The spoilage characteristics of Brochothrix thermosphacta and two psychrotolerant Enterobacteriacae in vacuum packed lamb and the comparison between high and low pH cuts

The spoilage potential of Brochothrix thermosphacta, Serratia proteamaculans and Rahnella aquatilis was investigated in vacuum packaged high (5.9 to 6.4) and low (5.4 to 5.8) pH lamb. Vacuum packaged fore shank (m. extensor carpi radialis) and striploins (m. longissimus dorsi) (n = 306) inoculated with ~ 100 CFU of individual bacteria were stored for twelve weeks at temperatures − 1.5, 0, 2 and 7 °C. Spoilage characteristics and bacterial numbers were recorded and analysed in comparison to un-inoculated control samples. All three bacterial species were shown to grow in vacuum packaged lamb of pH values between 5.4 and 6.4, when stored at chilled temperatures (− 1.5 to 7 °C) for up to 84 days. B. thermosphacta and S. proteamaculans caused spoilage to the meat under these conditions whilst R. aquatilis spoiled high pH meat at 7 °C. These results go against previous beef models stipulating that Brochothrix and Enterobacteriacae species cannot grow on or cause spoilage of low pH meat in the absence of oxygen.     

Development and validation of a real-time PCR assay specific for Clostridium estertheticum and C. estertheticum-like psychrotolerant bacteria

A new real-time PCR assay was developed targeted to the psychrotolerant spoilage bacteria, Clostridum estertheticum, a causative agent of 'blown-pack' spoilage of vacuum packaged meats during chilled storage. Further, a robust validation of the sensitivity and specificity in different meat processing related matrices was carried out. Results show that real-time PCR is a valid method for the detection of C. estertheticum spores as long as consideration is given to the matrix being tested and the sensitivity of detection required. For meat, hide, blood/drip and environmental swabs it was possible to detect low numbers of C. estertheticum spores (approx 3 spores per ml) by direct real-time PCR (without pre-enrichment of the samples). For faeces and soil matrices, a cold temperature enrichment step was required prior to DNA extraction and real-time PCR analysis to increase the ability to detect samples containing C. estertheticum spores; this was particularly important when the samples contained low numbers of spores (less than 3 spores per ml). For matrices with high levels of PCR inhibitors such as soil, it was necessary to dilute the extracted DNA sample 100 fold especially for detection of high levels of contamination (greater than 10(3)per ml) otherwise a pre-enrichment was required.     

Reduction of spoilage of chilled vacuum-packed lamb by psychrotolerant clostridia

Methods for the reduction of spoilage, of lamb, by psychrotolerant clostridia were investigated including exposure to air, hot and cold water spray washing and tyndallisation. Initially vegetative cells of psychrotolerant clostridia associated with spoilage of chilled vacuum-packed meat were exposed to aerobic cooked meat medium at room temperature (21 °C) to determine how long they remained viable. Survival of strains varied from 2 h to 3 days. Vegetative cells of Clostridium estertheticum subsp. estertheticum survived 7 days at 10 °C with little reduction in viable numbers. This ruled out exposure to air as a practical method for reducing spoilage. Trials were also carried out on chilled vacuum-packed lamb inoculated with spores of Cl. estertheticum subsp. estertheticum. The time until inoculated packs reached the loss of vacuum stage varied from 38 to 53 days. Hot and cold water washing extended the shelf life by 12 to 13 days in comparison to untreated packs.     

Total and pathogenic Vibrio parahaemolyticus in shrimp: Fast and reliable quantification by real-time PCR

