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Novel active packaging based on carboxymethyl cellulose-chitosan-ZnO NPs nanocomposite for increasing the shelf life of bread
Food Packaging and Shelf Life 11 (2017) 106–114
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Food Packaging and Shelf Life journal homepage: http://www.elsevier.com/locate/fpsl
Novel active packaging based on carboxymethyl cellulose-chitosan -ZnO NPs nanocomposite for increasing the shelf life of bread Nooshin Noshirvania,* , Babak Ghanbarzadeha , Reza Rezaei Mokarrama , Mahdi Hashemib a b
Faculty of Agriculture, University of Tabriz, Tabriz, Iran Faculty of Chemistry, Bu-Ali Sina University, Hamedan, Iran
A R T I C L E I N F O
Article history: Received 18 September 2016 Received in revised form 9 January 2017 Accepted 18 January 2017 Available online xxx Keywords: Wheat bread Active packaging Nano zinc oxide Chitosan Carboxymethyl cellulose
A B S T R A C T
In this study a new nanocomposite film and coating based on chitosan-carboxymethyl cellulose-oleic acid (CMC-CH-OL) incorporated with different concentrations (0.5, 1 and 2%) of zinc oxide nano particles (ZnO NPs) have been suggested as a packaging material to increase the shelf life (microbial and staling) of sliced wheat bread. Water vapor permeability (WVP) of the CMC-CH film was significantly (P < 0.05) decreased after incorporation of oleic acid as well as ZnO NPs (2%) from 8.27 10 7 to 5.28 10 7 and 1.96 10 7 g/m.h.pa, respectively. Results of moisture content and water activity (aw) showed better maintenance of moisture content for breads stored by active coatings compared to the control one (bread without coating). Differential scanning calorimetry (DSC) thermograms of control bread showed an endothermic peak corresponding to amylopectin retrogradation. Control exhibited the highest firmness over 15 days of storage in all other samples. The results of microbial tests revealed an increase in microbial shelf life of sliced wheat bread from 3 to 35 days for CMC-CH-OL-ZnO NPs 2% in compared to the control. All active coatings lessened the number of yeasts and molds in sliced bread during 15 days, and further improvement in antimicrobial properties obtained for coatings contains 1 and 2% ZnO NPs with no fungal growth during 15 days. © 2017 Elsevier Ltd. All rights reserved.
1. Introduction Bread is one of the oldest and main prepared food of most people all over the world. Due to increasing of the world’s population as well as reducing of the food sources, food preservation management plays an important role in reducing food loss (Fazeli, Shahverdi, Sedaghat, Jamalifar, & Samadi, 2004). Mold spoilage and staling are two main factors, which restrict the quality of bread. Normally, shelf-life of bread without any preservative method is about 3–4 days (Muizniece Brasava et al., 2012). Limited shelf life of bread causes great economic losses annually around the world (Baik & Chinachoti, 2000; Maga & Ponte, 1975). Fungi is the most common spoiler in bakery products and most of the times, fungal growth specifies shelf life of bread and bakery products. Due to the presence of about 40% of water, bread has a water activity (aw) of around 0.96, which makes it susceptible to mold growth (Cioban, Alexa, Sumalan, & Merce, 2010). Penicillium, Aspergillus, Monilia, Mucor, Endomyces,
* Corresponding author. E-mail addresses: [email protected], [email protected] (N. Noshirvani). http://dx.doi.org/10.1016/j.fpsl.2017.01.010 2214-2894/© 2017 Elsevier Ltd. All rights reserved.
Cladosporium, Fusarium, Alternaria and Rhizopus include the most popular fungi in bread and bakery products (Alhendi & Choudhary, 2013; Cioban et al., 2010; Dal Bello et al., 2007; Luciana Gerez, Torino, Rollan, & De Valdez, 2009; Muhianldin, Hassan, & Saari, 2013; Rodriguez, Nerin, & Batlle, 2008). Recently, it was mentioned that about 60% of spoilage in bakery products caused by Penicillium spp. and Aspergillus niger (Alhendi & Choudhary, 2013). Addition of organic acids (propionic acid and its salts) (Muhianldin et al., 2013) is a common method to inhibit fungal growth in bread. However, consumers are demanding for food products without any chemical preservatives (Gutierrez, Sanchez, Batlle, & Nerin, 2009). As a solution, application of active packaging is a promising approach to increase the microbial shelf life of bread without direct addition of chemical agents. In this regards, previous studies have shown that the microbial shelf life of bread can be increased by active packaging such as cellulose acetate films incorporated with sodium propionate (Soares, Rutishauser, Melo, Cruz, & Andrade, 2002); gliadin films incorporated with cinnamaldeyde (Balaguer, Lopez-Carballo, Catala, Gavara, & Hernandez-Munoz, 2013); methylcellulose films incorporated with clove and oregano essential oils (Otoni, Pontes, Medeiros, & Soares, 2014); and Ag/ TiO2-polyethylene packaging (Mihaly Cozmuta et al., 2014). In addition, other methods such as modified atmosphere packaging
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(MAP) (Del Nobile, Matoriello, Cavella, & Giudici, Masi, 2003; Rasmussen & Hansen, 2001); combination of both active packaging and MAP (Degirmencioglu et al., 2011; Gutierrez et al., 2009; Muizniece Brasava et al., 2012; Nielsen & Rios, 2000) have been used to increase the microbial shelf life of bread. Along with mold contamination, staling is another factor which limits the shelf life of bread. Staling is a complicated phenomenon that its exact mechanism is not completely founded. However, starch retrogradation which involves physicochemical changes in bread structure, is suggested as one main mechanism for bread staling. Moreover, other mechanisms such as cross linking between starch and protein, partial drying, glassy-rubbery change, and moisture transformation from crumb to crust of bread are involved in staling (Baik & Chinachoti, 2000; Besbes, Jury, Monteau, & Bail, 2014; Gray & Bemiller, 2003). Generally, staling is divided to crust staling and crump staling. Crust staling is attributed to migration of moisture from crumb to crust, but crump staling is related to physicochemical alteration in starch (Bhatt & Nagaraju, 2009). Some studies have been carried out on the effects of different hydrocolloids (Bhattacharya, Erazo-Castejon, Doehlert, & Mc Mullen, 2002; Guarda, Rosell, Benedito, & Galotto, 2004; Shalini & Laxmi, 2007), and anti-staling agents (Purhagen, Sjoo, & Eliasson, 2011; Purhagen, Sjoo, & Eliasson, 2012) on the bread staling. In addition, Licciardello, Cipri, & Muratore, (2014) studied the usefulness of packaging material on the control of bread staling. Ahmadi, Azizi, Abbasi, Hadian, and Sareminezhad (2011) exhibited retardation in bread staling by measuring mechanical and organoleptic tests in breads were kept by edible active coatings of HPMC-corn starch and sunflower oil. In another study, Salehifar, Beladi Nejad, Alizadeh, and Azizi (2013) studied on the effects of LDPE- multi walled carbon nanotube (MWCNT) nanocomposite films on the shelf life of Lavash bread. Increase in bread shelf life have reported by improvement in water and oxygen barrier properties of LDPE-MWCNT nanocomposites films. Application of biopolymers is a favorable approach to produce active biodegradable packaging. Chitosan (CH) is an amino polysaccharide polymer that shows superior antimicrobial properties (Mitelut, Tanase, Popa, & Popa, 2015). For reasons of biodegradability, having desirable film forming properties, high permeability to gases, and non-toxicity, chitosan is known as a promising polymer for active packaging (Kanatt, Rao, Chawla, & Sharma, 2012). Carboxymethyl cellulose (CMC), a derivative of cellulose, shows good barrier properties against oxygen and lipids, and it forms transparent films that is a desirable feature to consumer acceptance (Ebrahimzadeh, Ghanbarzadeh, & Hamishehkar, 2016; Ghanbarzadeh & Almasi, 2011). Since CMC is an ionic polymer, and CH is a cationic polymer, they are biocompatible by formation of strong ionic cross-linking bonds (Youssef, El-Sayed, El-Sayed, Salama, & Dufresne, 2016). Thus, in this study, to produce antimicrobial films with transparent views, chitosan and CMC are mixed and CMC-CH blend film is produced. On the other hand, decrease in water vapor permeability (WVP) of films is an important parameter in food packaging application. We hypothesize that reduction of water vapor transmission from packaging film may lead to a reduction in the rate of bread staling. Previous studies (Preda, Amica & Marcovich, 2012; Vargas, Albors, Chiralt, & Gonzalez-Martinez, 2009; Wang et al., 2014), have shown the effects of lipid compounds on WVP and hydrophobicity of films. Therefore, in this study oleic acid (OL) was added to the CMC-CH blend and an emulsified film was produced. Zinc oxide is an interesting agent in food and pharmacy fields due to its antimicrobial properties, high stability, non-toxicity, and good UV absorbance properties. Additionally, it is generally recognized as safe (GRAS) from FDA (182.8991 code) (Shahmohammadi Jebel & Almasi, 2016). The antimicrobial efficiency of ZnO NPs in active films, have reported by several studies (Arfat,
