CN104730161B - The detection method of content of triglyceride in microalgae - Google Patents
The detection method of content of triglyceride in microalgae Download PDFInfo
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Abstract
The invention discloses the detection method of content of triglyceride in a kind of microalgae, when carrying out microalgae grease extraction, have employed mixed solvent, after being mixed with microalgae powder by mixed solvent, carry out supersonic wave wall breaking extraction；Microalgae wall breaking rate is up to 99%, and broken time is the shortest, and the efficiency of whole procedure extraction microalgae intracellular glycerol three ester is high.In microalgae, the detection method of content of triglyceride uses vapor phase method, and vapor phase method can detect content of triglyceride more effectively, accurately.
Description
The application is Application No. 201310585841.9, filing date on November 20th, 2013, and invention and created name is the divisional application of the application for a patent for invention of " detection method of content of triglyceride in the extracting method of microalgae grease and microalgae ".
Technical field
The detection method of content of triglyceride contained in the present invention relates to the extracting method of oils and fats in a kind of microalgae cell and microalgae cell.
Background technology
World's crude output persistently reduces, it is contemplated that will settle to 7,500,000,000 barrels/year by 2018, just corresponds to nineteen fifty crude output.Along with the problems such as petroleum resources energy shortage the most exhausted, global and environmental pollution are day by day serious, exploitation is green, the bio-fuel of cleaning replaces conventional fossil fuel, it has also become international research focus.
Biodiesel refers to the organic aliphatic acid Ester obtained through ester exchange reaction by oils and fats in vegetable oil, Animal fat and extracellular microbial with short chain alcohol, makes it have the strongest competitiveness as renewable alternative energy source, environment friendly and recyclability.In numerous raw materials of biodiesel, microalgae has that photosynthetic efficiency is high, oil content high, growth cycle is short, oils and fats area yield high is it is considered to be the biomass resource of the most potential petroleum replacing.Microalgae directly utilizes simple nutrient substance such as sunlight, carbon dioxide and nitrogen, phosphorus etc. and synthesizes a large amount of oils and fatss in intracellular.Oils and fats composition predominantly triglyceride (content >=80%) and the C of most of microalgae intracellular14-C22Long-chain fatty acid, wherein fatty acid is with C16And C18Be fatty acid be main.
Owing to having tough and tensile cell wall outside microalgae cell, thus first frond should be carried out front broken wall treatment to before triglyceride extracts in microalgae cell.In microalgae cell, the extraction ratio of triglyceride crushes closely related the most completely with cell wall.Common wall-breaking method has grinding, acid heat method, frond from melting method, protein solvent degeneration methods, multigelation method, ultrasonic disruption etc..Wherein supercritical ultrasonics technology is suitable for most of microalgae cell, and ultrasound wave is relevant with cell category, time and concentration to cytoclastic efficiency.
After microalgae cell is broken, the method extracting oils and fats in microalgae cell is most important.Direct extraction method currently, with respect to micro-algae intracellular grease mainly has physical squeezing method, organic solvent extraction, supercritical extraction etc..Wherein, organic solvent extraction is current domestic and international a kind of wide variety of method.
Such as Chinese patent literature CN 102816636 A(application number
201210259778.5) extracting method of a kind of microalgae grease is disclosed, first centrifugal microalgae cell of collecting, lyophilised dry algae powder；Dry algae powder is mixed with ethanol, sonicated；Material after supersound process is added in apparatus,Soxhlet's, extract 3 to 4 hours in boiling water bath, be then passed through filter separation, concentrating under reduced pressure acquisition microalgae grease.
In general, selected organic solvent should make every effort to obtain high oil recovery during extracting, and avoids the solvent injury to human body simultaneously, and research shows, single solvent is difficult to extract micro-algae intracellular grease.
About mixed solvent extracting method, Chinese patent literature CN 102942991 A(application number 201210511744.0) disclose a kind of Microalgae grease extraction method, first Microalgae fermentation liquid is centrifuged, collects the microalgae cell after separating；The microalgae cell wash buffer that will obtain, then make stand-by algae solution；Above-mentioned algae solution is carried out 2 to 3 multigelations, while melting, adds ultrasound field；For the stock solution obtained after freeze thawing, use extractant glycerol with Soxhlet extraction device: ethanol: petroleum ether=0.5: extract at 6.5: 3；Extractant during after bake blows down the extraction mixed liquor containing oils and fats going to obtain.
