CN116042761A - Method for co-producing protein and lutein by using ultra-high density heterotrophic microalgae - Google Patents

Method for co-producing protein and lutein by using ultra-high density heterotrophic microalgae Download PDF

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CN116042761A
CN116042761A CN202211110076.0A CN202211110076A CN116042761A CN 116042761 A CN116042761 A CN 116042761A CN 202211110076 A CN202211110076 A CN 202211110076A CN 116042761 A CN116042761 A CN 116042761A
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谢友坪
刘心宇
马瑞娟
陈剑锋
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Abstract

The invention relates to a method for co-producing protein and lutein by using ultra-high density heterotrophic microalgae, which comprises the steps of culturing algae seeds into flat seeds, culturing the flat seeds into shake flask seeds, fermenting in a fermentation tank, and controlling the glucose concentration in a culture solution to be 1-5 g/L when the initial glucose concentration is exhausted; when the consumption of glucose in the culture solution reaches 30 g/L, supplementing the concentrated solution of other nutrient components to reach the respective initial concentration; when the biomass concentration of the algae reaches 160-200 g/L, glucose solution is fed in a pulse mode, so that dissolved oxygen rapidly and repeatedly fluctuates between 10% and 50%, the concentration of residual sugar in the culture solution is controlled to be close to zero, and the synthesis of protein and lutein is induced. The method not only realizes the ultra-high density heterotrophic culture of the microalgae, but also can enable the algae cells to synchronously accumulate high-content protein and lutein, has short fermentation period and simple production process, and can remarkably improve the industrial prospect of the co-production of the protein and lutein by the microalgae.

Description

Method for co-producing protein and lutein by using ultra-high density heterotrophic microalgae
Technical Field
The invention belongs to the field of fermentation processes, and particularly relates to a method for co-producing protein and lutein by using ultra-high density heterotrophic microalgae.
Background
Lutein is an oxygen-containing carotenoid, and is widely applied to industries such as feed, cosmetics, foods, health products, medicines and the like due to the strong coloring, antioxidation and anti-inflammatory effects. Microalgae are considered an emerging source of commercial lutein. In recent years, researchers have made a great deal of research on the production of microalgae-based lutein, and most of the algae used are green algae. Wherein, the cord Luo Jinxiao is chlorellaChlorella sorokiniana) The method has high growth speed, can adapt to different nutrition modes and wide culture conditions, and has great commercial application potential in various aspects of lutein production and the like.
In addition, microalgae can synthesize all types of amino acids, and the protein content of most microalgae is equivalent to or even higher than that of traditional protein source plants, so that the microalgae is an ideal strategic protein source. Numerous nutritional and toxicological evaluations have also shown that microalgae are suitable as a valuable feed additive or a replacement for traditional protein sources (e.g. soybean meal, fish meal). However, microalgae produced commercially at present are mainly obtained by culturing in an open runway pool in an photoautotrophic mode, and the wide application of microalgae as a protein source is greatly limited due to the problems of low production efficiency, high production cost and the like of the culture mode.
Heterotrophic culture is used as a more economical and efficient industrial production mode, and is a potential way for reducing the production cost of microalgae and improving the productivity. But the different source of the chlorella so Luo Jinxiao has obvious algae species specificity in the heterotrophic process and shows different growth and product accumulation characteristics. By cultivating microalgae in a heterotrophic system we have to consider not only the biomass yield that it can achieve, but also the algal protein and lutein content, since this will determine the final application value and market value of the heterotrophic microalgae as a protein source and lutein source.
However, the algae cell density, protein and lutein content are generally low under the traditional heterotrophic process, and are limited to production studies of single components of protein or lutein. In addition, in order to reduce the development and utilization cost of microalgae biomass, the development trend of microalgae biomass has been that microalgae cells are comprehensively utilized in multiple components and coupled to produce high-added-value products. However, up to now, no mature culture method is available which can realize the ultra-high density heterotrophic culture of microalgae and can synchronously accumulate high-content protein and lutein in the algae cells.
