CN112945926A - Method for determining fucoxanthin content in algae cells - Google Patents
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Abstract
The invention relates to the field of physicochemical detection, and discloses an analysis method of fucoxanthin content in algae cells, which comprises two ways, wherein one way is to establish a linear regression equation of the content measured by a chemical method in an algae cell sample extracting solution and a characteristic peak of the algae cell extracting solution measured by a Raman spectrum, and the other way is to establish a linear regression equation of the content of fucoxanthin measured by the chemical method in a single algae cell and the characteristic peak of the single algae cell measured by the Raman spectrum; detecting the content of the fucoxanthin in the single algae cell by the obtained linear regression equation. The method can rapidly and nondestructively detect the fucoxanthin content in the algal cells, does not need complex data processing and analysis, has high sensitivity and high accuracy, and can be used for screening fucoxanthin algae or analyzing the physiological and biochemical states of the algal cells.
Description
Technical Field
The invention relates to the field of fucoxanthin detection, in particular to a method for determining the content of fucoxanthin in algal cells.
Background
Fucoxanthin (Fucoxanthin) is also called Fucoxanthin and Fucoxanthin, and is an important carotenoid contained in brown algae, diatom, chrysophyceae and yellow-green algae, wherein the content of brown algae and diatom is more abundant. Fucoxanthin exists in algal cells in the form of a Fucoxanthin-chlorophyll a/c-protein complex (FCP complex), and plays a role of a light-capturing pigment to participate in photosynthesis. In addition, the molecular structure of the compound is special, and the compound also contains unusual allene bond and 5, 6-monoepoxy structure besides a long-chain conjugated double bond structure. Fucoxanthin has a unique molecular structure, and various powerful biological functions are endowed to the fucoxanthin, such as: anti-obesity, antioxidant, anti-tumor, anti-inflammatory, blood sugar/blood lipid lowering, and liver protecting effects. Therefore, the fucoxanthin source is a research hotspot and is widely applied in the fields of medicines, health care products, feed additives and the like, and the current market demand of the fucoxanthin is about 10 tons/year. In recent years, with the great improvement of the national living standard, the consumption requirements related to the field of human health are increasing day by day, so that the product containing fucoxanthin has wide application prospect and huge market demand.
At present, fucoxanthin is mainly produced from large brown algae such as kelp and undaria pinnatifida. However, the fucoxanthin content in the kelp is extremely low, and is more than 0.01-0.05%, and not more than 0.1% (based on the dry weight of the cells). Due to the low fucoxanthin content in kelp, only about 1 kg of fucoxanthin (10% purity) can be extracted from 2000 kg of kelp. Furthermore, the high cost of fucoxanthin extraction from kelp has led to a price of up to $ 200/kg of fucoxanthin in the market at a 1% content. In the current technology, the production of fucoxanthin from large brown algae is difficult to realize industrialization, under the condition, the development of a new source of fucoxanthin is the key point for realizing the large-scale production of the fucoxanthin, and how to screen the fucoxanthin-rich algae species is a necessary problem.
The existing analysis and determination methods for the fucoxanthin content in algal cells comprise High Performance Liquid Chromatography (HPLC), ultraviolet spectrophotometer (UV) and fluorescence detection methods. For example, CN108279204A discloses a method for rapidly and nondestructively detecting fucoxanthin in phaeodactylum tricornutum, which specifically uses a flow cytometer to detect the average fluorescence intensity value of algae cells under the emission wavelength of 533nm or 585nm, and makes linear regression on the logarithmic value of the fucoxanthin content of the phaeodactylum tricornutum suspension sample and the logarithmic value of the correspondingly measured average fluorescence intensity value to obtain R2The lower limit of the quantification of the intracellular fucoxanthin content of phaeodactylum tricornutum is 3.24mg/g dry algal powder, which is the linear regression equation of 0.9267. In the case of the HPLC detection method, the sample is subjected to pretreatment such as drying and pigment extraction, the analysis process takes a long time, and a large amount of organic reagents are consumed, so that the production process and quality control of fucoxanthin are time-consuming and labor-consuming. For the UV method, a fucoxanthin sample usually contains a large amount of other pigments, and the sample needs to be pretreated during the UV detection, and the pigment is extracted and then subjected to UV detection, so that the UV absorption possibly caused by other pigments and impurities cannot be eliminated because the high-purity fucoxanthin cannot be extracted during the pigment extraction process, and thus, the fucoxanthin cannot be quantitatively determined accurately and accurately. In the fluorescence detection method, since Fucoxanthin exists in the form of a Fucoxanthin-chlorophyll a/c-protein complex (Fucoxanthin-chlorophyl protein complex) in algal cells, the obtained fluorescence signal of Fucoxanthin is greatly affected by chlorophyll, and at different physiological stages of algal cells,the content of chlorophyll changes, so the fluorescence signal of fucoxanthin is affected, and the accuracy of the test result is affected. In addition, the above method requires a relatively large amount of algal cells. Therefore, it is necessary to develop a method for detecting fucoxanthin, which can improve the detection efficiency, simplify the detection process, reduce the interference of the impure pigments, and have high reliability.
Disclosure of Invention
The invention aims to overcome the problems of low detection efficiency, complex detection process and data processing, more interference of variegated pigment, low reliability and the like in the prior art, and provides a method for determining the content of fucoxanthin in algae cells.
