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.
Carbohydrate Polymers 246 (2020) 116609 Contents lists available at ScienceDirect Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol Review Kefiran-based films: Fundamental concepts, formulation strategies and properties T Luís Marangoni Júniora,*, Roniérik Pioli Vieirab, Carlos Alberto Rodrigues Anjosa a b Department of Food Technology, School of Food Engineering, University of Campinas, Campinas, São Paulo, Brazil Department of Bioprocesses and Materials Engineering, School of Chemical Engineering, University of Campinas, Campinas, São Paulo, Brazil ARTICLE INFO ABSTRACT Keywords: Exopolysaccharide Kefiran Biopolymer Edible film Material properties Concerns about plastic pollution have driven research into novel bio-derived and biodegradable polymers with improved properties Among the various classes of biopolymers studied, kefiran films only have gained emphasis in recent years Its film-forming ability and outstanding biological activities illustrate its potential for active packaging applications However, despite recent advances, the key challenge is still associated with obtaining high water vapor barrier and better mechanical properties In that fashion, this review highlights for the first time the cutting-edge advances in the preparation, characterization and enhancement of the packaging performance of kefiran-based films The fundamental concepts of the biopolymer production and chemical analysis are previously outlined to direct the reader to the structure-property relationship In addition, this research critically discusses the current challenges and prospects toward better material properties Introduction Food packaging and coatings have experienced impressive progress in recent decades, driven by growing demand for safe and high-quality foods Its primary function is the protection against external agents (microorganisms, water vapor, oxygen and light) In addition, it contributes to prevent loss of desirable compounds (flavor volatiles) and consequently extending the product's shelf life (Mohamed et al., 2020) Materials made of paper, metal, glass and plastics are frequently used as food packaging (Mahalik & Nambiar, 2010) Plastics (from fossil sources) are the most used due to low cost, low specific mass, high versatility, flexibility, transparency, good mechanical and barrier performance (Licciardello, 2017; Marangoni Júnior et al., 2020; Robertson, 2013) Plastic materials from fossil sources generate waste that needs a correct destination, such as landfills, reuse, recycling, among others Although these materials are consolidated in industries, their environmental aspects have raised concerns that are in growing discussion (Zhong et al., 2020) Linked to this, a growing consumer demand for materials that not degrade the environment, that are safe and non- toxic is increasingly present in society (Sharma et al., 2020) These facts motivate the search for renewable alternatives to these applications, such as the use of bio-derived polymers In general, the most used to form films and coatings are composed of polysaccharides, proteins and lipids (Sampathkumar et al., 2020; Vieira et al., 2011) Currently, there are two dominant classes of commercially viable biopolymers: alkyl polyesters (poly(lactic acid) and polyhydroxyalkonates) (Garlotta, 2002; Suriyamongkol et al., 2007), and starch-based plastics (Lu et al., 2009) However, when compared to petroleum-based counterparts, bio-based films are unable to supply all of their functionality The reason is the lower mechanical and barrier performance, in addition to water sensitivity (Azeredo & Waldron, 2016), which limits its use in many applications (Peelman et al., 2013) In that fashion, the search for polymeric alternatives and/or different film formulation strategies has been gaining more and more prominence Indeed, it is recognized that considerable improvements in properties have been reported with the production of nanocomposites, blends and by obtaining active films, mainly involving polysaccharide bases (Cazón et al., 2017; Ribeiro-Santos et al., 2017) Among this class of biopolymers, exopolysaccharides (EPS) have Abreviations: Al2O3, aluminum oxide; ATR-FTIR, attenuated total reflection Fourier-transform infrared spectroscopy; CMC, carboxymethylcellulose; CuO, copper oxide; DSC, differential scanning calorimetry; EO, essential oil; EPS, exopolysaccharides; FTIR, Fourier-transform infrared spectroscopy; HPLC, high performance liquid chromatography; MMT, montmorillonite; Mw, weight-average molecular weight; NC, nano-cellulose; NMR, nuclear magnetic resonance; OA, oleic acid; RSM, response surface methodology; SEM, scanning electron microscopy; Tg, glass transition; Tm, melting temperature; TGA, thermogravimetric analysis; TiO2, titanium oxide; UV, ultraviolet; WPI, whey protein isolate; WVP, water vapor permeability; ZnO, zinc oxide; XRD, x-ray diffraction; [η], intrinsic viscosity ⁎ Corresponding author at: Rua Monteiro Lobato, 80 - Cidade Universitária Zeferino Vaz, CEP: 13083-862, Campinas, São Paulo, Brazil E-mail address: marangoni.junior@hotmail.com (L Marangoni Júnior) https://doi.org/10.1016/j.carbpol.2020.116609 Received May 2020; Received in revised form 17 May 2020; Accepted 26 May 2020 Available online 20 June 2020 0144-8617/ © 2020 Elsevier Ltd All rights reserved Carbohydrate Polymers 246 (2020) 116609 L Marangoni Júnior, et al received remarkable attention Kefiran is an edible and biodegradable water-soluble EPS obtained during milk fermentation in the kefir production (Frengova et al., 2002; Kooiman, 1968; la Riviére et al., 1967; Moradi & Kalanpour, 2019) Kefiran apparently protects the microbiota inside the kefir granules (Badel et al., 2011) Moreover, it has been attributed numerous beneficial properties for human health, such as high antimicrobial and healing potential (Piermaria et al., 2008; Rodrigues, Caputo et al., 2005), anti-inflammatory activity (Rodrigues, Carvalho et al., 2005), anti aging properties (Sugawara et al., 2019), contribution to the reduction of blood pressure and cholesterol levels (Amorim et al., 2019; Maeda, Zhu, Mitsuoka, 2004), besides anticancer activity (Jenab et al., 2020; Medrano et al., 2011; Sharifi et al., 2017) In this context, kefiran has been incorporated in a broad range of applications in the food industry For exemple, as stabilizer, emulsifier, fat substitute and gelling agent (Moradi & Kalanpour, 2019; Moradi et al., 2019) In addition to these extraordinary biological activities, another highlight of kefiran is its considerable potential for the production of films and coatings Distinguished appearance and satisfactory mechanical and barrier properties were demonstrated (Ghasemlou et al., 2011a; Moradi & Kalanpour, 2019; Piermaria et al., 2011; ShahabiGhahfarrokhi et al., 2015) It is noted, however, that its film-forming potential began to be explored only in recent years, mostly in the last decade Hence, several studies in the literature have focused on the development of kefiran films employing distinct plasticizers In addition, the development of blends based on this EPS and other biopolymers, as well as incorporation of nanoparticles have also been reported However, despite the promising results, further research in the literature remains to be explored towards improving its properties for effective use in food packaging and coatings Therefore, this work aims to present readers with a bibliographic trend directed to the notable advances, challenges and future perspectives in the production of kefiran-based films This research initially outlines the chemical structure characterization, production, extraction and purification of the exopolysaccharide These fundamental concepts facilitate the films structure-property relationship subsequently discussed In addition, an extensive analysis of the formulation methods and evaluation of the film’s properties are presented The key desirable materials characteristics are also discussed To the best of our knowledge, this is the first time a review is presented with a focus on the production and properties evaluation of kefiran based-films Fig Kefiran chemical structure Chemical structure The extracellular polysaccharides or exopolysaccharides (EPSs) are produced by many bacteria, which secrete in the form of a capsule or slime layer around the bacterial cell (Nouha et al., 2018) Kefiran is the main exopolysaccharide produced from kefir grains (Moradi & Kalanpour, 2019) It is produced typically by bacteria of the type Lactobacillus kefiranofaciens, but also by several other unidentified species of Lactobacillus (Zajšek et al., 2013) Kefiran is a light or pale yellow viscous polysaccharide, water-soluble, containing approximately the equivalent amount of D-glucose and D-galactose (Badel et al., 2011; Kooiman, 1968; Pop et al., 2016) However, some authors have reported the possibility of small variations in these proportions Zajšek, Kolar & Goršek (2011) used electrophoresis to identify the residues of Dglucose and D-galactose in the proportion of 1:0.7 Chen et al (2015) identified by high performance liquid chromatography (HPLC) that the proportions of D-glucose and D-galactose in kefiran produced from a Tibetan kefir are 1:1.88, respectively Kefiran is a branched-structure carbohydrate (Fig 1), with a repeat of hexa or hepta-saccharide composed of a regular pentasaccharide unit, in which one or two sugar residues are randomly linked (Kooiman, 1968; Maeda, Zhu, Suzuki et al., 2004; Micheli et al., 1999) For the identification of the kefiran structure, it is possible to proceed with the analysis of nuclear magnetic resonance Fig 2(a) shows the kefiran 1H Fig Kefiran nuclear magnetic resonance spectra, (a) 1H NMR and (b) 13C NMR (Maeda, Zhu, Suzuki et al., 2004) Adapted with permission from the American Chemical Society, Copyright (2004) NMR in D2O (Maeda, Zhu, Suzuki et al., 2004), which it is verified that the region around 4.4–5.5 ppm of the spectrum contains seven typical signals (a1 – f1) The peak b1 at 4.61 ppm is attributed to (1 → 6) -β-DGalactose corresponding to a small proportion of 2,3,4-tri-O-methyl-Dgalactose (sugar on a side branch) The other peaks show three welldefined signals and three overlapping signals that are attributed to the Carbohydrate Polymers 246 (2020) 116609 L Marangoni Júnior, et al Fig Scanning electron microscopy (SEM) images with the surface morphology of kefiran produced from Tibetan kefir (Chen et al., 2015) Adapted with permission from Elsevier, Copyright (2015) hexasaccharide repeat unit The peak c1 at 5.14 ppm suggests the presence of α-hexapyranosyl The peaks f1 around 4.82 ppm (7.92 Hz), b1 at 4.68 ppm, e1 at 4.53 ppm (7.52 Hz), d1 at 4.53 ppm (7.52 Hz), and a1 at 4.49 ppm (7.92 Hz) are attributed to the pyranose ring forms in an β anomeric configuration (Maeda, Zhu, Suzuki et al., 2004) The results identified in the spectra of Fig 2(a) corroborate the structural characterization presented by other authors (Micheli et al., 1999; Radhouani et al., 2018; Staaf et al., 1996) The 13C NMR spectrum in Fig 2(b) shows six signals in the region around 95−110 ppm The signal c1 around 98.5 ppm indicates an α-hexapyranosyl residue and five β-hexapyranosyl residues at 105.7 ppm (a1, b1, d1, e1 and f1) In a complementary way, the analysis of Fourier-transform infrared spectroscopy (FTIR) is extremely relevant to identify the functional groups characteristic of kefiran or other EPS Numerous researches are available in literature with details of this characterization (Chen et al., 2015; Moradi & Kalanpour, 2019; Radhouani et al., 2018; Semeniuc, 2013) In all of them, some regions of the spectrum should be highlighted The prime region and usually the first to be evaluated represent the one with an intense and broad peak around 3400 cm−1 This signal corresponds to the intramolecular vibration of hydroxyl or intermolecular hydrogen bonding of the polysaccharide Weak absorption close to 2930 cm- is related to modes of asymmetric and symmetrical C–H stretching of the sugar chain (Parikh & Madamwar, 2006), that can be attributed to methylene groups Furthermore, another relatively notable peak in the region of 1100 to 1150 cm−1 indicates sections C–O–C and alcoholic groups in carbohydrates (Rodrigues, Carvalho et al., 2005; Rodrigues, Caputo et al., 2005) Finally, an existing peak at 900 cm−1 indicates a β-glycosidic configuration and also modes of glucose and galactose vibration (Davidović et al., 2015) It is relevant to note that EPS with β-glycosidic linkage was considered to retain the most extensive biological activity (Wu et al., 2009) The molecular weights reported for kefiran varied a lot, depending frequently on the conditions of isolation and purification, being in the range of 50 to 15,000 kDa (Ahmed et al., 2013; Exarhopoulos et al., 2018a; Ghasemlou, Khodaiyan, Jahanbin et al., 2012; Liou & Chen, 2009; Maeda, Zhu, Suzuki et al., 2004; Piermaria et al., 2008; Pop et al., 2016; Radhouani et al., 2018) Among this broad range of values found, Exarhopoulos et al (2018a) determined the weight-average molecular weight (Mw) by size exclusion chromatography, finding a Mw value equal to 614.4 kDa, with dispersity equal to 1.978, which indicates randomness of polymer chain sizes These authors also determined that kefiran is semi-crystalline, with a sharp peak around 2θ = 20.9° by Xray diffraction (XRD), and a 27 % crystallinity percentage In addition, the authors delved into the specific viscosity studies of the diluted aqueous solution of kefiran, which provided quite a fascinating structural information At low concentrations of kefiran, the specific viscosity increases linearly as a function of concentration However, at a particular concentration, considered a “critical concentration”, there is an abrupt shift in the gradient of the curve towards more elevated concentrations (Exarhopoulos et al., 2018a) The critical concentration indicates that the individual polymer molecules previously present as single entities in the diluted solution now exceeded the volume of the solution The result is an overlap of molecules (Morris et al., 1981) Exarhopoulos et al (2018a) identified a critical concentration of 0.53 g dL−1 for kefiran Conversely, Piermaria et al (2008) reported a critical concentration equal to 0.35 g dL−1 These and other researches provided the values of intrinsic viscosity ([η]) for the diluted solutions of kefiran by fitting the equations of Huggins and Kraemer In this context, molecular weight can be correlated as a function of intrinsic viscosity Some intrinsic viscosity values for diluted solutions of kefiran reported in the literature using Huggins and Kraemer equations, respectively, were: 600 mL g−1 and 595 mL g−1 (Mw = 10,000 kDa) (Piermaria et al., 2008), 584 mL g−1 and 553 mL g−1 (Mw = 1350 kDa) (Ghasemlou, Khodaiyan, Gharibzahedi, 2012; Ghasemlou, Khodaiyan, Jahanbin et al., 2012), 84.6 mL g−1 and 85.2 mL g−1 (Mw =706 kDa) (Exarhopoulos et al., 2018a) Through the analysis of these values, it is possible to notice the drastic reduction in the intrinsic viscosity of the solution due to the reduction in molecular weight Its intrinsic viscosity values can be considered relatively high However, previous work reported a much lower viscosity for kefiran when compared to other polysaccharides such as guar gum, locust bean gum and methylcellulose (Piermaría et al., 2016) The aforementioned works calculated the Huggins parameter (k’), and also the difference between the Huggins and Kraemer parameters (k’- k”) The reported values for k’ varied