Antimicrobial efficacy of mixtures of silver nanoparticles and polyhydric alcohols against health-promoting bacteria

Abstract

The safe production and storage of food is associated with a number of challenges with respect to controlling microbiological hazards and pathogens (Kulak et al., 2018; Nawrocka and Cieśla, 2013; Ognik et al., 2016). In recent years silver nanoparticles (AgNPs) have been used as antimicrobial agents for improving food safety especially in the field of food packaging (Bikiaris, and Triantafyllidis 2013). On the other hand, some authors consider that glycerol, and other polyalcohols are the best plasticizers to use for the production of food packaging (Chocyk et al., 2015; Oniszczuk et al., 2016; Toxqui-Teránet al., 2018). Increasingly, many consumer products with food packaging contain AgNPs. For that reason, it is possible for the nanoparticles to migrate into food matrices. However, it is well known that nanoparticles featuring novel chemical and physical properties that differ from normal macroparticles of the same composition may interact with food and with the human body after consumption (He and Hwang, 2016). Due to their small size, there may be an increased risk of nanoparticles penetrating human cells and causing cytotoxicity. For example, AgNPs may cross the cellular barrier © 2019 Institute of Agrophysics, Polish Academy of Sciences G. CZERNEL et al. 474 thereby leading to the formation of free radicals in the tissues and eventually causing oxidative damage to the cells and tissues (Pradhan et al., 2015). The oxidative stressdependent mechanism of toxicity of AgNPs described above have led to the conclusion that silver nanoparticles are genotoxic and even carcinogenic. Other important recent studies have evaluated the potential effect of AgNPs on intestinal microflora after ingestion (Bergin and Witzmann, 2013; Javurek et al., 2017). Karavolos and Holban (2016) reported the effect of dietary AgNPs, which preferentially kills microbiome components and, thus, causes the induction of dysbiosis. The connection between dysbiosis and disease has been well documented (Karavolos, 2015). However, at present, the mechanisms of action of silver nanoparticles against gut microbiota are not yet clearly understood. Nevertheless, some authors have described AgNPs binding to cell membranes that include sulphur-containing proteins and a sugar-phosphate backbone of DNA. As a result of this binding activity, the nanoparticles preferentially attack the respiratory chain, they also influence cell division, which may finally lead to cell death (Rai et al., 2009).The human intestinal microbiota comprises bacteria, fungi, viruses and archaea, which form a complex ecosystem and have a close relationship with the host. The Lactobacillus and Bifidobacterium genera constitute the majority of beneficial bacteria and have been used as probiotic dietary supplements to improve the activities of digestion, metabolism, and the immune system (Degnan, 2008). Apart from the positive effect of probiotics on general well-being, there are several particular clinical symptoms or conditions that are reported to be beneficially affected by the use of specific probiotics. Such conditions include diarrhoea, gastroenteritis, inflammatory bowel disease, irritable bowel syndrome, Crohn’s disease, and the alleviation of the symptoms of lactose intolerance (Degnan, 2008). As yet, there have been investigations regarding the antibacterial effects of AgNPs mainly using pathogenic bacteria; only a few studies have been carried out with beneficial bacteria (Gornicka et al., 2014; Tyagi et al., 2016). The increasing usage of AgNPs in connection with different types of plasticizers such as polyalcohols suggest that studies concerning the influence of AgNPs activity on a wider range of bacteria are necessary. In the current study, we investigated the antimicrobial potential of AgNPs toward health-promoting bacteria based on the example of Lactobacillus, Bifidobacterium, which inhabit the human intestine and are commonly used as probiotic dietary supplements. We conducted research to identify polyalcohols, which may have a protective effect on probiotic bacteria, with a view to their application in the food industry. MATERIAL AND METHODS Silver nitrate (AgNO3) trisodium citrate (C6H5O7Na3), sodium hydroxide NaOH and hydrochloric acid HCl (Sigma Aldrich) of analytical grade purity, were used as starting materials without further purification. Polyhydric alcohols (glycerol, erythritol, mannitol and xylitol) were purchased from Sigma-Aldrich. In all of the experiments double distilled water was used. The silver nanoparticles were prepared by using a chemical reduction method according to the description of Lee and Meisel (1982). Typically, 50 mL of 10M AgNO3 aqueous solution was heated up to the boiling temperature and the solution was stirred vigorously during the synthesis process. While stirring, a solution of 1% sodium citrate (5 mL) was added. The solution was kept at the boiling point for 