Application of the SureTect Detection Methods for <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> and <Emphasis Type="Italic">Listeria</Emphasis> spp. in Meat,Dairy, Fish,and Vegetable Products

The aim of this study was to evaluate the application of the Listeria monocytogenes and Listeria spp. SureTect detection methods for a rapid and sensitive detection in an ample range of food products: raw pork and poultry meat, Spanish chorizo, pate, smoked salmon, raw sheep milk cured cheese, and ready-to-eat lettuce salad. The combination of a 24-h enrichment in the 24 Listeria Enrichment Broth (LEB) coupled to a rapid bacterial DNA extraction and real-time polymerase chain reaction (RTi-PCR) using the specific SureTect methods detected down to 2–6 L. monocytogenes CFU per sample in less than 27 h on the food categories tested. Furthermore, the applicability of L. monocytogenes and Listeria spp. SureTect detection methods in real samples was assessed using 303 food samples, obtaining at least the same analytical performance as the international reference method ISO 11290-1.     

A New Multiplexed Real-Time PCR Assay to Detect <Emphasis Type="Italic">Campylobacter jejuni</Emphasis>, <Emphasis Type="Italic">C. coli</Emphasis>, <Emphasis Type="Italic">C. lari</Emphasis>, and <Emphasis Type="Italic">C. upsaliensis</Emphasis>

A new rapid method based on real-time PCR was developed to detect four thermophilic Campylobacter species (Campylobacter jejuni, Campylobacter coli, Campylobacter lari, and Campylobacter upsaliensis) in food samples. The assay targeted the bipA gene for C. upsaliensis and C. lari, whereas the gene encoding the ATP-binding protein CJE0832 was used to detect C. coli and C. jejuni. These genes were chosen for this assay due to their low variability and mutation rate at a species level. The multiplex PCR showed 100% inclusivity for all 25 thermophilic Campylobacter strains tested and 100% exclusivity for 38 non-targeted strains belonging to closely related species. The newly developed real-time PCR could detect down to 10² genomes/reaction and displayed efficiency above 97% for all species except for C. upsaliensis (90.1%). The method proved to be a reliable tool for food analysis, showing 100% sensitivity, 96% efficiency, and 92.45% specificity when validated against the gold standard method UNE-EN ISO 10272:2006 using 200 diverse food samples (meat, fish, fruits and vegetables, and raw milk). In artificially spiked samples, the detection limit of the method was 10 cfu/g in salad, 5 cfu/g in turkey meat, and 1 cfu/g in the rest of meat samples tested. Consequently, the newly designed molecular tool represents a quick and safe alternative to obtain reliable results concerning the presence/absence of the main thermophilic Campylobacter in any food sample.     

Analysis of the survival of <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> in food-grade lubricants

Listeria monocytogenes is a ubiquitously occurring Gram-positive bacterium, which is associated with raw ready-to-eat foods, in particular with smoked or graved salmon. Due to its tenacity and psychrotrophic growth properties, it may persist on the equipment and machinery used in food production. In this study, we analysed the survival of three L. monocytogenes strains in eight H1 lubricants, seven greases and one oil, applicable for food-processing machinery. None of the native lubricants contained Listeria spp. above the detection limit of 10³ cfu/g. In artificially contaminated lubricants, the viable counts of different L. monocytogenes strains decreased by more than 99.9% within 7 days, and the reduction rates were found to dependent on the composition of the respective lubricant as well as on the L. monocytogenes strain. Chemical binding of either benzoate or acetate to native grease 3 caused a similar anti-listerial activity, but a decreased effect was observed when stearic acid was used. When benzoate, acetate, salicylate or parabene was added at a concentration of 0.5% to native grease 1, similar anti-listerial effects were observed. The water content (0, 1 and 5%) and the age of the lubricant did not influence the results. Used lubricants originating from dismantled industrial machinery did not contain L. monocytogenes, though this microorganism was re-isolated from the lubricants following artificial contamination. Thus, there is no evidence that L. monocytogenes replicate or persist in the H1 lubricants used in this study.     

