International Journal of Biological Macromolecules 79 (2015) 934–942

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Development and characterization of an LDPE/chitosan composite antimicrobial film for chilled fish storage K.V. Reesha a , Panda Satyen Kumar b , J. Bindu c,∗ , T.O. Varghese a a b c

Centre for Bio-Polymer Science and Technology (CBPST), FACT Township, Eloor, Udyogamandal P.O., Cochin 683501, India Quality Assurance and Management Division, Central Institute of Fisheries Technology, Cochin 682029, Kerala, India Fish Processing Division, Central Institute of Fisheries Technology, Cochin 682029, Kerala, India

a r t i c l e

i n f o

Article history: Received 4 March 2015 Received in revised form 9 June 2015 Accepted 12 June 2015 Available online 16 June 2015 Keywords: Chitosan LDPE Packaging film Tilapia Chilled storage

a b s t r a c t An antimicrobial packaging material was developed by uniformly embedding 1, 3 and 5% chitosan (w/w) in low density polyethylene matrix using maleic anhydride grafted LDPE as a compatible agent. The materials were mixed by compounding and blown into monolayer films via blown film extrusion. The developed films showed good barrier properties against oxygen. Characterization of the composite films with Fourier transform infrared spectroscopy revealed that chitosan and LDPE interacted well with each other. Overall migration showed better release of chitosan adduct from the LDPE matrix which enhanced the antibacterial properties of the films. The interaction between the LDPE/CS and maleic anhydride grafted LDPE had a decreasing effect on the tensile strength and heat sealing properties. Investigation on antimicrobial properties of LDPE/CS films showed 85–100% inhibition of Escherichia coli. Efficacy of LDPE/CS films was evaluated by using them as packaging material for chilled storage of Tilapia (Oreochromis mossambicus). Analysis of storage quality indices (peroxide value, free fatty acid, total volatile base nitrogen and aerobic plate count) revealed good antibacterial property and extension of shelf life of Tilapia in the chitosan incorporated novel composite films compared to virgin LDPE film. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Packaging plays a very important role in today’s commerce and trade. The aim of packaging is to maintain the unique wholesomeness to produce right from manufacturing to storage, without altering the inherent characteristics of the product. Packaging has a significant impact on international trade of fish and fishery products which are important source of nutritional components like protein, essential vitamins, minerals and polyunsaturated fatty acids [1,2]. Fish is highly perishable due to the presence of high moisture and protein. It is vulnerable to various microbial and biochemical forms of deterioration throughout the production chain, due to free amino acids and highly oxidizable polyunsaturated fatty acids (PUFA) [3]. In order to extend shelf life, packaging has to be either combined with refrigeration to further reduce the growth of microorganisms or by antimicrobial agents incorporated

∗ Corresponding author. Tel.: +91 484 2666845; fax: +91 484 2668212; mobile: +91 9447648921. E-mail addresses: [email protected] (P. Satyen Kumar), [email protected] (J. Bindu). http://dx.doi.org/10.1016/j.ijbiomac.2015.06.016 0141-8130/© 2015 Elsevier B.V. All rights reserved.

in the packaging material that can prevent proliferation of spoilage microorganisms. Packaging materials are available mainly in the form of plastic films. They should have flexibility, good mechanical and barrier properties, and also should be easily recycled. In addition to this, they should be capable of heat sealing and withstand the strain of transportation and of low cost. Polyethylene packaging as sheets or films is suitable to wrap, seal and protect consumer goods. Polyethylene is considered superior to any other material because of its cheap available nature and its use is widespread in the flexible packaging market. Polyethylene polymers are both durable and resistant to environmental hazards. For the preservation of fresh fish, active packaging technologies can be used. The technology offers several benefits over standard passive packaging materials and can be designed to protect product quality such as freshness and storage stability. Antimicrobial packaging is a promising form of active packaging since it extends the lag phase and reduce growth phase of microorganisms to extend shelf life of product [4]. While preparing packaging materials, antimicrobial components can be either incorporated or coated on to the surface of packaging materials. Biodegradable packing materials can be produced from natural polymers such as chitosan, collagen, elastin and starch. Chitosan

