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Co-concentration effect of silane with natural extract on biodegradable polymeric films for food packaging
Accepted Manuscript Title: Co-concentration effect of silane with natural extract on biodegradable polymeric films for food packaging Authors: Anbreen Bashir, Sehrish Jabeen, Nafisa Gull, Atif Islam, Misbah Sultan, Abdul Ghaffar, Shahzad Maqsood Khan, Sadia Sagar Iqbal, Tahir Jamil PII: DOI: Reference:
S0141-8130(17)31539-8 http://dx.doi.org/doi:10.1016/j.ijbiomac.2017.08.025 BIOMAC 8017
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
28-4-2017 2-8-2017 3-8-2017
Please cite this article as: Anbreen Bashir, Sehrish Jabeen, Nafisa Gull, Atif Islam, Misbah Sultan, Abdul Ghaffar, Shahzad Maqsood Khan, Sadia Sagar Iqbal, Tahir Jamil, Co-concentration effect of silane with natural extract on biodegradable polymeric films for food packaging, International Journal of Biological Macromoleculeshttp://dx.doi.org/10.1016/j.ijbiomac.2017.08.025 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Co-concentration effect of silane with natural extract on biodegradable polymeric films for food packaging
Anbreen Bashira, Sehrish Jabeenb, Nafisa Gullb, Atif Islamb,1, Misbah Sultanc, Abdul Ghaffara*, Shahzad Maqsood Khanb, Sadia Sagar Iqbalb, Tahir Jamilb
a
Department of Chemistry, University of Engineering and Technology, Lahore, Pakistan.
b
Department of Polymer Engineering and Technology, University of the Punjab, Lahore,
Pakistan c
Institute of Chemistry, University of the Punjab, Lahore, Pakistan
d
College of Engineering and Emerging Technology, University of the Punjab, Lahore, Pakistan
1
Corresponding author: A Islam; Email: [email protected], +092-300-6686-506, A Ghaffar;
[email protected], +92-332-4858002.
1
Graphical abstract
Highlights
Natural polymer based silane crosslinked blended films along with natural extracts were developed
Biodegradable blended films were devised by solution casting technique
Physical, mechanical, morphological and optical properties of films were studied
Biodegradability and antioxidant activity recommended these films for food packaging
2
Abstract Novel biodegradable films were prepared by blending guar gum, chitosan and poly (vinyl alcohol) having mint (ME) and grapefruit peel (GE) extracts and crosslinked with nontoxic tetraethoxysilane (TEOS). The co-concentration effect of TEOS with natural extracts on the films was studied. FTIR analysis confirmed the presence of incorporated components and the developed interactions among the polymer chains. The surface morphology of the films by SEM showed the hydrophilic character due to porous network structure. The films having both ME and GE with maximum amount of crosslinker (100 µL), showed maximum swelling (58 g/g) and stability while the optical properties showed increased protection against UV light. This film sample showed compact network structure which enhanced the ultimate tensile strength (40.03 MPa) and elongation at break (104.8 %). ME/GE conferred the antioxidant properties determined by radical scavenging activity and total phenolic contents (TPC) as ME films have greater TPC compared to GE films. The soil burial test exhibited the degradation of films rapidly (6 days) confirming their strong microbial activity in soil. The lower water vapour transmission rate and water vapour permeability showed better shelf life; hence, these biodegradable films are environmental friendly and have potential for food and other packaging.
Key Words: Chitosan; Food packaging; Guar gum; PVA; Silane crosslinker.
1.
Introduction
Packaging is the most vital and fundamental factor of the present world. Recently, widespread concerns regarding disposal of plastic bags has led to the development of biodegradable food and
3
other packaging materials. The research work on biodegradable films has been increased to minimize the ecological impacts of synthetic packaging materials [1]. The films are thin layers of materials comprising of polymers that provide mechanical strength with thin structure. A significant research on these films has been made using biopolymers from renewable resources i.e. products or by-products derived from agriculture or from agroindustries. Usually, the films based on biopolymers are highly sensitive to the environmental conditions and generally present low mechanical resistance. Therefore, the films are blended with compatible synthetic polymers to enhance these properties [2, 3]. Guar gum is a low cost, highly abundant and easily available among various biopolymers. It is a polysaccharide consists of a chain of β-D-mannopyranosyl units joined by 1→4 linkages while a d-galactopyranosyl residue bound to the main chain by α (1→6) linkage. It is capable of film forming via solution casting method [4-8]. Poly(vinyl alcohol) (PVA) is a synthetic polymer with excellent mechanical properties and widely used in film preparation. PVA based films and membranes have characteristics like biocompatibility, non-carcinogenic, non-hazardous, high swelling in aqueous solutions with good thermal and physical properties. It is used for the preparation of membranes for reverse osmosis, waste water treatment, removal of heavy metals, controlled release of agriculture fertilizers and packaging. Chemical crosslinking is an appropriate method to alter the structure of natural polymers and to make them striking material for advanced applications [9, 10]. The crosslinking with glutaraldehyde, phosphate, urea formaldehyde and borax modified guar gum was practiced in several fields such as; controlled drug release [8], liquid pesticide, etc. [11]. But, TEOS is preferred due to its non-hazardous nature which is easy to synthesize and binds through condensation reaction with other polymers by its silanol groups. Chitosan is a linear
4
polysaccharide found in the exoskeleton of crustaceans and other invertebrates like insects and molluscs. It contains randomly dispersed deacetylated units of β-(1→4)-linked D-glucosamine and acetylated units of N-acetyl-D-glucosamine obtained by the partial deacetylation of chitin [12, 13]. It can also be obtained from fungal mycelia [14, 15]. In recent decades, chitosan and its derivatives attracted the attention of various researchers due to its suitability in numerous fields like; cosmetics, agriculture, pharmacy, food, biomedical and also in the biomaterials science due to its biological activities such as: wound healing activity, immunological properties, antibacterial behavior, drug delivery and tissue engineering [16-20]. It is valued in food packaging industry due to its film forming, biocompatibility, non-antigenic and nontoxic properties [21, 22]. It has been commonly categorized as safe by the US FDA in 2001 [23]. These characteristics make chitosan an exceptional option to take part in food active packaging. The plant extracts have received considerable attention due to having high concentrations of phenolic compounds that retain strong antioxidant activity. The previous studies have shown that the mint extract (ME) and pomegranate peel extract (PE) have good antioxidant properties [24]. These extracts
efficiently scavenged
2, 2-diphenyl-1-picryl-hydrazyl-hydrate
(DPPH),
superoxide and hydroxyl (-OH) radical and their scavenging capability was similar to the synthetic antioxidant i.e. butylated hydroxytoluene. ME/PE also had good iron chelation capacity and reducing power [25]. The reducing power is related to the antioxidant activity and serve as a significant reflection of the antioxidant activity. ME and GE due to having phenolic compounds assist as electron donor and hence exhibit reducing power [26]. The focus of this work was the development of a novel, biodegradable, environmental friendly and economical packaging material. The co-concentration effect of silane crosslinker with natural extracts (mint and grape fruit) on chitosan, PVA and guar gum blended films was
5
analyzed and reported. The functional group analysis, optical, mechanical and antioxidant properties of these films were investigated as well as their biodegradability in soil. The developed films were also examined for their food and other packaging applications. 2.
