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

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LWT - Food Science and Technology

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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
b
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

a r t i c l e i n f o a b s t r a c t

Article history: In this study, survival of four probiotic strains (Lactobacillus acidophilus, L. casei, L. rhamnosus and Bifi-
Received 22 December 2016 dobacterium bifidum) immobilized in edible films based on carboxymethyl cellulose (CMC) and physi-
Received in revised form cochemical properties of films were investigated during 42 days of storage at 4 and 25
C. Results showed
15 August 2017
a significant decrease in viability of bacterial cells during 42 days of storage at 25
C. However, viability of
Accepted 21 August 2017
Available online 23 August 2017
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 phys-
Keywords:
Carboxymethyl cellulose
icochemical changes were observed among probiotic films containing different strains. Therefore,
Edible film incorporation of some probiotic strains in edible coats and films could be their suitable carrier at
Probiotic refrigerated temperatures.
Viability © 2017 Elsevier Ltd. All rights reserved.

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 im-
mune 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: mortazvn@sbmu.ac.ir (A.M. Mortazavian), s_shojaee@sbmu.
ac.ir (S. Shojaee-Aliabadi).
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 (Altamirano-
Fortoul, Moreno-Terrazas, Quezada-Gallo, & Rosell, 2012). The
term “edible films” can be defined as a thin layer of natural poly-
mers 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
environment (Emmambux & Stading, 2007 and Go
mez-Guille
n
et al., 2009). The films protect the food products from deteriora-
tion and improve food quality due to roles of moisture barrier and
additive carrier. Therefore, they have been recommended as po-
tential vehicles for the delivery of functional compounds
(Emmambux & Stading, 2007;; Falguera, Quintero, Jime
nez, Mun
~ oz,
& 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,

http://dx.doi.org/10.1016/j.lwt.2017.08.066
0023-6438/© 2017 Elsevier Ltd. All rights reserved.
 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 limi-
tation of cellulose applications in food technology is water insolu-
bility. 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 car-
boxymethyl 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. Car-
boxymethyl 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 incu-
bated 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
Lo
mez-Estaca, Go
mez-Guille
n, & Montero, 2012).
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.
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 vor-
texed 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
55
agitation in a shaker incubator at 37
C for 1 h to release the bac-
teria. 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:
wi ewf
Percentage of residual water content ¼  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:
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
DE ¼ 2
ðDL* Þ þ ðDa* Þ þ ðDb* Þ
2 2
where, DL*, Da* and Db* are the luminosity, redness and yellowness
intensity difference from the standard color plate. All measure-
ments 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:
A550
Opacity ¼
thickness
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
56 B. Ebrahimi et al. / LWT - Food Science and Technology 87 (2018) 54e60
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:
WVTR
WVP ¼ 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 cryo-
fractured for evaluation of film cross-section with SEM (Cam-
bridge 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
Analysis of variance (ANOVA) followed by Duncan's post hoc
comparison were used for the analysis (p < 0.05). Differences be-
tween survivability of probiotics during storage were assessed us-
ing General Linear Model (GLM). Data were analyzed using SPSS
Software V.17.0 (SPSS Inc., USA).
3. Results and discussion
3.1. Viability of probiotics in films during storage
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 sig-
nificant 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 <3 log CFU decrease in viability at the end of storage at 4
C
and >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
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 signifi-
cantly 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 (approx-
imately 106 CFU/g) at the end of storage, except for B. bifidum.
Therefore, the highest rate of probiotic death at the end of refrig-
erated 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 re-
ported 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 ox-
ygen 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 over-
come limitations such as physicochemical and osmotic stress con-
ditions (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.
3.2. Physical properties
3.2.1. Thickness
Thickness is a critical parameter that determines the trans-
parency, 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 dif-
ference 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 pro-
biotics. Higher amounts of the total solids in the film-forming so-
lution 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.
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
 B. Ebrahimi et al. / LWT - Food Science and Technology 87 (2018) 54e60 57

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 Parameters

Thickness Residual water content WVP Opacity L* a* b* DE*

(mm) (g/100 g of film) (g s1 m1 Pa1  107)

Control 40.3 ± 1.0b 33.33 ± 0.57a 2.73 ± 0.27b 2.46 ± 0.42b 91.08 ± 0.15a 1.23 ± 0.31b 3.81 ± 0.60a 0.64 ± 0.15b
L. acidophilus 50.4 ± 5.0a 34.33 ± 0.52a 4.48 ± 0.47a 5.46 ± 0.43a 90.90 ± 0.25b 1.26 ± 0.29a 3.76 ± 0.84a 4.75 ± 0.10a
L. casei 50 ± 3.0a 34.33 ± 0.57a 4.51 ± 0.38a 5.63 ± 0.41a 90.89 ± 0.21b 1.25 ± 0.15a 3.76 ± 0.75a 4.76 ± 0.22a
L. rhamnosus 50 ± 4.0a 34 ± 0.50a 4.43 ± 0.45a 5.41 ± 0.40a 90.90 ± 0.17b 1.26 ± 0.21a 3.83 ± 0.72a 4.75 ± 0.18a
B.bifidum 50 ± 3.0a 34.66 ± 0.52a 4.40 ± 0.32a 5.50 ± 0.43a 90.91 ± 0.21b 1.24 ± 0.25a 3.83 ± 0.45a 4.78 ± 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 re-
actions such as lipid oxidation and loss nutritional value.
Acceptability of edible films and coats can be affected by
58 B. Ebrahimi et al. / LWT - Food Science and Technology 87 (2018) 54e60

unfavorable changes in color properties. Table 1 shows that L* value
(indicating lightness) of the control film (91.08 ± 0.15) was signif-
icantly 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,
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 pro-
biotic 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 result-
ing from the incorporation of probiotics was higher than WVP re-
sults 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

Table 2
Mechanical properties of carboxymethyl cellulose films*. Edible films should be adequately resistant to external stresses
to use as food packaging materials,. Furthermore, films must be
Treatment Tensile strength Elongation at break
flexible and strong during packaging and storage (Pranoto, Salokhe,
(MPa) (%)
& Rakshit, 2005).
Control 27.10 ± 0.04a 17.67 ± 0.58a
Tensile strength (TS) and elongation at break (EB) are two key
L. acidophilus 22.38 ± 0.11b 12.41 ± 0.41b
L. casei 22.42 ± 0.11b 12.43 ± 0.50b indicators of edible films used for packaging. The TS and EB of the
L. rhamnosus 22.30 ± 0.15b 12.30 ± 0.29b control and probiotic films are shown in Table 2. In the present
B.bifidum 22.39 ± 0.21b 12.38 ± 0.55b study, addition of probiotic cells into CMC films plasticized with
*Results are represented as mean ± standard deviation. Values with different su- glycerol significantly reduced tensile strength. This result is in
perscript letters in each column are significantly different (p < 0.05). 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.
 B. Ebrahimi et al. / LWT - Food Science and Technology 87 (2018) 54e60
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, indi-
cating 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 pro-
biotic 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. How-
ever, 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 pro-
biotics to these films on the sensory properties could be investi-
gated for their application in food packaging.
Acknowledgement
We are grateful to the Department of Food Science and Tech-
nology, National Nutrition and Food Technology Research Institute
(Shahid Beheshti University of Medical Sciences) for support of this
study.
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