Method for increasing pigment yield in diatom

Abstract

The invention belongs to the technical field of diatom culture and utilization, and particularly relates to a method for increasing the yield of pigments in diatom. According to the method disclosed by the invention, the yield of various pigments including fucoxanthin, violaxanthin, silcoxanthin and diatom yellow can be effectively increased by adjusting the light-dark period of the cultured diatom or performing ultraviolet radiation on the diatom, and particularly the yield of fucoxanthin in the diatom can be increased; and a technical support is provided for industrial production of fucoxanthin based on diatom.

Description

Technical Field

[0001] The present invention belongs to the technical field of diatom culture and utilization, and particularly relates to a method for increasing the pigment yield in diatoms. Background Art

[0002] Fucoxanthin, also known as fucoxanthol and fucoxanthin, is a natural pigment in the xanthophyll class of carotenoids. It accounts for more than 10% of the total amount of about 700 naturally occurring carotenoids, with a color ranging from light yellow to brown. It is a pigment contained in brown algae, diatoms, chrysophytes, and yellow-green algae, and is widely present in various algae, marine phytoplankton, aquatic shellfish and other animals and plants. It has various pharmacological effects such as anti-tumor, anti-inflammatory, antioxidant, weight loss, and neuroprotective effects, and is widely used in the markets of drugs, skin care and beauty products, and health care products.

[0003] Currently, the sources of industrially produced fucoxanthin are mostly large algae. However, the concentration of fucoxanthin in large algae is low, the growth is slow, the cultivation is mostly extensive, the cultivation area is large, and it is easily affected by environmental factors. Microalgae including diatoms are widely distributed, have a fast growth rate, a high fucoxanthin content (1 - 3 orders of magnitude higher than that of large algae), and the cultivation conditions are controllable. However, at present, the commercial degree of fucoxanthin from microalgae is not high, and there is no perfect extraction method for fucoxanthin. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for increasing the pigment yield in diatoms, which can effectively increase the yield of various pigments including fucoxanthin, violaxanthin, diadinoxanthin, and diatoxanthin in diatoms.

[0005] The present invention provides a method for increasing the pigment yield in diatoms, including the following steps: during the cultivation of diatoms, performing light treatment or ultraviolet radiation treatment on the diatoms, and collecting samples from the exponential phase to the stationary phase;

[0006] The light-dark cycle of the light treatment is 12 h light: 12 h dark or 2 h light: 2 h dark;

[0007] The ultraviolet radiation treatment includes UVB radiation treatment and / or UVA radiation treatment; the radiation intensity of the UVB radiation treatment is 1 W / m 2 ; the radiation intensity of the UVA radiation treatment is 30 W / m 2 ; the frequency of the ultraviolet radiation treatment is 1.5 h / day;

[0008] The pigments include one or more of fucoxanthin, violaxanthin, diadinoxanthin, and diatoxanthin.

[0009] Preferably, the light source of the light treatment is a D65 standard light source.

[0010] Preferably, the light intensity of the light treatment is 70-80 μmol·m -2 ·s -1 .

[0011] Preferably, the light intensity of the ultraviolet radiation treatment is 60-70 μmol·m -2 ·s -1 .

[0012] Preferably, the cultivation method of the diatoms is batch culture or semi-continuous culture.

[0013] Preferably, the culture medium for the batch culture or semi-continuous culture is f / 2 medium, and the culture temperature is 18-22 °C.

[0014] Preferably, the initial culture concentration of the diatoms is (7-9)×10 4 cells / mL.

[0015] Preferably, the diatoms include Phaeodactylum tricornutum.

[0016] Preferably, when the pigment is fucoxanthin, the light-dark cycle of the light treatment is 2 h light: 2 h dark; sampling is carried out from the late exponential phase to the stationary phase, and the late exponential phase is the 7th to 8th day of the exponential phase.

[0017] Preferably, when the pigment is fucoxanthin, the ultraviolet radiation treatment is UVB radiation treatment; sampling is carried out during the exponential phase.

[0018] Beneficial effects:

[0019] The present invention provides a method for increasing the pigment yield in diatoms, comprising the following steps: during the cultivation of diatoms, performing light treatment or ultraviolet radiation treatment on the diatoms, and sampling from the exponential phase to the stationary phase; the light-dark cycle of the light treatment is 12 h light: 12 h dark or 2 h light: 2 h dark; the ultraviolet radiation treatment includes UVB radiation treatment and / or UVA radiation treatment; the radiation intensity of the UVB radiation treatment is 1 W / m 2 ; the radiation intensity of the UVA radiation treatment is 30 W / m 2 ; the frequency of the ultraviolet radiation treatment is 1.5 h / day; the pigments include one or more of fucoxanthin, violaxanthin, diadinoxanthin and diatoxanthin. By adjusting the light-dark cycle or performing ultraviolet radiation on the cultivated diatoms, the present invention can effectively increase the yields of various pigments including fucoxanthin, violaxanthin, diadinoxanthin and diatoxanthin, and in particular can increase the yield of fucoxanthin in diatoms, providing technical support for the industrial production of fucoxanthin based on diatoms. Description of the drawings

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the accompanying drawings required for the embodiments.

