Sodium hydroxide (naoh)

Oranges (Citrus sinensis) and orange peel bagasse were purchased from a local supermarket in Campinas (Sao Paulo, Brazil). Hesperidin standard was bought from Sigma-Aldrich (St. Louis, MA, USA) at analytical grade (>97%). Calcium(II) chloride, copper(II) acetate, sodium hydroxide, and hydrochloric acid were from Labsynth (Diadema, Sao Paulo, Brazil) at practical grade. Deuterated solvent (DMSO-d₆) was purchased from Sigma-Aldrich (St. Louis, MA, USA). Methanol and phosphoric acid for the UHPLC assays were analytical grade (Avantor “Performance Materials”, Mexico^®, Ecatepec de Morelos, Mexico). ABIL^® Care 85 (Bis-PEG/PPg-16/16 PEG/PPG16/16 Dimethicone; Caprylic/Capric Triglyceride) were sampled from the EVONIK^® group (Essen, Germany). Muru muru (Astrocaryum murumuru Seed Butter), Cupuaçu butter (Theobroma grandiflorum Seed Butter), and Andiroba oil (Carapa Guianensis Seed Oil) were sampled from Amazon oil industry; Glyceryl monostearate, Mineral oil, Carbopol ULTREZ 10, Triethanolamine, and Vit E-acetate (α-Tocopheryl acetate) were bought from Fagron^® (Rotterdam, The Netherlands).

The pieces were subjected to demineralization in EDTA solution, a solution containing 4.13% tritiplex^® III (Merck KGaA, Hessen, Germany) and 0.44% sodium hydroxide (Labsynth, São Paulo, Brazil) with weekly changes of the solution for a period of approximately 40 days. Subsequently, semi-serial coronal sections were performed, considering the central region of the defect with the aid of the Leica^® RM2245 semi-automatic microtome (Leica Biosystems, Wetzlar, Germany). Sections 5 µm thick (six slides with four sections each) were made for hematoxylin-eosin and Masson’s trichrome staining. Two evaluators previously calibrated and blinded in relation to the groups and periods performed the constant analyses in the methodology.

To verify the stability of BLIS against different temperatures and pH, the method described by Todorov and Dicks³² was used. To this end, BLIS were subjected to heat treatments (30, 50, 70 or 90 °C for 1 h; 121 °C for 15 min) and pH treatments adjusted to pH 2, 4, 6, 8 or 10 with 1 N NaOH and HCl; Labsynth, Diadema, Brazil) at 30 °C for 1 h. To evaluate the proteinaceous nature of BLIS, samples were subjected to 1% (w/v) trypsin, pepsin, papain or pancreatin (Inlab, Alamar Tecno Científica Ltda, São Paulo, Brazil) and incubated at 30 °C for 2 h. After this period, the stability of BLIS was verified using the diffusion agar technique against L. monocytogenes.

The melanin content was quantified as previously described [30] (link). The B16-F10 cells were seeded in 96-well plates (1×10⁴ cells.mL⁻¹), and after 24 h, they were treated with 0.5 mM Tyr (Sigma-Aldrich Germany) and 10 mM NH₄Cl (Labsynth Brazil) for 48 h. After incubation, a portion of the cells was centrifuged and suspended in 1 M NaOH (Labsynth Brazil). The other portion was maintained in PBS (8 g/L NaCl, 0.20 g/L KCl, 1.15 g/L Na₂HPO₄, and 0.2 g/L KH₂PO₄) for protein quantification. Both aliquots were lysed in a Branson Sonifier 450 (USA) at 20 W for 30 sec. The melanin was quantified by measuring the absorption at 470 nm in a Tecan Infinite 200M plate reader using a standard curve for commercial melanin (Sigma Aldrich Germany) [30] (link). The total protein content was determined using the Bradford method [31] (link). Melanin is expressed as µg of melanin/mg of protein.