Arrested or limited fermentation process is a widespread method used for production of alcohol-free beer (AFB). However, it usually leads to worty off-flavour and a lack of pleasant fruity flavour/aroma. The aim of this study was to isolate spontaneous mutants of Saccharomyces pastorianus strain 2 resistant to 5,5,5-trifluoro-DL-leucine, since the resistance is related to overproduction of flavour active isoamyl alcohol (IAAL) and isoamyl acetate (IAAC). The mutants were the subject of selection during series of screening tests aimed at selecting the best producer of target compounds (IAAL and IAAC). Subsequently, the overproduction stability of the selected mutant strain was verified in four consecutive fermentations of AFBs. A higher pitching rate and increased fermentation temperature did not result in an overall improvement in the production of the target compounds. Sensory analysis proved the flavouring effect of the spontaneous lager yeast mutant on the AFB produced by arrested fermentation.

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Liquid chromatography-electrospray tandem mass spectrometry (LC/ESI-MS/MS) was used to analyze phospholipids from three species of the anaerobic beer-spoilage bacterial genus Pectinatus. Analysis of total lipids by HILIC (Hydrophilic Interaction Liquid Chromatography) column succeeded in separating diacyl- and plasmalogen phospholipids. Plasmalogens were then analyzed by means of the ESI-MS/MS and more than 220 molecular species of four classes of plasmalogens (PlsCho (choline plasmalogen), PlsEtn (ethanolamine plasmalogen), PlsGro (glycerol plasmalogen), and PlsSer (serine plasmalogen)) were identified. Major molecular species were c-p19:0/15:0 PlsEtn and PlsSer, which accounted for more than 4% of the total lipids.

Novel rhamnolipid-producing strains of three thermophilic bacteria, Thermus sp., T. aquaticus and Meiothermus ruber were identified that have not been previously described as rhamnolipid producers. Rhamnolipids were extracted from supernatant and further purified by thin-layer chromatography. Mass spectrometry with negative electrospray ionization revealed 77 rhamnolipid homologues varying in chain length and unsaturation. Tandem mass spectrometry identified mono-rhamnolipid and di-rhamnolipid homologues containing one or two 3-hydroxy-fatty acids, saturated, monounsaturated or diunsaturated, even- or odd-chain, up to unusual long chains with 24 carbon atoms. The stereochemistry of rhamnose was L and that of 3-hydroxy-fatty acids was R, the position of double bonds in monoenoic acids was cis ω-9. All three strains produced a rhamnolipid that differs in structure from Pseudomonas aeruginosa rhamnolipids and exhibits excellent surfactant properties. Importantly, in comparison to P. aeruginosa both strains, i.e., Thermus and Meiothermus, are Biosafety level 1 microorganisms and are not pathogenic to humans.

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