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Carbohydrate Polymers

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Rosin modified cellulose nanofiber as a reinforcing and co-antimicrobialagents in polylactic acid /chitosan composite film for food packaging

Xun Niua, Yating Liua, Yang Songa, Jinquan Hanb, Hui Pana,⁎

a College of Chemical Engineering, Nanjing Forestry University, Nanjing 210037, Chinab College of Material Science and Engineering, Nanjing Forestry University, Nanjing 210037, China

A R T I C L E I N F O

Keywords:Cellulose nanofiberRosinAntimicrobial compositeFood packaging

A B S T R A C T

Cellulose nanofiber (CNF) was modified by rosin and used as a reinforcement filler within a polylactic acid (PLA)matrix. The resulting film was then coated with chitosan (CHT) to prepare a two-layer composite film for an-timicrobial food packaging. The FT-IR spectra of rosin modified CNF (R-CNF) displayed a clear peak at1730 cm−1, which confirmed the successful esterification of CNF by rosin. The R-CNF showed a better dispersionin PLA matrix than CNF and the loading of R-CNF had a significant effect on the mechanical properties of theresulting film. A percolation network was formed when the R-CNF loading was 8%, where the composite filmdisplayed optimum mechanical properties. The antimicrobial test showed that the R-CNF/PLA/CHT compositefilm exhibited excellent antimicrobial performance against E. coli and B. subtilis, which could be attributed to thesynergistic antimicrobial effect of CHT and rosin.

1. Introduction

Petroleum-based plastics such as polyethylene (PE), polypropylene(PP), polystyrene (PS), polyethylene terephthalate (PET), polyvinyl al-cohol (PVA) are currently the most prevalent raw materials mainly dueto their low cost, good mechanical performance and good barrierproperties (Bouwmeester, Hollman, & Peters, 2015; Koelmans, Bakir,Burton, & Janssen, 2016; Carole, Pellegrino, & Paster, 2004). On theother hand, the large consumption of petroleum-based plastics hasposed serious environmental impacts due to the non-biodegradability ofthese plastics. Exploring bio-based and biodegradable plastics forpackaging applications is considered a promising method to solve thecurrent environmental problem causing by the disposal of non-biode-gradable plastics.

Polylactic acid (PLA) is one of the most attractive bio-based andbiodegradable polymers used as a short term film for food packaging(Ingrao et al., 2015) due to its high transparency, good processability,and comparable mechanical properties to its counterpart of fossil-basedpolymers such as polyethylene and polypropylene (Raquez, Habibi,Murariu, & Dubois, 2013; Gu et al., 2017). However, PLA also showshigh brittleness, low thermal stability, and poor barrier properties,which have limited its applications for food packaging. Various stra-tegies have been adopted to remedy these limitations. Incorporation ofnanosized reinforcements is one of the most feasible routes to enhancethe mechanical, barrier, and physical properties of PLA. Cellulose

nanofibers (CNF) possess high stiffness and low density. In addition,they are renewable and biodegradable. These features make CNF apromising candidate as reinforcement filler for polymers. Studies haveshown that the incorporation of CNF into the PLA matrix has improvedthe oxygen barrier and mechanical properties of PLA films (Zhu et al.,2016; Lin, Gèze, Wouessidjewe, Huang, & Dufresne, 2016; Rhim, Park,& Ha, 2013; Reddy, Vivekanandhan, Misra, Bhatia, & Mohanty, 2013).However, the inherent hydrophilicity of CNF is one of the main ob-stacles that hinder its compatibility with hydrophobic PLA. Never-theless, the large amount of hydroxyl groups on CNF also facilitates thesurface modification of CNF. Therefore, several chemical strategies,such as esterification (Espino-Perez, Domenek, Belgacem, Sillard, &Bras, 2014; Sato et al., 2016), salinization (Robles, Urruzola, Labidi, &Serrano, 2015), and polymer grafting (Hatton et al., 2016; Navarroet al., 2015) have been developed to tune the interfacial compatibilitybetween PLA and CNF.

