Survival of probiotic bacteria in carboxymethyl ...

LWT - Food Science and Technology 87 (2018) 54e60

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Survival of probiotic bacteria in carboxymethyl cellulose-based edible film and assessment of quality parameters Behzad Ebrahimi a, Reza Mohammadi b, Milad Rouhi b, Amir Mohammad Mortazavian a, *, Saeedeh Shojaee-Aliabadi a, **, Mohammad Reza Koushki c a

Department of Food Science and Technology, National Nutrition and Food Technology Research Institute, Faculty of Nutrition Sciences, Food Science and Technology, Shahid Beheshti University of Medical Sciences, P.O. Box 19395-4741, Tehran, Iran Department of Food Science and Technology, School of Nutrition Sciences and Food Technology, Kermanshah University of Medical Sciences, Kermanshah, Iran c Department of Food Technology Research, National Nutrition and Food Technology Research Institute, Faculty of Nutrition and Food Technology, Shahid Beheshti University of Medical Sciences, P.O. Box 19395-4741, Tehran, Iran b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 22 December 2016 Received in revised form 15 August 2017 Accepted 21 August 2017 Available online 23 August 2017

In this study, survival of four probiotic strains (Lactobacillus acidophilus, L. casei, L. rhamnosus and Bifidobacterium bifidum) immobilized in edible films based on carboxymethyl cellulose (CMC) and physicochemical properties of films were investigated during 42 days of storage at 4 and 25 C. Results showed a significant decrease in viability of bacterial cells during 42 days of storage at 25 C. However, viability of L. acidophilus and L. rhamnosus were in the range of recommended levels during the storage at 4 C (107 CFU/g). Probiotic films caused more water vapor permeability (WVP)and opacity, and less tensile strength (TS) and elongation at break (EB) compared to the control film. However, no significant physicochemical changes were observed among probiotic films containing different strains. Therefore, incorporation of some probiotic strains in edible coats and films could be their suitable carrier at refrigerated temperatures. © 2017 Elsevier Ltd. All rights reserved.

Keywords: Carboxymethyl cellulose Edible film Probiotic Viability

1. Introduction Probiotics are live microorganisms added to food products in certain numbers to improve the quality of the consumer health (Champagne, Ross, Saarela, Hansen, & Charalampopoulos, 2011). Some of the health effects associated to probiotic foods include improved gastrointestinal tract health, reduced lactose intolerance symptoms, reduced serum cholesterol levels and modulated immune system (Thushara, Gangadaran, Solati, & Moghadasian, 2016). To benefit from these health effects, the recommended intake count of the probiotics must be greater than 107 CFU/g of product (Gialamas, Zinoviadou, Biliaderis, & Koutsoumanis, 2010; Jankovic, Sybesma, Phothirath, Ananta, & Mercenier, 2010; Soukoulis, Singh, Macnaughtan, Parmenter, & Fisk, 2016). A wide range of detrimental factors due to food processing (osmotic,

* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (A.M. Mortazavian), s_shojaee@sbmu. ac.ir (S. Shojaee-Aliabadi). http://dx.doi.org/10.1016/j.lwt.2017.08.066 0023-6438/© 2017 Elsevier Ltd. All rights reserved.

mechanical and acid stresses) and storage (oxygen level, hydrogen peroxide and water vapor) has been found to reduce the viability of probiotics (Iaconelli et al., 2015 and Jankovic et al., 2010). One of the newest approaches to improve the survivability of probiotics is immobilization of them in edible films (AltamiranoFortoul, Moreno-Terrazas, Quezada-Gallo, & Rosell, 2012). The term “edible films” can be defined as a thin layer of natural polymers directly used onto the surface of materials which can be used to partly or totally substitute synthetic polymers for coating on foods or serving as a barrier between foods and the surrounding mez-Guille n environment (Emmambux & Stading, 2007 and Go et al., 2009). The films protect the food products from deterioration and improve food quality due to roles of moisture barrier and additive carrier. Therefore, they have been recommended as potential vehicles for the delivery of functional compounds nez, Mun ~ oz, (Emmambux & Stading, 2007;; Falguera, Quintero, Jime & Ibarz, 2011). Generally, edible films and coats are classified into three categories based on the components: 1) hydrocolloids such as proteins, polysaccharides and alginates, 2) lipids such as fatty acids, acylglycerols and waxes, and 3) composite films (Garavand, Rouhi,

