Sabouraud dextrose agar

Phosphate buffered saline (PBS), alginate, sulfuric acid, absolute ethanol (99.7%), trichloroacetic acid, sulforhodamine B, acetic acid and tris(hydroxymethyl)aminomethane (TRIS base) were purchased from Sigma-Aldrich^® (Darmstadt, Germany). Gram’s crystal violet solution, dimethyl sulfoxide (DMSO), deuterated dimethyl sulfoxide (DMSO-d6), anhydrous chloroform, deuterated chloroform (CDCl₃), methanol, ethyl acetate (AcOEt) and calcium chloride were bought from Merck^® (Lisboa, Portugal). Dehydroabietic acid was obtained from Wako^® (Neuss, Germany) (FUJIFILM Wako Pure Chemical Corporation; CAS RN^®: 1740-19-8, Molecular Formula: C20H28O2; Molecular Weight: 300.44). Mueller–Hinton Broth (MHB), Mueller–Hinton agar (MHA), Sabouraud dextrose broth (SDB), Sabouraud dextrose agar (SDA) and tryptic soy broth (TSB) were purchased from Biokar^® Diagnostics (Allonne, France).

In this work, the following microorganisms from Culture collection of Instituto Superior de Agronomia, Portugal, were used: C. albicans ISA 2289; C. krusei ISA 2290; L. monocytogenes ISA 4008; S. cerevisiae ISA 1028, Salmonella sp. ISA 4348, and E. coli ATCC 25922 from the American Type Culture Collection. The antimicrobial activity was assessed against the microorganisms described above. In order to prepare the working culture of the microorganisms, a subculture was prepared from the stock culture in plates with recommended media and incubation conditions for each microorganism, Luria-Bertani (LB) agar plates (10 g/L tryptone, 10 g/L NaCl, 5 g/L yeast extract, 20 g/L agar, pH was adjusted to 7.0 with 5 N NaOH) for E. coli ATCC 25922 and Salmonella sp. ISA 4348 and Brain Heart Infusion (BHI) (Biokar Diagnostics, Allonne, France) for L. monocytogenes ISA 4008 at 35 ± 2 °C, or Sabouraud Dextrose Agar (SDA) (Biokar Diagnostics, Allonne, France) for yeasts at 30 ± 2 °C [37 (link)].

All formulations’ organoleptic characteristics, such as appearance, color, odor, and pH, were evaluated. The pH values were determined at room temperature using a pH-meter (Mettler Toledo, Columbus, OH, USA) in the production day (T₀) and 6 days after production (T₆).
To evaluate the microbiological contamination of all formulations, microbiological control tests were carried out using a spread-plating method, always in sterile conditions. In this assay, Trypticase Soy Agar (TSA) (BIOKAR Diagnostics) and Sabouraud Dextrose Agar (SDA) (BIOKAR Diagnostics) were used as media. After autoclaving, the media were distributed by Petri plates with 90 cm in diameter (VWR). The formulations were diluted 1:10 and 1:1000 in sterile Buffered Peptone Water (BIOKAR Diagnostics) pH 7.0. The TSA and SDA media plates were inoculated with 100 μL of each sample dilution (n = 1) and were incubated at 30 °C for the SDA medium and at 37 °C for the TSA media, for 3–5 days. The number of colony-forming units (CFU) that developed during the incubation period (3–5 days) was evaluated and recorded. The count of total microorganisms corresponds to the average of CFU present in the TSA and SDA plates multiplied by the dilution factor.