Journal of Food Engineering 109 (2012) 520–524

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Journal of Food Engineering

journal homepage: www.elsevier .com/locate / j foodeng

Development of cellulose-based bactericidal nanocomposites containing silvernanoparticles and their use as active food packaging

Márcia R. de Moura a,b,⇑, Luiz H.C. Mattoso b, Valtencir Zucolotto a

a Nanomedicine and Nanotoxicology Laboratory, IFSC, University of Sao Paulo, P.O. Box 369, São Carlos/SP 13566-970, Brazilb National Nanotechnology Laboratory for Agriculture, EMBRAPA-CNPDIA, São Carlos/SP, Brazil

a r t i c l e i n f o a b s t r a c t

Article history:Received 22 June 2011Received in revised form 24 August 2011Accepted 30 October 2011Available online 7 November 2011

Keywords:Silver nanoparticlesHydroxypropyl methylcelluloseActive filmsPackaging

0260-8774/$ - see front matter � 2011 Elsevier Ltd. Adoi:10.1016/j.jfoodeng.2011.10.030

⇑ Corresponding author at: Nanomedicine and NanUniversity of Sao Paulo, P.O. Box 369, São Carlos/SP 12107 2800; fax: +55 16 2107 2902.

E-mail address: marciadqi@gmail.com (M.R. de M

The use of nanomaterials, including metallic as active fillers in polymeric nanocomposites for food pack-aging has been extensively investigated. Silver nanoparticles (AgNPs), in particular, have been exploitedfor technological applications as bactericidal agents. In this paper, AgNPs were incorporated into ahydroxypropyl methylcellulose (HPMC) matrix for applications as food packaging materials. The averagesizes of the silver nanoparticles were 41 nm and 100 nm, respectively. Mechanical analyses and watervapor barrier properties of the HPMC/AgNPs nanocomposites were analysed. The best results wereobserved for films containing smaller (41 nm) AgNPs. The antibacterial properties of HPMC/AgNPs thinfilms were evaluated based on the diameter of inhibition zone in a disk diffusion test against Escherichiacoli (E. coli) and Staphylococcus aureus (S. aureus). The disk diffusion studies revealed a greater bactericidaleffectiveness for nanocomposites films containing 41 nm Ag nanoparticles.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

Active packaging is an innovative concept in which the package,the product, and the environment interact to prolong the shelf-life,or to enhance safety or sensory properties, while maintaining thequality of the product. This concept, which is of special importancein the area of fresh and extended shelf-life foods (Labuza and Bre-ene, 1989) have recently benefited with the use of nanotechnolog-ical materials including nanocoatings and nanoparticles (Li et al.,2011; Sekhon, 2010). The antimicrobial properties of silver nano-particles (AgNPs), for example, have been increasingly exploitedin consumer products such as deodorants, clothing, bandages, aswell as in cleaning solutions and sprays (Chen and Schluesener,2008; Kumari et al., 2009) as antimicrobial agents (Suppakulet al., 2003). It has been reported that some antimicrobial agentsmay affect the physical properties, processability or machinabilityof the packaging material.

Cellulose-based materials are being widely used as they offeradvantages including edibility, biocompatibility, barrier properties,attractive appearance, non-toxicity, non-polluting and low cost(Imran et al., 2010). Hydroxypropyl methylcellulose (HPMC) is abiopolymer approved by FDA (Food and Drug Administration)and the EU (EC, 1995) for food uses (21 CFR 172.874). HPMC exhib-

ll rights reserved.

otoxicology Laboratory, IFSC,3566-970, Brazil. Tel.: +55 16

oura).

its good film-forming characteristic, as well as the ability to formthermally induced gelatinous coatings, allowing their use as mate-rials to retard oil absorption in deep frying food products (Bala-subramaniam et al., 1997; Kester and Fennema, 1986). Coatingand films are different; the coating is formed on the surface ofthe material that will protect. But the film is formed separately tothe material that will protect. HPMC’s versatility gives him the abil-ity to form both. Moreover, HPMC films are water-soluble, odorlessand tasteless, and present moderate resistance to moisture andoxygen permeability (Krochta and Mulder-Johnston, 1997). There-fore, technological applications of HPMC for packaging usually re-quire to be improved of mechanical and barrier properties.

