Culture method for improving yield of active substances of marine microalgae

Abstract

The invention discloses a culture method for improving the yield of active substances of marine microalgae, and relates to the technical field of algae culture, and the culture method comprises the following steps: S1, preparing a culture medium; s2, preparing a seed solution; and S3, adding an allogenic material and culturing. According to the method, the growth of phaeodactylum tricornutum and the accumulation of fucoxanthin are promoted by adding food-grade allogenic materials which are low enough in cost and are biologically safe.

Description

Technical Field

[0001] The invention relates to the technical field of algae cultivation, and in particular to a cultivation method for increasing the yield of active substances in marine microalgae by adding exogenous substances. Background Art

[0002] Fucoxanthin is a special type of carotenoid - allene carotenoid. This special configuration brings it many powerful biological activities, such as anti-inflammatory, anti-tumor, anti-obesity, anti-oxidation and anti-diabetes, and even has potential in the treatment of Alzheimer's disease. This series of biological activities makes fucoxanthin have great market potential in the fields of food, medicine, health products and cosmetics.

[0003] Fucoxanthin is a marine fat-soluble carotenoid, which can account for more than 10% of the total carotenoids in the ocean. In the ocean, fucoxanthin is mainly found in diatoms, golden algae and some brown algae. In the past, the source of fucoxanthin was basically the adult of large seaweeds such as kelp and Sargassum. Although the biomass was large, the fucoxanthin yield was extremely low, and the outdoor culture conditions were difficult to control. Various difficulties have led to the high price of fucoxanthin. In recent years, it has been found that compared with large algae such as Sargassum, kelp and kelp, the content of fucoxanthin in microalgae is higher, especially diatoms, such as: triangular brown finger algae.

[0004] Brown finger algae is a marine microalgae belonging to the phylum Bacillariophyceae, class Pinnata, family Phaeophyceae, order Phaeophyceales and genus Phaeophyceae. Domestic and foreign studies have reported that Brown finger algae contains a large amount of polyunsaturated fatty acids, fucoxanthin, golden algae kelp polysaccharide and other substances with good biological activity. In addition, because Brown finger algae is a microalgae, it has the advantages of fast growth and reproduction and easy control of culture conditions, which makes it very suitable for the production of fucoxanthin. However, although the fucoxanthin content of natural Brown finger algae is much higher than that of large seaweeds such as kelp, it is still insufficient compared to the market gap. At present, the optimization of conditions for the cultivation of microalgae still stays in common physical and chemical factors such as light, temperature, culture medium components, or the addition of chemical induction factors such as plant hormones, and the cultivation of Brown finger algae is also the same. For example, Cai Luyun et al. used β-cyclodextrin to increase the yield of active substances in marine microalgae. Liu Hao et al. used rapamycin to induce the increase of fucoxanthin. Zhu Shuaiqi and others used acetylsalicylic acid, arachidonic acid, ammonium cerium sulfate and methyl jasmonic acid to achieve the same goal. However, although these methods are effective to a certain extent, they are not ideal. The cost and biosafety of using chemical products such as plant hormones or rare earth elements also have certain problems.

[0005] Therefore, it is necessary to seek more effective cultivation methods to increase the production of active substances in marine microalgae. Summary of the invention

[0006] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a cultivation method for increasing the yield of active substances in marine microalgae, which promotes the growth of Phaeodactylum tricornutum and the accumulation of fucoxanthin by adding sufficiently low-cost and biosafe food-grade exogenous substances.

[0007] To achieve the above object, the technical solution adopted by the present invention is: a cultivation method for increasing the yield of active substances in marine microalgae, comprising the following steps:

[0008] Step S1, preparing a culture medium: preparing a culture medium according to a culture medium formula for culturing Phaeodactylum triangularis, and sterilizing the prepared culture medium for later use;

[0009] Step S2, seed liquid preparation: 2-10 mL of liquid seeds of Phaeodactylum triangularis for seed preservation are inoculated into a pre-sterilized conical flask, which contains 20-100 mL of sterilized culture medium. The whole inoculation process is carried out in an ultra-clean UV workbench; after inoculation, the seeds are placed in a light incubator and cultured until the exponential phase before entering the next stage;

[0010] Step S3, adding exogenous substances and culturing: adding exogenous substances to the culture medium for culturing P. triangularis, taking the first-level seed liquid expanded in the previous step and inoculating it into the culture medium added with the exogenous substances, the whole inoculation process should be carried out in an ultra-clean UV workbench, and culturing is carried out again after the inoculation is completed.

