CN107099564A - The method for producing fucoxanthin using the smooth rhombus algae of Heterotrophic culture - Google Patents

Abstract

The invention belongs to the cultivation of microorganisms, and in particular refers to a method for producing fucoxanthin by using heterotrophic cultivation of Nishina smoothis. Including the preparation of seed liquid, fermentation culture, extraction of fucoxanthin from the fermentation liquid after fermentation, etc., the present invention solves the problems of biomass concentration and fucoxanthin of Nitzkirella smoothis under heterotrophic conditions existing in the prior art. Problems such as low yield and fucoxanthin content. 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 using heterotrophic culture of Nitzschia smoothis

technical field

The invention belongs to the cultivation of microorganisms, and in particular refers to a method for producing fucoxanthin by using heterotrophic cultivation of Nishina smoothis.

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.

Studies have found that diatoms such as Navicula, Cyclotella, and Cylindrica can grow in heterotrophic culture. The designer in this application found that the biomass concentration is extremely low, and it is difficult to apply to high-density heterotrophic fermentation (Guo, et al., 2016). Nitzchia laevis is a single-celled alga belonging to the division Bacillariophyta, class Pennaeae, order Surirellales, genus Nitzschia, and has heterotrophic fermentation cultivated potential. There are few studies on the production of fucoxanthin by using Nitzschia smoothis. After searching, the applicant found that the patent literature with application number 201310329269.X disclosed a cultivation method for increasing the yield of fucoxanthin in diatoms. The patent optimized the medium and added tomato extract to promote the synthesis to a certain extent. Fucoxanthin was obtained, and light treatment was used in the later stage to finally make Cyclotella reach a fucoxanthin yield of 7.77mg/(L d). It was proposed that Nitzschia smoothis can be used for light culture to produce fucoxanthin, but this document Only the method, steps and process conditions for preparing fucoxanthin by using Cyclotella are disclosed. 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. The designers of the present application used heterotrophic methods to cultivate Nitzschia smoothis, the content of fucoxanthin was only about 0.4%, and the yield of fucoxanthin was only 0.1 mg/(L·d). However, Nitzschia laevis UTEX 2047 produces fucoxanthin under photoautotrophic conditions, and its content (accounting for dry cell weight) is as high as 0.7%, but the biomass concentration is <0.2g/L, and the yield is only 0.06mg /(L·d), and this paper did not optimize the culture conditions (Guo, et al., 2016). Therefore, it is necessary to improve the biomass concentration and fucoxanthin yield of Nitzschia platypus under heterotrophic 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 using heterotrophic culture of Nitzschia plastifolia, which can realize high-yield preparation of fucoxanthin by optimizing the culture conditions.

Overall technical idea of the present invention is:

The method for producing fucoxanthin by using heterotrophic cultivation of Nitzschia smoothis comprises the following steps:

A. Preparation of seed solution

Put the activated Nitzschia smoothii in a sterile seed medium for heterotrophic culture for 3-11 days to make a seed solution, so that the Nitzkiplankton cells are in the logarithmic growth phase;

B. Fermentation culture

The seed liquid in step A is transferred to the sterile fermentation medium according to the inoculum amount of 3%-20% by volume to carry out shaker culture to prepare the fermentation liquid, the culture temperature is 20°C-30°C, and the culture period is 3-11 days ;

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

NaCl 10g/L-32g/L; MgSO ₄ 7H ₂ O 1.09g/L-2.18g/L; CaCl ₂ 2H ₂ O 0.1g/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; organic carbon source 2g/L- 50g/L; Silicate 20mg/L-700mg/L; Nitrogen source 0.4g/L-7g/L; pH=6-9;

The organic carbon source includes but is not limited to: glucose, starch hydrolyzate, fructose syrup, sucrose or a combination thereof; the nitrogen source is selected from an organic nitrogen source, an inorganic nitrogen source or a combination thereof, wherein the organic nitrogen source includes but is not limited to : yeast extract, peptone, yeast extract, amino acid, urea, protein hydrolyzate or a combination thereof, inorganic nitrogen sources include but are not limited to potassium nitrate, sodium nitrate, ammonium chloride, ammonium bicarbonate or a combination thereof; the silicic acid Salts include, but are not limited to, sodium silicate, potassium silicate, ammonium silicate, or combinations thereof.

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 (NCMA for short), Nitzschia laevis (Nitzschia laevis) O-7 (purchased from Milford Laboratory Marine Microalgal Culture Collection), wherein Nitzschia laevis is preferred (Nitzschia laevis) UTEX 2047.

