Method for improving fucoxanthine yield by adopting nutrition mode switching

Abstract

The invention relates to a high-fucoxanthine algae cell culture method, which realizes rapid accumulation of fucoxanthine by adjusting the nitrogen-carbon ratio and starvation culture transition in the microalgae growth process and the fucoxanthine accumulation process, and finally obtains microalgae cells with high fucoxanthine content.

Description

Technical Field

[0001] The invention relates to a method for increasing the yield of fucoxanthin, specifically to a method for increasing the yield of microalgae fucoxanthin by controlling the nitrogen-carbon ratio. Background Technology

[0002] Fucoxanthin, also known as fucoxanthin, is a carotenoid abundant in nature, especially in marine environments. In cells, fucoxanthin forms a light-harvesting complex with chlorophyll a and chlorophyll c. Some algae, such as dinoflagellates and diatoms, exhibit a characteristic brownish-yellow color due to the presence of fucoxanthin. Therefore, fucoxanthin is of significant research importance.

[0003] Studies have found that fucoxanthin has many significant effects, such as antioxidant, antitumor, anticancer, skin protection, anti-angiogenic, cerebrovascular protection, and osteoporosis prevention. Therefore, fucoxanthin has a wide range of applications. In recent years, in particular, research has found that fucoxanthin has weight-loss, blood sugar-lowering, and lipid-lowering effects, further demonstrating its high application value.

[0004] At present, there are many raw materials that can be used to extract fucoxanthin, such as brown algae and diatoms. The earliest raw materials were mainly large seaweeds, including kelp (Laminaria japonica), Sargassum fusiforme and Undaria pinnatifida

[21] . However, there are many problems with the extraction of fucoxanthin from large seaweeds. On the one hand, the cultivation cost of large seaweeds is high and they have the characteristics of seasonal growth. On the other hand, the fucoxanthin of large seaweeds is only present in the surface cortical cells, so the content is low, and the fucoxanthin content of large seaweeds is different under fresh and dry conditions. These problems limit the development and utilization of fucoxanthin. In addition to large seaweeds, marine microalgae also contain a lot of fucoxanthin. Marine microalgae can be used to produce it, such as: brown finger algae, smooth rhomboid algae, etc.

[0005] However, using microalgae synthesis methods requires further improvement in yield, and the biggest problem at present is:

[0006] 1. There is a lack of effective means to significantly increase the yield of fucoxanthin.

[0007] 2. Existing methods are mostly very simple, such as adding a certain hormone, or using heterotrophic and commensal methods to improve.

[0008] The reasons for improving fucoxanthin were not considered at a deeper level.

[0009] Therefore, there is an urgent need to invent a method for increasing the yield of fucoxanthin in a high-yield and stable manner. Summary of the Invention

[0010] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for increasing the yield of fucoxanthin with high and stable yield.

[0011] The technical problem to be solved by the present invention is achieved through the following technical solution:

[0012] A method for improving the yield of fucoxanthin from microalgae includes the following steps:

[0013] 1. Algal cell proliferation stage;

[0014] 2. Photoautotrophic culture of algal cells;

[0015] 3. Facultative culture of algal cells under high nitrogen conditions;

[0016] Preferably, the operation of steps 1-3 is repeated 1-2 times after step 3.

[0017] Specifically, this invention relates to a method for increasing the yield of fucoxanthin from microalgae, comprising the following steps:

[0018] 1. Algal cell proliferation stage, wherein the proliferation stage is carried out by co-trophic culture, preferably, the culture process adopts a progressively increasing light mode;

[0019] 2. Photoautotrophic culture of algal cells, wherein the photoautotrophic process does not involve changing the culture device or replenishing the culture medium until the initial carbon and nitrogen are depleted, and starvation culture is continued for a period of time;

[0020] 3. Algal cells are cultured under high nitrogen conditions, wherein the high nitrogen conditions are achieved by adding exogenous nitrogen.

[0021] Preferably, the operation of steps 1-3 is repeated 1-2 times after step 3, preferably twice.

[0022] Preferably, the illumination in step 1 uses direct sunlight or LED lights, and the progressive enhancement is up to 30,000 lux.

[0023] Preferably, step 1 involves replenishing glycerol to maintain a glycerol concentration of 0.05 mol / L.

[0024] Preferably, the algal cells are selected from *Phaeodactylum triangularis*, *Rhomboidea smoothii*, and *Rhomboidea microcarpa*.

[0025] Preferably, the starvation culture time in step 2 is 1-5 days, preferably 2 days.

