TY - JOUR
AU - Hosainpour,, Laila
AB - Abstract We investigated bactericidal and fungicidal properties of chitosan extracted from adults and nymphs from both German cockroach, Blattella germanica (Blattodea: Blattellidae) and American cockroach, Periplaneta americana (Dictyoptera: Blattidae). The cuticle of adults and nymphs extracted from both cockroaches were dried and ground. The powders were demineralized and deproteinized followed by deacetylation using NaOH. Finally, the chitosan yields were examined for antibacterial and antifungal activities. The degree of deacetylation (DD) was different between adults and nymph stages. The antimicrobial effect of American cockroach chitosan (ACC) and German cockroach chitosan (GCC) was tested against four bacteria and four fungi. The extracted chitosans from American cockroach, Periplaneta americana and German Cockroach, Blattella germanica suppressed the growth of Gram-negative/positive bacteria except Micrococcus luteus. The growth of Aspergillus flavus and Aspergillus niger were notability inhibited by the extracted chitosans. The antimicrobial effect of the chitosan depended on the cockroach species, with chitosan of the American cockroach showing more inhibitory effect. This difference may be due to differences in the structure of chitin between the two cockroach species. chitosan, cockroach, antibacterial, antifungal Chitin and its deacetylated derivative chitosan are natural polymers which exhibit varied properties with wide range of applications particularly in biomedical science. Chitosan is the second largest amount of biopolymer after cellulose chitin found in arthropods (Periayah et al. 2016). This compound is a polysaccharide with fibrous structure that is abundantly found in animals. Crustacean and insect exoskeletons are largely constructed by chitosan (Goy et al. 2009). Chitosans have hydrophilic structure which is derived from polysaccharide of chitin. Chitosan adsorption is dependent on the charge density on the surface of the cell and by increasing the adsorption of chitosan, the permeability of the cell membrane is amplified and also the structure of bacteria membrane changed (Huang et al. 2017). This mode of action may describe the antibacterial activities of chitosan which depends on the host microorganism. In addition, it is stated that chitosan is able to penetrate into the nuclei of microorganisms and bind with DNA or inhibit mRNA activity and therefore interrupt protein synthesis. For example, chitosan was found inside of exposed Escherichia coli, and it interrupted bacterial development (Jarmila and Vavrikova 2011). Generally, chitosan has an amine group that chelates metal ions and may cause microbial growth to decrease (Goy et al. 2009, Ahmad et al. 2017). This positive ion bonding is more efficient at high pH because the electron pair on the amine group is donated to metal ions (Bhalkaran and Wilson 2016). Chitosan molecules generally have bactericidal and fungicidal properties (Jarmila and Vavrikova 2011). Molecular weight (MW) and degree of acetylation (DA) are important for the activity of chitosan (Jarmila and Vavrikova 2011, Perinelli et al. 2018). However, chitosan molecules exhibit varying ways of effectiveness on different microorganisms (Carrion-Granda et al. 2016, Mohammadi Amirabad et al. 2018). Generally, chitosan is considered to be bactericidal or bacteriostatic (a growth inhibitor). Chitosan from natural sources such as marine arthropods are widely available (Periayah et al. 2016). It has been shown that the extracted chitosan from the blue crab (Callinectes sapidus) has high bactericidal activity (Kaya et al. 2016b). It has also been remarked that the chitosan with low molecular weight possesses high antimicrobial and antifungal activities (Kaya et al. 2016a). As it has been shown, chitosan (with low or high molecular weight) affects bacteria