcus pluvialis by using dopamine according to claim 1, characterized in that: The light intensity in step (1) is 30 μmol·m -2 ·s -1 .

5. The method for improving the astaxanthin yield of Haematococcus pluvialis by utilizing dopamine according to claim 1, characterized in that: The light intensity in step (2) is 250 μmol·m -2 ·s -1 .

6. The method for increasing the astaxanthin yield of Haematococcus pluvialis by using dopamine according to claim 1, characterized in that: Step (2) The content of NaCl in the culture medium is 1 g·L -1 .

7. The method for increasing the astaxanthin yield of Haematococcus pluvialis by using dopamine according to claim 1, characterized in that: The number of days of cultivation in step (2) is 13 days.

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