Additionally, how oils and fats especially content of triglyceride in Accurate Determining microalgae cell, it is that to weigh extracting method the most effective crucial.Select quick, easy, efficient, sensitive assay method significant.
The most the most frequently used assay method is fluorimetry.Such as Chinese patent literature CN 103063631 A(application number 201210560247.X) method that discloses a kind of solvent extraction-fluoremetry microalgae grease content, first standard curve is made: take microalgae, collected after centrifugation precipitates, it is diluted to algae solution with PBS, as standard algae solution after cleaning up with PBS；Take 5 parts of 19.8mL standard algae solutions, the triolein solution of differently configured concentration；Respectively toward triolein solution being sequentially added into dimethyl sulfoxide and Nile red dyes 20 minutes, measure the fluorescence intensity of each triolein solution, with absorbance as vertical coordinate, triolein solution concentration as abscissa, draw standard curve and also calculate regression equation；Then microalgae grease is extracted；Take in solution to be measured addition 19.8mL standard algae solution prepared by 0.2mL and obtain sample algae solution, 0.2mL normal hexane is added in 19.8mL standard algae solution and obtains blank sample, net sample algae solution, blank sample are sequentially added into dimethyl sulfoxide respectively and Nile red dyes 20 minutes, measure sample algae solution, the fluorescence intensity of blank sample respectively, the fluorescence intensity of sample algae solution deducts the fluorescence intensity of blank sample and obtains the fluorescence intensity of solution to be measured, by the concentration of microalgae grease in regression equation calculation solution to be measured.
Summary of the invention
The technical problem to be solved be to provide the high microalgae grease of a kind of oils and fats extraction efficiency extracting method and a kind of more effectively, the accurate method of content of triglyceride in detection microalgae cell.
The technical scheme realizing an object of the present disclosure is the extracting method of a kind of microalgae grease, comprises the following steps:
1., after taking micro algae culturing liquid centrifugation, remove supernatant and the microalgae of acquisition is carried out lyophilization, taking freeze-dried algae powder to be extracted in test tube.
2. by oxolane: chloroform: normal hexane is that 1: 0.8～1.5: 1～1.2 preparation mixed solutions are stand-by by volume.
3. the mixed solution 2. prepared with pipet removing step, add step 1. in fill after lyophilizing in the test tube of algae powder.
4. algae powder step 3. obtained carries out the first shattering process with the mixed material of mixed solution in ultrasound field, and in the first shattering process, mixed material crushes 3～5 times in ultrasound field, and the persistent period is 30～60s each time, each interval time 10～30s.
Centrifugation after first shattering process, collects in the backward precipitation being centrifugally separating to obtain of supernatant and adds the mixed solution that 2. fresh step is prepared, carry out the second shattering process；In second shattering process, mixed material crushes 3～5 times in ultrasound field, and the persistent period is 30～60s each time, each interval time 10～30s.
Centrifugation after second shattering process, collects supernatant；Repeating the second shattering process 1～2 times, the supernatant being centrifugally separating to obtain after each shattering process being terminated merges pending.
5. the supernatant that 4. step merges is poured in preprepared receiving flask；Receiving flask carries out vacuum pumping, and mixed organic solvents separates with the oils and fats extracted from microalgae, and in receiving flask, remaining liquid is from microalgae the oils and fats extracted, thus completes the extraction of microalgae grease.
Above-mentioned steps 3. in algae powder after every 1g lyophilizing add the 20～60mL mixed solutions that 2. step is prepared.
When 5. above-mentioned steps carries out vacuum pumping to receiving flask, mixed organic solvents enters in the returnable bottle that connected of vacuum extractor outlet side after extracting out from receiving flask, and mixed organic solvents can be reused for step 2. in step extraction operation 4. after collecting.
Above-mentioned steps 4. in, during ultrasonication, the frequency of ultrasound wave is 20～30kHz.
The technical scheme realizing the present invention the second purpose is the detection method of content of triglyceride in a kind of microalgae, comprises the following steps:
(1) prepare to treat test sample solution, comprise the following steps:
1., after taking micro algae culturing liquid centrifugation, remove supernatant and the microalgae of acquisition is carried out lyophilization, taking freeze-dried algae powder 0.2g to be extracted in test tube.
2. by oxolane: chloroform: normal hexane is that 1: 0.8～1.5: 1～1.2 preparation mixed solutions are stand-by by volume.
3. pipette, with pipet, the mixed solution that 2. 5mL step is prepared, add step 1. in fill after lyophilizing in the test tube of algae powder.
