Gelatin-Based Film Integrated with Copper Sulfide Nanoparticles for Active Packaging Applications
Gelatin-Based Film Integrated with Copper Sulfide Nanoparticles for Active Packaging Applications
Roy, Swarup;Rhim, Jong-Whan
2021-07-08 00:00:00
applied sciences Article Gelatin-Based Film Integrated with Copper Sulfide Nanoparticles for Active Packaging Applications Swarup Roy and Jong-Whan Rhim * BioNanocomposite Research Center, Department of Food and Nutrition, Kyung Hee University, 26 Kyungheedae-ro, Dongdaemun-gu, Seoul 02447, Korea; swarup@khu.ac.kr * Correspondence: jwrhim@khu.ac.kr Abstract: Gelatin-based multifunctional composite films were prepared by reinforcing various amounts of copper sulfide nanoparticles (CuSNP, 0.0, 0.5, 1.0, and 2.0 wt %), and the effect of CuSNP on the film was evaluated by analyzing its physical and antibacterial properties. CuSNP makes a compatible film with gelatin. The inclusion of CuSNP significantly enhanced the UV blocking, mechanical strength, and water vapor barrier properties of the gelatin film. The inclusion of CuSNP of 1.0 wt % or less did not affect the transparency of the gelatin film. When 2.0 wt % of CuSNP was mixed, the hydrophilicity of the gelatin film did not change noticeably, but its thermal properties slightly increased. Moreover, the gelatin/CuSNP composite film presented effective antibacterial activity against E. coli and some activity against L. monocytogenes. Gelatin/CuSNP composite films with better functional and physical properties can be used for food packaging or biomedical applications. Keywords: gelatin; CuSNP; multifunctional film; UV-barrier property; mechanical property; antimi- crobial activity Citation: Roy, S.; Rhim, J.-W. Gelatin-Based Film Integrated with Copper Sulfide Nanoparticles for 1. Introduction Active Packaging Applications. Appl. With the essential packaging functions of protection, containment, communication, Sci. 2021, 11, 6307. https://doi.org/ and utility, food packaging plays a vital role in reducing food waste by ensuring food 10.3390/app11146307 safety, maintaining quality, and extending shelf life [1–4]. Commodity plastics (polyethy- lene, polypropylene and polystyrene) are widely used as food packaging materials, and Academic Editor: Adina these plastic packaging materials cause enormous environmental pollution due to their Magdalena Musuc non-biodegradability and improper waste management after use [5]. Therefore, the need to use biopolymer-based biodegradable packaging materials has emerged to reduce the Received: 2 June 2021 plastic waste problem and replace plastic packaging materials. Various biopolymers, such Accepted: 7 July 2021 as chitosan, cellulose, agar, carrageenan, and gelatin, are utilized for this purpose owing to Published: 8 July 2021 their excellent film-producing ability, bio-degradability, and biocompatibility [6–9]. Gelatin, a protein-based natural biopolymer, is an edible biodegradable material blended with Publisher’s Note: MDPI stays neutral functional ingredients to create efficient packaging films [10,11]. Gelatin is the denatured with regard to jurisdictional claims in and hydrolyzed product of collagen. Gelatin is frequently applied in the food industry published maps and institutional affil- due to its excellent gelling and thickening properties. Gelatins are prone to degrade under iations. irradiation as they produce free radicals from the tyrosine and phenylalanine moiety [12]. Prolyl-hydroxyproline and glycine are the major degradation products of gelatin. The gelatin-based film is commonly used in food packaging and has an adequate gas bar- rier and mechanical properties [13,14]. For added value utilization of biopolymer-based Copyright: © 2021 by the authors. films, they are usually reinforced with functional fillers to enhance their physicochemical Licensee MDPI, Basel, Switzerland. properties with additional features, such as antibacterial, antioxidant, and UV-blocking This article is an open access article properties [15–27]. Among these functional materials, various nanomaterials attract at- distributed under the terms and tention because they have a solid reinforcing effect in improving physical properties and conditions of the Creative Commons antibacterial properties. Attribution (CC BY) license (https:// In particular, to manufacture gelatin-based functional