The juice (with an efficiency of 25.8%) was passed through a 1 mm mesh filter and immediately transferred into a sterile glass container under sanitized conditions, sterilized in an autoclave at 121 °C for 15 min and refrigerated at 4 °C until processed.
Pre-cultures were prepared by transferring a colony from each plate containing L. plantarum (Section 2.2) into 10 mL of MRS broth, followed by incubation for 24 h at 37 °C.
The pre-cultures were then subcultured into 3L of the respective growth media and statically incubated for 8 h to reach the early stationary phase (to obtain loads of approximately 9.5 log cfu/mL).
The cells were spun in a refrigerated centrifuge (Sigma, 6K15, Germany) for 5 min at 4420×g. The pellets were washed by sterile buffered peptone water then resuspended in 500 mL of sterile orange juice aliquots to inoculate 29.5 L orange juice before packaging.
Samples were taken from final aliquot, plated on MRS agar and incubated for 48 h at 37 °C under CO2 atmosphere (5%) to determine initial microbial population.
Packages were prepared by a hand heat sealer using antimicrobial nanocomposite and pure LDPE films 15 × 10 cm in size, similar to Doypack packaging commonly used for packaging fruit juice.
The packages were immediately wrapped in aluminum foil and sanitized at 95 °C for 2 min.
After cooling and under a sterile laboratory hood, 175 mL of inoculated orange juice was poured into each package and sealed by the heat sealer.