阅读说明：本技术 培养雨生红球藻生产天然虾青素的方法 (Method for producing natural astaxanthin by culturing haematococcus pluvialis ) 是由 默罕默德·伊利亚斯·巴沙·卡奇 李健 于 2021-09-08 设计创作，主要内容包括：本发明涉及培养雨生红球藻生产天然虾青素的方法,属于微藻生物技术领域。培养雨生红球藻生产天然虾青素的方法,包括以下两个步骤：A、采用兼养培养模式在室内光生物反应器中培养雨生红球藻,生产红球藻绿色细胞种液；B、将步骤A培养的细胞种液经稀释后,接种到室外以自然光为光源的培养设施中,自养培养,诱导雨生红球藻生产虾青素。本发明能够使用低成本的室外开放式跑道培养池作为红球藻虾青素的主要生产设施,从而能够降低生产设施的固定资产投资,同时室内兼养培养的微藻种液具有生产效率高和品质高的优势,和传统的光自养两步法相比能够进一步降低红球藻虾青素的生产成本。(The invention relates to a method for producing natural astaxanthin by culturing haematococcus pluvialis, belonging to the technical field of microalgae biology. The method for producing natural astaxanthin by cultivating haematococcus pluvialis comprises the following two steps: A. culturing haematococcus pluvialis in an indoor photobioreactor in a mixotrophic culture mode to produce haematococcus pluvialis green cell liquid; B. and D, diluting the cell culture solution cultured in the step A, inoculating the diluted cell culture solution into an outdoor culture facility taking natural light as a light source, performing autotrophic culture, and inducing haematococcus pluvialis to produce astaxanthin. The method can use the outdoor open runway culture pond with low cost as a main production facility of the haematococcus pluvialis astaxanthin, thereby reducing the fixed asset investment of the production facility, simultaneously, the microalgae liquid cultured by indoor mixotrophic culture has the advantages of high production efficiency and high quality, and compared with the traditional photoautotrophic two-step method, the method can further reduce the production cost of the haematococcus pluvialis astaxanthin.)

1. The method for producing natural astaxanthin by culturing haematococcus pluvialis is characterized by comprising the following two steps:

A. culturing haematococcus pluvialis in an indoor photobioreactor in a mixotrophic culture mode to produce haematococcus pluvialis green cell liquid;

B. and D, diluting the cell culture solution cultured in the step A, inoculating the diluted cell culture solution into an outdoor culture facility taking natural light as a light source, performing autotrophic culture, and inducing haematococcus pluvialis to produce astaxanthin.

2. The method for producing natural astaxanthin from Haematococcus pluvialis as claimed in claim 1, wherein in step A, the photobioreactor is provided with LED as light source, and has white or red color spectrum and light quantum intensity of 20-160 μmol-m^-2·s^-1。

3. The method of cultivating Haematococcus pluvialis for the production of natural astaxanthin according to claim 1, wherein in step A, the mixotrophic culture is a continuous mixotrophic culture; preferably, the dilution rate of the continuous mixotrophic culture is 0.2-0.8 d^-1。

4. The method for producing natural astaxanthin by Haematococcus pluvialis according to any one of claims 1 to 3, wherein the culture medium used in the step A is a BBM-modified medium which is: BBM culture medium is used as basic culture medium, and the concentration of sodium nitrate is adjusted to be 0.1-1.0 g.L^-1And then adding acetate to ensure that the concentration of the acetate is 1-10 mmol/L and the content of the rest components is unchanged.

5. The method for producing natural astaxanthin from Haematococcus pluvialis according to any one of claims 1 to 3, wherein the temperature for mixotrophic culture of Haematococcus pluvialis in step A is 20 to 25 ℃.

6. The method of claim 3, wherein the aeration rate of the continuous mixotrophic culture in step A is 0.05-0.5 vvm, the gas is a mixture of carbon dioxide and air, and the ratio of carbon dioxide to air is 1-5%.

7. The method for producing natural astaxanthin from Haematococcus pluvialis according to any one of claims 1 to 3, wherein the dilution ratio in step B is 2 to 6.

8. The method for producing natural astaxanthin from Haematococcus pluvialis according to any one of claims 1 to 3, wherein in the step B, the autotrophic culture medium is a carbonate solution, and the concentration of carbonate is 0.1 to 1.0 mmol/L; the pH value of the culture medium is 6.0-8.0; preferably, by introducing CO₂To control the pH of the medium.

