Evaluation of non-volatile metabolites in beer stored at high temperature and utility as an accelerated method to predict flavour stability
Introduction
Flavour stability is vital to success in the brewing industry, as beer is stored, often for long periods of time and under variable conditions. Beer quality is known to be affected by storage conditions, as factors such as temperature and light can influence chemical processes related to taste, aroma, mouthfeel and appearance (Stewart, 2004, Vanderhaegen et al., 2006). Volatile compounds, such as aldehydes and sulphur-based compounds, are major contributors to an aged flavour profile (stale flavours, reviewed in (Vanderhaegen et al., 2006)). Additionally, non-volatile compounds, such as polyphenols (contribute to haze and astringent mouthfeel) and hop-derived iso-α-acids (bitter taste), can also vary during storage (Cooman et al., 2000, De Clippeleer et al., 2010, Mikyška et al., 2002).
Standard packaging and storage procedures (e.g., refrigeration, coloured glass bottles, and providing external carbonation) have been widely adopted in the beer industry to inhibit age-related flavour changes. Nevertheless, flavour stability is variable among beer types, and therefore manufacturers continue to refine brewing methods to inhibit flavour changes during storage. Examples of modifications to the brewing process that may improve beer flavour stability include the addition of antioxidants (e.g., use of glutathione (Gijs et al., 2004)) or oxygen-scavenging crown liners (Gohil and Wysock, 2014, Miltz and Perry, 2005)), chelation of pro-oxidant metals (Wietstock and Shellhammer, 2011, Zufall and Tyrell, 2008), and modified hopping schemes to affect polyphenolic and bitter acid content (Aron and Shellhammer, 2010, Karabín et al., 2014, Kunz et al., 2014).
In addition to modifying brewing techniques, there is a need to develop standard analytical procedures that can quickly and accurately measure small molecules and their association to flavour stability. For example, a method was described that estimated oxidative deterioration based on the trans/cis ratio of iso-α-acids (Araki, Takashio, & Shinotsuka, 2002), or volatile profiling combined with multivariate analysis (Rodrigues et al., 2011). These assays are direct measures of components that contribute to flavour; however, such methods require storing and testing the beer over several months for the analytical data to correlate with sensory changes in the beer. The need for analytical methods that can rapidly predict sensory shelf life are critical for brewers when developing new beers or making process changes to improve stability, evaluate new materials, or make process efficiency improvements. There are rapid predictive methods that measure individual components or classes of compounds (e.g., free radical formation measured by electron spin resonance spectroscopy). However, flavour is derived as an integration of many non-volatile and volatile compounds (e.g. aldehydes, bitter acids), and flavour stability is related to many types of chemical processes (e.g., oxidation, Maillard reactions). Therefore, measuring a single class of flavour compounds may fail to estimate the overall effect of brewing modifications to flavour.
Previously, we conducted a study that utilised a non-targeted metabolomics workflow encompassing ultra-performance liquid chromatography coupled to mass spectrometry (UPLC–MS) to profile the non-volatile metabolite content of beer stored cold or at room temperature (Heuberger et al., 2012). The putative non-volatile markers of aging have not been previously described to affect flavour, specifically the purines guanosine, guanine, deoxyadenosine, and 5-methylthioadenosine (5-MTA). Together, 5-MTA and other purines may act as indirect markers of beer quality. While these markers have potential utility for assessing overall flavour stability, the observed trends were evaluated over a traditional aging timeline of 16 weeks, which limits their application and advantage over sensory panel evaluation.
Here, we describe the development and validation of an accelerated aging model based on a purine marker. The method reduces the time required to evaluate flavour stability from 16 weeks to 3–5 days, thus enabling high throughput screening to evaluate the impact of experimental brewing techniques on flavour stability. To test the utility of the method, three brewing techniques that were hypothesised to improve the stability of two types of brews (an amber ale and an India pale ale) were evaluated. The amber ale was the same brew as previously reported (Heuberger et al., 2012) and an India pale ale was chosen to determine if similar aging trends are observed in a beer with very different flavour characteristics. Beer was aged under both traditional and accelerated time scales. Both sensory and molecular analysis was performed on beer stored in regular conditions (16 weeks), and non-volatile metabolite variation was measured in the beer aged under accelerated conditions. The data presented herein support that an accelerated aging model based on non-volatile molecular markers may expedite the evaluation of different brewing modifications to improve beer stability.
Section snippets
Brewing parameters and storage
Beer was brewed at New Belgium Brewing Company (Fort Collins, CO) to produce an amber ale (AA) or India pale ale (IPA) style beer. Modified brewing methods included (1) the use of antioxidant crowns (Pelliconi, Bologna, Italy) (2) late-hopping to reduce α-acid isomerisation and chelate iron (Wietstock & Shellhammer, 2011) and (3) adding hops with low (control), medium (MBhop1), or high plant content (MBhop2), determined by small, medium, or large particle size. Long-term cold storage (“regular
5-Methylthioadenosine is a marker for variation in flavour stability during regular storage
Three modified brewing (MB) methods were evaluated for an effect on flavour stability during regular storage (see Methods 2.1) on two beer types: an amber ale (AA) and an India pale ale (IPA). Different MB methods were used because AA and IPA contain distinct flavour profiles and an optimal MB method should reduce the effect on normal flavour characteristics of each beer-type. For the AA, antioxidant-containing crowns were used to inhibit oxidation products known to result in flavour
Acknowledgements
This work was supported by the New Belgium Brewing Company. We would like to thank members of the analytical and sensory team at New Belgium Brewing for providing materials and guidance critical to this investigation and manuscript.
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