[0026] Preferably, in step 2, the pH is controlled at 6-10, more preferably 8.0, and preferably, CO2 is introduced.

[0027] Preferably, the exogenous nitrogen in step 3 is peptone, and the amount added is 0.2-1.0 g / L, preferably 0.6 g / L.

[0028] Preferably, in step 3, the pH is controlled at 6-10, more preferably 8.0, and preferably, CO2 is introduced.

[0029] Preferably, step 3 lasts for 1-5 days, more preferably 1-3 days.

[0030] Specifically, this invention relates to a method for increasing the yield of fucoxanthin from microalgae, comprising the following steps:

[0031] (1) The stage of dual-trophic proliferation of algal cells;

[0032] The algal cells were inoculated into the initial culture medium at a concentration of 0.1-0.5 g / L, kept at a constant temperature of 30°C, and continuously illuminated with a light intensity of 10,000-20,000 lux (white light or natural light). The aeration rate was 0.1-0.2 VVM. 0.05 mol / L glycerol and 0.85 g / L urea were added.

[0033] Gradually increase the light intensity according to the cell mass, up to a maximum of 30,000 lux, or use direct sunlight. During this period, continue to supplement glycerol according to its consumption, maintaining a concentration of 0.01 mol / L. Intermittently control the pH between 6.0 and 10.0 using CO2. This stage of cultivation lasts 5-10 days.

[0034] The initial culture medium formulation is shown in Table 1.

[0035] Table 1 Initial Culture Medium Formulation Table

[0036] Element Mass (1L) Element Mass (1L) NaCl 20.00g <![CDATA[NaNO3]]> 75.00mg <![CDATA[NaH2PO4·H2O]]> 10.00mg <![CDATA[Na2SiO3·9H2O]]> 30.00mg <![CDATA[FeCl3·6H2O]]> 3.15mg <![CDATA[Na2EDTA·2H2O]]> 4.36mg <![CDATA[CuSO4·5H2O]]> 9.80ug <![CDATA[Na2MoO4·2H2O]]> 6.30ug <![CDATA[ZnSO4·7H2O]]> 22.00ug <![CDATA[CoCl2·6H2O]]> 10.00ug <![CDATA[MnCl2·4H2O]]> 180.00ug Thiamine HCl (VB1) 200.00ug Biotin (VH) 1.00ug Cyanocobalamin (VB12) 1.00ug

[0037] (2) Photoautotrophic stage with organic carbon deficiency and nitrogen starvation

[0038] No need to change the culture apparatus. Stop adding glycerol; at this point, the initially added nitrogen will be almost completely depleted. Keep other conditions unchanged and continue photoautotrophic culture under carbon and nitrogen starvation for 1-5 days. During this period, intermittently control the pH at 6.0-10.0 using CO2.

[0039] (3) Dutrophic stage with high nitrogen-to-carbon ratio

[0040] Add 0.2-1.0 g / L of peptone (which serves not only as a nitrogen source but also as an organic carbon source, and has a high nitrogen-to-carbon ratio). Keep other conditions constant, and intermittently control the pH between 6.0 and 10.0 using CO2. Incubate for 1-5 days for large-scale synthesis of fucoxanthin.

[0041] Steps 1-3 can be repeated 1-2 times.

[0042] The advantages of this invention are:

[0043] (1) This invention achieves rapid accumulation of fucoxanthin by adjusting the nitrogen-carbon ratio during microalgae cultivation, thereby obtaining algal cells with high fucoxanthin yield.

[0044] (2) Peptone was used as the nitrogen source during the fucoxanthin accumulation stage. It not only served as a nitrogen source but also provided a small amount of organic carbon source and had a high nitrogen-carbon ratio. This maintained the proliferation of algal cells while achieving rapid accumulation of fucoxanthin.

[0045] (3) The starvation culture stage enables the control of nitrogen and carbon sources in the culture medium, which is more stable and efficient than the initial addition / feeding addition culture method of the existing technology. At the same time, the combination of starvation and high nitrogen stress increases the yield of fucoxanthin.

[0046] (4) In order to repeatedly cause sudden high nitrogen-carbon ratios, the production of fucoxanthin can be increased multiple times by repeatedly switching between autotrophic and multitrophic. Attached Figure Description

[0047] Figure 1 Cell growth and fucoxanthin accumulation in Example 1;

[0048] Figure 2 Fucoxanthin standard curve;

[0049] Figure 3 Cell growth and fucoxanthin accumulation in Example 3;

[0050] Figure 4 Cell growth and fucoxanthin accumulation in Example 4;

[0051] Figure 5 Cell growth and fucoxanthin accumulation in Example 5. Detailed Implementation

[0052] To further illustrate the present invention, preferred embodiments will now be described. It should be understood that these preferred embodiments are provided for illustrative purposes and do not limit the scope of the invention.

