Food Chemistry 360 (2021) 129966

Contents lists available at ScienceDirect

Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Study of the effect of the addition of plasticizers on the physical properties

of biodegradable films based on kefiran for potential application as
food packaging
Lissette Montoille a, Constanza Morales Vicencio a, Daniel Fontalba a, J. Andrés Ortiz a, b,
Viviana Moreno-Serna a, Laura Peponi c, Silvia Matiacevich d, Paula A. Zapata a, *
a
Universidad de Santiago de Chile (USACH), Facultad de Química y Biología, Departamento de Ciencias del Ambiente, Grupo de Polímeros, Chile
b
Departamento de Ingeniería Química, Biotecnología y Materiales, Facultad de Ciencias Físicas y Matemáticas, Universidad de Chile, Avenida Beaucheff 851, Santiago,
Chile
c
Institute of Polymer Science and Technology, ICTP-CSIC, Madrid, Spain
d
Food Properties Research Group, Departamento de Ciencia y Tecnología de los Alimentos, Facultad Tecnológica, Universidad de Santiago de Chile, Obispo Manuel
Umaña 050, Estación Central, Santiago 9170201, Chile

A R T I C L E I N F O A B S T R A C T

Keywords: Spectroscopies analysis indicated that kefiran contains branched hexasaccharide repeating units. Neat kefiran
Biodegradable polymers films, 2 and 5% w/w of glycerol, D-glucitol, D-galactitol, D-mannitol, and D-limonene were incorporated as
Food packaging plasticizers. Neat and plasticized kefiran films were characterized by physical, thermal, mechanical, optical, and
Kefiran
water solubilization properties. Neat kefiran had a glass transition temperature (Tg) of − 20 ± 2 ◦ C and, with the
Plasticizers
addition of plasticizers between − 15 to − 17 ± 2 ◦ C. The values were close to the neat kefiran, and the results
could be attributed to a lower amount of plasticizer used. The solubility of the glycerol plasticized films increases
by 33% and decreased with the concentration of other plasticizers in comparison with the neat kefiran. D-glucitol
and D-galactitol decreased the microhardness and Young’s Modulus of films around 30% and 74% respectively,
obtaining more flexible kefiran films. Kefiran based films could find applications as potential materials in the
food-packaging industry.

1. Introduction polyhydroxyalkanoates (PHA) are widely studied (Roohi, Srivastava,

In the last decades, traditional plastics derived from fossil resources
have become indispensable materials for our society. Consequently, its
consumption has increased considerably in the last decades, with pro­
duction levels bordering the 360 million tons worldwide, being one of its
main uses in food packaging, generating an accumulation of waste in the
ecosystem (Lebreton & Andrady, 2019). For this reason, innovation in
the packaging industry is focused on the development of new polymeric
materials from natural sources such as biopolymers like proteins, lipid-
based natural polymers, alginate, pectin, and starch (Mohamed, El-
Sakhawy, & El-Sakhawy, 2020). The interest in biopolymers arises
from its advantages, such as good biodegradability, biocompatibility,
environmental friendliness, availability, and even edibility (Ili Balqis,
Nor Khaizura, Russly, & Nur Hanani, 2017). Currently, food packaging
using starch-based biopolymers, where polylactic acid (PLA) and
Bano, & Zaheer, 2018). However, these biopolymers from renewable
resources have the disadvantage of high production costs. Therefore, the
research and development of food packaging films from alternative
biopolymers such as kefiran are necessary. Kefiran is an attractive
polysaccharide substitute because it is a biomass material obtained from
milk fermentation, resulting in a possible material that can help solve
the mentioned environmental issues.
Kefiran is a high-temperature water-soluble extracellular branched
polysaccharide with many hydroxyl groups obtained from the lactic and
acetic acid bacteria present in kefir grains. This biopolymer in its mo­
lecular structure has a repetitive unit with roughly equal amounts of D-
glucose and D-galactose. It is a branched hexa- or hepta-saccharide
repeating unit composed of a regular pentasaccharide unit to which
one or two sugar residues are randomly attached (Moradi & Kalanpour,
2019). This polysaccharide presents unique properties as

* Corresponding author.
E-mail address: paula.zapata@usach.cl (P.A. Zapata).

