以液相層析質譜技術檢測進行酒花乾投之啤酒釀造過程中的苦味化合物

Abstract

啤酒花(Humulus lupulus L.)是釀造啤酒的基本原料之一，其中iso-humulones (iso-α-acids)和humulinones是主要苦味的來源。近年來許多酒廠會在啤酒發酵階段加入啤酒花以達到更具啤酒花特性的風味，也使得humulones (α-acids)產生更多不同的苦味化合物衍生物。在本實驗中，針對60種的啤酒花苦味化合物開發液相層析串聯質譜分析方法，以鹼性的甲酸銨溶液與甲醇/乙腈(70/30)作為層析條件能在25分鐘內將分析物分離。此方法線性範圍在0.053-3912 ng/mL之間，並且相關係數皆大於0.9938，iso-α-acids和humulinones的LOQ分別為0.26 ng/mL和0.053 ng/mL。本實驗也有良好的精密度(RSD< 0.5%)和準確度(recovery 86.3%-118.1%)。針對兩款啤酒Vienna Lager、Double-India Pale Ale在發酵的過程與儲存的過程進行監測，其定量結果顯示未經由酒花乾投(Dry-hopping)的Vienna Lager較傾向於產生質子催化、環化的的苦味衍生物，而有進行酒花乾投的DIPA較傾向由氧化產生的苦味衍生物。此結果也顯示釀造原料、釀造手法等對於啤酒花苦味化合物產生的傾向有不同的影響。Hops (Humulus lupulus L.) are essential raw materials for beer brewing, and the major contributors to beer bitterness are iso-humulones (iso-α-acids) and humulinones. In recent years, many breweries have focused on the production of hop-forward beer style by adding hops after or during the cold fermentation stage, which will tend to release humulinones or other hop-derived bitter compounds. In this study, a LC-MS/MS method was developed for quantification of 60 hop-derived bitter compounds in 25 minutes. A reverse phase chromatography with alkaline methanol/acetonitrile (70/30) mobile phase was used for the separation. The quantitative range were 0.053-3912 ng/mL with correlation coefficient r> 0.99, and the LOQ were 0.26 and 0.053 ng/mL for iso-α-acids and humulinones. Precision (RSD< 5.0%) and accuracy (recovery 86.3%-118.1%) were both satisfactory. The abundance of hop-derived bitter compounds in the dry-hopping beer Double-India Pale Ale and the non-dry-hopping beer (Vienna Lager) were monitored throughout the fermentation and storage stages, the formation of oxidation and cyclization products showed difference profiles between these two beers. The quantification results reveal how hop-derived bitter compounds change throughout the brewing process, as well as the influence of hops and brewing techniques on beer bitterness.