LWT - Food Science and Technology 73 (2016) 543e550

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LWT - Food Science and Technology

journal homepage: www.elsevier.com/locate/lwt

Edible lms as carrier for lactic acid bacteria

Soares, Se

rgio Sousa, Ana Raquel Madureira, Ana Gomes,
Joana Odila Pereira, Jose
Manuela Pintado*

rio Associado, Escola Superior de Biotecnologia, Universidade Cato

lica Portuguesa/Porto, Rua
CBQF e Centro de Biotecnologia e Qumica Fina e Laborato
~o Vital, Apartado 2511, 4202-401 Porto, Portugal
Arquiteto Loba

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 22 January 2016
Received in revised form
25 May 2016
Accepted 28 June 2016
Available online 29 June 2016

The use of edible coatings and lms formulated with bioactive compounds in food products in order to
convey new functionalities or extend shelf-life opens new possibilities as a carrier for functional lactic
acid bacteria. In this work the main objective was to study the stability of probiotic microorganisms, viz.
Bidobacterium animalis Bb-12 and Lactobacillus casei-01, in edible lm formulations based on whey
protein isolate (WPI).
The results demonstrated a loss of bacterial cell viability of ca. 3 log cycles (reaching 106 CFU/g lm)
until 60 d at both 23 and 4
C, noting that the most marked decrease was at 23
C for both strains.
Bidobacterium animalis Bb-12 remained viable for a longer period of time and with less decrease in its
cell numbers (108 CFU/g lm).
Physical properties, namely color, water activity, thickness, youngs modulus, tensile strength, elongation at break and the molecular structure of WPI lms were maintained stable throughout the storage
period at both temperatures tested.
Edible lms incorporated with probiotics can be good carriers for these to be ingested together with
food products.
2016 Elsevier Ltd. All rights reserved.

Keywords:
Edible coatings/lms
Whey protein isolate
Lactic acid bateria
Bidobacterium
Lactobacillus

1. Introduction
Edible coatings and lms are natural polymers used to retain the
appearance and physicochemical properties of foods during storage. When adsorbed on the surface of the food, they may promote
protection against moisture migration or oxidation and control of
microbial growth (Han, 2000; Naushad & Stading, 2007).
Several reviews have demonstrated that edible lms can be
prepared from different structural materials such as lipids
(Hambleton, Debeaufort, Bonnotte, & Voilley, 2009), poly
nez, Fabra, Talens, & Chiralt, 2013; Jridi et al.,
saccharides (Jime
2014) and proteins (Ramos, Fernandes, Silva, Pintado, & Malcata,
2011; Ramos et al., 2013; Ramos et al., 2012) or by combining two
or several of these compounds. Protein based lms have received
considerable attention because they have advantages over others,
in particular, due to their mechanical properties that are generally
better since proteins have a distinctive structure, which confers a
wider range of functional properties, (Cuq, Gontard, & Guilbert,
1995; Gennadios & Weller, 1990; Wittaya, 2012).
Edible packaging systems mainly deal with maintaining or
* Corresponding author.
E-mail address: mpintado@porto.ucp.pt (M. Pintado).
http://dx.doi.org/10.1016/j.lwt.2016.06.060
0023-6438/ 2016 Elsevier Ltd. All rights reserved.

increasing the quality and safety of packaged foods, extending their

shelf life. On the other hand, bioactive packaging systems (coating/
lms) are a new technology concept to assist in the production of
functional foods, where bioactive principles are designed to be
contained within coatings or coating materials (Korhonen, 2002) in
order to have a direct impact on consumer health, creating healthy
foods packed for a specic bioactive attribute (Han, Lederer,
McDaniel, & Zhao, 2005). A bioactive edible coating/lm is
dened as a protective coating applied to the surface of a food with
addition of functional compounds such as antioxidants, color
agents, avors, nutrients, probiotics, prebiotics and antimicrobial
agents that increase the functionality of the coating/lm (Min,
Harris, & Krochta, 2005; Pranoto, Rakshit, & Salokhe, 2005;
Salmieri & Lacroix, 2006). These coatings/lms have the function
of protecting and controlling the release rate at which functional
compounds are provided in the desired location (Pothakamury &

novas, 1995). To promote these characteristics, the
Barbosa-Ca
coating must be prepared in accordance with the incorporated
compound and the nature of the food. In this sense, the development of protein-based lms is considered suitable for use as the
carrier of functional ingredients and covers a wide range of foods.
Various studies have pointed out the benecial effects of probiotics (Daoud & Hani, 2013; Nagpal et al., 2012; Vasiljevic & Shah,

