# The Effect of Active Chitosan Films Containing Bacterial Cellulose Nanofiber and ZnO Nanoparticles on the Shelf Life of Loaf Bread

Authors: Mehrasa Bakouei; Jalal Sadeghizadeh Yazdi; Mohammad Hasan Ehrampoush; Mahdieh Salari; Farzan Madadizadeh
Journal: Journal of Food Quality
 (2023)

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## Abstract

Due to the disadvantages of synthetic packaging materials such as migration, environmental pollution, lack of easy recycling, and high production costs, natural polymers have received much attention as safe and biodegradable alternatives to plastics. The aim of this study was to investigate the effect of the active film of chitosan (CH) containing bacterial cellulose nanofiber (BCNF) and ZnO nanoparticles (ZnO NPCs) on the shelf life of loaf bread (toast, baguette, and sandwich-type bread). The results showed that ZnO NPCs significantly reduced the thickness and water vapor permeability (WVP) and increased the opacity of filmsp<0.05. CH-BCNF-ZnO 2% NPCs film had the lowest thickness and WVP and the highest opacity. Differential scanning calorimetric (DSC), thermal gravimetry analysis (TGA), and derivative thermogravimetry (DTG) showed that ZnO NPCs increased the thermal stability of chitosan films. CH-BCNF-ZnO 1% NPCs had the highest melting point (148.66°C) and melting enthalpy (ΔHm). Scanning electron microscopy (SEM) images showed the good distribution of ZnO NPCs in the chitosan film. The higher concentrations of ZnO NPCs formed aggregates in the polymer. ZnO NPCs had a significant effect on the physicochemical properties of bread. The highest moisture content and water activity aw were observed in CH-BCNF-ZnO 2% toast and control toast, respectively. CH toast showed high ash and insoluble ash. CH baguettes and control baguettes showed the highest pH. As the ZnO NPCs concentration increased, the nanoparticle migration increased. The highest migration was observed in CH-BCNF-ZnO 2% baguette. The highest and lowest hardness was observed in CH-BCNF-ZnO NPCs 2% baguette and CH-BCNF- ZnO NPCs 1% toast bread, respectively. Composite films decreased the microbial population in all bread samples except sandwich-type bread. It can be concluded that BCNF and ZnO NPCs improve the physical properties of chitosan film and can be suggested as active packaging in bread.


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## Body

## 1. Introduction

Bread is one of the most important food products in different parts of the world, which has long played a major role in human nutrition and provides energy, carbohydrates, proteins, and B vitamins to our diet. In addition, the intake of vitamins, iron, and calcium from bread is significant [1].The growth of mold and staling are the two main causes of bread spoilage, which causes huge economic losses annually. Chemical preservatives are one of the common methods to prevent the growth of mold in bread but due to the side effects of these compounds on human health and consumers’ demands for functional and healthy foods, researchers find suitable alternatives [1].Food packaging made from synthetic plastics is a serious environmental issue worldwide due to the production of nonbiodegradable waste. Since nonbiodegradable plastics are part of dry waste, biodegradable polymers are a good alternative to synthetic polymers and reduce environmental problems. Active and biodegradable packaging is a promising way to maintain food quality in which contains antimicrobials and antioxidants and increases the shelf life of the food by gradually releasing compounds during storage [2].Edible films and coatings increase the shelf life of food. They are usually composed of polysaccharides, proteins, and lipids. Chitosan (C6H11NO4) is a natural polysaccharide biopolymer found in the crustacean exoskeleton and fungal cell walls. Chitosan (CH) has functional properties such as antimicrobial, antifungal, and antioxidant properties, and it is an ideal polymer for the production of edible films due to its barrier properties to water vapor, oxygen, and carbon dioxide as well as nontoxicity, environmental compatibility, and film-forming ability [3, 4].Bacterial cellulose (BC) is a special type of cellulose produced byAcetobacter xyleneum and due to its low cost, biocompatibility, renewability, and biodegradability have many applications in food packaging and biological materials such as fibers, films, and membranes. BC (30–100 nm) has high water retention ability, high crystallization, and high purity [4–6].Metal oxide nanoparticles have many applications as catalysts, sensors, nutrient carriers, and adsorbents. Zinc oxide (ZnO) is a good biocompatible metal oxide semiconductor with a band gap of 3.47 eV with high antioxidant and antibacterial activity. Zinc oxide nanoparticles (ZnO NPCs) have various properties such as chemical stability, high catalytic activity, high coating, recoverability, antibacterial activity, and nontoxicity, which makes them used in the food industry [1, 5, 7].Noshirvani et al. [1] used nanocomposite film based on chitosan-carboxymethylcellulose-oleic acid containing ZnO nanoparticles in wheat bread. The results of moisture content, water activity, and microbial tests showed an increase in the shelf life of bread from 3 to 35 days for treated bread compared to the control sample. All active coatings reduced the mold and yeast counts in bread within 15 days, and more improvement of antimicrobial properties was achieved for coatings containing ZnO nanoparticles. In another report, Balaguer et al. showed that the active films containing gliadin and cinnamaldehyde inhibited Penicillium expansum and Aspergillus niger after 10 days and increased the shelf life of bread [8].The purpose of this study was to prepare an active film of chitosan (CH) containing bacterial cellulose nanofibers (BCNF) and ZnO nanoparticles (ZnO NPCs) and its effect on loaf bread shelf life.


## 2. Materials and Methods

### 2.1. Materials

Chitosan (1.60–185 kDa, deacetylation degree 80–85%) and glycerol were purchased from Sigma Aldrich (USA). Bacterial cellulose nanofiber was bought from Nano Novin Polymer Company (Iran). All microbial media were obtained from Merck (Germany). ZnO NPs (<20 nm) were purchased from Nemad Kala Company (Iran). All other chemicals were of analytical grade.


### 2.2. Preparation of Nanocomposite Films

The films were prepared using the solvent casting method. To prepare the nanocomposite chitosan film, aqueous acetic acid solution (2% v/v) was prepared and mixed with 0.5, 1, and 2% ZnO NPCs (w/w chitosan) and subjected to an ultrasound probe for 30 minutes [4]. Simultaneously, BCNF suspension was prepared at a concentration of 4% (w/w chitosan) and treated with ultrasound waves for 30 minutes. Then 30% glycerol (w/w chitosan) was added to the solution as a plasticizer and stirring was continued for 30 minutes. Then, the film-forming solution was poured into a plastic plate and dried in an oven at 25°C for 48 hours. Prior to analysis, all films were qualified in a desiccator containing saturated magnesium nitrate solution at a relative humidity of 50 ± 2% at 25 ± 1°C for 48 hours [1, 6].


### 2.3. Coating of Bread Surface

The coating formulation including CH, CH- BCNF, CH- BCNF- ZnO NPCs (0.5, 1, and 2%) was sterilized at 121°C for 20 minutes and was coated on the bread surface with a sterile brush, then were dried under aseptic conditions [1].


### 2.4. Nanocomposite Films Characterization

#### 2.4.1. Water Vapor Permeability (WVP)

Water vapor permeability was measured according to the procedures previously characterized by Noshirvani et al. [1]. The film samples were placed in the special vials containing CaSO4 and the vials were weighed and placed in a desiccator containing a saturated K2SO4 (RH = 97%). After the calculation of the water vapor transmission rate (WVTR), the water vapor permeability (WVP) was calculated using the following equations:(1)WVTRgm−2s−1=SA,WVPgm−1s−1Pa−1=WVPR×x∆P.S is the slope by a linear regression, A is the film surface, X is the average film thickness (m) and ΔP is vapor pressure (Pa).


#### 2.4.2. Light Transmission and Opacity

The light transmission through the films was measured at wavelengths between 200 and 800 nm using a UV-VIS spectrophotometer (DR6000 UV-VIS Laboratory Spectrophotometer-HACH, USA) and the opacity of the films was calculated using the following equation [1, 9]:(2)Opacity=A600d,where A600 is the absorbance at 600 nm and d is the film thickness (mm).