[0011] Preferably, the culture medium for culturing the triangular algae in step S1 is formulated as follows: 0.05-0.08 g / L sodium nitrate, 0.001-0.01 g / L sodium dihydrogen phosphate dihydrate, 0.01-0.1 g / L sodium silicate nonahydrate, 1-2 mL / L trace element solution, 0-100 mmol / L glycerol, and the balance is water.

[0012] Preferably, the trace element solution includes components with the following concentrations: 1-5 g / L disodium oxalate tetraacetate, 1-5 g / L ferric chloride hexahydrate, 0.01-1 g / L copper sulfate pentahydrate, 0.01-1 g / L zinc sulfate heptahydrate, 0.01-1 g / L cobalt chloride hexahydrate, 0.01-1 g / L manganese chloride tetrahydrate, and 0.001-0.1 g / L sodium molybdate hexahydrate.

[0013] Preferably, the final pH value of the culture medium for culturing Phaeodactylum tricornutum is 7.6-7.8, and the salinity is 20-30‰.

[0014] Preferably, the sterilization in step S1 is high temperature sterilization, the sterilization temperature is 120° C., and the time is 10-20 min.

[0015] Preferably, the temperature of the illumination incubator in step S2 is 15-30° C., and the light intensity is 1000-8000 Lux.

[0016] Preferably, the exogenous substance in step S3 is at least one of olive oil, lutein, sodium glutamate, and tomato extract.

[0017] Preferably, the exogenous substance in step S3 is diluted with a small amount of sterile water and filtered using a filter membrane with a pore size of 0.22-0.45 μm before being added.

[0018] Preferably, the added concentration of the exogenous substance in step S3 is 3-300 mg / L.

[0019] Preferably, in step S3, the volume ratio of the primary seed solution expanded in the previous step to the culture medium added with exogenous substances is 1:(10-30), and more preferably, the volume ratio is 1:10.

[0020] Preferably, the ambient temperature of the secondary cultivation in step S3 is 20-25° C., and the light intensity is 3000-5000 Lux.

[0021] Due to the application of the above technical solution, the present invention has the following beneficial effects: the present invention adds exogenous substances to the culture medium before the start of cultivation, and through a period of cultivation, the cultured triangular brown finger algae obtains a higher content or concentration of fucoxanthin in the required cultivation stage, thereby achieving the purpose of increasing the yield of fucoxanthin; through the reasonable selection of the culture medium composition formula, the type and amount of exogenous substances, the culture conditions, etc., after adding sufficiently low-cost and biosafe food-grade exogenous substances, the growth of triangular brown finger algae and the accumulation of fucoxanthin can be better promoted. The present invention proposes new insights into the cultivation technology of triangular brown finger algae. There are technical advances in biomass, fucoxanthin content and fucoxanthin concentration, which has opened up a wider market for the cultivation of triangular brown finger algae and the production and application of fucoxanthin. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Growth curves of Phaeodactylum triangularis after adding exogenous substances in Examples 1-4 of the present invention and Comparative Example 1;

[0023] Figure 2 Schematic diagram of fucoxanthin content and concentration after adding exogenous substances and culturing for 8 days in Examples 1-4 of the present invention and Comparative Example 1;

[0024] Figure 3 Schematic diagram of fucoxanthin content and concentration after adding exogenous substances and culturing for 16 days in Examples 1-4 and Comparative Example 1. DETAILED DESCRIPTION

[0025] The following description is used to disclose the present invention so that those skilled in the art can implement the present invention. The preferred embodiments described below are only examples, and those skilled in the art may think of other obvious variations.

[0026] Example 1

[0027] The formula of the culture medium is: sodium nitrate 0.075 g, sodium dihydrogen phosphate dihydrate 0.00565 g, sodium silicate nonahydrate 0.030 g, trace element solution 1 mL, glycerol 100 mmol L -1 , add water to make up to 1L.

[0028] Each liter of trace element solution contains the following ingredients: 4.16g disodium oxalate tetraacetate, 3.15g ferric chloride hexahydrate, 0.01g copper sulfate pentahydrate, 0.022g zinc sulfate heptahydrate, 0.01g cobalt chloride hexahydrate, 0.18g manganese chloride tetrahydrate, 0.006g sodium molybdate hexahydrate. The final pH value is 7.6 and the salinity is 20‰.

[0029] The prepared culture medium is dispensed into conical flasks, 200 mL per bottle. After being dispensed into conical flasks, the culture medium is sealed with a biological breathable sealing film and sterilized. A high-temperature steam sterilizer is used for sterilization at 120°C for 20 minutes. The sterilized culture medium is placed in a UV clean bench to cool to room temperature and wait for transfer.