B. Ebrahimi et al. / LWT - Food Science and Technology 87 (2018) 54e60

Razavi, Cacciotti, & Mohammadi, 2017; Jridi et al., 2014; Skurtys et al., 2010). Cellulose is the most widely used polysaccharide and the structural component of the cell wall associated with the structural integrity of the cell (Zugenmaier, 2006). The most important limitation of cellulose applications in food technology is water insolubility. Carboxymethyl cellulose (CMC) is one of the water soluble cellulosic derivatives with a wide variety of applications in food and non-food products; such as viscosity modifiers, lubricants, papers, pharmaceutical applications and edible films (Biswal & Singh, 2004). In this study, the survivability of probiotics and physical and mechanical properties of the probiotic edible films based on carboxymethyl cellulose were investigated. 2. Materials and methods 2.1. Materials and probiotic bacterial strains Strains of Lactobacillus acidophilus, L. casei, L. rhamnosus and Bifidobacterium bifidum were purchased as freeze-dried cultures from TakGene (Tehran, Iran) and kept at 80 C until used. Carboxymethyl cellulose (CMC) was supplied by Caragum Parsian (Tehran, Iran). Glycerol, Tween 80 (analytical grade), magnesium chloride (MgCl2), sodium chloride (NaCl), and magnesium nitrate (Mg(NO3)2 were purchased from Merck (Darmstadt, Germany). 2.2. Preparation of probiotic cells Probiotic bacteria were individually inoculated in MRS broth (de Man, Rogosa and Shape, Oxoid, Basingstoke, UK) and then incubated at 37 C for 48 h. Cell suspensions were transferred to 50 ml sterile tubes under aseptic conditions and centrifuged at 4000 g for 10 min. The supernatant was discarded and the cultured cells were washed twice using phosphate buffer saline (PBS with pH 7.0). The suspension were directly added to film forming solutions (De Lacey, pez-Caballero, Go mez-Estaca, Go mez-Guille n, & Montero, 2012). Lo 2.3. Preparation of probiotic CMC film Film solutions were prepared as described by Dashipour et al. (2015) with minor modifications. Solution (1% w/v) was prepared by the gradual addition of 1 g CMC powder to 100 ml distilled water at 70 C. Solution was mixed well using magnetic stirrer at 500 rpm on for 40 min to ensure uniform dispersion. Then, glycerol (50% of CMC weight) as plasticizer was added to the solution and stirred at 70 C for 20 min. Solution was heated to 80 C for 10 min to kill potential pathogens. Air bubbles were removed from the solution by vacuum. When the solution temperature was cooled down to 37 C, L. acidophilus, L. casei, L. rhamnosus and B. bifidum were added to film forming solutions to reach a final concentration of 109 CFU/ ml. Films were formed by casting 50 ml of the final solutions in the center of sterile glass plates and drying at 35 C for 15 h in a ventilated incubator. Then, the films were peeled off and stored in zipped bags. Non-probiotic films were prepared as control.

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agitation in a shaker incubator at 37 C for 1 h to release the bacteria. The serial dilutions were cultured on MRS agar and incubated at 36 C for 72 h. Enumeration of the bacteria on agar plates was carried out in triplicates using colony count technique (Champagne et al., 2011). The total count of viable bacteria was expressed as log colony forming units per gram (log CFU/g, CFU/g ¼ CFU/ plate dilution factor). 2.5. Physical properties of films 2.5.1. Thickness The thickness of edible films was measured using micrometer with an accuracy of 0.001 mm (Mituto, Tokyo, Japan). At least eight measurements were randomly carried out from different segments of the film and the average values were represented as the film thickness to ensure results consistency. 2.5.2. Moisture content The moisture content was assessed according to AACC method 44e1502. Pre-weighed aluminum pans containing edible films (approximately 0.7 g) were dried at 105 C in hot air oven until they reached to constant weight. The moisture content was calculated using the following equation:

Percentage of residual water content ¼

wi ewf 100 wi

where, wi and wf are the initial and final weight of the edible films, respectively. 2.5.3. Color characteristics Color characteristics of the films were assessed using Hunter lab colorimeter (Reston, USA). The CIE Lab color scale was used to assess L* (black to white), a* (red to green) and b* (yellow to blue) parameters. The total color difference (DE*) between the films and standard color plate was calculated using the following equation:

