Extracts were tested for their effect on two distinct skin enzymes inhibition: neutrophil elastase (NE), and tyrosinase. Samples were prepared in dH2O according to the following final concentrations in the wells: 20, 15, 7.5, 3.75, and 1.875 mg.mL− 1.
2.10.1. Elastase
The assay was performed using a commercial kit of neutrophil elastase inhibitory screening (fluorometric) (ab118971, ABCAM, Cambridge, UK), according to manufacturer’s instructions. Briefly, NE enzyme stock was first reconstituted in 220 µL of assay buffer and stored at -80 ºC. When testing, all reagents (assay buffer, substrate, NE solution, and inhibitor control (Succinyl-alanyl-alanyl-prolyl-valine chloromethyl ketone - SPCK) were equilibrated to room temperature. Then, NE enzyme stock solution, enzyme substrate, and inhibitor control were diluted 1/25, 2/25, and 1/25, respectively, in assay buffer, to required total volume. 50 µL of diluted NE solution was added to all wells. Then, 25 µL of sample, or assay buffer for blank (enzyme control), or inhibitor control were pipetted in duplicate, into each desired well of the microplate. The microplate was mixed and left incubating at 37 ºC, for 5 min. After incubation, 25 µL of diluted enzyme substrate were added to all wells and fluorescence was immediately measured at Ex/Em 400/505 nm at 37 ºC for 30 min, using a Synergy H1 microplate reader (BioTek, Vila Nova de Gaia, Portugal) in kinetic mode. The RFU of fluorescence is ∆RFU = R2 – R1, and the kinetic mode was used to choose the R1 and R2 at linear range. The percentage inhibition of this assay was calculated by Eq. (2):
$$\text{E}\text{n}\text{z}\text{y}\text{m}\text{e} \text{i}\text{n}\text{h}\text{i}\text{b}\text{i}\text{t}\text{i}\text{o}\text{n} \text{a}\text{c}\text{t}\text{i}\text{v}\text{i}\text{t}\text{y} \left(\text{%}\right)=\frac{{\Delta }\text{R}\text{F}\text{U} \text{s}\text{a}\text{m}\text{p}\text{l}\text{e}}{{\Delta }\text{R}\text{F}\text{U} \text{e}\text{n}\text{z}\text{y}\text{m}\text{e} \text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}}\times 100 \left(2\right)$$
2.10.2. Tyrosinase
The assay was performed using a commercial kit of tyrosinase inhibitory screening (colorimetric) (ab204715, ABCAM), according to manufacturer’s instructions. Briefly, tyrosinase substrate and lyophilized tyrosinase were dissolved in 220 µL of dH2O and assay buffer, respectively, and stored at -20 ºC. Inhibitor control (kojic acid) was prepared in dH2O to a 10 mM concentration and stored at -20 ºC. When testing, all reagents (assay buffer, tyrosinase substrate stock solution, tyrosinase stock solution, tyrosinase enhancer, and inhibitor control) were equilibrated to room temperature, prior to use. Then, tyrosinase enzyme was diluted 1/25 in assay buffer to required total volume. For diluted tyrosinase substrate solution, tyrosinase substrate and tyrosinase enhancer were diluted 2/30, and 5/30, respectively, together in assay buffer to required total volume. 20 µL of sample, inhibitor control, assay buffer for enzyme control, or solvent for solvent control, were pipetted in duplicate into each desired well of the microplate. Prior to use, inhibitor control was set to a 0.75 mM concentration. Then, 50 µL of diluted tyrosinase enzyme were added to all wells and the plate was left incubating at 25 ºC, for 10 min. After incubation, 30 µL of diluted tyrosinase substrate solution was added to all wells and the absorbance (abs) was recorded at 510 nm, every 2–3 min for 30 to 60 min, using a Synergy H1 microplate reader (BioTek, Vila Nova de Gaia, Portugal), in kinetic mode. Data was plotted as abs versus time for each sample. Two points (T1 and T2) were chosen in the linear range of the plot, and the corresponding values of absorbance were obtained (A1 and A2). The slope was calculated for all samples (S), inhibition control (IC) and enzyme control (EC) by dividing the net ΔA (A2 - A1) values with the time ΔT (T2 - T1). The percentage inhibition of this assay was calculated by Eq. (3):
$$\text{E}\text{n}\text{z}\text{y}\text{m}\text{e} \text{i}\text{n}\text{h}\text{i}\text{b}\text{i}\text{t}\text{i}\text{o}\text{n} \text{a}\text{c}\text{t}\text{i}\text{v}\text{i}\text{t}\text{y} \left(\text{%}\right)=\frac{\text{S}\text{l}\text{o}\text{p}\text{e} \text{o}\text{f} \text{E}\text{C}-\text{S}\text{l}\text{o}\text{p}\text{e} \text{o}\text{f} \text{S}}{\text{S}\text{l}\text{o}\text{p}\text{e} \text{o}\text{f} \text{E}\text{C}}\times 100 \left(3\right)$$
2.11. Cell culture
Human dermal fibroblasts (HDF) and immortalized human keratinocytes (HaCaT) were cultured in Dulbecco's Modified Eagle Medium (DMEM; Sigma-Aldrich), supplemented with 10% Fetal Bovine Serum (FBS; Thermo Fischer) and 1% antibiotic at 37 ºC, with 5% CO2 in a humidified atmosphere.
