Synthesis and characterization of Acacia lignin-gelatin film for its possible application in food packaging
© The Author(s) 2016
Received: 4 December 2015
Accepted: 11 May 2016
Published: 27 May 2016
The aim of the present investigation was to develop Acacia lignin-gelatin (LG) blended films using glycerol as plasticizer and to establish correlation between lignin contents and structure, thermal and mechanical properties of the film. Acacia lignin extracted by alkali method was used for the preparation of LG blended films by solution casting method.
Solubility and swelling tests of the films concluded that the lignin incorporation reduced water affinity of film. Lignin incorporation produces a noticeable plasticizing effect on the blended film, showing optimum values for film incorporated with 20 and 30 % (w/v) lignin, as deduced from their mechanical and thermal properties. Lignin blended film had lower glass transition temperatures (T g) as compared to control gelatin. Infrared spectroscopy (FTIR) analysis of films suggested that lignin interacts with gelatin by hydrogen bonding and hydrophobic interaction consequently creating conformational changes. Atomic force microscopic (AFM) study displays smooth surface of synthesized films. Light barrier properties of film revealed that the lignin addition improved barrier properties against UV light in the range of 280–350 nm. Furthermore, the lowest scavenging activity was observed in LG-E (111.10 µg/ml) trailed by LG-D (249.29 µg/ml) and LG-C (259.53 µg/ml).
KeywordsAcacia lignin Gelatin Edible film Antioxidant activity In vitro cytotoxicity
In the recent past, much attention has been focused to replace petroleum based products, with biodegradable materials owing to their cost-effective nature and good mechanical properties. Biopolymers are considered as the feasible alternative materials for the replacement of petrochemical based products. Gelatin is a protein and an important biopolymer with a wide range of functional properties, such as biodegradability, biocompatibility, film forming, and gelling (Cao et al. 2007). It is derived from the chemical degradation of collagen, and mainly, consist of glycine, proline and 4-hydroxyproline (Pena et al. 2010). It has a triple-helix structure stabilized mainly by the formation of inter-chain hydrogen bonds between carbonyl and amines groups (Rivero et al. 2010). It is a promising raw material for biodegradable packaging materials due to its good barrier properties against oxygen and aromas with intermediate relative humidity (Carvalho et al. 2008; Jongjareonrak et al. 2006). But several of the potential applications of gelatin films require improvements in some of the properties like mechanical, water and light barrier properties. Normally, gelatin is brittle in dry state with high moisture absorption due to tightly bound hydrogen bonds, hydrophobic interaction and the polar groups of amino acids, present in the gelatin structure (Karnnet et al. 2005). To overcome this problem, the addition of plasticizers and natural fillers having antioxidant and antibacterial properties, such as polyphenols would be significant to improve their physico-chemical and functional properties (Pena et al. 2010; Nunez-Flores et al. 2013). The incorporation of fillers aids, to reduce the intrinsic brittleness of the films by decreasing intermolecular forces, increasing the mobility of polymeric chains and improving their flexibility, and thus, will extend the functional properties of the film and provide an active packaging biomaterial (Nunez-Flores et al. 2013).
Lignin, a natural biopolymer, mostly derived from wood, is an enormous and renewable reservoir of latent polymeric materials and aromatic chemicals. It is also a waste product of paper and pulp industries wherein approximately 50 million tons of lignin are generated annually (Sivasankarapillai and McDonald 2011; Zeng et al. 2014; Saini et al. 2015). The complex polyphenolic structure and numerous functional groups of lignin are useful in their effective utilization for the development of polymers, adhesives, coating, additives, carbon fibers, activated carbon, foams and metal nanoparticles (Park et al. 2008; Wang et al. 2009; Aadil et al. 2016). The effective use of lignin in blends with different biopolymers, such as starch (Bhat et al. 2013), gelatin (Nunez-Flores et al. 2013) and synthetic polymers like poly(vinyl alcohol), poly(ethylene), poly(lactic acid), poly(vinyl chloride) have also been reported in the literature (Gordobil et al. 2014; Sahoo, et al. 2011). Being a natural and potent antioxidant, Acacia wood lignin is better suited for the development of safe, biodegradable and functional edible film as compared to other biopolymers (Aadil et al. 2014; Barapatre et al. 2015). However, there are limited reports on the effect of Acacia lignin on the film forming ability, and physical and chemical properties of the gelatin–lignin admixtures.