by the amino protonation and consequently cationic formation on its molecular side chain. These reactions cause the bacterial cells to die (Li et al. 2016). It has been proposed that the bactericidal mechanism of chitosan is based on the presence of a positively charged molecule which may interact with negatively charged membrane of microbial cell and cause ammonia group as a protonate molecule bound to the negative residues by electrostatic forces (Tsai and Su 1999). Therefore, electrostatic interaction of chitosan caused the permeability of bacteria wall to change, and due to internal osmotic imbalance, the growth of bacteria is inhibited (Goy et al. 2009). In addition, chitosan inhibits microorganism growth by other ways, and these molecules can penetrate into the nuclei of the cells and bind with DNA of microorganism and then inhibit mRNA activity and protein synthesis. The chitosan rings extracted from the body segments of a diplopod species (Julus terrestris) can interact with plasmid DNA (Kaya et al. 2016c). However, it has been proposed that chitosan functions by disrupting outer membranes rather than penetrating them (Helander et al. 2001). The aims of current study were to compare the amount of chitosans obtained from the adults and nymphs of the American cockroach, Periplaneta americana (Dictyoptera: Blattaria, Blattidae) or German cockroach, Blattella germanica (Dictyoptera: Blattellidae, Blattidae) and characterize some physicochemical properties of chitin and chitosan extracted from the adults of the American and German cockroach as an alternative chitin and chitosan source after. In addition, the antimicrobial and antifungal properties of the obtained chitosans from both cockroaches were compared. Materials and Methods The cockroaches were reared in an insectary at 25 ± 2°C with a 12h light/dark ratio, and fed on dried bread, date, and water. The adults and last stage of nymphs from colony of both German and American were starved for 48 h to empty the gut contents. The insects then were killed by freezing in −20°C and the remains were washed with water. The dead cockroaches were dried by heating at 50°C for 24 h and then the whole body mechanically grinded and filtered through 20 mesh. Finally, 5 g of powder from each cockroach species was used for chitosan extraction. Extraction of Chitin and Chitosan Initially, and for deproteinization, 5 g of powder from each cockroach were separately treated with 1M HCl at 100°C for 24 h and filtered through a 20-mesh sieve and washed with distilled water and then treated with 50 ml·1 g·100 ml−1 oxalic acid for 3 h at room temperature with moderate stirring. Subsequently, the demineralization procedure was carried out by filtration of the treated samples with a 20-mesh sieve and washed with distilled water. The procedure was followed by mixing each sample with 50 ml 1% sodium hypochlorite solution (1%, w/v), kept at room temperature for 3 h with moderate stirring to remove the color of the samples. The obtained chitins were filtered by 20-mesh sieve, washed with distilled water, dried overnight at 60°C, and then the dry weight was recorded. To remove acetyl group from chitins, the yields were treated with 50% w/w NaOH at 100°C with moderate stirring for 4 h and washed with distilled water and then ethanol. The obtained chitosans were left to dry at room temperature. The whole process of preparing chitin and chitosan of cockroach batches was performed three times. Infrared Spectra Analysis To characterize the composition of chitin and chitosan and the degree of acetylation (DA), the samples were analyzed by infrared (IR) spectrophotometry at 4,000–500 cm−1 with potassium bromide (KBr) pellets. Commercial chitin and chitosan from Sigma–Aldrich were used as standards. In addition, the degree of chitin deacetylation (DD) was measured using a Fourier-transform infrared spectroscopy (FTIR) spectrum (Perkin-Elmer, Norwalk, CA). The wavelength range was 4000–500 cm−1 at a resolution of 4 cm−1 and comparing absorbance of the peaks to that of the reference peak at A1655/A3450 (de Queiroz Antonino et al. 2017). X-Ray Diffraction Analysis XRD analysis was utilized to detect the crystallinity of extracted chitins and chitosan using a Bruker AXS D8 Advance X-ray diffractometer equipped with Ni-filtered Cu Kα radiation (λ = 1.5406 Å). The XRD measurement on the powder samples was done at 5°–40° in steps of 0.1°. The crystalline index (CrI; %) was obtained by the following equation: CrI020 = (I020 − Iam) × 100/I020. Bacterial and Fungal Strains The bacterial strains including Staphylococcus aureus, Pseudomonas aeruginosa, E. coli, and Micrococcus luteus were obtained by the Industrial Research Organization of Iran. The fungi, Candida albicans, Candida auris, Aspergillus flavus, and Aspergillus albican were taken from the laboratory of Mycology, within School of Public Health, Public Health Tehran University. Each bacteria was inoculated into an Erlenmeyer flasks containing 100 ml of sterile nutrient broth (peptone 1%, beef extract 0.5%, NaCl 0.5%, pH 6) and incubated at 37°C for 24 h. Sterile Mueller Hinton Agar (MHA, Himedia) medium was prepared in sterile Petri dishes, incubated at 37°C for 24 h, and used for antibacterial activity tests. Antibacterial and Antifungal Assays The antibacterial activity of the chitosan samples against individual bacteria species was evaluated using the punch diffusion method using Mueller–Hinton agar by measuring zones of inhibition as recommended (Balouiri et al. 2016). Initially the bacteria were taken from medium and resuspended in liquid nutrient broth (NB) medium at a concentration of about 1 × 106 bacteria per ml of NB medium. Then 50 µl of each suspended bacterium was incubated at 37°C for 24 h in the sterile Petri dishes containing Mueller–Hinton agar. Subsequently, 6-mm-diameter wells were punched over the medium plates. A drop of molten Mueller–Hinton agar medium containing 5% defibrinated sheep blood was poured into the bottom of each well to seal it (World Health Organization 2003). Then, 50 µl of the chitosan solution (at concentration of 10 mg/ml in 0.1% acetic acid, pH = 6) was poured into each well. These plates were kept at a temperature of 4°C, which allowed the materials in the wells to completely diffuse into the medium, and then the plates were incubated anaerobically at 37°C for 24 h. Finally, the diameter of the growth inhibition zones surrounding each disk was measured with a metric ruler as recommended previously (World Health Organization 2003, Balouiri et al. 2016, Shanmugam et al. 2016). The test was conducted in triplicate and the inhibition zone was recorded. The antifungal activity of chitosan solution was tested on C. albicans, C. auris, A. flavus, and A. albican. The fungi were harvested from Sabouraud Dextrose (4%) Agar contained Chloramphenicol and resuspended in physiological saline solution at a concentration of 1 × 106 cells/ml. Then 6-mm-diameter wells were punched over the medium plates using sterile Pasteur pipettes and 50 µl of the chitosan solution (at concentration of 10 mg/ml in 0.1% acetic acid, pH = 6) was loaded on each well. The inhibition zone diameters were observed and then measured in mm after 4–7 d of incubation at 30°C (Sanguinetti and Posteraro 2018). Results Extraction of Chitin and Chitosan The amount of chitin and chitosan obtained from 5-g dried cockroach powder varied based on species of cockroach and also the development stage of the insects. Comparatively, the amount of obtained chitosans was near half of the chitins yield in all samples (Table 1). Table 1. The yields of chitin and chitosan obtained from 5-g cockroach powder after deproteinization and deacetylation process Pure chitosan after deacetylation (g) Pure