Identification and Quantification of Volatile Chemical Spoilage Indexes Associated with Bacterial Growth Dynamics in Aerobically Stored Chicken.

Volatile organic compounds (VOCs) as chemical spoilage indexes (CSIs) of raw chicken breast stored aerobically at 4, 10, and 21 °C were identified and quantified using solid phase microextraction (SPME) combined with gas chromatography-mass spectrometry (GC-MS). The growth dynamics of total viable count (TVC), psychrotrophs, Pseudomonas spp., lactic acid bacteria (LAB), Brochothrix thermosphacta and H2 S producing bacteria were characterized based on maximum growth rates (µmax ), maximal microbial concentration (Nmax ) and at the moment of microbial shelf life (Svalues ), calculated from Gompertz-fitted growth curves. Pseudomonas spp. was predominant species, while B. thermosphacta was characterized by the highest µmax . The microbiological and sensory shelf lives were estimated based on TVC, Pseudomonas spp., and B. thermosphacta counts and sensory evaluation, respectively. Among 27 VOCs identified by GC-MS in spoiled chicken samples, ethanol (EtOH), 1-butanol-3-methyl (1But-3M), and acetic acid (C2 ) achieved the highest Pearson's correlation coefficients of 0.66, 0.61, and 0.59, respectively, with TVC, regardless of storage temperature. Partial least squares (PLS) regression revealed that the synthesis of 1But-3M and C2 was most likely induced by the metabolic activity of B. thermosphacta and LAB, while EtOH was attributed to Pseudomonas spp. The increase in concentration of selected volatile spoilage markers (EtOH, 1But-3M, and C2 ) in the headspace over spoiled chicken breast was found to be statistically significant (P < 0.05) with TVC growth. These findings highlight the possibility of analyzing the combination of 3 selected spoilage markers: EtOH, 1But-3M, and C2 as rapid evaluation for poultry quality testing using SPME-GC-MS.

Evaluation of real-time PCR coupled with immunomagnetic separation or centrifugation for the detection of healthy and sanitizer-injured Salmonella spp. on mung bean sprouts.

Fresh mung bean sprouts have been identified as a source of many Salmonella outbreaks worldwide. The aim of this study was to develop a rapid and accurate detection methodology for low levels of healthy and sanitizer-injured Salmonella on mung bean sprouts using real-time PCR coupled with either immunomagnetic separation (PCR-IMS) or centrifugation (PCR-cen). Initially, three parameters of IMS; specificity/sensitivity, bacterial concentration and bead incubation time were optimized. Secondly, limit of detection (LOD) was determined for the optimized PCR-IMS and PCR-cen. These two methods were compared against PCR alone (PCR) and the standard culture method (ISO) for their ability to detect Salmonella using inoculated and uninoculated sprouts. Under optimum IMS conditions (10(5)CFU/ml for 30 min), capture efficiency of Salmonella in sprout suspensions was lower than 40%, most probably due to the non-specific binding of the background microbiota. PCR-IMS and PCR-cen had a similar LOD at 10(3)CFU/ml, which was one log unit lower than PCR. Enrichment of 10h was sufficient to detect 100% of the inoculated sprouts with both PCR-IMS and PCR-cen, which was significantly faster compared to PCR and the ISO method. Moreover, the validation study using uninoculated sprouts revealed that PCR-IMS and PCR-cen were equally effective on Salmonella detection, showing 98.3% accuracy. These results suggest that PCR-cen would be the effective and less costly method for the detection of both healthy and sanitizer-injured Salmonella on mung bean sprouts.

Volatile chemical spoilage indexes of raw Atlantic salmon (Salmo salar) stored under aerobic condition in relation to microbiological and sensory shelf lives.