Irradiation Sensitivity of Planktonic and Biofilm-associated <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> and <Emphasis Type="Italic">L. innocua</Emphasis> as Influenced by Temperature of Biofilm Formation

The human pathogen Listeria monocytogenes forms biofilms that are relatively resistant to chemical sanitizing treatments. Ionizing radiation effectively inactivates planktonic Listeria, but no information is available on the relative efficacy of the process against biofilm-associated Listeria. The irradiation sensitivity of planktonic or biofilm cells was determined for L. monocytogenes ATCC 43256 and ATCC 49594 and a commonly used surrogate Listeria innocua ATCC 51742. Biofilms were formed on sterile glass slides incubated for 48 h at 22°C, 28°C, or 37°C. The cultures were gamma irradiated and the irradiation D ₁₀ value was calculated for each combination of isolate/culture/temperature. The effect of temperature of cultivation on the irradiation sensitivity of both planktonic cells and biofilm cells varied for each of the isolates. Depending on isolate and temperature, biofilm cells were equally sensitive or more sensitive (P < 0.05) to irradiation. D ₁₀ values overall tended to increase with temperature of cultivation for L. monocytogenes 49594 and L. innocua 51742, but tended to decrease with increasing temperature for L. monocytogenes 43256. The D ₁₀ values of the various culture/temperature combinations differed significantly among the isolates examined. Irradiation effectively eliminates both planktonic and biofilm-associated cells. The extent to which the biofilm habitat modifies the antimicrobial efficacy of irradiation is dependent on the specific isolate examined and the temperature at which it forms. This study is the first inquiry to show that biofilm Listeria cells are as sensitive or more sensitive to irradiation compared with planktonic cells and that this response is dependent on biofilm formation conditions.     

Polymerase chain reaction (PCR) for the detection of celery (<Emphasis Type="Italic">Apium graveolens</Emphasis>) in food

A method based upon polymerase chain reaction (PCR) for the detection of celery (Apium graveolens) in food was developed. The method involves DNA isolation by chaotropic or non-chaotropic solid-phase extraction and PCR with primers oriented to the sequence of the nuclear gene encoding mannitol dehydrogenase. The PCR method was shown to be specific for celery, producing a 279 bp fragment with four celery varieties and negative results with other species commonly present together with celery in food products (16 samples). The detection limit of PCR was 490–1530 pg DNA, which corresponds to 10² genome copies. When evaluated with model samples of celery in meat pâtés, a detection limit of 0.1% (w/w) was determined. When used to analyse food products from the market (dried vegetable seasonings, dehydrated bouillons), all four products declared to contain celery were correctly identified as positive and all three products in which celery was not declared were identified as negative.     

Sinapic acid derivatives in defatted Oriental mustard (<Emphasis Type="Italic">Brassica juncea</Emphasis> L.) seed meal extracts using UHPLC-DAD-ESI-MS<Superscript><Emphasis Type="Italic">n</Emphasis></Superscript> and identification of compounds with antibacterial activity

This study identified phenolic compounds from mustard seed meal and characterized their antibacterial activity. Phenolic compounds were extracted from defatted Oriental mustard (Brassica juncea L.) seed meal and characterized using ultra-high-performance liquid chromatography with diode array and electrospray ionization-mass spectrometric detection (UHPLC-DAD-ESI-MS ⁿ ). Sinapic acid and several sinapoyl conjugates were identified based on retention time, UV spectra, MS fragmentation pattern, and by comparison with the authentic sinapic acid reference substance. The crude extract and a purified phenolic fraction exhibited selective antibacterial effects against Gram-negative and Gram-positive spoilage bacteria including Staphylococcus aureus and Listeria monocytogenes; Lactobacillus plantarum was resistant. After alkaline hydrolysis, only sinapic acid could be detected, enabling quantification with the authentic reference substance. Alkaline hydrolysis released 2.66 ± 0.00 mg sinapic acid g⁻¹ dry matter defatted mustard seed meal. Minimum inhibitory concentrations of the hydrolyzed extract against Bacillus subtilis, Escherichia coli, L. monocytogenes, Pseudomonas fluorescens, and S. aureus were 0.1 g L⁻¹ or less. Growth of L. plantarum remained unaffected. Sinapic acid and sinapoyl esters are generally found in members of the Brassicaceae family. Methods for their fast identification will be useful in chemotaxonomic studies. The release of sinapic acid after alkaline hydrolysis not only allows for the quantification using the reference substances but also facilitates the standardization of the antibacterial activity of plant extracts for use as food preservative.     

