Antibacterial activity of freshwater lobster (Cherax quadricarinatus) shell chitosan gel preparation against Escherichia coli and Staphylococcus aureus

Materials

The materials used are chitosan from a freshwater lobster shell, Aquadest, waste of freshwater lobster shell, acetic acid (Merck), NaOH pa (Merck), HCl pa (Smart Lab), AgNO₃ (Merck), indicator phenolphthalein (Merck), Na-CMC, propylene glycol, and methylparaben, nutrient agar (Merck), Mueller-Hinton agar (MHA) (Merck), NaCl (Merck), and McFarland’s solution (Merck).

Sample preparation

Lobster shell waste was washed under running water and dried in the sun. After drying, the sample was mashed and sieved in a 60-mesh sieve to get a powder-like texture.

Demineralization process

The sample was placed in a glass beaker, and a 1.5 M HCl solution was added in the ratio of 1:15 (w/v) between the solvent and the sample. The mixture was then heated at 60°C–70°C for 4 hours while stirring at 50 rpm with a magnetic stirrer. The residue was separated, dried, and then weighed after cooling in a desiccator (Al Shaqsi et al., 2020).

Deproteination process

The demineralization residue was transferred to a new glass beaker, and a 3.5% NaOH solution was added in the ratio of 1:10 (w/v) between the solvent and the sample. The mixture was then heated at 60°C–70°C for 4 hours while stirring at 50 rpm with a magnetic stirrer. After drying, the residue was chilled in a desiccator before being weighed (Paul et al., 2014).

Depigmentation process

Chitin was placed in a glass beaker, followed by the addition of 60% NaOH in the ratio of 1:20 (w/v), and then heated at 100°C–110°C for 4 hours while stirring with a magnetic stirrer at a speed of 50 rpm. After drying, the residue was chilled in a desiccator before being weighed. The chitosan extracted from each sample was then qualitatively characterized (Boarin-Alcalde and Graciano-Fonseca, 2016).

Chitin deacetylation process

Chitin was placed in a glass beaker, followed by the addition of 60% NaOH in the ratio of 1:20 (w/v), and then heated at 100°C–110°C for 4 hours while stirring with a magnetic stirrer at a speed of 50 rpm. After drying, the residue was chilled in a desiccator before being weighed. The chitosan extracted from each sample was then qualitatively characterized (Vallejo-Domínguez et al., 2021).

Chitosan characterization

Examination of the characteristics of chitosan from the shells of freshwater lobster (Cherax quadricarinatus) includes organoleptic properties, yield, water content, and chitosan solubility (Tripathi et al., 2011).

Organoleptic examination

Organoleptic examination was carried out on chitosan from freshwater lobster (C. quadricarinatus) shells by paying attention to shape, smell, and color.

Chitosan yield

The yield of chitin to chitosan was calculated by dividing the weight of chitin by the percentage of chitosan produced (Tripathi et al., 2011) as follows:

$\text{Chitosan yield}=\frac{m}{w}\times 100\text{,}$

where m is the mass of isolated chitosan and w is the weight of chitin.

Determination of ash content

A total of 2 g of Simplicia powder was weighed carefully, put into a porcelain exchange that had been ignited and tempered, and then leveled. The sample was incandescent at a temperature of 600°C until the charcoal was exhausted, cooled, and weighed until a constant weight was obtained, and the ash content was calculated in percent of the material that had been dried in air (Liu, 2019).

Fourier Transform Infrared (FTIR) analysis

The functional groups of the chitosan formed were determined using an FTIR spectrophotometer at wavenumbers ranging from 4,000 to 400 cm⁻¹. Based on the FTIR data, the degree of deacetylation can be determined using the baseline method formulated by Domszy and Robert. The equation used is as follows:

$\mathrm{\%}\mathrm{D}\mathrm{D}=1(\frac{A1647}{A1647}\times \frac{1}{1.33}),$

where A₁₆₄₇ is the absorbance at a wavelength of 1,647 cm⁻¹ for the absorption of the NH group, A₃₄₁₀ is the absorbance at a wavelength of 3,410 cm⁻¹ for the absorption of the OH group, and 1.33 is the constant for the degree of complete deacetylation.

