Exploring aldehyde release in beer by 4-vinylpyridine and the effect of cysteine addition on the beer's pool of bound aldehydes

As a continuation of our previous work, which concerned the binding of aldehydes to bisulfite and cysteine, this article presents more results on the applicability of 4-vinylpyridine addition to beer prior to analysis aiming at release of aldehydes from these preformed bound states, thus making them quantifiable with the headspace solid-phase microextraction method combined with gas chromatography-mass spectrometry. This article also presents the first results on spiking beer samples with cysteine prior to forced-aging, pointing to the important role of cysteine in beer flavor stability. Both the levels in free and bound aldehydes show a relatively large degree of variation among different beers, but also between marker aldehydes. For some aldehydes (e.g., hexanal), the bound amount was shown to increase rather strongly upon forced beer aging, whereas for others (e.g., 2-methylpropanal), large amounts appeared to already be present in a bound state in the fresh samples. Spiking beer samples with cysteine prior to aging significantly lowered the aldehyde levels compared with nonspiked samples. Flavor stability of the cysteine-spiked beers was thereby greatly improved through aldehyde-cysteine adduct formation. It was further hypothesized that, in addition to efficient binding of aldehydes, cysteine also inhibits the formation of furfural during beer aging from Maillard reaction intermediates

On the contribution of malt quality and the malting process to the formation of beer staling aldehydes: a review

Despite decades of extensive research, beer flavour instability remains a challenge for both brewing and malting industries. Malt impacts the brewing process as well as the quality of the final beer. It also affects the stability of beer flavour, as it delivers to the brewing process various compounds with the potential to compromise the desired flavour characteristics of beer. These include staling aldehydes and their precursors, such as amino acids, reducing sugars, α-dicarbonyls and bound-state aldehydes. In general, the content of these compounds depends on barley variety and quality, the malting regime and final malt quality. Malt that represents a low potential for beer staling, i.e. that has low values of Kolbach Index, heat load, colour, LOX activity, Strecker aldehydes, transition metal ions and high antioxidative activity, leads to beer with enhanced flavour stability. However, the consistent production of malt with the desired quality remains challenging. Approaches to achieve this include adjustment of steeping and germination conditions, allowing control of grain modification and thus, the reservoir of aldehydes precursors. Also, the application of alternative kilning technologies may reduce the applied heat load, responsible for the formation of staling aldehydes and triggering development of the oxidising free radical species. This review provides an evaluation of current knowledge on the contribution of the malting process and malt quality to the formation of beer staling aldehydes. © 2021 The Authors. Journal of the Institute of Brewing published by John Wiley & Sons Ltd on behalf of The Institute of Brewing & Distilling

Unravelling formation and/or release of beer staling aldehydes

status: publishe

Beer flavour instability: Unravelling formation and/or release of staling aldehydes

