CN107119099A - The method for producing fucoxanthin using the smooth rhombus algae of illumination cultivation - Google Patents

Abstract

The invention belongs to the cultivation of microorganisms, in particular to a method for producing fucoxanthin by cultivating Nitzkiri algae with light. Including the steps of preparation of seed liquid, fermentation culture, and extraction of fucoxanthin from the fermentation liquid after fermentation, the present invention solves the problems in the prior art, such as the biomass concentration and fucoxanthin concentration of Nitzschia smoothis under phototrophic conditions. Yield, low content of fucoxanthin and other issues. The content of fucoxanthin in the prepared dry algae powder of Nishida smoothis is high, the yield of fucoxanthin is high, and it is safer than the fucoxanthin derived from large seaweeds. It improves production efficiency and reduces production costs at the same time , greatly reducing the risk of contamination during the cultivation process and other advantages.

Description

Method for producing fucoxanthin by cultivating Nitzpaschlae smoothii under light

technical field

The invention belongs to the cultivation of microorganisms, in particular to a method for producing fucoxanthin by cultivating Nitzkiri algae with light.

Background technique

Fucoxanthin (Fucoxanthin), also known as fucoxanthin and fucoidin, mainly comes from large algae and microalgae such as algae, diatoms, and golden algae. It is a natural carotenoid that participates in the photosystem of photosynthesis II's response. Because of its unique allene structure, fucoxanthin is an active molecule with strong antioxidant properties. In recent years, studies have found that fucoxanthin has a variety of functional activities in cells, animals and humans, including anti-oxidation, anti-inflammation, anti-cancer, anti-obesity, anti-diabetes, anti-angiogenesis and anti-malarial and other physiological activities, and It has a protective effect on organs such as liver, brain blood vessels, bones, skin and eyes. Based on the above functions, fucoxanthin is a natural product with broad prospects for health food and drug development. The market capacity of fucoxanthin is 500 tons, and the price of fucoxanthin extract with 10% content is as high as 40,000 yuan/kg , so it has a huge market value.

Fucoxanthin's significant role in weight loss has attracted more and more attention. It can achieve the effect of weight loss by inhibiting the formation of fat cells and accelerating fat catabolism. Fucoxanthin currently on the market is mainly extracted from large seaweeds such as Undaria pinnatifida and Laminaria japonica. However, the above-mentioned method has problems such as large seaweed having a thick cell wall, high polysaccharide content, difficult purification and marine pollution, and the content of fucoxanthin in the large seaweed is extremely low (only 0.01%-0.07% of the dry weight), To sum up, the product quality of fucoxanthin is difficult to guarantee to a certain extent, and the difficulty of downstream separation and purification is increased, because it has higher technical requirements for extraction, separation and purification, resulting in expensive high-purity fucoxanthin. Further limit the application of fucoxanthin.

Marine microalgae are a better alternative source of fucoxanthin than macroalgae. At present, the content of concentrated fucoxanthin extracted from macroalgae on the market is generally less than 1%, while the content of fucoxanthin in some marine microalgae cells is as high as 0.6%, which is nearly 100 times that of macroalgae. Diatoms are widely distributed marine microalgae that exist widely in oceans, fresh water and wet surfaces, and generally rely on light energy to synthesize the nutrients they need. Nitzchia laevis is a unicellular alga belonging to the division Bacillariophyta.

