**17. Antimicrobial activity**

The exact mechanism by which chitosan exerts its antimicrobial activity is currently unknown, it has been suggested that the polycationic nature of this biopolymer that forms from acidic solutions below pH 6.5 is a crucial factor. Thus, it has been proposed that the positively charged amino groups of the glucosamine units interact with negatively charged components in microbial cell membranes, altering their barrier properties, and thereby

preventing the entry of nutrients or causing the leakage of intracellular contents [95-100]. Another reported mechanism involves the penetration of low- molecular weight chitosan into the cell, binding to DNA and the subsequent inhibition of RNA and protein synthesis [101]. Chitosan has also been shown to activate several defence processes in plant tissues and it inhibits the production of toxins and microbial growth because of its ability to chelate metal ions [102,103].

EI-Ghaouth et. al.(1992) have proposed possible antibacterial actions of chitosan and its derivatives [104]. They asserted that chitosan reacted with the cell surface, altered cell permeability, and further prevented the entry of material or caused the leakage of material. However, no evidence has been provided to demonstrate the relationship between the antibacterial activity of chitosan and the surface characteristics of the bacterial cell wall. Antimicrobial activity of chitosan has been demonstrated against many bacteria, filamentous fungi and yeasts [105-108]. Chitosan has wide spectrum of activity and high killing rate against Gram-positive and Gram-negative bacteria, but lower toxicity toward mammalian cells [109,110]. Variations in chitosan's antimicrobial efficacy arise from various factors. These factors can be classified into four categories as follows: (1) microbial factors, related to microorganism species and cell age; (2) intrinsic factors of chitosan, including positive charge density, Mw, concentration, hydrophilic/hydrophobic characteristic and chelating capacity; (3) physical state, namely water-soluble and solid state of chitosan; (4) environmental factors, involving ionic strength in medium, pH, temperature and reactive time [111].

Although owning a broad spectrum of antimicrobial activity, chitosan exhibits different inhibitory efficiency against different fungi, Gram-positive and Gram-negative bacteria. Chitosan exerts an antifungal effect by suppressing sporulation and spore germination [112]. In contrast, the mode of antibacterial activity is a complicating process that differs between Gram positive and Gram-negative bacteria due to different cell surface characteristics.

Based on the available evidences, bacteria appear to be generally less sensitive to the antimicrobial action of chitosan than fungi. The antifungal activity of chitosan is greater at lower pH values [113]. For a given microbial species, age of the cell can influence antimicrobial efficiency.

Chitosan has a broad spectrum of unique biological activities, including its ability to induce resistance to viral infections in plants, inhibit viral infection in animal cells, and prevent the development of phage infection in infected microbial cultures. High polymeric chitosan, when added to a nutrient medium, prevents the accumulation of the infectious phage progeny in infected cultures of Gram-positive and Gram-negative organisms. The yield of infectious DNA containing phage can decrease by several orders of magnitude in the presence of chitosan [114, 115]. The observed effect also depends on the concentration, degree of polymerization, and molecular structure of chitosan. Thus, chitosans with a polymerization degree of 250 and higher were much more effective in inhibiting coliphage infection than their fragments with a polymerization degree of 15–19. On the other hand, chitosan oligomers were more effective than their polymeric precursor in inhibiting the replication of 1-97A phage in *Bacillus thuringiensis* cultures. Factors determining such strong differences are currently unclear. Anionic derivatives of chitosan, such as 6-O-sulfate and Nsuccinate-6-O-sulfate, caused no effect on phage infection [114]. This finding showed that the positive charge of a chitosan molecule is important for inhibition of phage infection.

16 The Complex World of Polysaccharides

metal ions [102,103].

time [111].

antimicrobial efficiency.

preventing the entry of nutrients or causing the leakage of intracellular contents [95-100]. Another reported mechanism involves the penetration of low- molecular weight chitosan into the cell, binding to DNA and the subsequent inhibition of RNA and protein synthesis [101]. Chitosan has also been shown to activate several defence processes in plant tissues and it inhibits the production of toxins and microbial growth because of its ability to chelate

