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首页> 外文期刊>Food microbiology >Next-generation multiparameter flow cytometry assay improves the assessment of oxidative stress in probiotics
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Next-generation multiparameter flow cytometry assay improves the assessment of oxidative stress in probiotics

机译：下一代多道次流式细胞术测定改善了益生菌中氧化应激的评估

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Stability of probiotic products' potency throughout shelf life is essential to ensure systematic delivery of the dosages required to provide clinically-proven health benefits. Due to the oxygen sensitivity of gut-derived microorganisms, methods for the rapid and accurate monitoring of oxidative stress in probiotics are greatly needed as they can be instrumental to both bioprocess optimization and quality control. This study introduces a next-generation flow cytometry method multiplexing the CellROX® Green and Propidium Iodide probes for the simultaneous measurement of free total reactive oxygen species (ROS) and membrane integrity, respectively. The multiparameter method was compared to the single-parameter assays, measuring either ROS or membrane integrity, for the ability to evaluate the fitness of Lactobacillus rhamnosus GG (LGG) after freeze drying, spray drying and H_2O_2-mediated oxidative stress. Each stand-alone assay detected only three cell populations, showing either differential membrane integrity (Syto 24 + /PI-, Syto 24 + /PI +, Syto 24-/PI +) or ROS levels (ROS-, low-ROS, high-ROS), and no correlation could be drawn between these groups. Conversely, the multiparameter method detected up to five physiologically distinct cell populations and allowed the integrated assessment of their membrane integrity and oxidative stress. It also revealed a much larger fitness heterogeneity in LGG as each group of low-ROS and high-ROS cells was found to be formed by a healthier population with an intact membrane (L-ROS/PI-, H-ROS/PI-) and a population with damaged membrane (L-ROS/PI +, H-ROS/PI +). As the CRG probe only detects free unreacted ROS, these populations are suggested to reflect the dynamic lifecycle of ROS formation, accumulation and reactive depletion leading to oxidative damage of macromolecules and consequent cell death. With the stand-alone CRG assay being unable to detect ROS lifecycle, the multiparameter method here presented delivers a superior profiling of the heterogeneity generated by oxidative stress in bacteria and enables a more correct interpretation of CRG fluorescence data. We provide recent examples from literature where the use of a single-parameter fluorescence approach may have led to misinterpret oxidative stress data and eventually draw erroneous conclusions.
机译：益生菌产品稳定性在保质期内的效力至关重要，以确保提供提供临床验证的健康益处所需的剂量。由于肠道衍生的微生物的含氧敏感性，因此极大地需要对益生菌中的氧化应激的快速和准确监测的方法，因为它们可以为生物过程优化和质量控制施用。本研究引入了下一代流式细胞术方法，复用Cellrox®绿色和碘化丙锭探针，分别同时测量自由的总反应性氧物质（ROS）和膜完整性。将MultiParameter方法与单参数测定进行比较，测量ROS或膜完整性，以便在冷冻干燥，喷雾干燥和H_2O_2介导的氧化应激后评估乳杆菌菌株GG（LGG）的适应性。每个独立的测定只检测到三种细胞群，显示差异膜完整性（Syto 24 + / Pi-，Syto 24 + / Pi +，Syto 24- / Pi +）或ROS水平（ROS-，低ROS，高-ROS），这些组之间无法绘制相关性。相反，多道琼布方法检测到多达五种生理学上不同的细胞群，并允许综合评估其膜完整性和氧化应激。它还揭示了LGG中的更大的适应性异质性，因为发现每组低ROS和高ROS细胞由具有完整膜（L-ROS / PI-，H-ROS / PI-）的更健康的群体形成和膜损坏的人群（L-ROS / PI +，H-ROS / PI +）。由于CRG探针仅检测自由未反应的ROS，因此建议这些人群反映ROS形成，积累和反应性耗尽的动态生命周期，导致大分子的氧化损伤和随后的细胞死亡。通过独立的CRG测定无法检测ROS生命周期，这里的多辐射测量方法呈现出通过细菌中氧化应激产生的异质性的优越谱，并且能够更正确地解释CRG荧光数据。我们提供了最近从文献中的使用，其中使用单参数荧光方法可能导致误解氧化应力数据并最终引起错误的结论。

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