Author:

Keywords:

influence of hop bitter acids on storage-induced appearance of aldehydes in beer, aldehyde formation and the role of hops in beer flavour instability

Abstract:

Beer is often distributed and stored at room temperature, at which stability in terms of brightness and foam is largely under control in contrast to the ‘flavour’ (combination of odour, aroma, taste and mouthfeel). Already in the early stages of storage of virtually all bottled beers, especially lagers, the disappearance of pleasant fresh beer flavour characteristics is associated with the accelerated development of unwanted ageing flavours. Given the increasing importance of export and the ever intensifying competition in the international market, a prolonged stability of the beer flavour is however a prerequisite. The distribution and particularly the export of beers can then be broadened while consumers are minimally confronted with changes in flavour and drinkability, even upon prolonged storage time prior to consumption. An optimised flavour stability is equally important for beers intended for the domestic market, as it is the best guarantee for maintaining the drinkability, which is with a direct impact on consumption very important in the light of a declining pale lager beer consumption. Some brewers do in fact operate a cold-chain, expensive though it may be. Consequently, prolonging the flavour stability of beer remains a major concern for each modern brewery.With their off-flavour character and very low thresholds, aldehydes play a key role in the sensory perception of aged beer flavours, since their concentration increase, even at μg/L levels, coincides with the appearance and intensity of organoleptic defects. Several reaction pathways give rise to these ageing compounds during beer production and storage, but knowledge on their proportional role is far from complete.Next to the significant decline in beer bitterness, the decomposition of in particular trans-iso-alfa-acids into a series of non-volatile cyclic compounds contributes to a harsh lingering bitterness upon ageing. The flavour shelf life of beers brewed with a strongly reduced level of trans-isomers can thus be extended with regard to bitterness intensity and quality. However, the impact that the iso-alfa-acids degradation has on the formation of ageing aldehydes is currently only exploited to a very limited extent. It remains unclear whether ageing aldehydes would develop less in beers with enhanced levels of cis-iso-alfa-acids, since the cis-isomers are distinctly less susceptible to degradation compared to their trans-counterparts.Against this background, this doctoral study aimed to generate new insights into the relationship between iso-alfa-acids degradation and the formation of ageing aldehydes, with focus on the marked instability of trans-iso-alfa-acids, in order to determine critical factors related to wort production and hopping for an improved beer flavour stability.A number of aldehydes were selected as beer flavour instability markers, i.e. 2-methylpropanal, 2-methylbutanal, 3-methylbutanal, methional, benzaldehyde, phenylacetaldehyde, furfural, hexanaland trans-2-nonenal. Accurate analytical measurement of these aldehydes in a complex matrix such as beer requires highly specific extraction and detection techniques to obtain the necessary sensitivity and selectivity. Based on the properties of the compounds of interest, headspace solid phase microextraction with on-fibre O-(2,3,4,5,6-pentafluorobenzyl)-hydroxylamine (PFBHA) derivatisation in combination with capillary gas chromatography-negative chemical ionisation mass spectrometry (CGC-NCI-MS) was the technique of choice in this PhD study. The unique characteristics of the NCI spectra of aldehyde PFBHA-oxime derivatives provided excellent selectivity and sensitivity for measurement of the selected marker aldehydes, even when present at trace levels. The developed methodology was successfully applied to study the formation of aldehydes in fresh and forced aged beers.Given the pronounced instability of trans-iso-alfa-acids compared to their cis-counterparts, the evaluation of their potential specific impact on the formation of ageing aldehydes is necessary. Therefore both trans- and cis-iso-alfa-acids were required in their pure form and separated from a commercial isomerised hop extract by formation of trans-iso-alfa-acids/ß-cyclodextrin (ß-CD) complexes. The implemented preparative-scale separation procedure yielded the required amounts of isolated trans- and cis-iso-alfa-acids in a purity of 94% and 98%, respectively, for addition to model solutions and repeated trials of beer.The possible formation of ageing aldehydes arising from iso-alfa-acids degradation upon dark storage, was initially investigated in model solutions containing bitter acids or/and amino acids or/and xylose. In this way, the huge complexity of the beer matrix was originally evaded, allowing us to reveal the proportional contribution of each of the constituents to the formation of