# Thai Rice Vinegars: Production and Biological Properties
## 3 Results and Discussion
### Biological Properties of Rice Vinegars
#### 3.3.4 Determination of Antioxidant Activity  
* ABTS method  
The ABTS (2,2\({}^{\prime}\)-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) assay depends on the blue-green color of ABTS\({}^{\bullet+}\) generated in the aqueous phase with a maximum absorption at 734 nm. The ABTS\({}^{\bullet+}\) was reduced by hydrogen donation by the antioxidant; the blue-green of ABTS\({}^{\bullet+}\) is converted to a colorless form and the absorption is decreased [43]. The remaining ABTS percentages in the rice vinegas are shown in Figure 4A. The blue-green of ABTS\({}^{\bullet+}\) was decolorized by black glutinous rice vinegar to 50% in less than 20 s, exhibiting the strongest antioxidant ability against ABTS\({}^{\bullet+}\). Subsequently, black fragment, polished and glutinous rice vinegar exhibited 50% ABTS scavenging activity over 30, 120 and 220 s, respectively. In contrast to commercial fermented rice vinegar, commercial distilled vinegar and 4% acetic acid are incapable of decolorizing ABTS\({}^{\bullet+}\). * DPPH method The DPPH (2,2-diphenyl-1-picrylhydrazyl) assay has been widely used to determine the antioxidant activity of foods. The DPPH\({}^{\bullet}\) radical is a stable organic nitrogen radical with a deep purple color and the greatest absorption at a wavelength of 515 nm. The purple color of the DPPH\({}^{\bullet}\) radical is reduced to the form DPPH-H by pairing with the hydrogen atom from antioxidant compounds, changing the color to pale yellow, which causes the absorption at 515 nm to decrease [43]. The DPPH\({}^{\bullet}\) radical was reduced by the tested Thai rice vinears. The results are shown in Figure 4B, wherein the DPPH\({}^{\bullet}\) radical was reduced by black glutinous and black fragment rice vinegar to 50% in less than 20 s. In contrast, polished and glutinous rice vinegar exhibited 50% DPPH scavenging activity for 50 and 70 s, respectively. Commercial fermented rice vinegar was unable to inhibit the DPPH\({}^{\bullet}\) radical to 50% in 5 min but to 79.88% in 5 min. In contrast, the DPPH\({}^{\bullet}\) radical was barely reduced by commercial distilled vinegar and 4% acetic acid. Based on the present results, black glutinous rice vinegar showed the strongest antioxidant activity against both ABTS and DPPH, followed by black fragment rice vinegar, polished rice vinegar and glutinous rice vinegar, respectively. The results demonstrated that all Thai rice vinegas had antioxidant activity of both ABTS\({}^{\bullet+}\) and DPPH\({}^{\bullet}\) radicals. However, commercial fermented rice vinegar had slight antioxidant activity against DPPH but was ineffective with ABTS. Commercial distilled vinegar and 4% acetic acid showed no antioxidant activity. L-ascorbic acid (vitamin C) was used as a positive control for antioxidant determination. Vitamin C is a water-soluble antioxidant, which is an important reducing agent and scavenger of free radicals in biological systems. The free radical was generated with donated electron from vitamin C for stability [44]. In this study, vitamin C exhibited powerful antioxidant activity against both ABTS and DPPH (Figure 4). Figure 4: Antioxidant activity of rice vinegas by the ABTS method (**A**) and the DPPH method (**B**). The total phenolic content of commercial fermented rice vinegar was 10.55 \(\pm\) 0.46 mg gallic acid/100 mL of rice vinegar, which was less than all Thai rice vinegas in this study. Thus, the total phenolic content was insufficient for inhibiting the ABTS\({}^{\bullet+}\) and DPPH\({}^{\bullet}\) radicle, while commercial distilled vinegar and 4% acetic acid were not detected. Thus, they showed no antioxidant activity. The antioxidant activity results were related to the total phenolic content of the vinegas. The two processes of vinegar production, include traditional and submerged (industrial), were studied, using the same type of raw material for vinegar production. The vinegar from the traditional method showed a higher total phenolic content compared with the submerged method.