## 3 Results and Discussion
### Volatile Flavor Pattern Profiling According to Fermentation Temperature and Multiple Starters
For AD, higher acetic acid levels were detected on day 18, which decreased on day 27 at 20 \({}^{\circ}\)C. For AB and AD starters, the titratable acidity was slightly higher when fermented at 30 \({}^{\circ}\)C, while the measured volatile compounds were higher when fermented at 20 \({}^{\circ}\)C. For the ABCD multiple starter, similar amounts of acetic acid were detected at 20 \({}^{\circ}\)C and 30 \({}^{\circ}\)C; however, a lower volatile compounds content was detected, compared to that in the AB and AD starters. Therefore, AB fermented at 30 \({}^{\circ}\)C for 18 days and AD fermented at 20 \({}^{\circ}\)C for 18 days were judged to have a high flavor component-producing ability. PLS-DA was used to identify the pattern of volatile compounds according to the fermentation temperature and multiple starters (Figure 5b). For AB, the factors at 20 \({}^{\circ}\)C moved to the upper part of the plot as fermentation progressed, and those at 30 \({}^{\circ}\)C were located in the 4 th quadrant of the plot at the beginning of fermentation and moved to the center of the plot as fermentation progressed. For AD, the factors at 20 \({}^{\circ}\)C moved upward in the plot according to the fermentation process, and the factors at 30 \({}^{\circ}\)C were in the center of the plot, regardless of the fermentation period. The ABCD factors at 30 \({}^{\circ}\)C were in the center of the plot, regardless of the fermentation period, and the factors at 20 \({}^{\circ}\)C moved to the lower part of the plot on day 27 of fermentation. Vinegar taste and flavor components originate from the unique characteristics of its raw materials; moreover, differences in  
Figure 5: (**a**) Heatmap and (**b**) partial least squares–discriminant analysis (PLS–DA) of vinegar samples fermented by multiple AAB according to the fermentation temperature and period (days). The blue color in the heatmap corresponds to low abundance, and red corresponds to high abundance, as shown in the color key legend on the right side of the figure. Low abundance indicates low detection of volatile compounds. Multivariate PLS–DA displays different colors according to complex AAB and fermentation temperature. The factors indicated by each color are indicated in the figure. Strains: A, _Acetobacter ascendens_ GV–8; B, _A. ascendens_ GV–12; C, _A. pasteurianus_ GV–17; D, _A. pasteurianus_ GV–22. volatile compounds appear depending on fermentation and aging methods [49]. In this study, the fermentation temperature and type of AAB affected the flavor composition. Changes in the Beta Diversity of Microbial Communities According to Fermentation Temperature and Multiple Starters  
As AAF progresses, the dominance between multiple AAB and microorganism diversity change, and microorganisms that worsen the fermentation quality can grow. Microbiome taxonomic profiling was performed to confirm the microbial diversity of co-cultured AAB according to the fermentation temperature and duration and to control the microbial community of grain vinegar through AAF [50]. The alpha diversity index determines whether sequencing data are sufficient for detecting all microbial species included in the sample [51]. Alpha diversity refers to the diversity \"within\" a sample; the higher the number of species (abundance) and the more evenly distributed (evenness) they are, the higher the species' diversity is [52]. In this study, alpha diversity showed similar values because only the starter was inoculated and fermented in a strictly controlled environment (Table S6). Alpha diversity analysis revealed no significant difference in the diversity within the sample. Therefore, beta diversity was analyzed to investigate the diversity of the samples. The distance metric of beta diversity measures the diversity between samples using the Generalized UniFrac method, where higher values indicate a more diverse microbiome composition [53; 54]. All samples at the beginning of fermentation were located at the center of the PCoA (Figure 6a).