The purpose of this investigation was to identify and quantify the volatile chemical spoilage indexes (CSIs) for raw Atlantic salmon (Salmo salar) fillets stored under aerobic storage conditions at 4, 10 and 21 °C in relation to microbial and sensory shelf lives. The volatile organic compounds (VOCs) were analyzed with SPME-GC-MS technique. Through multivariate chemometric method, hierarchical cluster analysis (HCA) and Pearson's correlations, the CSIs: trimethylamine (TMA), ethanol (EtOH), 3-methyl-1-butanol (3Met-1But), acetoin and acetic acid (C2) were selected from the group of 28 detected VOCs. At the moment of microbiological shelf life established at total viable count (TVC) of 7.0 log CFU/g, the CSIs achieved levels of 11.5, 38.3, 0.3, 24.0 and 90.7 µg/g of salmon for TMA, EtOH, 3M-1But, acetoin and C2, respectively. Pseudomonas spp. was found as major specific spoilage organism (SSOs), suitable for shelf life prediction using modified Gompertz model at the cut-off level of 6.5 log CFU/g. H2S producing bacteria and Brochothrix thermosphacta were considered as important spoilage microorganisms; however, they were not suitable for shelf life estimation. Partial least square (PLS) regression revealed possible associations between microorganisms and synthetized VOCs, showing correlations between Pseudomonas spp. and 3Met-1But and aldehydes synthesis, lactic acid bacteria were linked with EtOH, C2 and esters, and B. thermosphacta with acetoin formation.

Antibacterial effect and mechanism of high-intensity 405 ± 5 nm light emitting diode on Bacillus cereus, Listeria monocytogenes, and Staphylococcus aureus under refrigerated condition.

This study investigated the antibacterial effect of 405 ± 5 nm light emitting diode (LED) on Bacillus cereus, Listeria monocytogenes, and Staphylococcus aureus, and examined its antibacterial mechanism by determining the bacterial membrane and DNA damages. A 405 ± 5 nm LED illuminated the Gram-positive pathogens until 486 J/cm(2) at 4 °C. Weibull model was used to calculate reliable life (tR) to compare bacterial sensitivities to LED illumination. The membrane damage was determined by NaCl and LIVE/DEAD® assay, while comet assay and DNA ladder analysis were conducted to determine DNA degradation. The illumination resulted in 1.9, 2.1, and 1.0 log reductions for B. cereus, L. monocytogenes, and S. aureus at 486 J/cm(2), respectively. The comparison of tR values revealed that L. monocytogenes was identified as the most susceptible strain to LED illumination. The percentage of the bacterial sensitivity to NaCl remarkably increased in LED-illuminated cells compared to non-illuminated cells. Moreover, loss of membrane integrity was confirmed for LED-illuminated cells by LIVE/DEAD® assay, whereas no DNA breakage was indicated by comet assay and DNA ladder analysis. Thus, these findings suggest that the antibacterial effect of 405 ± 5 nm LED illumination on these pathogens might be due to physical damage to bacterial membrane rather than DNA degradation.

Growth of healthy and sanitizer-injured Salmonella cells on mung bean sprouts in different commercial enrichment broths.

The ability of nine commercial broths to enrich healthy and 90% sanitizer-injured Salmonella Typhimurium and Salmonella cocktail on mung bean sprouts was evaluated to select an optimum broth for detection. Results showed that S. Typhimurium multiplied faster and reached a higher population in buffered peptone water (BPW), Salmonella AD media (AD) and ONE broth-Salmonella (OB), compared with other broths. Healthy and 90% sanitizer-injured Salmonella at low concentrations increased by 4.0 log CFU/ml in these three broths. However, no Salmonella growth was observed in lactose broth (LB). Further investigation showed that during incubation, pH of LB dropped from 6.7 to 4.2, due to production of lactic (66 mM) and acetic acids (62 mM) by lactic acid bacteria that were identified as dominant microbiota in bean sprouts. Though no cell membrane damage was detected by propidium monoazide combined with real-time PCR, it was found that LB inhibited Salmonella growth, especially from low inoculum levels. This study suggests that in consideration of effectiveness and cost, BPW would be a suitable enrichment broth to use for isolating and detecting Salmonella on mung bean sprouts, while using LB might cause false negative results in Salmonella detection by either PCR or standard cultural method.

Evaluation of commercial kit based on loop-mediated isothermal amplification for rapid detection of low levels of uninjured and injured Salmonella on duck meat, bean sprouts, and fishballs in Singapore.