Antimicrobial Activity of Vanillin and Mixtures with Cinnamon and Clove Essential Oils in Controlling <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> and <Emphasis Type="Italic">Escherichia coli</Emphasis> O157:H7 in Milk

The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of vanillin against Listeria monocytogenes Scott A and Escherichia coli O157:H7 was determined in tripticase soy broth (TSB), pH 7 and 6, incubated at 35 °C/24 h and in semi-skim milk incubated at 35 °C/24 h and 7 °C/14 days. The influence of the fat content of milk on the antimicrobial activity of vanillin was tested in whole and skim milk incubated at 7 °C/14 days. Mixtures of clove and cinnamon with vanillin were also evaluated in semi skim milk incubated at 7 °C. The MICs for L. monocytogenes were 3,000 ppm in TSB (pH 7) and 2,800 ppm in TSB (pH 6). The MICs for E. coli O157:H7 were 2,800 ppm in TSB (pH 7) and 2,400 ppm in TSB (pH 6). The MBCs in TSB were 8,000 ppm for L. monocytogenes and 6,000 ppm for E. coli O157:H7. The pH values assayed did not influence significantly the MIC or MBC in TSB. The MICs in semi-skim milk for L. monocytogenes and E. coli O157:H7 were 4,000 and 3,000 ppm at 35 °C/24 h, and 2,500 and 1,000 ppm at 7 °C/7 days, respectively. The MBCs were 20,000 ppm for L. monocytogenes and 11,000 ppm for E. coli O157:H7. High incubation temperatures did not affect the MBC but increased the MIC of the vanillin in milk. This effect could be attributed to the increased membrane fluidity and to the membrane perturbing activity of vanillin at low temperatures. The fat in milk reduced significantly the antimicrobial activity of vanillin, probably due to effect protective of the fat molecules. Mixtures of clove and cinnamon leaves inhibited the growth of L. monocytogenes in a similar way that vanillin alone but had a synergistic effect on the E. coli O157:H7. Mixtures of cinnamon bark and vanillin had always a synergistic effect and some of the combination assayed showed bactericidal activity on the population of L. monocytogenes and E. coli O 157:H7.     

Improved Recovery of Stressed <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> from Frozen Foods

The Canadian reference method for the enumeration of Listeria monocytogenes (Health Canada method MFLP-74) was modified by the addition of a layer of non-selective tryptone soy agar (TSA) to the Oxford, Palcam, and (or) Rapid’L.mono selective agars, to improve the recovery of stressed L. monocytogenes from frozen foods. The performance of the standard selective agars versus the selective agars with an additional TSA agar layer (TAL agars) showed that for each of the frozen food items tested (vegetables, shrimp and meatballs), as well as for all data combined, L. monocytogenes counts were significantly higher (up to 20% higher) in TAL agars in relation to the standard agars (p < 0.0001, α = 0.05). A selectivity assessment showed that the 50 inclusivity L. monocytogenes strains tested produced true positive reactions on the TAL agars, and the 30 exclusivity bacterial strains tested produced true negative results on the TAL agars, indicating that the specificity of the selective agars was not altered by the additional non-selective TSA layer. In light of the 2016 Listeria outbreak linked to frozen fruits and vegetables in the USA, the results of this method investigation seem to be timely and particularly important for the regulatory testing of frozen foods.     

Application of magnetic immuno-polymerase chain reaction assay for detection of <Emphasis Type="Italic">Salmonella</Emphasis> spp. in chicken meats

Since contaminated chicken meats have been the principal foodborne source of the contamination of Salmonella to human beings and cultural detection methods are labor-intensive and time-consuming, a study evaluating the performance of the combination of two techniques that are immunomagnetic separation (IMS) and polymerase chain reaction (PCR) for the detection of Salmonella in chicken meats was conducted. The IMS and PCR assay combines selective extraction of Salmonella by specific antibodies with primer-specific (primer pair based on the sequence of invA gene) PCR amplification. Initially chicken meat samples, in which no Salmonella contamination had been determined by using ISO 6579 reference method, were inoculated with Salmonella Enteritidis culture and subsequently the shortest non-selective pre-enrichment time, that had been needed for the detection of approximately 1 or 10 CFU/mL chicken meat levels of target bacteria by magnetic immuno-PCR assay, was found by using 14, 12, 10 and 8-h periods. In conclusion, it was found that magnetic immuno-PCR assay was able to detect 1–10 CFU Salmonella/25 g chicken meat, after only incorporating a non-selective pre-enrichment period of 12 h. Therefore, an overall 16-h (magnetic immuno-PCR assay in conjunction with 12-h non-selective pre-enrichment) magnetic immuno-PCR assay statistically evaluated as sufficient (p = 0.182 > 0.05) for rapid and sensitive detection of approximately 1–10 CFU Salmonella from 25 g chicken meat samples. Accordingly, 16-h magnetic immuno-PCR assay can be promising for routine use in the detection of Salmonella in chicken meat samples, and it consequently may prevent the risk of Salmonella infections in regard to chicken meats.     

