development of starch-polyvinyl alcohol (pva

Development of starch-PVA antimicrobial film 1

RESEARCH ARTICLE

PREFACE API 2015

Development of Starch-Polyvinyl Alcohol (PVA) Biodegradable Film: Effect of Cross-Linking

Agent and Antimicrobials on Film Characteristics

Aniket Satish MoreIndian Institute of Technology, Kharagpur

[email protected]

Chandani SenIndian Institute of Technology, Kharagpur

[email protected]

Madhusweta Das Indian Institute of Technology, Kharagpur

[email protected]

ABSTRACT

To satisfy the need of developing eco-friendly flexible antimicrobial packaging film with minimum use of synthetic chemical ingredients, the present study examined the efficacy of citric acid (CA) as cross-linking agent and essential oils (EOs), viz., cinnamon essential oil (CEO) and oregano essential oil (OEO) as natural antimicrobials in corn starch-polyvinyl alcohol (CS-PVA) film. Compared to film prepared from filmogenic solution (FS) containing 75 kg CS+8.75 kg PVA+24.6 kg glycerol per m3 FS, film additionally containing CA at 0.07 kg/kg CS indicated 95% higher ultimate tensile strength (UTS) and 27% lower water vapor permeability (WVP). Film developed with incorporation of CEO and OEO at 1.875 m3 in 100 m3 FS (CS:PVA= 8.5:1) containing CA at 0.07 kg/kg CS exhibited antimicrobial action against Staphylococcus aureus. Added advantage was, both EOs could reduce WVP of film with no EO by about 50%, though CEO exhibited better antimicrobial action. Structural alteration in film matrix due to incorporation of EOs was evident from FTIR and SEM analyses. Thus, from the overall results, CEO (at 1.875 m3 /100 m3 FS) incorporated CS-PVA film cross-linked with CA, in prescribed amounts, was found to be the suitable antimicrobial film with appreciable mechanical properties (UTS ≈4 MPa, Elongation ≈50%) and water vapor permeability (≈0.5×10-6 kg.m.m-2.kPa-1.h-1).

KEY WORDS

Biodegradable starch film, Cross-linking agent, Citric acid, Natural antimicrobials, Cinnamon essential oil, Oregano essential oil

Journal of Applied Packaging Research 2

1. INTRODUCTION

Substantial increase in the use of petroleum based synthetic polymers particularly in disposable flexible packaging film sector has led to serious ecological problems due to their non-biodegradabil-ity. In the case of laminated multi-layer packaging, recycling is not feasible [1, 2] whereas, for single layer packaging recycling is possible but material property degrades after 3-4 times of recycling. Moreover, incineration of such polymers produces toxic greenhouse gases that lead to global warming. Therefore, there is a need to develop biodegradable alternatives which do not burden the environment.

A major fraction of flexible film market is shared by food packaging [3, 4]. Different natural biopoly-mers like starch, cellulose, chitosan, gelatin, etc., have been attempted so far for the development of biodegradable films in food packaging research [5]. Among these, starch is considered most promising due to its wide spread availability and relatively low cost [6]. However, to overcome the brittleness arising from high intermolecular forces in neat starch film, addition of a plasticizer is must [7], and glycerol, being compatible with amylose packing of starch, has been largely used for this purpose. In spite of that, the use of starch based films for flexible packag-ing has been limited because of their weak mechani-cal properties and high sensitivity to moisture [8]. Thus, there is a need to incorporate some synthetic polymer and a cross-linking agent, in addition to plasticizer, to impart desirable properties.

Among different synthetic polymers, polyvi-nyl alcohol (PVA) is one of the best options due to its water solubility and biodegradability [9], high thermal stability [10] and excellent compatibil-ity with starch [11, 12]. Cross-linking agent rein-forces the film matrix forming covalent bonds with hydroxyl (–OH) groups of starch and PVA, which act as the bridges between the polymers and reduce the hydrating character [13]. The various cross-linking

agents such as glutaraldehyde [14], boric acid [15], epichlorohydrin [16], etc., have been used in starch based film. But, addition of these chemicals may lead to residual effect in the product, thus it is always desirable to select some natural one, like citric acid (CA). CA, a non-toxic, three-carboxylic compound containing one –OH group, improves mechanical properties and water resistibility due to intermolecular covalent and hydrogen bonding that helps to block free -OH groups in the starch film [17, 18]. Several researchers developed starch-PVA-CA films, but the content of PVA in these works was ≥ starch content [5, 18, 19, 20, 21, 22]. However, con-sidering the cost and limited petroleum resources, its minimal use is desirable.

