CN104964957B - A kind of method of Fast nondestructive evaluation chlorella intracellular astaxanthin - Google Patents

A kind of method of Fast nondestructive evaluation chlorella intracellular astaxanthin Download PDF

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CN104964957B
CN104964957B CN201510388357.6A CN201510388357A CN104964957B CN 104964957 B CN104964957 B CN 104964957B CN 201510388357 A CN201510388357 A CN 201510388357A CN 104964957 B CN104964957 B CN 104964957B
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mobile phase
astaxanthin
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chlorella
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CN104964957A (en
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魏东
陈俊辉
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South China University of Technology SCUT
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Abstract

The present invention provides a kind of method of Fast nondestructive evaluation chlorella intracellular astaxanthin, including step：1) frustule being collected into from chlorella suspension known sample is resuspended with deionized water or phosphate buffer, is adjusted to cell density 6 × 105~2 × 106CFU/mL re-suspension liquid, with flow cytomery frustule re-suspension liquid in FL1 passages or the average fluorescent strength of FL2 passages；The logarithm value of the logarithm value of the content astaxanthin of chlorella suspension known sample and the average fluorescent strength value of the FL1 or FL2 passages accordingly measured is made into linear regression, obtains standard curve；2) prepare testing sample and the frustule being collected into from chlorella suspension to be measured is resuspended with deionized water or phosphate buffer, be adjusted to cell density 6 × 105~2 × 106CFU/mL frustule re-suspension liquid to be measured；3) according to the standard curve of step 1), its content astaxanthin is calculated to obtain in FL1 passages or the average fluorescent strength of FL2 passages with flow cytomery testing sample.The method of the present invention has the characteristics of detection efficiency is high.

Description

A kind of method of Fast nondestructive evaluation chlorella intracellular astaxanthin
Technical field
It is more particularly to a kind of to be based on detection technique of fluorescence the invention belongs to the Fast nondestructive evaluation technology of microalgae para chrome Chlorella intracellular astaxanthin fast non-destructive detection method.
Background technology
Astaxanthin (astaxanthin) is referred to as " super antioxidant ", oxidation resistance be other carotenoid such as β- More than 10 times of carrotene, luteole, lutein and canthaxanthin etc., have and remove free radical, prevent lipid peroxidation capacity, Play an important roll in terms of the age-related diseases such as prevention of arterial hardening, parkinsonism, macular degeneration, it is right In safeguarding eyes and central nervous system health, enhancing function of immune system, strengthen human body energetic supersession, anticancer, anti-infective etc. With multi-efficiency.
It is most important production method currently with Haematococcus pluvialis production natural astaxanthin, but it is high, raw to be faced with cost Produce the restriction of the technical bottlenecks such as low and unstable, the easy biological pollution of efficiency.Chlorella C.zofingiensis is a kind of unicellular Green alga, cell dia are 2-15 μm, and intracellular content astaxanthin can be significantly improved under bloom, low nitrogen inductive condition, are production A kind of important alternative microalge of natural astaxanthin.Recent study shows that chlorella C.zofingiensis can Heterotrophism high density fermentation is carried out using organic matter under dark condition, through inductive conditions such as bloom, high temperature, high salt, the limitations of nitrogen phosphorus Can efficient accumulation astaxanthin.Therefore, exploitation is examined for chlorella C.zofingiensis para chromes especially astaxanthin The technology of analysis is surveyed, for significant using carotenoid such as chlorella production astaxanthins.
The conventional method of present analysis measure astaxanthin is ultraviolet-visible spectrophotometer method (UV-vis), efficient liquid phase Chromatography (HPLC) etc..But its detection efficiency of both approaches ideal not enough, it is necessary to carry in advance to the astaxanthin in frustule Take, then detect content astaxanthin for extract, which not only adds process needed for detection, and required time compared with It is long, reduce operating efficiency.
Developing a kind of chlorella astaxanthin detection method for improving detection efficiency, simplifying detection process is highly desirable.
The content of the invention
In order to make up the deficiencies in the prior art, it is an object of the invention to provide a kind of Fast nondestructive evaluation chlorella intracellular The method of astaxanthin.