Disclosure of Invention
The invention aims to provide a method for co-producing protein and lutein by using ultra-high density heterotrophic microalgae, and the method is used for realizing the efficient co-production of protein and lutein by using the microalgae. The method solves the problems that the density of algae cells, the content of protein and lutein is generally lower under the traditional heterotrophic process, and is only limited to the production research of single components of protein or lutein, and can provide a new method for the high-efficiency coupling production of microalgae lutein and protein.
In order to achieve the above purpose, the invention adopts the following technical scheme:
a method for co-producing protein and lutein by using ultra-high density heterotrophic microalgae comprises the following steps:
(1) Algae species and their preservation: the algae isChlorella sorokinianaFZU60 the preservation number is CGMCC No. 45230, which is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) at the date of 08 and 01 of 2022, and the preservation address is North Chen Xili No. 1, 3 of the Korean area of Beijing, the algae cells are transferred into a freezing tube by using 20% glycerol sterile solution, and then are preserved in a refrigerator at-80 ℃.
(2) Plate culture: the algae liquid frozen in the refrigerator at the temperature of minus 80 ℃ is coated on a flat plate containing a solid culture medium and is placed in an incubator at the temperature of 25 ℃ to 35 ℃ for culturing for 4 days to 8 days in a dark place.
(3) Seed culture: selecting 1-4 ring algae from flat plate with inoculating loop, placing into shake flask containing seed culture medium, placing into 25-35deg.C shake flask, setting rotation speed at 50-300 r/min, and culturing in dark for 3-7 days.
(4) Fermentation culture:
1) The first stage: inoculating the seeds in the step (3) into a fermentation tank filled with a fermentation medium, controlling the initial concentration of cells to be 0.5-3.0 g/L, controlling the aeration rate to be 0.5-2.0 vvm, controlling the initial rotating speed to be 100-300 rpm, controlling the temperature to be 25-35 ℃ and controlling the pH to be 6.5-8.0;
2) And a second stage: when the initial glucose concentration is exhausted, starting to feed a glucose solution with the concentration of 750 g/L, and controlling the glucose concentration in the culture solution to be 1-5 g/L; each time the glucose consumption in the culture solution reaches 30 g/L, the concentrated solution of other nutrient components is supplemented to reach the respective initial concentration; the relevance of dissolved oxygen and the rotating speed is controlled to be 10-30 percent;
3) And a third stage: when the biomass concentration of the algae reaches 160-200 g/L, the maximum rotating speed of the fermentation tank is fixed, and 750 g/L of glucose solution is fed in a pulse mode instead, so that the dissolved oxygen rapidly and repeatedly fluctuates between 10% and 50%, the concentration of residual sugar in the culture solution is controlled to be close to zero, and the synthesis of protein and lutein is induced. The fermentation period is 6-8 days, the biomass of algae can reach 180-220 g/L, the protein content is 40-50%, and the lutein content is 5-7 mg/g.
In the steps (2) and (3), agar 15 g/L is additionally added on the basis that the solid culture medium is a seed culture medium; the seed culture medium is as follows: glucose 10 g/L, sodium nitrate 1.75 g/L, sodium chloride 5 g/L, magnesium sulfate heptahydrate 0.4 g/L, potassium chloride 0.6 g/L, calcium chloride dihydrate 0.15 g/L, dipotassium phosphate trihydrate 0.1 g/L, tris 0.5 g/L, EDTA.2Na 75mg/L, boric acid 15mg/L, ferrous sulfate heptahydrate 5mg/L, manganese chloride tetrahydrate 3.5 mg/L, zinc sulfate heptahydrate 0.825 mg/L, cobalt nitrate hexahydrate 0.0175mg/L, copper sulfate pentahydrate 0.005 mg/L.
In step (4), the fermentation medium is: glucose 30 g/L, urea 1.854 g/L, magnesium sulfate heptahydrate 1.2 g/L, calcium chloride dihydrate 0.45 g/L, dipotassium phosphate trihydrate 0.3 g/L, EDTA.2Na 213 mg/L, boric acid 45 mg/L, ferrous sulfate heptahydrate 15mg/L, manganese chloride tetrahydrate 10.5 mg/L, zinc sulfate heptahydrate 2.475 mg/L, cobalt nitrate hexahydrate 0.0525 mg/L, and copper sulfate pentahydrate 0.015 mg/L.
