Food Hydrocolloids for Health 2 (2022) 100065

Contents lists available at ScienceDirect

Food Hydrocolloids for Health

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

Development and characterization of sodium caseinate based probiotic

edible film with chia mucilage as a protectant for the safe delivery of
probiotics in functional bakery
Anupama Semwal, Kiran Ambatipudi∗, Naveen Kumar Navani∗
Department of Biosciences and Bioengineering, Indian Institute of Technology, Roorkee, Roorkee, Uttarakhand 247667, India

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

Keywords: The probiotic edible film is a promising method to deliver the probiotics safely to exert their health beneficial
Probiotic effects. However, supplementation with protectants is important for the enhanced viability of probiotics under
Edible film stress conditions during storage. The present study incorporated chia mucilage in sodium caseinate-based pro-
Sodium caseinate
biotic edible film as a protectant to investigate the survival of probiotic bacteria (Limosilactobacillus fermentum
Chia mucilage
NKN51 and Lactobacillus brevis NKN52) embedded in films stored at 25 °C and 4 °C. We observed significantly
Viability
Wheat bun (p < 0.001) enhanced survival of probiotic cells in the presence of chia mucilage. The inclusion of chia mucilage
in film conferred enhanced probiotics’ viability, improved flexibility, and decreased solubility due to interaction
between chia mucilage polysaccharides and protein matrix. Morphological examination of embedded probiotic
cells using atomic force and scanning electron microscopy showed the intact bacterial cells, proving the signif-
icance of sodium caseinate and chia mucilage. Subsequently, the probiotic edible film was assessed by coating
wheat buns where the probiotics’ viability was maintained for 3 weeks at 4 °C and 2 weeks at 25 °C, indicating
the effective application of developed probiotic film for delivering the probiotics on the bakery products. Our
study supports the applicability of chia mucilage as a promising protectant for enhanced probiotics’ viability in
edible films, which may act as a suitable probiotic carrier to deliver probiotics safely for consumption. Further-
more, our study provides the plant-based natural protectant alternative of synthetic compounds and chemicals
as protectants, making it a healthier way of preparing functional bakery products.

1. Introduction immune system, improving mood, enhancing the bioavailability of nu-

Probiotics are non-pathogenic gut-friendly live microorganisms that
confer healthful effects on the host in adequate numbers. Probiotic
bacteria have been isolated from various ecological niches, including
fermented food products. Probiotic bacteria provide numerous health
benefits, including a reduction in lactose intolerant symptoms, allevi-
ating the recurrence of infectious agents like Clostridium difficile and
rotavirus associated diarrhoea, reduction of irritable bowel syndrome
and inflammatory bowel diseases, enhancing mucosal immunity, pre-
venting the growth and adhesion of pathogen in the intestinal tract
(Reid et al., 2003; Saad et al., 2013). Lactobacilli and Bifidobacteria
have been widely studied and extensively consumed as dietary sup-
plements, functional foods, probiotic drinks, etc. (Linares et al., 2017;
O’Toole et al., 2017). Apart from satisfying hunger, functional foods
supplemented with probiotics are prominent and effective methods to
confer health benefits to consumers, including balancing gut microflora,
improving digestion, delivering bioactive compounds, strengthening the
trients in the gut, alleviating disease risk, and thus promoting overall
good health (Misra et al., 2021; Roberfroid, 2000).
In the wake of the current pandemic, functional food containing pro-
biotic bacteria has gained popularity in several probiotic products (e.g.,
yogurt, lassi, infant formula, kefir, Yakult, etc.). However, probiotics’
survival throughout food processing steps involving heat treatment, ox-
idative, osmotic stress, mechanical stress, etc., is a limiting factor in the
manufacture of probiotic food products (Tripathi & Giri, 2014). Sim-
ilarly, it is important to maintain the viability of probiotic cells ex-
posed to gastric juices after being released into the gut to exert health
benefits and modulate the gut microbiota. Currently, several methods
such as microencapsulation, freeze-drying, or spray drying, including
the use of polysaccharides, milk proteins, and fats to encapsulate the
probiotic cells, are being followed to maintain the bacterial viability
(Bustamante et al., 2017; Soukoulis et al., 2017; Tripathi & Giri, 2014).
Of these products, edible films have been considered as an efficient ve-
hicle to transport the probiotic bacterial cells viable in food and the

∗
Corresponding authors.
E-mail addresses: kiran.ambatipudi@bt.iitr.ac.in (K. Ambatipudi), naveen.navani@bt.iitr.ac.in (N.K. Navani).