There are few studies on the production of fucoxanthin by using Nitzschia smoothis. The applicant found through searching that the patent literature with application number 201310329269.X discloses a cultivation method for increasing the yield of fucoxanthin in diatoms. The patent has optimized the medium and added tomato extract to a certain extent Fucoxanthin was synthesized, and light treatment was used in the later stage to finally make Cyclotella reach a fucoxanthin yield of 7.77mg/(L d). The literature only discloses the method, steps and process conditions for preparing fucoxanthin by Cyclotella. It has not been announced that other strains can be used for the production of fucoxanthin, and there are no related culture conditions, light intensity, medium composition, biomass concentration and other culture results, as well as fucoxanthin content and yield. Series key technical information. Guo and the designer in this application obtained several strains of diatoms capable of synthesizing fucoxanthin through screening, and found that the fucoxanthin content of Cyclotella cryptica CCMP 333 can reach 0.77%, which is the highest among more than ten strains of microalgae. It is suitable for fucoxanthin producing algal strains, but its biomass concentration and fucoxanthin yield are low, and industrial application is still difficult. Under the culture conditions used, Nitzschia laevis UTEX 2047 produces fucoxanthin under photoautotrophic conditions, and its content (accounting for cell dry weight) is as high as 0.7%, but the biomass concentration is <0.2g/L, and the yield is only It was 0.06mg/(L·d), but the culture conditions were not optimized in this paper (Guo, et al., 2016). Therefore, it is necessary to improve the biomass concentration and fucoxanthin yield of Nitzschia platypus under phototrophic conditions by optimizing medium composition and culture conditions and other technical means, and further increase the fucoxanthin content at the same time.

Contents of the invention

The purpose of the present invention is to provide a method for producing fucoxanthin by cultivating Nitzbra smoothis under light, which can realize high-yield preparation of fucoxanthin by optimizing the culture conditions.

Overall technical idea of the present invention is:

1. The method for producing fucoxanthin by cultivating Nitzbra smoothis by light, is characterized in that it comprises the following steps:

A. Preparation of seed solution

Put the activated Nitzschia smooth in a sterile seed medium for heterotrophic culture for 3-9 days to make a seed solution, so that the cells of Nitzkiplank are in the logarithmic growth phase;

B. Fermentation culture

The seed solution in step A is transferred into the sterile fermentation medium according to the inoculation amount of 3%-20% by volume, and the fermentation solution is prepared by aeration under light conditions, and the medium loading is 20%-80%, The culture temperature is 20°C-30°C, and the culture period is 4-12 days;

Described seed culture medium and fermentation medium comprise the raw material of following content scope:

NaCl 10g/L-32g/L; Na ₂ SiO ₃ 9H ₂ O 30mg/L-700mg/L; MgSO ₄ 7H ₂ O 1.09g/L-2.18g/L; CaCl ₂ 2H ₂ O 0.1g/L L-0.27g/L; KH ₂ PO ₄ 0.031g/L-0.062g/L; K ₂ HPO ₄ 0.00375g/L-0.0075g/L; FeCl ₃ 6H ₂ O 0.291mg/L-0.582mg/L ; MnCl ₂ ·4H ₂ O 0.025mg/L-0.246mg/L; ZnCl ₂ 0.031mg/L-0.311mg/L; CoCl ₂ ·6H ₂ O 0.0114mg/L-0.0228mg/L; Na ₂ MoO ₄ · 2H ₂ O 0.012mg/L-0.024mg/L; H ₃ BO ₃ 3.06g/L-30.56g/L; (NH ₄ ) ₆ MO ₇ O ₂₄ 4H ₂ O 0.028mg/L-0.278mg/L; Tris-buffer 0.089g/L-0.892g/L; H ₂ SO ₄ 1.64μg/L-16.4μg/L; vitamin B ₁₂ 1.5g/L-15×10 ^-5 g/L; biotin 2.5g/L- 25×10 ^-5 g/L; nitrogen source 0.2g/L-7g/L; pH=6-9, the nitrogen source is organic nitrogen source, inorganic nitrogen source or a combination thereof, wherein the organic nitrogen source includes but not limited to : Yeast extract, peptone, yeast extract, amino acid, urea, protein hydrolyzate or a combination thereof, inorganic nitrogen sources include but not limited to potassium nitrate, sodium nitrate, ammonium chloride, ammonium bicarbonate or a combination thereof;

The light intensity is no more than 200μmol·m ^-2 ·s ^-1 ;

The smooth Nitzschia laevis is selected from Nitzschia laevis UTEX 2047 (purchased from the Microalgae Collection of the University of Texas at Austin, Culture Collection of Algae at The University of Texas at Austin, referred to as UTEX), smooth Nitzschia laevis Algae (Nitzschia laevis) CCMP559 (purchased from the National Center for Marine Algae and Microbiota, NCMA for short), or Nitzschia laevis CCMP 1092 (purchased from the American Marine Microalgae and Microbiota Collection Center, National Center for Marine Algae and Microbiota, referred to as NCMA), wherein Nitzschia laevis (Nitzschia laevis) UTEX 2047 is preferred.

