Guar gum benzoate nanoparticle reinforced gelatin films for enhanced thermal insulation, mechanical and antimicrobial properties

Carbohydrate Polymers 170 (2017) 89–98

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Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol

Guar gum benzoate nanoparticle reinforced gelatin films for enhanced thermal insulation, mechanical and antimicrobial properties Sonia Kundu, Aatrayee Das, Aalok Basu, Md. Farooque Abdullah, Arup Mukherjee ∗ Division of Pharmaceutical and Fine Chemical Technology, Department of Chemical Technology, University of Calcutta, 92, A.P.C. Road, Kolkata 700009, West Bengal, India

a r t i c l e

i n f o

Article history: Received 21 February 2017 Received in revised form 17 April 2017 Accepted 20 April 2017 Available online 23 April 2017 Chemical compounds studied in this article: Guar gum (PubChem CID: 44134661) Citric acid monohydrate (PubChem CID: 22230) Dimethyl sulphoxide (PubChem CID: 679) Dimethyl amino pyridine (PubChem CID: 14284) Benzoyl chloride (PubChem CID: 7412) Lithium chloride (PubChem CID: 433294) Glutaraldehyde (PubChem CID: 3485) Glycerol (PubChem CID: 753) Sodium hydroxide (PubChem CID: 14798) Sodium chloride (PubChem CID: 5234)

a b s t r a c t This work relates to guar gum benzoate self assembly nanoparticles synthesis and nano composite films development with gelatin. Guar gum benzoate was synthesized in a Hofmeister cation guided homogeneous phase reaction. Self assembly polysaccharide nanoparticles were prepared in solvent displacement technique. Electron microscopy and DLS study confirmed uniform quasi spherical nanoparticles with ␨-potential − 28.7 mV. Nanocomposite films were further developed in gelatin matrix. The film capacity augmenting due to nanoparticles incorporation was noteworthy. Superior barrier properties, reinforcing and thermal insulation effects were observed in films dispersed with 20% w/w nanoparticles. Detailed FTIR studies and thermal analysis confirmed nanoparticles interactions in the film matrix. The nanocomposite film water vapour permeability was at 0.75 g mm−1 kPa−1 h−1 , thermal conductivity 0.39 W m−1 K−1 and the tensile strength were recorded at 3.87 MPa. The final film expressed excellent antimicrobial properties against water born gram negative and gram positive bacteria. © 2017 Elsevier Ltd. All rights reserved.

Keywords: Guar gum benzoate gelatin nanocomposite films thermal insulation antimicrobial properties Hofmeister cation guided reaction

1. Introduction Associative interactions of polysaccharide and proteins are intriguing. Similar interactions are very useful for functionally superior and bio-safe materials design. Precise control over molecular association due to size, shape, conformations, van der Walls forces, ionic exchange or hydrophobic stacking interactions were experimented earlier for applications in pharmaceuticals, food packaging, environmental and biomedical areas (Dickinson, 2008; Ettelaie & Akinshina, 2014). There is a growing interest in recent years for complete understanding of similar macromolecular interaction (Semenova, 2016). Soft matter microstructures non-covalent

∗ Corresponding author. E-mail addresses: [email protected], [email protected] (A. Mukherjee). http://dx.doi.org/10.1016/j.carbpol.2017.04.056 0144-8617/© 2017 Elsevier Ltd. All rights reserved.

interactions are pronounced in polymer blends (Sarika, Pavithran, & James, 2015). Apparently dissimilar forces were conjoined intelligently for bio-based materials functional enhancements. Best effects can be achieved when at least one or both macromolecules are incorporated in large surface colloid phases. Guar gum (GG) is a galactomannan obtained directly from Cyamopsis tetragonoloba seed pericarp. GG blends well in protein helices and is frequently used in ice creams. Native GG exists in a three chain associative coil formation which hydrates hugely in water (Mukherjee & Basu, 2007). GG surface hydroxyl groups are however polar and facile hydrogen substitution products are known (Sharma, Kumar, & Soni, 2004). Different other GG derivatives are also known to take up un-restrictive formations in biopolymer blend environment (Bosio, Lopez, Mukherjee, Mechetti, & Castro, 2014). Some selective ones have found successful applications already in the commercial space. A cationic

