CN114214203B - Method for improving fucoxanthin yield by mixing and preserving of nitenpyram - Google Patents

Method for improving fucoxanthin yield by mixing and preserving of nitenpyram Download PDF

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CN114214203B
CN114214203B CN202111619731.0A CN202111619731A CN114214203B CN 114214203 B CN114214203 B CN 114214203B CN 202111619731 A CN202111619731 A CN 202111619731A CN 114214203 B CN114214203 B CN 114214203B
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fucoxanthin
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牟海津
孙翰
朱常亮
杨淑芳
付晓丹
朱琳
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Weihai Dipusen Biology Technology Co ltd
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Abstract

The application provides a method for improving the yield of fucoxanthin by mixing and preserving the health of the nitenpyram minor, which comprises the following steps: the first step: culturing activated Nicotiana microphylla to make it in logarithmic growth phase; and a second step of: taking the culture solution of the Nicotiana microphylla in the logarithmic growth phase in the first step as seed solution, and transferring the seed solution into the prepared culture medium according to the inoculation amount of 10-20% for shake culture. The application uses red light to induce the small crescent diamond algae to absorb low concentration glucose, to gradually increase the glucose concentration, to obtain small crescent diamond algae G5, to improve the biomass and fucoxanthin output, to be used in micro algae laboratory directed evolution, to realize micro algae growth mode transformation, to improve directed evolution efficiency, and the application can be used in mixed culture and domestication of other micro algae, to provide new research direction for micro algae high density culture.

Description

Method for improving fucoxanthin yield by mixing and preserving of nitenpyram
Technical Field
The application relates to the technical field of microbial fermentation, in particular to a method for improving the yield of fucoxanthin by mixing and preserving the yield of the nitenpyram.
Background
Fucoxanthin (Fucoxanthin), which is also known as Fucoxanthin, is a xanthophyll carotenoid, and Fucoxanthin is an orange carotenoid accounting for more than 10% of total carotenoid yield in nature. Fucoxanthin has a unique molecular structure, comprises 5, 6-monoepoxide and allene, and has multiple functional activities. In recent years, fucoxanthin has been found to have various physiological functions and biological characteristics, such as anti-obesity, anti-tumor, anti-diabetes, anti-oxidation, anti-inflammatory, liver protection, cardiovascular and cerebrovascular protection and the like. Fucoxanthin has a natural product of functional food and drug development, and the global market value of fucoxanthin in 2020 is $ 9,500 tens of thousands, and it is expected that it will reach $ 1.135 billion at the end of 2026.
Fucoxanthin is widely distributed in algae such as brown algae, diatom, golden algae, yellow green algae, and marine phytoplankton. At present, the phycocyanin source is mainly extracted from large-scale seaweeds such as kelp (Laminaria japonica) and undaria pinnatifida (Undaria pinnatifida). However, the fucoxanthin content of the large seaweed is low (about 0.01% -0.1% of dry weight), the extraction and purification process of the cell wall thickness of the seaweed is complex, and the quality of seasonal growth products is poor, so that the industrial development and utilization of the fucoxanthin are severely limited. Marine microalgae are a better source of fucoxanthin than large algae. The single-cell microalgae is rich in fucoxanthin, and the content of the fucoxanthin is 0.2% -2.6% of the dry weight, and is 10-100 times of that of the large-scale seaweed. The microalgae has high growth rate, can be controllably cultured in a bioreactor, has the yield not limited by seasons, and is favorable for solving the raw material problem of industrialized production of fucoxanthin.