between 0.3 and 0.8, indicating that water can be considered a good solvent The difference between the constants (k’ – k”) was near 0.5, suggesting a random coil shape for kefiran in aquous solution (Exarhopoulos et al., 2018a; Ghasemlou, Khodaiyan, Gharibzahedi, 2012; Ghasemlou, Khodaiyan, Jahanbin et al., 2012; Piermaria et al., 2008; Yang & Zhang, 2009) With regard to surface morphology, kefiran and other EPS exhibit attractive characteristics Fig illustrates the surface morphology of EPS produced from Tibetan kefir by scanning electron microscopy (SEM) analysis Arrows A and B indicate a grainy appearance and irregular surface under magnifications of 2000 and 5000 times However, under greater magnification (10,000 times) it appears that the material retains a compact structure, with smooth surfaces without the presence of pores (Chen et al., 2015) Comparatively, the kefiran surface is extremely similar to the appearance of L kefiranofaciens ZW3 EPS (Ahmed et al., 2013) The compact structure illustrated in the 10,000 times magnifications suggests this material displays significant potential for the production of plasticized films 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 Carbohydrate Polymers 246 (2020) 116609 L Marangoni Júnior, et al worth US$2154.9 mn by the end of 2025 (“Global Kefir Market,” 2017) The starter culture used to produce the drink consists of gelatinous irregular grain shapes with diameters ranging from to 15 mm (GüzelSeydim et al., 2000) These grains have a varied and complex microbial composition, which includes yeast species, lactic acid bacteria, acetic acid bacteria and mycelial fungi (Takizawa et al., 1998), all kept together by kefiran This exopolysaccharide is one of the lactic acid bacteria products, which can reach up to 50 % (w / w) of grains on a dry basis (Exarhopoulos et al., 2018b) Among the various microorganisms isolated from kefir grains, the following stand out: Lactobacillus kefir, Lactobacillus parakefir, Lactobacillus brevis, Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus kefirgranum, Lactobacillus kefiranofaciens, Lactobacillus sp KPB-167B and Lactobacillus casei (Bosch et al., 2006; Dertli & Çon, 2017; Jeong et al., 2017; Takizawa et al., 1998; Xing et al., 2017; Yokoi & Watanabe, 1992) Greater variability has been reported in the lactic acid Streptococcus population (443 %) than Lactobacillus (28 %) and yeasts (35 %), isolated from kefir grains (Ninane et al., 2005) In addition, it was also demonstrated that the lactic acid bacteria and yeasts present in kefir grains vary significantly, from 6.4 × 104 - 8.5 × 108 and 1.5 × 105 - 3.7 × 108 cfu / mL, respectively (Witthuhn et al., 2004) Most of the research directed to the production of kefiran used a pure culture of the species Lactobacillus sp KPB-167B (Yokoi & Watanabe, 1992), Lactobacillus kefirgranum sp nov and L parakefir (Takizawa et al., 1994), among others (Moradi & Kalanpour, 2019) The most prominent one has been the species Lactobacillus kefiranofaciens (Dailin et al., 2014; Jeong et al., 2017; Xing et al., 2017) On the other hand, the mixed culture of L kefiranofaciens and Saccharomyces cerevisiae has also been extensively studied (Cheirsilp & Radchabut, 2011; Cheirsilp et al., 2007; Cheirsilp, Shimizu et al., 2003; Cheirsilp, Shoji et al., 2003; Tada et al., 2007) The fundamental researches indicated that this alternative can significantly increase the production of kefiran in relation to the pure cultures For example, it was observed that with this mixed culture, under anaerobic conditions, the production rate of kefiran was 36 mg L−1 h−1 (Cheirsilp, Shimizu et al., 2003), which is 50 % higher than that obtained using pure culture (24 mg L−1 h−1) 3.3 Extraction and purification of kefiran It has been reported that kefiran can represent about 50 % or more of the dry mass of kefir grains (Exarhopoulos et al., 2018b) Thus, after choosing the optimum fermentation conditions, the kefiran isolation and purification steps are essential to guarantee a high purity polymer For this, several procedures have been described in the literature with some similarities (Micheli et al., 1999; Pais-Chanfrau et al., 2018; Piermaria et al., 2008; Pop et al., 2016; Zajšek et al., 2011) In general, the procedure consists of adding a certain amount of kefir grains in hot water, under agitation, temperature and fixed times Then, the mixture must be cooled and centrifuged to remove microbial cells and proteins The polysaccharide dissolved in the supernatant is then purified by freezing overnight, followed by slow thawing After that, the mixture undergoes cold centrifugation, and the kefiran-rich pellets undergo dissolution in hot distilled water The purification procedure is repeated twice to obtain a high purity kefiran solution Fig provides a simplified overview with the essential steps reported in the literature The first point to be highlighted in the procedure refers to the hot water extraction step Some authors have not specified the exact temperature used However, temperatures close to 100 °C have been reported to cause polymer degradation A recent study has shown that this initial hot water extraction phase influences considerably the quality of this polysaccharide (Pop et al., 2016) In this research, the authors evaluated the effects of temperature (from 60 to 100 °C) and time (from to h) on the rheological and structural characteristics of the kefiran It was exposed that the kefiran solution viscosity decreased as the temperature and residence time increased The more severe conditions led to obtaining polysaccharides with lower molecular weight Finally, the material was degradated during processing at 100 °C The polysaccharide with the most superior molecular weight (about 15,000 kDa) was obtained by extraction at 80 °C and 30 (Pop et al., 2016) Another pertinent point in the procedure reported in Fig is associated with the kefiran precipitation by cold ethanol Several researches used absolute ethanol (Dailin et al., 2016; Piermaria et al., 2008; Radhouani et al., 2018; Taniguchi et al., 2001) However, considering a possible scale-up, a recent study evaluated the effect of using 96 % ethanol, which provides a more reduced cost The results of kefiran yield in both procedures not present significant differences between them, suggesting this may be an economical alternative in the precipitation stage (Pais-Chanfrau et al., 2018) Finally, the centrifugation steps reported in the aforementioned researches varied in time (from 10−45 min) and the centrifugal force used (from 5,000 to 10,000 g) 3.2 Strategies for optimizing kefiran yield The primary operational parameters that must be evaluated to obtain an optimal yield in the production of kefiran are the temperature and pH In addition, other key parameters must equally be considered, such as the type and concentration of microorganisms and nutrients, and the high cost of producing kefiran is mainly related to sources of carbon and nitrogen (Moradi & Kalanpour, 2019) As follows, these factors can be assessed univariately or with the help of the response surface methodology (RSM), to identify the optimal production conditions Despite being a very effective tool, RSM has been little explored in the literature for maximizing kefiran yield Regarding the effects of these factors individually, it was reported that the highest production of kefiran was obtained in the range of 20–30 °C (Blandón et al., 2018; Dailin et al., 2015; Ghasemlou, Khodaiyan, Gharibzahedi, 2012; Montesanto et al., 2016; Zajšek et al., 2013) In parallel, most research has reported the ideal pH for maximum kefiran production is between the values of and (Cheirsilp, Shimizu et al., 2003; Ghasemlou, Khodaiyan, Gharibzahedi, 2012; Zajšek & Goršek, 2011; Zajšek et al., 2013) Once the culture of microorganisms has been chosen, it proceeds with the development of the most appropriate medium Thus, distinct types and concentrations of key nutrients, such as carbon sources (glucose, mannitol, sucrose, lactose), nitrogen sources (yeast extract, peptone, meat extract, casein hydrolyzate) have been investigated in the literature Some of these surveys are highlighted in Table Kefiran-based films Currently, kefiran-based materials are gaining prominence for possessing unique properties, including biodegradability, safety, biocompatibility, stabilizing and emulsifying effect, satisfactory mechanical and water vapor barrier properties (Jain et al., 2020) Moreover, kefiran films have good visual aspects and are effectively produced with edible plasticizers, such as glycerol Therefore, the use of kefiran in film production can lead to suitable packaging and specific protective coatings with improved properties It results in high-quality food and consequently contributing to an increase in shelf life (Moradi & Kalanpour, 2019) In the literature some researches have developed pure kefiran films, which have evaluated different concentrations and types of plasticizers, development of kefiran blends with other biopolymers, inclusion of essential oils and nanofillers in kefiran films and application of radiation to improve the films properties, as described below Carbohydrate Polymers 246 (2020) 116609 L Marangoni Júnior, et al Table Optimized conditions for kefiran production using L kefiranofaciens, pure or mixed with S cerevisiae Type of methodology for optimization and optimal operating conditions Response analyzed References Univariate optimization; fermentation medium: whey lactose; lactose concentration: 4%; yeast extract: 4%; pH: 5.5; temperature: 30 °C; time: 48 h Response surface optimization; fermentation medium: whey; lactose concentration: 88.4 g L−1; concentration of yeast extract: 21.3 g L−1; pH: 5.2; temperature: 20 °C Response surface optimization; fermentation medium: whey; lactose concentration: 67 g L−1; concentration of yeast extract: 13 g L−1; pH: 5.7; temperature: 25 °C, time: 24 h Response surface optimization; lactose concentration: 50 g L−1; yeast extract concentration: 12 g L−1; temperature: 30 °C Response surface optimization; fermentation medium: whey; glucose concentration: 15 %; temperature: 30 °C; Time: 10 h Response surface optimization; fermentation medium: whey lactose; sugar concentration: 2%; yeast concentration: g L−1; pH: 5.5; temperature: 35 °C time: 48 h Kefiran production rate: ∼ 53 mg L−1 h−1 Maximum grain increase: 81.34 % Kefiran production rate: 29.7 mg L−1 h−1 Kefiran production rate: 21 mg L−1 h−1 Kefiran production rate: 37.14 mg L−1 h−1 Kefiran production rate: ∼ 35 mg L−1 h−1 Cheirsilp and Radchabut (2011) Ghasemlou, Khodaiyan, Gharibzahedi (2012) Zajšek et al (2013) Dailin et al (2015) (Blandón et al., 2018) Cheirsilp et al (2018) film-forming solutions, and the glycerol proportions were 12.5–50.0 % (w/w) (based on kefiran weight) (Piermaria, Pinotti et al., 2009) Other studies have been carried out varying the glycerol content, for example, the study of Ghasemlou et al (2011d), with films containing 2% kefiran and glycerol concentrations of 15, 25 and 35 % (w/w) (based on kefiran weight); and the work of Coma, Peltzer, Delgado, & Salvay, (2019) that prepared a film with 3% Kefiran and 0, 10, 20, 30 % glycerol (w/w) (based on kefiran weight) Furthermore, the opportunity and the need to use other plasticizers have also been reported by Ghasemlou et al (2011a) with sorbitol (15, 25 and 35 % w/w based on kefiran weight), by Ghasemlou et al (2011c) with oleic acid (OA) (15, 25 and 35 % w/w based on the weight of kefiran) and Tween 80 as an emulsifier (1% of OA concentration), by Piermaria et al (2011) with polyols and sugars (galactose, glucose, sucrose, glycerol or sorbitol (25 g/100 g kefiran) and by Ghasemlou et al (2011b) with glycerol, sorbitol and oleic acid (OA) (25 % w/w based on the weight of kefiran), in the solutions with OA, Tween 80 emulsifier was added (1% of the concentration OA) 4.2 Kefiran blend films and nanocomposites Pure kefiran films demonstrate the potential to be applied as packaging However, it is necessary to improve the properties of the films Therefore, the mixture of kefiran with other biopolymers (polysaccharides, proteins, among others) aims to include better properties and in some cases make the material more attractive In addition, the incorporation of nanoparticles, essential oils and other active compounds should be considered, since these components can provide other functions to the films (light barrier, antioxidant and antimicrobial activities) Fig Simplified flowchart summarizing the main steps of the purification procedures described in the literature 4.2.1 Kefiran-based film with polysaccharides Polysaccharides are naturally occurring polymers, including starch, cellulose, pectin and chitosan, which remains why they are widely employed to prepare edible films and/or coatings (Hassan et al., 2018) Starch is the most widely used renewable polysaccharide for the development of edible films and coatings, because of its abundance, costbenefit ratio and excellent film forming skills Starch films possess good optical, organoleptic and gas barrier properties, however, they demonstrate limitations due to their hydrophilicity and are weak in mechanical properties (Ogunsona et al., 2018; Ojogbo et al., 2020; Thakur et al., 2019) Therefore, several mixtures and composites have been developed to overcome their sensitivity to humidity and mechanical properties (Jiang et al., 2020) In the literature, several authors work with kefiran blends with starch The study of Motedayen et al (2013) developed kefiran/starch blends with kefiran contents ranging from 7030 % In addition, other works developed composites based on kefiran/ starch added with zinc oxide (ZnO) (Babaei-Ghazvini et al., 2018; Shahabi-Ghahfarrokhi & Babaei-Ghazvini, 2019) and added titanium oxide (TiO2) and with solution exposed to UV-A radiation for up to 12 h 4.1 Plasticized kefiran films Films of pure kefiran, without the use or combination of another polymer, were developed and studied for packaging applications by several research groups However, for obtaining and forming films with good characteristics, the incorporation of other ingredients is necessary, including typically plasticizers and emulsifiers Hence, improving the flexibility of the films due to their stability and compatibility with the hydrophilic chains of the biopolymers, reducing the intermolecular forces and increasing the mobility of the polymer chains (Motedayen et al., 2013; Piermaria et al., 2011) In addition, it has been reported that films prepared without plasticizer exhibit brittle aspects and are difficult to obtain (Ghasemlou et al., 2011a) The first research with kefiran films was related to the necessary proportion of kefiran and plasticizer, to obtain films with good characteristics The kefiran concentrations were 0.5, 0.75 and 1.0 % in the Carbohydrate Polymers 246 (2020) 116609 L Marangoni Júnior, et al for physical modification and radical formation (Goudarzi & ShahabiGhahfarrokhi, 2018) Chitosan is a deacetylated derivative of chitin It is a functional versatile biopolymer due to the presence of amino groups responsible for the various polymer properties, including barrier properties and its antimicrobial activity (Priyadarshi & Rhim, 2020) In addition, it demonstrates excellent structural properties, which allow the formation of a continuous layer of food coatings, which is why it has been employed successfully in food applications (Devlieghere et al., 2004; Hassan et al., 2018) Blend films composed by kefiran/chitosan were developed by Sabaghi, Maghsoudlou, and Habibi (2015), using Kefiran (2%) and chitosan (2%) solutions, resulting in different film proportions with kefiran contents ranging from 32 to 78 % The objective was to exploit a by-product from the fishing industry to improve the properties of kefiran films, with the suggestion of implementing it as food packaging Cellulose itself is a polysaccharide composed of glucose units, being a water-insoluble polymer that can be chemically modified to form water-soluble cellulose ethers, for example, carboxymethylcellulose (CMC) (Fiori, Camani, Rosa, & Carastan, 2019) CMC is the most common cellulose-derived biopolymer for the preparation of films and coatings (Dashipour et al., 2015), as it presents good film formation skills However, it presents strictly limited mechanical properties and water vapor barrier, which restricts its use in potential food packaging applications (Fiori et al., 2019) Therefore, research aimed at improving the films properties was developed, such as the study by Hasheminya et al (2019a) with biocomposite films made from kefiran (1%), carboxymethylcellulose (CMC) (1%), glycerol (50 % of dry weight), essential oil (EO) from Satureja khuzestanica (0.0, 1.0, 1.5 and 2.0 % v/v) and Tween 80 emulsifier (0.5 % v/v based on EO) and the study by Hasheminya et al (2019b) that added copper oxide (CuO) nanoparticles and simultaneous addition of CuO and essential oil (EO) Satureja khuzestanica The development of kefiran blends with other polysaccharides, such as alginates, pullulan and pectin can still be explored for application in films and coatings In addition, the extraction of polysaccharides from food industry residues for this application should be considered, since an ingredient from the food industry itself will not be used have addressed the incorporation of essential oils for a specific purpose, such as antimicrobial activity, which is discussed in section 7.10 4.2.4 Kefiran-based nanocomposites and other developments Theoretically, the incorporation of nanoparticles in biopolymer films aims to reduce the effective permeation area of the films (Zhao et al., 2020) This characteristic is attributed to the change in the diffusion path of the molecules, which makes it more tenuous and long during the diffusion phenomenon This results in better barrier properties, provided that the nanoparticles are well dispersed throughout the polymeric matrix Moreover, the incorporation of these nanomaterials can improve thermal, physical and mechanical properties, besides in some cases adding specific functions, such as antimicrobial activity (Joshi et al., 2018) Research evaluating the incorporation of nanoparticles in kefiran films has been carried out in recent years The nanoparticles evaluated were aluminum oxide (Al2O3) (Moradi et al., 2019), zinc oxide (ZnO) (Shahabi-Ghahfarrokhi et al., 2015b) and nano-cellulose (NC) (Shahabi-Ghahfarrokhi et al., 2015a) In addition, titanium oxide (TiO2) and montmorillonite (MMT) were used in kefiran/WPI blends (Zolfi et al., 2014a, 2014b) and zinc oxide (ZnO) in kefiran/starch blends (Babaei-Ghazvini et al., 2018; ShahabiGhahfarrokhi & Babaei-Ghazvini, 2019) Other applications consisted of the application of UV-A radiation and γ irradiation in film solutions, to improve the its properties (Goudarzi & Shahabi-Ghahfarrokhi, 2018; Shahabi-Ghahfarrokhi et al., 2015) Properties of the film-forming solutions The rheological properties of film-forming solutions must be determined, since they will inform about the processing conditions for obtaining the films (Piermaria, Pinotti et al., 2009) Aqueous filmforming solutions of kefiran (10 g kg−1) added with glycerol plasticizer (0, 25 and 50 g/100 g of kefiran) presented pseudoplastic behavior The same was observed for kefiran films with oleic acid (Ghasemlou et al., 2011c) and sorbitol (Ghasemlou et al., 2011b) The viscosity values were less than 0.50 Pa s and without visible air bubbles, which results in a thin and level layer for casting In addition, the apparent viscosity of the solutions was uninfluenced by the different glycerol concentrations (Piermaria et al., 2009) The intrinsic viscosity provides a view of the hydrodynamic volume occupied by a given polymer and the length of the polymer chain (Piermaria et al., 2008; Shahabi-Ghahfarrokhi & Babaei-Ghazvini, 2019) The increase in the concentration of oleic acid in the kefiran solution produced an increase in viscosity (Ghasemlou et al., 2011c) The work by Shahabi-Ghahfarrokhi and Babaei-Ghazvini (2019) evaluated the intrinsic viscosity of kefiran/starch/ZnO solutions subjected to UV radiation (0, 1, and 12 h) It has been observed that viscosity decreases as radiation exposure time increases The authors attributed these results to the formation of free radicals that induced molecular changes and fragmentation Similar behavior was observed for kefiran/starch/ TiO2 solutions, attributed to the effect of radiation on the breaking of polymer chains in shorter chains (Goudarzi & Shahabi-Ghahfarrokhi, 2018) It is worth mentioning solutions with high viscosity are difficult to be homogenized, which can result in films with a certain heterogeneity In addition, air bubbles tend to get trapped in viscous solutions, which can result in defective films Moreover, low viscosity solutions can lead to the formation of films with reduced thickness, because of the significant dilution of the solutions (Piermaria et al., 2009) That is, it is necessary to find an equilibrium viscosity to obtain films without defects and with adequate thickness for the intended application Moreover, the storage module (G’) and loss module o (G”) help to interpret the viscoelastic behavior of polymeric solutions Piermaria 4.2.2 Kefiran-based film with proteins Distinct types of globular proteins, such as whey protein, have been investigated in the development of films and coatings Whey protein isolate (WPI) films are characterized by satisfactory mechanical properties and excellent barrier properties to gases, aromatics and fat However, due to the fact that whey protein is hydrophilic in nature, these films experience some moisture limitations (Hassan et al., 2018) Films composed of blends of kefiran and WPI were developed to improve the films properties (Gagliarini et al., 2019) In addition, other authors have incorporated titanium oxide (TiO2) nanoparticles and montmorillonite (MMT) in proportions of 1, and 5% to obtain films with more robust properties (Zolfi et al., 2014a, 2014b) Other proteins display the potential to develop blends with kefiran It can be cited as zeins, gelatin, wheat gluten and soy protein 4.2.3 Kefiran-based film with lipids Lipids represent excellent barriers to water vapor, and when blended with other biopolymers they can improve barrier and mechanical properties (Hassan et al., 2018) In addition, lipids are effective in blocking moisture release due to their low polarity (Hassan et al., 2018; Perez-Gago et al., 2002) The particular lipids applied in films and coatings are waxes, monoglycerides and surfactants Oleic acid (OA) is a fatty acid with the potential to improve the water vapor barrier of hydrophilic films In this context, Ghasemlou et al (2011c) prepared kefiran films with oleic acid (15–35 % w/w) and Tween 80 emulsifier (1% OA concentration), in order to intensify the water vapor barrier and the mechanical properties Other studies Carbohydrate Polymers 246 (2020) 116609 L Marangoni Júnior, et al et al (2008) reported that low frequency kefiran solutions (1 % w/v) presented a loss module greater than that storage module At higher frequencies, G’ surpassed G”, indicating that the inter-chain tangles did not have enough time to slide and behave like a gel A similar behavior was observed by Radhouani et al (2018) The authors evaluated the behavior at two concentrations of kefiran (1 and 10 % w/v) The 1% samples started with a viscous behavior and 10 % samples started with an elastic behavior Specifically, 1% kefiran samples crossed at approximately Hz, going from a phase angle of about 47° (viscous liquid) to approximately 31° (elastic/gel); while 10 % solutions showed a crossover at 1.6 Hz, going from a phase angle of about 25.7° (elastic/ gel) to an average of approximately 58° (viscous liquid) In that manner, the viscoelastic properties of kefiran indicate its potential application in tissue engineering and regenerative medicine For example, it could be employed in osteoarthritis treatment therapies to restore the viscoelastic properties of the joint synovial fluid Kefiran represents an economical alternative to the traditional hyaluronic acid (Radhouani et al., 2018) As follows, the solvent is removed by evaporation to decrease the distance between the polymer chains, favoring their interaction This interaction allows the formation of a polymer network that will be finalized with the film conformation (Coma et al., 2019; Felton, 2013; Priyadarshi & Rhim, 2020) However, this method can result in slight variations in the film properties, due to variations in the formulations In addition, this method is currently unused on an industrial scale, being uneconomical and time consuming (Priyadarshi & Rhim, 2020; Zhang et al., 2019) The processing conditions for obtaining kefiranbased films have peculiarities in each study Therefore, to better illustrate the differences among some kefiran-based film researches, these data are presented in Table 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 Manufacturing methods Coatings are applied in liquid form on food or on the surfaces of other packaging materials It can be made by immersing the product in a solution or by spraying, followed by drying to adhere the material to the product surface (Maringgal et al., 2020) A coating does not act as packaging, but it can limit intrinsic factors and reduce the barrier requirements of the packaging, and consequently extend the food shelf life (Ganiari et al., 2017; Nor & Ding, 2020) The use of coatings in food applications depends on several characteristics such as: cost, availability, functional attributes and their properties These characteristics are influenced by parameters such as the type of material used as the structural matrix, the processing conditions, the type and additives concentration added (Ganiari et al., 2017) Biopolymers have often been reported as excellent materials for the coating’s development The structural materials utilized in the construction of coatings are based on proteins, lipids and polysaccharides However, no reports were provided on the development and application of kefiran-based coatings The main reason for this is that the study of the kefiran film-forming potential is still in the beginning of its development, and it is doubtful whether researchers will use it for food coating or other related applications In this sense, this topic presents itself as a future trend with great potential to be assessed Biopolymer-based films are typically produced from a solution or dispersion of the film-forming agent, followed by methods that aim to separate it from the solvent (Ghasemlou et al., 2011b) Current techniques for preparing films based on biopolymers include direct casting, coating (Fig 5) and extrusion These methods can be implemented for films based on a single material or mixed materials (blends) The choice of the most effective method will depend on factors such as equipment availability, costs, efficiency, and application 6.1 Direct casting The direct casting method has been widely employed, as it is the simplest method for the preparation of biopolymers-based films based The films’ preparation by the casting method involves the use of at least one film-forming agent (biopolymers), a solvent and a plasticizer To form the film matrix, it is necessary to prepare a homogeneous, viscous film-forming solution containing biopolymers, which will undergo filtration, centrifugation or another method to eliminate insoluble particles and air bubbles, followed by dispersing the solution on a flat-sized surface and shape Fig 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) Constant stirring for 60 Kefiran (5% w/v) and WPI 5% (w/v) (50:50 v/v), added with MMT and nano-TiO2 (0, 1, 3, and 5% w/w) Kefiran 2% and starch 2% (100/0, 70/30, 50/50 and 30/70) Kefiran (10 g/kg) with galactose, glucose, sucrose, glycerol or sorbitol (25 g/100 g kefiran) Kefiran (2%) with 0, 15, 25 and 35 % glycerol (w/w based on kefiran weight) Kefiran (2%) with 0, 15, 25 and 35 % oleic acid (w/ w based on kefiran weight) Kefiran (10 g/kg) with and 25 % glycerol/100 g kefiran Constant stirring for 10 Constant stirring for 10 Constant stirring for 60 Kefiran (2%) and nano cellulose (0, 1, and 3% dry basis) Kefiran (2%) and nano ZnO (0, 1, and 3% dry basis) Kefiran (5% w/v) and WPI 5% (w/v) (50:50 v/v), added nano-TiO2 (0, 1, 3, and 5% w/w) Constant stirring for 15 Constant stirring for 15 Constant stirring Constant stirring for 15 Constant stirring Constant stirring for 15 Constant stirring Kefiran (2%) and chitosan (2%) in proportions (100/ 0, 68/32, 50/50 and 32/68) Kefiran films as vehicle for probiotic microorganisms By sonicator – 50 mL – Constant stirring at 90 °C for 30 Constant magnetic stirring at 50 °C for 1h Constant stirring for 15 Constant stirring for 15 Constant stirring for 15 Constant stirring for 120 Kefiran (2% w/v) and starch (5% w/v) (50:50 v/v), added with nano-ZnO (0, 1, 3, and 5% w/ w) Kefiran/starch/TiO2 exposed to UV-A radiation for 0, 1, and 12 h Kefiran/carboxymethyl cellulose added with copper oxide nanoparticles (1, 1.5 and 2%) kefiran/waterborne polyurethane blend film incorporated with Zataria multiflora and Rosmarinus officinalis EO (5, 10, 15 and 20% v/v) Kefiran (2%) treated at 3, and kGy – Rest until reaching 50 °C Rest until reaching 50 °C 1500 rpm at 70 °C for 1500 rpm at 70 °C for 50 mL – By sonicator – – 70 mL By sonicator By sonicator and Vacuum oven for 30 at 30 °C By sonicator and Vacuum oven for 30 at 30 °C Vacuum oven for 30 at 30 °C – Teflon plates Plastic petri (8.7 cm diameter) 50 g 25 g Under vacuum for – Teflon plates – Plastic petri (8.7 cm diameter) 25 g Rest Teflon plates Teflon plates Teflon plates (15 cm diameter) Plastic petri (10 cm diameter) Plastic petri (5 cm diameter) Teflon plates (15 cm diameter) Teflon plates (15 cm diameter) Teflon plates Glass plates (9 cm diameter) Teflon plates Polystyrene plates (15 cm diameter) Plastic petri Plastic petri (5 cm diameter) Plastic petri (12 cm diameter) Glass plates (9 cm diameter) Glass plates (9 cm diameter) Polystyrene plates (15 cm diameter) Plastic petri Plates material – 70 mL – 3.5 g – By sonicator 15 mL By sonicator – 50 mL – – 50 mL By sonicator 50 mL 50 mL 50 mL By sonicator Constant stirring for 15 Kefiran (2%) + starch (5%) + nano zinc oxide (1%) + glycerol (40 %) exposed to UV radiation for 0, 1, and 12 h Kefiran/CMC and EO by Satureja Khuzestanica (0.0, 1.0, 1.5 and 2.0% v/v) Kefiran/CMC and CuO and the combination of CuO and EO by Satureja Khuzestanica (0.0, 1.0, 1.5 and 2.0% v/v) Kefiran/WPI/glycerol (6%, 2% and 3.2 % w/w) with and without the addition of probiotics Kefiran (2%) with Al2O3 nanoparticles (1, and 5% w/w dry basis) Solution mass 17 g 15,000 rpm for Kefiran (3%) + glycerol (20 and 30 %) Dispersion and/or Degassing Vacuum for 30 Stirring Film material Table Kefiran-based films preparation by casting Room temperature and RH for 18 h 40 °C in a ventilated oven until reaching constant weight for h Room temperature and RH for 18 h Room temperature and RH for 18 h 40 °C in a ventilated oven until constant weight along h Room temperature and RH for 18 h