Action mechanisms involved in the bioprotective effect of Lactobacillus harbinensis K.V9.3.1.Np against Yarrowia lipolytica in fermented milk

Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.ijfoodmicro.2017.02.013

English. The use of lactic acid bacteria (LAB) as bioprotective cultures can be an alternative to chemical preservatives or antibiotic to prevent fungal spoilage in dairy products. Among antifungal LAB, Lactobacillus harbinensisK.V9.3.1Np showed a remarkable antifungal activity for the bioprotection of fermented milk without modifyingtheir organoleptic properties (Delavenne et al., 2015). The aim of the present study was to elucidate the actionmechanism of this bioprotective strain against the spoilage yeast Yarrowia lipolytica. To do so, yeast viability,membrane potential, intracellular pH (pHi) and reactive oxygen species (ROS) production were assessed using flow cytometry analyses after 3, 6 and 10 days incubation in cell-free supernatants. The tested supernatants were obtained after milk fermentation with yogurt starter cultures either in co-culture with L. harbinensis K.V9.3.1Np (active supernatant) or not (control supernatant). Scanning-electron microscopy (SEM) was used to monitor yeast cell morphology and 9 known antifungal organic acids were quantified in both yogurt supernatants using high-performance liquid chromatograph (HPLC). Yeast growth occurred within 3 days incubation in control supernatant,while itwas prevented for up to 10 days by the active supernatant. Interestingly, between 66 and 99% of yeast cellswere under a viable but non-cultivable (VNC) state despite an absence ofmembrane integrity loss. While ROS production was not increased in active supernatant, cell physiological changes including membrane depolarization and pHi decrease were highlighted. Moreover, morphological changes including membrane collapsing and cell lysis were observed. These effects could be attributed to the synergistic action of organic acids. Indeed, among the 8 organic acids quantified in active supernatant, five of them (acetic, lactic, 2-pyrrolidone-5-carboxylic, hexanoic and 2-hydroxybenzoic acids) were at significantly higher concentrations in the active supernatant than in the control one. In conclusion, this study has provided new information on the physiological mechanisms induced by an antifungal LAB that could be used as part of the hurdle technology to prevent fungal spoilage in dairy products.