Kefiran-based films: Fundamental concepts, formulation strategies and properties

Carbohydrate Polymers 246 (2020) 116609

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Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol

Review

Kefiran-based films: Fundamental concepts, formulation strategies and
properties

T

Luís Marangoni Júniora,*, Roniérik Pioli Vieirab, Carlos Alberto Rodrigues Anjosa
a
b

Department of Food Technology, School of Food Engineering, University of Campinas, Campinas, São Paulo, Brazil
Department of Bioprocesses and Materials Engineering, School of Chemical Engineering, University of Campinas, Campinas, São Paulo, Brazil

ARTICLE INFO

ABSTRACT

Keywords:
Exopolysaccharide
Kefiran
Biopolymer
Edible film
Material properties

Concerns about plastic pollution have driven research into novel bio-derived and biodegradable polymers with
improved properties. Among the various classes of biopolymers studied, kefiran films only have gained emphasis
in recent years. Its film-forming ability and outstanding biological activities illustrate its potential for active
packaging applications. However, despite recent advances, the key challenge is still associated with obtaining
high water vapor barrier and better mechanical properties. In that fashion, this review highlights for the first
time the cutting-edge advances in the preparation, characterization and enhancement of the packaging performance of kefiran-based films. The fundamental concepts of the biopolymer production and chemical analysis
are previously outlined to direct the reader to the structure-property relationship. In addition, this research
critically discusses the current challenges and prospects toward better material properties.

1. Introduction
Food packaging and coatings have experienced impressive progress
in recent decades, driven by growing demand for safe and high-quality
foods. Its primary function is the protection against external agents
(microorganisms, water vapor, oxygen and light). In addition, it contributes to prevent loss of desirable compounds (flavor volatiles) and
consequently extending the product's shelf life (Mohamed et al., 2020).
Materials made of paper, metal, glass and plastics are frequently used as
food packaging (Mahalik & Nambiar, 2010). Plastics (from fossil
sources) are the most used due to low cost, low specific mass, high
versatility, flexibility, transparency, good mechanical and barrier performance (Licciardello, 2017; Marangoni Júnior et al., 2020; Robertson,
2013).
Plastic materials from fossil sources generate waste that needs a
correct destination, such as landfills, reuse, recycling, among others.
Although these materials are consolidated in industries, their environmental aspects have raised concerns that are in growing discussion
(Zhong et al., 2020). Linked to this, a growing consumer demand for
materials that do not degrade the environment, that are safe and non-

toxic is increasingly present in society (Sharma et al., 2020). These facts
motivate the search for renewable alternatives to these applications,
such as the use of bio-derived polymers. In general, the most used to

form films and coatings are composed of polysaccharides, proteins and
lipids (Sampathkumar et al., 2020; Vieira et al., 2011).
Currently, there are two dominant classes of commercially viable
biopolymers: alkyl polyesters (poly(lactic acid) and polyhydroxyalkonates) (Garlotta, 2002; Suriyamongkol et al., 2007), and starch-based
plastics (Lu et al., 2009). However, when compared to petroleum-based
counterparts, bio-based films are unable to supply all of their functionality. The reason is the lower mechanical and barrier performance,
in addition to water sensitivity (Azeredo & Waldron, 2016), which
limits its use in many applications (Peelman et al., 2013). In that
fashion, the search for polymeric alternatives and/or different film
formulation strategies has been gaining more and more prominence.
Indeed, it is recognized that considerable improvements in properties
have been reported with the production of nanocomposites, blends and
by obtaining active films, mainly involving polysaccharide bases
(Cazón et al., 2017; Ribeiro-Santos et al., 2017).
Among this class of biopolymers, exopolysaccharides (EPS) have

Abreviations: Al2O3, aluminum oxide; ATR-FTIR, attenuated total reflection Fourier-transform infrared spectroscopy; CMC, carboxymethylcellulose; CuO, copper
oxide; DSC, differential scanning calorimetry; EO, essential oil; EPS, exopolysaccharides; FTIR, Fourier-transform infrared spectroscopy; HPLC, high performance
liquid chromatography; MMT, montmorillonite; Mw, weight-average molecular weight; NC, nano-cellulose; NMR, nuclear magnetic resonance; OA, oleic acid; RSM,
response surface methodology; SEM, scanning electron microscopy; Tg, glass transition; Tm, melting temperature; TGA, thermogravimetric analysis; TiO2, titanium
oxide; UV, ultraviolet; WPI, whey protein isolate; WVP, water vapor permeability; ZnO, zinc oxide; XRD, x-ray diffraction; [η], intrinsic viscosity
⁎
Corresponding author at: Rua Monteiro Lobato, 80 - Cidade Universitária Zeferino Vaz, CEP: 13083-862, Campinas, São Paulo, Brazil.
E-mail address: (L. Marangoni Júnior).
/>Received 3 May 2020; Received in revised form 17 May 2020; Accepted 26 May 2020
Available online 20 June 2020
0144-8617/ © 2020 Elsevier Ltd. All rights reserved.


Carbohydrate Polymers 246 (2020) 116609

L. Marangoni Júnior, et al.

received remarkable attention. Kefiran is an edible and biodegradable
water-soluble EPS obtained during milk fermentation in the kefir production (Frengova et al., 2002; Kooiman, 1968; la Riviére et al., 1967;
Moradi & Kalanpour, 2019). Kefiran apparently protects the microbiota
inside the kefir granules (Badel et al., 2011). Moreover, it has been
attributed numerous beneficial properties for human health, such as
high antimicrobial and healing potential (Piermaria et al., 2008;
Rodrigues, Caputo et al., 2005), anti-inflammatory activity (Rodrigues,
Carvalho et al., 2005), anti aging properties (Sugawara et al., 2019),
contribution to the reduction of blood pressure and cholesterol levels
(Amorim et al., 2019; Maeda, Zhu, Mitsuoka, 2004), besides anticancer
activity (Jenab et al., 2020; Medrano et al., 2011; Sharifi et al., 2017).
In this context, kefiran has been incorporated in a broad range of applications in the food industry. For exemple, as stabilizer, emulsifier, fat
substitute and gelling agent (Moradi & Kalanpour, 2019; Moradi et al.,
2019).
In addition to these extraordinary biological activities, another
highlight of kefiran is its considerable potential for the production of
films and coatings. Distinguished appearance and satisfactory mechanical and barrier properties were demonstrated (Ghasemlou et al.,
2011a; Moradi & Kalanpour, 2019; Piermaria et al., 2011; ShahabiGhahfarrokhi et al., 2015). It is noted, however, that its film-forming
potential began to be explored only in recent years, mostly in the last
decade. Hence, several studies in the literature have focused on the
development of kefiran films employing distinct plasticizers. In addition, the development of blends based on this EPS and other biopolymers, as well as incorporation of nanoparticles have also been reported.
However, despite the promising results, further research in the literature remains to be explored towards improving its properties for effective use in food packaging and coatings.
Therefore, this work aims to present readers with a bibliographic
trend directed to the notable advances, challenges and future perspectives in the production of kefiran-based films. This research initially
outlines the chemical structure characterization, production, extraction
and purification of the exopolysaccharide. These fundamental concepts
facilitate the films structure-property relationship subsequently discussed. In addition, an extensive analysis of the formulation methods

and evaluation of the film’s properties are presented. The key desirable
materials characteristics are also discussed. To the best of our knowledge, this is the first time a review is presented with a focus on the
production and properties evaluation of kefiran based-films.

Fig. 1. Kefiran chemical structure.

2. Chemical structure
The extracellular polysaccharides or exopolysaccharides (EPSs) are
produced by many bacteria, which secrete in the form of a capsule or
slime layer around the bacterial cell (Nouha et al., 2018). Kefiran is the
main exopolysaccharide produced from kefir grains (Moradi &
Kalanpour, 2019). It is produced typically by bacteria of the type Lactobacillus kefiranofaciens, but also by several other unidentified species
of Lactobacillus (Zajšek et al., 2013). Kefiran is a light or pale yellow
viscous polysaccharide, water-soluble, containing approximately the
equivalent amount of D-glucose and D-galactose (Badel et al., 2011;
Kooiman, 1968; Pop et al., 2016). However, some authors have reported the possibility of small variations in these proportions. Zajšek,
Kolar & Goršek (2011) used electrophoresis to identify the residues of Dglucose and D-galactose in the proportion of 1:0.7. Chen et al. (2015)
identified by high performance liquid chromatography (HPLC) that the
proportions of D-glucose and D-galactose in kefiran produced from a
Tibetan kefir are 1:1.88, respectively.
Kefiran is a branched-structure carbohydrate (Fig. 1), with a repeat
of hexa or hepta-saccharide composed of a regular pentasaccharide
unit, in which one or two sugar residues are randomly linked (Kooiman,
1968; Maeda, Zhu, Suzuki et al., 2004; Micheli et al., 1999). For the
identification of the kefiran structure, it is possible to proceed with the
analysis of nuclear magnetic resonance. Fig. 2(a) shows the kefiran 1H

Fig. 2. Kefiran nuclear magnetic resonance spectra, (a) 1H NMR and (b) 13C
NMR (Maeda, Zhu, Suzuki et al., 2004). Adapted with permission from the
American Chemical Society, Copyright (2004).

NMR in D2O (Maeda, Zhu, Suzuki et al., 2004), which it is verified that
the region around 4.4–5.5 ppm of the spectrum contains seven typical
signals (a1 – f1). The peak b1 at 4.61 ppm is attributed to (1 → 6) -β-DGalactose corresponding to a small proportion of 2,3,4-tri-O-methyl-Dgalactose (sugar on a side branch). The other peaks show three welldefined signals and three overlapping signals that are attributed to the
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L. Marangoni Júnior, et al.

Fig. 3. Scanning electron microscopy (SEM) images with the surface morphology of kefiran produced from Tibetan kefir (Chen et al., 2015). Adapted with permission
from Elsevier, Copyright (2015).

hexasaccharide repeat unit.
The peak c1 at 5.14 ppm suggests the presence of α-hexapyranosyl.
The peaks f1 around 4.82 ppm (7.92 Hz), b1 at 4.68 ppm, e1 at 4.53
ppm (7.52 Hz), d1 at 4.53 ppm (7.52 Hz), and a1 at 4.49 ppm (7.92 Hz)
are attributed to the pyranose ring forms in an β anomeric configuration (Maeda, Zhu, Suzuki et al., 2004). The results identified in the
spectra of Fig. 2(a) corroborate the structural characterization presented by other authors (Micheli et al., 1999; Radhouani et al., 2018;
Staaf et al., 1996). The 13C NMR spectrum in Fig. 2(b) shows six signals
in the region around 95−110 ppm. The signal c1 around 98.5 ppm
indicates an α-hexapyranosyl residue and five β-hexapyranosyl residues
at 105.7 ppm (a1, b1, d1, e1 and f1).
In a complementary way, the analysis of Fourier-transform infrared
spectroscopy (FTIR) is extremely relevant to identify the functional
groups characteristic of kefiran or other EPS. Numerous researches are
available in literature with details of this characterization (Chen et al.,
2015; Moradi & Kalanpour, 2019; Radhouani et al., 2018; Semeniuc,
2013). In all of them, some regions of the spectrum should be highlighted. The prime region and usually the first to be evaluated represent

the one with an intense and broad peak around 3400 cm−1. This signal
corresponds to the intramolecular vibration of hydroxyl or intermolecular hydrogen bonding of the polysaccharide. Weak absorption
close to 2930 cm- 1 is related to modes of asymmetric and symmetrical
C–H stretching of the sugar chain (Parikh & Madamwar, 2006), that can
be attributed to methylene groups. Furthermore, another relatively
notable peak in the region of 1100 to 1150 cm−1 indicates sections
C–O–C and alcoholic groups in carbohydrates (Rodrigues, Carvalho
et al., 2005; Rodrigues, Caputo et al., 2005). Finally, an existing peak at
900 cm−1 indicates a β-glycosidic configuration and also modes of
glucose and galactose vibration (Davidović et al., 2015). It is relevant to
note that EPS with β-glycosidic linkage was considered to retain the
most extensive biological activity (Wu et al., 2009).
The molecular weights reported for kefiran varied a lot, depending
frequently on the conditions of isolation and purification, being in the
range of 50 to 15,000 kDa (Ahmed et al., 2013; Exarhopoulos et al.,
2018a; Ghasemlou, Khodaiyan, Jahanbin et al., 2012; Liou & Chen,
2009; Maeda, Zhu, Suzuki et al., 2004; Piermaria et al., 2008; Pop et al.,
2016; Radhouani et al., 2018). Among this broad range of values found,
Exarhopoulos et al. (2018a) determined the weight-average molecular
weight (Mw) by size exclusion chromatography, finding a Mw value
equal to 614.4 kDa, with dispersity equal to 1.978, which indicates
randomness of polymer chain sizes. These authors also determined that
kefiran is semi-crystalline, with a sharp peak around 2θ = 20.9° by Xray diffraction (XRD), and a 27 % crystallinity percentage. In addition,
the authors delved into the specific viscosity studies of the diluted
aqueous solution of kefiran, which provided quite a fascinating structural information.
At low concentrations of kefiran, the specific viscosity increases
linearly as a function of concentration. However, at a particular concentration, considered a “critical concentration”, there is an abrupt shift
in the gradient of the curve towards more elevated concentrations

