CN111411069B - Method for increasing fucoxanthin content of phaeodactylum tricornutum by using photosynthesis promoter - Google Patents
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Abstract
The invention provides a method for improving fucoxanthin content of phaeodactylum tricornutum by using a photosynthesis promoter, belongs to the technical field of algae biology, and particularly relates to a method for improving fucoxanthin content of phaeodactylum tricornutum by using a photosynthesis promoter; according to the invention, the phaeodactylum tricornutum is cultured in a self-made culture medium, when the phaeodactylum tricornutum reaches the logarithmic growth phase, the photoinduced pigment PIF is added, the cell growth of the phaeodactylum tricornutum can be improved, the fucoxanthin related gene expression can be rapidly induced, and the rapid accumulation of fucoxanthin can be promoted, when the phaeodactylum tricornutum is cultured to reach the logarithmic growth phase, the photoinduced pigment is added, the cell amount of the phaeodactylum tricornutum is improved by 17.7%, the content of the fucoxanthin is improved by 44%, and the problem that the fucoxanthin is scarce in the prior art is successfully solved.
Description
Technical Field
The invention relates to the technical field of algae biology, in particular to a method for improving fucoxanthin content of phaeodactylum tricornutum by using a photosynthesis promoter.
Background
Fucoxanthin (Fucoxanthin) is a natural pigment of xanthophylls in carotenoids, and research shows that the Fucoxanthin has various health promotion characteristics, such as anti-obesity, anti-diabetes, blood pressure reduction, antioxidation, anticancer, neuroprotection, eye inflammation treatment and the like, and can be widely applied to the market as a medicine, a skin care and beauty product and a health care product; among them, the antioxidant properties of fucoxanthin are considered to be the main mechanism for exerting a health-beneficial effect.
Fucoxanthin is widely present in various algae, marine phytoplankton, aquatic shellfish and other animals and plants; the earliest substances for extracting fucoxanthin are mainly macroalgae, such as kelp, sargassum fusiforme and the like, and the development and utilization of the fucoxanthin are limited due to high culture cost, low extraction content and the like; researchers at home and abroad find that the phaeodactylum tricornutum also contains fucoxanthin and has the advantages of fast growth and reproduction, easy artificial culture and the like, but the fucoxanthin content in the phaeodactylum tricornutum is extremely low, so that the fucoxanthin price is extremely expensive, and therefore, a method for improving the fucoxanthin content in the phaeodactylum tricornutum is urgently needed.
Disclosure of Invention
The invention aims to provide a method for improving fucoxanthin content of phaeodactylum tricornutum by using a photosynthesis promoter, which overcomes the defects in the prior art.
In order to achieve the purpose, the invention provides the following technical scheme:
the invention provides a method for improving fucoxanthin content of phaeodactylum tricornutum by using a photosynthesis promoter, which specifically comprises the following steps:
(1) inoculating and culturing: inoculating Phaeodactylum tricornutum into improved F/2 culture solution, inoculating and culturing, and shaking the algae regularly during the culture process;
(2) and (3) growth determination: taking the phaeodactylum tricornutum algae liquid, measuring the absorbance of the phaeodactylum tricornutum algae liquid by using an ultraviolet spectrophotometer, converting the absorbance into the number of cells, and determining the growth cycle of the cells;
(3) adding of photosynthetic inducer: and when the growth of the phaeodactylum tricornutum reaches the logarithmic growth phase, adding the photosynthetic inducer, continuously culturing for 40-60 h, and determining the content of fucoxanthin in the phaeodactylum tricornutum.
Preferably, the composition of the modified F/2 culture solution is as follows: sodium nitrate 74.8 is red, sodium dihydrogen phosphate 4.4 are red light, 8.4~16.7 of sodium silicate are polished round, 1mL of F2 microelement solution, 1mL of F2 vitamin solution, disinfection seawater 1000 mL.
Preferably, the composition of the F/2 trace element solution is as follows: 23mg zinc sulfate, 10mg copper sulfate, 178mg manganese chloride, 3.9mg ferric citrate, 7.3mg sodium molybdate, 4.35mg sodium ethylene diamine tetracetate, 12mg cobalt chloride and 1000mL purified water.
