**4. Discussion**

XOS and high value-added compounds generated from agricultural residues have great potential for application in pharmaceutical, food, and fine chemical industries. It has been reported that the total world production of corn, a key agricultural crop, was estimated to exceed 875,226,630 tons [29]. CC, a by-product of corn is an attractive agricultural residue due to its high xylan content and the fact that it's readily available. The production of XOS could be achieved by application of chemical and hydrothermal pre-treatments. However, higher severity pre-treatment conditions can lead to increased decomposition of XOS to xylose and generation of unwanted by-products [14]. The application of low severity pre-treatment conditions to increase accessible area for enzymes, followed by enzymatic hydrolysis, is an effective method for XOS production. In this study, the synergistic interactions between two termite metagenome-derived FAEs and a xylanase, Xyn11, for the production of XOS, *p*-CA and FA from untreated and pre-treated CC was evaluated. WAX was used as a model substrate to optimize the enzyme cocktail as it maintains the ferulic acid cross-linkages in the native arabinoxylan. CC was subjected to two pre-treatment strategies, hydrothermal and dilute sulfuric acid pre-treatment. The results for composition analysis (Table 1) showed that there was an increase in recoverable sugars and that hydroxycinnamic acid content was maintained in pre-treated samples. However, the xylan content of all samples was much less than the values reported in the literature (28%) [30]. Biomass (morphology) characterization indicated that these changes were associated with increased surface area due to pre-treatment (Figure 1). It can be speculated that the increased sugar content of pre-treated CC samples (Table 1) may be due to these structural modifications which may include changes in lignin content. An increase in the production of reducing sugars was observed during enzymatic hydrolysis of pre-treated CC (Figure 3), indicating the success of the pre-treatment strategies selected for this study.

Regarding the enzymatic release of hydroxycinnamic acids from the substrates, the data presented above suggests that FAE5 or FAE6 could release significant amounts of FA and *p*-CA only in the presence of Xyn11. The inability of FAEs to release high quantities of hydroxycinnamic acids when incubated alone could be attributed to the type of bonds available on the substrate. FA or *p*-CA is usually found esterified to polysaccharides such as arabinoxylan and could also ether-link with lignin or dimerize with other hydroxycinnamic acid-linked polysaccharides, forming cross-linkages between these polymers. FAEs are known for specifically catalyzing the cleavage of ester bonds between hydroxycinnamic acids and polysaccharides, but not the ether-linkages. It is, therefore, possible that FAE5 and FAE6 couldn't act on these cross-linked complex structures. Previous studies have indicated that FAEs could release FA from substrates when co-incubated with xylanase [27,31]. Zhang and co-workers [32] reported that the combination of a xylanase and AfFaeA increased the amount of FA released from steam exploded corn stalk 13-fold. It appears that FAE activity can be enhanced by the addition of a xylanase - it appears as if the xylanase generates feruloylated XOS from the xylan main chain which are easily hydrolyzed by FAE in comparison to feruloylated xylan. In turn, FAE action allows the xylanase to be able to further hydrolyze XOS with a high degree of polymerization into shorter chain XOS products. The patterns observed in the production of reducing sugars from acid pre-treated CC, presented in Figure 3d, are indicative of such a relationship between xylanase and FAE. The activity of Xyn11 alone was drastically reduced, while the enzyme combinations resulted in the highest reducing sugar release. It is likely acid pre-treatment of CC generated XOS esterified with FA or *p*-CA, which may pose limitations on their accessibility (steric hindrance) for the Xyn11 to hydrolyze them to shorter chain XOS products. Therefore, the addition of FAEs resulted in the improvement of Xyn11 activity on these long, feruloylated XOS. There have been reports on oligosaccharides resulting from the pre-treatment of several natural substrates [33–35].

The hydrolysis patterns of individual enzymes and enzyme combinations were then also investigated. Xyn11 and a combination of Xyn11 with FAEs released XOS with a low degree of polymerization (DP 6–4 and DP 2, respectively) from WAX and released mainly xylobiose from untreated and pre-treated CC. The variations in product patterns observed could be due to the differences in the xylan structure between WAX and CC. The yield of xylobiose from the pre-treated substrates was significantly increased by enzyme combinations compared to individual enzymes. It has been reported that xylobiose rich XOS are the preferred products for prebiotic activity [36]. The product patterns demonstrated that the Xyn11: FAEs enzyme combination (cocktail) has the potential of releasing XOS with a high xylobiose content from CC, most notably when combined with hydrothermal or dilute sulfuric acid pre-treatments.
