title: Action mechanisms involved in the bioprotective effect of Lactobacillus harbinensis K.V9.3.1.Np against Yarrowia lipolytica in fermented milk.
abstract: 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 harbinensis K.V9.3.1Np showed a remarkable antifungal activity for the bioprotection of fermented milk without modifying their organoleptic properties (Delavenne et al., 2015). The aim of the present study was to elucidate the action mechanism 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 10days 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 3days incubation in control supernatant, while it was prevented for up to 10days by the active supernatant. Interestingly, between 66 and 99% of yeast cells were under a viable but non-cultivable (VNC) state despite an absence of membrane 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.
authors.affiliation: Université de Brest, EA3882 Laboratoire Universitaire de Biodiversité et Ecologie Microbienne, IBSAM, ESIAB, Technopôle Brest-Iroise, 29280 Plouzané, France.
authors.forename: Sophie
authors.initials: S
authors.lastname: Mieszkin
authors.affiliation: Université de Brest, EA3882 Laboratoire Universitaire de Biodiversité et Ecologie Microbienne, IBSAM, ESIAB, Technopôle Brest-Iroise, 29280 Plouzané, France.
authors.forename: Nolwenn
authors.initials: N
authors.lastname: Hymery
authors.affiliation: Université de Brest, EA3882 Laboratoire Universitaire de Biodiversité et Ecologie Microbienne, IBSAM, ESIAB, Technopôle Brest-Iroise, 29280 Plouzané, France.
authors.forename: Stella
authors.initials: S
authors.lastname: Debaets
authors.affiliation: Université de Brest, EA3882 Laboratoire Universitaire de Biodiversité et Ecologie Microbienne, IBSAM, ESIAB, Technopôle Brest-Iroise, 29280 Plouzané, France.
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Coton
authors.affiliation: Université de Brest, EA3882 Laboratoire Universitaire de Biodiversité et Ecologie Microbienne, IBSAM, ESIAB, Technopôle Brest-Iroise, 29280 Plouzané, France.
authors.forename: Gwenaelle
authors.initials: G
authors.lastname: Le Blay
authors.affiliation: STLO, Agrocampus Ouest, INRA, 35000 Rennes, France.
authors.forename: Florence
authors.initials: F
authors.lastname: Valence
authors.affiliation: Université de Brest, EA3882 Laboratoire Universitaire de Biodiversité et Ecologie Microbienne, IBSAM, ESIAB, Technopôle Brest-Iroise, 29280 Plouzané, France. Electronic address: jerome.mounier@univ-brest.fr.
authors.forename: Jérôme
authors.initials: J
authors.lastname: Mounier
mesh_terms: D019342:Acetic Acid / Q000378:metabolism; D000818:Animals; D000898:Antibiosis / Q000502:physiology*; D000935:Antifungal Agents / Q000494:pharmacology*; D002208:Caproates / Q000378:metabolism; D002264:Carboxylic Acids / Q000378:metabolism; D002462:Cell Membrane / Q000473:pathology; D002851:Chromatography, High Pressure Liquid; D018920:Coculture Techniques; D005285:Fermentation; D005520:Food Preservatives / Q000378:metabolism / Q000494:pharmacology*; D019344:Lactic Acid / Q000378:metabolism; D007778:Lactobacillus / Q000378:metabolism*; D008855:Microscopy, Electron, Scanning; D008892:Milk / Q000382:microbiology*; D019936:Probiotics / Q000494:pharmacology*; D011760:Pyrrolidinones / Q000378:metabolism; D020156:Salicylic Acid / Q000378:metabolism; D025062:Yarrowia / Q000254:growth & development*; D015014:Yogurt / Q000382:microbiology*
chemical_list: D000935:Antifungal Agents; D002208:Caproates; D002264:Carboxylic Acids; D005520:Food Preservatives; D011760:Pyrrolidinones; C037652:hexanoic acid; D019344:Lactic Acid; C028537:2-pyrrolidone; D020156:Salicylic Acid; D019342:Acetic Acid
keywords: Action mechanisms; Antifungal; Bioprotection; Dairy products; Lactobacillus harbinensis; Yarrowia lipolytica