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ABSTRACT: Kefir is a fermented dairy product with well recognized probiotic properties. Recently, consumer interest in fermented products with probiotic microorganisms has increased due to the accumulating evidence of the effects of kefir microorganisms on the modulation of gut microbiota and their antimicrobial activity. Although the health properties of kefir have been reviewed in other works, the present review addresses the antimicrobial effects of kefir microbiota and associated compounds. The antimicrobial activity of kefir microorganisms could derive from different mechanisms. The microorganisms' capacity to adhere to the intestinal epithelium, preventing the adhesion of pathogens, and their immunomodulation properties are among the mechanisms suggested. Bacteria and yeast isolated from kefir have been shown to have in vivo and in vitro antimicrobial activity against enteropathogenic bacteria and spoilage fungi. However, most reports have focused their approach on single-strain antimicrobial properties; evaluation of antimicrobial activity of cocultures of kefir microbiota and their potential mechanisms of action has been neglected. Kefir microbiota and associated compounds have shown promising antimicrobial effects; however, more research needs to be done to discern the mechanisms of action.

Active packaging films based on kefiran-gelatin were developed and characterized using Zhumeria majdae essential oil nanoemulsion (ZMEO-NE) at concentrations of 0 (control), 1, 2 and 3 %. The main compounds of the essential oil (EO) of Zhumeria majdae (ZM) plant were linalool (61.44 %) and camphor (20.67 %). Adding the ZMEO-NE to the films decreased permeability to water vapor (from 7.82 × 10 -7 to 4.09 × 10 -7 g·m/m 2 ·Pa·h), ultimate tensile strength (from 38.44 to 33.48 MPa), percentage of light transmission, and increased thickness (from 0.085 to 0.121 mm), opacity (from 2.11 to 2.79), and elongation at break (from 19.97 to 34.73 %), and changed color parameters. The establishment of hydrogen bonds between the ZMEO-NE and polymer network was confirmed. The ZMEO-NE decreased the storage modulus and glass transition temperature. Distinct variations in the films' surface morphology and a reduction in the crystalline structure were observed due to the presence of the ZMEO-NE. Elevating the concentration of the ZMEO-NE increased antioxidant capabilities of the films. The films incorporating the ZMEO-NE exhibited notable antibacterial efficacy against Staphylococcus aureus (reductions ≥4 log CFU/cm 2 ) and Escherichia coli (reductions ≥2 log CFU/cm 2 ) bacteria. The films also demonstrated suitable antifungal properties during storage of sponge cakes for 16 days., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The use of biopolymers in medicine and pharmacology is of great scientific, technological, and commercial interest. A possibility of biopolymer for use in pharmacological applications is the kefiran, an exopolysaccharide (EPS) produced by bacteria that form kefir grains during the milk fermentation process in which the grains are growing. The EPS extraction method can modify its concentration and structure. To establish the most appropriate kefiran extraction protocol and standardize this method, three different extraction processes were evaluated, which differ in the boiling times and in the presence (or not) of agitation. Kefiran obtained by different methods were characterized by thermogravimetric analysis (TGA), Fourier transform infrared spectroscopy (FTIR), electron scanning field emission microscopy (FESEM), and determination of antimicrobial potential. The samples showed differences in yield, chemical composition, degree of purity, thermal properties, and antimicrobial capacity. The results analyzed indicated that extraction protocol 1 (EP 1) with a longer heating time and constant agitation was able to provide the best thermal resistance, with a high degree of purity (93%), and yield (22.18 mg of kefiran obtained for each gram of kefir grains used). In addition, this method provides good biological activity as a potential antimicrobial agent and can be a potential biopolymer for pharmacological application.

