Vilcacundo Rubén, Méndez Pilar, Reyes Walter, Romero Herman, Pinto Adelita, Carrillo Wilman
Laboratory of Functional Foods, Faculty of Foods Sciences and Engineering, Technical University of Ambato, Av. Los Chasquis y Rio Payamino, Campus Huachi, Ambato 1801334, Ecuador.
Sciences & Education Faculty, Universidad Distrital, Bogotá 11021-110231588, Colombia.
Sci Pharm. 2018 Oct 30;86(4):E48. doi: 10.3390/scipharm86040048.
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 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 ( ATCC 25922). HEWL at 120 °C and pH 6.0 (1.0 mg/mL) inhibited 78.20% of the growth of . HEWL/DTT treatment for 4.0 h (1.0 mg/mL) inhibited 68.75% of the growth . 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 ( 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.
本研究的目的是通过热化学处理提高改性鸡蛋清溶菌酶（HEWL）对革兰氏阴性菌的抗菌谱。在15小时的培养试验中评估了热变性HEWL和化学变性HEWL对革兰氏阴性菌和革兰氏阳性菌的抗菌活性。通过在pH 6.0和pH 7.0下加热使HEWL变性，并使用DL-二硫苏糖醇（DTT）进行1.0、1.5、2.0和4.0小时的化学变性。采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）法对经热化学处理改性的HEWL进行表征。热变性HEWL的溶菌活性随着温度升高以及与化学试剂（DTT）孵育时间的增加而降低。改性HEWL中溶菌活性的丧失表明抗菌活性的作用机制不依赖于溶菌活性。对HEWL进行热化学处理能够产生寡聚体形式，并在更广泛的范围内提高抗菌活性。pH 6.0的热变性HEWL和化学变性HEWL扩大了HEWL对革兰氏阴性菌（ATCC 25922）的抗菌谱。120℃和pH 6.0（1.0mg/mL）的HEWL抑制了78.20%的[细菌名称未给出]生长。用HEWL/DTT处理4.0小时（1.0mg/mL）抑制了68.75%的[细菌名称未给出]生长。pH 6.0和pH 7.0的热变性HEWL以及化学变性HEWL（用DTT处理1.0、1.5、2.0和4.0小时）对革兰氏阳性菌（CECT 4491T）有活性。热变性和化学变性的HEWL导致细菌死亡并破坏细胞壁。对细胞膜进行荧光染料染色的LIVE/DEAD分析显示，与改性HEWL孵育后细菌的细胞膜受到扰动。使用电子显微镜观察到细胞壁被破坏。本研究获得的结果表明，pH 6.0的热变性HEWL和化学变性HEWL处理提高了HEWL对革兰氏阴性菌的抗菌活性。
