Green Synthesis, Characterization, and Antimicrobial Activity of Silver Nanoparticles from Water-Soluble Fractions of Brazilian Kefir

Green Synthesis, Characterization, and Antimicrobial Activity of Silver Nanoparticles from Water-Soluble Fractions of Brazilian Kefir | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Green Synthesis, Characterization, and Antimicrobial Activity of Silver Nanoparticles from Water-Soluble Fractions of Brazilian Kefir Lucas Matos Martins Bernardes, Serena Mares Malta, Ana Carolina Costa Santos, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4830503/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 27 Mar, 2025 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract Background: Nanotechnology offers innovative approaches to combat drug-resistant diseases. Silver nanoparticles (AgNPs) have emerged as potent antimicrobial agents in vitro and in vivo . Green synthesis methods, which leverage the bioactive components of the water-soluble fractions of Brazilian kefir (whole water-soluble fraction and smaller than 10 kDa fraction), provide sustainable alternatives to conventional nanoparticles production. However, despite the documented therapeutic benefits of kefir, its potential in nanomedicine remains underexplored. Results: The successful synthesis of silver nanoparticles using water-soluble fractions of kefir was confirmed by UV-Visible spectroscopy and Fourier-transform infrared analyses. The hydrodynamic radius of nanoparticles derived from the entire water-soluble fraction was 1300 nm, while those from the smaller than 10 kDa fraction displayed a radius of 400 nm. All synthesized AgNPs exhibited a zeta potential of -30 mV. The disk diffusion method demonstrated the antimicrobial efficacy of our AgNPs against a range of multidrug-resistant bacteria and Candida fungi (p<0.0001), with no observed toxicity on Drosophila melanogaster on a long-term treatment. Conclusion: This study highlights the potential of these AgNPs as effective antimicrobial agents, particularly against drug-resistant pathogens. Future research is needed to evaluate the minimum inhibitory concentrations of our AgNPs and enhance specificity through conjugation with other compounds. Additionally, further investigations into electron microscopy analysis and various applications, such as disinfectant solutions, wound healing, and antibiotic production, will advance the utilization of kefir-derived AgNPs in healthcare. Biological sciences/Biotechnology/Nanobiotechnology Biological sciences/Microbiology/Antimicrobials Physical sciences/Nanoscience and technology/Nanobiotechnology Physical sciences/Nanoscience and technology/Techniques and instrumentation/Characterization and analytical techniques Physical sciences/Nanoscience and technology/Techniques and instrumentation/Design synthesis and processing Silver nanoparticles kefir nanomedicine anti-bacterial agents antifungals Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. INTRODUCTION Nanotechnology is a rapidly advancing field that involves manipulating materials at the nanoscale, resulting in nanoparticles (NPs) ranging from 1 to 100 nm in size. These nanoparticles exhibit unique physicochemical properties and hold promise for addressing pressing health challenges [ 1 ]. In particular, the increase in the incidence of drug-resistant diseases poses a significant threat, with an estimated 700,000 deaths annually—a figure that is projected to increase to 10 million by 2050 if left unchecked [ 2 ]. Fungal infections are even more concerning, with more than 300 million people suffering from various fungal diseases, resulting in more than 2 million deaths annually from mycoses [ 2 ]. Silver nanoparticles (AgNPs) are particularly noteworthy in nanomedicine due to their exceptional antimicrobial efficacy and relatively low toxicity. AgNPs can potentially combat microbial resistance due to their unique properties, such as a high surface area-to-volume ratio and the ability to generate reactive oxygen species [ 2 , 3 ]. Therefore, exploring their antimicrobial activities could be a promising approach for addressing the growing threat of drug-resistant infections. One method for assessing the antimicrobial efficacy of AgNPs is through the disk diffusion technique, a reliable methodology that has been employed in numerous previous studies [ 4 – 7 ]. The NPs can be synthesized using physical, chemical, or biological methods. Although physical and chemical methods have traditionally been used for nanoparticle synthesis, their drawbacks, including cost, time consumption, and environmental concerns, have led to increased interest in biological synthesis methods [ 8 – 11 ]. Biological synthesis, also known as 'green synthesis,' uses nontoxic and eco-friendly reagents, offering a more streamlined approach to nanoparticle production. Organism-derived extracts, which are rich in biomolecules such as metabolites and proteins, play crucial roles as reducing and capping agents in this process [ 1 , 12 ]. This highlights the efficiency and versatility of biological synthesis. Studies have used cell-free extracts and derived substances from bacteria as precursors for nanoparticle synthesis [ 13 , 14 ]. In this context, kefir, a fermented beverage with well-documented antioxidant and antimicrobial properties, is a promising candidate for nanoparticle synthesis [ 15 – 18 ]. Although kefir has not been extensively studied in nanotechnology, our group has discovered significant antioxidant activity in the smaller than 10 kDa (< 10 kDa) fraction of the water-soluble component of kefir. Additionally, bioactive peptides have been identified from the this fraction [ 19 , 20 ]. Based on these findings, the water-soluble fraction (WSF) of kefir can be considered a promising agent for the green synthesis of nanoparticles. Controlling the characteristics of the resulting nanoparticles in biological synthesis is challenging due to various factors that can impact the process, such as pH, temperature, and concentration of precursor agents [ 21 ]. Therefore, rigorous characterization of the NPs properties, including size, morphology, and chemical composition, is essential. Techniques such as UV‒visible (UV‒Vis) spectroscopy, Fourier transform infrared (FT-IR) spectroscopy, dynamic light scattering (DLS), and zeta potential measurements are well-described in the literature and provide valuable insights into these properties [ 10 , 22 – 24 ]. The toxicity of nanoparticles can be evaluated through in vitro and in vivo assays. The fruit fly ( Drosophila melanogaster ) is a well-established model organism that offers several advantages, including a rapid life cycle, low cost of maintenance in laboratories, and a genome with significant human homology [ 25 ]. This model has been previously utilized for studies on the toxicity of AgNPs, examining aspects such as lifespan and gut morphology [ 26 – 28 ]. Consequently, it is a reliable model for assessing the safety of AgNPs. This study aimed to examine the potential of kefir for nanoparticle synthesis and its antimicrobial applications. First, we analyzed the water-soluble fraction of kefir as a reagent for nanoparticle synthesis. We then characterize the synthesized nanoparticles including size, morphology, and chemical composition. Additionally, we assessed their antimicrobial activity through disk diffusion against a set of multiresistant bacteria and fungi from the Candida genus. Moreover, we evaluated the toxicity of AgNPs by using fruit flies. This research aimed to contribute to the advancement of the use of nanotechnology in healthcare, more specifically the potential of kefir-derived silver nanoparticles in antimicrobial and antifungal activities. 2. MATERIALS AND METHODS 2.1 Kefir production and collection of its water-soluble fractions The kefir grains utilized in this study were donated by residents of the city of Uberlândia, Brazil. The grains were previously characterized through genetic fingerprinting by our group [ 20 ]. To produce kefir, whole cow's milk was fermented with a 10% (w/v) concentration of kefir grains. Fermentation occurred over a 24-hour period at a controlled temperature of 28°C, shielded from light. Following fermentation, the kefir was separated from the grains using a sieve. To obtain the water-soluble fraction (WSF), the kefir was centrifuged twice at 4900 g for 10 minutes at 4°C. After each centrifugation, the supernatant was collected, and the pellet was discarded. Then, the resulting supernatant was filtered through two layers of filter paper [ 20 ]. To ensure the suitability of the WSF for nanoparticle synthesis, we modified the protocol [ 20 ]. Specifically, we added ammonium hydroxide (NH 4 OH) to the WSF immediately after filtration at a concentration of 0.05% v/v. This addition aimed to alter the pH of the sample and precipitate proteins that might interfere with nanoparticle synthesis. Subsequently, we centrifuged the solution at 12000 g for 15 minutes at 4°C to pellet the proteins, which enabled us to collect the clarified supernatant for nanoparticle synthesis. The NH 4 OH is usually added during the nanoparticle synthesis process, but the protocol required adaption due to an unsuccessful first synthesis (explained in the Results section). Furthermore, the supernatant underwent further filtration using a vacuum pump and a 0.22 µm membrane. Subsequently, it was purified through a 10 kDa Amicon column to fractionate the WSF into the smaller than 10 kDa (< 10 kDa) fraction. 2.2 Green synthesis of silver nanoparticles from kefir fractions The silver nanoparticles synthesis method employed in this study was modified from a previous described green synthesis protocol [ 29 ]. First, we added 60 mL of each fraction (whole WSF and < 10 kDa), which already contained NH 4 OH, to individual flasks containing 0.18 g of silver nitrate (AgNO 3 ). The flasks were briefly shaken and then heated to boiling in a microwave oven. Upon reaching the boiling point, the solutions turned brown, indicating the formation of nanoparticles. Each solution was stirred using a magnetic stirrer for 15 minutes at medium speed. After the synthesis process, the nanoparticles were subjected to three cycles of centrifugation (16000 g , 15 minutes, 4°C). After each cycle, the supernatant was discarded, and the pellet containing the silver nanoparticles was resuspended in deionized water. The resulting solution was stored in a refrigerator set at 4°C. To determine the concentration of each solution, a portion was air-dried. The nanoparticles derived from the WSF and < 10 kDa fractions are referred to as WSF AgNPs and < 10 kDa AgNPs, respectively. 2.3 Characterization of silver nanoparticles Analytical techniques, including UV‒Vis, DLS, zeta potential analysis, and FT-IR were used to characterize the silver nanoparticles. UV‒Vis spectroscopy was employed to analyze characteristics of the nanoparticles and the efficiency of their synthesis based on the absorbance peak (typically 400–500 nm) of silver nanoparticles. In a 96-well plate, triplicate samples (100 µL each) of nanoparticle solutions, along with WSF and silver nitrate controls, were analyzed. Spectra were measured from 300 to 800 nm using a standardized UV‒Vis spectrophotometer. Analysis of the data included peak identification based on wavelength and intensity criteria. Triplicate samples were used to ensure the reliability of the results. The infrared spectra of the AgNPs and their precursors were obtained using an Agilent Cary 630 FTIR spectrophotometer in the range from 4000 to 650 cm − 1 . Sample analyses were performed in the solid state using the Attenuated Total Reflectance (ATR) accessory with a diamond crystal. The initial step of the DLS analysis involved optimizing the measurements for each sample. Nanoparticle solutions were prepared at a range of concentrations from 0.01 mg/mL to 5 mg/mL. Subsequently, size distribution, hydrodynamic radius, and polydispersity index of the nanoparticles were evaluated in quintuplicate. Each measurement was conducted at 25°C using 2 mL of sample. To ensure stable measurements, a 3-minute equilibrium time was implemented, allowing the nanoparticles to reach a steady state and match the equipment's temperature. The backscatter measurement angle (175°) was chosen due to its high sensitivity to minute particles and its ability to minimize the impact of multiple scattering, thus ensuring reliable and precise data on size distribution. The optimization process aimed to determine the nanoparticle concentration that would consistently yield reliable results in five replicates. We considered parameters provided by the DLS equipment (Litesizer 500, Anton Paar, UK), such as baseline, g1², and transmittance values, to ensure accurate measurements. Adhering to recommended criteria is crucial for obtaining reliable data. Our analysis focused on two parameters: transmittance levels and baseline values. Based on manufacturer guidelines, transmittance levels should ideally exceed 80%, with values above 60% considered acceptable. Baseline values should remain at 1 for optimal performance. The DLS equipment was also used for examining the zeta potential of the AgNPs, which was measured in quintuplicate at 0.1 mg/mL for WSF AgNPs and 0.05 mg/mL for the < 10 kDa AgNPs. The measurements were made using 200 V at 25°C with an equilibrium time of 3 minutes. The reasoning for selecting these concentrations for the analysis will be further discussed in the Results section. A scanning electron microscopy (SEM) analysis was conducted to corroborate the data obtained from the DLS analysis regarding the dimensions of the nanoparticles and to gather additional data on their morphology. To obtain SEM images, the dispersed samples were deposited onto a silicon substrate. A Vega 3 TESCAN scanning electron microscope, operated at 20 kV, was used, equipped with a secondary electron detector and an energy-dispersive X-ray (EDX) detector (Oxford Instruments, Bucks, England). A voltage of 20 kV was used for acquiring the EDX spectra and mapping. 