Kefiran is a water soluble polysaccharide produced by Lactobacillus kefiranofaciens ATCC 43761. It has wide potential applications in food, pharmaceutical and nutraceutical industries. To the best of our knowledge, there have been no previous reports on the effect of osmotic stress and ionic surfactants on kefiran production by L. kefiranofaciens ATCC 43761. Accordingly, the current work aimed at optimizing kefiran production as affected by osmotic stress and nonionic surfactants in submerged cultivation system. Afterwards, the work was extended to investigate cytotoxic as well as antioxidant potentials of kefiran. Firstly, different osmolarities, different ionic surfactants (Triton X-100, Tween 20, Tween 80) as well as their concentrations and addition time were evaluated. The kinetics of cell growth and kefiran production were evaluated before and after the addition of surfactants. Results clearly demonstrated that osmotic stress and surfactant addition had a stimulatory effect on kefiran production. Using the optimal medium osmolality, 550 mOsmol.kg−1, kefiran production was enhanced from 1.29 to about 1.38 g.L−1. Furthermore, Triton X-100 was found to be the best surfactant stimulating kefiran production when added at a concentration of 1.0 g.L−1 at the onset of cultivation process (0 h). This increased kefiran production from 1.38 g.L−1 to 1.62 g.L−1. To summarize, the maximal kefiran production can be enhanced using 550 mOsmol.kg−1 and by adding 1.0 g.L−1 of Triton X-100 at 0 h. The new optimized medium showed an increase of about 25.6% in kefiran production (1.29 up to 1.62 g.L−1). After this step, the process was further optimized in 16-L stirred tank bioreactor. Maximal kefiran production reached 2.32 g.L−1 and 1.87 g.L−1 in bioreactor under control and un-controlled pH conditions, respectively, corresponding to 72.9 and 45.0% increase from the initial production titer, respectively. The produced kefiran exhibited promising anticancer activity against breast cancer (MCF-7) cells, with an IC50 value of 193.89 μg.mL−1. Also, kefiran showed 96.58% radical scavenging activity at 100 μg/mL, with an ED50 recorded of 12.29 ± 0.98 μg.mL−1.

Lactobacillus kefiranofaciens is non-pathogenic gram positive bacteria isolated from kefir grains and able to produce extracellular exopolysaccharides named kefiran. This polysaccharide contains approximately equal amounts of glucose and galactose. Kefiran has wide applications in pharmaceutical industries. Therefore, an approach has been extensively studied to increase kefiran production for pharmaceutical application in industrial scale. The present work aims to maximize kefiran production through the optimization of medium composition and production in semi industrial scale bioreactor. The composition of the optimal medium for kefiran production contained sucrose, yeast extract and K2HPO4 at 20.0, 6.0, 0.25 g L−1, respectively. The optimized medium significantly increased both cell growth and kefiran production by about 170.56% and 58.02%, respectively, in comparison with the unoptimized medium. Furthermore, the kinetics of cell growth and kefiran production in batch culture of L. kefiranofaciens was investigated under un-controlled pH conditions in 16-L scale bioreactor. The maximal cell mass in bioreactor culture reached 2.76 g L−1 concomitant with kefiran production of 1.91 g L−1.

Over the recent years, there is a growing interest in electrospun hybrid scaffolds composed of synthetic and natural polymers that can support cell attachment and proliferation. In this work, the physical and biological properties of polylactic acid (PLA) electrospun mats coated with kefiran (Kef) were evaluated. Gravimetric, spectroscopic (FTIR-ATR) and morphological investigations via scanning electron microscopy confirmed the effective formation of a thin kefiran layer wrapped on the PLA fibers with an easy-tunable thickness. Air plasma pre-treatment carried out on PLA (P-PLA) affected both the morphology and the crystallinity of Kef coating as confirmed by differential scanning calorimetry and X-ray diffraction analyses. Scaffolds were mechanically characterized with tensile tests to evaluate the reinforcing action of the Kef coating. The water resistance of Kefiran coating in distilled water at 37 °C evaluated on both PLA/Kef and P-PLA/Kef was carried out by gravimetric and morphological analyses. Finally, cell culture assays with embryonic fibroblast cells were conducted on selected hybrid scaffolds to compare the cell proliferation, morphology, and collagen production with PLA and P-PLA electrospun scaffolds. Based on the results, we can demonstrate that direct coating of PLA from Kef/water solutions is an effective approach to prepare hybrid scaffolds with tunable properties and that the plasma pre-treatment enhances the affinity between PLA and Kefiran. In vitro tests demonstrated the great potential of PLA/Kef hybrid scaffolds for skin tissue engineering., (Copyright © 2021 Elsevier B.V. All rights reserved.)