9. The method for producing natural astaxanthin from Haematococcus pluvialis according to any one of claims 1 to 3, wherein in the step B, the cultivation facilities using natural light as a light source are outdoor open raceway cultivation ponds; preferably, the rotating speed of the stirring rotating wheel is 15-30 rpm.

10. The method for producing natural astaxanthin from Haematococcus pluvialis according to any one of claims 1 to 3, wherein the period of the autotrophic culture in the step B is 7 to 14 days.

Technical Field

The invention relates to a method for producing natural astaxanthin by culturing haematococcus pluvialis, belonging to the technical field of microalgae biology.

Background

Astaxanthin (Astaxanthin), a carotenoid species commonly found in aquatic animals. The chemical name is as follows: 3,3 ' -dihydroxy-beta, beta ' -carotene-4, 4 ' -dione with molecular formula C₄₀H₅₂O₄And the molecular weight is 596.86. The middle of the astaxanthin molecule is a conjugated double-bond long carbon chain, two ends of the astaxanthin molecule respectively contain a hydroxyl group and a ketone group, and the astaxanthin molecule belongs to ketone type carotenes, and the molecular structural formula is shown as follows:

astaxanthin appears red in color due to absorption of blue light by its molecular conjugated double bond. Due to the long carbon chain, the astaxanthin has fat solubility, is insoluble in water, is slightly soluble in ethanol, and is easily soluble in common organic solvents such as acetone. Astaxanthin is easily oxidized due to the conjugated unsaturated double bond system.

Astaxanthin is the strongest biological antioxidant which can be produced on a large scale at present, has various beneficial biological effects, is widely used for health food raw materials, cosmetic raw materials, feed additives and the like, has the market scale of more than 8 hundred million dollars at present, and is increased at a high speed. Recent market research reports predict that the market for astaxanthin will exceed $ 25 billion in 2025.

Astaxanthin can be produced by chemical synthesis method or biosynthesis method. The molecular structure of chemically synthesized astaxanthin is different from the chiral structure of astaxanthin molecules in the nature, and the analysis results of the biosynthetic astaxanthin and the natural astaxanthin are completely consistent, so the biosynthetic astaxanthin is also called natural astaxanthin and is used for distinguishing the chemically synthesized astaxanthin.

Chemically synthesized astaxanthin is currently only allowed for use in aquatic feed additives due to potential health and environmental hazards. The natural astaxanthin has no harm to health and environment, and the biological effect is far stronger than that of chemical astaxanthin. The natural astaxanthin not only can be used as an aquatic feed additive, but also can be used as a teaching aid raw material, a cosmetic raw material, a medicine raw material and the like.

Because of the huge market for natural astaxanthin, several companies developed production techniques for natural astaxanthin internationally since the 80 s of the last century. At the end of the last century, several companies have realized the large-scale production of natural astaxanthin by culturing Haematococcus pluvialis.

Haematococcus pluvialis is a freshwater microalgae widely distributed around the world and has a special biological habit. The algae can rapidly split and grow under the condition of proper external environment, and is green. Under the condition that the external environment is not suitable, the haematococcus stops splitting, a large amount of astaxanthin begins to be accumulated in the body, and the haematococcus turns red.

Haematococcus can be cultured in three culture modes: firstly, inorganic carbon sources and mineral salt nutrient components are provided for haematococcus, and the haematococcus performs photosynthesis growth under the illumination condition, and the culture mode is called autotrophic culture; secondly, organic carbon sources and other nutrient components are provided for haematococcus, and the haematococcus absorbs and utilizes the organic carbon sources to grow under the condition of no illumination, and the culture mode is called heterotrophic culture; thirdly, organic carbon sources and other nutrient components are provided for haematococcus, the haematococcus not carries out photosynthesis and absorbs and utilizes the organic carbon sources to grow under the condition of illumination, and the culture mode is called mixotrophic culture.