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derivative guar hydroxypropyltrimonium chloride, ‘Jaguar’ for example interacts favorably with hair keratin and is used widely in conditioning shampoos. Jaguar deposits on the hair are efficient on small doses and strengthen hair over time. GG hydrophobic derivatives can undergo interesting self assembly formations while polar substituted biopolymers are useful in hydrogel network formations. Gelatin is a food gelling protein obtained from partial hydrolysis of cattle bones. Gelatin dissolves in water and undergoes conformational transition when heated above 35 ◦ C. Collagen type coil helix structures of gelatin at 35 ◦ C provide enough cross-linking capacity and diffusion space in nanoscale. The protein is biocompatible, non-immunogenic, biodegradable film forming material. Gelatin was therefore investigated extensively in drug delivery, tissue engineering and food packaging areas (Badhe et al., 2017). Unfortunately, gelatin films express poor vapour barrier properties, high hydrophilicity and low mechanical properties. Attempts were made earlier for gelatin film property enhancement following polymer blending, oil emulsion incorporation, biocomposite design, chemical cross-linking (Mohajer, Rezaei, & Hosseini, 2017). Compatible nanomaterials induce specific advantages in biopolymer interfaces. Metal and metal-oxide nanoparticles were used earlier for functional and mechanical property enhancement of gelatin films. Films embedded with single wall carbon nanotubes were used for membrane separation and performance enhancements. Likewise, cerium oxide nanoparticles were incorporated in antioxidant type active gelatin films intended for cell growth and regenerative medicine applications (Shi et al., 2013; Marino et al., 2017). Water retardant galactomannan nanoparticles incorporation in gelatin film was considered as one attractive exploration for film property augmenting. This work intends to develop polysaccharide derived nanoparticles and gelatin protein interact bio-safe films for packaging materials applications. To our knowledge this is a first ever study on fully bio-based protein-polysaccharide nanocomposite films design for functional property enhancements. Furthermore, high DS hydrophobic GGB nanoparticles were developed in Hofmeister ion guided homogeneous phase reactions and facile ouzo solvent diffusion techniques. Nanocomposite films were developed and property evaluated for intended applications. 2. Materials and methods

stirred and a homogeneous solution was obtained. The solution was transferred to a 250 mL three necked round bottomed flask placed over an electrical heating mantle. The flask was equipped with a reflux condenser, a nitrogen purging unit and a mechanical stirrer. The reaction temperature was maintained at 30 ± 2 ◦ C throughout. DMAP (11.29 g) dissolved in 5 mL of DMSO was added and the reaction mixture was stirred at 80 rpm to equilibrate. Benzoyl chloride (12 mL) was then added dropwise under nitrogen purging (5 mL min−1 ). The reaction mixture was stirred for additional 3 h for reaction completion and the content poured into ice cold aqueous ethanol (50% w/v, 200 mL). White precipitate GGB was collected after filtration and the product washed with distilled water. GGB was further soxlated against methanol and the leachants checked free from aromatics in UV spectrophotometer (EVO300 PC, Thermo Fisher, U.S.A). The GGB product (4.75 g) was preserved in screw cap bottles and kept in vaccum dessicator until application. Different polymer to the acid halide ratio was used to obtain biopolymer derivatives with different degree of substitution. 2.3. Guar gum benzoate nanoparticles (GGBnp) Typically, 10 mL of GGB dissolved in DMSO (3 mg mL−1 ) was taken in a dialysis bag (MWCO 12400, Sigma, U.S.A.) and placed against running water at room temperature for solvent diffusion. After 72 h the dialysis bag content was transferred in a flask and lyophilized (Eyela FDU 1200, Japan) at −52.3 ◦ C and 19 Pa to powders for analysis. Alternatively, the bag content was transferred to screw cap bottles and preserved in refrigerators. 2.4. Nanocomposite films Nanocomposite films were cast from gelatin solutions dispersed proportionately with GGBnp. Typically, 200 mg of GGBnp was added in 20 mL of distilled water containing 1 g of gelatin and 200 mg CA. The mixture was heated to 80 ± 2 ◦ C under magnetic stirring at 200 rpm and a clear homogeneous solution was obtained. To this solution, 0.2 mL of glycerol (20% v/v) was added as plasticizer and the pH adjusted to 10 using drops of 1N NaOH. The mixture was heated for 10 min more and the solution poured onto polypropylene petridishes to cool for 24 h in an incubator maintained at 35 ± 2 ◦ C. The films formed were then peeled off and kept in dessicators before analysis. Films without GGBnp were prepared similarly.

