# Chitin and Chitosan in the Alcoholic and Non-Alcoholic Beverage Industry: An Overview
## 4 Chitosan Immobilization
### Chitosan Enzyme Immobilization  
Enzymes are functional biological macromolecules able to catalyze chemical reactions at very high rates and with high molecular precision. Enzymes are used by many winemakers to improve extraction, enhance aromas, and block malotactic fermentation (MLF). For instance, in white wines, monoterenes are the most important compounds responsible for the aroma, most of these compounds are present in grapes as nonvolatile odorless glycoconjugates. Therefore, enzymatic hydrolysis has long been proposed as an alternative for the efficient release of aromatic compounds at the mild conditions of winemaking [140]. Commercial preparations of soluble enzymes from _Apergillus niger_ with \(\alpha\)-l-arabinofuranosidase, \(\alpha\)-l-rhannosidase, and \(\beta\)G (\(\beta\)-D-glucosidase) activities have been used in alcoholic fermentation, usually added at the end of the fermentation stage [141]. These glycosases are quickly inactivated in winemaking conditions media, making them less efficient. Moreover, catalyst residues remain in the final wine, thus requiring the addition of a fining agent, to stop the reaction [142]. Due to these disadvantages, the co-immobilization of glycosases has been proposed, increasing the stability of the enzymes, and recovering the biocatalyst from the medium [143]. Figure 3: SEM image of _S. cerevisiae_ cells, entrapped in double-layer alginate–chitosan beads: (**A**) double-layer bead with alginate 2% (\(w/v\)) and chitosan 1.0% (\(w/v\)) (\(\times\)600); (**B**) external layer (chitosan 1.0% \(w/v\)) of a bead after deacidification, pH 3.12 (\(\times\)2500). For SEM analysis, double-layer alginate–chitosan beads, were dehydrated (5 min in 70 % \(v/v\) ethanol followed by 5 min in absolute ethanol) and dried using critical point dehydration. Beads were then mounted into aluminum stubs and coated with a small amount of gold/palladium in a Polaron Range SC7620 Sputter Coater (Quorum Technologies Ltd., Ashford, Kent, UK). SEM analysis was performed in a Philips-FEI/Quanta 400 scanning electron microscope (FEI, Hillsboro, OR, USA). Cross-linked enzyme aggregates (CLEAs) allow for the immobilization of enzymes in their macromolecular matrix [144]. This technique has emerged in recent years as a substitute for carrier-bound enzymes. A relevant characteristic of this method is that it permits the co-immobilization of different enzymes, giving rise to the so-called comb-CLEAs that can be effectively applied in cascade reaction systems [144; 145; 146]. Good results have been obtained in the co-immobilization of glycosylases as comb-CLEAs for the enhancement of aromatic compounds in white wine [147]. But the small and variable particle size (5 to 50 \(\upmu\)m) delays their recovery and prevents their application in industrial processes, namely winemaking. Therefore, the idea of entrapping the comb-CLEAs in a polymeric carrier will allow recovering the biocatalyst. [PERSON] et al. [148] studied the effect of crosslinking reagents and crosslinking time on the specific activity and stability of entrapped comb-CLEAs of \(\beta\)G and \(\alpha\)-L-arabinofuranosidase in polymeric chitosan beads, with an average diameter of 1.24 mm. The beads retained full activity after 91 days of incubation under winemaking conditions, making them suitable for aroma enhancement in wines. This interesting work of [PERSON] et al. [148] shows that chitosan, due to its properties (high biodegradability, affinity to proteins, non-toxicity, inert) is suitable for food production and due to its electrical charges [in aqueous acidic media at pH! 6.5, is positively charged (NH3+ groups)], reacts with polyanionic compounds [149], offering exceptional gel-forming properties to be used for protein immobilization [150; 151]. Enzyme immobilization can also be used for several other purposes. It can be used to protect wines and other fermented and non-fermented beverages from spoilage. [PERSON] et al.