Materials and methods
2.1
Materials
Guar gum (extra pure food grade; viscosity 5000 cps) was received from Dabur India Ltd. PVA (D-85662; Mw = 72,000 g/mol; degree of hydrolysis ≥98 %;) was supplied by Merck, Germany. Chitosan (Mw = 17103.41 g/mol, DDA = 90.28 %; viscosity 200 cps) was obtained from Biolog (GmbH) Trademark. TEOS (98.5 %) was provided by DAEJUNG chemical KOSDAQ Co. Ltd., Korea. Mint and grapefruit were purchased from local market. Acetic acid (Mw = 60.05 g/mol; 100 % purity) was obtained from Merck Germany. Ethanol (100% purity) was received from BDH laboratory suppliers England. All other chemicals were of analytical grade. 2.2
Methods
2.2.1. Preparation of mint and grapefruit peel extract The grapefruit peel and mint leaves (100 g each) were refluxed with distilled water (1000 mL) for 1 h to prepare their extracts. The extracts were cooled and stored at room temperature for further use. 2.2.2. Film preparation Guar gum (0.025 g) was dissolved in distilled water (30 mL) with 1 h stirring at room temperature. PVA (1.5 g) was added in 30 mL of preheated distilled water with constant stirring at 80 ˚C. Chitosan (0.1 g) was dissolved in 10 mL of 2% acetic acid solution with constant stirring on a hot plate and was then added into guar gum solution. After blending for 1 h, PVA 6
solution was added to this blend and stirred for 1 h at 50 ˚C and then 0.5 mL of each extract (Table 1) was added to the guar gum/ chitosan/ PVA blended solution. After 30 min of blending, TEOS (in 5 mL ethanol to prepare silanol) was added as crosslinker with varying ratios (Table 1) and further stirred for 1 h at 60 ˚C. The films were formed by casting the blended solution on petri plates and dried at room temperature. The dried films were peeled off and stored in polythene bags in a desiccator for further analysis. The controlled film samples without extracts and crosslinker while the film samples having both the extracts (ME and GE) were also prepared. The detailed compositions and codes of the film samples are given in Table 1. The pictorial view of the experimental protocol for the prepared blends is given in Figure 1. 3.
Characterization
3.1
Film thickness
The film thickness was measured by digital micrometer screw gauge. The measurement of the samples was taken at different positions and the average value was calculated. 3.2
FTIR spectroscopy
The FTIR spectra of all the sample films were recorded on Shimadzu IR-Prestige-21, Kyoto prefecture, Japan with attenuated total reflectance (ATR) mode and programmed range of 4000400 cm-1 at a resolution of 4 cm-1. 3.3
Scanning electron microscopy
Scanning electron microscopy (SEM; JEOL/EO JSM 6490A, Japan) was carried out to investigate the surface morphology and pore size of the blended films. The samples were swollen in distilled water for half an hour and then freeze dried at -80 ˚C. These prepared
7
samples then gold coated to make their surface conductive and analyzed under vacuum and at 20 kV electron beam. All the images were collected at 10 m magnification. 3.4
Swelling test
The swelling analysis of the prepared was performed in distilled water. The blended dried films were first cut into small pieces, weighed (20 mg) and then immersed in a glass vial contained 50 mL of distilled water. The samples were removed after preset time interval (5 min). The surface wetness was vigilantly detached by paper napkin and then the swollen samples were weighed. The experiment was revised at the same time interval until equilibrium was attained. The degree of swelling (DS) was calculated using Equation 1. DS (%) =
𝑊𝑠 − 𝑊𝑑 𝑊𝑑
× 100
[1]
Where, Ws is the swollen weight and Wd is the dry weight of the films. The results were reported as the average of three replicated tests. 3.5
Optical analysis
Each film sample was cut in a rectangular shape and positioned straight in a UV spectrophotometer cell. The spectrum of every film sample was achieved in the wavelength range of 200-800 nm. The analysis was quantified as percentage transmittance and average of three spectra was taken [27]. The transparency at 600 nm was attained by Equation 2. 𝑇600 =
− log 𝑇(%) 𝑏
[2]
Where, b is the film thickness (mm) and T is the percentage transmittance. The opacity of the film samples was calculated using Equation 3 defined by Gilbert and co-workers [28]. 𝑂𝑝𝑎𝑐𝑖𝑡𝑦 = 𝑎𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑎𝑡 500 𝑛𝑚 × film thickness (mm)
[3]
8
3.6
Mechanical properties
Ultimate tensile strength (UTS) of the films was measured according to ASTM D882-91. The films were cut into strips of 0.5 × 2.5 inches for UTS. Gauge length (1.60 inches) and cross-head speed were set at 5.0 mm/min. UTS was measured by dividing force (F) for breaking the film to the area (A) of the sample film and expressed in MPa. The percent elongation at break (% E) was also calculated and the average of at least three determinations of UTS and % E were calculated. 3.7
Biodegradability / Soil burial test
The film samples were buried in composted soil obtained from a flower head nursery. The experiment was performed according to the process published by Thakur et.al [29]. Different containers of capacity 10 L were filled with soil. The films were cut into pieces (30 × 50 mm) and suppressed in the soil at a depth of 10 cm. The humidity of the soil was well-maintained by sprinkling water in different time intervals. The extra water was drained out through a hole at the bottom of the container. The degradation of the films was measured carefully on daily basis by removing the films from the soil, washing it softly by distilled water in order to remove mud from the films and then dried up to one week. The soil burial test was performed by calculating the weight loss of the film [30] which was measured since the beginning day to one week and determined by Equation 4. Weight loss (%) =
𝑊𝑖 − 𝑊𝑑 𝑊𝑖
× 100
[4]
Where, Wd is the dry weight of the blended film after washing and Wi is the initial dry weight of the blended film. 3.8
Antioxidant activity of films
9
The films (5 cm2) were positioned in different flasks comprised of distilled water (10 mL). The flasks were then constantly shaken in an orbital shaker on two different temperatures (30 and 60 ˚C). Three groups of each film sample were engaged for both temperatures. The antioxidant potential of the film sample was observed using supernatant gained from every flask after different time pauses and evaluated for total phenolic content (TPC) and DPPH radical scavenging activity. 3.8.1. Total phenolic content (TPC) The TPC of the plant extracts was assessed by Folin Ciocalteu process [31]. The mixture of diluted sample and Folin Ciocalteu reagent obtained by mixing and preserved them for 5 min at room temperature. 0.75 mL of 6% sodium bicarbonate solution was then added to the blend and incubated for 90 min. The absorbance of the mixture was determined at a wavelength of 725 nm. The TPC was indicated as catechin equivalents. 3.8.2. DPPH radical scavenging activity The DPPH radical scavenging activity was measured by Yamaguchi method [32]. 1 mL of diluted sample was mixed with DPPH in ethanol (1 mL). The absorbance was determined at 517 nm after 20 min in dark at room temperature. The percent DPPH scavenging activity was calculated by Equation 5. 𝐶𝑜𝑛𝑡𝑟𝑜𝑙 𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 = 3.9
𝐸𝑥𝑡𝑟𝑎𝑐𝑡 𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝐶𝑜𝑛𝑡𝑟𝑜𝑙 𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒
× 100
[5]
Water vapor transmission rate (WVTR) and water vapor permeability (WVP)
The WVTR and WVP of the prepared films were analyzed according to the ASTM Method (E 96/E 96M-05) using water vapor transmittance tester SATRA Technology Center, UK. The circular test cups were filled with 30 mL distilled water to attain 100% relative humidity (RH) on 10
one side of the films and maintained at 28 °C. The films were cut according to the size of cup mouth and then placed on the cup rim which was sealed with sealant ring. The samples cup was initially weighed. The temperature and relative humidity were maintained at 28 °C and 54% RH, respectively. The sample cups were weighed for 8 h with 1 h time interval. The WVTR was calculated as the slope of the regression line drawn between time and weight change of the test cups [33, 34]. The WVTR and WVP of the films were calculated using Equations 6 and 7. 𝑊𝑉𝑇𝑅 = 𝑊𝑉𝑃 =
𝑊𝑉𝑇𝑅 ∆𝑃
=
𝐺 𝑡
[6]
𝐴 𝑊𝑉𝑇𝑅 𝑆(𝑅1 − 𝑅2 )
[7]
Where, G is weight change (g), t is the time (h), A is the test area (m2) and ΔP is vapor pressure difference (Pa). 4.
