Accepted Manuscript

Antibacterial properties of LDPE nanocomposite films in packaging of UF cheese

Faranak Beigmohammadi, Seyed Hadi Peighambardoust, Javad Hesari, Sodeif

Azadmard-Damirchi, Seyed Jamaleddin Peighambardoust, Nader Karimian
Khosrowshahi

PII: S0023-6438(15)30076-1
DOI: 10.1016/j.lwt.2015.07.059
Reference: YFSTL 4853

To appear in: LWT - Food Science and Technology

Received Date: 6 April 2015

Revised Date: 15 July 2015
Accepted Date: 21 July 2015

Please cite this article as: Beigmohammadi, F., Peighambardoust, S.H., Hesari, J., Azadmard-Damirchi,
S., Peighambardoust, S.J., Khosrowshahi, N.K., Antibacterial properties of LDPE nanocomposite films in
packaging of UF cheese, LWT - Food Science and Technology (2015), doi: 10.1016/j.lwt.2015.07.059.

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 ACCEPTED MANUSCRIPT
1 Antibacterial properties of LDPE nanocomposite films in packaging of UF cheese
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3 Faranak Beigmohammadi a, Seyed Hadi Peighambardoust a*, Javad Hesari a, Sodeif

4 Azadmard-Damirchi a, Seyed Jamaleddin Peighambardoust, b Nader Karimian Khosrowshahic

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a
6 Department of Food Science, College of Agriculture, University of Tabriz, Tabriz 5166616471, Iran
b
7 Faculty of Chemical and Petroleum Engineering, University of Tabriz, Tabriz 5166616471, Iran

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c
8 Deputy of Food and Drug, Tabriz University of Medical Sciences, Tabriz 5197617151, Iran

9 * peighambardoust@tabrizu.ac.ir , Tel: +989141003110

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10

11

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12 Abstract
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13 The purpose of this study was to investigate antibacterial potential of low-density

14 polyethylene (LDPE) packaging films incorporating silver (Ag), copper oxide (CuO) and zinc
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15 oxide (ZnO) nanoparticles in measuring of coliform amounts of ultra-filtrated (UF) cheese.

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16 The initial LDPE/nanoparticle composites were produced by melting extrusion followed by

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17 extruding the obtained composites through a die to achieve a film thickness of 45±5 µm. The

18 number of surviving coliform bacteria was decreased by 4.21 log cfu/g after 4 weeks of
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19 storage at 4 ± 0.5 °C, whilst pure LDPE films showed a reduction of only 1.04 log cfu/g. A

20 composition of 0% Ag, 1% CuO, 0 % ZnO gave an optimum point in combined design using
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21 Design Expert analysis. A suitable microbial model was suggested for retarding coliform
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22 bacteria growth in UF cheese. The difference between the optimum point of nanocomposite

23 film and its repeat was not significant (p<0.05) by one-way ANOVA analysis using SPSS

24 software, while the difference was significant for pure film. Migration of metallic

25 nanoparticles into a food stimulant was within the accepted safe level.

26 Keywords: active packaging; antibacterial; LDPE film; nanoparticle; UF cheese

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27 1. Introduction

28 Nowadays, packaging plays an increasingly important role in the whole food chain “from

29 the field to table". In recent years, active packaging systems for food have gained much

30 attention mainly due to increased demands on product safety, shelf life extension, cost

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31 efficiency, environmental issues, and consumer convenience (Ahvenainan, 2003). Polymer

32 nanocomposites are mixtures of polymers with inorganic or organic fillers with certain

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33 geometry. The use of fillers, which have at least one dimension in the nanometric range

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34 (nanoparticle) produces polymer nanocomposite (Alexandre & Dubois, 2000). Besides

35 reinforcing nanoparticles, whose main role is to improve mechanical and barrier

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36 characteristics of packaging materials, there are several kind of nanostructures responsible for
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37 other function, providing antimicrobial activity (Azerdo, 2009). One of the best ways to

38 reduce, inhibit, or retard the growth of microorganisms in packaged food or packaging

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39 material is the application of antimicrobial food packaging systems (Appendini & Hotchkiss,

40 2002). Antimicrobial packaging is a form of active packaging. This technology consists of

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41 interaction between the food, headspace, and the package to achieve any desired result
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42 (Brody et al., 2001 and Soares et al., 2009).