chitin after deproteinization (g) After demineralization (g) Dry weight (g) Stage of insect Cockroach species 0.13 ± 0.02 (2.6%) 0.27 ± 0.04 (5.4%) 3.40 ± 0.23 (68.0%) 5 Nymph Blattella germanica 0.14 ± 0.01 (2.8%) 0.31 ± 0.06 (6.2%) 3.27 ± 0.26 (65.4%) 5 Adult 0.20 ± 0.03 (4.0%) 0.42 ± 0.03 (8.4%) 2.88 ± 0.09 (57.6%) 5 Nymph Periplaneta americana 0.37 ± 0.04 (7.4%) 0.75 ± 0.06 (15.0%) 3.66 ± 0.19 (73.2%) 5 Adult Pure chitosan after deacetylation (g) Pure chitin after deproteinization (g) After demineralization (g) Dry weight (g) Stage of insect Cockroach species 0.13 ± 0.02 (2.6%) 0.27 ± 0.04 (5.4%) 3.40 ± 0.23 (68.0%) 5 Nymph Blattella germanica 0.14 ± 0.01 (2.8%) 0.31 ± 0.06 (6.2%) 3.27 ± 0.26 (65.4%) 5 Adult 0.20 ± 0.03 (4.0%) 0.42 ± 0.03 (8.4%) 2.88 ± 0.09 (57.6%) 5 Nymph Periplaneta americana 0.37 ± 0.04 (7.4%) 0.75 ± 0.06 (15.0%) 3.66 ± 0.19 (73.2%) 5 Adult The process of samples preparation was repeated three times and the numbers indicate the mean of amount yields ± SD. Open in new tab Table 1. The yields of chitin and chitosan obtained from 5-g cockroach powder after deproteinization and deacetylation process Pure chitosan after deacetylation (g) Pure chitin after deproteinization (g) After demineralization (g) Dry weight (g) Stage of insect Cockroach species 0.13 ± 0.02 (2.6%) 0.27 ± 0.04 (5.4%) 3.40 ± 0.23 (68.0%) 5 Nymph Blattella germanica 0.14 ± 0.01 (2.8%) 0.31 ± 0.06 (6.2%) 3.27 ± 0.26 (65.4%) 5 Adult 0.20 ± 0.03 (4.0%) 0.42 ± 0.03 (8.4%) 2.88 ± 0.09 (57.6%) 5 Nymph Periplaneta americana 0.37 ± 0.04 (7.4%) 0.75 ± 0.06 (15.0%) 3.66 ± 0.19 (73.2%) 5 Adult Pure chitosan after deacetylation (g) Pure chitin after deproteinization (g) After demineralization (g) Dry weight (g) Stage of insect Cockroach species 0.13 ± 0.02 (2.6%) 0.27 ± 0.04 (5.4%) 3.40 ± 0.23 (68.0%) 5 Nymph Blattella germanica 0.14 ± 0.01 (2.8%) 0.31 ± 0.06 (6.2%) 3.27 ± 0.26 (65.4%) 5 Adult 0.20 ± 0.03 (4.0%) 0.42 ± 0.03 (8.4%) 2.88 ± 0.09 (57.6%) 5 Nymph Periplaneta americana 0.37 ± 0.04 (7.4%) 0.75 ± 0.06 (15.0%) 3.66 ± 0.19 (73.2%) 5 Adult The process of samples preparation was repeated three times and the numbers indicate the mean of amount yields ± SD. Open in new tab A higher yield of chitin and chitosan (from 5-g dried body cockroach) was obtained from the American cockroach. Generally, adults of the cockroaches had more chitin and chitosan than nymphs. The ratio of chitosan per 5-g dried body in adults and nymphs of American and German cockroaches were 7.4, 4.0, 2.8, and 2.6% relatively (Table 1). Infrared Spectra Analysis The amount of chitin and chitosan obtained from German and American cockroaches are presented in Table 1. Comparatively, the amount of chitosan was near half of the weight of chitins yield in all samples (Table 1). The degree of deacetylation (DD) of chitin for nymph and adult of American cockroach was 39.2 and 37.3%, respectively, and for nymph and adult of German cockroach 31 and 32.1%, respectively (Table 2). Table 2. The degree of deacetylation of the cockroaches’ chitin using infrared spectra analysis at 4,000–500 cm−1 Cockroach species Cockroach stages A1655 A3450 DD Blattella germanica Nymph 0.146 0.177 39.2 Adult 0.155 0.183 37.3 Periplaneta americana Nymph 0.111 0.151 31.0 Adult 0.115 0.148 32.1 Cockroach species Cockroach stages A1655 A3450 DD Blattella germanica Nymph 0.146 0.177 39.2 Adult 0.155 0.183 37.3 Periplaneta americana Nymph 0.111 0.151 31.0 Adult 0.115 0.148 32.1 Open in new tab Table 2. The degree of deacetylation of the cockroaches’ chitin using infrared spectra analysis at 4,000–500 cm−1 Cockroach species Cockroach stages A1655 A3450 DD Blattella germanica Nymph 0.146 0.177 39.2 Adult 0.155 0.183 37.3 Periplaneta americana Nymph 0.111 0.151 31.0 Adult 0.115 0.148 32.1 Cockroach species Cockroach stages A1655 A3450 DD Blattella germanica Nymph 0.146 0.177 39.2 Adult 0.155 0.183 37.3 Periplaneta