Antimicrobial gelatin/sericin/clay films for packaging of hygiene products

2.2.1 Preparation of sericin

The silk cocoons were cut into very small pieces, and sericin was extracted using the high-temperature high-pressure degumming (HTHP) technique [40]. Briefly, aqueous extraction of sericin was carried out by boiling cut cocoons in autoclave at 120°C for 1 h, keeping the material-to-liquor ratio at 1:15. The sericin extracted aqueous solution was then filtered with muslin cloth to remove fibroin and other suspended impurities. Solid sericin was obtained by cooling down the sericin solution.

2.2.2 Preparation of the gelatin/sericin blend films

Gelatin/sericin (solid weight 10 wt.%) blend films were prepared by the solution casting method. The aqueous solution of gelatin/sericin in 1:3, 1:1 and 3:1 (w/w) ratios were prepared in hot water with continuous stirring. Glutaraldehyde (5%, w/w) and glycerol (30%, w/w) were added in the gelatin/sericin solution. These solutions were poured in petri dishes and dried at 40°C at least for 24 h. After drying, the films were heat treated at 120°C for 1 h. The average thickness of the blend film was 0.25±0.02 mm. The dry films were stored in plastic bags before all characterization.

2.2.3 Preparation of the gelatin/sericin/clay blend films

Aqueous solution containing gelatin/sericin (3:1 w/w ratios), 5% (w/w) glutaraldehyde and 30% (w/w) glycerol were prepared in hot water. Different concentrations of clays ranging from 1 to 5% (w/w) were dispersed in the solution. The solution was ultrasonicated for 30 min and poured in petri dishes. After drying, the films were heat treated at 120°C for 1 h. The dry films were stored in plastic bags before all subsequent characterization. The average thickness of the blend film was 0.25±0.02 mm.

2.2.4 Mechanical testing

The mechanical properties of the gelatin/sericin/clay blend films were determined using an Instron Universal Testing Machine (INSTRON model no 3369) running at a crosshead speed of 5 mm/min and a gauge length of 50 mm. The samples were cut into 70 mm×10 mm rectangular pieces. At least three samples were tested, and their stress-strain curves were recorded.

2.2.5 Thermal analysis

The thermal properties of the gelatin/sericin films were analyzed by thermal gravimetric analysis (TGA). TGA measures the change in the mass of a sample as a function of temperature or time in a controlled atmosphere. TGA was done using the TGA4000 (Perkin Elmer). Samples were analyzed at a heating rate of 10°C/min in the range of 30–900°C under nitrogen atmosphere.

2.2.6 Water vapor transmission

The water vapor transport rate of the gelatin/sericin/clay blend films was measured by the method described by Kang et al. with some modifications [41]. The specimens were placed in test containers with anhydrous distilled water, which were placed in a control chamber at a constant temperature (37°C). Water vapor transmission was calculated from the weight of the test container before and after the test.

WVT (water vapor transmission rate) = GtA

where

• G: weight change (g)

• t: time during which G occurred (h)

• A: test area (m²).

2.2.7 Antibacterial testing

The antibacterial activity of the samples was carried out using the disk diffusion method and was followed by the method described by Purwar et al. with slight modifications [9]. The antimicrobial activity of the gelatin/sericin and gelatin/sericin/clay films was investigated against the growth of Escherichia coli and Staphylococcus aureus bacteria, which were cultured in Miller’s Luria broth, followed by incubation in an incubator shaker for 24 h. Bacterial suspension (20 μl) was uniformly spread on the sterile petri dishes of Müller-Hinton agar using a sterile spreader, and pieces of antimicrobial films were placed on the bacterial culture. Plates were sealed and incubated at 37°C for 24 h. There was no zone of inhibition, but the film surface was free from bacterial growth. After incubation, the samples were placed in a 50-ml sterilized flask containing 10 ml of sterilized distilled water. The flask was shaken for 1 h at 150 rpm to release the bacteria from the samples. Serial dilutions of inoculants (up to 10⁻⁵ time) were made in the sterilized water. The diluted inoculants were spread on an agar plate, and the plates were incubated at 37°C for 24 h. After incubation, the bacterial colonies were counted. The antibacterial activity was calculated using the equation

Antibacterial activity (%)=No of colonies in control plates−No of colonies in sample platesNo of colonies in control plates×100

2.2.8 Biodegradability test

The biodegradable behavior of the gelatin/sericin/clay blend film samples was tested by the method described by Kermani and Esfandiary with some modifications [42]. Briefly, the gelatin/sericin and gelatin/sericin/clay films were cut into (7 cm×1 cm) small pieces, weighted and buried into the soil in a pot covered with a plastic net and exposed to atmospheric conditions. The degradation behavior of the films was evaluated by measurement of their weight loss at regular time interval of 48 h.

3.1.1 Effect of sericin on gelatin

The effect of sericin on tensile strength, elastic modulus and elongation at the break of the gelatin/sericin blend films is shown in Table 1. It is observed that as content of the sericin in the blend increases from 25 to 75%, the tensile strength and elongation at break decrease, but the tensile modulus slightly increases. The enhanced amorphous structure in the blend films is accountable to the decrease in tensile strength. The increase in tensile modulus and reduced elongation at break with increasing sericin content are due to the brittle nature of sericin. Kundu et al. has reported a similar effect of high sericin content on the compressive strength of sericin-gelatin scaffolds [39]. The results suggest that blend films with higher gelatin percentage (i.e. 75%) provide bulk mechanical strength to the gelatin/sericin blend films, so films with a gelatin/sericin blend ratio of 3:1 were chosen to be used for further experimentation.

3.1.2 Effect of nanoclays

Nanoclays, namely, Closite 30B and CuMMT, used in this study act as antimicrobial agents as well as reinforcing fillers. Closite 30B having quaternary ammonium structure and the presence of Cu ions in CuMMT are known to offer antibacterial activity. The effect of different concentrations of the nanoclays on the tensile strength, tensile modulus and elongation at the break of the gelatin/sericin/clay blend films is shown in Tables 2 and 3. In the case of Closite 30B, as the concentration of the filler increases, the tensile modulus of the blend films increases. The tensile strength and elongation at break increase up to 3% concentration of clay, and on further increase in concentration it decreases, which can be attributed to lower dispersion and agglomeration of clay in the gelatin/sericin matrix [43]. Similarly, in the case of CuMMT, the film shows high tensile strength, elongation at break and low tensile modulus at 1% concentration of clay, and as the clay concentration increases tensile strength, elongation at break decreases and tensile modulus increases. The combination of gelatin/sericin in blend ratio 3:1 with 3% Closite 30B showed a tensile strength of 8.1 MPa comparable to the low-density polyethylene (LDPE)/starch blend films reported in literature [44]. So, for further testing, optimized samples with 1% CuMMT and 3% Closite 30B with gelatin/sericin blend ratio of 3:1 have been used. The gelatin/sericin film without clay has been used as control.