C. extracting fucoxanthin from the fermented liquid after step B fermentation is finished.

Since the isolation of Nitzbrae cells from the fermentation broth and the extraction of fucoxanthin from Nitzbrae cells belong to the prior art, the applicant will not repeat them here.

The concrete technical scheme of each step among the present invention is as follows:

In order to facilitate the industrial fermentation process, the medium is placed in a bioreactor, and the bioreactor is a triangular flask or a column photobioreactor.

The light source for illumination can adopt various forms without departing from the essence of the present invention. The preferred technical solution is that the light source used for illumination includes but is not limited to sunlight, fluorescent light, LED light or a combination thereof.

The culture conditions for light culture are: when using a triangular flask, place it in a light shaker for shaking culture, and the shaker speed is 100 rpm-240 rpm; when using a column photobioreactor, the volume content of carbon dioxide is low Incubate in 5% sterile air with an aeration rate of 3 L/min.

In order to increase the yield and content of fucoxanthin, the preferred culture bar is that the nitrogen source in the fermentation medium is selected from the following raw materials: NaNO ₃ 1g/L; peptone 1g/L, and the light intensity is 30μmol·m ^-2 · s ^-1 .

A more preferred culture condition is that the seed culture medium and fermentation medium also include raw materials in the following content range: glucose 5-40g/L.

A further preferred technical solution is to select a column-type photobioreactor for aerated culture under the condition of light intensity of 5-200 μmol·m ^-2 ·s ^-1 .

The detection method involved in the present invention is as follows:

1. Determination of cell dry weight of Nitzschia smoothis

After inoculation, take 3 mL of fermentation broth every 24 hours, centrifuge at a speed of 3000 rpm for 5 minutes, wash with ddH ₂ O and centrifuge again, repeat twice; filter the fermentation broth onto pre-weighed filter paper, put Dry in a vacuum oven at 80°C until constant weight.

2. Detection of fucoxanthin

At present, the applicant has not found a national or enterprise standard for the detection of fucoxanthin. It is mainly detected by ultraviolet-visible absorption method (UV method) and high-performance liquid chromatography (HPLC). Due to the poor specificity of the UV method, it is easily interfered by other pigments. Therefore, HPLC method is currently a better detection method for fucoxanthin. This application refers to the research of Guo et al., and improves on the basis of it, as follows:

Weigh 20 mg of lyophilized algae powder, grind at low temperature, add 5 mL of absolute ethanol to shake and extract for 10 minutes, centrifuge (conditions: temperature 4°C, speed 3000 rpm, time 5 minutes) to collect the supernatant, add 3 mL to the precipitate Extract by shaking with absolute ethanol until the algae powder turns white. Collect the extract, centrifuge at 4°C and 12,000 rpm for 10 minutes, take the supernatant and dry it with nitrogen, then add 1 mL of absolute ethanol to dissolve the pigment, and pass through the membrane for high-performance liquid chromatography (HPLC) analysis , the whole process was carried out under dark conditions.

3. HPLC analysis method

The high-performance liquid chromatograph waters2695 is configured with a PDA detector, the detection wavelength is 450nm, and a C18 reversed-phase column (250mm×4.6mm×5mm) is selected. The mobile phase is: phase A is pure ethyl acetate, phase B is acetonitrile:methanol:water=84:2:14, phase C is pure methanol, gradient elution is adopted, and the mobile phases are HPLC grade.

