| Summary: | Rapid, sensitive and selective detection and identification of pathogens is required in the prevention and recognition of problems related to food security. Salmonella is one of the dangerous foodborne pathogens. The identification of specific volatile organic compounds (VOCs) produced by Salmonella may contribute in providing a fast and accurate detection method for Salmonella in food samples. In this study, VOCs liberated by Salmonella strains have been identified and quantified via head space-solid phase microextraction coupled to gas chromatography/mass spectrometry (HS-SPME GC/MS). The dominant chemical class of volatiles liberated from Salmonella strains was alcohol compounds. In addition, ester and ketone compounds were also detected. The most sensitive VOCs detected were ethyl octanoate (LOD = 62.0 ng/mL and LOQ = 207 ng/mL) and ethyl decanoate (LOD = 66 ng/mL and LOQ = 219 ng/mL) with the lowest LOD and LOQ when using Rappaport-Vassiliadis Soya peptone (RVS) broth media and polar SPME fiber with polar GC column. The type of culture medium was found to affect the liberated VOCs. For example, 2-heptanone was not detected when S. london and S. stanley were grown in TSB but they were detected and quantified when using BHI as growth media. Also, 1-octanol was detected and quantified in all strains when Salmonella grown in TSB and BHI, and did not detected in all strains when RVS was used as growth media. The research has been extended to include the addition of specific enzyme substrates to the culture medium (RVS). The enzyme substrates are either commercially available or have been synthesised to allow exogenous VOC detection. The specific enzymes targeted in Salmonella were α-galactosidase, C-8 esterase and pyrrolidonyl peptidase. The enzyme substrates used are phenyl α-D-galactopyranoside, 2-chlorophenyl octanoate and L-pyrrollidonyl fluoroanilide respectively. All, except pyrrolidonyl peptidase, are known to give a positive response to Salmonella. This developed methodology was initially applied to pure cultures of S. stanley to evaluate the feasibility of the approach. The developed approach shows potential for future application in food samples to detect and identify Salmonella species in food samples of a level as low as 100 CFU/mL within a 5 h incubation at 37 ºC by the detection of the liberated VOCs. Subsequently the methodology was applied to a range of food samples (milk, cheese, eggs and chicken). It was found that all food samples were Salmonella free; however, false positive was detected due to the presence of other pathogens in the food samples. Inhibition of some of these pathogens in milk and cheese samples was achieved with the addition of 5 mg/L vancomycin and 10 mg/L of novobiocin. To improve the method specificity, it was necessary to deviate from the standard method and use Salmonella selective RVS broth in pre-enrichment step than using non selective one (BPW). This results in a successful detection of Salmonella contamination on milk samples and cheddar cheese samples. However, failed in detect Salmonella in other cheeses. Inhibition of resistant pathogens (Streptococcus salivarius ssp. Thermophilus, Lactobacillus rhamnosus and Enterococcus faecalis) using another combination of selective agents (vancomycin 10 mg/L, novobiocin 10 mg/L, erythromycin 0.75 mg/L and lithium chloride 15 g/L) failed. This study highlighted the benefits of the use of specific enzyme substrates along with antibiotics into Salmonella VOC analysis to improve the specificity of Salmonella detection method. The results of VOC analysis of specific enzymes inherent within Salmonella could be extended to develop a selective portable sensor approach to be used in food production.
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