Carbohydrate Polymers 223 (2019) 115100

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

Kefiran, a branched polysaccharide: Preparation, properties and T

applications: A review
⁎
Zahra Moradia, Nastaran Kalanpourb,
a
Department of Food Science and Technology, Faculty of Agriculture, Urmia University, Urmia, 5756151818, Iran
b
Department of Nanochemistry, Nanotechnology Research Centre, Urmia University, Urmia, 5756151818, Iran

A R T I C LE I N FO A B S T R A C T

Keywords: Considering harmful effects of the environmental pollution and increased concerns about the synthetic polymers,
Kefiran kefiran has been introduced as a biodegradable polymer due to its nontoxicity. This edible biopolymer and its
Biodegradable structural derivations play specific roles in a large number of applications including scaffolds, the reserve ma-
Reserve materials terials, and the encapsulation for drug delivery, thanks to its amazing properties such as the protective features,
Non toxicity
the bifidogenic effects and the antimicrobial activity against some microorganisms. The aim of the present
review was to focus on the methods of producing kefiran, its properties and finally its applications, bringing
significant changes in various fields.

1. Introduction Given the amazing features of kefiran including antimicrobial,

The renewable resources are essential in the fields of medicine,
environment, food, economy etc. On the contrary, the non-renewable
resources lead to serious environmental problems, due to their non-
biodegradation. Nowadays, the use of the non-renewable resources is
limited, due to the increasing attention to the renewable resources and
their applications in various fields (Kaplan, 1998). Polysaccharides are
widely used in various industries as stabilizers, galling agents, thick-
eners and binders, attracting an increasing attention due to their ben-
eficial effects.
One of the most interesting polysaccharides is kefiran, an extra-
cellular polysaccharide with many hydroxyl groups, obtained from the
microorganisms present in kefir grains and produced by several
Lactobacillus species including: L. kefiranofaciens, L. kefirgranum, L.
parakefir, L. kefir and L. delbrueckii subsp. bulgaricus (Micheli et al.,
1999). This edible biopolymer, a water-soluble branched glucogalactan
with roughly equal amounts of D-glucose and D-galactose in a chain
sequence, has recently attracted a lot of attentions (Mukai et al., 1990),
due to its unique features such as rheological behavior (Rimada &
Abraham, 2006), biodegradability (Ghasemlou et al., 2011b), bio-
compatibility (Shahabi-Ghahfarrokhi et al., 2015b), safety (Micheli
et al., 1999; Piermaría et al., 2016), emulsifier effect, stabilizing effect
(Botelho et al., 2014), resistance against hydrolysis (Micheli et al.,
1999), barrier and mechanical properties, and water vapor perme-
ability.
wound-healing (Rodrigues, Gaudino Caputo, Tavares Carvalho,
Evangelista, & Schneedorf, 2005), antibacterial, antitumor, anti-in-
flammatory, antioxidant and biocidal properties and some highlighted
capabilities such as the modulation of gut immune-system, the reduc-
tion of hypertension-induced high blood pressure, the protection of
epithelial cells against microbial toxins (Blandón et al., 2018), and
hypotensive activities (Maeda et al., 2004a), it is seriously used in
medicine. In addition, as a film-forming agent, kefiran has good me-
chanical and barrier properties in the satisfactory production of the
films in food packaging and it can also be used as a food-grade additive
(Piermaria, de la Canal, & Abraham, 2008), gelling and texturing agent.
However, the barrier properties are mostly limited to high permeability
to water. In order to overcome water permeability properties, kefiran
films have been modified with hydrophobic compounds such as fatty
acids (Ghasemlou et al., 2011a).
Considering the fact that the advancement of nanotechnology and
discovery of new nanomaterials have led to wonderful changes in
various fields, biomaterials have been used in nanotechnological fields
as well. Hence, kefiran nanocomposites and kefiran nanofibers could be
effectively used in various scientific fields (Naghibzadeh & Adabi, 2014;
Sreenivasan et al., 2010).
The main objectives of the present review were as follow: to classify
the methods used for obtaining kefiran, prepare the kefiran nano-
composites and nanofibers, discuss some characteristics of kefiran in-
cluding thermal and rheological properties and review the approaches

⁎
Corresponding author.
E-mail addresses: Moradiz1369@gmail.com (Z. Moradi), n.kalanpour@yahoo.com (N. Kalanpour).

https://doi.org/10.1016/j.carbpol.2019.115100
Received 19 March 2019; Received in revised form 13 July 2019; Accepted 15 July 2019
Available online 19 July 2019
0144-8617/ © 2019 Elsevier Ltd. All rights reserved.
Z. Moradi and N. Kalanpour
of improving some of its properties, and investigate some of the recent
works done on the applications of kefiran in different fields.
2. Preparation of kefiran
Kefiran can be obtained by the extraction from kefir grains cultured
in the milk. However, the extraction procedure in an industrial scale is
difficult and low-yielded. The mass production of this biopolymer has
been widely studied.