In the present investigation, attempts were made to prepare the film based on Acacia lignin and gelatin as a safe and biodegradable packaging material. Gelatin has been used in this study due to its biocompatibility, biodegradability, film forming ability and its efficacy as surface coating film to protect food from drying and exposure to light and oxygen. To explore the applicability of prepared lignin–gelatin films in packaging materials, physico-chemical properties, antioxidant activity and in vitro cytotoxicity of these films were evaluated.
The Acacia wood powder was obtained from the local timber mill of Bilaspur, Chhattisgarh, India. The dried wood powder of Acacia was first dewaxed using toluene-ethanol (2:1, v/v) in a soxhlet extractor and dried in an oven at 60 °C before lignin extraction. 2, 2,-dipheny l-1-picrylhydrazyl (DPPH) and sulforhodamine B (SRB) were purchased from Sigma-Aldrich (USA). Commercial type-A gelatin, trichloroacetic acid (TCA) and acetic acid were purchased from Merck, India. Glycerol and sodium hydroxide were obtained from Hi-Media Pvt. Ltd., Mumbai, India. Millipore deionized grade water was used for all the experiments. All other reagents used were of analytical grade.
Isolation of lignin from Acacia wood powder
Lignin was extracted from dewaxed Acacia wood by alkali extraction method as described previously by Aadil et al. (2014). In brief, alkali extraction dried wood was treated with 0.2 N NaOH solution (solid/liquid ratio 1:15 (w/v)) at 120 °C for 45 min. The dark brown liquor was separated by filtration and concentrated in oven at 60 °C to reduce the volume. Dissolved hemicellulose fraction was removed through precipitation by reducing the pH of filtrate up to 5.5 with 5 N HCl followed by adding three volumes of 95 % ethanol. After removing hemicellulose fraction by filtration, soluble lignin fractions were obtained by re-precipitation of lignin at pH 1.5–2.0. To the end, the extracted lignin was washed thoroughly with deionized water to remove the residual impurities. The extracted alkali lignin was labeled as A2.
Preparation of lignin–gelatin film
The films were prepared by a solution casting method. Gelatin powder (4 % w/v) was first dissolved in Millipore deionized water at 60 °C for 30 min. Further, Lignin (1 % w/v in 0.1 N NaOH) was added in pre-solubilized gelatin in different ratios. To plasticize gelatin–lignin film, glycerol (0.6 %, w/v) was added in lignin–gelatin mixture. Thus, the films were prepared in various ratios of gelatin: lignin (100:0, 90:10, 80:20, 70:30, and 60:40) and labeled as LG-A, LG-B, LG-C, LG-D and LG-E. After stirring at 60 °C for 1 h, the filmogenic solution (40 ml) was cast on petri plates (13.5 cm diameter) precoated with aluminum foil and dried in an oven at 60 °C for 2 h. The film obtained was peeled off and kept in desiccator containing P2O5 maintained (at 25 ± 2 °C) at 0 % relative humidity (RH) until further characterization. Initial test was performed to define the most appropriate film thickness for the different reaction mixture containing varying concentration of the lignin and constant amount of glycerol as plasticizer.
Measurement of film thickness
The thickness of the films was measured using the manual digital micrometer (Mitutoyo Manufacturing, Japan) with an accuracy of 0.001 mm. Ten different positions were measured and the average thickness was calculated for each film.
Light barrier properties
Tensile strength (TS) and bursting strength (BS) of the films were determined according to ASTM standard method D882-01 (ASTM 2001) using a tensile strength and bursting strength tester (JSR Instruments, Roorkee, India).
FTIR analysis was carried using a Perkin-Elmer Spectrum One FTIR spectrophotometer at the resolution of 4 cm−1 in the wave number region 400–4000 cm−1. Spectra of samples were obtained from discs containing 1.0 mg sample in approximately 100 mg potassium bromide (KBr).
Differential scanning calorimetry (DSC) analysis
Calorimetric analysis was performed using a differential scanning calorimeter (Mettler Toledo DSC 822e), previously calibrated by running high purity indium. Samples of approximately 7 mg (±0.02 mg) were tightly encapsulated in aluminum pans and scanned under dry nitrogen (50 ml/min). An empty hermetic aluminum pan was used as reference. Freshly conditioned films were cooled to −50 or 0 °C, at 10 °C min−1 and scanned up to 300 °C at a heating rate of 10 °C/min. After cooling at the same rate down to the corresponding initial temperature, a second heating scan was run. Glass transition temperatures, T g (°C), were calculated by the inflection-midpoint method and usually reported on the first heating scans to thermally characterize the same material used in the rest of the analyses. The energetic parameter was normalized to a dry matter content of the corresponding film sample.