For the present invention to reach its purpose, the technical scheme of use is as follows：
A kind of method of Fast nondestructive evaluation chlorella intracellular astaxanthin, comprises the following steps：
1) standard curve is established --- chlorella suspension known sample known to multiple content astaxanthins is taken, uses deionization The frustule being collected into from the chlorella suspension known sample is resuspended in water or phosphate buffer, is adjusted to cell density 6 × 105~2 × 106Frustule re-suspension liquid between CFU/mL (individual/mL), with the stream equipped with 488nm argon ion lasers Formula cell instrument detects frustule re-suspension liquid in FL1 passages (533/30nm band logicals filter disc) or (the 585/40nm band logicals filter of FL2 passages Piece) average fluorescent strength value；By the logarithm value of the content astaxanthin of chlorella suspension known sample and the FL1 accordingly measured Or the logarithm value of the average fluorescent strength value of FL2 passages makees linear regression, obtains standard curve；Known to the multiple content astaxanthin Chlorella suspension known sample between content astaxanthin it is different.
2) prepare and detect testing sample --- it is resuspended with deionized water or phosphate buffer from chlorella suspension to be measured In the frustule that is collected into, be adjusted to cell density 6 × 105~2 × 106Frustule weight to be measured between CFU/mL (individual/mL) Suspension, as testing sample；
3) with the flow cytomery testing sample equipped with 488nm argon ion lasers in FL1 passages or FL2 passages Average fluorescent strength value, according to step 1) establish standard curve, calculate testing sample content astaxanthin.
When preferably, with flow cytomery sample, its sample introduction flow control is 14~100 μ L/min.
It is more highly preferred to, sample introduction flow control is 35 μ L/min.
Further, the concentration of the phosphate buffer is 10mM, and pH is 6.8~7.6.
Preferably, sample also has the aqueous phase filter membrane or organic phase with 5~40 μm of aperture before with flow cytomery The step of membrane filtration；The logarithm value is denary logarithm.
Further, the content astaxanthin of known sample described in step 1) passes through HPLC methods or UV-vis spectroscopy light Degree meter method is measured, and the existing HPLC methods of the art or ultraviolet-visible spectrophotometer method or other method can be used to survey .
Preferably, the content astaxanthin of known sample described in step 1) is measured by HPLC methods, and determination step includes：
A, prepared by HPLC methods testing sample --- and chlorella suspension known sample is freezed as algae powder, extracted from algae powder Astaxanthin extract is obtained, with methanol and MTBE (methyl tertiary butyl ether(MTBE)) according to 1:3~7:The solvent dissolving of 3 volume ratio mixing The astaxanthin extract；
B, HPLC methods testing conditions --- sample, Detection wavelength 480nm, chromatographic column used are detected using liquid chromatograph For YMCTMC30；Methanol, methyl tertiary butyl ether(MTBE) and water are respectively mobile phase A, B, C, are entered with mobile phase A, B, C eluent formed The percent by volume of row gradient elution, eluent gradient elution program and each mobile phase is：
0-6min, mobile phase A 95% → 80%, Mobile phase B 5 → 20%, mobile phase C 0%；
6-12min, mobile phase A 80 → 60%, Mobile phase B 20 → 38%, mobile phase C 0 → 2%；
12-28min, mobile phase A 60 → 50%, Mobile phase B 38 → 48%, mobile phase C 2%；
28-33min, mobile phase A 50%, Mobile phase B 48%, mobile phase C 2%；
33-35min, mobile phase A 50 → 95%, Mobile phase B 48 → 5%, mobile phase C 2 → 0%；
35-38min, mobile phase A 95%, Mobile phase B 5%, mobile phase C 0%；
C, HPLC method standard curves are established --- with methanol and MTBE according to 1:3~7:The solvent of 3 volume ratio mixing is prepared Astaxanthin standard solution, the mark of each concentration is detected according to step b testing conditions to the standard solutions of multiple concentration gradients The liquid phase peak area of quasi- product solution, liquid phase peak area and corresponding standard solution concentration are done into linear regression, draw HPLC methods Standard curve；
D, obtained sample detects to obtain liquid phase peak area through step b in step a, the HPLC then established according to step c Method standard curve, calculate the known sample content astaxanthin.
Preferably, in step b, when being detected with liquid chromatograph, column oven temperature is 25~35 DEG C, eluent flow rate control For 0.5~1.5mL/min；The liquid chromatograph is further equipped with PDA detectors, and in detection, PDA detectors are at 480nm pairs Astaxanthin is detected, while carries out full wavelength scanner to sample in 300~700nm.
