Browsing by Author "Muthuraman S"

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Air-liquid interface biofilm formation of pseudomonads and the impact of traditional clean-in-place on biofilm removal
(Elsevier Ltd, 2026-02-28) Muthuraman S; Palmer J; Flint S
Pseudomonads are common psychrotrophic spoilage bacteria associated with dairy, poultry, and meat processing environments. They can multiply at low temperatures, 4–7 °C, producing thermostable spoilage enzymes. Pseudomonads form strong biofilms by producing higher EPS (Extracellular polymeric substances) at low temperatures. This study focused on the biofilm formation of pseudomonads at the air-liquid interface and their EPS removal. Two strong biofilm-forming isolates, (Pseudomonas lundensis) 3SM and (Pseudomonas cedrina) 20SM were allowed to form biofilms on stainless steel coupons in a CDC reactor under a continuous flow of nutrients at 4 °C over a week. The cell counts reached approximately 7.5 log CFU/cm2. The biofilms formed at the air-liquid interface showed more visible biofilms, polysaccharides, and higher cell counts than those submerged in liquid. Cleaning the biofilms using 1 % NaOH at 70 °C resulted in viable bacterial cells below the detection limit. However, residual material termed biofilm “footprints” was present after cleaning and were analysed with SEM and FTIR. The SEM observations showed tightly packed robust biofilm cells before cleaning. Coupons treated with 55 °C water showed an upper layer of degraded cells. After treatment with 70 °C NaOH, organic material was still visible under SEM. Based on the FTIR observations, the EPS extracted from the control and treated coupons showed that the amount of biomolecules reduced after cleaning with NaOH, but the footprints still existed. The biofilm footprints led to the early appearance of biofilms at the air-liquid interface compared to new coupons exposed to strong biofilm-forming isolates. Cleaning with caustic can eliminate the cells, but the EPS from biofilms of pseudomonads is not completely removed, resulting in a possibility of regrowth when the new inoculum is introduced.
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 J
Pseudomonas 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.
Enzymatic dispersion of pseudomonad biofilms grown at psychrotrophic temperature
(Elsevier Ltd on behalf of Institution of Chemical Engineers, 2026-01-01) Muthuraman S; Palmer J; Flint S
Pseudomonads are robust biofilm formers in psychrotrophic temperatures, which can cause spoilage in dairy, poultry, and meat processing. This study screened eleven isolates for the biofilm-forming ability using the Congo Red Assay (CRA) and the crystal violet assay. Two isolates, 3SM and 20SM, showed significantly higher EPS production, cellulose synthesis and cell count at 4ºC and were selected for the enzymatic dispersion. Mature biofilms formed on the stainless-steel surface for 72 h at 4°C were treated with laboratory enzymes (Proteinase-K, Cellulase, and DNase I) and commercial enzymes (formulated cleaners, EnduroZyme, DualZyme, and TriZyme). Compared to laboratory enzymes, commercial enzymes were efficient in dispersing the biofilms (EnduroZyme- 62 %, DualZyme- 42 %, and TriZyme-32 % of biofilm removal), which was confirmed by cell counts, crystal violet assay, and microscopic observations. However, none of the treatments resulted in complete biofilm dispersion. These findings highlight the resilience of psychrotrophic pseudomonad biofilms and underscore the need for improved enzymatic strategies tailored for cold-chain environments.
Extracellular polymeric substances- the real target in eradicating pseudomonad biofilms
(Elsevier Ltd, 2025-09-01) Muthuraman S; Palmer J; Flint S
Pseudomonads are common psychrotrophic spoilage bacteria associated with dairy, poultry, and meat processing environments. Pseudomonads can form robust biofilms at cold temperatures and produce thermostable spoilage enzymes. This review discusses the biofilm formation aspects of pseudomonads, such as biofilm formation at the air-liquid interface, psychrotrophic temperatures, and distinct EPS production. The components of the EPS produced by pseudomonads and the potential of pseudomonads as a public good provider to other bacteria within the biofilm are highlighted. The elimination strategies available, other than conventional CIP methods, were discussed. The elimination strategies either target the cells or the EPS. When the cells were removed completely, the remaining EPS footprints encouraged the robust regrowth of the biofilms and strategies targeting only the EPS, such as enzymes, led to multiple colonisation possibilities from the dispersed aggregates. Combining the cell and EPS targeting strategies would result in complete biofilm removal. However, the cost-effective production, rapid removal and safety on food matrices need to be considered while designing the control strategies of pseudomonad biofilm removal.
Sequential treatment of psychrotrophic pseudomonad biofilms with sodium hydroxide and commercial enzyme cleaners
(Elsevier Ltd, United Kingdom, 2026-04) Muthuraman S; Palmer J; Flint S
Pseudomonads 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.