https://doi.org/10.1016/j.fhfh.2022.100065
Received 6 March 2022; Received in revised form 7 April 2022; Accepted 14 April 2022
2667-0259/© 2022 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/)
A. Semwal, K. Ambatipudi and N.K. Navani
gut (Ebrahimi et al., 2018; Namratha et al., 2020; Orozco-Parra et al.,
2020; Soukoulis et al., 2017). Entrapment of probiotics in edible films
is an inexpensive and simple method to deliver probiotics safely than
encapsulation. This includes efficient delivery of the probiotics to the
gut in multiple ways like a cover or coating on various food products
(e.g., fruits, bakery products, confectionery items, wraps, cereal bars,
etc.). Additionally, these could be used as edible strips to dissolve in
any drink to prepare different probiotic beverages (e.g., juices, dairy
products, etc.) or eaten with other foodstuff. Furthermore, edible films
can improve the nutrition profile of foodstuff by delivering nutritional
compounds, including enhancing the shelf life of food products due to
their moisture and gas barrier properties (Zoghi et al., 2020).
Edible films have been developed with various biomolecules like
polysaccharides, proteins, and lipids either alone or incorporated
together, defining their functional properties (Pavli et al., 2018;
Ramos et al., 2012;). The protein-based edible film possesses better me-
chanical and barrier properties than other biomolecules and improves
the nutritional value of the food product (Cao et al., 2007; Fabra et al.,
2008). In our study, sodium caseinate was used to form an edible film.
Sodium caseinate is considered in active food coatings and films due to
its biodegradable nature, thermal stability, easy commercially availabil-
ity, ability to easily transform the aqueous solution into films because of
forming extensive intermolecular hydrogen, electrostatic and hydropho-
bic bonds, and random coil nature of casein (Audic & Chaufer, 2005;
Fabra et al. 2007). Chia seed mucilage was used as a protectant for the
probiotics due to its dense polysaccharide network and high amount of
dietary fiber, including its usage as a stabilizing, thickening and emul-
sifying agent along with its biodegradable packaging and coating appli-
cations (Salehi, 2019; Soukoulis et al., 2018;).
In the present work, we aimed to investigate the protective effect of
chia mucilage (CM) on the probiotic bacteria in the edible film. There
is a dearth of studies that have explored the efficacy of chia mucilage as
a protectant in a probiotic, edible film instead of prebiotics. We studied
the shelf life of probiotic, edible film with and without chia mucilage at
4o and 25° C. After incorporating CM and probiotics, the edible film’s
physical, mechanical, and structural properties were also evaluated. We
also showed the application of developed probiotic, edible film on bak-
ery products and determined the shelf life of probiotic film coated on
selected food products.
2. Materials and Methods
2.1. Materials and chemicals
Sodium caseinate and Chia seeds were procured from local suppliers.
Probiotic strains (Limosilactobacillus fermentum NKN51 and Lactobacillus
brevis NKN52), previously characterized in our laboratory, were acti-
vated from glycerol stock in MRS (De Man, Rogosa and Sharpe) media
(BD Difco TM , USA). Deionized water was used for the film preparation.
Food grade Glycerol (>99%, FCC, FG, Sigma-Aldrich, Darmstadt, Ger-
many) was used as a plasticizer. Dulbecco’s Phosphate Buffered Saline
10X (Sigma-Aldrich, Darmstadt, Germany) was used to prepare a bacte-
rial suspension.
2.2. Mucilage extraction from chia seeds
Chia mucilage was extracted from the seeds following
Castejón et al. (2017) procedure with slight modifications. In brief,
chia seeds were soaked in deionized water in a 1:30 ratio, and the
solution was kept for heating at 50 °C for 2 h under stirring. Chia
mucilage was extracted from the seeds by sonication for 2 min at 30 %
amplitude using a Probe Sonicator (Branson, USA). Subsequently, the
solution was centrifuged at 10,000 rpm at 4 °C for 20 min, resulting in
a three-layered separation. The Middle layer contained chia mucilage,
the upper layer contained water, while the seeds were settled down at
2
Food Hydrocolloids for Health 2 (2022) 100065
the bottom. Chia mucilage was collected and filtered through a 0.22 μ
filter (Millipore, USA).
2.3. Preparation of bacterial suspension
L. fermentum NKN51 and L. brevis NKN52 strains were activated from
glycerol stock in MRS media. The overnight culture was cultivated in
MRS broth and incubated till their late log phase, i.e., 13 h and 15 h for
L. fermentum NKN51 and L. brevis NKN52, respectively. Bacterial cells
were pellet down after centrifugation at 5000 rpm for 10 min at 4 °C
and resuspended in sterilized 1 X PBS solution to be incorporated into
the film solution.
2.4. Film Preparation
Film solution was prepared by dissolving 5% (w/w) sodium ca-
seinate in deionized water following the procedure reported by
Ramos et al. (2013) with slight modifications. In brief, deionized wa-
ter was preheated at 50 °C, and sodium caseinate was added and kept
for stirring to dissolve sodium caseinate thoroughly. Glycerol (30% w/w
of protein content) and chia mucilage (1% w/w) were added to the so-
lution. The resulting solution was heated at 60 °C for 2 h under stir-
ring. Subsequently, the solution was heated at 90 °C for 30 min under
stirring to form the intermolecular network. This process results in a
flexible film that maintains its structural integrity under environmental
conditions. After heating, the film solution was allowed to cool down at
room temperature (RT). Bacterial suspension of both strains was added
to the solution and kept for stirring to ensure proper mixing. The solu-
tion was further kept for degassing for 20 min to remove the remaining
air. The film-forming solution was poured into glass Petri dishes (14 cm
diameter) and kept for air drying for 24 h at RT. The film was peeled
off from the Petri dish and conditioned at 54% ± 3% relative humidity
(RH) (maintained with a saturated magnesium nitrate Mg(NO3 )2 solu-
tion) for 72 h. All the experiments were performed in triplicate after
conditioning.
2.5. Enumeration of the bacteria
One mL of the probiotic film-forming solutions was suspended in
9 mL sterile PBS and vortexed to ensure proper mixing. For the recovery
of L. fermentum NKN 51 and L. brevis NKN 52 from the probiotic, edi-
ble films, the method adopted by Soukoulis, Behboudi-Jobbehdar et al.,
2014 was performed with slight modification. In brief, 1 g of the film
containing L. fermentum NKN 51 and L. brevis NKN 52 was mixed with
9 mL of PBS and vortexed to dissolve the film adequately. The total
bacterial count enumeration was performed in triplicate following the
standard plating methodology (Champagne et al., 2011), and the total
counts of the viable (TVC) bacteria were expressed as log colony forming
units per gram (log CFU/g).
L.fermentum NKN51 and L. brevis NKN52 inactivation upon storage
were expressed as the relative viability fraction (log Nt /N0 ). Viability
was fitted to a first-order reaction kinetics model as described by the
formula:
log Nt = log N0 – kT t (1)
Where N0 represents the initial number of viable bacteria and Nt the
number of viable bacteria after a specific time of storage (CFU/g), t
is the storage time (day), and kT is the inactivation rate constant (log
CFU/day) at temperature, T (°C).
2.6. Thickness
A digital micrometer (Mitutoyo Corporation Ltd., Kawasaki, Japan)
with a sensitivity of 0.001 mm was used to measure the film thickness.
Fifteen measurements were taken from different parts of the films to
A. Semwal, K. Ambatipudi and N.K. Navani
ensure consistency. The average value was taken further to analyse the
film’s water vapor permeability (WVP) and mechanical properties.
2.7. Moisture content
The moisture content of each film was determined according to the
procedure of Mei and Zhao (2003) with some modifications. In brief,
200 mg film specimens were cut, placed into aluminium petri dishes,
and then dried inside an oven at 105 °C for 24 h. Weight was taken before
and after drying using a weight balance. Measurements were obtained
in triplicates. The moisture content was calculated as,
Wi − Wf
%RM = × 100 (2)
Wi
Here Wi, Wf is the initial and final weight of the edible film.
2.8. Water solubility
Water solubility was measured according to the procedure reported
by Dick et al. (2015). In brief, water solubility was defined as the per-
centage of dried film matter dissolved in water after 24h immersion in
distilled water. The dried film obtained after moisture content analysis
was assigned as the initial dry weight (Wi ) of edible film and was im-
mersed in 40 mL distilled water, followed by stirring for 24 h at 25°C.
The solution was filtered through pre-weighed desiccated filter paper,
and undissolved fragments of the edible film obtained on filter paper
were dried in the oven at 105 °C for 24 h. Final dry weight (Wf ) was
measured. All the tests were performed in triplicate. The water solubil-
ity (%) was calculated according to the following equation:
𝑊 i − Wf
WS(%) ≡ × 100 (3)
Wf
2.9. Water activity
After preconditioning at 54% RH for 72 h, the water activity of the
edible films was determined using an AquaLab water activity meter
(AquaLab, 3 TE, Decagon, USA). The edible film was stored at 4 °C,
and water activity was measured on the 10th and 45th days to observe
the effect of storage conditions on this parameter of the film.
2.10. Water vapor permeability
The WVP of the probiotic, edible film was determined gravimetri-
cally, as reported by Soukoulis et al. (2017). In brief, pre-conditioned
edible films were placed on the top of the glass Petri-dish containing
silica gel (0% RH) to 1/6 of petri dish height; the exposed film area was
1.96 × 10−3 m2 . The glass Petri dishes were transferred to a desiccator
maintained at 100% RH (pure water) and 25 °C, and the WVP difference
was 3169 Pa. WVP was calculated according to the formula:
Δm ∗ e
WVP = (4)
A ∗Δt ∗Δp
Where WVP is water vapor permeability (g.mm.m−2 . h−1 . kPa−1 ),
Δm/Δt is the moisture uptake rate (g/h) from silica gel, A is the film
area exposed to moisture transfer (m2 ), e is the film thickness (mm),