[0053] Example 1

[0054] (1) The stage of dual-trophic proliferation of algal cells;

[0055] The *Phaeodactylum tricornutum* cells were inoculated into the initial culture medium at a concentration of 0.1 g / L, kept at a constant temperature of 30°C, and continuously illuminated with a light intensity of 10,000 lux (white light or natural light). The aeration rate was 0.2 VVM. 0.05 mol / L glycerol and 0.85 g / L urea were added.

[0056] Gradually increase the light intensity according to the cell mass, up to a maximum of 30,000 lux, or use direct sunlight. During this period, continue to supplement glycerol based on its consumption, maintaining a concentration of 0.01 mol / L. Intermittently control the pH at 8.0 using CO2. This stage of cultivation lasts 10 days.

[0057] The initial culture medium formulation is shown in Table 1 of the instruction manual.

[0058] (2) Photoautotrophic stage with organic carbon deficiency and nitrogen starvation

[0059] No need to change the culture apparatus. Stop adding glycerol. At this point, the initially added nitrogen is almost completely depleted. Keep other conditions unchanged and continue photoautotrophic culture under organic carbon deficiency and nitrogen starvation conditions for 2 days. During this period, maintain pH at 8.0 using CO2.

[0060] (3) Dutrophic stage with high nitrogen-to-carbon ratio

[0061] Add 0.6 g / L of peptone (which serves not only as a nitrogen source but also as an organic carbon source, and has a high nitrogen-to-carbon ratio). Keep other conditions constant, and intermittently control the pH at 8.0 with CO2. Incubate for 3 days for large-scale synthesis of fucoxanthin.

[0062] After completing steps 1-3, repeat steps 1-3 twice more. When repeating, change step (1) to culturing for 2 days.

[0063] The final cell dry weight was measured to be 5.1 g / L, the fucoxanthin content was 4.9%, and the yield was 253 mg / L (see [link to data]). Figure 1 ).

[0064] The detection of fucoxanthin employs high-performance liquid chromatography (HPLC), which utilizes specific elution conditions to detect the target peak at a specific wavelength. In this patent, samples of different concentrations were prepared using standards to establish conditions for HPLC detection of fucoxanthin, and a standard curve for fucoxanthin concentration was obtained. The specific method is as follows: Fucoxanthin standards were prepared into different concentrations (1, 2, 3, 4, 6, 8, and 10 mg / L) using methanol. -1The sample volume was fixed at 20 μL. The mobile phase consisted of water (A), methanol (B), and acetonitrile (C), with an initial ratio of 20:20:60. Gradient elution was used, linearly changing to 20:15:65 within 10 min. Between 10 and 13 min, the mobile phase ratio changed to 20:0:80. Finally, in the last 7 min, the mobile phase ratio was adjusted to 15:0:85. During the assay, the column temperature was controlled at 40℃, and the flow rate was 1 mL / min. -1 The detection wavelength was 448 nm. The HPLC model was an Agilent 1260 Infinity. The chromatographic column was a SiO2 column (Welch brand, specifications: 10×250 mm, 5 μm).

[0065] Figure 2 This establishes a linear relationship between fucoxanthin concentration and peak area at 448 nm. The R² value is 0.99708. The specific formula is as follows:

[0066] y = 0.00963x + 0.01937 (3)

[0067] Where x (mAU·min) is the peak area of ​​the sample, and y is the fucoxanthin concentration (mg / L) of the sample. -1 ).

[0068] In the fucoxanthin determination process, 10 mL of algal solution was centrifuged at 9000 rpm for 10 min, and the supernatant was discarded. The solution was then resuspended in RO water and centrifuged again, repeated 1-2 times to remove residual salts and nutrients from the algal surface. Chromatographically pure methanol was added, and the solution was sonicated for 10 min, followed by extraction in the dark for at least 2 h. The supernatant was collected again after centrifugation. The color of the algal residue was observed; if it remained green, fresh methanol was added and the extraction process was repeated until the residue turned grayish-white. The supernatants were combined, filtered through a 0.22 μm filter, and analyzed by liquid chromatography. The fucoxanthin concentration was calculated using a standard curve, and then converted into fucoxanthin content in the algal cells and fucoxanthin concentration in the algal solution based on the algal cell concentration data.

