Characterisation of barley-associated bacteria and their impact on wort separation performance Arja Laitila, 1 * Jenny Manninen, 1 Outi Priha, 1 Katherine Smart, 2 Irina Tsitko 1 and Sue James 2 Wort separation is one of the rate-limiting steps in the brewhouse. It is a complex process, influenced by barley components such as proteins, β-glucans, residual starch and lipids. Filtration performance may also be influenced by microbial biofilms forming on the outer layers of the grains. This study aimed to identify the main barley-associated bacteria influencing wort separation effi- ciency. Next-generation sequencing was applied to characterise indigenous bacterial communities associated with Overture bar- ley from different geographical locations as well as the bacterial population dynamics during laboratory-scale malting. In order to study the weakened filtration performance potentially caused by induced bacterial biofilm formation, a small portion of barley (5–12%) was subjected to mild husk damage prior to steeping. The bacterial communities were dominated by Gammaproteobacteria, accounting for >70% of the total bacterial population. Bacterial growth induction significantly decreased wort filtration performance. A content of ~12% of injured grains decreased the rate of wort separation by up to 25%, with over 10% lower extract yields. This study showed that bacteria associated with barley are one of the key factors influencing wort sep- aration. © 2018 The Institute of Brewing & Distilling Keywords: barley; malt; sequencing; wort separation; bacteria; biofilm; microbial diversity Introduction Wort filtration performance is regulated by the permeability of the filter bed formed in the lautering process. This is dependent on the ingredients used in mashing. Barley is the main ingredient in beer production. Before brewing, hard barley kernels are modified by malting into friable malt. Malting is a complex process involving the synthesis of various barley-derived hydrolytic enzymes to degrade the endosperm wall and solubilise starch and storage proteins (1,2). A poor wort filtration performance with its origin in malting can be traced back to various components in malt. Filtra- tion performance is dependent on the quality of barley and malt and on the reactions occurring during the mashing process (1). Comparatively small differences in malt quality can cause prob- lems in a brewery. Sometimes the wort run-off becomes slow or even ceases as the grain bed clogs. The high β-glucan and arabinoxylan contents of poorly modified malts are known to increase the wort viscosity and negatively affect extract recovery (wort filterability) (3). Furthermore, the complexes formed between poorly degraded proteins, carbohydrates and lipids may also slow down the filtration process (1). In this case, the native microbiota of barley may produce polysaccharides, with consequent gelling and layer forming potential during malting. Our previous studies have indicated that malt quality and wort filtration performance are significantly influenced by the growth of indigenous microbiota during the malting process (4–6). However, it is not known which barley-associated bacteria could influence wort separation performance in good quality barley, or the extent of impact on mashing. Bacteria and fungi colonise the space between the testa and outer epidermis in barley grains, forming biofilm structures during malting (6). Depending on the nature, number of microbes and their metabolites, the consequences for malt and beer quality may be either deleterious or beneficial (5). During the malting pro- cess, the total viable count of microbes increases substantially in steeping and eventually reaches its maximum in the germination stage (7). The microbial dynamics also change during malting. The microbiota of barley is numerically dominated by Gram- negative bacteria (5,8). The initial microbial populations, and inter- actions between various microbial groups in malting and malthouse operations, have an impact on the microbiota in pro- duced malt (7,9,10). The several different layers found in the barley covering act as a carrier for microbes (6,11). In intact barley grain the nutrient rich starchy endosperm is protected by the testa layer from the inva- sion of microbes. However, harsh weather conditions during the grain filling period may cause the ripening barley kernel to swell and split. Such split barley kernels, with husk damage invisible to the naked eye but large enough for microbes to enter, might be overlooked during the cleaning and grading operations and con- sequently not be separated from the good batches. Kernels may also be injured during the transport, cleaning and grading opera- tions before steeping. Split kernels germinate normally in the malting process and their water uptake is usually even slightly ac- celerated (4,12). However, severe mash filtration difficulties have * Correspondence to: Arja Laitila, VTT Technical Research Centre of Finland Ltd, Espoo, Finland. E-mail: arja.laitila@vtt.fi 1 VTT Technical Research Centre of Finland Ltd, Espoo, Finland 2 SABMiller Ltd, ABInBev House, Woking, UK J. Inst. Brew. 2018; 124: 314–324 © 2018 The Institute of Brewing & Distilling Research article Received: 13 November 2017 Revised: 25 March 2018 Accepted: 28 May 2018 Published online in Wiley Online Library: 26 July 2018 (wileyonlinelibrary.com) DOI 10.1002/jib.509 314