CN115074251B - Culture medium and culture method for improving fucoxanthin yield in Phaeodactylum tricornutum - Google Patents
Culture medium and culture method for improving fucoxanthin yield in Phaeodactylum tricornutum Download PDFInfo
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Abstract
The invention discloses a culture medium and a culture method for improving fucoxanthin yield in Phaeodactylum tricornutum. The culture medium is prepared by adding exogenous plant hormone into an f/2 culture medium. The culture method is to add exogenous plant hormone when Phaeodactylum tricornutum reaches the exponential culture phase. The invention not only can promote the growth of Phaeodactylum tricornutum and increase the accumulation speed of fucoxanthin, but also has the advantages of no additional equipment, simple operation, low cost and short production period, and effectively increases the yield of fucoxanthin.
Description
Technical Field
The invention relates to the technical field of microalgae culture, in particular to a culture medium and a culture method for improving fucoxanthin yield in Phaeodactylum tricornutum.
Background
Fucoxanthin (Fx), also known as fucoxanthin or fucoxanthin, is reddish brown, contains a propadiene skeleton, belongs to an oxygen-containing derivative of carotenoids, and is commonly found in brown algae and diatoms. Fucoxanthin is a natural active substance which has proven to be a safe and effective dietary supplement with antioxidant, anti-inflammatory, antitumor, anti-obesity, anti-diabetic effects without affecting the nervous system. Although fucoxanthin is a natural pigment with very high utilization value, the application of fucoxanthin is greatly limited due to the lack of germplasm resources, low extraction efficiency and difficult synthesis by chemical means.
Phaeodactylum tricornutum (Phaeodactylum tricornutum) belongs to one of the diatoms, belonging to the genus Phaeodactylum of the phylum diatom, the class Phaeodactylum. The Phaeodactylum tricornutum does not contain endotoxin, and has simple culture, low cost and high growth speed. However, fucoxanthin is not contained in Phaeodactylum tricornutum. At present, genetic information of Phaeodactylum tricornutum is changed by means of physical and chemical mutagenesis, genetic engineering breeding and the like, so that the yield of fucoxanthin in Phaeodactylum tricornutum is improved. However, the method has a large investment in the early stage, high research and development cost and long research and development period. Therefore, there is an urgent need for a method that is simple to operate, low in cost, and short in production cycle to increase fucoxanthin production in Phaeodactylum tricornutum.
The 5-aminolevulinic acid (5-aminolevulinic acid, 5-ALA) has physiological activity similar to that of plant hormone, is a novel photoactivation pesticide with high environmental compatibility and selectivity, is also one of plant growth regulators, and is widely applied in the agricultural field. 6-Benzylaminopurine (6-BAP) is a plant growth regulator and has been one of the more successful synthetic cytokinins to date. It can promote cell division and promote cell growth. The two hormones are creatively added into the Phaeodactylum tricornutum culture medium, so that the expression of fucoxanthin related genes in Phaeodactylum tricornutum can be obviously promoted, and the yield of fucoxanthin is improved.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention aims to provide a culture medium for improving the fucoxanthin yield in Phaeodactylum tricornutum by adding exogenous plant hormones. The invention further provides a culture method for improving the fucoxanthin yield in Phaeodactylum tricornutum, and specifically, exogenous plant hormone is added when Phaeodactylum tricornutum reaches an exponential growth phase, so that the growth of Phaeodactylum tricornutum is promoted, and the accumulation speed and the yield of fucoxanthin are improved.
The technical scheme is as follows: the culture medium for culturing Phaeodactylum tricornutum provided by the invention is an f/2 culture medium, wherein the f/2 culture medium contains 0-3mg/L of 6-benzyl aminopurine; or the culture medium is an f/2 culture medium, wherein the f/2 culture medium contains 0-3mg/L of 5-aminolevulinic acid; or the culture medium is an f/2 culture medium, wherein the f/2 culture medium contains 0-3mg/L of 6-benzyl amino purine and 0-3mg/L of 5-aminolevulinic acid.
