## 4 Discussion  
The main objective of this study was the exploitation of IHER as a substrate for the performance of the SSF process initiated by _Pleurotus ostreatus_ in order to evaluate the fermentation outcome as a proteinaceous used in animal feed. Because moisture constitutes a critical parameter for the successful implementation of SSF, the presence of high moisture content secures the ability of sufficiently supporting fungal growth and nutrient transfer. On the other hand, high moisture levels can cause adverse effects because the particles of the substrates are being covered by a thick water layer, which reduces the distance between the particles, thus limiting air diffusion. In addition, the presence of high moisture levels promotes contamination risk because the substrates facilitate the development of unfavorable microorganisms [20]. When the moisture level is lower than that required, the nutrients' solubility is hindered, resulting in their inadequate uptake by the fungi [20,21]. Regarding the closed SSF system, [PERSON] et al. [20] stated that a sufficient moisture content is closely connected with the substrate's lignocellulosic structure, whereas [PERSON] et. al. [22] reported that moisture content can be influenced by the properties of the substrate, such as porosity and particle size. [PERSON] et al. [23] reported that SSF's water balance can be affected by the required water content for starch hydrolysis, the metabolic water production, the intracellular water uptake thorough biomass production, and finally by the water's evaporation as an effect of the metabolic heat production. These \begin{table}
\begin{tabular}{c c c} \hline \hline
**Parameters** & **Day 0** & **Day 11** \\ \hline
**CFS** & 12.10 \(\pm\) 0.09 & 11.14 \(\pm\) 0.63 \\
**Cellulose** & 14.27 \(\pm\) 0.44 & 15.02 \(\pm\) 0.28 \\
** Lignin** & 6.39 \(\pm\) 0.28 [41] & 14.26 \(\pm\) 0.61 [42] \\
**Ash** & 3.14 \(\pm\) 0.06 [41] & 8.80 \(\pm\) 0.17 [42] \\ \hline \hline \end{tabular}  
* CPS, crude fiber substances. Statistical analysis was performed between Day 0 and Day 11. Different superscripts between the same letters indicate statistical significance, \(p\leq 0.05\).  
\end{table} 
Table 2: Impact of fermentation time on fiber substance content in the examined substrates. Figure 2: Evaluation of \(\beta\)-glucan content between Day 0 and Day 11. Different superscripts between the same letters indicate statistical significance, \(p\leq 0.05\). four significant factors could possibly explain the moisture content increase during the fermentation procedure. In the present work, our findings concerning moisture are in line with previous literature reports. More specifically, the moisture content at the baseline of fermentation (Day 0) was found to be 65.47%. Although moisture content was not considered as optimum, SSF was successfully performed, with the latter exceeding a proportion of 80% at the end of the procedure (Day 11). This increment can be rationalized by the lignocellulosic structure of the examined substrates. The crude protein content of the substrates constitutes the most important variable examined, because it is closely connected with their conversion efficiency into high-added nutritional protein materials. After the end of the SSF process initiated by _P. ostreatus_, the crude protein content was increased. This observation can be rationalized considering the report of [PERSON] et al. [24], who stated that such an increment could be resulted by a possible fungal biomass accumulation. According to [PERSON] et al. [25], a possible explanation for the observed increase of crude proteins could be connected with the potential to uptake excess nitrogen via aerobic fermentation. On the other hand, according to [PERSON] and [PERSON] [26], the increased crude protein content could be attributed to the secretion of specific proteinaceous extracellular enzymes, which are capable of degrading the materials during the fermentation process.