hop-derived aldehydes, Strecker aldehydes, and furfural, respectively. The involvement of different bitter acids (trans- vs. cis-iso-alfa-acids and iso-alfa-acids vs. dihydro- and tetrahydroiso-alfa-acids) in these reactions was evaluated, based upon their reported relative stability.The relationship between the bittering and the aldehyde formation was subsequently further explored in beer. In a comparative study, the impact of the bitter acids degradation on the aldehyde increase upon forced ageing in the dark was repeatedly studied in identically prepared pilot pale lager beers conventionally bittered (pellets, CO2-extract), bittered with iso-alfa-acids end boiling, and post-fermentation bittered with exclusively iso-alfa-acids, enriched trans-iso-alfa-acids, enriched cis-iso-alfa-acids, dihydroiso-alfa-acids and tetrahydroiso-alfa-acids, respectively, and compared with the aldehyde levels in the corresponding unhopped beers.For the first time, the differential behaviour upon ageing of trans- and cis-iso-alfa-acids was studied separately in model solution as well as in the beermatrix. Even though an increase in the aldehyde levels was monitored both in model solutions and aged beer samples, the suggested chemical pathways for aldehydes as degradation products of iso-alfa-acids were derived from the model experiments. The observed increase in the levels of marker aldehydes was only related to the conversion of iso-alfa-acids during storage in the model solutions. 2-Methylpropanal was identified as a volatile, direct degradation product of both cis- and trans-iso-alfa-acids without the involvement of amino acids and/or xylose in these model experiments. The conversion of both cis- and trans-iso-alfa-acids was unmistakably influenced by the oxygen levels of the model solutions. In the presence of amino acids, this led to the formation of 2-methylbutanal, 3-methylbutanal, methional, and phenylacetaldehyde. This has been reported for the first time, and is explained as a Strecker-like degradation of the amino acids by a non-volatile oxidation product of both cis- and trans-iso-alfa-acids, the hydroxy-allo-iso-alfa-acids. These alfa-unsaturated carbonyl compounds are postulated as reactants for the Strecker-like degradation of amino acids in model solution, delivering the corresponding Strecker aldehydes.However, the contribution of this reaction to the increase in Strecker aldehydes in the aged iso-bittered beers was found negligible. cis-Iso-alfa-acids showed increased stability in beer compared to the model solutions, indicating less oxidative degradation. Despite the fact that clear differences in the stability of the bitter compounds between the nine specific bittered beers were observed, it was shown that the increase in aldehydes is irrespective of the bittering. In unhopped beer in fact an at least equally high increase in aldehydes was observed upon storage, so that the presence and conversion of, in particular, trans-iso-alfa-acids in the beer matrix can not be considered as a critical factor for the formation of aldehydes during beer ageing. On the basis of beers made with another pale malt it was further demonstrated that the malt and wort production should be considered as critical factors for the flavour stability of beer, as deduced from the higher aldehyde levels in the fresh and aged beers.In conclusion, through a comparative study of the impact of different types of bitter acids on aldehyde formation upon beer storage, the opportunities of using highly enriched cis-iso-alfa-acids preparations in brewing practice to prolong beer flavour stability were explored. The results obtained in this study created attractive new insights into the degradation of iso-alfa-acids and proved that under oxidative conditions, both cis- and trans-iso-alfa-acids are a source of Strecker aldehydes through a postulated Strecker-like reaction between oxidative iso-alfa-acids degradation products, the hydroxy-allo-iso-alfa-acids, and amino acids. However, this reaction was of no or at least less importance in the beer matrixat low oxygen concentrations, since the aldehyde increase upon ageing was irrespective of the applied bittering. Still, enriched cis-iso-alfa-acids can be utilised in beer in view of an enhanced bitterness stability, in the absence of light and with low-oxygen bottling. Therefore, the upscaling of the enrichment of cis-iso-alfa-acids from a mixture of cis- and trans-iso-alfa-acids opens up new perspectives. Moreover, cis-iso-alfa-acids are more bitter than the corresponding trans-isomers, so for the same bitterness intensity, less cis-iso-alfa-acids will be needed, which also yields an additional economic advantage. This doctoral study argues for an improved quality control of the used malt with the aim of reducing the aldehyde content in fresh and aged beer, and suggests to the brewer some points to reduce aldehydes production during brewing and beer storage, such as mashing-in conditions, limiting heat load (rapid wort filtration) and oxygen-free bottling.