Since starch is food material, its films are sus-ceptible to microbial attack if left as such in contact with water vapor present in air, which can be checked if antimicrobials are added in the film formulation. The added antimicrobials also extend the shelf life of packaged food, to give the benefit of active pack-aging [23]. To achieve this target, various chemical preservatives such as sorbic acid [24], potassium sorbate [25], benzoic acid [26], and sodium pro-pionate [27] have been used. However, the natural antimicrobial agents extracted from plant materials, like essential oils (EOs) may be considered as good alternatives [28]. Carvacrol, eugenol, thymol, cin-namaldehyde, etc., present in various EOs, possess strong antibacterial and antifungal properties against foodborne pathogenic and spoilage micro-organisms [29, 30]. Additionally, these compounds exhibit antioxidant properties. The cinnamon essential oil (CEO) and oregano essential oil (OEO) are traditionally harvested and abundantly found in Asian countries. The principal constituents of these oils, cinnamaldehyde (CEO) and carvacrol (OEO), exhibit low minimum inhibitory concentrations [31]. Cellulose based edible films containing cinna-maldehyde and eugenol was found to be inhibitory against Saccharomyces cerevisiae, Escherichia


coli, and Staphylococcus aureus [31]. The mode of action is generally considered to be the distur-bance in the cytoplasmic membrane, active trans-port, and coagulation of cell contents [32]. Thus, the addition of these EOs in films allows improvements in food safety and shelf life by reducing or prevent-ing the growth of pathogenic and spoilage microor-ganisms. Recently, Li et al. [33] reviewed that OEO effectively reduces infectivity of foodborne Norovi-rus surrogates, and in this context, it is worth men-tioning that the feasibility of developing antiviral active packaging (silver-infused polylactide films) has been documented by Martínez-Abad et al. [34].

Essential oils, depending on their concentration and chemical characteristics as well as the ingre-dients in film matrix, can form inter-molecular complexes with polymers and modify film proper-ties [35, 36, 37]. It has been reported that the incor-poration of garlic oil in starch film reduces water vapor permeability due to its lipidic behavior [38]. However, report on the addition of EOs in starch-PVA film is limited.

Thus, the objectives of the present study were to investigate the effect of:

1. CA as cross-linking agent on tensile strength (TS), elongation (El) and water vapor permeability (WVP) of starch-PVA (8.5:1) film, and selection of optimum CA concentration for maximum TS and El, and minimum WVP.

2. CEO and OEO on TS, El, WVP, and struc-tural and antimicrobial characteristics of the above mentioned starch-PVA film contain-ing optimum amount of CA, followed by the selection of the suitable EO.

2. MATERIALS AND METHODS

2.1 Materials

The commercial grade corn starch (CS) powder was procured from Angel Starch and chemicals, Tamil Nadu, India. Cinnamon essential oil (CEO) (Cinnamaldehyde 65-75% and Eugenol 5-10%) and oregano essential oil (OEO) (Carvacrol 70%+) were purchased from Moksha Lifestyle Products, New Delhi, India, and used as natural antimicrobial agents. Polyvinyl alcohol (PVA) (molecular weight 1,25,000 Da) was purchased from Loba Chemie, Mumbai, India. Citric acid (CA) (molecular weight 210 Da) was purchased from S. D. Fine-Chem Ltd., Thane, India. Glycerol (GLY) (87%, GR) was purchased from Merck Specialities Private Ltd., Mumbai, India. Ethanol (99.9%) was obtained from Jiangsu Huaxi International Trade Co. Ltd, China. Staphylococcus aureus culture was obtained from MTCC, Chandigarh, India. Glass distilled water, used as solvent throughout the experiment, was prepared in the laboratory.