The gradient elution conditions are as follows:

time (min) A(%) B(%) C(%) Flow rate (mL/min) 0 0 100 0 0.8 15 32 0 68 0.8 30 32 0 68 0.8 35 0 100 0 0.8

The substantive features possessed by the present invention and the remarkable technical progress obtained are:

1. For the first time, the present invention proposes to utilize Nitzporella smoothis to produce fucoxanthin through a large-scale fermentation method. It has been confirmed by the applicant’s experiment that the content of fucoxanthin in the dried Nitzkidula smoothis prepared is high (at least up to 0.7- 1.0%, up to 1.38%, 97.1% higher than the prior art), the yield of fucoxanthin is high (at least up to 1.02mg/(L d), up to 9.88mg/(L d) , 163.7 times higher than the prior art).

2. The method provided by the present invention has the potential to be applied to industrial production of fucoxanthin. One is that the fucoxanthin content of Nitzschia smoothis is greatly increased after optimizing the culture conditions; the other is that the yield of fucoxanthin is much higher than that of all diatoms and other algae reported so far.

3. The smooth diamond-shaped algal powder obtained by the present invention can stably realize continuous industrial production without being restricted by external conditions, and can control common marine pollutants such as heavy metals and polychlorinated biphenyls from the source of the culture medium. Seaweed-derived fucoxanthin is safer.

4. Compared with other microalgae cultures to produce fucoxanthin, the production cycle is greatly shortened, and the fermentation can be completed in as little as 4 days. While improving production efficiency and reducing production costs, the risk of pollution during the cultivation process is greatly reduced.

Description of drawings

Fig. 1 is the growth curve of Nitzschia plaza under the initial concentration conditions of autotrophic (phototrophic without sugar) and polyculture (adding 5 g/L glucose in the medium under light conditions).

It can be seen from the figure that the biomass concentration of autotroph is extremely low, and the biomass is only 0.5g/L after 6 days, while the biomass under polyculture conditions is more than 5 times that of autotroph.

Fig. 2 is the comparison of the fucoxanthin content (i.e. the mass ratio of fucoxanthin/freeze-dried algal powder) and the yield of Nitzschia smoothis in autotrophic (phototrophic sugar-free) and initial concentration of 5g/L glucose .

It can be seen from the figure that A stands for phototrophic sugar-free, and M stands for phototrophic 5g/L glucose: polyculture not only significantly increases the biomass, but also significantly increases the content of fucoxanthin at the same time, up to 1.2%, while autotrophy is only 1.0%; the yield of fucoxanthin is also higher than the autotrophic condition (1.02mg/(L·d) for autotrophy), up to 4.59mg/(L·d).

Fig. 3 is the growth curve of Nitzschia plaza under different light intensities.

It can be seen from the figure that under polyculture conditions, low light intensity is more beneficial to the biomass of Nitzschia smoothis, and the biomass concentration is the highest when the light intensity is 5 μmol m ^-2 s ^-1 , and the biomass concentration decreases significantly with the increase of light intensity , when the light intensity is 70μmol·m ^-2 ·s ^-1 , the biomass is about 0.7g/L.

Fig. 4 is the effect of different light intensities on the content and yield of fucoxanthin in Nitzschia smoothis cells.

It can be seen from the figure that low light intensity is not only conducive to the growth of Nitzschia smoothis, but also to the accumulation of fucoxanthin. When the light intensity is 5μmol·m ^-2 ·s ^-11 , the content and yield of fucoxanthin are the highest, the highest concentration and yield are respectively 1.38% and 9.88mg/(L·d), compared with the prior art respectively The increase is 97.1% and 163.7 times, and it is also the maximum yield of fucoxanthin produced by the current microalgae culture known to the applicant.

detailed description

The embodiments of the present invention will be further described below in conjunction with the accompanying drawings, but this is not a limitation of the present invention. The scope of protection of the present invention is subject to the content recorded in the claims, and any equivalent technical means replacement made according to the specification will not depart from protection scope of the present invention.