https://doi.org/10.1016/j.foodchem.2021.129966
Received 13 December 2020; Received in revised form 8 April 2021; Accepted 24 April 2021
Available online 4 May 2021
0308-8146/© 2021 Elsevier Ltd. All rights reserved.
L. Montoille et al.
biodegradability, safety, emulsifier and stabilizing effect, resistance
against hydrolysis, barrier, and water vapor permeability (Moradi &
Kalanpour, 2019). Furthermore, compared to other polysaccharides,
kefiran has several significant advantages, such as antibacterial and
antifungal properties (Jeong et al., 2017). At once, kefir production is
obtained from a green synthesis and can be purified to produce biode­
gradable films. It only requires the prior preparation of kefir granules
with milk to produce its fermentation from milk, followed by physical
processes that do not generate dangerous or toxic substances (Rimada,
2001). Studies also show microorganisms degraded kefiran films in a
shorter time compared to low-density polyethylene (LDPE) or polylactic
acid (PLA) films (Ghasemlou, Khodaiyan, & Oromiehie, 2011a). How­
ever, kefiran films exhibit several disadvantages, such as a strong hy­
drophilic character and poor mechanical properties compared to
synthetic packaging films. In this sense, it is necessary to add innocuous
plasticizers to create a satisfactory food packaging application (Gha­
semlou, Khodaiyan, Oromiehie, & Yarmand, 2011a, 2011b; Piermaria
et al., 2011).
Plasticizers are low-volatility, low-molecular-weight molecules
incorporated into polymers that help decrease the inherent brittleness of
films by reducing intermolecular forces, increasing their flexibility, and
facilitating their processing (Sanyang, Sapuan, Jawaid, Ishak, & Sahari,
2015). A plasticizer can change and tailor the mechanical properties of
the polymer by reducing the viscosity, lowering the glass transition
temperature, or decreasing the elastic modulus (Jha, 2020). Ideally,
plasticizers used for modifying plastic polymers must be of low toxicity
and have good adaptability with the polymer used. Commonly used
plasticizers in polysaccharide-based film formations are polyols, mono,
di- and oligosaccharides due to their hydrophilic nature (Ballesteros-
Mártinez, Pérez-Cervera, & Andrade-Pizarro, 2020). One of the most
wide plasticizers used in polysaccharide film-making techniques is
glycerol due to its high-boiling-point, water-soluble, polar, and
nonvolatile properties (Orsuwan & Sothornvit, 2018). Preliminary
studies reported that glycerol in 10–35% w/w added to kefiran films
changed the stiffness and increased the mechanical properties (Gha­
semlou et al., 2011a; Piermaria, Pinotti, Garcia, & Abraham, 2009).
Meanwhile, plasticized kefiran films with sorbitol exhibited lower water
vapor permeability values than kefiran films containing glycerol (Gha­
semlou et al., 2011a). It was determined that both plasticizers could play
an essential role in improving film properties, being more suitable for
food-packaging technology applications. Besides, Piermaria et al. (2011)
obtained plasticized kefiran films using sugars, such as D-glucose, D-
galactose, sorbitol, sucrose, and glycerol, with 25% w/w plasticizer
content. The results concluded that the addition of the plasticizer
improved the mechanical properties and water vapor permeability of
kefiran films. However, those studies reported high loading of plasticizer
content, suggesting low miscibility and increased costs when applying
this compostable film in the food market.
Authors only have reported studies where plasticizer with high
loadings from 10 to 35% w/w have been incorporated into the kefiran
films (Coma, Peltzer, Delgado, & Salvay, 2019; Ghasemlou et al., 2011a;
Ghasemlou, Khodaiyan, Oromiehie, et al., 2011a; J. Piermaria et al.,
2011). The incorporation of a plasticizer lower than 10% had not yet
been studied. Therefore, in this work, it has been interesting to study the
addition of lower plasticizer contents for kefiran (2 and 5% w/w).
Considering the characteristics of kefiran and the requirements of the
food industry for the development films that are biodegradable and not
based on fossil resources, the aim of this work is determining the effect
of plasticizer type (hydrophilic or hydrophobic) in a low concentration,
as a polyol, aldose, alditol, and terpene on the thermal, mechanical,
opacity and water solubilizing physical–chemical properties compared
to neat kefiran films. Furthermore, this study explores the addition of
new plasticizers for kefiran such as D-limonene and D-mannitol that
improve the film properties in order to obtain a new potential biode­
gradable film.
2
Food Chemistry 360 (2021) 129966
2. Materials and methods
2.1. Materials
The product for the growth of the kefir granules was skim milk
(Colun, Chile). For the extraction of kefir, ethanol (Winkler, 96%) was
used. The reagents for the NMR characterization were deuterium oxide
with 3- (trimethylsilyl) propionic-d4 acid in technical grade (97%) and
deuterium oxide in analytical grade (99.9%) from Sigma-Aldrich. Plas­
ticizers glycerol (Omnipur 97%), D-glucitol, D-galactitol, D-mannitol, and
D-limonene 97% from Sigma-Aldrich were employed.
2.2. Growth and maintenance of kefir granules
For the growth and development of the kefir granules, daily main­
tenance was necessary. The grains were kept in skimmed milk at room
temperature, and the medium was exchanged daily for a new culture to
maintain the grains’ viability. It was washed twice a week with distilled
water and avoiding sunlight to prevent contamination of the granules.
After the culture was maintained for seven subsequent days, the grains
were considered active (Zannini, Waters, Coffey, & Arendt, 2016).
2.3. Isolation and purification of kefiran
The isolation and purification of kefiran were performed, as was
previously reported (Judith A Piermaria, de la Canal, & Abraham, 2008;
Piermaria et al., 2009). Briefly, active kefir grains were treated in
heating water (80 ◦ C) for 30 min. The mixture was centrifuged at
10,000 rpm for 20 min at room temperature (TG1650-WS, Bioridge,
Melbourne, Australia). The polysaccharides in the supernatant were
precipitated by the addition of cold ethanol and frozen at − 20 ◦ C for 12
h. The mixture was centrifuged for 20 min at 4 ◦ C. For further purifi­
cation, the process mentioned above was repeated. After this, the solvent
was evaporated, and the product was lyophilized to remove any
remaining water molecules (Christ Alpha 1–2 Freeze Dryer, Osterode am
Harz, Germany).
2.4. Preparation of films
According to previously reported methods, the kefiran-based films
were prepared by conventional solution-casting technique (Ghasemlou,
Khodaiyan, Oromiehie, et al., 2011b; Piermaria et al., 2011, 2009). To
study the effect of plasticizer on film properties, glycerol, D-glucitol, D-
galactitol, D-mannitol, and D-limonene were added to kefiran in water to
concentrations 2 and 5% w/w. Initially, aqueous solutions of lyophilized
kefiran and plasticizer were prepared under constant stirring for 15 min
at 30 ◦ C to dissolve the kefiran and homogenize the mixture. The
mixture was then poured into Petri dishes of 35 mm diameter and kept in
an oven at 70 ◦ C for 48 h.
2.5. Characterization of films.
2.5.1. Spectroscopic characterization
Infrared spectra of isolated and kefiran based films were recorded in
the range of 4000–450 cm− 1 on a Fourier-Transform Infrared Analyzer
(FTIR) (Spectrum Two, Perkin Elmer, Karlsruhe, Germany) equipped
with an attenuated total reflectance diamond module (ATR). All spectra
were obtained as an average of 30 scans with a 2 cm− 1 resolution. 1H
(400.13 MHz) and 13C (100.62 MHz) NMR spectra of isolated kefiran
were recorded in D2O after the isotopic exchange (3 × 0.75 mL) at 70 ◦ C
on a Bruker Avance DRX 400 spectrometer (Bruker, Coventry, UK) using
the sodium salt of 3-(trimethylsilyl)-1-propionic-2,2,3,3-d4 acid as an
internal reference. Two-dimensional experiments were performed using
a pulse-field gradient incorporated into the NMR pulse sequence. The
number of scans in each experiment depended on sample concentration
(30–45 mg/mL) (Matsuhiro, Barahona, Encinas, Mansilla, & Ortiz,
L. Montoille et al.
2014).
2.5.2. Differential scanning calorimetry (DSC)
Thermal transitions of the films were studied using a Differential
Scanning Calorimeter (DSC-1 Mettler Toledo equipment, Columbus, OH,
USA) with the STARe Evaluation software. The DSC equipment was
previously calibrated with Indium. The samples were heated in a range
of temperatures from − 30 ◦ C to 200 ◦ C with a heating rate of 5 ◦ C/min,
in order to obtain the glass transition temperature (Tg) and the melting
point (Tm) calculated as the temperature where the peak of the endo­
therm occurs. Approximately 6 mg of samples were placed into
aluminum pans of 40 μL, an empty pan was used as a control, and all
tests were performed in an inert nitrogen environment with a constant
flow at 2 mL/min.
2.5.3. Thermogravimetric analysis (TGA)
Mass loss in samples as a function of temperature was registered in a
TGA Q500 (TA instrument, New Castle, DE, USA). Approximately 5 mg
of each sample was weighed in a pan of 50 μL. The samples were heated
from 28 to 630 ◦ C, at 10 ◦ C⋅min− 1, to study the thermal degradation of
the films. Experiments were carried out under a nitrogen atmosphere
with a constant flow at 10 mL/min. The temperature at the maximum
degradation rate (Tmax) was determined from the peak of derivative
curves using the Advantage/Universal Analysis (UA) Software v.5.5.24.
2.5.4. X-ray diffraction
The crystallographic structure of kefiran films was analyzed using an
X-ray diffractometer. The crystalline phase study was carried out in a
Diffractometer model D5000 (Siemens, Aubrey, TX, USA) using Bragg’s
law, which relates the diffraction angle (θ) to the distance between the
planes. The scan was of a range 0◦ < 2θ > 80◦ , with λ = Cu.
2.5.5. Scanning electron microscopy (SEM)
A 10 mm × 7 mm size piece of each film was analyzed using a
scanning electron microscope XL30 Phillips (SEMTech Sc., North Bill­
erica, MA, USA) at 20 kV. A 7 mm × 7 mm piece of each film was taken
and mounted on aluminum pins, using a double copper contact tape. For
best viewing under a microscope, the samples were coated with a layer
of gold.
2.5.6. Mechanical properties
Mechanical properties were evaluated by tensile test in a QTest™1/L
Elite instrument equipped with a 100 N load cell at room temperature.
Strain rate and initial length between clamps were set at 10 mm.min− 1
and 10 mm, respectively. Samples of 10 mm length, 5 mm width were
measured, and results from five specimens were averaged. Young’s
modulus (obtained as the slope of the curve from 0 to 2% deformation)
maximum stress and elongation at break were calculated.
2.5.7. Microhardness properties
Microhardness measurements of kefiran films were carried out in
triplicate with an Indentor Vickers (Buehler, Lake Bluff, IL, USA) con­
nected to a Leiz microhardness tester (Spectrographic, Guiseley, UK).
Five measurements were made at different film points to know about the
mechanical support average for each plasticizer case, obtaining an
average value, and minimizing the deviation. The measurements were
made at a temperature of 23 ◦ C, under a force of 4.809 N, and with an
application time of 25 s. Microhardness (MH) were reported as the mean
value.
2.5.8. Solubility in water of films (SW)
In this study, solubility in water (SW) was defined as the ratio of the
water-soluble dry matter of film dissolved after immersion in distilled
water. Besides, the SW test for each type of film was carried out in three
replicates. A film sample was dried at 105 ◦ C for 24 h in an oven and
weighed to determine the initial dry weight. The solubility in water of
3
Food Chemistry 360 (2021) 129966
the different kefiran films with plasticizer was measured by immersion
in 50 mL of distilled with constant agitation at 250 rpm and room
temperature for six hours. The films were then taken out and dried again
at 105 ◦ C until constant weight (final dry weight). The percentage of
solubility (%SW) of the films in water was calculated using Eq. (1)
(Ghasemlou et al., 2011a):
initialdryweight − finaldryweight
%SW = x100
initialdryweight
2.5.9. Optical properties measurements.
The films were photographed inside a computer vision system
(LabVisionQ, Santiago, Chile) using a Rebel XS Canon digital camera at
30 cm of distance from the samples. The black box lighting system was
composed of four lamps (illuminant D65, 18 W, Phillips) at an angle of
90◦ to the sample. Three replicates were conducted on a black (Ob) and
white (Ow) background. Image analysis using Photoshop was performed
to calculate optical indexes from L*, a*, and b* parameters and the
tristimulus values XYZ from CIEL*a*b* and CIE color spaces, respec­
tively. The film opacity (O) was determined as the ratio between the L*
mean values on the standard black background (Ob) and the standard
white background (Ow) for each sample (Yam & Papadakis, 2004). The
results were expressed as a percentage (Eq. (2)). The yellowness index
(YI), whiteness index (WI), and Chrome (C) were calculated using Eq.
(3) (Gooch, 2011a), Eq. (4) (Gooch, 2011b), and Eq. (5) (Skurtys, O. &
Acevedo, C. & Pedreschi, Franco & Enrione, Javier & Osorio, Fernando
& Aguilera, 2010), respectively. The color differences (CIEDE2000)
between plasticized samples compared to neat kefiran film were calcu­
lated using the free CIEDE2000 excel program from Sharma, Wu, and
Dalal (2004).
Ob
%O = *100 (2)
Ow
YI =
(128X − 106Z)
(3)
Y
√̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅
WI = 100 − (100 − L* )2 + (a*2 + b*2 ) (4)
√̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅
C= (a*2 + b*2 ) (5)
2.6. Statistical analysis.
A multilevel 5x2 factorial design was performed with five qualitative
levels (plasticizer type) and two quantitative levels (plasticizer con­
centration, 2 and 5% w/w). Data were analyzed by the analysis of
variance (ANOVA) procedure using the Statgraphics Centurion XVI
software. A categorical factorial design was used since one of the factors
for the design has qualitative levels. A multiple range test was used to
discriminate between means using Fisher’s least significant difference
(LSD) procedure. Also, three complete replicates associated with this
factorial design were used. The response variables analyzed were water
solubility (SW), Young modulus, yield stress, deformation at break,
microhardness (MH), and optical properties.
3. Results and discussion
The purpose of this study was to evaluate the effect of the addition of
new plasticizers to kefiran such as D-galactitol, D-mannitol, and D-limo­
nene in the thermal, mechanical, opacity, and water solubilizing phys­
ical–chemical properties of kefiran. Kefiran films containing glycerol
and D-galactitol, previously reported in the literature, also were studied
by comparison. It was interesting to study the effect of plasticizer type
(hydrophilic or hydrophobic) in a low concentration considering (2 and
5% w/w) that there was not still report.
L. Montoille et al. Food Chemistry 360 (2021) 129966