Determination of water content

The amount of water in chitosan is one of the most important factors in determining its quality. The water content of chitosan is specified at 10% by Protan Biopolymer as the quality requirement (Abun and Haetami, 2016). The Association of Official Analytical Chemist method of heating can be used to perform the water content test. The sample was weighed in a porcelain cup or watch glass dish with a known weight of up to 0.5 g. The oven was preheated to 100°C–105°C, and the sample was baked for 1–2 hours, depending on the ingredients. Then, it was weighed after cooling in a desiccator for 30 minutes (Sun et al., 2017). The sample was reheated in the oven and chilled in a desiccator, and the process was repeated until the weight was constant. Calculation of water content can be done with the following formula:

$\mathrm{\%}\text{Water content}=\frac{a-b}{c}\times 100,$

where a is the weight of the cup and initial sample (g), b is the weight of the cup and sample after drying (g), and c is the initial sample weight (g).

Chitosan solubility

The solubility of chitosan is one of the parameters that may be used to measure its quality. The higher the chitosan solubility, the higher the quality of the chitosan generated. In this case, chitosan was dissolved in 1% glacial acetic acid at a ratio of 1:100 (g/ml) (Salamat et al., 2019).

Gel preparation formula

The formula for making hand sanitizer gel from freshwater lobster shell chitosan is shown in Table 1.

Gel hand sanitizer preparation procedure

The gel base was made by dissolving chitosan with 100 ml of 1% glacial acetic acid. The carboxy methyl celulose (CMC) was developed in a mortar previously soaked in hot water. After the CMC bloomed, chitosan and methylparaben were added, which had been previously dissolved in hot water and ground quickly and vigorously until a homogeneous and transparent mass was formed; then, glycerin was added and crushed until it became a homogeneous gel (Wang et al., 2021).

Evaluation of gel hand sanitizer preparations

Hand sanitizer gel antibacterial activity test against E. coli and S. aureus

An antibacterial test of freshwater lobster shell chitosan gel hand sanitizer against E. coli and S. aureus was conducted. A sterile swab was inserted into the inocula of E. coli and S. aureus, and the entire surface of the MHA medium was swabbed aseptically for approximately 5 minutes in laminar airflow. The paper disks were soaked with freshwater lobster shell chitosan hand sanitizer with various concentrations for 10 minutes, and then the paper disks were removed with sterile tweezers. The paper disks were placed on MHA media that had been inoculated by bacteria, and the media were placed in an incubator at 37°C for 24 hours. The diameter was measured with a caliper and recorded in mm (Harahap et al., 2021; Nasri et al., 2021).

Table 1. Formula for hand sanitizer gel preparation. [Click here to view]

Chitosan isolation

The isolation stage of chitin begins with the demineralization process, which is the initial step in removing inorganic salts or mineral content from chitin, particularly CaCO₃ present in the sample. The minerals in the sample react with HCl during the demineralization stage, causing the minerals to separate from the lobster shell. The creation of CO₂ gas in the form of air bubbles, as well as the dissolution of calcium and phosphate in the sample, as well as fixed and insoluble chitin compounds, characterizes the mineral separation process. In the separation of chitin, this demineralization process is critical. The sample is then taken to the next step after it has been mineral-free (Boarin-Alcalde and Graciano-Fonseca, 2016).

Results of chitosan characterization from freshwater lobster shells

The results shown in Table 2 indicate that the chitosan obtained met the standard values so it can be used for various applications. The resulting chitosan has low water content. The water content of chitosan is influenced by the method and duration of drying, the amount of chitosan being dried, and the surface area of the chitosan being dried. The ash content obtained was 0.93%. This indicates that the chitosan produced met the quality standard for chitosan ash content set by Protan Biopolymer, which is 2%. The results of the isolation of chitin from waste and chitosan from lobster shells are shown in Figure 1.