2.2 Methods

2.2.1 Preparation of film

The filmogenic solution (FS) containing previ-ously optimized [39] amount of CS (75 kg/m3 FS) and PVA (8.75 kg/m3 FS) was used to prepare the self-supporting CS-PVA film.

Initially, required amount of PVA was kept for dispersion in a 30×10-6 m3 portion of distilled water for 12 hours. Then the PVA solution was heated in boiling water bath for 5-7 minutes to obtain a clear solution. The required amount of CS and GLY was added in PVA solution followed by manual stirring for mixing. Finally, CA was added in different concentration (Table 1) in the polymer solution followed by adding water to make up the volume up to 80×10-6 m3. The mixture was kept for 3 hours


at the room temperature to initiate the cross-linking reaction, and finally, heated in a boiling water bath for 10-12 minutes for gelatinizing the starch. The FS thus obtained was immediately cast on polypro-pylene plate with a thin layer chromatography appli-cator maintaining a thickness of 2 mm, followed by drying of film at 40 °C under low air current. The dried film was peeled off from the plate, kept in paper flap, and stored at room temperature for ageing for stabilization of properties prior to testing [27].

Antimicrobial containing films were prepared by adding CEO and OEO in different amounts to maintain up to 2.5 m3 EO/100 m3 FS. For this, EOs

were dissolved in 1×10-6 m3 of 99.9% ethanol and added in gelatinized FS containing CS, PVA and the optimum amount of CA, and reheated for 2 min. The range of concentration was selected on the basis of literature reviews [6, 40] and the results of preliminary experiments. Finally, the same proce-dure as mentioned above was followed for casting and drying of the film. Henceforth, films with CEO will be called as CEO film whereas films with OEO will be called as OEO film. Storing and aging were similar as mentioned above [41, 42]. The following table presents the composition of all the FSs used for casting.

SI.No.

CS(kg/m3

FS)

PVA(kg/m3

FS)

GLY(kg/m3 FS)

CA(kg/kg

CS)

CEO(m3/100m3 FS)

OEO(m3/100m3 FS)

1* 75 8.75 24.6 0 - -

2 75 8.75 24.6 0.01 - -3 75 8.75 24.6 0.03 - -

4 75 8.75 24.6 0.05 - -5** 75 8.75 24.6 0.07 - -

6 75 8.75 24.6 0.10 - -7 75 8.75 24.6 0.07 0.625

1.2501.875

2.5000.625

1.2501.875

2.500

0.070.07

0.070.07

0.070.07

0.07

-

8 75 8.75 24.6 -9 75 8.75 24.6 -

10 75 8.75 24.6 -11 75 8.75 24.6 -

12 75 8.75 24.6 -13 75 8.75 24.6 -

14 75 8.75 24.6 -*, control for e�ect of CA; **, control for e�ects of EOs

Table 1: Composition of different filmogenic solutions (FS) used for casting


2.2.2 Measurement of properties

Prior to measurement of the properties, the films were cut in proper shape (wherever necessary) and conditioned at 50% relative humidity (RH) and 25 ºC for minimum 48 hours [43] to ensure equilib-rium. Thickness of cut films, at least at five differ-ent positions, was measured by hand dial thickness gauge (Model 7301, Mitutoyo Co., Japan).

Tensile strength and Elongation

Films were cut into a rectangular shape of 0.1 m × 0.01 m. The stress strain diagram was recorded using Instron universal testing machine (Model No. 5965 U 2597, USA) with the cross-head speed of 0.01 m/min following standard procedure [44] as described in the earlier publication [27]. The peak TS (UTS, MPa) and the elongation (El, %) at break were noted.

Water vapor permeability

WVP was determined [27] using gravimet-ric cup method [45]. Initially, the standard perspex cup was filled with distilled water occupying 50% of its volume. The test film sample (cut in the form of a circular disc of 0.05 m diameter) was placed at the top of the cup so as to cover its aperture, which maintained 100% RH inside the cup. A rubber washer and on this a collar lid was fixed on the cup. The assembly was placed in desiccator maintained at 50% RH, and the desiccator was kept at 25 °C. The water vapor transferred in 24 hours through the film was determined from weight loss and WVP calculated as-

where W1 = initial weight of cup (kg); W2 = final weight of cup (kg); h = average film thickness (m); PA1 = partial pressure (kPa) of water vapor at 100% RH; PA2 = partial pressures (kPa) of water vapor at

50% RH; A1 = area of exposed film for permeation (m2) and T1 = permeation time (hour).