2.4 In vitro susceptibility test (disk diffusion method) The anti-bacterial property of AgNPs was evaluated using a diffusion disk test against eight strains of multidrug-resistant bacteria, including Acinetobacter baumannii , Escherichia coli (MCR), Klebsiella pneumoniae carbapenemase , three strains of Pseudomonas aeruginosa (IMP, VIM, and SPM), and Staphylococcus aureus (MRSA), obtained from the Molecular Microbiology Laboratory of the Federal University of Uberlândia. Strain characterization was performed by bacterial genotyping by PCR and phenotyping on ampicillin and polymyxin supplemented BHI agar. The modified Kirby-Bauer disk diffusion method was used to assess antimicrobial activity [ 30 , 31 ]. For testing, bacteria were plated on agar plates at a concentration of 0.5 on the McFarland scale. Subsequently, 15 µL of AgNPs at three concentrations (10, 100 and 1000 µg/mL) were applied to antimicrobial susceptibility test discs in triplicate. The discs were incubated at 37°C for 24 hours and then examined for the presence of inhibition zones, with halo diameters measured using calipers. For yeasts, susceptibility testing was performed as standardized by M44-A2 [ 32 ]. Sterile filter paper discs impregnated with 10 µL of concentrations were placed on plates previously inoculated by the seeding technique with an inoculum adjusted to 0.5 McFarland in Mueller-Hinton agar supplemented with 2% glucose and 0.5 µg/mL methylene blue. Plates were incubated at 37°C and read after 24 hours, with an additional 24-hour incubation if no growth was observed. Prior to testing, strains were subcultured on Sabouraud dextrose agar for 24 hours. Strains evaluated included Candida albicans ATCC 90028, Candida glabrata ATCC 2001, and Candida krusei ATCC 6258. 2.5 Toxicity assessment A toxicity evaluation of AgNPs was conducted using fruit flies of the Drosophila melanogaster species, specifically the Canton S stock. Flies aged 0–2 days post-emergence (d.p.e.) were separated into groups of 20 individuals, with a 1:1 ratio of males to females maintained. Subsequently, the groups were placed in an incubator set at 25°C under a 12/12-hour light/dark cycle. The administration of treatments was conducted by hydrating 0.5 g of potato puree (comprising 75% instant potato puree, 15% yeast extract, 9.3% glucose, and 0.07% nipagin) with nanoparticle solutions at concentrations of 1, 10, and 100 µg/mL. A control group was hydrated with distilled water. The treatment media were replaced every two days, and the number of deceased individuals was recorded at each change. The total exposure time of the fruit flies to the nanoparticle-enriched diet was 14 days. 2.6 Statistical analysis The statistical analysis was performed using the GraphPad Prism 9.0 software. To evaluate data normalization, we used the Shapiro‒Wilk test. For DLS optimization, we carried out a one-way ANOVA with Tukey's multiple comparison or the Kruskal‒Wallis for continuous variables with normal or non-normal distribution, respectively. The statistical analysis for the disk diffusion assay was conducted using a two-way ANOVA with Tukey's multiple comparison test. The toxicity assay was analyzed using the Mantel-Cox statistical test. A significance of p ≤ 0.05 was applied for statistical threshold. 3. RESULTS 3.1 Green synthesis of silver nanoparticles from kefir fractions In our first attempt at synthesis, the protocol required the addition of NH 4 OH after mixing the WSF with AgNO 3 . However, this sudden change in pH caused the precipitation of large proteins from the WSF. As a result, during the subsequent washing process, these proteins formed a separate pellet on top of the AgNPs pellet, making it difficult to effectively wash and recover the AgNPs. To address the issue, we introduced NH 4 OH during the fractionation of the WSF, just before filtration through a 0.45 µM filter. This addition facilitated the precipitation of large proteins, which were then easily removed by centrifugation. By collecting the supernatant, the synthesis process could proceed smoothly avoiding challenges associated with washing. 3.2 Characterization of silver nanoparticles Following the green synthesis of kefir AgNPs, we performed the analysis of nanoparticle absorbance within the ultraviolet-visible spectra revealed a distinct absorbance peak between 400 and 500 nm, specifically at 470 nm, exclusively in the WSF AgNPs. No distinct absorbance peaks were observed in the AgNO 3 solution, the WSF, or the nanoparticles from both < 10 kDa fraction (Fig. 1 ). The results of the FT-IR analysis (Fig. 2 ) showed significant peaks in various regions. In the 3000–3500 cm − 1 range (labeled A), WSF exhibited a peak that corresponds to N-H and O-H functionalities. Additionally, within the 1500–1700 cm − 1 range (labeled B), distinct peaks indicated the presence of C = O stretching vibration and trivalent nitrogen, observed in both WSF and AgNPs spectra. The WSF displayed two peaks in the 1000–1300 cm − 1 range (labeled C), indicating the presence of C-O and C-N bonds. The analysis validates the successful synthesis of AgNPs using WSF as a precursor, as indicated by the matching peaks found in both WSF and the AgNPs. On the DLS optimization, the transmittance of the WSF AgNPs samples started to decrease at a concentration of 0.5 mg/mL, falling below the recommended threshold, and reached 0% at a concentration of 5 mg/mL (Fig. 3 A). Among the first three concentrations—those that had satisfactory transmittance values—the 0.1 mg/mL concentration was closest to achieving a baseline value of 1 (Fig. 3 B). During the analysis of < 10 kDa AgNPs, the transmittance started to decrease at a concentration of 0.05 mg/mL and reached 0% at a concentration of 1 mg/mL (Fig. 3 C), while the baseline stabilized at a concentration of 0.1 mg/mL, yielding a value closer to the recommended value (Fig. 3 D). Given that the DLS technique is dependent on light passing through the sample and reaching the detector, maintaining transmittance values above the recommended threshold was of primary importance. Therefore, for subsequent analyses of hydrodynamic radius, polydispersity index (PDI), and zeta potential, a concentration of 0.1 mg/mL was selected for WSF AgNPs, and 0.05 mg/mL was chosen for the < 10 kDa AgNPs, based on optimization data from DLS analysis. The DLS measurements reported that the WSF AgNPs exhibited only one peak between 500 and 1000 nm, indicating the presence of only nanoparticles within this size range in the sample. The < 10 kDa AgNPs showed a predominance of nanoparticles smaller than 100 nm in their samples but also a small quantity of larger nanoparticles (Fig. 4 A). The hydrodynamic radius of the WSF AgNPs was approximately 1201 nm, while for the < 10 kDa AgNPs it was approximately 481 nm (Fig. 4 B). For the WSF AgNPs, the PDI was approximately 50%, while for the < 10 kDa AgNPs, it was 28,63% (Fig. 4 C). Finally, the mean zeta potential for all nanoparticle samples was approximately − 30 mV (Fig. 4 D). The SEM analysis validated the findings observed in the DLS. The WSF AgNPs have a spherical-like shape and exhibited a size range of 500 to 1000 nm (Fig. 5 A), whereas the < 10 kDa AgNPs demonstrated a smaller size range of 100 to 200 nm, with some nanoparticles measuring approximately 400 nm, with a spherical-like shape, with the exception of the larger AgNPs, that have a cubic shape (Fig. 5 C). The EDX results revealed the presence of carbon, silicon, chloride, and silver atoms on both AgNPs (Fig. 5 B and 5 D). Given that silver is the primary material utilized in nanoparticle synthesis and silica was the substrate employed for the analysis, it can be inferred that the carbon and chloride originate from the molecules present in the water-soluble fraction of kefir. 3.3 In vitro susceptibility test (disk diffusion method) The investigation of in vitro susceptibility of bacteria through disk diffusion (Table 1 ) showed that only A. baumannii was inhibited by the WSF alone. The WSF AgNPs at 10 µg/mL did not inhibit the growth of any of the tested multidrug-resistant bacteria. Increasing the concentration to 100 µg/mL resulted in inhibitory effects observed on K. pneumoniae and A. baumannii (p < 0.0001). At a concentration of 1000 µg/mL, the WSF AgNPs exhibited inhibitory activity against all the tested bacterial strains. At a concentration of 10 µg/mL, there was no inhibition zone for the < 10 kDa AgNPs. However, at 100 µg/mL, inhibition zones were observed against A. baumannii , E. coli , K. pneumoniae , and S. aureus (p < 0.0001). Notably, complete growth inhibition was evident at a concentration of 1000 µg/mL against all multidrug-resistant bacteria evaluated. For fungi (Table 2 ), the WSF did not inhibit the growth of drug-resistant strains. The growth of C. albicans was not affected by any of the AgNPs. However, both AgNPs inhibited the growth of C. krusei at concentrations of 100 and 1000 µg/mL, and the growth of C. glabrata was inhibited at 1000 µg/mL (p < 0.0001). It is important to note that both C. krusei and C. glabrata are naturally resistant to Fluconazole, a commonly used antifungal. Table 1 Assessment of bacterial growth inhibition via the diffusion disk technique. The WSF AgNPs inhibited the growth of A. baumannii and K. pneumoniae at a concentration of 100 µg/mL. At a concentration of 1000 µg/mL, they inhibited the growth of all tested bacteria. The < 10 kDa AgNPs inhibited the growth of A. baumannii , E. coli , K. pneumoniae , and S. aureus at 100 µg/mL and inhibited the growth of all tested bacteria at 1000 µg/mL. Inhibition zones values of 0 indicate that they were absent. Lowercase letters indicate significant differences between AgNPs and WSF. The capital letters indicate significant differences among different concentrations of the same AgNPs. Symbols indicate significant differences among the same concentration of different AgNPs. Statistics were calculated by two-way ANOVA. All differences shown have a p value < 0.0001. Multiresistant microorganisms Inhibition Zone (mm ± SD) WSF WSF AgNPs (µg/mL) < 10 kDa AgNPs (µg/mL) 10 100 1000 10 100 1000 A. baumannii 8.87 ± 0.304 a 0 aA# 8.777 ± 0.240 aB# 14.193 ± 0.693 bC# 0 aA# 10.133 ± 0.506 bB* 12.903 ± 1.098 bC* E. coli 0 a 0 aA# 0 aA# 10.173 ± 0.456 bB# 0 aA# 6.98 ± 0.046 bB* 10.173 ± 0.482 bC# K. pneumoniae 0 a 0 aA# 8.373 ± 0.423 bB# 9.783 ± 0.446 bC# 0 aA# 7.213 ± 0.405 bB* 9.993 ± 0.444 bC# P. aeruginosa IMP 0 a 0 aA# 0 aA# 9.807 ± 0.514 bB# 0 aA# 0 aA# 8.510 ± 0.572 bB* P. aeruginosa SPM 0 a 0 aA# 0 aA# 9.717 ± 0.321 bB# 0 aA# 0 aA# 9.643 ± 0.179 bB# P. aeruginosa VIM 0 a 0 aA# 0 aA# 9.610 ± 0.937 bB# 0 aA# 0 aA# 8.627 ± 0.263 bB# S. aureus 0 a 0 aA# 0 aA# 9.037 ± 0.127 bB# 0 aA# 8.237 ± 0.585 bB* 11.617 ± 0.391 bC* Table 2 Assessment of fungi growth inhibition via the diffusion disk technique. WSF alone did not inhibit the growth of any of the tested fungi. None of the AgNPs inhibited the growth of C. albicans at any concentration. However, the WSF AgNPs at a concentration of 100 µg/mL did inhibit the growth of C. krusei . At a higher concentration of 1000 µg/mL, it also prevented the growth of C. krusei and C. glabrata . Similar results were observed for the < 10 kDa AgNPs. At a concentration of 100 µg/mL, it inhibited the growth of C. krusei . Additionally, at a concentration of 1000 µg/mL, it also inhibited the growth of C. glabrata . Inhibition zones values of 0 indicate that they were absent. Lowercase letters indicate significant differences between AgNPs and WSF. The capital letters indicate significant differences among different concentrations of the same AgNPs. Symbols indicate significant differences among the same concentration of different AgNPs. Statistics were calculated by two-way ANOVA. All differences shown have a p value < 0.0001. Resistant microorganisms Inhibition Zone (mm ± SD) WSF WSF AgNPs (µg/mL) < 10 kDa AgNPs (µg/mL) 10 100 1000 10 100 1000 C. albicans 0 a 0 aA# 0 aA# 0 aA# 0 aA# 0 aA# 0 aA# C. krusei 0 a 0 aA# 13.073 ± 0.834 bB# 14.873 ± 0.994 bC# 0 aA# 12.663 ± 0.375 bB# 15.597 ± 0.745 bC# C. glabrata 0 a 0 aA# 0 aA# 7.350 ± 0.352 bB# 0 aA# 0 aA# 11.633 ± 0.349 bB* 3.4 Toxicity assessment The tested concentrations of WSF AgNPs and < 10 kDa AgNPs did not result in toxicity in Drosophila melanogaster during long-term treatment (Fig. 6 ). There were no statistically significant differences in the survival probability of the flies compared to the control group throughout the 14-day analysis period, indicating that these nanoparticles are safe under the conditions tested. 