Results and discussion
4.1
Physical properties
The blended films prepared from guar gum, chitosan and PVA were apparently homogeneous, transparent and colorless with no stiff parts or bubbles. The films were readily peeled from the petri plates. The film thickness was a key factor to determine its physical properties. The average thickness of films was 0.2 mm. The thickness of the film was not affected by the addition of extracts owing to the low concentrations of extracts. 4.2
FTIR analysis
The FTIR spectra of pure guar gum, chitosan, PVA and their blended films (GCPm1, GCPg1 and GCPmg1) are shown in Figure 2 which confirmed the presence of incorporated components and the developed interactions. The FTIR spectrum of the pure and blended films displayed broad 11
band between 3541 to 3110 cm-1 correspond to -OH/ -NH2 stretching of inter- and intermolecular hydrogen bonds [35]. The strong bands of amide-I at 1653 cm-1 and at 1548 cm-1 of amide-II confirmed the characteristic of chitosan moiety. Moreover, the band at 834 cm-1 due to pyranose ring also confirmed the existence of chitosan and guar gum moieties. C—O stretching of C—O—C was observed at 1020 cm-1 while -CH stretching of alkyl group was observed at 2926 cm-1. The absorption bands at 1020 and 1085 cm-1 confirmed the presence of siloxane bond (Si—O—Si) which confirmed network structure by TEOS [36]. The absorption bands at 1415 and 1085 cm-1 were ascribed to the bending (-OH) and stretching (C—O) of pure PVA, respectively [37]. 4.3
SEM analysis
The morphological features of the blended films were investigated by SEM. The controlled film without extracts and crosslinker was uneven and irregular with visible depressions (Figure 3a). With the addition of ME and GE in the blended crosslinked films having 100 µL TEOS, the porosity was generated and numerous evenly distributed pores were observed on the film surface (Figure 3b, 3c and 3d). The observed pore size was increased with the decrease in the amount of TEOS (Figure 3e and 3f) confirmed the good hydrophilic character. The histogram was developed between the number of pores and pore size and shown in Figure 3 as well. 4.4
Swelling analysis
The swelling trend of all the blended films is shown in Figure 4. A gradual increase in swelling was observed with increase in time. The film (GCPmg1) having both mint and grapefruit peel extracts and maximum amount of crosslinker (100 µL), showed maximum swelling (58 g/g) and stability compared to the other films. The equilibrium time for GCPm1, GCPg1, and GCPmg1 was 25 min while for GCPmg2 and GCPmg3, it was 15 and 20 min, respectively. The films 12
(GCPm1, GCPg1 and GCPmg1) having higher amount of crosslinker (100 µL) were more stable as compared to GCPmg2 and GCPmg3 which contained lesser amount of crosslinker (75 µL) and (50 µL), respectively. The films were observed to be deformed after 25 min of swelling except GCPm1 and GCPmg1. Initially, GCPmg2 showed increased swelling in 15 min after which a decreasing trend was observed up to 25 min. GCPmg3, having the lowest amount of crosslinker showed maximum swelling in first 5 min then a steady increase was observed up to 20 min while the film sample was deformed after 25 min. 4.5
Optical analysis
One of the preferred properties of packaging is that it must be shielded from light, particularly UV radiation. The films were scanned at wavelength range of 200-800 nm to find the light transmission characteristics and the percent transmittance was calculated. Each film specimen exhibited different light transmission properties in UV light, especially the films showed low light transmission at wavelength of 300 nm (Figure 5). GCPg1 showed lower transmission percent at 300 nm as compared to GCPm1 having the same amount of crosslinker (100 µL). GCPmg1 (100 µL crosslinker) showed maximum percentage of transmittance as compared to GCPmg2 and GCPmg3 (75 and 50 µL crosslinker, respectively). Gómez-Guillén et al. have shown that the mixing of extract from murta with gelatin has increased the light barrier properties [38]. The opacity and transparency values showed higher transparency value with the lowest opacity (Table 2). There was a small difference in transparency due to low extract concentration.
4.6
Mechanical properties
13
In order to resist the stress that occur during storage and handling, packaging films require film reliability. The mechanical properties of the blend films are described in Table 3. The guar gum chitosan and PVA blended film (GCPmg1) with mint and grapefruit peel extract and higher concentration of crosslinker (100 µL) showed the highest UTS (40.03 MPa) and % E of 104.8 % exhibited that the greater force was required to break the sample. Kanatt et al. have shown that with the addition of extracts, there was a rise in UTS of the films particularly with higher concentration of PVA [39]. The phenolic compounds comprised of a number of -OH groups which form hydrogen bonding with chitosan and consequently there was an increase in UTS with the addition of extracts[32]. The improvement in UTS of chitosan films combined with green tea extract ascribed to the strong bonding between chitosan and phenolic compounds from green tea extract [40]. The integration of grape seed extract considerably increased the UTS of soy protein isolated blend films [41]. 4.7
Soil burial test/composting
All the blended films with and without extracts showed biodegradability in soil. There was a rapid degradation process with great weight loss of all the samples observed as shown in Figure 6 and 7. Almost all the films degraded rapidly in 6 days which exhibited that the blended films have strong microbial activity in soil. The fast degradation was due to the composting procedure which happened in two main steps; an active composting step and a curing period [30]. In the first stage, the temperature rose and remained elevated with the availability of oxygen which caused the strong microbial action while the temperature was decreased in second step but the film samples compost at a mild rate continuously. The compost was made of organic materials obtained from plant and animal substance that have been decayed mainly through aerobic breakdown. It was rich in nutrients and valuable for the land in various ways because of having
14
properties as a fertilizer and soil conditioner. The addition of vigorous humus also acts as natural pesticide for soil. The composting nutrients could decompose the blended film more speedily than the normal soil. 4.8
Antioxidant properties
The antioxidants in films permits nutritive and appealing characteristics to be improved without disturbing the quality of the product. The food might be kept at diverse temperatures in antioxidant packaging and therefore in guar gum/ chitosan/ PVA films having natural extracts, the release of phenolic and DPPH radical scavenging activity was determined. 4.8.1 Total phenolic content The phenolic compounds considered as secondary plant metabolites, active hydrogen donors and hold the best structural characteristics for scavenging free radical and therefore have remarkable antioxidant activity. To study the release mechanism of bioactive components, the blended films were submerged in distilled water while being polar the blended films started to hydrate with subsequent swelling producing relaxation of polymer chains. The blended film ultimately lost their structural stability and released the bioactive components. The films without any extract contained no significant phenolic content. The TPC of blended films comprising mint extract was greater than grapefruit peel extract. Additionally, the phenolic content released from the films varied significantly as TPC was monitored at two different temperatures. At higher temperature (37 ˚C), the release of phenolic content from the film sample was maximum than at 28 ˚C as shown in Figure 8. The integration of phenolic compounds such as catechin, p-hydroxy benzoic acid and gallic acid in films reduced the antioxidant activity of the films, subsequently a significant percentage of the phenolic component in the blended films was arised in soluble form [7]. 15
4.8.2 DPPH radical scavenging activity In order to find the antioxidant characteristic or to test the capability of compounds as hydrogen donors, DPPH radical has been commonly used. Figure 9 showed that the films containing extracts have DPPH radical scavenging activity. The control film sample having no extract did not display scavenging action. The films having mint and grapefruit extract had considerably higher antioxidant activity as compared to other films. The temperature was a main aspect in the release of natural extracts from the guar gum, chitosan and PVA films. At 37 ˚C, the release of natural extract was more from the blended films whereas at 28 ˚C, the antioxidant activity of the film sample were considerably reduced owing to the slow release of natural extracts from the film. When the film samples were kept at 37 ˚C, the release of extracts from the film in the 45 min and after that the release was fairly constant even incubated after 24 h. Correspondingly, the trend in gooseberry extract has been described by Devahastin et al [42]. 4.9
Water vapor transmission rate (WVTR) and water vapor permeability (WVP)
The WVTR and WVP of films are essential parameters for fruit coatings and packaging applications. The water barrier property of packaging materials is key factor to prevent the spoilage of food. Chitosan is also used as a food additive and act as an antibacterial agent and these properties not only help to retard microbial growth in fruit but also enhanced the shelf life of fruits and other food stuff [33, 43, 44]. The values for WVTR and WVP of films (GCP, GCPm1, GCPg1, GCPmg1, GCPmg2, GCPmg3) were 144.44, 150, 111.11, 150, 166.66, 166.66 (× 10-3) gh-1 m-2 and 1.42, 1.48, 1.09, 1.48, 1.64, 1.64 (× 10-6) gPa-1 h-1 m-2, respectively. All the blended films showed lower permeability values which proved that the material has the ability to extend the shelf life of food and has potential for packaging. 5.