In formulations of films, different polysaccharide matrices, suchas cellulose derivative materials, have been widely used. An exam-ple is the polysaccharide HPMC, which is a biodegradable andrenewable polymer, as well as an alternative for novel packagingmaterial, as it has been reported by Sánchez-González et al.(2011). In the latter paper, the authors found significant differencesin physicochemical properties, respiration rate and microbialcounts of organic table grapes in two different polysaccharide matri-ces, HPMC and chitosan, with or without bergamot essential oil.

The objective of this study was to investigate the properties ofHPMC and silver nanoparticles films and their possible applicationas active packaging. Different sizes of nanoparticles were investi-gated to optimize the performance of the composites. The effectsof nanoparticles on mechanical properties, water vapor permeabil-ity and antibacterial activity of HPMC films have also beeninvestigated.

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2. Materials and methods

2.1. Materials

Hydroxypropyl methylcellulose (Methocel E15) was obtainedfrom Dow Chemical Co. (Midland, MI, USA.). All reagents, includingAgNO3 and polyvinyl alcohol (PVA) were obtained from Aldrichand used without purification. Escherichia coli and Staphylococcusaureus were obtained from the culture collection of the EmbrapaAgricultural Instrumentation, Brazil. Deionized water was used toprepare all the solutions employed.

2.2. Preparation of silver nanoparticles (AgNPs)

Polyvinyl alcohol (PVA)-coated silver nanoparticles were synthe-sized by reduction of silver nitrate salts with sodium borohydride.The synthesis can be summarized as follows: 45 mM PVA was addedin 20 ml of a 79 mM silver nitrate solution and stirred for 5 min. Fol-lowing, 10 ml of a freshly prepared sodium borohydride solutionwas quickly added to the reactional medium. The solution immedi-ately turned yellow confirming the formation of silver nanoparticles.The solutions were then stored in dark bottles and were found to bestable for more than 2 months. Two different particle sizes withaverage diameter at 41 and 100 nm were obtained by using two dif-ferent stirring speeds during the synthesis (Neto et al., 2008).

2.3. Particle size distribution and zeta potential analyses

Particle size was evaluated using a Zetasizer Nano ZS (MalvernInstruments Inc., USA) which uses laser diffraction to measure bothparticle size distribution and zeta potential. All analyses were per-formed in triplicate.

2.4. AFM measurements

Morphological analyses of nanoparticles were performed di-rectly by Atomic Force Microscopy (AFM) (Dimension V from Veeco).AFM images were acquired in contact mode in random areas of5.0 lm � 5.0 lm using a conventional silicon-nitrite tip with springconstant of 0.06 N/m. Surface scans were analyzed using Gwyddionfree SPM data analysis software21 based on MS-Windows.

2.5. Thin film fabrication

HPMC films were obtained according to the procedure reportedby Moura et al. (2008). Neat HPMC films (control films) were ob-tained by casting from a solution containing 3.0 g of HPMC in100 ml of distilled water kept under magnetic stirring for 12 h.The HPMC:water ratio was kept at 3:97 (w:w) in all film-formingsolutions. The HPMC films containing silver nanoparticles were ob-tained by addition of 3.0 g of HPMC in 100 ml of nanoparticle solu-tion (recently synthesized) under magnetic stirring for 12 h. Aftersolutions preparation, the flasks were kept closed during 4 h toprevent microbubble formation. The solutions were poured intoan acrylic plate (30 � 30 cm) for film formation. Films were ob-tained at a wet thickness of 0.5 mm using casting bars and theplates were placed on a leveled surface at room temperature andlet dry for 25 h. After drying, the films were removed and condi-tioned in sealed plastic bags, stored at room temperature.

2.6. Microorganisms and culture media

S. aureus and E. coli strains were purchased from BacFar – CEFARDIAGNÓSTICA LTDA (S. aureus ATCC 25923, E. coli ATCC 25922). Thestrains from these genera were cultivated in tryptic soy agar (Neo-

gen, Lansing, MI, USA). Incubation was performed at 32 �C. Agarmedium was prepared by addition of 15 g L�1 of bacteriological agar.

2.7. Film characterization

Film thickness was measured using a model 7326, digitalmicrometer (Mitutoyo Manufacturing, Tokyo, Japan) at 5 randompositions on the film. The mean values were used to calculate watervapor permeability and mechanical properties (Rojas-Graü et al.,2007). FT-IR spectra of HPMC films were taken using a Paragon1000 Perkin Elmer Spectrum (Perkin Elmer Life and Analytical Sci-ences, Inc., Waltham, MA, USA) in the range from 4000 to400 cm�1. Powdered sample was prepared using KBr to form pellets.