Preferably, in step a, astaxanthin extract obtains as follows：Algae powder is added into the mixed solvent, shaken Swing, be placed in liquid nitrogen and cool down, supernatant is collected by centrifugation；Precipitation adds the in the mixed solvent again, vibrates, is placed in cold in liquid nitrogen But, supernatant is collected by centrifugation, repeats the step untill frustule is in colourless；Merge all supernatants, dried up with nitrogen；Institute It is the mixed solvent mixed by solvent orange 2 A and solvent B containing 0.01~0.2% (quality) additive to state mixed solvent, its Described in solvent orange 2 A be selected from dichloromethane, at least one of n-hexane, solvent B is selected from least one of methanol, ethanol, institute State additive be selected from BHT (2,6- di-tert-butyl-4-methy phenol), BHA (butylated hydroxy anisole), (tertiary butyl is to benzene by TBHQ At least one of diphenol).
Preferably, the volume ratio of the in the mixed solvent solvent orange 2 A and solvent B is 1:1~9:1.
Technical scheme provided by the invention has the advantages that：
The present invention realizes the Fast nondestructive evaluation of chlorella intracellular astaxanthin, greatly simplifies detecting step, is building After day-mark directrix curve, without carrying out extraction operation to sample when detecting sample, sample pre-treatments are simple, shorten detection time, The Fast nondestructive evaluation analysis of microalgae cell astaxanthin especially in chlorella cells is particularly suitable for simultaneously；
Flow cytometry (Flow Cytometry) can excite the astaxanthin of chlorella intracellular to launch fluorescence under 488nm, And average fluorescent strength of the chlorella cells under 533nm (FL1 passages) and 585nm (FL2 passages) launch wavelength is detected respectively. The average fluorescent strength that present inventor is found surprisingly that under the two specific emission wavelengths contains with chlorella intracellular astaxanthin High-positive correlation between amount be present.Match by determining the average fluorescent strength, and with HPLC measurement results, Ke Yizhun Really, quickly, nondestructively reflect astaxanthin accumulation situation.This method can greatly improve detection sensitivity compared with conventional method, Detection rates are also faster.Flow cytometry can carry out quick nondestructive analysis measure to the cell for launching fluorescence, fast in astaxanthin Fast context of detection has important application value.
Brief description of the drawings
Fig. 1 is biomass and astaxanthin accumulation rule in chlorella C.zofingiensis incubations；
In Fig. 2 each figure of A, B, C be culture originate, centre and at the end of chlorella cells light micrograph；
The streaming of FL1 (533nm) the vs. number of cells (Count) of chlorella cells is thin when Fig. 3 is culture starting and ending Born of the same parents' analysis chart；
The streaming of FL2 (585nm) the vs. number of cells (Count) of chlorella cells is thin when Fig. 4 is culture starting and ending Born of the same parents' art analysis chart；Dotted line represents chlorella cells when culture originates in Fig. 3, Fig. 4, and solid line represents the chlorella at the end of culture Cell.FL1/FL2 represents the average fluorescent strength value of flow cytometer FL1/FL2 passages, and Count represents number of cells.
FSC vs.FL1 (533nm) flow cytometry figure of chlorella cells when Fig. 5 is culture starting and ending；
FSC vs.FL2 (585nm) flow cytometry figure of chlorella cells when Fig. 6 is culture starting and ending；
Grey Point represents chlorella cells when culture originates in Fig. 5, Fig. 6, and black color dots represent the bead at the end of culture Frustule, FSC represent the measured value of forward angle light scatter light, and FL1 (533nm), FL2 (585nm) represent flow cytometer respectively The average fluorescent strength value of FL1, FL2 passage；
Fig. 7 is the changing rule of cell average fluorescent strength in chlorella C.zofingiensis incubations；
Fig. 8 is the liquid chromatogram of the chlorella C.zofingiensis intracellular astaxanthins of different incubation times；
Fig. 9 is that the average fluorescent strength FL1 of chlorella cells and the linear of the intracellular content astaxanthin of HPLC methods measure return Return analysis, MFI (Mean fluorescence intensity) represents average fluorescent strength；
Figure 10 is that the average fluorescent strength FL2 of chlorella cells and the linear of the intracellular content astaxanthin of HPLC methods measure return Return analysis, MFI (Mean fluorescence intensity) represents average fluorescent strength.
Embodiment
Technical scheme is described further below in conjunction with the accompanying drawings：
Embodiment 1
Present inventor is during technical solution of the present invention is developed to chlorella C.zofingiensis intracellular shrimps The variation tendency of blue or green cellulose content is studied.