#### 2.4.3. DSC, DTG, and TGA

The thermal properties of the films were determined using DSC (Mettler Toledo, Switzerland), TGA (Rheometric Scientific-STA 1500, United Kingdom), and DTG. 5 mg of the film was placed in a DSC pan. An empty aluminum pen was used as a reference. The analysis of samples were performed at 10°C/min between 30 and 600°C and nitrogen atmosphere flow rate of 20 ml/min. The enthalpy of melting was also calculated from the thermogram [1].


#### 2.4.4. Scanning Electron Microscopy (SEM)

The morphology of the films is examined using an SEM device (TESCAN-MIRA111, Czech Republic) at an accelerator voltage of 30 kV and a magnification of 10,000x [1].


### 2.5. Bread Coating Characterization

#### 2.5.1. Chemical Properties

The moisture content, ash, acid-insoluble ash, and pH were determined according to the AACC method (2000). Water activity (aw) was determined using aw meter (ROTRONIC HC2-AW-USB, Switzerland) at 25°C [10].


#### 2.5.2. Migration of Nanoparticles to Bread

After microwave digestion, the migration rate was measured using ICP-MS (Agilent7500, USA) at room temperature on day 10 [7, 11].


#### 2.5.3. Hardness

The hardness of the coated bread (with a thickness of 20 mm) was assessed using a penetration test by a texture analyzer (KOOPA.TA20, Iran) equipped with a cylindrical probe (36 mm) with a speed of 30 mm/min [1].


#### 2.5.4. Antimicrobial Activity

(1) Preparation of Fungal Suspension. Aspergillus Niger (PTCC 5298) was obtained from the culture collection at the Iran Institute of Industrial and Scientific Research (Tehran, Iran) and its suspension (106 cfu/ml) was prepared according to the method previously described by Noshirvani et al. [1].(2) Antifungal Activity of Composite Films in Bread. The toast, baguette, and sandwich-type bread were obtained from a local supermarket. Some bread were inoculated at three points with 5 μl Aspergillus Niger suspension. All bread samples (inoculated and noninoculated) were sandwiched with two pieces of film and packed in polyethylene bags (17 × 30 cm) and stored at 25°C for 60 days. Film-free breads were prepared as controls [1].(3) Antimicrobial Activity of Composite Coating in Bread. The number of yeasts and molds in bread samples was determined by Noshirvani et al. [1] method with some modifications. 10 g of bread was poured into a flask containing 90 ml of physiological serum (0.9% salt by weight/volume) under aseptic conditions and mixed for 5 minutes at 260 rpm. After peroration of serial dilution, the Petri dishes containing potato dextrose agar (PDA) were inoculated at 25°C for 5 days, and then the number of yeasts and molds was expressed as log cfu/g. Coliform and Escherichia coli were also counted according to the Iranian National Standard No. 19888 [12].


### 2.6. Statistical Analysis

After film production, physicochemical and microbiological tests were performed with three replications. A completely randomized statistical design was applied to the analysis of variance (ANOVA) using SPSS V.16 statistical software. The difference between the means was evaluated by Duncan’s multiple range test at an error level of 5%p<0.05.


## 2.1. Materials

Chitosan (1.60–185 kDa, deacetylation degree 80–85%) and glycerol were purchased from Sigma Aldrich (USA). Bacterial cellulose nanofiber was bought from Nano Novin Polymer Company (Iran). All microbial media were obtained from Merck (Germany). ZnO NPs (<20 nm) were purchased from Nemad Kala Company (Iran). All other chemicals were of analytical grade.


## 2.2. Preparation of Nanocomposite Films

The films were prepared using the solvent casting method. To prepare the nanocomposite chitosan film, aqueous acetic acid solution (2% v/v) was prepared and mixed with 0.5, 1, and 2% ZnO NPCs (w/w chitosan) and subjected to an ultrasound probe for 30 minutes [4]. Simultaneously, BCNF suspension was prepared at a concentration of 4% (w/w chitosan) and treated with ultrasound waves for 30 minutes. Then 30% glycerol (w/w chitosan) was added to the solution as a plasticizer and stirring was continued for 30 minutes. Then, the film-forming solution was poured into a plastic plate and dried in an oven at 25°C for 48 hours. Prior to analysis, all films were qualified in a desiccator containing saturated magnesium nitrate solution at a relative humidity of 50 ± 2% at 25 ± 1°C for 48 hours [1, 6].


## 2.3. Coating of Bread Surface

The coating formulation including CH, CH- BCNF, CH- BCNF- ZnO NPCs (0.5, 1, and 2%) was sterilized at 121°C for 20 minutes and was coated on the bread surface with a sterile brush, then were dried under aseptic conditions [1].


## 2.4. Nanocomposite Films Characterization

### 2.4.1. Water Vapor Permeability (WVP)

Water vapor permeability was measured according to the procedures previously characterized by Noshirvani et al. [1]. The film samples were placed in the special vials containing CaSO4 and the vials were weighed and placed in a desiccator containing a saturated K2SO4 (RH = 97%). After the calculation of the water vapor transmission rate (WVTR), the water vapor permeability (WVP) was calculated using the following equations:(1)WVTRgm−2s−1=SA,WVPgm−1s−1Pa−1=WVPR×x∆P.S is the slope by a linear regression, A is the film surface, X is the average film thickness (m) and ΔP is vapor pressure (Pa).


### 2.4.2. Light Transmission and Opacity

The light transmission through the films was measured at wavelengths between 200 and 800 nm using a UV-VIS spectrophotometer (DR6000 UV-VIS Laboratory Spectrophotometer-HACH, USA) and the opacity of the films was calculated using the following equation [1, 9]:(2)Opacity=A600d,where A600 is the absorbance at 600 nm and d is the film thickness (mm).


### 2.4.3. DSC, DTG, and TGA

The thermal properties of the films were determined using DSC (Mettler Toledo, Switzerland), TGA (Rheometric Scientific-STA 1500, United Kingdom), and DTG. 5 mg of the film was placed in a DSC pan. An empty aluminum pen was used as a reference. The analysis of samples were performed at 10°C/min between 30 and 600°C and nitrogen atmosphere flow rate of 20 ml/min. The enthalpy of melting was also calculated from the thermogram [1].


### 2.4.4. Scanning Electron Microscopy (SEM)

The morphology of the films is examined using an SEM device (TESCAN-MIRA111, Czech Republic) at an accelerator voltage of 30 kV and a magnification of 10,000x [1].


## 2.4.1. Water Vapor Permeability (WVP)

Water vapor permeability was measured according to the procedures previously characterized by Noshirvani et al. [1]. The film samples were placed in the special vials containing CaSO4 and the vials were weighed and placed in a desiccator containing a saturated K2SO4 (RH = 97%). After the calculation of the water vapor transmission rate (WVTR), the water vapor permeability (WVP) was calculated using the following equations:(1)WVTRgm−2s−1=SA,WVPgm−1s−1Pa−1=WVPR×x∆P.S is the slope by a linear regression, A is the film surface, X is the average film thickness (m) and ΔP is vapor pressure (Pa).


## 2.4.2. Light Transmission and Opacity

The light transmission through the films was measured at wavelengths between 200 and 800 nm using a UV-VIS spectrophotometer (DR6000 UV-VIS Laboratory Spectrophotometer-HACH, USA) and the opacity of the films was calculated using the following equation [1, 9]:(2)Opacity=A600d,where A600 is the absorbance at 600 nm and d is the film thickness (mm).


## 2.4.3. DSC, DTG, and TGA

The thermal properties of the films were determined using DSC (Mettler Toledo, Switzerland), TGA (Rheometric Scientific-STA 1500, United Kingdom), and DTG. 5 mg of the film was placed in a DSC pan. An empty aluminum pen was used as a reference. The analysis of samples were performed at 10°C/min between 30 and 600°C and nitrogen atmosphere flow rate of 20 ml/min. The enthalpy of melting was also calculated from the thermogram [1].


## 2.4.4. Scanning Electron Microscopy (SEM)

The morphology of the films is examined using an SEM device (TESCAN-MIRA111, Czech Republic) at an accelerator voltage of 30 kV and a magnification of 10,000x [1].


## 2.5. Bread Coating Characterization

### 2.5.1. Chemical Properties

The moisture content, ash, acid-insoluble ash, and pH were determined according to the AACC method (2000). Water activity (aw) was determined using aw meter (ROTRONIC HC2-AW-USB, Switzerland) at 25°C [10].