[0069] Example 2

[0070] (1) The stage of dual-trophic proliferation of algal cells;

[0071] The *Phaeodactylum tricornutum* cells were inoculated into the initial culture medium at a concentration of 0.3 g / L, kept at a constant temperature of 30°C, and continuously illuminated with a light intensity of 15000 lux (white light or natural light). The aeration rate was 0.2 VVM. 0.05 mol / L glycerol and 0.85 g / L urea were added.

[0072] Gradually increase the light intensity according to the cell mass, up to a maximum of 30,000 lux, or use direct sunlight. During this period, continue to supplement glycerol based on its consumption, maintaining a concentration of 0.01 mol / L. Intermittently control the pH at 7.0 using CO2. This stage of cultivation lasts for 8 days.

[0073] The initial culture medium formulation is shown in Table 1.

[0074] (2) Photoautotrophic stage with organic carbon deficiency and nitrogen starvation

[0075] No need to change the culture apparatus. Stop adding glycerol. At this point, the initially added nitrogen is almost completely depleted. Keep other conditions unchanged and continue photoautotrophic culture under organic carbon deficiency and nitrogen starvation conditions for 2 days. During this period, the pH is intermittently controlled at 7.0 using CO2.

[0076] (3) Dutrophic stage with high nitrogen-to-carbon ratio

[0077] Add 0.6 g / L of peptone (which serves not only as a nitrogen source but also as an organic carbon source, and has a high nitrogen-to-carbon ratio), keep other conditions constant, and intermittently control the pH at 7.0 with CO2. Incubate for 2 days for large-scale synthesis of fucoxanthin.

[0078] After completing steps 1-3, repeat steps 1-3 once more. When repeating, change step (1) to culturing for 2 days.

[0079] Example 3

[0080] (1) The stage of dual-trophic proliferation of algal cells;

[0081] The *Phaeodactylum tricornutum* cells were inoculated into the initial culture medium at a concentration of 0.1 g / L, kept at a constant temperature of 30°C, and continuously illuminated with a light intensity of 10,000 lux (white light or natural light). The aeration rate was 0.2 VVM. 0.05 mol / L glycerol and 0.85 g / L urea were added.

[0082] Gradually increase the light intensity according to the cell mass, up to a maximum of 30,000 lux, or use direct sunlight. During this period, continue to supplement glycerol based on its consumption, maintaining a concentration of 0.01 mol / L. Intermittently control the pH at 8.0 using CO2. This stage of cultivation lasts 10 days.

[0083] The initial culture medium formulation is shown in Table 1.

[0084] (2) Dutrophic stage with high nitrogen-to-carbon ratio

[0085] No need to change the culture apparatus. Skip the photoautotrophic phase of organic carbon deficiency and nitrogen starvation, and directly add 0.6 g / L of peptone. Keep other conditions constant, intermittently controlling the pH at 8.0 with CO2. Culture for 3 days for fucoxanthin synthesis.

[0086] After completing steps 1-2, repeat steps 1-2 twice more. Testing showed that the final cell dry weight reached 5.1 g / L, the fucoxanthin content was 3.8%, and the yield was 195 mg / L. (See...) Figure 3 )

[0087] Example 4

[0088] (1) The stage of dual-trophic proliferation of algal cells;

[0089] The *Phaeodactylum tricornutum* cells were inoculated into the initial culture medium at a concentration of 0.1 g / L, kept at a constant temperature of 30°C, and continuously illuminated with a light intensity of 10,000 lux (white light or natural light). The aeration rate was 0.2 VVM. 0.05 mol / L glycerol and 0.85 g / L urea were added.

[0090] Gradually increase the light intensity according to the cell mass, up to a maximum of 30,000 lux, or use direct sunlight. During this period, continue to supplement glycerol based on its consumption, maintaining a concentration of 0.01 mol / L. Intermittently control the pH at 8.0 using CO2. This stage of cultivation lasts 10 days.

[0091] The initial culture medium formulation is shown in Table 1.

[0092] (2) Photoautotrophic stage with organic carbon deficiency and nitrogen starvation

[0093] Once the cell count reaches its peak, there is no need to change the culture apparatus. Stop adding glycerol, as the initially added nitrogen is nearly depleted. Maintain all other conditions and continue photoautotrophic culture under carbon and nitrogen starvation for two days. During this period, intermittently control the pH at 8.0 using CO2.