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 and achieve a better fermentation effect, the preferred technical solution is that the rotating speed of the shaking table in the shaking bed cultivation process of step B is 100-240 rpm.

In order to improve the yield and content of fucoxanthin, the preferred culture strip is that the nitrogen source in the seed medium and the fermentation medium is selected from the following content range: peptone 1g/L; the organic carbon source is selected from the following content range: Glucose 5g/L.

Further preferred culture conditions are that the nitrogen source in the seed medium and the fermentation medium is selected from raw materials with the following content range: peptone 1g/L; the organic carbon source content range is as follows: 5g/L<organic carbon source content≤50g/L.

A further preferred technical solution is that the nitrogen source in the seed medium and the fermentation medium is selected from the following content range of raw materials: potassium nitrate or sodium nitrate 0.4g/L-2g/L; the organic carbon source is selected from the following content range of raw materials: glucose 5g/L.

A further preferred technical solution is that the nitrogen source in the seed medium and the fermentation medium is selected from the following content range: sodium nitrate 1g/L; the organic carbon source is selected from the following content range: glucose 5g/L.

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 large-scale fermentation method. It is confirmed by the applicant's experiment that the content of fucoxanthin in the dried Nitzkiplano smoothis powder prepared is high (at least up to 0.38%) , up to 1.15%, 187.5% higher than prior art), the productive rate of fucoxanthin (at least up to 1.1mg/(L d), up to 3.31mg/(L d), compared with The existing technology improves by 32 times).

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 3 days. While improving production efficiency and reducing production costs, it also greatly reduces the risk of pollution during the cultivation process.

Description of drawings

Figure 1 is the growth curve of Nitzschia smoothis under heterotrophic conditions.

As can be seen from the figure, the highest biomass after 5 days of cultivation is 1.1g/L, the concentration of fucoxanthin is 0.5%, and the maximum yield is 1.1mg/(L d), which is smoother than that of heterotrophic culture in the prior art. The highest content of fucoxanthin and the highest yield of fucoxanthin in Nitzschia are increased by 25% and 10 times respectively.

Fig. 2 is the growth curve of Nitzschia plaza under different initial concentrations of glucose.

Among them, H-5, H-10, H-20, and H-40 respectively represent that the initial concentration of glucose added in the culture medium is 5g/L, 10g/L, 20g/L, 40g/L, as can be seen from the figure, when glucose The specific growth rate was the highest when the concentration was 5g/L.

Fig. 3 is a comparison of the content of fucoxanthin (ie, the mass ratio of fucoxanthin/freeze-dried algal powder) and yield in algal powder obtained under different initial concentrations of glucose.

Among them, H-5, H-10, H-20, and H-40 respectively represent that the initial glucose concentration under heterotrophic conditions is 5g/L, 10g/L, 20g/L, and 40g/L. As can be seen from the figure, the glucose concentration is At 10g/L, the fucoxanthin content reaches the highest 0.72%, and the fucoxanthin yield reaches the highest 3.26mg/(L·d), which is the highest fucoxanthin content compared with the heterotrophic cultured Nitzschia smoothis in the prior art and the highest fucoxanthin yield were increased by 80% and 31.6 times.

Fig. 4 is the effect of different types of nitrogen elements on the content and yield of fucoxanthin in Nitzschia smoothis cells.

It can be seen from the figure that when the nitrogen source is nitrate, the content of fucoxanthin is significantly increased, and when the nitrogen source is sodium nitrate, the content of fucoxanthin is as high as 0.91%, which is 127.5% higher than that in the prior art heterotrophically cultured Nishiki. %.

Fig. 5 is a comparison of the content and yield of fucoxanthin in the algae powder obtained under the culture conditions of different initial concentrations of sodium nitrate.

As can be seen from the figure, when the sodium nitrate concentration was 0.25g/L, the fucoxanthin content was lower than 0.2%, and the productive rate was lower than 0.5mg/(L·d), and when the sodium nitrate concentration was increased to 0.5g/L , the yield and yield of fucoxanthin were greatly improved, which were greater than 0.4% and 1.1mg/(L·d) respectively. And when the concentration of sodium nitrate is 1g/L, the content of fucoxanthin is higher (1.06%), which is 165% higher than the highest content of fucoxanthin in the heterotrophically cultured Nishiki in the prior art.

