2.11 Toxicity of the colorants
The cytotoxicity of the colorants was evaluated in front of MRC-5 cells (human fibroblast)[24] grown in DMEM medium (Dulbecco’s Modified Eagle’s Medium), which was supplemented with 10% fetal bovine serum and kept in a CO2 incubator at 37 ºC and an atmosphere containing 5% CO2. Cells were seeded in 96-well plates (0.5 x 104 cells per well). After 24 hours, extracts were dissolved in DMSO and added to each well (10 μg/mL) and incubated for 48 hours. Doxorubicin (5 µg/mL) was used as a positive control. Negative controls (blanks) received the same amount of DMSO and had the same final DMSO concentrations as the samples (0.1%). Two hours before the end of the incubation, 10 μL of alamarBlueTM were added to each well. The fluorescence signal was monitored with a multiplate reader using a range of 530-560 nm excitation wavelength and 590 nm emission wavelength. The fluorescence signal generated from the assay was proportional to the number of live cells in the sample.
2.12 Textile dyeingWe conducted preliminary tests to evaluate the potential of crude colorant extracts for dyeing textiles. To prepare the coloring solutions, we dissolved the extracts from three studied isolates (Clonostachys rosea P2SO329, Penicillium gravinicaseiP3SO332, and Penicillium sclerotiorum P3SO224) in 10% methanol, resulting in a concentration of 0.1 mg/mL. A control solution without added colorant was also prepared. For the dyeing process, we used polyester, silk, and cotton fabric fragments measuring 5 x 5 cm. The textile fragments were immersed in beakers containing 50 mL of the colorant solutions and left at 35 °C for 1 hour, following the method previous described [25,26]. After dyeing, the samples were washed with warm distilled water at 35 °C to remove any unfixed colorants. Next, the fabric fragments were soaked in a 0.1% CuSO4 mordant solution for 10 minutes. Finally, the samples were rinsed with water and air dried at room temperature.
Stability was visually assessed by observing the loss of color after exposing the fabric fragments to natural light for a period of five days.
2.13 Evaluation of fungal and commercial colorants in cream and cosmetic formulationsA cream with a water-in-oil (W/O) emulsion was prepared using the following formulation. The oil phase consisted of 4 g of mineral oil and 10 g of a commercial base containing vegetal oil and an emulsifier, sourced from “Essências da Amazônia” (Manaus, AM-Brazil). The aqueous phase was prepared by combining 79 g of distilled water, 4 g of glycerin, and 2 mL of a commercial Aloe Vera extract, also obtained from Essências da Amazônia. To achieve emulsification, the oil phase was heated separately until it reached a temperature of 80 ºC. Subsequently, the water phase was gradually added to the oil phase under constant stirring, while cooling the mixture to approximately 40 °C. This process resulted in the formation of a homogeneous cream. Fungal and commercial colorants were incorporated into the cream at a concentration of 0.05 mg per gram of the formulation. The cream was fortified with 1mL of a preservative solution (methyl paraben, propylparaben and propylene glycol)[27,28].
Colorant 1, from the Penicillium sclerotiorum P3SO224 strain, showed satisfactory results in the incorporation of the cream base a more attractive coloring, and was thus selected for the soap development test.
The soap formulation consisted of 250 g of glycerin soap base (Nossa Terra; composition: sodium babassuate, sodium tallow, propylene glycol, glycerin, sodium laureth sulfate, alcohol, water, sucrose, tetrasodium EDTA, dipropylene glycol), 20 mL of lauryl, 2-5 mL of essence, 5 mL of glycolic extract and the respective fungal colorant and commercial colorant in a final content of 0.05 mg of colorant per g of soup. All components were placed a hot plate for the complete casting of the base. The formulation was added to a silicone mold and cooled to -20 °C for solidification.
The developed cream formulation was subjected to a centrifugation test (3,000 rpm/30 min, Cosmetic products stability guide) in order to assess whether the product would maintain the miscible water and oil emulsion phases. The pH, appearance, color and odor parameters were evaluated for a period of 7 days. A 1:10 preparation (0.5 g cream/4.5 mL distilled water) was used for pH determination with analysis at time zero and every 48 hours for 7 days.