Optimizing hops gradient plates for assessing bacterial beer-spoilage potential

Microbiology Session
Barry Ziola, University of Saskatchewan, Saskatoon, Saskatchewan, Canada
Co-author(s): Barry Bushell and Vanessa Pittet, University of Saskatchewan, Saskatoon, SK, Canada

ABSTRACT: Important to the brewing industry is the ability to determine if bacteria present in post-filtered beer are capable of growing in (i.e., spoiling) the product. A major factor for bacterial growth in beer is hop resistance, which we have previously shown can be assessed with hop extract (iso-alpha-acids) infused agar (HGA) plates with a bitterness gradient ranging from 0 to 29 BU. To make hop agar gradient plates more applicable to a brewery setting, we assessed various parameters, including the steepness of the BU gradient, effect of the presence of ethanol, and stability of hop extract over time. HGA plating consists of pouring a dual layer agar plate such that the bottom layer containing the maximum BU concentration forms a 10.5 degree wedge of hop-infused agar, which is then covered by a top layer of hop-free agar. Diffusion from the bottom agar layer creates a hops gradient at the agar surface. Lines of bacterial cultures are stamped upon the surface, and the distance of growth indicates the BU level at which growth is inhibited. The effect of ethanol on hop resistance can be assessed by including ethanol in both agar layers. Gradient plates containing four different ranges of hop concentrations were tested (0–29 BU, 1×; 0–58 BU, 2×; 0–87 BU, 3×; 0–116 BU, 4×). In addition, to assess hop stability, two samples of the same commercial hop preparation separated by 5 years in storage at 4°C were tested. In this study, 26 isolates (22 Lactobacillus and 4 Pediococcus) were assessed. Of these, 10 grew completely through the 1× gradient plates, and hop resistance could not be scored. With 2× hop-gradient plates only four isolates could not be scored. Ultimately, all four could be scored (grew only partially along the hop gradient) using a 4× plate. With 2× plates containing 5% ethanol, three isolates showed increased hop resistance—two of which we had previously described as having ethanol-enhanced hop resistance. This increased hop resistance in the presence of ethanol was verified for one of the three isolates by growth trials in liquid medium. Lastly, no difference in antimicrobial efficacy of the two hop extracts was found. Based on these results, the use of 2× hop gradient agar plates (0–58 BU) is recommended for routinely assessing bacterial hop resistance, with highly hop-resistant isolates requiring a 4× gradient plate (0–116 BU). As well, properly stored hop extracts can be used over extended periods of time in hop gradient plates.

Barry Ziola received a B.S. degree (with honors) in botany from McGill University, Montreal, in 1970. After completing a Ph.D. degree in biochemistry at the University of Alberta, Edmonton, in 1975, he undertook a three-year post-doctoral stint at the University of Turku, Turku, Finland. He has been at the University of Saskatchewan, Saskatoon, since 1978, with promotion to professor coming in 1986. His interest and continuing research in brewing spoilage bacteria dates to the mid-1980s.