It is prepared by 1.1 actication of culture and seed liquor
By the chlorella Chlorella zofingiensis strain transfers preserved in laboratory to equipped with improvement Basal trainings Support and cultivated on the inclined-plane of base, cultivation temperature is 28 DEG C, and illumination is 10 μm of ol m-2s-1, and observe chlorella growth situation.
With oese by chlorella algae tongue be inoculated into equipped with improvement Basal fluid nutrient mediums, temperature be 28 DEG C, illumination be 10μmol m-2s-1Under the conditions of cultivate 3~5 days and be used as seed liquor.
Wherein described improvement Basal culture mediums (pH6.1) composition is (unit mg/L) as shown in table 1 below：
Table 1
1.2 chlorella C.zofingiensis cultivate accumulation astaxanthin under the conditions of bloom, low nitrogen
1.2.1 chlorella C.zofingiensis bloom, low nitrogen condition of culture
Seed liquor is inoculated into improvement Basal culture mediums by 10% inoculum concentration and carries out chlorella C.zofingiensis trainings Support accumulation astaxanthin.Using improvement Basal culture mediums, the difference of the culture medium in itself and table 1 is fermentation medium：Its grape Sugar is 30g/L, with the addition of NaNO in addition3For 0.6g/L, pH 6.5.
Condition of culture is：Temperature is 28 DEG C, intensity of illumination is 80 μm of ol m-2s-1More than, cultivate 12 days.In incubation Chlorella cells biomass is measured by sampling (result is shown in Fig. 1)；To cultivating 146h, 168h, 192h, 216h, 240h, 264h, 288h Bead algae culturing liquid sampling it is standby；Cell is collected by centrifugation to some beads algae culturing liquid in sampling, frozen after centrifuge washing It is dry, be placed in -20 DEG C of refrigerators and carry out freezen protective, the freeze-dried algae powder sample of preservation include culture 146h, 168h, 192h, 216h, 240h, 264h, 288h freeze-dried algae powder, the analysis for follow-up chlorella para chrome content.
1.2.2 the HPLC detection methods of chlorella intracellular astaxanthin
1.2.2.1 the extracting method of chlorella intracellular astaxanthin
Freeze-dried algae powder 10mg accurately is weighed, is placed in the cryopreservation tube equipped with ceramic bead, addition contains 0.1% (quality) BHT Dichloromethane and methanol in the mixed solvent, the in the mixed solvent dichloromethane：The volume ratio of methanol is 3:1, vibration 1 at a high speed Minute, it is subsequently placed in liquid nitrogen and cools down rapidly, supernatant is collected by centrifugation；Precipitation adds two containing 0.1% (quality) BHT again The in the mixed solvent of chloromethanes and methanol, vibration, is placed in liquid nitrogen and cools down, and supernatant is collected by centrifugation, and repeats the step until algae Untill cell is in colourless.Merge all supernatants, after being dried up with nitrogen, with the methanol of chromatographically pure and MTBE mixed solvent (two The volume ratio of person is 1:1) solvent constant volume analyzes measure chlorella para chrome content to 1mL for follow-up HPLC.
1.2.2.2 the HPLC assay methods of astaxanthin
The HPLC detection methods of chlorella C.zofingiensis intracellular astaxanthins：Using outfit PDA detectors and YMCTM The liquid chromatograph of C30 chromatographic columns (4.6mm × 150mm, 3 μm) is analyzed, eluent be hplc grade methanol (mobile phase A), Methyl tertiary butyl ether(MTBE) MTBE (Mobile phase B) and water (mobile phase C).Gradient elution program is：0-6min, 95 → 80%A, 5 → 20%B, 0%C；6-12min, 80 → 60%A, 20 → 38%B, 0 → 2%C；12-28min, 60 → 50%A, 38 → 48%B, 2%C；28-33min, 50%A, 48%B, 2%C；33-35min, 50 → 95%A, 48 → 5%B, 2 → 0%C；35-38min, 95%A, 5%B, 0%C.
Column oven temperature is 30 DEG C, eluent flow rate 0.8mL/min, and sample size is 20 μ L, PDA detector Detection wavelengths Full wavelength scanner is carried out to determine absolution spectroscopy figure in 300-700nm, while is determined astaxanthin under 480nm wavelength and contained Amount.The qualitative analysis of astaxanthin is analyzed using retention time and abosrption spectrogram, and quantitative analysis is made using standard items Standard curve is analyzed.
