Estudo químico de lúpulos cultivados em diferentes localidades por RMN e CLAE - EM/EM e desenvolvimento de um método eletroanalítico para quantificação de iso-α-ácidos em cervejas

Abstract

Hops (Humulus lupulus) are a key raw material in beer production, responsible for important sensory characteristics such as aroma and, notably, bitterness. During wort boiling, the α-acids present in hops undergo thermal isomerization to form iso-α-acids compounds directly linked to the beverage's bitterness intensity. In this study, commercial hop varieties were characterized using Nuclear Magnetic Resonance (NMR) and High-Performance Liquid Chromatography coupled with Mass Spectrometry. NMR analysis provided structural information via fingerprints, enabling the identification of major metabolites in the hop extracts, while HPLC-ESI-MS/MS analysis allowed for the annotation of the primary metabolites present in the samples. Specifically, for the Sabro hop variety, the α-acid mixture was separated yielding 31% of the hop extract and subsequently isomerized, resulting in a 42.5% yield of the iso-α-acid mixture. Structural confirmation was achieved through ¹³C NMR spectroscopy; α-acids exhibited a characteristic signal at δ 79.2 ppm, whereas the cis and trans isomers of the iso-α-acids showed signals in the δ 205.1–207.3 ppm range. Subsequently, an electrochemical study using a graphite sheet as the working electrode was conducted to investigate the behavior of iso-α-acids and their relationship to beer bitterness. The voltammetrics profiles obtained for α-acids and iso-α-acids displayed distinct potential regions; consequently, the analysis focused on iso-α-acids, as they are the primary contributors to the beverage's bitterness. Direct analysis of commercial beers revealed the presence of interfering compounds, such as sodium metabisulfite, necessitating sample pre-treatment via isooctane extraction. The efficiency of this procedure was monitored using HPLC-ESI-MS/MS, confirming the presence of iso-α-acids following the extraction step. Finally, bitterness values expressed in IBU were determined using Square Wave Voltammetry; the measured values (BS1 = 48.1; BS2 = 41.6; BS3 = 17.6) were close to those declared by the manufacturers (BS1 = 46; BS2 = 40; BS3 = 18). The results demonstrated that the developed electroanalytical method offers a higher-performance alternative for determining iso-α-acids and for future applications in beer quality control.