Biotechnological Modification of Cider Brewing Processes for the Enhanced Production of 2-Phenylethanol

Consumers of Australian cider are currently trending towards higher-quality cider products. As a result, boutique and craft cider breweries are expected to experience a period of growth over the next five years. Supporting this trend and subsequent growth is paramount to rebuilding the cider industry post-COVID-19. Many current practices and procedures, such as must clarification and biomass reduction in cider brewing, have been adapted from the beer and wine industry. While these practices are beneficial to the quality of cider and often promote the production of favourable volatile organic compounds (VOCs), the targeted enhancement of specific VOCs has not been achieved. This work investigates the specific enhancement of 2-phenylethanol (2-PE), which is known to improve the organoleptic properties of cider and provide potential health benefits through its antioxidant properties. The effect of three levels of biomass reduction (90%, 80%, and 0%) and five levels of L-phenylalanine (L-phe) saturation (0.5, 1.0, 1.5, 2.0, and 2.5 g L⁻¹) for the enhanced production of 2-PE during cider fermentation were investigated. A high-performance liquid chromatography method was developed to accurately quantify the concentration of both 2-PE and L-phe, with a root-mean-square deviation (RSMD) of 0.41% and 1.60%, respectively. A significant increase in 2-PE production was achieved for all treatments, with 2-PE levels up to two orders of magnitude higher than respective controls. The highest 2-PE production was achieved by a moderate (80%) biomass reduction at a 2.5 g L⁻¹ L-phe spike. Additionally, the VOC profile of several of the ciders was quantitively determined, and subsequent data underwent extensive chemometric analysis. Principle component analysis (PCA) showed that 2-PE and its derivatives (2-phenylethyl pivalate and phenylacetaldehyde) were correlated with the 80% biomass reduction treatment at the highest L-phe spike. Additionally, it was observed that several acids and alkanes were negatively correlated with the production of 2-PE and its derivatives. Additionally, hierarchical cluster analysis (HCA) showed clustering between the 80% and 90% biomass reduction treatments at several L-phe spike concentrations. However, the 0% biomass reduction treatments only showed similarity with other treatments with 0% biomass reduction. This work provides insight into the production of 2-PE during apple cider fermentation while building the foundation for more targeted biotechnological production of favourable compounds to improve cider quality.