composite films, various filler creativecommons.org/licenses/by/ materials, such as zinc oxide nanoparticles (ZnONP), silver nanoparticles (AgNP), copper 4.0/). Appl. Sci. 2021, 11, 6307. https://doi.org/10.3390/app11146307 https://www.mdpi.com/journal/applsci Appl. Sci. 2021, 11, 6307 2 of 12 nanoparticles (CuNP), titanium dioxide (TiO ), nanochitin, melanin nanoparticles (MNP), montmorillonite (MMT), graphene oxide, nanoclay, corn oil, and curcumin, have been used [28–31]. Numerous studies have shown that the addition of nanofillers improved the physical (mechanical, thermal, and barrier) and functional properties of biopolymer films [32–36]. To this end, various metal or metal-oxide nanomaterials have been widely applied to provide additional functionality to the polymer film. However, few studies on the use of metal sulfide nanoparticles, such as copper sulfide nanoparticles (CuSNP), have been performed. CuSNPs are attracting substantial consideration today owing to their low toxicity, ability to absorb infrared radiation, and potential application in the biomedical field [33,35–38]. CuSNP was also recently used to make active packing films made of agar, carrageenan, alginate, and pullulan [38–41]. Their results showed that the incorporation of CuSNPs significantly increased the antibacterial, UV shielding, water vapor barrier, and tensile strength of these films. Moreover, CuSNP and polyurethane have recently been used to make a light-induced shape-memory polymer, which can be applied for medical device and packaging appli- cations [42]. CuSNP has various functional properties that are less toxic than other metal nanoparticles, making it a promising candidate as a functional nanofiller to produce active food packaging films. Therefore, we studied the effect of CuSNP as a protein gelatin-based film. The combination of gelatin and CuSNP can make excellent functional films given the excellent film properties of gelatin and the excellent properties (physical and functional) of CuSNP. So far, there have been no studies on the preparation of gelatin-based functional nanocomposite films integrated with CuSNP. The main aim of the current work was to use gelatin as a polymer matrix and CuSNP as a reinforcing agent to create a multifunctional packaging film that controlled microbial protection and improved the physical properties of active packaging applications. 2. Materials and Methods 2.1. Materials Gelatin (Type A, 200 Bloom) and corn starch were purchased from Gel Tec Co. Ltd. (Seoul, Korea). Glycerol, copper acetate, and thiourea were obtained from Daejung Chemi- cals & Metals Co., Ltd. (Siheung, Korea). CuSNP was prepared according to the method reported previously [40]. The corn starch stabilized CuSNP was fabricated with copper acetate and thiourea [40]. The structure of the CuSNP was detected using field emission transmission electron microscopy (JEM-2100F, JEOL, Ltd., Tokyo, Japan). 2.2. Fabrication of Gelatin/CuSNP Nanocomposite Films The films were fabricated by a solution casting process [43]. First, CuSNP suspensions were prepared by adding various quantities of CuSNP (0.0, 0.5, 1.0, and 2.0 wt % by weight of polymer) to 150 mL DI water with strong agitation for 1 h, and then sonicated for 20 min and shear mixed for 10 min at 5000 rpm. Subsequently, as a plasticizer, 1.2 g of glycerol (30% by weight of polymer) and then 4 g of gelatin were gradually mixed at 80 C for 20 min with continuous stirring at 500 rpm to prepare the film-forming solutions, cast on a Teflon film-coated glass plate and dried. The films were conditioned (25 C and 50% relative humidity (RH)) for 48 h before further testing. All film specimens were fabricated 0.5 1.0 2.0 in triplicate and designated Gelatin, Gel/CuS , Gel/CuS , and Gel/CuS , respectively, according to the content of CuSNP (Table 1). Table 1. Composition of gelatin/CuSNP nanocomposite films. Films Gelatin (g) Glycerol (g) CuSNP (mg) Gelatin 4 1.2 0 0.5 4 1.2 20 Gel/CuS 1.0 Gel/CuS 4 1.2 40 2.0 Gel/CuS 4 1.2 80 Appl. Sci. 2021, 11, 6307 3 of 12 2.3. Film Characterization and Properties 2.3.1. Optical Properties The surface color of the film sample was estimated using a Chroma meter (Konica Minolta, Tokyo CR-400, Japan) to measure Hunter color (L, a, b and DE) values [44]. The film’s light absorbance (UV–visible and near IR, 200–1100 nm) for the UV-barrier property and transparency of the film transmittance at 280 (T ) and 660 nm (T ) were 280 660 taken using a UV-vis spectrophotometer (Mecasys Optizen POP Series UV/Vis, Seoul, Korea) [44]. 