2.1. Materials Reagent grade gelatin, citric acid (CA, C6 H8 O7 ·H2 O), dimethyl sulphoxide (DMSO, 99.9%), glycerol (98%), sodium hydroxide (NaOH), sodium chloride (NaCl), potassium bromide (KBr, IR grade), fused calcium chloride (CaCl2 ), glutaraldehyde (25%) and uranyl acetate were from Merck (Mumbai, India). Dimethyl amino pyridine (DMAP), lithium chloride (LiCl) and benzoyl chloride (C6 H5 COCl) for synthesis were from Spectrochem (Mumbai, India). Guar gum (GG, CAS No. 9000-30-0) was received as gift from Nuevo Polymers (Gurgaon, India). Microbial growth media constituents beef extract, peptone and agar were from Himedia (Mumbai, India). Analytical grade methanol and ethanol used were from Merck. 2.2. Homogeneous phase synthesis for guar gum benzoate (GGB) Native GG was washed free of debris and oligomers before starting any reaction (McCleary & Nurthen, 1983). Typically, 5.0 g of pre-washed GG was taken in 100 mL of 50% (v/v) aqueous isopropanol and soaked for 24 h. The solvent was decanted off and the GG remnant added into 80 mL of DMSO under magnetic stirring. LiCl (2 g) dissolved previously in 20 mL of DMSO was added into it,

2.5. Characterization of guar gum benzoate and nanoparticles C,H,N combustion analysis for GG and GGB was carried out in an analyzer (CHNS-932, Leco Corp., U.S.A). The results were compared against acetophenone standard samples. FT-IR studies for GG and GGB were carried out in a FTIR spectrometer (Jasco 6300, Japan). Samples were ground-mixed with KBr, pressed into pellets and scanned in wavelength range of 400–4000 cm−1 with background corrections. The X-ray diffraction studies for GG, GGB and GGBnp were carried out in a X’Pert Pro X-Ray diffractrometer (PW 3050/60, ´˚ Philips, India) with Cu anode having K␣ radiation (␭ = 1.54060A) at 40 kV voltage and 30 mA current. The sample diffractions were recorded in 10◦ –80◦ (2␪) range, step size 0.03◦ . The thermal analysis was carried out in Pyris Diamond TG/DTA (Perkin Elmer, Singapore). Samples were placed in platinum crucibles and heated incrementally (10 ◦ C min−1 ) up to 450 ◦ C under N2 purging. The particle size, polydispersity index (PDI) and zeta-potential ® (␨) of nanoparticles were recorded in DLS (Zetasizer Nano ZS, Malvern Instrument Ltd., U.K). GGBnp in water were exposed to a 4 mW helium–neon laser beam, 633 nm, and the back scattering angle was at 173◦ . Analyses were carried out in triplicate and an

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average was recorded. The particle shape and morphology were examined in SEM (Zeiss EVO 18 Special Edition, ZEISS, Germany). Samples were drop cast onto cover slips, dried for 48 h, sputter coated with platinum at 15 kV and analyzed using Smart SEM software. TEM (Jeol Jem 2100, Japan) studies were carried out after uranyl acetate staining and particle distributions were recorded from three sample experiments. 2.6. Characterisation of nanocomposite films Thickness of each nanocomposite film was measured using a thickness guaze (Mitutoyo 7301, Japan). Three different positions in three film samples were recorded and the data averaged for analysis. The transparency value (T) was calculated for 3 cm × 0.5 cm film samples at 600 nm. T = Abs600 /d; Abs600 was the absorbance value of films at 600 nm, d, thickness in mm. Moisture content of each film was determined by measuring weight loss of films (1 cm × 4 cm) upon drying in an oven at 105 ± 1 ◦ C for 24 h. The moisture content was calculated as:MC(%) = (Mw − Md )/Mw × 100 Where Mw is the weight of the films conditioned in 75% RH and Md is dry weight of the films. In order to determine the total soluble matter (TSM) the dried films were immersed in 200 mL of distilled water for 24 h. Each film was dried thereafter in an oven for 24 h at 105 ± 1 ◦ C and the final weight (Mf ) was measured. TSM values were calculated as:TSM(%) = (Md − Mf )/Md × 100 Each experiment was run in triplicate and data averaged for comparative studies. Films were evaluated for water vapour permeability (WVP) following ASTME96 in Fisher/Payne permeability cups (Mariniello et al., 2007). 3 g of fused CaCl2 was placed inside each cup and circular films were attached at the top with o-ring and screw clamps. The film surface area exposed to water vapour transmission was 0.00274 m2 . The cups were accurately weighed and placed in 85% RH at 25 ± 1 ◦ C. The cups were weighed periodically until constant weight. Increase in cup weight versus time was plotted and used to derive WVP as: WVP = (WVTR × d)/P Where d, film thickness in mm; P, partial pressure difference of vapour across film and WVTR was slope/film area. Each film was analyzed in FT-IR (ATR PRO45O-S, Japan), the results stacked in Biorad Knowit All software for analysis. XRD and thermal studies were recorded in order to understand chemical intricacies. Film morphology was studied in SEM. 2.7. Film mechanical property The tensile strength (TS), elongation at break (EB) and Young’s Modulus (Y) of each film type were recorded using Llyod Instruments (LR10 K Plus, U.K) equipped with a tensile load cell of 10 kN. The cross-head speed was set at 50 mm min−1 and ASTM D170893 procedure followed with 14 mm width and 40 mm length film strips. Three film strips were studied in each case and the data averaged for evaluations. 2.8. Film thermal conductivity study Thermal conductivity of film was determined following Lee’s Disc method (Bendahou, Bendahou, Seantier, Grohens, & Kaddami, 2015). The apparatus contains two circular bell metal discs attached to thermocouples and one electrical heating device placed over a