Preferably, the composition of the F/2 trace element solution is as follows: 120.5mg of vitamin B, 0.5mg of vitamin H, 1000mL of vitamin B and pure water.
Preferably, the temperature of the inoculation culture is 20 +/-1 ℃, and the illumination intensity is 60-80 mu mol/(m) 2 S), the light and dark periods alternate, each period being 12 h.
Preferably, during the inoculation culture, the weight ratio of the phaeodactylum tricornutum to the improved F/2 culture solution is 1: 2-3.
Preferably, in the step (2), the method for converting the absorbance of the algal solution into the number of cells is as follows: measuring the absorbance value of the phaeodactylum tricornutum algae liquid at 680nm on a UV-5200 ultraviolet spectrophotometer by taking 4mL of the algae liquid, and obtaining the value according to the regression equation y of 124.14x-9.5138 (R) 2 0.9983), calculating the cell density of phaeodactylum tricornutum;
wherein x is the absorbance at 680nm and y is the cell density of Phaeodactylum tricornutum.
Preferably, the addition concentration of the photosynthetic inducer is 0.01-10 mug/L.
Preferably, the determination method of the fucoxanthin content in the phaeodactylum tricornutum comprises the following steps:
(1) taking 50mL of algae solution treated by the photosynthetic inducer, centrifuging at 4 ℃ and 5000rpm for 10min to obtain algae mud, and freeze-drying in vacuum for 24h to obtain solid algae;
(2) mixing solid algae and anhydrous ethanol at a ratio of 1:40(g/mL), extracting at 60 deg.C in dark for 1h, removing supernatant, adding equal volume of anhydrous ethanol, mixing, and repeatedly extracting for 1h to obtain extractive solution;
(3) centrifuging the leaching solution at 5000rpm for 10min, collecting supernatant, and measuring the concentration of the supernatant in A 445 The absorbance of (f) is represented by (C ═ 1000 XA) 445 Calculating the fucoxanthin content by multiplying N by V)/(A1% by multiplying cm by M by 100);
wherein N represents the dilution factor; v represents the volume of the extract; m is the sample mass; a1% cm is 1600.
The invention has the beneficial technical effects that:
the invention provides a method for improving fucoxanthin content of phaeodactylum tricornutum by using a photosynthetic accelerant, which comprises the steps of culturing phaeodactylum tricornutum in a self-made culture medium, adding a photosynthetic inducer PIF when the phaeodactylum tricornutum reaches a logarithmic growth phase, and adding the photosynthetic inducer as the photosynthetic accelerant, so that the cell growth of the phaeodactylum tricornutum can be improved, the expression of genes related to fucoxanthin can be quickly induced, and the quick accumulation of the fucoxanthin can be promoted; experimental results show that when the Phaeodactylum tricornutum culture reaches the logarithmic growth phase, the photosynthetic inducer is added, the cell amount of the Phaeodactylum tricornutum is increased by 17.7%, the content of fucoxanthin is increased by 44.2%, and a better technical effect is achieved.
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FIG. 1 is a graph showing the effect on the cell growth of Phaeodactylum tricornutum after treatment in examples and comparative examples;
FIG. 2 is a graph showing the effect of treatment of examples and comparative examples on the fucoxanthin content of Phaeodactylum tricornutum.
Detailed Description
The invention provides a method for improving fucoxanthin content of phaeodactylum tricornutum by using a photosynthetic accelerant, which specifically comprises the following steps:
(1) inoculating and culturing: inoculating Phaeodactylum tricornutum into improved F/2 culture solution, inoculating and culturing, and shaking the algae regularly during the culture process;
(2) and (3) growth determination: taking the phaeodactylum tricornutum algae liquid, measuring the absorbance of the phaeodactylum tricornutum algae liquid by using an ultraviolet spectrophotometer, converting the absorbance into the number of cells, and determining the growth cycle of the cells;
(3) adding of photosynthetic inducer: and when the growth of the phaeodactylum tricornutum reaches the logarithmic growth phase, adding the photosynthetic inducer, continuously culturing for 40-60 h, and determining the content of fucoxanthin in the phaeodactylum tricornutum.