and Δp is the water vapor pressure difference between the two sides of
the film (KPa).
2.11. Mechanical properties
A tensile tester (Bose) was used to characterize the mechanical prop-
erties of the tensile strength (TS), elongation percentage (% EB) at the
break, and Young’s modulus (YM) of the films. Pre-conditioned edible
films (54% RH, 25 °C for 72 h), cut in 20 × 5 mm of 0.11–0.14 mm
thickness of rectangular shapes, were placed between the tensile grips
giving a grip separation distance of 5 mm. A 400 N static load cell was
3
Food Hydrocolloids for Health 2 (2022) 100065
used for tensile tests with a cross-head speed of 0.1 mm/s. The following
properties were calculated from the stress-strain deformation curves:
Fmax
TS = (5)
𝐴
𝐿
%𝐸 = 100 × (6)
𝐿𝑜
Where Fmax is the force at break (N), A is the cross-sectional film area
(mm2 ), L0 is the initial film length (mm), L is the film length at break
(mm). Young’s modulus was estimated as the slope of the elastic region
in the stress-strain curve. Strain (𝜀) = (L-L0 )/L, stress (𝜎) = F/A (MPa).
2.12. Fourier transform infrared spectroscopy (FTIR)
FTIR analysis was used to study cross-linking amongst different
derivatives of the edible film. Fourier Transform Infrared spectrome-
ter recorded the chemical spectra of the edible film, sodium caseinate
powder, and chia mucilage. FTIR spectrometer (Nicolet NEXUS Agilent
1100 series) was used in 400–4000 cm−1 .
2.13. Atomic force microscopy
Atomic force microscopy (NT-MDT-INTEGRA) was used to observe
the surface morphology of edible film incorporated with chia mucilage,
L. fermentum NKN51, and L. brevis NKN52. A small glass slide was dipped
in edible film solution to form a thin layer of film on the glass slide. Glass
slide was kept for 3 days for drying at RT for AFM analysis.
2.14. Morphological characterization
Scanning electron microscope analysis (SEM) was performed using
Carl Zeiss Ultra Plus (Carl Zeiss, Germany) to visualize the morphology
of probiotic cells entrapped in the edible film. The images were acquired
using secondary electron imaging at an accelerating voltage of 5 kV.
Film specimens were carefully mounted on aluminium stubs and coated
with gold to improve the conductivity.
2.15. Statistical analysis
Graph Pad Prism 5 software was used for statistical analysis. All
experiments were performed in triplicate, and data were subjected to
analysis of variance (ANOVA) and Bonferroni multiple comparison test
(p ≤ 0.05) to estimate the significant effect of chia mucilage on micro-
biological, physicochemical, and mechanical properties of the film.
3. Results and discussion
3.1. Probiotics (L. fermentum NKN51 and L. brevis NKN52) survivability
throughout the drying process
The survival of probiotic cells during the drying process is shown
in Fig. 1. No acute toxic effect of the drying process at 25 °C for 24 h
was observed in the survival of bacterial cells in the presence and ab-
sence of CM. This result is in line with the findings reported earlier
(Piermaria et al., 2015; Soukoulis, Behboudi-Jobbehdar et al., 2014;
Soukoulis et al., 2017), indicating that the protection of bacterial cells
can be attributed to the low rate of water evaporation during drying and
the presence of protein matrix. At RT, the rate of water evaporation is
very low, giving sufficient time to the bacterial cells for adaptation to
transition in water activity of the carrier matrix from high to low value,
maintaining the viability of probiotic cells during the drying process.
The present study applied moderate drying temperature for water evap-
oration during film preparation, exposing the bacterial cells to osmotic
stress without any thermal stress. Of note, it has been shown that pro-
tein can support the survival of probiotics cells against osmotic stress
during drying by maintaining the cell membrane integrity through free
A. Semwal, K. Ambatipudi and N.K. Navani
Table 1
Inactivation rates of L. fermentum NKN51 and L. brevis NKN52 during storage at chilling (4 °C) and room (25 °C) temperature
conditions and estimated shelf life (day) (R2 indicates squared correlation coefficient)
Edible film K4 °C (R2 ) Shelf-life 4 °C
-CM 0.110 ± 0.006a (0.98) 37
+CM 0.071 ± 0.007b (0.94) 60
Numbers with different superscript letters are significantly different (p < 0.0001).
Fig. 1. Survival of total bacterial cells (L. fermentum NKN51 and L. brevis
NKN52) embedded in the films during air drying for 24h. Viable counts are
shown prior to drying (black) and after drying (light grey). Data are presented
as mean ± SD (n = 3).
radicals scavenging and providing micronutrients (Burgain et al., 2013;
Soukoulis, Yonekura et al., 2014). For instance, gelatin and sodium
caseinate have been shown to provide maximum protection to Lacto-
bacillus rhamnosus GG against osmotic and heat stress-driven injuries
(Soukoulis et al., 2016). In the present study, sodium caseinate acted
as an efficient protein-based drying substrate preventing detrimental ef-
fects on bacterial cells during the drying process.
3.2. Effect of chia mucilage on the storage stability of L. fermentum
NKN51 and L. brevis NKN52
The storage viability is essential for developing the probiotic edible
film. Inactivation curves of bacterial cells entrapped in sodium caseinate
film stored at 25 °C and 4 °C are shown in Fig. 2. The inactivation rate
of L. fermentum and L. brevis was significantly (p < 0.001) affected by
storage conditions and CM supplementation. Inactivation of L. fermen-
tum and L. brevis followed first-order kinetics (Table 1). At 4 °C, the
inactivation rates of L. fermentum NKN51 and L. brevis NKN52 were sig-
nificantly lower (0.092 log CFU day−1 ) in comparison to those stored at
room temperature (0.326 log CFU day−1 ), in accordance with previous
studies (Piermaria et al., 2015; Soukoulis, Behboudi-Jobbehdar et al.,
2014; Soukoulis et al., 2016; Soukoulis et al., 2017). At low tempera-
tures, bacterial cells possess slow metabolic activities and reduced bio-
chemical reactions like oxidation, which can have lethal effects. More-
over, any structural modifications in molecular interaction networks are
restricted, which helps prevent microorganisms’ exposure to external
stress factors like osmotic pressure and relative humidity.
Films fabricated with CM significantly (p < 0.001) enhanced the bi-
ological activity of probiotic cells (L. fermentum NKN51 and L. brevis
NKN52) entrapped in sodium caseinate film at both storage tempera-
tures 25 °C (0.077 log CFU day−1 and 0.309 log CFU day-1 with and
without CM respectively) and 4 °C (0.059 log CFU day−1 and 0.103 log
CFU day−1 for a matrix with and without the addition of CM respec-
tively). At RT storage for 10 days, the total viable count was reduced by
1 log in the presence of CM, and 3 log reductions occurred without CM.
4
Food Hydrocolloids for Health 2 (2022) 100065
K25 °C (R2 ) Shelf-life 25 °C
0.292 ± 0.023c (0.98) 10
0.059 ± 0.011d (0.91) 10
The storage study of edible film at RT was performed for 10 days, beyond
which the film showed lower solubility, possibly due to the metabolic
activity of probiotic cells. In contrast, at 4 °C, the viability of probiotic
strains in the film was followed for 60 days with 3 log reduction with
CM and 6 log reduction without CM (Fig. 2). The chemical composition
of CM constitutes protein, high dietary fibre, lipid, ash and moisture
(Avila-De La Rosa et al., 2015; Câmara et al., 2020; Felisberto et al.,
2015). CM is comprised of high molecular weight complex polysaccha-
rides ranging from 0.8 to 2 × 106 Da comprising of 𝛽-D-xylose, 𝛼-D-
glucose, and 𝛼-D-glucuronic acid in a ratio of 2:1:1(Lin et al., 1994). Sim-
ilarly, da Silveira Ramos et al. (2021) reported the xylose, glucose, ara-
binose, and galactose are components of CM. Goh et al. (2016) reported
that CM polysaccharides consist of glucose, xylose, galactose, mannose,
arabinose, and glucuronic acid, of which glucose, xylose, and arabinose
constitute the major fraction. More recently, Silva et al. (2022) also re-
ported xylose and arabinose as the major monosaccharides with smaller
amounts of fucose, rhamnose, mannose, and galactose. The protective
effect of CM on the survival of bacterial cells at two different temper-
atures could be due to the presence of heteropolysaccharides such as
xylose, glucose, mannose, galactose, arabinose, fucose, rhamnose, and
glucuronic acid (Bustamante et al., 2017) and their interaction with
bacterial membrane phospholipids preventing membrane damage and
maintaining fluidity (Carvalho et al., 2004; Gialamas et al., 2010). Ad-
ditionally, heteropolysaccharides present in CM retain water due to
their water holding capacity, providing better protection to the bacte-
rial cells (Bustamante et al., 2020). Furthermore, the CM retains the
moisture content in the film, protecting the bacterial cells from en-
vironmental stress during storage and maintaining the cells’ viability
(Rodrigues et al., 2020). Interestingly, Bustamante et al. (2017) have
reported that the incorporation of chia seed mucilage in juice powder
significantly (p < 0.0001) improved the probiotic viability, suggesting
that the chia-based edible film may be a viable alternative for entrap-
ping probiotic cells. The present work has shown that CM incorporation
and 4 °C storage enhanced the shelf life (herein described as the dura-
tion of approaching the minimum total viable count of 6 log CFU/g) of
probiotics edible film.
3.3. Physical characteristics
3.3.1. Thickness
Sodium caseinate films exhibited a thickness of 0.13 ± 0.02 mm.
CM and probiotic cells (L. fermentum NKN51 and L. brevis NKN52)
did not significantly influence the thickness of the developed films
(0.13 ± 0.03 mm and 0.12 ± 0.02 mm for probiotics with CM and only
CM, respectively). Film thickness was considered further to assess the
mechanical and functional properties of the film.
3.3.2. Residual moisture content
CM significantly (p < 0.005) increased the film’s moisture content,
as shown in Fig. 3. The hydrophilic property of CM could be due to
the heteropolysaccharides with hydrophilic groups responsible for the
good water holding property of CM, leading to an increased residual
moisture content of the film (Punia & Dhull, 2019). Similar results with
increased moisture content in the edible film were reported based on CM