544

J. Odila Pereira et al. / LWT - Food Science and Technology 73 (2016) 543e550

2008). Probiotics have key functionalities including: relief from

lactose intolerance, increased resistance to intestinal invasion by
pathogenic bacterial species, stimulation of the immune system
and possible protection against colon cancer (Zubillaga et al., 2001).
Another functionality is the antimicrobial capacity, through bacteriocins, or by competition in situ (Messaoudi et al., 2013). For
these reasons, the incorporation of probiotics into food products
has been increasing, to assure safe and healthy products.
Though a large number of genera and species of microorganisms
are considered as potential probiotics (Holzapfel, Haberer, Snel,
Schillinger, & Huis int Veld, 1998; Shah & Ravla, 2004), the most
common bacteria commercially available are from the genera
Lactobacillus and Bidobacterium. Probiotics incorporated into food
products made by fermentation of milk, cereals, fruits and vegetables and meat are currently receiving great attention (Gupta &
_
Abu-Ghannam, 2012; Koozyn-Krajewska
& Dolatowski, 2012;
le, Auty, Brunton,
Rouhi, Sohrabvandi, & Mortazavian, 2013; Ro
Gormley, & Butler, 2010). However, the main challenge for their
successful incorporation is ensuring their viability (Madureira,
~o, Gomes, Pintado, & Malcata, 2011).
Branda
The survival of probiotics during the production of foods can
lead to signicant losses of viability due to mechanical and/or heat
treatments, presence of oxygen and osmotic stress mechanisms

rquez, 2013; Fu & Chen, 2011), hence, it is important to
(Bustos & Bo
protect and incorporate them in coated foods. Preparation of such
bioactive coatings is a very innovative eld in the food industry and
therefore only a small number of studies on this topic are available

pez de Lacey, Lo

pez-Caballero, Go

mez(Kanmani & Lim, 2013; Lo

mez-Guille

n, & Montero, 2012; Soukoulis et al., 2014;
Estaca, Go
Tapia et al., 2007).
The rst study describes the incorporation of Bidobacterium
lactis Bb-12, in alginate-gellan coatings and lms for coating of
fresh-cut apple and papaya. Storage values of probiotic viable
cell numbers higher than 106 CFU/g were maintained for 10 d,
thus maintaining the viability of probiotics in lms applied to

pez de Lacey et al. (2012)
fresh fruit (Tapia et al., 2007). Lo
studied the incorporation of Lactobacillus acidophilus and Bidobacterium bidum into gelatin edible coatings applied to sh and
assessed its effect during storage. Lactic acid bacteria remained
viable (above 108 CFU/g) and the H2S producing microorganisms
were reduced in 2 log cycles. Additionally, this coated sh was
suited to treatment with high pressure (200 MPa/10 min/20
C)
which resulted in a reduction of total viable counts (<2 log cy
pez de Lacey et al., 2012). Kanmani
cles) after 13 d of storage (Lo
and Lim (2013) reported that starch-pullulan based edible
lms incorporated with probiotic strains, Lactobacillus
reuteri ATCC 55730, Lactobacillus plantarum GG ATCC 53103
and L. acidophilus DSM 20079 maintained the viability of the
selected strains after 30 d of storage at 4
C. More recently,
Soukoulis, Behboudi-Jobbehdar, et al. (2014) and Soukoulis,
Yonekura, et al. (2014) demonstrated the development of a probiotic pan bread by application of edible coatings based on sodium alginate or sodium alginate/whey protein concentrate

incorporated with Lactobacillus rhamnosus GG. Lactobacillus

rhamnosus GG viability was improved signicantly by the presence of whey protein concentrate throughout 7 d storage
(Soukoulis et al., 2014).
All the above studies represent innovations of great interest and
of promising nature when it comes to using edible coatings and lms
as carriers of probiotics, opening new possibilities for the development of novel functional foods. However, no study has considered
until now, the characteristics and bacterial cell viability of stable
dried edible whey protein lms incorporated with probiotics.
So, considering that there are very few studies on this topic, in
particular using protein-based lms, the objective of this study was
to evaluate the stability of two probiotic microorganisms in edible
lms based on whey protein formulations opening new possibilities for coating food products.
2. Material and methods
2.1. Bacterial strains, media and growth conditions
Probiotic strains, Bidobacterium animalis Bb-12 and Lactobacillus casei-01 obtained from Christian Hansen (Denmark) were
stored at 80
C in de ManeRogosaeSharpe (MRS) broth (Biokar
Diagnostics, France) supplemented with 30% (v/v) sterile glycerol.
The aforementioned microorganisms were reactivated, and precultures were prepared in MRS medium supplemented with
lter-sterilized 0.05% (w/v) L-cysteine$HCl (Fluka, St. Gallen,
Switzerland), in order to lower the redox potential, and incubated
at 37
C during 24 h under anaerobic conditions, in a plastic
anaerobic jar with an AnaeroGen sachet (an atmosphere generation
system, Oxoid, Basingstoke, England). Subsequently, grown cells
were harvested by centrifugation at 4000 rpm for 30 min, at 4
C.
The supernatant was discarded and the pellet was resuspended in a
0.9% (w/v) NaCl solution.
2.2. Formulation of the lms
The lm-forming solutions, one for each probiotic strain, were
prepared by slowly dissolving 10% (w/w) whey protein isolate

s, France) in
(WPI) powder (Armor Proteines, Saint Brice en Cogle

rez-Gago and Krochta (2002).
deionized water, according to Pe
Glycerol was added at 5% (w/w), as plasticizer, and the resulting
solutions were magnetically stirred for approximately 2 h. Subsequently, the solutions were heated in a water bath at 80
C, for
20 min under continuous agitation and cooled to room temperature for 1.5 h.
Afterwards, 5% (w/w) inoculum of each probiotic strain was
added to each lm solution to attain a nal concentration of
109 CFU/mL. To prepare the lms, the same amount (300 mL) of
each solution was poured onto level Teon plates (38  34 cm),
so as to control the lm thickness. The lm solutions were
allowed to dry at room conditions under a sterile environment
(in a vertical laminar-ow cabinet, ca. 23
C and 50% relative

