Desenvolvimento de métodos analíticos por técnicas de eletromigração, cromatográficas e espectroscópicas para detecção e determinação de α e β-ácidos, terpenos e ácidos graxos em amostras comerciais de Lúpulo (Humulus lupulus L.)

Abstract

Hop (Humulus lupulus L.) is a plant with a complex chemistry composition and the main hop application relies on brewing industry. In this work methodologies were developed to study the main hop metabolites that plays important roles in brewing process. The α-acids are precursors of the characteristic beer bitterness, the β-acids have bacteriostatic effects. Firstly, a methodology was optimized considering the extraction step followed by capillary zone electrophoresis analysis with ultraviolet detection, which enabled the separation and identification of four α and β-acids. From the electrophoretic profile of the different hop varieties, it was possible to classify aromatic and bitter hops without the use of analytical standards by employing principal component analysis. As a second step, it was optimized a method by cyclodextrin-modified micellar electrokinetic chromatography with ultraviolet detection, considering uni and multivariate approaches, to completely separate isomers and homologues of α and β-acids compounds, which occurred in a separation time of 7.8 minutes. The second metabolite studied was the terpene class. The hop essential oil is essentially formed by these compounds, which are responsible for beer aroma. They were identified by gas chromatography coupled to mass spectrometry, being employed a simple sample preparation by using a direct solvent extraction assisted by ultrasound. Eighteen terpenes, considering mono and sesquiterpenes, were identified. The different contents of thirteen terpenes were used to perform the chemometric modeling by principal component analysis and cluster analysis, which enabled the classification of different hop varieties, making possible differentiate aroma, bitter and dual purpose hops. Finally, fatty acids in hops were studied. These compounds are neglected in hops when compared to the aforementioned metabolites. They play important roles in brewing, as in fermentation step, however they may contributes to off flavors in beer. Different analytical methodologies were developed to determine fatty acids in hops. Fourteen fatty acids methyl esters were determined in thirty hop samples by gas chromatography with flame ionization detector after the lipid extraction carried out employing Folch and Stanley procedure and a base-catalyzed transesterification reaction. To the same hop sample, it was proposed the building of multivariate calibration models by proton nuclear magnetic resonance in association with partial least squares regression, with and without variable selection, to determine the fatty acids C16:0, C18:0, C18:1, C18:2, C20:1, C20:2 and C22:2. The results showed the matrix complexity, however, the approach is regarded as promising. Additionally, it was proposed an approach to determine the fatty acids C16:0, C18:0, C18:1 and C18:2 in hops by capillary zone electrophoresis with indirect ultraviolet detection. The sample preparation was optimized considering a direct sample saponification reaction and the method was properly validated taking into account the main validation parameters, such as: linearity, precision, accuracy, limits of detection and quantification and selectivity.