4. DISCUSSION The therapeutic effects of kefir have been explored in medicine because of its ability to treat many medical conditions, from neurological to gut diseases [ 33 – 35 ]. Although the existing literature extensively covers the therapeutic benefits of kefir, there is a gap in research regarding the utilization of nanotechnology to enhance kefir's effects through the production of nanoparticles. Our research aimed to assess the potential of kefir-derived silver nanoparticles in improving the therapeutic benefits of kefir, specifically its antimicrobial and antifungal activities. The AgNPs synthesized from both kefir fractions exhibited a spherical morphology, with the particles obtained from the < 10 kDa fraction being notably smaller in size. Both AgNPs demonstrated a -30 mV mean zeta potential, indicative of their stability, while exhibiting antimicrobial activity against all multi-drug resistant bacteria, C. krusei and C. glabrata . Furthermore, no toxicity was observed in Drosophila melanogaster following long-term treatment. The nanoparticles were comprehensively characterized using UV‒Vis spectroscopy, DLS, and FT-IR spectroscopy. Silver nanoparticles typically exhibit a distinct absorbance peak in the 400–500 nm range in UV-Vis spectra [ 36 – 39 ]. The results of our UV-Vis spectroscopy analysis demonstrated that the distinctive peak was exclusively present in our WSF AgNPs, but not in the < 10 kDa AgNPs. It is hypothesized that the < 10 kDa AgNPs, due to their smaller size, may exhibit a broader absorbance, resulting in the absence of a distinct peak. Additionally, it is possible that these nanoparticles are composed of silver oxide (Ag 2 O or AgO), which also exhibit a broader absorbance range, rather than metallic silver. Further investigation is required to explain the lack of distinct absorbance peaks in the < 10 kDa AgNPs. As previously reported by our group, the WSF of kefir is primarily made up of amino acids, peptides, carboxylic acids, and alcohols [ 19 , 20 ]. According to our FT-IR results, the synthesized nanoparticles are likely formed through the interaction between the amino acids and peptides in WSF and silver nitrate [ 40 ]. The absence of transmittance bands corresponding to alcohol and carboxylic acid functional groups in the AgNPs spectra indicates that these groups may have not participate in the synthesis [ 41 ]. Optimizing the parameters for DLS was crucial due to the limited insight provided by the literature and equipment manuals on key parameters and equipment settings [ 42 ]. For instance, the equipment manual recommends using backscatter measurements for concentrated samples, but it does not provide specific concentration guidelines. Furthermore, information on parameters such as transmittance and baseline are not well explored in the literature. These parameters are utilized to assess the reliability of the collected data, leaving room for further knowledge. To address this gap, our study conducted rigorous testing across various sample concentrations to determine the most effective parameters for reliable analysis of our samples. This empirical approach yielded valuable insights into the influence of concentration and equipment settings on measurement outcomes. The findings provide important guidance for future research aiming to accurately characterize silver nanoparticles based on sample properties. The hydrodynamic radius of our AgNPs (WSF AgNPs: 1201 nm and < 10 kDa AgNPs: 481,6 nm) is larger than that reported in previous syntheses [ 43 , 44 ]. However, improving the biological synthesis process of AgNPs—by testing different pH, precursor concentrations, temperature, and exposure to light—can affect their physical characteristics, such as size, shape, and PDI [ 45 ]. Future research will aim to optimize the synthesis process and analyze the outcomes resulting from varying these parameters. The PDI of nanoparticles is a critical factor in drug delivery efficacy and safety [ 46 ]. Notably, the synthesized < 10 kDa AgNPs fall within the acceptable PDI range (below 30%) [ 46 ], allowing for more precise studies of potential side effects and the optimization for drug delivery purposes. Furthermore, our AgNPs exhibited a zeta potential of around − 30 mV, indicating that they are stable and have minimal tendency to aggregate [ 47 ]. A number of studies have demonstrated that AgNPs synthesized via green methods, such as the use of plant extracts and pollen, display antimicrobial activity against bacterial and fungal pathogens [ 28 , 48 – 51 ], which supports our findings. However, it is worth mentioning that our disk diffusion assay is preliminary, and a concentration of 1 mg/mL is very high and would be expected to produce inhibition zones for all the bacteria and fungi tested. High concentrations of antimicrobials can be toxic to users, and lack of specificity can lead to microbial resistance [ 52 – 54 ]. In future research, determining the minimum inhibitory concentration of these NPs should be a priority in order to fully understand their potential as novel antibiotics and antifungals. The size and shape of nanoparticles can influence their uptake by cells and their interaction within biological systems. The WSF AgNPs exhibit a size range of 500 to 1000 nm, while the < 10 kDa AgNPs display a size range of 100 to 200 nm, with some nanoparticles measuring approximately 400 nm. Given that, according to the literature, smaller nanoparticles tend to have higher absorption rates, it is anticipated that the < 10 kDa AgNPs will exhibit a higher rate of absorption compared to the WSF AgNPs [ 55 , 56 ]. With regard to their shape, spherical nanoparticles can enter tissues more easily than other shapes. However, the data in the literature remains inconsistent, and further studies are needed to ascertain the effects of different nanoparticle shapes on their uptake [ 56 ]. Nevertheless, despite the enhanced bioavailability of our 10 kDa AgNPs due to their higher uptake, no long-term toxicity was observed in Drosophila melanogaster in comparison to the WSF AgNPs. These findings are consistent with those of previous research conducted by our group, in which a silver nanoparticle produced using pollen extract and employing the same methodology did not demonstrate any toxicity to the flies [ 28 ], and those produced by other groups using natural products as the source for their nanoparticle synthesis [ 26 , 27 ]. However, it should be noted that fruit flies serve only as a preliminary trial model organism, and further research is necessary on chordate animals to more accurately assess the toxicity of the nanoparticles produced from the water-soluble fraction of kefir. In future research, we plan to investigate the feasibility of incorporating our AgNPs into disinfectant solutions with the aim of achieving effective decontamination [ 57 ]. Moreover, we will undertake an examination of the impact of kefir derived-AgNPs on wound healing processes, with a particular emphasis on their capacity to facilitate superior skin regeneration and serve as a healing agent and antiseptic. Finally, we aim to conduct drug development tests to explore novel avenues for antibiotic production, capitalizing on the distinctive properties possessed by these AgNPs. It is important to acknowledge the limitations of this research. Firstly, the production of AgNPs from kefir, a natural product, may be subject to variability in the green synthesis method due to differences in the bioactive components of kefir derived from different sources or batches. Moreover, although no toxicity was observed in Drosophila melanogaster during the lifespan analysis, a comprehensive toxicity assessment, including the effects of AgNPs on brain and gut morphology, is imperative. Furthermore, additional in vivo studies in higher organisms and clinical trials are necessary to confirm the safety and efficacy of these AgNPs in humans. Finally, although antimicrobial and antifungal properties against the tested bacteria and fungi were observed, determining the minimum inhibitory concentrations for these pathogens is essential for practical applications. Our group plans to address this, along with a more comprehensive toxicity assessment in Drosophila melanogaster , in future research. 5. CONCLUSION In this study, we presented the synthesis and characterization of silver nanoparticles from the water-soluble fraction of kefir, with the aim of combating drug-resistant diseases. The successful synthesis of AgNPs was confirmed through UV-visible and FT-IR spectroscopy, while DLS optimization provided valuable information on their size distribution and stability. In vitro testing demonstrated the promising antimicrobial activity of WSF and < 10 kDa AgNPs against multidrug-resistant bacteria and Candida fungi. In vivo studies showed no toxicity to fruit flies during long-term treatment. Future research will delve deeper into toxicity assessments, including potential morphological changes to specific organs of the flies. Further investigations will also focus on determining the minimum inhibitory concentrations, enhancing specificity, and exploring applications of AgNPs in wound healing and drug development. Abbreviations AgNPs silver nanoparticles DLS dynamic light scattering FT-IR Fourier-transformed infra-red MIC Minimum inhibitory concentration NPs Nanoparticles PDI Polydispersity index UV‒Vis Ultraviolet-visible spectroscopy WSF Kefir’s whole water-soluble fraction Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. Authors’ information Genetics Laboratory, Institute of Biotechnology, Federal University of Uberlândia, 1004 Acre Street, Building 2E, 38405-319, Uberlândia, MG, Brazil. Lucas Matos Martins Bernardes, Serena Mares Malta, Ana Carolina Costa Santos, Rafael Alves da Silva, Tamiris Sabrina Rodrigues, Carlos Ueira-Vieira Laboratory of Nanobiotechnology, Institute of Biotechnology, Federal University of Uberlândia, 1004 Acre Street, Building 2E, 38405-319 Uberlândia, Brazil. Murillo Néia Thomaz da Silva Department of Psychiatry, College of Medicine, University of Saskatchewan, 103 Hospital Drive, Room 366 Ellis Hall Saskatoon, SK, Canada. Ana Paula Mendes-Silva Corresponding authors Lucas Matos Martins Bernardes [email protected] Carlos Ueira-Vieira [email protected] Funding This project was funded by the Research Support Foundation of the State of Minas Gerais (FAPEMIG APQ-02766-17, APQ-00269-22 and National Council of Scientific and Technological Development (CNPq, grant number: 403193/2022-2) and FAPEMIG (grant number: CBB-APQ-03613-17) for INCT -TeraNano. Author Contribution LMMB: conceptualization; data curation; methodology; formal analysis; writing—original draft. SMM: data curation. ACCS: data curation. RADS: data curation. TSR: data curation. MNTDS: data curation. APMS: conceptualization. CUV: conceptualization; methodology; funding acquisition; formal analysis; writing—review and editing. All authors reviewed the manuscript. Acknowledgement We deeply appreciate all the assistance provided by Dr. Luiz Ricardo Goulart Filho, who tragically became one of the millions of victims of COVID-19. Data Availability The datasets produced or analyzed during the current study are available from the corresponding author upon request. References Simon S, Sibuyi NRS, Fadaka AO, Meyer S, Josephs J, Onani MO, et al. Biomedical Applications of Plant Extract-Synthesized Silver Nanoparticles. Biomedicines. 2022;10. Rabiee N, Ahmadi S, Akhavan O, Luque R. Silver and Gold Nanoparticles for Antimicrobial Purposes against Multi-Drug Resistance Bacteria. Materials (Basel). 2022;15. More PR, Pandit S, Filippis A De, Franci G, Mijakovic I, Galdiero M. Silver Nanoparticles: Bactericidal and Mechanistic Approach against Drug Resistant Pathogens. Microorganisms. 2022;11:369. Aljeldah MM, Aboul-Soud MAM, Yassin MT, Mostafa AAF. 