americana Nymph 0.111 0.151 31.0 Adult 0.115 0.148 32.1 Open in new tab According to IR spectra graph, the chitin and chitosan molecules extracted from both cockroaches contain similar stretching and bending vibrations bands with different IR spectra graphs (Fig. 1) showing reduced peaks due to absorption, which indicates a loss of acetyl groups and the occurrence of chitin deacetylation. Three major amide bands were characterized by the absorption bands of spectra at 1,680 (amid I stretching in C=O), 1,580 (NH2 binding), and 1,330 cm−1 (amid III in C-N; Badawy and Mohamed 2015, Jin et al. 2017). In addition, the absorption band at 3,435 cm−1 for German cockroach chitosan and 3,450 cm−1 for American cockroach chitosan indicates that OH groups are present in the molecules (Fig. 1). Fig. 1. Open in new tabDownload slide IR spectra of chitin and chitosan structure from Blattela germanica (German cockroach) (A) and Periplaneta americana (American cockroach) (B). Fig. 1. Open in new tabDownload slide IR spectra of chitin and chitosan structure from Blattela germanica (German cockroach) (A) and Periplaneta americana (American cockroach) (B). To compare amide bands present in chitosan of both cockroaches, the spectra graph showed the presence of absorption bands at 2,920 cm−1, 2,890 cm−1 for German cockroach and at 2,950 cm−1 for American cockroach. These absorption bands may be caused by the stretching and bending vibrations of C–H presence in chitosan molecules of both cockroaches. The absorption band at 1,035 cm−1 also indicates C–O–C stretching vibrations presence in chitosan molecules (Fig. 1). The bands characteristic to α-chitin was decreased in the sample extracted from German cockroach with reduction in the amide component. Additionally, Fig. 1 shows the existence of a chitosan absorption band of the stretching as well as bending vibration of C-H of chitosan plus the stretching vibrations of C–O–C, especially in the sample of German cockroach. X-Ray Diffraction Analysis Figure 2 shows the XRD pattern for α-chitin, with high reflections at 9.0° and 19.9°, and slight reflections at 12.7°, 24.8° and 26.1° (Liu et al. 2012). Both cockroach samples had similar XRD patterns with appearance of additional small peak at 22.5°. This peak confirmed the presence of chitosan. Fig. 2. Open in new tabDownload slide X-ray diffraction pattern of Blattela germanica (German cockroach) (A), and Periplaneta americana (American cockroach) (B), and standard chitin (C). Fig. 2. Open in new tabDownload slide X-ray diffraction pattern of Blattela germanica (German cockroach) (A), and Periplaneta americana (American cockroach) (B), and standard chitin (C). Antibacterial and Antifungal Assays Bactericidal and fungicidal activities of obtained chitosan are represented in Tables 3 and 4. The results of these assays show that the yield of chitosan obtained from both cockroach species affects Staphylococcus aureus, Pseudomonas aeruginosa, and E. coli but not M. luteus. When comparing with commercial chitosan, the chitosans obtained from adults and nymphs of German cockroaches showed relatively high inhibition activity of more than 10-mm clear zone against E. coli and less inhibition zone of 7 to 10 mm against S. aureus and P. aeruginosa, whereas there was no activity against M. luteus (Table 3). The inhibition zone of more than 10 mm diameter was observed against E. coli, S. aureus, and P. aeruginosa in chitosans extracted from adults and nymphs of American cockroaches, whereas no inhibition activity was noticed against M. luteus (Table 3). Table 3. Antibacterial activities yield chitosan extracted from adults and fifth-instar stage of German and American cockroaches was measured based on diameter of growth inhibition zone (mm) Micrococcus luteus (+) Staphylococcus aureus (+) Pseudomonas aeruginosa (−) Escherichia coli (−) Stage of insect Cockroach species – 