These three culture modes can be respectively adopted in two modes, namely continuous culture and batch culture. The continuous culture is to continuously add culture medium into corresponding culture facilities and continuously harvest culture solution. Batch culture the culture medium is added into the culture facility at one time, and the culture solution is harvested at one time. Although continuous culture has an advantage over batch culture in that the culture facilities are not frequently cleaned and assembled, the requirement for aseptic handling during the culture process is high, and it is difficult to perform the culture under outdoor conditions.

Most of the processes for culturing astaxanthin by haematococcus have two steps according to the biological characteristics; providing proper production conditions, enabling haematococcus to rapidly split and grow, accumulating biomass and providing microalgae liquid to turn into a green culture period; the second part changes the culture environment of Haematococcus, stresses the Haematococcus to stop division and starts to accumulate astaxanthin, which is called red culture period.

Three enterprises for realizing the culture and production of astaxanthin by haematococcus pluvialis in the early stage mainly adopt different patent technologies respectively.

One is MeraPharma, USA, which adopts the culture patented technology (LEONARD A B P, HUNTLEY M E, NIILER P P, et al. method of control Haematococcus spp growth process: WO/1998/000559[ P ].1998 ]) combining an outdoor pipeline bioreactor and an outdoor open raceway pond, and the technology comprises the steps of firstly carrying out batch autotrophic culture on Haematococcus production strain in the outdoor pipeline reactor and then carrying out batch autotrophic culture on Haematococcus production strain in the outdoor open raceway pond to produce astaxanthin.

Secondly, Israel Algatech company adopts the culture patent technology of outdoor glass pipeline photobioreactor (BOUSSIBA S, VONSHAK A, COHEN Z, et al. Process for large-scale production of astaxanthin from Haematococcus.U.S. Pat. No.6,022,701[ P ].2000-02-08.) and the technology firstly produces the haematococcus algae liquid by the batch autotrophic culture in the outdoor glass pipeline bioreactor and then produces the astaxanthin by the batch autotrophic culture of the haematococcus algae in the same type of outdoor glass pipeline bioreactor.

Thirdly, the outdoor runway pool technique culture adopted by ParryPharma corporation of India, and the patented technique of using the red ball algae pao to provide seed liquid (SWATI S T, SWAMINATHAN K, NAGARA J B. Process to product astaxanthhin from Haematococcus biomass: US, WO/2003/027267[ P ].2003.) are that the red ball algae pao is firstly used to produce red ball algae seed liquid by the indoor autotrophic culture, and then the red ball algae is autotrophic cultured in batch mode in the outdoor open runway pool to produce astaxanthin as in MeraPharma corporation.

The above patent technologies have each been industrialized at the end of the last century, and companies possessing them have also had some commercial success. However, the cost of astaxanthin production by Haematococcus sp is still high today, and is much higher than that of astaxanthin produced by chemical industry (LI J, ZHU D, NIU J, et al. an environmental assessment of astaxanthin production by large scale culture of Biotechnology Advances,2011,29(6): 568-74.).

The main reason for the high cost of haematococcus astaxanthin is the high investment and running cost of the production facilities for cultivating haematococcus. The production facilities for culturing microalgae are mainly divided into two types, one is an open runway culture pond; one type is a closed photobioreactor. The open raceway pond has low manufacturing cost and running cost, but the culture failure caused by the pollution of foreign organisms when the open raceway pond is used for culturing the haematococcus pluvialis. The closed photobioreactor can well solve the problem of foreign biological pollution, but the manufacturing cost and the operation cost are very high.

The patented technology of ParryPharma, India, has succeeded at a certain cost in the early stage by using a low-cost open raceway pond as a main production facility, but the production technology of the haematococcus alga seed liquid is immature, and the production process is unstable for a long time, which finally leads to commercial failure.

The patent technology of Algatech company in Israel adopts an outdoor glass pipeline photobioreactor for culture of seed solution and accumulation of astaxanthin respectively, and can realize stable production, but because the construction and operation costs of the glass pipeline reactor are high, the production cost of all natural astaxanthin is high.

While MeraPharma, USA, uses open raceway ponds as the main production facility, Haematococcus liquid production is carried out in outdoor photobioreactors, which are expensive to manufacture and operate, and the total production cost is high. One important reason that the company technology can be industrialized is because its production site is located near the national energy laboratory in hawaii, usa, and can obtain cheap deep-sea cooling water, reducing the temperature control cost of outdoor photobioreactors. This convenience is not available in other parts of the world.