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Benjakul, Prodpran, Sumpavapol, & Songtipya, 2014; Pantani, Gorrasi, Vigliotta, Murariu, & Dubois, 2013; Sanuja, Agalya & Umapathym, 2015). It seems that, addition of ZnO NPs in active packaging is a promising method to inhibit fungal growth in bread. Additionally, according to the previous studies (Arfat et al., 2014; Mohammadi Nafchi, Alias, Mahmud, & Robal, 2012; ShahabiGhahfarrokhi, Khodaiyan, Mousavi, & Yousefi, 2015) ZnO NPs has positive effects on the mechanical and barrier properties of the films which is useful property in food packaging application. Due to our knowledge, there are scarce reports on the CMC-CH film formulation. One is reported very recently by Youssef et al. (2016); that showed reduction of microbial growth in cheese by CMC-CH-ZnO NPs films. Also, in another work a CMC-CH film was prepared using layer by layer method and used as a coating for preserving of citrus fruits (Arnon, Granit, Porat, & Poverenov, 2015). The third one is reported by Hu, Wang, and Wang (2016); for CMC and quaternized chitosan. As far as we know, there is no study with CMC-CH-OL-ZnO NPs formulation. Additionally, a few studies have been carried out on the application of active packaging on bread staling. Thus, the objective of current work is production of active emulsified packaging based on CMC-CH-OL incorporated by ZnO NPs at different concentrations by solution casting method and investigation of the effects of this type of packaging on the microbial and physicochemical properties of sliced wheat bread. 2. Material and methods 2.1. Materials Chitosan (low molecular weight, 20–300 cP 1, 50–190 kDa, deacetylation degree 75–85%) and carboxymethyl cellulose sodium salt (CMC) (high viscosity, 1500–3000 cP 1% in H2O 25 C) were bought from Sigma Aldrich. Analytical grade glycerol, oleic acid and tween 80 were obtained from Merck (Darmstadt, Germany), potato dextrose agar (PDA) were purchased from Biokar Diagnostics (Beauvais, France). ZnO NPs with average size of <25 nm was purchased from Sigma Aldrich. All other chemicals were of analytical grade. 2.2. Preparation of films Films were elaborated as explained by Vargas et al. (2009) with some modifications. The ratio of Chitosan and CMC was 1:2; briefly, chitosan (0.2 g) was dispersed in 50.0 mL acidic water (0.5% v/v acetic acid) and stirred overnight; the pH of dispersion was adjusted to 6.8 with NaOH solution (3.0 M). ZnO NPs powder at different concentrations (0.5, 1 and 2% based on solid matter) was dispersed in water (50.0 mL) and sonicated for 30 min in an ultrasonic bath (Sonorex-super, Bandelin, Germany, 40 KHz). Then, 0.4 g CMC was dissolved in ZnO NPs suspension and the solution was mixed by stirrer for 1 h. Later, chitosan and CMC solutions were mixed together. Then, tween 80 (0.2 mL) was added as an emulsifier. After stirring for 15 min, OL (0.3 mL) was added, and the mixture, which has become turbid and milky white at the end, was homogenized by ultrasound probe (Bandelin Sonopuls, Germany, 31 KJ) for 15 min. Glycerol (0.3 mL) was added to plasticizing the solution and the mixture was stirred again for 15 min. Finally, 50.0 mL of each solution were poured into the petri dishes (9.0 cm diameter) and were dried at 25 C and relative humidity (RH) of 50% for 72 h. 2.3. Preparation of coating on the surface of bread slices In order to have a maximum contact between the active compounds and bread slices, active coatings were prepared as a model system to better estimation of staling rate and microbial
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growth. The formulations for preparation of the coatings were similar to the films as described in the Section 2.2. The prepared suspensions were sterilized by an autoclave (Thermo Fisher Scientific, USA) at 121 C for 20 min. Then, 50.0 mL of each suspension was coated on the bread pieces by a sterilized paint brush and then the coats were dried under ventilation at aseptic condition for 1 h. 2.4. Scanning electron microscopy (SEM) Cross-section microstructure observations of the films were performed using SEM technique in a Quanta 200 (FEI, tungsten source, USA). Samples were observed using low vacuum 50 Pa, 3 KV and working distance 8–10 mm. 2.5. Opacity Light transmission through the films (10 mm 40 mm) was recorded between 200 and 800 nm on an ultraviolet-visible (UV– vis) spectrophotometer (Speccord 210, Analytik AG, Jena, Germany) and opacity of the films were determined by Eq. (1): Opacity = A600/X
(1)
Where A600 is absorbance value at 600 nm, and X is the film thickness (mm) (Han & Floros, 1997). 2.6. Water vapor permeability (WVP) Water vapor permeability analyses were carried out according to the method of our previous research (Noshirvani, Ghanbarzadeh, Fasihi, & Almasi, 2016) with some modifications. Special vials were prepared with an average diameter of 2.0 cm and a depth of 4.5 cm. Films were cut in the form of discs with a diameters lightly larger than the diameter of the vial. Each vial was covered with the films after introducing anhydrous CaSO4 (3.0 g) inside to produce RH 0% in the vial. The vials were put to a desiccator containing a saturated K2SO4 solution (RH = 97%), then were placed to an incubator at 25 C. The weight of vials were recorded every 24 h and the changes of weight were noted as a function of time and the slopes were calculated by a linear regression (weight change vs. time). The water vapor transmission rate (WVTR) was defined as the slope (g/h) divided by the transfer area (m2). WVP (g m 1 h 1 Pa 1) was calculated as Eq. (2): WVP = (WVTR/P (RH1
RH2)).X
(2)
Where P is the saturation vapor pressure of water (Pa) at 25 C, RH1 is 0, RH2 is 97 and X is the thickness (m) of film. Three replicates carried out for each sample. 2.7. Moisture content and water activity (aw) of coated bread slices Moisture content of coated bread slices with different formulations, were evaluated at 110 C based on initial weight and constant final weight of bread slices. After drying the samples, they were cooled in a desiccator at ambient temperature. Water activity was determined at 25 C using an aw meter (Aqua Lab Series 3 TE, Decagon Deuices Inc, Pullman, USA). About 5.0 g of the ground breads were used for each test. Three replications were done for each sample.
rate of 10 C/min between 150 and 150 C under a nitrogen flow. An empty aluminum pan was submitted as a reference. Data were analyzed using the Thermal Analysis Universal 2000 software. 2.9. Firmness determination Firmness of the coated breads was evaluated by using penetration test by a QTS-25 texture analyzer (CNS Farnell, Hertfordshire, UK). The probe was moved into the samples with the speed of 30 mm/min. Three replications were done for each sample. 2.10. Preparation of fungal suspensions Aspergillus niger (PTCC 5298) was purchased from the culture collection at Iran Institute of Industrial and Scientific Research (Tehran, Iran). Preparation of fungal suspensions was done as explained by Balaguer et al. (2013) with brief modifications. A Neubauer lam (Simax Kavalier, Germany) was used for the counting of spores and the concentration of fungal suspension was adjusted to 106 cfu/mL. 2.11. Determination of antifungal properties of active films in sliced wheat bread Free preservative soft wheat bread slices were purchased from a local supermarket (Hamedan, Iran). The composition of breads include: wheat flour (60%), water, canola oil, brown sugar, wheat gluten, cider vinegar, yeast, salt, barley flour, and roasted malt. Some bread slices were inoculated with Aspergilus niger suspension by the method of Balaguer et al. (2013). Three points of each bread slice were inoculated by 5 mL of Aspergilus niger suspension (106 cfu/mL). In addition, some bread slices were remained without fungal inoculation, in order to investigate the effect of active films on the natural microflora of bread. Then, bread samples, either inoculated or not, were sandwiched by two pieces of films and were packaged in poly ethylene bags (30 17 cm), then kept at 25 C until visual observation of fungal growth for two months. Three replicates were prepared for each sample. Preservative free breads without any film were prepared as control. 2.12. Determination of antifungal properties of active coatings in sliced wheat bread The prepared coated bread slices which explained at Section 2.3 were packed in the plastic bags (30 17 cm) then kept at 25 C for 15 days. The numbers of yeasts and molds in bread samples were counted according to the method of Otoni et al. (2014) with some modifications. 10.0 g of each sliced bread was aseptically weighted and poured into a flask containing 90 mL physiologic serum (NaCl 0.9% W/V), then mixed under aseptic condition for 5 min at 260 rpm. After that, serial dilutions (10 1–10 5) were prepared and were inoculated in to the petri dishes which containing about 15 mL solidified potato dextrose agar (PDA) medium. The plates were incubated at 25 C for 5 days, then the number of yeasts and molds were counted and expressed in a logarithm scale (log cfu/g). 2.13. Statistical analysis
2.8. Differential scanning calorimetry (DSC) DSC tests were achieved in duplicate using a DSCQ100- RCS (TA Instruments, USA). Around 5.0 mg of each coated bread was placed in a hermetically sealed aluminum pan and heated and cooled at a
A completely randomized design statistics were carried out with the analysis of variance (ANOVA) procedure in SPSS 16 (SPSS Inc., Chicago, IL0 software, USA). Differences among average values were detected by Duncan’s multiple range test (P < 0.05).
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Table 1 Opacity and water vapor permeability (WVP) of the films.