The sensory perception of food and beverages is expected to be pleasant, characteristic, and consistent at the moment of consumption. However, in particular, beer has a limited shelf life because of flavour deterioration during transport and storage. All beers are subject to so-called flavour instability: the instability of beer in terms of odour, aroma, taste and mouthfeel. Beer flavour instability is very important from an economic point of view as the brewing industry represents a gigantic market. Therefore, more specifically, a prolonged flavour stability is essential, as it may be critical for acceptance or rejection of the product by the consumer. Flavour instability of beer is characterized by physical and chemical changes over beer storage. Two phenomena are considered to be of utmost importance with respect to the ageing of beer: a decrease in the intensity and the quality of the typical beer bitterness and, an increase in the levels of ageing aldehydes (so-called beer staling aldehydes). This PhD is focused on the second issue, mainly the generation of staling aldehydes as a function of beer ageing. Aldehydes might be formed (in the beer bottle but equally well during the brewing process) de novo from precursors. In particular, three major routes for de novo formation of aldehydes have been proposed: I) fatty acid oxidation (resulting in e.g. trans-2-nonenal), II) Strecker degradation of amino acids (resulting in e.g. 2‑methylpropanal), and III) the Maillard reaction (resulting in e.g. furfural). Alternatively, the formation of non-volatile bound-state aldehydes during the malting and/or brewing process has been proposed as a potential pathway to explain increases in staling aldehydes during beer ageing. Indeed, pre-formed non-volatile bound‑state aldehydes may survive the brewing and fermentation processes, thereby ending up in the final beer and, via subsequent decomposition result in the release of aldehydes during beer ageing. Several previous studies suggested that bound-state aldehydes might be formed through reaction of aldehydes with bisulphite during fermentation or through reaction of aldehydes with amino acids, peptides, or proteins to form imines during malting and brewing. In addition, very recent work pointed to the bound form of aldehydes with cysteine, so-called cysteinylated aldehydes or 2-substituted 1,3-thiazolidine-4-carboxylic acids, as potentially responsible for the beer staling problem. The major objective of this PhD is to better understand the binding/release behaviour of the most relevant beer staling aldehydes and to further investigate the potential role of aldehyde adducts with cysteine and bisulphite in relation to beer ageing. The most important marker aldehydes to be considered in beer ageing are 2-methylpropanal, 2‑methylbutanal, 3-methylbutanal, methional, phenylacetaldehyde, furfural, hexanal, and trans‑2‑nonenal. Therefore, in the first stage of this PhD, from these particular aldehydes all adducts with cysteine and bisulphite, respectively, were synthesized. Structure verification of aldehyde adducts was obtained via NMR (1H-NMR and 13C-NMR) and high mass accuracy UHPLC-MS. Moreover, purity was assessed by high mass accuracy UHPLC-MS. Upon successful synthesis of all bound-state aldehydes, these compounds were used throughout the PhD as new reference material in order to be able to conduct all subsequent studies. To this end, first of all, a methodology to reliably quantify both free and bound-state aldehydes, based on HS-SPME-GC-MS and UHPLC-MS, respectively, was implemented. Validation comprised linearity, accuracy, limits of detection (LOD) and quantification (LOQ), and demonstrated that the methodologies to analyse both, volatiles and non‑volatiles, are appropriate for quantification purposes within the desired concentration range (µg/L) and, thus, represent a truly integrated analytical GC-MS/UHPLC-MS methodology. Upon synthesizing the most relevant adducts of beer staling aldehydes and establishing an optimized analytical methodology, these tools were then applied in studies on (1) model solutions and (2) real malting and brewery samples. In model solutions, the influence of pH and temperature on the behaviour of cysteinylated aldehydes was investigated. Free and cysteine-bound aldehydes were analysed via HS‑SPME‑GC‑MS and UPLC‑PDA, respectively. To monitor the behaviour of cysteinylated aldehydes under malting and brewery relevant conditions in model solutions over a time frame of 24 hours, pH values of 2.0, 4.4, 5.2, 6.0, and 9.0 and temperatures of 0 ºC, 20 ºC and 40 ºC were selected. Cysteinylated aldehydes showed degradation and concomitant release of corresponding free aldehydes at the pH values 4.4, 5.2 and 6.0, that are related to malting and brewing. Furthermore, stability of the adducts was observed at alkaline pH. A higher degradation rate was noticed at 40 ºC compared to 0 ºC. In summary, our study in model solutions points to the potential importance of cysteinylated aldehydes as a possible source of staling aldehydes during beer storage. Following the work in model solutions, the raw material malt, intermediate brewery samples, final beer and forced aged beer were analysed for both free and bound-state aldehydes by applying the integrated methodology developed previously. Free aldehydes were quantified in all tested samples (from malt to beer). Cysteinylated aldehydes were detected from malt until the start or end of wort boiling, depending on the nature of the aldehyde. However, bisulphite‑bound aldehydes were not found in any of the samples. Malt contained all free and cysteinylated aldehydes under study. Analysis of samples taken at the onset of the mash compared to malt samples, showed the highest levels of cysteinylated aldehydes at mashing‑in and highest levels of free aldehydes in malt, pointing to additional formation (next to formation in malting) of cysteine‑aldehyde adducts at mashing-in. Furthermore, decreasing concentrations of both free and cysteine-bound aldehydes were observed throughout the brewing process. An exception was found however for the free aldehyde furfural which increased in levels when strong heat-load was applied, i.e. during wort boiling and wort clarification. In fresh beer, only traces of free aldehydes were detected. However, levels in free aldehydes increased during beer ageing, in particular for 2‑methylpropanal and furfural. In contrast, cysteinylated aldehydes were not quantifiable in fresh beer samples, neither during beer ageing, with the exception of the cysteinylated form of 2‑methylpropanal. Although these findings do not designate cysteine‑ or bisulphite‑bound aldehydes as being directly responsible for beer flavour deterioration in the beer matrix, they add evidence to the concept of bound‑state aldehydes, and therefore to further consider and investigate the potential contribution of bound-state aldehydes in relation to flavour instability of food and beverages. Consequently, in the final experimental part of the PhD, to further investigate potential formation of bound-state aldehydes in the beer matrix, labelled aldehydes, i.e. 2‑methylbutanal-d3 and furfural-d3, were added in fresh commercial pale lager beer. Binding of the selected deuterated aldehydes could however not be demonstrated. Moreover, release of deuterated aldehydes upon beer ageing was also not detected. Lastly, an initial study of de novo formation of aldehydes from amino acids was performed in the beer matrix. To this end, the labelled amino acids valine-d8 and leucine-d3 were added to a fresh commercial pale lager beer and potential formation of deuterated aldehydes was monitored during beer ageing up to 3 months at 30 ºC. Increasing concentrations of both corresponding deuterated free aldehydes (2-methylpropanal and 3-methylbutanal) were found over ageing. This finding represents the first unambiguous evidence of de novo formation of aldehydes from amino acids in the beer matrix.status: accepte