The making of astaxanthin standard curve：Astaxanthin standard items accurately are weighed, using the methanol of chromatographically pure and mixing for MTBE Closing solution, (the two volume ratio is 1:1) constant volume is carried out, it is respectively 1,2,3,4,5,6 μ g/mL astaxanthins series mark to be configured to concentration Quasi- solution, it is measured using above-mentioned HPLC methods, using liquid phase peak area as ordinate, using standard concentration as abscissa, is carried out Linear regression analysis, so as to obtain the quantitation curves of astaxanthin：Y=1786.8X -0.2668 (R2=0.9975), wherein X Astaxanthin concentration (μ g/mL) is represented, Y represents liquid phase peak area.
1.2.3 the changing rule of bead algae biomass and intracellular content astaxanthin
By the HPLC in 1.2.2 measure method determine 1.2.1 chlorella cultivate 146h, 168h, 192h, 216h, The content astaxanthin of 240h, 264h, 288h chlorella sample, and be scaled relatively per the content astaxanthin of g dry algae powders, As a result reference can be made to Fig. 1.As shown in Figure 1, bead algae biomass is up to 3.37g/L, and intracellular astaxanthin is after cultivating by the 6th day Start gradually to increase, content is up to 2.3mg/g xeraphiums.
The correlation of the chlorella intracellular content astaxanthin of embodiment 2 and the average fluorescent strength of chlorella cells
Present inventor in chlorella C.zofingiensis incubation, it is found that chlorella is thin in embodiment 1 Born of the same parents become orange red by green, and this is coerced mainly due to intracellular chlorophyll and primary carotenoid in bloom, low nitrogen etc. Under the conditions of, it can induce and roll up secondary carotenoid, mainly the carotenoid such as astaxanthin.Bead under light microscope Frustule culture starting, in incubation and at the end of cellular morphology respectively as shown in A, B, C figure in Fig. 2.
The launch wavelength of maximum can be presented respectively after specific wavelength excites for astaxanthin, this and its containing in the cell Amount is related.The present embodiment is logical in FL1 (533/30nm) using BD Accuri C6 flow cytometers measure chlorella cells The average fluorescent strength of all cells in road and FL2 (585/40nm) passage, accumulated so as to study it with chlorella intracellular astaxanthin Relation between tired amount.
The detection parameters of 2.1 flow cytometers：
In terms of flow control (Fluidics) setting of flow cytometer, in order to realize best Detection results, it is necessary to Optimization is controlled to sample introduction flow velocity, sample introduction flow velocity can be set as between 14-100 μ L/min.When setting sample introduction flow velocity, need Otherwise the cell velocity for ensureing to detect can influence the accuracy of measured value for 2500/second (events/sec) and below. Sample continuous mode, to ensure the accuracy of experiment, fixed flow velocity can be set as 35 μ L/min.In detection threshold value (Threshold) in terms of setting, then the peak height parameter (FSC-H) of forward angle light scatter light is set as 80000.In color compensation (Set Color Compensation) setting in terms of, it is 0% that can set offset.(Run Settings) is controlled in sample introduction In terms of setting, cell number (Events), sample injection time (Time), sampling volume (Volumn) can be selected to be set respectively, protected The target cell number finally detected is demonstrate,proved more than 10000.
The pre-treating method of testing sample used in 2.2 flow cytomeries
Testing sample separates and collects bead algae culturing liquid or suspension before analysis, using the rotating speed of 3800 × g centrifugal force In frustule, then suspended using deionized water or phosphate-buffered concentration (10mM, pH 7.4), hanged again after being repeated twice It is floating.Manufactured re-suspension liquid will ensure that chlorella cells density is 6 × 105~2 × 106Between CFU/mL (individual/mL), on the one hand Meet that (per cell number in mL or granule number is 1 × 10 for the sample introduction requirement of flow cytometer3~5 × 106), on the other hand it is too high or Too low cell density can all influence the Accurate Determining of fluorescent value.Finally use 40 μm of aqueous phase membrane filtration cell suspension to be measured Afterwards, it is placed in 2mL centrifuge tubes and is used for sample measure, is shaken up before sample introduction, ensure the accuracy of sample detection result.
