Investigation of the Reaction Pathways and Origins of Geraniol Biotransformation Products Using a Deuterated Chemical Standard

Monoterpene alcohol biotransformation by yeast is a heavily studied brewing topic which lacks definitive confirmation of the reaction pathways and product origins specifically regarding geraniol. Using a deuterated (isotopically flagged) chemical standard, the biotransformation of geraniol can be more concretely understood. The objective is to investigate and clarify the reactions and product origins involved in the biotransformation of geraniol by yeast in model wort fermentation. Bench top brewing was utilized for this experiment and divided into 3 trials. A model wort was brewed in 2-liter flasks using dry malt extract, spent hop pellets and Iso 30%. On day 0 prior to yeast addition, each flask was dosed with 2µL (~2000 ppb) of geraniol chemical standard. For trial 1, each flask was pitched with a different strain of yeast (WLP001, K-97, Pomona™, and New England™ (Lallemand)) and allowed to ferment for 10 days at room temperature (68°F - 72°F). Trial 2 was a repeat of trial 1 using four different yeast strains (S-04, W-34/70, LoNa™ (Lallemand), Lutra® Kviek (Omega)). Trial 3 involved using 3 previously tested yeast strains (WLP001, W-34/70 and S-04) but a deuterated geraniol chemical standard was used for dosing. A fourth flask was prepared to investigate a potential hydrolysis reaction and was dosed with citronellyl acetate and geranyl acetate. For trials 1 and 2, each flask had samples taken pre and post dose, day 2, day 4 (day 7 for trial 2) and day 10 of fermentation for GC-MS analysis. For trial 3, samples were taken every 24 hours for 4 days, on day 6 and day 10 of fermentation for GC-MS analysis. All yeast strains produced three products: citronellol, citronellyl acetate and geranyl acetate. The reaction pathways involved are a reduction into citronellol and esterification into citronellyl and geranyl acetate. Evidence of these reactions was supported by using a deuterated geraniol chemical standard. Only a small amount of the dosed geraniol was converted into product compounds while a portion remained unconverted. It was observed that the reduction reaction is initially favored with the detection of citronellol within 24 hours of fermentation followed by the detection of citronellyl acetate and geranyl acetate between 24 and 48 hours of fermentation. A general trend was also observed that citronellyl and geranyl acetate concentrations peak within 48 hours of fermentation and then begin to decrease. Citronellol, however, begins to increase 48 hours into fermentation even though geraniol concentrations remain stable or increase slightly. This observation suggests a hydrolysis reaction taking place later in fermentation in which the acetate products are being converted back into their original compounds. This was supported by the secondary dosing trial using citronellyl acetate and geranyl acetate.