Antimicrobial food packaging can inhibit the growth of spoilagemicroorganisms and therefore enhance shelf-life for food productswhile maintaining quality and safety (Tiwari et al., 2009). Low mole-cular antiseptics (metal ion metallic oxide), such as Ag nanoparticles,SiO2, and TiO2, have been used as antimicrobial agents for food pre-servatives (Mukhopadhyay et al., 2010; Costa, Conte, Buonocore, & DelNobile, 2011). However, many studies have demonstrated that theymight have residual toxicity that might release into food (Reijnders,2009; Alkan & Yemenicioglu, 2015). The use of natural antimicrobial

https://doi.org/10.1016/j.carbpol.2017.11.079Received 20 August 2017; Received in revised form 18 October 2017; Accepted 22 November 2017

⁎ Corresponding author at: College of Chemical Engineering, Nanjing Forestry University, 159# Longpan Road, Nanjing 210037, China.E-mail addresses: hpan@njfu.edu.cn, cheermay@msn.com (H. Pan).

Carbohydrate Polymers 183 (2018) 102–109

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T

compounds provides a promising alternative method to extending theapplication of antimicrobial packaging while maintain human health.Chitosan (CHT) is a non-toxic, biodegradable, and biocompatiblepolymer industrially produced from chitin and the second most abun-dant polysaccharide in nature. Chitosan has shown antimicrobial ac-tivity against several food pathogens and has remarkable film-formingability and oxygen and moisture permeability (Rabea, Badawy, Stevens,Smagghe, & Steurbaut, 2003). Sébastien, Stéphane, Copinet, & Coma(2006) prepared a chitosan/PLA composite film by solution castingmethod in which PEG400 was added as a plasticizer. They reported thatthe resulting composite film had an inhibitory activity against threefungal strains. Bie et al. (2013) developed a PLA/starch/chitosan blendsantimicrobial material by the melting- extrusion process. This materialexhibited an effective and long lasting antimicrobial property against E.coli and S. aureus, which could be used as a potential packing materialfor fresh meat.

Rosin is a natural product of pine resin. Rosin and its derivativeshave been used as stabilizers (Jimenez, Lopez, Iannoni, & Kenny, 2001),plasticizers (Moustafa, Kissi, Abou-Kandil, Abdel-Aziz, & Dufresne,2017; Arrieta, Samper, López, & Jiménez, 2014) due to their highlyhydrogenated phenanthrene ring structures. Additionally, rosin exhibitsnontoxic and antimicrobial characteristics, which makes it a potentialaddictive for food packaging application. Fabrication of ecofriendlyrosin/PLA (Narayanana, Loganathanb, Valapac, Thomasb, &Vargheseca, 2017) and rosin/PLA/PBAT (Moustafa et al., 2017) bio-composite films have been reported in literature. Recently, researchersreported using rosin to surface modify cellulose nanocrystals (CNC) andthe resulting rosin modified CNC showed antibacterial activity againstGram-negative bacteria and Gram-positive bacteria (Castro, Bras,Gandini, & Belgacem, 2016). In this study, rosin was employed tosurface modify CNF to improve its compatibility and dispersion in PLA.Modified CNF worked as a reinforcement and a co-antimicrobial agentto prepare a composite film with PLA and CHT. To the best of ourknowledge, such approach has not been reported before. The resultingCNF/PLA film was combined with chitosan to form a ternary compositefilm using layer-by-layer (LBL) method. The mechanical property andmorphology of the composite films were characterized by tensile testand scanning electron microscope (SEM). The antimicrobial propertiesof the composite films were also evaluated.

2. Experimental

2.1. Materials

Cellulose nanofiber aqueous suspension (0.5%) was purchased fromYu Yue Nano-technology (Shanghai, China). PLA (2003D) was pur-chased from Nature Works LLC (Minnetonka, MN, USA). Rosin wassupplied by Sigma-Aldrich (colophony rosin, gum, St. Louis, MO, USA).Chitosan powder(molecular weight from 600 to 800 kDa, DDA≥ 90%)was purchased from Acros Organics Co (Waltham, MA, USA). Ethanol,dichloromethane, glacial acetic acid, and hydrochloric acid were ob-tained from Sinopharm Chemical Reagent Co.Ltd. (Shanghai, China).All chemicals were used without further purification.