Fourier transform infrared spectroscopy (FTIR)

FTIR spectra of films were studied at 25 ± 1 ºC using NICOLET 6700 (Thermo scientific, USA). Film samples (without and with EOs at the highest concentration) were loaded directly on the sample holder and the frequency range varied from 400 cm-1 to 4000 cm-1.

X-ray diffraction (XRD)

XRD patterns were recorded with X’Pert PRO PW3040/60 (PANalytical, Netherland) X-Ray Dif-fractometer with a ray of wavelength (λ) 1.5418 Å. The radiation was generated at 40 kV and 30 mA. Tape glued CEO and OEO film samples oriented on neutral glass sample holder were scanned for the range of 5 to 60o (2θ) using scan speed of 0.05º/sec.

Scanning Electron Microscopy (SEM)

The surface microstructure was determined using Scanning Electron Microscope (JEOL JSM-5800, USA). The film sample was mounted on bronze stub by using double-sided tape followed by gold coating (100 Å) in vacuum electro sputter at 10-2 to 10-3 torr and examined using an accelerating voltage of 20 kV, wavelength of incident rays 500 µm with a magnification of 800 (×800).

Antimicrobial assay

The disc diffusion assay [31] was carried out for assessing antimicrobial action against S. aureus using nutrient agar media. Films were cut into 0.01 m diameter discs and made sterile by using ultra-violet radiation, by turning upside down after about 30 min. Then, cut films were laid onto the inocu-lated plate’s surface followed by incubation at 37 °C for 24 hours. Observation of inhibitory zone sur-rounding the periphery of film discs was recorded.

WVP (kg. m/kPa. m2.h) = (W1 - W2)h

(PA1 - PA2)A1T1

... (1)


2.2.3 Statistical analysis

The number of replications of each measure-ment was case specific. However, in each case, the mean and standard deviation were evaluated. Means were analyzed statistically by Analysis of variance (ANOVA) at 1 and 5% levels of significance using single factor experiment with completely random-ized design with equal replications [46]. Further, the least significant differences (LSD) amongst the mean values of response, i.e., treatment means were estimated at 1 and 5% levels to ascertain any signif-icant difference among the pair of treatments. All these calculations were done using Microsoft Excel 2007 (Microsoft Corp., USA).

3. RESULTS AND DISCUSSION

3.1 Effect of CA on CS-PVA film

The following section describes the effect of CA on mechanical properties and WVP of the CS-PVA film.

Tensile strength and elongation

Ultimate tensile strength (UTS) and elongation at break (El) of films developed from FS contain-ing CS, PVA and different concentrations of CA are shown in Table 2. It is evident that with increasing concentration of CA up to 0.07 kg/kg CS, UTS of the film gradually (p<0.01/0.05) increased, finally reaching to 5.25 MPa; thus showing ≈95% increase compared to CS-PVA film containing no CA. Further increase to 0.10 kg CA/kg CS decreased the UTS from 5.25 MPa to 3.33 MPa, i.e., lowering by about 36%. Exactly similar behavior of initial increase of tensile strength, followed by decrease was reported in neat starch film plasticized by glycerol [47]. The enhancement in UTS due to the incorporation of increasing amount of multi-carboxylic CA up to 0.07 kg may be attributed to the increasingly higher degree of cross-linking [48]. The decrease in TS at 0.10 kg CA/kg CS may be because of the fact that at higher concentration, besides acting as cross-linker, CA renders plasticization [5]. According to Shi et al [5], the addition of CA in starch-PVA blend

Table 2: Effect of concentrations of CA on UTS, El, and WVP of films

Citric Acid(kg/kg CS)

Ultimate tensilestrength* (MPa)

Elongation atbreak* (%)