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43 The most common nanocomposites used as antimicrobial films for food packaging are

44 based on silver, which is well known for its strong toxicity to a wide range of
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45 microorganisms with high temperature stability and low volatility (Soares et al., 2004; Kumar
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46 & Munstedt, 2005; Gammarillo et al., 2011). An active film with nanosilver can be

47 successfully used for inhibiting or reducing the growth of Alicyclobacillus acidoterrestris, a

48 thermal resistant food spoilage microorganism, in acidic beverage (Del Nobile et al., 2004).

49 In a study, using packaging based on LDPE containing metallic nanoparticles led to a

50 significant reduction in microbial population in orange juice (Emamifar et al., 2011). Other

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51 studies also revealed that LDPE-silver nanocomposite films were effective against E. coli,

52 Staphylococcus aureus, and Candida albicans (Jokar et al., 2012). In the same way, copper

53 has been of particular interest because, unlike other antimicrobial metals, it presents a broad

54 spectrum of action against bacteria and molds. The efficacy of copper depends on

55 environmental conditions, the concentration of copper ions, and the type of microorganism

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56 (Cioffi et al., 2005). Copper has the ability to reduce the growth rate of E. coli by more than

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57 99.99% causing damage to cell walls and altering bacterial cell contents. A critical factor

58 responsible for the antimicrobial properties of copper is the ability to easily accept or donate

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59 electrons so that it has a high level of catalytic oxidation and a high reduction potential (Nan

60 et al., 2008). When copper is in the oxidation state Cu+2, it is highly effective against

61
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microbial cells due to the interaction with nucleic acids, enzyme active site and components
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62 of the cell membrane that cause the death of microbial cells (Lejon et al., 2010). Moreover,
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63 ZnO is an inorganic compound currently listed as a GRAS by FDA (Espita et al., 2012).

64 Packaging materials containing ZnO nanoparticles provide antimicrobial activity and

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65 improved barrier properties (Soares et al., 2009). Such systems provide favorable activity
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66 against both E. coli and Staphylococcus aureus (Li et al., 2010). The antibacterial activity of

67 ZnO considered being in one hand, due to the positive electricity of ion that penetrate into
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68 cell membrane, and on the other hand, the presence of hydrogen peroxide (H2O2) released
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69 from ZnO surfaces (Li et al., 2010).

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70 Nowadays, consumers are more concerned about food safety as well as food quality. The

71 high their attention to the food safety aspects cause to increased research interest in active

72 agent derived from metal source. Therefore, it is very important to assay the migration of

73 nanoparticles into the food or food stimulant. It is technically difficult to measure the

74 migration of a given active agent into the food, however, because most foodstuffs are

75 comprised of a complex mixture of substances such as water, carbohydrates, fats, lipids,

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76 proteins, vitamins, fibers, and minerals (Echegoyen & Nerin, 2013). For this reason,

77 migration studies are usually performed using food stimulants (Dopico et al., 2003). In

78 current European food packaging laws various food stimulants that can used for migration

79 testing identified. These include water (stimulant A), 3% (v/v) acetic acid in water (stimulant

80 B), 15% (v/v) ethanol in water (stimulant C), olive oil, sunflower oil, and synthetic fat

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81 stimulant HB 307 (stimulant D) where each stimulant is representative of a particular type of

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82 food (European Standard EN 1186, 1999).

83 UF cheese is most commonly consumed type of cheese in Iran. Manufacturers use salting

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84 on surface of the product to extend its microbial shelf life. However, nowadays consumers

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85 are aware of negative effects of salt in their daily diet. There is a trend to use active
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86 packaging to provide a safer way for preservation of food quality with reduced additives and

87 preservatives. Numerous antibacterial compounds have been evaluated to reduce the

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88 microbial count of cheese (Altieri et al., 2005; Gumiero et al., 2013). However, to our

89 knowledge, there is no report on investigating antibacterial properties of metallic active

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90 nanocomposite packaging films in packaging of UF cheese. Therefore, the aim of this work is
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91 to diminish of coliform counting of UF cheese by using nanocomposite films using Design

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92 Expert models for optimum point of nanocomposite film and present a microbial model to

93 consider the concentration of each nanoparticle on coliform growth. Finally, optimum point
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94 will be validated, too.