Conclusions 16
Novel biodegradable silane crosslinked guar gum/ chitosan/ PVA blended films were successfully fabricated incorporated with ME and GE. FTIR analysis confirmed the functional groups and the developed interactions in the blended films. Nanoporous network structure was observed by SEM analysis confirmed the hydrophilic character in the films. GCPmg1 sample with both ME/GE extracts and the highest of silane crosslinker (100 µL), exhibited the maximum stability and swelling (58 g/g) as compared to other blended films. All film samples with and without extracts showed transparency, opacity and increased protection against UV light. GCPmg1 showed the enhanced ultimate tensile strength and % E of 40.03 MPa and 104.8 %, respectively. ME/GE conferred the outstanding antioxidant properties of the blended films as the films having mint extract have greater TPC compared to the films having grapefruit peel extract and TPC released from the films was maximum at 37 ˚C. There was a rapid biodegradation process with a great weight loss of all the film samples and almost all the films were degraded rapidly in 6 days showing the strong microbial activity in soil. The lower WVT rate and WVP confirmed the improved shelf life and environmental friendly behaviour of the blended films. Hence, bioactive biopolymers incorporated with natural extracts have excessive applications for food and other packaging materials. Acknowledgements The author is highly obliged to the Applied Chemistry Department, PCSIR laboratories, Lahore, Pakistan for performing the water vapor transmission tests and also to the School of Chemical and Materials Engineering, NUST, Islamabad, Pakistan for performing SEM analysis.
17
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K. Kurita, Chitin and chitosan: Functional biopolymers from marine crustaceans, Marine Biotechnol. 8 (2006) 203-226.
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M. Rinaudo, Chitin and chitosan: properties and applications, Prog. Polym. Sci. 31 (2006) 603-632.
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M.A. Cerqueira, B.W.S. Souza, J.A. Teixeira, A.A. Vicente, Effects of interactions between the constituents of chitosanedible films on their physical properties, Food and Bioprocess Tech. 5 (2012) 3181-3192.
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S. Hirano, C. Itakura, H. Seino, Y. Akiyama, I. Nonaka, N. Kanbara, T. Kawakami, Chitosan as an ingredient for domestic animal feeds. J. Agric. Food Chem. 38 (1990) 1214-1217.
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S. Sagoo, R. Board, S. Roller, Chitosan inhibits growth of spoilage micro-organisms in chilled pork products, Food Microbiol. 19 (2002) 175-182.
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S.R. Kanatt, R. Chander, A. Sharma, Antioxidant potential of mint (Mentha spicataL.) in radiation processed lamb meat, Food Chem. 100 (2007) 451-458.
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S.R. Kanatt, M.S. Rao, S.P. Chawla, A. Sharma, Active chitosane polyvinyl alcohol films with natural extracts. Food Hydrocoll. 29 (2012) 290-297.
26.
P. Jayanthi, P. Lalitha, Reducing power of the solvent extracts of eichhornia crassipes (mart.) Solms. Int. J. Pharm. Pharm. Sci. 3 (2011) 1-3.
20
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J.H. Han, J.D. Floros, Casting antimicrobial packaging films and measuring their physical properties and antimicrobial activity. J. Plast. Film Sheeting 13 (1997) 287-298.
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S. Guilbert, N. Contard, L.G.M. Gorris, Prolongation of shelf-life of perishable food products using biodegradablefilms and coatings. LWT - Food Sci. Technol. 29 (1996) 1017.
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V.K. Thakur, M.K. Thakur, R.K. Gupta, Review: Raw natural fiber-based polymer composites. Int. J. Polym. Anal. Ch. 19 (2014) 256-271.
30.
N.A. Azahari, N. Othman, H. Ismail, Biodegradation studies of polyvinyl alcohol/corn starch blend films in solid and solution media. Journal of Physical Science, 22 (2011) 1531.
31.
V.L. Singleton, J.A. Rossi, Colorimetry of total phenolics with phosphomolybdicphosphotunstic acid reagents. Am. J. Enol. Viti. 16 (1965) 144-158.
32.
T. Yamaguchi, H. Takamura, T., Matoba, J. Terao, HPLC method for theevaluation of the free radical escavenging activity of foods by using 1,1-Diphenyl-2-picrylhydrazyl. Biosci. Biotechnol. Biochem. 62 (1998) 1201-1204.
33.
I. Bano, M.A. Ghauri, T. Yasin, Q. Huang, A. D’ S. Palaparthi, Characterization and potential applications of gamma irradiatedchitosan and its blends with poly(vinyl alcohol). Int. J. Biol. Macromol. 65 (2014) 81–88.
34.
R.S. Jagadish, K.N. Divyashree, P. Viswanath, P. Srinivas, B. Raja, Preparation of Nvanillyl chitosan and 4-hydroxybenzyl chitosan and their physico-mechanical, optical, barrier, and antimicrobial properties. Carbohydr. Polym. 87 (2012) 110-116.
21
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S-Y. Lee, G. Tae, Formulation and in vitro characterization of an in situ gelable, photopolymerizable Pluronic hydrogel suitable for injection. J. Control. Release 119 (2007) 313-319.
36.
N. Rasool, T. Yasin, J.Y.Y. Heng, Z. Akhter, Synthesis and characterization of novel pH, ionic strength and temperature-sensitive hydrogel for insulin delivery, Polymer 51 (2010) 1687-1693.
37.
S. Jabeen, A. Islam, A. Ghaffar, N. Gull, A. Hameed, T. Jamil, Bashir, A., Hussain, T. Development of a novel pH sensitive silane crosslinked injectable hydrogel for controlled release of neomycin sulfate, Int. J. Biol. Macromol. 97 (2017) 218-227.
38.
M.C. Gómez-Guillén, M. Ihl, V. Bifani, A. Silva, P. Montero, Ediblefilms made from tuna-fish gelatin with antioxidant extracts of two different murta ecotypes leaves (Ugni molinae Turcz), Food Hydrocoll. 21 (2007) 1133-1143.
39.
S.R. Kanatt, R. Chander, A. Sharma, Antioxidant and antimicrobial activity of pomegranate peel extract improves the shelf life of chicken products. Int. J. Food Sci. Technol. 45 (2010) 216-222.
40.
U. Siripatrawan, B.R. Harte, Physical properties and antioxidant activity of an active film from chitosan incorporated with green tea extract, Food Hydrocoll. 24 (2010) 770-775.
41.
T. Sivarooban, N.S. Hettiarachchy, M.G. Johnson, Physical and antimicrobial properties of grape seed extract, nisin, and EDTA incorporated soy protein ediblefilms. Food Res. Int. 41 (2008) 781-785.
42.
P. Mayachiew, S. Devahastin, B.M. Mackey, K. Niranjan, Effects of drying methods and conditions on antimicrobial activity of edible chitosan films enriched with galangal extract. Food Res. Int. 43 (2010) 125-132.
22
43.
N. Pitak, S.K. Rakshit, Physical and antimicrobial properties of banana flour/chitosan biodegradable and self sealing films used for preserving Fresh-cut vegetables. LWT Food Sci. Tech. 44 (2011) 2310-2315.
44.
M. Sabaghi, Y. Maghsoudlou, P. Habibi, Novel Kefiran-Polyvinyl Alcohol Composite Film: Physical, Mechanical and Rheological Properties. Nutr. Food Sci. Res. 2 (2015) 3946.