K.V. Reesha et al. / International Journal of Biological Macromolecules 79 (2015) 934–942

is an amino polysaccharide biopolymer which demands an important role in the world economy since it is an edible, biodegradable, antimicrobial compound with film forming ability. Chitosan (CS), the linear and partly acetylated (1–4)-2-amino-2-deoxy--dglucan, is easily obtained from chitin [5]. Chitosan is a weak base and is insoluble in water, but soluble in dilute aqueous acidic solutions like acetic acid and propionic acid. The direct combination of synthetic materials with biodegradable materials is one of the easier and economic ways for biomaterial production [6] and also enhances the expansion and functional properties of the materials. Extrusion and press moulding techniques are used in the production of chitosan and low density polyethylene (LDPE) films, which allow chitosan to perform antimicrobial activity on different bacterial strains [7,8]. Chitosan incorporation in a LDPE matrix or application by coating improves the barrier properties of LDPE and also confers antimicrobial characteristics, which makes it a very promising packaging material [9]. Chitosan film is used as an edible coating to prolong shelf life and preserve quality of fresh fish due to the antimicrobial action between the positively charged chitosan molecules and negatively charged microbial cell membranes [10]. This study was undertaken to develop a chitosan incorporated antimicrobial polyethylene film by blown film extrusion process and to optimize the chitosan concentration and mechanical properties of the packaging films for studying the antimicrobial activity of the films against Escherichia coli. The ultimate aim was to use the films for packaging of fresh Tilapia steaks and to determine its shelf life during chilled storage.

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It had a melt flow of 2.52 g/10 min (190 ◦ C/2.16 kg) as determined by ASTM D 1238-04c method [12]. 2.2. Blend preparation and film production Pre-drying of chitosan, LDPE and MA-g-LDPE was done in a hot air oven at temperature of 80 ◦ C for 2 h and further pre-mixing of weighed chitosan, LDPE, MA-g-LDPE with glycerol was done at room temperature. The compounding of LDPE/chitosan was done using a co-rotating twin screw extruder (ZV20). Composite blend formulations were prepared by mixing low density polyethylene as matrix material and low viscosity chitosan (74 cps) as filler material. MA-g-LDPE was used to improve compatibility between filler and matrix. For ease of processing glycerol was also used as plasticizer. Three different combinations of blends were prepared incorporating 1, 3 and 5% chitosan by w/w of LDPE. The compounding process was carried out at a maximum speed of 90 rpm and melt temperature of 185 ◦ C. The extrudates were pelletized separately using a pelletizer machine for each formulation. Slight variations of processing temperature and torque occurred for the different combinations. For LDPE/CS compounding the feed zone was maintained at 103, 107 and 99 ◦ C for 1%, 3% and 5% compounded mixture, respectively. The temperatures maintained in the compression zones 1 and 2 were 103 ◦ C and 132 ◦ C for 1%, 107 ◦ C and 129 ◦ C for 3%, 99 ◦ C and 138 ◦ C for 5% LDPE/CS blends. The melt temperature ranged from 176 to 184 ◦ C and the melt pressure maintained was between 5 and 6 bar. 2.3. Blown film manufacture

2. Materials and methods 2.1. Film preparation 2.1.1. Matrix Low density polyethylene (LDPE) resin grade (Lotrene FD 0474) was supplied by Qatar Petrochemical Company (QAPCO). The density of the polymer was 0.923 g/cm3 as determined by ASTM D 792-08 method [11] and temperature of melting was 140–150 ◦ C. Melt flow rates of granules were determined by melt flow indexer (M/s. Saumya machineries Pvt. Ltd., Ahmedabad) according to ASTM D 123-04c method [12]. After conditioning at laboratory at 23 ± 2 ◦ C and 50 ± 5% relative humidity, the different concentrated LDPE/chitosan (CS) granules were adopted to procedural conditioning of 190 ◦ C/2.16 kg/20 s. The apparatus consisted of a small die of 2 mm diameter inserted into the extruder. A small amount of granules were taken in sample enclosure. Proper packing of material inside the barrel was ensured to avoid formation of air pockets. The samples were preheated for specific time. A piston was introduced which applied pressure on to the molten granules and caused extrusion. The combined action of shear and pressure made the molten material to flow throughout the die. The melt samples were collected after desired period of time and weighed accurately. Melt flow index (MFI) was expressed as grams of polymer/10 min of flow time. 2.1.2. Filler and processing aid Low viscosity chitosan used as the filler material had a viscosity of 74 cps. The average moisture content was 3.04% and degree of deacetylation was 80.92%. Degradation temperature of chitosan was above 190 ◦ C. Glycerol (glycerine C3 H8 O3 ) procured from Sisco Research Laboratory (SRL) Pvt. Ltd. was used as plasticizer. 2.1.3. Compatibilizer Maleic anhydride grafted LDPE grade, MA-g-LDPE (Commercial name – OPTIM-E142) was purchased from Pluss Polymers Pvt. Ltd.