2.1. Operational conditions of kefiran preparation
2.1.1. Type of microorganism
Several efforts have been made to produce kefiran on an industrial
scale using Lactobacillus species pure culture as L. kefr (Kandler &
Kunath, 1983), KPB-167B (Yokoi et al., 1991), L. keirgranum and L.
parakefir (Takizawa et al., 1994), among which Lactobacillus kefir-
anofaciens has attracted more attention. Coculture of L. kefiranofaciens
with Saccharomyces cerevisiae has widely been studied, according to
results of which it can significantly enhance kefiran production, in
contrast to the pure cultures, Since L. kefiranofaciens is a homo-fer-
mentative lactic acid bacterium, producing lactic acid during the lac-
tose fermentation. Accumulated lactic acid inhibits the cell growth of L.
kefiranofaciens, but the yeast can consume lactic acid and overcome the
inhibition (2007, Cheirsilp & Radchabut, 2011; Cheirsilp et al., 2003;
Tada et al., 2007).
2.1.2. Fermentation conditions
2.1.2.1. pH. Optimizing pH in medium is an important parameter
affecting the cell growth and kefiran production by L. kefiranofaciens.
It has been reported that high lactic acid accumulation led to decreased
pH in fermentation broth and inhibited the cell growth and kefiran
production. In many researches, the maximum kefiran production were
achieved in pH 5 (Cheirsilp & Radchabut, 2011; Cheirsilp et al., 2001;
Wang & Bi, 2008; Yeesang et al., 2008; Yokoi & Watanabe, 1992).
2.1.2.2. Temperature. The highest kefiran concentration was in
temperature 30 °C. Increasing temperature led to a decrease in kefiran
production (Yokoi & Watanabe, 1992).
2.1.3. Composition of the culture media
Developing suitable medium (in terms of components availability
and cost) in shorter production time is the main challenge of producing
kefiran in high concentration. The high cost of kefiran production is
mainly related to carbon and nitrogen sources. For this aim, some re-
searchers have tested different carbon and nitrogen sources to choose
the most suitable nutrients for kefiran production by L. kefiranofaciens,
the results of which are summarized in Table 1.
3. Properties of kefiran
3.1. General properties of kefiran
Kefiran is a water-soluble exopolysaccharide, mainly produced by L.
kefiranofaciens (Ahmed et al., 2013a; Kim et al., 2015; Zajšek et al.,
2011). More than half of the dry kefir grain weight consists of kefiran
Table 1
Effect of carbon and nitrogen sources on kefiran production in optimal conditions.
Carbon source Nitrogen source
Maltose Yeast extract
Lactose Yeast extract
Molasses Spent yeast cells hydrolysate
Whey lactose Spent yeast cell hydrolysate
Carbohydrate Polymers 223 (2019) 115100
(Abraham & De Antoni, 1999; Garrote et al., 2001). While, Kooiman
(1968), and la Riviére et al. (1967) reported that kefiran had optical
rotation of [α]D =+68°, Micheli et al. (1999), and Maeda et al.
(2004b) reported [α]D=+64°. However, Mukai et al. (1990) reported
[α]D= +54°. Different Molecular weights have been reported for ke-
firan: 106 Da (Mukai et al., 1990, 1991), 1.35 × 106 Da (Ghasemlou
et al., 2012), 107Da (Piermaria et al., 2008), 5.5 × 104 Da (Ahmed
et al., 2013b), and 6.71 × 105 (Exarhopoulos et al., 2018a). Kefiran
formed a gel in the solutions containing ethanol, and the gel firmness of
kefiran was increased by ethanol concentration (Mukai et al., 1991).
The analysis of the kefiran chemical structure demonstrated that it
is a branched hexa- or hepta-saccharide repeating units, that itself is
composed of a regular pentasaccharide unit to which one or two sugar
residues are randomly attached (Fig. 1) (Kooiman, 1968; Micheli et al.,
1999; Mukai et al., 1990). According to the results of the structural
evaluation of kefiran, it had a backbone composed of (1 → 6)-linked
Glc, (1 → 3)-linked Gal, (1 → 4)-linked Gal, (1 → 4)-linked Glc and (1
→ 2, 6)-linked Gal, with branch attached to O-2 of Gal residues and Glc
residues situated in the structure end (Ahmed et al., 2013b; Ghasemlou
et al., 2012; Mukai et al., 1991). Due to the type of linkages that this
polysaccharide has, it cannot be hydrolyzed by digestive enzymes of the
human gastrointestinal tract but it can be fermented by colonic bacteria
as was described by several authors, among them Serafini et al (2014).
The Infrared radiation (IR) analysis for identifying the reactive
functional groups in kefiran is very important, since these functional
groups make it more flexible to many modifications. The IR spectra of
kefiran (Fig. 2) displayed an intense peak around 3430 cm−1, corre-
sponded to the hydroxyl groups. The region of 2930 cm−1 indicated the
vibration of CeH in sugar chain, which can be attributed to methyl and
methylene groups. The peak at 1700 cm−1 indicated the fluctuations of
OeH links. The peak of 1400 cm−1 was associated to CH2 and OH
groups. The stretches of CeOeC, CeO of alcohol groups in poly-
saccharide were indicated in the area of 1000–1200 cm−1. The existing
peak at 900 cm−1 showed β-configuration and also the presence of
glucose and galactose (Radhouani et al., 2018b). The first study of X-ray
diffractogram (XRD) to determine the crystallinity of kefiran was con-
ducted by Exarhopoulos et al. (2018a); they proved that kefiran in
powder state had semicrystalline nature with a sharp peak at around
2θ = 20.9°, and calculated the crystallinity percentage equal to
Xc = 27.0% (Fig. 3).