Fig. 6 is a comparison of the content and yield of fucoxanthin in algae flour obtained under the culture conditions of different initial silicate concentrations.

It can be seen from the figure that the silicate concentration has a significant impact on the yield and yield of fucoxanthin. When no sodium silicate was added, the fucoxanthin content was only 0.41%, while the yield was lower than 0.5mg/(L·d), and when the sodium silicate concentration was 30mg/L, the fucoxanthin content and productive rate are significantly improved, and when silicate concentration is 120mg/L, fucoxanthin content is as high as 1.15%, and fucoxanthin productive rate reaches 3.31mg/(L·d), compared with prior art heterotrophic culture The highest fucoxanthin content and the highest fucoxanthin yield of Nitzschia smoothis increased by 187.5% and 32.1 times, respectively.

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

Heterotrophic 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 at Austin, UTEX for short) was selected as the production strain in this example.

A. Preparation of seed solution

Put the activated Nitzschia smoothii in a sterile seed medium for heterotrophic culture for 3-11 days to make a seed solution, so that the Nitzkiplankton cells are in the logarithmic growth phase;

B. Fermentation culture

Transfer the seed liquid in step A into a sterile fermentation medium according to the volume ratio of 10% inoculum to prepare a fermentation liquid on a shaker, and cultivate it under the conditions of a culture temperature of 23°C and a rotation speed of 150 rpm 5 days;

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

NaCl 10g/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 16.4μg/L; vitamin B ₁₂ 15×10 ^-5 g/L; biotin 25×10 ^-5 g/L; organic carbon source 5g/L; nitrogen source 1g/L; silicate 0mg/L-700mg/L; pH=8.5;

The organic carbon source is glucose; the nitrogen source is peptone; the silicate includes but not limited to sodium silicate, potassium silicate, ammonium silicate or a combination thereof.

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

The effect analysis of embodiment 1:

Nitzschia plastifolia (UTEX 2047) grows better under heterotrophic conditions, with a biomass of up to 1.1g/L and a fucoxanthin content of 0.5%. The yield of fucoxanthin was calculated to be 1.1 mg/(L·d) under heterotrophic conditions. This yield is the highest fucoxanthin yield of Nitzschia platypus reported so far.

Embodiment 2: Heterotrophic cultivation of Nitzschia smoothis

With a 250mL Erlenmeyer flask as a culture vessel, 100mL of fermentation medium was transferred, and the inoculation amount of 10% by volume was inserted into the fermentation medium for shaker culture. The culture conditions are as follows: the rotation speed of the shaker is 150 rpm, the temperature is 23° C., and the culture time is 5 days.

The fermentation medium and seed medium also include raw materials with the following content: NaNO ₃ 1g/L, and the rest are the same as in Example 1.

The difference between Example 3-5 and Example 2 is that the initial glucose concentration in Example 3-5 is 10g/L, 20g/L and 40g/L respectively, and the culture time is extended to 7, 9, 11 days respectively.

The effect analysis of embodiment 2-5:

Referring to Fig. 2 and Fig. 3, different initial concentrations of glucose have a great influence on the growth and fucoxanthin content of Nitzschia smoothis. The maximum biomass concentration (5.33g/L) was obtained when the initial glucose concentration was 40g/L, and the highest biomass concentration was 1.84g/L when the glucose concentration was 5g/L. When the glucose concentration is 10g/L, the content of fucoxanthin is the highest (0.72%), and the yield of fucoxanthin at this time is also the highest (3.26mg/(L·d)), however, when the glucose concentration is 5g/L, The growth cycle was only 5 days, and the biotransformation rate of glucose was the highest (1.84g biomass/5g glucose). Therefore, the initial glucose concentration of 5 g/L was selected in the later optimization experiment.

Example 6-11

The difference between Examples 6-11 and Example 1 is that the nitrogen sources in Examples 6-11 are respectively 2g/L NaNO ₃ , KNO ₃ , NH ₄ Cl, urea, peptone and yeast extract. All the other contents are the same as in Example 1.

The effect analysis of embodiment 6-11:

Referring to Fig. 4, only 2 g/L of sodium nitrate is used to detect that the content of fucoxanthin in the freeze-dried algal powder can be as high as 0.91%, and the content of fucoxanthin is greatly increased. Nitrogen sources Only use inorganic nitrogen sources—sodium nitrate or potassium nitrate can get higher content of fucoxanthin, while the content of fucoxanthin obtained when only organic nitrogen sources peptone and yeast extract are used is lower than when only inorganic nitrogen sources are used Fucoxanthin content.