(Exarhopoulos et al., 2018a). The critical concentration indicates that

the individual polymer molecules previously present as single entities
in the diluted solution now exceeded the volume of the solution. The
result is an overlap of molecules (Morris et al., 1981). Exarhopoulos
et al. (2018a) identified a critical concentration of 0.53 g dL−1 for
kefiran. Conversely, Piermaria et al. (2008) reported a critical concentration equal to 0.35 g dL−1. These and other researches provided
the values of intrinsic viscosity ([η]) for the diluted solutions of kefiran
by fitting the equations of Huggins and Kraemer. In this context, molecular weight can be correlated as a function of intrinsic viscosity.
Some intrinsic viscosity values for diluted solutions of kefiran reported in the literature using Huggins and Kraemer equations, respectively, were: 600 mL g−1 and 595 mL g−1 (Mw = 10,000 kDa)
(Piermaria et al., 2008), 584 mL g−1 and 553 mL g−1 (Mw = 1350
kDa) (Ghasemlou, Khodaiyan, Gharibzahedi, 2012; Ghasemlou,
Khodaiyan, Jahanbin et al., 2012), 84.6 mL g−1 and 85.2 mL g−1 (Mw
=706 kDa) (Exarhopoulos et al., 2018a). Through the analysis of these
values, it is possible to notice the drastic reduction in the intrinsic
viscosity of the solution due to the reduction in molecular weight. Its
intrinsic viscosity values can be considered relatively high. However,
previous work reported a much lower viscosity for kefiran when compared to other polysaccharides such as guar gum, locust bean gum and
methylcellulose (Piermaría et al., 2016). The aforementioned works
calculated the Huggins parameter (k’), and also the difference between
the Huggins and Kraemer parameters (k’- k”). The reported values for k’
varied between 0.3 and 0.8, indicating that water can be considered a
good solvent. The difference between the constants (k’ – k”) was near
0.5, suggesting a random coil shape for kefiran in aquous solution
(Exarhopoulos et al., 2018a; Ghasemlou, Khodaiyan, Gharibzahedi,
2012; Ghasemlou, Khodaiyan, Jahanbin et al., 2012; Piermaria et al.,
2008; Yang & Zhang, 2009).
With regard to surface morphology, kefiran and other EPS exhibit
attractive characteristics. Fig. 3 illustrates the surface morphology of
EPS produced from Tibetan kefir by scanning electron microscopy
(SEM) analysis. Arrows A and B indicate a grainy appearance and irregular surface under magnifications of 2000 and 5000 times. However,
under greater magnification (10,000 times) it appears that the material

retains a compact structure, with smooth surfaces without the presence
of pores (Chen et al., 2015). Comparatively, the kefiran surface is extremely similar to the appearance of L. kefiranofaciens ZW3 EPS (Ahmed
et al., 2013). The compact structure illustrated in the 10,000 times
magnifications suggests this material displays significant potential for
the production of plasticized films.
3. Kefiran production, isolation and purification
3.1. Microorganisms culture
Kefir is a fermented milk drink, which a report by Transparency
Market Research forecasts its global market to expand at an annual
growth rate of 5.9 % between 2017 and 2025 for the market to become
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L. Marangoni Júnior, et al.

worth US$2154.9 mn by the end of 2025 (“Global Kefir Market,” 2017).
The starter culture used to produce the drink consists of gelatinous irregular grain shapes with diameters ranging from 1 to 15 mm (GüzelSeydim et al., 2000). These grains have a varied and complex microbial
composition, which includes yeast species, lactic acid bacteria, acetic
acid bacteria and mycelial fungi (Takizawa et al., 1998), all kept together by kefiran. This exopolysaccharide is one of the lactic acid
bacteria products, which can reach up to 50 % (w / w) of grains on a
dry basis (Exarhopoulos et al., 2018b).
Among the various microorganisms isolated from kefir grains, the
following stand out: Lactobacillus kefir, Lactobacillus parakefir,
Lactobacillus brevis, Lactobacillus plantarum, Lactobacillus acidophilus,
Lactobacillus kefirgranum, Lactobacillus kefiranofaciens, Lactobacillus sp.
KPB-167B and Lactobacillus casei (Bosch et al., 2006; Dertli & Çon,
2017; Jeong et al., 2017; Takizawa et al., 1998; Xing et al., 2017; Yokoi
& Watanabe, 1992). Greater variability has been reported in the lactic

acid Streptococcus population (443 %) than Lactobacillus (28 %) and
yeasts (35 %), isolated from kefir grains (Ninane et al., 2005). In addition, it was also demonstrated that the lactic acid bacteria and yeasts
present in kefir grains vary significantly, from 6.4 × 104 - 8.5 × 108
and 1.5 × 105 - 3.7 × 108 cfu / mL, respectively (Witthuhn et al.,
2004).
Most of the research directed to the production of kefiran used a
pure culture of the species Lactobacillus sp. KPB-167B (Yokoi &
Watanabe, 1992), Lactobacillus kefirgranum sp. nov. and L. parakefir
(Takizawa et al., 1994), among others (Moradi & Kalanpour, 2019). The
most prominent one has been the species Lactobacillus kefiranofaciens
(Dailin et al., 2014; Jeong et al., 2017; Xing et al., 2017). On the other
hand, the mixed culture of L. kefiranofaciens and Saccharomyces cerevisiae has also been extensively studied (Cheirsilp & Radchabut, 2011;
Cheirsilp et al., 2007; Cheirsilp, Shimizu et al., 2003; Cheirsilp, Shoji
et al., 2003; Tada et al., 2007). The fundamental researches indicated
that this alternative can significantly increase the production of kefiran
in relation to the pure cultures. For example, it was observed that with
this mixed culture, under anaerobic conditions, the production rate of
kefiran was 36 mg L−1 h−1 (Cheirsilp, Shimizu et al., 2003), which is
50 % higher than that obtained using pure culture (24 mg L−1 h−1).

3.3. Extraction and purification of kefiran
It has been reported that kefiran can represent about 50 % or more
of the dry mass of kefir grains (Exarhopoulos et al., 2018b). Thus, after
choosing the optimum fermentation conditions, the kefiran isolation
and purification steps are essential to guarantee a high purity polymer.
For this, several procedures have been described in the literature with
some similarities (Micheli et al., 1999; Pais-Chanfrau et al., 2018;
Piermaria et al., 2008; Pop et al., 2016; Zajšek et al., 2011). In general,
the procedure consists of adding a certain amount of kefir grains in hot
water, under agitation, temperature and fixed times. Then, the mixture

must be cooled and centrifuged to remove microbial cells and proteins.
The polysaccharide dissolved in the supernatant is then purified by
freezing overnight, followed by slow thawing. After that, the mixture
undergoes cold centrifugation, and the kefiran-rich pellets undergo
dissolution in hot distilled water. The purification procedure is repeated
twice to obtain a high purity kefiran solution. Fig. 4 provides a simplified overview with the essential steps reported in the literature.
The first point to be highlighted in the procedure refers to the hot
water extraction step. Some authors have not specified the exact temperature used. However, temperatures close to 100 °C have been reported to cause polymer degradation. A recent study has shown that
this initial hot water extraction phase influences considerably the
quality of this polysaccharide (Pop et al., 2016). In this research, the
authors evaluated the effects of temperature (from 60 to 100 °C) and
time (from 1 min to 8 h) on the rheological and structural characteristics of the kefiran. It was exposed that the kefiran solution viscosity
decreased as the temperature and residence time increased. The more
severe conditions led to obtaining polysaccharides with lower molecular weight. Finally, the material was degradated during processing at
100 °C. The polysaccharide with the most superior molecular weight
(about 15,000 kDa) was obtained by extraction at 80 °C and 30 min
(Pop et al., 2016).
Another pertinent point in the procedure reported in Fig. 4 is associated with the kefiran precipitation by cold ethanol. Several researches used absolute ethanol (Dailin et al., 2016; Piermaria et al.,
2008; Radhouani et al., 2018; Taniguchi et al., 2001). However, considering a possible scale-up, a recent study evaluated the effect of using
96 % ethanol, which provides a more reduced cost. The results of kefiran yield in both procedures do not present significant differences
between them, suggesting this may be an economical alternative in the
precipitation stage (Pais-Chanfrau et al., 2018). Finally, the centrifugation steps reported in the aforementioned researches varied in
time (from 10−45 min) and the centrifugal force used (from 5,000 to
10,000 g).

3.2. Strategies for optimizing kefiran yield
The primary operational parameters that must be evaluated to obtain an optimal yield in the production of kefiran are the temperature
and pH. In addition, other key parameters must equally be considered,
such as the type and concentration of microorganisms and nutrients,
and the high cost of producing kefiran is mainly related to sources of

carbon and nitrogen (Moradi & Kalanpour, 2019). As follows, these
factors can be assessed univariately or with the help of the response
surface methodology (RSM), to identify the optimal production conditions. Despite being a very effective tool, RSM has been little explored
in the literature for maximizing kefiran yield.
Regarding the effects of these factors individually, it was reported
that the highest production of kefiran was obtained in the range of
20–30 °C (Blandón et al., 2018; Dailin et al., 2015; Ghasemlou,
Khodaiyan, Gharibzahedi, 2012; Montesanto et al., 2016; Zajšek et al.,
2013). In parallel, most research has reported the ideal pH for maximum kefiran production is between the values of 5 and 6 (Cheirsilp,
Shimizu et al., 2003; Ghasemlou, Khodaiyan, Gharibzahedi, 2012;
Zajšek & Goršek, 2011; Zajšek et al., 2013). Once the culture of microorganisms has been chosen, it proceeds with the development of the
most appropriate medium. Thus, distinct types and concentrations of
key nutrients, such as carbon sources (glucose, mannitol, sucrose, lactose), nitrogen sources (yeast extract, peptone, meat extract, casein
hydrolyzate) have been investigated in the literature. Some of these
surveys are highlighted in Table 1.

4. Kefiran-based films
Currently, kefiran-based materials are gaining prominence for possessing unique properties, including biodegradability, safety, biocompatibility, stabilizing and emulsifying effect, satisfactory mechanical and water vapor barrier properties (Jain et al., 2020). Moreover,
kefiran films have good visual aspects and are effectively produced with
edible plasticizers, such as glycerol. Therefore, the use of kefiran in film
production can lead to suitable packaging and specific protective
coatings with improved properties. It results in high-quality food and
consequently contributing to an increase in shelf life (Moradi &
Kalanpour, 2019).
In the literature some researches have developed pure kefiran films,
which have evaluated different concentrations and types of plasticizers,
development of kefiran blends with other biopolymers, inclusion of
essential oils and nanofillers in kefiran films and application of radiation to improve the films properties, as described below.