Thermo-gravimetric analysis (TGA) and a derivative of TGA (DTG) of synthesized films was performed using a thermo-gravimetric analyzer (Diamond STA-6000, Perkin-Elmer, Shelton, USA) under a nitrogen atmosphere at a flow rate of 200 ml/min. Film samples (about 5 mg) were heated from room temperature to 700 °C at a heating rate of 10 °C/min to obtain individual spectra.
Scanning electron microscopy (SEM)
SEM analysis (ZEISS EVO Series SEM Model EVO 18) was performed for microstructural analysis of the film. Film samples were mounted on a metal stub and gold coated using sputter coating technique for 20 s to make them conducting. Images of the film were taken at 20 kV accelerating voltage at different magnifications.
Atomic force microscopy (AFM)
AFM imaging was accomplished for topographic and surface study of the film using an AFM, (SPM 1600, Shimadzu, Japan) operating in the dynamic mode with a silicon cantilever tip. A thin film of the sample was prepared on a glass slide by dropping 100 µl of the sample solution. The sample coated slide was kept on vacuum desiccator prior to analysis. The topographic images were obtained by scanning the area from 10 × 10 µm to 625 × 625 nm. The SPM online software was used to process the collected images.
DPPH radical scavenging activity assay
In vitro cytotoxicity
All assays were performed in triplicates and the results were validated statistically using one-way analysis of variance (ANOVA). All the tests were considered statistically significant at p < 0.05 and the means were compared using Turkey’s multiple comparison test. The analysis was carried out using Graph Pad Prism Software Version 5. Results were presented as mean value ± standard deviation (SD).
Results and discussion
Physical properties of lignin-gelatin blended film
Moisture content (%)
Water solubility (%)
Swelling property (%)
Bursting index (kg/cm2)
Tensile strength (MPa)
0.17 ± 0.02a
8.96 ± 0.68a
59.15 ± 0.54d
511.48 ± 1.10a
9.34 ± 2.02b
1.22 ± 0.31d
0.16 ± 0.01a
10.03 ± 0.61a
49.34 ± 0.64c
438.75 ± 1.02b
5.32 ± 0.93a
0.74 ± 0.17bc
0.12 ± 0.01a
14.3 ± 0.36b
43.98 ± 0.33b
432.86 ± 0.93c
7.57 ± 0.97ab
0.87 ± 0.11bd
0.12 ± 0.005b
16.43 ± 0.45c
33.37 ± 0.90a
425.80 ± 0.59d
4.96 ± 0.67a
0.46 ± .007a
0.13 ± 0.002a
16.2 ± 0.20c
32.57 ± 0.58a
417.13 ± 0.70e
5.91 ± 2.61a
0.28 ± 0.06a
Moisture content and water solubility
Moisture content of Acacia lignin blended gelatin film is summarized in Table 1. The moisture content ranged between 8.96 % (LG-A) to 16.43 % (LG-D) and 16.2 % (LG-E) among the film. The high moisture content was found in LG-D and LG-E. In this regard, it could be explained that water is not only linked with the film matrix, but is also retained due to the hydrophilic nature of glycerol.
Solubility of edible film in water is an essential asset and water resistance is typically mandatory for possible commercial applications of the edible film. After the 24 h incubation in distilled water, the control gelatin film consequently altered its shape, while the Acacia lignin-gelatin films were found to retain their integrity. Addition of lignin into gelatin film brought about a pronounced decrease in film solubility, from 59.15 (control) to 32.57 % (LG-E) (Table 1). The high water resistance property of lignin-gelatin blended film is possibly due to the interaction and miscibility of phenolic compounds of lignin with amino groups of gelatin. Similar behavior was observed for sago starch films incorporated with lignin isolated from oil palm black liquor waste (Bhat et al. 2013). Literature also suggests that the incorporation of lignin alters the helical structure of gelatin, thereby, subsequently reducing the water solubility of the film (Nunez-Flores et al. 2012; Pena et al. 2010).
The swelling ratios of lignin-gelatin blended films reduced with increase in concentration of lignin. Swelling ratio was found significantly higher in control (LG-A: 511.48 %) film, while the swelling percentage of LG-B, LG-C, LG-D and LG-E (417.13 %) film was recorded lower than control (Table 1). The decrease of swelling values might be due to the interaction between lignin and gelatin molecules by hydrophobic or hydrogen bonding, which reduced water uptake by gelatin meanwhile polar-side-chain groups become less exposed to water molecules (Bigi et al. 2002). Cao et al. (2007) reported that the degree of swelling, significantly decreased to 30.91 and 42.15 % as the concentration of ferulic acid and tannic acid increased. The total phenolic contents of Acacia lignin used in the study was found to be 73.01 ± 3.2 µg in terms of gallic acid equivalent per mg of extracted lignin fraction as reported in our previous study (Aadil et al. 2014).
