# Thai Rice Vinegars: Production and Biological Properties
## 3 Results and Discussion
### Solid State Fermentation of Rice Vinegar
Saccharified glucose of 200 g cooking rice showed a maximum content on the first day of alcohol fermentation. The saccharified glucose content of glutinous rice and black glutinous rice showed no statistically significant difference and was higher than the content of polished and black fragrant rice. Glucose was then converted to ethanol during this step, and the maximum ethanol production was detected after day 7 of alcohol fermentation. Previous studies have reported an effect of conditions on acetic acid bacteria growth. The glucose tolerance of acetic acid bacteria isolated from traditional balsamic vinegar has been studied, demonstrating that the general acetic acid bacterial growth was inhibited by 25% glucose. _A. pasteurianus_ was able to grow in 10% ethanol with 5% yeast extract medium [25]. The effect of ethanol on acetic acid bacterial growth in wine was evaluated and showed that the cells remained viable in the presence of 10% and 14% (\(v/v\)) ethanol. Acetic acid from ethanol oxidation is indicated as a limiting factor for acetic acid bacterial growth. The cells are affected by the high acetic acid concentration (approximately 12%) due to the decreased enzymatic activity of alcohol dehydrogenase [26]. The observed pH value change in the fermentation broth during fermentation were determined. After the addition of water in the first alcohol fermentation step, the pH was approximately 7 and then decreased to 3.06-3.51 at the end of fermentation. The optimized pH for growth of acetic acid bacteria is 5.0-6.5, although these bacteria can grow at lower pH values (3.0-4.0) such as in wine with high ethanol concentrations (10-15%). _A. pasteurianus_ has been reported to show increased pH sensitivity to a high concentration of ethanol (12.5%). In general, the optimum growth temperature of acetic acid bacteria ranges from 25-30 \({}^{\circ}\)C, but several studies have demonstrated that acetic acid bacteria from industrial vinegar production are thermotolerant and can oxidize ethanol at high temperatures (38-40 \({}^{\circ}\)C) [26]. A study of a mixed culture of _S. cerevisiae_ and _A. pasteurianus_ reported an optimum growth temperature of _S. cerevisiae_ of 28-30 \({}^{\circ}\)C and _A. pasteurianus_ of 34-36 \({}^{\circ}\)C with a culture pH varying from 3.8 to 4.3 for acetic acid production [27]. During the acetic acid fermentation step, the acetic acid content decreased due to overoxidation. The accumulated acetic acid was oxidized to CO\({}_{2}\) and H\({}_{2}\)O via the tricarboxylic cycle when ethanol was absent in the fermentation broth [28]. A summary of reducing sugar, ethanol, total acid and acetic acid contents in Thai rice vinegars is shown in Table 2. The reducing sugar and ethanol contents were retained in Thai rice vinegas even though fermentation was completed. There was no statistically significant difference. However glutinous rice vinegar exhibited the highest content of total acid and acetic acid with statistically significant difference from other rice vinegars, follow by black glutinous rice vinegar. Otherwise, there was no statistically significant variation in total acid level between black fragrant rice and polished rice vinegar, but acetic acid content of black fragrant rice vinegar more than polished rice vinegar was statistically significant. Glutinous rice vinegar exhibited the highest content of acetic acid at 6.68% (\(w\)/\(v\)), followed by black glutinous rice, black fragrant rice and polished rice with values of 5.33%, 4.06% and 3.78% (\(w\)/\(v\)), respectively. The presence of acetic acid was related to the total acid content of Thai rice vinegar because it is the major organic acid in vinegar. However, several organic acids were also detected, as described in some studies of rice wine vinegar [24]. The acidity of wine vinegar must be at least 6% (\(w\)/\(v\)), and the maximum residual ethanol allowed is 1.5% (\(r\)/\(v\)), as determined by the European Union.