The solubility of chitosan can be seen in Table 3. The solubility of chitosan in glacial acetic acid is one of the parameters that can be used as a standard for assessing its quality. The higher solubility of chitosan in 1% glacial acetic acid (1 g/100 ml) means that the quality of the chitosan produced is getting better. The resulting chitosan has perfect solubility in 1% glacial acetic acid. Chitosan is a compound that is insoluble in water; slightly soluble in HCl, HNO₃, and H₃PO₄; and insoluble in H₂SO₄. Chitosan is more soluble in dilute acids such as acetic acid, formic acid, and citric acid. Chitosan is also insoluble in some organic solvents, such as alcohol, acetone, dimethyl formamide, and dimethyl sulfoxide, but chitosan is very soluble in formic acid with a concentration of 0.2%–100% in water solvents.

Yield

In this study, the isolation stage of chitin started with the demineralization stage and continued with the deproteination stage, which lasted for 4 hours at a temperature of 40°C–50°C. This affected the %yield of chitin produced apart from the heating temperature factor and the stirring time. In addition, the influencing factor on the chitin yield is the sequence of stages of chitin isolation. In this study, the sequence of chitin isolation started from demineralization to deproteination. Isolation with the demineralization to deproteination sequence produced more yield than isolation with the demineralization to deproteination sequence. This is because the minerals form a hard barrier on the lobster skin; therefore, moving the minerals first will facilitate the protein removal process so that the %chitin yield is greater. The next step is deacetylation to get chitosan. From this study, the %yield of chitosan obtained was 68.75% (Table 4).

Functional group analysis results using FTIR

The results of the isolation using infrared spectrophotometry can be seen in Figures 2 and 3. The isolation results showed absorption at wavenumbers of 3,750–3,000 (stretching O-H and N-H amines), which was 3,421.72 cm⁻¹ in standard chitosan, while in lobster shell chitosan the wavenumbers were 3,410.15, 3,290.58, and 3,105.39 cm⁻¹. The absorption at wavenumbers 2,400–2,100 cm⁻¹ (stretch-C≡C, ≡CN) was 2,345.44, 2,310.72, and 2,144.84 cm⁻¹ in standard chitosan, while in lobster shell chitosan it was at 2,314.59 cm⁻¹ and 2,129.41 cm⁻¹. Furthermore, the absorption at wavenumbers 1,675–1,500 cm⁻¹ (strain C=C aliphatic, C=O amide, and C=N) was 1,651.07, 1,585.49, and 1,535.34 cm⁻¹, while in lobster shell chitosan the wavenumbers were 1,647.21 and 1,554.63 cm⁻¹. The presence of an absorption band at a wavenumber of 1,068 cm⁻¹ indicates the stretching vibration of the C-O group. Then, the absorption appeared at wavenumbers 1,475–1,300 cm⁻¹ (C-H bending), namely 1,423.47, 1,377.17, and 1,319.31 cm⁻¹, in raw chitosan, while in lobster shell chitosan, it appeared at 1,377.17 and 1,315.45 cm⁻¹. Then, there was absorption at wavenumbers 1,250–1,000 cm⁻¹ (CN stretching vibration), which was 1,145 cm⁻¹ in standard chitosan, while in lobster shell chitosan, it was 1,068.56 and 1,037.70 cm⁻¹. The absorption at wavenumbers 1,470–1,350 cm⁻¹ (CH bending vibration) was 1,377.17 cm⁻¹. Based on the results of the analysis of the chitosan functional group data, the value of the degree of deacetylation can be determined using the formula of Robert and Domzy. Based on the baseline method, the value of the degree of deacetylation is 67.93%. This proves that the conversion of chitin into chitosan has been completed.

