One of the odors in deteriorated beer is the cardboard odor, and trans-2-nonenal (T2N) is known to be an important indicator of this odor. The T2N production pathways include enzyme reactions and lipid autoxidation reactions, in which lipoxygenase (LOX) and hydroperoxide lyase (HPL) is involved. Much effort has been made to control LOX activity such as the development of new barley varieties, control techniques in the malting and brewing process, and malt processing techniques. However, there are fewer reports on the techniques to control HPL activity than that on LOX activity. Therefore, in order to control HPL activity, we developed an analytical method for HPL activity and evaluated the thermal stability of HPL activity in malt. An analytical method has been reported in which a reaction substrate, 9-HPOD, is reacted with an enzyme extract, 9-HPOD is measured by ultraviolet absorbance at 234 nm, and HPL activity is evaluated from the amount of 9-HPOD reduced. In this study, an analytical process was established using LC/MS/MS as a detector capable of trace analysis. As a result, we established an evaluation method that can be used for routine analysis while ensuring sufficient quantitative accuracy. We evaluated HPL activity during mashing with the established evaluation method. It was found that LOX activity was inactivated by heating at 65℃ in 20 minutes, whereas HPL activity was remained 20% after heating for 60 minutes. To evaluate the thermal stability of HPL activity quantitatively, congress tests were performed at various temperatures. HPL activity was measured and the reaction rate of enzyme activity at each temperature was calculated. The results suggested that the enzyme inactivation conditions differed significantly between LOX and HPL. Based on these results, to examine the conditions for inactivating HPL activity before mashing, we investigated the activity of LOX and HPL when malt was exposed to a heat stress of 110℃. As a result, LOX activity reduced to less than 10% by heating at 110℃ for 30 seconds, whereas HPL activity required heating for 13 minutes. Thus, a new technology to inactivate HPL activity efficiently is required to suppress deterioration and improve the quality at the point of consumption. We will further understand the characteristics of HPL and develop brewing technology to suppress beer deterioration.