### 2.5.2. Migration of Nanoparticles to Bread

After microwave digestion, the migration rate was measured using ICP-MS (Agilent7500, USA) at room temperature on day 10 [7, 11].


### 2.5.3. Hardness

The hardness of the coated bread (with a thickness of 20 mm) was assessed using a penetration test by a texture analyzer (KOOPA.TA20, Iran) equipped with a cylindrical probe (36 mm) with a speed of 30 mm/min [1].


### 2.5.4. Antimicrobial Activity

(1) Preparation of Fungal Suspension. Aspergillus Niger (PTCC 5298) was obtained from the culture collection at the Iran Institute of Industrial and Scientific Research (Tehran, Iran) and its suspension (106 cfu/ml) was prepared according to the method previously described by Noshirvani et al. [1].(2) Antifungal Activity of Composite Films in Bread. The toast, baguette, and sandwich-type bread were obtained from a local supermarket. Some bread were inoculated at three points with 5 μl Aspergillus Niger suspension. All bread samples (inoculated and noninoculated) were sandwiched with two pieces of film and packed in polyethylene bags (17 × 30 cm) and stored at 25°C for 60 days. Film-free breads were prepared as controls [1].(3) Antimicrobial Activity of Composite Coating in Bread. The number of yeasts and molds in bread samples was determined by Noshirvani et al. [1] method with some modifications. 10 g of bread was poured into a flask containing 90 ml of physiological serum (0.9% salt by weight/volume) under aseptic conditions and mixed for 5 minutes at 260 rpm. After peroration of serial dilution, the Petri dishes containing potato dextrose agar (PDA) were inoculated at 25°C for 5 days, and then the number of yeasts and molds was expressed as log cfu/g. Coliform and Escherichia coli were also counted according to the Iranian National Standard No. 19888 [12].


## 2.5.1. Chemical Properties

The moisture content, ash, acid-insoluble ash, and pH were determined according to the AACC method (2000). Water activity (aw) was determined using aw meter (ROTRONIC HC2-AW-USB, Switzerland) at 25°C [10].


## 2.5.2. Migration of Nanoparticles to Bread

After microwave digestion, the migration rate was measured using ICP-MS (Agilent7500, USA) at room temperature on day 10 [7, 11].


## 2.5.3. Hardness

The hardness of the coated bread (with a thickness of 20 mm) was assessed using a penetration test by a texture analyzer (KOOPA.TA20, Iran) equipped with a cylindrical probe (36 mm) with a speed of 30 mm/min [1].


## 2.5.4. Antimicrobial Activity

(1) Preparation of Fungal Suspension. Aspergillus Niger (PTCC 5298) was obtained from the culture collection at the Iran Institute of Industrial and Scientific Research (Tehran, Iran) and its suspension (106 cfu/ml) was prepared according to the method previously described by Noshirvani et al. [1].(2) Antifungal Activity of Composite Films in Bread. The toast, baguette, and sandwich-type bread were obtained from a local supermarket. Some bread were inoculated at three points with 5 μl Aspergillus Niger suspension. All bread samples (inoculated and noninoculated) were sandwiched with two pieces of film and packed in polyethylene bags (17 × 30 cm) and stored at 25°C for 60 days. Film-free breads were prepared as controls [1].(3) Antimicrobial Activity of Composite Coating in Bread. The number of yeasts and molds in bread samples was determined by Noshirvani et al. [1] method with some modifications. 10 g of bread was poured into a flask containing 90 ml of physiological serum (0.9% salt by weight/volume) under aseptic conditions and mixed for 5 minutes at 260 rpm. After peroration of serial dilution, the Petri dishes containing potato dextrose agar (PDA) were inoculated at 25°C for 5 days, and then the number of yeasts and molds was expressed as log cfu/g. Coliform and Escherichia coli were also counted according to the Iranian National Standard No. 19888 [12].


## 2.6. Statistical Analysis

After film production, physicochemical and microbiological tests were performed with three replications. A completely randomized statistical design was applied to the analysis of variance (ANOVA) using SPSS V.16 statistical software. The difference between the means was evaluated by Duncan’s multiple range test at an error level of 5%p<0.05.


## 3. Results and Discussion

### 3.1. Physical Properties of Films

The physical properties of the films are shown in Table1. The results showed that the opacity of CH and CH-BCNF films was significantly lower than other films p<0.05. The opacity of films increased with the addition of ZnO NPCs. The highest opacity was observed in CH-BCNF- ZnO NPCs 2% film p<0.05. The composite films had low light transmission and had excellent UV-barrier properties. The addition of ZnO NPCs resulted in thinner films. Film thickness varied from 0.02 to 0.07 mm. ZnO NPCs significantly reduced WVP p<0.05. WVP decreased with the increasing percentage of ZnO NPCs.Table 1
Physical properties of the films.
FilmsOpacity (nm/mm)Light transmission (%)Thickness (mm)WVP (g·m−1·d−1·pa−1)280 nm600 nm800 nmCH1.54 ± 0.07d7.68 ± 0.30a69.74 ± 1.16a78.83 ± 1.71a0.079 ± 0.005a2.94 ± 0.03aCH-BCNF1.70 ± 0.06d6.91 ± 0.32ab67.42 ± 0.96ab77.30 ± 1.71ab0.058 ± 0.007b1.94 ± 0.13bCH-BCNF-ZnO NPCs 0.5%2.02 ± 0.06c6.11 ± 0.29bc65.47 ± 0.73bc75.24 ± 1.08bc0.049 ± 0.007c1.74 ± 0.14bCH-BCNF-ZnO NPCs 1%2.31 ± 0.10b5.44 ± 0.32cd63.70 ± 0.88cd73.27 ± 0.86cd0.031 ± 0.002d1.06 ± 0.01cCH-BCNF-ZnO NPCs 2%2.80 ± 0.09a4.78 ± 0.46d61.63 ± 1.11d71.30 ± 1.10d0.022 ± 0.003e0.77 ± 0.18dThe different letters in each column are significantly differentp<0.05.The increase in opacity of films with ZnO NPCs can be associated to the presence of ZnO as a mineral that cannot be dissolved in the polymer matrix and also the light scattering effects of the film with heterogeneous network [1]. Our results are in agreement with the those obtained by Noshirvani et al. on the effect of ZnO NPCs on the opacity of chitosan, PLA, and fish gelatin films [1, 13, 14].WVP reduction of chitosan film after the addition of ZnO NPCs is related to the decrease in free hydrophilic groups (OH) due to the formation of hydrogen bonds between ZnO NPCs and biopolymer matrix, tortuous pathway formation for water molecules, biopolymer crystallinity increasing, lower permeability or hydrophilicity of ZnO NPCs compared to the polymer matrix, reduction chain mobility, and filling spaces between polymer chains [1, 15–18]. WVP reduction of nanocomposite layers containing ZnO NPCs in films based on polylactic acid [14, 19], chitosan [20]; Chitosan-carboxymethylcellulose [1]; kefiran [16]; Fish gelatin and starch [18]; modified starch and albumin [21] has been shown previously.