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Cite Share Download PDF Status: Published Journal Publication published 27 Mar, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 02 Oct, 2024 Reviews received at journal 28 Sep, 2024 Reviewers agreed at journal 17 Sep, 2024 Reviews received at journal 09 Sep, 2024 Reviewers agreed at journal 26 Aug, 2024 Reviewers invited by journal 25 Aug, 2024 Editor assigned by journal 17 Aug, 2024 Editor invited by journal 16 Aug, 2024 Submission checks completed at journal 16 Aug, 2024 First submitted to journal 30 Jul, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4830503","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":351699627,"identity":"54dd5188-cca5-4390-a0e0-a9c8cbe8a73d","order_by":0,"name":"Lucas Matos Martins Bernardes","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABJklEQVRIie3Qv0rEMBzA8YRAXfpnTSn4DD8pVES4F3EsOLUquFQKXrv0Fh/gbtFX0CU4tnTokuPWkw56FOp8uFzBwfQ8UGiLujnkuyRDPiS/ICST/cP0r626W1OEq+2GkKiPKLuVfifERggEwb8nirUlaIBQd7VuvNG1Ycyz6upxdK4Xkzq8CN5PjIkgm4B1yaltacyl5vTMPZhz92jGuVNOOfjTHEf4hpdd4iELM0JhqTpmnBCApaeUWgJ+JAjBSR8hTcPGFBb8sImTMcDza33ZkrtholCN5RRSz8FxkotbkENacj9E1No51lhhzsQs4mEFmNyzLZXb/oMgWc8sxp5bPTUsNNofW8dJCHpRrN7UYN+/XeTZyybokB9K/3heJpPJZJ99AEypcBrMUQJ5AAAAAElFTkSuQmCC","orcid":"","institution":"Federal University of Uberlândia","correspondingAuthor":true,"prefix":"","firstName":"Lucas","middleName":"Matos Martins","lastName":"Bernardes","suffix":""},{"id":351699630,"identity":"2c407d3d-c19b-466c-8d2b-4d529e15d5ea","order_by":1,"name":"Serena Mares Malta","email":"","orcid":"","institution":"Federal University of Uberlândia","correspondingAuthor":false,"prefix":"","firstName":"Serena","middleName":"Mares","lastName":"Malta","suffix":""},{"id":351699632,"identity":"4cedeedb-d65b-4250-8af1-085f449fc78e","order_by":2,"name":"Ana Carolina Costa Santos","email":"","orcid":"","institution":"Federal University of Uberlândia","correspondingAuthor":false,"prefix":"","firstName":"Ana","middleName":"Carolina Costa","lastName":"Santos","suffix":""},{"id":351699633,"identity":"3fc11632-17bf-423f-936b-31628a9a942c","order_by":3,"name":"Rafael Alves da Silva","email":"","orcid":"","institution":"Federal University of Uberlândia","correspondingAuthor":false,"prefix":"","firstName":"Rafael","middleName":"Alves da","lastName":"Silva","suffix":""},{"id":351699635,"identity":"164065cd-a78a-46a3-9872-c860f8362fab","order_by":4,"name":"Tamiris Sabrina Rodrigues","email":"","orcid":"","institution":"Federal University of Uberlândia","correspondingAuthor":false,"prefix":"","firstName":"Tamiris","middleName":"Sabrina","lastName":"Rodrigues","suffix":""},{"id":351699636,"identity":"8d2aec71-57b3-478a-9610-8271e8c049fa","order_by":5,"name":"Murillo Néia Thomaz da Silva","email":"","orcid":"","institution":"Federal University of Uberlândia","correspondingAuthor":false,"prefix":"","firstName":"Murillo","middleName":"Néia Thomaz da","lastName":"Silva","suffix":""},{"id":351699637,"identity":"e1c03349-f71a-41ef-a5c3-eee5bbd21c60","order_by":6,"name":"Ana Paula Mendes-Silva","email":"","orcid":"","institution":"University of Saskatchewan","correspondingAuthor":false,"prefix":"","firstName":"Ana","middleName":"Paula","lastName":"Mendes-Silva","suffix":""},{"id":351699639,"identity":"d2a911d9-35ec-4a21-bd18-69509ec90da4","order_by":7,"name":"Carlos Ueira-Vieira","email":"","orcid":"","institution":"Federal University of Uberlândia","correspondingAuthor":false,"prefix":"","firstName":"Carlos","middleName":"","lastName":"Ueira-Vieira","suffix":""}],"badges":[],"createdAt":"2024-07-30 17:36:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4830503/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4830503/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-95616-4","type":"published","date":"2025-03-27T15:56:59+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":64481892,"identity":"238bb66b-c6ec-4ecb-a197-bc6fd4e97388","added_by":"auto","created_at":"2024-09-13 16:35:48","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":6903528,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUV\u003c/strong\u003e‒\u003cstrong\u003evisible spectroscopy.\u003c/strong\u003e AgNO\u003csub\u003e3\u003c/sub\u003e and WSF did not exhibit any absorbance within this spectral range. The WSF AgNPs exhibited an absorbance peak at 470 nm, as anticipated for AgNPs. The \u0026lt;10 kDa AgNPs did not show an absorbance peak within this spectrum, but their absorbance was not insignificant. The samples were evaluated in triplicate.\u003c/p\u003e","description":"","filename":"Figure1.tif.png","url":"https://assets-eu.researchsquare.com/files/rs-4830503/v1/88bb108b40870021c79aef71.png"},{"id":64481895,"identity":"094855e9-4f9a-436c-9bd6-5b2309dd709b","added_by":"auto","created_at":"2024-09-13 16:35:48","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":12973895,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFT-IR spectroscopy.\u003c/strong\u003e During the FT-IR analysis, matching peaks were observed between the AgNPs and their precursors, AgNO\u003csub\u003e3\u003c/sub\u003e and WSF. Transmittance peaks were reported for WSF at the N-H and O-H functionalities region (A), C=O and trivalent nitrogen region (B) and C-O and C-N bonds region (C). Matching peaks were observed for the WSF and AgNPs on the region labeled B. These results suggest that the silver nanoparticles synthesis was successful, due to the precursor and the AgNPs sharing similar chemical bonds in their composition.\u003c/p\u003e","description":"","filename":"Figure2.tif.png","url":"https://assets-eu.researchsquare.com/files/rs-4830503/v1/73ca7447ec2aadc7ddfd73a5.png"},{"id":64481897,"identity":"10918a67-d8f0-4a0d-8918-176325e82ad6","added_by":"auto","created_at":"2024-09-13 16:35:48","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":27437316,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDLS Optimization.\u003c/strong\u003e When conducting DLS, it is important to ensure that the transmittance values are not below 60% and the baseline is close to 1. For WSF AgNPs, only concentrations of 0.01, 0.05, and 0.1 mg/mL (A) had acceptable transmittance values. However, the baseline was closer to the reference value at the 0.1 mg/mL concentration (B). For both the \u0026lt;10 kDa AgNPs, only concentrations of 0.01 and 0.05 mg/mL provided transmittance values above the reference value (C). However, the baseline values for the 0.05 mg/mL concentration group were closer to the ideal value (D). The data normalization was evaluated using the Shapiro‒Wilk test. Statistical differences were evaluated using one-way ANOVA after normalizing the data. If the data did not pass the normality test, Kruskal‒Wallis test was used to calculate significant differences. In both cases, multiple comparisons were made using Tukey's test. The concentrations that displayed the most appropriate values for each parameter are indicated by the arrows.\u003c/p\u003e","description":"","filename":"Figure3.tif.png","url":"https://assets-eu.researchsquare.com/files/rs-4830503/v1/8bd2bd21bad3482e8753adef.png"},{"id":64483077,"identity":"e5b6a191-6d57-4eb2-9b4c-c97afa7055bf","added_by":"auto","created_at":"2024-09-13 16:51:48","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":20403557,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDLS size measurements and zeta potential.\u003c/strong\u003e The size measurement analysis for the WSF AgNPs reported particles between 500 and 1000 nm being the only ones present in the sample. For the \u0026lt;10 kDa AgNPs, the majority had sizes less than 100 nm (A). The hydrodynamic radius of the WSF AgNPs was approximately 1201 nm, while for the \u0026lt;10 kDa AgNPs it was 481,6 nm (B). The WSF AgNPs had a polydispersity index of 50%, while the \u0026lt;10 kDa AgNPs had a PDI of 30% (C). The mean zeta potential for all nanoparticles was lower than -30 mV (D).\u003c/p\u003e","description":"","filename":"Figure4.tif.png","url":"https://assets-eu.researchsquare.com/files/rs-4830503/v1/114d21b61990b82aaebafa55.png"},{"id":64481891,"identity":"68b0317f-9df4-4dba-bfcd-7ad9225ac6c2","added_by":"auto","created_at":"2024-09-13 16:35:48","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":716693,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDX) analysis.\u003c/strong\u003e The SEM imaging revealed that the WSF AgNPs have sizes varying from 500 to 1000 nm (Fig. 5A), with the presence of silver, chloride, silicon, and carbon in the sample, as indicated by the EDX analysis (Fig. 5B). In comparison, the \u0026lt;10 kDa AgNPs are smaller, with sizes ranging from 100 to 200 nm, with some particles measuring approximately 400 nm (Fig 5C). The EDX analysis also detected the presence of silver, chloride, carbon, and silicon in these samples (Fig 5D). The samples were prepared by dispersing them onto a silicon substrate, and the SEM images were taken at 10,000x magnification.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4830503/v1/093b62512992c593f780675a.png"},{"id":64482422,"identity":"55bc83df-b476-4077-8276-1469fb1ddcf5","added_by":"auto","created_at":"2024-09-13 16:43:48","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":7956013,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eToxicity assessment.\u003c/strong\u003e A 14-day toxicity assay was conducted on Drosophila melanogaster (Canton S stock) with flies ranging from 0-2 d.p.e. The results demonstrated that neither the WSF AgNPs nor the \u0026lt;10 kDa AgNPs exhibited any toxicity at any concentration during the tested period.\u003c/p\u003e","description":"","filename":"Figure6.tif.png","url":"https://assets-eu.researchsquare.com/files/rs-4830503/v1/0ee531827ef4daac13696b74.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Green Synthesis, Characterization, and Antimicrobial Activity of Silver Nanoparticles from Water-Soluble Fractions of Brazilian Kefir","fulltext":[{"header":"1. INTRODUCTION","content":"\u003cp\u003eNanotechnology is a rapidly advancing field that involves manipulating materials at the nanoscale, resulting in nanoparticles (NPs) ranging from 1 to 100 nm in size. These nanoparticles exhibit unique physicochemical properties and hold promise for addressing pressing health challenges [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. In particular, the increase in the incidence of drug-resistant diseases poses a significant threat, with an estimated 700,000 deaths annually\u0026mdash;a figure that is projected to increase to 10\u0026nbsp;million by 2050 if left unchecked [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Fungal infections are even more concerning, with more than 300\u0026nbsp;million people suffering from various fungal diseases, resulting in more than 2\u0026nbsp;million deaths annually from mycoses [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSilver nanoparticles (AgNPs) are particularly noteworthy in nanomedicine due to their exceptional antimicrobial efficacy and relatively low toxicity. AgNPs can potentially combat microbial resistance due to their unique properties, such as a high surface area-to-volume ratio and the ability to generate reactive oxygen species [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Therefore, exploring their antimicrobial activities could be a promising approach for addressing the growing threat of drug-resistant infections. One method for assessing the antimicrobial efficacy of AgNPs is through the disk diffusion technique, a reliable methodology that has been employed in numerous previous studies [\u003cspan additionalcitationids=\"CR5 CR6\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe NPs can be synthesized using physical, chemical, or biological methods. Although physical and chemical methods have traditionally been used for nanoparticle synthesis, their drawbacks, including cost, time consumption, and environmental concerns, have led to increased interest in biological synthesis methods [\u003cspan additionalcitationids=\"CR9 CR10\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBiological synthesis, also known as 'green synthesis,' uses nontoxic and eco-friendly reagents, offering a more streamlined approach to nanoparticle production. Organism-derived extracts, which are rich in biomolecules such as metabolites and proteins, play crucial roles as reducing and capping agents in this process [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. This highlights the efficiency and versatility of biological synthesis. Studies have used cell-free extracts and derived substances from bacteria as precursors for nanoparticle synthesis [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this context, kefir, a fermented beverage with well-documented antioxidant and antimicrobial properties, is a promising candidate for nanoparticle synthesis [\u003cspan additionalcitationids=\"CR16 CR17\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Although kefir has not been extensively studied in nanotechnology, our group has discovered significant antioxidant activity in the smaller than 10 kDa (\u0026lt;\u0026thinsp;10 kDa) fraction of the water-soluble component of kefir. Additionally, bioactive peptides have been identified from the this fraction [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Based on these findings, the water-soluble fraction (WSF) of kefir can be considered a promising agent for the green synthesis of nanoparticles.