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95 2. Materials and Methods

96 2.1. Materials

97 Film grade LDPE resin pellets (LH0075, MFI 0.75 gr/10 min, density 0.921 gr/ml,

98 softening point 94°C) was obtained from Bandar-Imam Petrochemical Co. (Bandar-Imam,

99 Iran) and used for preparation of polymer matrix. Ag nanoparticles with an average size of

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100 ca. 35 nm and purity of ca. 99.5%, ZnO nanoparticles with an average particle size of 20-30

101 nm (TECNAN co., Spain) and purity of ca. 99.9%, and CuO nanoparticles with an average

102 size of ca. 50 nm and purity of ca. 99% were obtained from Nutrino Co. (Tehran, Iran). The

103 white mineral oil-heavy (polyolefin of C17-C30, highly purity, food grade, Unicorn

104 Petroleum Industry, Mumbai, India) was kindly obtained from Tabriz Petrochemical

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105 Complex (Tabriz, Iran).

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106 2.1.1. Final film production

107 Similar to procedure used by Emamifar et al. (2010) and (2011), and Panea et al. (2013) a

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108 combination of 0.1% (w/w) mineral oil,1 % metal nanoparticles and about 99 % LDPE

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109 pellets was introduced to a co-rotating twin screw extruder (SMPLATEK, TEK 25 Model,
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110 South Korea) with a screw diameter of 20 mm. The heating profile at different zones of the

111 twin-screw extruder was 125, 145, 155, 170, 185, 195, 95, and 200°C. The rotation speed of
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112 feeder and extruder were 20.7 and 140 rpm, respectively. The pressure of extruder was 12.5

113 bars. LDPE film producer based on experience and propose of producer of equipment
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114 because of optimum mechanical and thermal properties chose this condition. Heating profile
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115 for this nanocomposite is similar to pure LDPE with little different because of filler

(nanoparticles), in addition rotation speed of feeder and extruder depends on debit (m3/s) and
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117 retention time, respectively. After insurance of a clean path with LDPE pure granules, LDPE
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118 and metallic nanoparticles were fed into the extruder by feed hopper. Passing through the
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119 extruder, the mixture of the material melted and as a result of shear and pressure forces they

120 were thoroughly mixed together. The molten material left out in string form passing through

121 the basin of cold water followed by forming in granule shape. The prepared granules were

122 then added into another twin-screw extruder (Castiny Ghioldys, Italy) to produce the final

123 nanocomposite films with a thickness of 45±5 µm. The temperature profile of extrusion

124 process was maintained at 239, 239, 223, 223, 218, 215 and 185°C. The obtained films were

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125 chilled to ambient temperature. The films were cut in appropriate sizes for subsequent

126 application. The concentration of different nanometals in production of films is shown in

127 Table 3.

128 2.1.2. UF cheese preparation and packaging

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129 The UF cheese was produced in Manizan Dairy Co., Kermanshah, Iran. The whole milk

130 was pasteurized at 72˚C for 15 seconds, concentrated by ultrafiltration system (APV sw: koch

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131 HFK 131, Mesh: 0.01µm, Denmark) to about 20% original weight at a temperature of 50˚C

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132 and a pressure of 4 bars. The retentate was homogenized at 50 bars at a temperature of 50-

133 55˚C followed by pasteurization at 78˚C for 15 seconds. Retentate inoculated with DVS1

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134 starter in the process tank, mesophilic culture of Streptococcus lactis subsp. lactis and
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135 Streptococcus lactis subsp. cremoris (Mesophile Homofermentative Culture R-708, Item No.