Room temperature and RH for 18 h 25 °C 25 °C 37 °C for 11−14 h 30 °C for 24 h 25 °C for 48 h Room temperature at 48 h 25 °C for 72 h Room temperature at 48 h 25 °C for 48 h 35 °C for 24 h 37 °C for 16 h 25 °C for 72 h 25 °C for 72 h 40 °C and 40 % RH in a ventilated oven until 10−15% water content 25 °C for 48 h Solvent evaporation 21.4 and 21.9 62, 75, 71 and 79 58, 64, 62 and 67 23, 22, 25, 31, 22 and 22 74, 62, 59 and 57 ̴ 74 (MMT) and 74, ̴ 91 (TiO2) 74, 75, 75 and 76 80, 70, 70 and 70 – 13 - 19 34, 33, 32 and 31 80, 80, 60 and 50 140, 130, 120 and 110 100, 80, 80 and 80 90, 107, 110 and 117 90 - 150 14.5, 15.5, 17.9 and 28.8 133 - 143 90, 113, 133 and 153 90 - 161 110 60 Thickness (μm) 55 % 55 % 75 % 50 % 55 % 25 °C and 50 % RH 20 °C and 75 % RH 25 °C 20 °C and 75 % RH 25 °C 25 °C 25 °C and RH 25 °C and RH 20 °C and RH 25 °C and RH 25 °C and RH 25 °C 25 °C and 55 % RH 55 % RH 25 °C and 50−55% RH 50−55% RH 20 °C and 75 % RH 25 °C and 55 % RH 25 °C and 55 % RH 25 °C and 55 % RH – 22 °C and 43 % RH Storage condition Piermaria et al (2009) Ghasemlou et al (2011c) Ghasemlou et al (2011d) Piermaria et al (2011) Motedayen et al (2013) Zolfi et al (2014b) Shahabi-Ghahfarrokhi et al (2015a) Shahabi-Ghahfarrokhi et al (2015b) Zolfi et al (2014a) Piermaria et al (2015) Shahabi-Ghahfarrokhi et al (2015) Sabaghi et al (2015) Rad et al (2018) Babaei-Ghazvini et al (2018) Goudarzi and ShahabiGhahfarrokhi (2018) Hasheminya et al (2018) Moradi et al (2019) Gagliarini et al (2019) Hasheminya et al (2019b) Hasheminya et al (2019a) Shahabi-Ghahfarrokhi and Babaei-Ghazvini (2019) Coma et al (2019) References L Marangoni Júnior, et al Carbohydrate Polymers 246 (2020) 116609 Carbohydrate Polymers 246 (2020) 116609 L Marangoni Júnior, et al 6.3 Extrusion nanoparticles increased This behavior can be illustrated by Moradi et al (2019), that included Al2O3 in the kefiran films In addition, Zolfi et al (2014a) included TiO2 in kefiran/WPI films, Goudarzi & ShahabiGhahfarrokhi (2018) included TiO2 in kefiran/starch films, and also exposed it to UV-A radiation Finally, Shahabi-Ghahfarrokhi & BabaeiGhazvini (2019) included ZnO in Kefiran/starch films exposed to UV radiation Similar behavior was observed for films with essential oils included, such as Hasheminya et al (2019a), that used the Satureja khuzestanica EO in kefiran/CMC films; and the work of Hasheminya et al (2019b) that used CuO and EO Studies report that the reduction in moisture content is due to the increase in the hydrophobic phase in the film after adding EO Moreover, the incorporation of probiotics also led to a reduction in the moisture content of kefiran/WPI films (Gagliarini et al., 2019) On the other hand, the incorporation of ZnO in kefiran films (Shahabi-Ghahfarrokhi et al., 2015b) and in kefiran/starch films (Babaei-Ghazvini et al., 2018) did not influenced the moisture content, regardless of the concentration In addition, the incorporation of nano cellulose in kefiran films led to an increase in moisture content, adding greater hydrophilicity to the film (Shahabi-Ghahfarrokhi et al., 2015a) Extrusion is frequently used in the packaging raw material industry and in the packaging manufacturing industry, as is the case for packaging materials based on fossil source polymers The polymers are heated to the molten state by a combination of two fundamental parameters: heating and shear The screw forces the resin through a mold, manufacturing the resin in the desired shape After that, the extruded material is cooled and solidified as it is pulled by the die or water This technology can be effectively exploited for the preparation of bio-based films (Aider, 2010) However, the extrusion-based kefiran films are yet undeveloped In comparison with other biopolymers, the melting of the kefiran produced by Tibetan kefir took place at about 93 °C, lower than xanthan gum (153.4 °C) and guar gum (490.11 °C) The endothermic enthalpy change required to melt g of kefiran, xanthan and guar gums were 249.7, 93.2 and 192.9 J, respectively (Wang & Bi, 2008) In parallel, Ahmed et al (2013) carried out the thermogravimetric analysis for kefiran, xanthan and locust gums It was observed a most pronounced initial weight loss of kefiran between 40 and 90 °C, which might be attributed to the evaporation of moisture The decline in weights above 90 °C was ascribed to the degradation The onset of decomposition occurred at 261.4 °C The polymer weight loss decreased substantially around 300 °C In another research, the TGA of kefiran presented one event during the increasing of the temperature (40–106 °C) This event occurred with the maximum mass loss (approximately 9%) also associated with the moisture The critical mass loss (12–65 %) occurred in the second event (264–350 °C), which was attributed to degradation of kefiran polysaccharide structure (Radhouani et al., 2018) Other studies of exopolysaccharides showed approximated temperature of degradation, with the maximum between 300–350 °C (Botelho et al., 2014; Moradi et al., 2019) Therefore, for the successful application of kefiran films as food packaging, it is essential to develop researches to optimize the process, considering its thermal characteristics In this sense, this processing area is however lacking in information and can be exploited to maximize the large-scale production of kefiran films 7.2 Solubility The solubility of kefiran-based films reveals the possible applications of this material In some potential food applications, the ideal is that the film presents good insolubility in water, hence improving its integrity and increasing the shelf life of the film However, according to Ghasemlou et al (2011a) in some cases, the film's water solubility is desirable before consumption, especially for edible films This property is substantially influenced by the type and concentration of plasticizer used The solubility of the kefiran film added with glycerol (25 g/100 g of kefiran) increased significantly by increasing temperature, where, all samples were partially soluble at 25 °C and 37 °C, and totally solubilized at 100 °C (Piermaria et al., 2009) In addition, the increase in glycerol content resulted in films with greater solubility (Ghasemlou et al., 2011a, 2011d) According to Coma et al (2019) the increase in the concentration of glycerol resulted in an increase in the amount of hydration water in the kefiran films, consequently leading to a more significant free volume, suggesting the glycerol decreased the attractive forces between the polymeric chains and consequently allowed greater mobility of water molecules On the other hand, exposure of the filmforming solution to γ irradiation reduced the water absorption capacity and the solubility at doses up to kGy (Shahabi-Ghahfarrokhi, Khodaiyan, Mousavi, et al., 2015) The sorbitol plasticizer maintained the solubility similar to the film without plasticizer, regardless of the concentration (Ghasemlou et al., 2011a) While the oleic acid plasticizer reduced the solubility of kefiran films (Ghasemlou et al., 2011c) The blends of kefiran with other biopolymers showed that the film's solubility can vary depending on the nature of each biopolymer Where chitosan (Sabaghi et al., 2015), starch (Motedayen et al., 2013) and cellulose (Shahabi-Ghahfarrokhi et al., 2015a) reduced solubility as their concentration increased That is, the hydrophilic character of these biopolymers exerted a direct influence on the results Another relevant procedure that affects the kefiran-based film solubility is the incorporation of nanoparticles Generally, as the nanoparticle content increases, the solubility of the film decreases These results were observed with Al2O3 for kefiran films (Moradi et al., 2019), ZnO (Babaei-Ghazvini et al., 2018; Shahabi-Ghahfarrokhi et al., 2015b), ZnO in kefiran/starch blends followed by exposure to UV radiation (Shahabi-Ghahfarrokhi & Babaei-Ghazvini, 2019), TiO2 in kefiran/WPI blends (Zolfi et al., 2014a), TiO2 in kefiran/starch blends exposed to UV-A radiation (Goudarzi & Shahabi-Ghahfarrokhi, 2018) and CuO and EO for kefiran/CMC films (Hasheminya et al., 2019b) Characterization and properties of kefiran films 7.1 Moisture content The moisture content and water activity of kefiran films (10 g kg−1) with glycerol plasticizer (0, 12.5, 25.0, 37.5 and 50.0 g/100 g of kefiran) were 14.8–36.4% and 0.453 to 0.556, respectively (Piermaria et al., 2009) That is, as the glycerol concentration increased, there was an increase in the moisture content and water activity Similar behavior was observed by Ghasemlou et al (2011a, 2011d) These results were attributed to the water retention in the film caused by the plasticizer hydrophilicity Other plasticizers were used in kefiran films (galactose, glucose, sucrose or sorbitol 25 g/100 g of kefiran) (Piermaria et al., 2011) and sorbitol (Ghasemlou et al., 2011a) In both studies, the plasticizers did not influence the moisture content The oleic acid plasticizer reduced the moisture content of kefiran films from 17.9–12.3% (Ghasemlou et al., 2011c) Furthermore, the application of γ irradiation (3, and kGy) in kefiran film-forming solutions led to a reduction in the moisture content of the films, because of the improvement of the hydrophobic properties in the polymer with the use of γ irradiation (Shahabi-Ghahfarrokhi et al., 2015) Considering kefiran blends with other biopolymers, the moisture content increased with the incorporation of starch, justified by its greater hydrophilicity (Motedayen et al., 2013) In contrast, it decreased with the incorporation of chitosan, as this biopolymer causes an increase in the hydrophobic phase (Sabaghi et al., 2015) In addition, the incorporation of nanoparticles in kefiran blends and other biopolymers reduced the moisture content of the films as the concentration of Carbohydrate Polymers 246 (2020) 116609 L Marangoni Júnior, et al 7.3 Hydrophobicity the casting surface The gradual addition of plasticizer significantly increased the film's flexibility The microstructure of the film faces and cross sections proved to be continuous and homogeneous, without clusters, pores, flaws or perforations of film (Fig 6) Similar results were noted by Ghasemlou et al (2011d), Piermaria et al (2009) The use of other plasticizers such as polyols and sugars showed morphologies similar to that of films with glycerol (Piermaria et al., 2011) However, when oleic acid was used as a plasticizer, the film showed structural discontinuities associated with the formation of two phases (lipids/polymer) (Ghasemlou et al., 2011c) Regarding blends of kefiran with other biopolymers, nanoparticles addition or radiation exposure, in general, as they increased, morphological differences were observed Films of kefiran/starch/TiO2 submitted to UV-A radiation were rough and heterogeneous, reflecting the low miscibility of starch and kefiran However, the increased exposure time to UV-A produced free-radicals, exhibiting smoother morphology (Goudarzi & Shahabi-Ghahfarrokhi, 2018) The incorporation of EO in kefiran/CMC films exhibited a homogeneous structure without porosity (Hasheminya et al., 2019a) The same was observed for kefiran/CMC films incorporated with EO and CuO (Hasheminya et al., 2019b) Kefiran/WPI films were homogeneous and transparent However, they showed surface roughness, because of the interactions between proteins and polysaccharides (Gagliarini et al., 2019) The incorporation of and 3% of Al2O3 in kefiran films improved the microstructure, that is, there was good dispersion of the particles, resulting in low pores and cracks (Moradi et al., 2019) The surface of kefiran/starch films changed as the amount of starch increased The matrixes morphologies were rougher, related to the formation of channels and the state and structure of the starch granule However, they were flat and compact with remarkably small particles and without any phase separation (Motedayen et al., 2013) Kefiran/starch/ZnO films presented a smooth The water contact angle is performed to determine the hydrophobicity of the biopolymer films The decrease in the contact angle occurred with the increase in the glycerol content in kefiran films (Ghasemlou et al., 2011d) This phenomenon was also observed in kefiran/starch blends as the starch content increased (Motedayen et al., 2013) That is, glycerol and starch resulted in more hydrophilic films Conversely, the increase in the concentration of hydrophobic additives, as oleic acid (Ghasemlou et al., 2011c) and EO of Satureja Khuzestanica in Kefiran/CMC films (Hasheminya et al., 2019a), led to an increase in the contact angle Similar result was obtained by incorporation of nanoparticles, as ZnO in kefiran/starch films (BabaeiGhazvini et al., 2018), TiO2 in kefiran/starch films (Goudarzi & Shahabi-Ghahfarrokhi, 2018) and CuO in kefiran/CMC blends (Hasheminya et al., 2019b); and also by radiation exposure (Goudarzi & Shahabi-Ghahfarrokhi, 2018; Shahabi-Ghahfarrokhi & BabaeiGhazvini, 2019) It is evident the incorporation of these substances increased the water contact angle The consequent improvement in their hydrophobicity can have a direct impact on the other properties and materials shelf life 7.4 Morphological properties - Visual aspect and microstructure The morphological properties of the films can be measured through visual and microscopic analysis, taking into account the film's maneuverability, homogeneity and continuity Kefiran films with the addition of 0, 10, 20, 30 % glycerol were evaluated by Coma et al (2019) The kefiran films with glycerol exhibited a homogeneous aspect, without cracking and high transparency The authors verified that the samples with and 10 % glycerol were brittle, requiring care when peeling from Fig Scanning electron microscopy (SEM) observations of the cross sections and the surface of non-plasticized films (a) (c) and plasticized with 30 % glycerol (b) (d) (Coma et al., 2019) Adapted with permission from Elsevier, Copyright (2019) 10 Carbohydrate Polymers 246 (2020) 116609 L Marangoni Júnior, et al and homogeneous surface, without any crack or bubble However, as the concentration of ZnO increased, aggregates appeared on the film surface (Babaei-Ghazvini et al., 2018) the NC content (up to 2%) and decreased in the film containing 3% NC However, films with NC exhibited no color difference compared to the control In addition, there was no difference between the value of whiteness index of kefiran film and that of kefiran/NC composite films (up to 2%) (Shahabi-Ghahfarrokhi et al., 2015a) The incorporation of ZnO in kefiran/starch films resulted in a difference in luminosity properties, whiteness index and color difference in relation to the control film (Babaei-Ghazvini et al., 2018) Kefiran/chitosan films showed changes in luminosity, decreased from 28.6 to 22.3 with the increase in chitosan content In addition, there was an increase in the film's opacity resulting from the crosslinking between the molecules of the films (Sabaghi et al., 2015) On the other hand, the incorporation of Al2O3 in kefiran films did not affect the luminosity and color difference, attributed to the uniformity and small size of the nanoparticles (Moradi et al., 2019) 7.5 Instrumental color The transparency and color aspects of films based on biopolymers can influence the valuation of the final product The study by Piermaria et al (2009) evaluated the transparency of kefiran films, showing values of 2.71 A600/mm In addition, the authors emphasized the addition of glycerol did not change this property The addition of sugars and polyols produced a variation in the transparency of kefiran films