2.2. Modification of CNF by rosin

CNF was modified with rosin by means of a SolReact process fol-lowing the method used by Castro et al., (2016). In brief, 15 g CNFsuspension (0.5 wt%) was added to a three-neck flask and adjusted thepH to 4.0 with 1 wt% HCl in dropwise. The flask was equipped with acondenser and connected to a closed water distillation system. The flaskwith CNF suspension was heated in an oil bath to 130 °C under mag-netic stir. Rosin powder (10/1 to CNF, wt/wt) was then directly addedto the CNF suspension. The reaction temperature was 130 °C and thereaction was performed for 24 h under continuous stir and in nitrogenambient. After the reaction, the resulting rosin modified CNF (R-CNF)

was washed with ethanol several times to remove the excess rosin andthen collected by centrifugation. Finally, R-CNF was solvent-exchangedto dichloromethane for further characterization and preparation ofcomposite film.

2.3. Preparation of CNF reinforced PLA/CHT composite film

The layer of chitosan film was prepared by dissolving chitosanpowder (3 g) in 100 mL aqueous glacial acetic acid solution under roomtemperature for 12 h to make a 3% chitosan solution (Broek, Knoop,Kappen, & Boeriu, 2015). This clear viscos solution (10 mL) was thencasted onto a flat glass and dried at 80 °C. The prepared chitosan layeron the glass was then put into a desiccator for further use.

PLA in pellet form was dissolved in dichloromethane at room tem-perature at a concentration of 6 mg/mL. The obtained solution wasmixed with CNF or R-CNF at designed loadings (0 wt%, 2 wt%, 5 wt%,8 wt%, and 10 wt%) and sonicated in an ice bath under 100 w for10 min to degas as well as enhance the dispersion of CNF. The mixture(10 mL) was then cast onto the chitosan layer and dried in a fume hoodat room temperature overnight. The PLA/CHT composite films withoutreinforcement filler were prepared in a similar manner. To remove theresidue solvents, all composite films were vacuum dried at 40 °C foradditional 24 h after dried in the fume hood and then put in a de-siccator before further use. The thickness of the composite films wereestimated by measuring 10 random points on each film using a digitalvernier caliper and the average value was adopted. All obtained two-layer composite films were controlled having a thickness around 50 μm.

2.4. Characterization of CNF and R-CNF

The morphology of CNF and R-CNF was investigated by means oftransmission electron microscopy (TEM, JEM-1400, JEOL, Toyko,Japan). To prepare the TEM samples, a drop of diluted CNF/R-CNFsuspension (0.1 wt%) was placed on a 400 square mesh copper gridwith Formvar carbon film and dried at room temperature. The grid wasthen floated in phosphotungstic acid for negative staining before sub-jected to TEM characterization.

The changes in chemical structure of CNF before and after rosinmodification were characterized by Fourier transform infrared spec-trometer (FT-IR). Freeze-dried CNF/R-CNF powder were mixed withKBr to produce tablets for FT-IR measurement. FT-IR spectra were

Fig. 1. FTIR spectra of R-CNF and CNF.

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recorded by a NICOLET 380 FT-IR spectrometer (Thermo Electron,Waltham, MA USA). The defined range was from 4000 to 800 cm−1 and32 scans were recorded for each sample.

The contents of carbon, hydrogen, nitrogen and oxygen of CNF andR-CNF were analyzed by Vario Macro cube elemental analyzer(Elementar, Fulda, German) to determine the degree of substitution(DS) in R-CNF according to a method in literature (Seema, Naceur,Joana, Graziano, & Julien, 2015). The calculation of DS (on the basis ofthe carbon content of R-CNF) in Eq. (1) was as follows:

= −−

M x MM x M

DS %%

C AGU

R C

1

2 (1)

where MC1 is the mass of carbon in an anhydrous glucose unit(72.07 g mol−1); MAGU is the mass of an anhydrous glucose unit(162.14 g mol−1); MR is molecular mass of rosin (302.46 g mol−1), MC2

Scheme 1. Illustration of synthesis of rosin modified CNF.

Table 1Elemental analysis of CNF and R-CNF.