136 100098 50 U, 50 unit/500kg, Christian Hansen Co.) at a temperature of 30˚C to ferment

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137 completely to obtain a pH of 4.8-5. Salt (3% w/w) and the rennet (1gr/100 kg) was added into

138 base UF cheese. This procedure for manufacturing of UF cheese obtained by experience in
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139 Manizan Dairy Co., Kermanshah, Iran and there is similar to Miocinovic et al. (2011). To
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140 prevent contamination in production line, adding of pure coliform to cheese base for
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141 investigating antimicrobial effect of the films (primary microbial load was about 107 cfu/g)

142 was done in laboratory. Different LDPE/nanocomposite films (with a dimension of 100×120
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143 mm2) were used to package 100 g cheese sample. After packaging, samples were stored at
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144 30˚C for 5 h for aging followed by refrigerating to 4±0.5 ˚C. The chemical properties of UF

145 cheese before inoculating by coliforms was measured.

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-Direct Vat Set: DVS culture need no activation or other treatment prior to use, and inoculate in milk
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146 2.2. Antimicrobial activity

147 50 grams of UF cheese samples that packaged in the different nanocomposites was

148 dispersed with 450 ml of a sterile Ringer (Merck No. 1.15525.0001, tp: 1035525, Germany)

149 in a stomacher bag and mixed for 1 min with stomacher. Decimal dilutions of cheese

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150 homogenates were performed in the same solution for determination of total coliforms using

151 pour plating in violet red bile agar (Merck no. 1.01406.0500, batch vm 629306, Germany),

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152 with a covering layer of the same medium, incubated at 30˚C for 24 hours (ISIRI 2406).

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153 2.3. Migration test

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154 Migration of CuO nanoparticles from LDPE matrix was assessed using the stimulant B
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155 at 40˚C for 10 days (Dopico et al., 2003). Because of limit fund, migration test was did only

156 on optimum point, nanocomposite film with 1 % CuO. The nanocomposite films with a size
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157 of 100×120 mm2 filled with 200 ml of stimulant solution and the migration results normalized

158 to metal migrated/cm2. Absorbance measurements of copper elements were done by electro
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159 thermal atomic absorption spectrometry and auto sampler system was used for making
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160 standard solutions of calibration graph from a stock solution with a proper concentration.

161 Standard solutions of metal ions were prepared by diluting of stock standard solution (Merck
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162 Darmstadt, Germany) with acetic acid (3%, Merck, Darmstadt, Germany) as solvent. Three
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163 repetitions were done at each measurement and the average of them was used for following
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164 studies. An Analytikjena atomic absorption spectrometer model Nova 400(Jena, Germany)

165 was used for determination ions. The conditions of equipment at measurement of Cu were

166 brought in table1. A.O.A.C 974.27, 2002b was used for doing the test.

167 2.4. Statistical Analyses

168 The optimization point of nanometal concentrations for active packaging films in

169 microbial properties of UF cheese was analyzed using Design-Expert 7.1.5 software by

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170 applying combined design, D-Optimal procedure. Mean differences between optimum point

171 and control sample and repeat of optimum point for validation were determined using one-

172 way analysis of variance (ANOVA), Tukey HSD and Sheffe tests; significance was set at

173 p<0.05 performed using SPSS 17.0 (SPSS, Inc.).

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174 3. Results and Discussion

175 Chemical characteristics of UF cheese measured before packaging and shown in Table 2.

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176 3.1. Antimicrobial evaluation of nanocomposite films

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177 As can be seen in table 3 all active films with metal nanoparticles showed a declined trend

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178 for microbial load during the storage. At the beginning of the test, the surviving number of
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179 coliforms was 1.31×106 cfu/g. The cell load of bacteria decreased to 8×101 - 2.5×102 cfu/g in

180 different active films for a total storage period of 4 weeks, while the cell load of pure LDPE
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181 film only decreased to 1.2×105 cfu/g. According to Japanese Industrial standard JIS Z 2801 :
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182 2000, from which ISO 22196 : 2007 drives, an antimicrobial activity of R>2.0 log cfu/cm2 is
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183 required for nano food packaging to demonstrate antimicrobial efficacy, as R is the difference