Table 1
Viable cell numbers (Log CFU/g) of B. animalis Bb-12 and L. casei-01 incorporated in whey protein based lms stored under vacuum for 60 days at 23 1
C and 4 1
C.
23
C

4
C

B. animalis Bb-12
Film-forming solutions
0 days
3 days
5 days
10 days
40 days
60 days
Note:

a, b, c, d, e, f

9.06
8.98
8.81
7.90
7.17
6.66
6.09

0.03
0.01a
0.04b
0.01c
0.01d
0.01e
0.02f

L. casei-01
9.07
8.93
8.53
7.43
6.53
6.40
5.98

0.03
0.04a
0.04b
0.04c
0.05d
0.02e
0.01f

B. animalis Bb-12
9.06
8.94
8.64
8.56
8.41
8.07
7.90

0.03
0.03a
0.03b
0.03c
0.03d
0.04e
0.01f

Means within the same columns, labeled with the same letter, do not statistically differ from each other (p > 0.05).

L. casei-01
9.07
8.93
8.55
8.34
8.75
7.17
6.95

0.03
0.04a
0.03b
0.02c
0.02d
0.03e
0.01f

Table 2
Evolution of thickness (mm) and water activity (Aw) of control and probiotic lms incorporated with B. animalis Bb-12 and L. casei-01 during storage for 60 days at 23 1
C
(a) and 4 1
C (b).
B. animalis Bb-12

Control
Thickness (mm)
a)
0 days
3 days
5 days
10 days
40 days
60 days
b)
0 days
3 days
5 days
10 days
40 days
60 days
Note:

L. casei-01

Thickness (mm)

aw

aw

Thickness (mm)

aw

0.396
0.395
0.400
0.399
0.397
0.397

0.006a
0.003a
0.007a
0.008a
0.008a
0.001a

0.578
0.577
0.575
0.576
0.577
0.576

0.004a
0.003a
0.001a
0.001a
0.001a
0.001a

0.408
0.407
0.408
0.405
0.409
0.408

0.006a
0.001a
0.001a
0.002a
0.001a
0.003a

0.637
0.637
0.638
0.637
0.637
0.637

0.001b
0.001b
0.001b
0.002b
0.001b
0.001b

0.395
0.395
0.396
0.396
0.397
0.395

0.001a
0.001a
0.001a
0.002a
0.007a
0.002a

0.738
0.738
0.737
0.736
0.738
0.737

0.001c
0.002c
0.001c
0.003c
0.001c
0.001c

0.405
0.406
0.404
0.404
0.402
0.403

0.001a
0.001a
0.000a
0.001a
0.000a
0.001a

0.589
0.588
0.590
0.588
0.589
0.589

0.002a
0.002a
0.003a
0.001a
0.001a
0.001a

0.410
0.408
0.410
0.409
0.410
0.410

0.001a
0.000a
0.001a
0.001a
0.002a
0.001a

0.551
0.550
0.550
0.551
0.551
0.549

0.001b
0.001b
0.002b
0.001b
0.001b
0.003b

0.399
0.400
0.400
0.398
0.399
0.397

0.001a
0.002a
0.001a
0.001a
0.000a
0.002a

0.570
0.570
0.571
0.570
0.570
0.570

0.001c
0.002c
0.001c
0.001c
0.001c
0.000c

a, b, c

, Means within the same columns, labeled with the same letter, do not statistically differ from each other (p > 0.05).

humidity, RH) for 24 h, according to the procedure of Gounga, Xu,

and Wang (2007) and Oses, Fernandez-Pan, Mendoza, and Mate
(2009). Once formed, the lms were peeled off and conditioned
at 23 2
C and 50 2% RH, in a controlled temperature and
humidity storage room (Packaging Center, CBQF, Porto Portugal),
for at least 72 h prior to testing (ASTM, 2000). All lm physical
measurements described below were conducted also at 23 2
C
and 50 2% RH.
2.3. Enumeration of bacteria in the lms throughout storage
The viability of incorporated bacteria was studied in the lms
during 60 d of storage under vacuum at different temperature
conditions, i.e. 23
C and 4
C.
Three lms were studied, without bacteria (control F0), with B.
animalis Bb-12 (Fba) and with L. casei-01 (Flc).
Each lm was cut into circular discs with 1 cm of diameter and
stored in plastic bags for 60 d under abovementioned conditions
and sampled at 0, 3, 5, 10, 40 and 60 d. At each sampling point, the
disks were put in a sterile ask and 2 mL of sterile peptone water
(1 g/L) were added and homogenized by vortexing during 1 min to
completely dissolve the lm and extract the bacteria.
Appropriate sequential 10-fold dilutions were done in sterile
peptone water and plated onto MRS and incubated under anaerobic
conditions in a plastic anaerobic jar with an AnaeroGen sachet (an
atmosphere generation system, Oxoid, Basingstoke, England) at
37
C during 48 h.