3.1. Kefiran isolation and spectroscopic characterization
Aqueous extraction of kefir grains at 80 ◦ C, followed by purification
cycles by cold ethanol precipitation, produced a 2% yield. A lyophilized
purified kefiran sample was analyzed by ATR-FTIR, 1H, 13C, and 2D
NMR spectroscopies (Fig. 1). The ATR-FTIR spectrum (Fig. 1a) presents
a broad and intense band among 3600 and 3100 cm− 1 attributed to νs
O–H of alcoholic groups. In the 2800–2900 cm− 1 region, signals were
assigned to νs/ νas C–H vibrations of the kefiran backbone (Ortiz,
Catalina, Corrales, Soto, & Zapata, 2020). The weak band at 1645 cm− 1
corresponds to water bending vibration due to the water occlusion in the
polymer matrix. Furthermore, it presents a set of medium intensity
signals between 1430 and 1250 cm− 1, which correspond to δ C–H, δ
C–O–H vibration modes. The most prominent and robust band at
1120–940 cm− 1 is assigned to νas C–O–C of glycosidic linkage vibra­
tion with contributions of ν C–O (pyranosyl ring), δ C–C–O, ν C–C, δ
C–C–H, and δ C–C–C vibrations (Cardenas-Jiron, Leal, Matsuhiro, &
Osorio Roman, 2011; J. Piermaria et al., 2011). Moreover, the region

Fig. 1. Spectroscopic characterization of kefiran obtained from kefir grains. a) ATR-FTIR spectrum in the region 4000–450 cm− 1. b) 13C NMR, c) 13C/1H HSQC, and
d) 13C/1H HMBC 2D NMR spectra in D2O at 70 ◦ C. e) Repeating unit of kefiran denoting each sugar residue sequence according to the spectroscopic characterization.
* νs, symmetrical stretching. νas, asymmetrical stretching. δ, deformation.