Example 1

Autotrophic culture of Nitzschia smoothis

Nitzschia laevis UTEX 2047 (purchased from the Microalgae Collection of the University of Texas at Austin, Culture Collection of Algae at The University of Texas Austin, UTEX for short) was selected as the production strain.

The process steps are as follows:

A, inoculating the Nitzschia smoothis that produces strain after the activation is placed in the aseptic seed culture medium that is placed in the shake flask and cultured heterotrophically for 3 days to make a seed liquid, and the Nitzkiplano cells in the seed liquid are in the logarithmic growth phase;

B. Using a 250mL Erlenmeyer flask as a culture vessel, transfer to 100mL of fermentation medium and sterilize it. Put the seed solution in step A into a sterile fermentation medium with an inoculum size of 10% by volume to carry out autotrophic culture on a shaking table to prepare a fermentation liquid. The culture conditions are as follows: the light intensity is 30 μmol m ^-2 s ^-1 , the temperature is 23°C, the rotational speed is 150 rpm, and the culture period is 5 days.

C, extract fucoxanthin from the fermented liquid after step B ferments:

After the cultivation, the fermentation broth was collected, washed by centrifugation, and freeze-dried.

The seed medium includes raw materials in the following amounts:

NaCl 10g/L; Na ₂ SiO ₃ 9H ₂ O 60mg/L; MgSO ₄ 7H ₂ O 2.18g/L; CaCl ₂ 2H ₂ O 0.27g/L; KH ₂ PO ₄ 0.062g/L; K ₂ HPO ₄ 0.0075g/L; FeCl ₃ 6H ₂ O 0.582mg/L; MnCl ₂ 4H ₂ O 0.246mg/L; ZnCl ₂ 0.311mg/L; CoCl ₂ 6H ₂ O 0.0228mg/L; Na ₂ MoO ₄ 2H ₂ O 0.024mg/L; H ₃ BO ₃ 30.56g/L; (NH ₄ ) ₆ MO ₇ O ₂₄ 4H ₂ O 0.278mg/L; Tris-buffer 0.892g/L; H ₂ SO ₄ 16.4 μg/L; vitamin B ₁₂ 15×10 ^-5 g/L; biotin 25×10 ^-5 g/L.

On the basis of the seed medium, the fermentation medium also contains the following raw materials: NaNO ₃ 1g/L, peptone 1g/L, pH=8.5.

Example 2

Polyculture culture of Nitzschia smoothis

The difference between Example 2 and Example 1 is that the seed medium and fermentation medium in Example 2 also contain the following raw materials: 5 g/L of glucose, and the rest of the content is the same as that of Example 1.

The effect analysis of embodiment 1,2:

Compared under autotrophic and polyculture conditions, the growth state of polyculture culture is better, and the biomass of polyculture is up to 2.68g/L, while that of autotroph is only 0.70g/L (see Figure 1). As shown in Figure 2, the autotrophic fucoxanthin content is 1.0%, and the polyculture is up to 1.2%. It was calculated that the yield of fucoxanthin under polyculture conditions was higher than that under autotrophy, and the optimal yield was 4.59 mg/(L·d).

Example 3

Polyculture culture of Nitzschia smoothis

The difference between Example 3 and Example 2 is that the seed medium and the fermentation medium also contain the following raw materials: glucose 20g/L, and the production strain is Nitzschia laevis (Nitzschia laevis) CCMP 559 (purchased from U.S. Ocean Micro Algae and Microbiota Collection Center, National Center for Marine Algae and Microbiota, NCMA for short), and all the other contents are the same as in Example 2.

The effect analysis of embodiment 3:

The growth state of the production strain in this embodiment is better, and the biomass is up to 2.88g/L. The content of autotrophic fucoxanthin is 0.9%. It is calculated that the yield of fucoxanthin produced by fermentation of the production strains in this example is higher than that of autotrophy, and the optimal yield is 4.32 mg/(L·d).