UNDERSTANDING THE BEHAVIOUR OF CYSTEINE ADDUCTS OF BEER STALING ALDEHYDES IN MODEL SOLUTIONS

status: publishe

Influence of pH on the stability of 2-substituted 1,3-thiazolidine-4-carboxylic acids in model solutions

Upon beer storage, the levels of staling aldehydes increase, which coincides with the appearance of characteristic off-flavors. Bound-state aldehydes have been reported as potential sources of off-flavor appearance in aging beer. This study investigated the stability of cysteinylated aldehydes, also referred to as 2-substituted 1,3-thiazolidine-4-carboxylic adds, and the release of aldehyde compounds from cysteine adducts in model solutions at different pH values that are relevant in malting and brewing. The cysteinylated aldehydes were initially synthesized for their use as reference compounds in model solutions. Confirmation of their chemical structure was obtained by H-1-NMR. The results from the stability tests showed that degradation of cysteine-bound aldehydes results in concomitant release of the free aldehydes. The rate of degradation is highly dependent on the 2-substitution pattern of the thiazolidine ring, as well as on the pH of the model solution. At malting and brewing relevant pH values (pH 4.4, 5.2, 6.0), degradation of cysteine-bound aldehydes is observed, in particular at pH 4.4, which is representative of the beer pH

First time quantification of cysteinylated aldehydes in malt and brewery samples

Beer flavour is not stable during storage. The mechanisms of formation of aldehydes, which are considered as a major cause of off-flavours during beer aging, remain uncertain. Increase in aldehydes by de novo formation or the release from bound-state precursors have been suggested as the cause of aldehyde appearance. In a previous study, we proposed the bound form between cysteine and aldehydes, also called cysteine-bound aldehydes or 2-substituted 1,3-thiazolidine-4-carboxylic acids, as potential sources of the increasing aldehyde levels found during beer aging. In this study, cysteine-bound aldehydes of selected marker aldehydes were synthesized and used as reference compounds. Implementation of the methodology for the detection and quantification of these compounds was done by Liquid Chromatography-Mass Spectrometry (LC-MS). Malt samples and samples collected at different points during the brewing process (from the onset of mashing until the fresh beer, as well as during beer aging) were analysed for their free and cysteine-bound aldehydes content using HS-SPME-GC-MS (Headspace-Solid Phase Microextraction-Gas Chromatography-Mass Spectrometry) and LC-MS (Liquid Chromatography-Mass Spectrometry), respectively. In malt, we were able to quantify all selected aldehyde markers, both free and cysteine-bound. Furthermore, quantification of free aldehydes was possible in all brewing samples, whereas cysteine-bound aldehydes detection and quantification was dependent on the nature of the 2-substitution in the thiazolidine ring. In conclusion, even though cysteine-bound aldehydes were not identified unambiguously in the final beer, our experimental data support the general concept of bound-state aldehydes as potential contributors to increasing aldehyde levels and, therefore, to beer flavour deterioration

Validation of an ultra-high-performance liquid chromatography-mass spectrometry method for the quantification of cysteinylated aldehydes and application to malt and beer samples

This paper describes the method validation for the simultaneous determination of seven cysteinylated aldehydes, i.e. 2-substituted 1,3-thiazolidines-4-carboxylic acids, using ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS). Authentic reference compounds were first synthesized for identification and quantification purposes. Moreover, nuclear magnetic resonance (1H NMR and 13C NMR) was applied for verification of their structure, while ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS) was applied for estimation of the purity. The method for quantification of cysteinylated aldehydes in model solutions has been validated according to the criteria and procedures described in international standards. The synthesized compounds were successfully identified via UHPLC-MS by comparing retention time and MS spectra with the commercial reference compounds. Method validation revealed good linearity (R2 > 0.995) over the range of 0.4-2.2 µg/L to approximately 1000 µg/L, depending on the analyte. The limits of quantification varied from 0.9 to 4.3 µg/L depending on the nature of the compound. Furthermore, evaluation of the method showed good accuracy and stability of the standard solutions. Reported chromatographic recoveries ranged from 112 to 120%. Consequently, the currently described method was applied on malt and beer samples. For the first time, quantification of cysteinylated aldehydes was obtained in malt. In contrast, in fresh beers unambiguous identification of these compounds was not achieved.status: Published onlin