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

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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 signifcant [1].
Te 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 efects of these compounds on human health and consumers' demands for functional and healthy foods, researchers fnd 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 flms and coatings increase the shelf life of food.Tey are usually composed of polysaccharides, proteins, and lipids.Chitosan (C 6 H 11 NO 4 ) 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 flms due to its barrier properties to water vapor, oxygen, and carbon dioxide as well as nontoxicity, environmental compatibility, and flm-forming ability [3,4].
Bacterial cellulose (BC) is a special type of cellulose produced by Acetobacter xyleneum and due to its low cost, biocompatibility, renewability, and biodegradability have many applications in food packaging and biological materials such as fbers, flms, and membranes.BC (30-100 nm) has high water retention ability, high crystallization, and high purity [4][5][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 flm based on chitosan-carboxymethylcellulose-oleic acid containing ZnO nanoparticles in wheat bread.Te 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 flms containing gliadin and cinnamaldehyde inhibited Penicillium expansum and Aspergillus niger after 10 days and increased the shelf life of bread [8].
Te purpose of this study was to prepare an active flm of chitosan (CH) containing bacterial cellulose nanofbers (BCNF) and ZnO nanoparticles (ZnO NPCs) and its efect on loaf bread shelf life.

Preparation of Nanocomposite
Films.Te flms were prepared using the solvent casting method.To prepare the nanocomposite chitosan flm, 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.Ten 30% glycerol (w/w chitosan) was added to the solution as a plasticizer and stirring was continued for 30 minutes.Ten, the flm-forming solution was poured into a plastic plate and dried in an oven at 25 °C for 48 hours.Prior to analysis, all flms were qualifed in a desiccator containing saturated magnesium nitrate solution at a relative humidity of 50 ± 2% at 25 ± 1 °C for 48 hours [1,6].

Coating of Bread Surface.
Te 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].

Water Vapor Permeability (WVP).
Water vapor permeability was measured according to the procedures previously characterized by Noshirvani et al. [1].Te flm samples were placed in the special vials containing CaSO 4 and the vials were weighed and placed in a desiccator containing a saturated K 2 SO 4 (RH � 97%).After the calculation of the water vapor transmission rate (WVTR), the water vapor permeability (WVP) was calculated using the following equations: ( S is the slope by a linear regression, A is the flm surface, X is the average flm thickness (m) and ΔP is vapor pressure (Pa).

Light Transmission and Opacity.
Te light transmission through the flms 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 flms was calculated using the following equation [1,9]: 2 Journal of Food Quality where A 600 is the absorbance at 600 nm and d is the flm thickness (mm).

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

Scanning Electron Microscopy (SEM).
Te morphology of the flms is examined using an SEM device (TESCAN-MIRA111, Czech Republic) at an accelerator voltage of 30 kV and a magnifcation of 10,000x [1].

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].

Hardness.
Te 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].

Antimicrobial Activity
(1) Preparation of Fungal Suspension.Aspergillus Niger (PTCC 5298) was obtained from the culture collection at the Iran Institute of Industrial and Scientifc Research (Tehran, Iran) and its suspension (10 6 cfu/ml) was prepared according to the method previously described by Noshirvani et al. [1].
(2) Antifungal Activity of Composite Films in Bread.Te 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 flm 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.
Te number of yeasts and molds in bread samples was determined by Noshirvani et al. [1] method with some modifcations.10 g of bread was poured into a fask 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 flm 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.Te diference between the means was evaluated by Duncan's multiple range test at an error level of 5% (p < 0.05).Te increase in opacity of flms 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 efects of the flm with heterogeneous network [1].Our results are in agreement with the those obtained by Noshirvani et al. on the efect of ZnO NPCs on the opacity of chitosan, PLA, and fsh gelatin flms [1,13,14].

Results and Discussion
WVP reduction of chitosan flm 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 flling spaces between polymer chains [1,[15][16][17][18].WVP reduction of nanocomposite layers containing ZnO NPCs in flms based on polylactic acid [14,19], chitosan [20]; Chitosan-carboxymethylcellulose [1]; kefran [16]; Fish gelatin and starch [18]; modifed starch and albumin [21] has been shown previously.

