No abstract

Naturally occurring antimicrobial compounds could be applied as food preservatives to protect food quality and extend the shelf life of foods and beverages. These compounds are naturally produced and isolated from various sources, including plants, animals and microorganisms, in which they constitute part of host defense systems. Many naturally occurring compounds, such as nisin, plant essential oils, and natamycin, have been widely studied and are reported to be effective in their potential role as antimicrobial agents against spoilage and pathogenic microorganisms. Although some of these natural antimicrobials are commercially available and applied in food processing, their efficacy, consumer acceptance and regulation are not well defined. This manuscript reviews natural antimicrobial compounds with reference to their applications in food when applied individually or in combination with other hurdles. It also reviews the mechanism of action of selected natural antimicrobials, factors affecting their antimicrobial activities, and future prospects for use of natural antimicrobials in the food industry.

Over the last fifty years, microbiologists have developed reliable culture-based techniques to detect food borne pathogens. Although these are considered to be the "gold-standard," they remain cumbersome and time consuming. Despite the advent of rapid detection methods such as ELISA and PCR, it is clear that reduction and/or elimination of cultural enrichment will be essential in the quest for truly real-time detection methods. As such, there is an important role for bacterial concentration and purification from the sample matrix as a step preceding detection, so-called pre-analytical sample processing. This article reviews recent advancements in food borne pathogen detection and discusses future methods with a focus on pre-analytical sample processing, culture independent methods, and biosensors.

The need for pre-analytical sample processing prior to the application of rapid molecular-based detection of pathogens in food and environmental samples is well established. Although immunocapture has been applied in this regard, alternative ligands such as nucleic acid aptamers have advantages over antibodies such as low cost, ease of production and modification, and comparable stability. To identify DNA aptamers demonstrating binding specificity to Campylobacter jejuni cells, a whole-cell Systemic Evolution of Ligands by EXponential enrichment (SELEX) method was applied to a combinatorial library of FAM-labeled single-stranded DNA molecules. FAM-labeled aptamer sequences with high binding affinity to C. jejuni A9a as determined by flow cytometric analysis were identified. Aptamer ONS-23, which showed particularly high binding affinity in preliminary studies, was chosen for further characterization. This aptamer displayed a dissociation constant (K(d) value) of 292.8 +/- 53.1 nM with 47.27 +/- 5.58% cells fluorescent (bound) in a 1.48-microM aptamer solution. Binding assays to assess the specificity of aptamer ONS-23 showed high binding affinity (25-36%) for all other C. jejuni strains screened (inclusivity) and low apparent binding affinity (1-5%) with non-C. jejuni strains (exclusivity). Whole-cell SELEX is a promising technique to design aptamer-based molecular probes for microbial pathogens without tedious isolation and purification of complex markers or targets.

Human noroviruses (HuNoV) are the leading cause of acute viral gastroenteritis and an important cause of foodborne disease. Despite their public health significance, routine detection of HuNoV in community settings, or food and environmental samples, is limited, and there is a need to develop alternative HuNoV diagnostic reagents to complement existing ones. The purpose of this study was to select and characterize single-stranded (ss)DNA aptamers with binding affinity to HuNoV. The utility of these aptamers was demonstrated in their use for capture and detection of HuNoV in outbreak-derived fecal samples and a representative food matrix. SELEX (Systematic Evolution of Ligands by EXponential enrichment) was used to isolate ssDNA aptamer sequences with broad reactivity to the prototype GII.2 HuNoV strain, Snow Mountain Virus (SMV). Four aptamer candidates (designated 19, 21, 25 and 26) were identified and screened for binding affinity to 14 different virus-like particles (VLPs) corresponding to various GI and GII HuNoV strains using an Enzyme-Linked Aptamer Sorbant Assay (ELASA). Collectively, aptamers 21 and 25 showed affinity to 13 of the 14 VLPs tested, with strongest binding to GII.2 (SMV) and GII.4 VLPs. Aptamer 25 was chosen for further study. Its binding affinity to SMV-VLPs was equivalent to that of a commercial antibody within a range of 1 to 5 µg/ml. Aptamer 25 also showed binding to representative HuNoV strains present in stool specimens obtained from naturally infected individuals. Lastly, an aptamer magnetic capture (AMC) method using aptamer 25 coupled with RT-qPCR was developed for recovery and detection of HuNoV in artificially contaminated lettuce. The capture efficiency of the AMC was 2.5–36% with an assay detection limit of 10 RNA copies per lettuce sample. These ssDNA aptamer candidates show promise as broadly reactive reagents for use in HuNoV capture and detection assays in various sample types.