## 4 Large-Scale Procedure for the Production of Chitooligosaccharides  
The scale-up of COS synthesis offers several difficulties derived from the low aqueous solubility of the substrates, the heterogeneity of the reaction mixtures and the presence of high concentrations of salts. Based on previous works performed in our laboratory at a small scale, the commercial proteolytic preparation Neutrase 0.8 L [46] and the chitinase Chit42 from _T. harzianum_[39] were selected to scale-up the synthesis of several COS mixtures. Chitosans of different sizes and DD (Table 1) were hydrolyzed with one or both enzymes acting synergically. A common procedure was designed for the large-scale production of COS of different sizes and DD (Figure 6), which could illustrate the difficulties in the downstream processing of these mixtures. \begin{table}
\begin{tabular}{c c c} \hline
**Chitosan** & **Molecular Mass (kDa) 1** & **Deacetylation Degree (\%) 1** \\ \hline CHIT2 & 56 & 79 \\ QS1 & 98 & 81 \\ CHIT600 & 600–100 & \textgreater{}90 \\ \hline \end{tabular} \({}^{1}\) Data from the providers.  
\end{table} 
Table 1: Main properties of the chitosans employed in the scale-up of COS synthesis. Figure 5: Strategies for the preparation of different types of chitooligosaccharides. First, chitosan (1%, _w/v_) was dissolved in sodium acetate buffer (pH 5.0), and hydrolysis was carried out at 35-50 \({}^{\circ}\)C for 48-72 h. In the case of commercial Neutrase 0.8 L, the enzyme was previously dialyzed to remove low-molecular-weight components. Afterward, enzymes and the remaining chitosan were removed by ultrafiltration with a 10 kDa membrane. Acetone or ethanol precipitation is also an alternative to separate water-insoluble components [57]. To obtain a fraction of COS with low molecular weight  
Figure 6: Strategies for the preparation of different types of chitol oligosaccharides. (DP \(\leq\) 6), ultrafiltration with a 1 kDa membrane was applied to some mixtures (option 1). Size-exclusion chromatography, ion-exchange chromatography and tangential filtration are also alternatives to ultrafiltration to fractionate COS [58, 59]. In all cases, salts and small contaminants were eliminated by dialysis with 100 Da tubing, and COS fractions were freeze-dried. This common methodology (with slight modifications) was employed for the depolymerization of chitosan. Depending on the characteristics of the chitosan (DD, PA, DP) and the enzyme used, different reaction outcomes were obtained (Figure 6). Consequently, we were able to prepare a mixture of majorly _fd_COS with MW \(\leq\) 1 kDa, a mixture mainly containing _pa_COS with MW \(\leq\) 1 kDa, and a mixture of _fd_COS and _pa_COS with MW \(\leq\) 10 kDa. Enzyme immobilization is a powerful tool that can facilitate the scaling-up of many biocatalytic processes [60], including those involved in the production of bioactive oligosaccharides [61]. This technology makes enzyme recovery and the reuse of the biocatalysts easier, and it usually increases the operational stability of the enzymes [62]. In this context, we recently immobilized chitinate2 from _T. harzianum_ onto different carriers: (1) magnetic nanoparticles; (2) chitosan beads with glutaraldehyde or genipin linkers [63]. The immobilized biocatalysts showed improved operational stability and were applied to the synthesis of COS from colloidal chitin and several chitosan. We demonstrated that the biocatalysts were easy to handle and separate from the reaction mixture and thus could be reused. We have also investigated the continuous production of COS in packed-bed reactors. For such purpose, we selected a chitosanolytic activity present in a commercial \(\alpha\)-amylase preparation (BAN), which was covalently immobilized onto glyoxal agarose beads [51]. The immobilization did not change the product profile but increased the enzyme thermostability.