Samples Elemental analysis values Normalized values

C (%) H (%) N (%) O (%) C (%)

CNF 38.27 6.00 0.98 47.37 42.27R-CNF 43.95 6.09 1.56 48.76 47.92

Fig. 2. a) High-resolution XPS spectra of C 1 s peakin CNF and R-CNF and b) X-ray diffractograms ofCNF and R-CNF.

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is mass of carbon in rosin (240.23 g mol−1), and x% is the normalizedcarbon content in modified CNF determined by elemental analysis. TheDS corresponds to the number of grafted hydroxyl function moleculesper anhydroglucose unit within the bulk of material.

CNF and R-CNF were also characterized by X-Ray photoelectronspectroscopy (XPS). Measurements were performed with a KratosAnalytical AXIS Ultra electron spectrometer (Kyoto, Japan) withmonochromatic A1 Kα irradiation at 100 W and effective charge neu-tralization with slow thermal electrons. The reported elemental con-centrations were average values. The spectrum decomposition wasperformed using the XPS PEAK 41 program with Gaussian functionsafter subtraction of a Shirley background,

The crystallinity of the CNF and R-CNF were studied using Ultima IVX-ray diffractometer (RigaKu, Tokyo, Japan) at an acceleration voltageof 40 kV and a current of 30 mA. The crystallinity index (CI) of the CNFand R-CNF were calculated according to the Segal method (Segal,Creely, Martin, & Conrad, 1959) as shown in Eq. (2):

⎜ ⎟= ⎛⎝

− ⎞⎠

×C II

1 100%I1

2 (2)

where I1 is the intensity at the minimum (2θ = 18.8°) and I2 is theintensity associated with the crystalline region of cellulose(2θ = 22.7°).

2.5. Characterization of CNF/PLA/CHT composite films

The mechanical properties of the composite films were measuredusing a mechanical testing machine (Shenzhen, China). A gauge lengthof 10 mm, 500 N loading cell, and a crosshead speed of 10 mm/minwere used. Samples were cut from different positions of the compositefilms as 40 × 5 mm pieces. The tensile strength, tensile modulus andthe elongation to break were obtained directly from testing results. Tensamples of each composite film were tested and the average values werereported.

The fracture surfaces of the composite films were observed using ascanning electron microscope (SEM, JSM-7600F, JEOL, Tokyo, Japan).The samples were sputter coated with gold before subjected to SEMobservation.

2.6. Antimicrobial test of the composite films

Antimicrobial activities of neat PLA, R-CNF/PLA, PLA/CHT, CNF/PLA/CHT, and R-CNF/PLA/CHT composite films were evaluatedagainst the Gram-negative bacterium Escherichia coli (E. coli, ATCC9677) and the Gram-positive bacterium Bacillus subtilis (B. subtilis,ATCC 6633) using the Kirby−Bauer disk-diffusion method (Rajendran,

Dhineshbabu, Kanna, & Kaler, 2014). The composite films were ad-hered on the sterilized filter paper using double-side adhesive tape andcut into discs (1.2 cm in diameter). The PLA layer directly contactedwith the filter paper. The discs were sterilized by UV irradiation for15 min. The two tested bacteria were cultivated in Lysogeny broth (LB)in an incubator at 37 °C. Before subjected the cultures to test, the cellconcentration was standardized to approximately 1 × 106 CFU/mL.About 0.5 mL standardized cultures of the test bacteria was spread asuniformly as possible throughout the entire surfaces of solidified LBagar plates using sterile cotton swaps. The discs (4 replicates of eachfilm) were carefully placed onto the seeded agar plate (90 mm). Theplates were then incubated at 37 °C for 24 h. Antimicrobial activity ofthe films was evaluated by measuring the diameter of inhibition zone(ZOI, mm) using a digital caliper based on four replicates.