DE ¼

qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 2 2 2 ðDL* Þ þ ðDa* Þ þ ðDb* Þ

where, DL*, Da* and Db* are the luminosity, redness and yellowness intensity difference from the standard color plate. All measurements were performed in triplicates. 2.5.4. Opacity The opacity of films was evaluated based on a method described by Soukoulis, Behboudi-Jobbehdar, Yonekura, Parmenter, and Fisk (2014a). Edible Film specimen were cut into rectangle pieces and directly placed on plastic cuvettes. Absorbance was measured at 550 nm using spectrophotometer (BIOMATE-3S, Thermo Scientific, Waltham, MA, USA). An empty test cell was used as blank. Each film was assessed in three replicates. Film opacity was calculated using the following equation:

Opacity ¼

A550 thickness

2.4. Survival of probiotics in film forming solutions and films The viability of L. acidophilus, L. casei, L. rhamnosus and B. bifidum incorporated into the film forming solutions or films was based on a method proposed by De Lacey et al. (2012) with slight modification. Briefly, 1 ml of the solution was suspended in sterile PBS and vortexed for 30 s and then appropriate dilution series were prepared. For the films and before preparing dilution series, 1 g of film was transferred to 99 ml of sterile PBS and mixed gently by constant

2.5.5. Water vapor permeability (WVP) WVP of the films were assessed based on ASTM E96 gravimetric method. Film samples were attached tightly to the top of a cup filled with anhydrous calcium chloride using paraffin wax. The system was placed in a desiccator containing saturated sodium chloride solution (RH ¼ 75 ± 1%) and kept at 25 C. Weight changes in test cups were recorded periodically with an accuracy of

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Analysis of variance (ANOVA) followed by Duncan's post hoc comparison were used for the analysis (p < 0.05). Differences between survivability of probiotics during storage were assessed using General Linear Model (GLM). Data were analyzed using SPSS Software V.17.0 (SPSS Inc., USA).

decreased 3 log CFU, reaching 103 CFU/g film between 14 and 42 days of storage. The highest viability loss was observed in L. rhamnosus at the end of day 42, reaching 103 CFU/g film at 25 C. Similarly, Soukoulis et al. (2016) showed that starch-protein based edible films can adversely affect the survivability of L. rhamnosus GG at 25 C. In general, a decrease in water activity (increase in osmotic stress) in media can affect the viability of probiotics (Prasad, McJarrow, & Gopal, 2003). Viability of L. acidophilus and L. rhamnosus were in the range of recommended levels (107 CFU/g) during storage at 4 C. The viability of L. acidophilus was significantly higher than that of other probiotics at the end of storage. This may occur due to its higher tolerance to detrimental conditions of edible films during storage (Mortazavian et al., 2007). The least decrease in survivability belonged to L. acidophilus between days 14 and 28 of storage at 4 C and afterwards, L. casei on day 14. However, the least decrease in survival rate belonged to L. acidophilus between days 28 and 42 and then, L. casei on day 14 of storage at 25 C. On day 14, the viability of L. casei was higher than that of other probiotic bacteria and slightly reduced on day 42. The final counts of probiotic cells were nearly similar at 4 C (approximately 106 CFU/g) at the end of storage, except for B. bifidum. Therefore, the highest rate of probiotic death at the end of refrigerated storage belonged to B. bifidum. These findings are similar to the results of Rouhi, Mohammadi, Mortazavian, and Sarlak (2015). Homayouni, Azizi, Ehsani, Yarmand, and Razavi (2008) have reported that bifidobacteria are less responsive to low temperatures than lactobacilli. Probiotic bifidobacteria are generally obligate anaerobes. Despite the importance of their viability, surveys have shown poor viability of bifidobacteria. Several factors such as oxygen content and storage temperature have been found to reduce the viability of bifidobacteria (Akalin, Fenderya, & Akbulut, 2004). The ability of probiotic cells to interact with polysaccharides via electrostatic or hydrophobic interactions enables them to overcome limitations such as physicochemical and osmotic stress conditions (Burgain, Gaiani, Cailliez-Grimal, Jeandel, & Scher, 2013). The viable probiotic counts were in the range of currently most accepted value of 107 CFU/g at the end of day 28 at 4 C.

3. Results and discussion

3.2. Physical properties

3.1. Viability of probiotics in films during storage

3.2.1. Thickness Thickness is a critical parameter that determines the transparency, WVP and mechanical properties of the films improving the film ability to enhance the mechanical integrity of foods (Ghanbarzadeh & Almasi, 2011). Table 1 demonstrates the effects of probiotics on the thickness of CMC-based films. A significant difference was observed between the thickness of the control (40 mm) and probiotic edible films (50 mm). However, no correlation was observed between the thicknesses of edible films containing probiotics. Higher amounts of the total solids in the film-forming solution results in the film thickness. Similarly, Amankwaah (2013) reported that the thickness of the chitosan films significantly increased (p < 0.05) after the addition of green tea and grape seed extracts. Contrary to the results obtained in present study, Soukoulis et al. (2014a) reported no significant effect in thickness by addition of L. rhamnosus GG cells to prebiotic fibers. In another study, Soukoulis et al. (2014b) reported that the incorporation of probiotic bacterial cells into the film-forming solutions changed the film thickness.

