The thickness of the pullulan/graphene based bio nanocomposite coating on nano cellulose film with various proportions is shown in Table 1. The addition of graphene in pullulan did not make any major change in the coating thickness. A slight variation of thickness was obtained for all the samples and it was normalized to 24 µm. The normalized thickness of 24 µm was used for all the characterization and testing of films.
The surface colour of the films is most important in terms of appearance because it directly influences the acceptance of customer . The surface colour values of pullulan/graphene based bio nanocomposite coating on nano cellulose film with various concentrations are shown in Table 2.
The L* (whiteness), a* (red to green), b* (yellow to blue) values of nanocellulose film were 71.46, - 0.39 and 0.35. After the pullulan coating in NC film, the L*, b* value reduced to 69.84, 0.11 and a* value increased to 0.09. After the inclusion of 0.05 wt% of graphene, the L* was significantly reduced to 32.95 and the a*, b* value increased to 0.22, 0.88. Similarly, the L* reduction of 28.63, 26.14 and the increase of a* and b* to 0.28, 0.31 and 0.92, 0.99 was obtained for 0.1 wt% and 0.2 wt%. Likewise, the colour difference also got increased. The results clearly show that the graphene coating in NC film greatly influences the surface colour and colour difference of the films. It improved the light barrier properties of the material and blocked UV light transmission . The degree of colour change was increased at higher graphene content. The surface colour of the packaging films was greatly dependent on the type of nanofillers used .
The opacity of pullulan/graphene bio nanocomposite coating on the nanocellulose film was determined and it is shown in Fig.1.
The opacity for pure nano cellulose film was 16.9%. After the pullulan coating in NC film, the opacity was increased to 17.8%. Similarly, the value was increased to 22.5% by the incorporation of 0.05 wt% of graphene in pullulan. After the addition of 0.1 wt% of graphene, the opacity value was increased to 24.2% and the maximum opacity value of 29.9% was attained for 0.2 wt% of graphene in pullulan coating. This result shows that the opacity value got increased with an increase in graphene content in pullulan. This indicates that the graphene has been mixed well with pullulan and completely dispersed between the polymer matrices at a very low concentration . It is commonly noticed with well developed exfoliated nanocomposites . The exfoliation allowed for improved interaction between the pullulan and graphene . Also, there was a strong scattering of graphene in pullulan increasing the opacity of UV visible light . The opacity of the films provides extra information of dispersed particle size in the polymer matrix. The reinforced particle sizes are larger than the visible wavelength would block the light and leads to opaque films . The opacity is a desirable property in packaging material intended to be used for food packaging applications.
Contact angle analysis
The contact angle is a measure of surface wettability and to identify whether it is hydrophobic or hydrophilic. The surface contact angle which is less than 65° is identified as hydrophilic . The contact angle of pullulan/graphene bio nanocomposite coating on the nanocellulose film was determined and it is shown in Fig. 2.
The contact angle value of nano cellulose film was 42.2°. After the coating of pullulan in NC film, the contact angle value was increased to 68.6°. This shows the hydrophobic nature of the pullulan film. After the incorporation of 0.05 wt% of graphene in pullulan, the contact angle value was still increased to 70.8°. Similarly, for 0.1 and 0.2 wt% of graphene in pullulan, the contact angle value was increased to 73.4° and 75.5°. This result shows that the graphene is hydrophobic and the contact angle value of coating was increased by the addition of graphene. The hydrophobic nature of graphene was due to the density functional theory. As compared to the adsorption energies on the surface of graphene the binding energy between the molecules of water is large. This cause the water molecules to form clusters on the surface . Also, the other aspects such as the amount of loading, compatibility and the polymer matrix play a major role in hydrophobicity.
Coating adhesion Strength
The coating strength is the most important property to characterize the adhesion behaviour of and evaluating the quality of any coated material. The T – peel strength of the pullulan/graphene bio nanocomposite coating on the nanocellulose film was determined and it is shown in Fig. 3.
The adhesion strength of the uncoated nanocellulose was 3.61 N/cm. After the pullulan coating, the value was increased to 3.89 N/cm. Consequently, the adhesion strength was increased to 5.6 N/cm after the incorporation of 0.05 wt% of graphene in pullulan. Similarly, the value was increased to 6.3 N/cm for 0.1 wt% of graphene. A maximum of 7.9 N/cm was attained for 0.2 wt% of graphene incorporated pullulan coating. The results showed that the adhesion strength value was increased by the addition of graphene. This was due to the surface roughness and surface energy of the nanocellulose film and these both were the main key factor for the increase in adhesion. Moreover, the addition of graphene in pullulan showed a significant effect on adhesion strength. The reinforced graphene in pullulan made the mechanical interlocking of the biopolymer matrix into the pores, cavities and asperities of the nanocellulose surface and made the adhesion strength increase .
Tensile strength is one of the most important parameters used to calculate the maximum stress that the material can withstand before breaking . The tensile strength of the pullulan/graphene bio nanocomposite coating on the nanocellulose film was determined and it is shown in Fig. 4.