In order to achieve the above object, the present invention provides a method for analyzing the content of fucoxanthin in algal cells, the method comprising the steps of:
s1, taking a plurality of algae cell samples with different fucoxanthin contents;
s2, respectively measuring the content of the fucoxanthin in the extracting solution of the algae cell samples with different fucoxanthin contents by a chemical method;
s3-1, respectively measuring the total area of the characteristic peaks of the fucoxanthin in the extracting solution of the algae cell samples with different fucoxanthin contents by Raman spectroscopy;
s4-1, establishing a linear regression equation of the total area of the characteristic peaks of the fucoxanthin and the content of the fucoxanthin in the extracting solution of the algae cell samples with different fucoxanthin contents; or
S3-2, respectively measuring the total area of the characteristic peaks of the fucoxanthin in the single algae cell in the algae cell samples with different fucoxanthin contents by Raman spectroscopy;
s4-2, respectively measuring the densities of the algae cells in the algae cell samples with different fucoxanthin contents, respectively calculating the fucoxanthin contents in single algae cells according to the fucoxanthin contents obtained in the step S2, and establishing a linear regression equation of the total area of characteristic peaks of the fucoxanthin in the single algae cells and the fucoxanthin contents in the single algae cells in the algae cell samples with different fucoxanthin contents;
s5, measuring the total area of the characteristic peaks of the fucoxanthin in the extracting solution of the algae cell sample to be detected or the single algae cell by Raman spectroscopy, and calculating the content of the fucoxanthin in the extracting solution of the algae cell sample to be detected or the single algae cell according to the linear regression equation obtained in S4-1 or S4-2 to be used as the content of the fucoxanthin in the algae cell to be detected.
Preferably, the measurement conditions of the raman spectrum include: the laser power reaching the sample is 0.01-100mW, the integration time of single acquisition is 0.01-100s, and the objective lens is 10-100 times.
Preferably, in step S3-2, the raman spectroscopy method includes acquiring raman spectrograms of 1-100 algae cells in the algae cell sample by using a central point scanning mode or a whole cell scanning mode, and taking the average characteristic peak area as the total characteristic peak area of fucoxanthin in a single algae cell in several algae cell samples with different fucoxanthin contents.
It should be understood that each cell sample can obtain a Raman spectrum, the Raman spectra of a plurality of algae cells can be superposed and averaged to obtain an average Raman spectrum, and the average total characteristic peak area can be obtained from the average Raman spectrum and is used as the total characteristic peak area of fucoxanthin in a single algae cell in a plurality of algae cell samples with different fucoxanthin contents.
Wherein the number of algal cells can be 1, 2, 5, 10, 20, 40, 60, 80, 100 and any range comprised between any two values.
More preferably, the raman spectrum of the algal cell sample is collected using a whole cell scan mode.
The method utilizes the Raman spectrum technology to detect the fucoxanthin content of the algae cells, establishes the linear regression relationship between the fucoxanthin content of the algae cells and the fucoxanthin Raman spectrum characteristic peak, and makes the rapid and nondestructive quantitative detection of the fucoxanthin content in the algae cells possible.
When the method is used for detecting the sample, the cell structure is not required to be damaged, organic solvent extraction is not required, and the fucoxanthin content in the algal cells can be rapidly and quantitatively analyzed through scanning in a very short time and very simple data analysis, so that the method is convenient for screening the algal species with high fucoxanthin content.
Compared with the traditional method, the method can detect the Raman spectrum signal intensity of the fucoxanthin in the single algae cell and the correlation R between the Raman spectrum signal intensity and the content of the fucoxanthin measured by the HPLC method2Can reach 0.98, can be used for the quantitative analysis of fucoxanthin in single algae cells, and has high result reliability.
Compared with other detection methods (such as a fluorescence detection method), the result obtained by the technical scheme provided by the invention is basically not influenced by the physiological state of the cells, the system error caused by the change of the physiological state is eliminated, and the reliability of the result is improved.
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The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:
FIG. 1 is a Raman spectrum of a fucoxanthin standard substance according to the present invention;
FIG. 2 is a Raman spectrum of Phaeodactylum tricornutum UTEX640 collected in whole cell scan mode on different days of culture according to the present invention;
FIG. 3 is an image of a scan taken in a single algal cell whole cell scan mode;
FIG. 4 is a linear regression analysis of fucoxanthin characteristic peak area in Phaeodactylum tricornutum UTEX640 cells and the content of fucoxanthin in the algae cells determined by HPLC method under a whole-cell Raman scanning mode according to a preferred embodiment of the present invention;
FIG. 5 is a linear regression analysis of fucoxanthin characteristic peak area in Phaeodactylum tricornutum UTEX640 cells and the content of fucoxanthin in the algae cells determined by HPLC method in a center point Raman scanning mode according to a preferred embodiment of the present invention;
FIG. 6 is a Raman spectrum of Phaeodactylum tricornutum P.tricornutum3H from whole cell scan of the invention on different days;
FIG. 7 is a linear regression analysis of the fucoxanthin characteristic peak area in Phaeodactylum tricornutum3H cells and the fucoxanthin content in the algae cells determined by HPLC method in the whole-cell Raman scanning mode according to a preferred embodiment of the present invention;
fig. 8 is a linear regression analysis of the fucoxanthin characteristic peak area in phaeodactylum tricornutum3H cells and the fucoxanthin content in the algal cells determined by HPLC method in the center point raman scan mode according to a preferred embodiment of the present invention.
