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Item 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 SPseudomonads 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.Item 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 SPseudomonads 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.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 Conditional synergy: Impact of nutrient abundance on multispecies biofilm formation and sanitizer tolerance(Elsevier Ltd, 2026-03-01) Pant K; Palmer J; Flint SBacteria exist in varying nutrient conditions and complex microbial consortia. Pseudomonas fluorescens, Staphylococcus aureus, and Listeria monocytogenes are commonly occurring biofilm-formers, share a similar nutritional niche, and have been isolated from common surfaces in multispecies environments. Biofilm properties, including biomass (O.D590 nm), cell concentration (log CFU/cm2), exopolysaccharide content (μg/cm2), structure, and sanitizer tolerance (sodium hypochlorite), were observed under varying nutrient (full-strength TSB and 10 % TSB) conditions on polystyrene surfaces for single and multispecies biofilm. The synergistic interactions between the bacteria in multispecies biofilm were found to be nutrient-dependent, with significantly higher (p < 0.05) biofilm formation, exopolysaccharide content, and sanitizer tolerance in high nutrient conditions (TSB) compared with low nutrient conditions (10 % TSB). The cell concentrations in the biofilm (single and multispecies) were found to be comparable between TSB and 10 % TSB. All three bacteria involved showed increased tolerance against sanitizers in the multispecies arrangement compared to their single-species counterparts, with significantly higher survival for L. monocytogenes (5.3 log CFU/cm2) in a multispecies biofilm compared to its single-species counterpart (2.3 log CFU/cm2). A positive correlation was observed between exopolysaccharide concentration and sanitizer tolerance. This study highlights the importance of taking multiple bacteria and their growth environment into account when understanding sanitizer response, as it varies in multispecies biofilm setups and according to nutrient availability.Item Shear stress adaptation of Listeria monocytogenes in mono and dual-species biofilms(Elsevier Ltd, 2025-12-01) Pant K; Palmer J; Flint SWhile the impact of stress on L. monocytogenes associated with food processing has been recognized in planktonic conditions, the available research overlooks the response of this pathogen in the multi-species biofilm, commonly found in food processing and manufacture. The objective of this study was to understand the effect of shear stress on L. monocytogenes in single and dual-species (with P. fluorescens) biofilm formed in a continuous turbulent flow system. In the single-species biofilm, L. monocytogenes was able to form a biofilm under the turbulent flow with cell concentration reaching 5.1 log CFU/cm2 after 48 h, where filamentous cells (27.7 μm in length) were observed. In contrast, there were no visible filaments in the dual-species biofilm, and L. monocytogenes cell concentration was significantly higher (p < 0.001) at 8.7 log CFU/cm2. The cells harvested from single-species L. monocytogenes biofilm formed under turbulent flow showed significantly (p < 0.001) lower motility and higher adhesion compared with cells harvested from planktonic and static conditions. Gene expression analysis showed significant (p < 0.001) downregulation of motB (motility), sigB (stress), and cell division (ftsX and ftsW), and upregulation of mpl (adhesion) and rodA (rod shape), indicating L. monocytogenes adaptation to shear stress. This study provides fundamental information on the multi-species biofilm formation by L. monocytogenes under stress.Item Extracellular polymeric substances- the real target in eradicating pseudomonad biofilms(Elsevier Ltd, 2025-09-01) Muthuraman S; Palmer J; Flint SPseudomonads 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.Item The relationship between pH, pigments production, and citrinin synthesis by Monascus purpureus during red fermented rice fermentation(Elsevier Ltd, 2025-09-01) Farawahida AH; Palmer J; Flint SRed fermented rice (RFR) is consumed as a traditional medicine in many countries. Consumption of food contaminated with citrinin (CIT) has adverse effects on the liver cells and the kidneys. This study aimed to understand the relationship between pH, pigments, and CIT levels during the fermentation of RFR. The rice underwent soaking, steaming, and autoclaving before being inoculated with Monascus purpureus isolates (MF1 or MS1) and fermented at 30 °C for 30 days. The difference between these isolates was that MF1 and MS1 required 4 days and 7 days, respectively, to produce light blue fluorescence on Coconut Cream Agar (CCA), an indicator of the presence of CIT. The pH, pigments, and CIT levels were recorded during fermentation using a pH meter, spectrophotometer, and Ultra-High-Performance Liquid Chromatography with a Fluorescence Detector (UHPLC-FLD), respectively. The pH dropped from 6.8 to 5.3 after 9 days of fermentation. The CIT levels increased from day 4 until day 5 and then decreased during the fermentation period. The pigments increased after 5 days of fermentation, suggesting a relationship between pigments production and CIT reduction. Mixing the CIT standards and pigments extracted from MF1 and MS1 (a mixture of yellow, orange, and red pigments) showed that there was a reduction in CIT of 26–68 % and 16−45 %, respectively. It is suggested that future work should determine which pigments are responsible for CIT reduction. The optimization of pigments production with the control of pH at 5.5 may help to control CIT levels during the fermentation of RFR.Item Characterization, antibacterial activity, and stability of supercritical fluid extracted lemongrass nanoemulsion on Bacillus cereus(Elsevier Ltd, 2025-06) Mohd Daud IS; Mahmud Ab Rashid NK; Palmer J; Flint SNatural food preservation is a sustainable approach to extend shelf life, combat foodborne pathogens and enhance food safety. Bacillus cereus, a resilient contaminant, poses challenges due to its spore-forming ability and association with foodborne illnesses. This study investigates the characterization, antimicrobial activity, and stability of lemongrass (Cymbopogon citratus) nanoemulsions, extracted using supercritical fluid extraction (SFE), and their efficacy against B. cereus isolates (ATCC 14579, P4, and M2). Lemongrass oil was extracted at 85, 100, 200, and 300 bar, with the highest yield (0.815 %) obtained at 300 bar. Nanoemulsions were formulated with lemongrass extract and commercial citral, characterized for droplet size, polydispersity index (PDI), conductivity, and zeta potential, and assessed for antimicrobial activity. Lemongrass nanoemulsions initially had droplet sizes of 86.32 ± 0.66 nm, but increased over six months due to coalescence, with PDI values rising from 0.50 ± 0.00 to 0.81 ± 0.27, indicating reduced stability. Although zeta potential declined from −44.01 ± 1.69 mV to −33.63 ± 1.45 mV, it remained within the stable range (>±30 mV), maintaining sufficient electrostatic repulsion to prevent rapid aggregation. At 2.0 % concentration, nanoemulsions effectively suppressed B. cereus isolates (<1.00 CFU/mL), though efficacy declined after four months with increasing droplet size. Lemongrass nanoemulsions exhibited comparable antibacterial activity and stability trends to citral, suggesting that whole lemongrass extract retains its bioactivity as effectively as its major compound. Improved stabilization strategies, such as polymer encapsulation, could enhance shelf life, expanding applications in food preservation.Item Multispecies biofilm cities and the importance of the order of colonization(Elsevier Ltd, 2025-09) Pant K; Palmer J; Flint SBiofilms are sessile communities of cells embedded in the extracellular matrix of polysaccharides formed to protect themselves from adverse conditions and are attached to the biotic/abiotic surfaces. In the natural environment, the probability of bacteria existing in multispecies is higher than the bacteria existing in isolation. The first step to a stable multispecies biofilm formation is the attachment and colonization of the surface by one or more bacteria. This review aimed to understand the impact of sequential attachment in overall multispecies biofilm formation, its role in defining biofilm properties, and the possible challenges it could present during the removal and disinfection process. In several cases, the highest biofilm former attached to the surface first resulting in a stronger biofilm which explains the enhanced resistance to removal in multispecies biofilm. Following the formation of a stable biofilm, environmental variables (e.g. temperature, surface, nutrient availability), and metabolic exchange between the bacteria drive the properties of biofilm, finally resulting in sequential detachment, driven by the predominant bacteria. These insights are vital in understanding biofilm formation and spatial layering of pathogenic bacteria for efficient biocontrol and removal.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.