### 3.2. Thermal Properties

Thermal analysis of biopolymers and their nanobiocomposites is especially important in flexible food packaging. In the DSC thermograms, the melting point (Tm) appears as an endothermic peak and corresponds to the crystalline regions of the polymer (Figure 1). The maximum point at the first endothermic peak is considered the melting point. The addition of ZnO NPCs increases the Tm of chitosan films. In this case, CH-BCNF-ZnO NPCs 1% film had a higher melting point (Tm) and melting enthalpy (ΔHm) than other nanocomposites (Table 2).Figure 1
DSC thermograms of different films.Table 2
Thermal properties of the films.
FilmsTm (°C)ΔHm (J/g)CH128.1524.18CH-BCNF147.67144.78CH-BCNF-ZnO NPCs 0.5%139.38136.17CH-BCNF-ZnO NPCs 1%148.66168.50CH-BCNF-ZnO NPCs 2%136.93127.09In TGA thermograms, all films showed three thermal degradation stages (Figure2). According to the DTG curves (Figure 3), it was found that the maximum thermal decomposition of CH, CH-BCNF, CH-BCNF-ZnO NPCs 0.5%, CH-BCNF-ZnO NPCs 1% and CH-BCNF-ZnO NPCs 2% films occurred at 238, 250, 245, 285 and 242°C, respectively.Figure 2
TGA thermograms of different films.Figure 3
DTG thermograms of different films.The higherTm of CH-BCNF-ZnO NPCs 1% film can be considered as an advantage for these nanobiocomposites and can be useful in thermal applications. Tm is related to the properties of the polymer crystalline region. Due to the compacted chains, nanoparticles typically cannot easily penetrate the crystalline region but can affect the increase of arrangement and the transformation of amorphous regions into crystalline regions. Increasing Tm by adding nanoparticles is important in relation to increasing heterogeneous nucleation, facilitating crystallization, and modifying the orientation of polymer chains by nanoparticles, which results in chain arrangement and large crystals formation (larger crystals have higher thermodynamic stability) [22].ΔHm of films can be related to their structural arrangement [23]. Therefore, since the ΔHm of chitosan films increases due to the integration of nanoparticles into the chitosan polymer matrix, it can be concluded that these nanoparticles can help to enclose chitosan chains and further cross-links between them. According to the obtained results, it can be said that nanoparticles can improve the thermal properties of polymers by acting as fillers and increasing the interactions between polymer chains [24].As shown on TGA thermograms, all chitosan-based films have three stages of thermal decomposition. The first stage, in the temperature range of 25 to 150°C, is mainly related to the evaporation of adsorbed and bound water (hydrogen bonding) as well as the residual acetic acid. Significant weight loss in the second stage, around 150–420°C, is related to the depolymerization of chitosan chains through deacetylation and the breakdown of glycosidic bonds through dehydration and deamination, as well as the breakdown of glycerol. The third stage, in the temperature range of 420–600°C, can be due to the oxidative degradation of carbon residues formed in the second stage [25].The presence of a single peak mass degradation for the films reflects the compatibility between BCNF, CH, and ZnO NPCs and also partially confirms the uniform distribution as well as the physical and chemical bonding between the compounds (cellulose and chitosan). Therefore, it can be concluded that the addition of nanoparticles significantly increases the thermal stability of pure chitosan film; in this regard, the highest thermal stability is related to CH-BCNF-ZnO NPCs 1% film.The difference in weight loss between pure chitosan and nanocomposite is related to the presence of ZnO NPCs in the chitosan matrix. In fact, good dispersion of nanoparticles in the polymer matrix provides an improvement in thermal properties. One of the requirements for improving the thermal properties of polymer nanocomposites is the homogeneous distribution of nanoparticles in the polymer matrix. Another reason for the improvement of thermal properties is the effective functional groups in the structure of these compounds in the proper interaction with chitosan, which effectively protects the thermal structure of the polymer. In general, the improvement of the thermal stability is due to the nanoparticles acting as a barrier and preventing the release of gaseous products of combustion and the entry of oxygen into the system. As a result, the degradation temperature of the material increases. This process is best carried out with nanoparticles because they also have better barrier properties. As a result, they delay the degradation process and improve thermal stability [26].Studies by Pantani et al., Swaroop and Shukla, Asadi and Pirsa , and Girdthep et al. on the effect of adding ZnO NPCs, MgO NPCs, TiO2 NPCs, and graphene in increasing the thermal stability of Nanocomposite is consistent with the present study [14, 26–28]. In contrast, Noshirvani et al. showed that thermal stability was reduced after the addition of ZnO NPCs to active carboxymethylcellulose/chitosan nanocomposites [1].Balaguer et al., [8, 29–31] concluded the thermal properties of bio-nanocomposite films improved with the addition of cellulose nanocrystals (CNC).


### 3.3. Morphology

The microstructure of the films are shown in Figure4. CH film showed a homogeneous structure. BCNF was well dispersed in the chitosan matrix and showed a uniform structure. CH-BCNF film shows a regular, compact, and homogeneous structure with good dispersion. After adding ZnO NPCs 0.5% nanoparticles, the film structure changed to be irregular but showed a good distribution of ZnO NPCs in the film matrix. In other words, ZnO NPCs are homogeneously and uniformly dispersed in the chitosan matrix, creating a denser, compact, and more regular structure than CH film. With increasing ZnO NPCs, accumulations were observed in the polymer matrix. It seems that higher concentrations of ZnO NPCs can form aggregates in the polymer matrix.Figure 4
SEM images of films. (a) CH, (b) CH-BCNF, (c) CH-BCNF-ZnO NPCs 0.5%, (d) CH-BCNF-ZnO NPCs 1%, and (e) CH-BCNF-ZnO NPCs 2%.
(a)(b)(c)(d)(e)The phases of chitosan and cellulose are compatible due to the structural similarities of chitosan and cellulose, so the amino groups of chitosan and the carboxyl groups of cellulose form strong ionic bonds [1, 32].Our results were consentientby the findings obtained with Perumal et al., [29].


### 3.4. Chemical Properties of Coated Breads

The chemical properties of bread are shown in Table3. The results of the analysis of variance showed that ZnO NPCs changed the moisture content and aw of bread p<0.05. The highest moisture content was observed in CH-BCNF-ZnO NPCs 2% toast. The highest aw and ash were observed in control toast and CH toast, respectively p<0.05. CH toast, CH baguettes, CH sandwiches, CH-BCNF-ZnO NPCs 0.5% baguettes, and CH-BCNF-ZnO NPCs 0.5% sandwiches-type bread showed the highest acid-insoluble ash p<0.05. The highest and lowest pH was reported in the CH baguette and control baguette and CH-BCNF-ZnO NPCs 0.5% baguette p<0.05. As the concentration of ZnO NPCs increased, the nanoparticle migration rate increased as expected. The highest migration rate of nanoparticles was observed in CH-BCNF- ZnO NPCs 2% baguette p<0.05.Table 3
Chemical properties of the different breads.
BreadsMoisture (%)awAsh (%)Insoluble ash (%)pHZn migration (ppm)ToastCH20.89 ± 0.07g0.80 ± 0.02c2.06 ± 0.03a0 ± 0d4.84 ± 0.03e—CH-BCNF21.83 ± 0.05d0.72 ± 0.05d1.93 ± 0.03b0.07 ± 0a4.72 ± 0.03f—CH-BCNF- ZnO NPCs 0.5%23.16 ± 0.05c0.91 ± 0.03a0.77 ± 0.03k0.01 ± 0c4.47 ± 0.03i128.13 ± 0.56cCH-BCNF- ZnO NPCs 1%23.51 ± 0.04b0.88 ± 0.05ab0.74 ± 0.03k0.01 ± 0c4.48 ± 0.02i217.03 ± 0.42bCH-BCNF- ZnO NPCs 2%24.71 ± 0.05a0.84 ± 0.02b1.14 ± 0.02g0.07 ± 0a4.56 ± 0.02h318.11 ± 0.77aControl14.82 ± 0.03l0.92 ± 0.02a0.91 ± 0.02i0.03 ± 0b5.36 ± 0.03b10.66 ± 0.04kBaguetteCH15.82 ± 0.05k0.86 ± 0.03b0.95 ± 0.03h0 ± 0d5.40 ± 0.01a—CH-BCNF17.98 ± 0.03i0.85 ± 0.02b0.87 ± 0.02j0.07 ± 0a4.92 ± 0.01d—CH-BCNF- ZnO NPCs 0.5%19.88 ± 0.07h0.82 ± 0.04bc0.95 ± 0.03h0 ± 0d4.42 ± 0.01j19.80 ± 0.38iCH-BCNF- ZnO NPCs 1%20.94 ± 0.06g0.81 ± 0.04bc0.96 ± 0.02h0.03 ± 0b4.71 ± 0.04f39.47 ± 0.30gCH-BCNF- ZnO NPCs 2%21.70 ± 0.02e0.76 ± 0.04d0.96 ± 0.02h0.07 ± 0a4.70 ± 0.03f67.94 ± 0.59eControl11.71 ± 0.02n0.85 ± 0.03b0.92 ± 0.02i0.03 ± 0b5.40 ± 0.01a11.07 ± 0.48jSandwich-typeCH15.86 ± 0.06k0.73 ± 0.05d1.44 ± 0.01d0 ± 0d4.75 ± 0.04f—CH-BCNF16.12 ± 0.03j0.75 ± 0.04d1.35 ± 0.02e0.03 ± 0b4.73 ± 0.04f—CH-BCNF- ZnO NPCs 0.5%19.96 ± 0.06h0.88 ± 0.04ab1.23 ± 0.02f0 ± 0d4.49 ± 0.03i27.78 ± 1.05hCH-BCNF- ZnO NPCs 1%21.13 ± 0.03f0.81 ± 0.04bc1.22 ± 0.02f0.07 ± 0a4.57 ± 0.02h55.48 ± 0.33fCH-BCNF- ZnO NPCs 2%21.85 ± 0.05d0.83 ± 0.03b1.34 ± 0.02e0.07 ± 0a4.62 ± 0.01g98.84 ± 0.39dControl12.93 ± 0.04m0.85 ± 0.03b1.63 ± 0.03c0.07 ± 0a5.25 ± 0.04c8.15 ± 0.05lThe different letters in each column are significantly differentp<0.05.The active coating can help to retain water in the bread by limiting the moisture migration from the crumb to the crust. Due to the moisture effect on the bread freshness, water retention can reduce the staling in bread. Also, WVP results are consistent with the moisture andaw results. The water retention in bread with ZnO NPCs is due to the hygroscopic properties of ZnO NPCs and the improvement of WVP. In this study, the amount of moisture, ash, acid-insoluble ash, and pH of bread samples were in the range of the national standard of Iran (ISIRI, no 2338) [33].


