## 3 Results and Discussion

Table 3: EA concentrations of WPC (mg/g fresh matter) for the four fermentations as determined by HPLC–DAD analysis (mean \(\pm\) SD, n = 3). \begin{table}
\begin{tabular}{c c c c c c c c} \hline \hline \multirow{2}{*}{**Time (h)**} & \multicolumn{2}{c}{_S. thermophilus_} & \multicolumn{2}{c}{_A. orgaze_} & \multicolumn{2}{c}{_R. oligosporus_} & \multicolumn{2}{c}{_S. thermophilus_ **and R. oligosporus Co-Culture**} \\ \cline{2-9}  & **pH** & **a\({}_{\text{w}}\)** & **pH** & **a\({}_{\text{w}}\)** & **pH** & **a\({}_{\text{w}}\)** & **pH** & **a\({}_{\text{w}}\)** \\ \hline
0 & 5.95 \(\pm\) 0.02 & 0.95 & 5.90 \(\pm\) 0.02 & 0.96 & 5.88 \(\pm\) 0.02 & 0.96 & 5.99 \(\pm\) 0.02 & 0.96 \\
24 & 5.76 \(\pm\) 0.01 & 5.83 \(\pm\) 0.01 & 5.77 \(\pm\) 0.03 & 5.80 \(\pm\) 0.02 & 5.80 \(\pm\) 0.02 & \\
48 & 5.70 \(\pm\) 0.03 & 5.87 \(\pm\) 0.01 & 5.67 \(\pm\) 0.06 & 5.81 \(\pm\) 0.03 & 5.79 \(\pm\) 0.02 & 0.96 \\
72 & 5.61 \(\pm\) 0.02 & 0.94 & 5.85 \(\pm\) 0.03 & 0.95 & 5.61 \(\pm\) 0.07 & 0.93 & 5.79 \(\pm\) 0.02 & 0.96 \\ \hline \hline \end{tabular}
\end{table} 
Table 2: pH and a\({}_{\text{w}}\) of WPC fermentation after 0 h, 24 h, 48 h, and 72 h using either _S. thermophilus, A. orgaze, R. oligosporus, or _S. thermophilus_ and _R. oligosporus_ co-culture. Figure 2: Chromatograms of a standard solution with 50 mg/L of ellagic acid (Rt = 10.7 min) and urolithin A (Rt = 13.7 min) in red. Samples of the fermentation with _R. oligosporus_ at t = 0 h (green) and t = 72 h (blue). variations in handling and processing parameters may be expected. Though unlikely, the differences in the results obtained previously and those reported here may be due to these factors. In addition, the effect of immobilization systems in improving productivity and efficiency was also previously documented using other ellagitannin-rich substrates. SSF using _A. niger_ GH1 showed that fungal ellagitannase and ellagitannin acyl hydrolases (EAH) were able convert ellagitannins to EA within 18 h of culture using a polyurethane foam support. The conversion corresponded to fungal biomass production [16]. Similarly, _Aspergillus oryzae_, under optimum conditions, was able to convert ellagitannins from oak acorn fringe, yielding 17.7% EA. Yields were dependant on pH and incubation time [22]. Bioconversions using _R. oligosporus_ and cranberry pomace provided a maximum EA production of 400 mg/100 g of pomace. However, fungal fermentation lasted 12 d [28]. Cocultures of _A. niger_ and _R. oryzae_ produced 6.9 mg/g of pomegranate husk [29]. SSF from powdered pomegranate husk using _Saccharomyces cerevisiae_ yielded five-fold higher concentrations of EA than those obtained by _A. niger_ fermentations [30]. Comparison of the fermentations suggests that puncialagins (\(\alpha\) and \(\beta\)) are the targets of the microorganisms' hydrolase activity during formation of EA [30]. To further convert EA to urolithin A during fermentation, _S. thermophilus_ FAM2932 was used in the present study. A related strain, _S. thermophilus_ FUA329, isolated from human breast milk was recently shown to convert EA to urolithin A in vitro [25] _S. thermophilus_ FUA329 degraded EA during the exponential phase, with urolithin A being produced in the stationary phase. A maximum concentration of 7.3 umol/L of urolithin A was produced after 50 h of anaerobic culture in an ABB medium supplemented with 20 umol/L EA and 0.005% L-cysteine. Other bacteria, such as _Enterococcus fiecium_ FUA029 and _Lactococcus garviae_ FUA009, were also implicated in the production of urolithin A under similar growth conditions [31; 32; 33]. Urolith A and B from EA were produced by _Bifidobacterium pseudocatenulatum_ INIA P815 [34]. The organism was incubated anaerobically for 5 d at 37 \({}^{\circ}\)C in BHI containing 0.5 g/L L-cysteine and 20 mg/L EA. Unfortunately, in this study, even with the more sensitive HPLC-MS analysis, no urolithin A, with a limit of detection of 25 ng/g WPC, was detected in the fermented products. However, the existence of EA has been unequivocally confirmed in all WPC samples.