In this study, kefiran was esterified with octenyl succinic anhydride (OSA). The esterification reaction variables including pH (8.5), kefiran concentration (5% (w/w)), OSA concentration (12% (w/w)), temperature (~38 °C) and reaction time (~80 min) were found as optimum points to achieve the maximum degree of substitution (DS) (0.041 ± 0.002). Kefiran-OSA samples with DS of 0.021 (FDA suggested DS) and 0.041 (maximum DS) were prepared and compared with unmodified kefiran in all experiments. FTIR and 1 H NMR spectroscopies proved the grafting of OSA on kefiran structure. XRD analysis revealed that with increase in DS, the physical state of kefiran to be more amorphous. In addition, the esterification modification led to a decrease in the degradation temperature and an increase in the apparent viscosity based on the obtained data from thermal analysis and viscosity measurement. The results of the foaming and emulsifying properties confirmed the improvement in surface properties of the modified kefiran. The frequency sweep test illustrated that with an increase in DS, the viscoelastic behavior of the kefiran cryogels to be more viscous. It can finally be stated that the modification with OSA was a high potential strategy to extend the industrial applications of the kefiran., (Copyright © 2020. Published by Elsevier B.V.)

Kefiran is a water-soluble polysaccharide well recognized as a bioactive ingredient to enhance nutritional and health-promoting features. Also, some therapeutic properties have made this macromolecule an active ingredient in ointments and oral anti-inflammatory drugs. However, the details of the molecular and cellular aspects of these effects have not been addressed. In this study, lipopolysaccharides (LPS)-induced monocytes, lymphocytes, and monocyte-derived dendritic cells (MDDCs) as representative cells for both innate and adaptive immunity were treated with kefiran for 2 h. Kefiran had an anti-inflammatory effect on monocytes to reduce pro-inflammatory cytokines, interleukin 1 β (IL-1β) & tumor necrosis factor α (TNF-α), as well as nuclear factor kappa b (NF-kb). However, it did not affect lymphocytes. Overexpression of Toll-like receptor 4 (TLR4) in LPS-induced cells was not reduced after kefiran treatment. Kefiran balanced MDDCs secretion of pro/anti-inflammatory cytokines by reducing and enhancing the expression of IL-1β and interleukin 10 (IL-10), respectively. Also, kefiran decreased the number of apoptotic immature MDDCs and promoted dose-dependent phagocytosis capacity of MDDCs. According to the results of the current study, it may be concluded that the immunomodulatory effects of kefiran are due to antagonist against innate immune receptors especially TLR4. The results of this study can be used as a guide to developing kefiran-based non-aggressive anti-inflammatory drugs. Furthermore, understanding the immunobiological effects of kefiran on monocytes and lymphocytes was another outcome of this study., Competing Interests: Declaration of competing interest This work was financially supported by the grant from the University of Zanjan, Iran. The authors declare no conflicts of interest., (Copyright © 2020. Published by Elsevier B.V.)

In this study, the effect of UV-C light on starch-kefiran-ZnO (1%) primary solution in different exposure times (1, 6, and 12 h) was investigated. Starch-kefiran-ZnO (SKZ) solution was modified by UV irradiation in different time periods. Also, nano-ZnO (ZN) was used as a photo-initiator and reinforcement agent, simultaneously. Mechanical properties of the films were affected after the UV treatment. The tensile strength increased because of the enhanced interaction between the biopolymer mixture and nano filler but elongation at break was decreased. WVP decreased about 16% and dwindled to 2.08 × 10 -10  g m -1  s -1  Pa -1 . Water related properties (i.e. moisture content, moisture absorption, and solubility in water) of the films decreased by UV-C exposure. On the other hand, UV absorption and water contact angle increased because of the better distribution of the ZNs in polymer matrix after the UV exposure. Better compatibility of the ZNs and the biopolymer matrix after UV treatment was confirmed by the SEM micrographs. Comparison of FTIR spectra before and after UV exposure showed slight shifts. It was due to some formed or deformed bonds inside of the nanocomposite matrix. The modified SKZ by UV could be an appropriate process to sanitizing and food packaging concurrently. As well as UV can be used as a nano-ZnO compatibilizer in food packaging materials., (Copyright © 2018. Published by Elsevier B.V.)