### 3.5. Hardness of Coated Breads

The hardness of the bread samples is shown in Table4. The highest and lowest hardness was observed in CH-BCNF- ZnO NPCs 2% baguette and CH-BCNF- ZnO NPCs 1% toast, respectively, p<0.05.Table 4
Hardness of the different breads.
BreadHardness (g)ToastCH-BCNF- ZnO NPCs 0.5%804 ± 90iCH-BCNF- ZnO NPCs 1%17.74 ± 0.41lCH-BCNF- ZnO NPCs 2%1324 ± 64gControl1350 ± 79fBaguetteCH-BCNF- ZnO NPCs 0.5%6585 ± 489cCH-BCNF- ZnO NPCs 1%2786 ± 67dCH-BCNF- ZnO NPCs 2%9272 ± 693aControl2402 ± 127eSandwich-typeCH-BCNF- ZnO NPCs 0.5%924 ± 31hCH-BCNF- ZnO NPCs 1%229 ± 20kCH-BCNF- ZnO NPCs 2%701 ± 21iControl7652 ± 574bThe different letters in each column are significantly differentp<0.05.The hardness of the breadcrumb is one of the main causes of staling. Consistent with moisture content, water activity, WVP, and DSC results, hardness results indicate a significant effect of ZnO NPs on water migration reduction. Starch retrogradation and moisture migration can affect on the hardness of bread [1, 34, 35]. Water by the hydrogen bond formation between starch molecules and starch/gluten increases hardness [1].Noshirvani et al. showed that moisture content and water activity indicate better shelf life of bread maintained by the active coating of chitosan-carboxymethylcellulose-oleic acid nanocomposite containing different concentrations of oxide nanoparticles compared to control bread. The control sample showed the highest amount of hardness during 15 days [1].Our results are consistent with a study by Janjarasskul et al. which showed that high barrier properties limited water loss and reduced staling of sponge cake [36]. Therefore, moisture retention is an important factor. With increasing water absorption, moisture content increases and hardness decreases [37].


### 3.6. Microbial Properties of Bread

The microbial properties of bread are shown in Table5. The composite films showed antimicrobial properties against microorganisms. Only in the control sandwich-type bread, the population of mold and yeast increase up to 2 × 103 cfu/g. Tables 6 and 7 show visual observation of mold growth in bread (noninoculated and inoculated) wrapped with different films at 25°C during storage. In noninoculated bread, mold growth was observed only in control of the baguettes (days 7, 14, and 60) and control sandwich-type bread (day 60). In inoculated samples, mold growth was observed in control toast (days 14 and 60), control baguette (days 7, 14, and 60), control sandwich-type bread (days 7, 14, and 60), CH baguette (days 14 and 60), CH-BCNF baguette (day 60), and CH-BCNF sandwich-type bread (day 60) (Figure 5).Table 5
Microbial properties of the different breads stored at 25°C.
BreadMold and yeastE. coliColiformToastCH<10—<10CH-BCNF<10—<10CH-BCNF- ZnO NPCs 0.5%<10—<10CH-BCNF- ZnO NPCs 1%<10—<10CH-BCNF- ZnO NPCs 2%<10—<10ControlNDNDNDBaguetteCH<10—<10CH-BCNF<10—<10CH-BCNF- ZnO NPCs 0.5%<10—<10CH-BCNF- ZnO NPCs 1%<10—<10CH-BCNF- ZnO NPCs 2%<10—<10ControlNDNDNDSandwich-typeCH<10—<10CH-BCNF<10—<10CH-BCNF- ZnO NPCs 0.5%<10—<10CH-BCNF- ZnO NPCs 1%<10—<10CH-BCNF- ZnO NPCs 2%<10—<10Control2 × 103—<10ND: not detection; —: no growth.Table 6
Visual observation of mold growth in noninoculated bread at 25°C.
Non-inoculated breadDay 7Day 14Day 60ToastCH−−−CH-BCNF−−−CH-BCNF- ZnO NPCs 0.5%−−−CH-BCNF- ZnO NPCs 1%−−−CH-BCNF- ZnO NPCs 2%−−−Control−−−BaguetteCH−−−CH-BCNF−−−CH-BCNF- ZnO NPCs 0.5%−−−CH-BCNF- ZnO NPCs 1%−−−CH-BCNF- ZnO NPCs 2%−−−Control+++++++Sandwich-typeCH−−−CH-BCNF−−−CH-BCNF- ZnO NPCs 0.5%−−−CH-BCNF- ZnO NPCs 1%−−−CH-BCNF- ZnO NPCs 2%−−−Control−−+++−: no growth; +: low growth; ++: medium growth; +++: high growth.Table 7
Visual observation of mold growth in inoculated bread withAspergillus niger at 25°C.
Inoculated breadDay 7Day 14Day 60ToastCH−−−CH-BCNF−−−CH-BCNF- ZnO NPCs 0.5%−−−CH-BCNF- ZnO NPCs 1%−−−CH-BCNF- ZnO NPCs 2%−−−Control−++++BaguetteCH+++++CH-BCNF+++++CH-BCNF- ZnO NPCs 0.5%−−−CH-BCNF- ZnO NPCs 1%−−−CH-BCNF- ZnO NPCs 2%−−−Control++++++++Sandwich-TypeCH−−−CH-BCNF−−++CH-BCNF- ZnO NPCs 0.5%−−−CH-BCNF- ZnO NPCs 1%−−−CH-BCNF- ZnO NPCs 2%−−−Control++++++++−: no growth; +: low growth; ++: medium growth; +++: high growth.Figure 5
Visual appearance of noninoculated and inoculated bread after 60 days storage at 25°C. (a) noninoculated baguette, (b) noninoculated sandwich-type bread, (c) inoculated toast, (d) inoculated baguette, (e) CH-BCNF inoculated baguette, (f) inoculated sandwich-type bread, and (g) CH-BCNF inoculated sandwich-type bread.
(a)(b)(c)(d)(e)(f)(g)Chitosan is an antimicrobial agent against spoilage microorganisms, pathogens, molds, and yeasts. The polymer structure of chitosan is essential for its antimicrobial properties. The higher the amine groups, the greater the antimicrobial properties of chitosan. Chitosan penetrates the bacterial cell wall, binds to bacterial DNA and inhibits mRNA synthesis, proliferation, and transcription of bacterial DNA. Chitosan, with its polycation property, acts as a chelator and inhibits the growth of bacteria by binding to metals. By bond-forming with the cell wall anions damages the cell wall of microorganisms. It acts as a water-binding agent and therefore prevents the activity of some enzymes. At low concentrations, it adheres to the outer surface of the bacterium and causes coagulation. However, the main mechanism is related to the reaction of cationic chitosan with the anionic membrane, which causes membrane permeability, resulting in rupture and leakage of materials inside the bacterial cell [38].Metal oxide nanoparticles have antibacterial activity. The difference between the negative charge of a microorganism and the positive charge of nanoparticles, the oxidation of the surface molecules of microorganisms, the reaction of ions released from the nanomaterials with the thiol (SH-) groups of bacterial cell surface proteins, inhibition of the activity of bacterial dehydrogenase and periplasmic enzymes, inhibition of RNA, DNA, and protein synthesis and impermeability of the membrane are responsible for the cell death [39–41].Studies have shown that nanoparticles such as Zn and their oxides have high bactericidal properties [42]. The electrostatic bonding of Zn+2 ions to the cell surface of the microorganism, inactivation of respiratory enzymes, and H2O2 production are mechanisms of the antimicrobial activity of ZnO NPCs. Also, Zn+2 ions migrate to the inner layer of the polymer and form an antimicrobial coating that interacts with microorganisms in the upper space of the package as well as on the surface of bread slices [1, 13, 43].Noshirvani et al. reported an increase in the microbial shelf life of bread for chitosan-carboxymethylcellulose-oleicacid-zinc oxide nanoparticles compared to the control sample. All active coatings reduced the number of molds and yeasts in bread and improved the antimicrobial properties of coatings containing ZnO NPCs [1].The antifungal properties of activated nanocomposite films based on carboxymethylcellulose-chitosan combined with ZnO NPCs were reported againstAspergillus niger [9]. Swaroop and Shukla stabilized MgO NPCs in PLA biopolymer and showed that composite films have antibacterial effects and kill 46% of E. coli after 12 hours [19].The findings of Balaguer et al., [8, 29] suggest that the bionanocomposite film has good antimicrobial activity against food-borne pathogenic bacteria and postharvest pathogenic fungi and might be suitable for food packaging applications.