The mechanical properties of the composites were evaluated inrectangular pieces of the film with dimensions chosen in accor-dance to ASTM (1997) and conditioned at 24 �C for 48 h beforemeasurements. An EMIC INSTRUMENTS was used to determinethe maximum tensile strength (TS), elongation at break (%), andelastic modulus (or Young’s modulus). Films were stretched upusing a speed of 50 mm min�1. Tensile properties were calculatedfrom the plot of stress (tensile force/initial cross-sectional area)versus strain (extension as a fraction of the original length). Themechanical properties were analyzed as a function of particles sizeinsert in the film.

The water vapor permeability (WVP) was determined using amodification of the ASTM E96-92 gravimetric method to determinethe relative humidity (RH) at the film underside, according toMcHugh et al. (1993). Five films were cast from each formulation.After drying, one sample without defects was cut from each film,onto 5.5 cm internal diameter Teflon� plates. Distilled water wasdispensed into flat-bottom acrylic cups with wide rims. The filmwas sealed to the cup base with a ring using 4 screws symmetri-cally located around the cup circumference. The cups were placedin temperature-controlled cabinets at 25 �C, containing fans andheld at 0% RH using anhydrous calcium sulfate (W.A. HammondDrierite Co., Xenia, OH, USA). Weights were taken periodically aftersteady state was achieved and used to calculate the percentage ofRH at the film underside and the WVP.

Water vapor permeability (WVP) was calculated using the fol-lowing relation:

WVP ¼ WVTRp2 � p3

y ð1Þ

where WVTR was obtained from the slope of the weight loss ratethrough the film surface and p2 was the water vapor partial pressureon the film underside calculated according to McHugh et al. (1993). p3

is the water vapor partial pressure at the film underside and y is theaverage film thickness. Water vapor permeability of each film wasmeasured as the mean and standard deviations of 5 replicates.

2.8. Microbiological analysis

To investigate the antibacterial activity of films, 1 cm diameterdisks were cut from different composite bioactive films and placedon inoculated nutrient medium. The method was previously stan-dardized by adjusting the microbial inoculation rate and the vol-ume of the agar medium layer. Dishes were incubated at 37 �Cfor 24 h. Data was expressed as growth inhibitory zone diameter(cm) for three replicates (Kumar et al., 2010).

2.9. Statistical analysis

Analysis of variance (ANOVA) was applied using Minitab 14.2(Minitab Inc., State College, PA, USA) to determine significance ofdifferences between means.

Fig. 2. Representative FTIR transmittance spectra of AgNPs-PVA nanoparticles,HPMC film and HPMC/AgNPs films.

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3. Results and discussion

Nanoparticles obtained have visual characteristics similar toother studies published (Neto et al., 2008). The nanoparticle sizewas estimated by DLS measurements, and the results are shownin Table 1, as a function of stirring speed. The effect of stirringspeed on the AgNps sizes may be explained based on the fact thatan increase in the stirring speed, and consequently, in the agitationof the system, resulting in the formation of smaller nanoparticles.Getting smaller size is simply more dispersion of particles due tothe more stirring. The silver nanoparticles exhibited negative zetapotentials, at ca. �20 mV.

Fig. 1 shows the UV–Vis spectra of silver nanoparticles and theprecursor silver salt solution. The spectra exhibit an absorptionband at around 405 nm, which is a typical plasmon band, suggest-ing the formation of silver nanoparticles (Huang et al., 2004). Theinset of Fig. 1 depicts AFM images of a HPMC/AgNPs nanocompos-ite film. An average surface roughness was evaluated at 33 nm,similar to what had been reported for AgNPs systems (Muniz-Mir-anda et al., 2007). According to Gennadios et al. (1993), controlover film thickness is important to guarantee film uniformity,experiments repeatability, thus validating the comparisons amongthe properties of the edible films. The films reported here exhibitedan average thickness value of 0.04 mm, being practically the samein all analyses.