Water vaporpermeability#x106

(kg.m.m-2.kPa-1.h-1)

00.01

0.030.050.070.10

2.69 ± 0.3a

3.24 ± 0.36b

4.09± 0.84c

4.14± 0.86c

5.25± 0.58d

3.33± 0.33b

48.728 ± 9.62ac

59.983 ± 11.67b

41.740± 12.81c

44.685± 20.73c

43.857± 12.21d

52.833± 13.11ab

1.625 ± 0.09a

1.913 ± 0.17b

1.609± 0.12a

1.229± 0.11c

1.180± 0.13c

1.157± 0.13c

LSD .01

LSD .05

0.5720.431

13.29910.032

0.1860.139

*, mean of 16 replications; #, mean of 8 replications; Means with common superscript (a,b,c,d) denote no sig-nificant (p>0.05) difference.


containing glycerol initially increased the tensile strength of the resulting film and then decreased with further addition, whereas elongation continu-ally increased. The continual increase in elonga-tion and decrease in tensile strength with increase of CA was also reported by Priya et al. [21] and Park et al. [49] in starch- PVA film with no other plas-ticizer. Shi et al. [5] explained that CA acted both as cross-linker and plasticizer, and therefore, the different functions were controlled by different amounts of CA added. Surprisingly, some erratic trend is observed in the case of El in the present study. For the whole range of concentration, El% remains insensitive and an average value of about 48% is exhibited. As explained above, this may be due to multifaceted contribution by CA, viz., esteri-fication, hydrogen bonding, cross-linking as well as plasticization, in presence of low amount of PVA. Moreover, the acidity of citric acid at higher concen-tration may lead to fragmentation of starch, which affects the chain entanglements in the polymer to lower the interactions between the starch molecules, facilitating the slipping movement vis-à-vis higher elongation [50].

Water vapor permeability (WVP)

The WVP values of CS-PVA films incorpo-rated with different concentration of CA are shown in Table 2. As concentration of CA was increased up to 0.05 kg, the general trend was decreasing in WVP (p<0.01), but after that no significant dif-ference was observed. Thus at 0.05 kg CA and onwards, the WVP decreased by ≈27%, compared to CS-PVA film without CA. The formation of a more compact structure owing to cross-linking prevents the swelling of starch and restricts the movement of molecules, leading to a decrease in water vapor permeability [47]. Formation of compact structure to reduce the degree of swelling of starch-PVA film cross-linked by CA was reported earlier [5, 21, 49]. Very recently, Wu et al. [22] reported a decrease

in WVP of starch-PVA-GLY film cross-linked with CA. In the case of a neat starch film also, CA reduced WVP, as reported by Sarwono et al. [51].

Thus from the above discussion, it may be compiled that with the incorporation of CA at 0.07 kg/kg CS, there is ≈95% increase in UTS, and ≈27% decrease in WVP, compared to film without CA. Also, elongation of the film from CS-PVA blend at the chosen ratio was not very sensitive to CA con-centration. Hence, CA at 0.07 kg/kg CS was selected as the optimum concentration for further study.

3.2 Effect of EOs on CS-PVA film cross-linked with CA

Tensile strength and Elongation

Effect of incorporation of CEO and OEO on UTS, El and WVP of the CS-PVA-CA films, along with statistical analyses, are shown in Table 3. In the case of CEO, UTS significantly (p<0.05) increased to 5.88 MPa for the addition of 0.625 m3/100 m3 FS; after this, UTS decreased (p<0.01) to 4.18 MPa for at 1.875 m3 level, with no signifi-cant change thereafter.