The compounded pellets were fed into a twin screw extruder (Konark Plastomech Pvt. Ltd.) by a gravimetric hopper which was maintained at a melt temperature of 184 ◦ C and a melt pressure of 20 bar. 2.4. Film characterization 2.4.1. Thickness Thicknesses of all four types of blown films were measured as per IS: 2508 method [13] at 23 ◦ C and 64% saturated sodium nitrite RH. The thickness was determined at five positions of each sample using a gauge meter (Mitutoyo, Model no: 2046-08 Japan). 2.4.2. Transparency of film The percentage of transparency of the films was determined by a haze meter as per ASTM D 1003 method [14]. 2.4.3. Surface morphology of film Surface morphology was investigated by atomic force microscopy (AFM) performed at room temperature on a PARK systems XE 100 (Schaefer technologies GmbH) setup. Topographic and amplitude images, obtained over an area from 5 ␮m2 for each sample were recorded by using non-contact mode with silicone tip. 2.4.4. Tensile and heat strength The tensile strength elongation at break and heat seal strength of different concentrated LDPE/CS films were done using Lloyd instruments UK; model TA plus according to ASTM D 882-02 method [15]. Film pieces were stored in a desiccator at 23 ◦ C with 50% relative humidity for at least 24 h. Samples were cut in lengthwise and cross direction 50 mm × 15 mm. For tensile test, a load range and appropriate grip separation was used and the test specimens were placed in the grip of testing machine. The extensometer measured the load versus extension. The testing speed was at 500 mm/min with a load cell of 500 N. The distance between the two anchorages was 50 mm.

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The following formula was used in calculating the tensile strength.

Tensile strength (TS) =

maximum load width × thickness

The same tensile strength test procedure was adopted for heat seal strength. Unsealed film samples were heat sealed and specimen dimensions same as tensile test were used. The only difference was that the heat sealable portion of the film was placed in the centre of the grip. 2.4.5. Water vapour transmission rate (WVTR) The water vapour transmission rate was performed by a Lyssy water vapour permeability tester (PBI Dan sensor Denmark, Model L80-5000) as per ASTM E 398-03 method [16]. 2.4.6. Oxygen transmission rate (OTR) The oxygen transmission rate was determined by using oxygen transmission tester (OPT-5000) (PBI Dansensor, Denmark). The samples were prepared according to ASTM F 2622-08 method [17]. 2.4.7. Overall migration test The overall migration of different LDPE/CS films was done as per the USFDA 176:170 test procedure [18]. The film pouches were filled with 250 mL stimulating solvent (water) and conditioned at 49 ◦ C at 24 h. After exposure for specified duration, the solvent was transferred, concentrated and evaporated to dryness to measure the residues. Overall migration residue (OMR) in mg/L Mass of residue (mg) × 1000 = Volume of stimulant (mL)

2.4.8. Fourier transform infrared (FTIR) spectroscopy Fourier transform infrared spectra of different concentrated LDPE/CS films were carried out on a Thermo Nicolet, Avatar 370 FTIR spectrometer, at STIC (Sophisticated Test and Instrumentation Centre, CUSAT, Kochi). Scanning was performed at 4 cm−1 resolution. The measurements were recorded between 4000 and 400 cm−1 . 2.4.9. Film antimicrobial test Antimicrobial efficacy of virgin LDPE and LDPE/CS films were evaluated by exposing the films to E. coli. Virgin LDPE and LDPE/CS films were cut to pieces of 5 cm × 6 cm (length × breadth) sizes and sterilized overnight in an UV chamber. E. coli (ATCC 25922) was inoculated in BHI broth (Hi-media) and incubated for 18–24 h at 35 ± 2 ◦ C. The overnight grown culture was centrifuged, washed with sterile phosphate buffer and re-suspended in 9 mL of phosphate buffer. From this culture 100 ␮L was spread on to both films by using sterile disposable spreader. The initial inoculum level was 5.2 × 108 cfu/cm2 . The films were kept in separate petridishes and incubated at 35 ± 1 ◦ C. Films were drawn at 24, 72 and 120 h to analyze load of E. coli. The load of E. coli was estimated as per AOAC 991.14 [19] (Dry Rehydratable Film Method, PetrifilmTM E. coli/Coli form Count Plate). Briefly, both control and inoculated films were suspended in 100 mL phosphate buffer, vortexed and serially diluted. One milliliter of inoculum from each dilution was placed on E. coli petrifilms. The petrifilms were incubated at 35 ± 1 ◦ C for 24 h. Number of blue colonies with entrapped gas bubbles were taken as positive and multiplied with corresponding dilution to obtain final E. coli count.