Human monocytes THP-1 (ATCC TIB-202) were cultured in Roswell Park Memorial Institute (RPMI; Thermo Fischer) culture medium, supplemented with 10% FBS, 1% antibiotic, and 50 mM beta-mercaptoethanol, at the same conditions than HDF and HaCaT cells.
2.12. Cytotoxicity
Cytotoxicity of fermentation extracts was evaluated using PrestoBlue™ Cell Viability assay kit (Invitrogen), according to the manufacturer´s instructions. HDF and HaCaT cells were seeded at 1 × 104 cells/well in 96-well plates and incubated over-night to allow cells to adhere. Cells were then exposed to the fermentations extracts at desired concentrations (80, 40, 20, 10, 5, and 2.5 mg.mL− 1), in DMEM, for 24h, at 37 ºC, with 5% CO2 in a humidified atmosphere. Each sample dilution was tested in quadruplicate in two independent experiments. Briefly, 100 µL of sample, culture medium for positive control, or culture medium with 10% dimethyl sulfoxide (DMSO; Sigma-Aldrich) for negative control, were pipetted into each appropriate well. After 24h incubation, 10 µL of PrestoBlue™ Cell Viability Reagent (Invitrogen, A13262) were added to each well and the plate was left incubating at 37 ºC, with 5% CO2 in a humidified atmosphere, protected from the light, up to 3h. Finally, the fluorescence was recorded using a Synergy H1 microplate reader (BioTek, Vila Nova de Gaia, Portugal). Results are expressed in percentage of metabolic inhibition in comparison to positive control with an inhibition superior to 30% being considered cytotoxic in accordance with ISO 10993-5 standard.
2.13. Cytokeratin 14 quantification
HaCaT cells were seeded at \(2.5 \times {10}^{5}\) cells.mL− 1 (1 mL per well) in 12-well plates. Cells were exposed to the fermentation extract at desired concentration (15 mg.mL− 1 in DMEM), for 24h. Each sample was tested in duplicate in two independent experiments. Culture medium was used as negative control. After incubation, the growth medium was removed, and cells were washed twice with PBS. Cells were then harvested by the addition of ice-cold 1X cell extraction buffer PTR (Human Cytokeratin 14 SimpleStep ELISA® Kit, ABCAM, ab226895) directly to the plate and were mechanically scrapped into a microfuge tube. Cell lysates were incubated on ice for 15 minutes, centrifuged at 18000 x g for 20 minutes at 4 ºC and supernatants were collected. Total protein was quantified by the BCA method using the Pierce™ BCA Protein assay kit (Thermo Scientific), according to the manufacturer´s instructions. For cytokeratin 14 (CK14) quantification, 25 ng of total protein were used. CK14 abundance was determined by ELISA using the Human Cytokeratin 14 SimpleStep ELISA® Kit (ABCAM, ab226895), according to manufacturer’s instructions.