7–10 7–10 10–12 Nymph Blattella germanica – 7–10 7–10 10–12 Adult – 10–12 10–12 10–12 Nymph Periplaneta americana – 10–12 10–12 10–12 Adult – 10–12 10–12 7–10 Commercial chitosan Micrococcus luteus (+) Staphylococcus aureus (+) Pseudomonas aeruginosa (−) Escherichia coli (−) Stage of insect Cockroach species – 7–10 7–10 10–12 Nymph Blattella germanica – 7–10 7–10 10–12 Adult – 10–12 10–12 10–12 Nymph Periplaneta americana – 10–12 10–12 10–12 Adult – 10–12 10–12 7–10 Commercial chitosan The diameter of zone of inhibition (mm) was measured and recorded as recommended by World Health Organization 2003. The experiment was conducted in triplicate. Open in new tab Table 3. Antibacterial activities yield chitosan extracted from adults and fifth-instar stage of German and American cockroaches was measured based on diameter of growth inhibition zone (mm) Micrococcus luteus (+) Staphylococcus aureus (+) Pseudomonas aeruginosa (−) Escherichia coli (−) Stage of insect Cockroach species – 7–10 7–10 10–12 Nymph Blattella germanica – 7–10 7–10 10–12 Adult – 10–12 10–12 10–12 Nymph Periplaneta americana – 10–12 10–12 10–12 Adult – 10–12 10–12 7–10 Commercial chitosan Micrococcus luteus (+) Staphylococcus aureus (+) Pseudomonas aeruginosa (−) Escherichia coli (−) Stage of insect Cockroach species – 7–10 7–10 10–12 Nymph Blattella germanica – 7–10 7–10 10–12 Adult – 10–12 10–12 10–12 Nymph Periplaneta americana – 10–12 10–12 10–12 Adult – 10–12 10–12 7–10 Commercial chitosan The diameter of zone of inhibition (mm) was measured and recorded as recommended by World Health Organization 2003. The experiment was conducted in triplicate. Open in new tab All obtained chitosan form adults and nymphs of both cockroaches did not show fungicidal activities against C. albicans or C. auris. The chitosan of adults and nymphs of German cockroach showed inhibition activities of 7- to 10-mm clear zone against A. flavus and A. albican. The inhibition zone of 7 to 10 mm was noticed in chitosan extracted from nymphs of American cockroach, whereas in comparing with commercial chitosan, the adult chitosan showed relatively good inhibition activity of more than 10-mm clear zone against both A. flavus and A. albican (Table 4). Table 4. Antifungal activities yield chitosan extracted from adults and fifth-instar stage of German and American cockroaches was measured based on diameter of growth inhibition zone (mm) Aspergillus albican Aspergillus flavus Candida auris Candida albicans Stage of insect Cockroach species 7–10 7–10 – – Nymph Blattella germanica 7–10 7–10 – – Adult 7–10 7–10 – – Nymph Periplaneta americana 10–12 10–12 – – Adult 7–10 7–10 – – Commercial chitosan Aspergillus albican Aspergillus flavus Candida auris Candida albicans Stage of insect Cockroach species 7–10 7–10 – – Nymph Blattella germanica 7–10 7–10 – – Adult 7–10 7–10 – – Nymph Periplaneta americana 10–12 10–12 – – Adult 7–10 7–10 – – Commercial chitosan The diameter of zone of inhibition (mm) was measured and recorded as recommended by World Health Organization 2003. The experiment was conducted in triplicate. Open in new tab Table 4. Antifungal activities yield chitosan extracted from adults and fifth-instar stage of German and American cockroaches was measured based on diameter of growth inhibition zone (mm) Aspergillus albican Aspergillus flavus Candida auris Candida albicans Stage of insect Cockroach species 7–10 7–10 – – Nymph Blattella germanica 7–10 7–10 – – Adult 7–10 7–10 – – Nymph Periplaneta americana 10–12 10–12 – – Adult 7–10 7–10 – – Commercial chitosan Aspergillus albican Aspergillus flavus Candida auris Candida albicans Stage of insect Cockroach species 7–10 7–10 – – Nymph Blattella germanica 7–10 7–10 – – Adult 7–10 7–10 – – Nymph Periplaneta americana 10–12 10–12 – – Adult 7–10 7–10 – – Commercial chitosan The diameter of zone of inhibition (mm) was measured and recorded as recommended