Enumeration of <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> at Low Contamination Levels in Several Food Matrices Using a Membrane Filtration Method

For the enumeration of Listeria monocytogenes in cold-smoked salmon, a sensitive enumeration method, based on membrane filtration followed by transfer of the filter on a selective medium, has been recently developed. An evaluation of this method was performed with several categories of foods likely to be contaminated with L. monocytogenes. The results obtained with the technique were compared with those from the reference EN ISO 11290-2 method and found to provide more precise results in the enumeration of L. monocytogenes from both artificially and naturally contaminated products. In most cases, the filtration method enabled a greater quantity of food to be examined (from around 0.5 to 14 g, instead of 0.01 to 0.1 g with the reference EN ISO 11290-2 method), thus greatly improving the sensitivity of the enumeration.     

Comparative Study on the Inactivation and Photoreactivation Response of <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> Seafood Isolates and a <Emphasis Type="Italic">Listeria innocua</Emphasis> Surrogate after Pulsed Light Treatment

The inactivation and photoreactivation response of six seafood-isolated Listeria monocytogenes and one Listeria innocua strain after pulsed light (PL) treatment was evaluated. The lower inactivation levels found after exposure of treated samples to daylight during the first 90 min of storage confirmed that both L. innocua and L. monocytogenes have the capability to photorepair PL-induced DNA damage upon appropriate conditions. Photoreactivation levels from 0.2 to 2.1 log CFU cm^?2 were observed depending on treatment intensity (fluence) and Listeria strain. Complete photorepair of PL-caused damage was not found even after treatments inducing low inactivation levels. Photoreactivation increased up to 2.1 log with the applied fluence up to a threshold able to cause between 2.4 and 5.4 log reductions under dark storage. Photorepair was not avoided but lower photoreactivation was observed after higher fluence inducing more than 6 log reductions under dark storage. Both L. innocua and L. monocytogenes serotype 1/2b exhibited the highest photoreactivation levels whereas serotypes 1/2a showed the lowest ones. The overall inactivation and photoreactivation responses of tested Listeria strains were comparable indicating that L. innocua may be a good surrogate for the safe evaluation, optimization and validation of PL technology to control L. monocytogenes in food products and food processing facilities.     

Preliminary geoherbalism study of <Emphasis Type="Italic">Dendrobium officinale</Emphasis> food by DNA molecular markers

Dendrobium officinale is commonly used as a traditional valuable Chinese tonic hygienic food. In order to authenticate the quality of hygienic food in the market and geoherbalism in the process of GAP (good agriculture practice), sequences of chloroplast genes (matK and rbcL), the nuclear rDNA ITS region and mitochondria gene nad1 intron2 were used to survey eight populations of D. officinale. No difference was found in sequences of matK, rbcL and nad1 intron2 between populations, except for nine single nucleotide polymorphisms (SNPs) sites in rDNA ITS regions. Two pairs of allele-specific PCR primers based on SNPs were designed to authenticate two genuine populations (GSG and FSC populations). The AS-PCR (allele-specific PCR) fragments of GSG and FSC populations are about 600 and 560 bp, and annealing temperatures are 55–57 and 58–60°C, respectively. Dried “Fengdou” food samples from genuine populations could also be authenticated using two pairs of diagnostic primers. It is demonstrated that methods based on SNPs of rDNA ITS region and AS-PCR are simple, practical and effective for genuine germplasm authentication of D. officinale during the process of GAP and hygienic food quality control.     

