Method for rapidly and nondestructively detecting astaxanthin in C.zofingiensis cells

A non-destructive testing, chlorella technology, applied in the direction of material excitation analysis, fluorescence/phosphorescence, etc., can solve the problems of long time required, reduce work efficiency, increase the process required for testing, etc., to simplify testing steps and sample pretreatment. The effect of simplicity and shortened inspection time

Active Publication Date: 2015-10-07
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the detection efficiency of these two methods is not ideal enough. It is necessary to extract the astaxanthin in the algae cells in advance, and then detect the astaxanthin content of the extract, which not only increases the detection process, but also takes a long time , reducing work efficiency

Method used

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  • Method for rapidly and nondestructively detecting astaxanthin in C.zofingiensis cells
  • Method for rapidly and nondestructively detecting astaxanthin in C.zofingiensis cells
  • Method for rapidly and nondestructively detecting astaxanthin in C.zofingiensis cells

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Embodiment 1

[0047] In the process of developing the technical solution of the present invention, the inventors of the present application studied the variation trend of the intracellular astaxanthin content of the chlorella C. zofingiensis.

[0048] 1.1 Strain activation and seed liquid preparation

[0049] The strains of Chlorella zofingiensis preserved in the laboratory were transferred to the slant equipped with modified Basal medium for cultivation, the cultivation temperature was 28°C, and the light was 10 μmol m -2 the s -1 , and observe the growth of Chlorella.

[0050] Use an inoculation loop to inoculate the Chlorella algae lawn into the modified Basal liquid medium, at a temperature of 28 °C and a light of 10 μmol m -2 the s -1 Cultivate under certain conditions for 3 to 5 days as seed liquid.

[0051] The composition of the improved Basal medium (pH6.1) described therein is shown in Table 1 below (in mg / L):

[0052] Table 1

[0053]

[0054]

[0055] 1.2 Chlorel...

Embodiment 2

[0068] Example 2 Correlation between the intracellular astaxanthin content of Chlorella and the average fluorescence intensity of Chlorella cells

[0069] During the cultivation of Chlorella C. zofingiensis in Example 1, the inventors of the present application found that the cells of Chlorella changed from green to orange-red, which was mainly due to the chlorophyll and primary carotenoids in the cells under high light, low nitrogen, etc. Under stress conditions, it can induce the accumulation of secondary carotenoids, mainly astaxanthin and other carotenoids. The cell morphology of Chlorella cells under the optical microscope at the beginning, during and at the end of the culture are as follows: figure 2 Shown in A, B, and C diagrams.

[0070] Astaxanthin can exhibit the maximum emission wavelength after being excited by a specific wavelength, which is related to its content in the cell. In this example, a BD Accuri C6 flow cytometer was used to measure the average fluore...

Embodiment 3

[0078] Example 3 The linear regression analysis of the astaxanthin content measured by the average fluorescence intensity of chlorella cells and the HPLC method

[0079] In order to realize the rapid and accurate analysis of the astaxanthin content in the cells of Chlorella, the average fluorescence intensity of the cells was measured by flow cytometry in this experiment, and linear regression analysis was performed with the astaxanthin content in the cells of Chlorella to determine the Flow cytometry is a fast and accurate non-destructive detection method.

[0080] In Example 1, the chlorella intracellular astaxanthin content of the chlorella samples cultivated to the 146h, 168h, 192h, 216h, 240h, 264h, and 288h was measured by HPLC assay, and the results are as follows figure 1 shown. The common logarithm of the measured values ​​of these astaxanthin contents (in μg / g, representing the content of astaxanthin per gram of dry algal powder) is taken as the ordinate to the base...

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Abstract

The invention provides a method for rapidly and nondestructively detecting astaxanthin in C.zofingiensis cells. The method comprises the following steps of (1) re-suspending algal cells collected from a known C.zofingiensis suspension sample to regulate the known C.zofingiensis suspension sample into heavy suspension with cell density of 6*10(5) to 2*10<6>CFU / mL by virtue of de-ionized water or a phosphate buffer solution, detecting the average fluorescence intensity of the heavy suspension in an FL1 channel or an FL2 channel by virtue of a flow cytometer, and performing linear regression on a log value of the content of astaxanthin in the known C.zofingiensis suspension sample and a log value of the corresponding measured average fluorescence intensity in the FL1 or FL2 channel to obtain a standard curve; (2) preparing a sample to be detected: re-suspending algal cells collected from C.zofingiensis suspension to be detected to regulate the C.zofingiensis suspension to be detected into algal cell heavy suspension to be detected with cell density of 6*10(5) to 2*10<6>CFU / mL by virtue of de-ionized water or a phosphate buffer solution; (3) detecting the average fluorescence intensity of the sample to be detected in the FL1 channel or the FL2 channel by virtue of the flow cytometer, and calculating the content of astaxanthin in the sample to be detected according to the standard curve obtained in step (1). The method has the characteristic of high detection efficiency.

Description

technical field [0001] The invention belongs to the rapid nondestructive detection technology of intracellular pigments in microalgae, in particular to a rapid nondestructive detection method for intracellular astaxanthin in chlorella based on fluorescence detection technology. Background technique [0002] Astaxanthin (astaxanthin) is called a "super antioxidant", and its antioxidant capacity is more than 10 times that of other carotenoids such as β-carotene, zeaxanthin, lutein and canthaxanthin. Lipid peroxidation ability plays an important role in preventing age-related diseases such as arteriosclerosis, Parkinson's syndrome, retinal macular degeneration, etc. It is important for maintaining the health of the eyes and central nervous system, enhancing the function of the immune system, strengthening the body's energy metabolism, and anti-cancer , anti-infection, etc. have multiple effects. [0003] At present, the production of natural astaxanthin from Haematococcus pluv...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64
Inventor 魏东陈俊辉
Owner SOUTH CHINA UNIV OF TECH
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