and whey protein concentrate (Muñoz et al., 2012). The moisture con-
tent of edible film depends on the components used to fabricate a film
A. Semwal, K. Ambatipudi and N.K. Navani
Fig. 2. Effect of chia mucilage (CM) and storage temperature (A= 25°C, B= 4°C) on the inactivation of total bacterial cells (L. fermentum NKN51 and L. brevis NKN52)
embedded in the sodium caseinate films.
Fig. 3. Effect of chia mucilage (CM) and bacterial cells (B) on residual moisture
content of sodium caseinate films. Data are presented as mean ± SD (n = 3).
matrix. However, the incorporation of probiotics L. fermentum NKN51
and L. brevis NKN52 were found to have a non-significant effect on the
moisture content of the developed edible film.
3.3.3. Water activity
The water activity of the developed film was measured since it is
one of the important attributes leading to changes in food quality dur-
ing storage and distribution. The water activity of the edible film stored
at 4 °C was measured for 10 and 45 days, respectively (Fig. 4). Food
product having a water activity value close to 1 is sensitive to microbio-
logical growth resulting in less food stability, while food products with
a water activity value <0.85 are characterized as shelf-stable food with
no requirement for heat treatment (Rodríguez et al., 2020). The water
activity of sodium caseinate edible film was observed to be 0.52±0.01,
indicating good food stability. Similarly, Pereira et al. (2016) reported
the water activity value of whey protein isolate edible film contain-
ing probiotics varied from 0.549 to 0.738. Another study conducted by
Rodríguez et al. (2020) reported the water activity value of edible pa-
paya film ranging from 0.63±0.01 to 0.67±0.01, indicating good shelf-
life. The lower water activity value in the present study indicates that
the edible film developed is microbiologically stable and can be easily
stored. The presence of CM and bacterial cells didn’t show any signifi-
cant differences in the water activity of edible film. Water activity signif-
icantly decreased (p < 0.001) due to water evaporation during storage
time, possibly due to packing using normal plastic bags.
5
Food Hydrocolloids for Health 2 (2022) 100065
Fig. 4. Effect of storage time on water activity of edible films. Data are pre-
sented as mean ± SD (n = 3).
3.4. Solubility of the film
The solubility of developed edible films was examined and found
to be between 35% to 92%. The solubility of the developed sodium
caseinate film was influenced by CM as its incorporation significantly
(p < 0.005) reduced the solubility of edible films from 92.2% to 74.3%,
as shown in Fig. 5. This could be attributed to the cross-linking be-
tween CM polysaccharides and the protein network stabilizing the com-
plexes (Su et al., 2010). CM is comprised of heteropolysaccharides and
uronic acid (Coorey et al., 2014; Goh et al., 2016), which could facil-
itate the formation of the stabilized compact structure during film for-
mation (Luo et al., 2019). It has been reported that casein hydrolysis is
inhibited by a high degree of branching and enhanced exposure to ion-
izable uronic acid groups (Acton et al., 1982). Similarly, another study
reported wheat gluten film solubility decreased with the addition of xy-
lan depending on pH and drying conditions (Kayserilioğlu et al., 2003).
Interestingly, the addition of probiotic cells significantly (p < 0.0001)
reduced the solubility of the probiotic film (49% and 35.3 % solu-
bility in the probiotic film without and with CM, respectively). This
low solubility can be explained by low pH (pH5 - pH5.5) in probiotic
edible film because of the metabolic end product lactic acid released
by probiotic cells in film. As pH comes closer to the isoelectric point
(pI = 4.6) of casein, the net charges approach zero, leading to no electro-
static repulsion resulting in modified protein’s molecular conformation
favouring strong protein-protein interaction causing less protein solu-
bility (Folegatti et al., 1998).
A. Semwal, K. Ambatipudi and N.K. Navani
Fig. 5. Solubility of sodium caseinate films with and without incorporation of
chia mucilage (CM) and bacterial cells (B). Data are presented as mean ± SD
(n = 3).
Fig. 6. Effect of chia mucilage (CM) and bacterial cells (B) on water vapor per-
meability of sodium caseinate films at ambient temperature (25 °C) and 100%
RH gradient rate. Data are presented as mean ± SD (n = 3).
3.5. Water Vapor Permeability
The films developed with sodium caseinate exhibited a low value of
WVP (0.0061 g mm m−2 h−1 KPa−1 ) required to limit biochemical re-
actions imparting adverse effects on shelf life, microbial spoilage, and
structural complications shown in Fig. 6. Sodium caseinate film shows
strong ionic interaction between sodium ions and protein, reducing
moisture diffusion through film by improving the protein network and
stability and decreasing the WVP of caseinate films (Mei & Zhao, 2003).
CM did not significantly affect the WVP of sodium caseinate film (0.0065
g mm m−2 h−1 KPa−1 ). Incorporation of L. fermentum NKN51 and L. bre-
vis NKN52 in the film resulted in significantly (p < 0.0001) increased
WVP (0.01 g mm m−2 h−1 KPa−1 ). Bacterial cells embedded inside the
film matrix interfere with the protein-protein interaction, facilitating
the permeation of water molecules through edible films. Incorporation
of the probiotic cells results in discontinuities in the film matrix, caus-
ing an increased moisture diffusion rate through the edible film. This
agrees with other studies that reported the enhanced water vapor per-
meability of incorporating bacterial cells in the film matrix (Sánchez-
González et al., 2014; Ye et al., 2018).
6
Food Hydrocolloids for Health 2 (2022) 100065
3.6. Microstructural analysis of edible film using SEM and AFM
Scanning electron microscopy evaluated the visualization of bacte-
rial cells embedded in film and the surface homogeneity of sodium ca-
seinate and CM blend films. Microscopic images of the control film and
an edible film containing L. fermentum NKN51 and L. brevis NKN52 are
shown in Figs. 7 and 8. It is observed that films fabricated with CM lead
to a more compact structure with fewer micropores than control films,
as shown by SEM in Fig. 7. Similarly, the surface morphology of the ed-
ible film was examined through AFM, as shown in Fig. 7. Topographical
images revealed that sodium caseinate film without CM had multiple
projections while sodium caseinate film supplemented with CM exhib-
ited less projections and a smoother surface, possibly due to protein-
polysaccharide interaction. Sadeghi-Varkani et al. (2018) reported the
smooth surface of the film made from Balangu seed mucilage due to the
homogenous distribution of film-forming components. Additionally, it
can be seen that intact microbial cells rod-shaped L. fermentum and L.
brevis embedded in the sodium caseinate film matrix protects the bacte-
rial cells from external stress conditions. Glass slide coated with edible
film solution containing bacterial cells was observed under SEM analy-
sis, which showed imprints of the intact bacterial cells indicating that
this formulation could act as a carrier of bacterial cells coated on any
food surface to deliver probiotics. Control film without bacterial cells
had more compact and less micropores on the surface than the edible
film loaded with bacterial cells (Fig. 8a). Incorporating bacterial cells (L.
fermentum NKN51 and L. brevis NKN52) created voids in the film matrix
due to bacterial cells embedded inside the film matrix. The embedding of
bacterial cells likely interrupted the intermolecular interactions, leading
to increased intermolecular spaces. This result can be correlated with
increased WVP on adding bacterial cells to the film. Our results align
with previous studies that have reported that bacterial cells embedded
in the edible film lead to a less uniform film surface (Piermaria et al.,
2015; Soukoulis, Behboudi-Jobbehdar et al., 2014; Ye et al., 2018). SEM
micrographs of lyophilized chia mucilage shown in Fig. 8B revealed
the intensive nanoscale 3D network formed upon hydration of the chia
seeds as shown previously (Samateh et al., 2018). Fibrous structures of
polysaccharides with deposited clusters of raisin-like structures depict
mucilage protein fractions, as reported earlier (Goh et al., 2016).
3.7. Mechanical properties of the film
The mechanical aspects of films are shown in Fig. 9, demonstrat-
ing that sodium caseinate film exhibited a tensile strength of 3.7 MPa,
similar to previous findings (Ghosh et al., 2009; Mei & Zhao, 2003). In-
corporating CM and probiotic cells did not significantly affect the edible
film’s tensile strength. Young’s film modulus varied from 45.4 MPa to
69.9 MPa, while the CM significantly (p < 0.05) reduced Young’s mod-
ulus of the edible film. Bacterial cells significantly enhanced Young’s
modulus of the film (p < 0.0001 for CM-containing film and p < 0.005
for an edible film without CM). Young’s modulus denotes the mate-
rial’s stiffness with a higher value, indicating less material flexibility.
Based on our results, it can be inferred that CM improved the flexibil-
ity of sodium caseinate film, possibly due to the interaction between
mucilage and sodium caseinate, which is confirmed by the FTIR studies
and compact surface as depicted in SEM micrographs. Notably, under
heat treatment, heteropolysaccharides in CM may interact with dena-
tured protein chains replacing protein-protein interaction with protein-
polysaccharide crosslinking, leading to accelerated mobility of poly-
mer chains conferring higher flexibility. In addition, improved flexibil-
ity of edible film could be due to the hydrophilic nature of chia mu-
cilage leading to moisture retention. It has been reported that water
increases flexibility and acts as a plasticizer responsible for the ductile
nature of the film (Kanmani & Lim, 2013; Kristo et al., 2008). Kristo
et al. (2008) showed that pure pollulan film showed higher flexibil-
ity due to the hydrophilic nature of pollulan in the film. Kanmani and
Lim (2013) also showed that Young’s modulus of sodium caseinate
A. Semwal, K. Ambatipudi and N.K. Navani Food Hydrocolloids for Health 2 (2022) 100065