Vibrio parahaemolyticus is found in aquatic environments and is the leading cause of gastroenteritis due to seafood consumption worldwide. We evaluated a quantitative real-time PCR (Q-PCR) assay with hydrolysis probes, to determine whether this method could be used for the efficient counting of total, tdh and trh-positive V. parahaemolyticus in shrimps. We assessed the specificity of this assay, using 62 strains from 12 non target bacterial species of the Vibrio, Photobacterium, Shewanella and Aeromonas genera. Only V. parahaemolyticus with the appropriate target gene generated a fluorescent signal. We assessed the robustness of this assay, by analyzing spiked shrimps by Q-PCR and traditional culture methods, using most probable number (MPN)-PCR. After a 6 h enrichment period, the assay successfully detected total and pathogenic V. parahaemolyticus in shrimps samples spiked with less than 5 V. parahaemolyticus cells/g of shrimp. The Q-PCR results obtained were compared with those obtained by most probable number (MPN)-PCR format. An excellent correlation between the two methods was observed in all cases (R² > 0.9742). The application of this Q-PCR assay to 85 natural shrimp samples also resulted in the successful quantification of V. parahaemolyticus in this matrix, and the counts obtained were correlated with those obtained by (MPN)-PCR (P = 0.2598). Thus, this rapid and sensitive Q-PCR can be used to quantify V. parahaemolyticus in natural shrimp samples. This assay could be proposed, in response to the demands of the European Commission, as a tool for testing for the presence of vibrios in crustaceans, making it possible to legislate in this domain.     

Taqman real-time PCR for the detection and enumeration of Saccharomyces cerevisiae in wine

Saccharomyces cerevisiae is the main yeast species responsible for wine fermentation; however, its presence during maturing or barrel-ageing can sometimes result in a reduction in the quality of wine by refermentation. In this work, we developed a quantitative real-time PCR (QPCR) for the rapid detection and quantification of S. cerevisiae in wine. The primers and the hydrolysis probe (TaqMan^®) were designed from the sequence of a DNA fragment present only in S. cerevisiae and absent in other wine yeasts obtained from an RAPD-PCR analysis. The QPCR developed was highly reproducible, allowing the specific detection and quantification of this yeast in artificially contaminated wines, with a detection limit of 78 CFU/mL. Furthermore, the usefulness of the QPCR developed was evaluated through the quantification of the yeast in wine samples obtained from vineyards, confirming the quantitative capacity of the method. The methodology developed was specific, fast and a sensitive tool for the detection and enumeration of S. cerevisiae cells in wine.     

A filtration-based real-time PCR method for the quantitative detection of viable Salmonella enterica and Listeria monocytogenes in food samples

We developed a novel filtration-based method that can eliminate dead or severally damaged Salmonella enterica and Listeria monocytogenes in food samples. This new method can recover all viable bacteria in less than 30 min, and can be coupled with a subsequent bacterial DNA extraction and real-time PCR. No statically significant differences (p < 0.01) were found between real-time PCR results obtained separately from S. enterica and L. monocytogenes when different ratios of living and dead cells were used. The analytical sensitivity in both cases was 1 genome equivalent (GE), and the quantification was linear (R² > 0.9969) over a 5-log dynamic range with PCR efficiencies >0.9754. When compared with the standard microbiological methods for the detection of these foodborne pathogens, the relative accuracy was excellent ranging from 95.72% to 104.48%. Finally, we applied the pre-treatment method to the direct detection of viable forms of these foodborne pathogens in food samples using yogurt as a model, the results being similar to those obtained using pure cultures.     

Quantitative PCR for the specific quantification of Lactococcus lactis and Lactobacillus paracasei and its interest for Lactococcus lactis in cheese samples

The first objective of this work was to develop real-time quantitative PCR (qPCR) assays to quantify two species of mesophilic lactic acid bacteria technologically active in food fermentation, including cheese making: Lactococcus lactis and Lactobacillus paracasei. The second objective was to compare qPCR and plate counts of these two species in cheese samples. Newly designed primers efficiently amplified a region of the tuf gene from the target species. Sixty-three DNA samples from twenty different bacterial species, phylogenetically related or commonly found in raw milk and dairy products, were selected as positive and negative controls. Target DNA was successfully amplified showing a single peak on the amplicon melting curve; non-target DNA was not amplified. Quantification was linear over 5 log units (R² > 0.990), down to 22 gene copies/μL per well for Lc. lactis and 73 gene copies/μL per well for Lb. paracasei. qPCR efficiency ranged from 82.9% to 93.7% for Lc. lactis and from 81.1% to 99.5% for Lb. paracasei. At two stages of growth, Lc. lactis was quantified in 12 soft cheeses and Lb. paracasei in 24 hard cooked cheeses. qPCR proved to be useful for quantifying Lc. lactis, but not Lb. paracasei.     

