Analytical and Nanotechnological Methods for Detection of 3-OH Oxylipins and Cell Ultrastructure in Fermenting Yeasts
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While 3-hydroxy (OH) oxylipins, a class of hydroxy free fatty acids (FFA), have been previously presumed to play a role in the industrially important brewing yeast flocculation phenomenon, the exact biological function was not known. In earlier yeast 3-OH oxylipin investigations, these molecules were identified by diazomethane derivatization and then separation using gas chromatography-mass spectrometry (GC-MS). Unfortunately, this approach only allowed for qualitative analysis of 3-OH oxylipins in yeast. The difficulty associated with diazomethane use has also meant that lipid analysts must use alternate approaches that still target FFA specifically. A survey of the literature identified numerous approaches to detect FFA that did not require diazomethane. They were grouped as selective quantifications/extractions, purifications and alternate derivatizations. Here it is shown that 3-OH oxylipins can be detected in the SMA strain of Saccharomyces pastorianus grown in lab-scale fermentations without diazomethane by extracting cellular lipids with ethyl acetate, trimethylsilylating the total lipid extract and GC-MS identification using the diagnostic m/z 233 fragment. A second detection strategy was also devised where 3-OH oxylipins were separated from the total lipid extract using thin layer chromatography. Thereafter, 3-OH oxylipins were methylated using BCl3-MeOH and derivatized at the hydroxy group with heptaflouorobutyric anhydride. Detection of the heptafluorobuyrate methyl ester derivatives using negative chemical ionization-mass spectrometry allowed for the first quantitative analysis of 3-OH oxylipins in yeast. During growth of the SMA strain in lab scale fermentations, 3-OH decanoic acid ranged from 0.68 ± 0.22 - 4.82 ± 0.18 ng/mg dry cell mass. The discovery of CO2 bubbles inside fermentation yeasts using Auger-architechtomics has also necessitated more comprehensive studies of bubble formation and 3-OH oxylipin production. Using Nano scanning Auger microscopy, time-of-flight secondary ion mass spectrometry (TOF-SIMS) and immunofluorescence microscopy with 3-OH oxylipin specific antibodies, bubble formation and 3-OH oxylipin production were studied in fermenting and respiring SMA cells. Examinations showed networks of CO2 in fermenting cells that increased in size with fermentation duration. TOF-SIMS analysis also showed a compositional difference at the interior and exterior of fermenting and respiring cells, while immunofluorescence results suggested contrasting 3-OH oxylipin profiles in fermenting vs. respiring SMA cells.