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    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 S
    Natural 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.
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    Phenotypic properties and genotyping analysis of Bacillus cereus group isolates from dairy and potato products
    (Elsevier Ltd, 2021-04) Huang Y; Flint SH; Yu S; Ding Y; Palmer JS
    Bacillus cereus group (B. cereus sensu lato) are ubiquitously distributed in diverse environments. In this study, eight isolates including B. cereus, B. paranthracis and B. toyonensis species, from dairy and potato products, were assessed for biofilm formation, sporulation and genetic information including biofilm-related genes and toxin genes. The isolates varied in their ability to form biofilm (either at the stainless steel-liquid-air interface or floating pellicles). The amounts of biofilms of B. cereus s.l., were increased when incubated in agitation condition varied between isolates. Sporulation within the planktonic and biofilm modes of growth was compared, suggesting that biofilm is a favourable environment for B. cereus s.l. to form spores. Whole genome sequencing (WGS) was used to compare these B. cereus s.l. isolates. New sequence types (STs) of B. cereus were found in this study. Isolates that shared similar genomes had different biofilm-forming and sporulation abilities. Most of isolates tested, possessed biofilm-related genes. Different combinations of toxin-producing genes were identified in different isolates, with all isolates containing nhe while only some contained hbl and cytK. None of the food isolates contained the emetic ces gene. This study highlights the diversity of B. cereus s.l. in biofilm formation, sporulation and their genetic variables.
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    The spore formation and toxin production in biofilms of Bacillus cereus : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Technology at Massey University, Palmerston North, New Zealand
    (Massey University, 2021) Huang, Yiying
    Bacillus cereus (B. cereus) is a foodborne pathogen causing diarrhoea and emesis which are the consequences of enterotoxin and emetic toxin production, respectively. Sporulation and biofilm formation are used as survival strategies by B. cereus protecting cells from harsh environments. However, these survival strategies also make B. cereus more difficult to control in the food industry. The aim of this study is to investigate the spore formation and toxin production in the biofilm of B. cereus. In this study, higher sporulation and higher spore heat resistance were demonstrated in biofilms grown on stainless-steel (SS) compared to planktonic populations. The structure of coat in spores isolated from biofilms, the upregulated germination genes in planktonic cells and upregulated sigma factor B in biofilm cells are possible explanations for these observations. The levels of dipicolinic acid (DPA) did not affect the heat resistance of spores harvested from biofilms in this study. Haemolytic toxin (Hbl) was mainly secreted by cells into surrounding media while emetic toxin (cereulide) was associated with cells. Higher Hbl toxin was observed in the presence of biofilms grown on SS compared to either planktonic culture or biofilm grown on glass wool (GW) using the Bacillus cereus Enterotoxin Reverses Passive Latex Agglutination test (BCET-RPLA). This was supported by the significant (P < 0.05) increase in HblACD expression in biofilm cells on SS, using both real-time quantitative PCR (RT-qPCR) and RNA sequencing. The transcriptomic analysis also revealed that biofilms grown on SS had an upregulated secretion pathway, suggesting biofilms of B. cereus grown on SS are more pathogenic than planktonic cells. Unlike the Hbl toxin, cereulide was associated with biofilm cells/structures and attached to the biofilm-forming substrates including SS and GW used in this study. The expression of cerA and cerB was similar between biofilms and planktonic cells using RT-qPCR. This project highlights the importance of biofilms by B. cereus in food safety through the enhanced heat resistance of spores, the higher Hbl toxin production and attached cereulide toxin.