\u003c/p\u003e \u003cp\u003eControlling the characteristics of the resulting nanoparticles in biological synthesis is challenging due to various factors that can impact the process, such as pH, temperature, and concentration of precursor agents [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Therefore, rigorous characterization of the NPs properties, including size, morphology, and chemical composition, is essential. Techniques such as UV‒visible (UV‒Vis) spectroscopy, Fourier transform infrared (FT-IR) spectroscopy, dynamic light scattering (DLS), and zeta potential measurements are well-described in the literature and provide valuable insights into these properties [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe toxicity of nanoparticles can be evaluated through \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e assays. The fruit fly (\u003cem\u003eDrosophila melanogaster\u003c/em\u003e) is a well-established model organism that offers several advantages, including a rapid life cycle, low cost of maintenance in laboratories, and a genome with significant human homology [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. This model has been previously utilized for studies on the toxicity of AgNPs, examining aspects such as lifespan and gut morphology [\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Consequently, it is a reliable model for assessing the safety of AgNPs.\u003c/p\u003e \u003cp\u003eThis study aimed to examine the potential of kefir for nanoparticle synthesis and its antimicrobial applications. First, we analyzed the water-soluble fraction of kefir as a reagent for nanoparticle synthesis. We then characterize the synthesized nanoparticles including size, morphology, and chemical composition. Additionally, we assessed their antimicrobial activity through disk diffusion against a set of multiresistant bacteria and fungi from the \u003cem\u003eCandida\u003c/em\u003e genus. Moreover, we evaluated the toxicity of AgNPs by using fruit flies. This research aimed to contribute to the advancement of the use of nanotechnology in healthcare, more specifically the potential of kefir-derived silver nanoparticles in antimicrobial and antifungal activities.\u003c/p\u003e"},{"header":"2. MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Kefir production and collection of its water-soluble fractions\u003c/h2\u003e \u003cp\u003eThe kefir grains utilized in this study were donated by residents of the city of Uberl\u0026acirc;ndia, Brazil. The grains were previously characterized through genetic fingerprinting by our group [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. To produce kefir, whole cow's milk was fermented with a 10% (w/v) concentration of kefir grains. Fermentation occurred over a 24-hour period at a controlled temperature of 28\u0026deg;C, shielded from light. Following fermentation, the kefir was separated from the grains using a sieve.\u003c/p\u003e \u003cp\u003eTo obtain the water-soluble fraction (WSF), the kefir was centrifuged twice at 4900\u003cem\u003eg\u003c/em\u003e for 10 minutes at 4\u0026deg;C. After each centrifugation, the supernatant was collected, and the pellet was discarded. Then, the resulting supernatant was filtered through two layers of filter paper [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo ensure the suitability of the WSF for nanoparticle synthesis, we modified the protocol [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Specifically, we added ammonium hydroxide (NH\u003csub\u003e4\u003c/sub\u003eOH) to the WSF immediately after filtration at a concentration of 0.05% v/v. This addition aimed to alter the pH of the sample and precipitate proteins that might interfere with nanoparticle synthesis. Subsequently, we centrifuged the solution at 12000\u003cem\u003eg\u003c/em\u003e for 15 minutes at 4\u0026deg;C to pellet the proteins, which enabled us to collect the clarified supernatant for nanoparticle synthesis. The NH\u003csub\u003e4\u003c/sub\u003eOH is usually added during the nanoparticle synthesis process, but the protocol required adaption due to an unsuccessful first synthesis (explained in the Results section).\u003c/p\u003e \u003cp\u003eFurthermore, the supernatant underwent further filtration using a vacuum pump and a 0.22 \u0026micro;m membrane. Subsequently, it was purified through a 10 kDa Amicon column to fractionate the WSF into the smaller than 10 kDa (\u0026lt;\u0026thinsp;10 kDa) fraction.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Green synthesis of silver nanoparticles from kefir fractions\u003c/h2\u003e \u003cp\u003eThe silver nanoparticles synthesis method employed in this study was modified from a previous described green synthesis protocol [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. First, we added 60 mL of each fraction (whole WSF and \u0026lt;\u0026thinsp;10 kDa), which already contained NH\u003csub\u003e4\u003c/sub\u003eOH, to individual flasks containing 0.18 g of silver nitrate (AgNO\u003csub\u003e3\u003c/sub\u003e). The flasks were briefly shaken and then heated to boiling in a microwave oven. Upon reaching the boiling point, the solutions turned brown, indicating the formation of nanoparticles. Each solution was stirred using a magnetic stirrer for 15 minutes at medium speed.\u003c/p\u003e \u003cp\u003eAfter the synthesis process, the nanoparticles were subjected to three cycles of centrifugation (16000\u003cem\u003eg\u003c/em\u003e, 15 minutes, 4\u0026deg;C). After each cycle, the supernatant was discarded, and the pellet containing the silver nanoparticles was resuspended in deionized water. The resulting solution was stored in a refrigerator set at 4\u0026deg;C. To determine the concentration of each solution, a portion was air-dried. The nanoparticles derived from the WSF and \u0026lt;\u0026thinsp;10 kDa fractions are referred to as WSF AgNPs and \u0026lt;\u0026thinsp;10 kDa AgNPs, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Characterization of silver nanoparticles\u003c/h2\u003e \u003cp\u003eAnalytical techniques, including UV‒Vis, DLS, zeta potential analysis, and FT-IR were used to characterize the silver nanoparticles. UV‒Vis spectroscopy was employed to analyze characteristics of the nanoparticles and the efficiency of their synthesis based on the absorbance peak (typically 400\u0026ndash;500 nm) of silver nanoparticles. In a 96-well plate, triplicate samples (100 \u0026micro;L each) of nanoparticle solutions, along with WSF and silver nitrate controls, were analyzed. Spectra were measured from 300 to 800 nm using a standardized UV‒Vis spectrophotometer. Analysis of the data included peak identification based on wavelength and intensity criteria. Triplicate samples were used to ensure the reliability of the results.\u003c/p\u003e \u003cp\u003eThe infrared spectra of the AgNPs and their precursors were obtained using an Agilent Cary 630 FTIR spectrophotometer in the range from 4000 to 650 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Sample analyses were performed in the solid state using the Attenuated Total Reflectance (ATR) accessory with a diamond crystal.\u003c/p\u003e \u003cp\u003eThe initial step of the DLS analysis involved optimizing the measurements for each sample. Nanoparticle solutions were prepared at a range of concentrations from 0.01 mg/mL to 5 mg/mL. Subsequently, size distribution, hydrodynamic radius, and polydispersity index of the nanoparticles were evaluated in quintuplicate. Each measurement was conducted at 25\u0026deg;C using 2 mL of sample. To ensure stable measurements, a 3-minute equilibrium time was implemented, allowing the nanoparticles to reach a steady state and match the equipment's temperature. The backscatter measurement angle (175\u0026deg;) was chosen due to its high sensitivity to minute particles and its ability to minimize the impact of multiple scattering, thus ensuring reliable and precise data on size distribution.\u003c/p\u003e \u003cp\u003eThe optimization process aimed to determine the nanoparticle concentration that would consistently yield reliable results in five replicates. We considered parameters provided by the DLS equipment (Litesizer 500, Anton Paar, UK), such as baseline, g1\u0026sup2;, and transmittance values, to ensure accurate measurements. Adhering to recommended criteria is crucial for obtaining reliable data. Our analysis focused on two parameters: transmittance levels and baseline values. Based on manufacturer guidelines, transmittance levels should ideally exceed 80%, with values above 60% considered acceptable. Baseline values should remain at 1 for optimal performance.\u003c/p\u003e \u003cp\u003eThe DLS equipment was also used for examining the zeta potential of the AgNPs, which was measured in quintuplicate at 0.1 mg/mL for WSF AgNPs and 0.05 mg/mL for the \u0026lt;\u0026thinsp;10 kDa AgNPs. The measurements were made using 200 V at 25\u0026deg;C with an equilibrium time of 3 minutes. The reasoning for selecting these concentrations for the analysis will be further discussed in the Results section.\u003c/p\u003e \u003cp\u003eA scanning electron microscopy (SEM) analysis was conducted to corroborate the data obtained from the DLS analysis regarding the dimensions of the nanoparticles and to gather additional data on their morphology. To obtain SEM images, the dispersed samples were deposited onto a silicon substrate. A Vega 3 TESCAN scanning electron microscope, operated at 20 kV, was used, equipped with a secondary electron detector and an energy-dispersive X-ray (EDX) detector (Oxford Instruments, Bucks, England). A voltage of 20 kV was used for acquiring the EDX spectra and mapping.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 \u003cem\u003eIn vitro\u003c/em\u003e susceptibility test (disk diffusion method)\u003c/h2\u003e \u003cp\u003eThe anti-bacterial property of AgNPs was evaluated using a diffusion disk test against eight strains of multidrug-resistant bacteria, including \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e, \u003cem\u003eEscherichia coli\u003c/em\u003e (MCR), \u003cem\u003eKlebsiella pneumoniae carbapenemase\u003c/em\u003e, three strains of \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e (IMP, VIM, and SPM), and \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (MRSA), obtained from the Molecular Microbiology Laboratory of the Federal University of Uberl\u0026acirc;ndia. Strain characterization was performed by bacterial genotyping by PCR and phenotyping on ampicillin and polymyxin supplemented BHI agar. The modified Kirby-Bauer disk diffusion method was used to assess antimicrobial activity [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFor testing, bacteria were plated on agar plates at a concentration of 0.5 on the McFarland scale. Subsequently, 15 \u0026micro;L of AgNPs at three concentrations (10, 100 and 1000 \u0026micro;g/mL) were applied to antimicrobial susceptibility test discs in triplicate. The discs were incubated at 37\u0026deg;C for 24 hours and then examined for the presence of inhibition zones, with halo diameters measured using calipers.\u003c/p\u003e \u003cp\u003eFor yeasts, susceptibility testing was performed as standardized by M44-A2 [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Sterile filter paper discs impregnated with 10 \u0026micro;L of concentrations were placed on plates previously inoculated by the seeding technique with an inoculum adjusted to 0.5 McFarland in Mueller-Hinton agar supplemented with 2% glucose and 0.5 \u0026micro;g/mL methylene blue. Plates were incubated at 37\u0026deg;C and read after 24 hours, with an additional 24-hour incubation if no growth was observed. Prior to testing, strains were subcultured on Sabouraud dextrose agar for 24 hours. Strains evaluated included \u003cem\u003eCandida albicans\u003c/em\u003e ATCC 90028, \u003cem\u003eCandida glabrata\u003c/em\u003e ATCC 2001, and \u003cem\u003eCandida krusei\u003c/em\u003e ATCC 6258.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Toxicity assessment\u003c/h2\u003e \u003cp\u003eA toxicity evaluation of AgNPs was conducted using fruit flies of the \u003cem\u003eDrosophila melanogaster\u003c/em\u003e species, specifically the \u003cem\u003eCanton S\u003c/em\u003e stock. Flies aged 0\u0026ndash;2 days post-emergence (d.p.e.) were separated into groups of 20 individuals, with a 1:1 ratio of males to females maintained. Subsequently, the groups were placed in an incubator set at 25\u0026deg;C under a 12/12-hour light/dark cycle. The administration of treatments was conducted by hydrating 0.5 g of potato puree (comprising 75% instant potato puree, 15% yeast extract, 9.3% glucose, and 0.07% nipagin) with nanoparticle solutions at concentrations of 1, 10, and 100 \u0026micro;g/mL. A control group was hydrated with distilled water. The treatment media were replaced every two days, and the number of deceased individuals was recorded at each change. The total exposure time of the fruit flies to the nanoparticle-enriched diet was 14 days.