from 1.88 to 3.30 A600/mm (Piermaria et al., 2011) However, the increase in glycerol concentration can cause an increase in luminosity and degree of whiteness and a decrease in the total color difference compared to film without plasticizer (Ghasemlou et al., 2011d) Kefiran/starch/ZnO films exposed to UV radiation for 0, 1, and 12 h were evaluated for instrumental color by Shahabi-Ghahfarrokhi and Babaei-Ghazvini (2019) It was observed that exposure to radiation led to a decrease in the color difference in relation to the white plate In addition, the luminosity and the yellowing index also decreased simultaneously, the authors attributed to the color amend the production of free-radicals induced by UV radiation Similar results were found in kefiran/starch/TiO2 films submitted to UV-A (Goudarzi & ShahabiGhahfarrokhi, 2018) The luminosity and whiteness index of Kefiran/CMC films with EO Satureja Khuzestanica decreased significantly with the increase in EO concentration There was an increase in the total color difference, yellowing index, chroma and opacity The authors attributed these modifications to the phenolic compounds present in the EO (Hasheminya et al., 2019a) Similar results were experienced for kefiran/CMC films with CuO and simultaneous addition of CuO and EO (Hasheminya et al., 2019b) Kefiran/WPI films with or without the addition of probiotic microorganisms revealed no difference in luminosity However, the probiotics addition induced a slightly yellow appearance which generated a total color difference (Gagliarini et al., 2019) The incorporation of MMT and TiO2 in kefiran/WPI films resulted in a slight change in the transparency of the one containing MMT It indicates proper distribution of MMT in the polymer matrix However, for TiO2 films, as the concentration increased, the level of transparency decreased, because of its metallic nature (Zolfi et al., 2014b) The luminosity factor of kefiran films increased with the increase in 7.6 Thermal behavior and changes in the IR spectrum The thermal behaviors of the films can be observed utilizing the techniques of differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) The attenuated total reflection Fouriertransform infrared spectroscopy (ATR-FTIR), X-ray diffraction (XRD), among others techniques must equally be used in a complementary way to the analysis of thermal behavior Because when the films are obtained, some changes in the spectra discussed in item of this article may undergo some changes The results can be directly related to the mechanical and barrier performance of the films as well FTIR is an effective technique for detecting chemical bonds in materials In most cases, the FTIR spectra of kefiran films can be classified as follows: the primary region is attributed to hydroxyl groups (OH) due to water and carbohydrates (3000−3600 cm−1) The second region is related to the symmetrical and anti-symmetric stretching modes of CH in methyl (CH3) and methylene (CH2) functional groups (2800−3000 cm−1) The third region is designated to the OH flexion mode in water molecules (1580−1700 cm‐1) The fourth zone contains the peaks attributed typically to the ways of stretching the carbohydrate rings and side groups (C–O–C, C–OH, C–H) (900−1200 cm‐1) (Coma et al., 2019; Goudarzi & Shahabi-Ghahfarrokhi, 2018; Hasheminya et al., 2019a; Piermaria et al., 2011; Shahabi-Ghahfarrokhi & Babaei-Ghazvini, 2019) It's significant to highlight that, when added plasticizers, the first and third regions showed larger bands It is a result from the increase in water content induced by the plasticizer and also by the presence of the glycerol itself (Coma et al., 2019) Still in this context, when zinc oxide Table DSC results for kefiran films Film material Conclusions References Kefiran (2%) treated at 0, 3, and kGy The films showed Tg of -16, -15, -14 and −23 °C and Tm of 85, 87, 86 and 85 °C The Tg of the samples increased with an increase in the irradiation dose by up to kGy and then decreased in higher doses kGy The increase in Tg was attributed to the fact that the low dose of γ-irradiation increased the cross-links between the biopolymer’s chains, but at high doses, the plasticizing effect of mono and disaccharides in kefiran decreased The Tm of the film did not change with changes in the γ irradiation dose The films presented Tg of -16, -27, -30 and −38 °C and Tm of 85, 87, 88 and 86 °C The incorporation of nano cellulose drastically reduced the Tg of kefiran films The films showed Tg of -16, -20, -22 and -30 °C and Tm of 85, 86, 87 and 88 °C Tg decreased with increasing ZnO content and Tm increased Tg reduced from -12 to -14 °C and Tm reduced from 82 to 72 °C, after adding TiO2 This behavior was due to the interruption of the regularity of the chain structures in the biopolymers matrix and to the increase in spacing between the chains As the OA concentration increased, Tg reduced from -16 to −22 °C and Tm increased form 69–92 °C, attributed to the higher molecular weight and the more hydrophobic nature of OA As the glycerol concentration increased, Tg and Tm decreased (Tg -14 at −21 °C and Tm 73 at 68 °C), attributed to the inherent structural characteristics (chain mobility) and high hydrophilic nature Shahabi-Ghahfarrokhi et al (2015) Kefiran (2%) and nano cellulose (0, 1, and 3% (dry basis)) Kefiran (2%) and nano ZnO (0, 1, and 3% (dry basis)) 5% WPI (w/v) and Kefiran (5% w/v) (50:50 (v/v)), added nano-TiO2 (0, 1, 3, and 5% (w/w)) Kefiran (2%) with 0, 15, 25 and 35 % oleic acid (w/w based on kefiran weight) Kefiran (2%) with 0, 15, 25 and 35 % glycerol (w/w based on kefiran weight) Tg: glass transition temperature; Tm: melting temperature 11 Shahabi-Ghahfarrokhi et al (2015a) Shahabi-Ghahfarrokhi et al (2015b) Zolfi et al (2014a) Ghasemlou et al (2011c) Ghasemlou et al (2011d) Carbohydrate Polymers 246 (2020) 116609 L Marangoni Júnior, et al was included in the kefiran and starch film, lesser peaks of around 500 cm−1 were observed (Shahabi-Ghahfarrokhi & Babaei-Ghazvini, 2019) Characteristic differences in peaks in the 400 to 4000 cm−1 range were also observed after addition of CuO in kefiran/CMC films (Hasheminya et al., 2019b) Thermogravimetric analysis (TGA) of kefiran films with 0, 10, 20 and 30 % glycerol was performed by Coma et al (2019) A degradation of the plasticized samples was observed at more reduced temperatures than those not plasticized This is justified by the presence of glycerol in the film matrix, which increases the mobility of the chains and exposes the polymer chains even more to thermal degradation According to Moradi et al (2019), the thermal degradation of kefiran films with glycerol starts at around 220 °C, being more expressive at around 250−320 °C The X-ray diffraction patterns of non-plasticized and plasticized kefiran films with polyols and sugars revealed a semi crystalline structure The degrees of crystallinity obtained were less than 3.1 % (Piermaria et al., 2011) The incorporation of Al2O3 nanoparticles in kefiran film significantly affected the crystalline region of the films, where the peak intensity decreased with increasing Al2O3 concentration (Moradi et al., 2019) The findings from the DSC analysis of kefiran films are indicated in Table Predominantly, it was observed that as the exposure time to radiation or the concentration of plasticizer, nanocomposites and other biopolymers increases, the glass transition temperature was reduced In addition, changes in the melting temperature resulting from the different formulations were observed 7.7 Mechanical properties The mechanical properties of films are associated with their performance during the handling, storage and distribution conditions imposed for each type of application (Marangoni Júnior et al., 2019) Traditionally, biopolymer films possess reduced mechanical resistance Therefore, to improve this aspect, the development of blends and the incorporation of nanoparticles must be considered The results of mechanical properties of kefiran films found in the literature are described in Table Table Mechanical properties of kefiran films Film material Thickness (μm) Tensile strength (MPa) Elongation at break (%) Young's Module (MPa) Conclusions References Kefiran (3%) + glycerol (20 and 30 %) 60 13 - 2.5 - 275 900 - 54 Coma et al (2019) Kefiran (2%) + starch (5%) + nano zinc oxide (1%) + glycerol (40 %) exposed to UV radiation for 0, 1, and 12 h 110 6.7 – 8.2 155 - 31 31 – 35 Kefiran/CMC and EO by Satureja Khuzestanica (0.0, 1.0, 1.5 and 2.0% v/v) 90, 113, 133 and 153 3.2 – 4.2 80 - 65 – Kefiran/CMC and CuO and the combination of CuO and EO by Satureja Khuzestanica (0.0, 1.0, 1.5 and 2.0% v/v) Kefiran/WPI/glycerol (6%, 2% and 3.2 % w/w) with and without the addition of probiotics Kefiran (2%) with Al2O3 nanoparticles (1, and 5% w/ w dry basis) 90 - 161 3.2 – 4.5 3.2 – 5.0 80 – 61 80 - 56 – 133 and 143 1.8 – 1.8 80 -105 – The film with 30 % plasticizer showed a reduction in Young's module and tensile strength followed by an increase in elongation at break compared to the film with 20% plasticizer Exposure to UV light increased the tensile strength and the Young’s module of the films, consequently led to a reduction in elongation at break and there was no influence on elastic energy These results were due to the kefiran/starch interaction with the ZnO surface, which resulted in an increase in the density of crosslinks by a free radical content mechanism The incorporation of EO led to increased tensile strength and decreased elongation at break, attributed to the formation of new hydrogen bonds between the hydroxyl groups of kefiran/CMC with EO The tensile strength increased and the elongation at break was reduced after the addition of CuO and EO, showing good interaction between CuO and EO with kefiran and CMC The kefiran/WPI films showed intermediate values in the mechanical properties, compared to kefiran films and WPI films There was no influence of probiotics 14.5, 15.5, 17.9 and 28.8 26 – 7.6 1.4 – 0.8 – Moradi et al (2019) Kefiran (2% w/v) and starch (5% w/v) (50:50 v/v), added with nano-ZnO (0, 1, 3, and 5% w/ w) 140, 130, 120 and 110 4.9 - 6.3 163 - 152 21 – 34 Kefiran/starch/TiO2 exposed to UV-A radiation for 0, 1, and 12 h 100, 80, 80 and 80 3.5 – 4.0 129 - 87 33 – 25 Kefiran (2%) treated at 3, and kGy 80, 80, 60 and 50 6.4 - 19 18 -10 – Kefiran (2%) and chitosan (2%) in proportions (100/0, 68/32, 50/50 and 32/68) 34, 33, 32 and 31 0.7 – 2.3 60 - 89 – The elasticity module showed no difference between the films The tensile strength decreased as the concentration of Al2O3 increased The elongation at break was greater for the film with 1% Al2O3 and decreased as the concentration of and % of Al2O3 Tensile strength and young modulus increased, with an increase in ZnO content of up to 3%, while elongation at break decreased The origin of these changes is the interfacial interaction between the polymeric matrix and ZnO As the time of radiation exposure increased, the tensile strength increased, the elongation at break decreased, resulting from the creation of cross-links formed by UVA The mechanical properties were improved after the irradiation doses, one of the important reasons for increasing the mechanical properties by γ irradiation was the creation of crosslinking in the polymeric chain The evaluated properties improve with the incorporation of chitosan, resulting from the intermolecular forces of the chitosan biopolymer ShahabiGhahfarrokhi and Babaei-Ghazvini (2019) Hasheminya et al (2019a) Hasheminya et al (2019b) Gagliarini et al (2019) Babaei-Ghazvini et al (2018) Goudarzi and ShahabiGhahfarrokhi (2018) ShahabiGhahfarrokhi et al (2015) Sabaghi et al (2015) (continued on next page) 12 Carbohydrate Polymers 246 (2020) 116609 L Marangoni Júnior, et al Table (continued) Film material Thickness (μm) Tensile strength (MPa) Elongation at break (%) Young's Module (MPa) Conclusions References Kefiran (2%) and nano cellulose (0, 1, and 3% dry basis) – 6.4 – 8.1 18 - 252 382 - 70 ShahabiGhahfarrokhi et al (2015a) Kefiran (2%) and nano ZnO (0, 1, and 3% dry basis) 80, 70, 70 and 70 6.4 - 13 18 - 221 – Kefiran (5% w/v) and WPI 5% (w/ v) (50:50 v/v), added nanoTiO2 (0, 1, 3, and 5% w/w) 74, 75, 75 and 76 6.5 – 3.5 84 - 334 105 - 42 Kefiran (5% w/v) and WPI 5% (w/ v) (50:50 v/v), added with MMT and nano-TiO2 (0, 1, 3, and 5% w/w) ̴ 74 (MMT) and 74, ̴ 91 (TiO2) 6.5–11 6.5 – 3.5 84 – 45 84 - 334 105 – 315 105 - 42 Kefiran 2% and starch 2% (100/0, 70/30, 50/50 and 30/70) 74, 62, 59 and 57 4.8 - 5.6 57 - 140 – Kefiran (10 g/kg) with galactose, glucose, sucrose, glycerol or sorbitol (25 g/100 g kefiran) 23, 22, 25, 31, 22 and 22 34 – 7.5 2.0 - 130 – Kefiran (2%) with 0, 15, 25 and 35 % glycerol (w/w based on kefiran weight) Kefiran (2%) with 0, 15, 25 and 35 % oleic acid (w/w based on kefiran weight) 58, 64, 62 and 67 11 – 5.0 40 - 162 – The tensile strength was not significantly changed, regardless of the concentration of nano cellulose The elongation at break of the films with NC was greater than the control, however as the NC concentration increased it tended to reduce Young's module decreases as the concentration of NC increases, attributed to the plasticizing properties of mono and disaccharide residues in the NC Higher ZnO concentrations increased the tensile strength and elongation at break, due to the interfacial interaction between the loads and the matrix TiO2 decreased the tensile strength and Young's modulus and increased the elongation at break, attributed to the decrease in the crosslinking in the film matrix, to the increase in the mobility of the chains and to the preventive effect of nanoparticles in the hardening by deformation of the polymer chains MMT increased the tensile strength and Young's modulus and reduced the elongation at break TiO2 decreased the tensile strength and Young's modulus and increased the elongation at break This was due to differences in structure and the number of active hydroxyl groups The tensile strength values increased first with the addition of starch, then decreased with the increase in the starch content, due to the formation of intermolecular hydrogen bonds between the two main components and the plasticizer The elongation values at break were greater in the proportion of 50/50, that is, the flexibility of the film was better in this proportion The addition of plasticizers to the kefiran films reduced the tensile strength and the modulus of elasticity, while the elongation at break increased, as it interfered with the association of the polymeric chain, facilitating the sliding and increasing the flexibility of the film Glycerol-free films showed less tensile strength and greater elongation at break 62, 75, 71 and 79 6.2 – 3.3 100 - 40 – Ghasemlou et al (2011c) Kefiran (10 g/kg) with and 25 % glycerol/100 g kefiran 21.4 and 21.9 41 - 15 2.7 – 117 – The elongation decreased as the OA concentration increased The tensile strength of films with AO was lower than the control, but the difference in concentration had no influence on this property, probably because it weakened inter-molecular interactions The plasticizer reduced the tensile strength and increased the elongation at break Although the plasticizer improves the flexibility of the film, the high moisture content influenced the mobility of the polymer chains The mechanical properties of the films depend on the characteristics of each isolated material, as well as on the composition of ingredients used in the development of these films Kefiran blending with biopolymers such as starch, CMC, WPI and chitosan, as well as the incorporation of nanoparticles (MMT, ZnO, CuO