4. algae powder step 3. obtained carries out the first shattering process with the mixed material of mixed solution in ultrasound field, and in the first shattering process, mixed material crushes 3～5 times in ultrasound field, and the persistent period is 30～60s each time, each interval time 10～30s.
Centrifugation after first shattering process, collects in the backward precipitation being centrifugally separating to obtain of supernatant and adds the 5mL mixed solution that 2. fresh step is prepared, carry out the second shattering process, and the operation of the second shattering process is identical with the first shattering process；Centrifugation after second shattering process, collects supernatant；Repeat the second shattering process 1 time.
5. after the most each for step shattering process being terminated, the centrifugal supernatant collected is poured in preprepared receiving flask after merging.
If volume is less than 15mL after the most each time supernatant merges, then it is settled to 15mL with the mixed solution that 2. step is prepared.
(2) preparation inner mark solution, weighs 10mg octacosane and is dissolved in 1mL mixed solution, the mixed solution that 2. step when described mixed solution is to prepare sample solution to be measured is prepared.
(3) preparing standard solution, weighs 50 μ L heptadecanes, 50mg myristic acid, 50mg Palmic acid, 20mg stigmasterol and 250 μ L vegetable oil and is dissolved in 4 mL mixed solvents, make standard solution；The mixed solution that 2. step when described mixed solution is to prepare sample solution to be measured is prepared.
(4) standard curve is made, 10 μ L are pipetted with pipet respectively from the standard solution that step (3) is prepared, 25 μ L, 50 μ L, 75 μ L, 100 μ L standard solution are in GC pipe, and are settled to 1mL with mixed solution, are subsequently adding the inner mark solution that 10 μ L step (2) prepare and obtain five parts of standard curve solution；Measure the peak area value of triglyceride in five parts of standard curve solution respectively with gas chromatograph after, with triglyceride peak area for X value, with content of triglyceride as Y value, draw standard curve, calculate regression equation.
(5) measuring content of triglyceride in microalgae, take that step (1) prepares treats that test sample solution 1mL, in GC pipe, adds 10 microlitre internal standard octacosane solution, then carries out GC detection；Being substituted in regression equation as X value by the triglyceride peak area value that GC detection obtains, its calculated Y value is content of triglyceride in microalgae.
Test sample solution is treated in above-mentioned preparation, take the fresh micro algae culturing liquid growing to late log phase, after being centrifuged 10～20 minutes in 7000～8500r/min, remove supernatant and the microalgae of acquisition is carried out lyophilization, in freezing dry process, every 12h measures the change of microalgae weight, weighs algae powder after constant weight in test tube.
During above-mentioned preparation treats that the step of test sample solution is 4., during ultrasonication, the frequency of ultrasound wave is 20～30kHz.
When step (4) makes standard curve, the content of triglyceride in standard solution i.e. Y value is to be obtained by the content z of the triglyceride in the content of vegetable oil in standard solution × known vegetable oil.
The present invention has positive effect: when (1) present invention carries out microalgae grease extraction, have employed mixed solvent, carries out supersonic wave wall breaking extraction after being mixed with microalgae powder by mixed solvent；Microalgae wall breaking rate is up to 99%, and broken time is the shortest, and the efficiency of whole procedure extraction microalgae intracellular glycerol three ester is high.
(2) present invention extracts that the process of microalgae grease is carried out at normal temperatures, power consumption is little, easy realization is extensive and automated production；Compared with soxhlet extraction method, the oils and fats extraction time of the present invention is substantially reduced, thus saves resource, saves the energy.
(3) mixed solvent used during microalgae grease of the present invention extracts is collected when evacuation is dried, can be used for using when microalgae grease next time extracts, both avoid the organic solvent injury to operator and the pollution to environment, saved again material, reduce extraction cost.
(4) in the microalgae of the present invention, the detection method of content of triglyceride uses vapor phase method, and vapor phase method can detect content of triglyceride more effectively, accurately.
Accompanying drawing explanation
Fig. 1 is the gas chromatogram that embodiment 6 treats test sample solution.
Detailed description of the invention
(embodiment 1, the extracting method of microalgae grease)
The extracting method of the microalgae grease of the present embodiment comprises the following steps:
1. take grow to late log phase fresh microalgae (microalgae in the present embodiment is diatom Fructus Gleditsia acupuncture algae,Chaetoceros
gracilisThered is provided by Univ Minnesota-Twin Cities USA) culture fluid 1000mL, after 8000r/min is centrifuged 15 minutes, remove supernatant and the microalgae of acquisition is carried out lyophilization, in freezing dry process, every 12h measures the change of microalgae weight, and after constant weight, (microalgae powder constant weight after lyophilizing 72h in the present embodiment) weighs 0.2g algae powder in 15mL test tube.