to measure RH from 0 to 100 1.5%, and a platinum resistance

temperature detector with a precision of 0.3
C. When aw became
constant (which usually took less than 1 h), its value was recorded.
Calibration resorted to six saturated solutions of known aw (viz.
LiCl 0.114, MgCl2 0.329, K2CO3 0.443, Mg(NO3)2 0.536,
NaBr 0.653 and KCl 0.821). The tests were run in quadruplicate.
2.4.3. Color of lms
The color was evaluated using a portable Chroma meter CR-400
(from Minolta Chroma, Osaka, Japan). A CIELab color scale was
employed to measure the degree of lightness (L), redness (a) or
greenness (a), and yellowness (b) or blueness (b) of the lms,
under D65 (daylight). Film disks were measured, on the surface of
the white standard plate, with color coordinates Lstandard 97.7,
astandard 0.04 and bstandard 1.47. The color of the lms was
expressed as the total difference in color (DE), calculated according
to:

DE



Lfilm  Lstandard

2

2

afilm  astandard

2 1=2

bfilm  bstandard
For each condition, four samples were measuredeand, on each
lm disk, four readings were made on each side.

2.4. Film characterization

2.4.1. Thickness
The lm thickness was measured using a micrometer Model
m120 (from Adamel Lhomargy, Roissy en Brie, France), to the
nearest 0.001 mm. The mean thickness was calculated from ve
independent measurements, taken randomly at different locations
on a disk lm sample, at each sampling time.
2.4.2. Water activity
The water activity (aw) was measured using a HygroLab 2 (from
Rotronic, Bassersdrof, Germany). Pieces of lms (ca. 0.5 g) were
placed on the sample holder of the water activity device; a sealed
system was formed by placing the water activity probe on top of the
sample holder. The probe was equipped with a small fan to circulate
air inside the sample container, a thin lm capacitance sensor able

Young s modulus MPa

2.4.4. Texture analysis

The preparation of lm specimens and measurements were
done according to the ASTM D-882-02 standard (ASTM, 2002).
Texture analysis was performed using a texturometer (TA.XT plus
Texture Analyser, Stable Micro Systems, Cardiff, UK). Force calibration was performed with a weight of 5 Kg and height calibration
was performed for Mini Tensile Grips (Stable Micro Systems).
Evaluated parameters were youngs modulus (Eq. (1)), tensile
strength, and elongation at break (%). Tensile strength (MPa) stands
for the maximum tensile stress that the test sample was capable of
carrying. Elongation at break (%) was determined as the strain at
the fracture point which corresponds to the ratio of the change of
length of the specimen to initial length. Each sample was cut into
rectangular lm probes (100  15 mm). All measurements were
performed in ve lms for each formulation.

Force at corresponding strain

Cross  sectional area of the film  Corresponding strain

(1)

0.05a
0.05a
0.01a
0.02a
0.02a
0.00a

16.75
16.73
16.75
16.72
16.72
16.71
0.05a
0.06a
0.01a
0.02a
0.02a
0.01a

17.45
17.44
17.46
17.43
17.43
17.42
0.01c
0.01c
0.01c
0.01c
0.01c
0.00c

0.53
0.53
0.53
0.51
0.53
0.53
0.01c
0.01c
0.01c
0.01c
0.01c
0.01c

92.74
92.75
92.75
92.75
92.75
92.75
0.01b
0.15b
0.14b
0.08b
0.05b
0.04b

18.77
18.90
18.92
18.85
18.83
18.88
Note:

a, b, c

, Means within the same columns, labeled with the same letter, do not statistically differ from each other (p > 0.05).

0.04b
0.21b
0.14b
0.08b
0.04b
0.03b

18.97
19.10
19.11
19.05
19.04
19.10
0.02b
0.01b
0.01b
0.01b
0.01b
0.01b

0.61
0.59
0.59
0.59
0.59
0.59
0.06b
0.12b
0.01b
0.01b
0.02b
0.03b

90.93
90.92
90.88
90.93
90.95
90.97
0.00a
0.04a
0.01a
0.00a
0.00a
0.00a

17.00
17.00
16.99
17.00
17.00
17.01
0.01a
0.03a
0.01a
0.01a
0.01a
0.01a

17.39
17.38
17.38
17.39
17.39
17.39
0.00a
0.00a
0.01a
0.01a
0.01a
0.01a
0.75
0.76
0.75
0.76
0.76
0.76
0.02a
0.03a
0.01a
0.02a
0.01a
0.03a

91.78
91.78
91.78
91.78
91.79
91.77

15.28
15.29
15.31
15.30
15.30
15.30
0.94
0.94
0.96
0.94
0.94
0.92
0.16a
0.18a
0.09a
0.11a
0.03a
0.01a

a)
23
C
0 days
3 days
5 days
10 days
40 days
60 days
b)
4
C
0 days
3 days
5 days
10 days
40 days
60 days