In the present invention, the composition of the modified F/2 culture solution is preferably: 74.8 mg of sodium nitrate, 4.4 mg of sodium dihydrogen phosphate, 8.4-16.7 mg of sodium silicate, 1mL of F/2 trace element solution, 1mL of F/2 vitamin solution and 1000mL of disinfected seawater; wherein the composition of the F/2 trace element solution is preferably as follows: 23mg of zinc sulfate, 10mg of copper sulfate, 178mg of manganese chloride, 3.9mg of ferric citrate, 7.3mg of sodium molybdate, 4.35mg of sodium ethylene diamine tetracetate, 12mg of cobalt chloride and 1000mL of purified water; the composition of the F/2 trace element solution is preferably as follows: vitamin B120.5mg, vitamin H0.5mg, vitamin B 1 100mg and 1000mL of purified water; the culture solution is prepared by the inventor, is closer to the growth environment, can meet the growth requirement of Phaeodactylum tricornutum, and is added with various nutrient elements to provide rich nutrition for Phaeodactylum tricornutum;
in the culture and inoculation process, the temperature of inoculation culture is preferably 20 +/-1 ℃, and the illumination intensity is preferably 60-80 mu mol/(m) 2 S), more preferably 70. mu. mol/(m) 2 S), in the light culture, light and dark cycles are alternately carried out, and the time of each cycle is 12 h; in the culture process, shaking algae is carried out at variable time, so that the position of the phaeodactylum tricornutum algae is convenient to replace, and the shaking algae is preferably carried out for 5-9 times every day; during culture and inoculation, the weight ratio of the phaeodactylum tricornutum to the improved F/2 culture solution is preferably 1: 2-3, and more preferably 1:2.
In the invention, during the inoculation culture process, the growth of the algae liquid is monitored, the algae liquid of the phaeodactylum tricornutum algae is taken every day, the absorbance of the algae liquid is measured by a UV-5200 type ultraviolet spectrophotometer (Shanghai Yuan analytical instruments Co., Ltd.), and the regression equation y is 124.14x-9.5138(R 2 0.9983), calculating the cell density of phaeodactylum tricornutum, wherein x is the absorbance at 680nm and y is the cell density of phaeodactylum tricornutum.
When the number of cells of the phaeodactylum tricornutum algae is obtained through calculation, and the growth of the phaeodactylum tricornutum algae is judged to reach a logarithmic growth phase (about 4 days after the phaeodactylum tricornutum algae is inoculated to reach the logarithmic growth phase), adding the photosynthetic inducer, wherein the adding concentration of the photosynthetic inducer is 0.01-10 mu g/L, namely 0.01-10 mu g of the photosynthetic inducer is added into 1L of the culture medium; the phaeodactylum tricornutum in logarithmic growth phase grows vigorously and vitality, the number of cells is the largest, and the photosynthetic elicitor is added at the moment, so that fucoxanthin-related gene expression can be rapidly induced, fucoxanthin rapid accumulation can be promoted, and the fucoxanthin rapid accumulation can be realized in a short time; after the Phaeodactylum tricornutum is inoculated for 40-60 hours, preferably, the photosynthetic inducer is added for 48 hours, and the fucoxanthin content in the Phaeodactylum tricornutum is determined by the following method:
(1) taking 50mL of algae liquid treated by the photosynthetic inducer, carrying out centrifugal separation for 10min at 4 ℃ and 5000rpm to obtain algae mud, and carrying out vacuum freeze drying for 24h to obtain solid algae;
(2) mixing solid algae and anhydrous ethanol at a ratio of 1:40(g/mL), extracting at 60 deg.C in dark for 1h, removing supernatant, adding equal volume of anhydrous ethanol, mixing, and repeatedly extracting for 1h to obtain extractive solution;
(3) centrifuging the leaching solution at 5000rpm for 10min, collecting supernatant, and measuring the concentration of the supernatant in A 445 The absorbance of (E) was measured by using (C ═ 1000 XA) 445 Calculating the fucoxanthin content by multiplying N by V)/(A1% by multiplying cm by M by 100);
wherein the light absorption value at A445 is determined by double-beam ultraviolet visible spectrophotometer with model of UV-4802, and the manufacturer is Shanghai Eunikov instruments Co., Ltd;
and N represents the dilution factor; v represents the volume of the extract; m is the sample mass; a1% cm is 1600.