3.2. Thermal properties
Beside other attributes, thermal properties largely affect the ap-
plicability of exopolysaccharides in industry and commerce. Many
foods may be subjected to rather intensive heat treatments when they
are manufactured or prepared for consumption (Ahmed et al., 2013a).
As for the thermal properties of exopolysaccharides, the heat absorption
and emission are accompanied with the physical change by deformation
of polymer structure or the melting of crystalline polysaccharides.
Wang et al. (2008) evaluated the physicochemical properties of
kefiran exopolysaccharide produced by L. kefiranofaciens ZW3 isolated
from Tibet kefir and compared the results of thermal analysis of exo-
polysaccharide with xanthan gum and guar gum. They showed that
melting of ZW3 EPS took place at about 93.38 °C, lower than xanthan
gum (153.4 °C) and guar gum (490.11 °C). The endothermic enthalpy
Time (h) Kefiran (gL−1) References
72 2.25 (Wang & Bi, 2008)
60 1.29 (Dailin et al., 2015)
96 1.286 (Suksawang et al., 2016)
168 1.693 (Cheirsilp et al., 2018)
2
Z. Moradi and N. Kalanpour Carbohydrate Polymers 223 (2019) 115100

Fig. 1. Proposed chemical structure of kefiran (Micheli et al., 1999).

change (ΔH) required to melt 1 g of ZW3 EPS, xanthan and guar gums
were 249.7, 93.2 and 192.9, respectively.
Chen et al. (2015) reported that the melting point of 131.46 °C for
kefiran obtained from Tibetan kefir grains was higher than that of the
kefiran obtained from L. kefiranofaciens ZW3. They also reported that
the enthalpy change of kefiran used in their research was 209.6 J.
Ahmed et al. (2013a) carried out the thermogravimetric (TGA)
analysis for ZW3 EPS. Xanthan and locust gums were used as the re-
ference material (Fig. 4). The initial weight loss of polymer between 40
and 90 °C might be attributed to the evaporation of moisture. The de-
cline in weights above 90 °C was ascribed to the degradation of the
sample. The onset of decomposition occurred at 261.4 °C and the re-
corded mass loss was 10%. The polymer weight loss decreased dra-
matically around 300 °C. The temperatures of degradation for xanthan
and locust gums were 282.65 °C and 278.46 °C, respectively. The dif-
ferent thermogravimetric behaviors of polymers could be attributed to
their structure. The nature of ZW3 EPS was glucogalactan, while locust
gum was a linear polysaccharides, composed of mannose and galactose
Fig. 3. XRD diffractogram of kefiran (Exarhopoulos et al., 2018a).
(Dakia et al., 2008), Xanthan gum was a hetero-polysaccharide con-
taining D-glucose, D-mannose and D-glucoronic acid (García-Ochoa
et al., 2000). Therefore, the thermal stability of ZW3 was higher than
those of both xanthan and locust gums.

Fig. 2. Infrared spectrum of kefiran (Radhouani et al., 2018b).

3
Z. Moradi and N. Kalanpour
Fig. 4. TGA curves of ZW3 EPS, xanthan gum, locust gum. (A) ZW3 EPS, (B)
xanthan (C) locust gum (Ahmed et al., 2013a).
3.3. Rheological properties
For commercial manufacture of some products such as dairy pro-
ducts, the texture modification is very important. Polysaccharides play
an important role in improving the rheology properties of fermented
products. In addition, they can bind water as physical stabilizers and
limit syneresis. The polysaccharides from different sources used in food
systems are alginate, carrageenan, cellulose, pectins, starch (Duboc &
Mollet, 2001), xanthan from Xanthomonas campestris (García-Ochoa
et al., 2000), gellan from Pseudomonas elodea (Matsukawa & Watanabe,
2007) etc. In some countries, the amount of stabilizers used in food
products must not exceed a certain amount and the demand for pro-
ducts with fewer additives is increasing. The physical properties of
above polymers are not suitable for all applications; therefore, there is a
request for novel material with the functional properties to enhance the
rheological characteristic. The foods formulated with thickeners, sta-
bilizers etc. must be developed alongside the consumer demands for all-
natural, healthy products. In this respect, the production of new poly-
saccharides by food grade bacteria may be considered (Duboc & Mollet,
2001; Laws & Marshall, 2001). Recently, hydrocolloid of kefiran has
been considered as a potential texture modifier (Rimada & Abraham,
2006).
4
Carbohydrate Polymers 223 (2019) 115100
Viscosity and elasticity are two important rheological character-
istics of kefiran (Sebastiani & Zelger, 1998). Micheli et al. (1999), and
Piermaria et al. (2008) reported that the intrinsic viscosity of kefiran
determined at 25 °C using Huggins and Kramer was 6.0 dl/g and
5.95 dl/g, respectively. However, in previous studies of Pintado et al.