2.3 sample detections and analysis
During BD Accuri C6 flow cytomery cells in sample average fluorescent strengths, FL1 is detected respectively The average fluorescent strength of (533nm), cells in sample under FL2 (585nm) passage.The astaxanthin of chlorella intracellular is thin in streaming Born of the same parents' instrument is equipped with the fluorescence that can launch different wave length under the exciting of 488nm argon ion lasers, but the possible difference of fluorescence intensity is very Greatly.All chlorella cells are selected by drawing door, then create fluorescence channel FL1 and FL2 histogram, flow cytometer is certainly It is dynamic to calculate chlorella cells in FL1 passages and the average fluorescent strength of FL2 passages.
146h, 168h, 192h, 216h, 240h, 264h, 288h bead algae culturing liquid will be cultivated in embodiment 1 by above-mentioned 2.1~2.3 the step of and method prepare sample, and detect average fluorescent strength of each sample in FL1 and FL2 passages.Detection knot Fruit sees Fig. 7, from fig.7, it can be seen that started gradually to accumulate astaxanthin in intracellular from the 6th day in chlorella incubation, its cell Average fluorescent strength is also being continuously increased.The small of 146h, 168h, 192h, 216h, 240h, 264h, 288h is cultivated in embodiment 1 The component of the chlorella intracellular astaxanthin that ball algae sample is determined using the HPLC methods of embodiment 1 is as shown in figure 8, wherein astaxanthin Species mainly includes free astaxanthin, the ester of astaxanthin one and astaxanthin diester.Understood with reference to Fig. 1,2 analyses, chlorella intracellular The average fluorescent strength of content astaxanthin and chlorella cells is all in synchronous increase, between the two in the presence of certain correlation.
The linear regression analysis for the content astaxanthin that the chlorella cells average fluorescent strength of embodiment 3 measures with HPLC methods
In order to realize the quick and precisely analysis for chlorella intracellular content astaxanthin, this experiment is surveyed by flow cytometer Determine cell average fluorescent strength, and linear regression analysis is carried out with the content astaxanthin of chlorella intracellular, so that it is determined that streaming Cell art quick and precisely lossless detection method.
In embodiment 1, by HPLC determination methods measured culture to 146h, 168h, 192h, 216h, 240h, Its chlorella intracellular content astaxanthin of 264h, 288h chlorella sample, as a result as shown in Figure 1.These content astaxanthins are surveyed Being sat using 10 for the common logarithm value at bottom as vertical for (unit representing the content of astaxanthin in every gram of dry algae powder based on μ g/g) must be worth Mark, at the same respectively with the corresponding average fluorescent strength value in flow cytometry FL1 and FL2 passage that is measured in embodiment 2 Using 10 for bottom common logarithm value as abscissa, mapped, and carry out linear regression analysis, as a result as shown in Fig. 9,10. As shown in figure 9, for the average fluorescent strength value of FL1 passages, the linear regression curves of acquisition are Y=1.938X-5.6681 (R2 =0.8725), in formula Y represent chlorella intracellular content astaxanthin (μ g/g xeraphiums) common logarithm value (with 10 for bottom pair Number), X represents the common logarithm value (denary logarithm) of the average fluorescent strength of Flow Cytometry Assay FL1 passages；Such as figure Shown in 10, for the average fluorescent strength value of FL2 passages, the linear regression curves of acquisition are Y=1.5325X -3.0668 (R2= 0.9209), in formula Y represent chlorella intracellular content astaxanthin (μ g/g xeraphiums) common logarithm value (with 10 for bottom pair Number), X represents the common logarithm value (denary logarithm) of Flow Cytometry Assay passage FL2 average fluorescent strength.
From Fig. 9,10, between chlorella intracellular content astaxanthin and passage FL2 and FL1 cell average fluorescent strength Linear regression coeffficient respectively reaches 0.9209 and 0.8725, illustrates high-positive correlation between the two be present, wherein leading to from FL2 The linear dependence in road is better than FL1 passages.This shows that Flow Cytometry Assay FL2 passages, the cell of FL1 passages can be used Average fluorescent strength carrys out the content of quick nondestructive analysis detection chlorella C.zofingiensis intracellular astaxanthins, but to use The cell average fluorescent strength of Flow Cytometry Assay FL2 passages is more preferably.
The methodological study of the flow cytometric assays of the chlorella intracellular astaxanthin of embodiment 4
By embodiment 3, the cell average fluorescent strength using Flow Cytometry Assay passage FL2 contains with astaxanthin High correlation between amount be present, the equation of linear regression of foundation can be used for Fast nondestructive evaluation analysis chlorella intracellular shrimp green grass or young crops Cellulose content.The present embodiment further demonstrates the degree of accuracy and the precision of the flow cytometric assays.