2.5. Raw materials Live Tilapia (Oreochromis mossambicus) fishes were procured from a fish farm, washed and transported in ice to the laboratory for further processing. The fishes were washed, dressed, cleaned, cut into steaks and kept in iced condition before packing in the chitosan (1, 3 and 5%) incorporated LDPE films and heat sealed. Virgin LDPE pouches were also prepared for packing the control samples. The samples were then iced separately in different insulated boxes and kept in chill room at 2 ◦ C. 2.6. Biochemical and microbiological analysis The chilled steaks were drawn at periodic intervals to determine the biochemical and microbiological qualities. Total volatile base nitrogen (TVBN) content [20], free fatty acid content [21], peroxide value [22] and aerobic plate count (APC) [23] of Tilapia steaks packed in control and LDPE/CS films were estimated. 2.7. Statistical analysis Statistical analysis was performed using SPSS Statistics version 20 (IBM, Armonk, NY, USA). Post hoc analysis using pair wise comparison of Tukey’s test was carried out for microbial and film property analysis. 3. Results and discussions 3.1. Optimization of process for film development 3.1.1. Compounding and extrusion of LDPE/chitosan Three different concentrations (1%, 3% and 5%) of chitosan with LDPE and other compounds were compounded in twin screw extruder. The processing of LDPE/CS was difficult with increase in the concentration of chitosan mainly due to the viscoelastic characteristics of the component. According to Liang [24] the flow of polymer introduces a structural deformation or aggregation during extrusion process, which makes the viscoelastic properties of the blend more complicated, resulting in variation in the volume, thickness of the extruded material due to presence of aggregates and separation of blend components. Hence in this study MA-g-LDPE was used as a compatibilizer, and glycerol as plasticizer. According to Quiroz-Castillo et al. [25] 5% weight of MA-g-LDPE is necessary to increase the ductility of the LDPE/CS blend since this combination gives a positive effect to the blend and the glycerol/chitosan concentration kept constant at 2 g of glycerol per 100 g of chitosan. The compounding of the LDPE/CS was carried out in a twin screw extruder at temperatures varying from 103 to 185 ◦ C in the different zones. Slight variations could have occurred due to the differences in chitosan concentration. The extruded pellets were smooth, bubble-free and opaque in nature. The colour of the compounded granules differed for each concentration because of the chitosan adduction. The immiscible blends are characterized by two or more phase that are separated by interfaces and when interfacial tension goes to zero, the blend become miscible [26]. This is achieved by the addition of compatibilizer which has both hydrophilic and hydrophobic molecules. In this reaction MA-gLDPE react with hydrophobic non polar LDPE and the other polar end reacts with hydrophilic chitosan. In similar way, Liu et al. [27] have reported that the miscibility between granular corn starch and LDPE was improved by the addition of a commercially available compatibilizer, MA-g-LDPE. This was attributed to a chemical reaction between hydroxyl groups in starch and anhydride groups in MA-g-LDPE and the physical interaction between the PE in MAg-LDPE and LDPE.

K.V. Reesha et al. / International Journal of Biological Macromolecules 79 (2015) 934–942 Table 1 Density and melt flow index (MFI) of the chitosan incorporated extruded LDPE and virgin LDPE granules. Types of granules

Density (g/cc)

Virgin LDPE 1%LDPE/CS 3%LDPE/CS 5%LDPE/CS

0.923 0.945 0.950 0.951

± ± ± ±

0.03 0.01 0.04 0.03

MFI value (g/10 min) 3.57 2.94 2.58 2.37

± ± ± ±

0.01 0.04 0.02 0.08

The density of the extruded granules increased with chitosan adduction percentage (Table 1). By increasing the filler concentration the filler particles get attached to the matrix and form a blend. The extruded granules showed increased density, which resulted in increased molecular weight of granules and decreased MFI value. Results showed that there was no significant difference in density of extruded granules (P > 0.05) in 3% LDPE/CS and 5% LDPE/CS. 3.1.2. Melt flow Index of extruded granules The MFI values of LDPE/CS composites decreased as the content of chitosan increased (Table 1). Reduction in MFI values indicated increase in the viscosity of the composite. The virgin LDPE granules had an MFI value of 3.57 g/cc, whereas it decreased with the addition of chitosan. The MFI was respectively 2.94, 2.58 and 2.37 g/cc for 1, 3 and 5% chitosan incorporated LDPE. Similar findings have been reported in most filled thermoplastics and Rosa et al. [28] have reported a decrease in MFI of starch composite with increase in filler loading. In the present study, the reduction in MFI value could be due to the chitosan granules that still retained their shape and functioned as rigid particulate fillers when processed. With increase in chitosan content the interaction between the granules increased and this contributed to the higher viscosity. For higher loading of chitosan, the spaces between particles to particle were small. The matrix molecules become trapped in filler particles as the size of agglomerates rise and flow becomes confined. Analysis of MFI variability of different LDPE/CS granules indicates that chitosan incorporated granules can be processed like virgin LDPE, however, with slight modification in process conditions such as temperature and pressure. 3.2. Film formation and properties The three different LDPE/CS compounded and virgin LDPE granules were blown into a tubular film using a monolayer extruder. Films were extruded at 140–195 ◦ C. The uniform mixing and controlled blowing determined the chitosan distribution and thickness of the film. The LDPE and LDPE/CS films varied in thickness and morphology. All films showed good processability, except the 5% LDPE/CS film, because of increased amount of filler. As compared with 5% LDPE/CS, 3% LDPE/CS films showed good distribution due to proper interaction between the components. 3.2.1. Thickness The difference in thickness is given in Table 2. The results show that with increase in chitosan concentration the thickness of films