The antibacterial activities of the composite films were also eval-uated using a dynamic shake flask method in reference (Tomé et al.,2015; Yang et al., 2016). The experiments were carried out also using E.coli and B. subtilis. The two bacteria were firstly each cultured overnightat 37 °C in 20 mL of LB subjected to horizontal shaking at 100 rpm. Andthen the inoculated LB was quantified with phosphate buffer to an in-itial bacteria concentration of 1 × 106 CFU/mL for the following test. Acomposite film sample (3.24 cm2) was placed in a sterile glass tubecontaining 5 mL of the prepared LB with 1 × 106 CFU/mL of E. coli orB. subtilis and subjected to vigorous shaking throughout the duration ofthe test to ensure the best contact with the bacteria. Samplings from alltubes/thesis were carried out at 0, 1, 12 and 24 h (after bacterial in-oculation) and serial dilution were carried out and plated on platecount agar. After incubation, the numbers of bacterial colonies werecounted. Test samples of each composite film were prepared for 3 re-plications each at 4 inoculation times.

3. Results and discussion

3.1. Modification of CNF by rosin

The FT-IR spectra of untreated CNF and modified CNF are displayedin Fig. 1. In the spectrum of untreated CNF, typical absorption bands ofcellulose can be observed, including peaks around 3420 cm−1,2883 cm−1, 1423 cm−1，and 1165 cm−1 that correspond to OeHstretching, CeH symmetrical stretching, HeCeH in-plane bending vi-bration, and the CeOeC vibrations bonds on glycosidic bridges, re-spectively. In addition, the intensive peak at 1625 cm−1 was assignedto the HeOeH bending of the absorbed water, indicating that the CNFsample is highly hygroscopic (Chen, Zhu, Baez, Kitin, & Elder, 2016;Tang, Sisler, Grishkewich, & Tam, 2017).

Rosin consists of various resin acids, most commonly including

Fig. 3. TEM images of (a) CNF and (b) R-CNF.

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Abietic acids, Levopimaric acids and Pimaric acids (Scheme S1).Basically, the modification of CNF by rosin is the formation of estergroups between the eOH groups on CNF and the eCOOH groups on theresin acids of rosin (Scheme 1). The peaks presented at 1730 cm−1 onthe spectrum of R-CNF were attributed to the ester carbonyl groups,which confirmed the successful grafting of rosin on CNF. Given the si-milar peak intensities of the eCH stretching (2883 cm−1) of both un-treated CNF and R-CNF, the intensities of the eOH stretching of R-CNFwas significantly weaker than that of untreated CNF, which could alsoindicate that part of the eOH on CNF had been reacted with rosin. Inaddition, no peaks at 1700 cm−1, assigned to the carbonyl on the resinacids of rosin was observed on the spectrum of R-CNF, indicating thatthe residual rosin were completely removed by the washing step afterthe rosin grafting reaction.

The results of elemental analysis are shown in Table 1. Given thevery small amount of N from the elemental analysis, it could be comefrom the impurity or other contaminant on testing instrument or sam-ples. The higher content of carbon in R-CNF (47.92%) compared to thatof neat CNF (42.27%) should probably be due to the grafting of rosinmoiety on CNF. The grafting of rosin might also cause the slight de-crease of the oxygen to carbon ratio of R-CNF compared to unmodifiedCNF (Morelli et al., 2016). The DS of R-CNF was calculated based on theresults of elemental analysis and the value is only 0.05, indicating thatthe introducing of rosin moiety was merely on the surface of CNF. TheXPS results and deconvolution of C 1 s signal were presented in Fig. 2aand Table S1, respectively. The deconvolution carbon signals of CNFdisplayed 3 peaks that are attributed to C1 (CeC, B.E. 285.0 eV), C2(CeO, B.E. 286.6 eV), and C3 (OeCeO or C]O, B.E. 287.8 eV), whilethe carbon signals of R-CNF displayed an additional C4 peak (OeC]O,B.E. 289.2 eV), which confirmed the successful esterification of CNF byrosin (Vuoti, Talja, Johansson, Heikkinen, & Tammelin, 2013). In ad-dition, the slight increase in the C1 peak of R-CNF indicates a higheramount of aliphatic carbon in the R-CNF, which could be attributed tothe presence of rosin. And the C1/C3 ratio increased from 0.80 to 1.08also confirmed the increase in carbon per glucose unit, which should bedue to the graft of rosin on CNF (Seema et al., 2015).

