Source of materials
Nile perch scale wastes were obtained from a local fish market while Banana Weevils and edible mushroom were collected from National Agricultural Research Laboratories Kawanda. Carbapenem resistant E. coli and K. pneumoniae were a kind donation from Department of Microbiology, College of Health Sciences, Makerere University.
Study design and site
This was a laboratory-based study conducted from College of Veterinary Medicine Animal Resources and Biosecurity, Makerere University, iThemba LABs, Cape Town and University of South Africa (UNISA). Isolation of chitin and chitosan was performed from the Pharmacology Laboratory while chitosan antibacterial activity was evaluated from the Central Diagnostic Laboratory. Characterization of chitosan was conducted from iThemba LABs and UNISA.
Banana weevils, Nile perch scales and mushrooms were cleaned using running tap water and finally rinsed in distilled water. The cleaned weevils, scales and mushrooms were oven dried at 60°C for 1 week and then ground to powder using an electric miller. Chitin was extracted from the resultant powder following Musarrat et al.,  adjusted procedure. Demineralization was carried out by treatment of the banana weevil, Mushroom and Nile perch scale powders with 1.0M HCl solution at ambient temperature for 24 hours with a solution to solid ratio of 15 mL/g. This step was replicated ten times. The mixture was centrifuged at a speed of 4000 x g for 10 minutes using Thermo Scientific™ Fiberlite™F6-10x1000 LEX roto centrifuge. The resultant sediment was washed with distilled deionized water until neutral pH was achieved. The sediment was deproteinized by adding 15 mL of 1.0 M sodium hydroxide and then heated at 80°C for 8 hours. This treatment was repeated four times. The resultant chitin was then washed with distilled deionized water to neutrality. Finally, chitin was washed by boiling in hot absolute ethanol and later in absolute acetone in a water bath for 10 minutes to remove any impurities. The purified chitin was dried in a vacuum oven at 50°C to constant weight. The chitin content was determined by computing the weight differences between the raw materials and that of the chitin obtained after acid and alkaline treatments.
Chitin was treated with 50% NaOH (15 mL/g) at 90°C for 10 hours with continuous mixing using a magnetic stirrer after which the resultant mixture was centrifuged at 4000 x g for 10 minutes using a Thermo Scientific™ Fiberlite™F6-10x1000 LEX roto centrifuge. The residue was washed with hot distilled deionized water until neutrality. The obtained chitosan was dried in a vacuum oven at 40°C for 48 hours. All the chitosan samples were purified by dissolving in 1% acetic acid and reprecipitated in 20% NaOH solution followed by centrifugation at 6000 x g for 10 minutes using a Thermo Scientific™ Fiberlite™F6-10x1000 LEX roto centrifuge to sediment chitosan. The sedimented chitosan was washed with distilled deionized water until a neutral pH, lyophilized and stored at -20°C until further use. The percentage chitosan yield was computed as a fraction of weight of dry chitosan and dry chitin from which it was generated.
Characterization of chitosan
Estimation of the Ash Content Chitosan
The ash content of each chitosan sample was gravimetrically estimated after the pyrolysis of 1 g in a muffle furnace at 650°C for 5 hours. This procedure was done in triplicates and the mean ash content computed. The ash content was computed as a fraction of mass of the residue (MR) and mass of the sample (MS) using the formula that follows;
where MS and MR are the weights (in grams) of the initial sample of chitosan and residue respectively .
Moisture content of Chitosan
The water content of chitosan samples was assessed by gravimetric technique. This method involved drying of the samples until a constant mass in a vacuum oven at 105°C for 24 hours. This experiment was done thrice and the average moisture content was calculated. The water content was computed as the difference between the wet weight (WW) and dry weight (DW) of samples per gram using the formula that follows:
Where WW is the wet weight of samples and DW is the dry weight of samples after oven drying .