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L. Marangoni Júnior, et al.

Table 1
Optimized conditions for kefiran production using L. kefiranofaciens, pure or mixed with S. cerevisiae.
Type of methodology for optimization and optimal operating conditions

Response analyzed

References

Univariate optimization; fermentation medium: whey lactose; lactose concentration: 4%; yeast extract:
4%; pH: 5.5; temperature: 30 °C; time: 48 h.
Response surface optimization; fermentation medium: whey; lactose concentration: 88.4 g L−1;
concentration of yeast extract: 21.3 g L−1; pH: 5.2; temperature: 20 °C.
Response surface optimization; fermentation medium: whey; lactose concentration: 67 g L−1;
concentration of yeast extract: 13 g L−1; pH: 5.7; temperature: 25 °C, time: 24 h.
Response surface optimization; lactose concentration: 50 g L−1; yeast extract concentration: 12 g L−1;
temperature: 30 °C.
Response surface optimization; fermentation medium: whey; glucose concentration: 15 %; temperature:
30 °C; Time: 10 h.
Response surface optimization; fermentation medium: whey lactose; sugar concentration: 2%; yeast
concentration: 6 g L−1; pH: 5.5; temperature: 35 °C. time: 48 h.

Kefiran production rate: ∼ 53
mg L−1 h−1
Maximum grain increase: 81.34

%
Kefiran production rate: 29.7
mg L−1 h−1
Kefiran production rate: 21 mg
L−1 h−1
Kefiran production rate: 37.14
mg L−1 h−1
Kefiran production rate: ∼ 35
mg L−1 h−1

Cheirsilp and Radchabut (2011)
Ghasemlou, Khodaiyan,
Gharibzahedi (2012)
Zajšek et al. (2013)
Dailin et al. (2015)
(Blandón et al., 2018)
Cheirsilp et al. (2018)

film-forming solutions, and the glycerol proportions were 12.5–50.0 %
(w/w) (based on kefiran weight) (Piermaria, Pinotti et al., 2009). Other
studies have been carried out varying the glycerol content, for example,
the study of Ghasemlou et al. (2011d), with films containing 2% kefiran
and glycerol concentrations of 15, 25 and 35 % (w/w) (based on kefiran
weight); and the work of Coma, Peltzer, Delgado, & Salvay, (2019) that
prepared a film with 3% Kefiran and 0, 10, 20, 30 % glycerol (w/w)
(based on kefiran weight).
Furthermore, the opportunity and the need to use other plasticizers
have also been reported by Ghasemlou et al. (2011a) with sorbitol (15,
25 and 35 % w/w based on kefiran weight), by Ghasemlou et al.
(2011c) with oleic acid (OA) (15, 25 and 35 % w/w based on the weight

of kefiran) and Tween 80 as an emulsifier (1% of OA concentration), by
Piermaria et al. (2011) with polyols and sugars (galactose, glucose,
sucrose, glycerol or sorbitol (25 g/100 g kefiran) and by Ghasemlou
et al. (2011b) with glycerol, sorbitol and oleic acid (OA) (25 % w/w
based on the weight of kefiran), in the solutions with OA, Tween 80
emulsifier was added (1% of the concentration OA).
4.2. Kefiran blend films and nanocomposites
Pure kefiran films demonstrate the potential to be applied as
packaging. However, it is necessary to improve the properties of the
films. Therefore, the mixture of kefiran with other biopolymers (polysaccharides, proteins, among others) aims to include better properties
and in some cases make the material more attractive. In addition, the
incorporation of nanoparticles, essential oils and other active compounds should be considered, since these components can provide other
functions to the films (light barrier, antioxidant and antimicrobial activities).
Fig. 4. Simplified flowchart summarizing the main steps of the purification
procedures described in the literature.

4.2.1. Kefiran-based film with polysaccharides
Polysaccharides are naturally occurring polymers, including starch,
cellulose, pectin and chitosan, which remains why they are widely
employed to prepare edible films and/or coatings (Hassan et al., 2018).
Starch is the most widely used renewable polysaccharide for the development of edible films and coatings, because of its abundance, costbenefit ratio and excellent film forming skills. Starch films possess good
optical, organoleptic and gas barrier properties, however, they demonstrate limitations due to their hydrophilicity and are weak in mechanical properties (Ogunsona et al., 2018; Ojogbo et al., 2020; Thakur
et al., 2019). Therefore, several mixtures and composites have been
developed to overcome their sensitivity to humidity and mechanical
properties (Jiang et al., 2020). In the literature, several authors work
with kefiran blends with starch. The study of Motedayen et al. (2013)
developed kefiran/starch blends with kefiran contents ranging from 7030 %. In addition, other works developed composites based on kefiran/
starch added with zinc oxide (ZnO) (Babaei-Ghazvini et al., 2018;
Shahabi-Ghahfarrokhi & Babaei-Ghazvini, 2019) and added titanium
oxide (TiO2) and with solution exposed to UV-A radiation for up to 12 h

4.1. Plasticized kefiran films
Films of pure kefiran, without the use or combination of another
polymer, were developed and studied for packaging applications by
several research groups. However, for obtaining and forming films with
good characteristics, the incorporation of other ingredients is necessary,
including typically plasticizers and emulsifiers. Hence, improving the
flexibility of the films due to their stability and compatibility with the
hydrophilic chains of the biopolymers, reducing the intermolecular
forces and increasing the mobility of the polymer chains (Motedayen
et al., 2013; Piermaria et al., 2011). In addition, it has been reported
that films prepared without plasticizer exhibit brittle aspects and are
difficult to obtain (Ghasemlou et al., 2011a).
The first research with kefiran films was related to the necessary
proportion of kefiran and plasticizer, to obtain films with good characteristics. The kefiran concentrations were 0.5, 0.75 and 1.0 % in the
5


Carbohydrate Polymers 246 (2020) 116609

L. Marangoni Júnior, et al.

for physical modification and radical formation (Goudarzi & ShahabiGhahfarrokhi, 2018).
Chitosan is a deacetylated derivative of chitin. It is a functional
versatile biopolymer due to the presence of amino groups responsible
for the various polymer properties, including barrier properties and its
antimicrobial activity (Priyadarshi & Rhim, 2020). In addition, it demonstrates excellent structural properties, which allow the formation
of a continuous layer of food coatings, which is why it has been employed successfully in food applications (Devlieghere et al., 2004;
Hassan et al., 2018). Blend films composed by kefiran/chitosan were
developed by Sabaghi, Maghsoudlou, and Habibi (2015), using Kefiran (2%) and chitosan (2%) solutions, resulting in different film

proportions with kefiran contents ranging from 32 to 78 %. The objective was to exploit a by-product from the fishing industry to improve the properties of kefiran films, with the suggestion of implementing it as food packaging.
Cellulose itself is a polysaccharide composed of glucose units, being
a water-insoluble polymer that can be chemically modified to form
water-soluble cellulose ethers, for example, carboxymethylcellulose
(CMC) (Fiori, Camani, Rosa, & Carastan, 2019). CMC is the most
common cellulose-derived biopolymer for the preparation of films and
coatings (Dashipour et al., 2015), as it presents good film formation
skills. However, it presents strictly limited mechanical properties and
water vapor barrier, which restricts its use in potential food packaging
applications (Fiori et al., 2019). Therefore, research aimed at improving
the films properties was developed, such as the study by Hasheminya
et al. (2019a) with biocomposite films made from kefiran (1%), carboxymethylcellulose (CMC) (1%), glycerol (50 % of dry weight), essential oil (EO) from Satureja khuzestanica (0.0, 1.0, 1.5 and 2.0 % v/v)
and Tween 80 emulsifier (0.5 % v/v based on EO) and the study by
Hasheminya et al. (2019b) that added copper oxide (CuO) nanoparticles and simultaneous addition of CuO and essential oil (EO) Satureja khuzestanica.
The development of kefiran blends with other polysaccharides, such
as alginates, pullulan and pectin can still be explored for application in
films and coatings. In addition, the extraction of polysaccharides from
food industry residues for this application should be considered, since
an ingredient from the food industry itself will not be used.

have addressed the incorporation of essential oils for a specific purpose, such as antimicrobial activity, which is discussed in section
7.10.
4.2.4. Kefiran-based nanocomposites and other developments
Theoretically, the incorporation of nanoparticles in biopolymer
films aims to reduce the effective permeation area of the films (Zhao
et al., 2020). This characteristic is attributed to the change in the
diffusion path of the molecules, which makes it more tenuous and long
during the diffusion phenomenon. This results in better barrier properties, provided that the nanoparticles are well dispersed throughout
the polymeric matrix. Moreover, the incorporation of these nanomaterials can improve thermal, physical and mechanical properties, besides in some cases adding specific functions, such as antimicrobial
activity (Joshi et al., 2018). Research evaluating the incorporation of

nanoparticles in kefiran films has been carried out in recent years. The
nanoparticles evaluated were aluminum oxide (Al2O3) (Moradi et al.,
2019), zinc oxide (ZnO) (Shahabi-Ghahfarrokhi et al., 2015b) and
nano-cellulose (NC) (Shahabi-Ghahfarrokhi et al., 2015a). In addition,
titanium oxide (TiO2) and montmorillonite (MMT) were used in kefiran/WPI blends (Zolfi et al., 2014a, 2014b) and zinc oxide (ZnO) in
kefiran/starch blends (Babaei-Ghazvini et al., 2018; ShahabiGhahfarrokhi & Babaei-Ghazvini, 2019).
Other applications consisted of the application of UV-A radiation
and γ irradiation in film solutions, to improve the its properties
(Goudarzi & Shahabi-Ghahfarrokhi, 2018; Shahabi-Ghahfarrokhi et al.,
2015).
5. Properties of the film-forming solutions
The rheological properties of film-forming solutions must be determined, since they will inform about the processing conditions for
obtaining the films (Piermaria, Pinotti et al., 2009). Aqueous filmforming solutions of kefiran (10 g kg−1) added with glycerol plasticizer
(0, 25 and 50 g/100 g of kefiran) presented pseudoplastic behavior. The
same was observed for kefiran films with oleic acid (Ghasemlou et al.,
2011c) and sorbitol (Ghasemlou et al., 2011b). The viscosity values
were less than 0.50 Pa s and without visible air bubbles, which results
in a thin and level layer for casting.
In addition, the apparent viscosity of the solutions was uninfluenced
by the different glycerol concentrations (Piermaria et al., 2009). The
intrinsic viscosity provides a view of the hydrodynamic volume occupied by a given polymer and the length of the polymer chain (Piermaria
et al., 2008; Shahabi-Ghahfarrokhi & Babaei-Ghazvini, 2019). The increase in the concentration of oleic acid in the kefiran solution produced an increase in viscosity (Ghasemlou et al., 2011c). The work by
Shahabi-Ghahfarrokhi and Babaei-Ghazvini (2019) evaluated the intrinsic viscosity of kefiran/starch/ZnO solutions subjected to UV radiation (0, 1, 6 and 12 h). It has been observed that viscosity decreases
as radiation exposure time increases. The authors attributed these results to the formation of free radicals that induced molecular changes
and fragmentation. Similar behavior was observed for kefiran/starch/
TiO2 solutions, attributed to the effect of radiation on the breaking of
polymer chains in shorter chains (Goudarzi & Shahabi-Ghahfarrokhi,
2018).
It is worth mentioning solutions with high viscosity are difficult to
be homogenized, which can result in films with a certain heterogeneity. In addition, air bubbles tend to get trapped in viscous solutions, which can result in defective films. Moreover, low viscosity

solutions can lead to the formation of films with reduced thickness,
because of the significant dilution of the solutions (Piermaria et al.,
2009). That is, it is necessary to find an equilibrium viscosity to obtain
films without defects and with adequate thickness for the intended
application.
Moreover, the storage module (G’) and loss module o (G”) help to
interpret the viscoelastic behavior of polymeric solutions. Piermaria