The X-ray diffraction patterns of CNF and R-CNF are presented inFig. 2b. Both CNF and R-CNF presented peaks at 2θ around 14.9° and22.5° that were assigned to amorphous and crystalline cellulose, re-spectively (Park, Baker, Himmel, Parilla, & Johnson, 2010). It con-firmed that the R-CNF remained Cellulose I after rosin grafting (Fan &Huang, 2012). The CI was 59.91 ± 0.11% for the untreated CNF and63.42±0.27% for the R-CNF. The slight increase in CI of R-CNF waslikely due to the partial hydrolyzing of CNF in its amorphous regionunder the acidic condition during the reaction with rosin (Birgit & John,2009).

TEM images of CNF and R-CNF are displayed in Fig. 3. The dia-meters of CNF and R-CNF were evaluated using Image J software. It wasfound that the diameters of CNF were in the range of 10–60 nm whilethe diameters of most R-CNF were less than 10 nm. The smaller dia-meter of R-CNF could be attributed to the partial hydrolysis of the CNFduring the reaction with rosin. This finding was in agreement with theCI results. In addition, it can be seen from the TEM that the CNF had atendency of agglomeration (Fig. 3a) whereas the tendency of agglom-eration was lessened a lot in the TEM image of R-CNF (Fig. 3b). It isknown that the eOH groups on cellulose form strong inter and intra H-bonds that cause the cellulose fibrils to be interconnected as bundles.More interconnected fibril bundles in CNF were disintegrated after thechemical modification. Inter and intra H-bonds were somewhat reducedafter CNF reacted with rosin because some of the eOH converted toester groups with the resin acids (Liu, Yu, & Wu, 2013; Shi, Yang, Kuga,& Matsumoto, 2015). Therefore, less agglomeration and better disper-sion of individual fibrils of R-CNF was obtained.

3.2. Mechanical properties of films

The mechanical properties of the composite films are presented inFig. 4 and Fig. S1. All the composite films reinforced with R-CNF or CNFshowed improved mechanical properties than neat PLA/CHT film (0%R-CNF loading). The tensile strength of R-CNF reinforced films gradu-ally increased as the R-CNF loading increased and reached the max-imum value of 32.3 MPa when the R-CNF loading was 8% (Fig. 4a). Itdecreased when R-CNF loading further increased to 10%. The Young’smodulus and elongation at break of the composite films displayed si-milar trends as that of tensile strength though the loading of R-CNFexhibited a more profound effect than it did on tensile strength. TheYoung’s modulus and elongation at break of the composite film with 8%R-CNF load reached 962.0 MPa and 139.3%, which was 1.498 and1.217 times higher than those of neat PLA/CHT film, respectively. Theincorporation of R-CNF significantly improved the brittleness of the

Fig. 4. (a) Tensile strength (b) Young’s modulus (c) elongation at break of (0 wt%, 2 wt%,5 wt%, 8 wt%, 10 wt%) R-CNF and 8 wt% CNF loads of PLA/CHT nanocomposites.

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PLA matrix and increased the mechanical properties, especially theelongation at break of the composite film due to the high stiffness ofcellulose nanofibers (Eichhorn, 2012). The R-CNF reinforced compositefilms showed similar mechanical properties to acetylated CNF re-inforced PLA composite films reported by Abdulkhani and co-workers,where the tensile strength of 26 ± 3.9 Mpa and elongation at break of14.6 ± 3.5% were obtained with a 5% loading of acetylated CNF(Abdulkhani, Hosseinzadeh, Ashori, Dadashi, & Takzare, 2014).

The maximum improvement in properties of a composite occurswhen there are just enough filler in the matrix to form a continuousstructure. This is known as percolation threshold (Jin & Gerhardt,2014). As shown in Fig. 5a, a R-CNF percolation thresh old of 8% wasobtained, indicating that at this R-CNF loading, each fiber was in con-tact with two or more fibers (Clift et al., 2011). R-CNF loading beyondthe percolation threshold was expected to decrease the mechanicalproperties of the composite films, which could be attributed to the self-aggregation due to high loading of R-CNF and poor dispersion in thePLA matrix.