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disc. The heating device was first operated at constant voltage and the steady state temperature across thermocouples was recorded. Film sample was then placed between the discs and the falling temperature (T2) was recorded at an interval of 30 s. The thermal conductivity for each film type was recorded as ␭ = [ms(dT/dt)d]/[A(T1 − T2)] Where, m and s are the metal disc mass and specific heat; d, thickness and A surface area and T1, T2, temperature across film at steady state. The rate of cooling at temperature T2 was taken as dT/dt. 2.9. Antimicrobial activity Antimicrobial activity was determined against E.coli MTCC44 and S. aureus MTCC160 strains. Qualitative evaluations were carried out in nutrient agar disc and ciprofloxacin was used as standard. Typically, 0.6 cm diameter film discs were punched and placed onto agar plates seeded with individual organism (Rojo, Barcenilla, Vazquez, Gonzalez, & Roman, 2008). Seeded and unseeded plates without test sample were run as control. Growth inhibition zones were recorded after 24 h incubation at 37 ± 2 ◦ C. MIC and time kill assay experiments were run with GGBnp and Gel-GGBnpC film. MIC was determined in broth micro-dilution technique (Wayne, 2006). In time kill assay nanocomposite films 2.25 cm2 surfaces and GGBnp in MIC concentration were used. Samples were placed in contact with 5 mL 3 × 105 CFU mL−1 bacterial cell suspension in normal saline taken in 25 mL screw cap bottles (Graziano et al., 2015). Tightly closed bottles were placed over environmental shaker incubator (Orbitek LJE, Scingenics Biotech Pvt. Ltd., Chennai, India) maintained at 37 ± 2 ◦ C, 100 rpm. Samples (0.1 mL) were withdrawn at different time intervals, spread on nutrient agar plates and viable colonies were counted after 24 h of incubation. Each experiment was repeated thrice and killing kinetics curves developed from the time vs CFU mL−1 plots. SEM experiments were also conducted to understand film induced bacterial killing condition. E.coli cells (3 × 105 CFU mL−1 ) were exposed to 2.25 cm2 film samples. Cells were withdrawn for SEM experiments after 8 h of exposure, fixed with 3% (w/v) glutaraldehyde solution, dehydrated serially with ethanol-water in 15 min exposure steps before visualization. Control experiments without exposure were run similarly (Watson, McKee, & Merrell, 1979). 2.10. Statistical analysis The results were represented as mean ± standard deviation (SD). Analysis of the experimental data was conducted in GraphPad Prism 7.02 and Origin 6.0 Professional Software. For comparative studies, Student t-test was carried out and the difference was considered significant when p < 0.01. 3. Results and discussion 3.1. Synthesis of guar gum benzoate and nanoparticles GG 200 mesh grade was processed in a facile solvent water washing technique to wash off soluble seed amines, oligomers and microbial debris. The product recovery yield after washing was 52%. Washed GG was a constant viscosity product showing no bacterial ´ growth in agar plates (Szymanska et al., 2016). Washed GG was used throughout for derivatisations and analysis. The native GG, Brookfield viscosity (DV-II, Brookfield, U.S.A, Spindle LV-21) in 1% w/v aqueous solution was 19680 cP at 25 ± 1 ◦ C which decreased rapidly over time and was recorded at 9824 cP after 24 h in water.

