**3. Properties, antimicrobial and toxicity of fungi chitosan**

Chitosan has numerous applications in several areas, mainly biomedical and pharmaceutical fields, due to its specific properties Among their properties we highlight the excellent biocompatibility; almost any toxicity to human beings and animals; high bioactivity; biodegradability; reactivity of the group amino deacetylated; selective permeability; polyelectrolyte action; antimicrobial activity; ability to form gel and film; chelation ability and absorptive capacity [36-38]. These peculiar properties provide a variety of applications to the chitosan, such as: drug carrier of controlled release [36], anti-bacterial [37, 39] and anti-acid [38]; inhibition of the bacterial plaque formation and decalcification of dental enamel [2, 10]; promotes the osteogenesis [40]; and promotes the healing of ulcers and lesions [41, 42].

The applicability of chitosan is related to their physical-chemical properties considering the different sources (crustaceans, fungi, and mollusk's) and different processes for extraction and purification cause alterations in the degree of deacetylation, molecular weight, thermal stability and degree of crystallinity of the chitosan. Various derivatives of chitosan differ of the degree of deacetylation, molar weight, arrangement of residual N-acetyl groups in the chain considering the reaction of -NH 2 group at the carbon 2 (C2) or unspecific -OH group in position 3 carbons or 6 (C3 or C6) of the polymer with other functional groups are reported in the literature [15].

Chitosan is a weak base insoluble in water but soluble in dilute aqueous solutions of various acids, the most widely used is acetic acid [43]. The acid solubility is explained by the protonation of the free amino group, characteristic in the chitosan *in natura*, which change to NH2 to NH3+, whereas in alkaline condition, the hydro solubility is due to the formation of carboxylate, from the introduced carboxylic group [19, 44]. The possibility to obtain a variety of polymer derivatives with differences solubility, thermal stability, reactivity with other substances and specificity regarding the binding site, providing several biological applications of the chitosan [15]. Some applications of the chitosan, it is highligh it's the use in the pharmaceutical industry, more specifically related to dental clinic [43].

232 Practical Applications in Biomedical Engineering

*Cunninghamella elegans* Hesseltine and

*Aspergillus niger* Potato Dextrose

*Lentinus edodes* Potato Dextrose

*Zygosaccharomyces rouxii* Yeast Malt

*Candida albicans* Yeast Malt

*Rhizomucor miehei* Sabouraod

*cirnelloides* using yam bean as substrate.

reported in the literature [15].

Broth

Broth

Extract Broth

Extract Broth

dextrose (SDB)

**3. Properties, antimicrobial and toxicity of fungi chitosan** 

**Table 1.** Chitin and chitosan production by Mucorales strains compared with *Cunninghamella* 

promotes the osteogenesis [40]; and promotes the healing of ulcers and lesions [41, 42].

The applicability of chitosan is related to their physical-chemical properties considering the different sources (crustaceans, fungi, and mollusk's) and different processes for extraction and purification cause alterations in the degree of deacetylation, molecular weight, thermal stability and degree of crystallinity of the chitosan. Various derivatives of chitosan differ of the degree of deacetylation, molar weight, arrangement of residual N-acetyl groups in the chain considering the reaction of -NH 2 group at the carbon 2 (C2) or unspecific -OH group in position 3 carbons or 6 (C3 or C6) of the polymer with other functional groups are

Chitosan has numerous applications in several areas, mainly biomedical and pharmaceutical fields, due to its specific properties Among their properties we highlight the excellent biocompatibility; almost any toxicity to human beings and animals; high bioactivity; biodegradability; reactivity of the group amino deacetylated; selective permeability; polyelectrolyte action; antimicrobial activity; ability to form gel and film; chelation ability and absorptive capacity [36-38]. These peculiar properties provide a variety of applications to the chitosan, such as: drug carrier of controlled release [36], anti-bacterial [37, 39] and anti-acid [38]; inhibition of the bacterial plaque formation and decalcification of dental enamel [2, 10];

*Cunninghamella bertholletiae*


Microorganism Substrate Biomass

(g.L-1)