Existing studies show that most microalgae accumulate fucoxanthin in an autotrophic mode, and that the addition of organic carbon sources limits cell growth or fucoxanthin accumulation. The diamond algae (Nitzschia closterium f. Minissima) is a marine eukaryotic single-cell diatom belonging to the phylum diatom (Baciliariophyllta), the class of the phylum Diatom, the class of the Phyllostachys Pubescens (Pennaeae), the order of the tube and shell (Aulon diamond, the genus of the diamond algae (Nitzschia), and has the potential to autotrophically accumulate fucoxanthin. However, the diamond algae of the crescent moon cannot effectively absorb the organic carbon source, and is difficult to realize high-density culture, so that the industrial production of the fucoxanthin is limited. Glucose is used as a common organic carbon source, and is used for microalgae mixed culture or heterotrophic culture due to the highest energy conversion rate. At present, the smooth diamond algae (Nitzchia laevis) is the only reported microalgae which can effectively absorb and metabolize glucose to produce fucoxanthin, and the small crescent diamond algae which induce and domesticate different species have certain biological informatics support. The transformation from microalgae autotrophy to mixed culture production of fucoxanthin is realized by utilizing a laboratory directed evolution system (ALE), and the method has important significance for the industrial production of fucoxanthin. ALE applies pressure to the growth and metabolism process of microalgae, so that the microalgae are continuously adapted to new environment, and the genotype of the algae is changed towards the evolution of beneficial mutation. Unlike genetic engineering, ALE-producing mutants are generally considered safe (GRAS). The directional introduction of autotrophic microalgae into heterotrophic or mixotrophic microalgae is of great significance for high-density cell culture and high-added-value product production. Heterotrophic or polyculture modes reduce the dependence of microalgae cells on illumination, increase the biomass harvested, and thereby increase the yield of high value-added products such as fucoxanthin and EPA. The mutation period of microalgae in ALE is long (3 months to 2 years), and the design strategy shortens the ALE period and effectively improves the fucoxanthin production efficiency.
Therefore, it is necessary to design a high-efficiency laboratory directed evolution strategy to domesticate autotrophic small-moon diamond algae into a nutritional mode for producing fucoxanthin by mixed health maintenance, and the aim is to obtain a high-efficiency fucose Huang Suxiao crescent diamond algae strain.
Disclosure of Invention
The application aims to provide a method for improving the yield of fucoxanthin by mixed cultivation of the nitazopsis grossedentata, which realizes mixed cultivation growth of the nitazopsis grossedentata and improves biomass and the yield of fucoxanthin.
In order to achieve the above purpose, the present application proposes the following technical scheme: a method for improving the yield of fucoxanthin by mixed health maintenance of the nitenpyram minor comprises the following steps:
the first step: culturing activated Nicotiana microphylla to make it in logarithmic growth phase;
and a second step of: taking the culture solution of the Nitzschia closterium in the logarithmic growth phase in the first step as seed solution, transferring into the prepared culture medium according to the inoculation amount of 10-20% for shaking culture, adding glucose into the culture medium, setting the concentration of the added glucose to be a preset value and gradually increasing, culturing at 20-30 ℃, culturing at 100-240rpm of shaking table rotation speed for 9-12 days under the illumination condition, wherein the light is red light, and the illumination intensity is 0-200 mu mol m -2 s -1 The method comprises the steps of carrying out a first treatment on the surface of the Because the wild type of the microcephala is incapable of absorbing and metabolizing glucose, the red light is utilized to induce the microcephala G5, so that the microcephala G5 can absorb and metabolize glucose rapidly, the mixed culture is realized, and the bottleneck that the autotrophic microalgae cannot be cultured at high density is overcome.
And a third step of: collecting algae liquid, centrifugally washing, freeze-drying, and detecting the content of fucoxanthin.
Preferably, the composition of the medium is as follows:
30g/L sea salt, 750mg/L NaNO 3 ，5mg/L NaH 2 PO 4 ·H 2 O，3.15mg/L FeCl 3 ·6H 2 O，4.36mg/L Na 2 EDTA，0.0098mg/L CuSO 4 ·5H 2 O，0.0063mg/L Na 2 MoO 4 ·2H 2 O，0.022mg/L ZnSO 4 ·7H 2 O，0.01mg/L CoCl 2 ·6H 2 O，0.18mg/L MnCl 2 ·4H 2 O,0.001mg/L vitamin B 12 0.2mg/L vitamin B 1 0.001mg/L biotin.
Preferably, the light source of the illumination condition comprises at least one of sunlight, an LED lamp or fluorescent lamp light, and the light quality further comprises blue light or white light.
Preferably, 2g/L, 5g/L, 10g/L and 20g/L glucose are added to the medium.
Preferably, the Chlorella vulgaris in the first step is wild type, and the Chlorella vulgaris obtained in the second step is defined as G5.
Preferably, the pH of the medium is 8.5.