Development and validation of a novel real-time PCR method for the detection of celery (Apium graveolens) in food

The paper presents a novel real-time PCR method allowing the detection of traces of celery (Apium graveolens) in complex food matrices. The method is based on the amplification of a sequence of the gene coding for the Apium graveolens NADPH-dependent mannose-6-phosphate reductase. It allows the detection of three commonly used celery varieties, celery roots (Apium graveolens var. rapaceum), celery stalks (Apium graveolens var. dulce) and leaf celery (Apium graveolens var. secalinum) and does not show any cross-reactivity with 64 biological species, including ten members of the Apiaceae family. The limit of detection, determined by analysing serially diluted celery extracts, is 10 pg celery DNA for all three celery varieties. In spiked model sausages, the LOD is 0.005% celery. The real-time PCR method was applied to 26 commercial food products. Celery DNA was found in one out of ten samples without any information about the presence of celery.     

Authentication of Atlantic salmon (Salmo salar) using real-time PCR

A real-time PCR assay based on LNA TaqMan probe technology was developed for the detection and identification of Atlantic salmon (Salmo salar). Among the advantages it is worth highlighting simplicity, rapidity, highest potential for automation and minor risk of contamination of this technique. The TaqMan real-time PCR is the currently most suitable method for screening, allowing the detection of fraudulent or unintentional mislabelling of this species. The method can be applied to all kind of products, fresh, frozen and processed products, including those undergoing intensive processes of transformation.     

Development and evaluation of aptamer magnetic capture assay in conjunction with real-time PCR for detection of Campylobacter jejuni

A prototype method for the concentration and detection of Campylobacter jejuni was developed using a previously reported biotinylated DNA aptamer in conjunction with qPCR. The so-called aptamer-based magnetic capture-qPCR (AMC-qPCR) assay was compared to a similar immunomagnetic separation (IMS)-qPCR assay. In small volume experiments (300 μl) applied to serially diluted C. jejuni suspended in buffer containing a mixed culture of other common food borne pathogens, the lower detection limit of the AMC-qPCR method was 1.1 log₁₀/300 μl C. jejuni cells, one log₁₀ better (lower) than that of IMS-qPCR (2.1 log₁₀ CFU/300 μl). AMC-qPCR capture efficiency was 10–13% at assay detection limit. In 10 ml scale-up experiments, the lower detection limit of AMC-qPCR was 2.0 log₁₀ CFU/10 ml with corresponding capture efficiency of 4–7%. Nucleic acid aptamers are promising alternatives to antibodies for magnetic bead-based capture followed by qPCR detection.     

Influence of different shrinking temperatures and vacuum conditions on the ability of psychrotrophic Clostridium to cause 'blown pack' spoilage in chilled vacuum-packaged beef

This study determined the ability of psychrotrophic Clostridium strains isolated from vacuum-packaged beefs and abattoir environments to cause 'blown-pack' spoilage of vacuum-packaged beef stored at 2 and 15°C. The influence of shrinking temperatures (83, 84 and 87°C) and vacuum pressure (6 and 9mbar) on the occurrence of such spoilage as well as the effects of simulated transportation (500km) on the integrity of packages was determined. At 15°C and 2°C, twelve and six strains caused 'blown-pack' spoilage, respectively. The combination of vacuum pressure (9mbar) combined with shrinking temperature (87°C) retarded the occurrence of spoilage. The simulated transportation under the experimental conditions did not affect the integrity of packages. More studies that assess the factors that may contribute for the occurrence of 'blown-pack' spoilage should be performed to avoid the occurrence of such spoilage during its shelf-life.     

