The global beer market is significant, but sales of standard alcoholic beer are declining due to health concerns and consumer preferences for reduced alcohol consumption. The non-alcoholic beer (NAB) market is expanding rapidly, driving research into improving NAB quality and sensory attributes. Flavor release is crucial to beverage enjoyment and depends on volatile and non-volatile components' chemical and physicochemical properties. Beer contains various volatile (alcohols, esters, acids, etc.) and non-volatile components (ethanol, proteins, polysaccharides, etc.), and their impact on flavor release is not fully understood. Ethanol plays a crucial role in beer's sensory properties. Previous research using trained sensory panels or static headspace techniques showed ethanol's impact on aroma and flavor, but these methods lack the dynamic aspects of real-life consumption (saliva mixing, mastication, etc.). Understanding the dynamic changes in flavor release during oral processing, particularly the role of saliva, is vital for developing more palatable NABs. This study uses a novel combined approach of sensory evaluation, headspace analysis, and macromolecular hydrodynamics to explore the differences in aroma release and flavor perception between 0% and 5% ABV beers, considering the influence of the beer matrix (lager and stout).

Prior studies investigated ethanol's impact on beer sensory properties, with some finding enhanced mouthfeel, sweetness, and complexity at higher alcohol content. Others focused on the effect of ethanol concentration on the retention of specific aroma compounds, like aldehydes, influencing off-flavors. Static headspace techniques, like SPME-GC-MS, generally reveal a decrease in headspace concentration of aroma compounds with increasing ethanol concentration due to enhanced solubility in the beer matrix. However, static headspace methods don't fully capture the dynamic aspects of consumption. Research on the bolus (food in the mouth mixed with saliva) and its role in flavor release is growing, emphasizing saliva's influence through interactions with aroma molecules. Saliva's complex protein composition (α-amylase, mucins, proline-rich proteins) impacts viscoelasticity and aroma compound binding. Most studies on beer examine ethanol's impact on individual aroma compounds in simple solutions rather than the complex beer matrix. This research addresses the gap by studying flavor interactions during oral processing in real beer matrices, particularly NABs, driven by the increase in NAB sales and the need for improvements in palatability.

This study employed a combined approach using consumer sensory evaluation, headspace aroma analysis (GC-MS), and macromolecular hydrodynamics. **Consumer Sensory Analysis:** 101 beer consumers (53 men, 48 women) evaluated 0% and 5% ABV lager samples. Orthonasal aroma was assessed using Check-All-That-Apply (CATA), while retronasal perception (flavor, taste, mouthfeel) was evaluated using Temporal Check-All-That-Apply (TCATA) over 60 seconds. Statistical analysis involved Cochran’s Q test (CATA) and two-factor ANOVA with Tukey's HSD post-hoc test (TCATA). **Physiochemical Analysis:** 0% and 5% ABV lager and stout beers were prepared. Volatile compounds (aldehydes, esters, alcohols) were added to ensure adequate signal for GC-MS analysis. Samples with and without α-amylase were analyzed to simulate saliva effects. SPME-GC-MS was used for headspace analysis. Statistical analyses included one-way ANOVA and Tukey's post-hoc test to compare the effect of ethanol and α-amylase, and two-way ANOVA with Tukey's post-hoc test and Pearson's correlation for interactions. **Hydrodynamic Analysis:** The effect of ethanol on α-amylase conformation was investigated using analytical ultracentrifugation to measure sedimentation coefficient and Ostwald viscometry to determine intrinsic viscosity. The Scheraga-Mandelkern equation and the ELLIPS program were used to calculate the shape of α-amylase at different ethanol concentrations.

Consumer sensory evaluation showed no significant differences in orthonasal aroma perception between 0% and 5% ABV lager. Retronasal TCATA analysis, however, revealed significant differences: 0% lager was perceived as significantly maltier and less fruity, sweet, full-bodied, and with reduced alcohol warming sensation (p<0.05). GC-MS analysis of headspace aroma compounds revealed that ethanol (5% ABV) significantly reduced headspace intensity in both lager and stout styles compared to 0% ABV controls (p<0.05). The stout, with its higher macromolecular content, showed lower aroma release than the lager. The presence of α-amylase decreased aroma release, especially for hydrophobic compounds. The relative proportion of hydrophobic aroma compounds was higher in 5% ABV beers and lower in 0% ABV beers; this logP-dependent effect was observed in both beer styles. Hydrodynamic analysis showed that ethanol affected α-amylase conformation, shifting from globular to elongated structures at higher ethanol concentrations. This suggests that ethanol denatures α-amylase, reducing its capacity to bind hydrophobic aroma compounds and influencing the perceived flavor profile.

The findings demonstrate that orthonasal aroma alone is insufficient to distinguish between 0% and 5% ABV beers. Retronasal perception, heavily influenced by saliva interactions, reveals significant differences. The shift towards more hydrophilic compounds in 0% ABV beer and more hydrophobic compounds in 5% ABV beer aligns with the observed effects of α-amylase and its interaction with ethanol. Ethanol's denaturation of α-amylase leads to conformational changes, reducing its ability to bind hydrophobic aroma compounds. This interaction explains the observed differences in sensory attributes, especially the increased maltiness and reduced fruitiness in the 0% ABV beer. The results suggest that product reformulation requires a holistic approach considering both physiochemical and sensory data, recognizing the significant role of saliva in flavor perception.

This study used a multi-faceted approach to understand ethanol's role in beer flavor, highlighting the importance of considering both sensory perception and physiochemical interactions, particularly the effect of ethanol and saliva on α-amylase. The findings demonstrate that ethanol's function is not simply about solubility but also involves complex interactions with salivary proteins. Future work should investigate the roles of other salivary components (mucins, PRPs) and explore the potential of 'ethanol-mimics' or mucoadhesives for NAB reformulation.

The study focused on two specific beer styles (lager and stout) and a limited range of aroma compounds. While α-amylase was used as a model for saliva proteins, the complexity of saliva may warrant further investigation using more realistic saliva models. The consumer panel, although substantial, might not represent the full diversity of consumer preferences.