[0021] Figure 1 It is the change in the cell abundance of *Phaeodactylum tricornutum* under different light-dark cycles in Example 1;

[0022] Figure 2 It is the comparison of the intrinsic growth rates under different light-dark cycles in Example 1;

[0023] Figure 3 It is the change in the pigment content of the *Phaeodactylum tricornutum* culture system in the control group (24:0) in Example 1;

[0024] Figure 4 It is the change in the pigment content of the *Phaeodactylum tricornutum* culture system in the continuous long-light group (16:8) in Example 1;

[0025] Figure 5 It is the change in the pigment content of the *Phaeodactylum tricornutum* culture system in the intermittent long-light group (4:2) in Example 1;

[0026] Figure 6 It is the change in the pigment content of the *Phaeodactylum tricornutum* culture system in the continuous medium-light group (12:12) in Example 1;

[0027] Figure 7 It is the change in the pigment content of the *Phaeodactylum tricornutum* culture system in the intermittent medium-light group (2:2) in Example 1;

[0028] Figure 8 It is the change in the pigment content of the *Phaeodactylum tricornutum* culture system in the continuous medium-short light group (8:16) in Example 1;

[0029] Figure 9 It is the change in the pigment content of the *Phaeodactylum tricornutum* culture system in the intermittent short-light group (2:4) in Example 1;

[0030] Figure 10 It is the change in the content of diadinoxanthin in the *Phaeodactylum tricornutum* culture system in Example 1;

[0031] Figure 11 It is the change in the content of violaxanthin in the *Phaeodactylum tricornutum* culture system in Example 1;

[0032] Figure 12 It is the total amount of pigments in the system under different light-dark cycles in Example 1;

[0033] Figure 13 It is the intracellular pigment content in the long-light groups (16:8, 4:2) in Example 1;

[0034] Figure 14Intracellular pigment content in the medium light group (12:12, 2:2) in Example 1;

[0035] Figure 15 In Example 1, there was no significant difference in the effect of light-dark cycles on intracellular pigment content during the two growth stages of the short light group;

[0036] Figure 16 Intracellular pigment ratio during the plateau phase (4:2, 16:8) in Example 1;

[0037] Figure 17 Intracellular pigment ratio during the plateau phase (2:2, 12:12) in Example 1;

[0038] Figure 18 Intracellular pigment ratio during the plateau phase (2:4, 8:16) in Example 1;

[0039] Figure 19 Highest yield of fucoxanthin in the system under different light-dark cycles in Example 1;

[0040] Figure 20 Changes in the cell abundance of Phaeodactylum tricornutum under different ultraviolet radiations in Example 2;

[0041] Figure 21 Changes in the pigment content of the Phaeodactylum tricornutum culture system under different ultraviolet radiations (Blank, UVA) in Example 2;

[0042] Figure 22 Changes in the pigment content of the Phaeodactylum tricornutum culture system under different ultraviolet radiations (UVB, UVAB) in Example 2;

[0043] Figure 23 Intracellular pigment content under ultraviolet radiation during the exponential phase of the control group in Example 2;

[0044] Figure 24 Intracellular pigment content under ultraviolet radiation during the exponential phase of the UVA group in Example 2;

[0045] Figure 25 Intracellular pigment content under ultraviolet radiation during the exponential phase of the UVB group in Example 2;

[0046] Figure 26 Intracellular pigment content under ultraviolet radiation during the exponential phase of the UVAB group in Example 2;

[0047] Figure 27 Intracellular pigment content under ultraviolet radiation during the plateau phase of the control group in Example 2;

[0048] Figure 28 Intracellular pigment content under ultraviolet radiation during the plateau phase of the UVA group in Example 2;

[0049] Figure 29 Intracellular pigment content under plateau-phase UV radiation in the UVB group in Example 2;

[0050] Figure 30 Intracellular pigment content under plateau-phase UV radiation in the UVAB group in Example 2;

[0051] Figure 31 Variation in fucoxanthin yield in the Phaeodactylum tricornutum culture system under different UV radiations in Example 2. Detailed implementation method

[0052] The present invention provides a method for enhancing pigment production in diatoms, comprising the following steps: during the cultivation of diatoms, subject the diatoms to light treatment or UV radiation treatment, and collect samples from the exponential phase to the plateau phase;

[0053] The light-dark cycle of the light treatment is 12 h light: 12 h dark or 2 h light: 2 h dark;

[0054] The UV radiation treatment includes UVB radiation treatment and / or UVA radiation treatment; the radiation intensity of the UVB radiation treatment is 1 W / m 2 ; the radiation intensity of the UVA radiation treatment is 30 W / m 2 ; the frequency of the UV radiation treatment is 1.5 h / day;

[0055] The pigments include one or more of fucoxanthin, violaxanthin, diadinoxanthin, and diatoxanthin.