Rapid detection of Listeria monocytogenes in food using culture enrichment combined with real-time PCR

A rapid method for the detection of Listeria monocytogenes in foods combining culture enrichment and real-time PCR was compared to the ISO 11290-1 standard method. The culture enrichment component of the rapid method is based on the ISO standard and includes 24 h incubation in half-Fraser broth, 4 h incubation in Fraser broth followed by DNA extraction and real-time PCR detection of the ssrA gene of L. monocytogenes. An internal amplification control, which is co-amplified with the same primers as the L. monocytogenes DNA, was also included in the assay. The method has a limit of detection of 1–5 CFU/25 g food sample and can be performed in 2 working days compared to up to 7 days for the ISO standard. A variety of food samples from retail outlets and food processing plants (n = 175) and controls (n = 31) were tested using rapid and conventional methods. The rapid method was 99.44% specific, 96.15% sensitive and 99.03% accurate when compared to the standard method. This method has the potential to be used as an alternative to the standard method for food quality assurance providing rapid detection of L. monocytogenes in food.     

Quantitative detection of Listeria monocytogenes in raw milk and soft cheeses: Culture-independent versus liquid- and solid-based culture-dependent real time PCR approaches

A culture-independent and two culture-dependent real time (rt-) PCR approaches were developed to quantitatively identify Listeria monocytogenes in raw milk and soft cheeses. The optimised rt-PCR revealed 100% inclusivity and exclusivity. DNA- and cell-based standard curves showed a good linearity of response (R² ≥ 0.987 and R² ≥ 0.998, respectively) for five orders of magnitude (39 × 10⁵ – 3 × 10⁰ genome equivalents and 10⁶–10¹ CFU equivalents, respectively) with about 100% relative accuracy and inter-assay variability ≤0.90%. Up to 1 genome equivalent/and 10 CFU/reaction were quantified in the DNA and cell standard curves, respectively. The rt-PCR was then combined with a liquid- (MPN technique) or a solid- (ALOA and PALCAM) based enumeration. The diagnostic sensitivity of the different approaches was investigated in artificially contaminated raw milk and soft cheeses. The rt-PCR culture-independent approach performed well in raw milk and (with a lower sensitivity) in stracchino cheese-based standard curves. MPN/rt-PCR was the best approach to enumerate low levels of L. monocytogenes in raw milk and stracchino cheese, while the ALOA-based rt-PCR quantification was more effective than the PALCAM-based. These performances were confirmed when 23 real samples of raw milk and soft cheeses by both the rt-PCR approaches were assayed.     

Rapid and reliable identification of Staphylococcus aureus harbouring the enterotoxin gene cluster (egc) and quantitative detection in raw milk by real time PCR

A TaqMan and a SYBR Green real time PCR (rt-PCR) were developed for the reliable identification and quantitative detection of Staphylococcus (S.) aureus strains harbouring the enterotoxin gene cluster (egc) regardless of its variants. Both approaches revealed 100% specificity against a panel of 70 reference strains, including 29 clinical and foodborne S. aureus strains harbouring all the egc variants to date known, 4 egc⁻S. aureus strains and 37 strains of phylogenetically closely and distantly related species. Standard curves made by 10 fold dilutions of either genomic DNA or cells from an egc⁺S. aureus log-phase broth culture showed a good linearity of response (R² ≥ 0.993) for six orders of magnitude, with about 100% relative accuracy and a low inter-assay variability (CV ≤ 3.02). The overall limit of quantification (LOQ) for both rt-PCR assays (about 100% PCR efficiency; running time 30 min) was 10 cfu or 10 genome equivalents per reaction mixture although 1 cfu or 1 genome equivalent was detected with a 33.33% probability. These performances were confirmed in raw milk artificially contaminated with log-phase broth cultures of either a single egc⁺S. aureus strain or a mixture of S. aureus strains harbouring all the egc variants to date known. Similar results were also obtained with a raw milk based standard curve of the S. aureus egc⁺ mixture in the presence of 10⁶ cfu/mL of egc⁻S. aureus strains harbouring some of the commonest enterotoxin genes associated to the staphylococcal food poisoning. Nonetheless, the TaqMan based approach resulted in a lower sensitivity (LOQ = 100 cfu equivalents per reaction mixture) than the SYBR Green based assay (LOQ = 10 cfu equivalents per reaction mixture). When applied to real milk samples, both PCR assays provided a good response with 100% diagnostic specificity and 96-107% relative accuracy, as compared to conventional culture-based PCR approaches. Due to the high specificity, the wide dynamic range of detection and the high sensitivity demonstrated even in a complex and potentially highly contaminated raw milk matrix, the SYBR Green rt-PCR assay is a useful diagnostic tool for quick, high throughput and reliable routine screening of egc⁺S. aureus isolates. Moreover, the SYBR Green based quantitative detection of these pathogens in raw milk could remarkably contribute to clarify their actual role in staphylococcal food poisoning and other clinical syndromes associated with the consumption of milk and milk-based products.     