Fig. 7. Micrographs obtained by SEM (1μm scale bar) and AFM of (A) sodium caseinate film (B) sodium caseinate film incorporated with chia mucilage (C) glass
slide coated with edible film solution containing L. fermentum NKN51 and L. brevis NKN52.

Fig. 8. SEM visualization of (A) probiotic edible film containing L. fermentum NKN51 and L. brevis NKN 52 (B) nanoscale 3D network of chia mucilage (CM). Scale
bar = 1 μm.

film plasticized with sorbitol decreased with increasing relative hu-
midity. The results from the present study are further supported by
Muñoz et al. (2012), who reported that increased flexibility and strength
of whey protein concentrate film on the addition of chia mucilage could
be due to interaction between protein and polysaccharide.
The incorporation of probiotic cells enhanced the stiffness of the edi-
ble film, which could be due to the low pH in the film matrix released by
lactic acid produced by entrapped lactic acid bacteria. At low pH (near
pI of casein), the altered molecular conformation of the film is promoted,
leading to protein-protein solid interaction, which could be responsible
for reducing the flexibility of the probiotic film (Folegatti et al., 1998).
CM has no significant effect on elongation at break (EB) of edible films,
while the incorporation of bacterial cells significantly (p < 0.0001) en-
hanced the value of EB of the probiotic, edible film (74% and 55% in
presence and absence of probiotic cells, respectively). The change in
EB can be due to the low pH in probiotic films, which strengthens the
protein-protein interaction that interferes with protein-polysaccharide
interaction and reduces the free mobility of the polymer chains. It was
observed that the presence of bacterial cells reduced the elasticity of
the film but enhanced plasticity, although flexible, not brittle, which
enables the film to withstand more strain and increased extensibility
before breaking.
3.8. FTIR analysis
The FTIR spectrum (Fig. 10A) of CM revealed the characteristic
peaks for carbohydrates at 3434 cm−1 and 1124 cm−1 representing O-H
stretching of hydroxyl group and bending vibration of C-O and C-O-C

7
A. Semwal, K. Ambatipudi and N.K. Navani Food Hydrocolloids for Health 2 (2022) 100065

Fig. 9. (A) Tensile strength (B) Young’s modulus (C) elongation at break of sodium caseinate films with and without incorporation of chia mucilage (CM) and
bacterial cells (B). Data are presented as mean ± SD (n=3).

Fig. 10. FTIR spectra of (A) chia mucilage (CM) (B) sodium caseinate film without chia mucilage (SC) and with varying concentration (1% and 5%w/w) of chia
mucilage (CM) plotted with transmittance (%T) as a function of wavenumber (4000–500 cm−1 ).

in pyranose ring, similar to previous reports (Cerqueira et al., 2011;
Liu et al., 2017; Timilsena et al., 2016). CM contains uronic acid that
confers anionic nature to the macromolecule, confirmed by the peaks
observed at 1403 cm-1 and 1583 cm-1 , corresponding to the stretching
of the carboxylic group of uronic acid (de Campo et al., 2017; Goh et al.,
2016; Timilsena et al., 2016). An intense peak at 1632 cm−1 governed by
mannose and galactose ring stretching was further observed. A similar
peak was also detected in locust bean gum and guar gum (Freitas et al.,
2009; Wang & Somasundaran, 2007). Furthermore, a significant peak
elucidating the presence of polysaccharide compound was observed at
1006 cm−1 assigned to C-O-H bending and C-O-C stretching vibration
of 1→4 glycosidic bonds of hemicellulosic compounds (de Campo et al.,
2017; Mujtaba et al., 2019). The IR spectra also showed a peak at
840 cm−1 associated with the glycosidic linkages by glucopyranose and

8
A. Semwal, K. Ambatipudi and N.K. Navani Food Hydrocolloids for Health 2 (2022) 100065

Fig. 11. Survival of probiotics on coated wheat bun stored at (A) room temperature (RT) (B) chilling condition (4 °C). Data are presented as mean ± SD (n = 3).