In the step (4), the concentrated solution is divided into 3 parts, wherein the concentrated solution I is 11.25 g/L of calcium chloride dihydrate, the concentrated solution II is 247.33 g/L of urea and 160 g/L of magnesium sulfate heptahydrate, and the concentrated solution III is 40 g/L, EDTA.2Na30 g/L of dipotassium phosphate trihydrate, 6 g/L of boric acid, 2 g/L of ferrous sulfate heptahydrate, 1.4 g/L of manganese chloride tetrahydrate, 0.33 g/L of zinc sulfate heptahydrate, 7 mg/L of cobalt nitrate hexahydrate and 2 mg/L of copper sulfate pentahydrate.
In the step (4), the culturing time in the third stage is 1-2 days.
The invention has the remarkable advantages that: the microalgae used in the invention is a high-protein and high-lutein-content algae strain, and the heterotrophic fermentation process provided by the invention can realize the ultrahigh-density heterotrophic culture of the microalgae, and can synchronously accumulate high-content protein and lutein by the algae cells, so that the production efficiency is far higher than the reported results at home and abroad, and the industrialization prospect of producing lutein and nutrient protein by coupling the microalgae can be remarkably improved.
Drawings
FIG. 1 is a graph showing the change in the concentration of microalgae biomass and the concentration of glucose in the fermentation tank in example 1.
FIG. 2 is a graph showing the change in lutein content and protein content of microalgae in the fermentation tank in example 1.
FIG. 3 is a graph showing the change in the concentration of dissolved oxygen in the fermenter according to example 1.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to the attached drawings: the present example provides detailed embodiments and specific operation procedures on the premise of the technical scheme of the present invention, but the protection scope of the present invention is not limited to the following examples.
Example 1
(1) Algae species and their preservation: the algae isChlorella sorokinianaFZU60 (CGMCC No. 45230, china general microbiological culture Collection center) is prepared by transferring algal cells with 20% glycerol sterile solutionTransferring to a freezing tube, and storing in a refrigerator at-80deg.C.
(2) Plate culture: the frozen algae liquid at-80 ℃ is coated on a flat plate containing a solid culture medium and is placed in an incubator at 30 ℃ for culturing for 5 days in a dark place.
(3) Seed culture: the 4-ring algae are picked from the flat plate by using an inoculating loop and placed in a shake flask filled with seed culture medium, and are placed in a shaking incubator at 30 ℃ at the rotating speed of 200 r/min for 5 days of light-proof culture.
(4) Fermentation culture:
1) The first stage: inoculating the seeds in the step (3) into a fermentation tank filled with a fermentation medium, controlling the initial concentration of cells to be 2 g/L, controlling the aeration rate to be 1 vvm, controlling the initial rotating speed to be 200rpm, controlling the temperature to be 30 ℃, and controlling the pH to be 7.5;
2) And a second stage: when the initial glucose concentration is exhausted, starting to feed a glucose solution with the concentration of 750 g/L, and controlling the glucose concentration in the culture solution to be 1-5 g/L; each time the glucose consumption in the culture solution reaches 30 g/L, the concentrated solution of other nutrient components is supplemented to reach the respective initial concentration; the correlation between dissolved oxygen and rotating speed is controlled at 20%;
3) And a third stage: when the biomass concentration of the algae reaches 200 g/L, the maximum rotating speed of the fermentation tank is fixed, and 750 g/L of glucose solution is fed in a pulse mode instead, so that the dissolved oxygen rapidly and repeatedly fluctuates between 10% and 50%, the concentration of residual sugar in the culture solution is controlled to be close to zero, and the synthesis of protein and lutein is induced.
