Production of active electrospun nanofibers based on kefiran containing purified postbiotics derived from kefir microflora and silver nanoparticles

Introduction: Kefir is a dairy that contains lactic acid, carbon dioxide, and other volatile aromatics traditionally produced by the activity of kefir dunkers (Gaware et al., 2011). One of the constituent components of kefir grain are mucoid substances that are produced by the microbial flora of this grain, and exopolysaccharide called kefiran is one of them. Radhouani et al. (2018) showed that kefir is a suitable material for use in the field of biomaterials production, film and tissue formation. The most prominent biodegradable biopolymers used in food packaging from the group of biopolymers produced by microorganisms are bacterial cellulose and kefir (Rhim et al., 2013 and Sabatino et al., 2020). Kefiran is able to produce transparent films with relatively good mechanical resistance. Recently, the antimicrobial effect of postbiotics has attracted the attention of researchers. Postbiotics are actually a byproduct of the fermentation process done by probiotics (Moradi et al., 2019). Silver nanoparticle is one of the nanomaterials that has been mentioned in many studies for its antimicrobial effect as well as the effect of improving the mechanical and barrier properties of biodegradable films (Ajitha et al., 2021; Blosi et al., 2021; Li et al., 2023). One of the new methods in the production of biopolymer films that has recently attracted the attention of researchers is the electrospinning method. Electrospinning is a process in which continuous polymer fibers with diameters in the sub-micrometer range are produced through the action of a high-voltage electric field applied to a solution.

Material and methods: To cultivate kefir seeds, kefir grains were first cultured in fresh cow's milk for activation and growth. In order to increase the activation speed, greenhouse was used at a temperature of 27°C. To extract kefir from kefir grains, kefir grains were mixed with distilled water at a ratio of 1:10 at a temperature of 81°C and then the resulting mixture was centrifuged at a temperature of 20°C for 20 minutes. Then, kefirs using 96% ethanol were centrifuged at a temperature of 4°C for 20 minutes, and the obtained precipitates were mixed with distilled water at a temperature of 80°C in a ratio of 1:5. This washing step was repeated twice until a white deposit of kefir was obtained and then, was dried.

In order to extract the postbiotic extract, first, 5 g of kefir seeds were inoculated in the milk culture medium and placed in a greenhouse at 35°C for 48 hours. Then the culture medium was centrifuged and the supernatant was filtered with a needle filter. In this study, a concentration of 4% kefir was used to produce electrospun nanofibers. In this way, 0.04 g of kefir was added to 10 ml of postbiotic extract and electrospuned (Ethnaashri et al. 2024). To prepare the next treatments, three levels of silver nanoparticles (1, 2.5, and 4% by weight of kefir) were added to the previous solution and treated with an ultrasonic probe for 5 minutes (40 kHz, 100W).

Also, in order to study the properties of the nanofibers, FTIR, XRD, DSC and SEM analysis were performed and the contact angle, antioxidant activity and antimicrobial activity of them were studied.

Results and discussion: This research was designed and implemented with the aim of developing an active electrospun nanofiber substrate for use in food preservation. Kefiran nanofibers containing kefir postbiotics in constant concentration and silver nanoparticles in different concentrations were prepared and their structural, morphological and functional properties were evaluated. Comparison of FTIR spectra of nanofibers containing nanosilver shows that the addition of nanoparticles had no effect on the chemical structure of nanofibers. Due to the small amount of nanosilver in the formulation of nanofibers, the peak related to the nanoparticles itself was not observed. The lack of change in the spectrum of kefir chemical groups due to the addition of nanosilver means that the nanoparticles are physically bound in the polymer network of kefir nanofibers. SEM images show tha nanofibers had a uniform cylindrical shape with a smooth surface and no knots. The average diameter of kefiran nanofibers without nanosilver was about 245 nm. Also, by increasing the concentration of silver nanoparticles to 2.5%, the size of nanoparticle clusters on the surface of nanofibers increased and the average diameter of kefir nanofibers decreased to 205 nm. XRD analisys of kefiran nanofibers showed a sharp peak at the angle of 2θ=44.5°. The results indicate the semi-crystalline nature of kefir nanofibers. With the increase of silver nanoparticles, there is no change in the intensity and location of the main peak at the angle of 44.5°, but a new peak is created at the angle of 2θ = 1.38° and the highest intensity is observed in the sample containing 4% nanosilver. The control nanofibers of kefir containing postbiotics showed three ranges of changes in the DSC test. The main melting peak of this nanofiber was observed at 73°C. By adding silver nanoparticles, the thermal properties of kefir nanofibers were weakened. The results show that silver nanoparticles affected the thermal properties of kefir and reduced its resistance to temperature. The contact angle of nanofibers by adding of silver nanoparticles at a concentration of 1%, shwed no significant change (p>0.05). But when the amount of silver nanoparticles reached 2.5% and 4%, the contact angle decreased significantly (p<0.05) and in fact the hydrophilic property of nanofibers increased. The antibacterial activity of electrospun kefir nanofibers containing postbiotics and nanosilver was investigated against E. coli and S. aureus. By increasing the amount of silver nanoparticles, the antimicrobial property increased and the sample containing 4% nanosilver showed the highest antibacterial activity against both bacteria. In this research, kefir nanofibers containing postbiotic showed an antioxidant activity of 5.65%, which is due to the low amount of postbiotic in the nanofiber formulation. By increasing the concentration of silver nanoparticles, the antioxidant activity increased significantly, and the highest inhibitory activity was observed at a concentration of 4% with a power of 59.21%.

Conclusion: The results of the FTIR test proved the physical connection and the lack of chemical bonding between silver nanoparticles and nanofibers. Good and uniform surface morphology was observed in kefir nanofibers in SEM test, but silver nanoparticles were observed in aggregated form. The XRD test showed no effect of nanosilver on the structure of kefiran nanofibers, but in the DSC test, the weakening of the thermal properties of kefiran nanofibers was reported with an increase in the concentration of nanosilver. The control nanofibers of kefirs that had only postbiotics also had antioxidant and antimicrobial properties, but these functional properties were enhanced by increasing the concentration of silver nanoparticles, and nanofibers containing 4% nanosilver showed the best antimicrobial and antioxidant properties. In general, the results of this study showed that kefir nanofibers containing postbiotic kefir and silver nanoparticles have favorable physical and functional characteristics and can be used as active food packaging and can be used for the purposes of increasing the shelf life of food and preventing microbial and oxidative spoilage.