[0094] (3) Dutrophic stage with high nitrogen-to-carbon ratio

[0095] Add 0.6 g / L of peptone (which serves not only as a nitrogen source but also as an organic carbon source, and has a high nitrogen-to-carbon ratio), keep other conditions unchanged, and intermittently control the pH at 8.0 with CO2. Culture for 3 days for large-scale synthesis of fucoxanthin.

[0096] Steps 1-3 were not repeated after completion. Testing showed the final cell dry weight reached 3.5 g / L, fucoxanthin content was 3.8%, and yield was 137 mg / L. (See...) Figure 4 )

[0097] Example 5

[0098] (1) The stage of dual-trophic proliferation of algal cells;

[0099] The *Phaeodactylum tricornutum* cells were inoculated into the initial culture medium at a concentration of 0.1 g / L, kept at a constant temperature of 30°C, and continuously illuminated with a light intensity of 10,000 lux (white light or natural light). The aeration rate was 0.2 VVM. 0.05 mol / L glycerol and 0.85 g / L urea were added.

[0100] Gradually increase the light intensity according to the cell mass, up to a maximum of 30,000 lux, or use direct sunlight. During this period, continue to supplement glycerol based on its consumption, maintaining a concentration of 0.01 mol / L. Intermittently control the pH at 8.0 using CO2. This stage of cultivation lasts 10 days.

[0101] The initial culture medium formulation is shown in Table 1.

[0102] (2) Photoautotrophic stage with organic carbon deficiency and nitrogen starvation

[0103] Once the cell count reaches its peak, there is no need to change the culture apparatus. Stop adding glycerol; at this point, the initially added nitrogen is also nearly depleted. Maintain other conditions unchanged and continue photoautotrophic culture under carbon and nitrogen starvation for 2 days. During this period, intermittently control the pH at 8.0 using CO2.

[0104] (3) Dutrophic stage with high nitrogen-to-carbon ratio

[0105] Add 0.6 g / L urea (mainly as a nitrogen source). Keep other conditions unchanged, and intermittently control the pH at 8.0 with CO2. Incubate for 3 days for fucoxanthin synthesis.

[0106] After completing steps 1-3, repeat steps 1-3 twice more. During the repetition, step (1) is changed to culturing for 2 days. The final cell dry weight was measured to be 3.5 g / L, the fucoxanthin content was 4.8%, and the yield was 169 mg / L.

[0107] (See Figure 5 ).

Claims

1. A cell culture method with high fucoidan content, characterized in that, The culture method is as follows: Step 1. Cell proliferation stage; Step 2. Photoautotrophic culture of cells; Step 3. Facultative anaerobic culture of cells under high nitrogen conditions.

2. The cell culture method with high fucoxanthin content as described in claim 1, characterized in that, The cells are algal cells, and more preferably, the algal cells are brown algae or diatoms, and more preferably, *Phaeodactylum tricornutum*, *Rhomboidella sclerotium*, or *Rhomboidella scabra*.

3. The cell culture method with high fucoxanthin content as described in claim 2, characterized in that, After step 3 is completed, repeat steps 1-3 1-2 times.

4. The cell culture method with high fucoxanthin content as described in claim 3, characterized in that, The cell proliferation stage employs a combined culture method with progressively increasing light intensity.

5. The cell culture method with high fucoxanthin content as described in claim 4, characterized in that, The maximum light intensity of the progressively increasing light is 30,000 lux, and the glycerol is supplemented in the mixed diet to maintain the glycerol concentration at 0.05 mol / L.

6. The cell culture method with high fucoxanthin content as described in claim 3, characterized in that, The photoautotrophic culture process does not involve changing the culture device or replenishing the culture medium until the initial carbon and nitrogen are depleted, and then continues the starvation culture for a period of time.

7. The cell culture method with high fucoxanthin content as described in claim 6, characterized in that, The starvation culture time is 1-5 days, preferably 2 days.

8. The cell culture method with high fucoxanthin content as described in claim 3, characterized in that, The high-nitrogen culture uses peptone as the nitrogen source, and preferably, the amount of peptone added is 0.2-1 g / L.

9. The cell culture method with high fucoxanthin content as described in claim 8, characterized in that, The pH of the high-nitrogen culture was controlled by intermittent CO2 introduction, with a pH range of 6.0-10.0 and a culture period of 1-5 days.

10. Cells with high fucoidan content produced by the cell culture method for high fucoidan content as described in claims 1-9.

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