The meaning of life for lactic acid bacteria: how to measure life, stress and death in production and application

Research output: Book/ReportPh.D. thesis

  • Sebastian Bech-Terkilsen
Lactic acid bacteria (LAB) are widely industrialized in food and biotechnology, particularly in dairy, where fermentations transform milk to e.g. yoghurts, cheeses and fermented milks. In the production of starter cultures for such applications, LAB may experience significant stresses during the whole stream and during storage in frozen and freeze-dried form. Their applications can happen in stressful environments or they play a part in making it stressful. The gold standard method to determine the concentration of active LAB in cultures remains colony forming units (CFU) in which LAB are cultured on nutrient gel into colonies. Alternative fluorescent methods rely on membrane integrity (MI) alone or the generation of a membrane potential (MP) which provide a faster alternative to CFU. While these methods can do a decent job of indicating the amount of active cells in a culture, they can fall short in assessing more complex, dynamic and stressful cultures and processes. The major aim of this study was to give insights into these methods, as well as improve on them or device new methods. The application of Oenococcus oeni (O. oeni) as a starter culture for malolactic fermentation in wine was chosen as a focus because it is one of the most stressful, dynamic and complex applications. In a literature review on this subject, numerous significant stress mechanisms were reviewed, which O. oeni has to endure. Particularly sulphur dioxide (SO2) is a potent stress factor, for which the mechanisms were still not identified. We put forward a model of significant stress events, to provide a more simplified view of the sequential events an O. oeni culture experiences upon inoculation. These stress events were studied further using CFU, flow cytometry (FCM) and microscopy with carboxyfluorescein (cF). Using an FCM method, a significant mechanism of SO2’s stress impact on O. oeni was measured, the deterioration of the membrane. Viable but non-culturable (VBNC) cells were found widespread in fermentations of varying stress and to a significant degree (>50%). This study provides clear evidence that wine stress should be studied with more sophisticated methods and has provided several important observations. An image analysis method using convolutional neural networks (CNNs) was made which on dataset of microscopy images of >700,000 bacteria across 5 genera, was able to predict the genus at a single-cell level with a 95% accuracy, showing the potential of CNNs to shape the future of imaging in microbiology. An advanced method using automated microscopes which could offer unprecedented insights into cellular health is introduced, as it uses the aspects of fluorescent probes and instrumentation that offers the highest quality information.
Original languageEnglish
PublisherDepartment of Food Science, Faculty of Science, University of Copenhagen
Number of pages142
Publication statusPublished - 2021

ID: 273018335