The method for increasing fucoxanthin content of Phaeodactylum tricornutum provided by the present invention will be described in detail with reference to the following examples, but they should not be construed as limiting the scope of the present invention.
1) The Phaeodactylum tricornutum used in the invention is selected from the Phaeodactylum tricornutum cultured by the laboratory of Ningbo university;
2) the improved F/2 culture medium used in the inoculation culture is a self-made culture medium, and comprises 74.8 mg of sodium nitrate, 4.4 mg of sodium dihydrogen phosphate, 10mg of sodium silicate, 1mL of an F/2 trace element solution, 1mL of an F/2 vitamin solution and 1000mL of disinfected seawater; wherein the composition of the F/2 trace element solution is preferably as follows: 23mg of zinc sulfate, 10mg of copper sulfate, 178mg of manganese chloride, 3.9mg of ferric citrate, 7.3mg of sodium molybdate, 4.35mg of sodium ethylene diamine tetracetate, 12mg of cobalt chloride and 1000mL of purified water; the composition of the F/2 trace element solution is preferably as follows: vitamin B120.5mg, vitamin H0.5mg, vitamin B1100mg and 1000mL of purified water;
3) the content of fucoxanthin is determined in the following manner;
(1) taking 50mL of algae solution treated by the photosynthetic inducer, centrifuging at 4 ℃ and 5000rpm for 10min to obtain algae mud, and freeze-drying in vacuum for 24h to obtain solid algae;
(2) mixing solid algae and anhydrous ethanol at a ratio of 1:40(g/mL), extracting at 60 deg.C in dark for 1h, removing supernatant, adding equal volume of anhydrous ethanol, mixing, and repeatedly extracting for 1h to obtain extractive solution;
(3) centrifuging the leaching solution at 5000rpm for 10min, collecting supernatant, and measuring the concentration of the supernatant in A 445 The absorbance of (E) was measured by using (C ═ 1000 XA) 445 Calculating the fucoxanthin content by multiplying N by V)/(A1% by multiplying cm by M by 100);
the Phaeodactylum tricornutum, modified F/2 medium and determination of fucoxanthin content used in the following examples were all selected from the types or measurements provided above;
example 1:
the Phaeodactylum tricornutum (Phaeodactylum tricornutum) which is cultured in Ningbo university laboratory and is in logarithmic growth phase and well-grown is inoculated into improved F/2 culture solution in a weight ratio of 1:2 for inoculation culture, the culture temperature is 20 ℃, and the illumination intensity is 60 mu mol/(m 2 S) alternately culturing in light and dark periods, wherein each period is 12h, and the algae shaking operation is carried out occasionally in the culturing process for 5 times every day;
the cultured Phaeodactylum tricornutum algae solution is taken every day, and the absorbance value is measured by UV-5200 ultraviolet spectrophotometer produced by Shanghai Yuan analysis instruments Co., Ltd, and according to regression equation y of 124.14x-9.5138 (R) 2 0.9983) and the absorbance measured is converted to cell number, thereby determining the growth cycle of the cells;
when the Phaeodactylum tricornutum is inoculated and cultured for 4 days, namely the logarithmic growth phase of the Phaeodactylum tricornutum is reached, adding the photosynthetic inducer to ensure that the concentration of the photosynthetic inducer is 0.01 mu g/L (0.01 mu g of the photosynthetic inducer is contained in 1L of culture solution), and continuously culturing for 45 hours.
