2.5. Microbial Distribution in the Milk Matrix

One millilitre of milk-containing LGG WT or LGG ΔspaCBA was labelled with the LIVE/DEAD BacLight viability kit (1:200 v/v; the LIVE/DEAD BacLight viability kit was prepared according to the procedure described for the L13152 kit by ThermoFisher Scientific, Waltham, MA, USA). Lipids were stained using Nile red (5:200 v/v; Sigma-Aldrich, St. Louis, MO, USA; the solution was prepared in PEG 200 according to [29]. Two hundred microlitres of suspension-containing LGG were placed on chambered glass slides (Nunc Lab-Tek, ThermoFisher Scientific, Waltham, MA, USA). Confocal laser scanning microscopy (CLSM) images were taken using a Leica TCS SP5-X-AOBS confocal laser scanning microscope (Leica Microsystems CMS GmbH, Mannheim, Germany) equipped with WLL lasers. The objective lens used was a HCX PL APO CS 100 × 1.40 (oil immersion). Image acquisition was performed in sequential mode with the following excitation and emission wavelengths: (1) λ_ex1 = 488 nm and λ_em1 = 490–555 nm; (2) λ_ex2 = 488 nm and λ_em2 = 560–600 nm. The images were processed and exported using LAS X software, version 3.4.2.18368. Three independent repetitions were performed, and approximately 20 representative images were acquired for each repetition. Image sequences were acquired at frequency f = 2 Hz in order to investigate the movements of LGG in the milk over time. The bacterial dynamic was illustrated by calculating the cumulative area covered by the bacteria as a function of time. For each image, the area of all the bacteria was measured first by applying a threshold function and then an edge-detection filter. The cumulative area was calculated over time and ensemble average, and finally normalized according to the initial area:

where N is the number of images in the sequence and A(ti) the bacteria area for the image at time ti, t0 being the first image analysed in the sequence.