(1996), and Mukai et al. (1990), lower values were reported for the
intrinsic viscosity of kefiran, probably due to the difference in the de-
gree of polymerization of the polysaccharide used in each research,
because molar mass (M), stiffness and radius of gyration (Rg) were
factors effective on the intrinsic viscosity. Kefiran had intrinsic viscosity
lower than some polysaccharides such as locus bean gum (Pintado
et al., 1996) but it had intrinsic viscosity higher than some dextrans
(Armstrong et al., 2004).
Kefiran had a Newtonian behavior at low concentrations (up to 4 g/
l), while it was pseudoplastic at higher concentrations. At a defined
shear rate, with the increase of the biopolymer concentration, apparent
viscosity increased (Piermaria et al., 2008).
Storage modulus or elastic modulus (G') and loss modulus or viscous
modulus (G") are both functions of frequency, helping to understand the
structure of polymer solutions or gels. Piermaria et al. (2008) reported
that the kefiran solutions at low frequency had the loss modulus greater
than the storage modulus. However, at higher frequencies, G' overcame
G" and the inter-chain entanglements did not have enough time to slide
and behaved like a gel. They also reported that the rheological behavior
of kefiran solution from a liquid-like system to a cryogel form can be
changed after freeze–thaw treatment of the solution. Cryogels inter-
connected macropores and could diffuse nano- and microparticles,
suitable carriers for biomolecules and cells. The unique structure and
properties of cryogels made them attractive material for many appli-
cations in biomedical and biotechnological (Lozinsky et al., 2003). High
MW (Molecular weight) of kefiran may be one of the chemical char-
acteristics for forming cryogels. The storage modulus in cryogels was
very higher than the solution. In case of cryogels, G' and G" were almost
independent of frequency. A three dimensional sponge-like structure
was observed for kefiran cryogel, confirming its high water-holding
capacity. At temperatures about 37 °C, both moduli of kefiran cryogels
fell, so it can be melted at mouth temperature and is suitable for many
food formulations.
The addition of kefiran had a great effect on the rheological prop-
erties of whey protein isolate (WPI) gel.
Kazazi et al. (2017) reported that kefiran at low concentrations (0-
0.08 w/v) could increase G' of WPI gel about 60%. It also decreased the
gelling point and gelation pH of WPI gel. The mechanical resistance of
mixed gel was higher than that of WPI-only.
Kefiran gel could be formed at the presence of sugars (sucrose and
fructose) and these sugars had different effects on the rheological at-
tributes of kefiran gel. The sugars could hold water, modifying the
water-polymer and polymer-polymer interactions. Sucrose and fructose
actually acted like plasticizer, because they could easily make -inter-
changeable linkages (Zavala et al., 2015).
In a study done by Dimitreli et al. (2016), the impact of adding
different supplements (kefiran, Whey Proteins Concentrates (WPC) and
Sodium Caseinates (SCN)) and temperature (0–25 °C) on the rheological
virtues of glucono-δ-lactone induced milk gels were evaluated and
compared. The elasticity of the samples made with SCN addition in-
creased when temperature increased; however, kefiran and WPC had
opposite results, attributed to hydrophilic and hydrophobic properties
of supplements.
Many food processes often involve a component of large extensional
deformation, about which shear flow results can’t provide enough in-
formation. The extensional rheology of kefiran solution at 2% wt. was
evaluated by Piermaria et al. (2016) and compared to galactomannans
(locust bean and guar gums) and methylcellulose (Fig. 5).
The polymer filament passes through four phases including rest,
stretch, relaxation and breaking. The filament evolution basically is
controlled by surface tension and viscous/elastic forces. Viscous/elastic
Z. Moradi and N. Kalanpour
Fig. 5. Evolution of normalized filament diameter (Dmid/D0) along extensional
time for 2% wt. gum solutions. Kefiran (∎), methylcellulose (●), locust bean
gum (▲) and guar gum (▼) (Piermaría et al., 2016).
forces acts as stabilizing agent and the other as destabilizing one.
According Fig. 5 the shape of the filament diameter vs. extensional
time curve for kefiran was very different than three other gums studied,
showing that kefiran did not have significant extensional properties in
concentration 2% wt. and approached a Newtonian fluid behavior;
whereas methylcellulose showed the highest breakup time, confirming
that it was more elastic with significant extensional flow.
4. Applications of kefiran
In the recent years, due to the attractive characteristics of kefiran, a
wide variety of applications in different fields have been reported for it.
In food applications, due to its texture and gelling feature, kefiran is
used to prepare biopolymer films as a biodegradable food packaging
material (Bodea et al., 2016; Ghasemlou et al., 2011; Motedayen et al.,
2013). Since the use of scaffolds containing high porosity with inter-
calated pores is of critical importance in good cellular penetration and
cellular diffusion, kefiran was used as proper scaffolds in tissue en-
gineering (Montesanto et al., 2016). Some researchers showed that
kefiran was useful in biological and medical process as well (Jenab
et al., 2015; Maeda, Zhu, Omura, et al., 2004a).