[0056] During the cultivation of diatoms in the present invention, the diatoms are subjected to light treatment or UV radiation treatment, and samples are collected from the exponential phase to the plateau phase.

[0057] As an implementation method, the diatom is Phaeodactylum tricornutum.

[0058] As an implementation method, the cultivation method of the diatoms is batch culture or semi-continuous culture. As an implementation method, the medium for the batch culture or semi-continuous culture is f / 2 medium. As an implementation method, the initial cultivation concentration of the diatoms is (7 - 9)×10 4 cells / mL; as another implementation method, the initial cultivation concentration of the diatoms is 8×10 4 cells / mL. As an implementation method, the cultivation temperature of the diatoms is 18 - 22 °C; as another implementation method, the cultivation temperature of the diatoms is 20 °C. As an implementation method, the cultivation time of the diatoms in the present invention is 10 days; as another implementation method, the cultivation time of the diatoms is 10 days.

[0059] As an implementation method, the light source for the light treatment is a D65 standard light source. As an implementation method, the light intensity of the light treatment is 70-80 μmol·m -2 ·s -1 ; As another implementation method, the light intensity of the light treatment is 75 μmol·m -2 ·s -1 . The light-dark cycle of the light treatment in the present invention is 12 h light: 12 h dark or 2 h light: 2 h dark; As an implementation method, the light-dark cycle of the light treatment is 2 h light: 2 h dark. As an implementation method, when the diatom is Phaeodactylum tricornutum, the present invention samples at the late exponential phase to the stationary phase of diatom growth; As another implementation method, the late exponential phase is the 7th to 8th day of the exponential phase.

[0060] The ultraviolet radiation treatment in the present invention includes UVB radiation treatment and / or UVA radiation treatment. The radiation intensity of the UVB radiation treatment in the present invention is 1 W / m 2 . The radiation intensity of the UVA radiation treatment in the present invention is 30 W / m 2 . The frequency of the ultraviolet radiation treatment in the present invention is 1.5 h / day. As an implementation method, the ultraviolet radiation treatment is a continuous ultraviolet radiation treatment. For example, in a specific embodiment, the ultraviolet lamp is continuously turned on for irradiation for 1.5 h / day. As an implementation method, the light intensity of the ultraviolet radiation treatment is 60-70 μmol·m -2 ·s -1 ; As another implementation method, the light intensity of the light treatment is 65 μmol·m -2 ·s -1 . As an implementation method, when increasing the pigment yield in diatoms by ultraviolet radiation treatment, normal light is applied to the diatoms at other times except for the radiation treatment time; As an implementation method, the light source of the normal light is a D65 standard light source; the light time is 10.5 h. The light intensity is 65 μmol·m -2 ·s -1 . As an implementation method, when the pigment is fucoxanthin, the ultraviolet radiation treatment is a UVB radiation treatment; sampling is performed at the exponential phase.

[0061] The present invention uses artificial sunlight irradiation or ultraviolet radiation irradiation methods to treat diatoms, which can effectively increase the pigment content in diatoms. At the same time, through comparison of examples, it can be found that under the conditions of artificial sunlight irradiation or ultraviolet radiation defined in the present invention, the pigment yield can be increased more than other artificial sunlight irradiation or ultraviolet radiation conditions, especially the yield of fucoxanthin.

[0062] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0063] The term translations in the following embodiments are as follows:

[0064] Chlorophyllide a is chlorophyllide a; Violaxanthin is violaxanthin; Diadinoxanthin is diadinoxanthin; Diatoxanthin is diatoxanthin; Chlorophyll a is chlorophyll a and Fucoxanthin is fucoxanthin;

[0065] culture time is culture time; content is content; cell density is cell density.

[0066] Example 1

[0067] A method for increasing the pigment yield in Phaeodactylum tricornutum by adjusting the light-dark cycle, the steps are as follows:

[0068] I. Culture method

[0069] Select the model organism Phaeodactylum tricornutum for diatom research as the culture object. The selected algal strain number is CCMP2561, which comes from the National Centre for Marine Algae and Microbiota (Bigelow, USA), and the algal strain is isolated from the waters near Blackpool, England, in the North Atlantic Ocean (54°N, 4°W).

[0070] To explore the effects of the light-dark cycle on the growth and pigment synthesis of Phaeodactylum tricornutum, a total of 7 experimental groups were set up, namely: (1) control group: 24 h light: 0 h dark (24:0); (2) continuous long light group: 16 h light: 8 h dark (16:8); (3) continuous medium light group: 12 h light: 12 h dark (12:12); (4) continuous short light group: 8 h light: 16 h dark (8:16); (5) intermittent long light group: 4 h light: 2 h dark (4:2), and this light-dark ratio is cycled; (6) intermittent medium light group: 2 h light: 2 h dark (2:2), and this light-dark ratio is cycled; (7) intermittent short light group: 2 h light: 4 h dark (2:4), and this light-dark ratio is cycled. The experiment was carried out in an incubator at a temperature of 20 °C, using a 2 L PC bottle as the culture container, and the light intensity was 75 μmol·m -2 ·s -1; The light source for irradiation is a D65 standard light source.