2. by oxolane: chloroform: normal hexane is that 1: 1: 1 preparation 100mL mixed solution is stand-by by volume.
3. pipette, with pipet, the mixed solution that 2. 5mL step is prepared, add step 1. in fill in the test tube of 0.2g algae powder.
4. algae powder step 3. obtained carries out the first shattering process with the mixed material of mixed solution in ultrasound field, mixed material broken 3～5 times (being 5 times in the present embodiment) in ultrasound field in first shattering process, persistent period is for 45s in 30～60s(the present embodiment each time), each interval time 10～30s(is 15s in the present embodiment).
Material after first shattering process is centrifuged 1 minute in 3000r/min, collects in the backward precipitation being centrifugally separating to obtain of supernatant and adds the 5mL mixed solution that 2. fresh step is prepared, carries out the second shattering process；Mixed material broken 3～5 times (being 5 times in the present embodiment) in ultrasound field in second shattering process, the persistent period is for 45s in 30～60s(the present embodiment each time), each interval time 10～30s(is 15s in the present embodiment).
Material after second shattering process is centrifuged 1 minute in 3000r/min, collects supernatant；Repeat the second shattering process 1～2 times.
During ultrasonication, ultrasonic output is 120w, and the frequency of ultrasound wave is for 23kHz in 20～30kHz(the present embodiment).
5. after the most each for step shattering process being terminated, the centrifugal supernatant collected all is poured in preprepared receiving flask；Receiving flask carries out vacuum pumping, and mixed organic solvents separates with the oils and fats extracted from microalgae, and in receiving flask, remaining liquid is from microalgae the oils and fats extracted, thus completes the extraction of microalgae grease；The quality of the oils and fats extracted in the present embodiment is 0.083g.
Entering after wherein mixed organic solvents is extracted out from receiving flask in the returnable bottle that connected of vacuum extractor outlet side, mixed organic solvents can be reused for step 2. in step extraction operation 4. after collecting.
(embodiment 2, the extracting method of microalgae grease)
Remaining is same as in Example 1 for the extracting method of the microalgae grease of the present embodiment, and difference is:
When 2. step prepares mixed solution, oxolane: chloroform: normal hexane is 1: 1.5: 1 by volume.The quality of the oils and fats that 5. step is extracted is 0.076g.
(embodiment 3, the extracting method of microalgae grease)
Remaining is same as in Example 1 for the extracting method of the microalgae grease of the present embodiment, and difference is:
When 2. step prepares mixed solution, oxolane: chloroform: normal hexane is 1: 0.8: 1 by volume.The quality of the oils and fats that 5. step is extracted is 0.079g.
(embodiment 4, the extracting method of microalgae grease)
Remaining is same as in Example 1 for the extracting method of the microalgae grease of the present embodiment, and difference is:
1. step takes Porphyridium cruentum (Inst. of Hydrobiology, Chinese Academy of Sciences's algae kind storehouse, the numbering growing to late log phase
FACHB-744) medium centrifugal separates and lyophilizing obtains algae powder oils and fats to be extracted.The quality of the oils and fats that 5. step is extracted is 0.061g.
(embodiment 5, the extracting method of microalgae grease)
Remaining is same as in Example 1 for the extracting method of the microalgae grease of the present embodiment, and difference is:
1. step takes nostoc (Inst. of Hydrobiology, Chinese Academy of Sciences's algae kind storehouse, the numbering growing to late log phase
FACHB-133) medium centrifugal separates and lyophilizing obtains algae powder oils and fats to be extracted.The quality of the oils and fats that 5. step is extracted is 0.058g.
The present invention uses supersonic wave wall breaking to extract, with mixed solvent, the extracting mode combined, select oxolane, chloroform, normal hexane these three organic solvent, final microalgae wall breaking rate is up to 99%, and broken time is the shortest, and the efficiency of whole procedure extraction microalgae intracellular glycerol three ester is high.The process of whole extraction microalgae grease is carried out at normal temperatures, power consumption is little, easy realization extensive and automated production；Compared with the extraction time of several with soxhlet extraction method hours, the oils and fats extraction time of the present invention is substantially reduced, thus saves resource, saves the energy.Additionally, the mixed solvent that microalgae grease uses during extracting is collected when evacuation is dried, and can be used for using when microalgae grease next time extracts, had both avoided the organic solvent injury to operator and the pollution to environment, save again material, reduce extraction cost.
