Brain heart infusion broth

Agrimonia eupatoria L. (AG) plant was obtained (CHÁ HUNOS, Lda., Vila Nova de Gaia, Portugal), stored and used according to the supplier’s recommendations. Normal human dermal fibroblast (NHDF) cells were acquired from ATCC—American Type Culture Collection. Cotton fabric was obtained from James H. Heal & Co. Ltd. (Halifax, UK). Resazurin (7-hydroxy-3H-phenoxazin-3-one 10-oxide) dye, 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO), sodium bromide (NaBr), sodium hypochlorite (NaOCl), chitosan (CS) (low molecular weight), sodium hydroxide (NaOH), hydrochloric acid (HCl), sodium chloride (NaCl), Mueller-Hinton broth (MHB), tween 80, dimethyl sulfoxide (DMSO) anhydrous ≥ 99.9%, trypsin, and 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) were purchased from Sigma-Aldrich (Sintra, Portugal). Glacial acetic acid and ethanol absolute were acquired from Fisher Scientific (Porto Salvo, Portugal). Poly(vinyl alcohol) (PVA) (MW 115.000 g/mol) was purchased from VWR International (Carnaxide, Portugal). Nutrient agar (NA), nutrient broth (NB), and agar for microbiology were bought from Fluka (Sintra, Portugal). Brain Heart Infusion (BHI) broth was obtained from Panreac (Barcelona, Spain). Phosphate-buffered saline (PBS) was purchased from Alfa Aesar (Ward Hill, USA). All solvents were of analytical grade and used as received without further purification.

Chelidoniummajus L. (C. majus) was bought from a Portuguese botanic shop. Normal human dermal fibroblasts (NHDF) cells were acquired from ATCC (American Type Culture Collection, Manassas, VA, USA). Polycaprolactone (PCL) (MW 80,000 g/mol) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Polyvinyl Alcohol (PVA) (MW 115,000 g/mol) was obtained from VWR Chemicals (Geldenaaksebaan, Leuven, Belgium). Pectin (PEC) was purchased from Acros Organics (Fisher Scientific, Göteborg, Sweden). Chloroform (analytical grade), Dimethylformamide (DMF) (analytical grade), and ethanol absolute were acquired from Fisher Chemical (Leicestershire, UK). Nutrient agar (NA), Nutrient broth (NB), and agar for microbiology were bought from Fluka (Barcelona, Spain). Brain Heart Infusion (BHI) broth was purchased from Panreac (Barcelona, Spain). Sodium chloride (NaCl), Mueller-Hinton broth (MHB), tween 80, trypsin, fetal bovine serum (FBS), 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), and Dimethyl Sulfoxide (DMSO) anhydrous ≥99.9% were provided from Sigma-Aldrich. Phosphate-buffered saline (PBS) was purchased from Alfa Aesar (Kandel, Germany). All solvents were used as received without further purification.

The probiotic strain A. faecalis A12C was isolated from Argyrosomus regius gills at the Instituto de Sanidad Animal y Seguridad Alimentaria of the University of Las Palmas de Gran Canaria. The strain was identified at the Microbiology Department of the Hospital Universitario de Gran Canaria Dr Negrín by means of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) system (Vitek®MS, Biomerieux, Madrid, Spain). A. faecalis A12C strain cultures were stored at − 80 °C with 20% glycerol (v/v) addition in Brain Heart Infusion broth (PanReac-AppliChem, Darmstadt, Germany). Fresh cultures were made prior the assays, and the strain was aerobically incubated in Trypticase Soy with 5% sheep blood (Becton Dickinson, Franklin Lakes, New Jersey, USA) medium for 18 h at 37 °C with shaking (120 rpm) each 2 days. Before the challenge, the bacteria were centrifuged at 2500×g for 10 min and washed three times with sterile 0.9% saline solution. The bacteria were resuspended in dinking mineral water (Fonteide®), and bacterial concentration was measured with a spectrometer at 600 nm. Mineral water (Fonteide®) was used to adjust the suspension to 6 × 10⁸ CFU/mL. Finally, the suspension was stored at 4 °C and protected from light.