# Bivolarization of Grape Stalks as Animal Feed by Solid State Fermentation Using White-Rot Fungi
## 3 Results and Discussion
As in the delignification process, IVOMD was positively associated with the increase of the incubation period (Figure 2). By contrast, incubation with _P. eryngii_ did not affect the IVOMD of GS. Similar results were observed by other authors [24; 40; 47; 46], when evaluating the effects on digestibility after incubating different raw materials with distinct fungal  
Figure 2: The ratio of cellulose (C) to lignin (sa) (L) in the dry matter, IVOMD, and lignin of GS treated with different fungi for 28, 35, and 42 days. strains. [PERSON] et al. [48] observed an increase in the _in vitro_ gas production of wheat straw and oak wood chips, after being incubated with _L. edodes_ for 56 days. [PERSON] et al. [42] also observed an increase of _in vitro_ rumen fermentation after the incubation of four raw materials (maize stover, rice straw, oil palm fronds, and sugarcane bagasse) with four WRF. In our study, samples treated with _L. edodes_ increased the IVOMD throughout the fungal incubation period (0 to 42 days). By contrast, no effect of the fungal incubation period was observed for _P. eryngii_. These results support the low effect of _P. eryngii_ treatment in improving IVOMD, and this could be due to the irrelevant effect of this fungus on the delignification of GS (Table 1). IVOMD differences among the studied treatments can be explained by specific modifications in the cell wall structure instigated by distinct WRFs during the incubation period (Figure 2). Low digestibility values in raw materials could be linked to the presence of lignin. The linkages between lignin and cellulose and hemicellulose interfere in the access of rumen microbial enzymes to the structural carbohydrate present in cell walls [7]. The fungal enzymatic system, especially that of WRF, breaks down those lignin linkages and simultaneously converts polysaccharides into simple monomer components [49]. Thus, lignin degradation enhances digestibility through the release of the more accessible cellulose and hemicellulose, which can be easily digested by rumen microbiota [20; 50]. In fact, WRF enzyme activities are characterized by the production of several extracellular enzymes that directly degrade the lignin, cellulose, and hemicellulose of the plant cell wall. These enzymatic complexes seem to exert their effect on the cell wall structure due to a concerted action between the different types of enzymes [33], and this may promote different types of synergetic efficiency in the degradation processes. According to [PERSON] et al. [50] both the physiological conditions for lignin degradation and the expressed enzyme systems differ between fungal species. Differences may be linked to the taxonomic position of the fungi, their substrate specialization, and stage of degradation, among other characteristics [22; 51]. In this way, there has been a growing research interest to characterize and quantify these ligninolytic enzymes for their ability to solubilize the lignocellulosic complex [18; 52]. The enzymatic activities of seven enzymes: lactose (Lacc), manganese peroxidase (MnP), lignin peroxidase (LnP), carboxymethyl cellulase (CMCase), avicalase, xylanase, and feruloyl esterases (FAEs), were evaluated (Table 2) for the incubation of three different strains of fungi (_L. edodes, P. citrinopiletatus_, and _P. eryngii_) in six different incubation periods (IP; 7; 14; 21; 28; 35 and 42 days). These enzymes can be divided into three important classes: enzymes involved in ligninolysis, cellulolysis, and hemicellulose [50]. Regarding the enzymes involved in ligninolysis, there are three main oxidative enzymes secreted by WRF: peroxididases (LiP and MnP) and Lacc, responsible for decomposing a variety of refractory compounds ranging from simple phenolic molecules to xenobiotics [50; 52]. All fungi had an increasing production of Lacc, achieving the peak of production until 35 days of IP, with _L. edodes_ showing the highest production (3.62 U g\({}^{-1}\) DM; \(p\) < 0.001). The same trend was observed for MnP production kinetics with the exception of _L.