4
L. Montoille et al.
between 950 and 700 cm− 1 corresponds to the fingerprint or anomeric
region for carbohydrates. The band at 890 cm− 1 can be attributed to the
anomeric C–H of β residues with contributions of δ C–O–C and ν C–C
modes. Below the 700 cm− 1 regions, the observed peaks are attributed to
the endocyclic and exocyclic C–C–O and C–C–C vibration modes of
pyranosyl ring such as deformation, breathing, twisting, and wagging
deformation O–H (Matsuhiro et al., 2014). Additionally, the ATR-FTIR
kefiran spectrum does not exhibit potential charged functional groups
and amide vibrational modes associated with proteins.
The 13C NMR spectrum of kefiran in D2O (Fig. 1b) shows at least
three signals at a low field assigned to the β-hexapyranosyl
(106.1–105.7 ppm) and α-hexapyranosyl (98.7 ppm) anomeric carbons
residues. The integration ratio in the 1H NMR spectrum (figure not
shown) of the signals at 5.14 ppm (α anomeric proton) and 4.82–4.49
ppm (β anomeric protons) was 1.0:4.9. These results indicate that
kefiran contains hexasaccharide repeating units corroborated by the
13 1
C/ H HSQC 2D NMR spectrum (Fig. 1c). According to previous works
related to chemical structure determination of kefiran type poly­
saccharide, from the six sugar residues, the cross-peak/correlation at
98.5/5.14 ppm was assigned to the anomeric Cα/Hα of → 4)-α-D-Galp-
(1→ (C) residues. In contrast, the correlations at 106.1/4.69 and 105.7/
4.85 ppm were assigned to the anomeric Cβ/Hβ of → 2,6)-β-D-Galp-(1→
(B) β-D-Glcp-(1→ (F) residues, respectively. The overlapped correlation
that appears at 105.7/4.54 ppm correspond to → 6)-β-D-Glcp-(1→ (A),
→3)-β-D-Galp-(1→ (D) and → 4)-β-D-Glcp-(1 → residue (E) (Ghasemlou,
Khodaiyan, Jahanbin, Gharibzahedi, & Taheri, 2012). The glycosidic
linkages were confirmed by the 13C/1H HMBC 2D NMR technique
(Fig. 1d). It presents some relevant correlations assigned to the six
glycosidic linkages of the kefiran repetitive unit denoted in sequence as
[→6)-β-D-Glcp-(1 → 6)-β-D-Galp-(1→ (A1/B6), →2,6)-β-D-Galp-(1 → 4)-
α-D-Galp-(1→ (B1/C4), →4)-α-D-Galp-(1 → 3)-β-D-Galp-(1→ (C1/D3),
→3)-β-D-Galp-(1 → 4)-β-D-Glcp-(1→ (D1/E4), →4)-β-D-Glcp-(1 → 6)-β-D-
Glcp-(1→ (E1/A6)] and the β-D-Glcp-(1 → 2)-β-D-Galp-(1→ (F1/B2)
pendant residue (Fig. 1e). Similar results were reported for the chemical
structure determination of extracellular polysaccharide isolated from
Lactobacillus helveticus, Lactococcus lactis, and Lactobacillus kefir­
anofaciens (Maeda, Zhu, Omura, et al., 2004).
3.2. General appearance of kefiran films
The appearance of the films can influence the appreciation of the
final product. The mechanical stability of films is improved to the touch
when increased flexibility occurs. For potential application in the
packaging industry, the packaging films require a certain degree of
flexibility. The photographic images shown in Fig. 2 describe the visual
appearance of plasticized and neat kefiran films. The neat kefiran films
had a homogeneous presentation, high transparency, and no cracks, as
Fig. 2. Photographic images of a) neat kefiran film and kefiran films with different plasticizer type; b-c) glycerol, d-e) D-glucitol, f-g) D-galactitol, h-i) D-mannitol and
j-k) D-limonene at 2 and 5%, respectively.
5
Food Chemistry 360 (2021) 129966
seen in Fig. 2a. This observation agrees with the findings of Piermaria
et al. (2009) and Coma et al. (2019), who also prepared kefiran films.
The introduction of plasticizers in kefiran films renders them flexible
with a smooth and homogeneous surface, corroborated in the micro­
hardness properties measurements. Kefiran films with 2 and 5% w/w of
glycerol show a better appearance and mechanical stability. It means
that they were apparently more flexible and slightly opalescent when
compared to neat kefiran. This result may be due to a good dispersion of
the small glycerol molecule, which intercalates between polymer chains,
disrupts inter-polymer’ bonds, increasing the interactions between
glycerol and the kefiran backbone (Coma et al., 2019; Piermaria et al.,
2009).
Moreover, kefiran is a neutral polysaccharide that constitutes many
numbers of hydroxyl groups in its backbone. It confers a polar and hy­
drophilic nature to kefiran, making its interaction with water thermo­
dynamically more favorable than hydrophobic solvents. Therefore, with
the addition of a plasticizing agent constituted of hydroxyl groups and
employing water as solvent, the intermolecular hydrogen bonding plays
a significant role in the casting method’s plasticization (Piermaria et al.,
2011).
On the other hand, in films with D-glucitol, D-galactitol, D-limonene,
and D-mannitol (Fig. 2d–k), the flexibility increases as the plasticizer
concentration increases. However, films presented less transparency,
independent of the plasticizer type. This could indicate low dispersion of
plasticizers in the polymeric matrix as the concentration of the plasti­
cizer increases, unlike films containing glycerol.
Meanwhile, ATR-FTIR analysis was carried out to determine the in­
fluence of the plasticizer’s functional groups on the kefiran films’
structure to elucidate the interactions between the film-forming polymer
and the different plasticizers. Fig. 3a shows the ATR-FTIR spectra of the
kefiran films at different plasticizer concentrations. As is shown in
Fig. 3a, there are differences in the peaks’ relative height of OH groups’
hydrogen–oxygen bonding, being higher for the films loaded with 5%
w/w of the plasticizers D-galactitol and D-glucitol. The absorption of the
hydroxyl groups’ signal is highly influenced by the hydrogen–oxygen
bond’s dipole moment, the hydrogen bond’s attractive interaction, and
the Lambert-Beer law. Based on this trend, it can be inferred that the
hydroxyl groups of the long-chain polyols contribute to an increase of
the absorption of the hydrogen–oxygen stretching in the infrared spec­
trum (Silverstein, Bassler, & Morrill, 1991). Contrary to the films based
on kefiran and D-limonene at 5% w/w, there is no contribution in the
increase in the hydrogen–oxygen stretching mode compared to the neat
ATR-FTIR kefiran spectrum due to the hydrocarbon constitution of D-
limonene.
Similar studies suggest that the glycerol and sorbitol addition at 25%
w/w in kefiran films conduces to small dislocations for νs O–H of
alcoholic groups and νs/νas C–H vibrations. It was attributed to the
L. Montoille et al. Food Chemistry 360 (2021) 129966

Fig. 3. a) ATR-FTIR spectrum, b) DSC curves, and c) TGA thermogram of neat and plasticized kefiran films at 2 and 5% w/w. TGA curves exhibit zone I: water loss,
zone II: maximum temperature decomposition (Tmax), and zone III: depolymerization.

disruption of kefiran film’s structure by the presence of plasticizer

Table 1
molecules or the water molecules that they absorb (Ghasemlou, Kho­
Thermal properties of kefiran films with different plasticizer types.
daiyan, & Oromiehie, 2011b). However, these results are not compa­
rable with the present work due to the low concentration of plasticizer Plasticizer Plasticizer DSC analysis
type concentration (% w/
used. Tg (◦ C) Tm T10 Tmax
w)
(◦ C) (◦ C) (◦ C)