In the invention, exogenous plant hormone is added in the culture process of Phaeodactylum tricornutum, and one or two of 5-aminolevulinic acid and 6-benzyl amino purine are added, so that the growth speed of Phaeodactylum tricornutum and the yield of fucoxanthin are greatly improved.
As a preferred embodiment of the invention, the f/2 medium contains the following components: 75-100mg/L sodium nitrate, 0.5-1mg/L sodium dihydrogen phosphate and vitamin B 12 0.5-1 mug/L, 0.5-1 mug/L biotin, 100-200 mug/L thiamine hydrochloride, 10-20mg/L sodium metasilicate and 1mL/L f/2 trace elements (f/2 metals), the balance being sea water.
As a preferred embodiment of the present invention, the f/2metals include the following components: 4.4g/L EDTANa 2 ·2H 2 O、3.16g/L FeCl 3 ·6H 2 O、0.012g/L CoSO 4 ·7H 2 O、0.021g/L ZnSO 4 ·7H 2 O、0.18g/L MnCl 2 ·4H 2 O、0.007g/L CuSO 4 ·5H 2 O、0.007g/L NaMoO 4 ·2H 2 O, the balance being water.
As a preferred embodiment of the present invention, the formula of the f/2 medium (seawater) is as follows: naNO 3 :75mg/L，NaH 2 PO 4 ·H 2 O0.6 mg/L, vitamin B 12 (Vitamin B12) 0.5. Mu.g/L, biotin (Biotin) 0.5. Mu.g/L, thiamine HCl 100. Mu.g/L, na 2 SiO 3 ·9H 2 O10 mg/L, f/2 trace elements (f/2 metals) 1mL/L.
As a preferred embodiment of the present invention, the medium is an f/2 medium, wherein the f/2 medium contains 0.8-1.2 mg/L of 6-benzylaminopurine; or the culture medium is an f/2 culture medium, wherein the f/2 culture medium contains 0.8-1.2 mg/L of 5-aminolevulinic acid; or the culture medium is an f/2 culture medium, and the f/2 culture medium contains 0.8-1.2 mg/L of 6-benzyl amino purine and 0.8-1.2 mg/L of 5-aminolevulinic acid.
As a preferred embodiment of the present invention, the medium is an f/2 medium, and the f/2 medium contains 1.0mg/L of 6-benzylaminopurine; or the culture medium is an f/2 culture medium, and the f/2 culture medium contains 1.0mg/L of 5-aminolevulinic acid; or the culture medium is an f/2 culture medium, and the f/2 culture medium contains 1.0mg/L of 6-benzyl amino purine and 1.0mg/L of 5-aminolevulinic acid.
The invention further provides a method for improving the fucoxanthin yield in Phaeodactylum tricornutum by adding plant hormone, wherein the method is to add exogenous plant hormone when Phaeodactylum tricornutum reaches an index culture period, and the adopted culture medium is an f/2 culture medium; the exogenous plant hormone is 6-benzyl amino purine, 5-amino levulinic acid or a combination of 6-benzyl amino purine and 5-amino levulinic acid.
As a preferred embodiment of the present invention, the amount of the 5-aminolevulinic acid added is 0 to 3mg/L; the addition amount of the 6-benzyl amino purine is 0-3 mg/L.
As a preferred embodiment of the present invention, 1.0mg/L of 5-aminolevulinic acid and 1.0mg/L of 6-benzylaminopurine are added to the f/2 culture.
As a preferred embodiment of the present invention, the culture method of Phaeodactylum tricornutum comprises the following steps:
(1) Preparing algae liquid, and culturing Phaeodactylum tricornutum cells to reach an exponential growth phase;
(2) Inoculating 10% -20% of the culture medium to seawater according to the volume;
(3) Exogenous plant hormone is added for illumination and aeration culture.
