The long-term goal of this project is to develop a antibiofilm antibiofilm enzyme cocktail (EC) that can extract live microbes frombiofilms on food industry surfaces for collection and use in food safety pathogen detection systems improving their sensitivity and reliability. The central component of this system is the antibiofilm EC, and thus the major goal of this projectis to develop a product-ready prototypeECfrom the topECs from the Phase I project that effectivelyextractdiverse and relevant microbesfrom biofilms on typical food industry surfaces without significant loss of microbe viability, andin a form compatible with downstream detection systems. The following objectives will achieve this goal.Objective 1: Develop improvements in antibiofilm activity and finalize the Enzyme Cocktail(EC) protein composition. The biofilm assay protocol of Phase I will be refined to produce more standardized and consistent biofilms and improve cell release into supernatants. The top 3ECs from the Phase I project will be retested against the 7 biofilm models of PHI plus additional food industry relevant untested species using the optimized protocol. Finally, ECs will be tested against biofilms grown on 3 alternative surfacesObjective 2: Develop lead ECs that broadly disrupts bacterial biofilms with maximal viable bacteria release, in a product ready formulation. The lead ECs from TO1 will go through formulation optimization to convert the simple EC formulations into prototype products with increased activity and cell release, faster action, more stability, and product-like qualities. The antibiofilm activity and surface release of these optimized products and the original ECs will be re-assessed against the 4 most challenging model biofilms to gauge improved performance. Objective 3: Evaluate thelead EC ina multi-species biofilm model usingtwo downstream detection assays conducted byan independent laboratory. Finally, the lead EC from TO2 will be challenged by application to a more realistic multi-species biofilm model containing L. monocytogenes developed by an independent contract vendor using Association of Official Analytical Chemists (AOAC, a food industry certification provider) approved methods. For analysis, bacteria will be extracted from BioXpose treated or PBS-treated surfaces and analyzed using food industry utilized PCR-based and ELISA based detection products that identify L. monocytogenes.