Example 2:
the Phaeodactylum tricornutum which is cultured in the laboratory of Ningbo university and is in logarithmic growth phase and well-grown is inoculated into the improved F/2 culture solution for inoculation culture at the weight ratio of 1:2.5, the culture temperature is 21 ℃, and the illumination intensity is 70 mu mol/(m mol) 2 S) alternately culturing in light and dark periods, wherein each period is 12h, and the algae shaking operation is carried out occasionally in the culturing process for 6 times every day;
the cultured Phaeodactylum tricornutum algae solution is taken every day, and the absorbance value is measured by UV-5200 ultraviolet spectrophotometer produced by Shanghai Yuan analysis instruments Co., Ltd, and according to regression equation y of 124.14x-9.5138 (R) 2 0.9983) and the absorbance measured is converted to cell number, thereby determining the growth cycle of the cells;
when the Phaeodactylum tricornutum is inoculated and cultured for 4 days, namely the logarithmic growth phase of the Phaeodactylum tricornutum is reached, adding the photosynthetic inducer to ensure that the concentration of the photosynthetic inducer is 1 mu g/L (1 mu g of the photosynthetic inducer is contained in 1L of culture solution), and continuously culturing for 48 hours.
Example 3:
the Phaeodactylum tricornutum (Phaeodactylum tricornutum) which is cultured in Ningbo university laboratory and is in logarithmic growth phase and well-grown is inoculated into improved F/2 culture solution in a weight ratio of 1:3 for inoculation culture, the culture temperature is 19 ℃, and the illumination intensity is 80 mu mol/(m 2 S) alternately culturing in light and dark periods, wherein each period is 12h, and the algae shaking operation is carried out occasionally in the culture process for 8 times every day;
the cultured Phaeodactylum tricornutum algae solution is taken every day, and the absorbance value is measured by UV-5200 ultraviolet spectrophotometer produced by Shanghai Yuan analysis instruments Co., Ltd, and according to regression equation y of 124.14x-9.5138 (R) 2 0.9983) and conversion of the absorbance values measuredThe number of cells, thereby determining the growth cycle of the cells;
when the Phaeodactylum tricornutum is inoculated and cultured for 4 days, namely the logarithmic growth phase of the Phaeodactylum tricornutum is reached, the photosynthetic inducer is added to ensure that the concentration of the photosynthetic inducer is 10 mu g/L (10 mu g of the photosynthetic inducer is contained in 1L of culture solution), and the culture is continued for 48 hours.
Comparative example 1:
comparative example 1 compared with example 1, only the photosynthetic inducer was not added, and the rest of the procedure was the same as example 1;
comparative example 2:
comparative example 2 compared with example 2, only the culture solution composition of Phaeodactylum tricornutum is different, and the rest is the same as example 2;
the composition of the phaeodactylum tricornutum culture medium in comparative example 2 was: na (Na) 2 SiO 3 、CuSO 4 、ZnSO 4 、MnCl 2 、EDTANa 2 、Na 2 MoO 4 、CoCl 2 、FeCl 3 、Na 2 SeO 3 、VB 12 、VB 7 、VB 1 、H 3 BO 3 、KCl、KBr、NaF、CaCl 2 、SrCl 2 、MgCl 2 、NaNO 3 、NaH 2 PO 4 And water.
The growth of the cells of Phaeodactylum tricornutum cultured in examples 1-3 and comparative examples 1-2 was determined and is shown in FIG. 1, wherein the experimental results of comparative example 1 were replaced by D1, comparative example 2 by D2, example 1 by S1, example 2 by S2, and example 3 by S3; as shown in fig. 1, the improved F/2 culture solution prepared by the inventor is selected to culture phaeodactylum tricornutum, and the photosynthetic inducer is added during the logarithmic growth phase of phaeodactylum tricornutum, so that the rapid growth of phaeodactylum tricornutum cells can be promoted, and the cell density in example 2 is increased by about 17.7% compared with that in D1 and D2.