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Statistical analysis\u003c/h2\u003e \u003cp\u003eThe statistical analysis was performed using the GraphPad Prism 9.0 software. To evaluate data normalization, we used the Shapiro‒Wilk test. For DLS optimization, we carried out a one-way ANOVA with Tukey's multiple comparison or the Kruskal‒Wallis for continuous variables with normal or non-normal distribution, respectively. The statistical analysis for the disk diffusion assay was conducted using a two-way ANOVA with Tukey's multiple comparison test. The toxicity assay was analyzed using the Mantel-Cox statistical test. A significance of p\u0026thinsp;\u0026le;\u0026thinsp;0.05 was applied for statistical threshold.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. RESULTS","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Green synthesis of silver nanoparticles from kefir fractions\u003c/h2\u003e \u003cp\u003eIn our first attempt at synthesis, the protocol required the addition of NH\u003csub\u003e4\u003c/sub\u003eOH after mixing the WSF with AgNO\u003csub\u003e3\u003c/sub\u003e. However, this sudden change in pH caused the precipitation of large proteins from the WSF. As a result, during the subsequent washing process, these proteins formed a separate pellet on top of the AgNPs pellet, making it difficult to effectively wash and recover the AgNPs.\u003c/p\u003e \u003cp\u003eTo address the issue, we introduced NH\u003csub\u003e4\u003c/sub\u003eOH during the fractionation of the WSF, just before filtration through a 0.45 \u0026micro;M filter. This addition facilitated the precipitation of large proteins, which were then easily removed by centrifugation. By collecting the supernatant, the synthesis process could proceed smoothly avoiding challenges associated with washing.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Characterization of silver nanoparticles\u003c/h2\u003e \u003cp\u003eFollowing the green synthesis of kefir AgNPs, we performed the analysis of nanoparticle absorbance within the ultraviolet-visible spectra revealed a distinct absorbance peak between 400 and 500 nm, specifically at 470 nm, exclusively in the WSF AgNPs. No distinct absorbance peaks were observed in the AgNO\u003csub\u003e3\u003c/sub\u003e solution, the WSF, or the nanoparticles from both \u0026lt;\u0026thinsp;10 kDa fraction (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe results of the FT-IR analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) showed significant peaks in various regions. In the 3000\u0026ndash;3500 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e range (labeled A), WSF exhibited a peak that corresponds to N-H and O-H functionalities. Additionally, within the 1500\u0026ndash;1700 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e range (labeled B), distinct peaks indicated the presence of C\u0026thinsp;=\u0026thinsp;O stretching vibration and trivalent nitrogen, observed in both WSF and AgNPs spectra. The WSF displayed two peaks in the 1000\u0026ndash;1300 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e range (labeled C), indicating the presence of C-O and C-N bonds. The analysis validates the successful synthesis of AgNPs using WSF as a precursor, as indicated by the matching peaks found in both WSF and the AgNPs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOn the DLS optimization, the transmittance of the WSF AgNPs samples started to decrease at a concentration of 0.5 mg/mL, falling below the recommended threshold, and reached 0% at a concentration of 5 mg/mL (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Among the first three concentrations\u0026mdash;those that had satisfactory transmittance values\u0026mdash;the 0.1 mg/mL concentration was closest to achieving a baseline value of 1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eDuring the analysis of \u0026lt;\u0026thinsp;10 kDa AgNPs, the transmittance started to decrease at a concentration of 0.05 mg/mL and reached 0% at a concentration of 1 mg/mL (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC), while the baseline stabilized at a concentration of 0.1 mg/mL, yielding a value closer to the recommended value (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eGiven that the DLS technique is dependent on light passing through the sample and reaching the detector, maintaining transmittance values above the recommended threshold was of primary importance. Therefore, for subsequent analyses of hydrodynamic radius, polydispersity index (PDI), and zeta potential, a concentration of 0.1 mg/mL was selected for WSF AgNPs, and 0.05 mg/mL was chosen for the \u0026lt;\u0026thinsp;10 kDa AgNPs, based on optimization data from DLS analysis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe DLS measurements reported that the WSF AgNPs exhibited only one peak between 500 and 1000 nm, indicating the presence of only nanoparticles within this size range in the sample. The \u0026lt;\u0026thinsp;10 kDa AgNPs showed a predominance of nanoparticles smaller than 100 nm in their samples but also a small quantity of larger nanoparticles (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eThe hydrodynamic radius of the WSF AgNPs was approximately 1201 nm, while for the \u0026lt;\u0026thinsp;10 kDa AgNPs it was approximately 481 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). For the WSF AgNPs, the PDI was approximately 50%, while for the \u0026lt;\u0026thinsp;10 kDa AgNPs, it was 28,63% (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Finally, the mean zeta potential for all nanoparticle samples was approximately \u0026minus;\u0026thinsp;30 mV (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe SEM analysis validated the findings observed in the DLS. The WSF AgNPs have a spherical-like shape and exhibited a size range of 500 to 1000 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA), whereas the \u0026lt;\u0026thinsp;10 kDa AgNPs demonstrated a smaller size range of 100 to 200 nm, with some nanoparticles measuring approximately 400 nm, with a spherical-like shape, with the exception of the larger AgNPs, that have a cubic shape (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). The EDX results revealed the presence of carbon, silicon, chloride, and silver atoms on both AgNPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Given that silver is the primary material utilized in nanoparticle synthesis and silica was the substrate employed for the analysis, it can be inferred that the carbon and chloride originate from the molecules present in the water-soluble fraction of kefir.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.3 \u003cem\u003eIn vitro\u003c/em\u003e susceptibility test (disk diffusion method)\u003c/h2\u003e \u003cp\u003eThe investigation of \u003cem\u003ein vitro\u003c/em\u003e susceptibility of bacteria through disk diffusion (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) showed that only \u003cem\u003eA. baumannii\u003c/em\u003e was inhibited by the WSF alone. The WSF AgNPs at 10 \u0026micro;g/mL did not inhibit the growth of any of the tested multidrug-resistant bacteria. Increasing the concentration to 100 \u0026micro;g/mL resulted in inhibitory effects observed on \u003cem\u003eK. pneumoniae\u003c/em\u003e and \u003cem\u003eA. baumannii\u003c/em\u003e (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). At a concentration of 1000 \u0026micro;g/mL, the WSF AgNPs exhibited inhibitory activity against all the tested bacterial strains.\u003c/p\u003e \u003cp\u003eAt a concentration of 10 \u0026micro;g/mL, there was no inhibition zone for the \u0026lt;\u0026thinsp;10 kDa AgNPs. However, at 100 \u0026micro;g/mL, inhibition zones were observed against \u003cem\u003eA. baumannii\u003c/em\u003e, \u003cem\u003eE. coli\u003c/em\u003e, \u003cem\u003eK. pneumoniae\u003c/em\u003e, and \u003cem\u003eS. aureus\u003c/em\u003e (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Notably, complete growth inhibition was evident at a concentration of 1000 \u0026micro;g/mL against all multidrug-resistant bacteria evaluated.\u003c/p\u003e \u003cp\u003eFor fungi (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), the WSF did not inhibit the growth of drug-resistant strains. The growth of \u003cem\u003eC. albicans\u003c/em\u003e was not affected by any of the AgNPs. However, both AgNPs inhibited the growth of \u003cem\u003eC. krusei\u003c/em\u003e at concentrations of 100 and 1000 \u0026micro;g/mL, and the growth of \u003cem\u003eC. glabrata\u003c/em\u003e was inhibited at 1000 \u0026micro;g/mL (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). It is important to note that both \u003cem\u003eC. krusei\u003c/em\u003e and \u003cem\u003eC. glabrata\u003c/em\u003e are naturally resistant to Fluconazole, a commonly used antifungal.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003eAssessment of bacterial growth inhibition via the diffusion disk technique.\u003c/b\u003e The WSF AgNPs inhibited the growth of \u003cem\u003eA. baumannii\u003c/em\u003e and \u003cem\u003eK. pneumoniae\u003c/em\u003e at a concentration of 100 \u0026micro;g/mL. At a concentration of 1000 \u0026micro;g/mL, they inhibited the growth of all tested bacteria. The \u0026lt;\u0026thinsp;10 kDa AgNPs inhibited the growth of \u003cem\u003eA. baumannii\u003c/em\u003e, \u003cem\u003eE. coli\u003c/em\u003e, \u003cem\u003eK. pneumoniae\u003c/em\u003e, and \u003cem\u003eS. aureus\u003c/em\u003e at 100 \u0026micro;g/mL and inhibited the growth of all tested bacteria at 1000 \u0026micro;g/mL. Inhibition zones values of 0 indicate that they were absent. Lowercase letters indicate significant differences between AgNPs and WSF. The capital letters indicate significant differences among different concentrations of the same AgNPs. Symbols indicate significant differences among the same concentration of different AgNPs. Statistics were calculated by two-way ANOVA. All differences shown have a p value\u0026thinsp;\u0026lt;\u0026thinsp;0.0001.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eMultiresistant microorganisms\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"7\" nameend=\"c8\" namest=\"c2\"\u003e \u003cp\u003eInhibition Zone (mm\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eWSF\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e \u003cp\u003eWSF AgNPs (\u0026micro;g/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c8\" namest=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;10 kDa AgNPs (\u0026micro;g/mL)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1000\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1000\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eA. baumannii\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.304\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003csup\u003eaA#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8.777\u0026thinsp;\u0026plusmn;\u0026thinsp;0.240\u003csup\u003eaB#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e14.193\u0026thinsp;\u0026plusmn;\u0026thinsp;0.693\u003csup\u003ebC#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003csup\u003eaA#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e10.133\u0026thinsp;\u0026plusmn;\u0026thinsp;0.506\u003csup\u003ebB*\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e12.903\u0026thinsp;\u0026plusmn;\u0026thinsp;1.098\u003csup\u003ebC*\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eE. coli\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003csup\u003eaA#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003csup\u003eaA#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10.173\u0026thinsp;\u0026plusmn;\u0026thinsp;0.456\u003csup\u003ebB#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003csup\u003eaA#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e6.