The aim of this study was to increase the antibacterial spectrum of modified hen egg white lysozyme (HEWL) with thermal and chemical treatments against Gram-negative bacteria. The antibacterial activity of heat-denatured HEWL and chemical denatured HEWL against Gram-negative and Gram-positive bacteria was evaluated in 15 h of incubation tests. HEWL was denatured by heating at pH 6.0 and pH 7.0 and chemical denaturing was carried out for 1.0, 1.5, 2.0, and 4.0 h with DL-Dithiothreitol (DTT). HEWL modified by thermal and chemical treatments was characterized using the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis method. Heat-denatured HEWL lytic activity against Micrococcus lysodeikticus lessened with increasing temperature and time of incubation with the chemical agent (DTT). The loss of lytic activity in modified HEWL suggests that the mechanism of action of the antibacterial activity is not dependent on the lytic activity. Thermal and chemical treatments of HEWL enabled the production of oligoforms and increased antibacterial activity over a wider spectrum. Heat-denatured HEWL at pH 6.0 and chemically-denatured HEWL increased the HEWL antibacterial spectrum against Gram-negative bacteria (Escherichia coli ATCC 25922). HEWL at 120 °C and pH 6.0 (1.0 mg/mL) inhibited 78.20% of the growth of E. coli. HEWL/DTT treatment for 4.0 h (1.0 mg/mL) inhibited 68.75% of the growth E. coli. Heat-denatured HEWL at pH 6.0 and pH 7.0 and chemically-denatured HEWL (1.0, 1.5, 2.0, and 4.0 h with DTT) were active against Gram-positive bacteria (Staphylococcus carnosus CECT 4491T). Heat-denatured and chemical-denatured HEWL caused the death of the bacteria with the destruction of the cell wall. LIVE/DEAD assays of fluorescent dye stain of the membrane cell showed membrane perturbation of bacteria after incubation with modified HEWL. The cell wall destruction was viewed using electron microscopy. The results obtained in this study suggest that heat-denatured HEWL at pH 6.0 and chemical-denatured HEWL treatments increase the HEWL antibacterial activity against Gram-negative bacteria.

The aim of the study was to determine the profile of bioactive compounds in cocoa residues (mucilage and bean shells), and to evaluate their antioxidant activity in two cocoa varieties, Nacional X Trinitario type (Fine Aroma) and the variety CCN-51. The extraction of phytonutrients from the residues was carried out selectively. The characterization and quantification of the total polyphenol content (TPC), and the total flavonoid content (TFC) were determined by UV–VIS spectrophotometry. High-performance liquid chromatography (HPLC) was used to determine the phenolic profile and methylxanthines. The antioxidant activity was evaluated by the methods of 2-azinobis (3-ethyl-benzothiazoline-6-sulfonic acid) cation bleaching (ABTS), ferric-reducing antioxidant power (FRAP) and oxygen radical absorbance capacity (ORAC). The exudate mucilage samples from Nacional X Trinitario-type cocoa presented the highest content of TPC 105.08 mg gallic acid equivalents (GAE)/100 mL, TFC 36.80 mg catechin equivalents (CE)/100 mL, catechin (CAT) 35.44 mg/g, procyanidins (PCB2: 35.10; PCB1: 25.68; PCC1: 16.83 mg/L), epicatechin (EPI) 13.71 mg/L, caffeine (CAF) 0.90% and theobromine (TBR) 2.65%. In the cocoa bean shell, the variety CCN-51 presented a higher content of TPC (42.17 mg GAE/100 g) and TFC (20.57 mg CE/100 g). However, CAT (16.16 mg/g), CAF (0.35%) and TBR (1.28%) were higher in the Nacional X Trinitario cocoa type. The EPI presented no significant differences between the two samples studied (0.83 and 0.84 mg/g). The antioxidant activity values (ABTS, FRAP and ORAC methods) were higher in the samples of CCN-51 than in the Nacional X Trinitario type. The bean shell samples presented antioxidant values of 171.32, 192.22 and 56.87 mg Trolox equivalents (TE)/g, respectively, and the bean shell samples presented antioxidant values of 167.06, 160.06 and 52.53 mg TE/g, respectively. The antioxidant activity (ABTS, FRAP and ORAC) of the residues was correlated with the bioactive compounds of the mucilage and bean shells, showing a strong positive correlation (<0.99) with the procyanidins (B1, B2 and C1), EPI and CAT and a positive/moderate correlation (0.94) with methylxanthines.

Andean blackberries (Rubus glaucus Benth) are fruits rich in phytocomponents with high antioxidant activity. In this work, the changes in the total polyphenol content (TPC), the total flavonoid content (TFC), and the total anthocyanin content (TAC) of four blackberry varieties at three maturity stages (E1-25%, E2-50%, and E3-100%) were measured. The antioxidant activity (AA) was evaluated using the 2,2’azinobis-(3-ethylbenzthiazolin 6-sulphonic acid (ABTS) and ferric reducing antioxidant power (FRAP) methods. TPC and TFC content decreased with the increase in the maturity stage. The blackberry Brazos cultivar presented TPC values of 51.26, 38.16, and 31.59 mg of gallic acid equivalents (GAE)/g dry weight (DW) at E1, E2, and E3, respectively. The TAC and soluble solids increased with the increase in the maturity stage of the fruits. The Andimora variety at E3 presented a high TPC content, and the Colombiana variety presented a high TFC content. The blackberry Colombiana cultivar presented TAC values of 1.40, 2.95, and 12.26 mg cy-3-glu/100g DW at E1, E2, and E3, respectively. The blackberry Colombiana cultivar presented a high AA value at 1278.63 µmol TE/g DW according to the ABTS method and 1284.55 µmol TE/g DW according to the FRAP method. The TPC and TFC showed a high correlation with the AA according to the ABTS and the FRAP methods. The Pearson correlation between the TFC and AA/ABTS has a value of r = 0.92. The TFC and AA/FRAP present a value of r = 0.94.