[0071] The Phaeodactylum tricornutum (CCMP 2561) strain was provided by Dr. Xinwei Wang from the School of Marine Sciences, Ningbo University, China. This strain was originally isolated from the waters near Blackpool, England, in the North Atlantic Ocean (latitude 54°N, longitude 4°W), obtained from the National Center for Marine Algae and Microbiota (NCMA), and can also be purchased from the National Centre for Marine Algae and Microbiota (Bigelow, USA).

[0072] This strain was cultured in sterilized f / 2 medium, and each liter of the medium contained the following components: 74.8 mg of NaNO3, 5.0 mg of NaH2PO4·2H2O, 30.0 mg of Na2SiO3·9H2O, 5.84 μg of Na2MoO4·2H2O, 8.0 μg of CuSO4·5H2O, 18.4 μg of ZnSO4·7H2O, 9.6 μg of CoCl2·6H2O, 142.4 μg of MnCl2·4H2O, 4.41 mg of Na2EDTA·2H2O, 2.57 mg of FeCl2·6H2O, 80 μg of VB1, 0.4 μg of VB 12 and 0.4 μg of V H .

[0073] Before the formal experiment, a 10-day semi-continuous culture was carried out [(1) Medium f / 2, with the formula as above; (2) Other culture conditions were the same as those in the formal experiment, i.e., 7 experimental conditions for the light-dark cycle, the temperature was 20 °C, and the light intensity was 75 μmol·m -2 ·s -1 ; 4 experimental conditions for ultraviolet radiation, the temperature was 20 °C, and the light intensity was 65 μmol·m -2 ·s -1 ; (3) Semi-continuous culture experimental method: Every day, 2 mL of the algal solution was taken and its in vivo fluorescence value was measured with a fluorometer (Trilogy, Turner Design, USA). After calculating the dilution factor, the algal solution was diluted with freshly prepared f / 2 medium to keep the cell abundance and nutrient salt concentration in the culture system relatively constant at the initial conditions], as an acclimation experiment, and the chlorophyll fluorescence value was monitored daily. After the growth rate was stable, the algal solution in the exponential growth phase was inoculated into the f / 2 medium, and the initial concentration was 8×10 4cells / mL, a one-time culture experiment was conducted for 10 days. During the experiment, the growth stage of *Phaeodactylum tricornutum* was judged by combining the results of chlorophyll fluorescence values and cell counts, and the number of days in the exponential growth phase and the stationary phase was selected for subsequent analysis. Sampling of the parameters to be measured was carried out on every even day, and the same volume of f / 2 medium was used as a blank control during sampling.

[0074] II. Sampling method

[0075] The biomass of diatoms in the culture system was monitored by in vivo fluorescence using a Trilogy fluorometer (Turner Designs, USA). Every day, 1 mL of the culture was taken, fixed with Lugol's solution, and counted under a microscope using a 100 μL plankton counting chamber.

[0076] Microalgal cells were collected by filtering 10 mL of the culture through a GF / F membrane (Whatman, USA), snap-frozen in liquid nitrogen, and stored at -80 °C. The frozen filter membrane was cut into small pieces and placed in a centrifuge tube containing 4 mL of N,N-dimethylformamide (DMF). The samples were sonicated in an ice-water bath (JY92-IIN, Jingxin, 40 W, 1 minute), centrifuged at 3000 rpm for 3 minutes, and the supernatant was filtered through a 0.22 μm nylon filter membrane. 1 mL of the filtrate was transferred to a vial and mixed with 200 μL of Milli-Q water. Pigment analysis was performed using high-performance liquid chromatography (Shimadzu, Japan). Mobile phase A consisted of methanol, acetonitrile, and aqueous pyridine solution (50:25:25, v / v), and mobile phase B consisted of methanol, acetonitrile, and acetone (20:60:20, v / v). The chromatographic column used was ZORBAX Eclipse XDB-C8 (4.6×150 mm, 5 μm), with a Gemini C18 guard column (4×3.0 mm). The column temperature was maintained at 25 °C, and the flow rate was 1 mL / min. Gradient elution was carried out within 47 minutes as follows: 0 - 2 minutes, A: 95%, B: 5%; 2 - 22 minutes, A: 95% → 60%, B: 5% → 40%; 22 - 24 minutes, A: 60%, B: 40%; 24 - 30 minutes, A: 60% → 5%, B: 40% → 95%; 30 - 40 minutes, A: 5%, B: 95%; 40 - 42 minutes, A: 5% → 95%, B: 95% → 5%; 42 - 47 minutes, A: 95%, B: 5%.