0.0001 g. Slopes were calculated by linear regression (weight change versus time). Water vapor transmission rate (WVTR) was defined as the slope (g/h) divided by the transfer area (m2). WVP (g m1 h1 Pa1) was calculated as follows:

WVP ¼

WVTR X DP

where, DP is the difference of the vapor pressure between the two sides of films (1753.55 Pa) at 25 C (Shojaee-Aliabadi et al., 2014) and X is the film thickness (m). All measurements were performed in triplicate. 2.5.6. Tensile strength (TS) and elongation at break (EB) The mechanical properties of films were measured using texture analyzer (TA.XT Stable Micro System, UK). Films were conditioned in 50% relative humidity in a desiccator containing Mg(NO3)2 saturated solutions for 48 h and then cut into rectangular strips (1.5 cm 10 cm). Samples were placed between grips. The initial grip distance and the cross-head speed was set at 50 mm and 5 mm/min, respectively. To increase the reliability of assessments, each test was repeated five times. 2.5.7. Scanning electron microscopy (SEM) Film samples were deposited to liquid nitrogen and cryofractured for evaluation of film cross-section with SEM (Cambridge Scan-360). Samples were fixed in a sample holder and coated with gold particles. Micrographs were prepared using an accelerating voltage of 5 kV. 2.6. Statistical analysis

One of the most important effective factors on the viability of probiotics is temperature of distribution and storage conditions (Ferdousi et al., 2013). Fig. 1 shows the survival of probiotic strains incorporated in CMC films during storage at 4 and 25 C. No significant decreases were observed in probiotics’ viability during the drying process (day 0). As shown in Fig. 1, viability of L. acidophilus, L. casei, L. rhamnosus and B. bifidum includes a negative correlation with the storage conditions (p < 0.05). Films stored at 25 C showed significantly more reduction in viable number of the probiotic bacteria compared to those stored at 4 C (p < 0.05). The probiotics showed 4 log CFU at 25 C. These results indicate that the storage temperature plays an important role in loss of viability. Similarly, Odila Pereira et al. (2016), have found that the viability of probiotic B. animalis Bb-12 and L. casei 01 was higher in treatments stored at 4 C. Gialamas et al. (2010) have reported that low levels of bacterial metabolism at low temperatures are associated with a higher viability observed at 4 C. Moreover, some factors such as WVP, oxygen content, aw, heat-induced injuries, type of probiotic strain and mechanical stress can have an adverse effect on the viability of probiotics in films (Soukoulis et al., 2014b). Therefore, the viability of probiotics dropped from an initial population of 109 CFU/g film to 106 CFU/g film at the end of 14 days of storage at 25 C and

3.2.2. Moisture content (MC) The moisture content after drying not only affects the rate of viability decrease during long storage periods but it facilitates melting of edible films in the mouth (Kanmani & Lim, 2013). Therefore, the quantitative determination of MC in films is


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Fig. 1. Survivability of L. acidophilus, L. casei, L. rhamnosus and Bifidobacterium bifidum during storage at refrigerated (4 C) and room (25 C) temperatures.

Table 1 Physicochemical and optical properties of carboxymethyl cellulose films.* Treatment

Control L. acidophilus L. casei L. rhamnosus B.bifidum

Parameters Thickness (mm)

Residual water content (g/100 g of film)

WVP (g s1 m1 Pa1 107)

40.3 ± 1.0b 50.4 ± 5.0a 50 ± 3.0a 50 ± 4.0a 50 ± 3.0a

33.33 ± 0.57a 34.33 ± 0.52a 34.33 ± 0.57a 34 ± 0.50a 34.66 ± 0.52a

2.73 4.48 4.51 4.43 4.40

± ± ± ± ±

0.27b 0.47a 0.38a 0.45a 0.32a

Opacity 2.46 5.46 5.63 5.41 5.50

± ± ± ± ±

0.42b 0.43a 0.41a 0.40a 0.43a

L* 91.08 90.90 90.89 90.90 90.91

a* ± ± ± ± ±

0.15a 0.25b 0.21b 0.17b 0.21b

1.23 1.26 1.25 1.26 1.24

DE*

b* ± ± ± ± ±

0.31b 0.29a 0.15a 0.21a 0.25a

3.81 3.76 3.76 3.83 3.83

± ± ± ± ±

0.60a 0.84a 0.75a 0.72a 0.45a

0.64 4.75 4.76 4.75 4.78

± ± ± ± ±

0.15b 0.10a 0.22a 0.18a 0.26a

*Results are represented as mean ± standard deviation. Values with different superscript letters in each column are significantly different (p < 0.05).