Biodegradable kefiran films, containing different concentrations of Al 2 O 3 (alumina) (1, 3 and 5% w/w), were prepared by casting method and their physical, mechanical, and thermal properties were studied. Based on the results, the increase of the nano-Al 2 O 3 content led to a decrease in the water vapor permeability, moisture content, moisture absorption, and water solubility. The addition of nanoparticles did not affect the lightness of the films significantly (P > 0.05). The mechanical tests revealed that the addition of Al 2 O 3 nanoparticles to the kefiran films significantly increased both tensile strength and elastic modulus, while had no effect on the elongation at break (P > 0.05). Thermogravimetric analysis (TGA) indicated the improvement of the films' thermal stability by adding alumina. The results of X-ray diffraction (XRD) confirmed the effect of Al 2 O 3 on the semi-crystalline structure of kefiran. Scanning electron microscopy (SEM) images approved a uniform distribution of nanocomposites containing 1 and 3% w/w of Al 2 O 3 . The obtained results suggested that Al 2 O 3 was able to improve the characteristics of the kefiran-based films for using as food packaging material., (Copyright © 2018. Published by Elsevier B.V.)

This paper reports on an experiment in which starch/kefiran/TiO 2 (SKT)-based bio-nanocomposite films were developed and modified by photo-chemical reaction. In so doing, film-forming solutions were exposed to ultraviolet A (UV-A) for different times (1, 6, and 12 h). The obtained results indicated that increasing UV-A exposure time brought about an increase (≈14.9%) in the tensile strength of bio-nanocomposites. However, elongation at break and Young's modulus of irradiated film specimen decreased (≈32%, ≈12%, respectively) by increasing UV-A exposure time, and the moisture-sensitive parameters of specimen decreased using UV-A irradiation. According to the results, the functional properties of irradiated bio-nanocomposite are depended on the ratio of cross-linkages between polymer chains and the potentially produced mono and disaccharide by UV-A., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Ciprofloxacin is a broad-spectrum antibiotic associated with gastric and intestinal side effects after extended oral administration. Alginate is a biopolymer commonly employed in gel synthesis by ionotropic gelation, but unstable in the presence of biological metal-chelating compounds and/or under dried conditions. Kefiran is a microbial biopolymer able to form gels with the advantage of displaying antimicrobial activity. In the present study, kefiran-alginate gel microspheres were developed to encapsulate ciprofloxacin for antimicrobial controlled release and enhanced bactericidal effect against common pathogens. Scanning electron microscopy (SEM) analysis of the hybrid gel microspheres showed a spherical structure with a smoother surface compared to alginate gel matrices. In vitro release of ciprofloxacin from kefiran-alginate microspheres was less than 3.0% and 5.0% at pH 1.2 (stomach), and 5.0% and 25.0% at pH 7.4 (intestine) in 3 and 21h, respectively. Fourier transform infrared spectroscopy (FTIR) of ciprofloxacin-kefiran showed the displacement of typical bands of ciprofloxacin and kefiran, suggesting a cooperative interaction by hydrogen bridges between both molecules. Additionally, the thermal analysis of ciprofloxacin-kefiran showed a protective effect of the biopolymer against ciprofloxacin degradation at high temperatures. Finally, antimicrobial assays of Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella typhymurium, and Staphylococcus aureus demonstrated the synergic effect between ciprofloxacin and kefiran against the tested microorganisms., (Copyright © 2016 Elsevier B.V. All rights reserved.)

In this study, the effect of different γ-ray dosages (3, 6, and 9 kGy) on the functional properties of kefiran biopolymer was investigated. The obtained results showed that increasing γ-ray dosage brought about an increase in the tensile strength of film specimens up to three-times. However, elongation at break, and tensile energy to break of γ-irradiated kefiran films decreased in the wake of increasing γ-ray dosage. γ-Irradiation could improve surface hydrophobicity, sensitivity of kefiran film specimens to water, and water vapor permeability, but yellowness of films increased, simultaneously. XRD spectrum confirmed increased crystallinity of γ-irradiated films. Melting point of films was constant but glass transition temperature decreased drastically at high γ-ray dosage (9 kGy). ATR-FTIR analysis confirmed that γ-ray engendered no changes in chemical functional groups. According to the result, a mechanism was proposed to percept the effects of γ-irradiation on kefiran biopolymer and its role on the functional properties of kefiran film. Hence, the functional properties of kefiran films were depend on the ratio of cross-linkages between polymer chains and produced mono and disaccharide by γ-irradiation., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Environmental concern about synthetic polymers and nanoparticles bring about development of the green bionanocomposite. Nanocellulose (NC) as safe nanofiller was prepared from beer industrial residues by acid hydrolysis in this study. ATR-FTIR spectrum showed no change in chemical structure of kefiran and NC after mixing. However, mechanical, visual, and WVP properties of kefiran/NC films improved with NC, but thermal properties and water sensitivity of them declined, simultaneously., (Copyright © 2015 Elsevier B.V. All rights reserved.)

