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Monitoring Cider Aroma Development Throughout the Fermentation 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. The work presented herein is the result of a novel, ongoing collaboration between orchards, cideries, and chemists in techniquesSouthwest inColorado order to which create aims the highestto better quality understan cidersd the in an myriad efficient variables manner. that The contribute work presented to local hereincider production. is the result of a novel, ongoing collaboration between orchards, cideries, and chemists in SouthwestIt is well-established Colorado which that aims aroma to better and understand flavor define the cider myriad quality. variables These that variables contribute have to localbeen ciderthoroughly production. studied through sensory means [4,5], but further research is required to understand the molecularIt is well-established profiles responsible that aroma for and those flavor organoleptic define cider quality.properties These and variables the methods have been tied thoroughly to their studiedproduction through [6]. sensoryThere meansremains [4, 5little], but furtherconsensus research on what is required specific to understand cider-making the molecular practices, profiles apple responsiblecharacteristics, for those or yeast organoleptic strains lead properties to the desired and the aroma-active methods tied compounds to their production in the headspace [6]. There remainsof apple littlecider consensus or how best on whatto measure specific those cider-making compounds. practices, In this apple work, characteristics, we report orthe yeast optimal strains analytical lead to thepractices desired to aroma-activeaddress these compounds gaps in knowledge in the headspace of cider volatile of apple organic cider or compound how best (VOC) to measure extraction, those compounds.create a timeline In this of work, odorant we development report the optimal during analytical the process practices of cider to address fermentation, these gaps and in analyze knowledge the ofkey cider molecular volatile constituents organic compound of cider (VOC) fragranc extraction,e produced create using a timeline four different of odorant strains development of Saccharomyces during thecerevisiae process (previously of cider fermentation, unexplored and for analyze cider production). the key molecular The central constituents aim of of this cider work fragrance is to optimize produced a usingcider analysis four different method strains and then of Saccharomyces apply it in order cerevisiae to better(previously understand unexplored the development for cider of production). VOCs both Theduring central cider aim fermentation of this work and is to after optimize maturation. a cider analysis method and then apply it in order to better understandHeadspace the development solid phase microextraction of VOCs both during gas chromatography–mass cider fermentation and spectrometry after maturation. (HS-SPME GC- MS) Headspaceis a reliable solid technique phase microextractionfor the pre-concentrat gas chromatography–massion and determination spectrometry of the odorant (HS-SPME volatile GC-MS)composition is a of reliable beer and technique wine [7–9]. for Recent the experime pre-concentrationnts involving and cider determination headspace analysis of the have odorant also volatileutilized compositionsolid phase microextraction of beer and wine (SPME), [7–9]. incorporating Recent experiments fibers such involving as polydimethylsiloxane cider headspace analysis(PDMS) have[10,11], also utilizedpolydimethylsiloxane/divinylbenzene solid phase microextraction (SPME), (PDMS/DVB) incorporating fibers[12], suchand as polydimethylsiloxanedivinylbenzene/carboxen/polydimethylsiloxane (PDMS) [10,11], polydimethylsiloxane (DVB/CAR/PDMS)/divinylbenzene [6] over a (PDMSrange of/DVB) exposure [12], andtimes divinylbenzene and temperatures./carboxen These/polydimethylsiloxane publications reveal (DVB a current/CAR/ PDMS)lack of [6agreement] over a range on ofthe exposure optimal timesanalytical and temperatures.approach for These cider publications headspace revealanalysis a current. SPME lack fiber of agreement choice depends on the optimal entirely analytical on the approachcompounds for ciderdeemed headspace most responsible analysis. SPME for aroma, fiber choice which depends have been entirely reported on the to compounds be primarily deemed esters mostand higher responsible alcohols for aroma,[10] with which a multitude have been of reportedlower abundance to be primarily ketones, esters aldehydes, and higher and alcohols phenols [ 10[4].] withOf interest a multitude within of this lower work abundance are a series ketones, of esters, aldehydes, higher andalcohols, phenols and [ 4phenols,]. Of interest as shown within in this Figure work 1. areWe aseek series to ofnarrow esters, the higher list of alcohols, significant and contributo phenols, asrs shownto cider in fragrance Figure1. and We seekodor to defects narrow through the list the of significantoptimization contributors and application to cider of fragrancean efficient and extraction odor defects and chemical through analysis the optimization method for and these application organic ofstructures. an efficient extraction and chemical analysis method for these organic structures. FigureFigure 1.1. VolatileVolatile organic organic species species of of interest interest to tothis this study study include include esters esters (A), ( Ahigher), higher alcohols alcohols (B), (andB), andphenols phenols (C) with (C) withspecific specific functional functional groups groups color coded color codedabove. above.Esters (A Esters) that are (A) routinely that are routinelyobserved observedherein include herein ethyl include acetate ethyl (R = acetate CH3), ethyl (R = benzoateCH3), ethyl (R = benzoateC6H5), ethyl (R =hexanoateC6H5), ethyl(R = CH hexanoate3(CH2)4), (Rethyl= CH octanoate3(CH2)4 ),(R
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