184 in bacteria concentration (expressed in log cfu/cm2) between the non-treated and treated test
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185 specimens (Panea et al., 2013). R was calculated for all runs in table 3. Therefore, all active

186 films had antimicrobial effect at the end of storage time; however, run 5 with the equal ratio
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187 of metal nanoparticles showed the highest and nanocomposite film with composition 0.5%
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188 Ag, 0.5 % CuO, 0% ZnO nanoparticles showed the lowest antibacterial effect during 4 weeks

189 of storage. Most studies dealing with the antimicrobial nanocomposite films have focused on

190 a single metal nanoparticle or at least two nanoparticles (Cioffi et al., 2005, Li et al., 2010,

191 Zapata et al., 2011, Emamifar, et al., 2011, Bodaghi, et al., 2013); whereas in this study three

192 kinds of metal nanoparticle were used and reduction or removing of Ag nanoparticle was

193 important for researchers, if gains. Reduction of coliform growth in this study was

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194 comparable or even more than results obtained by other researchers on different cheese and

195 active packages (Gumiero et al. 2013, Panfil-Kuncewicz et al. 2006, Sinigaglia et al., 2008).

196 In response analysis, the suggested model is linear. The chosen model had a significant fit

197 (p<0.0001), a non-significant lack of fit (p=0.27), and adequate precision1 more than 4

(22.558) and explained a high proportion of the total variance, R2= 0.9329 (Table 4). In

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198

199 addition, interaction of each nanoparticle with time was significant, too. These agents are the

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200 prerequisite for choosing a suitable model. Therefore, linear model (Eq. 1) is the only model

201 for this design. However, other model such as quadratic or cubic model are significant too,

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202 but interaction of nanoparticles with time is not significant.

203
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Eq. 1 gives a description of the influence of investigated factors and the effect of binary
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204 interaction between these nanoparticles with time on coliform load. The effect of each

205 individual nanoparticle on decreasing coliform load is in the following order: CuO>
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206 ZnO>Ag. However Kubacka et al., (2011) optimized antimicrobial properties of metal Ag,
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207 CuO, Zn on polymer-TiO2 film and resulted the disinfection capability improvement is
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208 observed for Ag and Zn promoted film materials but not for the Cu-containing composite.

209 They concluded it could be related to better charge separation and transfer from the excited
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210 TiO2 to the organic component upon illumination. It is clear that Ag nanoparticle in

211 comparison other metal nanoparticles such as CuO nanoparticle has more antimicrobial
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212 effect, but the aim of this research is to introduce other metal nanoparticle with the same or
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213 close antimicrobial effect of Ag. Our result is in line with our objective for removing Ag

214 nanoparticle because of its toxicity and higher price, and using CuO nanoparticle, instead. In

215 binary mixture of nanoparticle with time, negative coefficients represented a decreased in

216 coliform load and ZnO nanoparticle had the most important role. However, individual effects

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. Adequate precision is a measure of the rang in predicted response relative to its associated error, in other
word a signal to noise ratio. It desired value is 4 or more.

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217 of nanoparticles is important for two reasons. First, they have larger coefficient on the

218 inhibitory effect of coliform; secondly, a rapid effect of nanoparticle is preferred, since

219 prolonged storage can affect sensory properties of the cheese.

220 Eq.1 Log10 (coliform cfu/g) = + 6.08×Ag-NP1

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221 + 5.10×CuO-NP

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222 + 5.84×ZnO-NP

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223 - 0.93×Ag-NP×Time

224 - 0.80×CuO-NP×Time

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225 - 1.00×ZnO-NP×Time

226 As contour plots of coliform load (fig. 1) shows, with decreasing Ag and increasing CuO
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227 concentrations, microbial load is falling from 5.925log at the first week to 1.992 log at the
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228 last week. There was decreasing trend at the second and third weeks, too (data was not
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229 shown). Our data show that inactivation rates with nanocomposite films during 4 weeks is

230 higher than those obtained in previous reports.