2.3.2. Surface Morphology and FTIR The morphological examination of the prepared films’ surface was performed using FESEM (FE-SEM, S-4800, Hitachi Co., Ltd., Matsuda, Japan) [44]. The films’ FTIR spectra were recorded in an attenuated total reflectance-FTIR spectrophotometer (TENSOR 37 Spectrophotometer, Billerica, MA, USA), and all spectra were measured with 32 scans and 4 cm resolution. [43]. 2.3.3. Thickness, Mechanical Properties, and Thermal Stability The film thickness was measured using a QuantuMike IP 65 digital micrometer (Japan). For mechanical properties, an Instron Universal Testing Machine (Model 5565, Instron Engineering Corporation, Canton, MA, USA) was used (ASTM method D 882-88) [40]. Thermogravimetric analysis (TGA) was performed in the temperature range of 30–600 C with a Hi-Res TGA 2950, TA Instrument thermogravimetric analyzer (New Castle, DE, USA) [44]. 2.3.4. Water Vapor Permeability (WVP) and Water Contact Angle (WCA) The WVP was measured according to the ASTM E96-95 standard method. In the WVP cup, distilled water (18 mL) was added, and then the test film (7.5 7.5 cm) was fixed on the top of the cup and sealed to prevent vapor leakage. The cup was kept in a humidity chamber (25 C and 50% RH), and the alteration in the weight of the cup was determined. The WVP (gm/m Pas) was calculated as per our earlier report [44]. The films’ wettability was measured using a WCA analyzer (Phoneix 150, Surface Electro Optics Co., Ltd., Kunpo, Korea) by depositing a drop of water onto the film surface [39]. 2.3.5. Antibacterial Activity The nanocomposite films’ antibacterial properties were tested using the colony count method [40]. The bacterial strain was grown overnight at 37 C. The inoculum was diluted and shifted to 50 mL of culture broth with ~100 mg of films and incubated at 37 C for 12 h at 110 rpm agitation. At different intervals, samples were removed and plated on agar plates after correct dilution to compute viable cell counts. Broth without film and with the control gelatin film was also checked for comparison. 2.3.6. Statistical Analysis Analysis of variance (ANOVA) was applied to compare the mean differences between the samples, and the significance was determined (p < 0.05) by Duncan’s multiple range test using a statistical analysis system using SPSS Inc. (Chicago, IL, USA). 3. Results and Discussion 3.1. Morphology of CuSNP Due to the low stability of CuSNPs in aqueous media, corn starch was used as a stabi- lizing agent to prevent the aggregation of these particles and increase their dispersibility in aqueous solutions. The morphology of the CuSNP was observed using FETEM, as shown in Figure 1, and the particle size was analyzed using ImageJ. CuSNP nanoparticles had Appl. Sci. 2021, 11, x FOR PEER REVIEW 4 of 12 Analysis of variance (ANOVA) was applied to compare the mean differences be- tween the samples, and the significance was determined (p < 0.05) by Duncan’s multiple range test using a statistical analysis system using SPSS Inc. (Chicago, IL, USA). 3. Results and Discussion 3.1. Morphology of CuSNP Due to the low stability of CuSNPs in aqueous media, corn starch was used as a sta- Appl. Sci. 2021, 11, 6307 4 of 12 bilizing agent to prevent the aggregation of these particles and increase their dispersibility in aqueous solutions. The morphology of the CuSNP was observed using FETEM, as shown in Figure 1, and the particle size was analyzed using ImageJ. CuSNP nanoparticles an irregular shape and size ranging from 2 to 10 nm with an average of 6.2 1.7 nm, had an irregular shape and size ranging from 2 to 10 nm with an average of 6.2 ± 1.7 nm, consistent with the previously described data of guar gum stabilized CuSNP [34]. consistent with the previously described data of guar gum stabilized CuSNP [34]. Appl. Sci. 2021, 11, x FOR PEER REVIEW 5 of 12 Figure 1. TEM image of CuSNP. T660 and T280 of the film depending on CuSNP concentration. As the concentration of Figure 1. TEM image of CuSNP. CuSNP increased, the reduction in the transmittance for visible and ultraviolet light was 3.2. Properties of the Nanocomposite Films 3.2. Properties of the Nanocomposite Films different; namely, the