CN112945926B - 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 modified polypropylene is special, and the modified polypropylene also contains unusual propylene diene 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 cost of fucoxanthin extraction from kelp is high, resulting in a price of up to $ 200/kg of fucoxanthin having a content of 1% in the market. 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 cells, and particularly discloses a method for rapidly and nondestructively detecting fucoxanthin in phaeodactylum tricornutum cellsDetecting the average fluorescence intensity value of algae cells under the emission wavelength of 533nm or 585nm by using a flow cytometer, and performing linear regression on the logarithmic value of the fucoxanthin content of the Phaeodactylum tricornutum suspension sample and the logarithmic value of the average fluorescence intensity value correspondingly measured to obtain R 2 =0.9267 linear regression equation, quantitative lower limit of intracellular fucoxanthin content of phaeodactylum tricornutum is 3.24mg/g dry algal powder. In the case of the HPLC detection method, the sample is subjected to pretreatment for detection, 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. For the fluorescence detection method, because Fucoxanthin exists in the algae cell in the form of a Fucoxanthin-chlorophyll a/c-protein complex (Fucoxanthin-chlorophyl protein complex), the obtained fluorescence signal of Fucoxanthin is greatly influenced by the chlorophyll, and the content of the chlorophyll changes along with the change of the fluorescence signal of the Fucoxanthin in different physiological stages of the algae cell, so that the accuracy of the test result is influenced to a certain extent. 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 algal cell samples with different fucoxanthin contents;
s2, respectively determining 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 characteristic peaks of the fucoxanthin in the single algal cell in the algal 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 comprises 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 a plurality of 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 characteristic peak of the Raman spectrum of the fucoxanthin, and enables the fucoxanthin content in the algae cells to be detected quantitatively in a rapid and lossless manner.
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 detected by an HPLC method 2 Can reach 0.98, canThe method is used for quantitative analysis of fucoxanthin in single algae cells, and the result reliability is high.
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 fucoxanthin-characterized peak area in Phaeodactylum tricornutum3H cells and the fucoxanthin content in the algae cells measured by HPLC method in whole-cell Raman scan mode according to a preferred embodiment of the present invention;
fig. 8 is a linear regression analysis of fucoxanthin content of algal cells determined by HPLC method and fucoxanthin characteristic peak area of phaeodactylum tricornutum3H cells in central 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 determining 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 is understood that the algae cell sample in step S1 and the algae cell sample to be tested 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 pretreating the plurality of 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 comprises 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 the person skilled in the art can select the method according to the actual needs, for example, the time of centrifugation is preferably 3-15min, and the rotation speed of centrifugation is preferably 3000-10000rpm.
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 multicellular algae, 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 plurality of fucoxanthin standard substance solutions with concentration gradients by using an organic reagent, determining liquid phase peak areas of the fucoxanthin standard substance solutions with the concentration gradients by using a liquid chromatograph, and performing linear regression on the liquid phase peak areas of the standard solutions and the corresponding concentrations of the standard substance solutions to obtain an HPLC method standard curve;
(b) Peak area determination of fucoxanthin content by HPLC method: drying the pretreated algae cell sample to prepare dry algae powder, extracting the dry algae powder to obtain 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 1.5mL/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 algal cell samples with several different fucoxanthin contents determined by the chemical method is divided by the algal cell density (the number of algal cells per g dry algal powder) to obtain the average fucoxanthin content in a single algal 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 785nm.
In the present invention, a characteristic peak of a raman spectrum of fucoxanthin can be determined using a fucoxanthin standard, and the method thereof may be a method conventionally used in the art. Preferably, preparing fucoxanthin standard solution with concentration of 100-1000 μ g/mL, performing Raman spectroscopy, and collecting 5-3000cm -1 A 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 -1 or-CH present in the vicinity 3 Horizontal vibration absorption peak at Raman shift 1155cm -1 Or the C-C bond stretching vibration absorption peak and Raman shift occurring in the vicinity thereof is 1525cm -1 Or is attached withC = C key expansion vibration absorption peak appearing near.