In the case of OEO, the change for up to 1.25 m3 addition to reach 4.72 MPa was insignificant. Following this, the strength significantly decreased to 3.17 MPa for the amount of 1.875 m3/100 m3 FS, with insignificant lowering for further addition to 2.5 m3. Thus, except for the increase of strength for 0.625 m3 CEO, both the oils lower the UTS amount-ing ≈20% for CEO and 39% for OEO at 1.875 m3/100 m3 level, as compared to no oil film. The reduction of TS may be due to the disturbance in the film network microstructure caused by the added EOs. Probably, incorporation of incompatible sub-stances causes a discontinuity in the film matrix, by virtue of which TS is reduced [52]. Decrease in TS with the incorporation of CEO and OEO in gelatin-chitosan and starch films without any cross-linking agent were also reported by Souza et al. [53], Wu et


al. [54] and Šuput et al. [55]. As explained by Fabra et al. [56], the presence of oil droplets in the film reduces homogeneity of film matrix resulting in the weaker bonding between polymer chains. However, the initial increase for 0.625 m3 CEO may be due to the cross-linking effect of cinnamaldehyde, the observation similar to Ojagh et al. [40] in chitosan film.

It is seen that up to 0.625 m3 addition (Table 3), elongation decreased (p<0.01) from 43.756% (no oil

film) to 18.366% for CEO and 23.577% for OEO, i.e., a higher reduction is observed in the case of the former. With ≥ 1.25 m3 OEO/100 m3 FS, El remained at an average value of 37%, i.e., decreased by 15% compared to no EO film. Following 0.625 m3, on the other hand, El increased for up to 1.875 m3/100 m3 FS for CEO, with insignificant change thereaf-ter; thus, the flexibility of the film was increased by about 13% by CEO at ≥ 1.875 m3 from that of film without CEO. The initial decrease for 0.625 m3 of EO

Table 3: Effect of incorporation of EOs on tensile properties and WVP of films

*,each data point represents mean of 20 replications; #, each data point represents mean of 10 replications

Concentrations(m3/100 m3 FS)

Ultimate tensilestrength* (MPa)

Elongation atbreak* (%)

Water vaporpermeability#x106

(kg.m.m-2.kPa-1h-1)

CEO 0.0 5.25 ± 0.52a 43.756 ± 10.96ad 1.183 ± 0.12a

CEO 0.625 5.88 ± 1.06b 18.366 ± 10.13b 1.136 ± 0.18a

CEO 1.250 5.62 ± 0.77ab 23.485 ± 9.80b 0.698 ± 0.21b

CEO 1.875 4.18 ± 0.84c 50.316 ± 8.07c 0.568 ± 0.07bc

CEO 2.500 3.85 ± 1.01c 49.142 ± 7.77c 0.529 ± 0.06c

OEO 0.0 5.25 ± 0.52a 43.756 ± 10.96ad 1.183 ± 0.12a

OEO 0.625 5.58 ± 1.98a 23.577 ± 11.56b 0.933 ± 0.12b

OEO 1.250 4.72 ± 2.67a 32.736 ± 15.84c 0.612 ± 0.10c

OEO 1.875 3.17 ± 1.43b 38.479 ± 13.89ac 0.573 ± 0.12c

CEO 2.500 2.64 ± 0.24b 39.829 ± 8.50ac 0.579 ± 0.11c

LSD0.01 0.737 8.059 0.178

LSD0.05 0.556 6.079 0.133

LSD0.01 1.403 10.630 0.145

LSD0.05 1.058 8.020 0.109


may be due to the cross-linking effect of cinnamal-dehyde [40] and carvacrol (phenolic compound). At higher concentrations, the EOs probably rendered some plasticization to increase the elongation from the value corresponding to 0.625 m3 of EO [55].

Water vapor permeability

WVP of the CS-PVA-CA film (Table 3) decreased (p<0.01) first for incorporation of 1.25 m3 and 0.625 m3 of CEO and OEO, respectively. With concentration increasing to still higher level, the next lowering (p<0.05) occurred at 2.5 m3 CEO and 1.25 m3 OEO. In the latter case, there was no further lowering. Thus at 1.25 m3/100 m3 FS and onwards, an average value of WVP of 0.598×10-6 kg.m.m-2.kPa-1.h-1 for CEO and 0.588×10-6 kg.m.m-2.kPa-1.h-1 for OEO was maintained, i.e., the reduction was ≈49% for CEO and ≈50% for OEO, indicating com-parable reduction for the addition of both the oils. Decrease in WVP due to the addition of OEO was also reported for modified corn starch containing guar-xanthan mixture and gelatin-chitosan films by other researchers [54, 55].