4.2.2. Kefiran-based film with proteins
Distinct types of globular proteins, such as whey protein, have been
investigated in the development of films and coatings. Whey protein
isolate (WPI) films are characterized by satisfactory mechanical properties and excellent barrier properties to gases, aromatics and fat.
However, due to the fact that whey protein is hydrophilic in nature,
these films experience some moisture limitations (Hassan et al., 2018).
Films composed of blends of kefiran and WPI were developed to improve the films properties (Gagliarini et al., 2019). In addition, other
authors have incorporated titanium oxide (TiO2) nanoparticles and
montmorillonite (MMT) in proportions of 1, 3 and 5% to obtain films
with more robust properties (Zolfi et al., 2014a, 2014b). Other proteins
display the potential to develop blends with kefiran. It can be cited as
zeins, gelatin, wheat gluten and soy protein.
4.2.3. Kefiran-based film with lipids
Lipids represent excellent barriers to water vapor, and when
blended with other biopolymers they can improve barrier and mechanical properties (Hassan et al., 2018). In addition, lipids are effective in blocking moisture release due to their low polarity (Hassan
et al., 2018; Perez-Gago et al., 2002). The particular lipids applied in
films and coatings are waxes, monoglycerides and surfactants. Oleic
acid (OA) is a fatty acid with the potential to improve the water
vapor barrier of hydrophilic films. In this context, Ghasemlou et al.
(2011c) prepared kefiran films with oleic acid (15–35 % w/w) and
Tween 80 emulsifier (1% OA concentration), in order to intensify the
water vapor barrier and the mechanical properties. Other studies

6


Carbohydrate Polymers 246 (2020) 116609

L. Marangoni Júnior, et al.

et al. (2008) reported that low frequency kefiran solutions (1 % w/v)
presented a loss module greater than that storage module. At higher
frequencies, G’ surpassed G”, indicating that the inter-chain tangles did
not have enough time to slide and behave like a gel. A similar behavior
was observed by Radhouani et al. (2018). The authors evaluated the
behavior at two concentrations of kefiran (1 and 10 % w/v). The 1%
samples started with a viscous behavior and 10 % samples started with
an elastic behavior. Specifically, 1% kefiran samples crossed at approximately 8 Hz, going from a phase angle of about 47° (viscous liquid) to approximately 31° (elastic/gel); while 10 % solutions showed a
crossover at 1.6 Hz, going from a phase angle of about 25.7° (elastic/
gel) to an average of approximately 58° (viscous liquid). In that
manner, the viscoelastic properties of kefiran indicate its potential application in tissue engineering and regenerative medicine. For example,
it could be employed in osteoarthritis treatment therapies to restore the
viscoelastic properties of the joint synovial fluid. Kefiran represents an
economical alternative to the traditional hyaluronic acid (Radhouani
et al., 2018).

As follows, the solvent is removed by evaporation to decrease the
distance between the polymer chains, favoring their interaction. This
interaction allows the formation of a polymer network that will be finalized with the film conformation (Coma et al., 2019; Felton, 2013;
Priyadarshi & Rhim, 2020). However, this method can result in slight
variations in the film properties, due to variations in the formulations.
In addition, this method is currently unused on an industrial scale,
being uneconomical and time consuming (Priyadarshi & Rhim, 2020;

Zhang et al., 2019). The processing conditions for obtaining kefiranbased films have peculiarities in each study. Therefore, to better illustrate the differences among some kefiran-based film researches, these
data are presented in Table 2. The agitation conditions of the final
formulation, the degassing and the dispersion method, the solution
mass poured into each plate, the type of plate material used, the solvent
evaporation conditions, the final thickness of the films and the storage
conditions were taken into account.
6.2. Coating

6. Manufacturing methods

Coatings are applied in liquid form on food or on the surfaces of
other packaging materials. It can be made by immersing the product in
a solution or by spraying, followed by drying to adhere the material to
the product surface (Maringgal et al., 2020). A coating does not act as
packaging, but it can limit intrinsic factors and reduce the barrier requirements of the packaging, and consequently extend the food shelf
life (Ganiari et al., 2017; Nor & Ding, 2020). The use of coatings in food
applications depends on several characteristics such as: cost, availability, functional attributes and their properties. These characteristics
are influenced by parameters such as the type of material used as the
structural matrix, the processing conditions, the type and additives
concentration added (Ganiari et al., 2017).
Biopolymers have often been reported as excellent materials for the
coating’s development. The structural materials utilized in the construction of coatings are based on proteins, lipids and polysaccharides.
However, no reports were provided on the development and application of kefiran-based coatings. The main reason for this is that the study
of the kefiran film-forming potential is still in the beginning of its development, and it is doubtful whether researchers will use it for food
coating or other related applications. In this sense, this topic presents
itself as a future trend with great potential to be assessed.

Biopolymer-based films are typically produced from a solution or
dispersion of the film-forming agent, followed by methods that aim to
separate it from the solvent (Ghasemlou et al., 2011b). Current techniques for preparing films based on biopolymers include direct casting,

coating (Fig. 5) and extrusion. These methods can be implemented for
films based on a single material or mixed materials (blends). The choice
of the most effective method will depend on factors such as equipment
availability, costs, efficiency, and application.
6.1. Direct casting
The direct casting method has been widely employed, as it is the
simplest method for the preparation of biopolymers-based films based.
The films’ preparation by the casting method involves the use of at least
one film-forming agent (biopolymers), a solvent and a plasticizer. To
form the film matrix, it is necessary to prepare a homogeneous, viscous
film-forming solution containing biopolymers, which will undergo filtration, centrifugation or another method to eliminate insoluble particles and air bubbles, followed by dispersing the solution on a flat-sized
surface and shape.

Fig. 5. Simplified production scheme for films and coatings by casting and spraying, respectively (The pear fruit in this case was used as an illustration of the
application of coatings).

7


8
Constant stirring for
60 min

Kefiran (5% w/v) and WPI 5% (w/v) (50:50 v/v),
added with MMT and nano-TiO2 (0, 1, 3, and 5%
w/w).
Kefiran 2% and starch 2% (100/0, 70/30, 50/50 and
30/70).
Kefiran (10 g/kg) with galactose, glucose, sucrose,
glycerol or sorbitol (25 g/100 g kefiran).

Kefiran (2%) with 0, 15, 25 and 35 % glycerol (w/w
based on kefiran weight).
Kefiran (2%) with 0, 15, 25 and 35 % oleic acid (w/
w based on kefiran weight).
Kefiran (10 g/kg) with 0 and 25 % glycerol/100 g
kefiran.

Constant stirring for
10 min
Constant stirring for
10 min
Constant stirring for
60 min

Kefiran (2%) and nano cellulose (0, 1, 2 and 3% dry
basis).
Kefiran (2%) and nano ZnO (0, 1, 2 and 3% dry
basis).
Kefiran (5% w/v) and WPI 5% (w/v) (50:50 v/v),
added nano-TiO2 (0, 1, 3, and 5% w/w).

Constant stirring for
15 min
Constant stirring for
15 min
Constant stirring

Constant stirring for
15 min

Constant stirring

Constant stirring for
15 min
Constant stirring

Kefiran (2%) and chitosan (2%) in proportions (100/
0, 68/32, 50/50 and 32/68).
Kefiran films as vehicle for probiotic microorganisms

By sonicator

–

50 mL

–

Constant stirring at
90 °C for 30 min
Constant magnetic
stirring at 50 °C for
1h
Constant stirring for
15 min
Constant stirring for
15 min
Constant stirring for
15 min
Constant stirring for

120 min

Kefiran (2% w/v) and starch (5% w/v) (50:50 v/v),
added with nano-ZnO (0, 1, 3, and 5% w/ w).
Kefiran/starch/TiO2 exposed to UV-A radiation for 0,
1, 6 and 12 h.
Kefiran/carboxymethyl cellulose added with copper
oxide nanoparticles (1, 1.5 and 2%)
kefiran/waterborne polyurethane blend film
incorporated with Zataria multiflora and
Rosmarinus officinalis EO (5, 10, 15 and 20% v/v)
Kefiran (2%) treated at 0 3, 6 and 9 kGy.

–

Rest until reaching 50
°C
Rest until reaching 50
°C

1500 rpm at 70 °C
for 5 min
1500 rpm at 70 °C
for 5 min

50 mL
–

By sonicator
–

–
70 mL

By sonicator
By sonicator and
Vacuum oven for 30
min at 30 °C
By sonicator and
Vacuum oven for 30
min at 30 °C
Vacuum oven for 30
min at 30 °C
–

Teflon plates
Plastic petri (8.7 cm
diameter)

50 g
25 g

Under vacuum for 5
min
–

Teflon plates

–

Plastic petri (8.7 cm
diameter)

25 g

Rest

Teflon plates

Teflon plates

Teflon plates (15 cm
diameter)
Plastic petri (10 cm
diameter)
Plastic petri (5 cm
diameter)
Teflon plates (15 cm
diameter)
Teflon plates (15 cm
diameter)
Teflon plates

Glass plates (9 cm
diameter)
Teflon plates

Polystyrene plates
(15 cm diameter)
Plastic petri

Plastic petri
(5 cm diameter)
Plastic petri
(12 cm diameter)

Glass plates (9 cm
diameter)
Glass plates (9 cm
diameter)

Polystyrene plates
(15 cm diameter)

Plastic petri

Plates material

–

70 mL

–

3.5 g

–
By sonicator

15 mL

By sonicator

–

50 mL

–

–

50 mL

By sonicator

50 mL

50 mL

50 mL

By sonicator

Constant stirring for
15 min

Kefiran (2%) + starch (5%) + nano zinc oxide (1%)
+ glycerol (40 %) exposed to UV radiation for 0,
1, 6 and 12 h.
Kefiran/CMC and EO by Satureja Khuzestanica (0.0,

1.0, 1.5 and 2.0% v/v).
Kefiran/CMC and CuO and the combination of CuO
and EO by Satureja Khuzestanica (0.0, 1.0, 1.5
and 2.0% v/v).
Kefiran/WPI/glycerol (6%, 2% and 3.2 % w/w) with
and without the addition of probiotics.
Kefiran (2%) with Al2O3 nanoparticles (1, 3 and 5%
w/w dry basis).

Solution
mass
17 g

15,000 rpm for 5
min

Kefiran (3%) + glycerol (20 and 30 %).

Dispersion and/or
Degassing
Vacuum for 30 min

Stirring

Film material

Table 2
Kefiran-based films preparation by casting.

Room temperature and RH

for 18 h
40 °C in a ventilated oven
until reaching constant
weight for 6 h
Room temperature and RH
for 18 h
Room temperature and RH
for 18 h
40 °C in a ventilated oven until
constant weight along 6 h.

Room temperature and RH
for 18 h

Room temperature and RH
for 18 h

25 °C

25 °C

37 °C for 11−14 h

30 °C for 24 h

25 °C for 48 h

Room temperature at 48 h

25 °C for 72 h

Room temperature at 48 h

25 °C for 48 h

35 °C for 24 h

37 °C for 16 h

25 °C for 72 h

25 °C for 72 h

40 °C and 40 % RH in a
ventilated oven until
10−15% water content
25 °C for 48 h

Solvent evaporation

21.4 and 21.9

62, 75, 71 and 79

58, 64, 62 and 67

23, 22, 25, 31, 22
and 22.