3. Results and discussion 3.1. Film microstructure SEM microstructures of cross section images of CMC-CH, CMCCH-OL and CMC-CH-OL-ZnO NPs 2% films are shown in Fig. 1. The CMC-CH film showed a regular, compact and homogenous structure, with good dispersion of chitosan and CMC phases. Because of structural similarities, chitosan and CMC are compatible, thus the amino groups of chitosan and the carboxyl groups of CMC form strong ionic bonds (Youssef et al., 2016). However, after incorporation of OL the structure of the CMC-CH-OL film changed to be irregular with some pores in the film structure, which can be related to reduction in strong polymer–polymer interactions after addition of OL. Although, after addition of ZnO NPs, the structure of CMC-CH-OL-ZnO NPs 2% changed to be compact, and with more regularity compared to the CMC-CH-OL film. 3.2. Opacity of films The results of the opacity study are shown in Table 1. The CMCCH blend film showed a transparent view with low opacity (3.27). After addition of OL and ZnO NPs the opacity of CMC-CH-OL and the CMC-CH-OL-ZnO NPs 2% films increased by 4.87 and 6.27, respectively (P < 0.05), associated to reduction in transparency of the films. This phenemon could be related to the presence of ZnO NPs as an inorganic material which couldn’t dissolve in polymer matrix (Bruna, Penaloza, Guarda, Rodriguez & Galotto, 2012), as
Films
Opacity (600 nm mm 1*) WVP (g/m.h.pa) 10
CMC-CH CMC-CH-OL CMC-CH-OL-ZnO NPs 0.5% CMC-CH-OL-ZnO NPs 1% CMC-CH-OL-ZnO NPs 2%
3.27 0.22d 4.87 0.71c 5.12 0.82b,c 5.86 0.99b 6.27 0.87a
7
8.27 1.54a 5.28 0.99b 3.58 0.35c 2.85 0.16d 1.96 0.17e
* Averages in each column with different letters indicate a significant difference (P < 0.05).
well as to the light scattering through the film with heterogeneous network (Arfat et al., 2014). The obtained results are in good agreement with those obtained by Arfat et al. (2014) and Pantani et al. (2013) for effect of ZnO NPs on opacity of PLA films and fish gelatin films, respectively. 3.3. Water vapor permeability (WVP) of the films WVP of the CMC-CH and CMC-CH-OL films with different concentrations of ZnO NPs (0–2%) are shown in Table 1. Addition of OL, as well as ZnO NPs significantly (P < 0.05) decreased the WVP of nanocomposite films compare to the control film (CMC-CH). Moreover, there was more reduction in WVP with increasing ZnO NPs loading (P < 0.05). Hydrophobic character of OL could affect the hydrophilic/hydrophobic equilibrium of the film (Ebrahimzadeh et al., 2016; Ojagh, Rezaei, Razavi, & Hosseini, 2010) and caused a reduction in WVP. Generally, water vapor transmission through a
Fig. 1. SEM microstructure of CMC-CH (A), CMC-CH-OL (B) and CMC-CH-OL-ZnO NPs 2% (C).
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film depends on solubility and diffusibility of water molecules in polymer matrix. Conversely to the SEM results, CMC-CH-OL film showed lower WVP compared to the CMC-CH film, despite having some pores in film structure. We hypothesize that, the pores in the CMC-CH-OL films were filled with OL. Also, obtained results demonstrated that the effect of presence of hydrophobic compound (OL) on the reduction of WVP was more efficient than the effect of film structure. More reduction in WVP, after addition of ZnO NPs is mainly attributed to several mechanisms: (1) formation of hydrogen bonds between ZnO NPs and biopolymer matrix which cause to decrease in free hydrophilic groups (OH), (2) formation of tortuous pathway thorough the film for water molecules (Mohammadi Nafchi et al., 2012), (3) increase in crystallinity of biopolymer, (4) less permeability or hydrophilicity of ZnO NPs in comparison to polymer matrix, (5) filling of empty spaces between polymer chains cause to decrease of chains mobility which in turn decrease diffusion rate of water vapor (Shahabi-Ghahfarrokhi et al., 2015; Shahmohammadi Jebel and Almasi, 2016; Yu, Yang, Liu, & Ma, 2009). Decrease in WVP of nanocomposite films incorporated with ZnO NPs have shown in poly lactic acid (Pantani et al., 2013); chitosan (Sanuja et al., 2015); Kefiran (Shahabi-Ghahfarrokhi et al., 2015); fish gelatin; and starch (Yu et al., 2009) based films. 3.4. Moisture content and water activity of breads Moisture content and aw of the bread samples which were stored by active coatings over 15 days of storage at 25 C are shown in Fig. 2(a and b). Both moisture content and aw were gradually decreased during the time for all samples, however this reduction was sharper for control compared to the other samples. The results exhibited moisture content and aw changed in parallel. Generally, retrogradation of amylopectin is the most mechanism for bread staling but several factors such as moisture migration, cross links between gluten and starch can affect the staling rate (Fik & Surowka, 2002). According to these results, presence of active coating could help to maintain water in bread by restricting the
migration of water vapor from the bread's crump to the outside. Considering to the effect of moisture content on freshness of bread, retention of water in the bread slices could help to decrease in the rate of staling. In addition, according to the obtained results from WVP, all films especially after incorporation of ZnO NPs to CMCCH-OL film showed a reduction in WVP, which is in accordance with results of moisture content and aw. The obtained results showed a significant effect of OL in film network, on the barrier properties of the films by restriction the diffusion of water vapor over the films and coatings. Additionally, the presence of ZnO NPs and increasing its content led to more preservation of water in bread slices which is attributed to the hygroscopic effect of ZnO NPs and its positive effect in improving WVP of the films as already mentioned in WVP results. 3.5. Differential scanning calorimetry (DSC) DSC thermograms of control sample as well as the bread coated with different formulations, after 15 days of storage at 25 C, are shown in Fig. 3. The control sample showed two endothermic peaks, the first one around 48 C is associated to the melting of retrograded amylopectin and it is referred to the staling endotherm (Barber, Ortola, Barber, & Fernandez, 1992). Baik and Chinachoti (2000) showed the first transition temperature of bread at about 60 C which corresponded to the amylopectin recrystallization. However, the second one around 118 C is associated to the melting point of starch complex, which corresponded to complexes of amylose with lipid compounds. DSC results showed a broad endotherm for all coated breads in comparison to the control one. This may confirm the less retrograded amylopectin content of coated breads compare to the control. However there was not any difference in the second endothermic peak for all tested samples, which indicated that there was not any difference in the amylose structure. Similarly, Russell (1983) showed that the first endothermic peak, was sharper for stale bread. Generally, staling is associated to the gradual aggregation of amylopectin, in which starch gradually change from amorphous state to crystalline state (Gray & Bemiller, 2003). In fact, since the crystalline state is more stable than the amorphous state, thus there is a tendency to change the amorphous state of starch to crystalline state by the formation of cross linking between long starch chains. The mechanism of active coating in slowing the rate of staling, could attributed to water retention in the bread's loaf. Thus in the presence of moisture, aggregation of amylose and amylopectin chains is inhibited or retarded. 3.6. Firmness
Fig. 2. Moisture content (%) (A), and aw (B) of bread samples storage with active coatings over 15 days of storage, control was bread without any coating.
Firmness of bread crumb is one of the major parameters for evaluating the staling. Firmness of all samples increased gradually during the storage time, however the increase was much higher for control sample compared to all other samples (Fig. 4). In addition, comparison between CMC-CH, CMC-CH-OL, and nanocomposite coatings in terms of firmness, represented lower firmness for breads coated by nanocomposites followed by the CMC-CH-OL and CMC-CH, respectively. In agreement with the WVP, water activity and moisture content results, the results of firmness indicate significant effect of OL as well as ZnO NPs on lowering water migration through the coatings. The control sample showed the highest firmness over 15 days of storage, which according to the moisture content results exhibited the lowest moisture content among all other samples. The results are in good agreement with DSC results which exhibited more crystalline state for control compared to all other samples. Crystalline form of starch is more rigid than the amorphous one, which causes more stiffness of stale bread (Maga & Ponte, 1975). Textural properties of bread could
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Fig. 3. DSC thermograms of control bread and the bread coated with different formulation, after 15 days of storage at 25 C.
3.7. Microbial tests
Fig. 4. Firmness of different bread samples over 15 days of storage at 25 C.
affect by two main mechanisms: starch retrogradation and moisture migration (Janjarasskul, Tananuwong, Kongpensook, Tantratian, & Kokpol, 2016). Previous studies have shown the relationship between amylopectin retrogradation and stiffness (Fik & Surowka, 2002). Distribution of water plays an important role in firmness of bread besides the retrogradation of amylopectin, as increasing the moisture content, cause to lower firmness of bread (Baik & Chinachoti, 2000; He & Hoseney, 1990). During retrogradation, moisture migrates from starch to other components of bread (Gray & Bemiller, 2003). Water acts as a plasticizer and decrease in moisture content in bread, leads to the formation of hydrogen bonds between starch molecules, also with starch and gluten, which in consequence cause an increase in stiffness (He & Hoseney, 1990; Hematian Sourki et al., 2010). Relationship between increasing the moisture content with increasing softness have shown by Bashford and Hartung (1976), Bechtel and Meisner (1954), He and Hoseney (1990) and Hematian Sourki et al. (2010). According to the obtained results from firmness, moisture content and DSC, both moisture content and amylopectin retrogradation could affect the firmness of bread. With restriction of moisture transfer, by application of active coating, the rate of stiffness was decreased. Our results are in agreement with the study of Janjarasskul et al. (2016), exhibited high barrier packaging limited water loss, and caused reduction in staling rate in sponge cake.
