Rapid determination method for haematococcus pluvialis spore wall-broken rate

A rapid detection technology for Haematococcus pluvialis, which is applied in the preparation of test samples, measurement devices, color/spectral characteristics measurement, etc. It can solve the problems of large interference in extraction efficiency, difficult quantitative analysis, small counting units, etc. , to achieve the effects of improving stability and accuracy, reducing analysis and testing costs, and reducing operating steps

Inactive Publication Date: 2017-06-06
骆野鸣
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  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, the methods for determining the wall breaking rate of Haematococcus pluvialis include: 1. Cell counting method: use the "plate counting frame" to count and count the cells before and after breaking the wall under a microscope, and then calculate the wall breaking rate according to the formula: R= (total number of cells - number of remaining cells after breaking the wall)/total number of cells; due to manual counting, the counting unit is small and must be carried out under a microscope, this method is likely to cause huge errors and is difficult to use for quantitative analysis
2. The patent "Method for Determination of Haematococcus Pluvialls Spore Breaking Rate" (Publication No. CN 101609039 A) discloses a solvent method determination method: the principle of this method is mainly to use dichloromethane-n-hexane mixed solution and Dimethyl sulfoxide (DMSO) solution has different solubility of astaxanthin in the broken and unbroken cells of Haematococcus pluvialis, so two measurements were carried out to compare the results and calculate the breaking rate of Haematococcus pluvialls; but The following problems exist: (1) The principle of the above-mentioned patent is mainly that dimethyl sulfoxide can extract astaxanthin from unbroken Haematococcus pluvialis, and the method needs to be based on the accurate determination of astaxanthin content However, in practice, it has been found that although methylene chloride, n-hexane, and dimethyl sulfoxide are all lipophilic organic solvents, they can penetrate the cell wall of unbroken Haematococcus pluvialis spores, but the intracellular More than 90% of natural astaxanthin exists in esterified form (about 70% monoester, 25% diester and 5% monomer), the esterified molecule is relatively large, and the complete spore wall is thick and strong. It is difficult to extract all the astaxanthin through

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Embodiment 1 (broken Haematococcus pluvialis slurry),

[0050] Scope: Commonly used in the detection and quality control of the wall breaking rate in the process of Haematococcus pluvialis wall breaking,

[0051] Source of sample: after filtering and dehydrating mature Haematococcus pluvialis spore cells (containing astaxanthin), adjust the concentration of the algae slurry to 10-15% with ethanol, and pass through a high-pressure homogenizer (GEA Ariete NS2006, Italy) 600bar cell wall breaking to obtain broken Haematococcus pluvialis pulp;

[0052] A, acetone extraction, determination of astaxanthin absorbance of known samples:

[0053] The analytical purity of acetone is ≥99.5%,

[0054] 1) The balance accurately weighs 0.105g, 0.102g and 0.101g of the sample algae slurry (3 parallel samples), and places them in 100ml volumetric flasks respectively. 2) Add 100ml of acetone to the three volumetric flasks to make up to the mark. Extract at room temperature (20-25°C) f...

Embodiment 2

[0060] Embodiment 2 (broken Haematococcus pluvialis powder),

[0061] Scope: It is commonly used in the quality inspection of broken Haematococcus pluvialis powder, which is the quality index of broken Haematococcus pluvialls powder products.

[0062] Source of sample: after filtering and dehydrating mature Haematococcus pluvialis spore cells (containing astaxanthin), adjust the concentration of the algae slurry to 10-15% with ethanol, and pass through a high-pressure homogenizer (GEA Ariete NS2006, Italy) 1200bar cell wall breaking to obtain the broken Haematococcus pluvialis pulp; the broken wall algal pulp is dried in an oven (spray, drum, microwave, freeze-drying and other drying methods, and the products processed by different drying methods have different extraction times of about 5-15 minutes) after dehydration, the broken Haematococcus pluvialis powder with moisture content ≤ 10% is obtained;

[0063] A, acetone extraction, determination of astaxanthin absorbance of k...

Embodiment 3

[0071] Embodiment 3 (haematococcus pluvialis sheet),

[0072] Scope: It is commonly used in the quality inspection and identification of tablet products made of broken Haematococcus pluvialis powder,

[0073] Source of sample: Haematococcus pluvialis tablets are obtained by grinding, sieving, batching, mixing, tableting, packaging and other processes using the broken Haematococcus pluvialis powder after cell wall breaking and drying. The specification of the algal flakes used this time is 0.5g / piece, and the content of astaxanthin is 0.6%;

[0074] Supplementary note: The content of astaxanthin in Haematococcus pluvialis tablets produced by different manufacturers is different. Therefore, when using a UV spectrophotometer to detect the absorbance, the dilution factor should be adjusted according to the concentration of astaxanthin contained in the algal tablets. If the If the measured absorbance value is greater than 1.25, the solution needs to be diluted appropriately so tha...

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PUM

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Abstract

The invention discloses a rapid determination method for haematococcus pluvialis spore wall-broken rate. The method includes the steps of: A. conducting acetone extraction and determining astaxanthin absorbance of a known sample; B. grinding the known sample and performing homogenizing to make the wall-broken rate reach 100%, then conducting acetone extraction, and determining astaxanthin absorbance of the 100% wall-broken sample; and C. conducting acetone extraction and determining astaxanthin absorbance (light absorption value A1) of the known sample, grinding the known sample and performing homogenizing to make the wall-broken rate reach 100%, then conducting acetone extraction, and determining the ratio of astaxanthin absorbance (light absorption value A2) of the 100% wall-broken sample, i.e. a spore wall-broken rate. The method provided by the invention only includes three steps, has the characteristics of simple operation, convenient calculation (calculation of the actual content of astaxanthin is unnecessary, and only through division of the absorbance A1 and A2 of the two samples can a corresponding proportional result be obtained), high efficiency and rapidity (the whole process is about 15-20min), thus being very suitable for real-time monitoring and quality control of a haematococcus pluvialis product production process.

Description

technical field [0001] The invention belongs to the biological technical field of microalgae detection, and in particular relates to a method for quickly measuring the wall breaking rate of Haematococcus pluvialis spores. Background technique [0002] Astaxanthin, the chemical name is 3,3'-dihydroxy-β,β-carotene-4,4'-dione, the molecular formula is C40H52O4, it is a ketone carotenoid, most of its natural products Existing in the form of ester, it is water-insoluble and lipophilic, and is easily soluble in organic solvents such as carbon disulfide, acetone, benzene and chloroform. Haematococcus pluvialis is recognized as the most rich astaxanthin raw material in the world. Its astaxanthin content can reach more than 5% of the dry weight of cells, and it is a concentrated product of natural antioxidants. In addition, Haematococcus pluvialis is rich in nutrients such as protein, polysaccharides, vitamins, trace elements and natural pigments, and has attracted worldwide attenti...

Claims

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Application Information

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IPC IPC(8): G01N21/31G01N1/34
CPCG01N1/34G01N21/314
Inventor 骆野鸣
Owner 骆野鸣
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