74, 62, 59 and 57

̴ 74 (MMT) and
74, ̴ 91 (TiO2)

74, 75, 75 and 76

80, 70, 70 and 70

–

13 - 19

34, 33, 32 and 31

80, 80, 60 and 50

140, 130, 120
and 110
100, 80, 80 and
80
90, 107, 110 and
117
90 - 150

14.5, 15.5, 17.9
and 28.8

133 - 143

90, 113, 133 and

153
90 - 161

110

60

Thickness (μm)

55 %

55 %

75 %

50 %

55 %

25 °C and 50 %
RH
20 °C and 75 %
RH

25 °C

20 °C and 75 %
RH

25 °C

25 °C

25 °C and
RH
25 °C and
RH
20 °C and
RH
25 °C and
RH
25 °C and
RH
25 °C

25 °C and 55 %
RH
55 % RH

25 °C and
50−55% RH
50−55% RH

20 °C and 75 %
RH
25 °C and 55 %
RH

25 °C and 55 %
RH

25 °C and 55 %
RH

–

22 °C and 43 %
RH

Storage
condition

Piermaria et al. (2009)

Ghasemlou et al. (2011c)

Ghasemlou et al. (2011d)

Piermaria et al. (2011)

Motedayen et al. (2013)

Zolfi et al. (2014b)

Shahabi-Ghahfarrokhi
et al. (2015a)
Shahabi-Ghahfarrokhi
et al. (2015b)
Zolfi et al. (2014a)

Piermaria et al. (2015)

Shahabi-Ghahfarrokhi
et al. (2015)
Sabaghi et al. (2015)

Rad et al. (2018)

Babaei-Ghazvini et al.
(2018)
Goudarzi and ShahabiGhahfarrokhi (2018)
Hasheminya et al. (2018)

Moradi et al. (2019)

Gagliarini et al. (2019)

Hasheminya et al. (2019b)

Hasheminya et al. (2019a)

Shahabi-Ghahfarrokhi and
Babaei-Ghazvini (2019)

Coma et al. (2019)

References

L. Marangoni Júnior, et al.

Carbohydrate Polymers 246 (2020) 116609

Carbohydrate Polymers 246 (2020) 116609

L. Marangoni Júnior, et al.

6.3. Extrusion

nanoparticles increased. This behavior can be illustrated by Moradi
et al. (2019), that included Al2O3 in the kefiran films. In addition, Zolfi
et al. (2014a) included TiO2 in kefiran/WPI films, Goudarzi & ShahabiGhahfarrokhi (2018) included TiO2 in kefiran/starch films, and also
exposed it to UV-A radiation. Finally, Shahabi-Ghahfarrokhi & BabaeiGhazvini (2019) included ZnO in Kefiran/starch films exposed to UV
radiation. Similar behavior was observed for films with essential oils
included, such as Hasheminya et al. (2019a), that used the Satureja
khuzestanica EO in kefiran/CMC films; and the work of Hasheminya
et al. (2019b) that used CuO and EO. Studies report that the reduction
in moisture content is due to the increase in the hydrophobic phase in
the film after adding EO. Moreover, the incorporation of probiotics also
led to a reduction in the moisture content of kefiran/WPI films
(Gagliarini et al., 2019).
On the other hand, the incorporation of ZnO in kefiran films
(Shahabi-Ghahfarrokhi et al., 2015b) and in kefiran/starch films
(Babaei-Ghazvini et al., 2018) did not influenced the moisture content,
regardless of the concentration. In addition, the incorporation of nano
cellulose in kefiran films led to an increase in moisture content, adding
greater hydrophilicity to the film (Shahabi-Ghahfarrokhi et al., 2015a).

Extrusion is frequently used in the packaging raw material industry
and in the packaging manufacturing industry, as is the case for
packaging materials based on fossil source polymers. The polymers are

heated to the molten state by a combination of two fundamental
parameters: heating and shear. The screw forces the resin through a
mold, manufacturing the resin in the desired shape. After that, the
extruded material is cooled and solidified as it is pulled by the die or
water. This technology can be effectively exploited for the preparation
of bio-based films (Aider, 2010).
However, the extrusion-based kefiran films are yet undeveloped. In
comparison with other biopolymers, the melting of the kefiran produced by Tibetan kefir took place at about 93 °C, lower than xanthan
gum (153.4 °C) and guar gum (490.11 °C). The endothermic enthalpy
change required to melt 1 g of kefiran, xanthan and guar gums were
249.7, 93.2 and 192.9 J, respectively (Wang & Bi, 2008). In parallel,
Ahmed et al. (2013) carried out the thermogravimetric analysis for
kefiran, xanthan and locust gums. It was observed a most pronounced
initial weight loss of kefiran between 40 and 90 °C, which might be
attributed to the evaporation of moisture. The decline in weights above
90 °C was ascribed to the degradation. The onset of decomposition
occurred at 261.4 °C. The polymer weight loss decreased substantially
around 300 °C.
In another research, the TGA of kefiran presented one event during
the increasing of the temperature (40–106 °C). This event occurred with
the maximum mass loss (approximately 9%) also associated with the
moisture. The critical mass loss (12–65 %) occurred in the second event
(264–350 °C), which was attributed to degradation of kefiran polysaccharide structure (Radhouani et al., 2018). Other studies of exopolysaccharides showed approximated temperature of degradation, with
the maximum between 300–350 °C (Botelho et al., 2014; Moradi et al.,
2019). Therefore, for the successful application of kefiran films as food
packaging, it is essential to develop researches to optimize the process,
considering its thermal characteristics. In this sense, this processing
area is however lacking in information and can be exploited to maximize the large-scale production of kefiran films.

7.2. Solubility

The solubility of kefiran-based films reveals the possible applications of this material. In some potential food applications, the ideal is
that the film presents good insolubility in water, hence improving its
integrity and increasing the shelf life of the film. However, according to
Ghasemlou et al. (2011a) in some cases, the film's water solubility is
desirable before consumption, especially for edible films. This property
is substantially influenced by the type and concentration of plasticizer
used.
The solubility of the kefiran film added with glycerol (25 g/100 g of
kefiran) increased significantly by increasing temperature, where, all
samples were partially soluble at 25 °C and 37 °C, and totally solubilized at 100 °C (Piermaria et al., 2009). In addition, the increase in
glycerol content resulted in films with greater solubility (Ghasemlou
et al., 2011a, 2011d). According to Coma et al. (2019) the increase in
the concentration of glycerol resulted in an increase in the amount of
hydration water in the kefiran films, consequently leading to a more
significant free volume, suggesting the glycerol decreased the attractive
forces between the polymeric chains and consequently allowed greater
mobility of water molecules. On the other hand, exposure of the filmforming solution to γ irradiation reduced the water absorption capacity
and the solubility at doses up to 6 kGy (Shahabi-Ghahfarrokhi, Khodaiyan, Mousavi, et al., 2015). The sorbitol plasticizer maintained the
solubility similar to the film without plasticizer, regardless of the concentration (Ghasemlou et al., 2011a). While the oleic acid plasticizer
reduced the solubility of kefiran films (Ghasemlou et al., 2011c).
The blends of kefiran with other biopolymers showed that the film's
solubility can vary depending on the nature of each biopolymer. Where
chitosan (Sabaghi et al., 2015), starch (Motedayen et al., 2013) and
cellulose (Shahabi-Ghahfarrokhi et al., 2015a) reduced solubility as
their concentration increased. That is, the hydrophilic character of
these biopolymers exerted a direct influence on the results.
Another relevant procedure that affects the kefiran-based film solubility is the incorporation of nanoparticles. Generally, as the nanoparticle content increases, the solubility of the film decreases. These
results were observed with Al2O3 for kefiran films (Moradi et al., 2019),
ZnO (Babaei-Ghazvini et al., 2018; Shahabi-Ghahfarrokhi et al.,
2015b), ZnO in kefiran/starch blends followed by exposure to UV radiation (Shahabi-Ghahfarrokhi & Babaei-Ghazvini, 2019), TiO2 in kefiran/WPI blends (Zolfi et al., 2014a), TiO2 in kefiran/starch blends

exposed to UV-A radiation (Goudarzi & Shahabi-Ghahfarrokhi, 2018)
and CuO and EO for kefiran/CMC films (Hasheminya et al., 2019b).

7. Characterization and properties of kefiran films
7.1. Moisture content
The moisture content and water activity of kefiran films (10 g kg−1)
with glycerol plasticizer (0, 12.5, 25.0, 37.5 and 50.0 g/100 g of kefiran) were 14.8–36.4% and 0.453 to 0.556, respectively (Piermaria
et al., 2009). That is, as the glycerol concentration increased, there was
an increase in the moisture content and water activity. Similar behavior
was observed by Ghasemlou et al. (2011a, 2011d). These results were
attributed to the water retention in the film caused by the plasticizer
hydrophilicity. Other plasticizers were used in kefiran films (galactose,
glucose, sucrose or sorbitol 25 g/100 g of kefiran) (Piermaria et al.,
2011) and sorbitol (Ghasemlou et al., 2011a). In both studies, the
plasticizers did not influence the moisture content. The oleic acid
plasticizer reduced the moisture content of kefiran films from
17.9–12.3% (Ghasemlou et al., 2011c). Furthermore, the application of
γ irradiation (3, 6 and 9 kGy) in kefiran film-forming solutions led to a
reduction in the moisture content of the films, because of the improvement of the hydrophobic properties in the polymer with the use of
γ irradiation (Shahabi-Ghahfarrokhi et al., 2015).
Considering kefiran blends with other biopolymers, the moisture
content increased with the incorporation of starch, justified by its
greater hydrophilicity (Motedayen et al., 2013). In contrast, it decreased with the incorporation of chitosan, as this biopolymer causes an
increase in the hydrophobic phase (Sabaghi et al., 2015). In addition,
the incorporation of nanoparticles in kefiran blends and other biopolymers reduced the moisture content of the films as the concentration of
9


Carbohydrate Polymers 246 (2020) 116609

L. Marangoni Júnior, et al.

7.3. Hydrophobicity

the casting surface. The gradual addition of plasticizer significantly
increased the film's flexibility. The microstructure of the film faces and
cross sections proved to be continuous and homogeneous, without
clusters, pores, flaws or perforations of film (Fig. 6). Similar results
were noted by Ghasemlou et al. (2011d), Piermaria et al. (2009). The
use of other plasticizers such as polyols and sugars showed morphologies similar to that of films with glycerol (Piermaria et al., 2011).
However, when oleic acid was used as a plasticizer, the film showed
structural discontinuities associated with the formation of two phases
(lipids/polymer) (Ghasemlou et al., 2011c).
Regarding blends of kefiran with other biopolymers, nanoparticles
addition or radiation exposure, in general, as they increased, morphological differences were observed. Films of kefiran/starch/TiO2 submitted to UV-A radiation were rough and heterogeneous, reflecting the
low miscibility of starch and kefiran. However, the increased exposure
time to UV-A produced free-radicals, exhibiting smoother morphology
(Goudarzi & Shahabi-Ghahfarrokhi, 2018). The incorporation of EO in
kefiran/CMC films exhibited a homogeneous structure without porosity
(Hasheminya et al., 2019a). The same was observed for kefiran/CMC
films incorporated with EO and CuO (Hasheminya et al., 2019b). Kefiran/WPI films were homogeneous and transparent. However, they
showed surface roughness, because of the interactions between proteins
and polysaccharides (Gagliarini et al., 2019). The incorporation of 1
and 3% of Al2O3 in kefiran films improved the microstructure, that is,
there was good dispersion of the particles, resulting in low pores and
cracks (Moradi et al., 2019). The surface of kefiran/starch films
changed as the amount of starch increased. The matrixes morphologies
were rougher, related to the formation of channels and the state and
structure of the starch granule. However, they were flat and compact
with remarkably small particles and without any phase separation

