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

Contents lists available atScienceDirect Carbohydrate Polymers journal homepage:www.elsevier.com/locate/carbpol

Review

Kefiran-based films: Fundamental concepts, formulation strategies and

properties

A R T I C L E I N F O

Keywords:

Exopolysaccharide

Kefiran

Biopolymer

Edible film

Material properties

A B S T R A C T 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 per-formance 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

con-tributes 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

per-formance (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

environ-mental 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 polyhydroxyalk-onates) (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 func-tionality 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

https://doi.org/10.1016/j.carbpol.2020.116609

Received 3 May 2020; Received in revised form 17 May 2020; Accepted 26 May 2020

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:marangoni.junior@hotmail.com(L Marangoni Júnior)

Available online 20 June 2020

0144-8617/ © 2020 Elsevier Ltd All rights reserved

T

received remarkable attention Kefiran is an edible and biodegradable

water-soluble EPS obtained during milk fermentation in the kefir

pro-duction (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

ap-plications 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

me-chanical and barrier properties were demonstrated (Ghasemlou et al.,

2011a; Moradi & Kalanpour, 2019; Piermaria et al., 2011;

Shahabi-Ghahfarrokhi 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

addi-tion, the development of blends based on this EPS and other

biopoly-mers, as well as incorporation of nanoparticles have also been reported

However, despite the promising results, further research in the

litera-ture remains to be explored towards improving its properties for

ef-fective 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

perspec-tives 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

dis-cussed 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

knowl-edge, this is the first time a review is presented with a focus on the

production and properties evaluation of kefiran based-films

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

Lac-tobacillus 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

re-ported the possibility of small variations in these proportions.Zajšek,

Kolar & Goršek (2011)used electrophoresis to identify the residues ofD

-glucose andD-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 kefiran1H

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-D -Galactose corresponding to a small proportion of 2,3,4-tri-O-methyl-D -galactose (sugar on a side branch) The other peaks show three well-defined signals and three overlapping signals that are attributed to the

Fig 1 Kefiran chemical structure.

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)

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

configura-tion (Maeda, Zhu, Suzuki et al., 2004) The results identified in the

spectra of Fig 2(a) corroborate the structural characterization

pre-sented by other authors (Micheli et al., 1999;Radhouani et al., 2018;

Staaf et al., 1996) The13C NMR spectrum inFig 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

high-lighted 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

inter-molecular hydrogen bonding of the polysaccharide Weak absorption

close to 2930 cm- 1is 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

X-ray 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

struc-tural information

At low concentrations of kefiran, the specific viscosity increases

linearly as a function of concentration However, at a particular

con-centration, 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−1for kefiran Conversely, Piermaria et al (2008) reported a critical con-centration 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, mo-lecular weight can be correlated as a function of intrinsic viscosity Some intrinsic viscosity values for diluted solutions of kefiran re-ported in the literature using Huggins and Kraemer equations, respec-tively, were: 600 mL g−1 and 595 mL g−1 (Mw = 10,000 kDa) (Piermaria et al., 2008), 584 mL g−1and 553 mL g−1(Mw = 1350 kDa) (Ghasemlou, Khodaiyan, Gharibzahedi, 2012; Ghasemlou, Khodaiyan, Jahanbin et al., 2012), 84.6 mL g−1and 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 com-pared 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 ir-regular 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

ex-tremely 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

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)

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

ir-regular grain shapes with diameters ranging from 1 to 15 mm (

Güzel-Seydim 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

to-gether 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

ad-dition, 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 × 108cfu / 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

cerevi-siae 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−1h−1(Cheirsilp, Shimizu et al., 2003), which is

50 % higher than that obtained using pure culture (24 mg L−1h−1)

3.2 Strategies for optimizing kefiran yield

The primary operational parameters that must be evaluated to

ob-tain 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

condi-tions 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

max-imum 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

mi-croorganisms 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,

lac-tose), nitrogen sources (yeast extract, peptone, meat extract, casein

hydrolyzate) have been investigated in the literature Some of these

surveys are highlighted inTable 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 4provides a sim-plified 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 tem-perature used However, temtem-peratures close to 100 °C have been re-ported 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 character-istics 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 mole-cular 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 inFig 4 is as-sociated with the kefiran precipitation by cold ethanol Several re-searches used absolute ethanol (Dailin et al., 2016; Piermaria et al.,