Examples 12-16

The difference between Examples 12-16 and Example 1 is that the nitrogen source in Examples 12-16 is sodium nitrate, and the concentrations are respectively 0.4g/L, 0.5g/L, 1.0g/L, and 1.5g/L and 2g/L. All the other contents are the same as in Example 1.

The effect analysis of embodiment 12-16:

Referring to Fig. 5, when the initial concentration of sodium nitrate is 1.0g/L, the content of fucoxanthin is the highest (1.06%), and the yield of fucoxanthin is 3.18 mg/(L·d).

Examples 17-20

The difference between Example 17-20 and Example 1 is that the nitrogen source in Example 17-20 is 1g/L sodium nitrate, the silicate is sodium silicate, and the concentrations are respectively: 0mg/L, 30mg/L , 60mg/L and 120mg/L.

Embodiment 17-20 effect analysis:

Referring to Fig. 6, the fucoxanthin content was the highest (1.15%) when the initial silicate concentration was 120 mg/L, and the fucoxanthin yield was 3.31 mg/(L·d).

Example 21

The difference between Example 21 and Example 1 is that the production strain is Nitzschia laevis (Nitzschia laevis) CCMP1092 (purchased from the American Marine Microalgae and Microbiota Collection Center, National Center for Marine Algae and Microbiota, referred to as NCMA), and the seed liquid is Transfer an inoculation amount of 3% into a sterile fermentation medium, the loading of the fermentation medium is 20%, the culture temperature is 25°C, and the culture period is 9 days; the seed medium and fermentation medium include the following content ranges Raw materials: sucrose with a concentration of 25g/L as carbon source; NaCl 32g/L; MgSO ₄ 7H ₂ O 2.18g/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; 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 3.5g/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 silicate is potassium silicate, the concentration is 350mg/L. All the other contents are the same as in Example 4.

Embodiment 21 effect analysis:

The content of fucoxanthin is 1.08%, and the yield of fucoxanthin is 3.1 mg/(L·d).

Example 22

The difference between Example 22 and Example 1 is that: Nitzschia laevis (Nitzschia laevis) CCMP559 (purchased from the National Center for Marine Algae and Microbiota, NCMA for short) was selected as the production strain, and the seed medium was mixed with The organic carbon source concentration in the fermentation medium is 2g/L, the organic carbon source is selected from starch hydrolyzate, the seed liquid is transferred into the sterile fermentation medium according to the inoculation amount of 20% by volume, and the fermentation medium load is 80% , the culture temperature is 30°C, and the culture period is 3 days; the seed medium and fermentation medium include raw materials in the following content ranges: NaCl 10g/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 0.00375g/L; FeCl ₃ 6H ₂ O 0.582mg/L; MnCl ₂ 4H ₂ O 0.112mg/L; ZnCl ₂ 0.311mg/L 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 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 silicate is ammonium silicate with a concentration of 700 mg/L. All the other contents are the same as in Example 1.

Embodiment 22 effect analysis:

The content of fucoxanthin is 1.11%, and the yield of fucoxanthin is 3.12 mg/(L·d).

Example 23

The difference between Example 23 and Example 1 is that Nitzschia laevis O-7 (purchased from Milford Laboratory Marine Microalgal Culture Collection), seed medium and fermentation medium were selected. The medium carbon source concentration is 50g/L, the carbon source is a mixture of sucrose and fructose syrup according to the volume of 1:1, and the seed liquid is transferred into the sterile fermentation medium according to the volume ratio of 14% inoculum. The content of the medium is 40%, the culture temperature is 20°C, and the culture period is 6 days; the seed medium and fermentation medium include raw materials in the following content range: NaCl 22g/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 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 0.451g/L; H ₂ SO ₄ 1.64μg/L; vitamin B ₁₂ 0.1g/L; biotin 2.5g/L; nitrogen source 0.4g/L; pH= 6-9, the nitrogen source is composed of the following raw materials in parts by mass: amino acid: protein hydrolyzate: sodium nitrate: ammonium chloride = 2:2:1:2; the silicate is sodium silicate with a concentration of 20 mg/ L.

Embodiment 23 effect analysis:

The content of fucoxanthin is 1.10%, and the yield of fucoxanthin is 3.08 mg/(L·d).