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also increased. Mechanical properties of the films are also associated with thickness of the film. Morphological differences due to the domain structure of immiscible polymer blend gave rise to films with different thickness [29]. 3.2.2. Transparency The transparency level for each formulation in LDPE/CS blends is shown in Table 2. The control of transparency is very important in order to achieve the desired visual effect. The percentage of transparency decreased with incorporation of chitosan. The transparency got reduced because the higher amount of chitosan distributed in the packing blend system making it more disordered and rough or uneven with higher free volume, thus more light could diffuse back or get reflected. Hence, the transparency of the film was found to be dependent on composition, mixing and the processing conditions of the film. 3.2.3. Tensile strength and elongation at break Variations of tensile strength and elongation at break of LDPE/CS composite films are shown in Table 2. Tensile strength and elongation at break of LDPE/CS blends decreased with increase in chitosan content. The tensile properties of films are influenced by the processing conditions (mixing, type and number of screws, rotor temperature and speed, and resident time) and the processing variables during blowing and cooling [30] apart from material characteristics. Similar findings have been reported by Quiroz-Castillo et al. [25] where tensile strength as well as elongation at break decreased with increase in chitosan content. The decrease in the tensile strength can be due to the uneven dispersion of chitosan in the LDPE matrix [31]. The tensile strength of 1% LDPE/CS decreased as compared to virgin LDPE. Similar to tensile strength, decrease of elongation at break occurred because of the weak interfacial adhesion between LDPE and chitosan. In synthetic polymer blends, the addition of the immiscible component to a ductile matrix generally decreases the elongation properties at break. The elongation would therefore depend on the state of the interface [32]. When stress is applied on materials with chitosan blends, loss of adhesion between the inter phase components takes place that results in formation of pores due to the unfolding of matrix fibres [30]. This is due to the immiscibility of chitosan (particulate filler) with LDPE. Thus, mechanical performance of a filled polymer depends on the strength and the filler module, which further explains the lower tensile strength shown by the extruded films in the present study. 3.2.4. Heat seal strength All LDPE/CS film showed decreased heat sealing strength when compared to virgin LDPE film (Table 2). Since LDPE itself is a good heat sealable film, addition of hydrophilic chitosan to LDPE film affected its sealing strength. Rahman et al. [33] observed similar results in starch films where it was shown that hydrophilic nature of starch was not compatible with the hydrophobic nature of synthetic polymers that resulted in weakness of interfacial adhesion and led to reduction in mechanical properties.

Table 2 Properties of LDPE/CS and virgin LDPE films. Properties of films

Virgin LDPE

Film thickness (mm) Transparency (%) Tensile strength (MPa) Elongation at break (%) machine direction Heat seal strength (MPa) Oxygen transmission rate (mL/m2 /day) Water vapour transmission rate (g/m2 day) Overall migration residue (mg/L)

0.13 87.00 9.62 279.79 8.38 2838 2.43 3.68

± ± ± ± ± ± ± ±

0.20 0.12 0.12 0.23 0.11 0.32 0.01 0.01

1% LDPE/CS 0.16 85.00 3.73 243.03 3.17 2343 3.59 7.14

± ± ± ± ± ± ± ±

0.70 0.20 0.12 0.20 0.11 0.20 0.32 0.05

3% LDPE/CS 0.20 74.60 2.26 157.60 1.71 2189 2.88 8.80

± ± ± ± ± ± ± ±

0.01 0.07 0.12 0.21 0.22 0.12 0.01 0.01

5% LDPE/CS 0.24 71.05 1.24 111.96 1.26 2487 4.19 8.85

± ± ± ± ± ± ± ±

0.01 0.23 0.01 0.21 0.32 0.03 0.02 0.01

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Fig. 1. Surface morphology of (a) virgin LDPE and (b) 1% LDPE/CS film.

