TBI, Université de Toulouse, CNRS, INRAE, INSA, 31400 Toulouse, France.
Anti Oxidant Power AOP, 31000 Toulouse, France.
Int J Mol Sci. 2023 Jul 22;24(14):11800. doi: 10.3390/ijms241411800.
As the use of antioxidant compounds in the domains of health, nutrition and well-being is exponentially rising, there is an urgent need to quantify antioxidant power quickly and easily, ideally within living cells. We developed an Anti Oxidant Power in Yeast (AOPY) assay which allows for the quantitative measurement of the Reactive Oxygen Species (ROS) and free-radical scavenging effects of various molecules in a high-throughput compatible format. Key parameters for were investigated, and the optimal values were determined for each of them. The cell density in the reaction mixture was fixed at 0.6; the concentration of the fluorescent biosensor (TO) was found to be optimal at 64 µM, and the strongest response was observed for exponentially growing cells. Our optimized procedure allows accurate quantification of the antioxidant effect in yeast of well-known antioxidant molecules: resveratrol, epigallocatechin gallate, quercetin and astaxanthin added in the culture medium. Moreover, using a genetically engineered carotenoid-producing yeast strain, we realized the proof of concept of the usefulness of this new assay to measure the amount of β-carotene directly inside living cells, without the need for cell lysis and purification.
随着抗氧化化合物在健康、营养和保健领域的应用呈指数级增长，人们迫切需要快速、简便地定量抗氧化能力，理想情况下是在活细胞内进行。我们开发了一种酵母抗氧化能力（AOPY）测定法，该方法允许以高通量兼容的格式定量测量各种分子的活性氧（ROS）和自由基清除效果。研究了的关键参数，并为每个参数确定了最佳值。反应混合物中的细胞密度固定在 0.6；荧光生物传感器（TO）的浓度被发现最佳为 64µM，并且在指数生长的细胞中观察到最强的响应。我们优化的程序允许准确地定量酵母中已知抗氧化分子的抗氧化效果：在培养基中添加白藜芦醇、表没食子儿茶素没食子酸酯、槲皮素和虾青素。此外，使用经过基因工程改造的生产类胡萝卜素的酵母菌株，我们证明了该新测定法用于测量活细胞内β-胡萝卜素含量的概念验证，而无需细胞裂解和纯化。