Detailed Description
The following detailed description of embodiments of the invention refers to the accompanying drawings. It should be understood that the detailed description and specific examples, while indicating the present invention, are given by way of illustration and explanation only, not limitation.
The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.
The invention provides a method for analyzing the content of fucoxanthin in algae cells, which comprises the following steps:
s1, taking a plurality of algae cell samples with different fucoxanthin contents;
s2, respectively measuring the content of the fucoxanthin in the extracting solution of the algae cell samples with different fucoxanthin contents by a chemical method;
s3-1, respectively measuring the total area of the characteristic peaks of the fucoxanthin in the extracting solution of the algae cell samples with different fucoxanthin contents by Raman spectroscopy;
s4-1, establishing a linear regression equation of the total area of the characteristic peaks of the fucoxanthin and the content of the fucoxanthin in the extracting solution of the algae cell samples with different fucoxanthin contents; or
S3-2, respectively measuring the total area of the characteristic peaks of the fucoxanthin in the single algae cell in the algae cell samples with different fucoxanthin contents by Raman spectroscopy;
s4-2, respectively measuring the densities of the algae cells in the algae cell samples with different fucoxanthin contents, respectively calculating the fucoxanthin contents in single algae cells according to the fucoxanthin contents obtained in the step S2, and establishing a linear regression equation of the total area of characteristic peaks of the fucoxanthin in the single algae cells and the fucoxanthin contents in the single algae cells in the algae cell samples with different fucoxanthin contents;
s5, measuring the total area of the characteristic peaks of the fucoxanthin in the extracting solution of the algae cell sample to be detected or the single algae cell by Raman spectroscopy, and calculating the content of the fucoxanthin in the extracting solution of the algae cell sample to be detected or the single algae cell according to the linear regression equation obtained in S4-1 or S4-2 to be used as the content of the fucoxanthin in the algae cell to be detected.
It can be understood that the algae cell sample in step S1 and the test algae cell sample in step S5 should be the same algae cell.
In the present invention, the "algal cell samples with different fucoxanthin contents" may include algal cell samples cultured on day 0, algal cell samples cultured on the last day, and algal cell samples cultured on at least any one of other days. For example, when the algal cell culture time is 10 days, the algal cell samples with different fucoxanthin contents may include algal cell samples collected on days 0 and 10, and at least one of days 1, 2, 3, 4, 5, 6, 7, 8, and 9.
In the present invention, before performing step S2, the method preferably further comprises pre-treating the several algal cell samples with different fucoxanthin contents respectively. Wherein, the pretreatment method preferably comprises the following steps: and respectively carrying out solid-liquid separation on the plurality of algal cell samples with different fucoxanthin contents to obtain algal mud, and re-suspending the algal mud in a phosphate buffer solution or a NaCl solution.
In the present invention, in step S5, before performing the raman spectroscopy, the method preferably further includes pretreating the algal cell sample to be tested. Wherein, the pretreatment method preferably comprises the following steps: and carrying out solid-liquid separation on the algae cell sample to be detected to obtain algae mud, and suspending the algae mud in a phosphate buffer solution or a NaCl solution.
Wherein, the solid-liquid separation can be performed by methods conventionally used in the field, such as centrifugation or filtration, and the like, and can be selected by those skilled in the art according to actual needs, such as the time of centrifugation is preferably 3-15min, and the rotation speed of centrifugation is preferably 3000-10000 rpm.
In the present invention, when the algal cell sample is dry algal powder, the suspension may be performed without performing solid-liquid separation by directly using a phosphate buffer solution or a NaCl solution.
In the present invention, the phosphate buffer and NaCl solution preferably have a pH of 6.5 to 7.5 and a concentration of 0.5 to 15 (w/v)%.
In the present invention, when the algal cell sample is a multicellular alga, it is preferable that the single algal cell is obtained by the enzymatic treatment and then the pretreatment is performed.
The enzymatic treatment may be a technique conventional in the art, and may be performed using an enzyme such as lysozyme, cellulase, glucanase, and mannanase.
In the present invention, the chemical method may be a chemical analysis method for analyzing the fucoxanthin content, which is conventional in the art, and may be at least one of liquid chromatography, spectrophotometry, thin layer chromatography, mass spectrometry, fluorescence detection method and capillary electrophoresis method, preferably High Performance Liquid Chromatography (HPLC) method.
Wherein the HPLC method preferably comprises the following steps:
(a) establishing a standard curve of an HPLC method: preparing a fucoxanthin standard substance into a fucoxanthin standard substance solution with a plurality of concentration gradients by using an organic reagent, determining the liquid phase peak area of the fucoxanthin standard substance solution with the plurality of concentration gradients by using a liquid chromatograph, and performing linear regression on the liquid phase peak area of the standard solution and the corresponding concentration of the standard substance solution to obtain a standard curve of an HPLC method;
(b) peak area determination of fucoxanthin content by HPLC method: drying the pretreated algal cell sample to prepare dry algal powder, extracting the dry algal powder to obtain a fucoxanthin extracting solution, and measuring the liquid phase peak area of the fucoxanthin extracting solution by using a liquid chromatograph;
(c) calculating the content of fucoxanthin in the algae cell sample according to the HPLC method standard curve established in the step (a).