91.11
91.33
91.38
91.34
91.34
91.36

0.01a
0.01a
0.00a
0.01a
0.01a
0.00a

19.31
19.35
19.36
19.36
19.39
19.51

0.08a
0.26a
0.06a
0.06a
0.03a
0.02a

19.05
19.04
19.00
19.03
19.04
19.15

0.02a
0.18a
0.09a
0.01a
0.01a
0.01a

93.66
93.63
93.64
93.63
93.62
93.61

0.04b
0.03b
0.02b
0.03b
0.01b
0.01b

0.75
0.76
0.71
0.73
0.73
0.75

0.02b
0.01b
0.01b
0.02b
0.00b
0.00b

16.56
16.60
16.59
16.61
16.61
16.59

0.02b
0.03b
0.01b
0.02b
0.01b
0.01b

15.65
15.69
15.67
15.70
15.70
15.69

0.01b
0.02b
0.02b
0.03b
0.01b
0.00b

93.57
93.58
93.61
93.59
93.57
93.55

0.03b
0.09b
0.01b
0.01b
0.03b
0.02b

0.68
0.66
0.63
0.65
0.66
0.64

0.01c
0.01c
0.01c
0.01c
0.01c
0.01c

16.17
16.17
16.21
16.19
16.18
16.16

0.00b
0.05b
0.01b
0.01b
0.02b
0.01b

DE
b*
a*
L*

L. casei-01

DE
b*
a*
L*

DE
b

Enumeration of viable cell numbers was attained in lms stored

for 60 d under vacuum at two different temperatures, 23 and 4
C,
in order to verify the viability and stability of the probiotic microorganisms tested as shown in Table 1.
According to Rodrigues et al. (2011), the high relative humidity,
high temperature and long storage periods are harmful to probiotics survival. Viability of both B. animalis Bb-12 and L. casei-01
was well correlated with storage temperature (p < 0.05). Viable cell
numbers of both probiotic species decreased, as expected, at
signicantly higher rates (p < 0.001) in the lm systems stored at
23
C. Extrinsic factors such as aw, temperature and presence of
oxygen are known to adversely inuence the viability of encapsulated probiotic viable cells (Soukoulis, Behboudi-Jobbehdar, Yonekura, Parmenter, & Fisk, 2014).
In this way, from an initial concentration of ca. 109 CFU/g lm,
a loss of viability of ca. 3 log cycles (reaching 106 CFU/g lm) was
observed within 10 d of storage at 23
C for both probiotics tested
and was maintained steady thereafter, with a very slight further
decrease upon 40 d of storage. This loss of viability is due to
storage temperature. Some studies demonstrate that probiotic
food products should preferably be stored at a temperature of
4e5
C (Mortazavian et al., 2007; Rodrigues et al., 2011; Sousa
et al., 2012; Tripathi & Giri, 2014). Indeed, at 4
C the loss of
viability was less pronounced, with ca. 1 and 2 log cycles reduction for B. animalis Bb-12 and L. casei-01, respectively, at the end
of the storage period. In addition, the viability of both species of
probiotics remained constant for 10 d. This is in agreement with
Soukoulis, Behboudi-Jobbehdar, et al. (2014) and Soukoulis,
Yonekura, et al. (2014), which conrm that storage at 4
C is
better to maintain the viability of a L. rhamnosus probiotic strain
in prebiotic edible lms. Mortazavian, Ehsani, et al. (2007) and
Mortazavian, Razavi, et al. (2007) also conrmed highest
viability of L. acidophilus LA-5 in yogurt for up to 20 d when

3.1. Viability of B. animalis Bb-12 and L. casei-01 in lms during

storage

3. Results and discussion

B. animalis Bb-12

Statistical analyses were performed using the Statistical Package

for Social Sciences, v. 17.0 (SPSS, Chicago IL, USA), via one-way
analysis of variance. The difference of means between pairs was
resolved via condence intervals, using a Tukey test. The signicance level was set at p < 0.05.

2.6. Statistical analyses

Film morphology was evaluated by Scanning Electron Microscopy (SEM) using a JSM-5600 Lv microscope (from JEOL, Tokyo,
Japan). The samples were coated with gold/palladium using a
Sputter Coater (Polaron, Bad Schwalbach, Germany). The cold-stage
(20
C) method was used to examine the samples. SEM was
operated at the low vacuum mode, using a spot size of 25e29 and a
potential of 16e23 kV.

2.5. Scanning electron microscopy

Control

2.4.5. FTIR-ATR analysis

The spectra of the whey protein lms were obtained with a
Fourier transform infrared spectrometer (FTIR), model ABB
MB3000 (ABB, Zrich, Switzerland), with a horizontal attenuated
total reectance (ATR) accessory (PIKE Technologies, Madison, WI,
USA) with a diamond/ZnSe crystal. All spectra were acquired with
32 scans and 4 cm1 resolution, in the region of 4000e600 cm1.
Three replicates were collected for each lm surface sample.