3.2.5. Oxygen transmission rate (OTR) Virgin LDPE films showed a higher OTR value when compared to LDPE/CS films (Table 2). OTR value of virgin LDPE film was found to be around 2838 mL/m2 /day, similar to earlier reports [34]. The incorporation of chitosan decreased the oxygen transmission rate, to 2343 mL/m2 /day in 1% LDPE/CS film and 2189 mL/m2 /day in 3% LDPE/CS film. Kurek et al. [35] observed that application of chitosan coatings on PE films resulted in more than two-order decrease in oxygen permeability. When compared to each composition, 3% LDPE/CS film had lower oxygen transmission rate than 1 and 5% LDPE/CS. 3.2.6. Water vapour transmission rate (WVTR) Polyethylene films are known to be highly hydrophobic and relatively not very permeable to water vapour. The WVTR value of pure LDPE film was 2.43 ± 0.01 g/m2 /day and showed good barrier property. By increasing the chitosan content permeability increased to 2.88 ± 0.01, 3.59 ± 0.32 and 4.19 ± 0.02 g/m2 /day for 1, 3 and 5% LDPE/CS films, respectively (Table 2). In the chitosan incorporated films the hygroscopic chitosan layer acted as a water reservoir on the PE surface, thus significantly promoting its water vapour permeability [35]. 3.2.7. Overall migration rate (OMR) The overall migration (OMR) of virgin LDPE and LDPE/CS films was within the stipulated upper limit of 60 mg/L as shown in Table 2. The virgin LDPE had value of 3.68 mg/L, a very low value, when compared to other films. By increasing chitosan concentration the values of migration increased to 7.14, 8.80 and 8.85 mg/L in 1, 3 and 5% LDPE/CS films, respectively. Considering the low migration rate, these films can be suitably used for food contact applications. If the chitosan migration further increases then it will enhance the antimicrobial properties of the films. 3.2.8. Surface morphology of virgin LDPE and LDPE/CS blown films The surface morphology of virgin LDPE and 1% LDPE/CS composite films are shown in Fig. 1(a) and (b). Comparison of the topography of surfaces shows less troughs and valleys in the virgin LDPE films. The average roughness of LDPE film was 0.681 nm due to the defects formed during processing stage. The 1% LDPE/CS films

showed much more troughs and valleys indicating more roughness. The images revealed the distribution and dispersion of chitosan in the matrix material. Chitosan was distributed in matrix with surface roughness of 2.490 nm. Surface roughness was due to the incorporation of hydrophilic chitosan into a hydrophobic matrix. Surface roughness enhanced the antimicrobial nature of film providing adherence of bacterial cell wall.

3.2.9. FTIR analysis FTIR spectra of virgin LDPE and LDPE/CS films in the wave number range of 4000–500 are given in Fig. 2. Virgin LDPE showed accentuated peaks at 2915–2848 cm−1 for (CH) stretching. The peaks of 722 cm−1 and 1464 represented the skeletal vibrations of CH2 . In general, absorption peaks of chitosan at 3440 cm−1 showed the stretching vibration of (NH2 ) and (OH) as well as inter and intra molecular hydrogen bonding. Peaks around 1082 and 1366 cm−1 were due to saccharide structure and around 1637 cm−1 was due to carbonyl groups, respectively [31]. The peak at 1637 cm−1 represented acetylated amino group of chitin, which indicated incomplete deacetylation of the sample. In case of 1% LDPE/CS film, absorption spectra shows widening between 3750 and 3000 cm−1 . The virgin LDPE film shows sharp CH stretching at 2915 and 2848 cm−1 , CH3 bending at 1643 cm−1 which is almost the same as in the absorption spectra of 1% LDPE/CS film. Additionally, appearance of 1041 cm−1 corresponds to the vibration of C O C groups in chitosan since chitosan also belongs to the aliphatic ethers. The 3% LDPE/CS spectra is much broad and intense in the range between 3750 and 3000 cm−1 because of the increase in chitosan particles. As compared to 1% LDPE/CS, 3% LDPE/CS showed less intense characteristic peak of CH3 bending at a range of 1465 cm−1 , which may be due to the reactions of MA-g-LDPE on both LDPE and chitosan. In the same way skeletal vibration peak of 721 cm−1 was less intense when compared to 1% LDPE/CS. The 5% LDPE/CS films showed broadened and small sharpening in the range of 3750–3000 cm−1 because of chitosan OH &NH stretching and bending. The overall spectra band of 5% LDPE/CS films showed that there was not much difference with 3% LDPE/CS incorporated films.

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Fig. 2. FTIR spectra of virgin LDPE and LDPE/CS films.

3.3. Determination of antimicrobial properties of films against E. coli Antimicrobial activity of virgin LDPE & LDPE/CS films against E. coli is shown in Table 3. Growth and survival of E. coli was effected with increasing concentration of chitosan. E. coli got reduced by 79.20% after 96 h exposure on virgin LDPE films, whereas it reduced to 84.83% of initial count in 1% chitosan incorporated films. There was 100% reduction of E. coli in both 3% and 5% chitosan–LDPE films after 96 h. Chitosan films exhibiting antimicrobial activity against E. coli and Lactobacillus plantarum have been reported [36]. Similarly, complete inhibition of E. coli has been reported in 1.4% and 2.1% chitosan lactate impregnated LDPE films after 12 h exposure [7]. 3.4. Changes in quality indices of Tilapia steaks during storage in ice 3.4.1. Changes in TVB-N values The initial TVB-N value of chilled fish was 0.70 ± 0.10 mg/100 g of fish (Fig. 3) which slowly increased during the storage period and reached 18 ± 0.20 mg/100 g of fish. For LDPE/CS 1%, 3%, 5% samples, the initial TVB-N value ranged between 0.77 ± 0.01 mg and 0.80 ± 0.30 mg/100 g sample and reached to a final value of 9.30 ± 0.12, 8.50 ± 0.31 and 14.50 ± 0.61 mg/100 g sample, respectively after 30 days of storage. TVBN content of LDPE/CS 3% was significantly lower (P < 0.05) than other films on 15th and 30th day of chilled storage. Maximum TVBN content of 35–40 mg% is usually regarded as limit of acceptability [37,38]. However, in the present

