beverages Article Monitoring Cider Aroma Development throughout the Fermentation Process by Headspace Solid Phase Microextraction (HS-SPME) Gas Chromatography–Mass Spectrometry (GC-MS) Analysis Matthew T. Bingman 1, Claire E. Stellick 1, Jordanne P. Pelkey 1, Jared M. Scott 2 and Callie A. Cole 1,* 1 Department of Chemistry and Biochemistry, Fort Lewis College, 1000 Rim Drive, Durango, CO 81301, USA; [email protected] (M.T.B.); [email protected] (C.E.S.); [email protected] (J.P.P.) 2 EsoTerra Cider, P.O. Box 156, Hesperus, CO 81326, USA; [email protected] * Correspondence: [email protected] Received: 19 April 2020; Accepted: 9 June 2020; Published: 16 June 2020 Abstract: Volatile organic compounds (VOCs) play a crucial role in cider quality. Many variables involved in the fermentation process contribute to cider fragrance, but their relative impact on the finished odor remains ambiguous, because there is little consensus on the most efficient method for cider volatile analysis. Herein, we have optimized and applied a headspace solid phase microextraction gas chromatography–mass spectrometry (HS-SPME GC-MS) method for the chemical analysis of cider VOCs. We determined that the 30 min exposure of a divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) solid phase microextraction (SPME) fiber at 40 ◦C yielded detection of the widest variety of VOCs at an extraction efficiency >49% higher than comparable fibers. As a proof-of-concept experiment, we utilized this method to profile cider aroma development throughout the fermentation process for the first time. The results yielded a very practical outcome for cider makers: a pre-screening method for determining cider quality through the detection of off-flavors early in the fermentation process. The aroma profile was found to be well established 72 h after fermentation commenced, with major esters varying by 18.6% 4.1% thereafter and higher alcohols varying by just 12.3% 2.6%. ± ± Lastly, we analyzed four mature ciders that were identically prepared, save for the yeast strain. Twenty-seven key VOCs were identified, off-flavors (4-ethylphenol and 4-ethyl-2-methoxyphenol) were detected, and odorants were quantified at desirable concentrations when compared to perception thresholds. VOCs varied considerably following fermentation with four novel strains of S. cerevisiae, evidencing the central importance of yeast strain to the finished cider aroma. Keywords: aroma; cider; fermentation; yeast; gas chromatography; solid phase microextraction 1. Introduction Cider is an alcoholic beverage that is produced through the fermentation of fresh or concentrated apple juice. Cider production in early America was extremely popular, but Prohibition brought the industry to a stand-still during the 1920s. Many orchards were razed or abandoned at this time, and apple varieties that were once prized for their cider-specific traits were widely forgotten [1]. Within the past decade, however, cider has experienced a massive revival, recently outpacing competitors as one of the fastest growing alcoholic beverage categories in the U.S. [2]. In Southwest Colorado, farmers have begun to restore historical orchards and incentivize the purchase of cider-specific apple varieties that still grow abundantly in the region through the Montezuma Orchard Restoration Project [3]. Small cider businesses that use these apples in their production require data-driven Beverages 2020, 6, 40; doi:10.3390/beverages6020040 www.mdpi.com/journal/beverages Beverages 2020, 6, x FOR PEER REVIEW 2 of 13 BeveragesProject 2020[3]., 6Small, 40 cider businesses that use these apples in their production require data-driven2 of 14 techniques in order to create the highest quality ciders in an efficient manner. 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