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231 Finally, Design-Expert software proposed run 11 (0% Ag nanoparticle, 1% CuO

232 nanoparticle, 0% ZnO nanoparticle) as optimum point. At optimum point, microbial load of
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233 coliforms was reduced from 6.11 log cfu/gr to 1.9 log cfu/g. Based on the this statistical
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234 design variables for optimization are: Ag, CuO and ZnO nanoparticles percent, time, Log10

235 (coliform) cfu/g and POE2 (Log10(coliform) cfu/g, that they have the same weight and

236 importance, but we can change their importance. Therefore, we define the optimum variables

237 as following:

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- NP: nanoparticles
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- Propagation of Error

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238 Ag%: minimize, CuO%: in rang, ZnO%: in rang, Time: equal to 4 weeks, Log10 (coliform)

239 cfu/g: minimize, POE (Log10 (coliform) cfu/g: minimize. Run 5 has the most antimicrobial

240 effect, while we introduce run 11 as optimum point. Because we used POE, that it reduces

241 error. On the other hand, with considering POE and lower error, run 11 is optimum, while run

242 5 has higher POE. Furthermore, we need a nanocomposite film with low percentage of Ag

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243 nanoparticles for safety and cost.

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244 Desirability equal 1 is the best situation for the combination of these nanoparticles in

245 LDPE polymer. In addition, one of the capabilities of Design-Expert software is that it can

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246 show the results in the form of Ramp chart (Fig. 2).

247
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Validation of optimum point was shown in Table 5. To validate this point, UF cheese
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248 packed in optimum nanocomposite film and tested for total coliform during 4 weeks, again.

249 The difference between optimum point and its repeat should not be significant. In addition,
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250 control run (pure LDPE) was compared with optimum point. To have antimicrobial effect of
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251 nanocomposite film, the difference of optimum point and control run should be significant.
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252 There is no significant difference between optimum point (sample No.1) and the replication

253 (sample No.2) in Tukey HSD and Sheffe tests, while the above samples have the significant
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254 differences with pure LDPE (sample No.3). All of these results related to the last day of UF

255 cheese storage.

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256 Antibacterial films containing a combination of Ag+CuO+ZnO nanoparticles did not show

257 synergistic effect when assessed in cheese, suggesting a stronger interaction between CuO

258 nanoparticle and polymer and therefore smaller diffusion into the cheese.

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259 3.2. Migration assay

260 Panea et al. (2013) reported the migration of Ag and ZnO nanoparticles using in the

261 packaging with the highest concentration into food simulating solution under incubation

262 conditions similar to those used in present work. Migration test was performed only on the

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263 optimum point. The CuO nanoparticle levels in the food stimulant comply with the EFSA1

264 legislation, which allows a maximum of 10 mg/kg (Llorens et al., 2012). The amount of CuO

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265 nanoparticle released from the nanocomposite film used in this study was 0.23 ±0.005 mg/Kg

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266 which was well below the accepted values.

267 4. Conclusions

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268 The developed active packaging proposed in this work include 1% w/w CuO nanoparticle in

269 LDPE polymer produced by melt mixing for packaging UF cheese, could be advantageously
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270 used to diminish coliform load of the cheese with no toxicity. Concluding, the application of

271 antimicrobial films in food packaging systems could diminish the harshness of food
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272 processing, the amount of additives and chemical preservatives in food industries. In
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273 addition, D-optimal, combined design in Design Expert software is a good method in

274 designing the experiment, modeling and optimizing the composition of metal nanoparticles in
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275 nanocomposite film LDPE base.

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276 Acknowledgments
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277 This research was a part of PhD Thesis accomplished in University of Tabriz, Tabriz, Iran.

278 We thank Manizan Dairy Co. (Kermanshah, Iran) for supplying cheese samples and

279 laboratory equipments.

280 References

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- European Food Safety Authority

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331 milk and milk products-Specification. 2nd. Revision.