EI-Ghaouth et. al.(1992) have proposed possible antibacterial actions of chitosan and its derivatives [104]. They asserted that chitosan reacted with the cell surface, altered cell permeability, and further prevented the entry of material or caused the leakage of material. However, no evidence has been provided to demonstrate the relationship between the antibacterial activity of chitosan and the surface characteristics of the bacterial cell wall. Antimicrobial activity of chitosan has been demonstrated against many bacteria, filamentous fungi and yeasts [105-108]. Chitosan has wide spectrum of activity and high killing rate against Gram-positive and Gram-negative bacteria, but lower toxicity toward mammalian cells [109,110]. Variations in chitosan's antimicrobial efficacy arise from various factors. These factors can be classified into four categories as follows: (1) microbial factors, related to microorganism species and cell age; (2) intrinsic factors of chitosan, including positive charge density, Mw, concentration, hydrophilic/hydrophobic characteristic and chelating capacity; (3) physical state, namely water-soluble and solid state of chitosan; (4) environmental factors, involving ionic strength in medium, pH, temperature and reactive

Although owning a broad spectrum of antimicrobial activity, chitosan exhibits different inhibitory efficiency against different fungi, Gram-positive and Gram-negative bacteria. Chitosan exerts an antifungal effect by suppressing sporulation and spore germination [112]. In contrast, the mode of antibacterial activity is a complicating process that differs between Gram positive and Gram-negative bacteria due to different cell surface characteristics.

Based on the available evidences, bacteria appear to be generally less sensitive to the antimicrobial action of chitosan than fungi. The antifungal activity of chitosan is greater at lower pH values [113]. For a given microbial species, age of the cell can influence

Chitosan has a broad spectrum of unique biological activities, including its ability to induce resistance to viral infections in plants, inhibit viral infection in animal cells, and prevent the development of phage infection in infected microbial cultures. High polymeric chitosan, when added to a nutrient medium, prevents the accumulation of the infectious phage progeny in infected cultures of Gram-positive and Gram-negative organisms. The yield of infectious DNA containing phage can decrease by several orders of magnitude in the presence of chitosan [114, 115]. The observed effect also depends on the concentration, degree of polymerization, and molecular structure of chitosan. Thus, chitosans with a polymerization degree of 250 and higher were much more effective in inhibiting coliphage infection than their fragments with a polymerization degree of 15–19. On the other hand, chitosan oligomers were more effective than their polymeric precursor in inhibiting the It has been suggested that chitosan can inhibit the replication of bacteriophages by several mechanisms: it can (a) decrease the viability of cultured bacterial cells, (b) neutralize the infectivity of mature phage particles in the inoculum and/or daughter phage particles, and (c) block the replication of the virulent phage [114].

The condition of the phage culture is known to be of paramount importance for the development of phage infection because phages can reproduce only in viable cells. However, there is evidence that chitosan displays a bactericidal activity toward many microbial species including *Escherichia coli* [116, 117] and species of the genus Bacillus [118]. Chitosan, because of its polycationic nature, binds to the external membrane of Gram negative microorganisms by electrostatic forces, which is demonstrated in experiments with core phosphate groups of lipid A, thereby decreasing the potency of endotoxin. It was suggested that the antibacterial effects of chitosan and many other cationic agents are based on their ability to increase the permeability of the outer membrane of Gram negative microorganisms to an extent incompatible with their viability [116-119].

Great amount of literature support the essential importance of polycationic structure in antimicrobial activity. A higher positive charge density leads to strong electrostatic interaction. Therein, the positive charge is associated with DD or degree of substitution (DS) of chitosan or its derivatives, which affect positive charge density. Concerning chitosan derivatives, antimicrobial activity mostly depends on DS of the grafting groups.

There are several works regarding the influence of the molecular weight of chitosan on its antimicrobial properties [120-126]. Some of them have demonstrated that COS (chitooligosaccharides), which are soluble in water, were the least effective in terms of biocide properties [124-126]. Recent work carried out by Qin et al. 2006, on the evaluation of chitosan solutions against the growth of *Candida albicans, Escherichia coli* and *Staphylococcus aureus*, it has shown that only water insoluble chitosan in organic acidic solutions, i.e. chitosonium salts, exhibit efficient biocide properties. On the other hand, a research performed by Fernandes et al, 2008 showed that the growth of *E. coli* was markedly inhibited by COS, and this inhibition decreased slightly as molecular weight increased. In another work performed by Fernandez-Saiz et al. 2009, changes in molecular weight of the chitosan materials tested, i.e. 310–375 and 50–190 kDa, did not lead to significant variations in biocide properties [127, 128,129].

Concerning DD, there are several works that consider this feature, and there is no doubt that the antimicrobial properties of chitosan increased with this variable. The antimicrobial performance tends to increase upon an increase in the DD of chitosan, which is related to an increase in the positive charge of the polymer [130-133].

**Figure 6.** Staphylococcus bacteria (a), covered with chitosan (b)