FT-IR spectra for HPMC, AgNps-PVA, and the composites filmsare shown in Fig. 2. HPMC systems presented a band at2922 cm�1 due to C–H stretching. The broad band located around3469 cm�1 present in all films was from O–H stretching and inter-molecular/intramolecular hydrogen bonds. The C@O carbonylstretching bands from the glucose of the cellulose appear at1645 cm�1 and a C–O–C stretching region at 1300–900 cm�1 is alsoobserved (Zaccaron et al., 2005). The main bands associated to theAgNPs-PVA complex are the following: 3400 cm�1 due to O–H

Table 1Influence of overhead stirrer on the nanoparticle size and zeta potential.

Nanoparticles Particle size (nm) Zeta potential (mV)

Overhead stirrer (500 rpm) 100 �17Overhead stirrer (1000 rpm) 41 �38

Fig. 1. UV–Vis absorption spectra of Ag nanoparticles (a) and silver salt (b) used forAgNPs formation. Inset: AFM micrographs of Ag colloidal nanoparticles in HPMCfilms.

stretching; 2900 cm�1 due to C–H stretching; 1650 cm�1 due tocarbonyl stretching bands; 1384 cm�1 was from C–H vibrationand 840 cm�1 due to C–H vibration. All the bands observed inAgNPs-PVA were from of PVA present in nanoparticle structure.According to the spectra, it was not possible to infer any specificinteraction occurring between the HPMC matrix and the nanopar-ticles. As it may be observed, the spectrum from HPMC/AgNPs rep-resents the sum of the constituents.

To be useful, edible films shall maintain integrity during pro-cessing, shipping, and handling. Tensile strength, elastic modulus,and elongation analyses describe how the mechanical propertiesof such film materials relate to their chemical structures (Ninne-mann, 1968). Fig. 3 shows the tensile strength of the HPMC/AgNPsnanocomposites. The incorporation of Ag nanoparticles increasedthe mechanical resistance, which is due to the partial replacementof the polymer by nanoparticles in the film matrix. The nanocom-posites presented high stress values when smaller nanoparticlesare included in HPMC film. The nanocomposites containing larger

15

20

25

30

35

40

45

50

55

60

c

b

a

NanoAg 41 nm

Tens

ile S

treng

th (M

Pa)

Differents NanoAg sizes in HPMC films

NanoAg 100 nmonly HPMC

Fig. 3. Tensile strength for neat HPMC films, and HPMC films containing AgNPswith different particle sizes. Columns show the means and error bars indicate thestandard deviations. Different letters within a column indicated significant differ-ence at P < 0.05.

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(100 nm) nanoparticles exhibited lower mechanical resistance, incomparison to those containing smaller nanoparticles. After incor-poration of 41 nm AgNPs in the films, a significant increase in thetensile strength values was noticed, from 28.3 ± 1.0 (HPMC with-out nanoparticles) to 51.0 ± 0.9 MPa. For films containing 100 nmAgNPs, the tensile strength increased from 28.3 ± 1.0 to38.5 ± 2.0 MPa. Two-way ANOVA confirmed that the addition ofnanoparticles into HPMC films increased TS of the films. The levelof significant difference between the tensile strength of the silvernanoparticles indicated significant difference at P < 0.05.

Such reinforcement effect has been described by Chang et al.(2010), who observed that chitosan nanoparticles incorporated instarch matrix had an obvious enforcement effect. Upon increasingchitosan nanoparticles content, the tensile strength of the compos-ites increased. For chitosan nanoparticles contents varying from 0to 6 wt.%, the tensile strength increased from 2.84 to 10.80 MPa.Kim et al. (2008) modified carbon nanotubes (CNTs) by introducingcarboxylic acid groups on their surfaces in order to enhance theirintermolecular interactions with the poly(ethylene-2,6-naphta-lene) (PEN) matrix. In this case, the presence of CNTs even at con-centrations as low as 0.1 wt.%, greatly improved tensile strength.

The percentage elongations and elastic modulus did not presentsignificant variation when different sizes of nanoparticles wereemployed, as shown in Table 2. On the other hand, addition ofthe nanoparticles decreased the percentage elongation of films.