Fucoxanthin in Phaeodactylum tricornutum cultured in examples 1-3 and comparative examples 1-2 was measured, and is shown in FIG. 2, wherein the experimental data of comparative example 1 was replaced with D1, comparative example 2 was replaced with D2, example 1 was replaced with S1, example 2 was replaced with S2, and example 3 was replaced with S3; as can be seen from FIG. 2, the inventors selectedThe Phaeodactylum tricornutum is cultured in a self-made improved F/2 culture medium, and the photosynthetic inducer is added in the logarithmic growth phase of the Phaeodactylum tricornutum, when the concentration of the photosynthetic inducer is 1 mu g/L, the content of fucoxanthin in the Phaeodactylum tricornutum is the highest, and compared with D1 and D2, the content of fucoxanthin is 2.59mg g -1 DW is improved by about 44 percent, great technical progress is made, and a wider market is developed for the application of fucoxanthin.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (6)
1. A method for increasing fucoxanthin content of Phaeodactylum tricornutum by using a photosynthesis promoter specifically comprises the following steps:
(1) inoculating and culturing: inoculating Phaeodactylum tricornutum into improved F/2 culture solution, inoculating and culturing, and shaking the algae regularly during the culture process;
(2) and (3) growth determination: taking the phaeodactylum tricornutum algae liquid, measuring the absorbance of the phaeodactylum tricornutum algae liquid by using an ultraviolet spectrophotometer, converting the absorbance into the number of cells, and determining the growth cycle of the cells;
(3) adding of photosynthetic inducer: when the growth of the phaeodactylum tricornutum reaches the logarithmic growth phase, adding a photosynthetic inducer, continuously culturing for 40-60 h, and determining the content of fucoxanthin in the phaeodactylum tricornutum;
the improved F/2 culture solution comprises the following components: performing water treatment on 74.8 mg of sodium nitrate, 4.4 mg of sodium dihydrogen phosphate, 8.4-16.7 mg of sodium silicate, 1mL of F/2 trace element solution, 1mL of F/2 vitamin solution and 1000mL of disinfected seawater;
the F/2 vitamin solution comprises the following components: 120.5mg of vitamin B, 0.5mg of vitamin H, 1000mL of vitamin B and pure water;
the addition concentration of the photosynthetic inducer is 0.01-10 mu g/L.
2. The method of claim 1, wherein the F/2 trace element solution comprises the following components: zinc sulfate 23, copper sulfate 10mg, manganese chloride 178mg, ferric citrate 3.9mg, sodium molybdate 7.3mg, sodium ethylene diamine tetracetate 4.35mg, cobalt chloride 12mg, pure water 1000 mL.
3. The method for increasing fucoxanthin content of Phaeodactylum tricornutum with the photosynthetic promoter as claimed in claim 1, wherein the temperature of said inoculation culture is 20 ± 1 ℃, and the illumination intensity is 60-80 μmol/(m) 2 S), the light and dark periods alternate, each period being 12 h.
4. The method for increasing fucoxanthin content of Phaeodactylum tricornutum with photosynthesis promoter as described in claim 1, wherein the ratio by weight of Phaeodactylum tricornutum to the modified F/2 culture solution in said inoculation culture is 1: 2-3.
5. The method for increasing fucoxanthin content of Phaeodactylum tricornutum with the photosynthesis promoter according to claim 1, wherein in the step (2), the method for converting the absorbance of the algal solution into the cell number is as follows: measuring absorbance of Phaeodactylum tricornutum algae liquid at 680nm with 4mL of algae liquid on UV-5200 ultraviolet spectrophotometer according to regression equation y =124.14x-9.5138 (R) 2 =0.9983), calculating the cell density of phaeodactylum tricornutum;
wherein x is the absorbance at 680nm and y is the cell density of Phaeodactylum tricornutum.
6. The method of claim 1, wherein the fucoxanthin content of Phaeodactylum tricornutum is determined by the following method:
(1) taking 50mL of algae solution treated by the photosynthetic inducer, centrifuging at 4 ℃ and 5000rpm for 10min to obtain algae mud, and freeze-drying in vacuum for 24h to obtain solid algae;
(2) mixing solid algae and anhydrous ethanol at a ratio of 1:40(g/mL), extracting at 60 deg.C in dark for 1h, removing supernatant, adding equal volume of anhydrous ethanol, mixing, and repeatedly extracting for 1h to obtain extractive solution;
(3) centrifuging the leaching solution at 5000rpm for 10min, collecting supernatant, and measuring the concentration of the supernatant in A 445 Absorbance of (b) was measured by using C = (1000 × A) 445 Calculating the fucoxanthin content by multiplying N by V)/(A1% cm by multiplying M by 100);
wherein N represents the dilution factor; v represents the volume of the extract; m is the sample mass; a1% cm is 1600.
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