Université de Brest, EA3882 Laboratoire Universitaire de Biodiversité et Ecologie Microbienne, IBSAM, ESIAB, Technopôle Brest-Iroise, 29280 Plouzané, France.
STLO, Agrocampus Ouest, INRA, 35000 Rennes, France.
Int J Food Microbiol. 2017 May 2;248:47-55. doi: 10.1016/j.ijfoodmicro.2017.02.013. Epub 2017 Feb 21.
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.
将乳酸菌（LAB）用作生物保护培养物可以替代化学防腐剂或抗生素，以防止乳制品中的真菌变质。在抗真菌 LAB 中，哈尔滨乳杆菌 K.V9.3.1Np 对发酵乳的生物保护表现出显著的抗真菌活性，而不会改变其感官特性（Delavenne 等人，2015 年）。本研究的目的是阐明该生物保护菌株对腐败酵母解脂耶氏酵母的作用机制。为此，使用流式细胞术分析在无细胞上清液中孵育 3、6 和 10 天后，评估酵母活力、膜电位、细胞内 pH（pHi）和活性氧（ROS）的产生。测试的上清液是在用酸奶发酵剂培养物发酵牛奶后获得的，要么与哈尔滨乳杆菌 K.V9.3.1Np 共培养（活性上清液），要么不共培养（对照上清液）。扫描电子显微镜（SEM）用于监测酵母细胞形态，并用高效液相色谱（HPLC）定量测定两种酸奶上清液中的 9 种已知抗真菌有机酸。在对照上清液中，酵母生长发生在孵育 3 天内，而在活性上清液中，酵母生长可被抑制长达 10 天。有趣的是，尽管没有膜完整性丧失，但在 66%至 99%的酵母细胞处于存活但不可培养（VNC）状态。虽然在活性上清液中没有增加 ROS 的产生，但强调了包括膜去极化和 pHi 下降在内的细胞生理变化。此外，观察到包括膜塌陷和细胞裂解在内的形态变化。这些影响可能归因于有机酸的协同作用。事实上，在活性上清液中定量的 8 种有机酸中，有 5 种（乙酸、乳酸、2-吡咯烷酮-5-羧酸、己酸和 2-羟基苯甲酸）的浓度明显高于对照上清液。总之，本研究提供了关于抗真菌 LAB 诱导的生理机制的新信息，这些信息可用于作为障碍技术的一部分，以防止乳制品中的真菌变质。