332 Jokar, M., Rahman, R., A., Ibrahim, N., A., Abdullah, L., C. & Tan, C., P. (2012) Melt
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333 production and antimicrobial efficiency of low-density polyethylene (LDPE) - silver

334 nanocomposite film. Food and Bioprocess Technology, 5, 719-728.
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335 Kubacka, A., Feror, M., Fernandez-Garcia, M., Serrano, C., Cerrada, M. L., Fernandez-
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336 Garcia, M. (2011). Tailoring polymer-TiO2 film properties by presence of metal (Ag, CuO,
337 Zn) species: optimization of antimicrobial properties. Applied Catalysis B: Environmental,
338 104, 346-352.

339 Kumar, R. & Munstedt, H. (2005). Silver ion release from antimicrobial polyamide silver
340 composites. Biomaterial, 26, 2081-2088.

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341 Lejon, D., P., H., Pascault, N. & Ranjard, L. (2010). Differential copper impact on density,
342 diversity, and resistance of adapted culturable, bacterial populations according to soil organic
343 status. European Journal of soil Biology, 46(2), 168-174.

344 Li, Sh., Li, B., & Qin, Z. (2010). The effect of nano-ZnO concentration on the mechanical
345 antibacterial and melt rheological properties of LLDPE/modified nano-ZnO composite films.
346 Polymer-Plastics Technology and Engineering, 49, 1334-1338.

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347 Llorens, A., Lloret, E., Picouet, P. A., Trbojevich, R., Fernandez, A. (2012). Metallic-based
348 micro and nanocomposites in food contact material and active food packaging. Trends in

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349 Food Science and Technology, 24, 19-29.
350 Miocinovic, J., Puda, P., Radulovic, Z., Pavlovic, V., Miloradovic, Z., Radovanovic, M.,

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351 Paunoviv, D. (2011). Development of low fat UF cheese technology. Mljekarstro, 61(1), 33-
352 44.

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353 Nan, L., Yang, W., C., Liu, Y., Q., Xu, H., Li, Y., Lu, M., Q. & Yang, K. (2008).
354 Antibacterial mechanism of copper- bearing antibacterial stainless steel against E. coli.
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355 Journal of Material Science and Technology, 24(2), 197-201.
356 Panea, B., Ripoll, G., Gonzalez, J., Fernandez-Cuello, A. & Alberti, P. (2013). Effect of
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357 nanocomposite packaging containing different proportion of ZnO and Ag on chicken breast
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359 Panfil-Kuncewicz, H., Stanieewski, B., Szpendowski, J. & Nowak, H. (2006). Application of
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360 active packaging to improve the shelf life of fresh white cheeses. Polish Journal of Food and
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362 Sinigaglia, M., Bevilacqua, A., Corbo, M., R., Pati, S. & Del Nobile, M., A. (2008). Use of
363 active compounds for prolonging the shelf life of mozzarella cheese. International Dairy
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366 C., Sondi, I. & Salopek-Sondi, B. (2004). Silver nanoparticles as antimicrobial agent: a case
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374 Figure captions

375 Fig. 1. Contour plots of coliform load in cheese packed with nanocomposite films at the first
376 (a) and the end of storage (4th weeks) (b) using combined design. Red spots on the
377 contour plots implies log10 cfu/g of coliform in nanocomposite films.

378 Fig. 2. The Ramp chart for optimum point obtained using combined design

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379 Table 1. The condition of atomic absorption spectrometer at measurement of Cu
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Main Slit HCl Pyrolys.temp Atomization.temp. Detection Sensibility reproducibility
line(nm) with current °C °C limit(µg/L) (µg/L)
(nm) (mA)

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324.8 0.8 3 1200 1900 0.13 0.23 2.6-4.3%
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382 Table 2. Composition of UF cheese produced

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Component Fat Salt Moisture pH Acidity Protein
Mean (%) 15 1.95 63.83 4.76 108º D 11.86
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384 Table 3. Experimental design and its response for nanocomposite films incorporated with
385 metal nanoparticle using combined design

factors responses
Run Ag% CuO% ZnO% Time Coliform R (cfu/g)
(weeks) (cfu/g)
1 1 0 0 0 1.31×106 0

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2 0 0 1 3 8×10 6.1172
3 0.5 0.5 0 4 2.5×102 6.1171

