## 3 Results and Discussion
### Characterization of Cold and Hot Brew Coffee
#### 3.3.2 Total Phenolic Content and Antioxidant Activity Comparison between Cold and Hot Brew Coffee  
The antioxidant capacities and total phenolic content of CBC were compared with those of its hot brew counterpart and are shown below (Table 7). The table above showed that the total phenolic content and antioxidant activities measured by FRAP and DPPH radical scavenging assays were similar between cold brew and hot brew coffee (\(p>0.05\)). This was predicted because the method of CBC preparation was optimized to result in similar TDS obtained for hot brew coffee. However, the CBC exhibited significantly higher antioxidant activity than its hot counterpart, as measured by the ORAC assay.  
\begin{table}
\begin{tabular}{c c c c} \hline \hline  & **Cold Brew Coffee** & **Hot Brew Coffee** & \(p\)**-Value** \\ \hline TDS (\%) & 1.28 \(\pm\) 0.02 & 1.21 \(\pm\) 0.02 & 0.0048 * \\ pH & 5.45 \(\pm\) 0.08 & 5.45 \(\pm\) 0.03 & 0.9663 \\ Titratable acidity, pH 6.0 (mL NaOH/20 mL coffee) & 0.63 \(\pm\) 0.02 & 0.41 \(\pm\) 0.02 & 0.0096 * \\ Titratable acidity, pH 8.0 (mL NaOH/20 mL coffee) & 1.99 \(\pm\) 0.14 & 1.50 \(\pm\) 0.02 & 0.00245 * \\ Titratable acidity, pH 8.20 (mL NaOH/20 mL coffee) & 2.19 \(\pm\) 0.12 & 1.67 \(\pm\) 0.03 & 0.0115 * \\ \hline \hline \end{tabular}  
* Statistically significant \(p<0.05\); results are the mean \(\pm\) standard deviation from triplicate extractions.  
\end{table}
Table 6: Physicochemical characteristics of cold and hot brew coffee.  
The total phenolic content was measured using the Folin-Ciocalteu (FC) Assay, which is a colorimetric ET-based method. This assay utilizes the transfer of electrons from phenolic compounds to the FC reagent (a mixture of phosphor-molybdate and phosphotungstate) to form a blue-colored complex [29].  
In plants, antioxidants work in two major pathways, i.e., hydrogen atom transfer (HAT) and single electron transfer (SET) [30]. While FRAP, TPC, and DPPH radical scavenging assays utilize the electron transfer reaction, the ORAC assay utilizes the HAT mechanism [31]. The ferric reducing antioxidant power (FRAP) assay is a colorimetric SET-based method. It measures the reduction of ferric ion (Fe\({}^{3+}\)) by antioxidants to form a blue-colored ferrous (Fe\({}^{2+}\)) complex. The intensity of the end product is then measured and quantified using a spectrophotometer to indicate the reducing power of the antioxidant tested [31].  
2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay is also a colorimetric SET-based assay used to estimate the radical scavenging capacity of plants. The principle of this method utilizes the electron donation of antioxidants to neutralize the DPPH radical (purple in color), resulting in a color change that is measured by the spectrophotometer [31]. The oxygen radical absorbance capacity (ORAC) assay is a fluorometric HAT-based assay that measures the decrease in the fluorescence of a target under a constant flux of peroxyl radical [29; 31].  
The CBC samples showed that they had higher antioxidant activities according to the HAT-based mechanism than the hot brew, whereas the antioxidant activities according to the SET-based mechanism were not significant between the two samples. However, further investigations are needed to look into the cause of the difference in antioxidant activity from the ORAC Assay.