Viability assessment of Bifidobacterium longum ATCC 15707 on non-dairy foods using quantitative fluorescence microscopy

Abstract

This study demonstrates an effective technique for separating and purifying viable bacteria from samples that interfere with viability staining. The viability of Bifidobacterium longum ATCC 15707 was assessed using Percoll Buoyant Density Gradient Centrifugation (PBDC) to separate bacteria from complex non-dairy food matrices and Quantitative Fluorescence Microscopy (QFM) to determine individual cells using LIVE/DEAD BacLight bacterial viability staining. Water agar (3%) was used to retain cells of B. longum and offered a lower fluorescence background with BacLight viability staining, compared with fixation on polycarbonate (PC) black membrane. The effect of drying temperatures and non-dairy foods on viability of B. longum was assessed. B. longum coated on oat, peanut or raisin was separated by filtration, low- and high-speed centrifugation, flotation and sedimentation buoyant density centrifugation. Purified cells were subsequently deposited on water agar for rehydration followed by LIVE/DEAD BacLight viability staining and enumeration. Conventional plate counting was also conducted to compare viability results. Finally, this method was applied to assess cell membrane damages of B. longum incorporated onto non-dairy foods during 24 h drying. Furthermore, viability assessment of B. longum coated onto oat, peanut, or raisin was much lower by plate counting compared to viability staining. Drying appeared to have a greater impact when viability was assessed by plate counting compared to viability staining. IMPORTANCE: Enumeration of viable beneficial bacteria from function foods presents a significant bottleneck for product development and quality control. Interference with microscopic and/or fluorescent techniques by ingredients, time required to incubate plated microbes, and the transient nature of the colony forming unit make rapid assessment of viable bacteria difficult. Viability assessment of Bifidobacterium longum ATCC 15707 by Percoll Buoyant Density Gradient Centrifugation with LIVE/DEAD BacLight viability staining on water agar (3%) was in agreement with serial dilution enumeration. Without the need for incubation viability assessment by staining provided a more rapid means to assess the impact of drying on the viability of B. longum coated onto oat, peanut or raisin.

Keywords: Fluorescent staining; Non-dairy foods; Percoll centrifugation; Probiotics; Viability.