important. The MC of the films is shown in Table 1. In the present study, the addition of probiotics caused no significant effect on the film moisture (p > 0.05). Similar results were found in previous studies (Soukoulis et al., 2014a). Glycerol in the film samples, as an effective humectant, can chemically maintain the water content and thereby inhibit the water evaporation (Enrione, Hill, & Mitchell, 2007). 3.2.3. Color and opacity Transparency (low opacity) is one of the common optical

properties of the films. Addition of probiotic cells resulted in a significant decrease (p < 0.05) in transparency of the edible films, compared to that of control film (Table 1). Addition of bacterial cells into the films could affect the light passing through the film, possibly due to increased light scattering (Kanmani & Lim, 2013). This finding is similar to that reported by Soukoulis et al. (2014b). Light absorbance by films could be considered as an advantage for packaged foods because of decrease in unwanted chemical reactions such as lipid oxidation and loss nutritional value. Acceptability of edible films and coats can be affected by

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unfavorable changes in color properties. Table 1 shows that L* value (indicating lightness) of the control film (91.08 ± 0.15) was significantly different from that of probiotic films (about 90.90 ± 0.25). These results showed that the clearness of the films decreased with the addition of probiotics. Films including probiotics showed a higher a* value (indicating greenness) and DE (indicating total color difference). Therefore, probiotic cells provided the films a darker appearance with a green tint which was not confirmed by visual observations. The b* value was not affected by the addition of probiotics. Ghanbarzadeh and Almasi (2011) reported a decrease in b* values caused by the addition of glycerol to film solutions. In contrast, Martins et al. (2012) reported that the moisture content values of films might change the reflection of light on the film surface (a* values decreased) leading to less red-colored films. Also, Ghanbarzadeh, Almasi, and Entezami (2010) reported an improvement in optical properties and reduction in yellowness by addition of CMC to starch films. 3.2.4. Water vapor permeability (WVP) Biopolymers are usually sensitive to absorption of moisture. WVP is one of the most important attributes of edible films that can be affected by factors such as integrity of the film, hydrophobic ratio, crystalline and amorphous ratio and thickness (Kanmani & Lim, 2013). WVP of films is important to avoid mass transfer from the food with surrounding environment (Bertuzzi, Vidaurre,

Table 2 Mechanical properties of carboxymethyl cellulose films*. Treatment

Tensile strength (MPa)

Control L. acidophilus L. casei L. rhamnosus B.bifidum

27.10 22.38 22.42 22.30 22.39

± ± ± ± ±

0.04a 0.11b 0.11b 0.15b 0.21b

Elongation at break (%) 17.67 12.41 12.43 12.30 12.38

± ± ± ± ±

0.58a 0.41b 0.50b 0.29b 0.55b

*Results are represented as mean ± standard deviation. Values with different superscript letters in each column are significantly different (p < 0.05).

Armada, & Gottifredi, 2007;; Kristo & Biliaderis, 2006). The WVP values of the films with or without probiotics are described in Table 1. As shown in this table, WVP of the CMC films increased up to 50% by the addition of probiotics, compared to control. Possible pins and holes created on the surface of films deteriorated the cohesion of the films that increased the moisture absorption. However, no significant difference was observed within the different probiotic films (p < 0.05). Although the WVP of probiotic CMC-based films was 4.48 ± 0.47 g s1 m1 Pa1, Soukoulis et al. (2014a) reported 0.681 ± 0.009 g s1 m1 Pa1 for WVP of probiotic pullulan/starch films. This might occur due to differences between types of biopolymers and methods of film preparation. Some studies investigated the effects of the other components on WVP of CMC-based films. Similarly, Dashipour et al. (2015) reported that the WVP of CMC films increased with increasing Zataria multiflora essential oil concentration. The increase in WVP resulting from the incorporation of probiotics was higher than WVP results usually reported for polymer mixtures applied in packaging. In contrast, Bifani et al. (2007) found that murta (Ugni molinae Turcz) leaves extract decreased WVP of CMC-based films due to the structural modification of the CMC network by the extract. Addition of secondary biopolymer could improve the structure and WVP of films. Ghanbarzadeh et al. (2010) reported that the WVP of starch/ CMC films decreased with the increase of CMC content used to improve the structure of films. 3.2.5. Mechanical properties of films Edible films should be adequately resistant to external stresses to use as food packaging materials,. Furthermore, films must be flexible and strong during packaging and storage (Pranoto, Salokhe, & Rakshit, 2005). Tensile strength (TS) and elongation at break (EB) are two key indicators of edible films used for packaging. The TS and EB of the control and probiotic films are shown in Table 2. In the present study, addition of probiotic cells into CMC films plasticized with glycerol significantly reduced tensile strength. This result is in agreement with results of Kanmani and Lim (2013); in which,