3.7.1. Inoculated breads with Aspergilus niger Antifungal properties of the inoculated breads with Aspergilus niger sandwiched with two pieces of films are shown in Fig. 5. The growth of yeasts and molds was evidently affected by the active packaging. For control (bread without film) fungal development was seen after 3 days of storage, however this time was increased in all other samples. Fig. 5, relates to 15 days after incubation of bread slices at 25 C in which the number of days after fungal observation is recorded bellow the images. After 15 days of storage, all samples showed fungal development except CMC-CH-OL-ZnO NPs 2%, in which fungal growth observed after 22 days for this sample. Comparison between moisture content of bread samples (data are not shown) showed higher moisture content for breads were packed with films compared to the control. That is due to the fact that water migrated from the films to bread slices caused to an increase in the moisture content of breads. With these observations, despite to have the lowest moisture content, the control sample showed earlier fungal growth among all other samples. Similarly, Balaguer et al. (2013) showed an increase in the microbial shelf life of breads were packed by gliadin films incorporated with cinnamaldehyde from 5 days (control) to 28 days at 23 C, moreover they reported no growth of Penicilium expansum up to 30 days of storage in breads were kept by active films. In addition, Soares et al. (2002) exhibited an increment in microbial shelf life of bread by cellulose acetate films containing sodium propionate. In another study, Rodriguez et al. (2008) showed 100% inhibition of Rhizopus stolonifer by paper-cinnamon 6% (w/w) active packaging in bread. 3.7.2. Non inoculated breads In order to study the effectiveness of active packaging on the natural microorganisms in bread slices, breads were sandwiched by active films and stored at 25 C until visual observation of mold growth (Table 2). Due to the absence of preservatives in bread formulation, fungal growth observed very early (after 3 days) for bread without any film (control). All active packaging samples showed antifungal properties. Fungal growth was seen after 7 days in breads packed with CMC-CH-OL films. However, this time was
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Fig. 5. Visual appearance of inoculated bread slices by Aspergilus niger after 15 days storage at 25 C.
Table 2 Evaluation of visual observation of mold growth in bread sandwiched with different film stored at 25 C. Type of packaging
Growth delay (days
Control CMC-CH-OL CMC-CH-OL-ZnO NPs 0.5% CMC-CH-OL-ZnO NPs 1% CMC-CH-OL-ZnO NPs 2% *
Intensity of fungal growth*
e
3 0.5 7 1.2d 16 1.5c 28 2.1b 35 3.43a
Day 7
Day 15
Day 28
Day 35
+ +
++ ++
+++ +++ + +
+++ +++ ++ + +
: No fungal growth, + fungal growth lower than 25% of plate, ++ fungal growth between 25 and 50% of plate surface, +++ fungal growth around 75% of plate surface.
increased significantly (P < 0.05) in active films incorporated with ZnO NPs and with increasing its loading, which fungal growth observed after 35 days of storage at 25 C in breads stored with CMC-CH-OL-ZnO NPs 2% films. Showing antimicrobial activity for CMC-CH-OL film is associated to antimicrobial properties of chitosan biopolymer, which is corresponded to the interaction between positive charges of chitosan and negative charges of residues of macromolecules on the surface of fungal cell, leading to leakage of cell components and results in cell death (Muzzarelli et al., 2001). After incorporation of ZnO NPs to the CMC-CH-OL film the antimicrobial activity was increased considerably which is related to the presence of ZnO NPs in polymer matrix. Antimicrobial properties of ZnO NPs can be attributed to different mechanisms. The most important mechanism is related to the electrostatic bonding of Zn+2 ions to the microorganism's cell surface which cause to changing in permeability of cell membrane, interaction with respiratory enzymes and inactivation of them,
reaction with cell components, depletion of cytoplasmic contents. In addition, production of active H2O2 in the presence of UV light which migrates into the package headspace and interacts with the microorganisms by oxidizing of cell membrane components, is another mechanism for antimicrobial activity of ZnO NPs. Also, Zn+2 ions migrates to the internal layer of polymer and develop a antimicrobial coating, which interacts with the microorganisms in the headspace of package as well as on the surface of bread slices (Arfat et al., 2014; Bhuyan, Mishra, Khanuja, Prasad, & Varma, 2015). 3.7.3. Antifungal properties of active coatings on bread The counts of yeasts and molds for coated breads throughout the storage time are illustrated in Table 3. As expected, there was a trend of increasing counts of yeasts and molds during storage, however the increase was much lower in the bread slices were coated by active coatings compared to the control. The CMC-CH-OL
Table 3 Numbers of yeast and molds (log cfu/g) during the time at 25 C incubation. Days
Control
CMC-CH-OL
CMC-CH-OL-ZNO NPS 0.5%
CMC-CH-OL-ZNO NPS 1%
CMC-CH-OL-ZNO NPS 2%
0 3 7 15
2.35 0.02d 5.0 0.03c 6.26 0.03b 7.46 0.04a
1.92 0.02c 4.33 0.03b 6.11 0.03a 6.71 0.04a
N.D 0.0c N.D 0.0c 1.25 0.02b 2.45 0.04a
N.D 0.0a N.D 0.0a N.D 0.0a N.D 0.0a
N.D 0.0a N.D 0.0a N.D 0.0a N.D 0.0a
N.D: No total counts detected. Averages in each column with different letters indicate a significant difference (P < 0.05).
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film showed higher numbers of yeast and molds in compared to the nanocomposite coatings but the numbers of yeasts and molds were much lower than the control one. The best results were obtained for nanocomposite coatings of ZnO NPs 1% and 2%, which exhibited no fungal growth after 15 days. The results confirmed the antimicrobial effects of active coatings on the shelf life of bread. 4. Conclusion Fungal growth and staling are two main problems for bread and bakery products which causes great economic losses annually around the world. As a solution, new active nanocomposite films and coatings based on CMC-CH-OL-ZnO NPs (0–2%) prepared to increase the microbial and physicochemical shelf life of sliced wheat bread. The results of firmness showed harder texture for control in all other samples. DSC results exhibited lower amylopectin retrogradation in breads were kept by active coatings in compared to the control. The moisture content and aw of coated breads were higher than the control, which showed better moisture maintenance in those samples. According to the WVP results of the films that revealed incorporation of OL and ZnO NPs decreased WVP of the films, it can be concluded that active coatings with restriction of moisture transmission could retard the aggregation of amylose and amylopectin macromolecules in bread, and slow down the rate of staling. Microbial tests exhibited increase in the shelf life of breads by active films and coatings and the best results were obtained for CMC-CH-OL-ZnO NPs 2%. Generally, the results of the current study showed that application of CMC-CH-OL-ZnO NPs nanocomposite films is a useful method to increase the microbial shelf life of bread. Additionally, CMC-CHOL-ZnO NPs nanocomposite coatings can retard the staling rate of bread. Acknowledgements The authors acknowledge the University of Tabriz, Bu Ali Sina University and Iran nanotechnology initiative council for their financial supports. Nooshin Noshirvani acknowledges the laboratory of RAC analytical chemistry for their supplementary parts of this work. References Ahmadi, E., Azizi, M. H., Abbasi, S., Hadian, Z., & Sareminezhad, S. (2011). Extending bread shelf-life using polysaccharide coatings containing potassium sorbate. Journal of Food Research, 21(2), 209–217. Alhendi, A., & Choudhary, R. (2013). Current practices in bread packaging and possibility of improving bread shelf life by nanotechnology. International Journal of Food Science and Nutrition Engineering, 3(4), 55–60. Arfat, Y. A., Benjakul, S., Prodpran, T., Sumpavapol, P., & Songtipya, P. (2014). Properties and antimicrobial activity of fish protein isolate/fish skin gelatin film containing basil leaf essential oil and zinc oxide nanoparticles. Food Hydrocolloids, 41, 265–273. Arnon, H., Granit, R., Porat, R., & Poverenov, E. (2015). Development of polysaccharides-based edible coatings for citrus fruits: A layer-by-layer approach. Food Chemistry, 166, 465–472. Baik, M. Y., & Chinachoti, P. (2000). Moisture redistribution and phase transitions during bread staling. Cereal Chemistry, 77(4), 484–488. Balaguer, M. P., Lopez-Carballo, G., Catala, R., Gavara, R., & Hernandez-Munoz, P. (2013). Antifungal properties of gliadin films incorporating cinnamaldehyde and application in active food packaging of bread and cheese spread foodstuffs. International Journal of Food Microbiology, 166, 369–377. Barber, B., Ortola, C., Barber, S., & Fernandez, F. (1992). Storage of packaged white bread. Z Lebensm Unters Forsch, 194, 442–449. Bashford, L. L., & Hartung, T. E. (1976). Rheological properties related to bread freshness. Journal of Food Science, 41, 446–447. Bechtel, W. G., & Meisner, D. F. (1954). Staling studies of bread made with flour fractions: III. Effect of crumb moisture and starch. Cereal Chemistry, 31, 176–181. Besbes, E., Jury, V., Monteau, J. Y., & Bail, A. L. (2014). Effect of baking conditions and storage with crust on the moisture profile, local textural properties and staling kinetics of pan bread? LWT – Food Science and Technology, 58(2), 1–9.