0.01b
0.02b
0.01b
0.01b
0.01b
0.01b

J. Odila Pereira et al. / LWT - Food Science and Technology 73 (2016) 543e550
Table 3
Color characteristics of control and probiotic lms incorporated with B. animalis Bb-12 and L. casei-01 during storage for 60 days at 23 1
C (a) and 4 1
C (b), viz. L* (blackewhite), a* (greenered), b* (blueeyellow) and DE
(color difference).

546

J. Odila Pereira et al. / LWT - Food Science and Technology 73 (2016) 543e550

547

Table 4
Texture characteristics of control and probiotic lms incorporated with B. animalis Bb-12 and L. casei-01 during storage for 60 days at 23 1
C (a) and 4 1
C (b), viz. Youngs
Modulus (MPa), Tensile Strength (MPa) and Elongation at Break (%).
B. animalis Bb-12

Control
Youngs
modulus
(Mpa)
a)
0 days
3 days
5 days
10 days
40 days
60 days
b)
4
C
0 days
3 days
5 days
10 days
40 days
60 days
Note:

Tensile strength
(Mpa)

Elongation at
break (%)

Youngs
modulus (Mpa)

L. casei-01

Tensile
strength (Mpa)

Elongation at
break (%)

Youngs
modulus
(Mpa)

Tensile strength
(Mpa)

Elongation at
break (%)

0.311
0.312
0.311
0.311
0.311
0.312

0.000a
0.001a
0.001a
0.001a
0.000a
0.001a

0.770
0.767
0.771
0.777
0.769
0.767

0.004a
0.007a
0.003a
0.003a
0.010a
0.010a

65.500
65.500
65.534
65.533
65.521
65.500

1.803a
1.803a
1.859a
1.790a
1.782a
1.803a

0.309
0.309
0.309
0.310
0.313
0.310

0.000a
0.000a
0.000a
0.001a
0.006a
0.000a

0.675
0.676
0.676
0.675
0.679
0.679

0.025b
0.025b
0.025b
0.025b
0.023b
0.025b

64.667
64.671
64.700
64.737
64.677
64.684

0.577a
0.570a
0.608a
0.553a
0.586a
0.547a

0.310
0.310
0.310
0.311
0.312
0.311

0.002a
0.002a
0.002a
0.002a
0.003a
0.003a

0.615
0.615
0.616
0.615
0.610
0.615

0.020c
0.020c
0.020c
0.026c
0.020c
0.023c

65.000
65.018
65.037
65.004
64.968
65.001

3.500a
3.500a
3.505a
3.505a
3.449a
3.499a

0.310
0.312
0.311
0.310
0.311
0.312

0.002a
0.001a
0.001a
0.001a
0.002a
0.001a

0.771
0.770
0.771
0.775
0.771
0.767

0.005a
0.004a
0.003a
0.006a
0.009a
0.012a

65.502
65.510
65.527
65.530
65.525
65.507

1.807a
1.810a
1.850a
1.795a
1.804a
1.806a

0.310
0.310
0.308
0.311
0.310
0.310

0.001a
0.002a
0.000a
0.002a
0.002a
0.000a

0.676
0.677
0.676
0.679
0.679
0.678

0.024b
0.023b
0.025b
0.024b
0.025b
0.025b

64.700
64.685
64.720
64.733
64.635
64.690

0.578a
0.547a
0.609a
0.554a
0.580a
0.548a

0.311 0.000a
0.10 0.002a
0.311 0.003a
0.312 0.002a
0.312 0.003a
0.310 0.000a

0.615
0.612
0.615
0.613
0.610
0.615

0.020c
0.020c
0.020c
0.025c
0.021c
0.022c

65.020
65.018
65.021
65.020
65.027
65.011

3.498a
3.497a
3.500a
3.500a
3.486a
3.489a

a, b, c

, Means within the same columns, labeled with the same letter, do not statistically differ from each other (p > 0.05).

stored at 2
C, whereas for Bidobacterium lactis Bb-12, the optimum storage temperature was 8
C (Mortazavian, Razavi,
Ehsani, & Sohrabvandi, 2007; Mortazavian et al., 2007; Tripathi
& Giri, 2014). Storage temperature of 20
C resulted in signicant reductions in viable counts of this species in the dried
products.
These results suggest that the stability of probiotics increases
with low temperatures. This phenomenon occurs as the microorganisms are kept in a latent state avoiding rearrangements in the
wall material, preventing thus inadequate exposure of microorganisms (Albertini et al., 2010). Temperatures near to 0
C improve
the cell viability rate, because low temperatures reduce chemical
reaction rates that are harmful for microorganisms, such as,
oxidation (Nag, Han, & Singh, 2011).
Moreover, addition of whey protein and glycerol in the lm
solutions which are known as protectants could help in protecting
the viability of probiotic cells (Tripathi & Giri, 2014).
Furthermore, whey protein has signicant positive effects on
the survival of probiotic microorganisms during storage
(Mohammadi, Mortazavian, Khosrokhavar, & da Cruz, 2011) by
providing nutrition for the cells, reducing redox potential of the
medium as well as increasing buffering capacity of the medium that
results in a smaller decrease in pH (Mortazavian et al., 2007;
Soukoulis et al., 2014).
Nevertheless, the viable cell numbers at the end of the storage
period for both temperatures and probiotics tested are still within
the minimum threshold necessary for intended biological function
in the human body, although the storage at 23
C after 10 d would
slightly compromise the current most accepted value of 107 CFU/g
lm (FAO/WHO, 2002).
In similarity to the studies mentioned above, the results of this
study were the most promising to maintain the viability of
probiotics.