Development and validation of a rapid real-time PCR based method for the specific detection of Salmonella on fresh meat

In this study, a combined enrichment/real-time PCR method for the rapid detection of Salmonella on fresh meat carcasses, was designed, developed and validated in-house following requirements outlined in ISO 16140:2003. The method included an 18 h non-selective enrichment in buffered peptone water (BPW) and a 6 h selective enrichment in Rappaport Vasilliadis Soya (RVS) broth, based on the traditional culture method, ISO 6579:2002. The real-time PCR assay included an internal amplification control (IAC), was 100% specific and was sensitive to one cell equivalent. The alternative method was validated against the traditional culture method and relative accuracy of 94.9%, sensitivity of 94.7% and specificity of 100% were determined using 150 fresh meat carcass swabs. This alternative method had a detection limit of 1–10 CFU/100 cm² for fresh meat carcass swabs and was performed in 26 h. Following further inter-laboratory studies, this alternative method could be suitable for implementation in testing laboratories for the analysis of carcass swabs.     

Multiplex conventional and real-time PCR for fish species identification of Bianchetto (juvenile form of Sardina pilchardus), Rossetto (Aphia minuta), and Icefish in fresh,marinated and cooked products

A conventional and a realtime multiplex PCR were developed to detect fraudulent substitutions of Bianchetto (juvenile form of Sardina philcardus) and Rossetto (Aphia minuta) with Icefish (Neosalanx spp.), which show similar morphological characteristics. Since it is the major by-catch species, Engraulis encrasicolus was also included in the analytical procedure. A common reverse primer and forward species-specific primer were designed on the mitochondrial cytochrome b gene to amplify sequences of different lengths by conventional PCR. Specific peaks were therefore also provided after melting temperature analysis in real-time PCR, thus enabling each species to be clearly differentiated. The two PCR methods were validated on fresh and processed products after preparing four typical dishes: two marinades (from raw or lightly boiled fish), a pasta sauce, and batter-fried fish cakes. All samples were correctly identified, although there was some DNA degradation after processing.     

Survey of Listeria monocytogenes, Salmonella spp. and Escherichia coli O157:H7 in raw ingredients and ready-to-eat products by commercial real-time PCR kits

Real-time PCR (RTiPCR) assays including enrichment stage were evaluated for the rapid detection of Listeria monocytogenes, Salmonella spp. and Escherichia coli O157:H7 in raw ingredients and ready-to-eat products using molecular beacon probes available as commercial kits (WARNEX Genevision, Canada & AES Chemunex detection system, France). The accuracy of the assays was evaluated analyzing 1032 naturally contaminated food samples in combination to the conventional cultural methods. Presence/absence testing of the above pathogens was performed in 25 g samples of each product. In case of L. monocytogenes of 39 positive RTiPCR samples, 37 were confirmed by the cultural method (based on McNemar's test the difference between the two methods is insignificant). The highest incidence of L. monocytogenes in food products was found in desserts and the second highest in frozen pastries. None of the samples were cultural positive but negative in the RTiPCR test. One among the 343 investigated samples was positive for Salmonella spp. by RTiPCR and the cultural method. Out of 333 samples analyzed for E. coli O157:H7 no positive sample was detected. RTiPCR-based methods proved to be powerful tools for fast, sensitive and accurate pathogen detection in raw food ingredients and ready-to-eat products.     

