Institut Universitaire de la Vigne et du Vin Jules Guyot, Université de Bourgogne, Rue Claude Ladrey, BP 27877, F-21078 Dijon Cedex, France.
Food Microbiol. 2012 Jun;30(2):438-47. doi: 10.1016/j.fm.2011.12.020. Epub 2012 Jan 8.
Although the viable but not culturable (VBNC) state has been studied in detail in bacteria, it has been suggested that maintenance of viability with loss of culturability also exists in eukaryotic cells, such as in the wine spoilage yeast Brettanomyces. To provide conclusive evidence for the existence of a VBNC state in this yeast, we investigated its capacity to become viable and nonculturable after sulfite stress, and its ability to recover culturability after stressor removal. Sulfite addition induced loss of culturability but maintenance of viability. Increasing the medium pH to decrease the concentration of toxic SO(2) allowed yeast cells to become culturable again, thus demonstrating the occurrence of a VBNC state in Brettanomyces upon SO(2) exposure. Relative to culturable Brettanomyces, VBNC yeast cells were found to display a 22% decrease in size on the basis of laser granulometry. Assays for 4-ethylguaiacol and 4-ethylphenol, volatile phenols produced by Brettanomyces, indicated that spoilage compound production could persist in VBNC cells. These morphological and physiological changes in VBNC Brettanomyces were coupled to extensive protein pattern modifications, as inferred by comparative two-dimensional electrophoresis and mass spectrometric analyses. Upon identification of 53 proteins out of the 168 spots whose abundance was significantly modified in treated cells relative to control, we propose that the SO(2)-induced VBNC state in Brettanomyces is characterized by a reduced glycolytic flux coupled to changes in redox homeostatis/protein turnover-related processes. This study points out the existence of common mechanisms between yeast and bacteria upon entry to the VBNC state.
虽然存活但不可培养（VBNC）状态在细菌中已被详细研究，但有人认为，在真核细胞中也存在维持生存能力但丧失可培养性的情况，例如在葡萄酒腐败酵母 Brettanomyces 中。为了为该酵母中存在 VBNC 状态提供确凿证据，我们研究了其在亚硫酸盐应激后恢复生存能力和非可培养性的能力，以及在应激消除后恢复可培养性的能力。亚硫酸盐的添加导致丧失可培养性但保持生存能力。增加培养基 pH 值以降低有毒 SO2 的浓度使酵母细胞能够再次可培养，从而证明在 SO2 暴露时 Brettanomyces 中存在 VBNC 状态。与可培养的 Brettanomyces 相比，发现 VBNC 酵母细胞在基于激光粒度法的基础上大小减小了 22%。测定由 Brettanomyces 产生的挥发性酚 4-乙基愈创木酚和 4-乙基苯酚的含量表明，在 VBNC 细胞中仍可继续产生腐败化合物。VBNC Brettanomyces 的这些形态和生理变化与广泛的蛋白质图谱修饰有关，这可以通过比较二维电泳和质谱分析推断得出。在鉴定出处理细胞中与对照相比丰度显著改变的 168 个斑点中的 53 个蛋白质后，我们提出 SO2 诱导的 Brettanomyces VBNC 状态的特征是降低的糖酵解通量与氧化还原稳态/蛋白质周转相关过程的变化相关联。这项研究指出了酵母和细菌在进入 VBNC 状态时存在共同的机制。
