Sabouraud dextrose broth (sdb)

All the chemicals required for experiments were of analytical grade and were purchased from Loba Chemie (Mumbai, India), SRL (Mumbai, India), and Sigma Aldrich (Germany). Nutrient agar, nutrient broth, sabouraud dextrose agar and sabouraud dextrose broth required for antimicrobial and preservative efficacy were obtained from Hi-media Laboratories. Streptomycin, ciprofloxacin, ampicillin and fluconazole were obtained as gift sample from Belco Pharma, Bahadurgarh, India. Microbial strains S. aureus MTCC 3160, P. aeruginosa MTCC 1934, E. coli MTCC 45, C. albicans MTCC 183 and A. niger MTCC 282 strains were purchased from MTCC, Chandigarh, India. Chemical reactions were monitored by TLC on silica gel plates in iodine and UV chambers. Sonar melting point apparatus in open capillary tube was used for the recording of melting points. ¹H NMR and ¹³C NMR spectra were confirmed in DMSO and deuterated CDCl₃ on Bruker Avance II 400 NMR spectrometer at a frequency of 400 MHz downfield to tetramethyl silane standard. FTIR spectra were recorded on Perkin Elmer FTIR spectrophotometer with the help of KBr pellets technique. Waters Micromass Q-ToF Micro instrument was used for Mass spectrum recording.

To evaluate the antifungal effectiveness of Saudi Sidr honey, the measurement of the MIC was made using the broth microdilution technique. The CLSI protocol (NCCLS-M27-A, 2002) was adapted for usage. To make the stock solution, 3 g of honey was briefly diluted in 1.5 mL of sterile water. To speed up dissolving, the liquid was heated at 50 °C for 15 min. In sterile water, a series of dilutions of honeys (2000, 1800, 1600, 1400, 1200, and 1000 mg/mL) were made. All concentration was aliquoted into a sterile, 96-well round-bottom plate at a volume of 100 µL. A quantity of 100 µL of a fungus suspension made in Sabouraud dextrose broth (HiMedia^®, Mumbai, India) (optical density of 0.08) was pipetted into each well and stirred. Each plate included two wells of positive controls (fungal suspension) and three wells of negative controls (broth on its own, sterile water, and diluted honey). After incubating for 24 h at 30 °C, growth was observed for visual turbidity. The MIC was defined as the lowest amount of honey that could not allow the test microorganisms to proliferate. Each MIC value is provided in µg/mL and three replicas of each experiment were performed.

Sabouraud dextrose agar (SDA), Sabouraud dextrose broth (SDB), and peptone were obtained from HiMedia (Mumbai, India). Certified standards of DON and ZEA were obtained from Sigma-Aldrich (Bengaluru, India). Immunoaffinity columns specific for DON and ZEA were procured from Vicam (Waters business, USA). The other chemicals used in the study were analytical grade and obtained from Merck Millipore (Bengaluru, India).

Minimum inhibitory concentrations (MICs) of SeNPs-BSA, SeNPs-Chit, SeNPs-Gluc, and Na₂SeO₃ against standard microbial strains, were determined by the broth microdilution method (CLSI, 2017 ). Each sample was diluted with microbial growth media to final concentrations ranging from 400 to 12.5 μg/mL and transferred into 96-well polystyrene microtiter plates (Sarstedt, Germany). The wells were further supplemented with bacterial suspensions (~10⁶ colony-forming unit per mL–CFU/mL), as well as C. albicans suspension (~10⁷ CFU/mL), and incubated for 24 h at 35°C. Mueller-Hinton broth (MHB; HiMedia, India) and Sabouraud Dextrose Broth (SDB; Torlak, Serbia) were used as growth media for bacteria and yeast, respectively. MICs were determined as the lowest concentrations in which there was no visible growth of microorganisms.

The antifungal activity of the extract was evaluated against a standard strain (CA INCQS 40006) and a clinical isolate (CA URM 5974) of Candida albicans. These strains were inoculated in Sabouraud Dextrose Agar (SDA, KASVI, Laboratorios Conda S.A., Spain) and incubated at 37 °C for 24 h. After this period, culture aliquots were transferred to test tubes containing 3 mL of saline solution (0.9% sodium chloride), and the concentrations were adjusted by comparing the inoculum turbidity with 0.5 on the McFarland scale [84 ] Microdilutions were performed using doubly concentrated Sabouraud dextrose broth (HiMedia, Mumbai, India). Depleted potato dextrose agar (PDA) (Becton Dickinson Rowa France, Le Pont de Claix, France) with added bacteriological agar was used for micromorphological analysis. The CA INCQS 40006 strain was obtained from the Reference Collection of Microorganisms in Sanitary Surveillance (CMRVS) of the National Institute of Health Quality Control (INCQS, FIOCRUZ), while the CA URM 5974 strain was obtained from the University Recife Mycology (URM) library of the Federal University of Pernambuco.