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Scheme 1. Synthesis of GGB.

Washed GG Brookfield viscosity was 7217 cP in identical condition which remained constant after 24 h. GG forms a thixotropic gel in aqueous medium due to H-bond within the polymer network. This chemical complexity restricts access of reagents and limits molecular degree of functionalization. Innumerable applications of GG were often constrained due to lack of surface functional groups. Alkali catalyzed substitution reactions carried out earlier have resulted in low DS products (Gong et al., 2012). GGB were synthesized with higher DS values following reactions as in Scheme 1. We were also successful earlier in synthesizing high DS guar gum propionates in similar reactive environment (Kundu et al., 2016). GG represent a colloidal dispersion in water and expresses limited solubility in water and common organic solvents (Picout, Ross-Murphy, Errington, & Harding, 2001). This was likely due to hydrated self assembly formations and low or no free molecular mobility. Functionalization reactions in GG were performed in homogeneous phase in LiCl/DMSO solvent system. Benzoyl chloride was used as the reagent and DMAP as catalyst. The reaction was fast and exothermic. LiCl ion pairs acted as chaotropes and increased GG chain disorderness facilitating the substitution reaction. Furthermore, the undissociated ion pairs of LiCl in aprotic solvent reacted with the GG hydroxyl group and barred the reformation of the intramolecular H-bonds (Li, Sun, Xu, & Sun, 2012). The reaction was optimized in several batch experiments. GGB DS increased with the proportional increase of benzoyl chloride addition (Table S1, Supplementary). Near complete recovery of GG was attained under the same conditions adding 50% (v/v) aqueous ethanol instead of the acid halide. An optimum DS achieved was 1.0 when the GG anhydrohexose:benzoyl chloride molar ratio was 1:3. Increasing reaction time beyond 4 h affected product physico-chemical properties and higher proportional addition of benzoyl chloride lead to coloration and inconsistent product. This was likely due to glycosidic link breakage under drastic contact conditions (Geissler, Biesalski, Heinze, & Zhang, 2014). The unreacted acid was removed by distilled water washings and the product further soxlated to clear off UV responsive leachants and ionic components. GGB was easily soluble in DMSO (10 mg mL−1 ) but insoluble in water and salt solutions. Water borne nanoparticles were prepared by a simple, energy efficient technology commonly known as ‘ouzo effect’. Quasi spherical nanoparticles were developed due to solvent diffusion; phase change and self assembly formations. Similar interactions were studied earlier for biopolymers functionalized with hydrophobic groups (Stainmesse, Orecchioni, Nakache, Puisieux, & Fessi, 1995). Nanoparticles were created through associative nucleation and growth mechanism (Hornig & Heinze, 2007). The governing factor

for particle sizing was GGB concentration. The GGB concentration in ouzo region was initially determined in phase diagram studies (Fig. S1B,Supplementary) (Beck-Broichsitter, Nicolas, & Couvreur, 2015). The average particle diameter and the PDI decreased with the increasing concentration and the lowest levels were attained when GGB concentration was at 3 mg mL−1 . Propagation of DMSO in water produced ‘Marangoni mass transfer effects’ which lead to biopolymer chain aggregations and self assembly nanoparticle formations in water. Diffusive solvent exchange in dialysis membrane bags with 3 mg mL−1 GGB in DMSO was used to generate GGBnp for all further studies (Fig. S1A, Supplementary).