(Motedayen et al., 2013). Kefiran/starch/ZnO films presented a smooth

The water contact angle is performed to determine the hydrophobicity of the biopolymer films. The decrease in the contact angle
occurred with the increase in the glycerol content in kefiran films
(Ghasemlou et al., 2011d). This phenomenon was also observed in
kefiran/starch blends as the starch content increased (Motedayen et al.,
2013). That is, glycerol and starch resulted in more hydrophilic films.
Conversely, the increase in the concentration of hydrophobic additives, as oleic acid (Ghasemlou et al., 2011c) and EO of Satureja
Khuzestanica in Kefiran/CMC films (Hasheminya et al., 2019a), led to
an increase in the contact angle. Similar result was obtained by incorporation of nanoparticles, as ZnO in kefiran/starch films (BabaeiGhazvini et al., 2018), TiO2 in kefiran/starch films (Goudarzi &
Shahabi-Ghahfarrokhi, 2018) and CuO in kefiran/CMC blends
(Hasheminya et al., 2019b); and also by radiation exposure (Goudarzi &
Shahabi-Ghahfarrokhi, 2018; Shahabi-Ghahfarrokhi & BabaeiGhazvini, 2019) It is evident the incorporation of these substances increased the water contact angle. The consequent improvement in their
hydrophobicity can have a direct impact on the other properties and
materials shelf life.
7.4. Morphological properties - Visual aspect and microstructure
The morphological properties of the films can be measured through
visual and microscopic analysis, taking into account the film's maneuverability, homogeneity and continuity. Kefiran films with the addition
of 0, 10, 20, 30 % glycerol were evaluated by Coma et al. (2019). The
kefiran films with glycerol exhibited a homogeneous aspect, without
cracking and high transparency. The authors verified that the samples
with 0 and 10 % glycerol were brittle, requiring care when peeling from

Fig. 6. Scanning electron microscopy (SEM) observations of the cross sections and the surface of non-plasticized films (a) (c) and plasticized with 30 % glycerol (b)
(d) (Coma et al., 2019). Adapted with permission from Elsevier, Copyright (2019).
10


Carbohydrate Polymers 246 (2020) 116609

L. Marangoni Júnior, et al.

and homogeneous surface, without any crack or bubble. However, as
the concentration of ZnO increased, aggregates appeared on the film
surface (Babaei-Ghazvini et al., 2018).

the NC content (up to 2%) and decreased in the film containing 3% NC.
However, films with NC exhibited no color difference compared to the
control. In addition, there was no difference between the value of
whiteness index of kefiran film and that of kefiran/NC composite films
(up to 2%) (Shahabi-Ghahfarrokhi et al., 2015a). The incorporation of
ZnO in kefiran/starch films resulted in a difference in luminosity
properties, whiteness index and color difference in relation to the
control film (Babaei-Ghazvini et al., 2018). Kefiran/chitosan films
showed changes in luminosity, decreased from 28.6 to 22.3 with the
increase in chitosan content. In addition, there was an increase in the
film's opacity resulting from the crosslinking between the molecules of
the films (Sabaghi et al., 2015). On the other hand, the incorporation of
Al2O3 in kefiran films did not affect the luminosity and color difference,
attributed to the uniformity and small size of the nanoparticles (Moradi
et al., 2019).

7.5. Instrumental color
The transparency and color aspects of films based on biopolymers
can influence the valuation of the final product. The study by Piermaria
et al. (2009) evaluated the transparency of kefiran films, showing values of 2.71 A600/mm. In addition, the authors emphasized the addition
of glycerol did not change this property. The addition of sugars and
polyols produced a variation in the transparency of kefiran films from
1.88 to 3.30 A600/mm (Piermaria et al., 2011). However, the increase in
glycerol concentration can cause an increase in luminosity and degree

of whiteness and a decrease in the total color difference compared to
film without plasticizer (Ghasemlou et al., 2011d).
Kefiran/starch/ZnO films exposed to UV radiation for 0, 1, 6 and 12
h were evaluated for instrumental color by Shahabi-Ghahfarrokhi and
Babaei-Ghazvini (2019). It was observed that exposure to radiation led
to a decrease in the color difference in relation to the white plate. In
addition, the luminosity and the yellowing index also decreased simultaneously, the authors attributed to the color amend the production
of free-radicals induced by UV radiation. Similar results were found in
kefiran/starch/TiO2 films submitted to UV-A (Goudarzi & ShahabiGhahfarrokhi, 2018).
The luminosity and whiteness index of Kefiran/CMC films with EO
Satureja Khuzestanica decreased significantly with the increase in EO
concentration. There was an increase in the total color difference,
yellowing index, chroma and opacity. The authors attributed these
modifications to the phenolic compounds present in the EO
(Hasheminya et al., 2019a). Similar results were experienced for kefiran/CMC films with CuO and simultaneous addition of CuO and EO
(Hasheminya et al., 2019b). Kefiran/WPI films with or without the
addition of probiotic microorganisms revealed no difference in luminosity. However, the probiotics addition induced a slightly yellow appearance which generated a total color difference (Gagliarini et al.,
2019). The incorporation of MMT and TiO2 in kefiran/WPI films resulted in a slight change in the transparency of the one containing
MMT. It indicates proper distribution of MMT in the polymer matrix.
However, for TiO2 films, as the concentration increased, the level of
transparency decreased, because of its metallic nature (Zolfi et al.,
2014b).
The luminosity factor of kefiran films increased with the increase in

7.6. Thermal behavior and changes in the IR spectrum
The thermal behaviors of the films can be observed utilizing the
techniques of differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA). The attenuated total reflection Fouriertransform infrared spectroscopy (ATR-FTIR), X-ray diffraction (XRD),
among others techniques must equally be used in a complementary way
to the analysis of thermal behavior. Because when the films are obtained, some changes in the spectra discussed in item 2 of this article
may undergo some changes. The results can be directly related to the

mechanical and barrier performance of the films as well.
FTIR is an effective technique for detecting chemical bonds in materials. In most cases, the FTIR spectra of kefiran films can be classified
as follows: the primary region is attributed to hydroxyl groups (OH) due
to water and carbohydrates (3000−3600 cm−1). The second region is
related to the symmetrical and anti-symmetric stretching modes of CH
in methyl (CH3) and methylene (CH2) functional groups (2800−3000
cm−1). The third region is designated to the OH flexion mode in water
molecules (1580−1700 cm‐1). The fourth zone contains the peaks attributed typically to the ways of stretching the carbohydrate rings and
side groups (C–O–C, C–OH, C–H) (900−1200 cm‐1) (Coma et al., 2019;
Goudarzi & Shahabi-Ghahfarrokhi, 2018; Hasheminya et al., 2019a;
Piermaria et al., 2011; Shahabi-Ghahfarrokhi & Babaei-Ghazvini,
2019). It's significant to highlight that, when added plasticizers, the first
and third regions showed larger bands. It is a result from the increase in
water content induced by the plasticizer and also by the presence of the
glycerol itself (Coma et al., 2019). Still in this context, when zinc oxide

Table 3
DSC results for kefiran films.
Film material

Conclusions

References

Kefiran (2%) treated at 0, 3, 6 and 9 kGy.

The films showed Tg of -16, -15, -14 and −23 °C and Tm of 85, 87, 86 and 85 °C. The Tg of
the samples increased with an increase in the irradiation dose by up to 6 kGy and then
decreased in higher doses 9 kGy. The increase in Tg was attributed to the fact that the low
dose of γ-irradiation increased the cross-links between the biopolymer’s chains, but at

high doses, the plasticizing effect of mono and disaccharides in kefiran decreased. The Tm
of the film did not change with changes in the γ irradiation dose.
The films presented Tg of -16, -27, -30 and −38 °C and Tm of 85, 87, 88 and 86 °C. The
incorporation of nano cellulose drastically reduced the Tg of kefiran films.
The films showed Tg of -16, -20, -22 and -30 °C and Tm of 85, 86, 87 and 88 °C. Tg
decreased with increasing ZnO content and Tm increased.
Tg reduced from -12 to -14 °C and Tm reduced from 82 to 72 °C, after adding TiO2. This
behavior was due to the interruption of the regularity of the chain structures in the
biopolymers matrix and to the increase in spacing between the chains.
As the OA concentration increased, Tg reduced from -16 to −22 °C and Tm increased form
69–92 °C, attributed to the higher molecular weight and the more hydrophobic nature of
OA.
As the glycerol concentration increased, Tg and Tm decreased (Tg -14 at −21 °C and Tm 73
at 68 °C), attributed to the inherent structural characteristics (chain mobility) and high
hydrophilic nature.

Shahabi-Ghahfarrokhi et al.
(2015)

Kefiran (2%) and nano cellulose (0, 1, 2 and 3% (dry
basis))
Kefiran (2%) and nano ZnO (0, 1, 2 and 3% (dry
basis))
5% WPI (w/v) and Kefiran (5% w/v) (50:50 (v/v)),
added nano-TiO2 (0, 1, 3, and 5% (w/w))
Kefiran (2%) with 0, 15, 25 and 35 % oleic acid (w/w
based on kefiran weight)
Kefiran (2%) with 0, 15, 25 and 35 % glycerol (w/w
based on kefiran weight)

Tg: glass transition temperature; Tm: melting temperature.
11

Shahabi-Ghahfarrokhi et al.
(2015a)
Shahabi-Ghahfarrokhi et al.
(2015b)
Zolfi et al. (2014a)
Ghasemlou et al. (2011c)
Ghasemlou et al. (2011d)


Carbohydrate Polymers 246 (2020) 116609

L. Marangoni Júnior, et al.

was included in the kefiran and starch film, lesser peaks of around 500
cm−1 were observed (Shahabi-Ghahfarrokhi & Babaei-Ghazvini, 2019).
Characteristic differences in peaks in the 400 to 4000 cm−1 range were
also observed after addition of CuO in kefiran/CMC films (Hasheminya
et al., 2019b).
Thermogravimetric analysis (TGA) of kefiran films with 0, 10, 20
and 30 % glycerol was performed by Coma et al. (2019). A degradation
of the plasticized samples was observed at more reduced temperatures
than those not plasticized. This is justified by the presence of glycerol in
the film matrix, which increases the mobility of the chains and exposes
the polymer chains even more to thermal degradation. According to
Moradi et al. (2019), the thermal degradation of kefiran films with
glycerol starts at around 220 °C, being more expressive at around
250−320 °C.

The X-ray diffraction patterns of non-plasticized and plasticized
kefiran films with polyols and sugars revealed a semi crystalline
structure. The degrees of crystallinity obtained were less than 3.1 %
(Piermaria et al., 2011). The incorporation of Al2O3 nanoparticles in
kefiran film significantly affected the crystalline region of the films,

where the peak intensity decreased with increasing Al2O3 concentration
(Moradi et al., 2019).
The findings from the DSC analysis of kefiran films are indicated in
Table 3. Predominantly, it was observed that as the exposure time to
radiation or the concentration of plasticizer, nanocomposites and other
biopolymers increases, the glass transition temperature was reduced. In
addition, changes in the melting temperature resulting from the different formulations were observed.
7.7. Mechanical properties
The mechanical properties of films are associated with their performance during the handling, storage and distribution conditions imposed for each type of application (Marangoni Júnior et al., 2019).
Traditionally, biopolymer films possess reduced mechanical resistance.
Therefore, to improve this aspect, the development of blends and the
incorporation of nanoparticles must be considered. The results of mechanical properties of kefiran films found in the literature are described
in Table 4.

Table 4
Mechanical properties of kefiran films.
Film material

Thickness (μm)

Tensile
strength
(MPa)

Elongation at
break (%)

Young's
Module
(MPa)

Conclusions

References

Kefiran (3%) + glycerol (20 and
30 %).

60

13 - 2

2.5 - 275

900 - 54

Coma et al. (2019)

Kefiran (2%) + starch (5%) +
nano zinc oxide (1%) +
glycerol (40 %) exposed to UV
radiation for 0, 1, 6 and 12 h.

110

6.7 – 8.2

155 - 31

31 – 35

Kefiran/CMC and EO by Satureja
Khuzestanica (0.0, 1.0, 1.5 and
2.0% v/v).