4.1 linear regression curves and lower limit of quantitation
Embodiment 3 is understood using the analysis of flow cytometry regression curve, is worth to using passage FL2 average fluorescent strength Linear regression curves be：Y=1.5325X -3.0668 (R2=0.9209), Y represents chlorella intracellular content astaxanthin in formula Common logarithm value (denary logarithm), X represent flow cytometer measure passage FL2 average fluorescent strengths common logarithm It is worth (denary logarithm).The equation of linear regression can illustrate this detection method very well, but it is not to fix to express equation 's.The coefficient R of the linear regression curves2=0.9209, this shows to determine astaxanthin to contain using average fluorescent strength value Measurer has high correlation.The minimum average fluorescent strength (FL2) of linear regression curves measure is 7988.11, then calculates The lower limit of quantitation of the chlorella intracellular content astaxanthin gone out is 0.82mg/g dry algae powders.
4.2 accuracy test
Accuracy test is carried out using the chlorella sample of known content astaxanthin, it is accurate according to similar average recovery measure The method of exactness is tested, as a result as shown in table 2.
Totally two known samples, it is that known content astaxanthin is the chlorella sample 1 of 0.99mg/g dry algae powders, shrimp green grass or young crops respectively The chlorella sample 2 of cellulose content 1.72mg/g dry algae powders.Detection parameters are set, according in embodiment 2 according in embodiment 2 2.1 2.2 pairs of samples are handled, and are tested and analyzed according to 2.3 pairs of samples in embodiment 2, take detection sample 1,2 in FL2 passages Average fluorescent strength value, then according to linear regression curves Y=1.5325X -3.0668 (R2=0.9209) astaxanthin is calculated to obtain The measured value of content, it is as a result as shown in table 2 below：
Table 2
Conclusion：Mean sample recovery rate is that 99.18%, RSD is 1.23% (n=5), and this shows the degree of accuracy symbol of the result Close and require.
4.3 precision test
Known content astaxanthin is used to carry out Flow Cytometry Assay analysis for the chlorella cells of 2.40mg/g dry algae powders, Specifically：Detection parameters are set according in embodiment 2 2.1, handled according to 2.2 pairs of samples in embodiment 2, according to embodiment 2.3 pairs of samples test and analyze in 2, the average fluorescent strength value for detecting sample in FL2 passages are taken, then according to linear regression Curve Y=1.5325X -3.0668 (R2=0.9209) calculate content astaxanthin measured value.5 progress of replication are accurate Degree experiment, result of the test are as shown in table 3.
Table 3
Precision test shows that good using the repeatability of flow cytometer determination method, assay method is more stable, accurate Degree is good.Experiment shows above, and the present invention can Fast nondestructive evaluation analysis chlorella intracellular shrimp using flow cytometric assays Blue or green cellulose content, its linear regression curves, accuracy test, precision test meet the requirements.
The above described is only a preferred embodiment of the present invention, any formal limitation not is done to the present invention, therefore All contents without departing from technical solution of the present invention, the technical spirit according to the present invention any are simply repaiied to made for any of the above embodiments Change, equivalent variations and modification (fluoroscopic examination included but is not limited to after autofluorescence detection and fluorescent dyeing), still belong to In the range of technical solution of the present invention.