[0030] Tomato extract was added to the culture medium at a concentration of 3 mg·L -1 ; The preparation steps of tomato extract are as follows: take 1000g of fresh ripe tomatoes, wash the surface and remove the fruit stems. Cut the tomatoes into pieces and use a press to crush and squeeze them into a solid-liquid mixture. Filter the solid-liquid mixture, collect the clear liquid, and concentrate it to obtain the tomato extract. The tomato extract is not sterilized with the culture medium, but is filtered in a UV clean bench using a 0.22μm filter membrane and then added to the culture medium cooled to room temperature to prevent contamination by miscellaneous bacteria. After adding the tomato extract, the inoculation operation is performed, and 20mL of seed solution is inoculated into the culture medium with the tomato extract added using a disposable pipette. After the inoculation is completed, the bottle mouth is burned on a flame and sealed with a sealing film.

[0031] After inoculation and sealing, place it in a light shaker with the following parameters: light intensity of 5000 Lux, temperature of 20 ° C, and speed of 100 rpm. Take samples on the 8th and 16th days to determine the biomass and fucoxanthin production of P. triangularis. The sampling operation method is the same as inoculation. Take out 20 mL each time and put it into a centrifuge tube for analysis. Pay attention to the aseptic operation requirements when sampling; after sampling, continue to seal and put it back into the shaker.

[0032] Biomass determination: Take 10mL of culture solution, centrifuge at 9000rpm for 10min, discard the supernatant and add about 20mL of ultrapure water again, mix well; repeat the centrifugation operation three times. Wash the centrifuged algae mud with a washing bottle filled with ultrapure water into a weighed flat weighing dish. The weighing dish has been dried to constant weight at 90℃ in advance and cooled to room temperature in a dryer. The weighing dish containing algae mud is placed in an oven and dried to constant weight. Subtract the weight of the weighing dish to obtain the dry biomass of P. triangularis in the taken volume of algae liquid.

[0033] Fucoxanthin was determined using high performance liquid chromatography. A standard curve of fucoxanthin concentration and peak area was established by using different concentrations of fucoxanthin standards. Fucoxanthin during the culture process was determined using methanol extraction. Fucoxanthin standards were prepared with methanol at different concentrations (1, 2, 3, 4, 6, 8, and 10 mg·L -1 ) sample, the injection volume was fixed at 20 μL. The fucoxanthin peak area was detected by HPLC at 448 nm, and the HPLC model was Agilent 1260 Infinity. The standard curve formula obtained was: y = 0.00963x + 0.01937 (R2 = 0.9971), where x (mAU·min) is the sample peak area, and y is the fucoxanthin concentration corresponding to the sample (mg·L -1 ).

[0034] Take 10mL of algae liquid during the culture process, centrifuge it three times as when measuring dry biomass to obtain algae mud, add methanol, ultrasonicate for 10 minutes, then extract in the dark for more than 2 hours; centrifuge again to collect the supernatant. Observe the color of the algae residue. If it is still green, add new methanol again and repeat the extraction process until the algae residue is observed to be grayish white. Combine the supernatants, filter the supernatants with a 0.22μm filter, enter the liquid chromatography analysis, substitute the standard curve to calculate the fucoxanthin concentration, and then convert it into the algae fucoxanthin content and the fucoxanthin concentration in the algae liquid according to the algae dry biomass or cell number data.

[0035] Example 2

[0036] The formula of the culture medium is: sodium nitrate 0.075 g, sodium dihydrogen phosphate dihydrate 0.00565 g, sodium silicate nonahydrate 0.030 g, trace element solution 1 mL, glycerol 100 mmol·L -1 , add water to make up to 1L.

[0037] Each liter of trace element solution contains the following ingredients: 4.16 g of disodium tetraacetate, 3.15 g of ferric chloride hexahydrate, 0.01 g of copper sulfate pentahydrate, 0.022 g of zinc sulfate heptahydrate, 0.01 g of cobalt chloride hexahydrate, 0.18 g of manganese chloride tetrahydrate, and 0.006 g of sodium molybdate hexahydrate. The final pH value is 7.7 and the salinity is 20‰.

[0038] The prepared culture medium is dispensed into conical bottles, 200 mL per bottle; after the culture medium is dispensed into the conical bottles, it is sealed with a biological breathable sealing film and sterilized. The sterilization is performed using a high-temperature steam sterilizer at 120°C for 20 minutes. The sterilized culture medium is placed in a UV clean bench to cool to room temperature and wait for transfer.