10 min until the colloids changed colour. The solution was heated continuously until the colour changes were apparent (pale green). At that point, heat is no longer applied and the solution is stirred continuously until it cools to room temperature. The size of the synthesized AgNPs was controlled by the pH of the solution during synthesis. After that, the AgNPs solution was mixed with polyhydric alcohols in order to obtain 20% (m m) mixtures, which were used in the experiment. Electronic absorption spectra at desired dilutions of silver colloids were recorded using a spectrophotometer Cary 300 Bio (Varian) equipped with a thermostatted cuvette holder with a 6×6 multicell Peltier block. The temperature was controlled with a thermocouple probe (Cary Series II from Varian) placed directly in the sample. The UV-Vis spectra were recorded from a base solution, which had been diluted tenfold. The size and morphology of the silver nanoparticles were determined by SEM. After washing in double distilled water and ultrasonication, the samples were placed on circular aluminium stubs, slightly dried and transferred into the chamber of a scanning electron microscope Quanta 3D FEG (FEI). Micrographs were taken using an ETD detector, at an accelerating voltage of 30 kV. The AgNPs diameter was measured using Nis-Elements Advanced Research software. Particle distribution was derived from a histogram considering 500 particles. Colloidal AgNPs were deposited on a glass plate and dried overnight under vacuum. After that, the dry nanoparticles were analysed using a powder X-ray diffraction technique with an Empyrean (PANalytical) diffractometer with a Cu anode as a source for CuKα x-ray radiation (λ = 1.5406 Å). All samples were measured over a 2θ range of 2 to 90 with a step size of 0.013 and an exposition time per step of 1s. The approximated mean size of the spherical nanoparticles was calculated according to the well-known Scherrer equation with the Scherrer shape constant of 0.9 (Scherrer, 1918). The peak broadening was ~0.03. ANTIMICROBIAL MIXTURES AGAINST HEALTH-PROMOTING BACTERIA 475 A statistical analysis was carried out with the use of the Origin 9.0 software. For the UV-Vis spectra, the full width at half maximum (FWHM) value, was calculated from the fits of the Voight function to every UV-Vis peak. The SEM histogram was described by the bigaussian function with a different width for the left and right shoulder. Data are presented as a mean and standard deviation (±SD). The standard deviation was calculated from the confidence interval of 24 nm, which consisted of more than 99% (± 3σ) of the measured particles. For the left shoulder σl = 2.7 nm and for the right shoulder σr = 5.3 nm. For the X-ray diffraction of the FWHM, the average diameter and standard deviation was calculated from the fits of the Voight function to the three peaks {111}, {200} and {220}(Table 1). Bifidobacterium adolescentis 15706, B. bifidum 29521, B. breve 15700, Lactobacillus paraplantarum 700211, obtained from American Type Culture Collection (Rockville, MD, USA); L. rhamnosus, from Biomed Serum and Vaccine Production Plant Ltd., (Lublin, Poland), were used for this study. The specific test strains of Llactobacilli and Bbifidobacteria were selected because they are either already established as probiotics and are used in pharmaceutical products or they have potential probiotic properties. The Lactobacillus and Bifidobacterium cultures were maintained at – 80C in MRS Broth (BTL, Łódź, Poland) containing 20% (wt/vol) glycerol. Cultivation tests were performed in a medium based on MRS Broth supplemented with 0.05 % L-cysteine HCl. The antibacterial properties of AgNPs in the presence of polyalcohols were determined using a Bioscreen C system (Labsystem, Helsinki, Finland). After a 24-h incubation, the bacterial cultures were centrifuged and removed from the medium. The bacterial cells were suspended in physiological saline, and an optical density of 0.5 was set at 600 nm. The analysed bacteria were grown in MRS with 10 μg mL of AgNPs and 2% of glycerol, erythritol, mannitol and xylitol. 350 μL of the media were transferred onto honeycomb 100-well plates in triplicate, and the wells were inoculated with 50 μL of bacterial suspension. The experiment was performed under anaerobic conditions by measuring the OD 600 nm every 2 for 48 h. Growth curve parameters (max specific growth rate, lag time, doubling time, etc.) were determined using the PYTHON script according to Hoeflinger et al. (2017). A statistical analysis was carried out with use of Origin 9.0 software. For UV-Vis spectra, the full width at half maximum (FWHM) value, was calculated from the fits of the Voight function to every UV-Vis peak. The SEM histogram was described by the bigaussian function with a different width for the left and right shoulder. Data are presented as a mean and standard deviation (±SD).The standard deviation was calculated from the confidence interval of 24 nm, which consisted of more than 99% (± 3σ) of the measured particles.