In recent years, there has been continued interest in the technology for the Production of natural Astaxanthin by Haematococcus pluvialis culture, and some new patent applications and grants (Xin Li, Xiaoqian Wang, Chuanlan Dun, Shasha Yi, Zhengquan Gao, Chaowen Xiao, Spiros N.Agathos, Guangce Wang, and Jian Li, Biotechnology Production of Astaxanthin om Microalga Haematococcus pluvias. Biotechnology Advances 43:107602 (2020)).

Related to the invention is a Chinese invention patent with the patent number of 201210264946. X. This patent provides a method for the production of astaxanthin by the cultivation of Haematococcus sp. The method is characterized in that fermentation equipment is used for heterotrophically culturing haematococcus green cells to serve as a seed solution, and then natural light is used for inducing autotrophic culture of the haematococcus in an open raceway pond or an outdoor photobioreactor to produce astaxanthin. The method avoids using expensive photo-bioreactor equipment in the process of culturing the haematococcus green cells, thereby possibly greatly reducing the liquid culture cost of the haematococcus.

Although the above method has the potential to reduce the production cost of astaxanthin from Haematococcus, there are some problems that are technically difficult to solve. First, direct light induction of heterotrophically cultured H.rubrum leads to massive cell death, requiring an additional adaptation process, which undoubtedly increases the production process costs (Zhang, Z., Wang, B., Hu, Q., Sommerfeld, M., Li, Y., and Han, D. (2016. A new param for producing astaxanthine from the underfiller green haem Haematococcus pluvialis.Biotechnol.Bioeng.113, 2088-2099.). Secondly, the Chlorophyll content of heterotrophically cultured Haematococcus cells is greatly reduced, which leads to the severe reduction of the astaxanthin accumulation capacity of the Haematococcus during the later photoinduction process, thereby reducing the astaxanthin production efficiency of the Haematococcus (Fang, l., Zhang, j., Fei, z., and Wan, M. (2019). Chlorophytal as key indicator to evaluation enzyme metabolism activity availability ability of Haematococcus pluviality, Bioresource, bioprocess, 6, 1-7.).

Disclosure of Invention

In order to solve the above problems, the present invention provides a novel method for producing astaxanthin from Haematococcus in combination of indoor mixotrophic culture and outdoor autotrophic culture.

The method for producing natural astaxanthin by cultivating haematococcus pluvialis comprises the following two steps:

A. culturing haematococcus pluvialis in an indoor photobioreactor in a mixotrophic culture mode to produce haematococcus pluvialis green cell liquid;

B. and D, diluting the cell culture solution cultured in the step A, inoculating the diluted cell culture solution into an outdoor culture facility taking natural light as a light source, performing autotrophic culture, and inducing haematococcus pluvialis to produce astaxanthin.

In one embodiment, in step A, the photobioreactor uses an LED as a light source, has a white or red characteristic spectrum, and has a photon intensity of 20-160 μmol · m^-2·s^-1。

In one embodiment, in step a, the mixotrophic culture is a continuous mixotrophic culture; preferably, the dilution rate of the continuous mixotrophic culture is 0.2-0.8 d^-1。

In one embodiment, in step a, the medium used for culturing is a modified BBM medium that is: BBM culture medium is used as basic culture medium, and the concentration of sodium nitrate in the BBM culture medium is adjusted to be 0.1-1.0 g.L^-1And then adding acetate to ensure that the concentration of the acetate is 1-10 mmol/L and the content of the rest components is unchanged.

In one embodiment, in the step A, the continuous mixotrophic culture temperature of haematococcus pluvialis is 20-25 ℃.

In one embodiment, in step A, the aeration rate of the continuous mixotrophic culture is 0.05 to 0.5vvm, and the gas is a mixed gas of carbon dioxide and air, and the ratio of carbon dioxide to air is 1 to 5%.

In one embodiment, in the step B, the dilution factor is 2-6.

In one embodiment, in step B, the culture medium for autotrophic culture is carbonate solution, and the concentration of carbonate is 0.1-1.0 mmol/L; the pH value of the culture medium is 6.0-8.0; preferably, by introducing CO₂To control the pH of the medium.