As a preferred embodiment of the present invention, the culture method is as follows: before the growth period, the Phaeodactylum tricornutum culture condition is that the illumination intensity is 155-2000lux, the light-dark ratio is 16-20h:4-8h, the culture temperature is 20-25 ℃ until the growth density reaches 1X 10 7 -2×10 7 And each mL.
As a preferred embodiment of the present invention, the culture method is as follows: after exogenous plant hormone is added, the culture condition of Phaeodactylum tricornutum is that the illumination intensity is 5000-5500lux, the light-dark ratio is 16-20h:4-8h, and the culture temperature is 20-25 ℃.
As a preferred embodiment of the present invention, the exogenous plant hormone is added to the f/2 medium at a final concentration of 0-3mg/L of 6-benzylaminopurine and a final concentration of 0-3mg/L of 5-aminolevulinic acid.
As a preferred embodiment of the present invention, the exogenous plant hormone is added to the f/2 medium at a final concentration of 1.0mg/L of 6-benzylaminopurine and a final concentration of 1.0mg/L of 5-aminolevulinic acid.
The beneficial effects are that: (1) According to the invention, exogenous plant hormones 6-BAP and 5-ALA or mixed hormones of 6-BAP and 5-ALA are added when Phaeodactylum tricornutum reaches an index culture period, so that the growth of Phaeodactylum tricornutum can be greatly improved; (2) According to the invention, when Phaeodactylum tricornutum reaches the index culture period, the mixed hormone of 6-BAP and 5-ALA is added, so that the accumulation speed of fucoxanthin is improved; (3) According to the invention, the fucoxanthin related gene expression can be induced by adding 6-BAP or the mixed hormone of 6-BAP and 5-ALA in the culture process of Phaeodactylum tricornutum, and the transcription level of ZEP2 can be improved by 8.74 times by adding the mixed hormone of 6-BAP and 5-ALA; (4) The invention has the characteristics of simple operation, low cost, short production period and the like, does not need additional equipment in the culture process, can improve the yield of fucoxanthin in Phaeodactylum tricornutum, and realizes the large-scale application of fucoxanthin.
Drawings
FIG. 1 is the effect of the addition of different hormones on fucoxanthin; wherein, a is the influence of 6-BAP group, 5-ALA group, mixed hormone group and blank control group on Phaeodactylum tricornutum X1 growth; panel b shows the effect of 6-BAP, 5-ALA, mixed hormone and blank on Fx accumulation and growth in Phaeodactylum tricornutum X1.
FIG. 2 is the effect of different hormone additions on the amount of gene expression associated with the formation of Fx key enzyme, wherein a is the effect of 6-BAP group on the key enzyme of Phaeodactylum tricornutum X1 to form Fx; b, the influence of the mixed hormone group of the two on the key enzyme forming Fx of Phaeodactylum tricornutum X1 is shown in the graph; panel c is the effect of the 5-ALA group on key enzymes of Phaeodactylum tricornutum X1 to form Fx, and the bold in FIG. 2 indicates changes in transcript levels.
Detailed Description
In order to make the technical scheme and advantages of the embodiments of the present invention clearer, the following detailed description is made in connection with the embodiments of the present invention.
The following non-limiting examples will enable those of ordinary skill in the art to more fully understand the invention and are not intended to limit the invention in any way.
The experimental methods, reagents required for the experiments, and the like used in the following examples were all conventionally conducted unless otherwise specified.