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Food Packaging and Shelf Life journal homepage: http://www.elsevier.com/locate/fpsl
Novel active packaging based on carboxymethyl cellulose-chitosan -ZnO NPs nanocomposite for increasing the shelf life of bread Nooshin Noshirvania,* , Babak Ghanbarzadeha , Reza Rezaei Mokarrama , Mahdi Hashemib a b
Faculty of Agriculture, University of Tabriz, Tabriz, Iran Faculty of Chemistry, Bu-Ali Sina University, Hamedan, Iran
A R T I C L E I N F O
Article history: Received 18 September 2016 Received in revised form 9 January 2017 Accepted 18 January 2017 Available online xxx Keywords: Wheat bread Active packaging Nano zinc oxide Chitosan Carboxymethyl cellulose
A B S T R A C T
In this study a new nanocomposite film and coating based on chitosan-carboxymethyl cellulose-oleic acid (CMC-CH-OL) incorporated with different concentrations (0.5, 1 and 2%) of zinc oxide nano particles (ZnO NPs) have been suggested as a packaging material to increase the shelf life (microbial and staling) of sliced wheat bread. Water vapor permeability (WVP) of the CMC-CH film was significantly (P < 0.05) decreased after incorporation of oleic acid as well as ZnO NPs (2%) from 8.27 10 7 to 5.28 10 7 and 1.96 10 7 g/m.h.pa, respectively. Results of moisture content and water activity (aw) showed better maintenance of moisture content for breads stored by active coatings compared to the control one (bread without coating). Differential scanning calorimetry (DSC) thermograms of control bread showed an endothermic peak corresponding to amylopectin retrogradation. Control exhibited the highest firmness over 15 days of storage in all other samples. The results of microbial tests revealed an increase in microbial shelf life of sliced wheat bread from 3 to 35 days for CMC-CH-OL-ZnO NPs 2% in compared to the control. All active coatings lessened the number of yeasts and molds in sliced bread during 15 days, and further improvement in antimicrobial properties obtained for coatings contains 1 and 2% ZnO NPs with no fungal growth during 15 days. © 2017 Elsevier Ltd. All rights reserved.
1. Introduction Bread is one of the oldest and main prepared food of most people all over the world. Due to increasing of the world’s population as well as reducing of the food sources, food preservation management plays an important role in reducing food loss (Fazeli, Shahverdi, Sedaghat, Jamalifar, & Samadi, 2004). Mold spoilage and staling are two main factors, which restrict the quality of bread. Normally, shelf-life of bread without any preservative method is about 3–4 days (Muizniece Brasava et al., 2012). Limited shelf life of bread causes great economic losses annually around the world (Baik & Chinachoti, 2000; Maga & Ponte, 1975). Fungi is the most common spoiler in bakery products and most of the times, fungal growth specifies shelf life of bread and bakery products. Due to the presence of about 40% of water, bread has a water activity (aw) of around 0.96, which makes it susceptible to mold growth (Cioban, Alexa, Sumalan, & Merce, 2010). Penicillium, Aspergillus, Monilia, Mucor, Endomyces,
* Corresponding author. E-mail addresses: [email protected], [email protected] (N. Noshirvani). http://dx.doi.org/10.1016/j.fpsl.2017.01.010 2214-2894/© 2017 Elsevier Ltd. All rights reserved.
Cladosporium, Fusarium, Alternaria and Rhizopus include the most popular fungi in bread and bakery products (Alhendi & Choudhary, 2013; Cioban et al., 2010; Dal Bello et al., 2007; Luciana Gerez, Torino, Rollan, & De Valdez, 2009; Muhianldin, Hassan, & Saari, 2013; Rodriguez, Nerin, & Batlle, 2008). Recently, it was mentioned that about 60% of spoilage in bakery products caused by Penicillium spp. and Aspergillus niger (Alhendi & Choudhary, 2013). Addition of organic acids (propionic acid and its salts) (Muhianldin et al., 2013) is a common method to inhibit fungal growth in bread. However, consumers are demanding for food products without any chemical preservatives (Gutierrez, Sanchez, Batlle, & Nerin, 2009). As a solution, application of active packaging is a promising approach to increase the microbial shelf life of bread without direct addition of chemical agents. In this regards, previous studies have shown that the microbial shelf life of bread can be increased by active packaging such as cellulose acetate films incorporated with sodium propionate (Soares, Rutishauser, Melo, Cruz, & Andrade, 2002); gliadin films incorporated with cinnamaldeyde (Balaguer, Lopez-Carballo, Catala, Gavara, & Hernandez-Munoz, 2013); methylcellulose films incorporated with clove and oregano essential oils (Otoni, Pontes, Medeiros, & Soares, 2014); and Ag/ TiO2-polyethylene packaging (Mihaly Cozmuta et al., 2014). In addition, other methods such as modified atmosphere packaging
N. Noshirvani et al. / Food Packaging and Shelf Life 11 (2017) 106–114
(MAP) (Del Nobile, Matoriello, Cavella, & Giudici, Masi, 2003; Rasmussen & Hansen, 2001); combination of both active packaging and MAP (Degirmencioglu et al., 2011; Gutierrez et al., 2009; Muizniece Brasava et al., 2012; Nielsen & Rios, 2000) have been used to increase the microbial shelf life of bread. Along with mold contamination, staling is another factor which limits the shelf life of bread. Staling is a complicated phenomenon that its exact mechanism is not completely founded. However, starch retrogradation which involves physicochemical changes in bread structure, is suggested as one main mechanism for bread staling. Moreover, other mechanisms such as cross linking between starch and protein, partial drying, glassy-rubbery change, and moisture transformation from crumb to crust of bread are involved in staling (Baik & Chinachoti, 2000; Besbes, Jury, Monteau, & Bail, 2014; Gray & Bemiller, 2003). Generally, staling is divided to crust staling and crump staling. Crust staling is attributed to migration of moisture from crumb to crust, but crump staling is related to physicochemical alteration in starch (Bhatt & Nagaraju, 2009). Some studies have been carried out on the effects of different hydrocolloids (Bhattacharya, Erazo-Castejon, Doehlert, & Mc Mullen, 2002; Guarda, Rosell, Benedito, & Galotto, 2004; Shalini & Laxmi, 2007), and anti-staling agents (Purhagen, Sjoo, & Eliasson, 2011; Purhagen, Sjoo, & Eliasson, 2012) on the bread staling. In addition, Licciardello, Cipri, & Muratore, (2014) studied the usefulness of packaging material on the control of bread staling. Ahmadi, Azizi, Abbasi, Hadian, and Sareminezhad (2011) exhibited retardation in bread staling by measuring mechanical and organoleptic tests in breads were kept by edible active coatings of HPMC-corn starch and sunflower oil. In another study, Salehifar, Beladi Nejad, Alizadeh, and Azizi (2013) studied on the effects of LDPE- multi walled carbon nanotube (MWCNT) nanocomposite films on the shelf life of Lavash bread. Increase in bread shelf life have reported by improvement in water and oxygen barrier properties of LDPE-MWCNT nanocomposites films. Application of biopolymers is a favorable approach to produce active biodegradable packaging. Chitosan (CH) is an amino polysaccharide polymer that shows superior antimicrobial properties (Mitelut, Tanase, Popa, & Popa, 2015). For reasons of biodegradability, having desirable film forming properties, high permeability to gases, and non-toxicity, chitosan is known as a promising polymer for active packaging (Kanatt, Rao, Chawla, & Sharma, 2012). Carboxymethyl cellulose (CMC), a derivative of cellulose, shows good barrier properties against oxygen and lipids, and it forms transparent films that is a desirable feature to consumer acceptance (Ebrahimzadeh, Ghanbarzadeh, & Hamishehkar, 2016; Ghanbarzadeh & Almasi, 2011). Since CMC is an ionic polymer, and CH is a cationic polymer, they are biocompatible by formation of strong ionic cross-linking bonds (Youssef, El-Sayed, El-Sayed, Salama, & Dufresne, 2016). Thus, in this study, to produce antimicrobial films with transparent views, chitosan and CMC are mixed and CMC-CH blend film is produced. On the other hand, decrease in water vapor permeability (WVP) of films is an important parameter in food packaging application. We hypothesize that reduction of water vapor transmission from packaging film may lead to a reduction in the rate of bread staling. Previous studies (Preda, Amica & Marcovich, 2012; Vargas, Albors, Chiralt, & Gonzalez-Martinez, 2009; Wang et al., 2014), have shown the effects of lipid compounds on WVP and hydrophobicity of films. Therefore, in this study oleic acid (OL) was added to the CMC-CH blend and an emulsified film was produced. Zinc oxide is an interesting agent in food and pharmacy fields due to its antimicrobial properties, high stability, non-toxicity, and good UV absorbance properties. Additionally, it is generally recognized as safe (GRAS) from FDA (182.8991 code) (Shahmohammadi Jebel & Almasi, 2016). The antimicrobial efficiency of ZnO NPs in active films, have reported by several studies (Arfat,