and TiO2) and exposure to radiation doses were efficient to improve the mechanical properties Therefore, the incorporation of additives in kefiran films must be taken into account For example, there is an enormous number of nanomaterials that have not been explored yet, for example, functionalized carbonaceous materials and other silicates on a nanometer scale ShahabiGhahfarrokhi et al (2015b) Zolfi et al (2014a) Zolfi et al (2014b) Motedayen et al (2013) Piermaria et al (2011) Ghasemlou et al (2011d) Piermaria et al (2009) In addition, the evaluation of distinct plasticizers on the moisture barrier was reported, as well as the incorporation of essential oils and nanoparticles In general, the incorporation of these ingredients added more moisture barrier to kefiran films According to Goudarzi and Shahabi-Ghahfarrokhi (2018), the application of UV-A radiation in kefiran/starch/TiO2 films reduces the size of the biopolymer chains, forming possible oligo, di and monosaccharide, consequently resulting in a compact structure which will make the moisture path more tenuous, resulting in a more significant barrier (Fig 7) In addition to the water vapor barrier properties, identifying the light barrier of the films based on biopolymers is essential, since the incidence of light on packaged foods can trigger degradation reactions of macro and micro molecules in food Light absorption spectra of Kefiran/starch and ZnO films exposed to UV radiation for 0, 1, and 12 h have been reported by Shahabi-Ghahfarrokhi and Babaei-Ghazvini (2019) A strong absorption peak at 364 nm was clearly observed in all samples, indicating protection in the UV region The light transmission was significantly lower in kefiran/CMC films added with EO, because of 7.8 Barrier properties Predominantly, biopolymer films present a substantial barrier to gases, however the most substantial difficulty in employing these materials as food packaging is their deficiency in the moisture barrier Table presents the results of the water vapor permeability rate of kefiran-based films, as well as blends of kefiran with other biopolymers 13 Carbohydrate Polymers 246 (2020) 116609 L Marangoni Júnior, et al Table Water vapor permeability (WVP) of kefiran-based films Film material Thickness (μm) WVP −10 Kefiran (3%) + glycerol (0, 10, 20, 30 %) Kefiran/starch/ZnO exposed to UV radiation for 0, 1, and 12 h 60 Kefiran/CMC and EO by Satureja Khuzestanica (0.0, 1.0, 1.5 and 2.0% v/v) 90, 113, 133 and 153 5.75, 4.63, 3.74 and 3.04 × 10−7 (g.m/ m2.Pa.h) Kefiran/CMC and CuO and the combination of CuO and EO by Satureja Khuzestanica (0.0, 1.0, 1.5 and 2.0% v/v) Kefiran (2%) with Al2O3 nanoparticles (1, and 5% w/w dry basis) Kefiran/starch/TiO2 exposed to UV-A radiation for 0, 1, and 12 h Kefiran (2% w/v) and starch (5% w/v) and (50:50 v/v, added with nano-ZnO (0, 1, 3, and 5% w/w) Kefiran (2%) treated at 3, and kGy 90 to 161 5.75 to 107 × 10−7 (g.m/m2.Pa.h) 14.5, 15.5, 17.9 and 28.8 5.83, 3.63, 2.88 and 4.87 × 10−10 (g/m s Pa) 1.59, 1.31, 1.24 and 1.22 × 10−10 (g/m s Pa) ̴ 3.10 to 2.00 × 10−10 (g/m s Pa) 110 100, 80, 80 and 80 140, 130, 120 and 110 80, 80, 60 and 50 1.30 to 3.30 × 10 (g/m s Pa) 2.47, 2.38, 2.20 and 2.08 × 10−10 (g/m s Pa) 2.19, 2.55, 1.70 and 2.00 × 10−10 (g/m s Pa) 7.94, 7.83, 4.70 and 3.52 × 10−10 (g/m s Pa) 2.19, 1.73, 1.83 and 2.37 × 10−10 (g/m s Pa) Conclusions References Water vapor permeability increased linearly with increasing glycerol concentration The exposure to UV radiation for up to 12 h, significantly decreased WVP, due to the better distribution of ZnO in the polymeric matrix In addition to the interactions between nonaggregated biopolymers and ZnO, which lead to a decrease in the hydrophilic character of biopolymers The increase in the EO concentration resulted in an increase in the thickness and a reduction in the WVP of the films In addition, EO contains monoterpene hydrocarbons, as a hydrophobic phase, which led to the discontinuity of the hydrophilic phase of kefiran/ CMC The addition of CuO and EO reduced the WVP of the films The film containing 2% CuO and 2% EO showed the lowest WVP, resulting from the uniform distribution of CuO in the polymeric matrix, filling the cavities, restricting the diffusion of water vapor WVP decreased for films containing Al2O3 However, there was an increase as a consequence of the increase in nanoparticles, however, it was still smaller than that of the control film Exposure to radiation reduced the permeability of the films Result of the improvement of the hydrophobic properties in the biocomposite with the use of UV-A The WVP of the films was lower with the addition of ZnO It appears that the formation of hydrogen bonds between the ZnO surface and the biopolymer matrix causes the formation of a coherent network that reduces WVP Doses above 6kGy reduced the WVP of the films, due to the increase in the crystalline region induced by irradiation Coma et al (2019) Sabaghi et al (2015) Shahabi-Ghahfarrokhi and Babaei-Ghazvini (2019) Hasheminya et al (2019a) Hasheminya et al (2019b) Moradi et al (2019) Goudarzi and ShahabiGhahfarrokhi (2018) Babaei-Ghazvini et al (2018) Shahabi-Ghahfarrokhi et al (2015) Kefiran (2%) and chitosan (2%) in proportions (100/0, 68/32, 50/50 and 32/68) Kefiran (2%) and nano cellulose (0, 1, and 3% dry basis) 34, 33, 32 and 31 Kefiran (2%) and nano ZnO (0, 1, and 3% dry basis) 80, 70, 70 and 70 2.19, 2.34, 1.83 and 1.81 × 10−10 (g/m s Pa) Kefiran (5% w/v) and WPI (5% w/v) (50:50 v/v), added nano-TiO2 (0, 1, 3, and 5% w/w) Kefiran (5% w/v) and WPI (5% w/v) (50:50 v/v), added with MMT and nano-TiO2 (0, 1, 3, and 5% w/w) Kefiran 2% and starch 2% (100/0, 70/30, 50/50 and 30/70) 74, 75, 75 and 76 3.39, 2.83, 2.80 and 2.87 × 10−11 (g/m s Pa) With the increase of the chitosan content, the WVP decreased, due to the residual hydrophobic acetyl group of the chitosan, which acts as a barrier to the transport of water vapor The WVP of kefiran/NC nanocomposites decreased with the increase in the NC content (up to 2%), attributed to the increase in the tortuosity of the polymer and the cohesion of the polymeric matrix and the nano cellulose The increase in WVP of films with 3% NC was due to the heterogeneous distribution of NC in the polymer matrix The WVP decreases after a concentration of and 3% of ZnO, indicating a positive effect on the polymeric matrix, attributed to the formation of hydrogen bonds with the oxygen atoms of Zn, consequently a reduction in the diffusion of water molecules TiO2 decreases the WVP of the films, as it showed a homogeneous distribution in the polymer matrix, besides adding hydrophobicity to the film 74, ̴ 91 (TiO2) and ̴ 74 (MMT) 3.39, 1.51 (5% MMT) and 2.87 (5% TiO2) x 10−11 (g/m s Pa) A decrease in the WVP values of the films containing MMT and TiO2 was observed, due to its good dispersion in the film, making a tortuous way to diffuse molecules Zolfi et al (2014b) 74, 62, 59 and 57 3.13, 2.95, 2.78 and 3.88 × 10−11 (g/m s Pa) Motedayen et al (2013) 23, 22, 25, 31, 22 and 22 ̴ 7.50 to 3.00 × 10−11 (g/m s Pa) 58, 69, 72 and 74 4.95, 4.11, 3.61 and 3.67 × 10−11 (g/m s Pa) 4.95, 5.04, 5.55 and 5.88 × 10−11 (g/m s Pa) ̴ 5.50 to 3.80 × 10−11 (g/m s Pa) The WVP of the films decreased with the increase in the amounts of starch (30 % and 50 %) and increased with the additional addition of starch (70 %) The interactions between kefiran and starch molecules have the effect of preventing water molecules from diffusing through films However, in a higher concentration of starch, the dispersion is not sufficient in the matrix to block water vapor The addition of plasticizers to kefiran films improved the water vapor barrier Glucose was the most effective in reducing WVP compared to non-plasticized films The plasticized film with sorbitol exhibited lower WVP values The cyclic conformation of the sorbitol molecules was responsible for decreasing the permeability The increase in the concentration of glycerol resulted in an increase in WVP, attributed to the molecular mobility that glycerol added to the film and, consequently, greater free volume As the concentration of OA increased, the WVP of the films decreased, therefore, the presence of a hydrophobic phase, introduces discontinuities in the hydrophilic phase, thus decreasing the WVP The addition of plasticizer reduced the WVP of the films, attributed to the development of a more compact structure in plasticized films Kefiran (10 g/kg) with galactose, glucose, sucrose, glycerol or sorbitol (25 g/100 g) Kefiran (2%) with 0, 15, 25 and 35 % sorbitol (w/w based on kefiran weight) Kefiran (2%) with 0, 15, 25 and 35 % glycerol (w/w based on kefiran weight) Kefiran (2%) with 0, 15, 25 and 35 % oleic acid (w/w based on kefiran weight) Kefiran (10 g/kg) with and 25 % glycerol/100 g kefiran – 58, 64, 62 and 67 62, 75, 71 and 79 21.4 and 21.9 5.73 and 4.09 × 10−11 (g/m s Pa) 14 Shahabi-Ghahfarrokhi et al (2015a) Shahabi-Ghahfarrokhi et al (2015b) Zolfi et al (2014a) Piermaria et al (2011) Ghasemlou et al (2011a) Ghasemlou et al (2011d) Ghasemlou et al (2011c) Piermaria et al (2009) Carbohydrate Polymers 246 (2020) 116609 L Marangoni Júnior, et al Fig a) scheme of starch modification and kefiran structures during UV-A radiation, b) macroscopic changes of biopolymers during UV-A radiation (Goudarzi & Shahabi-Ghahfarrokhi, 2018) Adapted with permission from Elsevier, Copyright (2018) hydrogen ion of the solution (Sabaghi et al., 2015) The incorporation of active compounds in films based on biopolymers may incorporate antioxidant activity in these materials Hasheminya et al (2019a) has incorporated Satureja Khuzestanica EO into kefiran/CMC films The increase in EO concentration resulted in an increase in antioxidant activity and phenolic compounds, the main compound present in EO was carvacrol The incorporation of natural antioxidants in biopolymer films is being extensively explored in starch films, gelatin and other biopolymers This aspect still requires more attention and investigation for kefiran-based films and its blends 7.10 Antimicrobian activity Studies with the incorporation of antimicrobial substances in films based on biopolymers are increasing, since they include more functions to the material Kefiran/CMC films were incorporated with EO by Satureja Khuzestanica The results showed that with the increase of the EO concentration, the antimicrobial activity was intensified for the bacteria Staphylococcus aureus and Escherichia coli, being more effective for S aureus Antimicrobial activity was attributed to the presence of carvacrol, eugenol and thymol in EO (Hasheminya et al., 2019a) Similar behavior was observed with kefiran/CMC films incorporated with CuO nanoparticles and the combination of CuO nanoparticles with EO Satureja Khuzestanica However, the decrease in E coli count was more expressive (Hasheminya et al., 2019b) Fig a) ZnO-Kefiran film, (b) UV visible absorption spectra of kefiran and ZnO-kefiran film (Shahabi-Ghahfarrokhi et al., 2015b) Adapted with permission from Elsevier, Copyright (2015) the appropriate dispersion of the lipid droplets in the film (Hasheminya et al., 2019a) A decrease in the light transmission of the films in the UV and visible bands was also observed as the CuO concentration increased in the Kefiran/CMC films (Hasheminya et al., 2019b) The incorporation of ZnO in kefiran films resulted in films with excellent transparent and visual properties (Fig 8a) In addition, ZnO concentrations greater than 1% induced UV filtration in the biopolymer, adding light barrier (Fig 8b) (Shahabi-Ghahfarrokhi et al., 2015b) Similar results were experienced for kefiran/starch films added with ZnO (Babaei-Ghazvini et al., 2018) Conclusions and outlook The main objective of this review was to highlight the state-of-theart advances in the preparation of kefiran-based films This EPS possesses excellent biological properties that have been explored for over thirty years However, its use as a polymeric film became more prominent only in the last decade, indicating there is still a long way to be explored in this area Obtaining films of pure kefiran or blended with other biopolymers is reasonably simple, and the materials produced exhibit satisfactory properties The inclusion of additives (inorganic nanoparticles and essential oils) has substantially improved the mechanical and barrier properties of the films, in addition to maximizing 7.9 Antioxidant activity and total phenolic content Kefiran/chitosan films were evaluated for their antioxidant activity The results showed that the antioxidant activity increased as the proportion of chitosan increased This result was attributed to the chitosanfree amino group reacting with free-radicals to form stable macromolecular radicals Hence, forming ammonium groups absorbing the 15 Carbohydrate Polymers 246 (2020) 116609 L Marangoni Júnior, et al the antimicrobial and antioxidant characteristics However, there are still numerous nanomaterials and bioactive molecules that can contribute to obtaining even higher properties For example, considerable property improvements in starch films, chitosan, etc have been reported using different types of silicates or carbonaceous materials on a nanometer scale These examples have yet been unexplored for kefiran films Moreover, considering the composite films already produced, investigations about improving the compatibility between the load and matrix could also contribute to improving the results Together with this, the present research identified that the potential for producing food coatings from kefiran is yet unconsolidated This area could be a possible research line in growth in the next decade Nevertheless, despite the fact that innumerable human health properties have been attributed to pure kefiran, information about the biological activities of its films, such as studies of cytotoxicity, anti-inflammatory and anticancer activities are still scarce Hence, it is believed that directing research to this area could expand the potential for application of films, for example, in the medical and pharmaceutical fields Finally, the evaluation of production processes on a larger scale, such as continuous casting and extrusion; as well as biodegradation studies are equally necessary for the consolidation of this material applicability 1016/J.BCDF.2018.02.001 Bosch, A., Golowczyc, M A., Abraham, A G., Garrote, G L., De Antoni, G L., & Yantorno, O (2006) Rapid discrimination of lactobacilli isolated from kefir grains by FT-IR spectroscopy International Journal of Food Microbiology, 111(3), 280–287 https:// doi.org/10.1016/j.ijfoodmicro.2006.05.010 Botelho, P S., Maciel, M I S., Bueno, L A., Marques, M., de, F F., Marques, D N., Sarmento Silva, T M (2014) Characterisation of a new exopolysaccharide obtained from of fermented kefir grains in soymilk Carbohydrate Polymers, 107, 1–6 https:// doi.org/10.1016/j.carbpol.2014.02.036 Cazón, P., Velazquez, G., Ramírez, J A., & Vázquez, M (2017) Polysaccharide-based films and coatings for food packaging: A review Food Hydrocolloids, 68, 136–148 https://doi.org/10.1016/j.foodhyd.2016.09.009 Cheirsilp, B., & Radchabut, S (2011) Use of whey lactose from dairy industry for economical kefiran production by Lactobacillus kefiranofaciens in mixed cultures with yeasts New Biotechnology, 28(6), 574–580 https://doi.org/10.1016/J.NBT.2011.01 009 Cheirsilp, B., Shimizu, H., & Shioya, S (2007) Kinetic modeling of kefiran production in mixed culture of Lactobacillus kefiranofaciens and Saccharomyces cerevisiae Process Biochemistry, 42(4), 570–579 https://doi.org/10.1016/J.PROCBIO.2006.11.003 