Example 1: culture of Phaeodactylum tricornutum
Phaeodactylum tricornutum strain X1 is isolated from Qingdao coastal waters and stored in liquid nitrogen. The activated X1 strain was cultured as seeds in f/2 medium. The formula of the f/2 culture medium is as follows: naNO 3 :75mg/L,NaH 2 PO4·H 2 O0.6 mg/L, vitamin B 12 (Vitamin B 12 ) 0.5. Mu.g/L Biotin (Biotin) 0.5. Mu.g/L Thiamine HCl 100. Mu.g/L Na 2 SiO 3 ·9H 2 O:10mg/L，f/2metals:1mL。f/2metals(EDTANa 2 ·2H 2 O:4.4g/L,FeCl 3 ·6H 2 O:3.16g/L,CoSO 4 ·7H 2 O:0.012g/L,ZnSO 4 ·7H 2 O:0.021g/L,MnCl 2 ·4H 2 O:0.18g/L,CuSO 4 ·5H 2 O:0.007g/L,NaMoO 4 ·2H 2 O:0.007g/L,dH 2 O)。
Culturing at 23deg.C under light-dark ratio of 16h to 8h and light intensity of 2000lux to cell density of 1.5X10 7 And each mL. Then, the seeds were transferred to a 500mL flask, 300mL of f/2 medium was filled, and culture of Phaeodactylum tricornutum was performed at the same temperature to produce fucoxanthin with a 16h:8h light-dark ratio and 5500lux light intensity. The seed culture is immediately followed by pre-experiments with different plant hormones (including melatonin, 4-aminobutyric acid, 6-BAP and 5-ALA), observing the growth state of Phaeodactylum tricornutum, and selecting hormone with good growth effect for the next experiment. The culture conditions of the Phaeodactylum tricornutum control group are different from those of the experimental group, and no phytohormone is added.
Example 2: culture of Phaeodactylum tricornutum X1 by adding plant hormone
2.1 Pre-experiment was completed, 6-BAP and 5 were selectedALA was further tested and the optimal concentration was determined by hormone addition at a concentration gradient of 1.0 mg/L. Culturing Phaeodactylum tricornutum X1 to cell density of 1.5X10 7 As seed liquid, subculture was then carried out, and three experimental groups and a control group were set, wherein 1.0 mg/L6-BAP, 1.0 mg/L5-ALA, and 1.0 mg/L6-BAP and 1.0 mg/L5-ALA mixed hormone were added to the medium, and the culture was carried out for 10 days, and sampling was carried out every day to carry out data measurement.
2.2 experimental method: continuous sampling and determination of Phaeodactylum tricornutum X1
(1) Measuring the change in biomass during the growth of Phaeodactylum tricornutum: 40mL of algae liquid was taken, centrifuged at 8000rpm for 8min, the supernatant was discarded, and the algae mud was dried in an oven (85 ℃) overnight, and then the dry weight of the dry algae cells was weighed.
biomass=m/V (g/L)
(2) Measuring the change of fucoxanthin content in the growth process of Phaeodactylum tricornutum: subculturing Phaeodactylum tricornutum reaching the growth index stage, adding 6-BAP and 5-ALA and mixed hormone thereof respectively, culturing for 10d under proper conditions, and sampling; and sampling the test group containing 6-BAP and 5-ALA, and the mixed hormone and the control group without phytohormone for different time periods.
50mL of the algae liquid was centrifuged at 8000 Xg and then lyophilized. The biomass was extracted with ULTRA-TURRAX Tube Drive using 10mL ethanol. The supernatant was centrifuged and concentrated to 4mL at 65℃with a rotary vacuum concentrator. The fucoxanthin solution was filtered using a 0.22 μm filter and run in a High Performance Liquid Chromatography (HPLC) system. The column was C18, the mobile phase was acetonitrile: ultrapure water=85: 15 (V: V). The sample volume of each sample was 10 μl and specific data was recorded for analysis.
(3) Preparation of fucoxanthin standard curve: different quality fucoxanthin standard substances are diluted by absolute ethyl alcohol to prepare different concentrations of 1.8 mug/mL, 2.2 mug/mL, 2.7 mug/mL, 3.7 mug/mL, 5.5 mug/mL, 11 mug/mL and 22 mug/mL, then the fucoxanthin standard substances are operated in a High Performance Liquid Chromatography (HPLC) system, the peak area is taken as an ordinate, the fucoxanthin concentration is taken as an abscissa, a standard curve is drawn, and the fucoxanthin concentration in the sample is calculated according to the standard curve.