Wherein the organic reagent is preferably at least one of ethanol, methanol and acetonitrile, more preferably ethanol, and in the preferred case, a better linear regression can be obtained.
In the present invention, the conditions in the detection using the HPLC method can be selected within a wide range, and preferably, the temperature of the column oven is 25 to 35 ℃ and the flow rate of the eluent is 0.5 to 1.5 mL/min.
In the present invention, the mobile phase in the HPLC method for detecting fucoxanthin preferably contains at least one of dichloromethane, methanol, acetonitrile and water.
In the present invention, the drying treatment may be any means conventionally used in the art, and may be any of freeze drying, spray drying, air drying, oven drying, and the like.
In a preferred embodiment of the present invention, the fucoxanthin content in the extract of the algae cell sample with several different fucoxanthin contents determined by the chemical method is divided by the density of the algae cells (the number of algae cells per g of dry algae meal) to obtain the average fucoxanthin content in a single algae cell; then, a linear regression equation of the average fucoxanthin content in the single algal cell and the total area of the characteristic peak of the fucoxanthin in the single algal cell is established by linear regression fitting in the plurality of algal cell samples with different fucoxanthin contents.
In a preferred embodiment of the invention, the total area of the fucoxanthin characteristic peaks of a plurality of algae cells in the algae cell samples with different fucoxanthin contents is averaged according to the number of the algae cells to obtain the average total area of the fucoxanthin characteristic peaks of each algae cell, and the total area of the characteristic peaks is multiplied by the density of the algae cells (the number of the algae cells contained in each g of dry algae powder) to obtain the total area of the fucoxanthin characteristic peaks corresponding to each g of the algae cells; then, a linear regression equation of the average content of fucoxanthin in the individual algal cells and the total area of characteristic peaks of fucoxanthin in the individual algal cells was obtained by linear regression fitting.
In the present invention, the method for measuring the density of the algal cells may be a method conventionally used in the art, and may be, for example, an optical microscopy method, a flow cytometry method or a dilution-coating plate counting method.
In the present invention, the apparatus for Raman spectroscopy is not particularly limited, and may be any apparatus capable of Raman spectroscopy, and preferably, a living single Cell Raman Sorter prototype (RACS) of national institute of science, celand and energy and process research is used.
In the present invention, the step of collecting the raman spectrogram of the algal cell sample is not particularly limited, and may include, for example, placing the algal cell sample on a slide glass for measurement.
In the present invention, the condition of the raman spectrum is not particularly limited, and preferably, the measurement condition of the raman spectrum includes: the laser power reaching the sample is 0.01-100mW, the integration time of single acquisition is 0.01-100s, and the objective lens is 10-100 times.
In the present invention, the wavelength of the laser used in the Raman spectroscopy may be a wavelength conventionally used in the art, and preferably, the wavelength of the laser is 532nm, 633nm or 785 nm.
In the present invention, fucoxanthin Raman can be determined using fucoxanthin standardsCharacteristic peaks of the spectrum, which may be by methods conventionally used in the art. Preferably, the fucoxanthin standard solution with the concentration of 100--1A Raman signal within the range, thereby obtaining a Raman shift of a characteristic peak of a Raman spectrum of the fucoxanthin.
The reagent used for preparing the fucoxanthin standard solution can be a reagent conventionally used in the field, such as preferably chromatographic grade absolute ethyl alcohol.
In the present invention, the characteristic peak of fucoxanthin is contained in Raman shift 1011cm-1or-CH present in the vicinity3Horizontal vibration absorption peak at Raman shift 1155cm-1Or the C-C bond stretching vibration absorption peak and Raman shift occurring in the vicinity thereof is 1525cm-1Or a C ═ C bond stretching vibration absorption peak appearing in the vicinity.
Preferably, in the Raman spectrogram of the algae cell sample, at 1011cm-1、1155cm-1And 1525cm-1Or the peak height ratio of the resonance absorption peak appearing in the vicinity thereof is 1: 2-4: 4-5. Under the preferred conditions, the correlation of the obtained linear regression equation is high.
In a preferred embodiment of the present invention, the ratio of the peak heights of the resonance absorption peaks appearing at or near 1011cm-1, 1155cm-1 and 1525cm-1 is 1: 2.8: 4.3, R of the linear regression equation it finally obtains20.9822 is reached.
In the present invention, in step S3-2, the raman spectroscopy method preferably includes collecting raman spectrograms of 1 to 100 algae cells in the algae cell sample using a center point scan mode or a whole cell scan mode, and using the average raman spectrum thereof as modeling data.
Preferably, a whole-cell scanning mode is adopted to acquire a Raman spectrogram of the algae cell sample, and the content of the fucoxanthin in the algae cell sample measured by the Raman spectroscopy can be more accurate by using the whole-cell scanning mode, so that the system error is reduced.
In the present invention, the center point scanning mode may refer to performing center area scanning on algal cellsAnd randomly selecting and collecting Raman spectrum data, detecting Raman spectrum signals, and enabling the collected data to be in normal distribution along with more and more samples to be detected, so that the statistical significance is met. Specifically, the method preferably comprises selecting a region with a geometric symmetry center of algae cells as the selected region, and scanning the selected region at 5-3000cm-1Spectrogram in Raman shift range.