It is also worth noting that, when at the same filler loading of 8%,the composite films exhibited significantly enhanced mechanicalproperties reinforced with R-CNF than those with CNF, particularly inelongation at break (Fig. 4c). This result implied the R-CNF possessedbetter hydrophobicity than CNF, which entailed a better compatibilityof R-CNF with the PLA matrix than CNF. An optimized cellulose na-nofiber-PLA matrix interface would allow for an adequate stresstransfer from the matrix to the reinforcing nanofiber (Soman, Chacko, &Prasad, 2017). The tensile strength and Young’s modulus of CNF re-inforced composite films were similar to that of neat PLA/CHT film(Fig. 4a, b), while the elongation at break of the composite film de-creased even with the addition of 8% CNF. The reinforced CNF might beable to impart stiffness to the composite structure, but is not able toimpart toughness to the PLA matrix (Dong et al., 2015). In addition, theelongation at break is affected by the dispersion of reinforcing nano-fiber in the matrix, the interaction between the reinforcement and thematrix, and the volume fraction of the reinforcement (Fortunati et al.,2012). The interfacial interactions between the hydrophobic PLA ma-trix and the hydrophilic unmodified CNF were weak. Therefore, the

reduced elongation at break of the composite films with unmodifiedCNF was expected.

3.3. Morphology of the composite films

Fig. 5 shows the fractured surfaces of the composite films in-vestigated by SEM. All films showed typical bilayer structure and noobvious gaps were observed between the PLA layer and the CHT layer.In Fig. 5a, it can be clearly seen that the top PLA layer without anyaddition of CNF displayed a relatively smooth and clean fracturedsurface that was consistent with any brittle material. A rougher frac-tured surface was detected for the composites as the concentration of R-CNF increased (Fig. 5b, c). However, aggregates appeared when theload of R-CNF increased to 10% (Fig. 5c). The interfacial adhesionbetween fibers within the aggregates was expected to be much weakerdue to insufficient binding effect, which contributed to the lower me-chanical properties of the films when the R-CNF loading increased to10%.

By comparison, composite film contained 8% unmodified CNF asthe R-CNF percolation threshold was prepared and subjected to SEMobservation. A better compatibility of R-CNF than unmodified CNF withPLA can be seen from the SEM images. The large particle aggregation ofCNF, defects and voids in the films is evident in Fig. 5d. In addition,large numbers of CNF pulled out of the fractured surface and indicatedpoor dispensability in the PLA matrix due to its incompatibility withhydrophobic polymers.

3.4. Antimicrobial properties of films

The antibacterial activity of the composite films was investigatedusing a disk-diffusion method and a dynamic shake flask method. Theresults are shown in Fig. S2 and Fig. 6, respectively. Overall, in the caseof pure PLA film, no inhibition activity were observed (Fig. S2a, S2f),demonstrating that pure PLA has no ability to inhibit the bacteria intested stains. With the presence of R-CNF in the composites, a smallinhibition effeteness appeared surrounding the specimens (Fig. S2b,S2g). Meanwhile, the ZOI of CNF/PLA/CHT films (8.91/6 mm against

Fig. 5. Scanning electron microscope (SEM) on crosssection micrographs of R-CNF/PLA/CHT compositefilms with (a) 0 wt%, (b) 8 wt%, (c) 10 wt% R-CNFloadings and, (d) CNF/PLA/CHT with 8 wt% CNFloading.

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E. coli, 8.20/6 mm against B. subitilis) were slightly wider than those ofthe R-CNF/PLA films (8.43/6 mm against E. coli, 8.09/6 mm against B.subitilis, Fig. S2c, S2 h). Among all the composite films tested, the R-CNF/PLA/CHT films exhibited the largest ZOI of 10.88/6 and 8.47/6 mm against E. coli and B. subitilis (Fig. 6c), respectively, indicatedmore effective antibacterial activity with the addition of R-CNF in thePLA matrix than the sole activity of CHT.