The objective of this study was to evaluate performance of the commercial kit based on loop-mediated isothermal amplification (LAMP) in comparison with the International Organization for Standardization method for detecting uninjured and sublethally injured Salmonella cells artificially inoculated at levels of 10(0) and 10(1) CFU/25 g on raw duck wing, raw mung bean sprouts, and processed fishballs. Injured cells were prepared by a heat treatment for duck wings and fishball samples and a chlorine treatment for bean sprout samples. Additionally, a validation study was performed on naturally contaminated food samples sold in Singapore. A total of 110 samples of each commodity were analyzed in this study. Regardless of inoculum levels, the detection by the commercial LAMP kit showed 100% sensitivity and specificity for both inoculated and uninoculated samples compared with the International Organization for Standardization method, with the exception of bean sprout samples. Only 20% of bean sprout samples inoculated with 10(0) CFU/25 g injured Salmonella cells were positive by using the commercial LAMP-based kit. However, all negative samples became positive following a secondary enrichment in Rappaport-Vassiliadis medium with soy broth or after concentration by centrifugation. These results suggest that secondary enrichment or centrifugation should be considered as an additional step to increase the sensitivity of the commercial LAMP-based kit with low numbers of injured target cells in samples with high background microflora (such as mung bean sprouts). The validation study also showed that the commercial LAMP-based kit provided 91% sensitivity and 95% specificity for naturally contaminated samples. Thus, this study demonstrates that the commercial LAMP-based kit might be a cost-effective method, as this system could provide rapid, accurate detection of both uninjured and injured Salmonella cells on raw duck wings, raw mung bean sprouts, and processed fishballs in less than 26 h.

Real-time PCR method combined with immunomagnetic separation for detecting healthy and heat-injured Salmonella Typhimurium on raw duck wings.

Conventional culture detection methods are time consuming and labor-intensive. For this reason, an alternative rapid method combining real-time PCR and immunomagnetic separation (IMS) was investigated in this study to detect both healthy and heat-injured Salmonella Typhimurium on raw duck wings. Firstly, the IMS method was optimized by determining the capture efficiency of Dynabeads(®) on Salmonella cells on raw duck wings with different bead incubation (10, 30 and 60 min) and magnetic separation (3, 10 and 30 min) times. Secondly, three Taqman primer sets, Sal, invA and ttr, were evaluated to optimize the real-time PCR protocol by comparing five parameters: inclusivity, exclusivity, PCR efficiency, detection probability and limit of detection (LOD). Thirdly, the optimized real-time PCR, in combination with IMS (PCR-IMS) assay, was compared with a standard ISO and a real-time PCR (PCR) method by analyzing artificially inoculated raw duck wings with healthy and heat-injured Salmonella cells at 10(1) and 10(0) CFU/25 g. Finally, the optimized PCR-IMS assay was validated for Salmonella detection in naturally contaminated raw duck wing samples. Under optimal IMS conditions (30 min bead incubation and 3 min magnetic separation times), approximately 85 and 64% of S. Typhimurium cells were captured by Dynabeads® from pure culture and inoculated raw duck wings, respectively. Although Sal and ttr primers exhibited 100% inclusivity and exclusivity for 16 Salmonella spp. and 36 non-Salmonella strains, the Sal primer showed lower LOD (10(3) CFU/ml) and higher PCR efficiency (94.1%) than the invA and ttr primers. Moreover, for Sal and invA primers, 100% detection probability on raw duck wings suspension was observed at 10(3) and 10(4) CFU/ml with and without IMS, respectively. Thus, the Sal primer was chosen for further experiments. The optimized PCR-IMS method was significantly (P=0.0011) better at detecting healthy Salmonella cells after 7-h enrichment than traditional PCR method. However there was no significant difference between the two methods with longer enrichment time (14 h). The diagnostic accuracy of PCR-IMS was shown to be 98.3% through the validation study. These results indicate that the optimized PCR-IMS method in this study could provide a sensitive, specific and rapid detection method for Salmonella on raw duck wings, enabling 10-h detection. However, a longer enrichment time could be needed for resuscitation and reliable detection of heat-injured cells.

Patterns of survival and volatile metabolites of selected Lactobacillus strains during long-term incubation in milk.

The focus of this study was to monitor the survival of populations and the volatile compound profiles of selected Lactobacillus strains during long-term incubation in milk. The enumeration of cells was determined by both the Direct Epifluorescent Filter Technique using carboxyfluorescein diacetate (CFDA) staining and the plate method. Volatile compounds were analysed by the gas-chromatography technique. All strains exhibited good survival in cultured milks, but Lactobacillus crispatus L800 was the only strain with comparable growth and viability in milk, assessed by plate and epifluorescence methods. The significant differences in cell numbers between plate and microscopic counts were obtained for L. acidophilus strains. The investigated strains exhibited different metabolic profiles. Depending on the strain used, 3 to 8 compounds were produced. The strains produced significantly higher concentrations of acetic acid, compared to other volatiles. Lactobacillus strains differed from one another in number and contents of the volatile compounds.