Fig. 2. SEM cross-section image of CMC edible films: (a) control, (b) L. acidophilus, (c) L. casei, (d) L. rhamnosus and (e) B. bifidum.


addition of bacterial cells into pure pullulan films reduced the TS (3.76 ± 0.68 MPa). In contrast, Gialamas et al. (2010) reported that mechanical properties of the sodium caseinate films were not affected by the bacterial cells. In the current study, addition of probiotics to film matrix was caused to significantly less EB, indicating the less flexibility of films. However, TS and EB showed no significant difference within probiotic films (p > 0.05). With addition of probiotic cells, cohesiveness of the polymer chains was interrupted. Therefore, the high glycerol concentration was used in film samples. Glycerol as a plasticizer is one of the most important effective factors in mechanical properties. It reduces the intermolecular forces between polymers, which reduces TS and increases EB. This phenomenon is a consequence of the increase in molecular mobility and free volume in polymer chains (Hosseini, Rezaei, Zandi, & Ghavi, 2013; Rouhi, Razavi, & Mousavi, 2017). 3.2.6. Scanning electron microscopy (SEM) In this study, uniformity and microstructural characterizations of edible films were assessed using scanning electron microscopy (SEM). Results of cross-section micrographs from control and probiotic films are shown in Fig. 2. The SEM results showed that the structure of control CMC film was homogeneous, uniform and compact with no micropores. However, probiotic films showed a higher number of holes than control films. The bacterial cells were embedded in the film matrix (tiny rod-like shapes) that could result in increasing cell-protective effects of films. These results was confirmed by increasing the WVP of probiotic films (Table 1) and decreasing their flexibility and tensile strength (Table 2). This is in contrary to the findings of Odila Pereira et al. (2016) who reported that the effect of microorganism incorporation on the structural conformation of films was insignificant. 4. Conclusions In the present study, probiotic edible films stored at 5 C showed significantly the higher viability of probiotics than 25 C. Among probiotic strains, L. acidophilus exhibited higher viability during storage. Significant increase in thickness, opacity and WVP and significant decrease in TS, EB and cohesion of the film structure were observed in probiotic films compared to control film. However, the probiotic strains showed no significant effect on the physical and mechanical properties of the edible films. CMC-based edible films could act as a suitable carrier for some probiotic strains in food packaging during refrigerated temperatures. Future studies could be aimed to improve the mechanical strength and/or WVP by blending biopolymers and crosslinking agents with the probiotic CMC-based edible film. Moreover, the effects of addition of probiotics to these films on the sensory properties could be investigated for their application in food packaging. Acknowledgement We are grateful to the Department of Food Science and Technology, National Nutrition and Food Technology Research Institute (Shahid Beheshti University of Medical Sciences) for support of this study. References Akalin, A. S., Fenderya, S., & Akbulut, N. (2004). Viability and activity of bifidobacteria in yoghurt containing fructooligosaccharide during refrigerated storage. International Journal of Food Science and Technology, 39, 613e621. Altamirano-Fortoul, R., Moreno-Terrazas, R., Quezada-Gallo, A., & Rosell, C. M. (2012). Viability of some probiotic coatings in bread and its effect on the crust mechanical properties. Food Hydrocolloids, 29(1), 166e174. Amankwaah, C. (2013). Incorporation of selected plant extracts into edible chitosan