The tensile strength of the pure cellulose film was 7.4 MPa. After the coating of pullulan in the NC film, the tensile strength was increased to 8.9 MPa. The tensile strength was increased to 12.8 MPa after the incorporation of 0.05 wt% of graphene in pullulan. Similarly, the tensile strength was increased to 15.7 and 19.9 MPa for 0.1 and 0.2 wt% of graphene. These results showed that the tensile strength increases by an increase in graphene concentration. The increase in tensile strength was due to the exfoliated structure and the possible strain induces alignment of graphene in pullulan matrix . It also confirmed the excellent compatibility between the pullulan and graphene . The graphene could be efficiently used as the reinforcement in polymer composites which can provide good dispersion and improve the strengthening of the interface. Therefore, the force act on the pullulan/graphene may effectively be transferred to the graphene, results in a considerable increase in tensile strength . The increase in tensile strength of the coated bio nanocomposite films was due to the physical attraction between the coating and NC film. The adhesion strength showed good results for the attraction between NC film and pullulan/graphene coating.
Oxygen Transmission Rate:
The oxygen transmission rate (OTR) of the pullulan and pullulan/graphene bio nanocomposite coating on the nanocellulose film was determined and it is shown in Fig. 5.
The OTR for the nanocellulose film was 1984.25 cc/m2.day.atm. After the pullulan coating on NC film, it was decreased to 1854.64 cc/m2.day.atm. Then the value was significantly reduced to 1120.56 cc/m2.day.atm by the incorporation of 0.05 wt% of graphene. Similarly, the OTR value was still decreased to 987.47 cc/m2.day.atm for 0.1 wt% and the minimum value of 851.32 cc/m2.day.atm was achieved for 0.2 wt% for graphene. The mechanism for the increase in barrier property is usually due to the formation of a tortuous path against the gas molecules to cross through the film . The orientation and highest exfoliation level of graphene in pullulan was also the reason decrease in OTR. The various factors to improve the barrier properties of the bio nanocomposite films are the effect of nanofiller, crystallization behaviour of the polymer nanocomposites and the interfacial interaction between the matrix and nanofillers . Lee et al.  presented the lower barrier properties because of poor interfacial adhesion between PVC and GO nanocomposites. In this work, there was a large reduction in OTR after the addition of graphene in pullulan and it was due to the creation of a more tortuous path in the diffusing molecules and it can move around the impermeable fillers .
Watervapour Transmission Rate
The main aim of the food packaging material is to decrease the watervapour transfer between food and atmosphere and the WVTR should be low as possible . The watervapour transmission rate (WVTR) of the pullulan and pullulan/graphene bio nanocomposite coating on the nanocellulose film was determined and it is shown in Fig. 6.
The WVTR for the pure nanocellulose film was 14.21 g/m2.day. After the coating of pullulan in NC film, the WVTR value was decreased to 12.12 g/m2.day. The WVTR value of the film was decreased to 8.3 g/m2.day by the incorporation of 0.05 wt% of graphene. The WVTR value was still reduced by adding the 0.1 wt% of graphene in pullulan and the value was 6.1 g/m2.day. The minimum value of 3.9 g/m2.day was achieved for 0.2 wt% of graphene incorporated pullulan coating. From these results, it was noticed that the WVTR decreased by an increase in graphene in pullulan. The addition of graphene in pullulan is effectively improved the WVTR of the coating. These results might be attributed to the concept that the graphene was properly dispersed in the pullulan formed a tortuous path for transmission of watervapour and increased the effective path length of diffusion . The hydrophobic behaviour of graphene was also the reason for the reduction in WVTR . Also, the hydrogen bond formation between pullulan and graphene increased the force of adhesion made tougher for the water molecules to transmit through the film.
The pathogenic microorganisms and spoilage growth is the major reason for spoilage of food and reason for foodborne diseases . Therefore, antibacterial activity is needed for food packaging. The antibacterial activity of the coated films was evaluated by the colony count method against gram-positive S.aureus and gram-negative E.coli. The total number of colonies was 100 CFU/g and the reason for the selection of S.aureus and E.coli was being the major microorganisms causing foodborne diseases to humans . Fig. 7 presents the antibacterial activity of pullulan/graphene coating on nanocellulose film.
The nanocellulose film and pullulan did not show any antibacterial activity against both the microorganism. By the incorporation of 0.05 wt% of graphene in pullulan coating, the number of colonies was reduced to 73 for E.coli and 75 for S.aureus. Similarly, for 0.1 wt% of graphene, the colonies were still reduced to 61 for E.coli and 62 for S.aureus. The maximum reduction was obtained for 0.2 wt% of graphene and the values were 49 for E.coli and 51 for S.aureus. From these results, it was proved that graphene was responsible for the strong antibacterial activity and it reduces the growth of gram-negative bacteria and gram-positive bacteria. The possible antibacterial mechanisms of graphene are oxidative stress, wrapping isolation and stress of membrane . In the oxidative stress mechanism, it involves various reactive oxygen species such as hydroxyl radical, hydrogen peroxide and superoxide radicals . The membrane lipids, proteins and nucleic acid of bacteria were exposed to oxidative stress and it will be oxidized, degraded and lead to the destruction of cell . In the wrapping isolation, the bacterial cells are enclosed in graphene and separated from the medium of growth. The graphene blocks the nutrients to pass through cell membranes and obstructs the growth . In membrane stress, graphene nanoparticles destroy the bacteria by piercing the membrane and extract the phospholipids . Finally, the addition of graphene in pullulan improved the antibacterial activity of the coated films.