Solvent-extraction-and-fluorescence determination method of microalgae lipid content

A technology for the determination of microalgae oil and fluorescence, which is applied in the direction of fluorescence/phosphorescence, material excitation analysis, etc., which can solve the problems of dangerous operation, large sample volume required, unsuitable non-uniform sample measurement, etc., and achieve the effect of small environmental pollution

Inactive Publication Date: 2013-04-24
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Although the organic solvent extraction method is widely used, easy to operate, and low in cost, it requires a large amount of sample, and the obtained total lipid content is low. When the amount of microalgae is relatively small, the oil cannot be extracted or the extracted oil cannot be weighed. It is easy to volatilize when removing organic solvents, the volatile solvents are toxic and easy to pollute the environment, and the operation is time-consuming and laborious; the Soxhlet extraction method is more accurate in determining the total lipid content of microalgae. Volatile and pollute the environment, and the extraction time is long; fluorescent dye determination method, Fourier transform infrared spectroscopy, vanillin phosphate method, copper reagent method, Sudan black staining method and nuclear magnetic resonance method require less sample amount and shorter time , but there are still deficiencies
Among them, the fluorescent dye method does not need to extract oil and has high sensitivity, but it is easily affected by various factors such as incomplete dyeing and chlorophyll fluorescence peaks; Fourier transform infrared spectroscopy and nuclear magnetic resonance methods do not damage the sample and do not require or Only a little pretreatment is required, the latter has good repeatability, but the cost is high; the vanillin phosphoric acid assay is relatively stable, but it needs to use a strong acid such as sulfuric acid, and the operation is more dangerous; the copper reagent method is low in cost, but it is rarely reported. less; the Sudan black staining method is simple to operate, does not need to break the algae cells and chemical extractio

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] (1) Preparation of standard curve: take 50mL of fresh microalgae grown to the end of logarithm, centrifuge at 4000r / min for 10min, take the precipitate, wash it with PBS buffer, and then dilute it with 50mL of PBS buffer to form an algae liquid as a standard algae liquid (The absorbance of the standard algae solution is 0.470); take five 30mL brown reagent bottles, labeled 1 to 5, add 19.8mL of the standard algae solution, and then add 200μL isopropanol, 180μL isopropanol+20μL triolein, 160 μL isopropanol + 40 μL triolein, 140 μL isopropanol + 60 μL triolein, 120 μL isopropanol + 80 μL triolein, prepare triolein solutions with different concentrations; Methyl sulfoxide and 0.8mL10μgmL Nile red were stained for 20 minutes, and the fluorescence intensity of each triolein solution was measured (excitation wave wavelength 488nm, divergent light range 400-700nm, slit width 10nm, voltage value 400V), and triolein The fine concentration is the ordinate, the fluorescence is the...

Embodiment 2

[0035] (1) Preparation of standard curve: take 50mL of fresh microalgae grown to the end of the logarithm, centrifuge at 4000r / min for 10min, take the precipitate, wash it with PBS buffer, and then dilute it with 40mL of PBS buffer to form an algae liquid as a standard algae liquid (The absorbance of the standard algae solution is 0.636); take five 30mL brown reagent bottles, labeled 1 to 5, add 19.8mL of the standard algae solution, and then add 200μL isopropanol, 180μL isopropanol+20μL triolein, 160 μL isopropanol + 40 μL triolein, 140 μL isopropanol + 60 μL triolein, 120 μL isopropanol + 80 μL triolein, prepare triolein solutions with different concentrations; Methyl sulfoxide and 0.8mL10μgmL Nile red were stained for 20 minutes, and the fluorescence intensity of each triolein solution was measured (excitation wave wavelength 488nm, divergent light range 400-700nm, slit width 10nm, voltage value 400V), and triolein The fine concentration is the ordinate, the fluorescence is...

Embodiment 3

[0039] (1) Preparation of the standard curve: take 50 mL of fresh microalgae grown to the end of the logarithm, centrifuge at 4000 r / min for 10 min, take the precipitate, wash it with PBS buffer, and then dilute it with 50 mL of PBS buffer to form an algae liquid as a standard algae. (the absorbance of the standard algae liquid is 0.470); take five 30mL brown reagent bottles, labeled No. 1 to No. 5, add 19.8mL of the standard algal liquid, and then add 200 μL isopropanol, 180 μL isopropanol + 20 μL triolein , 160 μL isopropanol + 40 μL triolein, 140 μL isopropanol + 60 μL triolein, 120 μL isopropanol + 80 μL triolein, prepare triolein solutions with different concentrations; respectively add 5 ml to the triolein solution in sequence Dimethyl sulfoxide and 0.8mL10μgmL Nile red were stained for 20 minutes, and the fluorescence intensity of each triolein solution was measured (excitation wave wavelength 488nm, divergent light range 400-700nm, slit width 10nm, voltage value 400V), ...

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Abstract

The invention discloses a solvent-extraction-and-fluorescence determination method of microalgae lipid content, and belongs to the technical field of microalgae crude lipid determination. The solvent-extraction-and-fluorescence determination method of the microalgae lipid content solves the problems of a fluorimetry in the prior art that dyeing with direct use of Nile red fluorescent staining is halfway and disturbance of microalgae body chlorophyll fluorescence peaks and the like occur when an ultrasound method or a dimethyl sulfoxide (DMSO)-processing-Nile-red-dyeing fluorescence determination method is in use. The solvent-extraction-and-fluorescence determination method of microalgae lipid content comprises the following steps of preparing a standard curve by using microalgae to prepare standard microalgae liquid, extracting microalgae lipid and determining content of the microalgae lipid by using fluorescence. Due to the fact that in a determination process, operation steps are free from evaporation organics, and pollution to the environment is little. The solvent-extraction-and-fluorescence determination method of the microalgae lipid content has the advantages that the solvent-extraction-and-fluorescence determination method of the microalgae lipid content is rapid, simple, effective and sensitive, and determination results are free from influence of retention periods of microalgae samples.

Description

technical field [0001] The invention belongs to the technical field of measuring microalgae crude oil, in particular to a solvent extraction-fluorescence method for measuring the content of microalgae oil. Background technique [0002] With the development of the economy, human beings have higher and higher requirements for energy, and microalgae are considered to be ideal raw materials for the preparation of biodiesel fuel. Algae has the advantages of high photosynthetic efficiency, zero net carbon value, easy cultivation, short growth cycle, high oil hydrocarbon content, etc., and does not compete with farmers for land, does not compete with people for food, and does not affect global energy distribution. As a bioenergy source, it has caused Governments and scholars of various countries pay close attention to it. The research process of producing biodiesel from microalgae includes screening of oil-producing microalgae, microalgae culture system, collection and separation ...

Claims

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Application Information

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IPC IPC(8): G01N21/64
Inventor 张小平王晓晨覃业霞
Owner SOUTH CHINA UNIV OF TECH
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