In one embodiment, in step B, the cultivation facility using natural light as a light source is an outdoor open raceway cultivation pond; preferably, the rotating speed of the stirring rotating wheel is 15-30 rpm.

In one embodiment, in step B, the period of autotrophic culture is 7-14 days.

The invention has the beneficial effects that:

1. the invention adopts the outdoor open runway culture pond which takes natural light as a light source as a main production facility for the haematococcus pluvialis to accumulate the astaxanthin, and has low equipment manufacturing cost and operation cost, thereby reducing the production cost of the haematococcus pluvialis astaxanthin.

2. The method adopts the mixotrophic culture method to provide the haematococcus liquid for the outdoor raceway pond, can ensure the stable production process, does not need an adaptation process when the green haematococcus liquid produced by the method is transferred to a red culture stage, and can reduce the process cost. The chlorophyll content of the algae liquid cell produced by the method is far higher than that of the heterotrophically cultured haematococcus cell and is similar to the chlorophyll content level of the autotrophic cultured haematococcus cell, so that the photosynthesis of the cell and the astaxanthin accumulation capacity are ensured when the cell is shifted to a red culture stage.

3. The invention adopts the photobioreactor which takes the LED as the light source indoors as the culture facility of the haematococcus pluvialis seed solution, the temperature control and the sterile operation of the reactor are easy to realize, and the continuous mixotrophic method can be ensured to stably provide high-quality haematococcus pluvialis green seed solution for the outdoor runway pool, thereby further reducing the production cost of the haematococcus pluvialis liquid.

Drawings

FIG. 1 is a schematic view of a cultivation method according to the present invention;

FIG. 2 is a graph showing the change in biomass growth and astaxanthin accumulation in the reaction tank of Haematococcus in examples and comparative examples.

Detailed Description

The method for producing natural astaxanthin by cultivating haematococcus pluvialis comprises the following two steps:

A. culturing haematococcus pluvialis in an indoor photobioreactor in a mixotrophic culture mode to produce haematococcus pluvialis green cell liquid;

B. and D, diluting the cell culture solution cultured in the step A, inoculating the diluted cell culture solution into an outdoor culture facility taking natural light as a light source, performing autotrophic culture, and inducing haematococcus pluvialis to produce astaxanthin.

In one embodiment, in step A, the photobioreactor uses an LED as a light source, has a white or red characteristic spectrum, and has a photon intensity of 20-160 μmol · m^-2·s^-1。

Wherein, the light quantum intensity needs to be controlled within 20-160 mu molm^-2·s^-1If the light intensity is too weak, it is equivalent to no light; too strong light intensity can cause haematococcus to turn red, stop splitting and affect green growth.

In one embodiment, in step a, the mixotrophic culture is a continuous mixotrophic culture; preferably, the dilution rate of the continuous mixotrophic culture is 0.2-0.8 d^-1。

In one embodiment, in step a, the medium used for culturing is a modified BBM medium that is: BBM culture medium is used as basic culture medium, and the concentration of sodium nitrate is adjusted to be 0.1-1.0 g.L^-1And then adding acetate to ensure that the concentration of the acetate is 1-10 mmol/L and the content of the rest components is unchanged.

The components of the used basal medium BBM medium are as follows: NaNO₃ 250mg、KH₂PO₄ 175mg、K₂HPO₄75mg、MgSO₄·7H₂O 75mg、CaCl₂·2H₂O 25mg、NaCl 25mg、EDTA 50mg、KOH 31mg、FeSO₄·7H₂O 4.98mg、H₃BO₃ 11.42mg、ZnSO₄·7H₂O 8.82mg、MnCl₂ 1.44mg、MoO₃ 0.71mg、CuSO₄·5H₂O 1.57mg、Co(NO₃)₂·6H₂0.49mg of O, and the distilled water is added to make up for 1000mL, 15-20 g of agar can be added to the components, and the distilled water is made up for 1L to prepare a solid culture medium.

In a specific embodiment, the acetate is sodium acetate.

Wherein the concentration of sodium nitrate is controlled to 0.1-1.0 g.L during continuous culture^-1The concentration of the sodium nitrate is adjusted to adjust the concentration of the sodium nitrate in the runway pool during outdoor culture; too low a concentration of sodium nitrate will result in no loss of microalgae biomass, and too high a concentration will result in a slow accumulation of astaxanthin by Haematococcus sp.

