# Chitin and Chitosan in the Alcoholic and Non-Alcoholic Beverage Industry: An Overview
## 4 Chitosan Immobilization
### Chitosan Microorganism's Immobilization  
Microorganisms' immobilization consists of the physical confinement of intact cells to a region of space with conservation of biological activity. The use of these methodologies for alcoholic fermentation offers many advantages over the use of the conventional free yeast cell method. The most studied methods for yeast immobilization include the use of organic supports, mainly alginate. Some advantages of the yeast-immobilization systems include high cell densities, product yield improvement, lowered risk of microbial contamination, better control, and reproducibility of the processes, as well as reuse of the immobilization system for batch fermentation and continuous fermentation technologies [130; 131]. According to [PERSON] and [PERSON] [132], for alcoholic beverage production, the cell carrier or immobilization matrix, must fulfill certain requirements: (i) Large surface, with chemical groups favoring cells to adhere; (ii) Easy to handle and regenerate; (iii) High and retained cell viability and operational stability; (iv) Does not affect catalytic activity; (v) Uniform and controllable porosity to allow the free exchange of substrates, products, cofactors, and gases; (vi) Mechanical, chemical, thermal, and biological stability; (vii) Easy to handle, cost-effective, and amenable to scale-up immobilization technique; (viii) Does not affect product quality. Chitosan can be used in one single layer [133] or double-layer alginate-chitosan beads. Double layer beads have been used for the entrapment of bacterial and yeast cells in batch and continuous fermentation [134], alcoholic fermentations [135], sparkling wine fermentation by the traditional method [136], and in the reduction of the volatile acidity of acidic wines [137]. The presence of an outer chitosan layer improves the mechanical and chemical stabilities of the beads during fermentation and prevents cell leakage from the beads into the medium [137]. In a work performed by [PERSON] et al. [137] concerning the reduction of volatile acidity of acidic wines, immobilized \(S\). _cerevisiae_ cells (8.0 \(\times\) 10\({}^{7}\) cells/mL; double-layer alginate-chitosan beads) were made by ionic gelation method and used in a biological deacidification process. For the cell's immobilization procedure, pre-treated cells were centrifuged (5500 rpm at 4 \({}^{\circ}\)C for 10 min) and resuspended in an aqueous alginate solution (alginic acid sodium salt from brown algae, 2% \(w/v\)). The alginate cell suspension was then dropped into a CaCl\({}_{2}\) solution (0.18 M), with a needle attached to a 10-mL syringe, cooled at 4 \({}^{\circ}\)C, and homogenized by magnetic agitation (100 rpm). The beads remained in the CaCl\({}_{2}\) solution for 1 h (4 \({}^{\circ}\)C) and were then transferred to an Erlenmeyer flask (1000 mL) with 250 mL of low-molecular-weight chitosan (1 or 1.5% \(w/v\)) and incubated for 24 h at 25 \({}^{\circ}\)C with magnetic agitation (100 rpm). Lastly, beads were washed twice with sterilized water and transferred to 250-mL Erlenmeyer flasks. Approximately 100 beads were used in 230 mL of acidic wine. After a successful deacidification process, the beads were analyzed by scanning electron microscopy (SEM). It was possible to verify that the beads had an ellipsoidal shape (Figure 3A) and an average diameter of 500-2000 \(\upmu\)m. SEM analysis also showed that the beads had an irregular surface, and some yeasts were observed near the bead surface, however, the external membrane maintained its integrity (Figure 3B) and there was no cell leakage to the deacidified wine [137]. Coating the alginate beads with chitosan (1.0% \(w/v\)) led to better external membrane integrity after the deacidification process. However, external chitosan integrity was pH-dependent. Total integrity was observed at pH 3.12 while at pH 3.50 some cell leakage occurred [137]. [PERSON] et al. [138] immobilized _Rhodotorula mucilaginous_, producing the ethyl carbamate (EC)-degrading enzyme, urethanase.