## 3 Results and Discussion
### Optimal Inoculation Rate and Order of Multiple AAB  
To induce high acid production through the interaction of multiple AAB, the inoculation rate of multiple AAB was evaluated. The former and following strains were mixed at ratios of 1:1, 1:2, and 2:1, inoculated into a liquid medium, and fermented at 30 \({}^{\circ}\)C. The six AAB were as follows: AB (_A. ascendens_ GV-8 and _A. ascendens_ GV-12), AC (_A. ascendens_ GV-8 and _A. pasteiranus_ GV-17), AD (_A. ascendens_ GV-8 and _A. pasteiranus_ GV-22), BC (_A. ascendens_ GV-12 and _A. pasteiranus_ GV-17), BD (_A. ascendens_ GV-12 and _A. pasteiranus_ GV-22), and CD (_A. pasteiranus_ GV-17 and _A. pasteiranus_ GV-22). The increase in titratable acidity and growth showed a similar trend (Figure 3). On day 27, the titratable acidity of AB, AC, and AD was the highest at a ratio of 1:1 (5.59 \(\pm\) 0.23%, 5.62 \(\pm\) 0.14%, and 5.64 \(\pm\) 0.02%, respectively), and the titratable acidity at a ratio of 1:2 was higher than that at a 2:1 ratio, while both strains showed similar acid-producing effects. BC showed the highest titratable acidity of 5.55 \(\pm\) 0.37% at a ratio of 2:1, followed by that at 1:1 and 1:2. In other words, the B (GV-12) strain contribution to the increase in titratable acidity was greater. BD showed the highest titratable acidity of 5.94 \(\pm\) 0.12% at  
Figure 2: Changes in titratable acidity (**a–c**) and growth (**d–f**) under multiple starters according to initial pH and fermentation period. Initial pH: (**a,d**) pH 2.0; (**b,e**) pH 3.0; (**c,f**) pH 4.0. Symbols: A, _Acetobacter ascendens_ GV-8; B, _A. ascendens_ GV-12; C, _A. pasteiranus_ GV-17; D, _A. pasteiranus_ GV-22. The initial acidity was increased by decreasing pH (acidity as pH decreases: pH 2.0, 5.24%; pH 3.0, 3.24%; pH 4.0, 0.35%). For example, when the pH 2.0 treatment group showed titratable acidity of 5.53% and 5.60% on the 0 th and 15 th days of fermentation, Table 4 indicates them as 0.02% and 0.09%, respectively. a ratio of 2:1, followed by 1:2 and 1:1. B and D may inhibit each other and interfere with growth when they are co-cultured. The titratable acidity of the CD starter was the highest at 5.25 \(\pm\) 0.22% at a ratio of 1:1, followed by that at 2:1 and 1:2, showing that C and D may have a similar effect on the increase in titratable acidity. The optimal inoculation ratio for each AAB was as follows: AB, 1:1, AC, 1:1, AD, 1:1, BC, 2:1, BD, 2:1; and CD, 1:1. Multiple AAB strains were inoculated in different succession to identify the optimal inoculation order (Figure 4). For simultaneous inoculation, the former and following strains were simultaneously inoculated on day 0 (Symbol in Figure 4: 0). For pre-inoculation, the former strain was inoculated on day 0, and the following strain was inoculated on day 12 (Symbol in Figure 4: 1 st). Post-inoculation reversed the positions of each strain in pre-inoculation (Symbol in Figure 4: 2 nd). AB, AC, AD, and CD starters inoculated at a ratio of 1:1 showed the highest titratable acidity post-inoculation (5.73 \(\pm\) 0.12%, 6.38 \(\pm\) 0.11%, 6.59 \(\pm\) 0.11%, 6.82 \(\pm\) 0.11%, respectively), followed by that registered during simultaneous inoculation and pre-inoculation. BC and BD inoculated at a ratio of 2:1 showed the highest titratable acidity post-inoculation (7.01 \(\pm\) 0.14% and 6.55 \(\pm\) 0.14%, respectively), followed by that registered during pre-inoculation and simultaneous inoculation. This implies that multiple AAB are advantageous when the two strains are inoculated at different times. It can be interpreted that metabolites produced by the first inoculated strain had a beneficial effect on the latter inoculated strain. Figure 3: Changes in titratable acidity (solid line) and growth (dotted line) under multiple starters according to the AAB inoculation ratio and fermentation period. Inoculation ratios: 1:1, 1:2, and 2:1. Multiple starters: (**a**) AB; (**b**) AC; (**c**) AD; (**d**) BC; (**e**) BD; (**f**) CD.