In the steps (2) and (3), the solid culture medium is additionally added with agar 15 g/L on the basis of the seed culture medium; the seed culture medium is as follows: glucose 10 g/L, sodium nitrate 1.75 g/L, sodium chloride 5 g/L, magnesium sulfate heptahydrate 0.4 g/L, potassium chloride 0.6 g/L, calcium chloride dihydrate 0.15 g/L, dipotassium phosphate trihydrate 0.1 g/L, tris 0.5 g/L, EDTA.2Na 75mg/L, boric acid 15mg/L, ferrous sulfate heptahydrate 5mg/L, manganese chloride tetrahydrate 3.5 mg/L, zinc sulfate heptahydrate 0.825 mg/L, cobalt nitrate hexahydrate 0.0175mg/L, copper sulfate pentahydrate 0.005 mg/L.
In step (4), the fermentation medium is: glucose 30 g/L, urea 1.854 g/L, magnesium sulfate heptahydrate 1.2 g/L, calcium chloride dihydrate 0.45 g/L, dipotassium phosphate trihydrate 0.3 g/L, EDTA.2Na 213 mg/L, boric acid 45 mg/L, ferrous sulfate heptahydrate 15mg/L, manganese chloride tetrahydrate 10.5 mg/L, zinc sulfate heptahydrate 2.475 mg/L, cobalt nitrate hexahydrate 0.0525 mg/L, and copper sulfate pentahydrate 0.015 mg/L.
In the step (4), the concentrated solution is divided into 3 parts, wherein the concentrated solution I is 11.25 g/L of calcium chloride dihydrate, the concentrated solution II is 247.33 g/L of urea and 160 g/L of magnesium sulfate heptahydrate, and the concentrated solution III is 40 g/L, EDTA.2Na30 g/L of dipotassium phosphate trihydrate, 6 g/L of boric acid, 2 g/L of ferrous sulfate heptahydrate, 1.4 g/L of manganese chloride tetrahydrate, 0.33 g/L of zinc sulfate heptahydrate, 7 mg/L of cobalt nitrate hexahydrate and 2 mg/L of copper sulfate pentahydrate.
In the step (4), the culture time in the third stage was 2 days.
During the whole fermentation culture process, the biomass concentration, the glucose concentration, the lutein content and the protein content are sampled and measured at regular intervals. The biomass concentration is determined by a cell dry weight method, the glucose concentration is determined by a DNS colorimetric method, the lutein content and carotenoid composition are determined by a high performance liquid chromatography method, and the protein content is determined by a protein extraction and analysis kit. As can be seen from FIGS. 1-3, the biomass of algae can reach 220 g/L, the protein content is 50%, and the lutein content is 7 mg/g in 7 days of fermentation period.
Example 2
(1) Algae species and their preservation: the algae isChlorella sorokinianaFZU60 (CGMCC No. 45230, deposited in China general microbiological culture Collection center) the algae cells are transferred to a freezing tube with 20% glycerol sterile solution, and then stored in a refrigerator at-80deg.C.
(2) Plate culture: the frozen algae liquid at-80 ℃ is coated on a flat plate containing a solid culture medium and is placed in an incubator at 25 ℃ for culturing for 4 days in a dark place.
(3) Seed culture: the 1-ring algae is selected from the flat plate by using an inoculating loop, placed in a shake flask filled with a seed culture medium, placed in a shaking incubator at 25 ℃, set at a rotating speed of 300 r/min, and cultivated for 7 days in a dark place.
(4) Fermentation culture:
1) The first stage: inoculating the seeds in the step (3) into a fermentation tank filled with a fermentation medium, controlling the initial concentration of cells to be 0.5 g/L, controlling the aeration rate to be 1.5 vvm, controlling the initial rotating speed to be 200rpm, controlling the temperature to be 30 ℃, and controlling the pH to be 7.0;
2) And a second stage: when the initial glucose concentration is exhausted, starting to feed a glucose solution with the concentration of 750 g/L, and controlling the glucose concentration in the culture solution to be 1-5 g/L; each time the glucose consumption in the culture solution reaches 30 g/L, the concentrated solution of other nutrient components is supplemented to reach the respective initial concentration; the correlation between dissolved oxygen and rotating speed is controlled at 10%;
3) And a third stage: when the biomass concentration of the algae reaches 180 g/L, the maximum rotating speed of the fermentation tank is fixed, and 750 g/L of glucose solution is fed in a pulse mode instead, so that the dissolved oxygen rapidly and repeatedly fluctuates between 10% and 30%, the concentration of residual sugar in the culture solution is controlled to be close to zero, and the synthesis of protein and lutein is induced.
