## 3 Results and Discussion  
In this study, we adapted the fermentation conditions presented by [PERSON] et al. (2007) and performed SSF of 100 g portions of walnut press cake using inocula of either _S. thermophilus, A. ozyzae, R. oligosporus_, or S. thermophilus and _R. oligosporus_ in coculture. The WPC was examined visually after 0, 24, 48, and 72 h (Figure 1) [21]. As the optimal fermentation time for ellagic acid liberation was after 72 h of fermentation, we also stopped our assays at that time [22]. For fermentations with _A. ozyzae_, _R. oligosporus_ visible mycelium formation was observed after 48 h. Similarly, in cocultures with _R. oligosporus_ and _S. thermophilus_, visible mycelium formation by _R. oligosporus_ was also observed after 48 h. The densest mycelium formation appeared in fermentation with _A. oyzae_ after 72 h. The pH of the fermentations was monitored every 24 h. The water activity (a\({}_{\mathrm{w}}\)) of the WPC was evaluated at the beginning and the end of the fermentation (Table 2). Despite visible mycelium growth and a more than 300-fold increase in _S. thermophilus_ concentration within the first 48 h of incubation, neither the pH, water content, nor the a\({}_{\mathrm{w}}\) showed any significant changes throughout the incubation time. The presence of EA was confirmed through HPLC-DAD analysis (Figure 2). Nonetheless, concentrations of EA in all fermentations remained constant, with only marginal variations observed from the initial concentrations, which were attributed to raw material heterogeneity (Table 3). The absence of EA liberation is unexpected, considering the results reported in a recent article involving SSF under comparable conditions [24]. Figure 1: WPC fermentation after 0 h, 24 h, 48 h, and 72 h using (**a**) _S. thermophilus_, (**b**) _A. ozyzae_, (**c**) _R. oligosporus_, and (**d**) _S. thermophilus_ and _R. oligosporus_ coculture. The liberation of EA from WPC could potentially be influenced by insufficient moisture content. Based on the WPC preparation and the original humidity of the WPC, an approximate 48% water content was estimated in the fermentation setups at the beginning. For SSF using fungi, a moisture content of 60-70% is optimal to allow for adequate interaction of the mycelium with the substrate [11; 24]. Due to adequate levels of water activity (a\({}_{\text{w}}\)) the growth of neither the two fungi nor the _S. thermophilus_ was hindered. The results obtained contrast with previous data using the raw material source and using identical methods, where the liberation of EA from ellagitannins in WPC was achieved using either _A. orgaze_ or _R. oligosporus_[24]. However, as the studies where more than a year apart, the nut press cake used in this study did not originate from the same batch of production as before and may have been processed differently. Previous studies concluded that while ellagitannins are stable in acidic conditions, they are rapidly degraded in neutral and basic environments at elevated temperatures (60-80 \({}^{\circ}\)C). Ellagitannins may also oxidize to form compounds with a dehydrohexahydroylphenoyl (DHHDP) group such as maltotusinic acid [27]. Unfortunately, the authors of this study did not have control over any parameters involved in the production or storage of the walnut press cake before its procurement. Hence, even though the press cake was purchased at the same oil mill, \begin{table}
\begin{tabular}{c c c c c c c} \hline \hline
**Termation** & **0 h** & **24 h** & **48 h** & **72 h** \\ \hline _S. thermophilus_ & 0.14 \(\pm\) 0.01 & 0.13 \(\pm\) 0.00 & 0.14 \(\pm\) 0.00 & 0.17 \(\pm\) 0.03 \\ _A. orgaze_ & 0.15 \(\pm\) 0.04 & 0.18 \(\pm\) 0.03 & 0.14 \(\pm\) 0.04 & 0.13 \(\pm\) 0.02 \\ _R. oligosporus_ & 0.26 \(\pm\) 0.01 & 0.21 \(\pm\) 0.13 & 0.22 \(\pm\) 0.14 & 0.28 \(\pm\) 0.07 \\ _S. thermophilus and R._ & 0.19 \(\pm\) 0.03 & 0.21 \(\pm\) 0.06 & 0.20 \(\pm\) 0.02 & 0.16 \(\pm\) 0.02 \\ _oligosporus_ co-culture & & & & & \\ \hline \hline \end{tabular}
\end{table}