2019 Project Grant
University of Leuven, Belgium
Development of a Rapid Method for the Quantitative Detection of Hydrophobins in Barley and Malt
‘Gushing’, a process in which a carbonated beverage intensively over-foams upon opening the container (without previous shaking), is responsible for significant economic as well as reputational damage of maltings and breweries. It can be caused by two fundamentally different factors: the presence of small surface-active proteins, mainly hydrophobins, in malt (‘primary gushing’) or by careless brewing practices resulting in suspended particles (‘secondary gushing’)1. While secondary gushing is easily avoidable by good brewing practices, hydrophobins on malts are not easily detectable and therefore lead to major problems in industry. Hydrophobins are small (7–10 kDa), surface-active proteins which are produced by fungal species, mostly filamentous fungi, which infect cereals on the field. More specifically, F. graminearum, F. culmorum and F. tricinctum were identified to be the primary drivers of primary gushing2. While several studies have tried to reduce Fusarium infections in the field, all these strategies have their own downsides, either technical or economical, because of which hydrophobin-free malt can never be guaranteed3. The current method for hydrophobin detection in barley lots is the ‘Modified Carlsberg Test’, standardized by MEBAK. Unfortunately, this test takes several days, and generally has a low signal-to-noise ratio4. Hence, a strategy to directly detect hydrophobins is necessary. Cutting-edge techniques, e.g. HPLC/MS/MS methods, were suggested, but these are difficult to implement in the workflow of a malting or brewery.
1. Gjertsen et al. (1967) Brew. Dig. 2. Virkajärvi et al. (2017) JASBC 3. Postulkova et al. (2016) Trends Food Sci. Technol 4. Haikara et al. (2005) JIB.
Principal Investigator
Dr. Kevin Verstrepen
Project Objectives
As the complete avoidance of hydrophobins in malt is shown to be inevitable, there is an urgent but unmet need to directly detect hydrophobins in barley and malt. Therefore, this interdisciplinary, application-driven project has a general research objective to develop a rapid, reliable and cheap detection method which is easily implementable in a brewery or a malting. We will use state-of-the-art (bio)technological approaches to ultimately develop a sensor that is: - Quick (turnaround time of <15 min) - Simple (no high-tech equipment necessary) - Cheap (< $30 / sample) - Easy to operate - Quantitative - Accurate: o No false positives (= limited noise) o No false negative (= it covers a broad spectrum of hydrophobins) To reach this ambitious goal, we will pursue the following specific objectives: (1) Production and purification of hydrophobins of the three main gushing-related fungal species using Pichia pastoris as a protein production host (2) Obtain monoclonal antibodies (mAbs) that are highly specific for hydrophobins (3) Use these antibodies to develop an indirect competitive enzyme-linked immunosorbent assay (icELISA) test (4) Validation of the detection method in breweries and maltsters Although the main goal of this project is very application-driven, it will also provide fundamental knowledge on hydrophobin-related gushing. Moreover, the constructs and strains developed in WP1 (which we will distribute free of charge to the scientific community) will be a great tool for all researchers studying gushing. Indeed, efficiently obtaining high-quality, pure hydrophobins from all main gushing-related fungi is pivotal for any gushing-related study, but is notoriously difficult to acquire using Fusarium spp.
Project Final Report
As a sponsorship benefit, a final report will be submitted in the fall of 2020 to the ASBC Research Council.