3.3. Characterization of kefiran plasticized films. Neat kefiran – − 20 84 184 306

Kefiran + 2 − 16 82 241 312
glycerol 5 − 17 89 248 311
3.3.1. Differential scanning calorimetry (DSC)
Kefiran + D- 2 − 15 80 271 312
The determination of glass transition (Tg) and melting temperature glucitol 5 − 16 80 271 315
(Tm) by DSC analysis is important because it affects the thermo- Kefiran + D- 2 − 15 80 230 316
mechanical properties and physical and chemical stability of the poly­ galactitol 5 − 17 82 249 315
meric films. In this study, Tg and Tm values of the plasticized and neat Kefiran + D- 2 − 17 80 250 309
mannitol 5 − 17 89 261 313
kefiran films were obtained to study the effect of polyols type and Kefiran + D- 2 − 17 84 217 313
concentration on these parameters. The DSC thermograms (Fig. 3b) limonene 5 − 17 84 254 307
shown in all cases only two transitions, one associated with the glass PLAa – 60 167 350 370
transition and the other with endothermic peak related to kefiran LDPE b – − 125–− 55 107 352 417
melting (Shahabi-Ghahfarrokhi, Khodaiyan, Mousavi, & Yousefi,
2015a). The glass transition shows only a single peak which indicates
Tm: melting transition temperature. a PLA, poly(lactic acid), and bLDPE,
the miscibility between plasticizer and kefiran (Burgos, Martino, &
Low-Density Polyethylene, were inserted for comparison. Equipment
Jiménez, 2013). Table 1 shows the Tg and Tm values for all films ob­
error ± 2. DSC: Differential scanning calorimetry. TGA: Thermogravi­
tained from the DSC thermograms of kefiran films with and without
metric analysis.
plasticizers.
Plasticized films had a slightly higher Tg than neat kefiran film. The
T10: decomposition temperature at 10% weight loss. Tmax: temper­
neat kefiran presented a Tg value of − 20 ± 2 ◦ C and shifted between
ature for maximum rate of weight loss. Tg: glass transition temperature.

6
L. Montoille et al.
− 17 ± 2 ◦ C under the addition of plasticizers. This behavior is different
from that reported by Ghasemlou et al. (2011b). They reported a Tg
value around − 15 ◦ C based on kefiran films that decreased to − 20 ◦ C
when the glycerol amount increased to 35% w/w. Therefore, this study’s
obtained results could be attributed to a lower amount of plasticizer
used compared to Ghasemlou et al. (2011b). A similar result was found
when a polyol as sorbitol (10% w/w) was incorporated into the kefiran.
The Tg did not show a significant change compared to the neat kefiran
obtained by DMA analysis (Ghasemlou et al., 2011a). In general, Tg
values (approximately − 17 ◦ C), regardless of the plasticizer types,
indicate that kefiran films are in a rubbery state at room temperature,
just like synthetic films of polypropylene (-18 ◦ C) and low-density
polyethylene (LDPE) (-125 to − 55 ◦ C) (Robertson, 1993). Further­
more, this indicates that their chains have more movement than PLA
biopolymer, which has a Tg close to 60 ◦ C and is brittle at room tem­
perature (Pillin, Montrelay, & Grohens, 2006) (Table 1). The low Tg
values of kefiran films could be attributed to their inherent structural
characteristics (high chain mobility) and their relatively high hydro­
philicity (Ghasemlou, Khodaiyan, Oromiehie, et al., 2011b). Therefore,
kefiran films are competitive with synthetic films for possible applica­
tions in the food industry at room temperature.
The DSC thermogram of neat kefiran film (Fig. 3b) exhibited one
broad endothermic peak at 84 ◦ C attributed to the kefiran melting
temperature due to having a semi-crystalline form, which is confirmed
by the literature (Shahabi-Ghahfarrokhi et al., 2015a). The Tm value of
neat kefiran was affected by the type and concentration of plasticizer, as
indicated in Table 1. In general, a slight decrease in the Tm is presented
in most of the films containing 2% w/w plasticizers, except those con­
taining D-limonene (2 and 5% w/w), where there was no change in the
Tm value in comparison with the neat kefiran. This result could once
again indicate weak interactions present between D-limonene and the
chemical structure of kefiran. On the other hand, a slight increase of the
Tm was observed with the incorporation of the D-glycerol and D-mannitol
content at 5% w/w (Table 1) in comparison with neat kefiran. Different
studies indicate that the addition and increase in the plasticizer con­
centration induces a decrease in the melting temperature of poly­
saccharides films (Ghasemlou et al. (2011a, 2011b); Gheribi et al.
(2018)). However, the slight increase in melting temperature observed
in this study suggests the forming of a molecular interaction between the
polymer and the plasticizer (Katopodis et al., 2020). On the other hand,
Ili Balqis et al. (2017) observed an increase in Tm of κ-carrageenan films
plasticized with sorbitol than other plasticizers such as glycerol much
higher concentrations of plasticizers than those used in this study (be­
tween 10 and 60% w/w).
However, Ili Balqis et al. (2017) observed an increase in Tm of
κ-carrageenan films plasticized with sorbitol compared with other
plasticizers such as glycerol, using much higher concentrations of plas­
ticizers than those used in this study (between 10 and 60% w/w). This
behavior was explained due to the lower tendency of the film to absorb
water, where water acts as a mobility enhancer, thus imparting less
flexibility in the films (Ghasemlou, Khodaiyan, Oromiehie, et al.,
2011a).
3.3.2. Thermogravimetric analysis (TGA)
TGA analysis was applied to evaluate the thermal stability of the
kefiran films with different plasticizers. Table 1 and Fig. 3c displays the
decomposition temperature values at 10% mass loss (T10) and the
temperature for the maximum mass loss rate (Tmax). These values were
obtained from the differential thermogravimetric (DTG) curves of
plasticized and neat kefiran films (Fig. S1, supplementary section).
Values indicate that the highest thermal decomposition, starting around
260–370 ◦ C, corresponds to the decomposition of the kefiran polymeric
structure. After this, the kefiran films’ depolymerization was observed at
600 ◦ C (Moradi & Kalanpour, 2019). The plasticizer incorporation (2
and 5% w/w) into kefiran films was given an increase in T10 around 38%
compared to the neat kefiran, and higher values were obtained using D-
7
Food Chemistry 360 (2021) 129966
glucitol as a plasticizer at the same concentration added (Table 1).
Additionally, it is essential to note that the Tmax of all kefiran films
increased by adding plasticizers. This result was consistent with the TGA
profile obtained by other authors, where they explained that the in­
crease in the thermal stability is due to the incorporation and increase of
plasticizer concentrations (Cerqueira, Souza, Teixeira, & Vicente, 2012;
Razavi, Mohammad Amini, & Zahedi, 2015). The improved thermal
stability could be explained by strong hydrogen interaction between the
polyols and the polymeric matrix at low plasticizer concentrations. In
the case of D-limonene, this interaction could be attributed to the
interaction of London dispersion forces between the plasticizer and the
kefiran polymer matrix through C–C bonds. These results suggest that
the plasticizer’s nature and concentration influence the thermal stability
of kefiran films, which is close to that of PLA biopolymers (Table 1).
Therefore, plasticized kefiran films are competitive at the thermal
properties level with synthetic films for possible food industry
applications.
3.3.3. X-ray diffraction
X-ray diffraction (XRD) allowed analyzing the semi-crystalline
structure of the neat and plasticized kefiran films. XRD patterns of the
studied kefiran films are displayed in Fig. S2 (supplementary section). In
neat kefiran, XRD analysis revealed a peak at 2θ ~ 8◦ that indicates the
kefiran’s semi-crystalline structure (Exarhopoulos, Raphaelides, &
Kontominas, 2018), confirming results obtained by DSC. The analysis
agrees with those published by Piermaria et al. (2011) and Shahabi-
Ghahfarrokhi et al. (2015). They indicated that the peak close at 8◦ is
associated with a small percentage of crystallinity in the kefiran struc­
ture’s complexity. The XRD patterns of the kefiran films containing
various plasticizer types were like those without plasticizer films. The
intensity of the diffraction peak did not change with the plasticizer
incorporation into the polymer matrix. This result indicates that the
addition of plasticizers does not alter the semi-crystalline polymeric
structure of kefiran, which would be explained by the intermolecular
interactions between polysaccharides and the plasticizer due to the good
miscibility of the plasticizer with the polymeric matrix.
3.3.4. Solubility in water
Solubility in water is an essential property of films with potential use
in food packaging because the film must satisfy some requirements in
order to protect the integrity of the product and to guarantee water
resistance during processing and storage. In this sense, the decrease in
the solubility upon addition of plasticizer would be beneficial for
improving the stability of packaged food products (Ghasemlou et al.,
2011a; Ghasemlou, Khodaiyan, Oromiehie, et al., 2011b). Fig. 4a shows
that the solubilized films in water presented different behaviors.
Glycerol-plasticized kefiran films were the most soluble in water, and an
increase of 33% in the solubility is presented as the plasticizer concen­
tration increased. This result concurs with the findings of Ghasemlou
et al. (2011a) and Piermaria et al. (2009). They reported that kefiran-
based films plasticized with glycerol (10–25% w/w) were the most
soluble compare with other sorbitol-plasticized ones. It can be explained
that glycerol presents the lowest molecular weight than the other pol­
yols used. It can be easily integrated between polymer chains and
thereby act as a water-retaining agent due to the hydrophilic nature
(Gheribi et al., 2018). On the other hand, glycerol plasticizer can in­
crease film solubility and affinity to water by decreasing interactions
between biopolymer chains in favor of polymer-plasticizer interactions
(Razavi et al., 2015).
In the case of films containing D-glucitol, D-galactitol, and D-
mannitol, the solubility decreased as the plasticizer concentration
increased. This behavior is due to the molecular structure of the plas­
ticizers and the interaction strength with the polymeric matrix since all
three plasticizers possess the same amount of hydroxyl groups and
molecular weight, differing only in the disposition of the OH groups in
the carbonate chain. It could be expected that in the case of polyols, the
L. Montoille et al. Food Chemistry 360 (2021) 129966