Preferably, the specific process of the third step is as follows:
centrifuging the culture solution, washing, re-centrifuging, repeating for 2 times, filtering the algae solution onto pre-weighed filter paper, putting into a vacuum drying oven at 80 ℃ for drying to constant weight, freezing, weighing the freeze-dried algae powder, grinding at low temperature, adding absolute ethyl alcohol, vibrating and extracting, centrifuging to collect supernatant, adding absolute ethyl alcohol again in precipitation for vibrating and extracting until the algae powder is white, collecting extracting solution, centrifuging the extracting solution, drying the supernatant with nitrogen, adding absolute ethyl alcohol for dissolving pigment, and analyzing by a high performance liquid chromatograph after membrane filtration, wherein the whole process is performed under a dark condition.
Preferably, the high performance liquid chromatograph is waters2695, a PDA detector is configured, the detection wavelength is 450nm, a C18 reverse phase column is selected, and the mobile phase is: phase A is pure ethyl acetate, phase B is acetonitrile: methanol: water=84:2:14, pure methanol for phase c, gradient elution, HPLC grade for mobile phase.
Preferably, in the first step, the activated Nicotiana microphylla is cultured for 6-12 days.
The beneficial effects are that the technical scheme of the application has the following technical effects:
1. the red light is used for inducing the small crescent diamond algae to absorb low-concentration glucose, so that the glucose concentration is gradually increased, the mixed culture growth of the small crescent diamond algae is realized, the biomass and the fucoxanthin yield are improved, the red light can be used for laboratory directed evolution of microalgae, the change of the growth mode of the microalgae is realized, the directed evolution efficiency is improved, the mixed culture growth of the small crescent diamond algae is obtained, and in addition, the method provided by the application can be used for mixed culture domestication of other microalgae, and a new research direction is provided for the high-density culture of the microalgae.
2. The method comprises the steps of optimizing wild-type conditions of the small crescent diamond algae, screening light and glucose concentration in directed evolution, obtaining and detecting fucoxanthin which is an intracellular product, and the like, wherein the obtained small crescent diamond algae has the potential of producing fucoxanthin industrially, and the yield of fucoxanthin is greatly improved after directed evolution; secondly, the obtained diamond-shaped algae powder of the application can control marine common pollutants such as heavy metals from the source of the culture medium, and the obtained fucoxanthin is safer.
It should be understood that all combinations of the foregoing concepts, as well as additional concepts described in more detail below, may be considered a part of the inventive subject matter of the present disclosure as long as such concepts are not mutually inconsistent.
The foregoing and other aspects, embodiments, and features of the present teachings will be more fully understood from the following description, taken together with the accompanying drawings. Other additional aspects of the application, such as features and/or advantages of the exemplary embodiments, will be apparent from the description which follows, or may be learned by practice of the embodiments according to the teachings of the application.
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The drawings are not intended to be drawn to scale. In the drawings, each identical or nearly identical component that is illustrated in various figures may be represented by a like numeral. For purposes of clarity, not every component may be labeled in every drawing. Embodiments of various aspects of the application will now be described, by way of example, with reference to the accompanying drawings, in which:
FIG. 1 is the final biomass and fucoxanthin content of the Nicotiana microphylla (wild type) under autotrophic (glucose 0) and polyculture (glucose concentration gradient increasing) conditions of example 1.
FIG. 2 is a graph of the growth curve of the Nitzschia closterium (wild type) of example 2 under white, blue and red light.
FIG. 3 is the final biomass of the Nicotiana microcephala (wild type) of example 3 under evolution of red light directed laboratory, domesticated to obtain Nicotiana microcephala G5.
FIG. 4 shows the beta-carotene and fucoxanthin yields of example 4 after 12 days of culture of Nicotiana microphylla (wild type) and G5 in red light.
Detailed Description
For a better understanding of the technical content of the present application, specific examples are set forth below, along with the accompanying drawings.
Aspects of the application are described in this disclosure with reference to the drawings, in which are shown a number of illustrative embodiments. The embodiments of the present disclosure need not be defined to include all aspects of the present application. It should be understood that the various concepts and embodiments described above, as well as those described in more detail below, may be implemented in any of a number of ways, as the disclosed concepts and embodiments are not limited to any implementation. Additionally, some aspects of the disclosure may be used alone or in any suitable combination with other aspects of the disclosure.