Preferably, in the Raman spectrogram of the algae cell sample, at 1011cm -1 、1155cm -1 And 1525cm -1 Or 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 obtains 2 Reach 0.9822.
In the present invention, in step S3-2, the raman spectroscopy method preferably includes acquiring raman spectrograms of 1-100 algae cells in the algae cell sample by using a center point scanning mode or a whole cell scanning 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 invention, the central point scanning mode can be used for randomly selecting and collecting Raman spectrum data in the central area of the algae cell and detecting Raman spectrum signals, and the collected data are normally distributed along with more and more samples to be detected and accord with statistical significance. Specifically, the method preferably comprises selecting a geometrically symmetric center region of algae cells as a selected region, and scanning the selected region at 5-3000cm -1 Spectrogram 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 Phaeodactylum algae (Phaeodactylum) belonging to the class of Diatom, preferably Phaeodactylum tricornutum.
Wherein the algal cell may be one of the genera of brown algae such as Undaria pinnatifida (Undaria) and Laminaria japonica (Laminaria), preferably Undaria pinnatifida and Laminaria japonica.
Wherein the algae cell can be one of the genus of dinoflagellate (Ochromonas) of Chrysophyceae, preferably the genus of dinoflagellate.
The algae cell may be one of Coccolithospermum (Pleurochrysis) genus, isochrysis (Isochrysis) genus or Schiff algae (Pavlova) genus of Dioscoreaceae, preferably Lu Shiba Schiff algae or Schiff algae such as Zhanjiang.
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 × 4.6 mm), flow rate of 1.0mL/min, column temperature of 35 ℃, sample loading of 10 μ L. The PDA detector is used for full-wavelength scanning at the detection wavelength of 300-800 nm to determine the absorption spectrum of the pigment, and the fucoxanthin content is determined 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 ocular lens.
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
And (3) 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 10 6 One cell/mL, and the light intensity of the culture is 30 mu mol.m -2 ·s -1 ，CO 2 The aeration rate is 1.5-2 vol% (the rest is nitrogen).
The cultures collected on days 0, 2, 4 and 8 were collected 3000g,5000rpm 5min in turn, centrifuged to remove the supernatant, resuspended using 10% NaCl (m/v) solution, and used for the next detection analysis.
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 Medium microelement (1L) mother liquor formula
| FeCl 3 .6H 2 O | _ | 3.15g | 1.17*10 -5 |
| Na 2 EDTA.2H 2 O | _ | 4.36g | 1.17*10 -5 |
| CuSO 4 .5H 2 O | 9.8g/L H 2 O | 1mL | 3.93*10 -8 |
| Na 2 MoO 4 .2H 2 O | 6.3g/L H 2 O | 1mL | 2.60*10 -8 |
| ZnSO 4 .7H 2 O | 22.0g/L H 2 O | 1mL | 7.65*10 -8 |
| CoCl 2 .6H 2 O | 10.0g/L H 2 O | 1mL | 4.20*10 -8 |
| MnCl 2 .4H 2 O | 180.0g/L H 2 O | 1mL | 9.10*10 -7 |
TABLE 1C modified f/2 stock solution formulation of vitamins (1L) in seawater culture medium
| Vitamin B1 | _ | 200mg | 2.96*10 -7 |
| Biotin | 1.0g/L H 2 O | 1mL | 2.05*10 -9 |
| Vitamin B12 | 1.0g/L H 2 O | 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 CaF 2 Measuring its Raman spectra on a glass slideAnd obtaining a Raman spectrum chart of the fucoxanthin standard product, which is shown in a figure 1.