2.14. Collagen I α1 quantification
HDF cells were seeded at \(2.5 \times {10}^{5}\) cells.mL− 1 (1 mL per well) in 12-well plates. Cells were exposed to the fermentation extract at desired concentration (6 mg.mL− 1 in unsupplemented DMEM), for 24h. Unsupplemented DMEM was used, as the presence of FBS is reported to inhibit collagen synthesis [41]. Each sample was tested in duplicate in two independent experiments. Cells with only media and with palmitoyl tetrapeptide-3 (GenScript, New Jersey, USA) were used as negative and positive controls, respectively. Total protein quantification was performed as described in the previous section. For collagen I α1 quantification, 100 ng of total protein were used. Collagen I α1 abundance was determined by ELISA, using the Human Pro-Collagen 1 alpha 1 CatchPoint® SimpleStep ELISA® Kit (ABCAM, ab229389), according to the manufacturer’s instructions.
2.15. Production of IL-6 by macrophages
THP-1 cells were seeded at \(3 \times {10}^{5}\) cells/well in 24-well microplates and differentiated into macrophages by treatment with 50 nM of phorbol 12-myristate 13-acetate (Sigma-Aldrich), for 48 h. Cells were exposed to the fermentations extracts (10 and 1 mg.mL− 1) for 24h, in the presence or absence of lipopolysaccharides form E. coli O111:B4 (LPS, Sigma-Aldrich) to induce inflammation. For anti-inflammatory control, macrophages were treated with 20 nM of betamethasone (Sigma-Aldrich). After 24h, supernatants were collected and the level of proinflammatory cytokine IL-6 was determined by ELISA, using the ELISA MAX™ Deluxe Set Human IL-6 kit (Biolegend), according to manufacturer’s instructions. For total protein quantification, cells were lysed with water, and BCA method was performed, as previously described. The results were expressed in pg of cytokine.ug− 1 of total protein.
2.16. Evaluation of the impact of extracts on skin microbiota
A study protocol for the evaluation of the effect of the developed extracts in skin microbiota modulation was established. This protocol was validated by the Commission of Ethics for Health of Universidade Católica Portuguesa before its execution. Additionally, the team members which proceed with the trials are certified with ICH good clinical practice E6 (R2) recognized by the Global Health Training Centre. Nine female volunteers without diagnosed dermatological diseases were included on the study. On the day of the collection, the selected volunteers could not perform any skincare routine. Facial skin microbiota was collected from those volunteers following the protocol of Carvalho et al. [42].
The microbial DNA was purified using the PureLink™ Microbiome DNA Purification Kit (Invitrogen) and it was quantified using the Qubit™ 1X dsDNA HS (High Sensitivity) Assay Kit (Invitrogen) according to the manufacturer’s instructions. For quantitative real-time PCR (qPCR), it was used universal and specific primers to quantify the total load of bacteria or fungi, and the relative abundance of specific microbial genera, and species [43–50].
qPCR reactions were prepared to a final volume of 10 µL, containing 1x NZYSupreme qPCR Green Master Mix (NZYtech, Lisbon, Portugal), 0.5 to 1 µM of forward and reverse primers (Integrated DNA Technologies, IDT, Heverlee, Belgium), 2 µL of Microbial DNA-Free Water (Qiagen, Hilden, Germany) and 1 µL of DNA. The qPCR was performed in a qTOWER³ G (Analytik-Jena, Germany) with the following conditions: 10 minutes at 95°C, followed by 40 cycles of denaturation at 95°C for 15 seconds and annealing/extension at 60°C for 1 minute. The amplification steps are followed by a melt dissociation step to check for nonspecific product formation. The samples were tested in triplicate.
The relative standard curve method was used to quantify the total microbial load and the specific microbial genus or species. To create standard curves, dilution series of known microbial CFU number were used to create a standard curve for each pair of primers, by plotting the log10 of each known CFU number in the dilution series against the determined threshold cycle (Ct) value. For each genus and species, the relative abundance was calculated by log10 ratio between the CFU number determined for the genus- or specie-specific assay and the CFU number determined for the universal assay. To reduce the inter-individuality, for each volunteer it was calculated a ratio between the condition test and its control condition (fold-difference or fold-change).
2.17. Statistical Analysis
For statistical analysis, it was used the IBM® SPSS® Statistics 26 software. Data was first analysed for normality distribution (Shapiro-Wilk test, n < 50, or Kolmogorov-Smirnov test, n > 50). Afterwards, a one-way ANOVA test (normal distribution) with Tukey’s HSD post hoc test, or a Kruskal-Wallis test (non-normal distribution) were applied to determine differences between more than two groups. In case of a two-group comparison, a student’s t-test (normal distribution) or a Mann-Whitney test (non-normal distribution) were performed. In general, the significance level was set at 0.05.