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Benjakul, Prodpran, Sumpavapol, & Songtipya, 2014; Pantani, Gorrasi, Vigliotta, Murariu, & Dubois, 2013; Sanuja, Agalya & Umapathym, 2015). It seems that, addition of ZnO NPs in active packaging is a promising method to inhibit fungal growth in bread. Additionally, according to the previous studies (Arfat et al., 2014; Mohammadi Nafchi, Alias, Mahmud, & Robal, 2012; ShahabiGhahfarrokhi, Khodaiyan, Mousavi, & Yousefi, 2015) ZnO NPs has positive effects on the mechanical and barrier properties of the films which is useful property in food packaging application. Due to our knowledge, there are scarce reports on the CMC-CH film formulation. One is reported very recently by Youssef et al. (2016); that showed reduction of microbial growth in cheese by CMC-CH-ZnO NPs films. Also, in another work a CMC-CH film was prepared using layer by layer method and used as a coating for preserving of citrus fruits (Arnon, Granit, Porat, & Poverenov, 2015). The third one is reported by Hu, Wang, and Wang (2016); for CMC and quaternized chitosan. As far as we know, there is no study with CMC-CH-OL-ZnO NPs formulation. Additionally, a few studies have been carried out on the application of active packaging on bread staling. Thus, the objective of current work is production of active emulsified packaging based on CMC-CH-OL incorporated by ZnO NPs at different concentrations by solution casting method and investigation of the effects of this type of packaging on the microbial and physicochemical properties of sliced wheat bread. 2. Material and methods 2.1. Materials Chitosan (low molecular weight, 20–300 cP 1, 50–190 kDa, deacetylation degree 75–85%) and carboxymethyl cellulose sodium salt (CMC) (high viscosity, 1500–3000 cP 1% in H2O 25 C) were bought from Sigma Aldrich. Analytical grade glycerol, oleic acid and tween 80 were obtained from Merck (Darmstadt, Germany), potato dextrose agar (PDA) were purchased from Biokar Diagnostics (Beauvais, France). ZnO NPs with average size of <25 nm was purchased from Sigma Aldrich. All other chemicals were of analytical grade. 2.2. Preparation of films Films were elaborated as explained by Vargas et al. (2009) with some modifications. The ratio of Chitosan and CMC was 1:2; briefly, chitosan (0.2 g) was dispersed in 50.0 mL acidic water (0.5% v/v acetic acid) and stirred overnight; the pH of dispersion was adjusted to 6.8 with NaOH solution (3.0 M). ZnO NPs powder at different concentrations (0.5, 1 and 2% based on solid matter) was dispersed in water (50.0 mL) and sonicated for 30 min in an ultrasonic bath (Sonorex-super, Bandelin, Germany, 40 KHz). Then, 0.4 g CMC was dissolved in ZnO NPs suspension and the solution was mixed by stirrer for 1 h. Later, chitosan and CMC solutions were mixed together. Then, tween 80 (0.2 mL) was added as an emulsifier. After stirring for 15 min, OL (0.3 mL) was added, and the mixture, which has become turbid and milky white at the end, was homogenized by ultrasound probe (Bandelin Sonopuls, Germany, 31 KJ) for 15 min. Glycerol (0.3 mL) was added to plasticizing the solution and the mixture was stirred again for 15 min. Finally, 50.0 mL of each solution were poured into the petri dishes (9.0 cm diameter) and were dried at 25 C and relative humidity (RH) of 50% for 72 h. 2.3. Preparation of coating on the surface of bread slices In order to have a maximum contact between the active compounds and bread slices, active coatings were prepared as a model system to better estimation of staling rate and microbial
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growth. The formulations for preparation of the coatings were similar to the films as described in the Section 2.2. The prepared suspensions were sterilized by an autoclave (Thermo Fisher Scientific, USA) at 121 C for 20 min. Then, 50.0 mL of each suspension was coated on the bread pieces by a sterilized paint brush and then the coats were dried under ventilation at aseptic condition for 1 h. 2.4. Scanning electron microscopy (SEM) Cross-section microstructure observations of the films were performed using SEM technique in a Quanta 200 (FEI, tungsten source, USA). Samples were observed using low vacuum 50 Pa, 3 KV and working distance 8–10 mm. 2.5. Opacity Light transmission through the films (10 mm 40 mm) was recorded between 200 and 800 nm on an ultraviolet-visible (UV– vis) spectrophotometer (Speccord 210, Analytik AG, Jena, Germany) and opacity of the films were determined by Eq. (1): Opacity = A600/X
(1)
Where A600 is absorbance value at 600 nm, and X is the film thickness (mm) (Han & Floros, 1997). 2.6. Water vapor permeability (WVP) Water vapor permeability analyses were carried out according to the method of our previous research (Noshirvani, Ghanbarzadeh, Fasihi, & Almasi, 2016) with some modifications. Special vials were prepared with an average diameter of 2.0 cm and a depth of 4.5 cm. Films were cut in the form of discs with a diameters lightly larger than the diameter of the vial. Each vial was covered with the films after introducing anhydrous CaSO4 (3.0 g) inside to produce RH 0% in the vial. The vials were put to a desiccator containing a saturated K2SO4 solution (RH = 97%), then were placed to an incubator at 25 C. The weight of vials were recorded every 24 h and the changes of weight were noted as a function of time and the slopes were calculated by a linear regression (weight change vs. time). The water vapor transmission rate (WVTR) was defined as the slope (g/h) divided by the transfer area (m2). WVP (g m 1 h 1 Pa 1) was calculated as Eq. (2): WVP = (WVTR/P (RH1
RH2)).X
(2)
Where P is the saturation vapor pressure of water (Pa) at 25 C, RH1 is 0, RH2 is 97 and X is the thickness (m) of film. Three replicates carried out for each sample. 2.7. Moisture content and water activity (aw) of coated bread slices Moisture content of coated bread slices with different formulations, were evaluated at 110 C based on initial weight and constant final weight of bread slices. After drying the samples, they were cooled in a desiccator at ambient temperature. Water activity was determined at 25 C using an aw meter (Aqua Lab Series 3 TE, Decagon Deuices Inc, Pullman, USA). About 5.0 g of the ground breads were used for each test. Three replications were done for each sample.
rate of 10 C/min between 150 and 150 C under a nitrogen flow. An empty aluminum pan was submitted as a reference. Data were analyzed using the Thermal Analysis Universal 2000 software. 2.9. Firmness determination Firmness of the coated breads was evaluated by using penetration test by a QTS-25 texture analyzer (CNS Farnell, Hertfordshire, UK). The probe was moved into the samples with the speed of 30 mm/min. Three replications were done for each sample. 2.10. Preparation of fungal suspensions Aspergillus niger (PTCC 5298) was purchased from the culture collection at Iran Institute of Industrial and Scientific Research (Tehran, Iran). Preparation of fungal suspensions was done as explained by Balaguer et al. (2013) with brief modifications. A Neubauer lam (Simax Kavalier, Germany) was used for the counting of spores and the concentration of fungal suspension was adjusted to 106 cfu/mL. 2.11. Determination of antifungal properties of active films in sliced wheat bread Free preservative soft wheat bread slices were purchased from a local supermarket (Hamedan, Iran). The composition of breads include: wheat flour (60%), water, canola oil, brown sugar, wheat gluten, cider vinegar, yeast, salt, barley flour, and roasted malt. Some bread slices were inoculated with Aspergilus niger suspension by the method of Balaguer et al. (2013). Three points of each bread slice were inoculated by 5 mL of Aspergilus niger suspension (106 cfu/mL). In addition, some bread slices were remained without fungal inoculation, in order to investigate the effect of active films on the natural microflora of bread. Then, bread samples, either inoculated or not, were sandwiched by two pieces of films and were packaged in poly ethylene bags (30 17 cm), then kept at 25 C until visual observation of fungal growth for two months. Three replicates were prepared for each sample. Preservative free breads without any film were prepared as control. 2.12. Determination of antifungal properties of active coatings in sliced wheat bread The prepared coated bread slices which explained at Section 2.3 were packed in the plastic bags (30 17 cm) then kept at 25 C for 15 days. The numbers of yeasts and molds in bread samples were counted according to the method of Otoni et al. (2014) with some modifications. 10.0 g of each sliced bread was aseptically weighted and poured into a flask containing 90 mL physiologic serum (NaCl 0.9% W/V), then mixed under aseptic condition for 5 min at 260 rpm. After that, serial dilutions (10 1–10 5) were prepared and were inoculated in to the petri dishes which containing about 15 mL solidified potato dextrose agar (PDA) medium. The plates were incubated at 25 C for 5 days, then the number of yeasts and molds were counted and expressed in a logarithm scale (log cfu/g). 2.13. Statistical analysis
2.8. Differential scanning calorimetry (DSC) DSC tests were achieved in duplicate using a DSCQ100- RCS (TA Instruments, USA). Around 5.0 mg of each coated bread was placed in a hermetically sealed aluminum pan and heated and cooled at a
A completely randomized design statistics were carried out with the analysis of variance (ANOVA) procedure in SPSS 16 (SPSS Inc., Chicago, IL0 software, USA). Differences among average values were detected by Duncan’s multiple range test (P < 0.05).
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Table 1 Opacity and water vapor permeability (WVP) of the films.
