Contents lists available at ScienceDirect Journal of Cereal Science journal homepage: www.elsevier.com/locate/jcs Development of an automatable method for the measurement of endo-1,4-β- xylanase activity in barley malt and initial investigation into the relationship between endo-1,4-β-xylanase activity and wort viscosity David Mangan a,* , Claudio Cornaggia a , Agnija Liadova a , Anna Draga a , Ruth Ivory a , D. Evan Evans b , Barry V. McCleary a a Megazyme u.c., IDA Business Park, Southern Cross Road, Bray, Co, Wicklow, Ireland b The Tassie Beer Dr Consulting, 15 Rianna Road, Lindisfarne, Tasmania, 7015, Australia ARTICLE INFO Keywords: endo-1,4-β-xylanase Activity assay Barley malt Wort viscosity ABSTRACT Stages of the brewing process, such as mash separation to produce wort and beer filtration, can in certain cases prove problematic due to the increased viscosity caused by high levels of the non-starch polysaccharides, pri- marily β-glucan and arabinoxylan. Of these two polysaccharides, β-glucan has been extensively studied, but arabinoxylan has been somewhat overlooked. The concentration of arabinoxylan present during these process stages is principally expected to be inversely related to the malt endo-1,4-β-xylansase activity that is available to degrade these polysaccharides. The development of a novel method for the measurement of endo-1,4-β-xylansase activity in barley malt extracts is described herein. The method was characterised by two analysts in terms of repeatability (single analyst CVs = 2.2% and 2.3%, n = 8; interanalyst CV = 4.8%, n = 16) and sensitivity (LOD = 10 U/kg, LOQ = 34 U/kg). The assay procedure was then applied to the measurement of xylanase activity in a series of eight standard barley malts and the results obtained were compared with their associated Congress wort viscosities as measured using the conventional EBC Method 4.8, wort viscosity. A highly statis- tically significant relationship between xylanase activity and wort viscosity was found with a Pearson's corre- lation coefficient of -0.82 (p-value of 0.007). 1. Introduction In 2014, Hordeum vulgare L. (malting barley) was ranked 4th in cereal grains in terms of global production at 144 million tonnes (Food and Agriculture Organization of the United Nations (FAOSTAT), 2014). The majority of this usage is in the brewing industry where malted barley is one of the key ingredients for the production of beer or whiskey. In barley, the cell walls of the aleurone tissue are constituted primarily of arabinoxylan (Bacic and Stone, 1981) while the starchy endosperm cell walls are composed of ∼20% arabinoxylan with ∼75% of the remainder being made up of mixed linkage (1,3; 1,4)-β-glucan (Fincher, 1975). Traditionally, the brewing fitration issues associated with β-glucan have been more widely studied in brewing science (Narziss, 1993). However, the importance of arabinoxylan has also been demonstrated previously with respect to production issues such as poor mash separation, beer filterability and haze formation (Barrett et al., 1975; Coote and Kirsop, 1976; Li et al., 2015; Lu and Li, 2006). As endo-1,4-β-xylanase (xylanase, EC 3.2.1.8) is known to readily degrade the arabinoxylan present in barley (Debyser et al., 1997; Viëtor et al., 1994), it clearly has the potential to significantly effect both wort composition and also the processing efficiency and quality of the fin- ished beer product. A number of isoforms of barley xylanase have been purified pre- viously and characterised (Benjavongkulchai and Spencer, 1986; Dashek and Chrispeels, 1977; Kanauchi et al., 2013; Slade et al., 1989). Reported molecular weights range from ∼29,000–67,000. Isoelectric points have been observed between 4.6 and 6.5, with a pH optima appearing to vary between ∼5.0–6.7. However, all previous studies seem to be in agreement that the predominant forms exhibit a broad pH range with > 75% activity maintained from approximately pH 4.5–7. It had been reported that the largest of these enzymes (Mr = 61500) was an inactive form that needed to be processed by endogenous proteolytic machinery to produce active forms with lower molecular weight (Mr = 34,000 and 41,000) (Caspers et al., 2001), but in fact the larger isoform was later shown to be active following recombinant expression by Van Campenhout et al. (2007), a conclusion that has been https://doi.org/10.1016/j.jcs.2018.10.003 Received 24 August 2018; Received in revised form 5 October 2018; Accepted 12 October 2018 * Corresponding author. E-mail address: David@megazyme.com (D. Mangan). Journal of Cereal Science 84 (2018) 90–94 Available online 13 October 2018 0733-5210/ © 2018 Elsevier Ltd. All rights reserved. T