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films and the effect on the antiviral, antibacterial and mechanical properties of the material. The Ohio State University. Bertuzzi, M., Vidaurre, E. C., Armada, M., & Gottifredi, J. (2007). Water vapor permeability of edible starch based films. Journal of Food Engineering, 80(3), 972e978. Bifani, V., Ramírez, C., Ihl, M., Rubilar, M., García, A., & Zaritzky, N. (2007). Effects of murta (Ugni molinae Turcz) extract on gas and water vapor permeability of carboxymethylcellulose-based edible films. LWT-Food Science and Technology, 40(8), 1473e1481. Biswal, D. R., & Singh, R. P. (2004). Characterisation of carboxymethyl cellulose and polyacrylamide graft copolymer. Carbohydrate Polymers, 57(4), 379e387. Burgain, J., Gaiani, C., Cailliez-Grimal, C., Jeandel, C., & Scher, J. (2013). Encapsulation of Lactobacillus rhamnosus GG in microparticles: Influence of casein to whey protein ratio on bacterial survival during digestion. Innovative Food Science & Emerging Technologies, 19, 233e242. Champagne, C. P., Ross, R. P., Saarela, M., Hansen, K. F., & Charalampopoulos, D. (2011). Recommendations for the viability assessment of probiotics as concentrated cultures and in food matrices. International Journal of Food Microbiology, 149(3), 185e193. Dashipour, A., Razavilar, V., Hosseini, H., Shojaee-Aliabadi, S., German, J. B., Ghanati, K., et al. (2015). Antioxidant and antimicrobial carboxymethyl cellulose films containing Zataria multiflora essential oil. International Journal of Biological Macromolecules, 72(0), 606e613. Emmambux, M. N., & Stading, M. (2007). In situ tensile deformation of zein films with plasticizers and filler materials. Food Hydrocolloids, 21(8), 1245e1255. Enrione, J. I., Hill, S. E., & Mitchell, J. R. (2007). Sorption behavior of mixtures of glycerol and starch. Journal of Agricultural and Food Chemistry, 55(8), 2956e2963. nez, A., Mun ~ oz, J. A., & Ibarz, A. (2011). Edible films Falguera, V., Quintero, J. P., Jime and coatings: Structures, active functions and trends in their use. Trends in Food Science & Technology, 22(6), 292e303. Ferdousi, R., Rouhi, M., Mohammadi, R., Mortazavian, A. M., Khosravi-Darani, K., & Rad, A. H. (2013). Evaluation of probiotic survivability in yogurt exposed to cold chain interruption. Iranian Journal of Pharmaceutical Research: IJPR, 12(Suppl), 139. Garavand, F., Rouhi, M., Razavi, S. H., Cacciotti, I., & Mohammadi, R. (2017). Improving the integrity of natural biopolymer films used in food packaging by crosslinking approach: A review. International Journal of Biological Macromolecules, 104, 687e707. Ghanbarzadeh, B., & Almasi, H. (2011). Physical properties of edible emulsified films based on carboxymethyl cellulose and oleic acid. International Journal of Biological Macromolecules, 48(1), 44e49. Ghanbarzadeh, B., Almasi, H., & Entezami, A. A. (2010). Physical properties of edible modified starch/carboxymethyl cellulose films. Innovative Food Science & Emerging Technologies, 11(4), 697e702. Gialamas, H., Zinoviadou, K. G., Biliaderis, C. G., & Koutsoumanis, K. P. (2010). Development of a novel bioactive packaging based on the incorporation of Lactobacillus sakei into sodium-caseinate films for controlling Listeria monocytogenes in foods. Food Research International, 43(10), 2402e2408. mez-Guille n, M., Pe rez-Mateos, M., Go mez-Estaca, J., Lo pez-Caballero, E., Go nez, B., & Montero, P. (2009). Fish gelatin: A renewable material for Gime developing active biodegradable films. Trends in Food Science & Technology, 20(1), 3e16. Homayouni, A., Azizi, A., Ehsani, M., Yarmand, M., & Razavi, S. (2008). Effect of microencapsulation and resistant starch on the probiotic survival and sensory properties of synbiotic ice cream. Food Chemistry, 111(1), 50e55. Hosseini, S. F., Rezaei, M., Zandi, M., & Ghavi, F. F. (2013). Preparation and functional properties of fish gelatinechitosan blend edible films. Food Chemistry, 136(3), 1490e1495. Iaconelli, C., Lemetais, G., Kechaou, N., Chain, F., Bermúdez-Humar an, L. G., Langella, P., et al. (2015). Drying process strongly affects probiotics viability and functionalities. Journal of Biotechnology, 214, 17e26. Jankovic, I., Sybesma, W., Phothirath, P., Ananta, E., & Mercenier, A. (2010). Application of probiotics in food productsdchallenges and new approaches. Current Opinion in Biotechnology, 21(2), 175e181. Jridi, M., Hajji, S., Ayed, H. B., Lassoued, I., Mbarek, A., Kammoun, M., et al. (2014). Physical, structural, antioxidant and antimicrobial properties of gelatinechitosan composite edible films. International Journal of Biological Macromolecules, 67, 373e379. Kanmani, P., & Lim, S. T. (2013). Development and characterization of novel probiotic-residing pullulan/starch edible films. Food Chemistry, 141(2), 1041e1049. Kristo, E., & Biliaderis, C. G. (2006). Water sorption and thermo-mechanical properties of water/sorbitol-plasticized composite biopolymer films: Caseinateepullulan bilayers and blends. Food Hydrocolloids, 20(7), 1057e1071. pez-Caballero, M., Go mez-Estaca, J., Go mez-Guille n, M., & De Lacey, A. L., Lo Montero, P. (2012). Functionality of Lactobacillus acidophilus and Bifidobacterium bifidum incorporated to edible coatings and films. Innovative Food Science & Emerging Technologies, 16, 277e282. Martins, J. T., Cerqueira, M. A., Bourbon, A. I., Pinheiro, A. C., Souza, B. W., & Vicente, A. A. (2012). Synergistic effects between k-carrageenan and locust bean gum on physicochemical properties of edible films made thereof. Food Hydrocolloids, 29(2), 280e289. Mortazavian, A., Ehsani, M., Mousavi, S., Rezaei, K., Sohrabvandi, S., & Reinheimer, J. (2007). Effect of refrigerated storage temperature on the viability of probiotic