3.2. Guar gum benzoate and nanoparticle characterization Elemental analysis was performed by the combustion procedure and the DS was evaluated from the percentage carbon composition (Yoo & Youngblood, 2016). The high DS GGB elemental compositions were C, 44.0%, H, 5.58% while that for GG, was C, 44.4% and H, 6.17%. The degree of substitution recorded in that case was 1.0 (Table S1, Supplementary). The FTIR studies of GG and GGB were presented in Fig. 1A. The surface OH groups in GG appeared hydrogen bonded and were recorded at 3430 cm−1 . The CH2 stretching and bending vibrations in GG were observed at 2922 cm−1 & 1418 cm−1 . Characteristic glycosidic linkage C6 O C1 was observed at 1079 cm−1 and the main chain sugar ether appeared at 1072 cm−1 (Risica, Dentini, & Crescenzi, 2005). In case of GGB, the band intensity of the OH group was reduced and recorded at 3400 cm−1 . This was due to benzoate substitution and lack of hydrogen bond network. New bands at 1790 cm−1 , 1416 cm−1 and 1645 cm−1 appeared in GGB due to carbonyl group and aromatic group stretching vibrations. The X-ray diffractogram for GG, GGB and GGBnp were presented in Fig. 1B. GG 2␪ value was at 19.98◦ . The intricate H-bonds in GG presented a typical crystallinity. In case of GGB, the 2␪ was at 19.75◦ and that in case of GGBnp was at 25.12◦ . TGA analysis recorded biopolymer characteristics against incremental temperature changes. In GG, the mass loss was initiated at 55 ◦ C for escape of water molecules. Rapid polymer decomposition was observed at 278 ◦ C which terminated at 340 ◦ C (Fig. 1C). Mass loss was continuous thereafter to nil due to carbon escape. Intra-molecular H-bonding in GG surface OH groups was the reason for structural robustness. In case of GGB, the water mass loss was almost continuous and initiated at 50 ◦ C. The polymer decomposition appeared at 250 ◦ C which ended at 330 ◦ C. Total decomposition continued thereafter due to carbon escape. The water molecules were unbound in GGB and the thermal stability was lowered due to interposed ester groups. In GGBnp, the thermal characteristic was distinctive. A rapid first phase evap-

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Fig. 1. (A) FTIR spectra; (B) XRD studies; (C) TGA studies.

orative mass loss for escape of water molecules appeared in the beginning and that was followed by a continuous phase of decomposition starting at 200 ◦ C. Total decomposition of the nanoparticles was recorded near 305 ◦ C. The hydrodynamic diameter of GGBnp was 320 nm in DLS and the PDI was 0.259. The PDI parameters claimed stable colloidal dispersions in water. The zeta-potential was recorded at −28.7 ± 2.3 mV. Well distributed, compact, granular nanoparticles were observed in SEM studies (Fig. 2A). The particles were spheroids with an average diameter of 185 nm. GGBnp appeared similar in morphology in TEM (Fig. 2B). Individual nanoparticles presented aggregation regions apparently due to inter and intra molecular associations in nanoscale. Spontaneous self assembly aggregations and micellar structures were recorded in glucan fatty acid esters (Chang et al., 2017).

3.3. Guar gum benzoate gelatin nanocomposite films Films were developed in evaporation, cross-linking and casting method. Neat gelatin and GGBnp reinforced nanocomposite films were prepared for comparative evaluations. Negative charged GGBnp were readily dispersed in gelatin sol. Different proportional dispersions of GGBnp (5, 10 and 20% w/w) were used in film preparations. The neat gelatin and nanocomposite films were free standing, smooth surfaced, flexible and translucent in appearance. CA was used as a cross-linker. Unlike the commonly used crosslinkers glutaraldehyde and formaldehyde, CA is a biobased cross-linker and food component. Nucleophilic substitution reactions predominate for gelatin amine interactions with citrate carboxyl groups while cross-linking (Xu, Shen, Xu, & Yang, 2015). Very low concentration of glycerol was useful as a plasticizer. Cellulose nanocrystals were used earlier for gelatin film mechanical property enhancements (Alves, Dos Reis, Menezes, Pereirac, & Pereira, 2015). Unlike the cellulose nanocrystals the GGBnp were well dispersed spheroids carrying large surface negative charge.

Nanocomposites with uniquely similar structural interactions were considered for multifunctional films development.