2008;Radhouani et al., 2018;Taniguchi et al., 2001) However, con-sidering a possible scale-up, a recent study evaluated the effect of using

96 % ethanol, which provides a more reduced cost The results of ke-firan 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 cen-trifugation 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)

4 Kefiran-based films

Currently, kefiran-based materials are gaining prominence for pos-sessing unique properties, including biodegradability, safety, bio-compatibility, stabilizing and emulsifying effect, satisfactory mechan-ical 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 radia-tion to improve the films properties, as described below

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

char-acteristics The kefiran concentrations were 0.5, 0.75 and 1.0 % in the

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 ofGhasemlou 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 byGhasemlou 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 byGhasemlou

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 (poly-saccharides, 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 com-pounds should be considered, since these components can provide other functions to the films (light barrier, antioxidant and antimicrobial ac-tivities)

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 de-velopment of edible films and coatings, because of its abundance, cost-benefit ratio and excellent film forming skills Starch films possess good optical, organoleptic and gas barrier properties, however, they de-monstrate limitations due to their hydrophilicity and are weak in me-chanical 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 ofMotedayen et al (2013) developed kefiran/starch blends with kefiran contents ranging from

70-30 % 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

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. Kefiran production rate: ∼ 53mg L −1 h −1 Cheirsilp and Radchabut (2011 ) 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. Maximum grain increase: 81.34% Ghasemlou, Khodaiyan,Gharibzahedi (2012 )

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. Kefiran production rate: 29.7mg L −1 h −1 Zajšek et al (2013 )

Response surface optimization; lactose concentration: 50 g L −1 ; yeast extract concentration: 12 g L −1 ;

temperature: 30 °C. Kefiran production rate: 21 mgL −1 h −1 Dailin et al (2015 )

Response surface optimization; fermentation medium: whey; glucose concentration: 15 %; temperature:

30 °C; Time: 10 h. Kefiran production rate: 37.14mg L −1 h −1 ( Blandón et al., 2018 )

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: ∼ 35mg L −1 h −1 Cheirsilp et al (2018 )

Fig 4 Simplified flowchart summarizing the main steps of the purification

procedures described in the literature

for physical modification and radical formation (Goudarzi &

Shahabi-Ghahfarrokhi, 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

de-monstrates excellent structural properties, which allow the formation

of a continuous layer of food coatings, which is why it has been

em-ployed 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

Ke-firan (2%) and chitosan (2%) solutions, resulting in different film

proportions with kefiran contents ranging from 32 to 78 % The

ob-jective was to exploit a by-product from the fishing industry to

prove the properties of kefiran films, with the suggestion of

im-plementing 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 byHasheminya

et al (2019a)with biocomposite films made from kefiran (1%),

car-boxymethylcellulose (CMC) (1%), glycerol (50 % of dry weight),

es-sential 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)

nano-particles and simultaneous addition of CuO and essential oil (EO)

Sa-tureja 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

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

prop-erties and excellent barrier propprop-erties 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

im-prove 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

me-chanical properties (Hassan et al., 2018) In addition, lipids are

ef-fective 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

have addressed the incorporation of essential oils for a specific pur-pose, 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 prop-erties, provided that the nanoparticles are well dispersed throughout the polymeric matrix Moreover, the incorporation of these nanoma-terials can improve thermal, physical and mechanical properties, be-sides 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 ke-firan/WPI blends (Zolfi et al., 2014a,2014b) and zinc oxide (ZnO) in kefiran/starch blends (Babaei-Ghazvini et al., 2018; Shahabi-Ghahfarrokhi & 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 de-termined, since they will inform about the processing conditions for obtaining the films (Piermaria, Pinotti et al., 2009) Aqueous film-forming 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 occu-pied by a given polymer and the length of the polymer chain (Piermaria