by World Health Organization 2003. The experiment was conducted in triplicate. Open in new tab Discussion In the current study, chitin and chitosan of adults and nymphs of American and German cockroach were prepared and partially characterized and the DD calculated. Subsequently, bactericidal and fungicidal activities of the chitosan yield was investigated. Physicochemical properties as well as rheological characteristics and surface morphology of chitosans derived from cicada slough, silkworm chrysalis, mealworm, and grasshopper have been compared with shrimp shell chitosan and the results indicated that the activities of insects’ chitosan are quite different from shrimp chitosan (Luo et al. 2019). In the current study, we also found that the antimicrobial activity of extracted chitosans was different between cockroach species. In addition, the degree of polymerization (Fig. 1) and crystallinity of chitin and chitosan (Fig. 2) of American and German cockroaches were different. The viscosity and flow behavior of chitosan are dependent on chitosan DD. On the other hand, the chitosan antimicrobial activities are depended on DD (Omura et al. 2003, Ju Jung et al. 2010). In this regard, it has been shown that insect chitosan is more viscous than shrimp shell chitosan, which has a high degree of deacetylation. Generally, partial of bacteriostatic and bactericidal activity of chitosan is dependent on viscosity, with low viscosity being more effective (Dragland et al. 2016, Luo et al. 2019). Because the chitosan DD obtained from both cockroaches was different (Table 2), this may be at least partially the cause of the difference in antibacterial properties of chitosan. The primary component of the insect integument has generally been recognized as an effective alternative source with organic materials such as chitin, particularly the cuticle which has low level of inorganic materials (Kaya et al. 2018). In comparison with commercial chitosan extraction from shrimp (de Queiroz Antonino et al. 2017), the yields of chitosan obtained from the cockroaches during demineralization and deacetylation procedure were high. Although insects such as cockroaches can be rich and available sources of chitin and chitosan, rearing them may be a limiting factor for industrialization. However, the chitins of both cockroach species showed similar physiological properties, and it seems they are suitable for chitosan production. The DD of chitin from all cockroaches was relatively high as the weight of obtained chitosans from all samples was near half of the chitins (Table 1). Although the chitin and chitosan yield of the American cockroach was relatively higher, the DD of chitin to chitosan was slightly more in the samples from the German cockroach. These results may be due to the difference between molecular weight of chitosan extracted from both cockroaches. A similar result was previously reported by Badawy and Mohamed (Badawy and Mohamed 2015). In the current study, 5 g of powder from adults and nymph of each cockroach was used to extract chitosan. The results showed that the yield and purity of chitosan extracted from 5 g of chitin from the American cockroach was dependent on the stage of insect development, whereas no significantly difference was found between the amount of chitosan obtained from 5 g of chitin from adults and nymphs of the German cockroach. These results may be due to the structure of chitin in the cuticle of cockroaches. The previous study on larva and adult Colorado potato beetles (Leptinotarsa decemlineata) also showed that the chitosan yield and purity of chitin from adults was higher than that in the larvae. Therefore, the antimicrobial and antioxidant activities of adults chitosan were higher (Kaya et al. 2014). In the present study, we found that the extracted chitosan from both cockroaches suppressed the growth of Gram-positive