## 3 Results and Discussion  
### Cider VOC Development throughout the Fermentation Process  
Despite the many variables that are routinely altered in the cider preparation process, from apple type to fermentation temperature and yeast strain [10; 20], few analytical methods have been developed to monitor exactly how these variables develop the aroma profile of the cider over time during fermentation. Carefully controlling these parameters throughout the cider preparation process in order to measure the released volatile species can reveal which inputs yield the desired final product and at what point in production a finished aroma has been established. Figure 4 illustrates the results of such an approach. Specific gravity was measured regularly by a hydrometer to monitor the fermentation stage (Figure 4A), and the corresponding HS-SPME-GC-MS data were gathered to observe the volatiles released pre- (Figure 4B, red) and post-inoculation (Figure 4B, purple and green) as well as in the finished cider (Figure 4B, blue). For this proof-of-concept experiment, local Double Red Delicious apple juice (Teal Cider, Dolores, CO, USA with 1.064 starting specific gravity) was inoculated with Lalvin ICV D47 yeast at room temperature (\(23\pm 1\)\({}^{\circ}\)C). All chromatographic peaks are labelled in Figure 4, unless they were of negligible abundance (\(<\)0.002 relative peak area) or they corresponded to siloxanes that originated from the SPME fiber itself [27]. Prior to inoculation, the Red Delicious apple juice headspace contained primarily esters with various organic side chain lengths (major species in decreasing order of abundance: 2-methylbutyl acetate, hexyl acetate, ethyl-2-methyl butyrate, ethyl hexanoate, ethyl butyrate, and butyl acetate). Two higher alcohols, 1-hexanol and 2-methyl-1-butanol, were also observed in the pre-inoculation juice (Figure 4B, red), alongside estragole, which has previously been observed in Golden and Red Delicious apple juices [28]. Seventy-two hours after inoculation (Figure 4B, purple), the notable rise of six longer chain esters (in decreasing order of abundance: ethyl octanoate, ethyl decanoate, ethyl dodecanoate, 3-methylbutyl acetate, ethyl-3-methylbutyrate, and 3-methylbutyl octanoate) and one higher alcohol, 2-phenylethyll alcohol, were observed as by-products of the initiation of yeast metabolism [13]. These molecules have previously been identified as key contributors to cider aroma [10], with the esters generating a fruity smell and the higher alcohols creating a honey or whiskey fragrance [21; 29]. Upon inoculation, ethyl octanoate and ethyl decanoate dominated the chromatogram for the remainder of the fermentation. Interestingly, only one new volatile, ethyl-9-decenoate, was generated after the 72 h point in the fermentation, and those species that were present did not change significantly in their abundance. Esters collectively increased by 18.6% \(\pm\) 4.1% from 72 h (Figure 4B, purple) to the completion of fermentation (Figure 4B, blue), and higher alcohols collectively increased by 12.3% \(\pm\) 2.6% during this time frame, based on the chromatographic peak areas. This suggests that under the conditions described herein, the aroma profile was largely determined within the first 3 days of fermentation, after the specific gravity has dropped to 1.056 with an estimated alcohol by volume content of 1.05%. When comparing fermentation at day 8 (at a specific gravity of 1.004 and an estimated alcohol by volume content of 7.88%) to the final cider, esters and higher alcohols varied by an even smaller figure (2.6% \(\pm\) 0.6% and 1.6% \(\pm\) 0.3%, respectively). For cider makers, this can inform practical and strategic chemical analyses of a cider early in its development in order to determine whether the beverage is on or off track to achieving the desired product characteristics. The VOCs present in the apple juice significantly changed in abundance during fermentation.