transfer resistance and low WVP (McHugh, Huxsoll, & Krochta,

1996).
The results presented in Table 2 show that all developed lms,
with or without microorganisms, showed thickness of ca.
0.400 0.010 mm (0.395e0.410 mm) and no effects (p > 0.05) of
the probiotics strains on the lm thickness were observed.
Furthermore, the lm thickness, during the storage period, at
both temperatures, did not change signicantly in both control
lms (without microorganisms) and lms with probiotic strains
(p > 0.05), probably because all lms were made with the same
~ iga, Osorio, and
amount of solution, as described by Rubilar, Zn
Pedreschi (2015) and the microorganisms did not impact on this
property.
The lms were also evaluated for their aw during storage. The aw
ranged from 0.549 to 0.738, as shown in Table 2. No differences
(p > 0.05) in terms of aw were observed during the storage period in
the lms produced, probably due to the fact that the lms are
sealed in vacuum high barrier plastic bags, without contact with

3.2. Film proprieties

The effect microorganisms incorporation on the thickness of
those lms was evaluated. The lm thickness was measured at 0, 3,
5, 10, 40 and 60 d.
The thickness control is an important variable in studies of
barrier properties, and in this case to protect microorganisms. With
the increased thickness, there is a reduction in the water vapor

Fig. 1. FTIR absorbance spectra of whey protein based lms without microorganism
(control), with B. animalis Bb-12 at 0 days of storage at 23 1
C (a), with B. animalis
Bb-12 at 60 days of storage at 23 1
C (b), with B. animalis Bb-12 at 0 days of
storage at 4 1
C (c) and with B. animalis Bb-12 at 60 days of storage at 4 1
C (d).

548

J. Odila Pereira et al. / LWT - Food Science and Technology 73 (2016) 543e550

Fig. 2. Scanning electron microscopy visualization of the surface (a, b and c) and cross-section (d and e) of whey protein based lms without microorganism (a and d) and with B.
animalis Bb-12 or L. casei-01 (b, c and e). Arrows indicate bacterial cells.

the environment thus minimizing any unpredictable loss.

The visual aspect is related to the color and transparency of the
whey protein lms and was evaluated by color variation between
the three types of lm. The color characteristics of the lms are
given in Table 3.
All lms showed high brightness values (L*  90.88) showing
that they are light-colored. The results are in agreement with Rhim,
Gennadios, Weller, Cezeirat, and Hanna (1998) who obtained values
of L*  89.80 in lms made by in lms made by soy protein isolate.
Brindle and Krochta (2008) also obtained translucent lms with
glycerol, whey protein isolate and hydroxypropylmethylcellulose
blended lms. According to Ramos et al. (2013), whey proteins are
important polymers used in the preparation of transparent and
exible biodegradable lms.
All the samples maintained stable values of lightness (L*),
redness (a*) and yellowness (b*) throughout the storage period
(p > 0.05).
Many authors have reported WPI lms to be tasteless, colorless,
transparent, odorless and exible (Fairley, Monahan, German, &
Krochta, 1996; Perez-Gago, Rojas, & Del Rio, 2003; Perez-Gago,
Serra, Alonso, Mateos, & Del Ro, 2005), which is in agreement
with our results and no changes in color were observed during the
storage period highlighting the high stability of the lms.
Mechanical properties of whey protein lms are important to
maintain their integrity during storage and handling. Youngs
modulus (YM), tensile strength (TS) and elongation at break (EB)
are parameters that describe the lm behavior under different
conditions and expose changes in the lm microstructure.
Data pertaining to the tensile properties of WPI lms, with
different probiotic bacteria, are shown in Table 4.

No signicant differences (p > 0.05) during the storage period

for all the parameters studied, viz. YM, TS and EB, were observed in
the lms with or without bacteria.
Youngs modulus was around 0.310 0.002 MPa (Table 4) for all
the different lms tested and were similar to those reported for
similar whey protein based lms (Ramos et al., 2013). The addition
of probiotics did not affect the YM (p > 0.05) values. A similar trend
was observed by S

anchez-Gonz

alez, Quintero Saavedra, and Chiralt
(2014) with incorporation of L. acidophilus in glycerol plasticized
sodium caseinate lms. In addition, Piermaria, Diosma, Aquino,
Garrote, and Abraham (2015) studied glycerol plasticized lms
with inclusion of both, lactobacilli and yeasts, and demonstrated
that no apparent changes in the YM value were observed
(Piermaria et al., 2015).
WPI lms showed TS ranging between 0.777 0.003 and
0.610 0.020 MPa (Table 4). These results are in agreement with
Ramos et al. (2013) who obtained similar values in edible WPI lms.
The incorporation of probiotic cells in lms lead to a slight
decrease in TS parameter value (p < 0.05). Similar results were
achieved by Kanmani and Lim (2013), through addition of probiotic
cells into the pullulan/starch lm which resulted in a reduction of
TS.
The results for EB of WPI lms ranging between 65.534 1859
and 64,968 3449% are depicted in Table 4. These ndings are in
agreement with those of Ramos et al. (2013), who obtained analogous values in edible WPI lms. No signicant differences
were observed in EB values throughout the storage period for
both probiotic bacteria at each of the temperatures tested
(p > 0.05). Both temperature and the microorganisms addition
appeared to not affect the elasticity of the lms. In agreement with