xylopyranose units and 𝛽-anomeric C-H configuration (Cerqueira et al.,
2011; Punia & Dhull, 2019; Timilsena et al., 2016).
FTIR analysis confirmed the elucidation of structural changes in pro-
tein film and the interactions of proteins with CM, as shown in Fig. 10B.
The spectra of protein film incorporated with CM (1%w/w and 5%w/w
concentration) showed the broadband in the 3000-3500 cm−1 spectral
region due to the free and bound O-H from chia mucilage overlapping
with N-H groups from the protein. CM comprises hemicellulosic com-
pounds, protein macromolecules, and sugars providing -NH2 , -COOH,
and -OH functional groups. Gradual supplementation of the chia mu-
cilage in protein film contributed to more functional groups forming
intermolecular H bonds with protein, resulting in a wider -OH band.
Due to the hydrophilic nature of CM, the high moisture content is also
responsible for the wider -OH band (Punia & Dhull, 2019). Due to in-
creased supplementation (5% w/w) of CM, the OH peak shifted from
3429 cm−1 to 3430 cm−1 , associated with the strong interaction between
protein and chia mucilage (Liu et al., 2017). The wider and less intense
band observed at 1010 cm−1 could be due to incorporating 1% w/w CM
but disappeared on further supplementing CM at 5%w/w concentration.
The band’s disappearance could be due to the lack of free functional
groups in mucilage, which indicated the interaction between the func-
tional groups of polysaccharide and protein. A decrease in the intensity
of other bands at 1264 cm−1 , 1334 cm−1 , and 700-1000 cm−1 was due to
the adding 5% w/w CM that inferred structural modification in sodium
caseinate and strongly indicated the intermolecular interaction between
CM and protein film. Similarly, Diak et al. (2007) explained the interac-
tion between casein and oleic acid in the film leading to modifications
or disappearance of bands in FTIR spectra. Furthermore, a wider band
at 1403 cm-1 with the gradual incorporation of CM could be associ-
ated with an increased carboxylate group from protein and uronic acid
of CM. These findings strongly support the successful incorporation of
CM in the sodium caseinate film. FTIR spectrum of the probiotic edible
film (Supplementary Fig. S1) showed that the incorporation of bacterial
cells did not change the spectra of the film matrix, which is in line with
previously reported studies (Pereira et al., 2016; Ye et al., 2018).
3.9. The probiotic edible coating on bakery product
We coated the wheat bun with our developed probiotic film to in-
vestigate the feasibility of our developed film formulation as a car-
rier of probiotic cells, as shown in Fig. 12. It was observed that the
shelf-life of probiotic edible coating on the bun surface lasted for 22
days at 4 °C storage (8.07 log CFU/g) and 13 days at RT (6.35 log
CFU/g), as shown in Fig. 11, fulfilling the criteria of probiotic bread
(Soukoulis, Yonekura et al., 2014). However, it is challenging to develop
probiotic bakery products by directly inoculating the cells as the high
temperature applied during baking is a limiting factor for the survival
of the cells. It can be concluded that the developed probiotic edible film
is a suitable matrix that helps maintain the viability of bacterial cells
on the food surface. The probiotic, edible coating can be a promising

Fig. 12. Photographic images of wheat bun (A) uncoated (B) coated with probiotic film solution containing L. fermentum NKN 51 and L. brevis NKN 52.