23
Figure 1. The pictorial view of the preparation of chitosan/ guar gum/ PVA/ extracts blends.
24
Figure 2. The FTIR spectra of pure guar gum, chitosan, PVA films and GCPm1, GCPg1, GCPmg1 treated with natural extract and TEOS.
25
Figure 3. SEM analysis of the prepared blended films with pore size histogram a) GCP, b) GCPm1, c) GCPg1, d) GCPmg1, e) GCPmg2, f) GCPmg3.
26
Figure 4. The time (min) vs swelling (g/g) of GCPm1, GCPg1 and GCPmg1 blended films at room temperature.
27
Figure 5. The transmittance of guar gum, chitosan and PVA films along with mint and grapefruit extract.
28
Figure 6. The photographs of samples GCPm1 (a, b) and GPCg1 (c, d) before and after buried in soil.
29
Figure 7. Weight loss of samples with varying ratio of extract and TEOS.
30
Figure 8. TPC of guar gum, chitosan PVA films integrated with ME/GE (a) 28 ˚C (b) 37 ˚C
31
Figure 9. DPPH radical scavenging activity of guar gum, chitosan and PVA films integrated with ME/GE (a) 28 ˚C (b) 37 ˚C
32
Table 1. The codes and composition of guar gum, chitosan and PVA (GCP) films. Sr.
Sample
Guar
Chitosan
PVA
Mint
Grapefruit TEOS
No
Codes
gum (g)
(g)
(g)
extract
peel
(mL)
extract
(µL)
(mL) 1
GCP
0.025
0.1
1.5
0
0
0
2
GCPm1
0.025
0.1
1.5
0.5
0
100
3
GCPg1
0.025
0.1
1.5
0
0.5
100
4
GCPmg1
0.025
0.1
1.5
0.5
0.5
100
5
GCPmg2
0.025
0.1
1.5
0.5
0.5
75
6
GCPmg3
0.025
0.1
1.5
0.5
0.5
50
33
Table 2. The opacity and transparency of chitosan, guar gum PVA films integrated with ME/GE. Sample codes
Opacity
Transparency
GCP
0.060 ± 0.0001
9.0 ± 0.1
GCPm1
0.040 ± 0.0001
9.0 ± 0.1
GCPg1
0.055 ± 0.0002
8.9 ± 0.2
GCPmg1
0.036 ± 0.0001
9.0 ± 0.1
GCPmg2
0.065 ± 0.0002
8.8 ± 0.2
GCPmg3
0.070 ± 0.0003
8.7 ± 0.3
34
Table 3. UTS and % E of chitosan/guar gum/PVA films incorporated with mint and grapefruit extracts. Sample Codes
Ultimate
Tensile Elongation at break
strength (MPa)
(%)
GCP
15.3 ± 0.1
40.65 ± 0.1
GCPm1
32.99 ± 0.3
97.33 ± 0.3
GCPg1
29.39 ± 0.2
89.31 ± 0.2
GCPmg1
40.03 ± 0.1
104.8 ± 0.1
GCPmg2
28.88 ± 0.2
88.74 ± 0.2
GCPmg3
28.44 ± 0.1
86.18 ± 0.1
35
S0141-8130(17)31539-8 http://dx.doi.org/doi:10.1016/j.ijbiomac.2017.08.025 BIOMAC 8017
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
28-4-2017 2-8-2017 3-8-2017
Please cite this article as: Anbreen Bashir, Sehrish Jabeen, Nafisa Gull, Atif Islam, Misbah Sultan, Abdul Ghaffar, Shahzad Maqsood Khan, Sadia Sagar Iqbal, Tahir Jamil, Co-concentration effect of silane with natural extract on biodegradable polymeric films for food packaging, International Journal of Biological Macromoleculeshttp://dx.doi.org/10.1016/j.ijbiomac.2017.08.025 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Co-concentration effect of silane with natural extract on biodegradable polymeric films for food packaging
Anbreen Bashira, Sehrish Jabeenb, Nafisa Gullb, Atif Islamb,1, Misbah Sultanc, Abdul Ghaffara*, Shahzad Maqsood Khanb, Sadia Sagar Iqbalb, Tahir Jamilb
a
Department of Chemistry, University of Engineering and Technology, Lahore, Pakistan.
b
Department of Polymer Engineering and Technology, University of the Punjab, Lahore,
Pakistan c
Institute of Chemistry, University of the Punjab, Lahore, Pakistan
d
College of Engineering and Emerging Technology, University of the Punjab, Lahore, Pakistan
1
Corresponding author: A Islam; Email: [email protected], +092-300-6686-506, A Ghaffar;
[email protected], +92-332-4858002.
1
Graphical abstract
Highlights
Natural polymer based silane crosslinked blended films along with natural extracts were developed
Biodegradable blended films were devised by solution casting technique
Physical, mechanical, morphological and optical properties of films were studied
Biodegradability and antioxidant activity recommended these films for food packaging
2
Abstract Novel biodegradable films were prepared by blending guar gum, chitosan and poly (vinyl alcohol) having mint (ME) and grapefruit peel (GE) extracts and crosslinked with nontoxic tetraethoxysilane (TEOS). The co-concentration effect of TEOS with natural extracts on the films was studied. FTIR analysis confirmed the presence of incorporated components and the developed interactions among the polymer chains. The surface morphology of the films by SEM showed the hydrophilic character due to porous network structure. The films having both ME and GE with maximum amount of crosslinker (100 µL), showed maximum swelling (58 g/g) and stability while the optical properties showed increased protection against UV light. This film sample showed compact network structure which enhanced the ultimate tensile strength (40.03 MPa) and elongation at break (104.8 %). ME/GE conferred the antioxidant properties determined by radical scavenging activity and total phenolic contents (TPC) as ME films have greater TPC compared to GE films. The soil burial test exhibited the degradation of films rapidly (6 days) confirming their strong microbial activity in soil. The lower water vapour transmission rate and water vapour permeability showed better shelf life; hence, these biodegradable films are environmental friendly and have potential for food and other packaging.
Key Words: Chitosan; Food packaging; Guar gum; PVA; Silane crosslinker.
1.