*Mucor circinelloides* Yam bean 20.70 500 64 33 *Cunninghamella elegans* Yam bean 24.30 440 66 32

> Anderson added of 5%NaCl and 6%glucose

Chitin (mg.g-1)

Sugar cane juice 7.70 - 128 30

24.40 388 70 29

9.00 - 107 34

1.4 - 33 34

4.4 - 36 34

1.8 - 44 34

4.1 - 13.67 24

Chitosan (mg.g-1)

Reference

Chitosan has a recognized antimicrobial activity, being this, one of the main properties of the polysaccharide. Several researchers demonstrated that this polysaccharide has antimicrobial action in a great variety of microorganisms, included gram-positive bacteria and various species of yeast [21, 45]. In the literature is described that chitosan acts in the cellular wall of the microorganism modifying the electric potential of the cellular membrane [46]. This polysaccharide also acts potentiating other inhibition drugs, as the chlorhexidine gel, once it increases the drug permanence time action place [47, 48].

In reference [39] the authors report that chitosan has demonstrated low toxicity and the resistance development have not occurred. The antimicrobial action of the chitosan and its derivatives suffers influence from factors, which depending on the performed role may be classified in four main categories: 1. Microbial factors as species, age of the cell); 2. Intrinsic factors of the chitosan as: positive charge density, molecular weight, hydrophobic and hydrophilic characteristics, chelation capacity; 3. Physical state factors (soluble and solid state), and 4. Environmental factors (pH, ionic forces, temperature, and time).

The antimicrobial action mechanism of the chitosan is not yet fully elucidated, being several mechanisms are suggested by the literature. Some authors suggested the amino groups of the chitosan when in contact with physiological fluids are protonated and bind to anionic groups of the microorganisms, resulting in the agglutination of the microbial cells, and growth inhibition [49, 50]. On the other hand, reference [49] report that when interacting with the bacterial cell, the chitosan, promotes displacement of Ca++ of the anionic sites of the membrane, damaging them. Another postulate is the interaction between the positive load of the chitosan and the negative load of the microbial cell wall, because it causes the rupture and loss of important intracellular constituent of the microorganism life. Chitosan with low molecular weight penetrates in the cell and is linked to the microorganism DNA inhibiting the transcription and consequently the translation, whereas the chitosan of high molecular weight acts as a chelate agent, binding to the cell membrane [16].

Authors in reference [47] investigated the relation between antimicrobial activity of the chitosan and the characteristics of the cellular wall of bacteria. They verified that the chitosan is antibacterial agent more efficient to Gram-negative bacteria due the composition of phospholipids and carboxylic acids of the bacterial cellular wall. These results suggest that the effects of the chitosan are distinct in the two types of bacteria: in the case of the gram-positive, the hypothesis is that chitosan of high molecular mass may form films

around the cell that inhibit the absorption of nutrients, while chitosan of low molecular mass penetrates more easily in gram-negative bacteria, causing riots in the metabolism of these microorganisms.

Microbiological Chitosan: Potential Application as Anticariogenic Agent 235

evaluate the potential for irritation according to the method of HET-CAM. Thus, biocompatibility of chitosan was evaluated including the search for signs of inflammation,

3,5 x 106 1.25 5.0 1.25 7.5 1.25 5.0 1.25 5.0

90DA% 3,5 x 106 1.25 2.5 1.25 5.0 1.25 2.5 1.25 2.5

90DA% 3,1 x 104 0.625 1.25 0.625 2.5 0.625 1.25 0.625 1.25

80% DA 2,72x 106 1.25 2.5 1.25 2.5 1.25 2.5 1.25 2.5

90% DA 2,72x 106 1.25 2.5 1.25 2.5 1.25 2.5 1.25 2.5

90% DA 2,3x 104 0.625 1.25 0.625 1.25 0.625 1.25 0.625 1.25

demonstrating the Minimum Inhibitory Concentration (CIM) and Minimum Bactericidal Concentration (CBM) of the chitosan (derivatives from different origins, 80 and 90% of deacetylation degree (DA),