90, 113, 133
and 153

3.2 – 4.2

80 - 65

–

Kefiran/CMC and CuO and the
combination of CuO and EO
by Satureja Khuzestanica (0.0,
1.0, 1.5 and 2.0% v/v).
Kefiran/WPI/glycerol (6%, 2%
and 3.2 % w/w) with and
without the addition of
probiotics.
Kefiran (2%) with Al2O3
nanoparticles (1, 3 and 5% w/

w dry basis).

90 - 161

3.2 – 4.5
3.2 – 5.0

80 – 61
80 - 56

–

133 and 143

1.8 – 1.8

80 -105

–

The film with 30 % plasticizer showed a reduction in
Young's module and tensile strength followed by an
increase in elongation at break compared to the film
with 20% plasticizer.
Exposure to UV light increased the tensile strength and
the Young’s module of the films, consequently led to a
reduction in elongation at break and there was no
influence on elastic energy. These results were due to
the kefiran/starch interaction with the ZnO surface,
which resulted in an increase in the density of crosslinks by a free radical content mechanism.

The incorporation of EO led to increased tensile strength
and decreased elongation at break, attributed to the
formation of new hydrogen bonds between the hydroxyl
groups of kefiran/CMC with EO.
The tensile strength increased and the elongation at
break was reduced after the addition of CuO and EO,
showing good interaction between CuO and EO with
kefiran and CMC.
The kefiran/WPI films showed intermediate values in
the mechanical properties, compared to kefiran films
and WPI films. There was no influence of probiotics.

14.5, 15.5,
17.9 and 28.8

26 – 7.6

1.4 – 0.8

–

Moradi et al.
(2019)

Kefiran (2% w/v) and starch (5%
w/v) (50:50 v/v), added with
nano-ZnO (0, 1, 3, and 5% w/
w).

140, 130, 120

and 110

4.9 - 6.3

163 - 152

21 – 34

Kefiran/starch/TiO2 exposed to
UV-A radiation for 0, 1, 6 and
12 h.

100, 80, 80 and
80

3.5 – 4.0

129 - 87

33 – 25

Kefiran (2%) treated at 0 3, 6 and
9 kGy.

80, 80, 60 and
50

6.4 - 19

18 -10

–

Kefiran (2%) and chitosan (2%) in
proportions (100/0, 68/32,
50/50 and 32/68).

34, 33, 32 and
31

0.7 – 2.3

60 - 89

–

The elasticity module showed no difference between the
films. The tensile strength decreased as the
concentration of Al2O3 increased. The elongation at
break was greater for the film with 1% Al2O3 and
decreased as the concentration of 3 and 5 % of Al2O3
Tensile strength and young modulus increased, with an
increase in ZnO content of up to 3%, while elongation at
break decreased. The origin of these changes is the
interfacial interaction between the polymeric matrix
and ZnO.
As the time of radiation exposure increased, the tensile
strength increased, the elongation at break decreased,
resulting from the creation of cross-links formed by UVA.
The mechanical properties were improved after the

irradiation doses, one of the important reasons for
increasing the mechanical properties by γ irradiation
was the creation of crosslinking in the polymeric chain.
The evaluated properties improve with the
incorporation of chitosan, resulting from the
intermolecular forces of the chitosan biopolymer.

ShahabiGhahfarrokhi and
Babaei-Ghazvini
(2019)

Hasheminya et al.
(2019a)
Hasheminya et al.
(2019b)
Gagliarini et al.
(2019)

Babaei-Ghazvini
et al. (2018)

Goudarzi and
ShahabiGhahfarrokhi
(2018)
ShahabiGhahfarrokhi et al.
(2015)
Sabaghi et al.
(2015)

(continued on next page)

12


Carbohydrate Polymers 246 (2020) 116609

L. Marangoni Júnior, et al.

Table 4 (continued)
Film material

Thickness (μm)

Tensile
strength
(MPa)

Elongation at
break (%)

Young's
Module
(MPa)

Conclusions

References

Kefiran (2%) and nano cellulose
(0, 1, 2 and 3% dry basis).

–

6.4 – 8.1

18 - 252

382 - 70

ShahabiGhahfarrokhi et al.
(2015a)

Kefiran (2%) and nano ZnO (0, 1,
2 and 3% dry basis).

80, 70, 70 and
70

6.4 - 13

18 - 221

–

Kefiran (5% w/v) and WPI 5% (w/
v) (50:50 v/v), added nanoTiO2 (0, 1, 3, and 5% w/w).

74, 75, 75 and
76

6.5 – 3.5

84 - 334

105 - 42

Kefiran (5% w/v) and WPI 5% (w/
v) (50:50 v/v), added with
MMT and nano-TiO2 (0, 1, 3,
and 5% w/w).

̴ 74 (MMT) and
74, ̴ 91 (TiO2)

6.5–11
6.5 – 3.5

84 – 45
84 - 334

105 – 315
105 - 42

Kefiran 2% and starch 2% (100/0,
70/30, 50/50 and 30/70).

74, 62, 59 and
57

4.8 - 5.6

57 - 140

–

Kefiran (10 g/kg) with galactose,
glucose, sucrose, glycerol or
sorbitol (25 g/100 g kefiran).

23, 22, 25, 31,
22 and 22.

34 – 7.5

2.0 - 130

–

Kefiran (2%) with 0, 15, 25 and 35
% glycerol (w/w based on
kefiran weight).
Kefiran (2%) with 0, 15, 25 and 35
% oleic acid (w/w based on
kefiran weight).

58, 64, 62 and
67

11 – 5.0

40 - 162

–

The tensile strength was not significantly changed,
regardless of the concentration of nano cellulose. The
elongation at break of the films with NC was greater
than the control, however as the NC concentration
increased it tended to reduce. Young's module decreases
as the concentration of NC increases, attributed to the
plasticizing properties of mono and disaccharide
residues in the NC.
Higher ZnO concentrations increased the tensile
strength and elongation at break, due to the interfacial
interaction between the loads and the matrix.
TiO2 decreased the tensile strength and Young's
modulus and increased the elongation at break,
attributed to the decrease in the crosslinking in the film
matrix, to the increase in the mobility of the chains and
to the preventive effect of nanoparticles in the
hardening by deformation of the polymer chains.
MMT increased the tensile strength and Young's
modulus and reduced the elongation at break. TiO2
decreased the tensile strength and Young's modulus and
increased the elongation at break. This was due to
differences in structure and the number of active
hydroxyl groups.
The tensile strength values increased first with the
addition of starch, then decreased with the increase in
the starch content, due to the formation of intermolecular hydrogen bonds between the two main

components and the plasticizer. The elongation values
at break were greater in the proportion of 50/50, that is,
the flexibility of the film was better in this proportion.
The addition of plasticizers to the kefiran films reduced
the tensile strength and the modulus of elasticity, while
the elongation at break increased, as it interfered with
the association of the polymeric chain, facilitating the
sliding and increasing the flexibility of the film.
Glycerol-free films showed less tensile strength and
greater elongation at break.

62, 75, 71 and
79

6.2 – 3.3

100 - 40

–

Ghasemlou et al.
(2011c)

Kefiran (10 g/kg) with 0 and 25 %
glycerol/100 g kefiran.

21.4 and 21.9

41 - 15

2.7 – 117

–

The elongation decreased as the OA concentration
increased. The tensile strength of films with AO was
lower than the control, but the difference in
concentration had no influence on this property,
probably because it weakened inter-molecular
interactions.
The plasticizer reduced the tensile strength and
increased the elongation at break. Although the
plasticizer improves the flexibility of the film, the high
moisture content influenced the mobility of the polymer
chains.

The mechanical properties of the films depend on the characteristics
of each isolated material, as well as on the composition of ingredients
used in the development of these films. Kefiran blending with biopolymers such as starch, CMC, WPI and chitosan, as well as the incorporation of nanoparticles (MMT, ZnO, CuO and TiO2) and exposure
to radiation doses were efficient to improve the mechanical properties.
Therefore, the incorporation of additives in kefiran films must be taken
into account. For example, there is an enormous number of nanomaterials that have not been explored yet, for example, functionalized
carbonaceous materials and other silicates on a nanometer scale.

ShahabiGhahfarrokhi et al.
(2015b)
Zolfi et al. (2014a)

Zolfi et al. (2014b)

Motedayen et al.
(2013)

Piermaria et al.
(2011)

Ghasemlou et al.
(2011d)

Piermaria et al.
(2009)

In addition, the evaluation of distinct plasticizers on the moisture
barrier was reported, as well as the incorporation of essential oils and
nanoparticles. In general, the incorporation of these ingredients added
more moisture barrier to kefiran films. According to Goudarzi and
Shahabi-Ghahfarrokhi (2018), the application of UV-A radiation in
kefiran/starch/TiO2 films reduces the size of the biopolymer chains,
forming possible oligo, di and monosaccharide, consequently resulting
in a compact structure which will make the moisture path more tenuous, resulting in a more significant barrier (Fig. 7).
In addition to the water vapor barrier properties, identifying the
light barrier of the films based on biopolymers is essential, since the
incidence of light on packaged foods can trigger degradation reactions
of macro and micro molecules in food. Light absorption spectra of
Kefiran/starch and ZnO films exposed to UV radiation for 0, 1, 6 and 12
h have been reported by Shahabi-Ghahfarrokhi and Babaei-Ghazvini
(2019). A strong absorption peak at 364 nm was clearly observed in all
samples, indicating protection in the UV region. The light transmission
was significantly lower in kefiran/CMC films added with EO, because of

7.8. Barrier properties
Predominantly, biopolymer films present a substantial barrier to
gases, however the most substantial difficulty in employing these materials as food packaging is their deficiency in the moisture barrier.
Table 5 presents the results of the water vapor permeability rate of
kefiran-based films, as well as blends of kefiran with other biopolymers.
13


Carbohydrate Polymers 246 (2020) 116609

L. Marangoni Júnior, et al.

Table 5
Water vapor permeability (WVP) of kefiran-based films.
Film material

Thickness (μm)

WVP
−10

Kefiran (3%) + glycerol (0, 10,
20, 30 %).
Kefiran/starch/ZnO exposed to
UV radiation for 0, 1, 6 and
12 h.

60

Kefiran/CMC and EO by Satureja

Khuzestanica (0.0, 1.0, 1.5
and 2.0% v/v).

90, 113, 133 and
153

5.75, 4.63, 3.74 and
3.04 × 10−7 (g.m/
m2.Pa.h)

Kefiran/CMC and CuO and the
combination of CuO and EO
by Satureja Khuzestanica (0.0,
1.0, 1.5 and 2.0% v/v).
Kefiran (2%) with Al2O3
nanoparticles (1, 3 and 5%
w/w dry basis).
Kefiran/starch/TiO2 exposed to
UV-A radiation for 0, 1, 6 and
12 h.
Kefiran (2% w/v) and starch (5%
w/v) and (50:50 v/v, added
with nano-ZnO (0, 1, 3, and
5% w/w).
Kefiran (2%) treated at 0 3, 6 and
9 kGy.

90 to 161

5.75 to 107 × 10−7

(g.m/m2.Pa.h)

14.5, 15.5, 17.9
and 28.8

5.83, 3.63, 2.88 and
4.87 × 10−10 (g/m s
Pa)
1.59, 1.31, 1.24 and
1.22 × 10−10 (g/m s
Pa)
̴ 3.10 to 2.00 × 10−10
(g/m s Pa)

110

100, 80, 80 and
80
140, 130, 120 and
110
80, 80, 60 and 50

1.30 to 3.30 × 10
(g/m s Pa)
2.47, 2.38, 2.20 and
2.08 × 10−10 (g/m s
Pa)

2.19, 2.55, 1.70 and
2.00 × 10−10 (g/m s

Pa)
7.94, 7.83, 4.70 and
3.52 × 10−10 (g/m s
Pa)
2.19, 1.73, 1.83 and
2.37 × 10−10 (g/m s
Pa)

Conclusions

References

Water vapor permeability increased linearly with increasing
glycerol concentration.
The exposure to UV radiation for up to 12 h, significantly
decreased WVP, due to the better distribution of ZnO in the
polymeric matrix. In addition to the interactions between nonaggregated biopolymers and ZnO, which lead to a decrease in the
hydrophilic character of biopolymers.
The increase in the EO concentration resulted in an increase in the
thickness and a reduction in the WVP of the films. In addition, EO
contains monoterpene hydrocarbons, as a hydrophobic phase,
which led to the discontinuity of the hydrophilic phase of kefiran/
CMC.
The addition of CuO and EO reduced the WVP of the films. The
film containing 2% CuO and 2% EO showed the lowest WVP,
resulting from the uniform distribution of CuO in the polymeric
matrix, filling the cavities, restricting the diffusion of water vapor.
WVP decreased for films containing Al2O3. However, there was an
increase as a consequence of the increase in nanoparticles,
however, it was still smaller than that of the control film.