Determination of chitosan solubility
A 1% solution of chitosan was constituted by adding 0.1 g (W1) of each chitosan sample previously dried at 105°C for 24 hours into 10 ml of 1% acetic acid in 15 ml falcon tube. The tubes were sealed and placed in an overhead shaker running at 60 rpm for 24 hours. The solution was centrifuged at10,000 x g for 15 minutes using a Thermo Scientific™ Fiberlite™F6-10x1000 LEX roto centrifuge. The liquid phase was poured off and the sedimented residue was washed with 10 ml of distilled deionized water and centrifuged at 10,000 rpm for 15 minutes. The supernatant was decanted and the residue dried at 105°C for 24 hours (W2). This experiment was done three times and mean dry residue calculated. The dry residue was weighed and the percentage of solubility was determined using the formula that follows;
Where; W1 was the initial weight of dry chitosan and W2 was the weight of the dried residue
Fourier Transform Infra-Red Spectroscopy (FTIR)
Three milligrams (3 mg) of each chitosan sample and 5 g of Potassium bromide (KBr) were dried at 60°C and 120°C respectively under reduced pressure for 12 hours. Each dried chitosan sample was homogenized with 100mg of KBr and then compressed to form very thin discs of approximately 0.2 mm thickness. The chitosan samples were examined at 4000–400 cm-1 Wavenumber range using a PerkinElmer FT-IR Spectrometer. The spectrometer was set to perform at least 64 scans per sample. A KBr disc was used as reference. Functional group assigning to the generated FTIR spectra bands was done using documented literature [29-35].
Determination of the Degree of Deacetylation (DD%)
The acetylation and deacetylation percentage of chitosan samples was determined by Fourier Transform Infrared Spectroscopy (FTIR). This was done through the correlation of some absorbance bands linked to some of amide, methyl and hydroxyl bands registered by the FTIR spectra. Vilar Junior et al. [ 17] used the amide I band with a wavenumber of 1655 cm-1 and the hydroxyl group band at 3450cm-1 using the formula that follows to determine the degree of acetylation (DA) and then the DD ;
where A1655 was the absorbance at 1655 cm-1 of the amide-I band which is measure of the N-acetyl group content, A3450 was the absorbance at 3450 cm-1 corresponding to the hydroxyl band as an internal standard to correct for disc thickness, factor 1.33 is the ratio of A1655 and A3450 for fully N-acetylated chitosan.
X-Ray diffraction Analysis (XRD)
X-Ray diffraction was used to determine the crystallinity of the isolated chitosan where 500mg of chitosan powder were analyzed employing BRUKER AXS diffractometer, D8 Advance (Germany) fitted with Cu-Kα radiation (λKα1=1.5406Å) from 2θ = 0.5° to 130°, with increment D2J: (0.034°), voltage of 40 kV, current of 40 mA, power of 1.6 kW and counting time of 0.5 sec/step. Generated data was analyzed by OriginPro Version 8.5 and resultant peaks 2θ values were compared with the commercial shrimp chitosan from Sigma Aldrich.
Chitosan susceptibility assay
Antibacterial activity of the chitosan was evaluated using standardized inocula of 1 X107 CFU/mL with 0.5 McFarland standards streaked onto the surface of sterile agar plates. Carbapenem resistant E. coli and K. pneumoniae suspended in Brain Heart Infusion Broth were inoculated onto the Mueller Hinton Agar plates and round wells of diameter 6 mm, depth 3 mm were prepared using a sterile cork borer in which 25µl of chitosan solution (0.25, 0.5, 0.75, 1, 1.5 and 2mg/ml in 1% acetic) were pipetted. Meropenem disks were used as the positive control while 1% acetic acid as negative control. The plates were incubated at 37°C for 72 hours. Zones of bacterial growth inhibition were measured and record in millimeter (mm).
Data analysis was done using Graph Pad Prism version 7.01. Comparisons of chitin yield, chitosan yield, ash content, moisture content, solubility and DD among the chitosan samples isolated from BW, MSR and NS were performed using One-way analysis of variance (ANOVA) followed by Tukey's multiple comparisons test. A P-value of ≤ 0.05 indicated substantial statistical variance.