In one embodiment, in the step A, the continuous mixotrophic culture temperature of haematococcus pluvialis is 20-25 ℃.

In one embodiment, in step A, the aeration rate of the continuous mixotrophic culture is 0.05 to 0.5vvm, and the gas is a mixed gas of carbon dioxide and air, and the ratio of carbon dioxide to air is 1 to 5%.

The method adopts the introduction of carbon dioxide to supplement the inorganic carbon source, and avoids the influence of inorganic salt carbon source such as sodium bicarbonate and the like on the pH value of the culture medium along with the consumption of the carbon dioxide, thereby influencing the growth of the green cell liquid. The air is introduced to remove oxygen generated by photosynthesis, and to keep algae cells in a uniform suspension state to avoid precipitation.

The ratio of carbon dioxide to air is controlled to be 1-5%, if the concentration of carbon dioxide is high, the carbon dioxide is wasted firstly, the pH value of the culture solution is reduced, the pH value is reduced, and the growth of the green cell culture solution is also influenced.

In one embodiment, in the step B, the dilution factor is 2-6.

In one embodiment, in step B, the culture medium for autotrophic culture is carbonate solution, and the concentration of carbonate is 0.1-1.0 mmol/L; the pH value of the culture medium is 6.0-8.0; preferably, by introducing CO₂To control the pH of the medium.

Wherein, the outdoor autotrophic culture solution can also be a mixed solution of carbonate and chloride.

In one embodiment, in step B, the cultivation facility using natural light as a light source is an outdoor open raceway cultivation pond; preferably, the rotating speed of the stirring rotating wheel is 15-30 rpm.

In one embodiment, in step B, the period of autotrophic culture is 7-14 days.

The following examples are provided to further illustrate the embodiments of the present invention and are not intended to limit the scope of the present invention.

Measuring method

1. The method for measuring microalgae biomass uses a glass fiber filter membrane for filtration and drying.

(1) Drying a glass fiber filter membrane which is marked firstly and has the diameter of 3cm and the micropore diameter of 5 mu M in a blast drier for one day at 75 ℃, then taking out the dried filter paper, quickly weighing the filter paper by using an electronic balance with ten-thousandth precision, and reading out the mass M1 of the glass fiber filter membrane after the filter paper is stabilized;

(2) weighing a certain volume of algae liquid V (generally 50-500 mL), performing suction filtration by using a weighed glass fiber membrane, and washing once by using deionized water. After the filtration is finished, putting the membrane loaded with the biomass into an electrothermal drying oven for drying for 24 hours;

(3) after drying for one day, the petri dish with the filter paper is taken out, weighing is carried out quickly, and mass data M2 are recorded after the reading of the electronic balance is stable.

(4) The concentration of haematococcus biomass in the algae solution is calculated by the following formula:

DW＝(M2-M1)/V；

wherein DW is dry weight concentration of Haematococcus pluvialis cell (in gL)^-1) M1 is the mass of the filter paper (in g), M2 is the total mass of the filter paper and cells (in g), and V is the sample volume (in L).

2. The measurement of astaxanthin content and other pigments in Haematococcus biomass is carried out with reference to the following technical literature with slight modifications: pan X.S., Wang B.B., Duan R., et al.engineering an antibiotic accumulation in Xanthophyllomyces dendrorhous by a phytohormomone: microbiological and gene expression profiles [ J ]. microbiological Biotechnology, 2020, 13 (5).

The method comprises the following specific steps:

(1) firstly, 15mL of algae solution is weighed and centrifuged for 10min under the condition of 4000r/min, supernatant is poured out, and cell sediment is reserved. Adding 5mL preheated DMSO into the cell precipitate, shaking with a mixer to obtain homogeneous state, placing in digital display constant temperature water bath at 70 deg.C, and extracting pigment for 10 min.

(2) Centrifuging at 4000r/min for 10min again after the water bath is finished to realize solid-liquid separation, and collecting the extracted supernatant for later use. If the lower layer precipitate still presents green or red, the operations of DMSO extraction, water bath and centrifugation can be repeated for many times.