Introduction
Packaging is the most vital and fundamental factor of the present world. Recently, widespread concerns regarding disposal of plastic bags has led to the development of biodegradable food and
3
other packaging materials. The research work on biodegradable films has been increased to minimize the ecological impacts of synthetic packaging materials [1]. The films are thin layers of materials comprising of polymers that provide mechanical strength with thin structure. A significant research on these films has been made using biopolymers from renewable resources i.e. products or by-products derived from agriculture or from agroindustries. Usually, the films based on biopolymers are highly sensitive to the environmental conditions and generally present low mechanical resistance. Therefore, the films are blended with compatible synthetic polymers to enhance these properties [2, 3]. Guar gum is a low cost, highly abundant and easily available among various biopolymers. It is a polysaccharide consists of a chain of β-D-mannopyranosyl units joined by 1→4 linkages while a d-galactopyranosyl residue bound to the main chain by α (1→6) linkage. It is capable of film forming via solution casting method [4-8]. Poly(vinyl alcohol) (PVA) is a synthetic polymer with excellent mechanical properties and widely used in film preparation. PVA based films and membranes have characteristics like biocompatibility, non-carcinogenic, non-hazardous, high swelling in aqueous solutions with good thermal and physical properties. It is used for the preparation of membranes for reverse osmosis, waste water treatment, removal of heavy metals, controlled release of agriculture fertilizers and packaging. Chemical crosslinking is an appropriate method to alter the structure of natural polymers and to make them striking material for advanced applications [9, 10]. The crosslinking with glutaraldehyde, phosphate, urea formaldehyde and borax modified guar gum was practiced in several fields such as; controlled drug release [8], liquid pesticide, etc. [11]. But, TEOS is preferred due to its non-hazardous nature which is easy to synthesize and binds through condensation reaction with other polymers by its silanol groups. Chitosan is a linear
4
polysaccharide found in the exoskeleton of crustaceans and other invertebrates like insects and molluscs. It contains randomly dispersed deacetylated units of β-(1→4)-linked D-glucosamine and acetylated units of N-acetyl-D-glucosamine obtained by the partial deacetylation of chitin [12, 13]. It can also be obtained from fungal mycelia [14, 15]. In recent decades, chitosan and its derivatives attracted the attention of various researchers due to its suitability in numerous fields like; cosmetics, agriculture, pharmacy, food, biomedical and also in the biomaterials science due to its biological activities such as: wound healing activity, immunological properties, antibacterial behavior, drug delivery and tissue engineering [16-20]. It is valued in food packaging industry due to its film forming, biocompatibility, non-antigenic and nontoxic properties [21, 22]. It has been commonly categorized as safe by the US FDA in 2001 [23]. These characteristics make chitosan an exceptional option to take part in food active packaging. The plant extracts have received considerable attention due to having high concentrations of phenolic compounds that retain strong antioxidant activity. The previous studies have shown that the mint extract (ME) and pomegranate peel extract (PE) have good antioxidant properties [24]. These extracts
efficiently scavenged
2, 2-diphenyl-1-picryl-hydrazyl-hydrate
(DPPH),
superoxide and hydroxyl (-OH) radical and their scavenging capability was similar to the synthetic antioxidant i.e. butylated hydroxytoluene. ME/PE also had good iron chelation capacity and reducing power [25]. The reducing power is related to the antioxidant activity and serve as a significant reflection of the antioxidant activity. ME and GE due to having phenolic compounds assist as electron donor and hence exhibit reducing power [26]. The focus of this work was the development of a novel, biodegradable, environmental friendly and economical packaging material. The co-concentration effect of silane crosslinker with natural extracts (mint and grape fruit) on chitosan, PVA and guar gum blended films was
5
analyzed and reported. The functional group analysis, optical, mechanical and antioxidant properties of these films were investigated as well as their biodegradability in soil. The developed films were also examined for their food and other packaging applications. 2.
Materials and methods
2.1
Materials
Guar gum (extra pure food grade; viscosity 5000 cps) was received from Dabur India Ltd. PVA (D-85662; Mw = 72,000 g/mol; degree of hydrolysis ≥98 %;) was supplied by Merck, Germany. Chitosan (Mw = 17103.41 g/mol, DDA = 90.28 %; viscosity 200 cps) was obtained from Biolog (GmbH) Trademark. TEOS (98.5 %) was provided by DAEJUNG chemical KOSDAQ Co. Ltd., Korea. Mint and grapefruit were purchased from local market. Acetic acid (Mw = 60.05 g/mol; 100 % purity) was obtained from Merck Germany. Ethanol (100% purity) was received from BDH laboratory suppliers England. All other chemicals were of analytical grade. 2.2
Methods
2.2.1. Preparation of mint and grapefruit peel extract The grapefruit peel and mint leaves (100 g each) were refluxed with distilled water (1000 mL) for 1 h to prepare their extracts. The extracts were cooled and stored at room temperature for further use. 2.2.2. Film preparation Guar gum (0.025 g) was dissolved in distilled water (30 mL) with 1 h stirring at room temperature. PVA (1.5 g) was added in 30 mL of preheated distilled water with constant stirring at 80 ˚C. Chitosan (0.1 g) was dissolved in 10 mL of 2% acetic acid solution with constant stirring on a hot plate and was then added into guar gum solution. After blending for 1 h, PVA 6
solution was added to this blend and stirred for 1 h at 50 ˚C and then 0.5 mL of each extract (Table 1) was added to the guar gum/ chitosan/ PVA blended solution. After 30 min of blending, TEOS (in 5 mL ethanol to prepare silanol) was added as crosslinker with varying ratios (Table 1) and further stirred for 1 h at 60 ˚C. The films were formed by casting the blended solution on petri plates and dried at room temperature. The dried films were peeled off and stored in polythene bags in a desiccator for further analysis. The controlled film samples without extracts and crosslinker while the film samples having both the extracts (ME and GE) were also prepared. The detailed compositions and codes of the film samples are given in Table 1. The pictorial view of the experimental protocol for the prepared blends is given in Figure 1. 3.
Characterization
3.1
Film thickness
The film thickness was measured by digital micrometer screw gauge. The measurement of the samples was taken at different positions and the average value was calculated. 3.2
FTIR spectroscopy
The FTIR spectra of all the sample films were recorded on Shimadzu IR-Prestige-21, Kyoto prefecture, Japan with attenuated total reflectance (ATR) mode and programmed range of 4000400 cm-1 at a resolution of 4 cm-1. 3.3
Scanning electron microscopy
Scanning electron microscopy (SEM; JEOL/EO JSM 6490A, Japan) was carried out to investigate the surface morphology and pore size of the blended films. The samples were swollen in distilled water for half an hour and then freeze dried at -80 ˚C. These prepared
7
samples then gold coated to make their surface conductive and analyzed under vacuum and at 20 kV electron beam. All the images were collected at 10 m magnification. 3.4
Swelling test
The swelling analysis of the prepared was performed in distilled water. The blended dried films were first cut into small pieces, weighed (20 mg) and then immersed in a glass vial contained 50 mL of distilled water. The samples were removed after preset time interval (5 min). The surface wetness was vigilantly detached by paper napkin and then the swollen samples were weighed. The experiment was revised at the same time interval until equilibrium was attained. The degree of swelling (DS) was calculated using Equation 1. DS (%) =
𝑊𝑠 − 𝑊𝑑 𝑊𝑑
× 100
[1]
Where, Ws is the swollen weight and Wd is the dry weight of the films. The results were reported as the average of three replicated tests. 3.5
Optical analysis
Each film sample was cut in a rectangular shape and positioned straight in a UV spectrophotometer cell. The spectrum of every film sample was achieved in the wavelength range of 200-800 nm. The analysis was quantified as percentage transmittance and average of three spectra was taken [27]. The transparency at 600 nm was attained by Equation 2. 𝑇600 =
