Molecular Mechanisms of Basil (Ocimum basilicum L.) Polyphenol Extracts as Bio-Based Cryoprotectants for...

Natural plant extracts rich in polyphenols are increasingly being studied as multifunctional food ingredients with antioxidant and stabilizing properties. In this study, Ocimum basilicum L. extracts were evaluated as biological cryoprotective agents for Streptococcus thermophilus. The extracts contained high levels of phenolic compounds (~1350-2200 mg GAE equivalents/L) and exhibited strong antioxidant activity (up to 5.6 mM Trolox equivalents). Density functional theory calculations showed low O-H bond dissociation energies (~72-74 kcal/mol in ethanol) for key components, including luteolin and rosmarinic acid. These calculations indicate a high hydrogen donation capacity comparable to or exceeding that of ascorbic acid. Molecular dynamics simulations demonstrated the preferential localization of major phenolic compounds at the membrane-water interface in a POPC bilayer membrane. The interaction of molecules with POPC increased membrane thickness and formed stable hydrogen-bond networks with lipid head groups. Experiments showed that systems based on basil extract significantly increased the survival of bacteria after storage at -25 °C, with the number of viable cells reaching (1.5-2.75) × 10⁸ CFU/mL. This effect was observed in comparison with control groups that used saline or sucrose. Fluorescent analysis of live/dead cells confirmed the improvement in cell membrane preservation. At the same time, no signs of metabolic inhibition were detected. Taken together, the experimental and computational results support the hypothesis that the cryoprotective effect may involve complementary antioxidant and membrane-associated interactions. However, direct biophysical validation of the proposed membrane mechanism is still required. These results emphasize that polyphenol extracts are promising natural functional ingredients for improving the stability and shelf life of probiotic and starter cultures in food systems.