Termal Properties. Termal analysis of biopolymers
and their nanobiocomposites is especially important in fexible food packaging.In the DSC thermograms, the melting point (T m ) appears as an endothermic peak and corresponds to the crystalline regions of the polymer (Figure 1).Te maximum point at the frst endothermic peak is considered the melting point.Te addition of ZnO NPCs increases the T m of chitosan flms.In this case, CH-BCNF-ZnO NPCs 1% flm had a higher melting point (T m ) and melting enthalpy (ΔH m ) than other nanocomposites (Table 2).
Te higher T m of CH-BCNF-ZnO NPCs 1% flm can be considered as an advantage for these nanobiocomposites and can be useful in thermal applications.T m 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 afect the increase of arrangement and the transformation of amorphous regions into crystalline regions.Increasing T m 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].
ΔH m of flms can be related to their structural arrangement [23].Terefore, since the ΔH m of chitosan flms 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 fllers and increasing the interactions between polymer chains [24].
As shown on TGA thermograms, all chitosan-based flms have three stages of thermal decomposition.Te frst 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.Signifcant 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.Te 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].
Te presence of a single peak mass degradation for the flms refects the compatibility between BCNF, CH, and ZnO NPCs and also partially confrms the uniform distribution as well as the physical and chemical bonding between the compounds (cellulose and chitosan).Terefore, it can be concluded that the addition of nanoparticles signifcantly increases the thermal stability of pure chitosan flm; in this regard, the highest thermal stability is related to CH-BCNF-ZnO NPCs 1% flm.
Te diference 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 efective functional groups in the structure of these compounds in the proper interaction with chitosan, which efectively 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.Tis 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 efect of adding ZnO NPCs, MgO NPCs, TiO 2 NPCs, and graphene in increasing the thermal stability of Nanocomposite is consistent with the present study [14,[26][27][28].In contrast, Noshirvani et al.
showed that thermal stability was reduced after the addition of ZnO NPCs to active carboxymethylcellulose/chitosan nanocomposites [1].

Morphology.
Te microstructure of the flms are shown in Figure 4. CH flm showed a homogeneous structure.BCNF was well dispersed in the chitosan matrix and showed a uniform structure.CH-BCNF flm shows a regular, compact, and homogeneous structure with good dispersion.After adding ZnO NPCs 0.5% nanoparticles, the flm structure changed to be irregular but showed a good distribution of ZnO NPCs in the flm 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 flm.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.
Te 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 fndings obtained with Perumal et al., [29].3. Te results of the analysis of variance showed that ZnO NPCs changed the moisture content and a w of bread (p < 0.05).Te highest moisture content was observed in CH-BCNF-ZnO NPCs 2% toast.Te highest a w 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).Te 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.Te highest migration rate of nanoparticles was observed in CH-BCNF-ZnO NPCs 2% baguette (p < 0.05).

Chemical Properties of Coated Breads. Te chemical properties of bread are shown in Table
Te 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 efect on the bread freshness, water retention can reduce the staling in bread.Also, WVP results are consistent with the moisture and a w results.Te 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, acidinsoluble ash, and pH of bread samples were in the range of the national standard of Iran (ISIRI, no 2338) [33].Te 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 signifcant efect of ZnO NPs on water migration reduction.Starch retrogradation and moisture migration can afect on the hardness of bread [1,34,35].Water by the hydrogen bond formation between starch molecules and starch/gluten increases hardness [1].

Hardness of Coated
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 diferent concentrations of oxide nanoparticles compared to control bread.Te 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].Terefore, moisture retention is an important factor.With increasing water absorption, moisture content increases and hardness decreases [37].
3.6.Microbial Properties of Bread.Te microbial properties of bread are shown in Table 5. Te composite flms showed antimicrobial properties against microorganisms.Only in the control sandwich-type bread, the population of mold and yeast increase up to 2 × 10 3 cfu/g.Tables 6 and 7 show visual observation of mold growth in bread (noninoculated  6 Journal of Food Quality inoculated) wrapped with diferent flms 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 sandwichtype bread (days 7, 14, and 60), CH baguette (days 14 and 60), CH-BCNF baguette (day 60), and CH-BCNF sandwichtype bread (day 60) (Figure 5).
Chitosan is an antimicrobial agent against spoilage microorganisms, pathogens, molds, and yeasts.Te polymer structure of chitosan is essential for its antimicrobial properties.Te 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.Te diference 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][40][41].
Studies have shown that nanoparticles such as Zn and their oxides have high bactericidal properties [42].Te electrostatic bonding of Zn +2 ions to the cell surface of the microorganism, inactivation of respiratory enzymes, and H 2 O 2 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-carboxymethylcelluloseoleicacid-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].
Te antifungal properties of activated nanocomposite flms based on carboxymethylcellulose-chitosan combined with ZnO NPCs were reported against Aspergillus niger [9].Journal of Food Quality Swaroop and Shukla stabilized MgO NPCs PLA biopolymer and showed that composite flms have antibacterial efects and kill 46% of E. coli after 12 hours [19].
Te fndings of Balaguer et al., [8,29] suggest that the bionanocomposite flm has good antimicrobial activity against food-borne pathogenic bacteria and postharvest     Journal of Food Quality pathogenic fungi and might be suitable food packaging applications.

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

Table 2 :
Termal properties of the flms.

Table 3 :
Chemical properties of the diferent breads.

Table 4 :
Hardness of the diferent breads.

Table 5 :
Microbial properties of the diferent breads stored at 25 °C.

Table 6 :
Visual observation of mold growth in noninoculated bread at 25 °C.

Table 7 :
Visual observation of mold growth in inoculated bread with Aspergillus niger at 25 °C.