3. Results and discussion 3.1. Film microstructure SEM microstructures of cross section images of CMC-CH, CMCCH-OL and CMC-CH-OL-ZnO NPs 2% films are shown in Fig. 1. The CMC-CH film showed a regular, compact and homogenous structure, with good dispersion of chitosan and CMC phases. Because of structural similarities, chitosan and CMC are compatible, thus the amino groups of chitosan and the carboxyl groups of CMC form strong ionic bonds (Youssef et al., 2016). However, after incorporation of OL the structure of the CMC-CH-OL film changed to be irregular with some pores in the film structure, which can be related to reduction in strong polymer–polymer interactions after addition of OL. Although, after addition of ZnO NPs, the structure of CMC-CH-OL-ZnO NPs 2% changed to be compact, and with more regularity compared to the CMC-CH-OL film. 3.2. Opacity of films The results of the opacity study are shown in Table 1. The CMCCH blend film showed a transparent view with low opacity (3.27). After addition of OL and ZnO NPs the opacity of CMC-CH-OL and the CMC-CH-OL-ZnO NPs 2% films increased by 4.87 and 6.27, respectively (P < 0.05), associated to reduction in transparency of the films. This phenemon could be related to the presence of ZnO NPs as an inorganic material which couldn’t dissolve in polymer matrix (Bruna, Penaloza, Guarda, Rodriguez & Galotto, 2012), as
Films
Opacity (600 nm mm 1*) WVP (g/m.h.pa) 10
CMC-CH CMC-CH-OL CMC-CH-OL-ZnO NPs 0.5% CMC-CH-OL-ZnO NPs 1% CMC-CH-OL-ZnO NPs 2%
3.27 0.22d 4.87 0.71c 5.12 0.82b,c 5.86 0.99b 6.27 0.87a
7
8.27 1.54a 5.28 0.99b 3.58 0.35c 2.85 0.16d 1.96 0.17e
* Averages in each column with different letters indicate a significant difference (P < 0.05).
well as to the light scattering through the film with heterogeneous network (Arfat et al., 2014). The obtained results are in good agreement with those obtained by Arfat et al. (2014) and Pantani et al. (2013) for effect of ZnO NPs on opacity of PLA films and fish gelatin films, respectively. 3.3. Water vapor permeability (WVP) of the films WVP of the CMC-CH and CMC-CH-OL films with different concentrations of ZnO NPs (0–2%) are shown in Table 1. Addition of OL, as well as ZnO NPs significantly (P < 0.05) decreased the WVP of nanocomposite films compare to the control film (CMC-CH). Moreover, there was more reduction in WVP with increasing ZnO NPs loading (P < 0.05). Hydrophobic character of OL could affect the hydrophilic/hydrophobic equilibrium of the film (Ebrahimzadeh et al., 2016; Ojagh, Rezaei, Razavi, & Hosseini, 2010) and caused a reduction in WVP. Generally, water vapor transmission through a
Fig. 1. SEM microstructure of CMC-CH (A), CMC-CH-OL (B) and CMC-CH-OL-ZnO NPs 2% (C).
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film depends on solubility and diffusibility of water molecules in polymer matrix. Conversely to the SEM results, CMC-CH-OL film showed lower WVP compared to the CMC-CH film, despite having some pores in film structure. We hypothesize that, the pores in the CMC-CH-OL films were filled with OL. Also, obtained results demonstrated that the effect of presence of hydrophobic compound (OL) on the reduction of WVP was more efficient than the effect of film structure. More reduction in WVP, after addition of ZnO NPs is mainly attributed to several mechanisms: (1) formation of hydrogen bonds between ZnO NPs and biopolymer matrix which cause to decrease in free hydrophilic groups (OH), (2) formation of tortuous pathway thorough the film for water molecules (Mohammadi Nafchi et al., 2012), (3) increase in crystallinity of biopolymer, (4) less permeability or hydrophilicity of ZnO NPs in comparison to polymer matrix, (5) filling of empty spaces between polymer chains cause to decrease of chains mobility which in turn decrease diffusion rate of water vapor (Shahabi-Ghahfarrokhi et al., 2015; Shahmohammadi Jebel and Almasi, 2016; Yu, Yang, Liu, & Ma, 2009). Decrease in WVP of nanocomposite films incorporated with ZnO NPs have shown in poly lactic acid (Pantani et al., 2013); chitosan (Sanuja et al., 2015); Kefiran (Shahabi-Ghahfarrokhi et al., 2015); fish gelatin; and starch (Yu et al., 2009) based films. 3.4. Moisture content and water activity of breads Moisture content and aw of the bread samples which were stored by active coatings over 15 days of storage at 25 C are shown in Fig. 2(a and b). Both moisture content and aw were gradually decreased during the time for all samples, however this reduction was sharper for control compared to the other samples. The results exhibited moisture content and aw changed in parallel. Generally, retrogradation of amylopectin is the most mechanism for bread staling but several factors such as moisture migration, cross links between gluten and starch can affect the staling rate (Fik & Surowka, 2002). According to these results, presence of active coating could help to maintain water in bread by restricting the
migration of water vapor from the bread's crump to the outside. Considering to the effect of moisture content on freshness of bread, retention of water in the bread slices could help to decrease in the rate of staling. In addition, according to the obtained results from WVP, all films especially after incorporation of ZnO NPs to CMCCH-OL film showed a reduction in WVP, which is in accordance with results of moisture content and aw. The obtained results showed a significant effect of OL in film network, on the barrier properties of the films by restriction the diffusion of water vapor over the films and coatings. Additionally, the presence of ZnO NPs and increasing its content led to more preservation of water in bread slices which is attributed to the hygroscopic effect of ZnO NPs and its positive effect in improving WVP of the films as already mentioned in WVP results. 3.5. Differential scanning calorimetry (DSC) DSC thermograms of control sample as well as the bread coated with different formulations, after 15 days of storage at 25 C, are shown in Fig. 3. The control sample showed two endothermic peaks, the first one around 48 C is associated to the melting of retrograded amylopectin and it is referred to the staling endotherm (Barber, Ortola, Barber, & Fernandez, 1992). Baik and Chinachoti (2000) showed the first transition temperature of bread at about 60 C which corresponded to the amylopectin recrystallization. However, the second one around 118 C is associated to the melting point of starch complex, which corresponded to complexes of amylose with lipid compounds. DSC results showed a broad endotherm for all coated breads in comparison to the control one. This may confirm the less retrograded amylopectin content of coated breads compare to the control. However there was not any difference in the second endothermic peak for all tested samples, which indicated that there was not any difference in the amylose structure. Similarly, Russell (1983) showed that the first endothermic peak, was sharper for stale bread. Generally, staling is associated to the gradual aggregation of amylopectin, in which starch gradually change from amorphous state to crystalline state (Gray & Bemiller, 2003). In fact, since the crystalline state is more stable than the amorphous state, thus there is a tendency to change the amorphous state of starch to crystalline state by the formation of cross linking between long starch chains. The mechanism of active coating in slowing the rate of staling, could attributed to water retention in the bread's loaf. Thus in the presence of moisture, aggregation of amylose and amylopectin chains is inhibited or retarded. 3.6. Firmness
Fig. 2. Moisture content (%) (A), and aw (B) of bread samples storage with active coatings over 15 days of storage, control was bread without any coating.
Firmness of bread crumb is one of the major parameters for evaluating the staling. Firmness of all samples increased gradually during the storage time, however the increase was much higher for control sample compared to all other samples (Fig. 4). In addition, comparison between CMC-CH, CMC-CH-OL, and nanocomposite coatings in terms of firmness, represented lower firmness for breads coated by nanocomposites followed by the CMC-CH-OL and CMC-CH, respectively. In agreement with the WVP, water activity and moisture content results, the results of firmness indicate significant effect of OL as well as ZnO NPs on lowering water migration through the coatings. The control sample showed the highest firmness over 15 days of storage, which according to the moisture content results exhibited the lowest moisture content among all other samples. The results are in good agreement with DSC results which exhibited more crystalline state for control compared to all other samples. Crystalline form of starch is more rigid than the amorphous one, which causes more stiffness of stale bread (Maga & Ponte, 1975). Textural properties of bread could
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Fig. 3. DSC thermograms of control bread and the bread coated with different formulation, after 15 days of storage at 25 C.
3.7. Microbial tests
Fig. 4. Firmness of different bread samples over 15 days of storage at 25 C.
affect by two main mechanisms: starch retrogradation and moisture migration (Janjarasskul, Tananuwong, Kongpensook, Tantratian, & Kokpol, 2016). Previous studies have shown the relationship between amylopectin retrogradation and stiffness (Fik & Surowka, 2002). Distribution of water plays an important role in firmness of bread besides the retrogradation of amylopectin, as increasing the moisture content, cause to lower firmness of bread (Baik & Chinachoti, 2000; He & Hoseney, 1990). During retrogradation, moisture migrates from starch to other components of bread (Gray & Bemiller, 2003). Water acts as a plasticizer and decrease in moisture content in bread, leads to the formation of hydrogen bonds between starch molecules, also with starch and gluten, which in consequence cause an increase in stiffness (He & Hoseney, 1990; Hematian Sourki et al., 2010). Relationship between increasing the moisture content with increasing softness have shown by Bashford and Hartung (1976), Bechtel and Meisner (1954), He and Hoseney (1990) and Hematian Sourki et al. (2010). According to the obtained results from firmness, moisture content and DSC, both moisture content and amylopectin retrogradation could affect the firmness of bread. With restriction of moisture transfer, by application of active coating, the rate of stiffness was decreased. Our results are in agreement with the study of Janjarasskul et al. (2016), exhibited high barrier packaging limited water loss, and caused reduction in staling rate in sponge cake.