As can be seen in FIG. 1, fucoxanthin is at 1011.2cm -1 、1155.5cm -1 、1265.3cm -1 、1525.7cm -1 There 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: water =25:28:42.5:4.5 (v/v); the separation and elution procedures are 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 CaF 2 The 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, the whole cell scanning Raman spectrogram of the 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 -1 And 1525cm -1 Raman 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 peak in the single algae cell as an abscissa, and performing linear regression analysis to obtain a linear regression equation y =0.0013x-1.8643, R 2 =0.9709, see fig. 4.
Example 2
This example illustrates the establishment of a method for analysis of 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 0.05s.
The resulting linear regression equation y =2E-05x-2.5179 2 =0.8008, 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 in the algae cell sample was measured by the 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 fucoxanthin in the extracting solution and the total area of the fucoxanthin characteristic peak in the extracting solution to obtain a linear regression equation y =0.0483x +0.0949, R 2 ＝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 =0.0017x-1.0598 2 =0.9687, 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-05x-6.1847 2 =0.8658, 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 invention 2 0.9709, 0.8008, 0.9908, 0.9687 and 0.8658, and the lowest detection limit is 0.39, 0.49, 0.5, 0.2 and 0.2pg/cell, respectively, which shows that the technical scheme of the invention can achieve the technical effects of high sensitivity and 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 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 a single algae cell, 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 various combinations of the specific features, 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, 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, respectively measuring the density of the algae cells in the algae cell samples with different fucoxanthin contents, respectively calculating the content of the fucoxanthin in a single algae cell according to the content of the fucoxanthin 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 cell and the content of the fucoxanthin in the single algae cell in the algae cell samples with the different fucoxanthin contents;
s5, measuring the total area of the characteristic peaks of the fucoxanthin in the single algae cell of the algae cell sample to be detected by Raman spectroscopy, and calculating the content of the fucoxanthin in the single algae cell of the algae cell sample to be detected according to the linear regression equation obtained in S4 to serve as the content of the fucoxanthin in the algae cell to be detected;
in the step S3, the Raman spectrum method comprises the steps of collecting Raman spectrograms of 10-100 algae cells in the algae cell sample by adopting a whole cell scanning mode, and taking the average characteristic peak area as the total area of the characteristic peaks of fucoxanthin in a single algae cell in a plurality of algae cell samples with different fucoxanthin contents;
the whole-cell scanning mode comprises the steps of assuming a focusing plane as a plane, taking the direction parallel to the focusing plane as an x axis and a y axis, sequentially carrying out Raman spectrum scanning along the directions of the x axis and the y axis in steps of 0.1-5 mu m, and then carrying out superposition imaging processing on the spectrograms obtained by scanning.
2. The method according to claim 1, wherein before performing 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 carrying out Raman spectrum measurement, the method also comprises the steps of pretreating the algae cell sample to be measured;
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 of claim 1, wherein in step S2, the chemical method is at least one of liquid chromatography, spectrophotometry, thin layer chromatography, mass spectrometry, and capillary electrophoresis.
4. The method of claim 3, wherein in step S2, the chemical method is high performance liquid chromatography.
5. The method of claim 1, wherein the fucoxanthin has a characteristic peak at a Raman shift of 1011cm -1 or-CH present in the vicinity 3 Horizontal vibration absorption peak at Raman shift 1155cm -1 Or the C-C bond stretching vibration absorption peak and Raman shift occurring in the vicinity thereof is 1525cm -1 Or C = C bond stretching vibration absorption peak appearing in the vicinity.
6. 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.
7. The method of claim 6, wherein the laser wavelength is selected from 532nm, 633nm, or 785nm.
8. The method of claim 1, wherein the algal cell is one of the class diatoms, class phaeophyceae, class chrysophyceae, class xanthophyceae, and class dinoflagellates.
9. The method of claim 1, wherein the algal cell is one of the genus Phaeodactylum of the class Diatom.
10. The method of claim 1, wherein the algal cell is phaeodactylum tricornutum.
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