Light barrier properties
The result showed that as the lignin content in the film increased, tensile (TS) and bursting strength (BS) decreased. TS of the film ranged between 0.28 anbd 1.2 MPa, and the highest value was exhibited for LG-A, while the addition of lignin significantly decreased the TS of the film (Table 1). The highest bursting strength was observed in LG-A, whereas the lowest was observed in LG-D. Acacia lignin incorporation created an apparent plasticizing effect in gelatin film, as assumed from significant decrease in both tensile and bursting strength. It has been reported that lignin provides miscibility with other polymers and act as a plasticizing agent in blend films, but only when added in moderate concentrations (Nunez-Flores et al. 2013). The decrease in TS and BS in the lignin–gelatin blended film can be associated with high moisture content of LG-D and LG-E and deceptive plasticizing effect of lignin leading to a decrease in intermolecular attraction forces between polymer chains (Cuq et al. 1997).
The broad bands at 3421 cm−1 were characteristic of hydroxyl groups in phenolic and aliphatic structures. The band recorded at 2930 cm−1 indicates the C–H group, this band intensity slightly decreased on addition of lignin. The band at 1040 cm−1 (LG-A) could be attributed to the interactions arising between plasticizers (C–O stretch of glycerol) (Bergo and Sobral 2007; Hoque et al. 2011). This band shifted marginally towards higher wavenumber at 1048 cm−1 in the LG-D and LG-E. The most pronounced changes in the films was in the range of 1634–865 cm−1 indicating strong intrusion caused by the lignin in the hydrogen bonding between water and imide residues (Nunez-Flores et al. 2013). This result is also consistent with the report of Cao et al. (2007). Initially, hydrophobic groups of polyphenol interact with the hydrophobic region of protein via hydrophobic interaction followed by the hydrogen bonding between phenolic hydroxyl groups of polyphenols and polar group of protein. Based upon the above mechanism and FTIR data, it might be hypothesized that the hydroxyl and carboxyl group of lignin interact with amino acids of gelatin via hydrogen bonding and hydrophobic interaction. The consequent cross-linked network improved the physical and mechanical properties of the LG films.
Differential scanning calorimetry
Thermo-gravimetric analysis (TGA)
DPPH radical scavenging activity
In vitro cytotoxicity
To use lignin as active food packaging material, it is of importance to study its possible cytotoxic effects. The IC50 value obtained for A2 (149.67 µg/ml) and LG-D (229.34 µg/ml) reveals that it has cytotoxic effects, but only at moderate concentrations (Fig. 8b). Earlier report on lignin cytotoxicity suggested that carbohydrates content and polydispersity affect for cytotoxicity of lignin (Ugartondo et al. 2008). Lignins with low carbohydrate contents and high polydispersity are the most cytotoxic. In our study, the cytotoxic effect of A2 and LG-D exhibited IC50 values lower to those reported for lignin powder (631 µg/ml). Lignin derivatives have been shown to be effective antioxidants at concentration that are not toxic to normal cells, hence extending their possible application in the preparation of active food packaging material (Nunez-Flores et al. 2013; Ugartondo et al. 2008).
The Acacia lignin is a useful component in preparation of gelatin based films. The blended film is flexible, stable and eco-friendly. Addition of lignin significantly affected the water solubility, swelling properties and tensile strength of the film. Structural analysis suggested that the lignin interacts with gelatin by hydrogen and hydrophobic interaction. Lignin blended film might show potential application in food packaging industries due to good UV light absorption capacity and antioxidant activity. In addition, lignin blended gelatin film could also be useful for biomedical and domestic applications, such as coating, lamination, and packaging of non-food material.
KRA was involved in the synthesis and characterization of lignin-gelatin film and the preparation of the manuscript. AB helped in the performing the antioxidant assay. HJ was involved in the design of hypothesis and concept. All the authors are involved in the drafting and revision of the manuscript. All the authors read and approved the final manuscript.
The authors are grateful to the University Grant Commission (UGC), New Delhi, India for funding the project vide- F. No. 41-543/2012 (SR). Dr. Abhishek Kumar Singh for editing the manuscript and Head, Department of Biotechnology, GGV for his support and encouragement. We also grateful to Dr. Goverdhan Reddy Turpu, Department of Physics, GGV for SEM analysis. SAIF, IIT-Madras, Chennai and SAIF, STIC- Cochin is acknowledged for sample analysis.
The authors declare that they have no competing interests.
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