In the present invention, the whole-cell scanning mode refers to performing raman scanning on the whole cell, and superimposing the obtained raman spectrograms. As the algae cells are in a stereo form, the focusing plane is assumed to be a plane, the direction parallel to the focusing plane is taken as an x axis and a y axis, Raman spectrum scanning is sequentially carried out along the directions of the x axis and the y axis in 0.1-5 mu m step length, and then the spectrogram obtained by scanning is subjected to superposition imaging processing.
In the present invention, the algal cell may be one of diatoms, phaeophyceae, chrysophyceae and dinoflagellates.
Wherein the algae cell may be one of the genus Phaeodactylum (Phaeodactylum) of the class Diatom, preferably Phaeodactylum tricornutum.
Wherein the algal cell may be one of genus Undaria pinnatifida (Undaria) or Laminaria (Laminaria) of Phaeophyceae, preferably Undaria pinnatifida or Laminaria japonica.
Wherein the algae cell can be one of the genus of dinoflagellate (Ochromonas) of Chrysophyceae, preferably the genus of dinoflagellate.
The algal cell may be one of the genus Coccolithospermum (Pleurochrysis), Isochrysis (Isochrysis) or Pavlova (Pavlova), and is preferably Coccolithospermum ledeb or Hedgehog.
Further preferably, the algal cells are phaeodactylum tricornutum (p.tricornutum).
The present invention will be described in detail below by way of examples.
In the following examples, the establishment of an analytical method was carried out using Phaeodactylum tricornutum as a model organism;
the Phaeodactylum tricornutum strains are P.tricornutum UTEX640 and P.tricornutum3H, which are purchased from fresh water Algae seed repository (Freshwater Algae Culture Collection of Hydrobiology, FACHB);
in the following examples, the HPLC method used was a Waters e2696 high performance liquid chromatograph, column: symmetry C18(5 mm; 150 x 4.6mm), flow rate of 1.0mL/min, column temperature of 35 ℃ and sample size of 10. mu.L. And carrying out full-wavelength scanning at the detection wavelength of 300-800 nm by using a PDA (personal digital Assistant) detector to determine a pigment absorption spectrogram, and simultaneously determining the content of fucoxanthin at the wavelength of 440 nm.
The instrument for Raman spectroscopy is a living single Cell Raman Sorter prototype (RACS) developed by Qingdao bioenergy and process research institute of Chinese academy of sciences, wherein the wavelength of excitation light used for detecting fucoxanthin is 532nm, the power of the light source is 100mw, the light source is filtered by 1%, and the observation is carried out under a 100x eyepiece.
The fucoxanthin standard substance is obtained from Sigma, and the purity is more than or equal to 98%.
Preparation example 1
This preparation example is illustrative of the preparation of a cell sample from Phaeodactylum tricornutum
Algae breeding: tricornutum UTEX640 and p.tricornutum 3H;
culture medium: modified f/2 seawater medium (Table 1A-1C);
the culture conditions are as follows: initial inoculation concentration of algal cells 5x 106One cell/mL, and the light intensity of the culture is 30 mu mol.m-2·s-1，CO2The aeration rate is 1.5-2 vol% (the rest is nitrogen).
3000g of the culture on days 0, 2, 4 and 8 were collected in this order, centrifuged at 5000rpm for 5min to remove the supernatant, and resuspended in 10% NaCl (m/v) solution for further assay.
Wherein, the algae cell samples of the 0 th, 2 th, 4 th and 8 th days of the P.tricornutum UTEX640 are sequentially called as U640-0 day, U640-2 day, U640-4 day and U640-8 day; the samples of algal cells of the 0 th, 2 th, 4 th and 6 th days of tricornutum3H are referred to as 3H-0 day, 3H-2 day, 3H-4 day and 3H-6 day in this order.
TABLE 1A modified f/2 seawater culture Medium formulation
TABLE 1B modified f/2 seawater culture Medium microelement (1L) mother liquor formula
| FeCl3.6H2O | _ | 3.15g | 1.17*10-5 |
| Na2EDTA.2H2O | _ | 4.36g | 1.17*10-5 |
| CuSO4.5H2O | 9.8g/L H2O | 1mL | 3.93*10-8 |
| Na2MoO4.2H2O | 6.3g/L H2O | 1mL | 2.60*10-8 |
| ZnSO4.7H2O | 22.0g/L H2O | 1mL | 7.65*10-8 |
| CoCl2.6H2O | 10.0g/L H2O | 1mL | 4.20*10-8 |
| MnCl2.4H2O | 180.0g/L H2O | 1mL | 9.10*10-7 |
TABLE 1C modified f/2 seawater culture Medium vitamin (1L) stock solution formula
| Vitamin B1 | _ | 200mg | 2.96*10-7 |
| Biotin | 1.0g/L H2O | 1mL | 2.05*10-9 |
| Vitamin B12 | 1.0g/L H2O | 1mL | 3.69*10-10 |
Test example 1
This test example is intended to illustrate the acquisition of a Raman spectrum of a fucoxanthin standard substance according to the present invention
Dripping 500 μ g/mL fucoxanthin standard ethanol solution in CaF2The Raman spectrum signals of the glass slide are measured to obtain a Raman spectrum chart of the fucoxanthin standard product, which is shown in figure 1.
As can be seen from FIG. 1, fucoxanthin is at 1011.2cm-1、1155.5cm-1、1265.3cm-1、1525.7cm-1There is a significant raman shift.