9
A. Semwal, K. Ambatipudi and N.K. Navani
approach for delivering the probiotics to bakery products and can also
be applied to other food products.
4. Conclusion
In the present study, we developed a food-grade CM-sodium ca-
seinate composite probiotic film embedded with L. fermentum NKN51
and L. brevis NKN52. The investigations into the microbiological and
physical parameters of the probiotic film demonstrated substantial ev-
idence in delivering the probiotics to a wheat bun. Sodium caseinate
film supplemented with CM showed suitable water barrier property with
WVP as 0.0063 g mm m−2 h−1 KPa. It proved to be a suitable carrier
of probiotic cells, indicating that incorporating CM in sodium caseinate
film improves the storage stability of embedded bacterial cells (L. fer-
mentum NKN51 and L. brevis NKN52) for 2 months during storage at
4 °C. It is noteworthy to highlight that the viability of L. fermentum
NKN51 and L. brevis NKN52 improved by 3 log cycles in the presence
of chia mucilage in the film when stored at 4 °C. Furthermore, results
showed that incorporating CM didn’t alter the film’s tensile property,
thickness, and water barrier property of sodium caseinate film but im-
proved the flexibility of the probiotic film due to interaction between
protein-polysaccharide chains confirmed by the FTIR. There are very
few studies that focus on applying mucilage as a protectant to enhance
the survival of probiotics in films. We have successfully shown that CM
enhances the survival of probiotic bacterial strains entrapped inside the
edible film matrix. As confirmed by SEM analysis, an edible film solution
can be used as a coating solution to deliver the cells on the coated sub-
strate. Thus, considering the encouraging results obtained in this study,
sodium caseinate can be used as a suitable matrix for the entrapment
of probiotic strains with enhanced viability supported by CM as a pro-
tectant. Moreover, the developed probiotic edible film has been demon-
strated as an efficient carrier of probiotics to deliver bacterial cells on
a selected bakery product successfully. Multidirectional application of
developed probiotic film on different food matrices such as fruits, food
wraps, beverages, etc., can also be further explored, including applica-
tions in the pharmaceutical industry. Additionally, structural character-
ization and physicochemical properties of CM can also be performed to
explore its beneficial properties.
Declaration of Competing Interest
The authors declare no conflict of interests.
Acknowledgment
AS acknowledges financial support from MHRD, Govt of India.
NKN acknowledges Biotechnology Industry Research Assistance Council
(BIRAC) (BT/AIR0519/PACE-15/18_IITR) for funding support.
Supplementary materials
Supplementary material associated with this article can be found, in
the online version, at doi:10.1016/j.fhfh.2022.100065.
References
Acton, J. C., Breyer, L., & Satterlee, L. D. (1982). Effect of dietary fiber constituents on
the in vitro digestibility of casein. Journal of Food Science, 47(2), 556–560.
Audic, J. L., & Chaufer, B. (2005). Influence of plasticizers and crosslinking on the prop-
erties of biodegradable films made from sodium caseinate. European Polymer Journal,
41(8), 1934–1942.
Avila-De La Rosa, G., Alvarez-Ramirez, J., Vernon-Carter, E. J., Carrillo-Navas, H., &
Pérez-Alonso, C. (2015). Viscoelasticity of chia (Salvia hispanica L.) seed mucilage
dispersion in the vicinity of an oil-water interface. Food Hydrocolloids, 49, 200–207.
Burgain, J., Gaiani, C., Cailliez-Grimal, C., Jeandel, C., & Scher, J. (2013). Encapsulation
of Lactobacillus rhamnosus GG in microparticles: Influence of casein to whey pro-
tein ratio on bacterial survival during digestion. Innovative Food Science & Emerging
Technologies, 19, 233–242.
10
Food Hydrocolloids for Health 2 (2022) 100065
Bustamante, M., Laurie-Martínez, L., Vergara, D., Campos-Vega, R., Rubilar, M., &
Shene, C. (2020). Effect of three polysaccharides (inulin, and mucilage from chia and
flax seeds) on the survival of probiotic bacteria encapsulated by spray drying. Applied
Sciences, 10(13), 4623.
Bustamante, M., Oomah, B. D., Rubilar, M., & Shene, C. (2017). Effective Lactobacillus
plantarum and Bifidobacteriuminfantis encapsulation with chia seed (Salvia hispanica
L.) and flaxseed (Linumusitatissimum L.) mucilage and soluble protein by spray dry-
ing. Food Chemistry, 216, 97–105.
Cao, N., Fu, Y., & He, J. (2007). Preparation and physical properties of soy protein isolate
and gelatin composite films. Food Hydrocolloids, 21(7), 1153–1162.
Carvalho, A. S., Silva, J., Ho, P., Teixeira, P., Malcata, F. X., & Gibbs, P. (2004). Effects of
various sugars added to growth and drying media upon thermotolerance and survival
throughout storage of freeze-dried Lactobacillus delbrueckii ssp. bulgaricus. Biotechnol-
ogy Progress, 20(1), 248–254.
Castejón, N., Luna, P., & Señoráns, F. J. (2017). Ultrasonic removal of mucilage for pres-
surized liquid extraction of omega-3 rich oil from chia seeds (Salvia hispanica L.).
Journal of Agricultural and Food Chemistry, 65(12), 2572–2579.
Câmara, A. K. F. I., Okuro, P. K., da Cunha, R. L., Herrero, A. M., Ruiz-Capillas, C., &
Pollonio, M. A. R. (2020). Chia (Salvia hispanica L.) mucilage as a new fat substitute
in emulsified meat products: Technological, physicochemical, and rheological char-
acterization. Lwt, 125, Article 109193.
Cerqueira, M. A., Souza, B. W., Simões, J., Teixeira, J. A., Domingues, M. R. M., Coim-
bra, M. A., & Vicente, A. A. (2011). Structural and thermal characterization of
galactomannans from non-conventional sources. Carbohydrate Polymers, 83(1), 179–
185.
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), 185–193.
Coorey, R., Tjoe, A., & Jayasena, V. (2014). Gelling properties of chia seed and flour.
Journal of Food Science, 79(5), E859–E866.
de Campo, C., dos Santos, P. P., Costa, T. M. H., Paese, K., Guterres, S. S., de Oliveira
Rios, A., & Flôres, S. H. (2017). Nanoencapsulation of chia seed oil with chia mucilage
(Salvia hispanica L.) as wall material: Characterization and stability evaluation. Food
Chemistry, 234, 1–9.
Diak, O. A., Bani-Jaber, A., Amro, B., Jones, D., & Andrews, G. P. (2007). The manufacture
and characterization of casein films as novel tablet coatings. Food and Bioproducts
Processing, 85(3), 284–290.
Dick, M., Costa, T. M. H., Gomaa, A., Subirade, M., de Oliveira Rios, A., &
Flôres, S. H. (2015). Edible film production from chia seed mucilage: Effect of glyc-
erol concentration on its physicochemical and mechanical properties. Carbohydrate
Polymers, 130, 198–205.
Ebrahimi, B., Mohammadi, R., Rouhi, M., Mortazavian, A. M., Shojaee-Aliabadi, S., &
Koushki, M. R. (2018). Survival of probiotic bacteria in carboxymethyl cellulose-based
edible film and assessment of quality parameters. LWT, 87, 54–60.
Fabra, M. J., Talens, P., & Chiralt, A. (2008). Tensile properties and water vapor perme-
ability of sodium caseinate films containing oleic acid–beeswax mixtures. Journal of
Food Engineering, 85(3), 393–400.
Felisberto, M. H. F., Wahanik, A. L., Gomes-Ruffi, C. R., Clerici, M. T. P. S., Chang, Y. K.,
& Steel, C. J. (2015). Use of chia (Salvia hispanica L.) mucilage gel to reduce fat in
pound cakes. LWT-Food Science and Technology, 63(2), 1049–1055.
Folegatti, M. I., Antunes, A. J., & Marcondes, J. A. (1998). Mechanical and permeability
properties of milk protein films. Brazilian Archives of Biology and Technology, 41(3)
0-0.
Freitas, F., Alves, V. D., Pais, J., Costa, N., Oliveira, C., Mafra, L., & Reis, M. A. (2009).
Characterization of an extracellular polysaccharide produced by a Pseudomonas strain
grown on glycerol. Bioresource Technology, 100(2), 859–865.
Ghosh, A., Ali, M. A., & Dias, G. J. (2009). Effect of cross-linking on microstructure and
physical performance of casein protein. Biomacromolecules, 10(7), 1681–1688.
Gialamas, H., Zinoviadou, K. G., Biliaderis, C. G., & Koutsoumanis, K. P. (2010). Develop-
ment of a novel bioactive packaging based on the incorporation of Lactobacillus sakei
into sodium-caseinate films for controlling Listeria monocytogenes in foods. Food Re-
search International, 43(10), 2402–2408.
Goh, K. K. T., Matia-Merino, L., Chiang, J. H., Quek, R., Soh, S. J. B., & Lentle, R. G. (2016).
The physico-chemical properties of chia seed polysaccharide and its microgel disper-
sion rheology. Carbohydrate Polymers, 149, 297–307.
Kanmani, P., & Lim, S. T. (2013). Development and characterization of novel probiotic-re-
siding pullulan/starch edible films. Food Chemistry, 141(2), 1041–1049.
Kayserilioğlu, B. Ş., Bakir, U., Yilmaz, L., & Akkaş, N. (2003). Use of xylan, an agricultural
by-product, in wheat gluten based biodegradable films: Mechanical, solubility and