3.7.1. Inoculated breads with Aspergilus niger Antifungal properties of the inoculated breads with Aspergilus niger sandwiched with two pieces of films are shown in Fig. 5. The growth of yeasts and molds was evidently affected by the active packaging. For control (bread without film) fungal development was seen after 3 days of storage, however this time was increased in all other samples. Fig. 5, relates to 15 days after incubation of bread slices at 25 C in which the number of days after fungal observation is recorded bellow the images. After 15 days of storage, all samples showed fungal development except CMC-CH-OL-ZnO NPs 2%, in which fungal growth observed after 22 days for this sample. Comparison between moisture content of bread samples (data are not shown) showed higher moisture content for breads were packed with films compared to the control. That is due to the fact that water migrated from the films to bread slices caused to an increase in the moisture content of breads. With these observations, despite to have the lowest moisture content, the control sample showed earlier fungal growth among all other samples. Similarly, Balaguer et al. (2013) showed an increase in the microbial shelf life of breads were packed by gliadin films incorporated with cinnamaldehyde from 5 days (control) to 28 days at 23 C, moreover they reported no growth of Penicilium expansum up to 30 days of storage in breads were kept by active films. In addition, Soares et al. (2002) exhibited an increment in microbial shelf life of bread by cellulose acetate films containing sodium propionate. In another study, Rodriguez et al. (2008) showed 100% inhibition of Rhizopus stolonifer by paper-cinnamon 6% (w/w) active packaging in bread. 3.7.2. Non inoculated breads In order to study the effectiveness of active packaging on the natural microorganisms in bread slices, breads were sandwiched by active films and stored at 25 C until visual observation of mold growth (Table 2). Due to the absence of preservatives in bread formulation, fungal growth observed very early (after 3 days) for bread without any film (control). All active packaging samples showed antifungal properties. Fungal growth was seen after 7 days in breads packed with CMC-CH-OL films. However, this time was
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Fig. 5. Visual appearance of inoculated bread slices by Aspergilus niger after 15 days storage at 25 C.
Table 2 Evaluation of visual observation of mold growth in bread sandwiched with different film stored at 25 C. Type of packaging
Growth delay (days
Control CMC-CH-OL CMC-CH-OL-ZnO NPs 0.5% CMC-CH-OL-ZnO NPs 1% CMC-CH-OL-ZnO NPs 2% *
Intensity of fungal growth*
e
3 0.5 7 1.2d 16 1.5c 28 2.1b 35 3.43a
Day 7
Day 15
Day 28
Day 35
+ +
++ ++
+++ +++ + +
+++ +++ ++ + +
: No fungal growth, + fungal growth lower than 25% of plate, ++ fungal growth between 25 and 50% of plate surface, +++ fungal growth around 75% of plate surface.
increased significantly (P < 0.05) in active films incorporated with ZnO NPs and with increasing its loading, which fungal growth observed after 35 days of storage at 25 C in breads stored with CMC-CH-OL-ZnO NPs 2% films. Showing antimicrobial activity for CMC-CH-OL film is associated to antimicrobial properties of chitosan biopolymer, which is corresponded to the interaction between positive charges of chitosan and negative charges of residues of macromolecules on the surface of fungal cell, leading to leakage of cell components and results in cell death (Muzzarelli et al., 2001). After incorporation of ZnO NPs to the CMC-CH-OL film the antimicrobial activity was increased considerably which is related to the presence of ZnO NPs in polymer matrix. Antimicrobial properties of ZnO NPs can be attributed to different mechanisms. The most important mechanism is related to the electrostatic bonding of Zn+2 ions to the microorganism's cell surface which cause to changing in permeability of cell membrane, interaction with respiratory enzymes and inactivation of them,
reaction with cell components, depletion of cytoplasmic contents. In addition, production of active H2O2 in the presence of UV light which migrates into the package headspace and interacts with the microorganisms by oxidizing of cell membrane components, is another mechanism for antimicrobial activity of ZnO NPs. Also, Zn+2 ions migrates to the internal layer of polymer and develop a antimicrobial coating, which interacts with the microorganisms in the headspace of package as well as on the surface of bread slices (Arfat et al., 2014; Bhuyan, Mishra, Khanuja, Prasad, & Varma, 2015). 3.7.3. Antifungal properties of active coatings on bread The counts of yeasts and molds for coated breads throughout the storage time are illustrated in Table 3. As expected, there was a trend of increasing counts of yeasts and molds during storage, however the increase was much lower in the bread slices were coated by active coatings compared to the control. The CMC-CH-OL
Table 3 Numbers of yeast and molds (log cfu/g) during the time at 25 C incubation. Days
Control
CMC-CH-OL
CMC-CH-OL-ZNO NPS 0.5%
CMC-CH-OL-ZNO NPS 1%
CMC-CH-OL-ZNO NPS 2%
0 3 7 15
2.35 0.02d 5.0 0.03c 6.26 0.03b 7.46 0.04a
1.92 0.02c 4.33 0.03b 6.11 0.03a 6.71 0.04a
N.D 0.0c N.D 0.0c 1.25 0.02b 2.45 0.04a
N.D 0.0a N.D 0.0a N.D 0.0a N.D 0.0a
N.D 0.0a N.D 0.0a N.D 0.0a N.D 0.0a
N.D: No total counts detected. Averages in each column with different letters indicate a significant difference (P < 0.05).
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film showed higher numbers of yeast and molds in compared to the nanocomposite coatings but the numbers of yeasts and molds were much lower than the control one. The best results were obtained for nanocomposite coatings of ZnO NPs 1% and 2%, which exhibited no fungal growth after 15 days. The results confirmed the antimicrobial effects of active coatings on the shelf life of bread. 4. Conclusion Fungal growth and staling are two main problems for bread and bakery products which causes great economic losses annually around the world. As a solution, new active nanocomposite films and coatings based on CMC-CH-OL-ZnO NPs (0–2%) prepared to increase the microbial and physicochemical shelf life of sliced wheat bread. The results of firmness showed harder texture for control in all other samples. DSC results exhibited lower amylopectin retrogradation in breads were kept by active coatings in compared to the control. The moisture content and aw of coated breads were higher than the control, which showed better moisture maintenance in those samples. According to the WVP results of the films that revealed incorporation of OL and ZnO NPs decreased WVP of the films, it can be concluded that active coatings with restriction of moisture transmission could retard the aggregation of amylose and amylopectin macromolecules in bread, and slow down the rate of staling. Microbial tests exhibited increase in the shelf life of breads by active films and coatings and the best results were obtained for CMC-CH-OL-ZnO NPs 2%. Generally, the results of the current study showed that application of CMC-CH-OL-ZnO NPs nanocomposite films is a useful method to increase the microbial shelf life of bread. Additionally, CMC-CHOL-ZnO NPs nanocomposite coatings can retard the staling rate of bread. Acknowledgements The authors acknowledge the University of Tabriz, Bu Ali Sina University and Iran nanotechnology initiative council for their financial supports. Nooshin Noshirvani acknowledges the laboratory of RAC analytical chemistry for their supplementary parts of this work. References Ahmadi, E., Azizi, M. H., Abbasi, S., Hadian, Z., & Sareminezhad, S. (2011). Extending bread shelf-life using polysaccharide coatings containing potassium sorbate. Journal of Food Research, 21(2), 209–217. Alhendi, A., & Choudhary, R. (2013). Current practices in bread packaging and possibility of improving bread shelf life by nanotechnology. International Journal of Food Science and Nutrition Engineering, 3(4), 55–60. Arfat, Y. A., Benjakul, S., Prodpran, T., Sumpavapol, P., & Songtipya, P. (2014). Properties and antimicrobial activity of fish protein isolate/fish skin gelatin film containing basil leaf essential oil and zinc oxide nanoparticles. Food Hydrocolloids, 41, 265–273. Arnon, H., Granit, R., Porat, R., & Poverenov, E. (2015). Development of polysaccharides-based edible coatings for citrus fruits: A layer-by-layer approach. Food Chemistry, 166, 465–472. Baik, M. Y., & Chinachoti, P. (2000). Moisture redistribution and phase transitions during bread staling. Cereal Chemistry, 77(4), 484–488. Balaguer, M. P., Lopez-Carballo, G., Catala, R., Gavara, R., & Hernandez-Munoz, P. (2013). Antifungal properties of gliadin films incorporating cinnamaldehyde and application in active food packaging of bread and cheese spread foodstuffs. International Journal of Food Microbiology, 166, 369–377. Barber, B., Ortola, C., Barber, S., & Fernandez, F. (1992). Storage of packaged white bread. Z Lebensm Unters Forsch, 194, 442–449. Bashford, L. L., & Hartung, T. E. (1976). Rheological properties related to bread freshness. Journal of Food Science, 41, 446–447. Bechtel, W. G., & Meisner, D. F. (1954). Staling studies of bread made with flour fractions: III. Effect of crumb moisture and starch. Cereal Chemistry, 31, 176–181. Besbes, E., Jury, V., Monteau, J. Y., & Bail, A. L. (2014). Effect of baking conditions and storage with crust on the moisture profile, local textural properties and staling kinetics of pan bread? LWT – Food Science and Technology, 58(2), 1–9.
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