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

Xylanase producing bacteria during malting

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Shifts in microbial community structure during malting with emphasis on xylanase producing bacteria

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Characterization of microbial communities in industrial malting to enhance malt processing quality

Description of topic - Optimization of malt quality in terms of fast filtration is a critical factor in the malting and brewing industry. Since malt quality is inversely related to the concentration of endosperm cell wall components, management of cell wall degrading microorganisms may result in enhanced processing. An essential step in microflora management during malting, however, requires the understanding of the microbial community structure as well as the population dynamics in the processing. Although most studies undertaken so far focus on specific microbial populations using culture-dependent techniques, in this study an integrated approach of different technologies for microbial community profiling is presented as a basis for process optimization. Materials and methods - Both culture dependent and culture independent molecular techniques will be used to characterize the microbial (bacterial) communities in different industrial malting settings, generating malt of different quality. 16S ribosomal DNA-targeted Terminal Restriction Fragment Length Polymorphism (T-RFLP) analysis, supported by sequencing of clone libraries, will be used to screen and compare the microbial communities in the different maltings at different stages of the malting process, i.e. from barley up to kilned malt. In addition, the microflora present in used irrigation water and the air will be monitored. Specific patterns associated with high-quality malt will be further investigated, aiming at obtaining cultures responsible for the malt quality. Subsequently, promising bacterial strains will be screened for their cell wall degrading capacity using agar plate assays. Finally, one or more bacterial strains will be selected to evaluate their performance in practice using inoculation trials on a representative pilot scale malting facility. In addition, based on an off line closed-circuit perfusion approach, efforts will be made to manage the microbial community in such a way that desired populations will be stimulated and can be re-inoculated in the process. Novelty - With the increased demand for ‘clean-label’ technology, the malting and brewing industry is seeking for novel approaches to optimize the use of in-house microflora in the malting process. This study may result in the discovery of promising, process-borne microorganisms which may enhance malt processability, or the malting and brewing process in general. Most presumably, this research will reveal previously unidentified species as novel detection technologies will be used to unravel the microbial communities. Altogether, this project might be the basis for further process optimization such as the improvement of the filtration step.status: publishe

Molecular characterization of the microflora during malting to enhance malt quality and processability

Background - Optimization of malt quality with regard to a better lautering performance is a critical factor in the malting and brewing industry. Since malt quality is inversely related to the concentration of endosperm cell wall components such as arabinoxylans, management of cell wall degrading microorganisms may result in enhanced processing. An essential step in microflora management during malting requires the understanding of the microbial community structure as well as the population dynamics in the processing. Objectives – The objective of this study was to characterize the microbial (bacterial) communities in different industrial malting settings and relate these to malt quality and processability. Methods and Conclusions - Both culture-dependent and culture-independent molecular techniques were used to characterize the bacterial communities in different industrial malting settings, generating malt of different quality. 16S ribosomal DNA-targeted Terminal Restriction Fragment Length Polymorphism (T-RFLP) analysis, supported by sequencing of clone libraries, were used to screen and compare the microbial communities at different stages of the malting process, i.e. from barley up to kilned malt. In addition, emphasis is put on the xylanase producing microflora. Apart from isolation and identification, the diversity of the xylanase genes was explored by amplifying xylanase genes from the glycosyl hydrolase (GH) family 10 and 11. Specific patterns associated with high-quality malt will be further investigated, aiming at obtaining cultures important for the malt quality. Ultimately, this study may result in the discovery of promising, process-borne microorganisms which may enhance malt processability, or the malting and brewing process in general.status: publishe

Flavour instability of pale lager beers : determination of analytical markers in relation to sensory ageing

Flavour changes of six Belgian pate lager beers were studied in order to estimate the importance of different parameters and reactions in relation to the ageing process. An attempt was made to link analytical data with sensory evaluation using multivariate statistical analysis. Partial least squares regression techniques (PLSR) were employed on the analytical and sensory data. As apparent from the PLSR model, significant indicators of lager beer ageing are aldehyde markers (especially total aldehydes, furfural, hexanal, 2-methylpropanal, 2-methylbutanal, and 3-methylbutanal), cold and permanent haze, and beer colour. Conversely, compounds or parameters that load negatively in the PLSR model for beer ageing are trans-isohumulones, cis-isohumulones, total bitterness, the T/C-ratio, polyphenolic markers (especially proanthocyanidins), the flavanoid content, and, to a lesser extent, the TB-index and reducing power (TRAP). The integrated analytical-sensorial methodology is proposed as a useful tool for evaluation of the flavour instability of pale lager beers

Molecular characterisation of microbial communities associated with barley during malting, with an emphasis on lactic acid bacteria

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Zooming in on Lactic Acid Bacteria during industrial malting

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The influence of very thick and fast mashing conditions on wort composition

The mashing process is of highest technological relevance for all following processes of wort and beer production. However, it is time consuming and therefore cost intensive. The influence of important mashing parameters on wort composition was investigated in order to shorten the mashing process by applying the thickest mashes without a negative influence on wort composition. The thickest mash leads to lower energy and water consumption in the brewhouse. On the other hand, the shortest mashing scheme results in the highest throughput. Finally, fine milling, mashing-in above gelatinization temperature, decreased mashing-in pH of 5.2, very thick mash of 1:2.3 malt:water ratio, and a holding time of 30-40 min at 64 degrees C followed by 15-20 min at 72 degrees C guarantees a high extract yield, normal attenuation limit, sufficient FAN level, required buffering capacity, and a reduced fatty acid oxidation. Lowering the pH to 5.2 at mashing-in results in lower glucose levels but the sum of fermentable sugar is not influenced due to a higher level of maltotriose and similar maltose levels. However, not all yeast strains assimilate maltotriose well in industrial fermentations. A mash pH of 5.2, positive for beer flavor stability, may result in a somewhat lower alcohol potential

Bacterial community dynamics during industrial malting, with an emphasis on lactic acid bacteria

Characterization of the microflora during malting is an essential step towards process management and optimization. Up till now, however, microbial characterization in the malting process has mostly been done using culture-dependent methods, probably leading to biased estimates of microbial diversity. The aim of this study was to characterize the bacterial communities using two culture-independent methods, including Terminal Restriction Fragment Length Polymorphism (T-RFLP) and 454 pyrosequencing, targeting the 16S rRNA gene. Studied samples originated from two harvest years and two malting houses malting the same batch of barley. Besides targeting the entire bacterial community (T-RFLP), emphasis was put on lactic acid bacteria (LAB) (T-RFLP and 454 pyrosequencing). The overall bacterial community richness was limited, but the community structure changed during the process. Zooming in on the LAB community using 454 pyrosequencing revealed a total of 47 species-level operational taxonomic units (OTUs). LAB diversity appeared relatively limited since 88% of the sequences were covered by the same five OTUs (representing members of Weissella, Lactobacillus and Leuconostoc) present in all samples investigated. Fluctuations in the relative abundances of the dominant LAB were observed with the process conditions. In addition, both the year of harvest and malting house influenced the LAB community structure.publisher: Elsevier articletitle: Bacterial community dynamics during industrial malting, with an emphasis on lactic acid bacteria journaltitle: Food Microbiology articlelink: http://dx.doi.org/10.1016/j.fm.2013.10.010 content_type: article copyright: Copyright © 2013 Elsevier Ltd. All rights reserved.status: publishe