The reduction in WVP may be due to increase in film hydrophobicity resulting from incorporation of EOs in the matrix, which reduces the affinity towards water. The presence of a hydrophobic disperse phase, even at a small ratio, limits water vapor transfer through the film [57]. The hydropho-bic characteristic of the oil incorporated film is also evident from the FTIR study as described below.

Fourier transform infrared spectroscopy (FTIR)

FTIR spectra for control film (no EO) and films incorporated with CEO and OEO at 2.5 m3/100 m3 FS are depicted in Figure 1.

The spectral characteristics in terms of wave-number and intensities of full scale region for all the three types of films are also presented in tabular form (Table 4).

Figure 1: Full scale FTIR spectra of control film and films containing CEO and OEO at 2.5 m3/ 100 m3 FS.


In Table 4, peaks at 1694.27 and 1717.97 in CEO and OEO film, respectively, representing C=O stretching vibration indicates the presence of the carbonyl radical having ester functional group of triglycerides, associated with the presence of oil in the film matrix, which is not seen in control film. The peak at 1230.70 in OEO film refers to the C-O-H stretching of the phenolic compound, car-vacrol. Also, the peak observed at 2914.67 repre-senting stretch vibration of -CH3 and =CH2 in OEO incorporated film indicates the presence of a hydro-phobic group (lipid) in the film [58]. Therefore, the addition of OEO and CEO into CS-PVA-CA film

enhanced the hydrophobic group which could lead to increase in hydrophobicity and decrease the WVP of the film.

X-ray diffraction (XRD)

X-ray diffraction patterns of films containing CEO and OEO at higher concentrations, i.e., 1.875 m3/100 m3 FS and 2.5 m3/100 m3 FS are shown in Figure 2 and 3 respectively.

3500-2800 cm-1 1700-600 cm-1

Contorl �lm

3353.01 (0.006) 2336.75(0.005)

1077.57 (0.024) 653.63 (0.073) 1147.74 (0.019) 685.58 (0.057) 1622.13 (0.006) 753.63 (0.036)

858.05 (0.031) 992.87 (0.049)

CEO�lm

3348.02 (0.073) 2336.48(0.0036)

1078.33 (0.33) 654.43 (0.085) 1149.88 (0.024) 695.87 (0.067) 1332.99 (0.013)

1551.97 (0.007) 1694.27 (0.005)

759.60 (0.047)

856.13 (0.036) 921.08 (0.037) 990.08 (0.072)

OEO�lm

3254.05 (0.012) 2335.94(0.006)2914.67(0.006)

1076.00 (0.037) 650.69 (0.087)1147.32 (0.031) 697.29 (0.058)1230.70 (0.017)

1329.01 (0.018)1540.60 (0.008)1717.97 (0.007)

857.58 (0.041)

919.91 (0.051)989.03 (0.075)

Table 4: Spectral characteristics and intensities (in brackets) at various absorption frequencies for control, CEO and OEO films


In Figures 2 and 3, CS-PVA-CA film containing CEO at both the concentrations showed sharp peaks between 15o to 25o (2θ) compared to films contain-ing OEO, indicating the occurrence of a higher degree of crystallization in the former. The higher crystallinity in case of CEO incorporated film is in corroboration with its higher UTS (Table 3).

As explained by Liu et al. [50], a disintegration of the structure of the native starch granules occurs during gelatinization of aqueous starch suspension, and a new semi-crystalline structure comprised

of crystallized amylose molecules develops in dry films. Perhaps, OEO in the film matrix resists the reorganization of amylose domain to indicate lower crystallinity vis-à-vis the less intensity.

Scanning Electron Microscopy (SEM)

The SEM images (×800 magnification) showing the surface microstructure of CS-PVA-CA film without EO (control) and containing EOs are presented in Figure 4. With the incorporation of EOs at 0.625 m3/100 m3 FS, distinct oil droplets are not seen, but the surfaces (Figures 4B and 4C) become smoother compared to control (Figure 4A). However, the presence of an uneven distribution of oil at 2.5 m3/100 m3 FS is clearly visible. It is inter-esting to mention that presence of free oil at 2.5 m3/100 m3 of FS was also evident from FTIR study as mentioned above.