Fig. 4. a) Solubility in water (SW), b) deformation at break (ƐBreak), c) Young’s modulus (E), and e) microhardness (MH) of kefiran films with different types and
concentrations of plasticizers.

solubility of the films increases with the increase in the concentration of (23% of opacity), the other optical properties (YI, WI, and C) of neat
plasticizer due to its hydrophilic nature, giving the polymer molecules a kefiran films were slightly significantly different (p < 0.05). The low
higher affinity to attract water. However, in this study, as was observed, opacity obtained indicated transparency on the films, attributed to the
solubility decreased with increased concentration of plasticizer. There­ plasticizers’ good dispersion and compatibility in the film (Garcı ́a &
fore, the strong hydrogen bonding between the polyols and the poly­ Sobral, 2005). This result has appreciated commercial importance. Both
meric matrix and the higher molecular weight compared to glycerol yellowness index and chrome values slightly increased; meanwhile, the
affect the retention of water and the solubility of kefiran films (Gha­ whiteness index diminished. These optical changes could be attributed
semlou et al., 2011b). Kefiran films with D-limonene presented a similar to the intermolecular interaction between the film’s different compo­
behavior. It showed a greater decrease in the solubility of neat kefiran of nents, as previously explained in section 3.2. However, color differences
ca. 27% with an increase in plasticizer concentration compared with the (DE2000) between plasticized and neat kefiran films were calculated
other plasticizers (Fig. 4a). It may be due to the hydrophobic nature of D- considering the L*a*b* space. The color differences value was similar (p
limonene, and therefore presents low solubility in water. > 0.05) between plasticized films (1 < CIEDE2000 < 1.5). This low
CIEDE2000 value indicates that the plasticized films showed a color
3.3.5. Optical properties difference slightly visually detected due to the plasticizer addition,
The optical properties of the materials are directly related to the independently of its concentration and type (Sharma et al., 2004).
acceptability of the packaged product by consumers. The transparency Different letters (a, b) indicate significant differences (p < 0.05)
of the films allows consumers to inspect food before purchasing while between neat kefiran and plasticized films for each optical property.
also allowing for a better visual presentation of the products. Factors Due to the lower molecular weight of glycerol, it could enter with
that can alter the films’ opacity are the size of the added particles, their greater ease into the polymeric matrix’s intermolecular spaces, and at
spatial arrangement, or dispersion of the plasticizer in the matrix low concentrations of glycerol, H-bonding interaction strong can be
(Bierhalz, da Silva, & Kieckbusch, 2012). Table 2 shows each optical conducted (Farhan & Hani, 2017). Also, results showed that an increase
parameter measured for neat kefiran film and all plasticized samples’ in the concentration of D-glucitol, D-galactitol, D-mannitol, and D-limo­
mean values. These mean values are calculated considering that statis­ nene increased the opacity of kefiran films (low degree of transparency)
tical analysis (Table 2) showed no significant differences (p > 0.05) (Table 2). The results of opacity were similar to those published by
between plasticized samples, independently of type and concentration Piermaria et al. (2011), who reported that glycerol decreases opacity by
of plasticizers for all the optical parameters measured. Meanwhile, 9% in comparison with the neat kefiran films, while the addition of the
plasticizers’ addition did not change (p > 0.05) opacity of the samples plasticizer D-glucitol increased the opacity by 4%, with a similar trend to
those shown in this work. The opacity would be related on the one hand
to the size of the molecule, the higher molecular weight increases, more
Table 2 opacity, and therefore there is, more significant light scattering. On the
Optical properties of neat and plasticized kefiran films. other hand, one might think that the disposition of the plasticizer OH
Optical Neat Films with Statistical analysis (p-value) groups plays an essential role in the interaction with the polymer matrix.
properties kefiran plasticizers So, the interactions of D-glucitol, D-galactitol, and D-mannitol with the
A: B: Plasticizer
Plasticizer concentration
polymer matrix may be lower than with glycerol. With less interaction or
type greater repulsion, the interlacing of the plasticizer in the matrix is
smaller, the particle size is bigger, and therefore there is greater light
Opacity (O) 22.6 ± 24.6 ± 0.9a 0.0893 0.4321
1.2a scattering. Bierhalz et al. (2012) reported an increase in film opacity due
Yellowness − 13.2 − 15.3 ± 0.4b 0.7676 0.6270 to the addition of hydrophobic substances, therefore the hydrophobicity
index (YI) ± 0.9a of D-limonene also may be related to an increase in the opacity of the
b
Whiteness index 84.9 ± 83.6 ± 0.3 0.2904 0.6622
kefiran films.
(WI) 0.4a
Chrome (E) 10.7 ± 11.3 ± 0.2b 0.4773 0.4729
0.5a 3.3.6. Mechanical properties
Color variation – 1.2 ± 0.2 0.4815 0.7923 Table 3 shows the values of Young’s modulus (E), yield stress (σy),
(CIEDE2000) deformation at break (ƐBreak), and microhardness for the neat and

