Rapid Detection of the Activity of Lacticaseibacillus Casei Zhang by Flow Cytometry

Abstract

Food processing, e.g., freeze-drying, exerts strong pressure on bacteria in the food matrix, decreasing their viability/activity and even forcing them to become viable but unculturable (VBNC), which are often underestimated by traditional plate count. The strict standards of bacterial viability in probiotic products require accurate cell viability/activity enumeration. We developed a staining (5(6)-carboxyfluorescein diacetate succinimide ester, propidium iodide)-based flow cytometry rapid method for detecting the viability/activity of Lacticaseibacillus (Lb.) casei Zhang, a widely used probiotic in the dairy industry in China. We optimized the procedural and instrumental parameters for generating results comparable to that of standard plate counts. This method was also applied to freeze-dried Lb. casei Zhang, yielding 7.7 × 1011 CFU/g, which was non-significantly higher than the results obtained by plate count (6.4 × 1011 CFU/g), possibly due to the detection of VBNC cells in the freeze-dried powder. We anticipated that this method can be used for detecting lactic acid bacteria in other probiotic food/beverages.

Probiotic and postbiotic analytical methods: a perspective of available enumeration techniques

“…The difference between the two groups is expressed as Active Fluorescent Units (AFU) which represents the viable cells (cells with intact membranes) based on this protocol. Total Fluorescent Units (TFU) provide information on the total number of cells in the sample, whereas the difference between TFU and AFU (TFU-AFU), termed as non-AFUs (n-AFUs), represents the dead, likely irreparable, bacterial population ( Fallico et al, 2020 ; Ma et al, 2023 ). The enzymatic activity protocol is based on fluorescence generated by the non-fluorescent dye Carboxylfluorescein diacetate succinimidyl ester (cFDA) when it is cleaved by cellular esterase (a proxy of cellular viability), meanwhile the membrane potential is based on DiOC 2 that binds the membrane with a green fluorescence emission; when cells are activated the maximum fluorescence is then red-shifted.…”

“…Apart from the fluorescence techniques, ISO 19344 has been validated using a broad array of probiotic species, including Lactobacillus spp., Bidifobacterium spp., and Streptococcus spp., which emphasizes the method’s capability to be utilized for enumerating any strain belonging to the validated species, thereby offering a generalized approach which overcomes the intrinsic limitations of cultivability methods that rely on specific protocols that can vary according to the taxonomical species under examination. These technical advantages make FCM a more accurate and faster technique compared to PC enumeration, as FCM directly enumerates each single cell in a given sample and provides information on the bacterial population heterogeneity based on the fluorochrome used ( Sielatycka et al, 2021 ; Ma et al, 2023 ).…”

“…Beyond the ISO 19344 IDF 232 protocol, other fluorescent dyes with alternative properties can be used, such as carboxyfluorescein diacetate succinimide ester (CFDA), which binds to intracellular proteins of intact cells ( Ma et al, 2023 ), or CellROX ® Green Reagent, a DNA-binding cell-permeant dye that exhibits bright green fluorescence when oxidized by reactive oxygen species (ROS) ( Fallico et al, 2020 ).…”

“…This is because PC methods may potentially underestimate viable cell numbers, as they fail to detect VBNC cells ( Jackson et al, 2019 ; Fusco et al, 2022 ). Recent advancements in enumeration techniques, such as Imaging Flow Cytometry (IFC),staining-based flow cytometry and viability qPCR, have shown promise in capturing the VBNC population more accurately ( Ma et al, 2023 ; Pereira et al, 2023 ; Shehata et al, 2023 ).…”

Metabolomic Differences between Viable but Nonculturable and Recovered Lacticaseibacillus paracasei Zhang

“…Lacticaseibacillus paracasei Zhang ( L. paracasei Zhang) is a strain of lactic acid bacteria with good probiotic properties. It was isolated from a koumiss sample collected in Inner Mongolia, China [ 24 ]. This probiotic strain shows strong resistance to acid and bile salt stress [ 25 ].…”