[0039] Lutein (commercially available food grade) was added to the culture medium at an amount of 20 mg / L -1 Lutein is not sterilized along with the culture medium, but is filtered through a 0.22μm filter membrane in a UV clean bench and then added to the culture medium that has been cooled to room temperature to prevent contamination by foreign bacteria. After adding lutein, inoculation is performed, using a disposable pipette to inoculate 20mL of seed solution into the culture medium with lutein added; after inoculation, the bottle mouth is burned over a flame and sealed with a sealing film.

[0040] After inoculation and sealing, place it in a light shaker with the following shaker parameters: light intensity of 5000 Lux, temperature of 20 °C, and speed of 100 rpm. Take samples on the 8th and 16th days to determine the biomass and fucoxanthin production of P. triangularis. The sampling operation method is the same as inoculation. Take out 20 mL each time and put it into a centrifuge tube for distribution; pay attention to the aseptic operation requirements when sampling; after sampling, continue to seal and put it back into the shaker.

[0041] Biomass determination: Take 10mL of culture medium, centrifuge at 9000rpm for 10min, discard the supernatant and add about 20mL of ultrapure water again, mix well. Repeat the centrifugation operation three times. Wash the centrifuged algae mud with a washing bottle filled with ultrapure water into a weighed flat weighing dish. The weighing dish has been dried to constant weight at 90℃ in advance and cooled to room temperature in a dryer. The weighing dish containing algae mud is placed in an oven and dried to constant weight. Subtract the weight of the weighing dish to obtain the dry biomass of P. triangularis in the taken volume of algae liquid.

[0042] Fucoxanthin was determined using high performance liquid chromatography. A standard curve of fucoxanthin concentration and peak area was established by using different concentrations of fucoxanthin standards. Fucoxanthin during the culture process was determined using methanol extraction. Fucoxanthin standards were prepared with methanol at different concentrations (1, 2, 3, 4, 6, 8, and 10 mg·L -1) sample, the injection volume was fixed at 20 μL. The fucoxanthin peak area was detected by HPLC at 448 nm, and the HPLC model was Agilent 1260 Infinity. The standard curve formula obtained was: y = 0.00963x + 0.01937 (R2 = 0.9971), where x (mAU·min) is the sample peak area, and y is the fucoxanthin concentration corresponding to the sample (mg·L -1 ).

[0043] Take 10mL of algae liquid from the culture process, centrifuge it three times as when measuring the dry biomass to obtain algae mud. Add methanol, sonicate for 10 minutes, and then extract in the dark for more than 2 hours. Centrifuge again to collect the supernatant. Observe the color of the algae residue. If it is still green, add new methanol again and repeat the extraction process until the algae residue is observed to be grayish white. Combine the supernatants, filter the supernatants with a 0.22μm filter, enter the liquid chromatography analysis, substitute the standard curve to calculate the fucoxanthin concentration, and then convert it into the algae fucoxanthin content and the fucoxanthin concentration in the algae liquid according to the algae dry biomass or cell number data.

[0044] Example 3

[0045] The formula of the culture medium is: sodium nitrate 0.075 g, sodium dihydrogen phosphate dihydrate 0.00565 g, sodium silicate nonahydrate 0.030 g, trace element solution 1 mL, glycerol 100 mmol·L -1 , add water to make up to 1L.

[0046] Each liter of trace element solution contains the following ingredients: 4.16 g of disodium oxalate tetraacetate, 3.15 g of ferric chloride hexahydrate, 0.01 g of copper sulfate pentahydrate, 0.022 g of zinc sulfate heptahydrate, 0.01 g of cobalt chloride hexahydrate, 0.18 g of manganese chloride tetrahydrate, and 0.006 g of sodium molybdate hexahydrate.

[0047] The final pH value is 7.7 and the salinity is 20‰. The prepared culture medium is dispensed into conical flasks, 200 mL per bottle. After the culture medium is dispensed into conical flasks, it is sealed with a biological breathable sealing film and sterilized. Sterilization uses a high-temperature steam sterilizer at 120°C for 20 minutes. The sterilized culture medium is placed in a UV clean bench to cool to room temperature and wait for transfer.