One of the most important properties of an edible film is thewater vapor permeability. Most edible films, apart from waxesand modified natural polymers, are characterized by a high watervapor permeation, which makes them inappropriate for severalapplications (Torres, 1994). True permeability consists of a solu-tion/diffusion process in which the vapor penetrates in one sideof the film, then diffusing to the other side. Polysaccharide filmsare generally rather poor water barriers, due to their hydrophilicnature. The effect of addition of silver nanoparticles on WVP isshown in Table 3 for HPMC films. The relative humidity at the filmunderside was not significant different (71.2 RH) for the differentfilms, as indicated in Table 3. Upon addition of nanoparticles inHPMC matrix, a decrease in the WVP values was observed. Nano-particles with smaller sizes induced a decrease in WVP values.The WVP values varied from 0.480 ± 0.05 g mm K�1 Pa�1 h�1 m�2

for HPMC films containing 41 nm AgNPs to 0.556 ±0.02 g mm K�1 Pa�1 h�1 m�2 for films containing 100 nm nanopar-ticles. The presence of nanoparticles reduced the intermolecularspacing within the films, thus reducing the WVP through of film.This fact occurs because small size nanoparticles have more ability

Table 2Effect of AgNPs size on elastic modulus and elongation parameters of HPMC films.

Film type Elastic modulus (MPa) Elongation (%)

Only HPMC 900 ± 15a 8.1 ± 0.7a

Nanocomposite (41 nm) 1020 ± 10a 7.9 ± 0.2a

Nanocomposite (100 nm) 989 ± 22a 4.8 ± 0.1b

a,b,c Different letters within a column indicated significant difference at P < 0.05.

Table 3Water vapor permeability of hydroxypropyl methylcellulose (HPMC) film withdifferent AgNPs sizes.

Particle size (nm) WVP (g mm K�1 Pa�1 h�1 m�2) RH at filmunderside (%)

(No nanoparticle) 0.800 ± 0.04a 71.9 ± 0.7a

100 0.556 ± 0.02b 70.5 ± 0.3a

41 0.480 ± 0.05c 71.1 ± 0.5a

a,b,c Different letters within a column indicated significant difference at P < 0.05.

in occupying the empty spaces of the porous HPMC film matrix,making difficult the diffusion of water into the film. Two-way AN-OVA showed that the presence of nanoparticles in the films de-creased the WVP of the HPMC films. Important information isthat a decrease on the concentration of nanoparticles, in HPMCfilms decreased the WVP values.

Epidemiological studies have demonstrated that the number offood-related diseases caused by pathogenic microorganisms has

Fig. 4. The antibacterial activity of nanocomposite with AgNPS revealed by discdiffusion method: (a) control; (b) E. coli essay with nanocomposite disc containing41 nm AgNPs; and (c) S. aureus essay with nanocomposite disc containing 41 nmAgNPs.

Table 4Inhibition zone of nanocomposites active films.

Bacterial strains HPMC + silver nanoparticles(100 nm)

HPMC + silvernanoparticles (41 nm)

E. coli ATCC25923

1.05 ± 0.10a 2.75 ± 0.20a

S. aureus ATCC25922

1.35 ± 0.30b 3.11 ± 0.10b

a,b Different letters within a column indicated significant difference at P < 0.05.

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increased in recent years. As a form to introduce active agents toincrease food preservation, the area of antimicrobial packaginghas become one of the major areas of research in food packaging(Coma, 2008; Friedman et al., 2002).

The antibacterial activity of HPMC films containing 100 nmAgNPs was determined by disc diffusion method for E. coli and S.aureus (Fig. 4). The largest inhibition halos were observed for theGram positive bacterium (S. aureus), while the smallest ones wereobserved for the Gram-negative bacterium (E. coli). Films contain-ing 41 nm AgNPs also exhibited antibacterial activity, as revealedby the formation of larger inhibition halos (Table 4). Smaller parti-cles can be better dispersed into the nanocomposites surface, thusfavoring their interactions with the culture medium.

4. Conclusions

HPMC/AgNPs nanocomposites films exhibiting good mechanicaland barrier properties were successfully obtained. The presence ofAgNPs in the HPMC matrix promoted an increase in the tensilestrength of the films, from 28.3 ± 1.0 (neat HPMC) to51.0 ± 0.9 MPa (HPMC/41 nm AgNPs). The decrease observed inthe WVP values for the HPMC/AgNPs system, in comparison to sys-tems containing chitosan nanoparticles is promising, as a means toimprove final product quality and shelf stability. We also demon-strated the bactericidal potential of the HPMC/AgNPs films againstsome bacteria, in which nanoparticle size plays an important role.The latter is indicative that the HPMC/AgNPs nanocomposites canbe used in food packaging as active antimicrobial internal coatings.

Acknowledgments

The authors would like to acknowledge FINEP/MCT, FAPESP,CNPq and EMBRAPA.

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