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Item Sequential treatment of psychrotrophic pseudomonad biofilms with sodium hydroxide and commercial enzyme cleaners(Elsevier Ltd, United Kingdom, 2026-04) Muthuraman S; Palmer J; Flint SPseudomonads are psychrotrophic spoilage bacteria that can form biofilms at the air-liquid interface. Food processing utensils and equipment often facilitate the air-liquid interface biofilm formation. Pseudomonads produce thermostable enzymes and pigments that affect the organoleptic quality of perishable food products. In this study, Pseudomonas lundensis, Pseudomonas cedrina were allowed to form biofilms at 4 °C under continuous flow of nutrients in a CDC reactor (CBR 90; Biosurface Technologies, USA). The mature biofilms were treated with commercial enzyme cleaners, EnduroZyme (protease), DualZyme (protease and lipase), and TriZyme (protease, amylase, and cellulase). The dispersion with EnduroZyme was significantly (p < 0.05) higher than the other enzyme cleaners. Then the biofilms were treated with hot water and sodium hydroxide, and enzyme cleaners (sequential treatment). The cell counts after sodium hydroxide + Enzyme cleaners were below the detection limit. The microscopic observations with epifluorescence microscopy showed that the coupons had less fluorescence after the sequential treatment. FTIR observations showed that the extracellular polymeric substances (EPS) isolated after sodium hydroxide + enzyme cleaners differed from the untreated and sodium hydroxide-only-treated EPS. Biofilm regrowth was significantly (p < 0.05) lower in the biofilms treated with sodium hydroxide + EnduroZyme compared to acid-treated control coupons. The sequential treatment with sodium hydroxide and enzyme cleaners reduced the biofilm footprints, representing a better clean than enzyme treatment alone or sodium hydroxide-only cleaning.Item Characterization of the extracellular polymeric substances matrix of Pseudomonas biofilms formed at the air-liquid interface(Elsevier Ltd, 2025-01-27) Muthuraman S; Flint S; Palmer JPseudomonas are common psychotropic food spoilage organisms that affect the quality of aerobically chilled food products. Biofilm formation of these bacteria on food contact surfaces can provide a continuous contamination source, leading to food spoilage. Pseudomonas produce proteolytic and lipolytic enzymes which lead to organoleptic degradation of stored food products. The biofilm extracellular polymeric substances matrix (EPS) protects the bacterial cells from CIP (Cleaning-In-Place) chemicals and adverse conditions. Studies on the composition of the EPS matrix and the molecules present in the EPS matrix are limited. In this study, the EPS composition of mono-species biofilms of Pseudomonas lundensis and Pseudomonas cedrina on polystyrene and stainless-steel surfaces was characterized by chemical analysis and microscopical observations. The biofilms were allowed to grow on polystyrene and stainless-steel surfaces with half-strength TSB for 2 weeks at 30 °C and cold chain temperatures of 7 °C and 4 °C. The EPS was extracted by sonication and centrifugation and chemically analysed for cellulose, total polysaccharides, total proteins, and eDNA. Pseudomonas isolates in this study formed biofilms at the air-liquid interface. The formation of ring-like structures of cells was observed on the polystyrene surface. eDNA formed as a thread-like structure on a polystyrene surface while it formed channels on a stainless-steel surface. The amount of EPS varied at different temperatures. More EPS was formed at 4 °C than 30 °C. Flagellin, Clp protease, Arginine deiminase, and ATP-Binding Cassette (ABC) transporter substrate-binding proteins were the key proteins identified in the biofilm matrix of P. lundensis.