Example 1: autotrophic and mixotrophic culture studies of Nicotiana microphylla (wild type)
A250 mL conical flask was used as a culture vessel, and 100mL of medium was transferred. The activated Nicotiana microcystis (wild type) is autotrophically cultured in a shake flask, and Nicotiana microcystis cells in a logarithmic growth phase are obtained after 6 days and are used as experimental seed liquid. Inoculating 10% (v/v) of the culture medium to culture in shaking table autotrophy (glucose is 0), mixing with culture medium (glucose concentration gradient is increased), adding glucose into culture medium, and adjusting glucose gradient concentration to 0, 2g/L, 5g/L, 10g/L and 20g/L, pH to 8.5, and using 80 μm -2 s -1 Is a lighting source of the light source. The conical flasks were placed in a thermostatted shaker at 150rpm, 23℃and a incubation time of 12 days. The algae liquid was collected on day 12, centrifugally washed, freeze-dried, and tested for the content of fucoxanthin in the algae powder.
The composition of the culture medium is as follows: 30g/L sea salt, 750mg/L NaNO 3 ，5mg/L NaH 2 PO 4 ·H 2 O，3.15mg/L FeCl 3 ·6H 2 O，4.36mg/L Na 2 EDTA，0.0098mg/L CuSO 4 ·5H 2 O，0.0063mg/L Na 2 MoO 4 ·2H 2 O，0.022mg/L ZnSO 4 ·7H 2 O，0.01mg/L CoCl 2 ·6H 2 O，0.18mg/L MnCl 2 ·4H 2 O,0.001mg/L vitamin B 12 0.2mg/L vitamin B 1 0.001mg/L biotin
Determination of cell dry weight of Nicotiana microphylla: taking 3mL of culture solution, centrifuging at 3000rpm5min，ddH 2 Re-centrifuging after O washing, and repeating for 2 times; filtering the algae liquid onto pre-weighed filter paper, and drying in a vacuum drying oven at 80 ℃ to constant weight.
Detection of fucoxanthin: weighing 20mg of freeze-dried algae powder, grinding at low temperature, adding 5mL of absolute ethyl alcohol, shaking and extracting for 10min, centrifuging (at 4 ℃ and 3000rpm for 5 min), collecting supernatant, and adding 3mL of absolute ethyl alcohol again into the precipitate, shaking and extracting until the algae powder is white. Collecting the extractive solution, centrifuging at 12000rpm for 10min at 4deg.C, blow drying the supernatant with nitrogen, adding 1mL anhydrous alcohol to dissolve pigment, and performing High Performance Liquid Chromatography (HPLC) analysis after film coating under dark condition.
HPLC analysis method: high performance liquid chromatograph waters2695 is equipped with PDA detector, and the detection wavelength is 450nm, and C18 reverse phase column (250 mm×4.6mm×5 mm) is selected. The mobile phase is: phase A is pure ethyl acetate, phase B is acetonitrile: methanol: water=84:2:14, pure methanol for phase c, gradient elution, HPLC grade for mobile phase.
Gradient elution conditions were as follows:
example 1 effect analysis: compared with the autotrophic (glucose is 0) and the mixotrophic (glucose concentration gradient is increased) condition of the Chlorella, the autotrophic culture growth state of the wild type Chlorella is better, and the autotrophic final biomass is up to 0.35g/L as shown in figure 1. Glucose is added to inhibit the growth of the diamond algae, and the final biomass of the mixed culture is respectively 0.26g/L, 0.16g/L and 0.14g/L under the conditions of 2g/L, 5g/L, 10g/L and 20g/L of glucose; next, the highest fucoxanthin content in the autotrophic lower fucose of the Nicotiana microphylla is 7.6mg/g, and 5.4mg/g, 5.6mg/g, 5.0mg/g and 4.2mg/g of glucose are respectively obtained at 2g/L, 5g/L, 10g/L and 20 g/L.
Example 2: autotrophic and mixotrophic culture studies of Chlorella (wild type) under white light, red light and blue light culture conditions
Example 2 was identical to example 1 except that the illumination conditions in example 2 were white lightThe light intensity of the red light and blue light LED lamps is kept at 80 mu mol m -2 s -1 Unchanged, glucose was not added.
Effect analysis of example 2: the red LED lamp improves the autotrophic growth of the small crescent diamond algae, as shown in figure 2, and the wild small crescent diamond algae grows in white light, blue light and red light; on the 4 th day of cultivation, the biomass of the Nicotiana microcystis in red light is 0.18g/L, which is 1.69 times and 1.48 times of white light and blue light respectively; on day 12 of cultivation, the biomass of the Nicotiana microphylla under red light reaches 0.70g/L, which is 2.06 times and 1.27 times that of white light and blue light respectively.