− log 𝑇(%) 𝑏
[2]
Where, b is the film thickness (mm) and T is the percentage transmittance. The opacity of the film samples was calculated using Equation 3 defined by Gilbert and co-workers [28]. 𝑂𝑝𝑎𝑐𝑖𝑡𝑦 = 𝑎𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑎𝑡 500 𝑛𝑚 × film thickness (mm)
[3]
8
3.6
Mechanical properties
Ultimate tensile strength (UTS) of the films was measured according to ASTM D882-91. The films were cut into strips of 0.5 × 2.5 inches for UTS. Gauge length (1.60 inches) and cross-head speed were set at 5.0 mm/min. UTS was measured by dividing force (F) for breaking the film to the area (A) of the sample film and expressed in MPa. The percent elongation at break (% E) was also calculated and the average of at least three determinations of UTS and % E were calculated. 3.7
Biodegradability / Soil burial test
The film samples were buried in composted soil obtained from a flower head nursery. The experiment was performed according to the process published by Thakur et.al [29]. Different containers of capacity 10 L were filled with soil. The films were cut into pieces (30 × 50 mm) and suppressed in the soil at a depth of 10 cm. The humidity of the soil was well-maintained by sprinkling water in different time intervals. The extra water was drained out through a hole at the bottom of the container. The degradation of the films was measured carefully on daily basis by removing the films from the soil, washing it softly by distilled water in order to remove mud from the films and then dried up to one week. The soil burial test was performed by calculating the weight loss of the film [30] which was measured since the beginning day to one week and determined by Equation 4. Weight loss (%) =
𝑊𝑖 − 𝑊𝑑 𝑊𝑖
× 100
[4]
Where, Wd is the dry weight of the blended film after washing and Wi is the initial dry weight of the blended film. 3.8
Antioxidant activity of films
9
The films (5 cm2) were positioned in different flasks comprised of distilled water (10 mL). The flasks were then constantly shaken in an orbital shaker on two different temperatures (30 and 60 ˚C). Three groups of each film sample were engaged for both temperatures. The antioxidant potential of the film sample was observed using supernatant gained from every flask after different time pauses and evaluated for total phenolic content (TPC) and DPPH radical scavenging activity. 3.8.1. Total phenolic content (TPC) The TPC of the plant extracts was assessed by Folin Ciocalteu process [31]. The mixture of diluted sample and Folin Ciocalteu reagent obtained by mixing and preserved them for 5 min at room temperature. 0.75 mL of 6% sodium bicarbonate solution was then added to the blend and incubated for 90 min. The absorbance of the mixture was determined at a wavelength of 725 nm. The TPC was indicated as catechin equivalents. 3.8.2. DPPH radical scavenging activity The DPPH radical scavenging activity was measured by Yamaguchi method [32]. 1 mL of diluted sample was mixed with DPPH in ethanol (1 mL). The absorbance was determined at 517 nm after 20 min in dark at room temperature. The percent DPPH scavenging activity was calculated by Equation 5. 𝐶𝑜𝑛𝑡𝑟𝑜𝑙 𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 = 3.9
𝐸𝑥𝑡𝑟𝑎𝑐𝑡 𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝐶𝑜𝑛𝑡𝑟𝑜𝑙 𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒
× 100
[5]
Water vapor transmission rate (WVTR) and water vapor permeability (WVP)
The WVTR and WVP of the prepared films were analyzed according to the ASTM Method (E 96/E 96M-05) using water vapor transmittance tester SATRA Technology Center, UK. The circular test cups were filled with 30 mL distilled water to attain 100% relative humidity (RH) on 10
one side of the films and maintained at 28 °C. The films were cut according to the size of cup mouth and then placed on the cup rim which was sealed with sealant ring. The samples cup was initially weighed. The temperature and relative humidity were maintained at 28 °C and 54% RH, respectively. The sample cups were weighed for 8 h with 1 h time interval. The WVTR was calculated as the slope of the regression line drawn between time and weight change of the test cups [33, 34]. The WVTR and WVP of the films were calculated using Equations 6 and 7. 𝑊𝑉𝑇𝑅 = 𝑊𝑉𝑃 =
𝑊𝑉𝑇𝑅 ∆𝑃
=
𝐺 𝑡
[6]
𝐴 𝑊𝑉𝑇𝑅 𝑆(𝑅1 − 𝑅2 )
[7]
Where, G is weight change (g), t is the time (h), A is the test area (m2) and ΔP is vapor pressure difference (Pa). 4.
Results and discussion
4.1
Physical properties
The blended films prepared from guar gum, chitosan and PVA were apparently homogeneous, transparent and colorless with no stiff parts or bubbles. The films were readily peeled from the petri plates. The film thickness was a key factor to determine its physical properties. The average thickness of films was 0.2 mm. The thickness of the film was not affected by the addition of extracts owing to the low concentrations of extracts. 4.2
FTIR analysis
The FTIR spectra of pure guar gum, chitosan, PVA and their blended films (GCPm1, GCPg1 and GCPmg1) are shown in Figure 2 which confirmed the presence of incorporated components and the developed interactions. The FTIR spectrum of the pure and blended films displayed broad 11
band between 3541 to 3110 cm-1 correspond to -OH/ -NH2 stretching of inter- and intermolecular hydrogen bonds [35]. The strong bands of amide-I at 1653 cm-1 and at 1548 cm-1 of amide-II confirmed the characteristic of chitosan moiety. Moreover, the band at 834 cm-1 due to pyranose ring also confirmed the existence of chitosan and guar gum moieties. C—O stretching of C—O—C was observed at 1020 cm-1 while -CH stretching of alkyl group was observed at 2926 cm-1. The absorption bands at 1020 and 1085 cm-1 confirmed the presence of siloxane bond (Si—O—Si) which confirmed network structure by TEOS [36]. The absorption bands at 1415 and 1085 cm-1 were ascribed to the bending (-OH) and stretching (C—O) of pure PVA, respectively [37]. 4.3
SEM analysis
The morphological features of the blended films were investigated by SEM. The controlled film without extracts and crosslinker was uneven and irregular with visible depressions (Figure 3a). With the addition of ME and GE in the blended crosslinked films having 100 µL TEOS, the porosity was generated and numerous evenly distributed pores were observed on the film surface (Figure 3b, 3c and 3d). The observed pore size was increased with the decrease in the amount of TEOS (Figure 3e and 3f) confirmed the good hydrophilic character. The histogram was developed between the number of pores and pore size and shown in Figure 3 as well. 4.4
Swelling analysis
The swelling trend of all the blended films is shown in Figure 4. A gradual increase in swelling was observed with increase in time. The film (GCPmg1) having both mint and grapefruit peel extracts and maximum amount of crosslinker (100 µL), showed maximum swelling (58 g/g) and stability compared to the other films. The equilibrium time for GCPm1, GCPg1, and GCPmg1 was 25 min while for GCPmg2 and GCPmg3, it was 15 and 20 min, respectively. The films 12
(GCPm1, GCPg1 and GCPmg1) having higher amount of crosslinker (100 µL) were more stable as compared to GCPmg2 and GCPmg3 which contained lesser amount of crosslinker (75 µL) and (50 µL), respectively. The films were observed to be deformed after 25 min of swelling except GCPm1 and GCPmg1. Initially, GCPmg2 showed increased swelling in 15 min after which a decreasing trend was observed up to 25 min. GCPmg3, having the lowest amount of crosslinker showed maximum swelling in first 5 min then a steady increase was observed up to 20 min while the film sample was deformed after 25 min. 4.5
Optical analysis
One of the preferred properties of packaging is that it must be shielded from light, particularly UV radiation. The films were scanned at wavelength range of 200-800 nm to find the light transmission characteristics and the percent transmittance was calculated. Each film specimen exhibited different light transmission properties in UV light, especially the films showed low light transmission at wavelength of 300 nm (Figure 5). GCPg1 showed lower transmission percent at 300 nm as compared to GCPm1 having the same amount of crosslinker (100 µL). GCPmg1 (100 µL crosslinker) showed maximum percentage of transmittance as compared to GCPmg2 and GCPmg3 (75 and 50 µL crosslinker, respectively). Gómez-Guillén et al. have shown that the mixing of extract from murta with gelatin has increased the light barrier properties [38]. The opacity and transparency values showed higher transparency value with the lowest opacity (Table 2). There was a small difference in transparency due to low extract concentration.