2.3 experimental results
As a result, as shown in FIG. 1, it was confirmed that the addition of 6-BAP and 5-ALA, and the addition of the mixed hormone was optimal. As can be seen from graph a in FIG. 1, the biomass of Phaeodactylum tricornutum alone or 5-ALA alone increased most from 2d to 4d in culture, and the biomass of 6-BAP group 4d increased to 0.57g/L and the biomass of 5-ALA group 4d increased to 0.54g/L. As can be seen from the corresponding graph b in FIG. 1, the fucoxanthin yield was 17.20mg/L, which is almost 1.6 times that of the control. And the added mixed hormone increases the amount most from 2d culture to 4d culture, and 4d can reach the biomass to increase to 0.62g/L. The corresponding fucoxanthin yield was 19.63mg/L, almost 2 times that of the control. The results show that 6-BAP can significantly promote growth and fucoxanthin accumulation. As shown in FIG. 1, the first 4d, fucoxanthin accumulation was significant for both experimental groups with 6-BAP alone or 5-ALA alone, and the accumulation rate was rapidly slowed from day 6 to day 10. The biomass of the mixed hormone experimental group steadily increases in the whole culture process, and the accumulation speed tends to increase along with the 5 th day, while the accumulation speed of the fucoxanthin of the experimental group added with the hormone singly starts to decrease, and as can be seen from the experimental result of fig. 1, the accumulation speed of the fucoxanthin can be increased by adding the mixed hormone.
Example 3: transcriptome analysis
Phaeodactylum tricornutum X1 was cultured for 4d in f/2 medium with or without 6-BAP and mixed hormone of both, and cells were collected by centrifugation. Total RNA was extracted with TRIZOL reagent and reverse transcribed into cDNA. The cDNA was sent to transcriptome sequencing.
Primers were designed for qRT-PCR (the kits used were RNA extraction kit, reverse transcription kit and real-time fluorescence quantification kit, from Takara doctor technology (Beijing) Co., ltd.) based on the coding sequences of DXS, PSY, PDS, PDS2, ZDS, LCYB, ZEP, ZEP2, ZEP3, VDE, FCPb. The gene encoding β -actin was selected as an internal reference. 3 replicates of qRT-PCR were performed using SYBR PCR master mix. The relative transcriptional level changes of the genes in the phytohormone-containing medium were analyzed by CT values. Samples cultured with original f/2 medium were used as controls.
Primer sequences 5' -3:
DXS 5’：AGCCAATTCTGGACTCGGTG，DXS 3’：GCAAGGCAACAGTGAGTTCG；PSY 5’：CCACGCCGAACATGCTTTAG，PSY 3’：GACTTCTTGCACTTGTGCCG；
PDS1 5’：TTCTCCACGACACTCAAGGC，PDS1 3’：CCGGTTTCGATCCAGTCTCC；
PDS2 5’：GTGTTCTCGGTGGCAGTCTT，PDS2 3’：GAGCCGACGCTAGAGAAGTC；
ZDS 5’：TTGGACTCGATGGAAGGTGC，ZDS 3’：CCGCTTTCCTCTTTCGCTTG；
LCYB 5’：GCATTGCGACGTACATGGTC，LCYB 3’：TCGTCGAGCTTCACTCTTGG；
ZEP1 5’：GGCACTCGAACGCATCAATC，ZEP1 3’：TCGAAGCGTACCAACCAGTC；
ZEP2 5’：ATACACCGTCTTTGCGGGAG，ZEP2 3’：CCATCACCGACATCACTCGT；
VDE 5’：TTCCATCAAGGCGCAAAAGC，VDE 3’：GCTGGGAGGTTTCTCGTTCA；
FCPb 5’：AGCACCGCTTGGATTCTACG，FCPb 3’：TGCCAAGTATCCAGCAACGG；
β-actin5’：GACTCCACCTTCCAGACCATTA，β-actin3’：GACCCTCCAATCCAAACAGAG；
ITS 5’：TCCGTAGGTGAACCTGCGG，ITS 3’：TCCTCCGCTTATTGATATGC。
