Abstract Enzyme-based treatments could therefore be used to complement regular cleaning processes. Most studies using enzymes as anti-biofilms strategy are focused on their outcome in mono-species biofilms. Nevertheless, in real environments mixed biofilms are prevalent. In this work, seven types of dual-species biofilms were selected to serve as targets for enzymatic treatments carrying different environmental strains of L. monocytogenes and accompanying bacteria isolated from dairy, meat and seafood processing plants. The effectiveness of nine commercial enzymatic preparations, including pronase, cellulase, pectinase, DNase I, lysozyme, phospholipase, peroxidase, β-glucanase and chitinase, was evaluated. For this, residual attached viable cells of both L. monocytogenes and its partners were enumerated through swabbing and colony plate counting following the action of each enzyme. Moreover, Confocal Laser Scanning Microscopy (CLSM) images were analyzed pre and after enzymatic treatments in order to quantify changes in biofilm thickness, covered area and volume. The viable attached population of L. monocytogenes was almost unaffected by all of the enzymes here tested, being eliminated on average just the 90% of the initially attached population (around 1 Log10 cfu cm−2 reduction). Nevertheless, some of the partner species (Escherichia coli and Staphylococcus saprophyticus) were sometimes protected from enzymatic detachment when in dual-species biofilms, depending on the enzyme tested and the accompanying L. monocytogenes strain. CLSM images showed important changes in biofilm covered area and volume after DNase I, pronase and pectinase treatments. These results demonstrate that enzymes can greatly weaken dual-species biofilms structure. Nevertheless, it cannot be disregarded that detached cells from these treatments would still be viable. Thus, a control of cell viability after an enzymatic procedure in the food industry must be always considered before designing an efficient disinfection treatment.

中文翻译：

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携带单核细胞增生李斯特菌和其他相关食品工业细菌的双物种生物膜的酶促分散

摘要 因此，基于酶的处理可用于补充常规清洁过程。大多数使用酶作为抗生物膜策略的研究都集中在它们在单一物种生物膜中的结果。然而，在真实环境中，混合生物膜很普遍。在这项工作中，选择了七种双物种生物膜作为酶处理的目标，这些生物膜携带不同环境菌株的单核细胞增生李斯特菌和从乳制品、肉类和海鲜加工厂分离的伴随细菌。评估了九种商业酶制剂的有效性，包括链霉蛋白酶、纤维素酶、果胶酶、DNase I、溶菌酶、磷脂酶、过氧化物酶、β-葡聚糖酶和几丁质酶。为此，两种 L 的残留附着活细胞。在每种酶的作用后，通过拭子和菌落平板计数对单核细胞增生李斯特菌及其伙伴进行计数。此外，共聚焦激光扫描显微镜 (CLSM) 图像在酶处理前后进行分析，以量化生物膜厚度、覆盖面积和体积的变化。单核细胞增生李斯特菌的可行附着种群几乎不受此处测试的所有酶的影响，平均仅消除了最初附着种群的 90%（减少约 1 Log10 cfu cm-2）。然而，根据测试的酶和伴随的单核细胞增生李斯特氏菌菌株，一些伙伴物种（大肠杆菌和腐生葡萄球菌）有时在双物种生物膜中不会被酶促分离。CLSM 图像显示在 DNase I、链霉蛋白酶和果胶酶处理后生物膜覆盖面积和体积发生了重要变化。这些结果表明酶可以大大削弱双物种生物膜结构。然而，不能忽视的是，从这些治疗中分离出来的细胞仍然是可行的。因此，在设计有效的消毒处理之前，必须始终考虑在食品工业中的酶促过程后对细胞活力的控制。