In the steps (2) and (3), agar 15 g/L is additionally added on the basis that the solid culture medium is a seed culture medium; the seed culture medium is as follows: glucose 10 g/L, sodium nitrate 1.75 g/L, sodium chloride 5 g/L, magnesium sulfate heptahydrate 0.4 g/L, potassium chloride 0.6 g/L, calcium chloride dihydrate 0.15 g/L, dipotassium phosphate trihydrate 0.1 g/L, tris 0.5 g/L, EDTA.2Na 75mg/L, boric acid 15mg/L, ferrous sulfate heptahydrate 5mg/L, manganese chloride tetrahydrate 3.5 mg/L, zinc sulfate heptahydrate 0.825 mg/L, cobalt nitrate hexahydrate 0.0175mg/L, copper sulfate pentahydrate 0.005 mg/L.
In step (4), the fermentation medium is: glucose 30 g/L, urea 1.854 g/L, magnesium sulfate heptahydrate 1.2 g/L, calcium chloride dihydrate 0.45 g/L, dipotassium phosphate trihydrate 0.3 g/L, EDTA.2Na 213 mg/L, boric acid 45 mg/L, ferrous sulfate heptahydrate 15mg/L, manganese chloride tetrahydrate 10.5 mg/L, zinc sulfate heptahydrate 2.475 mg/L, cobalt nitrate hexahydrate 0.0525 mg/L, and copper sulfate pentahydrate 0.015 mg/L.
In the step (4), the concentrated solution is divided into 3 parts, wherein the concentrated solution I is 11.25 g/L of calcium chloride dihydrate, the concentrated solution II is 247.33 g/L of urea and 160 g/L of magnesium sulfate heptahydrate, and the concentrated solution III is 40 g/L, EDTA.2Na30 g/L of dipotassium phosphate trihydrate, 6 g/L of boric acid, 2 g/L of ferrous sulfate heptahydrate, 1.4 g/L of manganese chloride tetrahydrate, 0.33 g/L of zinc sulfate heptahydrate, 7 mg/L of cobalt nitrate hexahydrate and 2 mg/L of copper sulfate pentahydrate.
In the step (4), the culture time in the third stage was 1 day.
The detection method is the same as in example 1. The fermentation period is 8 days, the biomass of the algae can reach 200 g/L, the protein content is 40%, and the lutein content is 5 mg/g.
Example 3
(1) Algae species and their preservation: the algae isChlorella sorokinianaFZU60 (CGMCC No. 45230, deposited in China general microbiological culture Collection center) the algae cells are transferred to a freezing tube with 20% glycerol sterile solution, and then stored in a refrigerator at-80deg.C.
(2) Plate culture: the frozen algae liquid at-80 ℃ is coated on a flat plate containing a solid culture medium and is placed in an incubator at 35 ℃ for light-proof culture for 8 days.
(3) Seed culture: the 4-ring algae are picked from the flat plate by using an inoculating loop and placed in a shake flask filled with seed culture medium, and are placed in a shake flask at 35 ℃ and are cultured for 3 days in a dark place at the rotating speed of 300 r/min.
(4) Fermentation culture:
1) The first stage: inoculating the seeds in the step (3) into a fermentation tank filled with a fermentation medium, controlling the initial concentration of cells to be 3.0 g/L, controlling the aeration rate to be 2.0 vvm, controlling the initial rotating speed to be 200rpm, controlling the temperature to be 35 ℃, and controlling the pH to be 7.5;
2) And a second stage: when the initial glucose concentration is exhausted, starting to feed a glucose solution with the concentration of 750 g/L, and controlling the glucose concentration in the culture solution to be 1-5 g/L; each time the glucose consumption in the culture solution reaches 30 g/L, the concentrated solution of other nutrient components is supplemented to reach the respective initial concentration; the correlation between dissolved oxygen and rotating speed is controlled at 30%;
3) And a third stage: when the biomass concentration of the algae reaches 160 g/L, the maximum rotating speed of the fermentation tank is fixed, and 750 g/L of glucose solution is fed in a pulse mode instead, so that the dissolved oxygen rapidly and repeatedly fluctuates between 10% and 50%, the concentration of residual sugar in the culture solution is controlled to be close to zero, and the synthesis of protein and lutein is induced.