Example 3: research on obtaining Nicotiana microcephala G5 by mixotrophic culture of Nicotiana microcephala (wild type)
Example 3 the same conditions as in example 1, except that in example 3 the medium was first added with 0.5g/L glucose and the illumination was 80. Mu. Mol m -2 s -1 Culturing for 3 growth cycles to enable the cells to stably grow and passaged; increasing the concentration gradient of glucose in the culture medium to 1G, 2G/L and 5G/L, repeating the steps, enabling the cells to grow stably and passaged in 5G/L glucose finally, and obtaining the algae which is the rhombus trichomonas gingivalis G5 after directed evolution.
Final biomass of the wild type of the moon cake in the red light directed laboratory evolution polyculture; in the culture period of 0.5g/L glucose, the diamond-shaped algae gradually adapt to the concentration of 0.5g/L glucose, and the biomass reaches 0.91g/L; inoculating the domesticated algae strain into 1g/L directed evolution, wherein the final biomass is 1.10g/L; sequentially inoculating 2G/L and 5G/L of glucose into the algae seeds for directed evolution, wherein the final biomass is 1.33G/L and 1.80G/L respectively, and the cells can stably grow and passage to finally obtain the Nicotiana microcephala G5.
Effect analysis of example 3: the biomass of the Nicotiana microphylla is gradually increased in red light and low-concentration glucose domestication. As shown in FIG. 3, the biomass of the domesticated rhombohedral algae G5 under 5G/L glucose is up to 1.8G/L, which is improved by 2.35 times compared with that of the rhombohedral algae (wild type).
Example 4: culture study of Chlorella (wild type) autotrophic and polyculture (Chlorella G5)
Example 4 the same conditions as in example 1, except that example 4 was illuminated with 80. Mu. Mol m -2 s -1 Red LED lamp. Wherein, the moon cake is wild type in autotrophic mode; the culture of the mixed culture is that the nitzschia closterium G5 is cultivated, and the glucose concentration is 5G/L.
Detection of beta carotene: taking 20mg of freeze-dried algae powder, fully grinding the freeze-dried algae powder with a mortar under the protection of liquid nitrogen, extracting with chromatographic grade acetone, fully oscillating, centrifuging at 10000rpm and 4 ℃ for 5min, collecting algae residues, extracting twice with chromatographic grade acetone until the algae residues become colorless, combining acetone extract liquid of 3 times, blow-drying with nitrogen, fully dissolving pigment in 1mL of chromatographic grade acetone, analyzing by a High Performance Liquid Chromatograph (HPLC) after film coating, and carrying out the whole process under the condition of avoiding light.
HPLC analysis method: the same as in example 1.
Gradient elution conditions were as follows:
effect analysis of example 4: beta carotene has functions of coloring, antioxidant and nutrition enhancing, and is also important precursor for fucoxanthin synthesis, as shown in figure 4, beta carotene yield in the G5 of the Nicotiana microcephala is 0.11mg L -1 d -1 4.26 times that of the wild type; the yield of the G5 fucoxanthin of the Nicotiana microcephala is 1.0mg L -1 d -1 Is 1.79 times that of the wild type. The red light and glucose concentration gradient increase can improve the laboratory directed evolution efficiency of the Nicotiana microcrack G5, and the obtained Nicotiana microcrack G5 has great potential for industrial production of fucoxanthin.
The embodiment of microbial fermentation, in particular to a laboratory directed evolution method for producing fucoxanthin by utilizing red LED lamps and glucose concentration gradient increase domestication of wild mixed health of the Nicotiana microphylla. Comprises the process steps of optimizing wild conditions of the moon diamond algae, screening light quality and glucose concentration in directed evolution, obtaining and detecting fucoxanthin which is an intracellular product, and the like;
the embodiment fully verifies that the increase of the concentration gradient of the red LED lamp and the glucose improves the directed evolution efficiency of the mixed culture of the small crescent diamond algae, and the obtained small crescent diamond algae G5 has higher biomass and fucoxanthin yield which are 2.35 times and 1.79 times of wild type respectively, improves the fucoxanthin production efficiency and provides a new source for the industrial production of the fucoxanthin. The directed evolution method in the laboratory provided by the application can be applied to the mixed culture evolution of other microalgae.