4.6
Mechanical properties
13
In order to resist the stress that occur during storage and handling, packaging films require film reliability. The mechanical properties of the blend films are described in Table 3. The guar gum chitosan and PVA blended film (GCPmg1) with mint and grapefruit peel extract and higher concentration of crosslinker (100 µL) showed the highest UTS (40.03 MPa) and % E of 104.8 % exhibited that the greater force was required to break the sample. Kanatt et al. have shown that with the addition of extracts, there was a rise in UTS of the films particularly with higher concentration of PVA [39]. The phenolic compounds comprised of a number of -OH groups which form hydrogen bonding with chitosan and consequently there was an increase in UTS with the addition of extracts[32]. The improvement in UTS of chitosan films combined with green tea extract ascribed to the strong bonding between chitosan and phenolic compounds from green tea extract [40]. The integration of grape seed extract considerably increased the UTS of soy protein isolated blend films [41]. 4.7
Soil burial test/composting
All the blended films with and without extracts showed biodegradability in soil. There was a rapid degradation process with great weight loss of all the samples observed as shown in Figure 6 and 7. Almost all the films degraded rapidly in 6 days which exhibited that the blended films have strong microbial activity in soil. The fast degradation was due to the composting procedure which happened in two main steps; an active composting step and a curing period [30]. In the first stage, the temperature rose and remained elevated with the availability of oxygen which caused the strong microbial action while the temperature was decreased in second step but the film samples compost at a mild rate continuously. The compost was made of organic materials obtained from plant and animal substance that have been decayed mainly through aerobic breakdown. It was rich in nutrients and valuable for the land in various ways because of having
14
properties as a fertilizer and soil conditioner. The addition of vigorous humus also acts as natural pesticide for soil. The composting nutrients could decompose the blended film more speedily than the normal soil. 4.8
Antioxidant properties
The antioxidants in films permits nutritive and appealing characteristics to be improved without disturbing the quality of the product. The food might be kept at diverse temperatures in antioxidant packaging and therefore in guar gum/ chitosan/ PVA films having natural extracts, the release of phenolic and DPPH radical scavenging activity was determined. 4.8.1 Total phenolic content The phenolic compounds considered as secondary plant metabolites, active hydrogen donors and hold the best structural characteristics for scavenging free radical and therefore have remarkable antioxidant activity. To study the release mechanism of bioactive components, the blended films were submerged in distilled water while being polar the blended films started to hydrate with subsequent swelling producing relaxation of polymer chains. The blended film ultimately lost their structural stability and released the bioactive components. The films without any extract contained no significant phenolic content. The TPC of blended films comprising mint extract was greater than grapefruit peel extract. Additionally, the phenolic content released from the films varied significantly as TPC was monitored at two different temperatures. At higher temperature (37 ˚C), the release of phenolic content from the film sample was maximum than at 28 ˚C as shown in Figure 8. The integration of phenolic compounds such as catechin, p-hydroxy benzoic acid and gallic acid in films reduced the antioxidant activity of the films, subsequently a significant percentage of the phenolic component in the blended films was arised in soluble form [7]. 15
4.8.2 DPPH radical scavenging activity In order to find the antioxidant characteristic or to test the capability of compounds as hydrogen donors, DPPH radical has been commonly used. Figure 9 showed that the films containing extracts have DPPH radical scavenging activity. The control film sample having no extract did not display scavenging action. The films having mint and grapefruit extract had considerably higher antioxidant activity as compared to other films. The temperature was a main aspect in the release of natural extracts from the guar gum, chitosan and PVA films. At 37 ˚C, the release of natural extract was more from the blended films whereas at 28 ˚C, the antioxidant activity of the film sample were considerably reduced owing to the slow release of natural extracts from the film. When the film samples were kept at 37 ˚C, the release of extracts from the film in the 45 min and after that the release was fairly constant even incubated after 24 h. Correspondingly, the trend in gooseberry extract has been described by Devahastin et al [42]. 4.9
Water vapor transmission rate (WVTR) and water vapor permeability (WVP)
The WVTR and WVP of films are essential parameters for fruit coatings and packaging applications. The water barrier property of packaging materials is key factor to prevent the spoilage of food. Chitosan is also used as a food additive and act as an antibacterial agent and these properties not only help to retard microbial growth in fruit but also enhanced the shelf life of fruits and other food stuff [33, 43, 44]. The values for WVTR and WVP of films (GCP, GCPm1, GCPg1, GCPmg1, GCPmg2, GCPmg3) were 144.44, 150, 111.11, 150, 166.66, 166.66 (× 10-3) gh-1 m-2 and 1.42, 1.48, 1.09, 1.48, 1.64, 1.64 (× 10-6) gPa-1 h-1 m-2, respectively. All the blended films showed lower permeability values which proved that the material has the ability to extend the shelf life of food and has potential for packaging. 5.
Conclusions 16
Novel biodegradable silane crosslinked guar gum/ chitosan/ PVA blended films were successfully fabricated incorporated with ME and GE. FTIR analysis confirmed the functional groups and the developed interactions in the blended films. Nanoporous network structure was observed by SEM analysis confirmed the hydrophilic character in the films. GCPmg1 sample with both ME/GE extracts and the highest of silane crosslinker (100 µL), exhibited the maximum stability and swelling (58 g/g) as compared to other blended films. All film samples with and without extracts showed transparency, opacity and increased protection against UV light. GCPmg1 showed the enhanced ultimate tensile strength and % E of 40.03 MPa and 104.8 %, respectively. ME/GE conferred the outstanding antioxidant properties of the blended films as the films having mint extract have greater TPC compared to the films having grapefruit peel extract and TPC released from the films was maximum at 37 ˚C. There was a rapid biodegradation process with a great weight loss of all the film samples and almost all the films were degraded rapidly in 6 days showing the strong microbial activity in soil. The lower WVT rate and WVP confirmed the improved shelf life and environmental friendly behaviour of the blended films. Hence, bioactive biopolymers incorporated with natural extracts have excessive applications for food and other packaging materials. Acknowledgements The author is highly obliged to the Applied Chemistry Department, PCSIR laboratories, Lahore, Pakistan for performing the water vapor transmission tests and also to the School of Chemical and Materials Engineering, NUST, Islamabad, Pakistan for performing SEM analysis.
17
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Figure 1. The pictorial view of the preparation of chitosan/ guar gum/ PVA/ extracts blends.
24
Figure 2. The FTIR spectra of pure guar gum, chitosan, PVA films and GCPm1, GCPg1, GCPmg1 treated with natural extract and TEOS.
25
Figure 3. SEM analysis of the prepared blended films with pore size histogram a) GCP, b) GCPm1, c) GCPg1, d) GCPmg1, e) GCPmg2, f) GCPmg3.
26
Figure 4. The time (min) vs swelling (g/g) of GCPm1, GCPg1 and GCPmg1 blended films at room temperature.
27
Figure 5. The transmittance of guar gum, chitosan and PVA films along with mint and grapefruit extract.
28
Figure 6. The photographs of samples GCPm1 (a, b) and GPCg1 (c, d) before and after buried in soil.
29
Figure 7. Weight loss of samples with varying ratio of extract and TEOS.
30
Figure 8. TPC of guar gum, chitosan PVA films integrated with ME/GE (a) 28 ˚C (b) 37 ˚C
31
Figure 9. DPPH radical scavenging activity of guar gum, chitosan and PVA films integrated with ME/GE (a) 28 ˚C (b) 37 ˚C
32
Table 1. The codes and composition of guar gum, chitosan and PVA (GCP) films. Sr.
Sample
Guar
Chitosan
PVA
Mint
Grapefruit TEOS
No
Codes
gum (g)
(g)
(g)
extract
peel
(mL)
extract
(µL)
(mL) 1
GCP
0.025
0.1
1.5
0
0
0
2
GCPm1
0.025
0.1
1.5
0.5
0
100
3
GCPg1
0.025
0.1
1.5
0
0.5
100
4
GCPmg1
0.025
0.1
1.5
0.5
0.5
100
5
GCPmg2
0.025
0.1
1.5
0.5
0.5
75
6
GCPmg3
0.025
0.1
1.5
0.5
0.5
50
33
Table 2. The opacity and transparency of chitosan, guar gum PVA films integrated with ME/GE. Sample codes
Opacity
Transparency
GCP
0.060 ± 0.0001
9.0 ± 0.1
GCPm1
0.040 ± 0.0001
9.0 ± 0.1
GCPg1
0.055 ± 0.0002
8.9 ± 0.2
GCPmg1
0.036 ± 0.0001
9.0 ± 0.1
GCPmg2
0.065 ± 0.0002
8.8 ± 0.2
GCPmg3
0.070 ± 0.0003
8.7 ± 0.3
34
Table 3. UTS and % E of chitosan/guar gum/PVA films incorporated with mint and grapefruit extracts. Sample Codes
Ultimate
Tensile Elongation at break
strength (MPa)
(%)
GCP
15.3 ± 0.1
40.65 ± 0.1
GCPm1
32.99 ± 0.3
97.33 ± 0.3
GCPg1
29.39 ± 0.2
89.31 ± 0.2
GCPmg1
40.03 ± 0.1
104.8 ± 0.1
GCPmg2
28.88 ± 0.2
88.74 ± 0.2
GCPmg3
28.44 ± 0.1
86.18 ± 0.1
35