## 3 Results and Discussion
### Volatile Flavor Pattern Profiling According to Fermentation Temperature and Multiple Starters  
In general, the flavor of vinegar is produced by the complex action of acids, aldehydes, alcohols, ketones, and ester compounds, including acetic acid, owing to the interaction between fermenting microorganisms, resulting in various volatile components via raw material processing, fermentation, and maturation [41]. Analysis of volatile components in vinegar prepared at 20 and 30 \({}^{\circ}\)C helped us detect 16 types of volatile components (Figure 5 and Tables S3-S5). The main volatile components were acetic acid and alcohol, and the former was most abundant in AD-20 \({}^{\circ}\)C. As AAF progresses, AAB consume alcohol to produce acetic acid; therefore, the alcohol content decreases, and the acetic acid content increases. Total volatile compounds were the highest when fermented at a low temperature (20 \({}^{\circ}\)C), rather than at a medium temperature (30 \({}^{\circ}\)C) when fermentation ends. AB, AD, and ABCD showed the highest tittatable acidity on days 12, 18, and 18, respectively. The levels of decanal, which has a citrus flavor, and nonanal (y-nonanalactone), which has a coconut- and almond-like flavor, increased as the fermentation progressed [42; 43]. Decanal and nonanal showed the highest increase at ABCD-30 \({}^{\circ}\)C, and AD-30 \({}^{\circ}\)C, respectively. Octanal, 6-methyl-5-hepten-2-one, and 2-ethylhexyl acetate (acetic acid, 2-ethylhexyl ester), which have a citrus flavor, and 2-octanone (octanoic acid), which has a sweet flavor, were detected in trace amounts in all samples [44; 45; 46; 47]. A heatmap was used to visualize the profile of volatile compounds produced by the multiple starters according to the fermentation temperature (Figure 5a). Red color in the heatmap indicates a large amount detected in GC-MS quantitative analysis, and blue color indicates a small amount. A large amount of acetic acid was detected in AB at 30 \({}^{\circ}\)C on day 18 compared to day 27 of fermentation, consistent with its acid-producing ability. Acetic acid is produced during the bioconversion of ethanol through two reactions catalyzed by membrane-bound pyrroloquinoline quinone-dependent alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) [48]. ADH oxidizes ethanol to acetaldehyde, which  
Figure 4: Changes in tittatable acidity (solid line) and growth (dotted line) under multiple starters according to the AAB inoculation order and fermentation period. Inoculation order: 0, simultaneous inoculation; 1 st, former inoculation; 2 nd, following inoculation. Multiple starters: (**a**) AB; (**b**) AC; (**c**) AD; (**d**) BC; (**e**) BD; (**f**) CD. Symbols: A, _Acetobacter ascendens_ GV–8; B, _A. ascendens_ GV–12; C, _A. pasteariums_ GV–17; D, _A. pasteariums_ GV–22. is then converted to acetic acid by ALDH and released into the surrounding environment. These two dehydrogenase complexes are strictly connected to the respiratory chain, which transfers electrons to oxygen through ubiquinone and acts as the final electron acceptor. Acetic acid produced via partial ethanol oxidation can be further oxidized in the cytosol by a series of soluble NaD(P)\({}^{+}\)-dependent dehydrogenases (ADH and ALDH) via the tricarboxylic acid cycle, resulting in acetate oxidation (peroxidation). _Acetobacter_ spp. are known for their acetate peroxidation activity that can oxidize acetic acid, in addition to their ability to accumulate acetic acid [5]. It seems that volatile acetic acid was lost as the fermentation period elapsed in the AB-30 \({}^{\circ}\)C treatment group.