4.1. Food industry
Kefiran has many distinctive functional properties. This natural
polysaccharide was sufficiently used as food additives (Rimada &
Abraham, 2006). In addition, it was able to form gels at low tempera-
tures, so it was used as gelling agent in the food applications (Zavala
et al., 2015). Besides, the healing properties of kefiran such as anti-
fungal (Cevikbas et al., 1994), antibacterial (Rodrigues et al., 2005),
antioxidant activity (Sabaghi et al., 2015), epithelium protection and
immunomodulation (Vinderola et al., 2006) have attracted attention as
a potential food preservative. Therefore, the use of kefiran in the pro-
duction of edible and composite films can lead to suitable packaging
and specific protective coatings with good mechanical and physical
properties, in turn causing increased shelf-life, enhanced food quality,
positive health effects and satisfactory physicochemical properties
(Piermaria et al., 2009, 2011; Zolfi et al., 2015).
4.1.1. Stabilizer
Some recent studies showed the flow properties of kefiran, while
utilizing it as a thickener. However, when it was in high shear rates, it
behaved like a Newtonian one. In addition, due to the thermal treat-
ment of kefiran solutions by freeze-thaw proses, the texture of the
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Carbohydrate Polymers 223 (2019) 115100
kefiran systems remained virtually unchanged. Therefore, kefiran
showed a potential to be used in frozen desserts such as ice cream due
to its stabilizing properties (Exarhopoulos et al., 2018b).
4.1.2. Additive
Kefiran also was applied as a natural food additive in bakery in-
dustry, since it could create good rheological properties in dough and
ideal processing conditions (Soleimani Fard et al., 2017).
4.1.3. Film-forming agent
Ghasemlou et al. (2011) studied a new edible film extracted from
kefiran, in which glycerol and sorbitol were used as plasticizers. Ac-
cording to their results, kefiran played a basis role in these edible films
through its film-forming agent. Since, natural edible films of kefiran
had poor mechanical and physical properties, high concentration of
glycerol and sorbitol caused phase separation in the films and glycerol
was more effective than sorbitol as the plasticizer to improve the
properties of kefiran films.
Since, the produced films of kefiran were strong hydrophilic, in
order to eliminate its drawback Ghasemlou et al. (2011a) demonstrated
a significant improvement of the water barrier properties in edible
composite films with low affinity to water by film-forming solutions of
kefiran, as matrix, and oleic acid (OA), as hydrophobic component,
using emulsification. The barrier impact of the emulsified films could be
related to lipid aggregates obtained in both internal and external sur-
face of the films.
Hedayati Rad et al. (2017) prepared new biodegradable films based
on kefiran and waterborne polyurethane (WPU) as the soft segment
with relative humidity of 55%. Based on their results, when the content
of kefiran/WPU blends was lower than 30 wt%, some new features such
as homogeneous structure, higher tensile strength, miscibility and
thickness were observed. Due to the hydrophilic nature of kefiran, it
had high tendency to adsorb moisture. Hence, kefiran/ WPU blend films
improved barrier moisture and decreased water vapor permeability
rather than kefiran pure films.
Piermaria et al. (2009) demonstrated the pseudoplastic behavior in
the formed edible transparent films of kefiran with water vapor per-
meability barrier properties. In addition, an extraordinarily higher
flexibility was observed in the properties of kefiran films containing
glycerol, compared to the ones without it.
Motedayen et al. (2013) investigated the effects of adding starch,
containing different amounts of starch (0%–50% (v/v)), by blending
kefiran and corn starch at various concentrations on the water-vapor
permeability properties of formed new biocomposite edible films. In
this study, starch addition led to good water vapor barrier properties.
The addition of starch caused considerable extensibility and extra-
ordinary tensile strength. Moreover, results showed that in these bio-
composite films, the glass transition temperature increased and their
statures were homogeneous. Meanwhile, with increasing starch con-
tent, both mechanical properties and water vapor barrier decreased
significantly. These results were ascribed to the interaction between
kefiran and starch and their compatibility.
Piermaria et al. (2011) developed an edible film made of kefiran,
polyols (sorbitol and glycerol) and sugars (galactose, glucose and su-
crose) as plasticizers. It was claimed that the use of all plasticizers
improved film properties; glucose as plasticizer displayed the best water
vapor barrier, whereas the incorporation of glycerol as plasticizer re-
sulted in the excellent mechanical properties.
The study on the biopolymer films formed from kefiran and mod-
ified using γ -irradiation to boost the functional properties indicated
that use of γ -irradiation improved the mechanical properties and water
sensitivity of kefiran biopolymer films (Shahabi-Ghahfarrokhi et al.,
2015a).
As the newest application of kefiran, the edible films produced by
whey protein-kefiran acted out as a delivery system of probiotic mi-
croorganisms. The research showed that the driver system not only had
Z. Moradi and N. Kalanpour
the ability to protect probiotics during stress, but it also made the
targeted delivery feasible (Gagliarini et al., 2019).
4.1.4. Gelling agent
Piermaria et al. (2008) studied the rheological behavior of cryogels
formed from kefiran and evaluated their physicochemical properties.