Claims (6)

1. the method for producing fucoxanthin using the smooth rhombus algae of Heterotrophic culture, it is characterised in that comprise the following steps：

A, seed liquor preparation

The smooth rhombus algae activated is placed in Heterotrophic culture in aseptic seed culture medium and seed liquor is made within 3-11 days, makes smooth water chestnut Shape frustule is in exponential phase；

B, fermented and cultured

Seed liquor in step A is transferred according to volume ratio for 3%-20% inoculum concentration and shaken in no bacteria fermentation culture medium Bed culture prepares zymotic fluid, 20 DEG C -30 DEG C of cultivation temperature, cultivation cycle 3-11 days；

Described seed culture medium and fermentation medium include the raw material of following content range：

NaCl 10g/L-32g/L；MgSO₄·7H₂O 1.09g/L-2.18g/L；CaCl₂·2H₂O 0.1g/L-0.27g/L； KH₂PO₄0.031g/L-0.062g/L；K₂HPO₄0.00375g/L-0.0075g/L；FeCl₃·6H₂O 0.291mg/L- 0.582mg/L；MnCl₂·4H₂O 0.025mg/L-0.246mg/L；ZnCl₂0.031mg/L-0.311mg/L；CoCl₂·6H₂O 0.0114mg/L-0.0228mg/L；Na₂MoO₄·2H₂O0.012mg/L-0.024mg/L；H₃BO₃3.06g/L-30.56g/L； (NH₄)₆MO₇O₂₄·4H₂O 0.028mg/L-0.278mg/L；Tris-buffer 0.089g/L-0.892g/L；H₂SO₄ 1.64μ g/L-16.4μg/L；vitamin B₁₂ 1.5g/L-15×10^-5g/L；biot in 2.5g/L-25×10^-5g/L；Organic carbon source 2g/L-50g/L；Nitrogen source 0.4g/L-7g/L；Silicate 20mg/L-700mg/L；PH=6-9；

The organic carbon source includes but is not limited to：Glucose, glucidtemns, fructose syrup, sucrose or its combination；The nitrogen Source is from organic nitrogen source, inorganic nitrogen-sourced or its combination, and wherein organic nitrogen source includes but is not limited to：Yeast extract, peptone, yeast are carried Thing, amino acid, urea, protolysate or its combination are taken, it is inorganic nitrogen-sourced to include but is not limited to from potassium nitrate, sodium nitrate, chlorine Change ammonium, ammonium hydrogen carbonate or its combination；The silicate includes but is not limited to sodium metasilicate, potassium silicate, silicic acid ammonia or its combination；

Described smooth rhombus algae is selected from smooth rhombus algae (Nitzschia laevis) UTEX 2047, smooth rhombus algae (Nitzschia laevis) CCMP 559, smooth rhombus algae (Nitzschia laevis) CCMP 1092, smooth rhombus algae (Nitzschia laevis)O-7；

C, from step B fermentation terminate after zymotic fluid in extract fucoxanthin.

2. the method that the smooth rhombus algae of utilization Heterotrophic culture according to claim 1 produces fucoxanthin, it is characterised in that Shaking speed is 100-240 revs/min during described step B shaking table cultures.

3. the method that the smooth rhombus algae of utilization Heterotrophic culture according to any one of claim 1 or 2 produces fucoxanthin, It is characterized in that nitrogen source selects the raw material of following content range in seed culture medium and fermentation medium：Peptone 1g/L；It is organic Carbon source selects the raw material of following content range：Glucose 5g/L.

4. the method that the smooth rhombus algae of utilization Heterotrophic culture according to any one of claim 1 or 2 produces fucoxanthin, It is characterized in that nitrogen source selects the raw material of following content range in seed culture medium and fermentation medium：Peptone 1g/L；It is organic Carbon source content range is as follows：5g/L ＜ organic carbon sources content≤50g/L.

5. the method that the smooth rhombus algae of utilization Heterotrophic culture according to any one of claim 1 or 2 produces fucoxanthin, It is characterized in that nitrogen source selects the raw material of following content range in seed culture medium and fermentation medium：Potassium nitrate or sodium nitrate 0.4g/L-2g/L；Organic carbon source selects the raw material of following content range：Glucose 5g/L.

6. the method that the smooth rhombus algae of utilization Heterotrophic culture according to any one of claim 1 or 2 produces fucoxanthin, It is characterized in that nitrogen source selects the raw material of following content range in seed culture medium and fermentation medium：Sodium nitrate 1g/L；It is organic Carbon source selects the raw material of following content range：Glucose 5g/L.