Food Microbiology, Wageningen University and Research, Wageningen, Netherlands.
Food and Biobased Research, Wageningen University and Research, Wageningen, Netherlands.
J Microbiol Methods. 2022 Oct;201:106561. doi: 10.1016/j.mimet.2022.106561. Epub 2022 Aug 27.
Thiamine is an essential vitamin for most living organisms, of which yeasts are a rich nutritional source. In this study we developed a thiamine extraction and determination method to detect thiamine in fresh yeast biomass. The thiamine determination method combines the derivatization of thiamine to a highly fluorescent product, with chromatographic separation (HPLC) and fluorescence detection. The method specifically detects free thiamine (T), thiamine phosphate (TP), and thiamine pyrophosphate (TPP). It has a high sensitivity of 2 ng/ml for TPP and TP, and 1 ng/ml for T, excellent instrumental repeatability, and low day-to-day variation in retention time of the different phosphate forms. We demonstrated the robustness of the method by proving that the fluorescence signals of the derivatised samples are stable for at least 82 h after derivatization, and by showing that the final pH of the samples does not influence the fluorescent response. In addition, we developed and validated a thiamine extraction method consisting of beads beating the fresh yeast biomass in 0.1 M HCl using a lysing matrix composed of 0.1 mm silica spheres. The performance of this method was compared to extraction via heat treatment at 95 °C for 30 min, and a combination of beads beating and heat treatment carried out in different order. We demonstrated that thiamine extraction via beads beating is the only method that prevents the biologically active form thiamine pyrophosphate to be degraded to thiamine phosphate, therefore, the extraction method developed and described in this study is preferred when the different thiamine vitamers need to be detected in their actual proportions. The combination of the extraction via beads beating, the conversion of all vitamers to the thiochrome derivatives, and the separation of these compounds on the reversed phase HPLC with a fluorescence detector, yielded a sensitive, specific, repeatable, and robust method for extraction and determination of vitamin B in fresh yeast biomass.
硫胺素是大多数生物体必需的维生素，酵母是其丰富的营养来源。在本研究中，我们开发了一种从新鲜酵母生物质中提取和测定硫胺素的方法。该方法将硫胺素衍生化为高荧光产物，结合色谱分离（HPLC）和荧光检测。该方法专门检测游离硫胺素（T）、硫胺素磷酸（TP）和硫胺素焦磷酸（TPP）。它对 TPP 和 TP 的检测灵敏度高达 2ng/ml，对 T 的检测灵敏度为 1ng/ml，仪器重复性好，不同磷酸盐形式的保留时间日间变化小。我们通过证明衍生样品的荧光信号在衍生后至少 82 小时内稳定，以及证明样品的最终 pH 值不影响荧光响应，证明了该方法的稳健性。此外，我们开发并验证了一种硫胺素提取方法，该方法包括使用由 0.1mm 二氧化硅球组成的裂解基质在 0.1M HCl 中剧烈搅拌新鲜酵母生物质。该方法的性能与在 95°C 下热处理 30 分钟进行比较，并比较了不同顺序的珠磨和热处理的组合。我们证明，只有通过珠磨进行硫胺素提取才能防止生物活性形式的硫胺素焦磷酸降解为硫胺素磷酸，因此，当需要以实际比例检测不同的硫胺素维生素时，本研究中开发和描述的提取方法是首选。通过珠磨提取、将所有维生素转化为噻唑衍生物，以及在反相 HPLC 上用荧光检测器分离这些化合物的组合，为从新鲜酵母生物质中提取和测定维生素 B 提供了一种灵敏、特异、重现性好且稳健的方法。