## 3.1. Physical Properties of Films

The physical properties of the films are shown in Table1. The results showed that the opacity of CH and CH-BCNF films was significantly lower than other films p<0.05. The opacity of films increased with the addition of ZnO NPCs. The highest opacity was observed in CH-BCNF- ZnO NPCs 2% film p<0.05. The composite films had low light transmission and had excellent UV-barrier properties. The addition of ZnO NPCs resulted in thinner films. Film thickness varied from 0.02 to 0.07 mm. ZnO NPCs significantly reduced WVP p<0.05. WVP decreased with the increasing percentage of ZnO NPCs.Table 1
Physical properties of the films.
FilmsOpacity (nm/mm)Light transmission (%)Thickness (mm)WVP (g·m−1·d−1·pa−1)280 nm600 nm800 nmCH1.54 ± 0.07d7.68 ± 0.30a69.74 ± 1.16a78.83 ± 1.71a0.079 ± 0.005a2.94 ± 0.03aCH-BCNF1.70 ± 0.06d6.91 ± 0.32ab67.42 ± 0.96ab77.30 ± 1.71ab0.058 ± 0.007b1.94 ± 0.13bCH-BCNF-ZnO NPCs 0.5%2.02 ± 0.06c6.11 ± 0.29bc65.47 ± 0.73bc75.24 ± 1.08bc0.049 ± 0.007c1.74 ± 0.14bCH-BCNF-ZnO NPCs 1%2.31 ± 0.10b5.44 ± 0.32cd63.70 ± 0.88cd73.27 ± 0.86cd0.031 ± 0.002d1.06 ± 0.01cCH-BCNF-ZnO NPCs 2%2.80 ± 0.09a4.78 ± 0.46d61.63 ± 1.11d71.30 ± 1.10d0.022 ± 0.003e0.77 ± 0.18dThe different letters in each column are significantly differentp<0.05.The increase in opacity of films with ZnO NPCs can be associated to the presence of ZnO as a mineral that cannot be dissolved in the polymer matrix and also the light scattering effects of the film with heterogeneous network [1]. Our results are in agreement with the those obtained by Noshirvani et al. on the effect of ZnO NPCs on the opacity of chitosan, PLA, and fish gelatin films [1, 13, 14].WVP reduction of chitosan film after the addition of ZnO NPCs is related to the decrease in free hydrophilic groups (OH) due to the formation of hydrogen bonds between ZnO NPCs and biopolymer matrix, tortuous pathway formation for water molecules, biopolymer crystallinity increasing, lower permeability or hydrophilicity of ZnO NPCs compared to the polymer matrix, reduction chain mobility, and filling spaces between polymer chains [1, 15–18]. WVP reduction of nanocomposite layers containing ZnO NPCs in films based on polylactic acid [14, 19], chitosan [20]; Chitosan-carboxymethylcellulose [1]; kefiran [16]; Fish gelatin and starch [18]; modified starch and albumin [21] has been shown previously.


## 3.2. Thermal Properties

Thermal analysis of biopolymers and their nanobiocomposites is especially important in flexible food packaging. In the DSC thermograms, the melting point (Tm) appears as an endothermic peak and corresponds to the crystalline regions of the polymer (Figure 1). The maximum point at the first endothermic peak is considered the melting point. The addition of ZnO NPCs increases the Tm of chitosan films. In this case, CH-BCNF-ZnO NPCs 1% film had a higher melting point (Tm) and melting enthalpy (ΔHm) than other nanocomposites (Table 2).Figure 1
DSC thermograms of different films.Table 2
Thermal properties of the films.
FilmsTm (°C)ΔHm (J/g)CH128.1524.18CH-BCNF147.67144.78CH-BCNF-ZnO NPCs 0.5%139.38136.17CH-BCNF-ZnO NPCs 1%148.66168.50CH-BCNF-ZnO NPCs 2%136.93127.09In TGA thermograms, all films showed three thermal degradation stages (Figure2). According to the DTG curves (Figure 3), it was found that the maximum thermal decomposition of CH, CH-BCNF, CH-BCNF-ZnO NPCs 0.5%, CH-BCNF-ZnO NPCs 1% and CH-BCNF-ZnO NPCs 2% films occurred at 238, 250, 245, 285 and 242°C, respectively.Figure 2
TGA thermograms of different films.Figure 3
DTG thermograms of different films.The higherTm of CH-BCNF-ZnO NPCs 1% film can be considered as an advantage for these nanobiocomposites and can be useful in thermal applications. Tm is related to the properties of the polymer crystalline region. Due to the compacted chains, nanoparticles typically cannot easily penetrate the crystalline region but can affect the increase of arrangement and the transformation of amorphous regions into crystalline regions. Increasing Tm by adding nanoparticles is important in relation to increasing heterogeneous nucleation, facilitating crystallization, and modifying the orientation of polymer chains by nanoparticles, which results in chain arrangement and large crystals formation (larger crystals have higher thermodynamic stability) [22].ΔHm of films can be related to their structural arrangement [23]. Therefore, since the ΔHm of chitosan films increases due to the integration of nanoparticles into the chitosan polymer matrix, it can be concluded that these nanoparticles can help to enclose chitosan chains and further cross-links between them. According to the obtained results, it can be said that nanoparticles can improve the thermal properties of polymers by acting as fillers and increasing the interactions between polymer chains [24].As shown on TGA thermograms, all chitosan-based films have three stages of thermal decomposition. The first stage, in the temperature range of 25 to 150°C, is mainly related to the evaporation of adsorbed and bound water (hydrogen bonding) as well as the residual acetic acid. Significant weight loss in the second stage, around 150–420°C, is related to the depolymerization of chitosan chains through deacetylation and the breakdown of glycosidic bonds through dehydration and deamination, as well as the breakdown of glycerol. The third stage, in the temperature range of 420–600°C, can be due to the oxidative degradation of carbon residues formed in the second stage [25].The presence of a single peak mass degradation for the films reflects the compatibility between BCNF, CH, and ZnO NPCs and also partially confirms the uniform distribution as well as the physical and chemical bonding between the compounds (cellulose and chitosan). Therefore, it can be concluded that the addition of nanoparticles significantly increases the thermal stability of pure chitosan film; in this regard, the highest thermal stability is related to CH-BCNF-ZnO NPCs 1% film.The difference in weight loss between pure chitosan and nanocomposite is related to the presence of ZnO NPCs in the chitosan matrix. In fact, good dispersion of nanoparticles in the polymer matrix provides an improvement in thermal properties. One of the requirements for improving the thermal properties of polymer nanocomposites is the homogeneous distribution of nanoparticles in the polymer matrix. Another reason for the improvement of thermal properties is the effective functional groups in the structure of these compounds in the proper interaction with chitosan, which effectively protects the thermal structure of the polymer. In general, the improvement of the thermal stability is due to the nanoparticles acting as a barrier and preventing the release of gaseous products of combustion and the entry of oxygen into the system. As a result, the degradation temperature of the material increases. This process is best carried out with nanoparticles because they also have better barrier properties. As a result, they delay the degradation process and improve thermal stability [26].Studies by Pantani et al., Swaroop and Shukla, Asadi and Pirsa , and Girdthep et al. on the effect of adding ZnO NPCs, MgO NPCs, TiO2 NPCs, and graphene in increasing the thermal stability of Nanocomposite is consistent with the present study [14, 26–28]. In contrast, Noshirvani et al. showed that thermal stability was reduced after the addition of ZnO NPCs to active carboxymethylcellulose/chitosan nanocomposites [1].Balaguer et al., [8, 29–31] concluded the thermal properties of bio-nanocomposite films improved with the addition of cellulose nanocrystals (CNC).