[0048] Olive oil (commercially available food grade) is added to the transferred culture medium, with an addition amount of 1 mL per bottle. The olive oil is not sterilized along with the culture medium, but is filtered in a UV clean bench using a 0.22 μm filter membrane and then added to the culture medium that has been cooled to room temperature to prevent contamination by bacteria. After the olive oil is added, the inoculation operation is performed, and 20 mL of seed solution is inoculated into the culture medium with olive oil added using a disposable pipette. After the inoculation is completed, the bottle mouth is burned over a flame and sealed with a sealing film.

[0049] After inoculation and sealing, place it in a light shaker with the following parameters: light intensity of 5000 Lux, temperature of 20 ° C, and speed of 100 rpm. Take samples on the 8th and 16th days to determine the biomass and fucoxanthin production of P. triangularis. The sampling operation method is the same as inoculation. Take out 20 mL each time and put it into a centrifuge tube for analysis. Pay attention to the aseptic operation requirements when sampling. After sampling, continue to seal and put it back into the shaker.

[0050] Biomass determination: Take 10mL of culture solution, centrifuge at 9000rpm for 10min, discard the supernatant and add about 20mL of ultrapure water again, mix well, repeat the centrifugation operation three times, wash the algae mud after centrifugation with a washing bottle filled with ultrapure water into a weighed flat weighing dish. The weighing dish has been dried to constant weight at 90℃ in advance and cooled to room temperature in a dryer. The weighing dish containing algae mud is placed in an oven and dried to constant weight. Subtract the weight of the weighing dish to obtain the dry biomass of P. triangularis in the taken volume of algae liquid.

[0051] Fucoxanthin was determined using high performance liquid chromatography. A standard curve of fucoxanthin concentration and peak area was established by using fucoxanthin standards of different concentrations. The methanol extraction method was used to determine fucoxanthin during the culture process. The fucoxanthin standard was prepared into samples of different concentrations (1, 2, 3, 4, 6, 8 and 10 mg L-1, respectively) using methanol, and the injection volume was fixed at 20 μL. The fucoxanthin peak area was detected at 448 nm by high performance liquid chromatography, and the high performance liquid chromatography model was Agilent 1260 Infinity. The formula for the standard curve obtained is: y = 0.00963x + 0.01937 (R2 = 0.9971), where x (mAU·min) is the sample peak area, and y is the fucoxanthin concentration corresponding to the sample (mg·L -1 ).

[0052] Take 10mL of algae liquid during the culture process, centrifuge it three times as when measuring dry biomass to obtain algae mud, add methanol, ultrasonicate for 10 minutes, then extract in the dark for more than 2 hours, centrifuge again to collect the supernatant; observe the color of the algae residue, if it is still green, add new methanol again and repeat the extraction process until the algae residue is observed to be grayish white. Combine the supernatants, filter the supernatants with a 0.22μm filter, enter the liquid chromatography analysis, substitute the standard curve to calculate the fucoxanthin concentration, and then convert it into the algae fucoxanthin content and the fucoxanthin concentration in the algae liquid according to the algae dry biomass or cell number data.

[0053] Example 4

[0054] Preferably,

[0055] The formula of the culture medium is: sodium nitrate 0.075 g, sodium dihydrogen phosphate dihydrate 0.00565 g, sodium silicate nonahydrate 0.030 g, trace element solution 1 mL, glycerol 100 mmol·L -1 , add water to make up to 1L.

[0056] Each liter of trace element solution contains the following ingredients: 4.16 g of disodium oxalate tetraacetate, 3.15 g of ferric chloride hexahydrate, 0.01 g of copper sulfate pentahydrate, 0.022 g of zinc sulfate heptahydrate, 0.01 g of cobalt chloride hexahydrate, 0.18 g of manganese chloride tetrahydrate, and 0.006 g of sodium molybdate hexahydrate.

[0057] The final pH value was 7.8, and the salinity was 20‰; the prepared culture medium was dispensed into conical flasks, 200 mL per bottle; after being dispensed into conical flasks, the culture medium was sealed with a biological breathable sealing film and sterilized using a high-temperature steam autoclave at 120°C for 20 minutes. The sterilized culture medium was placed in a UV clean bench to cool to room temperature and wait for transfer.

[0058] The transfer medium was supplemented with sodium glutamate (commercially available food grade) at a concentration of 400 mg / L -1 . Sodium glutamate is not sterilized along with the culture medium, but is filtered in a UV clean bench using a 0.22 μm filter membrane and then added to the culture medium that has been cooled to room temperature to prevent contamination by foreign bacteria. After adding sodium glutamate, inoculation is performed, and 20 mL of seed solution is inoculated into the culture medium with sodium glutamate added using a disposable pipette. After inoculation, burn the bottle mouth over a flame and seal it with a sealing film.