Example 1
This example illustrates the establishment of a method for analyzing the fucoxanthin content in phaeodactylum tricornutum UTEX640 cells.
(1) HPLC method for detecting fucoxanthin content in algae cell sample
Freeze-drying the algal cell sample prepared in preparation example 1;
adding 10mg of phaeodactylum tricornutum freeze-dried algae powder and 1mL of chromatographic grade absolute ethyl alcohol into a brown glass bottle;
placing the glass bottle in a metal bath for fucoxanthin extraction at 45 deg.C for 2 hr. Vortex shaking the extract every 30min for 1-2min to ensure sufficient extraction;
all extracts were filtered through a 0.22 μm filter into HPLC brown loading vials to be detected;
carrying out qualitative and quantitative analysis on fucoxanthin in the pigment components by using a high performance liquid chromatograph, wherein the mobile phase comprises the following components: phase A is dichloromethane: methanol: acetonitrile: water 5: 85: 5.5: 4.5 (v/v); phase B is dichloromethane: methanol: acetonitrile: 25 parts of water: 28: 42.5: 4.5 (v/v); the separation and elution procedure is shown in table 2;
the fucoxanthin is confirmed according to the retention time of pigment separation and the difference of the spectrogram, and the fucoxanthin content in the sample is quantitatively analyzed according to the established fucoxanthin standard curve.
The preparation of the fucoxanthin standard solution is shown in table 3.
TABLE 2 separation and elution procedure
| Time (minutes) | Flow rate (mL/min) | ％ | ％B | |
| 0 | 1.00 | 100.0 | 0.0 | |
| 8 | 1.00 | 100.0 | 0.0 | |
| 14 | 1.00 | 0.0 | 100.0 | |
| 38 | 1.00 | 0.0 | 100.0 | |
| 40 | 1.00 | 100.0 | 0.0 | |
| 45 | 1.00 | 100.0 | 0.0 |
TABLE 3 preparation of fucoxanthin standard solution
| Mother liquor | Volume of mother liquor/mL | Ethanol volume (mL) | Final concentration (μ g mL)-1) |
| #1 | - | - | 500 |
| #2 | 100#1 | 900 | 50 |
| #3 | 500#2 | 500 | 25 |
| #4 | 400#3 | 600 | 10 |
| #5 | 500#4 | 500 | 5 |
| #6 | 200#5 | 800 | 1 |
| #7 | 500#6 | 500 | 0.5 |
(2) Determination of algal cell density
The cell density of the algal cell sample prepared in preparation example 1 was measured by RACS.
(3) Raman spectroscopy in whole-cell scanning mode
The algal cell samples obtained in preparation example 1 for U640-0 days, U640-2 days, U640-3 days and U640-8 days were respectively dropped on CaF2The raman spectral signals were sequentially measured on the slides.
The method for determining each sample of algal cells comprises: respectively scanning 100 algae cells in the algae cell sample in a whole cell scanning mode, wherein the specific parameter is 532nm laser, the power of the laser reaching the sample is 0.1mW, the integration time is 0.05s, and the step length is 1 mu m. And (3) obtaining a Raman spectrogram of a single algae cell through scanning, then carrying out superposition average treatment to obtain a Raman spectrogram of the fucoxanthin of the average single algae cell (see figure 2), and calculating the total area of the fucoxanthin characteristic peaks in the average single algae cell. At the same time, a whole-cell scanning Raman spectrogram of an average single algae cell is also obtained (see figure 3).
The results showed that Raman shift was detected by 1011cm in algal cells containing fucoxanthin-1、1155cm-1And 1525cm-1Raman shift appears near the position, and the peak height of the Raman shift is recorded; the peak height ratio of the three is 1: 3.2: 4.3, calculating the total area of the characteristic peaks.
4) Establishment of Linear regression equation
Calculating according to the fucoxanthin content of the dry algae powder obtained in the step 1) and the cell density of the algae obtained in the step 2) to obtain the average content of the fucoxanthin in the single algae cell.
Drawing by taking the obtained average content of the fucoxanthin in the single algae cell as an ordinate and the total area of the fucoxanthin characteristic peaks in the single algae cell as an abscissa, and performing linear regression analysis to obtain a linear regression equation y of 0.0013x-1.8643, wherein R is20.9709, see fig. 4.
Example 2
This example illustrates the establishment of a method for analyzing the fucoxanthin content in phaeodactylum tricornutum UTEX640 cells.
The method for determining the content of fucoxanthin is established according to the method described in the embodiment 1, except that during Raman spectrum scanning, a central point scanning mode is selected, the specific parameter is 532nm laser, the power of reaching the sample is 0.1mW, and the integration time is 0.05 s.
The resulting linear regression equation, y-2E-05 x-2.5179, R20.8008, see fig. 5.
Example 3
This example illustrates the establishment of a method for analyzing the fucoxanthin content in phaeodactylum tricornutum UTEX640 cells.
(1) HPLC method for detecting fucoxanthin content in algae cell sample
The fucoxanthin content of the algal cell sample was measured by HPLC method according to the method described in step (1) of example 1.
