Department of Chemical Engineering, University of Virginia, Charlottesville, Virginia, USA.
Department of Civil, Architectural, and Environmental Engineering, Drexel University, Philadelphia, Pennsylvania, USA.
Appl Environ Microbiol. 2021 Jun 11;87(13):e0026521. doi: 10.1128/AEM.00265-21.
Biofilm formation is often attributed to postharvest bacterial persistence on fresh produce and food handling surfaces. In this study, a predicted glycosyl hydrolase enzyme was expressed, purified, and validated for the removal of microbial biofilms from biotic and abiotic surfaces under conditions used for chemical cleaning agents. Crystal violet biofilm staining assays revealed that 0.1 mg/ml of enzyme inhibited up to 41% of biofilm formation by Escherichia coli O157:H7, E. coli 25922, Salmonella enterica serovar Typhimurium, and Listeria monocytogenes. Furthermore, the enzyme was effective at removing mature biofilms, providing a 35% improvement over rinsing with a saline solution alone. Additionally, a parallel-plate flow cell was used to directly observe and quantify the impact of enzyme rinses on E. coli O157:H7 cells adhering to spinach leaf surfaces. The presence of 1 mg/liter enzyme resulted in nearly 6-times-higher detachment rate coefficients than a deionized (DI) water rinse, while the total cells removed from the surface increased from 10% to 25% over the 30-min rinse time, reversing the initial phases of biofilm formation. Enzyme treatment of all 4 cell types resulted in significantly reduced cell surface hydrophobicity and collapse of negatively stained E. coli 25922 cells imaged by electron microscopy, suggesting potential polysaccharide surface modification of enzyme-treated bacteria. Collectively, these results point to the broad substrate specificity and robustness of the enzyme for different types of biofilm stages, solution conditions, and pathogen biofilm types and may be useful as a method for the removal or inhibition of bacterial biofilm formation. In this study, the ability of an engineered enzyme to reduce bacterial adhesion and biofilm formation of several foodborne pathogens was demonstrated, representing a promising option for enhancing or replacing chlorine and other chemical sanitizers in food processing applications. Specifically, significant reductions of biofilms of the pathogens Escherichia coli O157:H7, Salmonella Typhimurium, and Listeria monocytogenes are observed, as are reductions in initial adhesion. Enzymes have the added benefits of being green, sustainable alternatives to chemical sanitizers, as well as having a minimal impact on food properties, in contrast to many alternative antimicrobial options such as bleach that aim to minimize food safety risks.
生物膜的形成通常归因于收获后细菌在新鲜农产品和食品处理表面的持续存在。在这项研究中，一种预测的糖苷水解酶被表达、纯化并验证，用于在用于化学清洁剂的条件下从生物和非生物表面去除微生物生物膜。结晶紫生物膜染色测定表明，0.1mg/ml 的酶抑制了大肠杆菌 O157:H7、大肠杆菌 25922、肠炎沙门氏菌血清型 Typhimurium 和单核细胞增生李斯特菌生物膜形成的 41%。此外，该酶在去除成熟生物膜方面也很有效，与单独用生理盐水冲洗相比，生物膜去除率提高了 35%。此外，平行板流动池用于直接观察和量化酶冲洗对菠菜叶表面附着的大肠杆菌 O157:H7 细胞的影响。1mg/l 酶的存在导致附着系数比去离子（DI）水冲洗高近 6 倍，而在 30 分钟冲洗时间内，从表面去除的总细胞从 10%增加到 25%，逆转了生物膜形成的初始阶段。该酶处理所有 4 种细胞类型均导致细胞表面疏水性显著降低，并且电子显微镜下对负染大肠杆菌 25922 细胞的观察表明，酶处理细菌的多糖表面可能发生了修饰。总的来说，这些结果表明该酶对不同类型的生物膜阶段、溶液条件和病原体生物膜类型具有广泛的底物特异性和稳健性，可能作为一种去除或抑制细菌生物膜形成的方法很有用。在这项研究中，展示了一种工程酶降低几种食源性病原体粘附和生物膜形成的能力，这代表了增强或替代食品加工应用中氯和其他化学消毒剂的有前途的选择。具体而言，观察到病原体大肠杆菌 O157:H7、肠炎沙门氏菌和单核细胞增生李斯特菌的生物膜显著减少，并且初始粘附减少。与许多替代抗菌剂（如旨在最大限度降低食品安全风险的漂白剂）相比，酶具有作为化学消毒剂的绿色、可持续替代品的额外好处，并且对食品特性的影响最小。