(3) The absorbance values of the extract at 480nm, 530nm, 650nm and 666nm were measured by sampling with an ultraviolet spectrophotometer, and then the concentrations of total carotenoids (T-Car), chlorophyll a (Chl a), chlorophyll b (Chl b) and astaxanthin (asta) in the extract were determined according to the following empirical calculation formula in the literature. The total mass of astaxanthin in the 15mL sample was obtained by multiplying the concentration of astaxanthin in the extract by the measured dilution ratio and the total volume of the extract.

C_{Chl a}(mg·L^-1)＝13.34×A₆₆₆-4.85×A₆₅₀：

C_{Chl b}(mg·L^-1)＝24.58×A₆₅₀-6.65×A₆₆₆：

C_asta(mg·L^-1)＝(A₅₃₀-0.0107)/0.148；

C_T-Car(mg·L^-1)＝(1000×A₄₈₀-1.29×C_{Chl a}-53.76×C_{chl b})/220。

(4) And finally, calculating the content of the astaxanthin in the biomass of the haematococcus pluvialis according to the mass concentration measurement data of the haematococcus pluvialis and the total mass of the astaxanthin in the sample, wherein the content is expressed by percent.

Examples

A FACHB721 sterile strain of freshwater algae bank of Chinese academy of sciences was used as an experimentally cultured strain, and was subjected to indoor scale-up culture using BBM medium, and inoculated into a 10L flask reactor as shown in FIG. 1 for continuous mixotrophic culture to produce a green Haematococcus liquid (total of 2 groups, 5 flasks per group, total of 100L). The medium in the 10L flask was modified BBM medium and was characterized by reducing sodium nitrate to 0.2gL^-1Adding 5mmol/L sodium acetate as organic carbon source; the light source is LED white light, and the light quantum intensity is 160 mu mol.m^-2·s^-1(ii) a The dilution rate of continuous mixotrophic culture of Haematococcus is 0.5day^-1Pumping the mixotrophic culture medium into a culture medium by using a metering pump through a sterilizing filter; the temperature of the culture is controlled at 23 ℃; compressed air was introduced into each reactor for 1 L.min^-1Sterile air containing 3% carbon dioxide. After continuous culture for about 7-8 days, the concentration of algae liquid in the reactor reaches the balance, and is measured to be 0.42 g.L^-1. Collecting effluent of the reactor above 40L as seed liquid for outdoor inoculation.

Small open runway for outdoor cultivationThe length of the culture pond is about 2.0m, the width of the culture pond is about 0.76m, and the depth of the culture pond is about 0.4 m. For the cultivation of Haematococcus in the raceway pond, about 200L of tap water and 1L of 10% disinfectant were first added for sterilization and washing, followed by rinsing twice with tap water. And secondly, adding 160L of tap water, introducing ozone for disinfection for 30min, and then adjusting the rotating speed of the runway pool runner to 20r/min to enable the runway pool runner to run for more than 5 hours. Finally, 4g NaHCO was added to the culture pond₃And 400g NaCl, starting to calibrate the pH controller after dissolution, setting the pH value of carbon dioxide switched on by the pH controller to be 7.8, and then switching on the carbon dioxide on-off valve.

The indoor liquid 40L is pumped into the outdoor runway pool by a hose pump for inoculation, 200L (160L water +40L algae liquid) is cultured in each runway pool, and a 200L water level line is marked by a waterproof marker so as to replenish the evaporation loss water to the water level line during sampling. On the day of inoculation, 1L was sampled, followed by sampling in the evening every day, and sampling was continued for 14 days for one culture period for mass concentration measurement and astaxanthin content analysis.

The data of the examples are shown in Table 1, which shows that the process method of the invention can effectively culture haematococcus pluvialis to produce astaxanthin in outdoor open raceway ponds.

Comparative example

The experimental conditions of the comparative example are the same as the algae species and facilities used in the examples, and the outdoor culture conditions of the two are also the same. However, in the indoor culture part, the difference was only that sodium acetate was not added as an organic carbon source in the comparative example medium.

The examples and comparative example data demonstrate that the mixotrophic culture broth is as effective as the autotrophic culture broth for outdoor astaxanthin production by Haematococcus and performs even better than the autotrophic culture broth, as shown in Table 1 and FIG. 2, and that the mixotrophic culture broth produces slightly better biomass accumulation and astaxanthin accumulation than the autotrophic culture broth.

TABLE 1