water vapor transfer rate properties. Bioresource Technology, 87(3), 239–246.
Kristo, E., Koutsoumanis, K. P., & Biliaderis, C. G. (2008). Thermal, mechanical and water
vapor barrier properties of sodium caseinate films containing antimicrobials and their
inhibitory action on Listeria monocytogenes. Food Hydrocolloids, 22(3), 373–386.
Lin, K. Y., Daniel, J. R., & Whistler, R. L. (1994). Structure of chia seed polysaccharide
exudate. Carbohydrate Polymers, 23(1), 13–18.
Linares, D. M., Gómez, C., Renes, E., Fresno, J. M., Tornadijo, M. E., Ross, R. P., & Stan-
ton, C. (2017). Lactic acid bacteria and bifidobacteria with potential to design natural
biofunctional health-promoting dairy foods. Frontiers in Microbiology, 8, 846.
Liu, L., Lin, W. J., Liu, H. Z., Shi, A. M., Hu, H., Nasir, M. N., Deleu, M., & Wang, Q. (2017).
Effect of xylose on the structural and physicochemical properties of peanut isolated
protein based films. RSC Advances, 7(83), 52357–52365.
Luo, M., Cao, Y., Wang, W., Chen, X., Cai, J., Wang, L., & Xiao, J. (2019). Sustained-release
antimicrobial gelatin film: Effect of chia mucilage on physicochemical and antimicro-
bial properties. Food Hydrocolloids, 87, 783–791.
Mei, Y., & Zhao, Y. (2003). Barrier and mechanical properties of milk protein-based edi-
ble films containing nutraceuticals. Journal of Agricultural and Food Chemistry, 51(7),
1914–1918.
A. Semwal, K. Ambatipudi and N.K. Navani
Misra, S., Pandey, P., & Mishra, H. N. (2021). Novel approaches for co-encapsulation of
probiotic bacteria with bioactive compounds, their health benefits and functional food
product development: A review. Trends in Food Science & Technology.
Mujtaba, M., Akyuz, L., Koc, B., Kaya, M., Ilk, S., Cansaran-Duman, D., Martinez, A. S.,
Cakmak, Y. S., Labidi, J., & Boufi, S. (2019). Novel, multifunctional mucilage com-
posite films incorporated with cellulose nanofibers. Food Hydrocolloids, 89, 20–28.
Muñoz, L. A., Aguilera, J. M., Rodriguez-Turienzo, L., Cobos, A., & Diaz, O. (2012). Char-
acterization and microstructure of films made from mucilage of Salvia hispanica and
whey protein concentrate. Journal of Food Engineering, 111(3), 511–518.
Namratha, S., Sreejit, V., & Preetha, R. (2020). Fabrication and evaluation of physico-
chemical properties of probiotic edible film based on pectin–alginate–casein compos-
ite. International Journal of Food Science & Technology, 55(4), 1497–1505.
Orozco-Parra, J., Mejía, C. M., & Villa, C. C. (2020). Development of a bioactive synbiotic
edible film based on cassava starch, inulin, and Lactobacillus casei. Food Hydrocolloids,
104, Article 105754.
O’Toole, P. W., Marchesi, J. R., & Hill, C. (2017). Next-generation probiotics: The spectrum
from probiotics to live biotherapeutics. Nature Microbiology, 2(5), 1–6.
Pavli, F., Tassou, C., Nychas, G. J. E., & Chorianopoulos, N. (2018). Probiotic incorpora-
tion in edible films and coatings: Bioactive solution for functional foods. International
Journal of Molecular Sciences, 19(1), 150.
Pereira, J. O., Soares, J., Sousa, S., Madureira, A. R., Gomes, A., & Pintado, M. (2016).
Edible films as carrier for lactic acid bacteria. LWT, 73, 543–550.
Piermaria, J., Diosma, G., Aquino, C., Garrote, G., & Abraham, A. (2015). Edible kefi-
ran films as vehicle for probiotic microorganisms. Innovative Food Science & Emerging
Technologies, 32, 193–199.
Punia, S., & Dhull, S. B. (2019). Chia seed (Salvia hispanica L.) mucilage (a heteropolysac-
charide): Functional, thermal, rheological behaviour and its utilization. International
Journal of Biological Macromolecules, 140, 1084–1090.
Ramos, Ó. L., Fernandes, J. C., Silva, S. I., Pintado, M. E., & Malcata, F. X. (2012). Edible
films and coatings from whey proteins: A review on formulation, and on mechanical
and bioactive properties. Critical Reviews in Food Science and Nutrition, 52(6), 533–552.
Ramos, Ó. L., Reinas, I., Silva, S. I., Fernandes, J. C., Cerqueira, M. A., Pereira, R. N.,
Vicente, A. A., Poças, M. F., Pintado, M. E., & Malcata, F. X. (2013). Effect of whey
protein purity and glycerol content upon physical properties of edible films manufac-
tured therefrom. Food Hydrocolloids, 30(1), 110–122.
da Silveira Ramos, I. F., Magalhães, L. M., do O Pessoa, C., Ferreira, P. M. P., dos Santos
Rizzo, M., Osajima, J. A., & Costa, M. P. (2021). New properties of chia seed mu-
cilage (Salvia hispanica L.) and potential application in cosmetic and pharmaceutical
products. Industrial Crops and Products, 171, Article 113981.
Reid, G., Jass, J., Sebulsky, M. T., & McCormick, J. K. (2003). Potential uses of probiotics
in clinical practice. Clinical Microbiology Reviews, 16(4), 658–672.
Roberfroid, M. B. (2000). Concepts and strategy of functional food science: The European
perspective. The American Journal of Clinical Nutrition, 71(6), 1660S–1664S.
Rodrigues, F. J., Cedran, M. F., Bicas, J. L., & Sato, H. H. (2020). Encapsulated probi-
otic cells: Relevant techniques, natural sources as encapsulating materials and food
applications–A narrative review. Food Research International, 137, Article 109682.
Rodríguez, G. M., Sibaja, J. C., Espitia, P. J., & Otoni, C. G. (2020). Antioxidant active pack-
aging based on papaya edible films incorporated with Moringaoleifera and ascorbic
acid for food preservation. Food Hydrocolloids, 103, Article 105630.
11
Food Hydrocolloids for Health 2 (2022) 100065
Saad, N., Delattre, C., Urdaci, M., Schmitter, J. M., & Bressollier, P. (2013). In An overview
of the last advances in probiotic and prebiotic field: 50 (pp. 1–16). LWT-Food Science
and Technology.
Sadeghi-Varkani, A., Emam-Djomeh, Z., & Askari, G. (2018). Physicochemical and mi-
crostructural properties of a novel edible film synthesized from Balangu seed mu-
cilage. International Journal of Biological Macromolecules, 108, 1110–1119.
Salehi, F. (2019). Characterization of new biodegradable edible films and coatings based
on seeds gum: A review. Journal of Packaging Technology and Research, 3(2), 193–201.
Samateh, M., Pottackal, N., Manafirasi, S., Vidyasagar, A., Maldarelli, C., &
John, G. (2018). Unravelling the secret of seed-based gels in water: The nanoscale
3D network formation. Scientific Reports, 8(1), 7315.
Sánchez-González, L., Saavedra, J. I. Q., & Chiralt, A. (2014). Antilisterial and physical
properties of biopolymer films containing lactic acid bacteria. Food Control, 35(1),
200–206.
Silva, L. A., Sinnecker, P., Cavalari, A. A., Sato, A. C. K., & Perrechil, F. A. (2022). Extrac-
tion of chia seed mucilage: Effect of ultrasound application. Food Chemistry Advances,
Article 100024.
Soukoulis, C., Behboudi-Jobbehdar, S., Yonekura, L., Parmenter, C., & Fisk, I. D. (2014).
Stability of Lactobacillus rhamnosus GG in prebiotic edible films. Food Chemistry, 159,
302–308.
Soukoulis, C., Behboudi-Jobbehdar, S., Macnaughtan, W., Parmenter, C., &
Fisk, I. D. (2017). Stability of Lactobacillus rhamnosus GG incorporated in ed-
ible films: Impact of anionic biopolymers and whey protein concentrate. Food
Hydrocolloids, 70, 345–355.
Soukoulis, C., Gaiani, C., & Hoffmann, L. (2018). Plant seed mucilage as emerging biopoly-
mer in food industry applications. Current Opinion in Food Science, 22, 28–42.
Soukoulis, C., Singh, P., Macnaughtan, W., Parmenter, C., & Fisk, I. D. (2016). Composi-
tional and physicochemical factors governing the viability of Lactobacillus rhamnosus
GG embedded in starch-protein based edible films. Food Hydrocolloids, 52, 876–887.
Soukoulis, C., Yonekura, L., Gan, H. H., Behboudi-Jobbehdar, S., Parmenter, C., &
Fisk, I. (2014). Probiotic edible films as a new strategy for developing functional
bakery products: The case of pan bread. Food Hydrocolloids, 39, 231–242.
Su, J. F., Huang, Z., Yuan, X. Y., Wang, X. Y., & Li, M. (2010). Structure and properties of
carboxymethyl cellulose/soy protein isolate blend edible films crosslinked by Maillard
reactions. Carbohydrate Polymers, 79(1), 145–153.
Timilsena, Y. P., Adhikari, R., Kasapis, S., & Adhikari, B. (2016). Molecular and functional
characteristics of purified gum from Australian chia seeds. Carbohydrate Polymers, 136,
128–136.
Tripathi, M. K., & Giri, S. K. (2014). Probiotic functional foods: Survival of probiotics
during processing and storage. Journal of Functional Foods, 9, 225–241.
Wang, J., & Somasundaran, P. (2007). Study of galactomannose interaction with solids
using AFM, IR and allied techniques. Journal of Colloid and Interface Science, 309(2),
373–383.
Ye, J., Ma, D., Qin, W., & Liu, Y. (2018). Physical and antibacterial properties of sodium al-
ginate—sodium carboxymethylcellulose films containing lactococcuslactis. Molecules,
23(10), 2645.
Zoghi, A., Khosravi-Darani, K., & Mohammadi, R. (2020). Application of edible films con-
taining probiotics in food products. Journal of Consumer Protection and Food Safety,
15, 307–320.