## 4 Functional Properties of Chitosan and Derivatives
### Antimicrobial Functions  
CS was shown to inhibit the growth of many microbial species bacteria, yeasts, or other fungi: pathogens, phytopathogens, and spoilage species, for food, medical, or agricultural applications. It displays a high antiseptic spectrum and a high activity compared to other molecules. As a result, it can be used to eliminate microbial contaminants in planktonic or biofilm form, or to simply prevent their multiplication or adhesion in bioactive and antiseptic materials (to wrap foods or seeds, for instance, to immobilize lytic enzymes, to encapsulate vaccines); in solutions to clean material or teeth; to treat plants and crops; or thanks to its high biocompatibility, directly in liquid foods such as fruit juices or wine (Table 2). Depending on the aim of CS employment, the mode and duration of CS treatment, and the total experiment, the medium of the test and the measured effects vary a lot. Minimal inhibitory or minimal lethal concentrations (MIC < MLC) are often determined in liquid or solid media, inhibition diameters are also frequently measured on agar plates, and biofilm prevention or elimination is tested via microplate assays or even directly on medical material, through microbial sedimentation (Table 2). The type of microorganism present (yeast, bacteria, genera, species, and even strain) and their concentration or way of life (biofilm or planktonic) will greatly change the efficient CS concentration needed [18; 87; 89; 100; 107]. Furthermore, the origin, Mw, and DA of the CS or CS derivatives and formulations (nanoparticles, gels or grafted CS) used vary a lot and the conclusions drawn are sometimes conflicting. As a result, the antimicrobial mode of action of CS in liquid media is still highly hypothetical. Microbial inhibition by CS may be the result of a sequence of molecular mechanisms which altogether lead to cell inhibition and death [79; 86; 90; 108; 109]. Besides, some report that CS activity is mostly growth inhibitory and resistant subpopulations exist [110]. Most studies agree that the cationic nature of solubilized CS interferes with the negatively charged residues of the bacterial surface (Figure 6). The subsequent (sometimes controversial) reported effects are:  
1. The formation of a physico-chemical barrier (towards oxygen for example) by adhesion to the cell wall, especially on Gram positive bacteria [18; 111]. As a result, the microbial envelope, which is known to be highly variable depending on the species and strain, particularly with bacteria, plays an important role in CS initial activity. All the elements, such as teichoic acids or external polysaccharides, that can be negatively charged will favor the interactions with CS. However, the exact nature of the surface components that interact with CS has not been accurately defined [83; 103]. Species that contain chitin in their membrane would be less sensitive [82]. The membrane may not be the direct target as liposomes are poorly affected by CS [103; 112]. Proteins or elements emerging from the membrane or the wall seem to be more likely to be recognized. However, the membrane composition and fluidity may influence the subsequent consequences of CS treatment [97; 112];
2. Some studies suggest a subsequent separation of the cell wall from the cell membrane, whilst others only mention a morphological change. Interaction with the membrane leads to altered cell permeability and may disrupt energy generation pathways [89; 108; 112; 113; 114; 115; 116; 117; 118];
3. CS also causes agglutination and precipitation of the undesired microorganisms [77; 106]. Indeed, _E. coli_ was shown to protect itself by forming aggregates in the presence of chitol oligosaccharides