et al., 2008;Shahabi-Ghahfarrokhi & Babaei-Ghazvini, 2019) The in-crease in the concentration of oleic acid in the kefiran solution pro-duced an increase in viscosity (Ghasemlou et al., 2011c) The work by Shahabi-Ghahfarrokhi and Babaei-Ghazvini (2019)evaluated the in-trinsic viscosity of kefiran/starch/ZnO solutions subjected to UV ra-diation (0, 1, 6 and 12 h) It has been observed that viscosity decreases

as radiation exposure time increases The authors attributed these re-sults to the formation of free radicals that induced molecular changes and fragmentation Similar behavior was observed for kefiran/starch/ TiO2solutions, 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 hetero-geneity In addition, air bubbles tend to get trapped in viscous solu-tions, 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

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

ap-proximately 8 Hz, going from a phase angle of about 47° (viscous

li-quid) 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

ap-plication 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)

6 Manufacturing methods

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

tech-niques 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

fil-tration, centrifugation or another method to eliminate insoluble

parti-cles and air bubbles, followed by dispersing the solution on a flat-sized

surface and shape

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 fi-nalized 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 kefiran-based films have peculiarities in each study Therefore, to better illus-trate 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

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 re-quirements 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, avail-ability, 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 con-struction of coatings are based on proteins, lipids and polysaccharides However, no reports were provided on the development and applica-tion 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 de-velopment, 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

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)

Solution mass

Storage condition

Al2

O3

Shahabi-Ghahfarrokhi etal.

Shahabi-Ghahfarrokhi etal.

Shahabi-Ghahfarrokhi etal.

6.3 Extrusion

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

pro-duced 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

poly-saccharide structure (Radhouani et al., 2018) Other studies of

exopo-lysaccharides 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

max-imize the large-scale production of kefiran films

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

ke-firan) 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 byGhasemlou 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

im-provement 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

de-creased 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

biopo-lymers reduced the moisture content of the films as the concentration of

nanoparticles increased This behavior can be illustrated byMoradi

et al (2019), that included Al2O3in the kefiran films In addition,Zolfi

et al (2014a)included TiO2in kefiran/WPI films,Goudarzi & Shahabi-Ghahfarrokhi (2018) included TiO2 in kefiran/starch films, and also exposed it to UV-A radiation Finally,Shahabi-Ghahfarrokhi & Babaei-Ghazvini (2019)included ZnO in Kefiran/starch films exposed to UV radiation Similar behavior was observed for films with essential oils included, such asHasheminya et al (2019a), that used the Satureja

khuzestanica EO in kefiran/CMC films; and the work ofHasheminya

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)

7.2 Solubility

The solubility of kefiran-based films reveals the possible applica-tions of this material In some potential food applicaapplica-tions, 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 solubi-lized 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 toComa 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 film-forming solution to γ irradiation reduced the water absorption capacity and the solubility at doses up to 6 kGy (Shahabi-Ghahfarrokhi, Kho-daiyan, Mousavi, et al., 2015) The sorbitol plasticizer maintained the solubility similar to the film without plasticizer, regardless of the con-centration (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 so-lubility is the incorporation of nanoparticles Generally, as the nano-particle content increases, the solubility of the film decreases These results were observed with Al2O3for 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 ra-diation (Shahabi-Ghahfarrokhi & Babaei-Ghazvini, 2019), TiO2in ke-firan/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)

7.3 Hydrophobicity

The water contact angle is performed to determine the

hydro-phobicity 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

ad-ditives, 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

in-corporation of nanoparticles, as ZnO in kefiran/starch films (

Babaei-Ghazvini 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 &

Babaei-Ghazvini, 2019) It is evident the incorporation of these substances

in-creased 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

maneu-verability, homogeneity and continuity Kefiran films with the addition

of 0, 10, 20, 30 % glycerol were evaluated byComa 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

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 byGhasemlou et al (2011d),Piermaria et al (2009) The use of other plasticizers such as polyols and sugars showed morpholo-gies 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, morpho-logical differences were observed Films of kefiran/starch/TiO2 sub-mitted 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) Ke-firan/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 Al2O3in 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

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)