In this study, we investigated the effect of ZnO nanoparticles (ZN) as a UV-protective agent of kefiran biopolymers. Our results showed that with increasing ZN content, the tensile strength, elongation at break, and tensile energy to break the kefiran film and nanocomposites also increased. Kefiran nanocomposites with a ZN content higher than 2% produced a UV-protective film with good visual properties, low sensibility to water, and low water-vapor permeability. The thermal properties of all specimens, analyzed by DSC, showed that the ZN content had a negative effect on Tg and a positive effect on nanocomposites' melting point. TEM, SEM micrography and XRD spectrum analysis confirmed the hypothesis that ZNs act like a ball bearing, making movement of kefiran chains easier and increasing elongation at break, while simultaneously decreasing the Tg of kefiran nanocomposites., (Copyright © 2014 Elsevier B.V. All rights reserved.)

In this study, we report the first successful production of kefiran nanofibers through electrospinning process using distilled water as solvent. For this purpose, kefiran was extracted from cultured kefir grains, and homogenous kefiran solutions with different concentrations were prepared and then electrospun to obtain uniform nanofibers. The effect of main process parameters, including applied voltage, tip-to-collector distance, and feeding rate, on diameter and morphology of produced nanofibers, was studied. Scanning electron microscopy (SEM) and attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy were used to characterize electrospun mats. Rheological behavior of the kefiran solution was evaluated via a cone and plate rheometer too. The results exhibited that diameter of kefiran nanofibers increased with increasing polymer concentration, applied voltage, and polymer feeding rate, while tip-to-collector distance did not have significant effect on nanofiber diameter. ATR-FTIR spectra showed that kefiran has maintained its molecular structure during electrospinning process. Flow curves also demonstrated shear thinning behavior for kefiran solutions., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Biodegradable kefiran-whey protein isolate (WPI)-titanium dioxide (TiO2) blend films were developed and characterized as a function of incorporating amount of TiO2 nanoparticles (1, 3 and 5% wt.). Results showed that the water vapor permeability, moisture content, moisture absorption and water solubility decreased by increasing the nano-TiO2 content. Mechanical tests revealed the plasticizing effect of TiO2 nanoparticles on the kefiran-WPI-TiO2 film. Addition of TiO2 nanoparticles to kefiran-WPI films significantly decreased tensile strength and Young's modulus, while increased its elongation at break. Differential scanning calorimetry data indicated that the glass transition temperature significantly changed by adding nano-TiO2. X-ray diffraction analysis also demonstrated that crystal type in kefiran-WPI was not affected by incorporation of TiO2 nanoparticles. A uniform distribution at 1 and 3% wt. loading levels of TiO2 nanoparticles was observed using scanning electron microscopy (SEM) micrographs., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The biological and technological characteristics of kefiran as well as its importance in grain integrity led us to analyze the microbial kefir grain consortium with focus on Lactobacillus kefiranofaciens. The presence of L. kefiranofaciens in the nine kefir grains studied was demonstrated by denaturing gradient gel electrophoresis. By culture dependent methods applying a methodology focused on the search of this species, 22 isolates with typical morphology were obtained and identified applying a combination of SDS-PAGE of whole cell proteins, (GTG)5-PCR and sequence analysis of the housekeeping gene encoding the α-subunit of bacterial phenylalanyl-tRNA synthase (pheS). This polyphasic approach allowed the reliable identification of 11 L. kefiranofaciens, 5 Lactobacillus paracasei, 4 Lactobacillus kefiri and 2 Lactobacillus parakefiri isolates. Isolated L. kefiranofaciens strains produced polysaccharide in strain-dependent concentrations and EPS produced by them also differed in the degree of polymerization. The isolation and accurate identification of L. kefiranofaciens is relevant taking into account the important role of this microorganism in the grain ecosystem as well as its potential application as starter in food fermentations., (Copyright © 2013 Elsevier Ltd. All rights reserved.)