Rapid determination method for haematococcus pluvialis spore wall-broken rate

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...

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 the cell wall. Therefore, the above-mentioned patent method cannot completely extract the astaxanthin in the intact Haematococcus cells at 70°C, which is likely to cause large errors; (2) Dimethyl sulfoxide extraction The efficiency is greatly disturbed by the quality of Haematococcus pluvialls, and the measurement results of different batches of Haematococcus pluvialls are unstable, and it is difficult to adapt to the reliability of quality detection in large-scale production; (3) Haematococcus pluvialls cells In addition to astaxanthin, it also contains a large amount of impurities such as chlorophyll and yellow pigment, which are easily extracted by solvents such as dimethyl sulfoxide, and the absorbance during its measurement is close to that of astaxanthin, and its measurement results are easily affected by these impurities. Interference causes measurement error; (4) The above-mentioned patent uses two solvents (dichloromethane-n-hexane mixed solution and dimethyl sulfoxide). These two solvents have different absorption wavelengths for astaxanthin, but they all use the same DMSO does not match the absorbance measured at 530nm as the control, which will easily lead to errors in the final calculation of the wall breaking rate, which is not conducive to the accuracy and effectiveness of the quality control process; (5) organic solvents can degrade natural astaxanthin through isomerization, and Under alkaline conditions, irreversible denaturation is prone to occur; (6) n-hexane-dichloromethane is non-polar and insoluble in water. If the water content in the sample raw material is high, it cannot fully contact with the sample and the pigment cannot be extracted

However, the actual wall-breaking process contains a lot of water. If it is dried and then analyzed, the processing cost will be increased, and the significance of real-time quality control will be lost.