T660 decreased linearly with increasing CuSNP concentration (C) 3.2.1. Optical Properties 3. such 2.1. Op that T tica 660 l = Prop −25 ert .371C ies + 89.3 (R = 0.9929), while the T280 decreased exponentially such −1.494C 2 The appearance of the neat gelatin and its nanocomposite films is shown in Figure 2a. that T280 = 19.535e (R = 0.9901). In other words, the clarity of the film decreased line- The appearance of the neat gelatin and its nanocomposite films is shown in Figure The neat gelatin film is colorless and has high transparency, but the gelatin film with arly, while the UV-light blocking property of the film decreased exponentially with 2a. The neat gelatin film is colorless and has high transparency, but the gelatin film with CuSNP added has a green color depending on the concentration of the nanofiller. The color CuSNP concentration. These results imply that the UV-blocking phenomena of the film CuSNP added has a green color depending on the concentration of the nanofiller. The properties of the films are presented in Table 2. When CuSNP was included in the gelatin improved significantly with less sacrificing visibility of the film. At 2 wt % of CuSNP color properties of the films are presented in Table 2. When CuSNP was included in the film, the L- and a-values of the film were reduced, and the b-value increased, demonstrating added gelatin film, ultraviolet rays were almost completely blocked. However, the trans- gelatin film, the L- and a-values of the film were reduced, and the b-value increased, that the brightness of the film was reduced, and the greenness and yellowness of the film parency of the film was still high enough to see through the film with a T660 of 39.5%, demonstrating that the brightness of the film was reduced, and the greenness and yellow- improved. The total color difference (DE) also increased noticeably compared to the control which is a useful property in UV-barrier packaging applications. A similar conclusion of ness of the film improved. The total color difference (ΔE) also increased noticeably com- film, mainly due to the green color of CuSNP. A similar color alteration was detected for CuSNP on the optical properties of agar and alginate-based films has recently been de- pared to the control film, mainly due to the green color of CuSNP. A similar color altera- the agar-based film to which CuSNP was added [40]. scribed [40,44]. tion was detected for the agar-based film to which CuSNP was added [40]. The UV–visible light transmittance spectra of the films are shown in Figure 2b. The control gelatin film presented high UV and visible light transmittance. In contrast, the transmittance of gelatin/CuSNP composite films reduced reliance on the CuSNP content Gelatin due to the obstruction of the light passageway via light opaque nanoparticles. An increase in the UV blocking properties of gelatin/CuSNP films was primarily due to CuSNP’s UV absorption capacity [40]. The UV-barrier and transparency properties of the film are also provided in Table 2. The control gelatin film was extremely transparent with some UV 0.5 Gel/CuS blocking properties with 89.5% of T660 and 18.2% of T280. The control film’s UV protection properties were due to the UV absorption ability of the aromatic amino acids of gelatin, such as tyrosine and phenylalanine [7]. The addition of CuSNP significantly reduced the 1.0 Gel/CuS 2.0 Gel/CuS 200 400 600 800 1000 Wavelength (nm) (a) (b) Figure 2. Appearance (a) and UV-vis transmittance spectra (b) of Gel/CuSNP films. Figure 2. Appearance (a) and UV-vis transmittance spectra (b) of Gel/CuSNP films. Table 2. Optical properties of gelatin/CuSNP films. Films L a b ΔE T280 (%) T660 (%) d a a a d d Gelatin 91.1 ± 0.1 −0.7 ± 0.1 6.6 ± 0.2 2.3 ± 0.3 18.2 ± 0.1 89.5 ± 0.3 0.5 c b b b c c Gel/CuS 83.7 ± 1.0 −1.3 ± 0.2 9.5 ± 0.6 10.0 ± 1.1 11.0 ± 0.9 78.1 ± 0.5 1.0 b c c c b b Gel/CuS 66.5 ± 3.0 −2.1 ± 0.3 18.2 ± 1.6 29.2 ± 3.4 3.9 ± 0.8 61.3 ± 1.8 2.0 a b c d a a Gel/CuS 55.7 ± 4.0 −1.35 ± 0.3 19.1 ± 0.7 39.3 ± 4.0 1.0 ± 0.1 39.5 ± 2.1 The matching superscript letters in each column indicate that they are not significantly dissimilar (p > 0.05) from Duncan’s multiple range tests. 