3.4. Film properties In case of gelatin film (GelF), thickness was 0.345 mm when 5% (w/v) gelatin was used. Nanocomposite films with different GGBnp (5, 10 and 20% w/w) mass incorporation were developed. Perceptible difference in film thickness was only very minor (Table 1). Film transparency is one important consumer acceptability parameter (Chen, Kuo, & Lai, 2010). Gel-GGBnpC nanocomposite film showed significantly higher transparency than GelF. This was due to strong protein-polysaccharide interactions and nanoparticles homogeneous dispersion. Similar observations were recorded earlier in cellulose nanoparticle based films (Chen, Liu, Chang, Cao, & Anderson, 2009). GelF TSM was however significantly lower than the nanocomposite films. In Gel-GGBnpC films the MC was also reduced (Table 1). The WVP parameter was decreased but was within reasonable limits in case of Gel-GGBnpC . Lower WVP and MC are very enabling characteristic for renewable material film applications. Hydrophobic GGBnp packed structure and uniform dispersion in gelatin film matrix was reasoned for that. Similar correlations were also recorded earlier (Han et al., 2011). Film mechanical properties like TS, EB and Y were recorded in each case (Fig. 3). The TS and Y of the films were higher but the EB parameter was lowered with increasing amount of GGBnp. Interaction between GGBnp and the gelatin matrix was possibly stronger than the gelatin chain interactions (Ibarguren, C´ıeliz, D´ııaz, Bertuzzi, Daz, & Audisio, 2015). This increase in TS values was due to GGBnp non-covalent interactions with the gelatin protein and simultaneous stacking interactions among GGBnp incorporated. Film thermal conductivity (␭) observations were presented in Table 1. Thermal conductivity was clearly influenced by the GGBnp incorporation. GelF showed very low insulation capacity. Thermal conductivity value for GelF was 2.69 W m−1 K−1 and that data was compara-

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Fig. 2. (A) SEM and (B) TEM studies for GGBnp.

Table 1 Film properties of gelatin (GelF) and nanocomposite films (Gel-GGBnpA , Gel-GGBnpB , Gel-GGBnpC ).

a

Sample Type

Gel:GGBnp (% w/w)

Thickness (mm)

Thermal Conductivity (␭, W m−1 K−1 )

Transparency

MC (%)

TSM (%)

WVP (g mm−1 kPa−1 h−1 )

GelF Gel-GGBnpA Gel-GGBnpB Gel-GGBnpC

– 5.0 10.0 20.0

0.345 ± 0.14 0.357 ± 0.13 0.369 ± 0.15 0.392 ± 0.18

2.68 ± 0.03 0.83 ± 0.031a 0.65 ± 0.035 a 0.39 ± 0.036 a

2.08 ± 0.04 2.0 ± 0.03a 2.11 ± 0.01a 4.69 ± 0.15a

20.605 ± 0.15 14.986 ± 0.28 11.965 ± 0.19 9.284 ± 0.15

36.52 ± 1.05 20.765 ± 0.28 16.987 ± 0.17 12.841 ± 0.20

2.26 ± 0.12 1.08 ± 0.15 a 0.97 ± 0.21 a 0.75 ± 0.17 a

p < 0.01, n = 3.

ble to marble. This value was nearly one seventh in Gel-GGBnpC . Increasing mass incorporation of GGBnp added air pockets and nanoparticle void regions which were apparently responsible for favorable effects in film thermal conductivity. Electron microscopy studies on film fine structures have also confirmed that observation (Fig. 5). The SEM morphology in case of GelF shows the presence of fibrous networks throughout the surface and cross-section (Fig. 4A1,A2). This also accounted for the mechanical properties and high WVP of gelatin films. Incorporation of GGBnp resulted in loss of gelatin fibrillar orientation which was likely one reason for decrease in WVP (Fig. 4B1–D1, B2–D2). Moreover, increase in GGBnp mass incorporation, film porosity was increased and nanoparticle voids and air pockets appeared throughout the nanocomposite films cross-section. Nanocomposite films enhanced thermal insulation was apparently due to perceptible changes in films physical characteristics. Gelatin fibrillar distortion was prominent upon GGBnp incorporation. However that effect was compensated partly due to negatively charged polysaccharide nanoparticles non-covalent interactions in film matrix. No much change was therefore recorded in nanocomposite films tensile properties. Similar observations were recorded earlier in case of interactive protein films (Chambi & Grosso, 2006). GGBnp noncovalent interactions throughout have contributed significantly in enhancement of Gel-GGBnpC film Young’s modulus over GelF. In TGA, GelF water mass loss appeared from the beginning with a peak at 70 ◦ C. The first decomposition phase appeared at 180 ◦ C and continued rapidly for carbon escape. Nanocomposite films (GelGGbnpA , Gel-GGBnpB , Gel-GGBnpC ) were deficient of free water molecules and no marked mass loss was recorded in the beginning (Fig. 5A). The first decomposition peak was shifted to 155 ◦ C