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.046\u003csup\u003ebB*\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e10.173\u0026thinsp;\u0026plusmn;\u0026thinsp;0.482\u003csup\u003ebC#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eK. pneumoniae\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003csup\u003eaA#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8.373\u0026thinsp;\u0026plusmn;\u0026thinsp;0.423\u003csup\u003ebB#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9.783\u0026thinsp;\u0026plusmn;\u0026thinsp;0.446\u003csup\u003ebC#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003csup\u003eaA#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e7.213\u0026thinsp;\u0026plusmn;\u0026thinsp;0.405\u003csup\u003ebB*\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e9.993\u0026thinsp;\u0026plusmn;\u0026thinsp;0.444\u003csup\u003ebC#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eP. aeruginosa\u003c/b\u003e \u003cb\u003eIMP\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003csup\u003eaA#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003csup\u003eaA#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9.807\u0026thinsp;\u0026plusmn;\u0026thinsp;0.514\u003csup\u003ebB#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003csup\u003eaA#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003csup\u003eaA#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e8.510\u0026thinsp;\u0026plusmn;\u0026thinsp;0.572\u003csup\u003ebB*\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eP. aeruginosa\u003c/b\u003e \u003cb\u003eSPM\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003csup\u003eaA#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003csup\u003eaA#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9.717\u0026thinsp;\u0026plusmn;\u0026thinsp;0.321\u003csup\u003ebB#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003csup\u003eaA#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003csup\u003eaA#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e9.643\u0026thinsp;\u0026plusmn;\u0026thinsp;0.179\u003csup\u003ebB#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eP. aeruginosa\u003c/b\u003e \u003cb\u003eVIM\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003csup\u003eaA#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003csup\u003eaA#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9.610\u0026thinsp;\u0026plusmn;\u0026thinsp;0.937\u003csup\u003ebB#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003csup\u003eaA#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003csup\u003eaA#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e8.627\u0026thinsp;\u0026plusmn;\u0026thinsp;0.263\u003csup\u003ebB#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eS. aureus\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003csup\u003eaA#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003csup\u003eaA#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9.037\u0026thinsp;\u0026plusmn;\u0026thinsp;0.127\u003csup\u003ebB#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003csup\u003eaA#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e8.237\u0026thinsp;\u0026plusmn;\u0026thinsp;0.585\u003csup\u003ebB*\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e11.617\u0026thinsp;\u0026plusmn;\u0026thinsp;0.391\u003csup\u003ebC*\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003eAssessment of fungi growth inhibition via the diffusion disk technique.\u003c/b\u003e WSF alone did not inhibit the growth of any of the tested fungi. None of the AgNPs inhibited the growth of \u003cem\u003eC. albicans\u003c/em\u003e at any concentration. However, the WSF AgNPs at a concentration of 100 \u0026micro;g/mL did inhibit the growth of \u003cem\u003eC. krusei\u003c/em\u003e. At a higher concentration of 1000 \u0026micro;g/mL, it also prevented the growth of \u003cem\u003eC. krusei\u003c/em\u003e and \u003cem\u003eC. glabrata\u003c/em\u003e. Similar results were observed for the \u0026lt;\u0026thinsp;10 kDa AgNPs. At a concentration of 100 \u0026micro;g/mL, it inhibited the growth of \u003cem\u003eC. krusei\u003c/em\u003e. Additionally, at a concentration of 1000 \u0026micro;g/mL, it also inhibited the growth of \u003cem\u003eC. glabrata\u003c/em\u003e. Inhibition zones values of 0 indicate that they were absent. Lowercase letters indicate significant differences between AgNPs and WSF. The capital letters indicate significant differences among different concentrations of the same AgNPs. Symbols indicate significant differences among the same concentration of different AgNPs. Statistics were calculated by two-way ANOVA. All differences shown have a p value\u0026thinsp;\u0026lt;\u0026thinsp;0.0001.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eResistant microorganisms\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"7\" nameend=\"c8\" namest=\"c2\"\u003e \u003cp\u003eInhibition Zone (mm\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eWSF\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e \u003cp\u003eWSF AgNPs (\u0026micro;g/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c8\" namest=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;10 kDa AgNPs (\u0026micro;g/mL)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1000\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1000\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eC. albicans\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003csup\u003eaA#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003csup\u003eaA#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003csup\u003eaA#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003csup\u003eaA#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003csup\u003eaA#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003csup\u003eaA#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eC. krusei\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003csup\u003eaA#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e13.073\u0026thinsp;\u0026plusmn;\u0026thinsp;0.834\u003csup\u003ebB#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e14.873\u0026thinsp;\u0026plusmn;\u0026thinsp;0.994\u003csup\u003ebC#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003csup\u003eaA#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e12.663\u0026thinsp;\u0026plusmn;\u0026thinsp;0.375\u003csup\u003ebB#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e15.597\u0026thinsp;\u0026plusmn;\u0026thinsp;0.745\u003csup\u003ebC#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eC. glabrata\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003csup\u003eaA#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003csup\u003eaA#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7.350\u0026thinsp;\u0026plusmn;\u0026thinsp;0.352\u003csup\u003ebB#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003csup\u003eaA#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003csup\u003eaA#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e11.633\u0026thinsp;\u0026plusmn;\u0026thinsp;0.349\u003csup\u003ebB*\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Toxicity assessment\u003c/h2\u003e \u003cp\u003eThe tested concentrations of WSF AgNPs and \u0026lt;\u0026thinsp;10 kDa AgNPs did not result in toxicity in \u003cem\u003eDrosophila melanogaster\u003c/em\u003e during long-term treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). There were no statistically significant differences in the survival probability of the flies compared to the control group throughout the 14-day analysis period, indicating that these nanoparticles are safe under the conditions tested.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. DISCUSSION","content":"\u003cp\u003eThe therapeutic effects of kefir have been explored in medicine because of its ability to treat many medical conditions, from neurological to gut diseases [\u003cspan additionalcitationids=\"CR34\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Although the existing literature extensively covers the therapeutic benefits of kefir, there is a gap in research regarding the utilization of nanotechnology to enhance kefir's effects through the production of nanoparticles. Our research aimed to assess the potential of kefir-derived silver nanoparticles in improving the therapeutic benefits of kefir, specifically its antimicrobial and antifungal activities. The AgNPs synthesized from both kefir fractions exhibited a spherical morphology, with the particles obtained from the \u0026lt;\u0026thinsp;10 kDa fraction being notably smaller in size. Both AgNPs demonstrated a -30 mV mean zeta potential, indicative of their stability, while exhibiting antimicrobial activity against all multi-drug resistant bacteria, \u003cem\u003eC. krusei\u003c/em\u003e and \u003cem\u003eC. glabrata\u003c/em\u003e. Furthermore, no toxicity was observed in \u003cem\u003eDrosophila melanogaster\u003c/em\u003e following long-term treatment.\u003c/p\u003e \u003cp\u003eThe nanoparticles were comprehensively characterized using UV‒Vis spectroscopy, DLS, and FT-IR spectroscopy. Silver nanoparticles typically exhibit a distinct absorbance peak in the 400\u0026ndash;500 nm range in UV-Vis spectra [\u003cspan additionalcitationids=\"CR37 CR38\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. The results of our UV-Vis spectroscopy analysis demonstrated that the distinctive peak was exclusively present in our WSF AgNPs, but not in the \u0026lt;\u0026thinsp;10 kDa AgNPs. It is hypothesized that the \u0026lt;\u0026thinsp;10 kDa AgNPs, due to their smaller size, may exhibit a broader absorbance, resulting in the absence of a distinct peak. Additionally, it is possible that these nanoparticles are composed of silver oxide (Ag\u003csub\u003e2\u003c/sub\u003eO or AgO), which also exhibit a broader absorbance range, rather than metallic silver. Further investigation is required to explain the lack of distinct absorbance peaks in the \u0026lt;\u0026thinsp;10 kDa AgNPs.\u003c/p\u003e \u003cp\u003eAs previously reported by our group, the WSF of kefir is primarily made up of amino acids, peptides, carboxylic acids, and alcohols [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. According to our FT-IR results, the synthesized nanoparticles are likely formed through the interaction between the amino acids and peptides in WSF and silver nitrate [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. The absence of transmittance bands corresponding to alcohol and carboxylic acid functional groups in the AgNPs spectra indicates that these groups may have not participate in the synthesis [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOptimizing the parameters for DLS was crucial due to the limited insight provided by the literature and equipment manuals on key parameters and equipment settings [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. For instance, the equipment manual recommends using backscatter measurements for concentrated samples, but it does not provide specific concentration guidelines. Furthermore, information on parameters such as transmittance and baseline are not well explored in the literature. These parameters are utilized to assess the reliability of the collected data, leaving room for further knowledge.\u003c/p\u003e \u003cp\u003eTo address this gap, our study conducted rigorous testing across various sample concentrations to determine the most effective parameters for reliable analysis of our samples. This empirical approach yielded valuable insights into the influence of concentration and equipment settings on measurement outcomes. The findings provide important guidance for future research aiming to accurately characterize silver nanoparticles based on sample properties.\u003c/p\u003e \u003cp\u003eThe hydrodynamic radius of our AgNPs (WSF AgNPs: 1201 nm and \u0026lt;\u0026thinsp;10 kDa AgNPs: 481,6 nm) is larger than that reported in previous syntheses [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. However, improving the biological synthesis process of AgNPs\u0026mdash;by testing different pH, precursor concentrations, temperature, and exposure to light\u0026mdash;can affect their physical characteristics, such as size, shape, and PDI [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Future research will aim to optimize the synthesis process and analyze the outcomes resulting from varying these parameters.\u003c/p\u003e \u003cp\u003eThe PDI of nanoparticles is a critical factor in drug delivery efficacy and safety [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Notably, the synthesized\u0026thinsp;\u0026lt;\u0026thinsp;10 kDa AgNPs fall within the acceptable PDI range (below 30%) [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e], allowing for more precise studies of potential side effects and the optimization for drug delivery purposes. Furthermore, our AgNPs exhibited a zeta potential of around \u0026minus;\u0026thinsp;30 mV, indicating that they are stable and have minimal tendency to aggregate [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eA number of studies have demonstrated that AgNPs synthesized via green methods, such as the use of plant extracts and pollen, display antimicrobial activity against bacterial and fungal pathogens [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan additionalcitationids=\"CR49 CR50\" citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e], which supports our findings. However, it is worth mentioning that our disk diffusion assay is preliminary, and a concentration of 1 mg/mL is very high and would be expected to produce inhibition zones for all the bacteria and fungi tested. High concentrations of antimicrobials can be toxic to users, and lack of specificity can lead to microbial resistance [\u003cspan additionalcitationids=\"CR53\" citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. In future research, determining the minimum inhibitory concentration of these NPs should be a priority in order to fully understand their potential as novel antibiotics and antifungals.