Fabrication, characterization, and application of pea protein-based edible film enhanced by oregano essential oil (OEO) micro- or nano-emulsion.

Pea protein isolate (PPI)-based active films were prepared by incorporating 0.5 %, 1.0 %, or 2.0 % of oregano essential oil (OEO), either in the form of micro-emulsion (MOEO) or nano-emulsion (NOEO). The particle size and polydispersity index of OEO droplets were 2755.00 nm and 0.63 for MOEO, and 256.30 nm and 0.20 for NOEO. The surface and cross-sectional SEM results revealed the presence of holes and internal pores within the film upon the addition of OEO. The molecular interaction between PPI and OEO was confirmed by FTIR. The addition of OEO significantly increased film thickness, decreased water contact angle, and imparted a more yellow color. At a low concentration (0.5 %), the addition of OEO significantly improved the water vapor barrier and mechanical properties of the film. However, at higher concentrations, these film properties were significantly weakened. Additionally, the film antimicrobial properties were assessed after OEO addition. In vitro inhibition zone results indicated that a 2.0 % addition of OEO significantly suppressed the growth of three Salmonella strains [Salmonella Typhimurium (ATCC14028), Salmonella Infantis 94-1, and Salmonella Enteritidis PT-30]. Application of pea protein-based film with 2.0 % OEO on chicken breast demonstrated significant reduction in microbial count. Our results further showed that reducing the particle size of OEO from micrometer-scale to nanometer-scale in the PPI film matrix did not significantly alter film properties or antimicrobial activities. The study demonstrated that the antibacterial film based on pea protein and OEO is an innovative food packing material for prohibiting bacteria growth on poultry products.

Real-Time PCR High-Resolution Melting Assays for the Detection of Foodborne Pathogens.

Real-time PCR high-resolution melting assays are a method for the identification of single nucleotide polymorphisms (SNPs). The assay is performed by amplifying a short DNA fragment using a specific primer pair flanking a target SNP in the presence of a high-resolution melting dye. The HRM analysis of amplicons groups the samples based on the differences in the melting temperature and the shape of the melt curves, facilitating a convenient genotyping of samples. This chapter describes the steps and considerations of real-time PCR HRM assay standardization.

Rapid PCR-lateral flow assay for the onsite detection of Atlantic white shrimp.

The Atlantic white shrimp (Litopenaeus setiferus) is of great economic importance to the United States and risk being substituted with imported species due to a shortage in domestic production. To improve the current methods used for the identification of the Atlantic white shrimp species, we designed and validated a robust multiplex PCR-lateral flow assay for the onsite identification of L. setiferus. The standardized assay was validated using a miniaturized, low-cost PCR instrument with 68 shrimp, prawn, and fish samples, spread over fourteen seafood species. L. setiferus was simultaneously amplified by the multiplex assay to give three visual bands, which distinguished it from other species having either one or two bands on the dipstick. The standardized assay showed 100% inclusivity for target L. setiferus samples, 100% exclusivity for non-target samples and can be completed in less than two hours. The assay standardized in this study can be used for onsite testing of L. setiferus samples at processing facilities, restaurants, and wholesalers' facilities.

Universal hydrolysis probe-based approach for specific detection and genotyping of foodborne pathogens.

Real-time PCR assays are the method of choice for the specific detection of DNA targets. Multiple real-time PCR chemistries are used for developing pathogen detection assays. Among them, a hydrolysis probe is a preferred choice for pathogen detection assays. Two known limitations of hydrolysis probes are high cost and limited storage life. Therefore, this study aimed to develop and validate a universal hydrolysis probe (UHP)-based approach with high-resolution melt (HRM) analysis capabilities. The approach can be used for the detection and genotyping of target DNA. The approach described in this study was validated by standardizing nine UHP assays for detecting seven Shiga toxin-producing Escherichia coli serogroups, Listeria monocytogenes, and Salmonella strains. These nine assays were validated with 141 pure culture bacterial strains. Additionally, the HRM capability of the developed approach was validated for three UHP assays targeting E. coli O26, O111, and O121 using 96 DNAs isolated from enriched food samples. The nine assays specifically detected the target bacterial strains, and the three assays showed single nucleotide polymorphism (SNP) identification capability and no cross-reactivity with non-target strains. The developed approach can be performed in singleplex or multiplex format and combined with HRM analysis. The data from this study demonstrate that the UHP real-time PCR approach is a robust method for detecting any deoxyribonucleic acid target.

Iron-enriched Aspergillus oryzae as an alternative to iron sulphate to limit iron accumulation, growth and motility of the enteric pathogen S. Typhimurium.

Excess unabsorbed iron in the gastrointestinal tract may select for enteric pathogens and increase the incidence and severity of infectious disease. Aspergillus oryzae (Ao) is a filamentous fungus that has the ability to accumulate and store large amounts of iron, and when used as a supplement or fortificant, has similar absorption to ferrous sulphate (FeSO4) in humans. The objective of this study was to determine the effect of iron-enriched Ao (Ao iron) compared with FeSO4 on iron accumulation, growth and motility of the Gram-negative enteric pathogen, S. Typhimurium. S. Typhimurium was cultured in media containing no added iron or 1 µM elemental iron as either Ao iron or FeSO4. S. Typhimurium cultured with FeSO4 accumulated more iron than those cultured with Ao iron. Genes regulated by the iron-activated transcriptional repressor, Fur, did not differ between control and Ao iron, but decreased in S. Typhimurium cultured with FeSO4 compared with both groups. Growth of S. Typhimurium was greater when cultured with FeSO4 compared with Ao iron and control. S. Typhimurium swam faster, had greater acceleration and travelled further when cultured with FeSO4 compared with Ao iron and control; swim speed, acceleration and distance travelled did not differ between Ao iron and control. These findings provide evidence that Ao iron reduces the virulence of a common enteric pathogen in vitro. Further research is required to determine whether iron-enriched Ao is a suitable iron supplement to improve iron delivery in areas with a high infection burden.