The number of bacterial cells is currently recognized as the most important parameter for the efficacy and quality of finished probiotic or live biotherapeutic products (LBP). Cell enumeration is generally performed by culture-dependent methodologies like plate count (PC). These techniques are able to reveal the number of viable cells able to replicate and generate a colony. However, they are limited by their dependence on the combination of culture conditions (e.g. nutrients, temperature) selected for cell recovery. Additionally, they do not provide information on the heterogeneity of a bacterial culture, namely they do not detect the cells in a viable but not cultivable (VBNC) status. Flow-cytometry (FC) is a culture-independent methodology having the potential to enumerate selectively live and damaged or dead cells. FC relies on the use of specific probes for different cell targets (e.g. membrane, enzymes) to unveil information on the cell structure and physiological statuses within a bacterial population. In this context, we monitored three batches of freeze-dried Lactobacillus rhamnosus GG (ATCC 53103) during a 3 year of storage at different conditions of temperature and relative humidity, according to ICH guidelines, by means of PC and FC. The Arrhenius model was applied to assess the suitability of the model to predict the mortality of probiotic cells in finished products. The higher destruction rate (k) obtained by PC data compared to FC data suggests a faster reduction of cultivability compared to membrane integrity, probably representing a dynamic shift of the bacterial population into a VBNC/dormant status during storage time. Interestingly, this mechanistic approach works both for PC and FC methodologies increasing the chances to monitor biological phenomenon within a mathematical modelling. The combined use of PC and FC shed lights on the true bacterial potency within a closed system like a finished product and the complexity of its heterogeneity.

目前公认细菌细胞的数量是成品益生菌或活生物治疗产品（LBP）的功效和质量的最重要参数。细胞计数通常通过依赖培养的方法进行，例如平板计数（PC）。这些技术能够揭示能够复制并产生菌落的活细胞的数量。但是，它们受到对细胞回收所选择的培养条件（例如营养物，温度）的依赖的限制。此外，它们不提供有关细菌培养物异质性的信息，即它们不检测处于存活但不可培养（VBNC）状态的细胞。流式细胞仪（FC）是一种独立于培养物的方法，具有选择性枚举活细胞和受损或死细胞的潜力。FC依赖于针对不同细胞靶标（例如膜，酶）的特定探针的使用来揭示有关细菌种群内细胞结构和生理状态的信息。在这种情况下，我们监测了三批冻干鼠李糖乳杆菌GG（ATCC 53103）根据ICH指南，通过PC和FC在不同的温度和相对湿度条件下存放3年。Arrhenius模型用于评估该模型的适用性，以预测成品中益生菌细胞的死亡率。与膜数据相比，通过PC数据获得的更高的破坏率（k）表明，与膜的完整性相比，可培养性的降低更快，这可能表示在存储期间细菌群体向VBNC /休眠状态的动态转移。有趣的是，这种机械方法适用于PC和FC方法，从而增加了在数学建模中监视生物现象的机会。