[0077] III. Experimental results

[0078] 3.1 Cell abundance

[0079] The changes in the cell abundance of *Phaeodactylum tricornutum* under different light-dark cycles are as Figure 1As shown (error bars represent standard deviation, n = 3), the growth stage can be divided into: lag phase (day 0 - day 2), exponential phase (day 3 - day 6), and stationary phase (day 7 - day 10). The results show that there are certain differences in the cell abundance of *Phaeodactylum tricornutum* under different light - dark cycles. The average cell abundance in the exponential growth phase generally shows the trend of control group ≈ long - light group > medium - light group > short - light group. After fitting with the integral form of the logistic equation, the intrinsic growth rate (r) of different treatment groups was obtained. The results are as Figure 2 shown (error bars represent standard deviation, n = 3; letters are significance markers, and completely different combinations of letters represent significant differences between groups). It can be seen that the intrinsic growth rate of the 4:2 group is significantly higher than that of the 2:4 group and the 16:8 group (p < 0.05).

[0080] 3.2 Pigments

[0081] 3.2.1 Pigment composition of the culture system

[0082] The changes in the pigment content of the *Phaeodactylum tricornutum* culture system in the control group (24:0) are as Figure 3 shown (error bars represent standard deviation, n = 3). It can be seen that the changes in the pigment content in the control group system over the culture time are as follows: At the initial stage of culture, the pigments in the control group system increased rapidly. As the culture progressed, the increase rate of the total pigment content decreased significantly. Specifically, in the early exponential phase (day 4), the proportion of chlorophyll a reached a peak, accounting for 45.92%. In the remaining growth stages, this proportion stabilized at about 39%. The proportion of diadinoxanthin reached 2.9%, about twice that of other stages. At the same time, the proportion of fucoxanthin was relatively low in the early exponential phase, accounting for 39.21%, and stabilized at about 42% in the remaining stages. In addition, the high proportion (17.5%) of chlorophyllide a in the lag phase (day 2) reflects its accumulation as an intermediate, and the decrease in its proportion (11.31%) on day 4 indicates that it has been largely converted into chlorophyll a.

[0083] The changes in the pigment content of the *Phaeodactylum tricornutum* culture system in the long - light groups (16:8, 4:2) are as Figures 4 - 5Shown (error bars represent standard deviation, n = 3). It can be seen that in the initial stage of cultivation, the pigment growth rate of the 4:2 group system is faster; at all growth stages, the total pigment content of the 16:8 group is lower than that of the 4:2 group. In the early exponential phase (day 4), the proportion of chlorophyll a in the 4:2 group reached the peak (50.4%), while the proportions of fucoxanthin and chlorophyllide a decreased to the lowest values (36.89% and 10.48% respectively), and these change trends are similar to those of the control group. Relatively, these changes in the 16:8 group all occurred in the stationary phase (day 2), that is, the peak proportion of chlorophyll a was reached on day 2, and at the same time, the proportions of fucoxanthin and chlorophyllide a were the lowest. It should be noted that the proportion of fucoxanthin in the 16:8 group generally showed an upward trend and reached the highest value at the end of cultivation; and the proportion of diadinoxanthin in the 16:8 group was higher than that of the 4:2 group.

[0084] The changes in the pigment content of the Phaeodactylum tricornutum culture system in the medium light groups (12:12, 2:2) are as Figures 6 - 7 shown (error bars represent standard deviation, n = 3). It can be seen that the pigment increase in the 2:2 group is larger and reaches the highest value until the plateau phase. In the early exponential phase (day 4), the proportion of chlorophyll a in the 2:2 group reached the maximum value, while the proportions of fucoxanthin and chlorophyllide a were the lowest. Whether it is the 12:12 group or the 2:2 group, there is always an inverse relationship between chlorophyll a and chlorophyllide a. The final proportion of fucoxanthin in the continuous light group is higher, and this phenomenon is similar to that of the long light group.

[0085] The changes in the pigment content of the Phaeodactylum tricornutum culture system in the short light groups (8:16, 2:4) are as Figures 8 - 9 shown (error bars represent standard deviation, n = 3). It can be seen that there is no significant difference in the total pigment content between the 2:4 group and the 8:16 group. In both groups, at the end of cultivation, the proportion of fucoxanthin accumulated in the system reached the maximum value; the proportion of chlorophyllide a increased day by day; while the proportion of chlorophyll a generally showed a decreasing trend.

[0086] The results of the changes in the contents of violaxanthin and diatoxanthin in the Phaeodactylum tricornutum culture system are as Figures 10 - 11 shown (error bars represent standard deviation, n = 3). It can be seen that the contents of violaxanthin and diatoxanthin in the culture system are always low, accounting for less than 1% of the system pigment content. The proportion of diatoxanthin in the system pigment generally shows an increasing trend, while the content of violaxanthin shows a decreasing trend.

[0087] The results of the influence of the light-dark cycle on the total pigment accumulation in the Phaeodactylum tricornutum culture system are as Figure 12Shown (error bars represent standard deviation, n = 3; letters are significance markers, and completely different combinations of letters represent significant differences between groups). In the medium light group, intermittent light significantly promoted pigment synthesis (p < 0.05), and the total pigment synthesis in the 2:2 group reached 2025.2553 ± 144.2963 mg / L; in the long light group and the short light group, the average total pigment in the intermittent light group was higher than that in the continuous light group, but no significant difference was observed.