Exposure to radiation reduced the permeability of the films. Result
of the improvement of the hydrophobic properties in the
biocomposite with the use of UV-A.
The WVP of the films was lower with the addition of ZnO. It
appears that the formation of hydrogen bonds between the ZnO
surface and the biopolymer matrix causes the formation of a
coherent network that reduces WVP.
Doses above 6kGy reduced the WVP of the films, due to the
increase in the crystalline region induced by irradiation.

Coma et al. (2019)

Sabaghi et al. (2015)

Shahabi-Ghahfarrokhi
and Babaei-Ghazvini
(2019)
Hasheminya et al.
(2019a)

Hasheminya et al.
(2019b)
Moradi et al. (2019)
Goudarzi and ShahabiGhahfarrokhi (2018)
Babaei-Ghazvini et al.
(2018)
Shahabi-Ghahfarrokhi
et al. (2015)

Kefiran (2%) and chitosan (2%) in

proportions (100/0, 68/32,
50/50 and 32/68).
Kefiran (2%) and nano cellulose
(0, 1, 2 and 3% dry basis).

34, 33, 32 and 31

Kefiran (2%) and nano ZnO (0, 1,
2 and 3% dry basis).

80, 70, 70 and 70

2.19, 2.34, 1.83 and
1.81 × 10−10 (g/m s
Pa)

Kefiran (5% w/v) and WPI (5%
w/v) (50:50 v/v), added
nano-TiO2 (0, 1, 3, and 5%
w/w).
Kefiran (5% w/v) and WPI (5%
w/v) (50:50 v/v), added with
MMT and nano-TiO2 (0, 1, 3,
and 5% w/w).
Kefiran 2% and starch 2% (100/0,
70/30, 50/50 and 30/70).

74, 75, 75 and 76

3.39, 2.83, 2.80 and

2.87 × 10−11 (g/m s
Pa)

With the increase of the chitosan content, the WVP decreased, due
to the residual hydrophobic acetyl group of the chitosan, which
acts as a barrier to the transport of water vapor.
The WVP of kefiran/NC nanocomposites decreased with the
increase in the NC content (up to 2%), attributed to the increase in
the tortuosity of the polymer and the cohesion of the polymeric
matrix and the nano cellulose. The increase in WVP of films with
3% NC was due to the heterogeneous distribution of NC in the
polymer matrix.
The WVP decreases after a concentration of 2 and 3% of ZnO,
indicating a positive effect on the polymeric matrix, attributed to
the formation of hydrogen bonds with the oxygen atoms of Zn,
consequently a reduction in the diffusion of water molecules.
TiO2 decreases the WVP of the films, as it showed a homogeneous
distribution in the polymer matrix, besides adding hydrophobicity
to the film.

74, ̴ 91 (TiO2) and
̴ 74 (MMT)

3.39, 1.51 (5% MMT)
and 2.87 (5% TiO2) x
10−11 (g/m s Pa)

A decrease in the WVP values of the films containing MMT and
TiO2 was observed, due to its good dispersion in the film, making a
tortuous way to diffuse molecules.

Zolfi et al. (2014b)

74, 62, 59 and 57

3.13, 2.95, 2.78 and
3.88 × 10−11 (g/m s
Pa)

Motedayen et al.
(2013)

23, 22, 25, 31, 22
and 22.

̴ 7.50 to 3.00 × 10−11
(g/m s Pa)

58, 69, 72 and 74

4.95, 4.11, 3.61 and
3.67 × 10−11 (g/m s
Pa)
4.95, 5.04, 5.55 and
5.88 × 10−11 (g/m s
Pa)
̴ 5.50 to 3.80 × 10−11
(g/m s Pa)

The WVP of the films decreased with the increase in the amounts

of starch (30 % and 50 %) and increased with the additional
addition of starch (70 %). The interactions between kefiran and
starch molecules have the effect of preventing water molecules
from diffusing through films. However, in a higher concentration
of starch, the dispersion is not sufficient in the matrix to block
water vapor.
The addition of plasticizers to kefiran films improved the water
vapor barrier. Glucose was the most effective in reducing WVP
compared to non-plasticized films.
The plasticized film with sorbitol exhibited lower WVP values. The
cyclic conformation of the sorbitol molecules was responsible for
decreasing the permeability.
The increase in the concentration of glycerol resulted in an
increase in WVP, attributed to the molecular mobility that glycerol
added to the film and, consequently, greater free volume.
As the concentration of OA increased, the WVP of the films
decreased, therefore, the presence of a hydrophobic phase,
introduces discontinuities in the hydrophilic phase, thus
decreasing the WVP.
The addition of plasticizer reduced the WVP of the films,
attributed to the development of a more compact structure in
plasticized films.

Kefiran (10 g/kg) with galactose,
glucose, sucrose, glycerol or
sorbitol (25 g/100 g).
Kefiran (2%) with 0, 15, 25 and
35 % sorbitol (w/w based on
kefiran weight).
Kefiran (2%) with 0, 15, 25 and

35 % glycerol (w/w based on
kefiran weight).
Kefiran (2%) with 0, 15, 25 and
35 % oleic acid (w/w based
on kefiran weight).
Kefiran (10 g/kg) with 0 and 25 %
glycerol/100 g kefiran.

–

58, 64, 62 and 67
62, 75, 71 and 79

21.4 and 21.9

5.73 and 4.09 × 10−11
(g/m s Pa)

14

Shahabi-Ghahfarrokhi
et al. (2015a)

Shahabi-Ghahfarrokhi
et al. (2015b)
Zolfi et al. (2014a)

Piermaria et al. (2011)
Ghasemlou et al.
(2011a)

Ghasemlou et al.
(2011d)
Ghasemlou et al.
(2011c)
Piermaria et al. (2009)


Carbohydrate Polymers 246 (2020) 116609

L. Marangoni Júnior, et al.

Fig. 7. a) scheme of starch modification and kefiran structures during UV-A radiation, b) macroscopic changes of biopolymers during UV-A radiation (Goudarzi &
Shahabi-Ghahfarrokhi, 2018). Adapted with permission from Elsevier, Copyright (2018).

hydrogen ion of the solution (Sabaghi et al., 2015). The incorporation
of active compounds in films based on biopolymers may incorporate
antioxidant activity in these materials. Hasheminya et al. (2019a) has
incorporated Satureja Khuzestanica EO into kefiran/CMC films. The increase in EO concentration resulted in an increase in antioxidant activity and phenolic compounds, the main compound present in EO was
carvacrol. The incorporation of natural antioxidants in biopolymer
films is being extensively explored in starch films, gelatin and other
biopolymers. This aspect still requires more attention and investigation
for kefiran-based films and its blends.
7.10. Antimicrobian activity
Studies with the incorporation of antimicrobial substances in films
based on biopolymers are increasing, since they include more functions
to the material. Kefiran/CMC films were incorporated with EO by
Satureja Khuzestanica. The results showed that with the increase of the
EO concentration, the antimicrobial activity was intensified for the
bacteria Staphylococcus aureus and Escherichia coli, being more effective
for S. aureus. Antimicrobial activity was attributed to the presence of

carvacrol, eugenol and thymol in EO (Hasheminya et al., 2019a). Similar behavior was observed with kefiran/CMC films incorporated with
CuO nanoparticles and the combination of CuO nanoparticles with EO
Satureja Khuzestanica. However, the decrease in E. coli count was more
expressive (Hasheminya et al., 2019b).

Fig. 8. a) ZnO-Kefiran film, (b) UV visible absorption spectra of kefiran and
ZnO-kefiran film (Shahabi-Ghahfarrokhi et al., 2015b). Adapted with permission from Elsevier, Copyright (2015).

the appropriate dispersion of the lipid droplets in the film (Hasheminya
et al., 2019a). A decrease in the light transmission of the films in the UV
and visible bands was also observed as the CuO concentration increased
in the Kefiran/CMC films (Hasheminya et al., 2019b).
The incorporation of ZnO in kefiran films resulted in films with
excellent transparent and visual properties (Fig. 8a). In addition, ZnO
concentrations greater than 1% induced UV filtration in the biopolymer, adding light barrier (Fig. 8b) (Shahabi-Ghahfarrokhi et al.,
2015b). Similar results were experienced for kefiran/starch films added
with ZnO (Babaei-Ghazvini et al., 2018).

8. Conclusions and outlook
The main objective of this review was to highlight the state-of-theart advances in the preparation of kefiran-based films. This EPS possesses excellent biological properties that have been explored for over
thirty years. However, its use as a polymeric film became more prominent only in the last decade, indicating there is still a long way to be
explored in this area. Obtaining films of pure kefiran or blended with
other biopolymers is reasonably simple, and the materials produced
exhibit satisfactory properties. The inclusion of additives (inorganic
nanoparticles and essential oils) has substantially improved the mechanical and barrier properties of the films, in addition to maximizing

7.9. Antioxidant activity and total phenolic content
Kefiran/chitosan films were evaluated for their antioxidant activity.
The results showed that the antioxidant activity increased as the proportion of chitosan increased. This result was attributed to the chitosanfree amino group reacting with free-radicals to form stable macromolecular radicals. Hence, forming ammonium groups absorbing the
15

Carbohydrate Polymers 246 (2020) 116609

L. Marangoni Júnior, et al.

the antimicrobial and antioxidant characteristics.
However, there are still numerous nanomaterials and bioactive
molecules that can contribute to obtaining even higher properties. For
example, considerable property improvements in starch films, chitosan,
etc. have been reported using different types of silicates or carbonaceous materials on a nanometer scale. These examples have yet been
unexplored for kefiran films. Moreover, considering the composite films
already produced, investigations about improving the compatibility
between the load and matrix could also contribute to improving the
results.
Together with this, the present research identified that the potential
for producing food coatings from kefiran is yet unconsolidated. This
area could be a possible research line in growth in the next decade.
Nevertheless, despite the fact that innumerable human health properties have been attributed to pure kefiran, information about the biological activities of its films, such as studies of cytotoxicity, anti-inflammatory and anticancer activities are still scarce. Hence, it is
believed that directing research to this area could expand the potential
for application of films, for example, in the medical and pharmaceutical
fields. Finally, the evaluation of production processes on a larger scale,
such as continuous casting and extrusion; as well as biodegradation
studies are equally necessary for the consolidation of this material applicability.

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CRediT authorship contribution statement
Luís Marangoni Júnior: Conceptualization, Data curation, Formal
analysis, Investigation, Writing - original draft, Writing - review &
editing. Roniérik Pioli Vieira: Conceptualization, Data curation,

Formal analysis, Investigation, Writing - original draft, Writing - review
& editing. Carlos Alberto Rodrigues Anjos: Supervision, Writing review & editing.
Declaration of Competing Interest
The authors declare no conflicts of interest.
Acknowledgements
The authors would like to thank the Council for Scientific and
Technological Development (CNPq) for the PhD scholarships. This
study was partly funded by the Coordination for the Improvement of
Higher Education Personnel – Brazil (CAPES) – Financial Code 001.
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