study TVB-N values for all the samples were within the suggested limit throughout the storage period. TVB-N are products of bacterial spoilage and their contents are often used as an index to assess the keeping quality and shelf life of fish products [39]. 3.4.2. Changes in peroxide value (PV) In the present study, the peroxide values increased progressively in all samples during the storage period (Fig. 4). A value of 15.01 ± 0.30 milli equivalents (meq) per kg was reported on 30th day of storage for Tilapia packed in virgin LDPE pouches. Similarly, for the fish samples packed in LDPE/CS 1%, 3%, 5%, the initial PV values were 0.52 ± 0.04 meq/kg which increased to 7.70 ± 0.21, 6.50 ± 0.10, 14.00 ± 0.01, respectively on 30th day of storage. PV content of LDPE/CS 3% was significantly lower (P < 0.05) than other films throughout the storage duration. PV value in the range 18–20 meq/kg of fish sample is usually taken as the limit of acceptability. The gradual increase in PV is due to the breakdown of unsaturated fat into primary products of lipid oxidation which further changes to secondary lipid oxidation products like malonaldehyde [40]. 3.4.3. Changes in free fatty acid value The initial FFA content of samples packed in virgin LDPE was 2.00 ± 0.01 mg % oleic acid and it gradually increased to 15.20 ± 0.01 mg % oleic acid at the final day of storage (Fig. 5). The presence of free fatty acids is due to the oxidation and hydrolysis of lipids and is undesirable since the fatty acids may be converted to odorous volatiles [40]. The FFA content of all the samples increased during the storage period. In LDPE/CS 1%, 3% and 5% packed

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Table 3 Percentage reduction of Escherichia coli on virgin LDPE and LDPE/CS films. Duration (h)

Reduction (%) of Escherichia coli Virgin LDPE

24 48 72 96

10.07 53.84 62.18 79.20

± ± ± ±

0.55 1.36 1.98 2.63

1% LDPE/CS 11.82 58.72 67.80 84.83

± ± ± ±

3% LDPE/CS

1.26 2.63 3.12 2.12

12.91 60.74 69.67 100.00

± ± ± ±

0.96 1.95 3.11 0.00

5% LDPE/CS 11.34 62.18 71.10 100.00

± ± ± ±

0.33 1.12 2.56 0.00

Fig. 3. Changes in TVBN values of Tilapia during storage in LDPE and LDPE–chitosan films.

Fig. 4. Changes in free peroxide value (PV) of Tilapia during storage in LDPE and LDPE–chitosan films.

samples the initial FFA content ranged from 1.00 ± 0.50 mg % oleic acid which reached 7.00 ± 0.01, 6.00 ± 0.01and 11.00 ± 0.01 mg % oleic acid during the storage period. FFA content of LDPE/CS 3% was significantly lower (P < 0.05) than other films on 15th

and 30th day of chilled storage. The values observed for chilled fish samples in the present study were less than the reported values for different fishes under chilled storage condition [41,42].

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Fig. 5. Changes in free fatty acid content (FFA) of Tilapia during storage in LDPE and LDPE–chitosan films.

3.4.4. Changes in aerobic plate count The aerobic plate count of chilled Tilapia packed in various films was carried for 15 days of storage. A significant difference (P < 0.05) was observed in APC of Tilapia packed in different films on 7 and 15 days of chilled storage (Fig. 6). A linear increase in APC was observed in sampled packed with virgin LDPE films, where it increased from an initial 4.87 log10 cfu/g to 6.34 log10 cfu/g at the end of 15 days of chilled storage. Similarly, APC of Tilapia packed in 1% LDPE/CS films crossed 106 cfu/g on 15th day of storage life. However, there was moderate increase in APC values of Tilapia packed in 3% and 5% LDPE/CS films and samples remained acceptable even beyond 15 days. Although there was no significant difference in APC values of

virgin LDPE, LDPE/CS 1% and LDPE/CS 3% on 15th day, the lower APC values of 5.89 and 4.12 log10 cfu/g obtained in 3% and 5% LDPE/CS samples must be due to antimicrobial action of released chitosan on the spoilage microflora. Similar antimicrobial activity of chitosan has been reported by [7], where total viable log populations on fresh red meat applied with chitosan-incorporated LDPE film were lower than control, but no significant difference was observed. Coating consisting of a blend of chitosan dissolved in acetic acid and gelatine exerting inhibitory effect on the Gram-negative flora of fish patties has been reported [43]. Tsai et al. [44] have reported that a 3 h pre-treatment of fish fillets (Oncorhynchus nerka) with 1% chitosan solution could retard the increase in mesophilic count.