Example 4

The difference between Example 4 and Example 2 is that the bioreactor is a 250mL column photobioreactor, and at the same time, in step B, it is supplemented with light with a light intensity of 5 μmol m ^-2 s ^-1 and an air flow of 3 L/min , the rest of the content is the same as in Example 2.

Example 5-8

The difference between Examples 5-8 and Example 4 is that the light intensity in Step B of Example 5-8 is 15, 30, 50 and 70 μmol·m ^-2 ·s ^-1 respectively, and the culture period in Step B is 4 Day; All the other contents are the same as in Example 4.

The effect analysis of embodiment 4-8:

Referring to Fig. 3 and Fig. 4, light treatment was carried out to assist culture, and the light intensity was 5-200 μmol·m ^-2 ·s ^-1 . Light treatment can further increase the content of fucoxanthin, so that the further increase of fucoxanthin, when the light intensity is 5μmol·m ^-2 ·s ^-1 , the content of fucoxanthin is up to 1.38%. The highest yield can reach 9.88mg/(L·d), and the highest yield is 863.9% higher than that before optimization.

Example 9

The difference between embodiment 9 and embodiment 4 is: the production strain in embodiment 9 selects Nitzschialaevis (Nitzschialaevis) CCMP1092 (purchased from the American Marine Microalgae and Microbial Collection Center, National Center for Marine Algae and Microbiota, referred to as NCMA), the steps In B, the aeration rate is 1 liter/minute, and the seed liquid is transferred into the aseptic fermentation medium according to the inoculation amount of 3% according to the volume ratio, and the fermentation liquid is prepared by aeration under light conditions, and the medium loading is 80%. The temperature is 25°C, and the culture period is 12 days; the seed medium and fermentation medium include raw materials in the following content ranges: NaCl 32g/L; Na ₂ SiO ₃ 9H ₂ O 410mg/L; MgSO ₄ 7H ₂ O 2.18 g/L; CaCl ₂ 2H ₂ O 0.1g/L; KH ₂ PO ₄ 0.031g/L; K ₂ HPO ₄ 0.0055g/L; FeCl ₃ 6H ₂ O 0.291mg/L; MnCl ₂ 4H ₂ O 0.025mg/L; ZnCl ₂ 0.031mg/L; CoCl ₂ 6H ₂ O 0.0180mg/L; Na ₂ MoO ₄ 2H ₂ O 0.012mg/L; H ₃ BO ₃ 16.1g/L; (NH ₄ ) ₆ MO ₇ O ₂₄ ·4H ₂ O 0.278mg/L; Tris-buffer 0.089g/L; H ₂ SO ₄ 9.4μg/L; vitamin B ₁₂ 15×10 ^-5 g/L; biotin 0.21g/L; nitrogen source 0.2g/L; pH=6, the nitrogen source is composed of the following raw materials in parts by mass: yeast extract: ammonium bicarbonate: peptone: urea=1:2:1:2; the light intensity is 200 μmol·m ^-2 ·s ^-1 , the light source is LED. All the other contents are the same as in Example 4.

Embodiment 9 effect analysis:

The content of fucoxanthin is 1.21%, and the yield of fucoxanthin is 9.1 mg/(L·d).

