Ahlborn Gene, Sheldon Brian W
Department of Food Science, North Carolina State University, Raleigh 27695, USA.
J Food Prot. 2006 Apr;69(4):729-38. doi: 10.4315/0362-028x-69.4.729.
The biological activity (D-value determination) of eggshell membrane (ESM) was examined to determine the membrane components and mechanisms responsible for antibacterial activity. Biological and enzymatic activities (i.e., beta-N-acetylglucosaminidase [beta-NAGase], lysozyme, and ovotransferrin) of ESM denatured with trypsin, lipases, or heat were compared with those of untreated ESM. Trypsin-treated ESM lost all biological activity (D-values at 54 degrees C were 5.12 and 5.38 min for immobilized and solubilized trypsin, respectively) but showed no significant loss of enzymatic activities. Treatments with porcine lipase and a lipase cocktail did not impact biological or enzymatic activities. Heat denaturation of ESM (at 80 and 100 degrees C for 15 min) resulted in significant decreases in biological activity (D-values of 3.99 and 4.43 min, respectively) and loss of beta-NAGase activity. Lysozyme and ovotransferrin activities remained but were significantly reduced. Purified ESM and hen egg white components (i.e., beta-NAGase, lysozyme, and ovotransferrin) were added to Salmonella Typhimurium suspensions (in 0.1% peptone water) at varying concentrations to evaluate their biological activity. D-values at 54 degrees C were 4.50 and 3.68 min for treatment with lysozyme or beta-NAGase alone, respectively, and 2.44 min for ovotransferrin but 1.47 min for a combination of all three components (similar to values for ESM). Exposure of Salmonella Typhimurium cells to a mixture of ovotransferrin, lysozyme, and beta-NAGase or ESM resulted in significant increases in extracellular concentrations of Ca2+, Mg2+, and K+. Transmission electron microscopic examination of Salmonella Typhimurium cells treated with a combination of ovotransferrin, lysozyme, and beta-NAGase revealed membrane disruption and cell lysis. The findings of this study demonstrate that ovotransferrin, lysozyme, and beta-NAGase are the primary components responsible for ESM antibacterial activity. The combination of these proteins and perhaps other ESM components interferes with interactions between bacterial lipopolysaccharides, sensitizing the outer bacterial membrane to the lethal affects of heat and possibly pressure and osmotic stressors.
对蛋壳膜（ESM）的生物活性（D值测定）进行了检测，以确定负责抗菌活性的膜成分和机制。将经胰蛋白酶、脂肪酶或加热变性的ESM的生物活性和酶活性（即β-N-乙酰氨基葡萄糖苷酶[β-NAGase]、溶菌酶和卵转铁蛋白）与未处理的ESM进行比较。经胰蛋白酶处理的ESM失去了所有生物活性（固定化胰蛋白酶和可溶性胰蛋白酶在54℃时的D值分别为5.12和5.38分钟），但酶活性没有显著损失。用猪脂肪酶和脂肪酶混合物处理对生物活性或酶活性没有影响。ESM的热变性（在80℃和100℃下15分钟）导致生物活性显著降低（D值分别为3.99和4.43分钟）以及β-NAGase活性丧失。溶菌酶和卵转铁蛋白活性仍然存在，但显著降低。将纯化的ESM和鸡蛋清成分（即β-NAGase、溶菌酶和卵转铁蛋白）以不同浓度添加到鼠伤寒沙门氏菌悬液（在0.1%蛋白胨水中）中，以评估它们的生物活性。在54℃时，单独用溶菌酶或β-NAGase处理的D值分别为4.50和3.68分钟，卵转铁蛋白为2.44分钟，但三种成分组合时为1.47分钟（与ESM的值相似）。将鼠伤寒沙门氏菌细胞暴露于卵转铁蛋白、溶菌酶和β-NAGase或ESM的混合物中导致细胞外Ca2+、Mg2+和K+浓度显著增加。对经卵转铁蛋白、溶菌酶和β-NAGase组合处理的鼠伤寒沙门氏菌细胞进行透射电子显微镜检查，发现膜破裂和细胞裂解。本研究结果表明，卵转铁蛋白、溶菌酶和β-NAGase是负责ESM抗菌活性的主要成分。这些蛋白质以及可能的其他ESM成分的组合会干扰细菌脂多糖之间的相互作用，使细菌外膜对热以及可能的压力和渗透应激源的致死作用敏感。