(2) Establishment of Raman spectrum and linear regression equation
Performing Raman spectroscopy on the extracting solution obtained in the step (1) and filtered by the filter membrane of 0.22 mu m, collecting a Raman spectrogram, and determining the total area of the fucoxanthin characteristic peaks in the extracting solution; performing linear regression fitting on the content of the fucoxanthin in the extracting solution and the total area of the fucoxanthin characteristic peaks in the extracting solution to obtain a linear regression equation (y is 0.0483x + 0.0949), wherein R is2＝0.9908。
Example 4
This example illustrates the establishment of a method for analysis of fucoxanthin content in phaeodactylum tricornutum3H cells.
The procedure was followed as described in example 1 except that the algal species was P.tricornutum3H, wherein the Raman spectroscopy gave a Raman spectrum of fucoxanthin as an average of individual algal cells as shown in FIG. 6.
The resulting linear regression equation, y, is 0.0017x-1.0598, R20.9687, see fig. 7.
Example 5
This example illustrates the establishment of a method for analysis of fucoxanthin content in phaeodactylum tricornutum3H cells.
The procedure was as described in example 2, except that the algal species was p.
The resulting linear regression equation, y-3E-05 x-6.1847, R20.8658, see fig. 8.
As can be seen from the data in examples 1-5, R of the linear regression equation established using the method of the present invention20.9709, 0.8008, 0.9908, 0.9687 and 0.8658 respectively, and the minimum detection limits are 0.39, 0.49, 0.5, 0.2 and 0.2pg/cell respectively, which shows that the technical scheme of the invention can realize the technical effects of high sensitivity and high accuracy.
Test example 2
The test example is used for verifying the accuracy of the linear regression equation established by the invention
The method comprises the steps of adopting P.tricornutum UTEX640 dry algae powder with known fucoxanthin content as a sample to be detected, measuring the cell density of the sample, and calculating the actual value of the fucoxanthin content in the average single algae cell sample.
According to the method and the linear regression equation described in the example 1, the total area of the fucoxanthin characteristic peaks in the sample to be detected is detected, and the calculated value of the average fucoxanthin content in the single algal cell is calculated.
The results are shown in Table 4.
TABLE 4 accuracy test results
Test example 3
The test example is used for verifying the accuracy of the linear regression equation established by the invention
The procedure was carried out as described in test example 2, except that the algal species was P.tricornutum3H, and the experimental results are shown in Table 5.
TABLE 5 accuracy test results
From the data of the test examples 2 and 3, the analysis method obtained by the technical scheme of the invention has high accuracy and low detection limit, and can be used for measuring the content of fucoxanthin in single algae cell and algae liquid or algae powder sample.
The preferred embodiments of the present invention have been described in detail above with reference to the accompanying drawings, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, many simple modifications can be made to the technical solution of the invention. Including each of the specific features, are combined in any suitable manner. The invention is not described in detail in order to avoid unnecessary repetition. Such simple modifications and combinations should be considered within the scope of the present disclosure as well.
Claims (10)
1. A method for analyzing the content of fucoxanthin in algae cells, which comprises the following steps:
s1, taking a plurality of algae cell samples with different fucoxanthin contents;
s2, respectively measuring the content of the fucoxanthin in the extracting solution of the algae cell samples with different fucoxanthin contents by a chemical method;
s3-1, respectively measuring the total area of the characteristic peaks of the fucoxanthin in the extracting solution of the algae cell samples with different fucoxanthin contents by Raman spectroscopy;
s4-1, establishing a linear regression equation of the total area of the characteristic peaks of the fucoxanthin and the content of the fucoxanthin in the extracting solution of the algae cell samples with different fucoxanthin contents; or
S3-2, respectively measuring the total area of the characteristic peaks of the fucoxanthin in the single algae cell in the algae cell samples with different fucoxanthin contents by Raman spectroscopy;
s4-2, respectively measuring the densities of the algae cells in the algae cell samples with different fucoxanthin contents, respectively calculating the fucoxanthin contents in single algae cells according to the fucoxanthin contents obtained in the step S2, and establishing a linear regression equation of the total area of characteristic peaks of the fucoxanthin in the single algae cells and the fucoxanthin contents in the single algae cells in the algae cell samples with different fucoxanthin contents;
s5, measuring the total area of the characteristic peaks of the fucoxanthin in the extracting solution of the algae cell sample to be detected or the single algae cell by Raman spectroscopy, and calculating the content of the fucoxanthin in the extracting solution of the algae cell sample to be detected or the single algae cell according to the linear regression equation obtained in S4-1 or S4-2 to be used as the content of the fucoxanthin in the algae cell to be detected.
2. The method according to claim 1, wherein before the step S2, the method further comprises, respectively pre-treating the plurality of algal cell samples with different fucoxanthin contents;
wherein the pretreatment method comprises the following steps: respectively carrying out solid-liquid separation on the plurality of algal cell samples with different fucoxanthin contents to obtain algal mud, and re-suspending the algal mud in a phosphate buffer solution or a NaCl solution; and/or
In step S5, before performing raman spectroscopy, the method further includes, pre-treating the algal cell sample to be detected;
wherein the pretreatment method comprises the following steps: and carrying out solid-liquid separation on the algae cell sample to be detected to obtain algae mud, and suspending the algae mud in a phosphate buffer solution or a NaCl solution.
3. The method as claimed in claim 1, wherein, in step S2, the chemical method is at least one of liquid chromatography, spectrophotometry, thin layer chromatography, mass spectrometry, capillary electrophoresis;
preferably high performance liquid chromatography.