3.2.2. Morphology and FTIR Figure 3 shows the FESEM surface and cross-section micrographs of gelatin-based films. The control gelatin film was integral and smooth, deprived of cracks or defects. The nanocomposite film with a low content of CuSNP formed a well-dispersed and homoge- neous film without any aggregation. However, when a high amount of CuSNP (1.0 and 2.0 wt %) was added, the film showed an accumulation of nanoparticles. The cross-section image also showed good compatibility of nanofillers in the polymer matrix. The neat gel- atin film has a relatively soft morphology, but with the addition of nanofillers, it became somewhat rough. Previously, an analogous accumulation of particles at higher CuSNP content was reported for agar/CuSNP and carrageenan/CuSNP nanocomposite film [40,41]. Transmittance (%) Appl. Sci. 2021, 11, 6307 5 of 12 Table 2. Optical properties of gelatin/CuSNP films. Films L a b DE T (%) T (%) 280 660 d a a a d d Gelatin 0.7 0.1 6.6 0.2 2.3 0.3 91.1 0.1 18.2 0.1 89.5 0.3 0.5 c b b b c c Gel/CuS 83.7 1.0 1.3 0.2 9.5 0.6 10.0 1.1 11.0 0.9 78.1 0.5 1.0 b c c c b b 2.1 0.3 18.2 1.6 29.2 3.4 Gel/CuS 66.5 3.0 3.9 0.8 61.3 1.8 2.0 a b c d a a Gel/CuS 55.7 4.0 1.35 0.3 19.1 0.7 39.3 4.0 1.0 0.1 39.5 2.1 The matching superscript letters in each column indicate that they are not significantly dissimilar (p > 0.05) from Duncan’s multiple range tests. The UV–visible light transmittance spectra of the films are shown in Figure 2b. The control gelatin film presented high UV and visible light transmittance. In contrast, the transmittance of gelatin/CuSNP composite films reduced reliance on the CuSNP content due to the obstruction of the light passageway via light opaque nanoparticles. An increase in the UV blocking properties of gelatin/CuSNP films was primarily due to CuSNP’s UV absorption capacity [40]. The UV-barrier and transparency properties of the film are also provided in Table 2. The control gelatin film was extremely transparent with some UV blocking properties with 89.5% of T and 18.2% of T . The control film’s UV protection 660 280 properties were due to the UV absorption ability of the aromatic amino acids of gelatin, such as tyrosine and phenylalanine [7]. The addition of CuSNP significantly reduced the T and T of the film depending on CuSNP concentration. As the concentration of 660 280 CuSNP increased, the reduction in the transmittance for visible and ultraviolet light was different; namely, the T decreased linearly with increasing CuSNP concentration (C) such that T = 25.371C + 89.3 (R = 0.9929), while the T decreased exponentially such 660 280 1.494C 2 that T = 19.535e (R = 0.9901). In other words, the clarity of the film decreased linearly, while the UV-light blocking property of the film decreased exponentially with CuSNP concentration. These results imply that the UV-blocking phenomena of the film improved significantly with less sacrificing visibility of the film. At 2 wt % of CuSNP added gelatin film, ultraviolet rays were almost completely blocked. However, the transparency of the film was still high enough to see through the film with a T of 39.5%, which is a useful property in UV-barrier packaging applications. A similar conclusion of CuSNP on the optical properties of agar and alginate-based films has recently been described [40,44]. 3.2.2. Morphology and FTIR Figure 3 shows the FESEM surface and cross-section micrographs of gelatin-based films. The control gelatin film was integral and smooth, deprived of cracks or defects. The nanocomposite film with a low content of CuSNP formed a well-dispersed and ho- mogeneous film without any aggregation. However, when a high amount of CuSNP (1.0 and 2.0 wt %) was added, the film showed an accumulation of nanoparticles. The cross-section image also showed good compatibility of nanofillers in the polymer matrix. The neat gelatin film has a relatively soft morphology, but with the addition of nanofillers, it became somewhat rough. Previously, an analogous accumulation of particles at higher CuSNP content was reported for agar/CuSNP and carrageenan/CuSNP nanocomposite film [40,41]. Appl. Sci. 2021, 11, 6307 6 of 12 Appl. Sci. 2021, 11, x FOR PEER REVIEW 6 of 12 Figure 3. FESEM micrographs (surface and cross-section) of Gel/CuSNP films. Figure 3. FESEM micrographs (surface and cross-section) of Gel/CuSNP films. The FTIR spectra of the gelatin and gelatin/CuSNP films are presented in Figure 4a,b. The FTIR spectra of the gelatin and gelatin/CuSNP films are presented in Figure 4a,b. −