[0059] After inoculation and sealing, place it in a light shaker with the following parameters: light intensity of 5000 Lux, temperature of 20 ° C, and speed of 100 rpm. Take samples on the 8th and 16th days to determine the biomass and fucoxanthin production of P. triangularis. The sampling operation method is the same as inoculation. Take out 20 mL each time and put it into a centrifuge tube for analysis. Pay attention to the aseptic operation requirements when sampling. After sampling, continue to seal and put it back into the shaker.

[0060] Biomass determination: Take 10mL of culture medium, centrifuge at 9000rpm for 10min, discard the supernatant and add about 20mL of ultrapure water again, mix well. Repeat the centrifugation operation three times. Wash the centrifuged algae mud with a washing bottle filled with ultrapure water into a weighed flat weighing dish. The weighing dish has been dried to constant weight at 90℃ in advance and cooled to room temperature in a dryer. The weighing dish containing algae mud is placed in an oven and dried to constant weight. Subtract the weight of the weighing dish to obtain the dry biomass of P. triangularis in the taken volume of algae liquid.

[0061] Fucoxanthin was determined using high performance liquid chromatography. A standard curve of fucoxanthin concentration and peak area was established by using different concentrations of fucoxanthin standards. Fucoxanthin during the culture process was determined using methanol extraction. Fucoxanthin standards were prepared with methanol at different concentrations (1, 2, 3, 4, 6, 8, and 10 mg·L -1 ) sample, the injection volume was fixed at 20 μL. The fucoxanthin peak area was detected by HPLC at 448 nm, and the HPLC model was Agilent 1260 Infinity. The standard curve formula obtained was: y = 0.00963x + 0.01937 (R2 = 0.9971), where x (mAU·min) is the sample peak area, and y is the fucoxanthin concentration corresponding to the sample (mg·L -1 ).

[0062] Take 10mL of algae liquid from the culture process, centrifuge it three times as when measuring the dry biomass to obtain algae mud. Add methanol, sonicate for 10 minutes, and then extract in the dark for more than 2 hours. Centrifuge again to collect the supernatant. Observe the color of the algae residue. If it is still green, add new methanol again and repeat the extraction process until the algae residue is observed to be grayish white. Combine the supernatants, filter the supernatants with a 0.22μm filter, enter the liquid chromatography analysis, substitute the standard curve to calculate the fucoxanthin concentration, and then convert it into the algae fucoxanthin content and the fucoxanthin concentration in the algae liquid according to the algae dry biomass or cell number data.

[0063] Comparative Example 1

[0064] Preferably,

[0065] Preferably,

[0066] The formula of the culture medium is: sodium nitrate 0.075 g, sodium dihydrogen phosphate dihydrate 0.00565 g, sodium silicate nonahydrate 0.030 g, trace element solution 1 mL, glycerol 100 mmol·L -1 , add water to make up to 1L.

[0067] Each liter of trace element solution contains the following ingredients: 4.16 g of disodium oxalate tetraacetate, 3.15 g of ferric chloride hexahydrate, 0.01 g of copper sulfate pentahydrate, 0.022 g of zinc sulfate heptahydrate, 0.01 g of cobalt chloride hexahydrate, 0.18 g of manganese chloride tetrahydrate, and 0.006 g of sodium molybdate hexahydrate.

[0068] The final pH value is 7.6 and the salinity is 20‰. The prepared culture medium is dispensed into conical bottles, 200 mL per bottle. After the culture medium is dispensed into the conical bottles, it is sealed with a biological breathable sealing film and sterilized. Sterilization uses a high-temperature steam sterilizer at 120°C for 20 minutes. The sterilized culture medium is placed in a UV clean bench to cool to room temperature and wait for transfer.

[0069] No exogenous substances are added to the transferred culture medium. After cooling to room temperature, inoculate the culture medium with 20 mL of seed solution using a disposable pipette. After inoculation, burn the bottle mouth over a flame and seal it with a sealing film.

[0070] After inoculation and sealing, place it in a light shaker with the following shaker parameters: light intensity of 5000 Lux, temperature of 20 °C, and speed of 100 rpm. Take samples on the 8th and 16th days to determine the biomass and fucoxanthin production of P. triangularis. The sampling operation method is the same as inoculation. Take out 20 mL each time and put it into a centrifuge tube for analysis. Pay attention to the aseptic operation requirements when sampling. After sampling, continue to seal and put it back into the shaker.