8
L. Montoille et al. Food Chemistry 360 (2021) 129966

Table 3
Solubility in water and mechanical properties of neat and plasticized kefiran films.
Films Plasticizer concentration (% w/w) SW (%) E (MPa) σmax (Mpa) ƐBreak (%) MH (MPa)
Neat kefiran – 21.7 ± 3.1 1208 ± 196 30.1 ± 11 5.6 ± 0.4 1387 ± 54
Kefiran + glycerol 2 26.3 ± 1.2b 1463 ± 14a 29.9 ± 1.9a 6.0 ± 3.7a 1529 ± 26ab
Kefiran + glycerol 5 33.3 ± 2.9a 1237 ± 76a 3.6 ± 2.7a 8.2 ± 1.5b 1565 ± 54a
Kefiran + D-glucitol 2 30.0 ± 5.0c 904 ± 70b 21.3 ± 24a 3.2 ± 1.1ab 1279 ± 27ab
Kefiran + D-glucitol 5 19.7 ± 2.6ab 603 ± 231b 12.8 ± 2.3a 21.0 ± 0.1c 993 ± 41a
Kefiran + D-galactitol 2 27.7 ± 2.9c 890 ± 250b 18.8 ± 3.5a 7.1 ± 1.3ab 940 ± 58bc
Kefiran + D-galactitol 5 19.7 ± 4.2ab 549 ± 204b 3.8 ± 3.8a 7.9 ± 2.7c 1130 ± 82b
Kefiran + D-mannitol 2 29.0 ± 0.8d 1004 ± 471b 10.0 ± 4.7a 12.0 ± 5.7ab 1494 ± 60
Kefiran + D-mannitol 5 23.7 ± 1.2bc 1103 ± 43b 16.8 ± 2.1a 12.9 ± 4.0c 1325 ± 29c
Kefiran + D-limonene 2 18.3 ± 1.2e 704 ± 108b 6.8 ± 4.7a 8.4 ± 2.1d 2148 ± 25e
Kefiran + D-limonene 5 17.0 ± 1.6 1044 ± 188b 15.3 ± 4.4a 18.0 ± 2.8 2414 ± 240d

plasticized kefiran films. Fig. 4 shows the microhardness values, Young’s
modulus, and deformation at break (ƐBreak) obtained for films based on
kefiran with and without plasticizers. In general, the plasticized kefiran
films showed a decrease of Young’s modulus ca. 74% and yield stress in
ca. 87% when D-galactitol with 5% w/w was incorporated and compared
with the neat kefiran. Deformation at break also increased with the
amount of the plasticizer kefiran ca. 73% when D-glucitol was incorpo­
rated into the kefiran. This work studies the incorporation of a low
amount of plasticizer (2 and 5% w/w), unlike other works reported in
the literature that used a high amount of up to 35% of polyols.
Different correlative letters (a-e) are reported in each column to
indicate that there is a significant difference between the corresponding
groups using P < 0.05. Data reported are mean values ± SD. SW (%):
solubility in water percent. MH: Microhardness. E = Young’s modulus;
σmax = Yield stress; εBreak = deformation at break.
The authors have reported that kefiran film with 30% plasticizer
showed a reduction of around 94% in Young’s modulus and 85% in
tensile strength, followed by an increase in elongation at break
compared to the film with 20% plasticizer (Coma et al., 2019). On the
other hand, similar behavior has been reported for other films incor­
porating plasticizers obtained from different biopolymers (Ili Balqis
et al., 2017). This trend is attributed to the plasticizers’ ability to
enhance the segmental mobility, decrease intermolecular forces, soften
the mechanical stability of the film’s structure, and increase polymer
mobility (Ghasemlou et al., 2011b). Piermaria et al. explained this
behavior because most plasticizers contain hydroxyl groups that will
form hydrogen bridges with biopolymers and increase the flexibility of
the film matrix. The plasticizer increases polymer chains’ movement
ability (Piermaria et al., 2009).
The hardness of a polymeric material can be defined as a measure­
ment of the resistance to permanent deformation under the action of an
indenter. This deformation is ruled by the viscoelastic and plastic
components that also govern the mechanical properties of the material.
Microhardness involves a complex combination of properties (elastic
modulus, yield strength, strain hardening, toughness) (Baltá Calleja F.J.,
2000). Therefore, it can also be expected that microhardness is related to
structural parameters since mechanical properties are structure-
dependent (Baltá Calleja F.J., 2000).
Thus, the behavior of the microhardness and Young’s modulus de­
pends on the polymer structure, and therefore they should show the
same trend (Ostafinska et al., 2017).
MH values allow to check the flexibility of the films and ensure the
durability and ability to enhance the mechanical integrity of foods. The
microhardness values change according to the type and concentration of
plasticizer, and no definite trend is observed as the concentration of
plasticizer increases. It could be due to the low concentration of plasti­
cizer used. However, the size, the shape, and the number of oxygen
atoms in the plasticizer molecules affecting the plasticizer action, and
different trends might be found in mechanical properties for different
polymer-plasticizer pairs (Cerqueira et al., 2012; J. Piermaria et al.,
2011, 2009). In a general way, the results indicated that under the
plasticizer concentration range studied, the addition of D-glucitol and D-
galactitol significantly decreased the microhardness of kefiran film (P <
0.05) in 28% (with 5% w/w of D-glucitol) and 32% (with 2% w/w of D-
galactitol) shown the same tendency that Young modulus (Table 3). In
this way, the diastereomers D-glucitol and D-galactitol are alditols with
the same six-carbon chain length. These polyols have one hydroxyl
group attached to each carbon. Contrary to monosaccharides, in water,
the alditols do not exist as hemiacetals. Thus, all its hydroxyl groups are
available for the attractive noncovalent forces as hydrogen bonds be­
tween kefiran chains and these polyols. On the other hand, glycerol is a
polyol molecule of three-carbon chain length constituted of three hy­
droxyl groups. As a result, glycerol confers less hydrogen bonding per
molecule than the six-carbon chain length alditols, leading to better
properties of plasticized films based on kefiran with D-glucitol and D-
galactitol than with glycerol.
On the other hand, with the D-limonene incorporation, the Micro­
hardness increased in comparison with the neat kefiran. Previous reports
based on PLA and PLA/D-limonene blends demonstrated a good plasti­
cization in the mechanical properties evidenced in a decrease of Young’s
modulus, loading D-limonene in the PLA matrix (Arrieta, López,
Ferrándiz, & Peltzer, 2013; Brüster et al., 2019). The authors concluded
that these blends could find potential applications in food packaging.
Therefore, these previous results motivated the study of D-limonene as a
potential plasticizer in kefiran films. When the D-limonene was incor­
porated into the kefiran, the microhardness values increased, and the
Young’s modulus decreased slightly with the 5% w/w of D-limonene
incorporation. This behavior could be due to that D-limonene was not
homogeneously distributed in the kefiran matrix. Some zones with an
agglomeration of D-limonene could increase the polymer’s rigidity
increased the microhardness, and it could be due to the hydrophobic
character of the D-limonene and the hydrophilic character of the kefiran
(Hasheminya et al., 2019). The compatibility mainly governs this
property at molecular levels between the polymeric matrix and the
plasticizer.
The flexibility of polymeric films with hydrophobic plasticizer has
been discussed in the literature (Ghasemlou, Khodaiyan, Oromiehie,
et al., 2011a; Srinivasa, Ramesh, & Tharanathan, 2007). For example,
Ghasemlou et al. (2011a) explained that the heterogeneity structure of
the kefiran film, when oleic acid (OA) is added, probably led to weak­
ened intermolecular interactions and a consequent decrease in the
flexibility of films. The interactions between non-polar OA molecules
and polar polymer molecules are much lower than those between polar
glycerol molecules and polar polymer molecules. These results indicate
that the nature and amount of plasticizer had a significant effect on the
mechanical properties of kefiran films.
3.3.7. Scanning electron microscopy (SEM)
The microstructure of the film surfaces was examined to determine
the difference in surface morphology caused by different plasticizer
types and concentrations. Fig. 5 shows the SEM images of the kefiran
films with and without plasticizers. SEM images indicate that the neat
kefiran film showed smooth and uniform surfaces without pores or
cracks. On the other hand, it could be observed the higher the