Due to gelling properties of kefiran, it was suggested that kefiran
cryogels supported their own weight (self-supporting) and were trans-
lucence and cohesive. Moreover, formed gels from kefiran by addition
of ethanol for producing gels with high strength by Mukai et al (1991)
and formed porous cryogels from kefiran by using sugars (sucrose and
fructose) in order to modification of polysaccharide networks by Zavala
et al (2015) reported. This results demonstrated that the gels could hold
high concentration of water and could use in order to cryogenic treat-
ment such as defrosting and freezing as a potential alternative in food
formulations.
4.2. Antioxidant activity
Since the antioxidant activity of antioxidants was via retarding the
oxidation, they could preserve foods as the scavenge radicals (Kumar,
2011; Yilmaz-Ersan et al., 2016). Therefore, kefiran was used as a
natural antioxidative supplements, thanks to its antioxidative, anti-
microbial and anticancer activity (Thyagarajan et al., 2017).
One of other notable applications of kefiran is the use of the com-
bination of chitosan and kefiran, as composite films, in the evaluation
of antioxidative activity and structural properties (including mechan-
ical, physical features and water vapor permeability). Studies have
shown that the addition of chitosan to these films improves the anti-
oxidative activity, the physical features, the water-vapor permeability
and the optical properties, besides enhancing the elongation at break,
tensile strength, puncture deformation and puncture strength (Sabaghi
et al., 2015).
4.3. Antimicrobial
Kefiran can be used as an antimicrobial and cicatrizing agent
against several highly resistant pathogenic strains. Recent investiga-
tions on the antimicrobial activity of kefiran in Staphylococcus aureus
and Wistar rats with induced skin lesions have revealed that both
showed high antimicrobial, cicatrizing and anti-inflammatory activity
against all organisms. In this study, the highest antimicrobial activity of
kefiran was reported against Streptococcus pyogenes (Rodrigues et al.,
2005). Likewise, the studies showed that kefiran increased the anti-
fungal activity against several filamentous fungal species and anti-
microbial activity against bacterial species. The excellent antimicrobial
function of kefiran was reported against Fusarium graminearum CZ1 and
Streptococcus faecalis KR6. In addition, due to the antifungal activity of
kefiran, it prevented the production of aflatoxin B1 by Aspergillus flavus
AH3 (Ismaiel et al., 2011; Vijayendra & Shamala, 2013).
Fig. 6. Structure of kefiran and encapsulated of platelet-kefiran were shown by light microscope. The Crystals made from kefiran and polysaccharide organized and
neatly regulated shown by light microscope (Fig. 2a). The connection of platelets and kefiran by optical microscope showed (Fig. 2b) (Jenab et al., 2015).
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Carbohydrate Polymers 223 (2019) 115100
The bifidogenic activity of kefiran made it an oral prebiotic in-
gredient in food applications. The health beneficial activity of kefiran
could increase the intestinal bifidobacteria (Hamet et al., 2016;
Hongpattarakere et al., 2012).
The ability of Kefiran, as a carrier material (Micheli et al., 1999) or
capsular polysaccharide, to release active molecules was shown.
Therefore, the microspheres produced from kefiran/alginate gel via the
encapsulation of ciprofloxacin could cause the controlled antimicrobial
delivery and increase the antibacterial activity against pathogens
(García, 2016).
In addition, kefiran was used in industry such as dairy food (Duboc
& Mollet, 2001), given its antimicrobial properties. Since some bacteria
are resistant against antibiotics, the objective of using kefiran with
antimicrobial peptides is to fight against pathogenic bacteria. There-
fore, in the production of new antibacterial drugs, the kefiran with the
increased amount of antimicrobial peptides was used (Jenab et al.,
2015).
4.4. Biological and medical activities
Kefiran also attracted considerable attention as a novel biological
and medical material, due to its biocompatibility and water sensitivity
(Leite et al., 2013; Radhouani et al., 2018b). It was found out that
kefiran could be effective on the cholesterol metabolism (Xiao et al.,
2003), the prevention of several cancer (Elsayed et al., 2017), and the
antitumor activities by oral administration. Kefiran could also be used
to increase interferon production from the animal cells (Kabayama
et al., 1997).
Considering the anti-proliferative characteristics of kefiran, the ke-
firan produced in vitro by L. kefiranaciens presented good anticancer
activity against many cancer cells such as cervical and hepatocellular
carcinoma (Elsayed et al., 2017).
Similarly, the biological activity of kefiran on the Bacillus cereus
strain B10502 by using cultured human enterocytes (Caco-2 cells) and
human red blood cells demonstrated that kefiran could obstruct re-
ceptors on the cell surface, prevent cytolytic effects and trigger some
cellular functions, opposite to that obtained by B.cereus. Therefore,
kefiran, with prebiotic effects, could be used as additives in food ap-
plications (Medrano et al., 2008). As such, kefiran could be used in the
medicine, because it had some healing effects in disease therapies such
as antitumor activity (Shiomi et al., 1982), anti-inflammatory agent
(Rodrigues et al., 2016), and intestinal infections against B. cereus ob-
tained (Magalhães-Guedes et al., 2016). Researches have shown that
the encapsulation and drug delivery of platelets, as biological cells, in
kefiran biopolymer and also prebiotic kefiran for immobilized platelet
could be applied in surface bleeding treatment (Fig. 6) (Jenab et al.,
2015).