Figure 2: XRD analysis of CEO and OEO films at 1.875 m3/100 m3 FS

Figure 3: XRD analysis of CEO and OEO films at 2.5 m3/100 m3 FS


Antimicrobial activity

From the above discussion, it is evident that WVP, the most significant constraint of starch based film, is reduced to the maximum extent (≈49-50%) for the addition of ≥ 1.25 m3 EOs/100 m3 FS. Besides this, the flexibility of the film is also increased up to the incorporation of 1.875 m3 CEO. Antimicrobial efficacy, therefore, was investigated with the film containing 1.875 m3 EOs/100 m3 FS.

Figure 5 shows the antimicrobial inhibitory effect of control film and film containing OEO and CEO. Films with EO generated a zone of inhi-bition by absence of bacterial growth around the periphery of the cut films (Figures 5B and 5C), while in Figure 5A, it was not there. The average zone of inhibition (four replications) for CEO con-taining film was 3.5 ± 1.1 mm, whereas for OEO film it was found to be 1 ± 0 mm.

Incorporation of CEO in CS-PVA-CA cross-linked film at 1.875 m3/100 m3 FS exhibited a higher

inhibitory zone against the tested microorganism (Staphylococcus aureus). This is because, CEO contains a high concentration of trans-cinnamalde-hyde, a well-known antimicrobial compound with linalool, eugenol and other phenolic compounds [59], which work against the possible microbial growth. Ojagh et al. [40] opined that CEO in coating solution possessed significant antibacterial effects. Compared to CEO film (Figure 5C), lower zone of inhibition exhibited by OEO film (Figure 5B) may be due to the fact that gram-positive bacteria have a thick peptidoglycan layer that may function as a protective barrier against the essential oil com-pounds [32]. Thus, it could be stated that CEO con-taining film was more effective against Staphylo-coccus aureus than that of the OEO film.

A

D E

B C

Figure 4: SEM surface images (× 800 magnification) of films: A) Control, B) CEO at 0.625 m3/100 m3, C) OEO at 0.625 m3/100 m3, D) CEO at 2.5 m3/100 m3, and E) OEO at 2.5 m3/100 m3


4. CONCLUSIONS

Use of starch to develop flexible biodegradable food packaging film possessing desirable mechani-cal properties and water vapor resistance is a chal-lenge. To accomplish, several attempts were docu-mented by mixing starch with PVA, water soluble synthetic polymer, and CA, a three-carboxylic compound containing one –OH group, as cross-linking agent; mostly, the amount of PVA used remained ≥ starch. However, considering the limi-tation of petroleum resources, minimum use of PVA is desirable. In addition to biodegradability, the concept of extension of shelf life of packaged food by embedded chemicals in the film itself, i.e., the concept of active packaging is recent inclusion in the field of food packaging research. In the first phase of this study, films were developed with varying amount of CA in a pre-optimized starch-PVA (8.5:1) blend. Compared to control film (without CA), maximum benefit was obtained with CA at 0.07 kg/kg CS, which improved UTS by 95% and reduced WVP by 27%, with no adverse effect on elonga-tion. In the next phase, films were prepared using this CS-PVA-CA formulation but with variable con-centration of EOs (CEO and OEO) as antimicrobial agents. Incorporating 1.875 m3 EOs/100 m3 FS, the film containing CEO exhibited appreciable inhi-bition to Staphylococcus aureus than that of OEO film. Added benefits for the addition of EOs were

reduction in WVP by about 49-50%, and improve-ment in flexibility exclusively for CEO film. Perhaps due to the disturbance in the film microstructure, both the essential oils lowered the UTS amounting ≈20% for CEO and 39% for OEO at 1.875 m3/100 m3 FS, as compared to no oil film. The relatively higher strength of CEO film was also confirmed by its higher crystallinity than that of film containing OEO. Thus, based on the overall results, CEO at 1.875 m3/100 m3 FS may be selected as the suitable antimicrobial agent in CS-PVA film cross-linked with CA, in prescribed amount, against Staphylo-coccus aureus.

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