Table 2. Parameters of chitosan. [Click here to view]

Figure 1. The results of chitin isolated from waste and chitosan from lobster shells. [Click here to view]

Table 3. Solubility of chitosan. [Click here to view]

Table 4. Chitosan yield. [Click here to view]

Figure 2. Results of raw chitosan FTIR spectra. [Click here to view]

Evaluation results of gel hand sanitizer preparations

The active ingredient used in this gel is chitosan, with additional ingredients consisting of Na-CMC, glycerin, methylparaben, Aquadest, and perfume. In making this gel, four were made: the first formula was made without the addition of chitosan; the second formula was made with the addition of chitosan, with a concentration of 1.5%; the third formula was made with the addition of chitosan, with a concentration of 3%; and the fourth formula was made with the addition of chitosan with a concentration of 4.5%. After the gel (Fig. 2) was formed, a physical evaluation was carried out with testing parameters, including organoleptic observations, homogeneity tests, pH measurements, viscosity tests, and physical stability tests. The gels were stored at room temperature, and observations were made during week 0–3.

Organoleptic test results for gel hand sanitizer preparations

In the results of the organoleptic evaluation of the hand sanitizer gel from lobster shell chitosan, the smell, shape, and color of the gel preparation gave quite good results because there were no visible changes in them starting from week 0 to week 3. The aroma is given the aroma of apples because the essence of apples is added to the preparation. Turbidity in the gel preparation is caused by the addition of chitosan in each hand sanitizer gel preparation. The higher the concentration of the chitosan used, the more cloudy the color of the hand sanitizer gel (Fig. 4) (Kaban et al., 2022).

Test results for homogeneity of gel hand sanitizer preparations

The homogeneity test (Fig. 5) shows that the gel preparation does not form coarse grains. It can be concluded that the hand sanitizer gel preparation is said to be physically homogeneous and the gel ingredients used in the formulation are dissolved and completely mixed.

Figure 3. FTIR spectra results of freshwater lobster shell chitosan. [Click here to view]

Table 5. Testing dispersion. [Click here to view]

Table 6. Data of pH measurement results. [Click here to view]

Table 7. Viscosity test results. [Click here to view]

Results of testing the spreadability of gel hand sanitizer preparations

The dispersion test on the gel gave results in the form of semiliquid dispersion in each formula (Table 5). The hand sanitizer gel preparation still meets the dispersion requirements, where the application requirements on the skin are between 5 and 7 cm.

Results of pH measurement of gel hand sanitizer preparations

The pH test was carried out to measure the pH (degree of acidity) of the preparation and to determine whether the preparation meets the pH requirements according to the pH of the skin, namely 4–8 (Table 6). The pH observations carried out every week for 3 weeks resulted in a gel that has stable pH, so it is safe to use and does not cause irritation to the skin.

Viscosity test results for gel hand sanitizer preparations

A viscosity test was carried out using a Brookfield spindle L-4 viscometer with a speed of 20 rpm. Viscosity testing was carried out two times, namely at the beginning and after storage for 3 weeks at room temperature. The results of the study showed that the hand sanitizer gel meets the requirements of a good gel (Table 7), which is in the range of 2,000–4,000 cP.

Irritation test results on volunteers

The irritation test on volunteers who observed the irritation test of the hand sanitizer gel from lobster shell chitosan did not show any irritation reactions, such as redness, itching, and crusting on the skin. Therefore, it can be concluded that the hand sanitizer gel preparation is safe to use.

Table 8. Antibacterial test against E. coli for gel hand sanitizer. [Click here to view]

Table 9. Antibacterial test against S. aureus for gel hand sanitizer. [Click here to view]

Figure 4. Preparation of gel hand sanitizer with chitosan concentrations of 1.5%, 3%, and 4.5%. [Click here to view]

Gel hand sanitizer antibacterial activity results against E. coli and S. aureus

The results of the antibacterial activity (Tables 8 and 9) showed that the inhibition zone formed from the hand sanitizer gel from freshwater lobster shell chitosan was categorized as having medium inhibition. According to Davis and Stout (1971), the inhibitory zone formed on the media can be categorized into four categories, namely weak inhibition is the inhibition zone of less than 5 mm, medium inhibition is the inhibition zone of 5–10 mm, strong inhibition is the inhibition zone of 10–19 mm, and very strong inhibition is the inhibition zone of values more than 20 mm (Silaban et al., 2022).

Figure 5. Homogeneity test. [Click here to view]