Example 10

The difference between embodiment 10 and embodiment 4 is: the production strain in embodiment 9 selects Nitzschialaevis (Nitzschialaevis) CCMP1092 (purchased from the United States Marine Microalgae and Microbial Collection Center, National Center for Marine Algae and Microbiota, referred to as NCMA), the seeds The glucose concentration in the culture medium and the fermentation medium is 40g/L, the seed liquid is transferred into the sterile fermentation medium according to the inoculation amount of 20% by volume, and the fermentation liquid is prepared by aeration culture under light conditions, and the aeration rate is 3L/min (wherein air: the ratio of carbon dioxide is 95:5), the medium load is 20%, the culture temperature is 30°C, and the culture period is 4 days; the described seed medium and fermentation medium include raw materials in the following content range: NaCl 10g/ L; Na ₂ SiO ₃ 9H ₂ O 30mg/L; MgSO ₄ 7H ₂ O 1.09g/L; CaCl ₂ 2H ₂ O 0.16g/L; KH ₂ PO ₄ 0.062g/L; K ₂ HPO ₄ 0.00375 g/L; FeCl ₃ 6H ₂ O 0.582mg/L; MnCl ₂ 4H ₂ O 0.112mg/L; ZnCl ₂ 0.031mg/L; CoCl ₂ 6H ₂ O 0.0228mg/L; Na ₂ MoO ₄ 2H ₂ O 0.024mg/L; H ₃ BO ₃ 3.06g/L; (NH ₄ ) ₆ MO ₇ O ₂₄ ·4H ₂ O 0.140mg/L; Tris-buffer 0.892g/L; H ₂ SO ₄ 16.4μg/L ;vitamin B ₁₂ 1.5g/L; biotin 25×10 ^-5 g/L; nitrogen source 0.2g/L-7g/L; pH=6-9, the nitrogen source is composed of the following raw materials in parts by mass: peptone : yeast extract: amino acid: potassium nitrate = 1:1:1:2; the light intensity is no more than 5 μmol·m ^-2 ·s ^-1 , and the light source is sunlight adjusted for shading.

Embodiment 10 effect analysis:

The content of fucoxanthin is 1.11%, and the yield of fucoxanthin is 7.8 mg/(L·d).

Example 11

The difference between Example 11 and Example 10 is that the concentration of glucose in the seed culture medium and the fermentation medium is 20g/L, and the seed liquid is transferred into the sterile fermentation medium according to the volume ratio of 14% inoculum. Under aerated culture, the fermented liquid was prepared, the aeration rate was 3L/min (wherein the ratio of air: carbon dioxide was 98:2), the medium load was 40%, the culture temperature was 20°C, and the culture period was 7 days; the seed culture medium and fermentation The medium includes raw materials in the following content ranges: NaCl 22g/L; Na ₂ SiO ₃ 9H ₂ O 700mg/L; MgSO ₄ 7H ₂ O 1.66g/L; CaCl ₂ 2H ₂ O 0.27g/L; KH ₂ PO ₄ 0.045g/L; K ₂ HPO ₄ 0.0075g/L; FeCl ₃ 6H ₂ O 0.431mg/L; MnCl ₂ 4H ₂ O 0.246mg/L; ZnCl ₂ 0.151mg/L; CoCl ₂ 6H ₂ O 0.0114mg/L; Na ₂ MoO ₄ 2H ₂ O 0.018mg/L; H ₃ BO ₃ 30.56g/L; (NH ₄ ) ₆ MO ₇ O ₂₄ 4H ₂ O 0.028mg/L; Tris-buffer 0.451 g/L; H ₂ SO ₄ 1.64μg/L; vitamin B ₁₂ 0.1g/L; biotin 2.5g/L; nitrogen source 0.2g/L-7g/L; pH=6-9, the nitrogen source is selected as follows Raw material composition in parts by mass: amino acid: protein hydrolyzate: sodium nitrate: ammonium chloride = 2:2:1:2; the light intensity is no more than 100 μmol·m ^-2 ·s ^-1 , and the light source is a fluorescent lamp.

Embodiment 11 effect analysis:

The content of fucoxanthin is 1.11%, and the yield of fucoxanthin is 7.8 mg/(L·d).