and the mass loss was sluggish thereafter. DTA observations corroborated well with the TGA results. In GelF, the first endotherm appeared at 70 ◦ C and a second endotherm was recorded at 180 ◦ C. The final decomposition endotherm was recorded at 260 ◦ C. In case of nanocomposite films DTA endotherm was first recorded at 155 ◦ C and the second endotherm was recorded at 325 ◦ C (Fig. 5B). Nanocomposites contributed favorably in thermal durability of the films. Gelatin peaks in FT-IR were recorded at 1632 cm−1 for C O stretching, 1527 cm−1 for N H bending and 1238 cm−1 for C N stretching vibrations (Fig. S2, Supplementary). In GelF, peaks for gelatin amide I and II were conjoined and appeared at 1580 cm−1 . The broad band for O H and NH2 near 3000 cm−1 was split into two at 3444 cm−1 and 3260 cm−1 due to cross-linking (Uranga, Leceta, Etxabid, Guerrero, & Caba, 2016). In the nanocomposite films (Gel-GGbnpA , Gel-GGBnpB , Gel-GGBnpC ) the GGBnp carbonyl peak at 1790 cm−1 was shrouded for gelatin non-covalent interactions. The GGBnp aromatic peak at 1645 cm−1 was shifted to 1641 cm−1 in the nanocomposite films indicated stacking interactions with gelatin (Fig. 5C). XRD studies for GelF recorded a broad diffraction peak 2␪ 22.4◦ (Fig. 5D). This was due to cross-linked gelatin secondary structure (Morsy, Hosny, Reicha, & Elnimr, 2017). The film XRD pattern was not much affected due to GGBnp incorporation. 3.5. Antibacterial activity Bacterial growth remained unabated in GelF. Inhibition zones were however recorded in case of Gel-GGBnpA , Gel-GGBnpB and Gel-GGBnpC films (Fig. S3, Supplementary). GGBnp MIC value was recorded as 500 ␮g mL−1 in E.coli and 1000 ␮g mL−1 in S.aureus. Bacterial contact killing was very pronounced in GGBnp and Gel-

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Fig. 3. (A) Tensile strength (MPa), (B) Elongation at break (%) and (C) Young’s modulus (MPa) of gelatin (GelF) and nanocomposite films (Gel-GGBnpA , Gel-GGBnpB , GelGGBnpC ).

Fig. 4. (A1-D1) SEM surface studies and (A2-D2) cross-sections of gelatin and nanocomposite films [(A) GelF, (B) Gel-GGBnpA , (C) Gel-GGBnpB and (D) Gel-GGBnpC ].

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Fig. 5. (A) TGA analysis, (B) DTA studies, (C) FTIR and (D) XRD of gelatin (GelF) and nanocomposite films (Gel-GGBnpA , Gel-GGBnpB , Gel-GGBnpC ).

Fig. 6. Antimicrobial activity of GGBnp and nanocomposite films. (A) E.coli and (B) S.aureus.

GGBnpC films. E.coli growth was terminated and quantitative killing was recorded after 4 h contact time (Fig. 6A). Strong hydrophobic and the benzoate groups interactions appeared as the responsible factors for that. Similar killing interactions were observed in case of S.aureus strains (Fig. 6B). Widespread antibacterial potency of Gel-GGBnpC films was one very useful attribute for packaging applications of edibles. SEM studies were conducted with E.coli strains to understand the growth inhibition effects. E.coli growth and cell multiplications were clearly recorded in GelF exposure (Fig. 7B). When the organism was exposed to Gel-GGBnpC films cell death and membrane collapse was marked (Fig. 7C). Gel-GGBnpC film therefore extended a bacteria growth retardant surface due to porous access and GGBnp incorporation.

4. Conclusions Guar gum benzoate was synthesized for the first time in Hofmeister cation guided homogeneous phase reactions. The biopolymer DS varied with the proportional addition of benzoyl chloride and high DS derivatives were synthesized. Guar gum benzoate self assembly ouzo nanoparticles were further prepared in a facile solvent diffusion technique. Favorable protein-polysaccharide interactions between guar gum benzoate nanoparticles and gelatin was used to develop bio-based castable nanocomposite films. Film transparency, thermal insulation, antimicrobial and mechanical properties were augmented due to guar gum benzoate nanoparticles interactions in gelatin.

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Fig. 7. SEM studies of E.coli: (A) control; (B) gelatin film (GelF) treated; (C) nanocomposite film (Gel-GGBnpC ) treated.

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