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micro-organisms in yogurt. International Journal of Dairy Technology, 60(2), 123e127. Odila Pereira, J., Soares, J., Sousa, S., Madureira, A. R., Gomes, A., & Pintado, M. (2016). Edible films as carrier for lactic acid bacteria. LWT - Food Science and Technology, 73, 543e550. Pranoto, Y., Salokhe, V. M., & Rakshit, S. K. (2005). Physical and antibacte rial properties of alginate-based edible film incorporated with garlic oil. Food Research International, 38(3), 267e272. Prasad, J., McJarrow, P., & Gopal, P. (2003). Heat and osmotic stress responses of probiotic Lactobacillus rhamnosus HN001 (DR20) in relation to viability after drying. Applied and Environmental Microbiology, 69(2), 917e925. Rouhi, M., Mohammadi, R., Mortazavian, A., & Sarlak, Z. (2015). Combined effects of replacement of sucrose with d-tagatose and addition of different probiotic strains on quality characteristics of chocolate milk. Dairy Science & Technology, 95(2), 115e133. Rouhi, M., Razavi, S. H., & Mousavi, S. M. (2017). Optimization of crosslinked poly (vinyl alcohol) nanocomposite films for mechanical properties. Materials Science and Engineering: C, 71, 1052e1063. Shojaee-Aliabadi, S., Hosseini, H., Mohammadifar, M. A., Mohammadi, A., Ghasemlou, M., Hosseini, S. M., et al. (2014). Characterization of k-carrageenan films incorporated plant essential oils with improved antimicrobial activity.

Carbohydrate Polymers, 101, 582e591. Skurtys, O., Acevedo, C., Pedreschi, F., Enrione, J., Osorio, F., & Aguilera, J. (2010). Food hydrocolloid edible films and coatings. Food Hydrocolloids: Characteristics, Properties. Nova Science Publishers, Inc. Soukoulis, C., Behboudi-Jobbehdar, S., Yonekura, L., Parmenter, C., & Fisk, I. D. (2014a). Stability of Lactobacillus rhamnosus GG in prebiotic edible films. Food Chemistry, 159, 302e308. Soukoulis, C., Singh, P., Macnaughtan, W., Parmenter, C., & Fisk, I. D. (2016). Compositional and physicochemical factors governing the viability of Lactobacillus rhamnosus GG embedded in starch-protein based edible films. Food Hydrocolloids, 52, 876e887. Soukoulis, C., Yonekura, L., Gan, H.-H., Behboudi-Jobbehdar, S., Parmenter, C., & Fisk, I. (2014b). Probiotic edible films as a new strategy for developing functional bakery products: The case of pan bread. Food Hydrocolloids, 39, 231e242. Thushara, R. M., Gangadaran, S., Solati, Z., & Moghadasian, M. H. (2016). Cardiovascular benefits of probiotics: A review of experimental and clinical studies. Food & Function, 7(2), 632e642. Zugenmaier, P. (2006). Materials of cellulose derivatives and fiber-reinforced cellulose-polypropylene composites: Characterization and application. Pure and Applied Chemistry, 78(10), 1843e1855.