The prebiotic effect of kefiran and its interaction with a lipid bilayer
membrane, as biomembrane, showed that it had biological activity with
health benefits. In this procedure, the carbohydrate of kefiran reacted
Z. Moradi and N. Kalanpour
with phosphate head group of the phospholipid to obtain hydrogen
bonding. This interaction led to the disruption of membrane based on
the concentration of kefiran (Fig. 7) (Barbosa et al., 2011).
Kefiran could be extensively used in different therapeutic fields,
such as the treatment of Osteoarthritis (OA). According to researches
done by Radhouani et al (2018a), the specific antioxidant activity of the
kefiran biopolymer reduced and scavenged the superoxide radicals.
Also, due to this anti-inflammatory activity, it could scavenge the nitric
oxide radicals (NO). Therefore, regarding these features, the kefiran
helped in treating Osteoarthritis (OA), the most common form of ar-
thritis disease (Fig. 8) (Radhouani, Goncalves, Maia, Oliveira, & Reis,
2018a).
It has been clarified that kefiran has many applications in diverse
areas. In biological area, the existence of kefiran could cause a decrease
in the blood pressure (Maeda, Zhu, Omura, et al., 2004a). Beyond that,
due to the unique features of kefiran, orally-administrated kefiran
presented hypocholesterolemic, hypotensive, and atherosclerosis and
immunomodulatory effects. Studies demonstrated that kefiran could be
effective in inhibiting and healing allergic disorders and inflammatory
diseases. Considering the important role of the activation of mast cells
Fig. 8. Applications of kefiran in tissue engineering and medical (Radhouani, Goncalves, Maia, Oliveira, & Reis, 2018a).
7
Carbohydrate Polymers 223 (2019) 115100
Fig. 7. The carpet-like mechanism proposed for
kefiran-membrane interaction. Oligosaccharide
molecules line up on the membrane surface
until a critical concentration is reached and a
detergent-like effect takes place. At this stage,
oligosaccharides from kefiran and membrane
components form aggregates that leave the
membrane cause disruption (Barbosa et al.,
2011).
in the allergic and inflammatory disorders, Kefiran could present anti-
inflammatory activity through the inhibition of mast cells degranula-
tion and cytokine creation (Furuno & Nakanishi, 2012).
The application of kefiran in prophylaxis and therapy area is due to
its effect on the immune cells and its oral administration utilization in
intestinal pathologies. In addition, the immunomodulation feature of
kefiran in the form of oral administration to modify the balance of
immune cells in intestinal mucosa revealed that due to probiotic ac-
tivities of kefiran, it could be effective in the therapy of intestinal
pathologies (Medrano et al., 2011).
The investigation of biopolymers, especially kefiran biopolymer in
order to make three-dimensional porous scaffolds structure in biome-
dical fields revealed that the kefiran could be utilized to produce
scaffolds with high porosity structure, satisfactory in terms of biome-
dical applications (Toscano et al., 2018).
4.5. Nanotechnology
Nowadays, nanotechnology is a branch of science, applicable widely
in different life fields (Niska et al., 2018). As a promising option, the
Z. Moradi and N. Kalanpour
nanocomposites can increase significantly composites properties. Due
to amazing characteristics of nanocomposites such as mechanical, op-
tical, barrier, and thermal features (Zolfi et al., 2015), they are in-
creasingly used in industrial packaging, automotive industry, elec-
tronic, biotechnological, aerospace and drug-delivery. The use of
nanocomposites to improve many properties makes them satisfactory
for some applications. Generally, the poor mechanical and weak barrier
properties and also sensibility to moisture of biomaterials in the natural
packaging films are limiting factors in their utilization (Babaei-
Ghazvini et al., 2018) To overcome these defects, the use of nano-
composites and nanofillers based on the biomaterials such as kefiran
could improve mechanical and physical properties, leading to its de-
veloped use in different fields in nanotechnology (Niska et al., 2018;
Zafar et al., 2016; Zolfi et al., 2014a). The utilization of nanoparticles in
food packaging to create the bionanocomposite films, due to introdu-
cing valuable attributes, can be very effective in eliminating the defi-
ciencies, improving properties such as strength, mechanical attributes,
physical and chemical features, and protecting against microorganisms,
barrier and water-vapor permeability (WVP) properties.
4.5.1. Kefiran-based bio-nanocomposites
Zolfi et al. (2014b) produced the bionanocomposites films from
kefiran- whey protein (WPI), as matrix, using 5% (w/w) of montmor-
illonite (MMT) nano-clay and TiO2 nanoparticles, as nanofilers, to im-
prove the features of kefiran- whey protein biocomposites.
Another study was done by Babaei-Ghazvini et al. (2018) on UV-
protective starch/kefiran/ZnO biodegradable nanocomposite films.
They fabricated bionanocomposites films using starch and kefiran, as a
bio-matrix, and ZnO nanoparticles with particle diameter of 20–30 nm,
as nanofiller.
As part of their studies on the biodegradable films, Zolfi et al.
(2014a) developed the blend films from kefiran-whey protein isolate-
TiO2 nanoparticles. Due to high-nutritional value of kefiran and whey
protein, they were used in these blend films, to which TiO2 nano-
particles with a diameter of about 20 nm were added.
