Characterisation of microbial communities and xylanolytic bacteria during malting of barley: impact on malt quality

Non-starch polysaccharides, such as beta-glucans and arabinoxylans, present in the cell walls of barley grains, are important contributors to wort filtration problems due to their viscous nature. Since nowadays it is the current practice to produce well-modified malts in the malting plant, the role of beta-glucans in reduced filtration rate is of minor importance because of their advanced degradation during malting. Conversely, a lot of the filtration problems to date are associated with arabinoxylans which are not extensively degraded during malting, as the endogenous enzymes that hydrolyse arabinoxylans, i.e. xylanases, are produced relatively late in the germination process. Furthermore, because of the relatively low temperature stability of endogenous xylanases, arabinoxylans are insufficiently degraded during the brewing process. In addition, it is well-known that a substantial part of the malt xylanolytic activity originates from the microbial community present on the barley kernels, representing a second metabolically active compound in the malting ecosystem. Against this background, this doctoral study aimed to generate insights into the xylanase-producing bacterial community present during barley malting. Special emphasis was put on the bacterial glycoside hydrolase (GH) family 10 xylanases, since they degrade arabinoxylans very effectively and are resistant to xylanase inhibitors present in barley.As traditional culture-dependent approaches are known to underestimate the microbial species diversity, molecular culture-independent 454 amplicon pyrosequencing was successfully applied to investigate the structure and dynamics of the bacterial communities associated with industrial malting. The bacterial community structure in the malting ecosystem was found to be complex and community dynamics appeared to differ from year to year and to be influenced by the malting process steps. Although many bacteria were found to occur from the harvested barley kernel up to the end of the malting process, the observed differences between two harvest years could not be explained by a different microbial load initially present on the barley kernels. Environmental parameters such as varying seasonal weather conditions and, as a consequence of this, adjustments of the air circulation pattern in the maltings, could potentially explain the differences in bacterial community structure of germinated barley and kilned malt samples between both investigated years. The bacterial community structure was also found to change along the malting process, but eventually the kilned malt samples were more similar to their corresponding harvested barley sample than to the germinating barley samples. Also at the phylum level, clear differences could be observed between both harvest and malting years.In order to explore the arabinoxylan-degrading microbial community present during barley malting, the genetic diversity and distribution of GH10 xylanase genes during malting were examined using 454 amplicon pyrosequencing. Most of the GH10 xylanases detected in the malting barley samples showed a high identity with known xylanases. The GH10 xylanase sequences obtained in this study were mainly related to xylanases from the phylum Bacteroidetes, in particular from the genus Sphingobacterium.The second part of this study focuses on the availability of xylanolytic bacteria and their exploitation during malting. Therefore, the diversity of the arabinoxylan-degrading bacterial community during barley malting was assessed by microbial isolation, cultivation on different media enriched with arabinoxylan, and identification. As such, 33 species-level operational taxonomic units belonging to 25 genera were found. Most of the arabinoxylan-hydrolysing bacteria isolated during malting could be assigned to Sphingobacterium species. Genetic fingerprinting revealed shifts in S. multivorum populations during the process, especially during germination. Furthermore, the xylanase produced by an isolate showing the highest activity (identified as Cellulomonas flavigena) was partially purified and characterised with respect to temperature stability, revealing a relatively thermostable enzyme.A filtration assay which is relevant to current brewing practices, was successfully applied to study the lautering performance of kilned malts. Addition of commercially available, thermostable xylanases at mashing-in was highly efficient to increase the wort filtration rate, especially when GH10 xylanases were applied. Besides direct application in mashing, the addition of exogenous GH10 xylanase during barley germination also resulted in improved lautering performance. Furthermore, the addition of two xylanolytic bacteria (Cellulomonas flavigena or Enterococcus casseliflavus), originating from malting barley, into the steeping or germination process resulted in an increased wort filtration rate without negative effects on malting performance or on standard malt quality characteristics. This knowledge opens up new perspectives for the application of xylanolytic bacteria during steeping or germination on an industrial scale.In conclusion, this doctoral study contributes to the existing body of knowledge of bacterial diversity during malting and leads to a better understanding of the role of bacteria in the malting process. In particular, this study may provide a basis for microbiota management during malting, aiming at an increased mash filtration rate since introducing more xylanase activity in the malting/brewing process shows high potential in this respect. As improved wort filtration is linked to lower heat load and reduced production of aldehydes, it may ultimately also result in enhanced beer flavour quality, including flavour stability.nrpages: 184status: publishe