Fig. 6. Changes in aerobic plate count of Tilapia during storage in LDPE and LDPE–chitosan films.

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4. Conclusions The study revealed that LDPE/CS antimicrobial blown films can be prepared by process optimization and incorporation of chitosan up to 3% level since good processability was found only up to 3% chitosan addition into the LDPE film. The extruded granules showed increased density with increasing chitosan concentration but 5% LDPE/CS granules did not show significant variation with 3% LDPE/CS films. The films showed varying processing characteristics which brought about changes in the mechanical properties. LDPE/CS film shows good oxygen permeability properties. But in the case of water vapour transmission rate the hydrophilic chitosan negatively influenced the barrier properties of the LDPE films. The overall migration rates of films were within the limit of contact applications and the slight increase in the migration was due to increase in the chitosan concentration which enhanced the antimicrobial properties of the film. The antimicrobial assay against E. coli proved that LDPE/CS films were highly efficient than virgin LDPE films. Virgin LDPE and 1%, 3% and 5% LDPE/CS films tested as packaging films for chill stored tilapia showed that samples packed in LDPE films were rejected by 7th day whereas fish packed in 1%, 3% were remained acceptable up to 15 days. The study revealed that 3% LDPE/CS films had better physical and antimicrobial property and enhanced the keeping quality of Tilapia steaks during chilled storage when compared to the other films used in the study. Acknowledgements The authors gratefully acknowledge the laboratory facilities provided by Director, Central Institute of Fisheries Technology, Cochin, India for film testing and the Officer-In-Charge, Centre for Bio-Polymer Science and Technology, CIPET, Cochin India for film extrusion and characterization. References [1] R.G. Ackman, Fatty acids, in: R. Ackman (Ed.), Marine Biogenic Lipids, Fats and Oils, CRC Press, Boca Raton, USA, 1989, pp. 103–137. [2] I.N.A. Ashie, J.P. Smith, B.K. Simpson, Spoilage and shelf life extension of fresh fish and shellfish, Crit. Rev. Food Sci. 36 (1996) 87–121. [3] C.O. Mohan, C.N. Ravishankar, T.K.S. Gopal, Active packaging of fishery products: a review, Fish. Technol. 47 (2010) 1–18. [4] J.H. Han, Antimicrobial food packaging, Food Technol. 54 (3) (2004) 56–65. [5] R.A.A. Muzzarelli, J. Boudrant, D. Meyer, N. Manno, M. DeMarchis, M.G. Paoletti, Current views on fungal chitin/chitosan, human chitinases, food preservation, glucans, pectins and inulin: a tribute to Henri Braconnot, precursor of the carbohydrate polymer science on the chitin bicentennial, Carbohyd. Polym. 87 (2012) 995–1012. [6] S.Z. Rogovina, C.V. Alexanyan, E.V. Prut, Biodegradable blends based on chitin and chitosan: production, structure and properties, J. Appl. Polym. Sci. 121 (2011) 1850–1859. [7] S.I. Park, K.S. Marsh, P. Dawson, Application of chitosan-incorporated LDPE film to sliced fresh red meats for shelf life extension, Meat Sci. 85 (2010) 493–499. [8] H.Z. Zhang, Z.C. He, G.H. Liu, Y.Z. Qiao, Properties of different chitosan/low-density polyethylene antibacterial plastics, J. Appl. Polym. Sci. 113 (2009) 2018–2021. [9] C. Vasile, R.N. Darie, A. Sdrobis, E. Pâslaru, G. Pricope, A. Baklavaridis, S.B. Munteanu, I. Zuburtikudis, Low density polyethylene–chitosan composites, Compos.: Part B, Cellulose Chem. Technol. 48 (3–4) (2014) 325–336. [10] R.C. Goy, D. Britto de, O.B.G. Assis, A review of the antimicrobial activity of chitosan, Polím.: Ciênc. Tecnol. 19 (2009) 241–247. [11] ASTM D 792-08, Standard Test Methods for Density and Specific Gravity (Relative Density) of Plastics by Displacement, 2008. [12] ASTM D 1238-04c, Standard Test Method for Melt Flow Rates of Thermoplastics by Extrusion Plastometer, 2004. [13] Indian Standard Institute, Specification for Low Density Polyethylene Films. New Delhi, India. IS: 2508, 1984. [14] ASTM D 1003, Standard Test Method for Haze and Luminous Transmittance of Transparent Plastics, 2000. [15] ASTM D 882-02, Standard Test Method for Tensile Properties of Thin Plastic Sheeting, 2002.

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chitosan composite antimicrobial film for chilled fish storage.

An antimicrobial packaging material was developed by uniformly embedding 1, 3 and 5% chitosan (w/w) in low density polyethylene matrix using maleic an...
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