(detection method of content of triglyceride in embodiment 6, microalgae)
In the microalgae of the present embodiment, the detection method of content of triglyceride comprises the following steps:
(1) prepare to treat test sample solution, comprise the following steps:
1. take grow to late log phase fresh microalgae (microalgae in the present embodiment is diatom Fructus Gleditsia acupuncture algae,Chaetoceros
gracilisThered is provided by Univ Minnesota-Twin Cities USA) culture fluid 1000mL, after 8000r/min is centrifuged 15 minutes, remove supernatant and the microalgae of acquisition is carried out lyophilization, in freezing dry process, every 12h measures the change of microalgae weight, and after constant weight, (microalgae powder constant weight after lyophilizing 72h in the present embodiment) weighs 0.2g algae powder in 15mL test tube.
2. by oxolane: chloroform: normal hexane is that 1: 1: 1 preparation 100mL mixed solution is stand-by by volume.
3. pipette, with pipet, the mixed solution that 2. 5mL step is prepared, add step 1. in fill in the test tube of 0.2g algae powder.
4. algae powder step 3. obtained carries out the first shattering process with the mixed material of mixed solution in ultrasound field, in first shattering process, mixed material crushes 3～5 times in ultrasound field, persistent period is 30～60s each time, after once with previous interval time 10～30s.
Material after first shattering process is centrifuged 1 minute in 3000r/min, collects in the backward precipitation being centrifugally separating to obtain of supernatant and adds fresh 5mL mixed solution, carries out the second shattering process, and the operation of the second shattering process is identical with the first shattering process；Material after second shattering process is centrifuged 1 minute in 3000r/min, collects supernatant；Repeat the second shattering process 1 time.For 23Hz during during ultrasonication, the frequency of ultrasound wave is 20～30Hz(the present embodiment).
5. after the most each for step shattering process being terminated, the centrifugal supernatant collected all is poured in the graduated receiving flask of preprepared band.
If volume is less than 15mL after 6. three supernatant merge, then it is settled to 15mL with the mixed solution that 2. step is prepared.
(2) preparation inner mark solution.Weigh 10mg octacosane to be dissolved in 1mL mixed solution；The mixed solution that 2. step when described mixed solution is to prepare sample solution to be measured is prepared.
(3) preparing standard solution.Weigh 50 μ L heptadecanes, 50mg myristic acid, 50mg Palmic acid, 20mg stigmasterol and 250 μ L vegetable oil to be dissolved in 4 mL mixed solvents, make standard solution.Described vegetable oil is Fructus Maydis oil；The mixed solution that 2. step when described mixed solution is to prepare sample solution to be measured is prepared.
(4) standard curve is made.From the standard solution that step (3) is prepared, pipette 10 μ L, 25 μ L, 50 μ L, 75 μ L, 100 μ L standard solution in GC pipe with pipet respectively, and be settled to 1mL with mixed solution；It is subsequently adding inner mark solution four parts of standard curve solution of acquisition that 10 μ L step (2) prepare, then carries out standard curve making.
Concrete curve plotting step is: after five parts of standard curve solution measure respectively the peak area value obtaining wherein triglyceride with gas chromatograph, with triglyceride peak area value for X value, with content of triglyceride as Y value, draw standard curve, calculating regression equation, the regression equation obtained is Y=5.8637x+0.1192(R2=0.995).
In vegetable oil used by the present embodiment preparing standard solution, the content of triglyceride is 96%, and in the most above-mentioned five parts of standard curve solution, the content i.e. Y value of triglyceride is to be obtained by content × 96% of vegetable oil in standard solution.
In concrete operations, when selecting different vegetable oil, the content z of the triglyceride in each vegetable oil is known, and therefore in standard solution, the content × z of vegetable oil in standard solution only need to be obtained by the content of triglyceride.
(5) content of triglyceride in microalgae is measured.Take that step (1) prepares treats that test sample solution 1mL, in GC pipe, adds 10 microlitre internal standard octacosane solution, then carries out GC detection, and Fig. 1 is shown in by GC collection of illustrative plates.
The triglyceride peak area value value that GC detection obtains being substituted in the regression equation of step (4), calculating microalgae intracellular glycerol three ester content is 41.2%.