## 3.3. Morphology

The microstructure of the films are shown in Figure4. CH film showed a homogeneous structure. BCNF was well dispersed in the chitosan matrix and showed a uniform structure. CH-BCNF film shows a regular, compact, and homogeneous structure with good dispersion. After adding ZnO NPCs 0.5% nanoparticles, the film structure changed to be irregular but showed a good distribution of ZnO NPCs in the film matrix. In other words, ZnO NPCs are homogeneously and uniformly dispersed in the chitosan matrix, creating a denser, compact, and more regular structure than CH film. With increasing ZnO NPCs, accumulations were observed in the polymer matrix. It seems that higher concentrations of ZnO NPCs can form aggregates in the polymer matrix.Figure 4
SEM images of films. (a) CH, (b) CH-BCNF, (c) CH-BCNF-ZnO NPCs 0.5%, (d) CH-BCNF-ZnO NPCs 1%, and (e) CH-BCNF-ZnO NPCs 2%.
(a)(b)(c)(d)(e)The phases of chitosan and cellulose are compatible due to the structural similarities of chitosan and cellulose, so the amino groups of chitosan and the carboxyl groups of cellulose form strong ionic bonds [1, 32].Our results were consentientby the findings obtained with Perumal et al., [29].


## 3.4. Chemical Properties of Coated Breads

The chemical properties of bread are shown in Table3. The results of the analysis of variance showed that ZnO NPCs changed the moisture content and aw of bread p<0.05. The highest moisture content was observed in CH-BCNF-ZnO NPCs 2% toast. The highest aw and ash were observed in control toast and CH toast, respectively p<0.05. CH toast, CH baguettes, CH sandwiches, CH-BCNF-ZnO NPCs 0.5% baguettes, and CH-BCNF-ZnO NPCs 0.5% sandwiches-type bread showed the highest acid-insoluble ash p<0.05. The highest and lowest pH was reported in the CH baguette and control baguette and CH-BCNF-ZnO NPCs 0.5% baguette p<0.05. As the concentration of ZnO NPCs increased, the nanoparticle migration rate increased as expected. The highest migration rate of nanoparticles was observed in CH-BCNF- ZnO NPCs 2% baguette p<0.05.Table 3
Chemical properties of the different breads.
BreadsMoisture (%)awAsh (%)Insoluble ash (%)pHZn migration (ppm)ToastCH20.89 ± 0.07g0.80 ± 0.02c2.06 ± 0.03a0 ± 0d4.84 ± 0.03e—CH-BCNF21.83 ± 0.05d0.72 ± 0.05d1.93 ± 0.03b0.07 ± 0a4.72 ± 0.03f—CH-BCNF- ZnO NPCs 0.5%23.16 ± 0.05c0.91 ± 0.03a0.77 ± 0.03k0.01 ± 0c4.47 ± 0.03i128.13 ± 0.56cCH-BCNF- ZnO NPCs 1%23.51 ± 0.04b0.88 ± 0.05ab0.74 ± 0.03k0.01 ± 0c4.48 ± 0.02i217.03 ± 0.42bCH-BCNF- ZnO NPCs 2%24.71 ± 0.05a0.84 ± 0.02b1.14 ± 0.02g0.07 ± 0a4.56 ± 0.02h318.11 ± 0.77aControl14.82 ± 0.03l0.92 ± 0.02a0.91 ± 0.02i0.03 ± 0b5.36 ± 0.03b10.66 ± 0.04kBaguetteCH15.82 ± 0.05k0.86 ± 0.03b0.95 ± 0.03h0 ± 0d5.40 ± 0.01a—CH-BCNF17.98 ± 0.03i0.85 ± 0.02b0.87 ± 0.02j0.07 ± 0a4.92 ± 0.01d—CH-BCNF- ZnO NPCs 0.5%19.88 ± 0.07h0.82 ± 0.04bc0.95 ± 0.03h0 ± 0d4.42 ± 0.01j19.80 ± 0.38iCH-BCNF- ZnO NPCs 1%20.94 ± 0.06g0.81 ± 0.04bc0.96 ± 0.02h0.03 ± 0b4.71 ± 0.04f39.47 ± 0.30gCH-BCNF- ZnO NPCs 2%21.70 ± 0.02e0.76 ± 0.04d0.96 ± 0.02h0.07 ± 0a4.70 ± 0.03f67.94 ± 0.59eControl11.71 ± 0.02n0.85 ± 0.03b0.92 ± 0.02i0.03 ± 0b5.40 ± 0.01a11.07 ± 0.48jSandwich-typeCH15.86 ± 0.06k0.73 ± 0.05d1.44 ± 0.01d0 ± 0d4.75 ± 0.04f—CH-BCNF16.12 ± 0.03j0.75 ± 0.04d1.35 ± 0.02e0.03 ± 0b4.73 ± 0.04f—CH-BCNF- ZnO NPCs 0.5%19.96 ± 0.06h0.88 ± 0.04ab1.23 ± 0.02f0 ± 0d4.49 ± 0.03i27.78 ± 1.05hCH-BCNF- ZnO NPCs 1%21.13 ± 0.03f0.81 ± 0.04bc1.22 ± 0.02f0.07 ± 0a4.57 ± 0.02h55.48 ± 0.33fCH-BCNF- ZnO NPCs 2%21.85 ± 0.05d0.83 ± 0.03b1.34 ± 0.02e0.07 ± 0a4.62 ± 0.01g98.84 ± 0.39dControl12.93 ± 0.04m0.85 ± 0.03b1.63 ± 0.03c0.07 ± 0a5.25 ± 0.04c8.15 ± 0.05lThe different letters in each column are significantly differentp<0.05.The active coating can help to retain water in the bread by limiting the moisture migration from the crumb to the crust. Due to the moisture effect on the bread freshness, water retention can reduce the staling in bread. Also, WVP results are consistent with the moisture andaw results. The water retention in bread with ZnO NPCs is due to the hygroscopic properties of ZnO NPCs and the improvement of WVP. In this study, the amount of moisture, ash, acid-insoluble ash, and pH of bread samples were in the range of the national standard of Iran (ISIRI, no 2338) [33].


## 3.5. Hardness of Coated Breads

The hardness of the bread samples is shown in Table4. The highest and lowest hardness was observed in CH-BCNF- ZnO NPCs 2% baguette and CH-BCNF- ZnO NPCs 1% toast, respectively, p<0.05.Table 4
Hardness of the different breads.
BreadHardness (g)ToastCH-BCNF- ZnO NPCs 0.5%804 ± 90iCH-BCNF- ZnO NPCs 1%17.74 ± 0.41lCH-BCNF- ZnO NPCs 2%1324 ± 64gControl1350 ± 79fBaguetteCH-BCNF- ZnO NPCs 0.5%6585 ± 489cCH-BCNF- ZnO NPCs 1%2786 ± 67dCH-BCNF- ZnO NPCs 2%9272 ± 693aControl2402 ± 127eSandwich-typeCH-BCNF- ZnO NPCs 0.5%924 ± 31hCH-BCNF- ZnO NPCs 1%229 ± 20kCH-BCNF- ZnO NPCs 2%701 ± 21iControl7652 ± 574bThe different letters in each column are significantly differentp<0.05.The hardness of the breadcrumb is one of the main causes of staling. Consistent with moisture content, water activity, WVP, and DSC results, hardness results indicate a significant effect of ZnO NPs on water migration reduction. Starch retrogradation and moisture migration can affect on the hardness of bread [1, 34, 35]. Water by the hydrogen bond formation between starch molecules and starch/gluten increases hardness [1].Noshirvani et al. showed that moisture content and water activity indicate better shelf life of bread maintained by the active coating of chitosan-carboxymethylcellulose-oleic acid nanocomposite containing different concentrations of oxide nanoparticles compared to control bread. The control sample showed the highest amount of hardness during 15 days [1].Our results are consistent with a study by Janjarasskul et al. which showed that high barrier properties limited water loss and reduced staling of sponge cake [36]. Therefore, moisture retention is an important factor. With increasing water absorption, moisture content increases and hardness decreases [37].