as a result, as shown in fig. 2, the transcription levels of DXS and PSY were 1.07 times and 6.80 times, respectively, that of the control group in the 6-BAP group, and 1.76 times and 7.68 times that of the control group in the mixed hormone group, respectively, indicating that the mixed hormone group induced more carbon flux-shifted pigment formation, particularly carotenoid biosynthesis. Other genes in the fucoxanthin formation step are also commonly up-regulated. As shown in figure 2 b, the transcriptional levels of ZEP1, ZEP2 and ZEP3 were significantly up-regulated upon addition of both mixed hormones. Of these genes, ZEP2 is most sensitive to its response. In the two mixed hormone groups, the transcription level of ZEP2 is improved by 8.74 times. As shown in panel c of FIG. 2, the transcript levels of ZEP1, ZEP2 and ZEP3 are also generally up-regulated in the 5-ALA group. As can be seen from the results of FIG. 2, the up-regulation of gene expression associated with fucoxanthin biosynthesis pathway suggests that the addition of 5-ALA and 6-BAP in Phaeodactylum tricornutum culture significantly promotes fucoxanthin accumulation.
The above embodiments are only for illustrating the technical scheme of the present invention, and are not intended to limit the present invention. It will be apparent to those skilled in the art that many modifications and adaptations of the invention using the foregoing disclosure are possible and can be made by way of illustration to the inventive concepts herein without departing from the scope of the invention.
Claims (4)
1. A method for improving fucoxanthin yield in Phaeodactylum tricornutum by adding phytohormone is characterized in that exogenous phytohormone is added when Phaeodactylum tricornutum reaches an index culture period, and a culture medium is f/2 culture medium; the exogenous plant hormone is a combination of 6-benzyl amino purine and 5-amino levulinic acid; the f/2 culture medium contains 1mg/L of 6-benzyl amino purine and 1mg/L of 5-aminolevulinic acid; the f/2 medium contains the following components: 75-100mg/L of sodium nitrate, 0.5-1mg/L of sodium dihydrogen phosphate, 0.5-1 mug/L of vitamin B, 0.5-1 mug/L of biotin, 100-200 mug/L of thiamine hydrochloride, 10-20mg/L of sodium metasilicate, 1mL/L of f/2 trace elements and the balance of seawater; the f/2 trace elements comprise the following components: 4.4g/L EDTANa 2 ·2H 2 O、3.16g/L FeCl 3 ·6H 2 O、0.012g/L CoSO 4 ·7H 2 O、0.021g/L ZnSO4·7H 2 O、0.18g/L MnCl 2 ·4H 2 O、0.007g/L CuSO 4 ·5H 2 O、0.007g/LNaMoO4·2H 2 O, the balance being water.
2. The method according to claim 1, comprising the steps of:
(1) Preparing algae liquid, and culturing Phaeodactylum tricornutum cells to reach an exponential growth phase;
(2) Inoculating 10% -20% of the culture medium to the seawater according to the volume;
(3) Exogenous plant hormone is added for illumination and aeration culture.
3. The method according to claim 2, wherein the culture conditions of Phaeophyllum tricornutum are light intensity of 155-2000lux and light-dark ratio of (16-20) h (4-8) h before reaching growth period, and the culture temperature is 20-25 ℃ until the growth density reaches 1 x 10 7 ~2×10 7 And each mL.
4. The method according to claim 3, wherein after the exogenous plant hormone is added, the culture condition of the Phaeodactylum tricornutum is that the illumination intensity is 5000-5500lux, the light-dark ratio is (16-20) h (4-8) h, and the culture temperature is 20-25 ℃.
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