\u003c/p\u003e \u003cp\u003eThe size and shape of nanoparticles can influence their uptake by cells and their interaction within biological systems. The WSF AgNPs exhibit a size range of 500 to 1000 nm, while the \u0026lt;\u0026thinsp;10 kDa AgNPs display a size range of 100 to 200 nm, with some nanoparticles measuring approximately 400 nm. Given that, according to the literature, smaller nanoparticles tend to have higher absorption rates, it is anticipated that the \u0026lt;\u0026thinsp;10 kDa AgNPs will exhibit a higher rate of absorption compared to the WSF AgNPs [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. With regard to their shape, spherical nanoparticles can enter tissues more easily than other shapes. However, the data in the literature remains inconsistent, and further studies are needed to ascertain the effects of different nanoparticle shapes on their uptake [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNevertheless, despite the enhanced bioavailability of our 10 kDa AgNPs due to their higher uptake, no long-term toxicity was observed in \u003cem\u003eDrosophila melanogaster\u003c/em\u003e in comparison to the WSF AgNPs. These findings are consistent with those of previous research conducted by our group, in which a silver nanoparticle produced using pollen extract and employing the same methodology did not demonstrate any toxicity to the flies [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], and those produced by other groups using natural products as the source for their nanoparticle synthesis [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. However, it should be noted that fruit flies serve only as a preliminary trial model organism, and further research is necessary on chordate animals to more accurately assess the toxicity of the nanoparticles produced from the water-soluble fraction of kefir.\u003c/p\u003e \u003cp\u003eIn future research, we plan to investigate the feasibility of incorporating our AgNPs into disinfectant solutions with the aim of achieving effective decontamination [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. Moreover, we will undertake an examination of the impact of kefir derived-AgNPs on wound healing processes, with a particular emphasis on their capacity to facilitate superior skin regeneration and serve as a healing agent and antiseptic. Finally, we aim to conduct drug development tests to explore novel avenues for antibiotic production, capitalizing on the distinctive properties possessed by these AgNPs.\u003c/p\u003e \u003cp\u003eIt is important to acknowledge the limitations of this research. Firstly, the production of AgNPs from kefir, a natural product, may be subject to variability in the green synthesis method due to differences in the bioactive components of kefir derived from different sources or batches. Moreover, although no toxicity was observed in \u003cem\u003eDrosophila melanogaster\u003c/em\u003e during the lifespan analysis, a comprehensive toxicity assessment, including the effects of AgNPs on brain and gut morphology, is imperative. Furthermore, additional \u003cem\u003ein vivo\u003c/em\u003e studies in higher organisms and clinical trials are necessary to confirm the safety and efficacy of these AgNPs in humans. Finally, although antimicrobial and antifungal properties against the tested bacteria and fungi were observed, determining the minimum inhibitory concentrations for these pathogens is essential for practical applications. Our group plans to address this, along with a more comprehensive toxicity assessment in \u003cem\u003eDrosophila melanogaster\u003c/em\u003e, in future research.\u003c/p\u003e"},{"header":"5. CONCLUSION","content":"\u003cp\u003eIn this study, we presented the synthesis and characterization of silver nanoparticles from the water-soluble fraction of kefir, with the aim of combating drug-resistant diseases. The successful synthesis of AgNPs was confirmed through UV-visible and FT-IR spectroscopy, while DLS optimization provided valuable information on their size distribution and stability. \u003cem\u003eIn vitro\u003c/em\u003e testing demonstrated the promising antimicrobial activity of WSF and \u0026lt;\u0026thinsp;10 kDa AgNPs against multidrug-resistant bacteria and \u003cem\u003eCandida\u003c/em\u003e fungi. \u003cem\u003eIn vivo\u003c/em\u003e studies showed no toxicity to fruit flies during long-term treatment. Future research will delve deeper into toxicity assessments, including potential morphological changes to specific organs of the flies. Further investigations will also focus on determining the minimum inhibitory concentrations, enhancing specificity, and exploring applications of AgNPs in wound healing and drug development.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAgNPs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003esilver nanoparticles\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDLS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003edynamic light scattering\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFT-IR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFourier-transformed infra-red\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMIC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMinimum inhibitory concentration\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNPs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNanoparticles\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePDI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePolydispersity index\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eUV‒Vis\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eUltraviolet-visible spectroscopy\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eWSF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eKefir\u0026rsquo;s whole water-soluble fraction\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":" \u003cp\u003e \u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eAuthors\u0026rsquo; information\u003c/h2\u003e \u003cp\u003eGenetics Laboratory, Institute of Biotechnology, Federal University of Uberl\u0026acirc;ndia, 1004 Acre Street, Building 2E, 38405-319, Uberl\u0026acirc;ndia, MG, Brazil.\u003c/p\u003e \u003cp\u003eLucas Matos Martins Bernardes, Serena Mares Malta, Ana Carolina Costa Santos, Rafael Alves da Silva, Tamiris Sabrina Rodrigues, Carlos Ueira-Vieira\u003c/p\u003e \u003cp\u003eLaboratory of Nanobiotechnology, Institute of Biotechnology, Federal University of Uberl\u0026acirc;ndia, 1004 Acre Street, Building 2E, 38405-319 Uberl\u0026acirc;ndia, Brazil.\u003c/p\u003e \u003cp\u003eMurillo N\u0026eacute;ia Thomaz da Silva\u003c/p\u003e \u003cp\u003eDepartment of Psychiatry, College of Medicine, University of Saskatchewan, 103 Hospital Drive, Room 366 Ellis Hall Saskatoon, SK, Canada.\u003c/p\u003e \u003cp\u003eAna Paula Mendes-Silva\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCorresponding authors\u003c/strong\u003e \u003cp\u003eLucas Matos Martins Bernardes\u003c/p\u003e \u003cp\[email protected]\u003c/p\u003e \u003cp\u003eCarlos Ueira-Vieira\u003c/p\u003e \u003cp\[email protected]\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis project was funded by the Research Support Foundation of the State of Minas Gerais (FAPEMIG APQ-02766-17, APQ-00269-22 and National Council of Scientific and Technological Development (CNPq, grant number: 403193/2022-2) and FAPEMIG (grant number: CBB-APQ-03613-17) for INCT -TeraNano.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eLMMB: conceptualization; data curation; methodology; formal analysis; writing\u0026mdash;original draft. SMM: data curation. ACCS: data curation. RADS: data curation. TSR: data curation. MNTDS: data curation. APMS: conceptualization. CUV: conceptualization; methodology; funding acquisition; formal analysis; writing\u0026mdash;review and editing. All authors reviewed the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe deeply appreciate all the assistance provided by Dr. Luiz Ricardo Goulart Filho, who tragically became one of the millions of victims of COVID-19.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets produced or analyzed during the current study are available from the corresponding author upon request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSimon S, Sibuyi NRS, Fadaka AO, Meyer S, Josephs J, Onani MO, et al. Biomedical Applications of Plant Extract-Synthesized Silver Nanoparticles. 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Silver nanoparticles and their antibacterial applications. Int J Mol Sci. 2021;22.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":false,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Silver nanoparticles, kefir, nanomedicine, anti-bacterial agents, antifungals","lastPublishedDoi":"10.21203/rs.3.rs-4830503/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4830503/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eNanotechnology offers innovative approaches to combat drug-resistant diseases. Silver nanoparticles (AgNPs) have emerged as potent antimicrobial agents \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. Green synthesis methods, which leverage the bioactive components of the water-soluble fractions of Brazilian kefir (whole water-soluble fraction and smaller than 10 kDa fraction), provide sustainable alternatives to conventional nanoparticles production. However, despite the documented therapeutic benefits of kefir, its potential in nanomedicine remains underexplored.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e The successful synthesis of silver nanoparticles using water-soluble fractions of kefir was confirmed by UV-Visible spectroscopy and Fourier-transform infrared analyses. The hydrodynamic radius of nanoparticles derived from the entire water-soluble fraction was 1300 nm, while those from the smaller than 10 kDa fraction displayed a radius of 400 nm. All synthesized AgNPs exhibited a zeta potential of -30 mV. The disk diffusion method demonstrated the antimicrobial efficacy of our AgNPs against a range of multidrug-resistant bacteria and \u003cem\u003eCandida \u003c/em\u003efungi (p\u0026lt;0.0001), with no observed toxicity on \u003cem\u003eDrosophila melanogaster\u003c/em\u003eon a long-term treatment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003eThis study highlights the potential of these AgNPs as effective antimicrobial agents, particularly against drug-resistant pathogens. Future research is needed to evaluate the minimum inhibitory concentrations of our AgNPs and enhance specificity through conjugation with other compounds. Additionally, further investigations into electron microscopy analysis and various applications, such as disinfectant solutions, wound healing, and antibiotic production, will advance the utilization of kefir-derived AgNPs in healthcare.\u003c/p\u003e","manuscriptTitle":"Green Synthesis, Characterization, and Antimicrobial Activity of Silver Nanoparticles from Water-Soluble Fractions of Brazilian Kefir","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-09-13 16:35:43","doi":"10.21203/rs.3.rs-4830503/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-10-03T02:41:28+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-09-28T13:20:37+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"219862759243542412408406913579654785689","date":"2024-09-17T08:31:47+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-09-09T19:19:34+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"250772634522752565941466522454895438684","date":"2024-08-26T19:01:22+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-08-25T07:20:53+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-08-17T06:45:52+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-08-17T03:16:35+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-08-17T03:11:55+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-07-30T17:33:43+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c6a7d0a0-fbda-42fd-aa0a-6d341154b627","owner":[],"postedDate":"September 13th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":37347832,"name":"Biological sciences/Biotechnology/Nanobiotechnology"},{"id":37347833,"name":"Biological sciences/Microbiology/Antimicrobials"},{"id":37347834,"name":"Physical sciences/Nanoscience and technology/Nanobiotechnology"},{"id":37347835,"name":"Physical sciences/Nanoscience and technology/Techniques and instrumentation/Characterization and analytical techniques"},{"id":37347836,"name":"Physical sciences/Nanoscience and technology/Techniques and instrumentation/Design synthesis and processing"}],"tags":[],"updatedAt":"2025-03-31T15:59:44+00:00","versionOfRecord":{"articleIdentity":"rs-4830503","link":"https://doi.org/10.1038/s41598-025-95616-4","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-03-27 15:56:59","publishedOnDateReadable":"March 27th, 2025"},"versionCreatedAt":"2024-09-13 16:35:43","video":"","vorDoi":"10.1038/s41598-025-95616-4","vorDoiUrl":"https://doi.org/10.1038/s41598-025-95616-4","workflowStages":[]},"version":"v1","identity":"rs-4830503","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4830503","identity":"rs-4830503","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}