Claims (7)

1. utilize light to cultivate the method for smooth nitzia to produce fucoxanthin, it is characterized in that comprising the steps: A. Preparation of seed solution Put the activated Nitzschia smooth in a sterile seed medium for heterotrophic culture for 3-9 days to make a seed solution, so that the cells of Nitzkiplank are in the logarithmic growth phase; B. Fermentation culture The seed solution in step A is transferred into the sterile fermentation medium according to the inoculation amount of 3%-20% by volume, and the fermentation solution is prepared by aeration under light conditions, and the medium loading is 20%-80%, The culture temperature is 20°C-30°C, and the culture period is 4-12 days; Described seed culture medium and fermentation medium comprise the raw material of following content scope: NaCl 10g/L-32g/L; Na ₂ SiO ₃ 9H ₂ O 30mg/L-700mg/L; MgSO ₄ 7H ₂ O 1.09g/L-2.18g/L; CaCl ₂ 2H ₂ O 0.1g/L L-0.27g/L; KH ₂ PO ₄ 0.031g/L-0.062g/L; K ₂ HPO ₄ 0.00375g/L-0.0075g/L; FeCl ₃ 6H ₂ O 0.291mg/L-0.582mg/L ; MnCl ₂ ·4H ₂ O 0.025mg/L-0.246mg/L; ZnCl ₂ 0.031mg/L-0.311mg/L; CoCl ₂ ·6H ₂ O 0.0114mg/L-0.0228mg/L; Na ₂ MoO ₄ · 2H ₂ O 0.012mg/L-0.024mg/L; H ₃ BO ₃ 3.06g/L-30.56g/L; (NH ₄ ) ₆ MO ₇ O ₂₄ 4H ₂ O 0.028mg/L-0.278mg/L; Tris-buffer 0.089g/L-0.892g/L; H ₂ SO ₄ 1.64μg/L-16.4μg/L; vitamin B ₁₂ 1.5g/L-15×10 ^-5 g/L; biotin 2.5g/L- 25×10 ^-5 g/L; nitrogen source 0.2g/L-7g/L; pH=6-9, the nitrogen source is organic nitrogen source, inorganic nitrogen source or a combination thereof, wherein the organic nitrogen source includes but not limited to : Yeast extract, peptone, yeast extract, amino acid, urea, protein hydrolyzate or a combination thereof, inorganic nitrogen sources include but not limited to potassium nitrate, sodium nitrate, ammonium chloride, ammonium bicarbonate or a combination thereof; The light intensity is no more than 200μmol·m ^-2 ·s ^-1 ; The smooth Nitzschia laevis is selected from Nitzschia laevis UTEX 2047, Nitzschia laevis CCMP559, or Nitzschia laevis CCMP 1092; C. extracting fucoxanthin from the fermented liquid after step B fermentation is finished. 2. the method for cultivating Nitzbra smoothis by light according to claim 1 to produce fucoxanthin is characterized in that the fermentation medium in the described step B is placed in the bioreactor, and the bioreactor selects a triangular flask or Column photobioreactor. 3. The method according to claim 1, wherein the method for producing fucoxanthin by cultivating Nitzbra smoothis by light is characterized in that the light source used for light includes but not limited to sunlight, fluorescent light, LED light or a combination thereof. 4. the method for producing fucoxanthin by cultivating Nitzbra smoothis according to claim 2 is characterized in that it is placed in a light shaker for shaking culture when using a triangular flask, and the shaker speed is 100 revs/min- 240 revolutions/minute; When using a column photobioreactor, sterile air is introduced into the culture, and the ventilation volume is 1-3 liters/minute. 5. the method for producing fucoxanthin according to claim 1, characterized in that the nitrogen source in the seed culture medium and the fermentation medium is selected from the raw materials of the following content: NaNO ₃ 1g/ L; peptone 1g/L, and the light intensity in step B is 30 μmol·m ^-2 ·s ^-1 . 6. The method for producing fucoxanthin by cultivating Nitzschia smoothis according to claim 1, characterized in that said seed culture medium and fermentation medium also include raw materials in the following content range: glucose 5-40g/ L. 7. The method for producing fucoxanthin by cultivating Nitzschia smoothis according to claim 6, characterized in that the fermentation medium in step B is selected from a column photobioreactor, and the light intensity is 5-200 μmol m ^-2 · s ^-1 under the condition of aeration culture.