[0088] 3.2.2 Intracellular pigment content

[0089] The results of the intracellular pigment content in the long light group (16:8, 4:2) are as Figure 13 shown (error bars represent standard deviation, n = 3, ***p < 0.001, *p < 0.05). The 6th day was selected as the representative day of the exponential phase, and the 10th day was selected as the representative day of the stationary phase. It can be seen that in the exponential phase, the fucoxanthin content in the 4:2 group was 0.1813 ± 0.0023 pg·cell -1 , while the fucoxanthin content in the 16:8 group was 0.1404 ± 0.0133 pg·cell -1 , and intermittent light significantly increased the intracellular fucoxanthin content of Phaeodactylum tricornutum (p < 0.05). In the stationary phase, intermittent light significantly increased the intracellular chlorophyll a content (p < 0.001). The chlorophyll a content in the 4:2 group was 0.1615 ± 0.0221 pg·cell -1 , while that in the 16:8 group was 0.1167 ± 0.0084 pg·cell -1 ; meanwhile, the intracellular violaxanthin content in the 4:2 group was also significantly higher than that in the 16:8 group (p < 0.05).

[0090] The results of the intracellular pigment content in the medium light group (12:12, 2:2) are as Figure 14 shown (error bars represent standard deviation, n = 3, ***p < 0.001, *p < 0.05). The 6th day was selected as the representative day of the exponential phase, and the 10th day was selected as the representative day of the stationary phase. It can be seen that in the stationary phase, intermittent light significantly reduced the intracellular diatoxanthin content (p < 0.05). The diatoxanthin content in the 2:2 group was 0.0095 ± 0.0001 pg·cell -1 , while that in the 12:12 group was 0.0120 ± 0.0012 pg·cell -1 ; meanwhile, the intracellular diatoxanthin content in the intermittent light group was also significantly lower than that in the continuous light group (p < 0.001). In addition, intermittent light significantly increased the chlorophyll a content in the stationary phase (p < 0.001). The chlorophyll a content in the 2:2 group was 0.2251 ± 0.0001 pg·cell -1, while the 12:12 group was 0.1022 ± 0.0001 pg·cell -1 .

[0091] The results of the intracellular pigment content in the short-daylight groups (8:16, 2:4) are as Figure 15 shown (error bars represent standard deviation, n = 3). The 6th day was selected as the representative day of the exponential phase, and the 10th day was selected as the representative day of the stationary phase. It can be seen that there was no significant difference in the effect of the light-dark cycle on the intracellular pigment content during the two growth stages of the short-daylight groups.

[0092] 3.2.3 Intracellular pigment ratio in the stationary phase

[0093] The pigment ratio in Phaeodactylum tricornutum can reflect its photosynthetic physiological state. The pigment ratio reflects the physiological mechanism of adapting to light changes. For example, an increase in the ratio of photosynthesis-related pigments to photoprotective pigments means that photosynthesis in diatoms is relatively more active than photoprotection at this node, and vice versa. For example, the activation state of the diadinoxanthin cycle can be represented by the de-epoxidation state (DEPS), DEPS = Dt / (Dt + Dd), where Dt is diatoxanthin and Dd is diadinoxanthin. In addition, the ratio of photosynthesis-related pigments to photoprotective pigments can also be calculated by the formula (Chla + Chlide + FX) / (Dd + Dt + V), where Chla is chlorophyll a, Chlide is chlorophyllide a, FX is fucoxanthin, and V is violaxanthin. The Chla / FX ratio indicates the change in the pigment ratio used for photosynthesis in the cell, while FX / POC and Chla / POC reflect the relationship between photosynthetic pigments and particulate organic carbon production, where POC is particulate organic carbon.

[0094] The results of the intracellular pigment ratio in the stationary phase are as Figures 16 - 18As shown (error bars represent standard deviation, n = 3, ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05). It can be seen that the Chla / FX in the 4:2 group was significantly higher than that in the 16:8 group (p < 0.0001); meanwhile, the ratios of Chla / POC (p < 0.001) and (Chla + Chlide + FX) / (Dd + Dt + V) (p < 0.01) were significantly higher than those in the 16:8 group; the three ratios of Chla / FX, (Chla + Chlide + FX) / (Dd + Dt + V), and Chla / POC in the 2:2 group were all significantly higher than those in the 12:12 group (p < 0.0001), while the DEPS (p < 0.01) and FX / POC (p < 0.05) in the intermittent light group were significantly lower than those in the continuous light group; the three ratios of Chla / FX, FX / POC, and Chla / POC in the 2:4 group were all significantly lower than those in the 8:16 group, while (Chla + Chlide + FX) / (Dd + Dt + V) was significantly higher than that in the 8:16 group (p < 0.05).

[0095] 3.2.4 Fucoxanthin production

[0096] The results of fucoxanthin production under different light cycles are as Figure 19 shown (error bars represent standard deviation, n = 3; letters are significance markers, and completely different combinations of letters represent significant differences between groups). It can be seen that intermittent light significantly promoted pigment synthesis. Especially for the medium light group (2:2), the production of fucoxanthin also reached the highest value; for the short light group, the effect of intermittent light on intracellular pigment synthesis was not significant.