In the steps (2) and (3), agar 15 g/L is additionally added on the basis that the solid culture medium is a seed culture medium; the seed culture medium is as follows: glucose 10 g/L, sodium nitrate 1.75 g/L, sodium chloride 5 g/L, magnesium sulfate heptahydrate 0.4 g/L, potassium chloride 0.6 g/L, calcium chloride dihydrate 0.15 g/L, dipotassium phosphate trihydrate 0.1 g/L, tris 0.5 g/L, EDTA.2Na 75mg/L, boric acid 15mg/L, ferrous sulfate heptahydrate 5mg/L, manganese chloride tetrahydrate 3.5 mg/L, zinc sulfate heptahydrate 0.825 mg/L, cobalt nitrate hexahydrate 0.0175mg/L, copper sulfate pentahydrate 0.005 mg/L.
In step (4), the fermentation medium is: glucose 30 g/L, urea 1.854 g/L, magnesium sulfate heptahydrate 1.2 g/L, calcium chloride dihydrate 0.45 g/L, dipotassium phosphate trihydrate 0.3 g/L, EDTA.2Na 213 mg/L, boric acid 45 mg/L, ferrous sulfate heptahydrate 15mg/L, manganese chloride tetrahydrate 10.5 mg/L, zinc sulfate heptahydrate 2.475 mg/L, cobalt nitrate hexahydrate 0.0525 mg/L, and copper sulfate pentahydrate 0.015 mg/L.
In the step (4), the concentrated solution is divided into 3 parts, wherein the concentrated solution I is 11.25 g/L of calcium chloride dihydrate, the concentrated solution II is 247.33 g/L of urea and 160 g/L of magnesium sulfate heptahydrate, and the concentrated solution III is 40 g/L, EDTA.2Na30 g/L of dipotassium phosphate trihydrate, 6 g/L of boric acid, 2 g/L of ferrous sulfate heptahydrate, 1.4 g/L of manganese chloride tetrahydrate, 0.33 g/L of zinc sulfate heptahydrate, 7 mg/L of cobalt nitrate hexahydrate and 2 mg/L of copper sulfate pentahydrate.
In the step (4), the cultivation time in the third stage was 1.5 days.
The detection method is the same as in example 1. The fermentation period is 6 days, the biomass of the algae can reach 180 g/L, the protein content is 45%, and the lutein content is 6.5 mg/g.
The foregoing description is only of the preferred embodiments of the invention, and all changes and modifications that come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

Claims (6)

1. A method for co-producing protein and lutein by using ultra-high density heterotrophic microalgae is characterized by comprising the following steps: the method comprises the following steps:
(1) Algae species and their preservation: the algae is chlorella, 20% glycerol sterile solution is used to transfer algae cells into a freezing tube, and then the freezing tube is placed in a refrigerator at-80 ℃ for preservation;
(2) Plate culture: coating the frozen algae liquid on a plate containing a solid culture medium at-80 ℃ and placing the plate in an incubator at 25-35 ℃ for light-proof culture for 4-8 days;
(3) Seed culture: selecting 1-4 ring algae from the flat plate by using an inoculating loop, placing into a shake flask filled with seed culture medium, placing into a shake flask at 25-35deg.C, setting rotation speed at 50-300 r/min, and culturing in dark for 3-7 days;
(4) Fermentation culture:
1) The first stage: inoculating the seeds in the step (3) into a fermentation tank filled with a fermentation medium, controlling the initial concentration of cells to be 0.5-3.0 g/L, controlling the aeration rate to be 0.5-2.0 vvm, controlling the initial rotating speed to be 100-300 rpm, controlling the temperature to be 25-35 ℃ and controlling the pH to be 6.5-8.0;
2) And a second stage: when the initial glucose concentration is exhausted, starting to feed a glucose solution with the concentration of 750 g/L, and controlling the glucose concentration in the culture solution to be 1-5 g/L; each time the glucose consumption in the culture solution reaches 30 g/L, the concentrated solution of other nutrient components is supplemented to reach the respective initial concentration; the relevance of dissolved oxygen and the rotating speed is controlled to be 10-30 percent;
3) And a third stage: when the biomass concentration of the algae reaches 160-200 g/L, the maximum rotating speed of the fermentation tank is fixed, and 750 g/L of glucose solution is fed in a pulse mode instead, so that the dissolved oxygen rapidly and repeatedly fluctuates between 10% and 50%, the concentration of residual sugar in the culture solution is controlled to be close to zero, and the synthesis of protein and lutein is induced; the total fermentation period is 6-8 days.