Assays Chmw Clmw SLS 1% Vasoconstriction 0.0±0.0 0.0±0.0 6.0 ±1.0 Hemorrhage 0.0±0.0 0.0±0.0 48 ±3.0 Coagulation 0.0±0.0 0.0±0.0 63 ±3.0 Irritation potential 0.0±0.0 0.0±0.0 17,74 ± 0,4

**Table 4.** Test of chitosan to high (Chmw) and low (Clmw) molecular weight against vasoconstriction,

Assays Inflammation edema neovascularization

1 0.0 0.0 0.0 0.0 0.0 0.0 2 0.0 0.0 0.0 0.0 0.0 0.0 3 0.0 0.0 0.0 0.0 0.0 0.0 4 0.0 0.0 0.0 0.0 0.0 0.0 5 0.0 0.0 0.0 0.0 0.0 0.0 **Table 5.** Test of chitosan to high (ChHMW) and low (ChLMW) molecular weight for inflammation,

Chmw Clmw Chmw Clmw Chmw Clmw

**Table 3.** Studies *in vitro* performed by Microbiology laboratory, UFPB (João Pessoa-PB, Brasil)

molecular weight (MW) g/mL and solubility) to *Streptococcus* species

Non-irritating: 0-0.9 ; slightly irritating: 1-4.9; Irritating: 5-8.9 and Severely irritating: 9-21

hemorrhage and coagulation. Positive control: sodium lauryl sulfate 1% (SLS1%).

*S. mutans S. sanguis S. oralis S. mitis* CIM CBM CIM CBM CIM CBM CIM CBM

Chitosan *Streptococcus* species

edema or neovascularization [55].

Crustaceans 65DA%

edema and neovascularization.

Crustaceaus

Crustaceans

Fungi

Fungi

Fungi

Chitosan from *M. circineloides* was obtained as described the literature [33]. The chitosan with different molecular weight was obtained by methods of extraction. Hight molecular weight of chitosan was obtained by methodology described by Stamford et al [32], and Fai et al.[ 33], and low molecular weight of chitosan by methodology described by Hu et al.[54]. The degree of deacetylation for chitosan was determined by infrared spectroscopy, the molecular weights by viscosity described by Stamford et al [32] and Fai et al.[33]. However, the chitosan with low molecular weight showed antimicrobial activity to Gram positive and negative bacteria than chitosan of high molecular weight (Table 2)


**Table 2.** Minimum Inhibitory Concentration (mg.mL-1) of chitosan from *Mucor circineloides* with different molecular weight: Low molecular weight chitosan with 3.2 x 103 g/mol (ChLMW) and higher molecular weight chitosan 2.72 x 105 g/mol (ChHMW), against pathogenic bacteria

Studies *in vitro* were carried out in the Microbiology laboratory of UFPB to determine the Minimum Inhibitory Concentration (CIM) and the Minimum Bactericidal Concentration (MBC) of chitosan from different origins, and varying the parameters deacetylation degree and molecular weight to oral Streptococcus species. The results showed greater influence of the molecular weight of the polymer on antibacterial activity. The low molecular weight of chitosan have shown higher antimicrobial activity to CIM and CBM, when compared with chitosan gel (soluble in acetic acid 1%), and with higher molecular weight, as described in the Table 3.

In addition, chick embryo chorioallantoic membrane (CAM), is a rapid and inexpensive method for determination of tissue reactions to biomaterials, and was used to evaluate the chitosan toxicity and physiological compatibility. Positive control was used as sodium lauryl sulfate 1%. The method allows prediction of the potential irritation by chitosan was studied.

Fertile hen's eggs at 10º days of incubation at 37°C, obtained from Guaraves Guarabira Aves Ltda, were used in the tests. Five eggs were used for each chitosan solution assayed. On day 10 of incubation, the egg shell above the air space was removed. The exposed membrane was moistened with a drop of 0.9% physiological saline and the saline was removed, uncovering the CAM. An aliquot of 200 µl of chitosan solution was applied on the CAM. Signs of vasoconstriction, hemorrhage and coagulation for 5 minutes were observed to


evaluate the potential for irritation according to the method of HET-CAM. Thus, biocompatibility of chitosan was evaluated including the search for signs of inflammation, edema or neovascularization [55].

