Origins of microorganisms and volatile organic compounds during lambic beer production

Scriptie/masterproef: Master's Thesis


Lambic beers are the product of a spontaneous fermentation and maturation conducted in wooden barrels that can last up to three years. Spontaneous inoculation of yeasts and bacteria from the brewery air and environment initiate the fermentation process, which is in contrast with the use of well-defined starter cultures for most other beer types. Traditionally, lambic beers are brewed around the Senne river valley, and it has long been assumed that specific air microbiota in this environment are responsible for the inoculation of the wort during the cooling in an open vessel (coolship). When the cooled, inoculated wort is transferred into wooden barrels, stored at ambient temperature, the wort starts to ferment. During this fermentation process, a unique microbial succession occurs when diverse groups of microorganisms become metabolically active. Typically, four different phases are distinguished during a lambic beer production process: an early enterobacterial and wild yeast phase followed by the main alcoholic fermentation phase by Saccharomyces species, an acidification phase primarily by Pediococcus spp., which partly overlaps with the final maturation phase dominated by Dekkera yeasts. In modern brewery practices, the wort is often acidified artificially, skipping the early phase of Enterobacteriaceae, to avoid deviations during the beginning of the fermentation. The characteristic sour taste of lambic beers can be linked to typical aroma compounds, originating from the metabolic activities of various yeasts, lactic acid bacteria (LAB), and acetic acid bacteria (AAB), whereby ethyl lactate and ethyl acetate are reported as the most influential ones.
Although the brewery air is regarded as the main source for inoculation, it is not yet completely clear what the origin of the lambic beer-related microorganisms is, since previous reports are sometimes contradictory. This knowledge is however imperative to avoid fermentation deviations and to assess the influence of the barrels on the final quality of the end-products. Therefore, the present study aimed to map the microbial diversity of the inner surfaces of different barrel types. Six barrels (three cask-type and three foeder-type) were sampled during their cleaning procedures between consecutive batches. By means of culture-dependent (selective plating and incubation) and culture-independent techniques (amplicon sequencing) the bacterial and fungal diversities were assessed. It was found that foeders harbored more bacteria in comparison with the casks. Also, the inner surfaces of casks showed greater bacterial and fungal diversities that increased significantly during the cleaning procedure. Foeders displayed a much lower diversity and little changes were found during the cleaning procedure. It was hypothesized that the greater porosity of the casks allowed more development of environmental contamination such as Aspergillus and Penicillium. More importantly, for both casks and foeders, Dekkera, Pichia, Saccharomyces and Pediococcus were found at the end of the cleaning procedures, suggesting that these barrels could act as an additional inoculation source for the wort. But since no viable counts were obtained at the end of the cleaning procedures, more research is needed to investigate if these microorganisms are in a viable but non-culturable (VBNC) state that can be reversed when new wort is added to the casks and foeders.
Also a lambic beer fermentation process was followed as a function of time to assess the bacterial and fungal diversities using amplicon sequencing of specific marker genes of genomic DNA. The profiles obtained were in correspondence with earlier research. The main bacterial species identified from the fermentation were Pediococcus and Acetobacter, whereas for the fungal species Saccharomyces followed by Pichia and Dekkera were found. An important finding during this fermentation process was that the wort already harbored fermentation-relevant microorganisms before its transfer to the casks, indicating that inoculation already occurred when the wort came into contact with brewery surfaces. More environmental sampling is however needed to identify which brewery environments are the most influential inoculation sources. Also, since the fermentation followed, was conducted in a cask-type barrel, it was not possible to make predictions about the influence of the barrel type on the fermentation profile during the present study.
A second objective of the present study was to quantify and unravel the source of targeted volatile organic compounds (VOCs) found within the lambic beer matrix during the fermentation/maturation phases. Since these compounds are typically present in very low concentrations, first a method optimization was performed, to enable accurate quantification using gas chromatography coupled with mass spectrometry. This method was previously reported as a sensitive method for aroma analysis. For all VOCs analyzed, a good quantification was obtained based on the optimized method and standard addition to avoid changing matrix effects during the lambic beer production process. 4-Ethylphenol (4-EP) and 4-ethylguaiacol (4-EG), metabolites linked to the Dekkera metabolism, were identified as possible biomarkers to monitor the progression of the maturation phase. Also, the acetion and isoamyl acetate concentration peaks could be used as markers for the transition of the main fermentation phase to the maturation phase.
In conclusion, the present study showed that the barrel type had a clear influence on the microbial diversity of its inner surfaces and that these barrels could act as an additional inoculation source. Via an optimized method for GC-MS/MS, 4-EP and 4-EG were identified as possible biomarkers together with isoamyl acetate and acetoin for the progression of the lambic beer production process.
Datum Prijs23 nov 2017
BegeleiderLuc De Vuyst (Promotor)

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