While the application has been described with reference to preferred embodiments, it is not intended to be limiting. Those skilled in the art will appreciate that various modifications and adaptations can be made without departing from the spirit and scope of the present application. Accordingly, the scope of the application is defined by the appended claims.

Claims (7)

1. A method for improving the yield of fucoxanthin by mixed health maintenance of the small crescent diamond algae is characterized by comprising the following steps: the method comprises the following steps:
the first step: culturing activated Nicotiana microphylla (Nitzschia closterium f. Minissima) to make it in logarithmic growth phase;
and a second step of: taking the culture solution of the Nitzschia closterium in the logarithmic growth phase in the first step as seed solution, transferring into the prepared culture medium according to the inoculation amount of 10-20% for shake culture, adding glucose into the culture medium, setting the concentration of the added glucose to be a preset value and gradually increasing, culturing at 20-30 ℃, culturing at 100-240rpm of the shaking table, culturing under the illumination condition, wherein the light is red light, and the illumination intensity is 80-200 mu mol m -2 s -1 ；
And a third step of: collecting algae liquid, centrifugally washing, freeze-drying, and detecting the content of fucoxanthin;
the method comprises the steps that the rhomboid algae of the small moon in the first step is wild type, and the rhomboid algae of the small moon obtained in the second step is defined as G5;
in the second step, glucose was added to the medium at 0.5g/L, 1g/L, 2g/L and 5g/L, and each glucose concentration was cultured for 3 growth cycles.
2. The method for improving the yield of fucoxanthin by mixing and preserving the yield of the nitenpyram as claimed in claim 1, which is characterized by comprising the following steps: the composition of the culture medium is as follows:
30g/L sea salt, 750mg/L NaNO 3 ，5mg/L NaH 2 PO 4 ·H 2 O，3.15mg/LFeCl 3 ·6H 2 O，4.36mg/L Na 2 EDTA，0.0098mg/L CuSO 4 ·5H 2 O，0.0063mg/L Na 2 MoO 4 ·2H 2 O，0.022mg/L ZnSO 4 ·7H 2 O，0.01mg/L CoCl 2 ·6H 2 O，0.18mg/L MnCl 2 ·4H 2 O,0.001mg/L vitamin B 12 0.2mg/L vitamin B 1 0.001mg/L biotin.
3. The method for improving the yield of fucoxanthin by mixing and preserving the yield of the nitenpyram as claimed in claim 1, which is characterized by comprising the following steps: the light source of the illumination condition is an LED lamp or fluorescent lamp light.
4. The method for improving the yield of fucoxanthin by mixing and preserving the yield of the nitenpyram as claimed in claim 1, which is characterized by comprising the following steps: the pH of the medium was 8.5.
5. The method for improving the yield of fucoxanthin by mixing and preserving the yield of the nitenpyram as claimed in claim 1, which is characterized by comprising the following steps: the specific process of the third step is as follows:
centrifuging the culture solution, washing, re-centrifuging, repeating for 2 times, filtering the algae solution onto pre-weighed filter paper, putting into a vacuum drying oven at 80 ℃ for drying to constant weight, freezing, weighing the freeze-dried algae powder, grinding at low temperature, adding absolute ethyl alcohol, vibrating and extracting, centrifuging to collect supernatant, adding absolute ethyl alcohol again in precipitation for vibrating and extracting until the algae powder is white, collecting extracting solution, centrifuging the extracting solution, drying the supernatant with nitrogen, adding absolute ethyl alcohol for dissolving pigment, and analyzing by a high performance liquid chromatograph after membrane filtration, wherein the whole process is performed under a dark condition.
6. The method for improving the yield of fucoxanthin by mixed health maintenance of the small crescent diamond algae according to claim 5, which is characterized in that: the high performance liquid chromatograph is waters2695, a PDA detector is configured, the detection wavelength is 450nm, a C18 reverse phase column is selected, and the mobile phase is: phase A is pure ethyl acetate, phase B is acetonitrile: methanol: water=84:2:14, pure methanol for phase c, gradient elution, HPLC grade for mobile phase.
7. The method for improving the yield of fucoxanthin by mixing and preserving the yield of the nitenpyram as claimed in claim 1, which is characterized by comprising the following steps: in the first step, the activated Nicotiana microcephala is cultured for 6-12 days.
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