2. The method for co-producing protein and lutein from ultra-high density heterotrophic microalgae according to claim 1, wherein the method comprises the following steps: the chlorella isChlorella sorokinianaFZU60 the preservation number is CGMCC No. 45230, and the CGMCC No. 45230 is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) in the year 08 and 01.
3. The method for co-producing protein and lutein from ultra-high density heterotrophic microalgae according to claim 1, wherein the method comprises the following steps: in the steps (2) and (3), agar 15 g/L is additionally added on the basis of the solid culture medium serving as a seed culture medium; the seed culture medium is as follows: glucose 10 g/L, sodium nitrate 1.75 g/L, sodium chloride 5 g/L, magnesium sulfate heptahydrate 0.4 g/L, potassium chloride 0.6 g/L, calcium chloride dihydrate 0.15 g/L, dipotassium phosphate trihydrate 0.1 g/L, tris 0.5 g/L, EDTA.2Na 75mg/L, boric acid 15mg/L, ferrous sulfate heptahydrate 5mg/L, manganese chloride tetrahydrate 3.5 mg/L, zinc sulfate heptahydrate 0.825 mg/L, cobalt nitrate hexahydrate 0.0175mg/L, copper sulfate pentahydrate 0.005 mg/L.
4. The method for co-producing protein and lutein from ultra-high density heterotrophic microalgae according to claim 1, wherein: in the step (4), the fermentation medium is: glucose 30 g/L, urea 1.855 g/L, magnesium sulfate heptahydrate 1.2 g/L, calcium chloride dihydrate 0.45 g/L, dipotassium phosphate trihydrate 0.3 g/L, EDTA.2Na 213 mg/L, boric acid 45 mg/L, ferrous sulfate heptahydrate 15mg/L, manganese chloride tetrahydrate 10.5 mg/L, zinc sulfate heptahydrate 2.475 mg/L, cobalt nitrate hexahydrate 0.0525 mg/L, copper sulfate pentahydrate 0.015 mg/L.
5. The method for co-producing protein and lutein from ultra-high density heterotrophic microalgae according to claim 1, wherein: in the step (4), the concentrated solution is divided into 3 parts, wherein the concentrated solution I is 11.25/g/L of calcium chloride dihydrate, the concentrated solution II is 247.33 g/L of urea and 160/g/L of magnesium sulfate heptahydrate, and the concentrated solution III is 40 g/L, EDTA.2Na30g/L of dipotassium phosphate trihydrate, 6 g/L of boric acid, 2 g/L of ferrous sulfate heptahydrate, 1.4 g/L of manganese chloride tetrahydrate, 0.33 g/L of zinc sulfate heptahydrate, 7/mg/L of cobalt nitrate hexahydrate and 2 mg/L of copper sulfate pentahydrate.
6. The method for co-producing protein and lutein from ultra-high density heterotrophic microalgae according to claim 1, wherein: in the step (4), the culture time in the third stage is 1-2 days.
CN202211110076.0A 2022-09-13 2022-09-13 Method for co-producing protein and lutein by using ultra-high density heterotrophic microalgae Pending CN116042761A (en)

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