(detection method of content of triglyceride in comparative example 1, microalgae)
In order to detect the detection method of content of triglyceride in the microalgae of the present invention accurately and effectiveness, we weigh the microalgae powder of lyophilizing in 0.2g embodiment 6 simultaneously, after conventional milled processed, with 95% ethanol as Extraction solvent, using soxhlet extraction to extract the total oils and fats of microalgae intracellular, extraction time is 8h, calculates fat content by weight change before and after extracting, the total fat content of result microalgae intracellular is 39.6%, and effective, the accuracy of the inventive method are described.
Claims (4)
1. the detection method of content of triglyceride in a microalgae, it is characterised in that comprise the following steps:
(1) prepare to treat test sample solution, comprise the following steps:
1., after taking micro algae culturing liquid centrifugation, remove supernatant and the microalgae of acquisition is carried out lyophilization, taking freeze-dried algae powder 0.2g to be extracted in test tube；
2. by oxolane: chloroform: normal hexane is that 1: 0.8～1.5: 1～1.2 preparation mixed solutions are stand-by by volume；
3. pipette, with pipet, the mixed solution that 2. 5mL step is prepared, add step 1. in fill after lyophilizing in the test tube of algae powder；
4. algae powder step 3. obtained carries out the first shattering process with the mixed material of mixed solution in ultrasound field, and in the first shattering process, mixed material crushes 3～5 times in ultrasound field, and the persistent period is 30～60s each time, each interval time 10～30s；
Centrifugation after first shattering process, collects in the backward precipitation being centrifugally separating to obtain of supernatant and adds the 5mL mixed solution that 2. fresh step is prepared, carry out the second shattering process, and the operation of the second shattering process is identical with the first shattering process；Centrifugation after second shattering process, collects supernatant；Repeat the second shattering process 1 time；
5. after the most each for step shattering process being terminated, the centrifugal supernatant collected is poured in preprepared receiving flask after merging；
If volume is less than 15mL after the most each time supernatant merges, then it is settled to 15mL with the mixed solution that 2. step is prepared；
(2) preparation inner mark solution, weighs 10mg octacosane and is dissolved in 1mL mixed solution, the mixed solution that 2. step when described mixed solution is to prepare sample solution to be measured is prepared；
(3) preparing standard solution, weighs 50 μ L heptadecanes, 50mg myristic acid, 50mg Palmic acid, 20mg stigmasterol and 250 μ L vegetable oil and is dissolved in 4 mL mixed solutions, make standard solution；The mixed solution that 2. step when described mixed solution is to prepare sample solution to be measured is prepared；
(4) standard curve is made, 10 μ L are pipetted with pipet respectively from the standard solution that step (3) is prepared, 25 μ L, 50 μ L, 75 μ L, 100 μ L standard solution are in GC pipe, and are settled to 1mL with mixed solution, are subsequently adding the inner mark solution that 10 μ L step (2) prepare and obtain five parts of standard curve solution；Measure the peak area value of triglyceride in five parts of standard curve solution respectively with gas chromatograph after, with triglyceride peak area for X value, with the content of triglyceride in standard solution as Y value, draw standard curve, calculate regression equation；
(5) measuring content of triglyceride in microalgae, take that step (1) prepares treats that test sample solution 1mL, in GC pipe, adds 10 microlitre internal standard octacosane solution, then carries out GC detection；Being substituted in regression equation as X value by the triglyceride peak area value that GC detection obtains, its calculated Y value is content of triglyceride in microalgae.
The detection method of content of triglyceride in microalgae the most according to claim 1, it is characterized in that: prepare to treat test sample solution, take the fresh micro algae culturing liquid growing to late log phase, after being centrifuged 10～20 minutes in 7000～8500r/min, remove supernatant and the microalgae of acquisition is carried out lyophilization, in freezing dry process, every 12h measures the change of microalgae weight, weighs algae powder after constant weight in test tube.
The detection method of content of triglyceride in microalgae the most according to claim 1, it is characterised in that: in preparing to treat that the step of test sample solution is 4., during ultrasonication, the frequency of ultrasound wave is 20～30kHz.
The detection method of content of triglyceride in microalgae the most according to claim 1, it is characterized in that: when step (4) makes standard curve, the content of triglyceride in standard solution i.e. Y value is to be obtained by the content z of the triglyceride in the content of vegetable oil in standard solution × known vegetable oil.
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