and Gram-negative bacteria as well as molds. Generally, several factors may have affected the strength of the bactericidal and fungicidal activity of chitosan including the molecular weight of chitosan, deacetylation degree, concentration of chitosan in solution, and pH of medium culture (Jarmila and Vavrikova 2011). The chitosan oligomers with molecular weight less than 5000 kDa showed the antimicrobial effect (Varun et al. 2017), and the molecular weight of American cockroach chitosan has been reported to be between 212 and 230 kDa (Kim et al. 2017). It has been stated that the antibacterial effect of chitosan depends more on its molecular weight rather than the DD (Li et al. 2016), and low molecular weight showed more inhibitory effect on Gram-positive and Gram-negative bacteria as well as the yeast (Tikhonov et al. 2006). In the present study, although the DD of ACC was lower than GCC, the ACC showed slightly more growth inhibitory effect on P. aeruginosa and S. aureus in comparison with GCC and these may be due to the difference in the molecular structure of chitosan of the cockroach species. However, the antimicrobial and antifungal properties of chitosan are dependent on different factors and this may lead to varying results expressed by different authors. Therefore, the bactericidal and fungicidal activity of chitosan is somewhat controversial. It is stated that chitosan has more bactericidal activity on Gram-positive bacteria than Gram-negative bacteria (Goy et al. 2009, Zhao et al. 2018). In contrast, some authors stated that due to hydrophilicity of chitosan, Gram-negative bacteria are more sensitive to chitosan than Gram-positive bacteria (Helander et al. 2001, Jarmila and Vavrikova 2011). In the present study, among the tested bacteria, Gram-negative bacteria were very sensitive to the cockroach chitosans. This may be attributed to the the high degree of deacetylation of extracted chitosans. We found inhibition activity of extracted chitosans against A. flavus and A. albican but not the yeasts, C. albicans and C. auris. The fungistatic activity of chitosan against different Aspergillus species has been reported previously (Cota-Arriola et al. 2011, Dias et al. 2018, Shehata et al. 2018). Generally, the chitosan has been considered an effective fungistatic against plant fungus (Lopez-Moya and Lopez-Llorca 2016). Chitosan molecules penetrate inside hyphae of fungi and hamper the activity of essential enzymes which are essential for the fungus growth (Park et al. 2008, Devarayan et al. 2015). The important factors on degradation are the intensity of chitosan activities on fungal cells which are pH, chitosan concentration, and the degree of deacetylation (Jarmila and Vavrikova 2011). Conclusion In the current study, we found relatively high degree of deacetylation of chitosan obtained from German cockroach, Blattela germanica and American cockroach, Periplaneta americana. The weight of chitosan yield as well as the degree of deacetylation depended on the stage of the insects and species. We found that cockroach chitosan has stronger activity against bacteria than fungi. Moreover, the bactericidal and fungicidal activity of chitosan, and the yield, was considerable, and thus, cockroach chitosan could be used as a powerful and safe natural antimicrobial agent. Acknowledgments We are grateful to the Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences. 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TI - Antibacterial/Antifungal Activity of Extracted Chitosan From American Cockroach (Dictyoptera: Blattidae) and German Cockroach (Blattodea: Blattellidae)
JF - Journal of Medical Entomology
DO - 10.1093/jme/tjz082
DA - 2019-09-03
UR - https://www.deepdyve.com/lp/oxford-university-press/antibacterial-antifungal-activity-of-extracted-chitosan-from-american-h9Qeph1sMW
SP - 1208
VL - 56
IS - 5
DP - DeepDyve
ER -