The results of dynamic shake flask method showed similar trends as

the disk diffusion method (Fig. 6a, b). The thesis with pure PLA has thehighest bacteria concentration along the whole inoculation time, in-dicating no antibacterial activity of PLA. On the other hand, bacterialgrowth was significantly inhibited in the thesis with R-CNF/PLA/CHTfilms. In particular, a significant bacteria reduction was recorded after1 h of inoculation in the thesis with R-CNF/PLA/CHT film(5.81 ± 0.27 log CFU of E.coli and 5.80 ± 0.24 log CFU of B. subitilis).The antibacterial activity of this film continued effectively during 24 hinoculation time compared to other 4 types of composite films.

Resin acids of rosin have a characteristic bulky hydrophenanthrene,which provides resin acids with substantial hydrophobicity (Wanget al., 2012). It has been reported that resin acid-derived cationiccompounds exhibit high antimicrobial activities against a broad spec-trum of bacteria that due to the hydrophobicity and unique structure ofresin acids (Wang et al., 2012). Resin acids may also bind to othercellular components, for example, enzymes, and thereby be lethal to thecell (Soderberg, Gref, Holm, Elmros, & Hallmans, 1990). Although R-CNF/PLA film provided smaller ZOI than those of the CNF/PLA/CHTfilm, it is hard to draw the conclusion that rosin has lower antimicrobialactivity than chitosan giving the relative low DS of rosin modificationand low concentration of R-CNF in the R-CNF/PLA film. In addition,CNF with surface modified by rosin had a homogenous dispersion inPLA and more effective interaction between OeH groups in PLA andNeH groups in CHT, increasing the contact area between microorgan-isms and CHT. This could also lead to enhanced antimicrobial activitiesof the R-CNF/PLA/CHT composite films.

While the R-CNF/PLA/CHT composite films exhibited the effectiveantibacterial activity against both E. coli and B. subitilis, the ZOI of E.coli was apparently wider and the bacterial growth was slower thanthose of B. subitilis. This result was attributed to the better antibacterialactivity of chitosan against the Gram- negative (e.g., E. coli) bacteriumthan the Gram-positive bacteria (e.g., B. subitilis). Previous studiesshowed that the electrostatic forces between the cationic groups on thesurface of chitosan and the anionic groups of Gram-negative bacteriacould result in the damage of cell membranes (Robles, Salaberria,Herrera, Fernandes, & Labidi, 2016; Yu et al., 2013; Yang et al., 2016).Therefore, chitosan and rosin provided a synergistic effect on inhibitingantimicrobial performance against E. coli and B. subtilis.

4. Conclusion

PLA/CHT based composite films reinforced with functionalizedcellulose (R-CNF) for potential food packaging, were successfully de-veloped and characterized. To improve the compatibility of hydrophilicCNF with hydrophobic PLA, natural resource rosin was used to func-tionalize CNF with a green solvent-free reaction route. Rosin modifiedCNF was used as a reinforcing filler for PLA. The R-CNF reinforced filmwas then coated with CHT to prepare a two-layer composite film by LBLmethod. The modification of CNF increased its hydrophilicity and im-proved CNF dispersion in the PLA matrix. The increase in mechanicalproperties was observed for samples with R-CNF reinforcement. Theoptimum mechanical properties of the composite film were achievedwith a percolation threshold of 8% of R-CNF loading. The R-CNF/PLA/CHT composite film exhibited excellent antimicrobial performanceagainst E. coli and B. subtilis attributed to the synergistic antimicrobialeffect of CHT and rosin.

Acknowledgment

The authors are grateful for the financial support by the ForestryIndustry Research Special Funds for Public Welfare Projects[201504602]; the Jiangsu Specially-Appointed Professor program ofthe State Minister of Education of Jiangsu Province; and the project ofthe Priority Academic Program Development of Jiangsu HigherEducation Institutions [PAPD].

Fig. 6. Antibacterial activity of different composite films on the multiplication of (a)E.coli and (b) B. subitilis with initial concentration of 1 × 106 CFU/mL. (c) Average dia-meters of inhibition zones of R-CNF/PLA/CHT, CNF/PLA/CHT, R-CNF/PLA, PLA/CHTand neat PLA films, includes disk diameter of 6 mm.

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Appendix A. Supplementary data

Supplementary data associated with this article can be found, in theonline version, at https://doi.org/10.1016/j.carbpol.2017.11.079.

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