## 3.6. Microbial Properties of Bread

The microbial properties of bread are shown in Table5. The composite films showed antimicrobial properties against microorganisms. Only in the control sandwich-type bread, the population of mold and yeast increase up to 2 × 103 cfu/g. Tables 6 and 7 show visual observation of mold growth in bread (noninoculated and inoculated) wrapped with different films at 25°C during storage. In noninoculated bread, mold growth was observed only in control of the baguettes (days 7, 14, and 60) and control sandwich-type bread (day 60). In inoculated samples, mold growth was observed in control toast (days 14 and 60), control baguette (days 7, 14, and 60), control sandwich-type bread (days 7, 14, and 60), CH baguette (days 14 and 60), CH-BCNF baguette (day 60), and CH-BCNF sandwich-type bread (day 60) (Figure 5).Table 5
Microbial properties of the different breads stored at 25°C.
BreadMold and yeastE. coliColiformToastCH<10—<10CH-BCNF<10—<10CH-BCNF- ZnO NPCs 0.5%<10—<10CH-BCNF- ZnO NPCs 1%<10—<10CH-BCNF- ZnO NPCs 2%<10—<10ControlNDNDNDBaguetteCH<10—<10CH-BCNF<10—<10CH-BCNF- ZnO NPCs 0.5%<10—<10CH-BCNF- ZnO NPCs 1%<10—<10CH-BCNF- ZnO NPCs 2%<10—<10ControlNDNDNDSandwich-typeCH<10—<10CH-BCNF<10—<10CH-BCNF- ZnO NPCs 0.5%<10—<10CH-BCNF- ZnO NPCs 1%<10—<10CH-BCNF- ZnO NPCs 2%<10—<10Control2 × 103—<10ND: not detection; —: no growth.Table 6
Visual observation of mold growth in noninoculated bread at 25°C.
Non-inoculated breadDay 7Day 14Day 60ToastCH−−−CH-BCNF−−−CH-BCNF- ZnO NPCs 0.5%−−−CH-BCNF- ZnO NPCs 1%−−−CH-BCNF- ZnO NPCs 2%−−−Control−−−BaguetteCH−−−CH-BCNF−−−CH-BCNF- ZnO NPCs 0.5%−−−CH-BCNF- ZnO NPCs 1%−−−CH-BCNF- ZnO NPCs 2%−−−Control+++++++Sandwich-typeCH−−−CH-BCNF−−−CH-BCNF- ZnO NPCs 0.5%−−−CH-BCNF- ZnO NPCs 1%−−−CH-BCNF- ZnO NPCs 2%−−−Control−−+++−: no growth; +: low growth; ++: medium growth; +++: high growth.Table 7
Visual observation of mold growth in inoculated bread withAspergillus niger at 25°C.
Inoculated breadDay 7Day 14Day 60ToastCH−−−CH-BCNF−−−CH-BCNF- ZnO NPCs 0.5%−−−CH-BCNF- ZnO NPCs 1%−−−CH-BCNF- ZnO NPCs 2%−−−Control−++++BaguetteCH+++++CH-BCNF+++++CH-BCNF- ZnO NPCs 0.5%−−−CH-BCNF- ZnO NPCs 1%−−−CH-BCNF- ZnO NPCs 2%−−−Control++++++++Sandwich-TypeCH−−−CH-BCNF−−++CH-BCNF- ZnO NPCs 0.5%−−−CH-BCNF- ZnO NPCs 1%−−−CH-BCNF- ZnO NPCs 2%−−−Control++++++++−: no growth; +: low growth; ++: medium growth; +++: high growth.Figure 5
Visual appearance of noninoculated and inoculated bread after 60 days storage at 25°C. (a) noninoculated baguette, (b) noninoculated sandwich-type bread, (c) inoculated toast, (d) inoculated baguette, (e) CH-BCNF inoculated baguette, (f) inoculated sandwich-type bread, and (g) CH-BCNF inoculated sandwich-type bread.
(a)(b)(c)(d)(e)(f)(g)Chitosan is an antimicrobial agent against spoilage microorganisms, pathogens, molds, and yeasts. The polymer structure of chitosan is essential for its antimicrobial properties. The higher the amine groups, the greater the antimicrobial properties of chitosan. Chitosan penetrates the bacterial cell wall, binds to bacterial DNA and inhibits mRNA synthesis, proliferation, and transcription of bacterial DNA. Chitosan, with its polycation property, acts as a chelator and inhibits the growth of bacteria by binding to metals. By bond-forming with the cell wall anions damages the cell wall of microorganisms. It acts as a water-binding agent and therefore prevents the activity of some enzymes. At low concentrations, it adheres to the outer surface of the bacterium and causes coagulation. However, the main mechanism is related to the reaction of cationic chitosan with the anionic membrane, which causes membrane permeability, resulting in rupture and leakage of materials inside the bacterial cell [38].Metal oxide nanoparticles have antibacterial activity. The difference between the negative charge of a microorganism and the positive charge of nanoparticles, the oxidation of the surface molecules of microorganisms, the reaction of ions released from the nanomaterials with the thiol (SH-) groups of bacterial cell surface proteins, inhibition of the activity of bacterial dehydrogenase and periplasmic enzymes, inhibition of RNA, DNA, and protein synthesis and impermeability of the membrane are responsible for the cell death [39–41].Studies have shown that nanoparticles such as Zn and their oxides have high bactericidal properties [42]. The electrostatic bonding of Zn+2 ions to the cell surface of the microorganism, inactivation of respiratory enzymes, and H2O2 production are mechanisms of the antimicrobial activity of ZnO NPCs. Also, Zn+2 ions migrate to the inner layer of the polymer and form an antimicrobial coating that interacts with microorganisms in the upper space of the package as well as on the surface of bread slices [1, 13, 43].Noshirvani et al. reported an increase in the microbial shelf life of bread for chitosan-carboxymethylcellulose-oleicacid-zinc oxide nanoparticles compared to the control sample. All active coatings reduced the number of molds and yeasts in bread and improved the antimicrobial properties of coatings containing ZnO NPCs [1].The antifungal properties of activated nanocomposite films based on carboxymethylcellulose-chitosan combined with ZnO NPCs were reported againstAspergillus niger [9]. Swaroop and Shukla stabilized MgO NPCs in PLA biopolymer and showed that composite films have antibacterial effects and kill 46% of E. coli after 12 hours [19].The findings of Balaguer et al., [8, 29] suggest that the bionanocomposite film has good antimicrobial activity against food-borne pathogenic bacteria and postharvest pathogenic fungi and might be suitable for food packaging applications.


## 4. Conclusion

Despite all the advantages of chitosan in biodegradable film production, it has poor mechanical properties and sensitivity to water. For this reason, in this study, the active film of chitosan containing bacterial cellulose nanofiber and ZnO nanoparticles was prepared and its effect on the shelf life of loaf bread was investigated. The results showed that ZnO NPCs decreased the thickness and WVP and increased the opacity and thermal stability of chitosan films. ZnO NPCs had an effect on the physicochemical properties of bread. With increasing concentration, the migration of nanoparticles increased. Composite films showed good antimicrobial properties in all bread samples except sandwich-type bread. Therefore, their use as active packaging in bread is recommended.



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*Source: 7470296-2023-03-11.xml*