[0097] The following conclusions can be drawn from the above results:

[0098] (1) Under the condition of equal total light duration, the highest pigment synthesis amount in the system: intermittent group > continuous group, and the total pigment value in the 2:2 group was significantly higher than that in other treatment groups;

[0099] (2) The proportion of fucoxanthin in the exponential phase was lower than that in other growth stages, while the proportion of chlorophyll a was higher than that in other growth stages;

[0100] (3) Intermittent light significantly promoted pigment synthesis. Especially for the medium light group (2:2), the production of fucoxanthin also reached the highest value; for the short light group, the effect of intermittent light on intracellular pigment synthesis was not significant;

[0101] (4) As the culture stage advanced, the proportion of violaxanthin in different treatment groups generally showed a downward trend, while the proportion of diadinoxanthin generally showed an upward trend;

[0102] (5) Intermittent light significantly increased the ratio of photosynthesis-related pigments to photoprotective pigments compared with continuous light, indicating that intermittent light is beneficial to the improvement of the photosynthetic efficiency of *Phaeodactylum tricornutum*;

[0103] (6) From the perspectives of productivity and energy utilization, it is recommended that the light-dark cycle for industrial production preferably be 2 h light: 2 h dark,

[0104] the second choice is 12 h light: 12 h dark, and the sampling time is selected at the late exponential phase / plateau phase. The combined application of by-products violaxanthin, diadinoxanthin, and diatoxanthin can be considered.

[0105] Example 2

[0106] A method for increasing the pigment yield in *Phaeodactylum tricornutum* by ultraviolet irradiation, the steps are as follows:

[0107] I. Cultivation method

[0108] Select the model organism of diatom research, *Phaeodactylum tricornutum*, as the cultivation object. The selected algal strain number is CCMP2561, which comes from the National Centre for Marine Algae and Microbiota (Bigelow, USA), and the algal strain was isolated from the waters near Blackpool, England, in the North Atlantic Ocean (54°N, 4°W).

[0109] To explore the effects of ultraviolet radiation on the growth and pigment synthesis of *Phaeodactylum tricornutum*, a total of 4 experimental groups were set up, namely: (1) Control group: PAR (photosynthetically active radiation); (2) UVA group: radiation intensity 30 W / m 2 ; (3) UVB group: radiation intensity 1 W / m 2 ; (4) UVAB group: 30 W / m 2 UVA + 1 W / m 2 UVB. The experiment was carried out in an incubator at a temperature of 20 °C. A quartz bottle with a volume of 500 mL was used as the culture container. The light intensity was 65 μmol·m -2 ·s -1 . The ultraviolet lamp was turned on at 12:00 noon to 13:30 every day for ultraviolet radiation treatment; normal light was used at other times. The normal light used a D65 standard light source. The total light time was 12 h (including 1.5 h of ultraviolet lamp irradiation and 10.5 h of normal light), and the light intensity was 65 μmol·m -2 ·s -1 , and the remaining 12 h was dark treatment.

[0110] II. Sampling method

[0111] Same as Example 1.

[0112] III. Experimental Results

[0113] 3.1 Cell Abundance

[0114] The results of cell abundance are as Figure 20 shown. It can be seen that there are certain differences in the cell abundance of *Phaeodactylum tricornutum* under different ultraviolet radiations. Generally, the cell abundance shows the trend of control group > UVA > UVB > UVAB.

[0115] 3.2 Pigments

[0116] 3.2.1 Pigment Composition in the Culture System

[0117] The changes in the pigment content in the *Phaeodactylum tricornutum* culture system under different ultraviolet radiations are as Figures 21 - 22 shown (the error bars represent the standard deviation, n = 3).

[0118] 3.2.2 Intracellular Pigment Content

[0119] The results of the intracellular pigment content in the exponential phase under different ultraviolet radiations are as Figures 23 - 26 shown (the error bars represent the standard deviation, n = 3, ***p < 0.001, *p < 0.05). It can be seen that there is no difference in the content of each pigment before and after irradiation in the control group (the "before and after" here refers to before and after the ultraviolet lamp irradiation on the day of sample collection, and the same applies to others); UVA and UVB irradiations significantly increased the production of fucoxanthin in *Phaeodactylum tricornutum*, and the effect of UVB on the yield increase is better; UVA, UVB, and UVAB irradiations significantly increased the synthesis of violaxanthin; UVA promoted the synthesis of diadinoxanthin, and UVB promoted the synthesis of diatoxanthin.

[0120] The results of the intracellular pigment content in the stationary phase under different ultraviolet radiations are as Figures 27 - 30 shown (the error bars represent the standard deviation, n = 3, ***p < 0.001, *p < 0.05). It can be seen that there is no difference in the content of each pigment before and after irradiation in the control group; after the stationary phase, there is no significant difference in the production of fucoxanthin before and after UVA and UVB irradiations; UVA, UVB, and UVAB irradiations significantly increased the synthesis of diatoxanthin; UVB significantly promoted the accumulation of chlorophyll a, and UVAB significantly reduced the yield of chlorophyllide a.