Hasheminya et al. (2018) incorporated the kefiran with carbox-
ymethyl cellulose (CMC) polysaccharide along with CuO nanoparticles
with particle diameter of 40 nm. In this research, kefiran and carbox-
ymethyl cellulose were used as improvers of quality degradation and
shelf life in the bionanocomposite films. Studying the films produced
with various concentrations of CuO nanoparticles (1, 1.5, and 2% CuO
nanoparticles) showed that in high concentrations, films properties
changed and contact angle and ultraviolet light transmission features
were improved.
The nanocompsites of kefiran alongside Al2O3 nanoparticles with
particle diameter of 20 nm were produced in research done by Moradi
et al. (2019) and the impact of adding three different concentrations of
Al2O3 nanoparticles (1, 3 and 5% w/w) was evaluated on the properties
of the kefiran films.
Shahabi-Ghahfarrokhi et al. (2015) developed a new bio-degradable
nanocomposite composed of kefiran, as matrix, and cellulose nano-
crystal (NC(with particle diameter of 97 ± 18 nm, as non-toxic nano-
fillers. Their produced bionanocomposite was compatible with the en-
vironment and could be used in food packaging applications.
The preparation of biodegradable WPI/clay nanocomposite films
was carried out using exopolysaccharide kefiran by Zolfi et al. (2015).
In this research, the addition of montmorillonite (MMT) nanoclay to
reinforcement of matrix into kefiran-whey protein isolate (WPI) matrix
led to the formation of kefiran-WPI bio-nanocomposite films.
In recent reports, due to notable properties suggested for kefiran, it
was used as a natural matrix in combinations with several essential
metal nanoparticles and metal oxide nanoparticles as nanofillers to
generate bionanocomposites. Compared to other biopolymers, kefiran
could be used to fabricate biodegradable bionanocomposites and edible
films in the food packaging. Nonetheless, the use of biopolymers such as
kefiran in extensive food packaging applications is limited, considering
8
Carbohydrate Polymers 223 (2019) 115100
their poor performance in some properties due to their weak mechan-
ical and barrier properties (Babaei-Ghazvini et al., 2018), sensibility to
moisture and brittleness (Zolfi et al., 2014a). Thus, recent studies fo-
cused to removing these restrictions through using nanoparticles as
nanofillers in polymeric structures. Generally, the results demonstrated
that the addition of nanomaterials including: MMT nano clay, TiO2,
ZnO, CuO, Al2O3 nanoparticles and cellulose nanocrystals into poly-
meric structures not only improved and modified mechanical and
physical properties, but they also, in some cases, ameliorated other
properties such as enhancing its transparency (Zolfi et al., 2014b), in-
creasing thermal stability (Babaei-Ghazvini et al., 2018) and enhancing
thickness (Hasheminya et al., 2018).
4.5.2. Kefiran based nanofibers
Nanofibers are an example of rapidly development of nano-
technology. Indeed, due to good properties of nanofibers such as sig-
nificant porosity and high surface area to volume ratio, they can be
used in many applications including tissue engineering, medical, sen-
sing, security application, and energy generation etc (Elsabee et al.,
2012; Naghibzadeh & Adabi, 2014).
Esnaashari et al. (2017) prepared kefiran nanofibers using electro-
spinning by the modelling artificial neural networks (ANN). Their in-
vestigations confirmed that the result of modelling ANN was correlated
with the experimental data.
Jenab et al. (2017) produced electrospun kefiran/poly ethylene
oxide (PEO) nanofibers (diameter of 607.5 nm) with antimicrobial ac-
tivity as a biocontrol agent against Rhizoctonia, Pseudomonas sp and S.
aureus. They confirmed that kefiran/PEO nanofibers with the oxidizing
functional groups inhibited the growth of microorganisms. Indeed, due
to the antimicrobial properties of kefiran, kefiran/PEO nanofibers could
be used as a biocontrol agent in food packaging applications, such as
inhibition of food spoilage.
In other dissemination, Esnaashari et al. (2014) reported the fabri-
cation of uniform kefiran nanofibers with the minimum size about
235 nm and the maximum size about 294 nm using electrospinning
method. They demonstrated that in kefiran molecular structure no
changes were made until the end of electrospinning process.
5. Conclusion
The range and richness of researches done on the macromolecule of
kefiran during recent years have been amazing and it is continuing
quickly. This review attempted to illustrate a wide vista of the scientific
advancements and interesting researches in different fields up to the
present. The particular structure and the astonishing abilities of kefiran
have caused the further transformations. Its nature allows its applica-
tion in various fields, as a composition capable of creating impressive
changes in a system such as gelling ability in a food system or capsular
polysaccharide ability in a drug delivery system. The overall gamut of
applications for macromolecule of kefiran is virtually limitless espe-
cially in medical and biologic fields. So, currently, kefiran can be an
opportunity for the new advancements and discoveries in different
areas.
Funding
This research did not receive any specific grant from funding
agencies in the public, commercial, or not-for-profit sectors.
Acknowledgment
We are thankful to the financial aid provided by Urmia University.
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