染色法构建对超临界CO 2 提取沙棘籽油的过程分析

Construction of Staining Protocol for Process Analysis to Supercritical CO 2 Extraction of Hippophae rhamnoid L.Seed Oil

在沙棘籽油提取过程中,经粉碎干燥后的籽料颗粒是一种开放体系,脂滴以游离形式存在,采用油红O(Oil Red O,ORO)对其进行染色时,常规方法一次性滴加染液的方式以及后续的洗脱步骤均会导致脂滴迁移、过染等问题出现。研究构建了逐级染色法,采用少量多次滴加染液的方式,并优化了染色间隔时间为1.5 min,染色级数和染液浓度可根据样品的实际油含量进行调整,因而能够客观地显影脂滴在籽料颗粒内的分布状态。以此构建的面积占比法可用于精确测定籽料中的油脂含量,其准确度与萃取质量法相当,两者的相关系数为0.998,并具有较高重现性,相对标准偏差约为2.49%。

The dry ground seed particle of Hippophae rhamnoides L.can be classified as an open system during the extraction,therefore,the internal lipid droplets(LD)inside the particle can be movable in free way.It was found that when seed particles were stained by Oil Red O(ORO),some procedures in the traditional protocol,such as one-batch dye addition and the subsequent elution,etc.might probably lead to undesirable problems,including LD migration,improper staining and so forth.Aiming to address these problems,a modified method named as the sequential multi-stage staining protocol(SMSP)was established upon the principle of adding dye in both micro-amount and multi-batch way.Firstly,the incubation interval of SMSP was optimized with the favourable time of 1.5 min.Two key operating parameters of new method,namely the total staining times and the dye concentration,can be flexibly manipulated based on the actual oil content in the unknown samples obtained from the extraction,so that the distribution of LDs within the porous layer of slices would be visualized objectively.Consequently,the quantitative method on basis of SMSP for determining oil content in the seed samples did not only have similar accuracy comparable to the traditional weighing method with the correlation coefficient of 0.998,but also showed high reproducibility with the relative standard deviation(RSD)of about 2.49%.