Skim Milk as a Multifunctional Cryoprotectant for Fish Probiotic Enterococcus spp.: Impact on Viability During...

This study investigated the efficacy of different cryoprotectants (maltodextrin, skim milk, trehalose, sucrose, fructose, and dextrose) in protecting probiotic cultures isolated from the microbiota of arapaima (Arapaima gigas) (Enterococcus faecium CRBP46 and Enterococcus gallinarum CRBP19) during lyophilization, storage (-25 °C, 4 °C, and 25 °C for 120 days), and exposure to simulated gastrointestinal fluids (SGF). Cell surface hydrophobicity and the ultrastructural aspects of the coating matrices were also evaluated. Skim milk, trehalose, and dextrose (only for E. gallinarum) protected Enterococcus spp. against the negative effects of lyophilization, resulting in minimal viability loss (≤0.03 log CFU/g) and ≥99.50% survival. All cryoprotectants promoted stability (≥8.87 log CFU/g) for both bacteria when stored at refrigeration and freezing temperatures for 120 days. However, only skim milk maintained high viability (≥6.83 log CFU/g) for Enterococcus spp. during 120 days of storage at room temperature. Additionally, Enterococcus spp. lyophilized with skim milk demonstrated stability in SGF, with high cell viability (≥8.97 log CFU/g) and survival over 97%. Skim milk also significantly increased the cell adhesion capacity of Enterococcus spp., making them more hydrophobic. Scanning electron microscopy showed that Enterococcus cells were incorporated into the skim milk matrix and that its lower porosity directly contributed to the preservation of cell viability. Therefore, we conclude that skim milk is the most effective cryoprotectant under the tested conditions for E. faecium and E. gallinarum, as it ensured stability and high viability for both bacteria throughout all post-lyophilization challenges, maintaining bacterial concentrations above those suggested for probiotic formulations. Our findings provide unprecedented insights into the development of long-term stable, dry autochthonous probiotics, aiming to strengthen a more sustainable aquaculture for the arapaima, the Amazon's giant fish.