Cheirsilp, B., Suksawang, S., Yeesang, J., & Boonsawang, P (2018) Co-production of functional exopolysaccharides and lactic acid by Lactobacillus kefiranofaciens originated from fermented milk, kefir Journal of Food Science and Technology, 55(1), 331–340 https://doi.org/10.1007/s13197-017-2943-7 Cheirsilp, B., Shimizu, H., & Shioya, S (2003) Enhanced kefiran production by mixed culture of Lactobacillus kefiranofaciens and Saccharomyces cerevisiae Journal of Biotechnology, 100(1), 43–53 https://doi.org/10.1016/S0168-1656(02)00228-6 Cheirsilp, B., Shoji, H., Shimizu, H., & Shioya, S (2003) Interactions between Lactobacillus kefiranofaciens and Saccharomyces cerevisiae in mixed culture for kefiran production Journal of Bioscience and Bioengineering, 96(3), 279–284 https:// doi.org/10.1016/S1389-1723(03)80194-9 Chen, Z., Shi, J., Yang, X., Nan, B., Liu, Y., & Wang, Z (2015) Chemical and physical characteristics and antioxidant activities of the exopolysaccharide produced by Tibetan kefir grains during milk fermentation International Dairy Journal, 43(April), 15–21 https://doi.org/10.1016/j.idairyj.2014.10.004 Coma, M E., Peltzer, M A., Delgado, J F., & Salvay, A G (2019) Water kefir grains as an innovative source of materials : Study of plasticiser content on fi lm properties European Polymer Journal, 120(August), 109234 https://doi.org/10.1016/j eurpolymj.2019.109234 Dailin, D J., Elsayed, E A., Othman, N Z., Malek, R A., Ramli, S., Sarmidi, M R., El Enshasy, H A (2015) Development of cultivation medium for high yield kefiran production by Lactobacillus kefiranofaciens International Journal of Pharmacy and Pharmaceutical Sciences, 7(3), 159–163 Dailin, D J., Elsayed, E A., Othman, N Z., Malek, R., Phin, H S., Aziz, R., El Enshasy, H A (2014) Bioprocess development for kefiran production by Lactobacillus kefiranofaciens in semi industrial scale bioreactor Saudi Journal of Biological Sciences https://doi.org/10.1016/j.sjbs.2015.06.003 Dailin, D J., Elsayed, E A., Othman, N Z., Malek, R., Phin, H S., Aziz, R., El Enshasy, H A (2016) Bioprocess development for kefiran production by Lactobacillus kefiranofaciens in semi industrial scale bioreactor Saudi Journal of Biological Sciences, 23(4), 495–502 https://doi.org/10.1016/J.SJBS.2015.06.003 Dashipour, A., Razavilar, V., Hosseini, H., Shojaee-Aliabadi, S., German, J B., Ghanati, K., Khaksar, R (2015) Antioxidant and antimicrobial carboxymethyl cellulose films containing Zataria multiflora essential oil International Journal of Biological Macromolecules, 72, 606–613 https://doi.org/10.1016/j.ijbiomac.2014.09.006 Davidović, S Z., Miljković, M G., Antonović, D G., Rajilić-Stojanović, M D., & Dimitrijević-Branković, S I (2015) Water Kefir grain as a source of potent dextran producing lactic acid bacteria Hemijska Industrija, 69(6), 595–604 https://doi.org/ 10.2298/HEMIND140925083D Dertli, E., & Çon, A H (2017) Microbial diversity of traditional kefir grains and their role on kefir aroma LWT - Food Science and Technology, 85, 151–157 https://doi.org/10 1016/j.lwt.2017.07.017 Devlieghere, F., Vermeulen, A., & Debevere, J (2004) Chitosan: antimicrobial activity, interactions with food components and applicability as a coating on fruit and vegetables Food Microbiology, 21, 703–714 https://doi.org/10.1016/j.fm.2004.02.008 Exarhopoulos, S., Raphaelides, S N., & Kontominas, M G (2018a) Conformational studies and molecular characterization of the polysaccharide kefiran Food Hydrocolloids, 77, 347–356 https://doi.org/10.1016/J.FOODHYD.2017.10.011 Exarhopoulos, S., Raphaelides, S N., & Kontominas, M G (2018b) Flow behavior studies of kefiran systems Food Hydrocolloids, 79, 282–290 https://doi.org/10.1016/J FOODHYD.2017.12.030 Felton, L A (2013) Mechanisms of polymeric film formation International Journal of Pharmaceutics, 457(2), 423–427 https://doi.org/10.1016/j.ijpharm.2012.12.027 Fiori, A P S., de, M., Camani, P H., Rosa, D., dos, S., & Carastan, D J (2019) Combined effects of clay minerals and polyethylene glycol in the mechanical and water barrier properties of carboxymethylcellulose fi lms Industrial Crops and Products, 140(December 2018), 111644 https://doi.org/10.1016/j.indcrop.2019.111644 Frengova, G I., Simova, E D., Beshkova, D M., & Simov, Z I (2002) Exopolysaccharides produced by lactic acid bacteria of kefir grains Zeitschrift Fur Naturforschung - Section C Journal of Biosciences, 57(9–10), 805–810 https://doi.org/10.1515/znc-2002-91009 Gagliarini, N., Diosma, G., Piermaria, J., Garrote, G L., & Abraham, A G (2019) Whey protein-kefiran films as driver of probiotics to the gut LWT - Food Science and Technology, 105(February), 321–328 https://doi.org/10.1016/j.lwt.2019.02.023 Ganiari, S., Choulitoudi, E., & Oreopoulou, V (2017) Edible and active fi lms and coatings as carriers of natural antioxidants for lipid food Trends in Food Science & Technology, 68, 70–82 https://doi.org/10.1016/j.tifs.2017.08.009 CRediT authorship contribution statement Luís Marangoni Júnior: Conceptualization, Data curation, Formal analysis, Investigation, Writing - original draft, Writing - review & editing Roniérik Pioli Vieira: Conceptualization, Data curation, Formal analysis, Investigation, Writing - original draft, Writing - review & editing Carlos Alberto Rodrigues Anjos: Supervision, Writing review & editing Declaration of Competing Interest The authors declare no conflicts of interest Acknowledgements The authors would like to thank the Council for Scientific and Technological Development (CNPq) for the PhD scholarships This study was partly funded by the Coordination for the Improvement of Higher Education Personnel – Brazil (CAPES) – Financial Code 001 References Ahmed, Z., Wang, Y., Anjum, N., Ahmad, A., & Khan, S T (2013) Characterization of exopolysaccharide produced by Lactobacillus kefiranofaciens ZW3 isolated from Tibet kefir – Part II Food Hydrocolloids, 30(1), 343–350 https://doi.org/10.1016/J FOODHYD.2012.06.009 Aider, M (2010) Chitosan application for active bio-based films production and potential in the food industry: Review LWT - Food Science and Technology, 43(6), 837–842 https://doi.org/10.1016/j.lwt.2010.01.021 Amorim, F G., Coitinho, L B., Dias, A T., Friques, A G F., Monteiro, B L., de Rezende, L C D., Quinton, L (2019) Identification of new bioactive peptides from Kefir milk through proteopeptidomics: Bioprospection of antihypertensive molecules Food Chemistry, 282, 109–119 https://doi.org/10.1016/J.FOODCHEM.2019.01.010 Azeredo, H M C., & Waldron, K W (2016) Crosslinking in polysaccharide and protein fi lms and coatings for food contact e A review Trends in Food Science & Technology, 52, 109–122 https://doi.org/10.1016/j.tifs.2016.04.008 Babaei-Ghazvini, A., Shahabi-Ghahfarrokhi, I., & Goudarzi, V (2018) Preparation of UVprotective starch/kefiran/ZnO nanocomposite as a packaging film: Characterization Food Packaging and Shelf Life, 16(January), 103–111 https://doi.org/10.1016/j.fpsl 2018.01.008 Badel, S., Bernardi, T., & Michaud, P (2011) New perspectives for Lactobacilli exopolysaccharides Biotechnology Advances, 29(1), 54–66 https://doi.org/10.1016/J BIOTECHADV.2010.08.011 Blandón, L M., Noseda, M D., Islan, G A., Castro, G R., de Melo Pereira, G V., ThomazSoccol, V., Soccol, C R (2018) Optimization of culture conditions for kefiran production in whey: The structural and biocidal properties of the resulting polysaccharide Bioactive Carbohydrates and Dietary Fibre, 16, 14–21 https://doi.org/10 16 Carbohydrate Polymers 246 (2020) 116609 L Marangoni Júnior, et al Garlotta, D (2002) A Literature Review of Poly (Lactic Acid) Journal OfPolymers and the Environment, 9(2), 1–10 Ghasemlou, M., Khodaiyan, F., & Oromiehie, A (2011a) Physical, mechanical, barrier, and thermal properties of polyol-plasticized biodegradable edible film made from kefiran Carbohydrate Polymers, 84(1), 477–483 https://doi.org/10.1016/j.carbpol 2010.12.010 Ghasemlou, M., Khodaiyan, F., & Oromiehie, A (2011b) Rheological and structural characterisation of film-forming solutions and biodegradable edible film made from kefiran as affected by various plasticizer types International Journal of Biological Macromolecules, 49(4), 814–821 https://doi.org/10.1016/j.ijbiomac.2011.07.018 Ghasemlou, M., Khodaiyan, F., Oromiehie, A., & Saeid, M (2011c) Characterization of edible emulsified films with low affinity to water based on kefiran and oleic acid International Journal of Biological Macromolecules, 49(3), 378–384 https://doi.org/10 1016/j.ijbiomac.2011.05.013 Ghasemlou, M., Khodaiyan, F., Oromiehie, A., & Saeid, M (2011d) Development and characterisation of a new biodegradable edible film made from kefiran, an exopolysaccharide obtained from kefir grains Food Chemistry, 127(4), 1496–1502 https:// doi.org/10.1016/j.foodchem.2011.02.003 Ghasemlou, M., Khodaiyan, F., & Gharibzahedi, S M T (2012) Enhanced production of Iranian Kefir grain biomass by optimization and empirical modeling of fermentation conditions using response surface methodology Food and Bioprocess Technology, 5(8), 3230–3235 https://doi.org/10.1007/s11947-011-0575-x Ghasemlou, M., Khodaiyan, F., Jahanbin, K., Gharibzahedi, S M T., & Taheri, S (2012) Structural investigation and response surface optimisation for improvement of kefiran production yield from a low-cost culture medium Food Chemistry, 133(2), 383–389 https://doi.org/10.1016/J.FOODCHEM.2012.01.046 Global Kefir Market Retrieved from Transparency market research website: https://www transparencymarketresearch.com/kefir-market.html Goudarzi, V., & Shahabi-Ghahfarrokhi, I (2018) Development of photo-modified starch/ kefiran/TiO2 bio-nanocomposite as an environmentally-friendly food packaging material International Journal of Biological Macromolecules, 116, 1082–1088 https:// doi.org/10.1016/j.ijbiomac.2018.05.138 Güzel-Seydim, Z B., Seydim, A C., Greene, A K., & Bodine, A B (2000) Determination of organic acids and volatile flavor substances in kefir during fermentation Journal of Food Composition and Analysis, 13(1), 35–43 https://doi.org/10.1006/JFCA.1999 0842 Hasheminya, S., Rezaei, R., & Ghanbarzadeh, B (2018) Physicochemical, mechanical, optical, microstructural and antimicrobial properties of novel ke firan-carboxymethyl cellulose biocomposite fi lms as in fl uenced by copper oxide nanoparticles (CuONPs) Food Packaging and Shelf Life, 17(May), 196–204 https://doi.org/10.1016/j.fpsl 2018.07.003 Hasheminya, S.-M., Mokarram, R R., Ghanbarzadeh, B., Hamishekar, H., Kafil, H S., & Dehghannya, J (2019a) Development and characterization of biocomposite films made from kefiran, carboxymethyl cellulose and Satureja khuzestanica essential oil Food Chemistry, 289(November 2018), 443–452 https://doi.org/10.1016/j foodchem.2019.03.076 Hasheminya, S.-M., Mokarram, R R., Ghanbarzadeh, B., Hamishekar, H., Kafil, H S., & Dehghannya, J (2019b) Influence of simultaneous application of copper oxide nanoparticles and Satureja khuzestanica essential oil on properties of kefiran – Carboxymethyl cellulose films Polymer Testing, 73(December 2018), 377–388 https://doi.org/10.1016/j.polymertesting.2018.12.002 Hassan, B., Chatha, S A S., Hussain, A I., & Zia, K M (2018) Recent advances on polysaccharides, lipids and protein based edible films and coatings: A review International Journal of Biological Macromolecules, 109, 1095–1107 https://doi.org/ 10.1016/j.ijbiomac.2017.11.097 Jain, R., Shetty, S., & Yadav, K S (2020) Unfolding the electrospinning potential of biopolymers for preparation of nano fi bers Journal of Drug Delivery Science and Technology, 57(January), 101604 https://doi.org/10.1016/j.jddst.2020.101604 Jenab, A., Roghanian, R., Ghorbani, N., Ghaedi, K., & Emtiazi, G (2020) The Efficacy of Electrospun PAN/Kefiran Nanofiber and Kefir in Mammalian Cell Culture: Promotion of PC12 Cell Growth, Anti-MCF7 Breast Cancer Cells Activities, and Cytokine Production of PBMC International Journal of Nanomedicine, 15, 717–728 https://doi org/10.2147/IJN.S232264 Jeong, D., Kim, D H., Kang, I B., Kim, H., Song, K Y., Kim, H S., Seo, K H (2017) Characterization and antibacterial activity of a novel exopolysaccharide produced by Lactobacillus kefiranofaciens DN1 isolated from kefir Food Control, 78, 436–442 https://doi.org/10.1016/j.foodcont.2017.02.033 Jiang, T., Duan, Q., Zhu, J., Liu, H., & Yu, L (2020) Starch-based biodegradable materials: Challenges and opportunities Advanced Industrial and Engineering Polymer Research, 3(1), 8–18 https://doi.org/10.1016/j.aiepr.2019.11.003 Joshi, M., Adak, B., & Butola, B S (2018) Polyurethane nanocomposite based gas barrier films, membranes and coatings: A review on synthesis, characterization and potential applications Progress in Materials Science, 97(May), 230–282 https://doi.org/10 1016/j.pmatsci.2018.05.001 Kooiman, P (1968) The chemical structure of kefiran, the water-soluble polysaccharide of the kefir grain Carbohydrate Research, 7(2), 200–211 https://doi.org/10.1016/ S0008-6215(00)81138-6 la Riviére, J W M., Kooiman, P., & Schmidt, K (1967) Kefiran, a novel polysaccharide produced in the kefir grain by Lactobacillus brevis Archiv Für Mikrobiologie, 59(1–3), 269–278 https://doi.org/10.1007/BF00406340 Licciardello, F (2017) Packaging, blessing in disguise Review on its diverse contribution to food sustainability Trends in Food Science & Technology, 65, 32–39 https://doi org/10.1016/j.tifs.2017.05.003 Liou, R.-M., & Chen, S.-H (2009) CuO impregnated activated carbon for catalytic wet peroxide oxidation of phenol Journal of Hazardous Materials, 172(1), 498–506 https://doi.org/10.1016/j.jhazmat.2009.07.012 Lu, D R., Xiao, C M., & Xu, S J (2009) Starch-based completely biodegradable polymer materials Express Polymer Letters, 3(6), 366–375 https://doi.org/10.3144/ expresspolymlett.2009.46 Maeda, H., Zhu, X., & Mitsuoka, T (2004) Effects of an exopolysaccharide (Kefiran) from Lactobacillus kefiranofaciens on blood glucose in KKAy mice and constipation in SD rats induced by a low-fiber diet Bioscience and Microflora, 23(4), 149–153 https:// doi.org/10.12938/bifidus.23.149 Maeda, H., Zhu, X., Suzuki, S., Suzuki, K., & Kitamura, S (2004) Structural characterization and biological activities of an exopolysaccharide kefiran produced by Lactobacillus kefiranofaciens WT-2B T Journal of Agricultural and Food Chemistry, 52(17), 5533–5538 https://doi.org/10.1021/jf049617g Mahalik, N P., & Nambiar, A N (2010) Trends in food packaging and manufacturing systems and technology Trends in Food Science & Technology, 21(3), 117–128 https://doi.org/10.1016/j.tifs.2009.12.006 Marangoni Júnior, L., Cristianini, M., Padula, M., & Anjos, C A R (2019) Effect of highpressure processing on characteristics of flexible packaging for foods and beverages Food Research International, 119(August 2018), 920–930 https://doi.org/10.1016/j foodres.2018.10.078 Marangoni Júnior, L., De Oliveira, L M., Dantas, F B H., Cristianini, M., Padula, M., & Anjos, C A R (2020) Influence of high-pressure processing on morphological, thermal and mechanical properties of retort and metallized flexible packaging Journal of Food Engineering, 273(August 2019), https://doi.org/10.1016/j.jfoodeng 2019.109812 Maringgal, B., Hashim, N., Sya, I., Amin, M., Tengku, M., & Mohamed, M (2020) Recent advance in edible coating and its effect on fresh / fresh-cut fruits quality Trends in Food Science & Technology, 96(December 2019), 253–267 https://doi.org/10.1016/j tifs.2019.12.024 Medrano, M., Racedo, S M., Rolny, I S., Abraham, A G., & Pérez, P F (2011) Oral administration of kefiran induces changes in the balance of immune cells in a murine model Journal of Agricultural and Food Chemistry, 59(10), 5299–5304 https://doi org/10.1021/jf1049968 Micheli, L., Uccelletti, D., Palleschi, C., & Crescenzi, V (1999) Isolation and characterisation of a ropy Lactobacillus strain producing the exopolysaccharide kefiran Applied Microbiology and Biotechnology, 53(1), 69–74 https://doi.org/10.1007/ s002530051616 Mohamed, S A A., El-Sakhawy, M., & El-Sakhawy, M A.