4. The method of claim 1, wherein the fucoxanthin has a characteristic peak at a Raman shift of 1011cm-1or-CH present in the vicinity3Horizontal vibration absorption peak at Raman shift 1155cm-1Or the C-C bond stretching vibration absorption peak and Raman shift occurring in the vicinity thereof is 1525cm-1Or a C ═ C bond stretching vibration absorption peak appearing in the vicinity.
5. The method of claim 1, wherein the Raman spectroscopy measurement conditions comprise: the laser power reaching the sample is 0.01-100mW, the integration time of single acquisition is 0.01-100s, and the objective lens is 10-100 times;
preferably, the laser wavelength is selected from 532nm, 633nm or 785 nm.
6. The method of claim 1, wherein in step S3-2, the raman spectroscopy method comprises using a center point scan mode or a whole cell scan mode to collect raman spectrograms of 1-100 algae cells in the algae cell sample, and using the average characteristic peak area as the total area of the characteristic peaks of fucoxanthin in the individual algae cells in the algae cell samples with several different fucoxanthin contents;
preferably, the raman spectrum of the algal cell sample is acquired using a whole cell scan mode.
7. The method of claim 6, wherein the center point scan pattern comprises a selected area of the geometric center of symmetry of algal cells, the area being scanned between 5-3000cm-1Spectrogram in Raman shift range.
8. The method of claim 6, wherein the whole-cell scanning mode comprises dividing the whole cell into multiple collection points along x and y axis, scanning each point in 5-3000cm in 0.1-5 μm steps-1And (3) performing spectral diagram in a Raman shift range, and then performing superposition imaging processing on all the obtained Raman spectral diagrams in the x-axis direction and the y-axis direction.
9. The method of claim 1, wherein the algal cell is one of the class diatoms, class phaeophyceae, class chrysophyceae, class xanthophyceae, and class dinoflagellates.
10. The method of claim 1, wherein the algal cell is one of the genus Phaeodactylum of the class Diatom;
preferably Phaeodactylum tricornutum.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113984659A (en) * | 2021-10-13 | 2022-01-28 | 自然资源部第二海洋研究所 | Aerobic non-oxygen-producing photosynthetic bacterium detection method based on single-cell Raman spectrum |
| CN117106592A (en) * | 2023-08-18 | 2023-11-24 | 德默特生物科技(珠海)有限公司 | Phaeodactylum tricornutum mutant strain and screening method of quick-growth Phaeodactylum tricornutum mutant strain |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102753959A (en) * | 2009-12-04 | 2012-10-24 | 纽约市哥伦比亚大学信托人 | Spectral and temporal laser fluorescence analysis such as for natural aquatic environments |
| CN103398999A (en) * | 2013-08-08 | 2013-11-20 | 浙江大学 | Detection method for distribution of carotene in Isochrysis galbana based on Raman spectroscopy |
| CN107941783A (en) * | 2017-12-14 | 2018-04-20 | 中国科学院重庆绿色智能技术研究院 | A kind of water environment based on the scattering of frustule Characteristic Raman disturbs appraisal procedure |
| CN108279204A (en) * | 2017-12-29 | 2018-07-13 | 华南理工大学 | A kind of method of Fast nondestructive evaluation Phaeodactylum tricornutum intracellular fucoxanthin |
| CN108663350A (en) * | 2018-06-04 | 2018-10-16 | 上海理工大学 | A kind of assay method of index of unsaturated fatty acid in microalgae cell |
-
2019
- 2019-12-11 CN CN201911266842.0A patent/CN112945926B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102753959A (en) * | 2009-12-04 | 2012-10-24 | 纽约市哥伦比亚大学信托人 | Spectral and temporal laser fluorescence analysis such as for natural aquatic environments |
| CN103398999A (en) * | 2013-08-08 | 2013-11-20 | 浙江大学 | Detection method for distribution of carotene in Isochrysis galbana based on Raman spectroscopy |
| CN107941783A (en) * | 2017-12-14 | 2018-04-20 | 中国科学院重庆绿色智能技术研究院 | A kind of water environment based on the scattering of frustule Characteristic Raman disturbs appraisal procedure |
| CN108279204A (en) * | 2017-12-29 | 2018-07-13 | 华南理工大学 | A kind of method of Fast nondestructive evaluation Phaeodactylum tricornutum intracellular fucoxanthin |
| CN108663350A (en) * | 2018-06-04 | 2018-10-16 | 上海理工大学 | A kind of assay method of index of unsaturated fatty acid in microalgae cell |
Non-Patent Citations (1)
| Title |
|---|
| 李文军等: "岩藻黄素与岩藻黄素-叶绿素复合体(FCP)研究进展", 《食品研究与开发》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113984659A (en) * | 2021-10-13 | 2022-01-28 | 自然资源部第二海洋研究所 | Aerobic non-oxygen-producing photosynthetic bacterium detection method based on single-cell Raman spectrum |
| CN117106592A (en) * | 2023-08-18 | 2023-11-24 | 德默特生物科技(珠海)有限公司 | Phaeodactylum tricornutum mutant strain and screening method of quick-growth Phaeodactylum tricornutum mutant strain |
| CN117106592B (en) * | 2023-08-18 | 2024-05-28 | 德默特生物科技(珠海)有限公司 | Phaeodactylum tricornutum mutant strain and screening method of quick-growth Phaeodactylum tricornutum mutant strain |
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