J. Odila Pereira et al. / LWT - Food Science and Technology 73 (2016) 543e550

these results, no change in this parameter was reported by

nchez-Gonza

lez et al. (2014) for methylcellulose and hydroxSa
ypropylmethylcellulose lms.
The FTIR spectra of WPI lms and the potential changes in the
molecular structure of WPI lms upon incorporation of probiotics
cells were studied using FTIR-ATR.
The spectra of WPI control, with B. animalis Bb-12 at 0 days of
storage at 23 1
C (a), with B. animalis Bb-12 at 60 days of storage
at 23 1
C (b), with B. animalis Bb-12 at 0 days of storage at
4 1
C (c) and with B. animalis Bb-12 at 60 days of storage at
4 1
C (d) are displayed together in Fig. 1.
In the spectrum of WPI (Fig. 1 control) the main transmittance
peaks were located in the spectral range: (i) 800e1150 cm1transmittance bands of glycerol; (ii) 1200e1350 cm1-combination
of NeH in-plane bending with CeN stretching vibrations (amide
III); (iii) 1400e1550 cm1-NeH bending (amide II); (iv)
1600e1700 cm1-stretching vibration of C]O and CeN groups
(amide I); (v) 2850e2980 cm1-CeH stretching; and (vi)
3000e3600 cm1-free and bound OeH and NeH groups (Bagheri,
Madadlou, Yarmand, & Mousavi, 2013; Ramos et al., 2013).
The temperature and the storage time did not affect the molecular structure of the lms. Moreover, the incorporation of microorganisms did not modify the infrared spectra, as observed in
Fig. 1 (a, b, c and d), which may indicate that there are no interactions between the probiotics and the carry material.
Some studies have also demonstrated that microorganisms did
not affect the FTIR spectra of WPI and denatured WPI microcapsules (Rajam & Anandharamakrishnan, 2014) and the WPC cap
pez-Rubio, Sanchez, Wilkanowicz, Sanz, & Lagaron,
sules (Lo
2012).
The WPI lms containing microorganisms revealed a very slight
difference compared to WPI lms without probiotics only among
TS values. The bacteria once integrated into the lm structure, may
make the lm slightly less brittle and more malleable. Nevertheless,
these small differences are further conrmed by FTIR-ATR results
where no changes in the lms structures are reported.
3.3. Morphology of lms by SEM
A scanning electron microscopy analysis of the WPI based lms
allowed visualization of B. animalis Bb-12 and L. casei-01 (Fig. 2b, c
and e). The incorporation of probiotic strains in the edible lm did
not confer any noticeable modication to the structural conformation of the lms (Fig. 2a), apart from the presence of the bacterial cells embedded (tiny rod-like shapes as indicated by the
arrows) in the plasticized protein matrix. This incorporation of the
cells within the protein matrix can have a protective effect upon cell
viability, has it has been shown in studies where protein matrices
were utilized to produce microcapsules with probiotics, enhancing
probiotic resistance to environmental conditions (Rodrigues et al.,
2011; Tripathi & Giri, 2014). The different temperatures did not
result in detectable changes in the microstructure of the probiotic
lms (results not shown). In all cases, the lms retained their
structure, having non-uniform and reticular characteristics, these
images are in agreement with previous studies, in which similar
results are observed (Shakila, Jeevithan, Varatharajakumar,
Jeyasekaran, & Sukumar, 2012).
4. Conclusions
Taking into account the results, we conclude that WPI edible
lms could act as a suitable matrix to incorporate probiotic lactic
acid bacteria and support their viability throughout shelf-life. The
probiotics remained viable during 60 days maintaining high viable
cell number levels.

549

Storage of the edible lms under refrigerated temperature was

the best condition to maintain cellular viability of B. animalis BB12 and L. casei-01. It is also noteworthy that no structural changes
in the lm during the study period were observed, providing these
lms with high durability and with a high shelf-life allowing good
solutions for food applications.
Thus, incorporation of probiotics in edible lms could be a good
carrier for these bacteria to be ingested together with food and
simultaneously exert specic activities in food. Further investigations are warranted to demonstrate these potential
applications.
Acknowledgements
The authors gratefully acknowledged the Funda~
ao para a
^ncia e a Tecnologia (FCT) (SFRH/BD/88383/2012), Portugal for
Cie
providing PhD scholarship ref. SFRH/BD/88383/2012 to Joana Odila
Pereira. This work was supported by National Funds from FCT
through project UID/Multi/50016/2013.
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