[0071] Biomass determination: Take 10mL of culture solution, centrifuge at 9000rpm for 10min, discard the supernatant and add about 20mL of ultrapure water again, mix well; repeat the centrifugation operation three times; wash the algae mud after centrifugation with a washing bottle filled with ultrapure water into a weighed flat weighing dish. The weighing dish has been dried to constant weight at 90℃ in advance and cooled to room temperature in a dryer; the weighing dish containing algae mud is placed in an oven and dried to constant weight, and the weight of the weighing dish is subtracted to obtain the dry biomass of P. triangularis in the taken volume of algae liquid.

[0072] Fucoxanthin was determined by high performance liquid chromatography. A standard curve of fucoxanthin concentration and peak area was established by using different concentrations of fucoxanthin standards. Fucoxanthin during the culture process was determined by methanol extraction. Fucoxanthin standards were prepared with methanol at different concentrations (1, 2, 3, 4, 6, 8, and 10 mg·L -1 ) sample, the injection volume was fixed at 20 μL. The fucoxanthin peak area was detected by HPLC at 448 nm, and the HPLC model was Agilent 1260 Infinity. The standard curve formula obtained was: y = 0.00963x + 0.01937 (R2 = 0.9971), where x (mAU·min) is the sample peak area, and y is the fucoxanthin concentration corresponding to the sample (mg·L -1 ).

[0073] Take 10mL of algae liquid during the culture process, centrifuge it three times as when measuring dry biomass to obtain algae mud; add methanol, ultrasonicate for 10 minutes, and then extract in the dark for more than 2 hours; centrifuge again to collect the supernatant, observe the color of the algae residue, if it is still green, add new methanol again and repeat the extraction process until the algae residue is observed to be grayish white. Combine the supernatants, filter the supernatants with a 0.22μm filter, enter liquid chromatography analysis, substitute the standard curve to calculate the fucoxanthin concentration, and then convert it into the algae fucoxanthin content and the fucoxanthin concentration in the algae liquid according to the algae dry biomass or cell number data.

[0074] The triangular phycodactylum cultured in Examples 1-4 and Comparative Example 1 was cultured for 16 days, and samples were taken during the culture process to measure the biomass during the culture process. Figure 1 .

[0075] Depend on Figure 1 It can be seen that during the culture process, the biomass of the triangular algae of all the examples and the comparative examples showed a continuous upward trend. However, it is obvious that from the beginning to the end of the culture, the biomass of the examples 1-4 was significantly higher than that of the comparative example. From the culture results, the biomass of the triangular algae of each example reached 0.8 g·L after 16 days of culture. -1 The highest value was in Example 4, which was 1.07±0.05 g·L -1 , much higher than that of Comparative Example 1. After 8 days of cultivation, the biomass of the triangular phytosalis of Examples 1-4 increased by about 76.04%, 103.13%, 66.67% and 114.58% respectively compared with the biomass of the triangular phytosalis of Comparative Example 1. The smallest difference between each example and Comparative Example 1 was 0.21 g·L in Example 3. -1 After 16 days of cultivation, the relative gap between Examples 1-4 and Comparative Example 1 was narrowing. For example, the biomass of the triangular phytodactyl algae in Example 3 was only increased by 45.56% compared with that in Comparative Example 1. However, as can be seen from Table 1, the minimum absolute gap between the biomass of the triangular phytodactyl algae in Examples 1-4 and that of the triangular phytodactyl algae in Comparative Example 1 was 0.21 g·L at 8 days of cultivation. -1 Increased to 0.25 g·L -1 .

[0076] Figure 2 and Figure 3 The fucoxanthin content of the triangular phycodactylum cultured in Examples 1-4 and Comparative Example 1 after 8 and 16 days of culture and the concentration of fucoxanthin in the algae solution. Figure 2 and Figure 3 It can be clearly observed that Examples 1-4 are significantly higher than Comparative Example 1 in all aspects, and have a significant improvement. Specifically, after 8 days of cultivation, the fucoxanthin content of the triangular flavonoids cultured in Examples 1-4 increased by 60.43%, 96.76%, 52.52% and 82.01% respectively compared with Comparative Example 1, and after 16 days of cultivation, the fucoxanthin content in the triangular flavonoids cultured in each Example was the highest in Example 4, which was 21.20±0.80 mg·g -1 , which is 1.89 times that of comparative example 1.