[0121] The results of the yield change of fucoxanthin in the exponential phase of the *Phaeodactylum tricornutum* culture system under different ultraviolet radiations are as Figure 31 shown (the error bars represent the standard deviation, n = 3). It can be seen that UVB has a significant promoting effect on the yield of fucoxanthin in the exponential phase, and there is no difference between groups in the stationary phase. 3.3

[0123] On the 2nd day (lag phase), 4th day (before exponential phase), 6th day (exponential phase), 8th day (after exponential phase), and 10th day (stationary phase) of the ultraviolet radiation experiment, samples were taken twice respectively, that is, samples were taken before and after turning on the ultraviolet radiation lamp on the same day. Immediately after the first sampling, the ultraviolet lamp was turned on, and immediately after turning off the ultraviolet lamp, the second sampling was carried out. The results showed that if the sampling time was too long after turning off the lamp, there was no difference in pigment content between groups, and the diatoms would have physiological behaviors similar to recovery. Therefore, it was necessary to immediately collect samples after turning off the ultraviolet lamp on the sample collection day.

[0124] The following conclusions can be drawn from the above results:

[0125] (1) Generally speaking, ultraviolet radiation will reduce photosynthetic efficiency and biomass to a certain extent;

[0126] (2) UVB irradiation during the exponential phase has a significant promoting effect on the production of fucoxanthin. When the culture reaches the stationary phase at the end, ultraviolet radiation has no significant effect on fucoxanthin synthesis;

[0127] (3) Both UVA and UVB can significantly increase the production of fucoxanthin during the exponential phase, but there is still no significant difference in fucoxanthin content between groups during the stationary phase;

[0128] (4) Phaeodactylum tricornutum responds rapidly to pigment synthesis under ultraviolet radiation, and it is related to the culture stage. The reason may be related to the growth state of diatoms and pigment synthesis strategies; it may also be that Phaeodactylum tricornutum has strong adaptability to ultraviolet radiation.

[0129] (5) From the perspective of industrial production, UVB radiation can significantly increase the production of fucoxanthin in Phaeodactylum tricornutum during the exponential phase. If this method is selected to promote fucoxanthin synthesis, semi-continuous culture is selected to ensure that the culture system always remains in the exponential phase to ensure that the diatoms are in a relatively active growth state. The synthesis of fucoxanthin is very sensitive to ultraviolet radiation, and samples can be collected immediately after ultraviolet radiation to shorten the recovery period.

[0130] It can be concluded from the above examples that the present invention can effectively improve the pigment yield in Phaeodactylum tricornutum by appropriate light-dark cycles or ultraviolet radiation treatments.

[0131] Although the above examples have described the present invention in detail, they are only a part of the embodiments of the present invention, not all embodiments. People can also obtain other embodiments based on these embodiments without creative work, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for increasing the pigment yield in diatoms, characterized in that: The method comprises the following steps: during the cultivation process of diatoms, subjecting the diatoms to light treatment or ultraviolet radiation treatment, and collecting samples from the exponential phase to the plateau phase; The light-dark cycle of the light treatment is 12h light: 12h dark or 2h light: 2h dark; The ultraviolet radiation treatment includes UVB radiation treatment and / or UVA radiation treatment; The radiation intensity of the UVB radiation treatment is 1 W / m 2 ; The radiation intensity of the UVA radiation treatment is 30W / m 2 ; The frequency of the ultraviolet radiation treatment is 1.5h / day; The pigment includes one or more of fucoxanthin, violaxanthin, diatomaxanthin and diatomaxanthin.

2. The method according to claim 1, characterized in that The light source for the illumination treatment is a D65 standard light source.

3. The method according to claim 1, characterized in that The illumination intensity of the illumination treatment is 70 to 80 μmol·m -2 ·s -1 .

4. The method according to claim 1, characterized in that: The illumination intensity of the ultraviolet radiation treatment is 60 to 70 μmol·m -2 ·s -1 .

5. The method according to claim 1, characterized in that The diatom culture method is one-time culture or semi-continuous culture.

6. The method according to claim 5, characterized in that The culture medium for the one-time culture or semi-continuous culture is f / 2 culture medium, and the culture temperature is 18-22°C.

7. The method according to claim 1, characterized in that The initial concentration of the diatom culture is (7-9)×10 4 cells / mL.

8. The method according to any one of claims 1 to 7, characterized in that: The diatoms include Phaeophyta tricornuta.

9. The method according to claim 8, characterized in that When the pigment is fucoxanthin, the light-dark cycle of the light treatment is 2h light: 2h dark; samples are collected from the late exponential phase to the plateau phase, and the late exponential phase is the 7th to 8th day of the exponential phase.

10. The method according to claim 8, characterized in that When the pigment is fucoxanthin, the ultraviolet radiation treatment is UVB radiation treatment; and the samples are collected during the exponential phase.

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