-M (2020) Polysaccharides, Protein and Lipid -Based Natural Edible Films in Food Packaging: A Review Carbohydrate Polymers, 238(February), 116178 https://doi.org/10.1016/j.carbpol 2020.116178 Montesanto, S., Calò, G., Cruciata, M., Settanni, L., Brucato, V B., & La Carrubba, V (2016) Optimization of environmental conditions for kefiran production by kefir grain as scaffold for tissue engineering Chemical Engineering Transactions, 49, 607–612 https://doi.org/10.3303/CET1649102 Moradi, Z., & Kalanpour, N (2019) Kefiran, a branched polysaccharide: Preparation, properties and applications: A review Carbohydrate Polymers, 223, 115100 https:// doi.org/10.1016/J.CARBPOL.2019.115100 Moradi, Z., Esmaiili, M., & Almasi, H (2019) Development and characterization of kefiran - Al2O3 nanocomposite films: Morphological, physical and mechanical properties International Journal of Biological Macromolecules, 122, 603–609 https://doi org/10.1016/j.ijbiomac.2018.10.193 Morris, E R., Cutler, A N., Ross-Murphy, S B., Rees, D A., & Price, J (1981) Concentration and shear rate dependence of viscosity in random coil polysaccharide solutions Carbohydrate Polymers, 1(1), 5–21 https://doi.org/10.1016/01448617(81)90011-4 Motedayen, A A., Khodaiyan, F., & Salehi, E A (2013) Development and characterisation of composite films made of kefiran and starch Food Chemistry, 136(3–4), 1231–1238 https://doi.org/10.1016/j.foodchem.2012.08.073 Ninane, V., Berben, G., Romnee, J M., & Oger, R (2005) Variability of the microbial abundance of a kefir grain starter cultivated in partially controlled conditions Biotechnology, Agronomy and Society and Environment, 9(3), 191–194 Nor, S., & Ding, P (2020) Trends and advances in edible biopolymer coating for tropical fruit: A review Food Research International, 134(April), 109208 https://doi.org/10 1016/j.foodres.2020.109208 Nouha, K., Kumar, R S., Balasubramanian, S., & Tyagi, R D (2018) Critical review of EPS production, synthesis and composition for sludge flocculation Journal of Environmental Sciences (China), 66(August), 225–245 https://doi.org/10.1016/j.jes 2017.05.020 Ogunsona, E., Ojogbo, E., & Mekonnen, T (2018) Advanced material applications of starch and its derivatives European Polymer Journal, 108(September), 570–581 https://doi.org/10.1016/j.eurpolymj.2018.09.039 Ojogbo, E., Ogunsona, E O., & Mekonnen, T H (2020) Chemical and physical modi fi cations of starch for renewable polymeric materials Materials Today Sustainability, 7–8, 100028 https://doi.org/10.1016/j.mtsust.2019.100028 Pais-Chanfrau, J M., Acosta, L D C., Cóndor, P M A., Pérez, J N., & Guerrero, M J C (2018) Small-scale process for the production of kefiran through culture optimization by use of central composite design from Whey and Kefir granules Current Topics in Biochemical Engineering, 19 https://doi.org/10.5772/57353 Parikh, A., & Madamwar, D (2006) Partial characterization of extracellular polysaccharides from cyanobacteria Bioresource Technology, 97(15), 1822–1827 https:// doi.org/10.1016/J.BIORTECH.2005.09.008 Peelman, N., Ragaert, P., De Meulenaer, B., Adons, D., Peeters, R., Cardon, L., Devlieghere, F (2013) Application of bioplastics for food packaging Trends in Food Science & Technology, 32(2), 128–141 https://doi.org/10.1016/J.TIFS.2013.06.003 Perez-Gago, M B., Rojas, C., & Del Rio, M A (2002) Effect of Lipid Type and Amount of Edible Composite Coatings Used to Protect Postharvest Quality of Mandarins cv Fortune Journal of Food Science, 67(8) 17 Carbohydrate Polymers 246 (2020) 116609 L Marangoni Júnior, et al aging effects and heat stress tolerance in nematodes via DAF-16 Bioscience, Biotechnology, and Biochemistry, 83(8), 1484–1489 https://doi.org/10.1080/ 09168451.2019.1606696 Suriyamongkol, P., Weselake, R., Narine, S., Moloney, M., & Shah, S (2007) Biotechnological approaches for the production of polyhydroxyalkanoates in microorganisms and plants—A review Biotechnology Advances, 25(2), 148–175 https:// doi.org/10.1016/J.BIOTECHADV.2006.11.007 Tada, S., Katakura, Y., Ninomiya, K., & Shioya, S (2007) Fed-batch coculture of Lactobacillus kefiranofaciens with Saccharomyces cerevisiae for effective production of kefiran Journal of Bioscience and Bioengineering, 103(6), 557–562 https://doi.org/ 10.1263/JBB.103.557 Takizawa, S., Kojima, S., Tamura, S., Fujinaga, S., Benno, Y., & Nakase, T (1994) Lactobacillus kefirgranum sp nov and Lactobacillus parakefir sp nov., two new species from kefir grains International Journal of Systematic Bacteriology, 44(3), 435–439 https://doi.org/10.1099/00207713-44-3-435 Takizawa, S., Kojima, S., Tamura, S., Fujinaga, S., Benno, Y., & Nakase, T (1998) The composition of the Lactobacillus flora in kefir grains Systematic and Applied Microbiology, 21(1), 121–127 https://doi.org/10.1016/S0723-2020(98)80015-5 Taniguchi, M., Nomura, M., Itaya, T., & Tanaka, T (2001) Kefiran production by Lactobacillus kefiranofaciens under the culture conditions established by mimicking the existence and activities of yeast in kefir grains Food Science and Technology Research, 7(4), 333–337 https://doi.org/10.3136/fstr.7.333 Thakur, R., Pristijono, P., Scarlett, C J., Bowyer, M., Singh, S P., & Vuong, Q V (2019) Starch-based films : Major factors affecting their properties International Journal of Biological Macromolecules, 132, 1079–1089 https://doi.org/10.1016/j.ijbiomac 2019.03.190 Vieira, M G A., da Silva, M A., dos Santos, L O., & Beppu, M M (2011) Natural-based plasticizers and biopolymer films: A review European Polymer Journal, 47(3), 254–263 https://doi.org/10.1016/j.eurpolymj.2010.12.011 Wang, M., & Bi, J (2008) Modification of characteristics of kefiran by changing the carbon source ofLactobacillus kefiranofaciens Journal of the Science of Food and Agriculture, 88(5), 763–769 https://doi.org/10.1002/jsfa.3136 Witthuhn, R C., Schoeman, T., & Britz, T J (2004) Isolation and characterization of the microbial population of different South African kefir grains International Journal of Dairy Technology, 57(1), 33–37 https://doi.org/10.1111/j.1471-0307.2004.00126.x Wu, M., Wu, Y., Zhou, J., & Pan, Y (2009) Structural characterisation of a water-soluble polysaccharide with high branches from the leaves of Taxus chinensis var Mairei Food Chemistry, 113(4), 1020–1024 https://doi.org/10.1016/J.FOODCHEM.2008 08.055 Xing, Z., Tang, W., Geng, W., Zheng, Y., & Wang, Y (2017) In vitro and in vivo evaluation of the probiotic attributes of Lactobacillus kefiranofaciens XL10 isolated from Tibetan kefir grain Applied Microbiology and Biotechnology, 101(6), 2467–2477 https://doi.org/10.1007/s00253-016-7956-z Yang, L., & Zhang, L.-M (2009) Chemical structural and chain conformational characterization of some bioactive polysaccharides isolated from natural sources Carbohydrate Polymers, 76(3), 349–361 https://doi.org/10.1016/J.CARBPOL.2008 12.015 Yokoi, H., & Watanabe, T (1992) Optimum culture conditions for production of kefiran by Lactobacillus sp KPB-167B isolated from kefir grains Journal of Fermentation and Bioengineering, 74(5), 327–329 https://doi.org/10.1016/0922-338X(92)90069-7 Zajšek, K., & Goršek, A (2011) Experimental assessment of the impact of cultivation conditions on kefiran production by the mixed microflora imbedded in kefir grains Chemical Engineering Transactions, 24(April), 481–486 https://doi.org/10.3303/ CET1124081 Zajšek, K., Goršek, A., & Kolar, M (2013) Cultivating conditions effects on kefiran production by the mixed culture of lactic acid bacteria imbedded within kefir grains Food Chemistry, 139(1–4), 970–977 https://doi.org/10.1016/J.FOODCHEM.2012 11.142 Zajšek, K., Kolar, M., & Goršek, A (2011) Characterisation of the exopolysaccharide kefiran produced by lactic acid bacteria entrapped within natural kefir grains International Journal of Dairy Technology, 64(4), 544–548 https://doi.org/10.1111/j 1471-0307.2011.00704.x Zhang, Y., Liu, B., Wang, L., Deng, Y., & Zhou, S (2019) Preparation, structure and properties of acid aqueous solution plasticized thermoplastic chitosan Polymer, 11(818), 1–10 Zhao, D L., Japip, S., Zhang, Y., Weber, M., Maletzko, C., & Chung, T (2020) Emerging thin- film nanocomposite (TFN) membranes for reverse osmosis : A review Water Research, 173, Article 115557 https://doi.org/10.1016/j.watres.2020.115557 Zhong, Y., Godwin, P., Jin, Y., & Xiao, H (2020) Biodegradable polymers and greenbased antimicrobial packaging materials : A mini-review Advanced Industrial and Engineering Polymer Research, 3(1), 27–35 https://doi.org/10.1016/j.aiepr.2019.11 002 Zolfi, M., Khodaiyan, F., Mousavi, M., & Hashemi, M (2014a) Development and characterization of the kefiran-whey protein International Journal of Biological Macromolecules, 65, 340–345 https://doi.org/10.1016/j.ijbiomac.2014.01.010 Zolfi, M., Khodaiyan, F., Mousavi, M., & Hashemi, M (2014b) The improvement of characteristics of biodegradable films made from kefiran – Whey protein by nanoparticle incorporation Carbohydrate Polymers, 109, 118–125 https://doi.org/10 1016/j.carbpol.2014.03.018 Piermaria, J A., Canal, M L D L., & Abraham, A G (2008) Gelling properties of kefiran, a food-grade polysaccharide obtained from kefir grain Food Hydrocolloids, 22, 1520–1527 https://doi.org/10.1016/j.foodhyd.2007.10.005 Piermaria, J., Diosma, G., Aquino, C., Garrote, G., & Abraham, A (2015) Edible kefiran films as vehicle for probiotic microorganisms Innovative Food Science & Emerging Technologies, 32, 193–199 https://doi.org/10.1016/j.ifset.2015.09.009 Piermaria, J A., Pinotti, A., Garcia, M A., & Abraham, A G (2009) Films based on kefiran, an exopolysaccharide obtained from kefir grain : Development and characterization Food Hydrocolloids, 23, 684–690 https://doi.org/10.1016/j.foodhyd 2008.05.003 Piermaria, J., Bosch, A., Pinotti, A., Yantorno, O., Alejandra, M., & Graciela, A (2011) Kefiran films plasticized with sugars and polyols : Water vapor barrier and mechanical properties in relation to their microstructure analyzed by ATR / FT-IR spectroscopy Food Hydrocolloids, 25(5), 1261–1269 https://doi.org/10.1016/j.foodhyd 2010.11.024 Piermaría, J., Bengoechea, C., Abraham, A G., & Guerrero, A (2016) Shear and extensional properties of kefiran Carbohydrate Polymers, 152, 97–104 https://doi.org/10 1016/j.carbpol.2016.06.067 Pop, C R., Salanţă, L., Rotar, A M., Semeniuc, C A., Socaciu, C., & Sindic, M (2016) Influence of extraction conditions on characteristics of microbial polysaccharide kefiran isolated from kefir grains biomass Journal of Food and Nutrition Research, 55(2), 121–130 Priyadarshi, R., & Rhim, J (2020) Chitosan-based biodegradable functional films for food packaging applications Innovative Food Science & Emerging Technologies, 102346 https://doi.org/10.1016/j.ifset.2020.102346 Rad, F H., Sharifan, A., & Asadi, G (2018) Physicochemical and antimicrobial properties of kefiran/waterborne polyurethane film incorporated with essential oils on refrigerated ostrich meat LWT - Food Science and Technology, 97(May), 794–801 https://doi.org/10.1016/j.lwt.2018.08.005 Radhouani, H., Maia, F R., & Oliveira, J M (2018) Kefiran biopolymer: Evaluation of its physicochemical and biological properties Bioactive and Compatible Polymers, 33(August), 461–478 https://doi.org/10.1177/0883911518793914 Ribeiro-Santos, R., Andrade, M., de Melo, N R., & Sanches-Silva, A (2017) Use of essential oils in active food packaging: Recent advances and future trends Trends in Food Science & Technology, 61, 132–140 https://doi.org/10.1016/j.tifs.2016.11.021 Robertson, G (2013) Food packaging: Principles and practice Rodrigues, K L., Caputo, L R G., Carvalho, J C T., Evangelista, J., & Schneedorf, J M (2005) Antimicrobial and healing activity of kefir and kefiran extract International Journal of Antimicrobial Agents, 25(5), 404–408 https://doi.org/10.1016/J IJANTIMICAG.2004.09.020 Rodrigues, K L., Carvalho, J C T., & Schneedorf, J M (2005) Anti-inflammatory properties of kefir and its polysaccharide extract Inflammopharmacology, 13(5–6), 485–492 https://doi.org/10.1163/156856005774649395 Sabaghi, M., Maghsoudlou, Y., & Habibi, P (2015) Enhancing structural properties and antioxidant activity of kefiran films by chitosan addition Food Structure, 5, 66–71 https://doi.org/10.1016/j.foostr.2015.06.003 Sampathkumar, K., Tan, K X., & Loo, S C J (2020) Developing nano-delivery systems for agriculture and food applications with nature-derived polymers ISCIENCE, 23(5), Article 101055 https://doi.org/10.1016/j.isci.2020.101055 Semeniuc, C (2013) FTIR spectroscopic characterization of a new biofilm obtained from kefiran Journal of Agroalimentary Processes and Technologies, 19(2), 157–159 Retrieved from http://www.researchgate.net/publication/278678223_FTIR_ spectroscopic_characterization_of_a_new_biofilm_obtained_from_kefiran Shahabi-Ghahfarrokhi, I., & Babaei-Ghazvini, A (2019) Using photo-modification to compatibilize nano-ZnO in development of starch-kefiran-ZnO green nanocomposite as food packaging material International Journal of Biological Macromolecules, 124, 922–930 https://doi.org/10.1016/j.ijbiomac.2018.11.241 Shahabi-Ghahfarrokhi, I., Khodaiyan, F., Mousavi, M., & Yousefi, H (2015) Effect of yirradiation on the physical and mechanical properties of kefiran biopolymer film International Journal of Biological Macromolecules, 74, 343–350 https://doi.org/10 1016/j.ijbiomac.2014.11.038 Shahabi-Ghahfarrokhi, I., Khodaiyan, F., & Mousavi, M (2015a) Green bionanocomposite based on kefiran and cellulose nanocrystals produced from beer industrial residues International Journal of Biological Macromolecules, 77, 85–91 https://doi.org/ 10.1016/j.ijbiomac.2015.02.055 Shahabi-Ghahfarrokhi, I., Khodaiyan, F., & Mousavi, M (2015b) Preparation of UVprotective kefiran / nano-ZnO nanocomposites: Physical and mechanical properties International Journal of Biological Macromolecules, 72, 41–46 https://doi.org/10 1016/j.ijbiomac.2014.07.047 Sharifi, M., Moridnia, A., Mortazavi, D., Salehi, M., Bagheri, M., & Sheikhi, A (2017) Kefir: A powerful probiotics with anticancer properties Medical Oncology, 34(11), 1–7 https://doi.org/10.1007/s12032-017-1044-9 Sharma, R., Jafari, S M., & Sharma, S (2020) Antimicrobial bio-nanocomposites and their potential applications in food packaging Food Control, 112(January), 107086 https://doi.org/10.1016/j.foodcont.2020.107086 Staaf, M., Widmalm, G., Yang, Z., & Huttunen, E (1996) Structural elucidation of an extracellular polysaccharide produced by Lactobacillus helveticus Carbohydrate Research, 291, 155–164 https://doi.org/10.1016/S0008-6215(96)00166-8 Sugawara, T., Furuhashi, T., Shibata, K., Abe, M., Kikuchi, K., Arai, M., Sakamoto, K (2019) Fermented product of rice with Lactobacillus kefiranofaciens induces anti- 18 ... and 74, ̴ 91 (TiO2) 74, 75, 75 and 76 80, 70, 70 and 70 – 13 - 19 34, 33, 32 and 31 80, 80, 60 and 50 140, 130, 120 and 110 100, 80, 80 and 80 90, 107, 110 and 117 90 - 150 14.5, 15.5, 17.9 and. .. 113, 133 and 153 90 - 161 110 60 Thickness (μm) 55 % 55 % 75 % 50 % 55 % 25 °C and 50 % RH 20 °C and 75 % RH 25 °C 20 °C and 75 % RH 25 °C 25 °C 25 °C and RH 25 °C and RH 20 °C and RH 25 °C and RH... RH 25 °C and RH 25 °C and RH 25 °C 25 °C and 55 % RH 55 % RH 25 °C and 50−55% RH 50−55% RH 20 °C and 75 % RH 25 °C and 55 % RH 25 °C and 55 % RH 25 °C and 55 % RH – 22 °C and 43 % RH Storage condition