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4 0.333 0 0.667 0 1.31×106 0
5 0.333 0.333 0.333 4 8×101 6.1172

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6 0 0.5 0.5 4 9×101 6.1172
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7 0 0.5 0.5 2 9×10 6.1142
2.2×102

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8 0.5 0 0.5 4 6.1171
9 0.5 0.5 0 1 3.2×103 6.1162
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10 0.667 0.167 0.167 0 1.31×106 0
11 0 1 0 4 102 6.1172
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12 0 0.667 0.333 3 3.1×102 6.1171

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13 1 0 0 0 1.31×10 0
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14 0.5 0 0.5 2 3.4×103 6.1161

15 0 0.333 0.667 1 9×103 6.1142
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16 0.5 0.5 0 0 1.31×106 0

17 1 0 0 0 1.31×106 0
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18 0 0 1 4 102 6.1172
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19 0.5 0.5 0 2 5.1×10 6.1155
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20 0.333 0 0.667 4 1.1×102 6.1169

8×101
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22 0 0.5 0.5 2 9×103 6.1142
23 0 0.5 0.5 2 9×103 6.1142
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24 0.5 0 0.5 0 1.31×10 0
25 0.5 0.5 0 4 2.5×102 6.1171
26 0.667 0.167 0.167 2 3×103 6.1162
27 0 1 0 1 1.1×104 6.1136
28 0 0.667 0.333 0 1.31×106 0

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29 1 0 0 2 7×104 6.0934
30 0.5 0 0.5 2 3.4×103 6.1161
31 0 0.333 0.667 2 3.6×103 6.1160
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32 0.5 0.5 0 2 5.1×10 6.1155
386 The LDPE% in all runs is 99.

387 R is the difference in bacteria concentration (expressed in log cfu/cm2) between the non-treated

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388 (1.31×106 cfu/g) and treated test specimens in log.

389

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390 Table 4. ANOVA of the selected model fit

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Source Sum of Df Mean F p-value
Squares Squares Value prob> F

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Linear Mixture 15.37 2 7.69 40.65 <0.0001
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AD 10.58 1 10.58 57.35 <0.0001
BD 6.71 1 6.71 35.49 <0.0001
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CD 9.95 1 9.95 52.61 <0.0001

Residual 4.92 26 0.19
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Lack of Fit 4.92 18 0.27

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Pure Error 0.000 8 0.000

Cor Total 73.30 31
391 A=Ag nanoparticles, B=CuO nanoparticles, C=ZnO nanoparticles D=Time
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392 Df=degree of freedom

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394 Table 5. One-way ANOVA of the optimum point, validation of optimum point and pure
395 LDPE

Mean Std. error Sig. % 95 confidence level

difference Lower Upper
I(X) (J)X (I-J) bound bound
1 .00000 .00611 1.000 -0.189 0.0189
2 -3.08603* .00611 .000 -3.1049 -3.0671

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Tukey 3
HSD
2 .00000 .00611 1.000 -0.0189 0.0189
1 -3.08603* .00611 .000 -3.1049 -3.0671
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3 3.08603* .00611 .000 3.0671 3.1049

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1 3.08603* .00611 .000 3.0671 3.1049
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1 .00000 .00611 1.000 -0.0198 0.0198
2 -3.08603* .00611 .000 -3.1058 -3.0663

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Sheffe 2 .00000 .00611 1.000 -0.0198 0.0198
1 -3.08603* .00611 .000 -3.1058 -3.0663
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3 3.08603* .00611 .000 3.0668 3.1058
1 3.08603* .00611 .000 3.663 3.1058
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396 1: optimum point, 2: validation of optimum point, 3: pure LDPE
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397 * The mean difference is significant at p<0.05

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Highlights

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• A microbial model was suggested for antibactierial effect of active
LDPE films in UF cheese.
• All films had antibacterial effects on colimforms after 4 weeks.

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• LDPE film with Cu-Zno and with no Ag nanoparticles provided optimum
antibacterial effect.

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• Suggested statistical model verified optimum points obtained in
microbial tests.

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