Wort Substrate Consumption and Metabolite Production During Lambic Beer Fermentation and Maturation Explain the Successive Growth of Specific Bacterial and Yeast Species

Abstract

The present study combined high-throughput culture-dependent plating and culture-independent amplicon sequencing with a metabolite target analysis to systematically dissect the identity, evolution, and role of the microorganisms, substrates, and metabolites during the four-phase fermentation and maturation process of lambic beer production. This led to the following new insights. The changing physicochemical parameters and substrate and metabolite compositions of the fermenting wort and maturing lambic beer provoked several transitions between microbial species and explained the four-step production process. Manual wort acidification with lactic acid shortened the enterobacterial phase and thus kept biogenic amine formation by enterobacteria present during the early stages of fermentation at a minimum. Growth advantages during the alcoholic fermentation phase caused a transition from the prevalence by Hanseniaspora uvarum and Kazachstania species to that by Saccharomyces cerevisiae and later on Saccharomyces kudriavzevii, due to changing environmental parameters. During the acidification phase, Pediococcus damnosus was prevalent and performed a malolactic fermentation. Acetobacter pasteurianus produced acetic acid and acetoin. Upon maturation, Dekkera species appeared, together with P. damnosus and Pichia membranifaciens, thereby contributing to acetic acid production, depending on the oxygen availability. Moreover, the Dekkera species consumed the acetoin produced by the acetic acid bacteria for redox balancing. The breakdown of maltooligosaccharides seemed to be independent of the occurrence of Dekkera species and started already early in the fermentation process.

Temporal Shotgun Metagenomics Revealed the Potential Metabolic Capabilities of Specific Microorganisms During Lambic Beer Production

“…Further, P. damnosus was most probably responsible for biogenic amine production during the lambic beer production process examined, as this LAB species possessed the necessary decarboxylase genes for the production of histamine and tyramine. Moreover, biogenic amines are produced during the acidification phase of a lambic beer production process, as has been shown before for the process examined (De Roos et al, 2018a). Further, the partial lysine decarboxylase gene belonging to Klebsiella species was in accordance with the low concentrations of cadaverine produced during the first week of the lambic beer production process of the present study (De Roos et al, 2018a).…”

“…In particular, the most recent studies have hypothesized possible reasons behind particular community dynamics of AAB, LAB, and yeasts during the main fermentation, acidification, and maturation phases of lambic beer production processes. Moreover, some new fermentation characteristics have been shown, such as the occurrence of a malolactic fermentation most likely carried out by P. damnosus, acetoin production by Acetobacter species, the possible consumption of acetoin by Brettanomyces species, and the simultaneous breakdown of maltooligosaccharides probably by yeast species present at low counts (De Roos et al, 2018a). However, to strengthen these results and to clarify some of the hypotheses proposed in said studies, a temporal metagenomic analysis of a lambic beer production process that has been investigated microbiologically and metabolically in detail before was performed to target both the taxonomy and the functional properties of the different microorganisms involved.…”

“…With regard to the LAB, the presence of P. damnosus in high relative abundances could be hold responsible for a fast consumption of malic acid during the acidification phase, as this has been shown phenotypically before (De Roos et al, 2018a). However, culture-dependent analysis is limited in addressing sub-dominant microbial groups, therefore making it difficult to address malic acid consumption to P. damnosus solely.…”

“…The concentrations of D-lactic acid and L-lactic acid were measured by high-performance liquid chromatography with ultraviolet detection (HPLC-UV) and external calibration, as described previously (De Roos et al, 2018a).…”

Bottle Conditioning: Technology and Mechanisms Applied in Refermented Beers

“…After approximately 4 months, the acidification by LAB (Pediococci and Lactobacilli), acetic acid bacteria, and Brettanomyces takes place. During further maturation, bacterial and yeast counts progressively decline [93,94]. In Gueuze, the final stage of bottle conditioning is induced by blending of lambics aged for different periods of time prior to bottling.…”