Then, 30 mL of the solution was aseptically transferred
using a serologic pipette to sterile petri dishes (inner diameter 15.6 cm; Sarstedt Ltd., Leicester, UK). The cast solutions were air dried at 37 °C for 15 h in a ventilated incubator (Sanyo Ltd., Japan) in order to obtain films that could be easily peeled off and had acceptable mechanical properties (absence of brittleness and adequate flexibility/extensibility). After drying, the probiotic edible films were peeled intact from the petri dishes and conditioned at room (25 ± 1 °C) selleck chemicals llc or chilled temperature (4 ± 1 °C) under controlled relative humidity conditions (54% RH) in desiccators containing saturated magnesium nitrate solution. One mL of the probiotic film forming solution was suspended in sterile PBS and vortexed for 30 s to ensure adequate mixing using the method described by Lopéz de Lacey et al. (2012) with minor modifications.
More specifically, individual 1 g film samples containing L. rhamnosus GG were transferred to 9 mL of sterile PBS and left to hydrate and dissolve under constant agitation in an orbital incubator at 37 °C for 1 h. The complete dissolution of the edible films had been previously been tested using edible films without probiotics and no residual insoluble material could be identified. In both cases, the resulting solutions were subjected to serial dilutions using phosphate buffer saline. Each dilution was pour plated on a MRS agar
(MRS Agar, Oxoid Ltd., Basingstoke, UK) and the selleck plates were stored at 37 °C for 72 h under anaerobic conditions to allow colonies to grow. Enumeration of the bacteria on agar plates was performed in triplicates by colony counting ( Champagne, Ross, Saarela, Hansen, & Charalampopoulos, 2011) and the total counts of the viable bacteria were expressed as log colony forming units per gram (log CFU/g, CFU/g = CFU/plate × dilution factor). The survival rate of the bacteria throughout the film forming solution drying process was calculated according to the following equation: equation(1) %viability=100×NN0where N0, N represent the number of viable bacteria prior and after the implemented drying process ( Behboudi-Jobbehdar et al., 2013). L. rhamnosus GG inactivation upon storage data was expressed as Oxalosuccinic acid the value of the relative viability fraction N/N0. The viability data were fitted to a first order reaction kinetics model as described by the formula: equation(2) Nt/N0=1-kTtNt/N0=1-kTtwhere N0 represents the initial number of the viable bacteria and Nt the number of viable bacteria after a specific time of storage (in CFU/g), t is the storage time (in day), and kT is the inactivation rate constant at T temperature (day−1). A digital micrometre with a sensitivity of 0.001 mm was used for the measurement of the thickness of the probiotic edible films. Eight measurements were taken from different parts of the films to ensure results consistency.