[0077] Since the biomass and fucoxanthin content of the triangular phycodactylum cultured in Examples 1-4 were much higher than those in Comparative Example 1, the concentration of fucoxanthin in each Example was surprisingly improved. Figure 2It can be observed that after 8 days of cultivation, the fucoxanthin concentrations in Examples 1-4 increased by 5.40 mg·L -1 、8.88mg·L -1 , 4.57mg·L -1 and 8.63 mg·L -1 In particular, Examples 2 and 4 were both more than 290% higher than Comparative Example 1. After 16 days of cultivation, the concentration of fucoxanthin in Examples 1-4 reached 17.41±0.05 mg·L -1 、19.51±0.07mg·L -1 , 12.88±0.60mg·L -1 and 22.65±1.78mg·L -1 , compared with comparative example 1, the maximum improvement is 258.98%.

[0078] Table 1 Maximum algal cell concentration, maximum fucoxanthin content and maximum fucoxanthin concentration during the culture process

[0079]

[0080] The above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with this technology to understand the contents of the present invention and implement them accordingly. They cannot be used to limit the protection scope of the present invention. All equivalent changes or modifications made according to the spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for increasing the yield of active substances in marine microalgae, characterized in that: The steps include: Step S1, preparing a culture medium: preparing a culture medium according to a culture medium formula for culturing Phaeodactylum triangularis, and sterilizing the prepared culture medium for later use; Step S2, seed liquid preparation: 2-10 mL of liquid seeds of Phaeodactylum triangularis for seed preservation are inoculated into a pre-sterilized conical flask, which contains 20-100 mL of sterilized culture medium. The whole inoculation process is carried out in an ultra-clean UV workbench; after inoculation, the seeds are placed in a light incubator and cultured until the exponential phase before entering the next stage; Step S3, adding exogenous substances and culturing: adding exogenous substances to the culture medium for culturing P. triangularis, taking the first-level seed liquid expanded in the previous step and inoculating it into the culture medium added with the exogenous substances, the whole inoculation process should be carried out in an ultra-clean UV workbench, and culturing is carried out again after the inoculation is completed.

2. The method for cultivating marine microalgae to increase the yield of active substances according to claim 1, characterized in that: The formula of the culture medium for culturing the triangular algae in step S1 is: 0.05-0.08 g / L of sodium nitrate, 0.001-0.01 g / L of sodium dihydrogen phosphate dihydrate, 0.01-0.1 g / L of sodium silicate nonahydrate, 1-2 mL / L of trace element solution, 0-100 mmol / L of glycerol, and the balance is water.

3. The method for cultivating marine microalgae to increase the yield of active substances according to claim 2, characterized in that: The trace element solution includes components with the following concentrations: 1-5 g / L disodium oxalate tetraacetate, 1-5 g / L ferric chloride hexahydrate, 0.01-1 g / L copper sulfate pentahydrate, 0.01-1 g / L zinc sulfate heptahydrate, 0.01-1 g / L cobalt chloride hexahydrate, 0.01-1 g / L manganese chloride tetrahydrate, and 0.001-0.1 g / L sodium molybdate hexahydrate.

4. The method for cultivating marine microalgae to increase the yield of active substances according to claim 1, characterized in that: The final pH value of the culture medium for culturing the triangular phycodactylum is 7.6-7.8, and the salinity is 20-30‰.

5. The method for cultivating marine microalgae to increase the yield of active substances according to claim 1, characterized in that: The sterilization in step S1 is high temperature sterilization, the sterilization temperature is 120° C., and the time is 10-20 minutes.

6. The method for cultivating marine microalgae for increasing the yield of active substances according to claim 1, characterized in that: The temperature of the illumination incubator in step S2 is 15-30° C., and the light intensity is 1000-8000 Lux.

7. The method for cultivating marine microalgae to increase the yield of active substances according to claim 1, characterized in that: The exogenous substance in step S3 is at least one of olive oil, lutein, sodium glutamate, and tomato extract.

8. The method for increasing the yield of active substances in marine microalgae according to claim 1, characterized in that: The exogenous substance in step S3 is diluted with a small amount of sterile water before addition and filtered using a filter membrane with a pore size of 0.22-0.45 μm; the added concentration of the exogenous substance in step S3 is 3-300 mg / L.

9. The method for increasing the yield of active substances in marine microalgae according to claim 1, characterized in that: In step S3, the volume ratio of the primary seed solution expanded in the previous step to the culture medium added with exogenous substances is 1:(10-30), and more preferably, the volume ratio is 1:

10.

10. The method for cultivating marine microalgae to increase the yield of active substances according to claim 1, characterized in that: The ambient temperature of the secondary culture in step S3 is 20-25° C., and the light intensity is 3000-5000 Lux.

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