Journal Articles
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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.Item Evaluation of single and multispecies biofilm formed in the static and continuous systems.(Elsevier B.V., 2024-12-19) Pant K; Palmer J; Flint SBiofilms consisting of multiple species of bacteria compared to biofilms of single species are common in natural environments including food contact surfaces. The objective of this study was to understand the biofilm formation and the efficiency of sodium hypochlorite (50 ppm/5 mins) on the single and multiple species biofilm of Pseudomonas fluorescens, Staphylococcus aureus, and Listeria monocytogenes formed on stainless steel surfaces in static and continuous systems. The cell concentration of Listeria in the dual and triple species biofilm in the continuous system (7.3-8.4 log CFU/cm2) was higher compared to the static system (4.7-4.9 log CFU/cm2) while the concentration remained consistent in the single species biofilm (6.4-6.7 log CFU/cm2) for both systems. Biofilm formed in the static system was significantly (p < 0.001) more susceptible to sodium hypochlorite than biofilm formed in the continuous system. This observation agrees with the exopolysaccharide concentration which was found to be higher in the continuous system (8.0-15.6 μg/cm2) than in the static system (3.2-6.3 μg/cm2) indicating a positive correlation between EPS production and sanitizer resistance. Epifluorescence microscopy images showed the formation of interstitial voids within the three-species biofilm and filaments in the single and dual species Listeria biofilms in the continuous system which were absent in the static system. Overall, results showed that the biofilm formation and sanitizer resistance vary due to multispecies interaction and the presence of flow and should be considered an important variable in multispecies sanitizer resistance studies.Item Addition of selected cereal grains as non-dairy ingredients to dairy products: A microbiological risk assessment approach(Elsevier Ltd., 2024-08-01) Fatima F; Pramularsih I; Kyere EO; Lindsay D; Abernethy G; Laing S; Flint SThe addition of cereal grains to dairy products in the dairy industry has the potential to contaminate final products with pathogenic and spoilage microorganisms. In this study, the microbial risks involved in the addition of cereal grains to dairy products with low, intermediate, and high-water activity/moisture content were assessed using a semi-quantitative risk assessment method. The results showed that the most critical microbiological hazard in the selected cereal grains is Bacillus cereus (B. cereus) due to its ability to form spores and persist in cereal grains. The addition of cereal grains to dairy products with high water activity/moisture content such as liquid breakfast products were found to pose the highest theoretical risk, and processing mitigations, such as UHT, would need to be implemented. The results of this study have identified some knowledge gaps in conducting risk assessments and have also provided background information about the microbial risks involved in the addition of cereal grains to dairy products.Item The effect of variations in cold plasma conditions on the detoxification of Aflatoxin M1 and degradation products(Elsevier B.V., 2024-10-16) Nguyen T; Palmer J; Pedley J; Petcu M; Newson HL; Keener K; Flint SThe aim of this study was to explore the chemical reactive species of different operating gases, and their effect on the degradation of aflatoxin M1 (AFM1) by cold plasma by measuring the reactive species concentration. Helium, at 80, 90 or 95%, was used mixed with oxygen, nitrogen and air. The efficacy of cold plasma on aflatoxin M1 (AFM1) reduction was improved when decreasing the ratio of helium in the gas mixture. The ratio of the gas mixtures changed the cold plasma chemistry believed to be due to the differences in the concentrations of the reactive species. The degradation products of AFM1 after cold plasma treatment using a helium/air gas mixture and the degradation pathway were identified by LCMS. AFM1 was oxidised by reactive species in the cold plasma to produce degradant products with, theoretically, lower toxicity than AFM1.