9
L. Montoille et al.
Fig. 5. Scanning electron microscopy surface for a) neat kefiran films and with the incorporation of 5 and 2% w/w plasticizers: b-c) glycerol, d-e) D-glucitol, f-g) D-
galactitol, h-i) D-mannitol and j-k) D-limonene.
concentration of plasticizers, the smoother the surface compared to a
lower concentration of plasticizer. This indicates that the plasticizers
were distributed homogeneously in the kefiran matrix. It results from
the formation of stronger and more stable hydrogen bonds of plasticizers
with kefiran and consequently led to the formation of a more uniform
surface.
The surface microstructure of films prepared with 2 and 5% w/w D-
limonene concentration (Fig. 5b) shows a smooth and uniform surface.
This indicates that due to the low concentration of D-limonene the film-
forming solution had a stable emulsion system and that its stability was
maintained during drying of the solution (Tongnuanchan, Benjakul, &
Prodpran, 2012). In similar research on the effects of the addition of
essential oil to kefiran films, Hasheminya et al. (2019) pointed out a
uniform surface in the polymer matrix. However, when the D-limonene
was incorporated into the kefiran, the microhardness values increased.
This behavior could be due to that D-limonene was not homogeneously
distributed in the kefiran matrix. Some zones with an agglomeration of
D-limonene could increase the polymer’s rigidity, and it could be due
weak interaction of London dispersion forces between the plasticizer
and the kefiran polymer matrix through C–C bonds.
As shown above, the addition of plasticizers helps in solubilizing the
kefiran polymeric matrix, enhancing the smoothness and cohesiveness
of the film surface microstructure (Coma et al., 2019). It demonstrates
the effective integrity structure of the homogeneous matrix due to
plasticizer interactions with kefiran through intermolecular hydrogen
bonds or weakened interactions (Piermaria et al., 2009). This effect had
been observed previously on the surface of plasticized kefiran films
containing glycerol and sorbitol (Ghasemlou et al., 2011b; Piermaria
et al., 2011).
Due to the decrease of 74% in Young’s modulus (E), 87% for yield
strength (σy), 30% in the microhardness, and the solubility observed in
10
Food Chemistry 360 (2021) 129966
kefiran films with 5% w/w D-galactitol, it can be deduced that D-galac­
titol is a more effective plasticizer in kefiran films. Finally, it could be
concluded that the most suitable food packaging combination is kefiran
with 5% w/w D-galactitol.
4. Conclusions
In the present work, the spectroscopic analysis of the isolated exo­
polysaccharide from kefir grains showed that this polysaccharide is
constituted by branched glucogalactosylhexasaccharide repeating units
described as kefiran. The addition of different types and concentrations
of plasticizers (hydrophilic or hydrophobic) in a low concentration as
glycerol, D-glucitol, D-galactitol, D-mannitol, and D-limonene plasticizers
had an influence on the physical properties of films based on kefiran.
Results indicated that all plasticizers present good efficiency in plasti­
cizing kefiran films in terms of their visual appearance, thermal prop­
erties, solubility, optical properties, structural, surface integrity, and
mechanical properties. The presence of plasticizers increased the Tg of
neat kefiran ca. 15% compared to the neat kefiran, and a decrease in the
Tm is presented in films containing low concentrations of hydrophilic
plasticizers. These results could be attributed to a lower amount of
plasticizer used.
Furthermore, the thermal stability of all kefiran films increased by
adding plasticizers, and the thermal stability of films containing 2 and
5% of D-galactitol and D-glucitol was higher than other plasticized films
by strong hydrogen interaction between these polyols and the polymeric
matrix. On the other hand, it was observed that raising glycerol con­
centration leads to highly soluble films. However, the solubility
decreased with an increased concentration of D-glucitol, D-galactitol, D-
mannitol, and D-limonene. The low opacity obtained indicated trans­
parency on the films. Meanwhile, the other optical properties (YI, WI,
L. Montoille et al.
and C) of neat kefiran films were slightly significantly different (p <
0.05) to plasticized films, attributed to the intermolecular interaction
between the film’s diverse components. The microhardness of kefiran
films decreased significantly ca. 30% with the addition of D-glucitol and
D-galactitol of 2 and 5% concentrations; however, a drastic increase of
74% was obtained using D-limonene due to the weak interactions be­
tween plasticizer and the polymeric matrix. Moreover, D-glucitol and D-
galactitol decreased the microhardness (MH) and Young’s Modulus of
films ca. 30% and 74% respectively, obtaining more flexible kefiran
films. Scanning electron microscopy (SEM) also showed that the surface
structure of films incorporated with plasticizers was homogeneous
without porosities or cracks. These results were attributed to the strong
hydrogen bonding between the polyols and the polymeric matrix.
Kefiran based films could find applications in the food-packaging
industry, being the best plasticizers with D-glucitol and D-galactitol.
CRediT authorship contribution statement
Lissette Montoille: Methodology, Data curation, Investigation.
Constanza Morales Vicencio: Methodology, Data curation, Investiga­
tion. Daniel Fontalba: Conceptualization, Methodology. J. Andrés
Ortiz: Formal analysis, Data curation, Visualization. Viviana Moreno-
Serna: Writing - original draft, Formal analysis, Visualization. Laura
Peponi: Methodology, Data curation. Silvia Matiacevich: Writing -
review & editing. Paula A. Zapata: Funding acquisition, Resources,
Formal analysis, Supervision, Writing - review & editing.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgments
P. Zapata acknowledges the support of FONDECYT Project 1170226.
V. Moreno-Serna acknowledges the financial support under DICYT
project 022041ZR_POSTDOCT. J.A. Ortiz would like to thank FONDE­
CYT Postdoctorado 3200296 Universidad de Chile for their financial
support. L. Peponi acknowledges the funding for the project by the
Spanish Ministry of Economy, Industry and Competitiveness MAT2017-
88123-P, and S. Matiacevich would like to thank DICYT project 08197M
from the University of Santiago de Chile for their financial support.
Thanks to the ANID Fondequip USACH project and the nuclear magnetic
resonance laboratory for the study of samples with the Bruker Avance
DRX 400 NMR spectrometer.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.foodchem.2021.129966.
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