234 Practical Applications in Biomedical Engineering

these microorganisms.

the Table 3.

around the cell that inhibit the absorption of nutrients, while chitosan of low molecular mass penetrates more easily in gram-negative bacteria, causing riots in the metabolism of

Chitosan from *M. circineloides* was obtained as described the literature [33]. The chitosan with different molecular weight was obtained by methods of extraction. Hight molecular weight of chitosan was obtained by methodology described by Stamford et al [32], and Fai et al.[ 33], and low molecular weight of chitosan by methodology described by Hu et al.[54]. The degree of deacetylation for chitosan was determined by infrared spectroscopy, the molecular weights by viscosity described by Stamford et al [32] and Fai et al.[33]. However, the chitosan with low molecular weight showed antimicrobial activity to Gram positive and

> Chitosan Chitosan ChLMW ChHMW ChLMW ChHMW

Minimum bactericidal concentration (mg/mL)

negative bacteria than chitosan of high molecular weight (Table 2)

(mg/ml)

molecular weight chitosan 2.72 x 105 g/mol (ChHMW), against pathogenic bacteria

*S. aureus* 1.25 2.5 2.5 5.0 *E. coli* 1.25 2.5 2.5 5.0 *P. aeroginosa* 0.625 1.25 1.5 5.0 *S. mutans* 0.625 1.25 1.5 5.0 **Table 2.** Minimum Inhibitory Concentration (mg.mL-1) of chitosan from *Mucor circineloides* with different molecular weight: Low molecular weight chitosan with 3.2 x 103 g/mol (ChLMW) and higher

Studies *in vitro* were carried out in the Microbiology laboratory of UFPB to determine the Minimum Inhibitory Concentration (CIM) and the Minimum Bactericidal Concentration (MBC) of chitosan from different origins, and varying the parameters deacetylation degree and molecular weight to oral Streptococcus species. The results showed greater influence of the molecular weight of the polymer on antibacterial activity. The low molecular weight of chitosan have shown higher antimicrobial activity to CIM and CBM, when compared with chitosan gel (soluble in acetic acid 1%), and with higher molecular weight, as described in

In addition, chick embryo chorioallantoic membrane (CAM), is a rapid and inexpensive method for determination of tissue reactions to biomaterials, and was used to evaluate the chitosan toxicity and physiological compatibility. Positive control was used as sodium lauryl sulfate 1%. The method allows prediction of the potential irritation by chitosan was studied.

Fertile hen's eggs at 10º days of incubation at 37°C, obtained from Guaraves Guarabira Aves Ltda, were used in the tests. Five eggs were used for each chitosan solution assayed. On day 10 of incubation, the egg shell above the air space was removed. The exposed membrane was moistened with a drop of 0.9% physiological saline and the saline was removed, uncovering the CAM. An aliquot of 200 µl of chitosan solution was applied on the CAM. Signs of vasoconstriction, hemorrhage and coagulation for 5 minutes were observed to

Bacteria Minimum inhibitory concentration

**Table 3.** Studies *in vitro* performed by Microbiology laboratory, UFPB (João Pessoa-PB, Brasil) demonstrating the Minimum Inhibitory Concentration (CIM) and Minimum Bactericidal Concentration (CBM) of the chitosan (derivatives from different origins, 80 and 90% of deacetylation degree (DA), molecular weight (MW) g/mL and solubility) to *Streptococcus* species


Non-irritating: 0-0.9 ; slightly irritating: 1-4.9; Irritating: 5-8.9 and Severely irritating: 9-21

**Table 4.** Test of chitosan to high (Chmw) and low (Clmw) molecular weight against vasoconstriction, hemorrhage and coagulation. Positive control: sodium lauryl sulfate 1% (SLS1%).


**Table 5.** Test of chitosan to high (ChHMW) and low (ChLMW) molecular weight for inflammation, edema and neovascularization.