Claims (9)

  1. A kind of 1. method of Fast nondestructive evaluation chlorella intracellular astaxanthin, it is characterised in that comprise the following steps：
    1) establish standard curve --- take chlorella suspension known sample known to multiple content astaxanthins, with deionized water or The frustule being collected into from the chlorella suspension known sample is resuspended in phosphate buffer, is adjusted to cell density 6 ×105~2 × 106Frustule re-suspension liquid between CFU/mL, with the flow cytometer equipped with 488nm argon ion lasers Average fluorescent strength value of the frustule in FL1 passages or FL2 passages in detection re-suspension liquid；By chlorella suspension known sample Being averaged under the FL2 channel emission wavelengths of the logarithm value of content astaxanthin and the 533nm accordingly measured FL1 passages or 585nm The logarithm value of fluorescence intensity level makees linear regression, obtains standard curve；The content astaxanthin of the known sample is surveyed by HPLC methods , determination step includes：
    A, prepared by HPLC methods testing sample --- and chlorella suspension known sample is freezed as algae powder, extracts and obtains from algae powder Astaxanthin extract, with methanol and methyl tertiary butyl ether(MTBE) according to 1：3~7：The solvent of 3 volume ratio mixing dissolves the astaxanthin Extract；
    B, HPLC methods testing conditions --- sample, Detection wavelength 480nm are detected using liquid chromatograph, chromatographic column used is YMCTM C30；Methanol, methyl tertiary butyl ether(MTBE) and water are respectively mobile phase A, B, C, are entered with mobile phase A, B, C eluent formed The percent by volume of row gradient elution, eluent gradient elution program and each mobile phase is：
    0-6min, mobile phase A 95% → 80%, Mobile phase B 5 → 20%, mobile phase C 0%；
    6-12min, mobile phase A 80 → 60%, Mobile phase B 20 → 38%, mobile phase C 0 → 2%；
    12-28min, mobile phase A 60 → 50%, Mobile phase B 38 → 48%, mobile phase C 2%；
    28-33min, mobile phase A 50%, Mobile phase B 48%, mobile phase C 2%；
    33-35min, mobile phase A 50 → 95%, Mobile phase B 48 → 5%, mobile phase C 2 → 0%；
    35-38min, mobile phase A 95%, Mobile phase B 5%, mobile phase C 0%；
    C, HPLC method standard curves are established --- with methanol and methyl tertiary butyl ether(MTBE) according to 1:3~7:The solvent of 3 volume ratio mixing Astaxanthin standard solution is prepared, each concentration is detected according to step b testing conditions to the standard solution of multiple concentration gradients Standard solution liquid phase peak area, liquid phase peak area and corresponding standard solution concentration are done into linear regression, drawn HPLC method standard curves；
    D, obtained sample detects to obtain liquid phase peak area through step b in step a, the HPLC method marks then established according to step c Directrix curve, calculate the known sample content astaxanthin；
    2) testing sample is prepared --- it is resuspended what is be collected into from chlorella suspension to be measured with deionized water or phosphate buffer Frustule, cell density is adjusted to 6 × 105~2 × 106Frustule re-suspension liquid to be measured between CFU/mL, as treating test sample Product；
    3) with the flow cytomery testing sample equipped with 488nm argon ion lasers in the flat of FL1 passages or FL2 passages Equal fluorescence intensity level, according to step 1) establish standard curve, calculate testing sample content astaxanthin.
  2. 2. according to the method for claim 1, it is characterised in that during with flow cytomery sample, its sample introduction flow velocity control It is made as 14~100 μ L/min.
  3. 3. according to the method for claim 2, it is characterised in that sample introduction flow control is 35 μ L/min.
  4. 4. according to the method for claim 1, it is characterised in that the concentration of the phosphate buffer is 10mM, pH 6.8 ~7.6.
  5. 5. according to the method for claim 1, it is characterised in that sample also has before with flow cytomery uses aperture The step of 5~40 μm of aqueous phase or organic phase filter membrane filtering；The logarithm value is denary logarithm.
  6. 6. according to the method for claim 1, it is characterised in that the content astaxanthin of known sample described in step 1) may be used also To be measured by ultraviolet-visible spectrophotometer method.
  7. 7. according to the method for claim 1, it is characterised in that in step b, when being detected with liquid chromatograph, column temperature box temperature Spend for 25~35 DEG C, eluent flow rate control is 0.5~1.5mL/min；The liquid chromatograph is further equipped with PDA detectors, In detection, PDA detectors carry out the detection of astaxanthin in 480nm, while carry out all-wave length to sample in 300~700nm and sweep Retouch.
  8. 8. according to the method for claim 1, it is characterised in that in step a, astaxanthin extract obtains as follows ：Algae powder is added into the mixed solvent, vibration, is placed in liquid nitrogen and cools down, supernatant is collected by centrifugation；Precipitation adds described mixed again In bonding solvent, vibration, it is placed in liquid nitrogen and cools down, supernatant is collected by centrifugation, repeats the step untill frustule is in colourless；Close And all supernatants, dried up with nitrogen；It containing mass percent is 0.01~0.2% additive by molten that the mixed solvent, which is, The mixed solvent that agent A and solvent B are mixed, wherein the solvent orange 2 A is selected from least one of dichloromethane, n-hexane, it is molten Agent B is selected from least one of methanol, ethanol, and the additive is selected from least one of BHT, BHA, TBHQ.
  9. 9. according to the method for claim 8, it is characterised in that the volume ratio of the in the mixed solvent solvent orange 2 A and solvent B is 1:1~9:1.
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