Identification of two licorice species, Glycyrrhiza uralensis and Glycyrrhiza glabra, based on separation and identification of their bioactive components

The purpose of this study was to identify two licorice species, Glycyrrhiza uralensis and Glycyrrhiza glabra. A simple method including HPLC and LC/MS was developed to simultaneously separate, identify, and quantify 15 bioactive components of Glycyrrhiza species. G. uralensis was found to contain quercetin but G. glabra did not. G. glabra provided by Brion Research Institute of Taiwan contained uralsaponin B, but G. glabra brought from herbal shops did not. The DPPH radical scavenging activity of G. uralensis and G. glabra achieved approximately 72–75% if 10 mg/mL or more licorice extract was used. G. uralensis had slightly better radical scavenging activity than G. glabra. G. uralensis also showed higher reducing ability than G. glabra.     

Microbiological spoilage of vacuum and modified atmosphere packaged Vietnamese Pangasius hypophthalmus fillets

This study investigated the identity, growth and metabolite production of micro-organisms causing spoilage of Pangasius hypophthalmus fillets packaged in air, vacuum and modified atmospheres (MAP) (MAP 1: 50%CO(2)-50%N(2) and MAP 2: 50%CO(2)-50%O(2)) during storage at 4 °C. Based on the time it took for psychrotrophic total colony counts to exceed 7 log cfu g(-1), the shelf life of the fillets packaged in air, vacuum, MAP 1 and MAP 2 was estimated to be 7, 10, 12 and 14 days respectively. The longest lag phases were observed in the samples packaged in MAP 2 (50%CO(2)-50%O(2)). In the fillets packaged in air and under vacuum, the dominant flora identified by partial 16S rDNA sequencing at the end of the shelf life generally consisted of Gram-negative bacteria mostly belonging to the genera Serratia and Pseudomonas. In contrast, lactic acid bacteria (Carnobacterium maltaromaticum and Carnobacterium divergens) and Brochothrix thermosphacta were identified as the dominant spoilage flora in the samples packaged under the two MAPs investigated. By means of solid-phase microextraction gas chromatography mass spectrometry (SPME GC-MS) analysis, volatile organic compounds in the headspace of the samples at the end of the shelf life were identified for each packaging condition. Based on these results, a selective ion flow tube mass spectrometry (SIFT-MS) method was developed to quantify the production of volatile metabolites during storage of the fillets. The results of these analyses indicated that several compounds contributed to the bacterial spoilage of Pangasius fillets e.g., ethanol, 2,3-butanediol, diacetyl, acetoin, ethyl acetate, acetic acid and sulfur compounds. It also emerged that the production of these compounds was dependent on the packaging condition applied.     

Effect of sample preparation and bacterial concentration on Salmonella enterica detection in poultry meat using culture methods and PCR assaying of preenrichment broths

We evaluated the sensitivity of a PCR assay in the detection of Salmonella enterica at the broth preenrichment step of poultry meat. A total of 162 retail poultry meat samples, which were prepared by manual massaging, stomacher or no homogenization were compared for Salmonella recovery. Using these homogenization methods, the PCR assay at the broth preenrichment step detected Salmonella in, respectively, 48.9%, 62.2% and 50.0% of meat and giblet samples detected as Salmonella-positive using the culture method. In ground chicken, however, Salmonella was detected in 21.7% of samples treated by stomacher homogenization, compared to 40.7% and 48% of untreated and hand-massaged samples, respectively. These results suggest that stomaching of ground chicken causes excessive effusion of food constituents, which affects PCR results. Using the most probable number (MPN) technique, Salmonella was detected at under 1.0 CFU/g in 12 ground chicken samples and under 10³ CFU/ml of broth in seven of the 12 broth-enriched samples, which considered the minimum concentration detectable by PCR assay. These results show that Salmonella detection using routine PCR assays is difficult in poultry meat, and in particular ground chicken, due to low amounts of Salmonella and the presence of inhibitors.