A Pilot Study for Identification of <Emphasis Type="Italic">Salmonella</Emphasis> in Food Processing Plants by Real-Time PCR Screening

Salmonella remains a major public health concern worldwide. Microbiological methods are the gold standard for Salmonella detection. These methods are highly specific, but their sensitivity is variable. Moreover, they are lengthy, labour intensive and not always consistent with the speed of food manufacturing processes. Thus, in the food industry, there is the need for more rapid, sensitive and accurate detection methods. The purpose of this study is to describe a Salmonella-monitoring scheme in different food processing plants based on a screening approach by a commercial real-time polymerase chain reaction (PCR) kit and subsequent confirmation of positive molecular results by the reference microbiological method. This scheme was tested on a total of 4,693 samples, 90 of which were positive with the real-time PCR screening; 52 of the positive samples were eventually confirmed by the microbiological method. The real-time PCR kit was tested in comparison to the microbiological method in order to evaluate its performances and drawbacks. The comparison between cycle threshold (Ct) values of real-time PCR and the microbiological results (Wilcoxon rank sum test) showed a statistically significant difference between the Ct values of bacteriological positive and bacteriological negative samples (p value, <0.05). Furthermore, receiver operating characteristic curve analysis was used to identify the Ct value ensuring the lowest level of misclassification between Salmonella-positive and negative samples. The present study confirms that the real-time PCR kit tested could be used as a screening tool, leading to a rapid and sensitive identification of Salmonella and confining bacteriological confirmation to samples previously identified as positive.     

Tyramine production of technological important strains of <Emphasis Type="Italic">Lactobacillus</Emphasis>, <Emphasis Type="Italic">Lactococcus</Emphasis> and <Emphasis Type="Italic">Streptococcus</Emphasis>

The aim of this paper was to study the biogenic amines (histamine, tyramine, putrescine, cadaverine, agmatine, spermine and spermidine) production of selected technological important lactic acid bacteria (strains of the genera Lactococcus, Lactobacillus and Streptococcus). Three methods (ion-exchange chromatography (IEC), PCR and cultivation method with pH indicator) were used. Within the 39 strains of lactic acid bacteria tested, the production of tyramine (formed by tyrosine decarboxylase) was detected in eight strains (3 strains of Lactococcus lactis subsp. lactis, three strains of Lactococcus lactis subsp. cremoris, 1 strain of Streptococcus thermophilus and 1 strain of Lactobacillus delbrueckii subsp. bulgaricus). The other tested biogenic amines were not detected. Cultivation in decarboxylation broth seems to be the least accurate method for the detection of biogenic amines due to enhanced risk of false-positive reactions. Therefore, in order to detect bacteria producing biogenic amines, the combination of PCR and chromatographic methods (e.g. IEC) can be recommended.     

<Emphasis Type="Italic">In silico</Emphasis> phylogenetic analysis of lactic acid bacteria and new primer set for identification of <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> in food samples

Lactic acid bacteria (LAB) include species very closely related both physiologically and genotypically. Therefore, the identification of this bacteria group using conventional phenotypic methods is ambiguous and cumbersome. In this study, we have analyzed a recA gene fragment from 30 bacteria, including LAB and species common in the human gastrointestinal tract, aiming to evaluate the gene conservation among them and the development of primers and PCR conditions able to discriminate Lactobacillus plantarum strains from LAB closely related. The fragment with 995 bp of recA gene has grouped LAB, enterobacteria and bifidobacteria, in different clusters. A novel primer pair, LPrecAF and LPrecAR with 23 and 18 bp, respectively, has allowed the single amplification of a 108 bp fragment of L. plantarum strains contained in culture broth and fermented dairy samples. The observed detection limit for food samples and for cultures broth were 1 × 10³ and 7 × 10² CFU mL⁻¹, respectively. This approach proved to be a simple and efficient method for the identification and monitoring of L. plantarum in food, feeds, and culture broth. Moreover, the assay could be used in the studies from human or environmental microbiota.     

Quantitative determination of <Emphasis Type="Italic">Lactobacillus</Emphasis> spp. in milk using a series piezoelectric quartz crystal sensor

A series piezoelectric quartz crystal (SPQC) sensor was developed for quantitative determination of Lactobacillus spp. populations in milk. When the electrodes were immersed in a reaction cell with bacterial inoculum, the change of frequency was caused by the impedance change of the microbial metabolism. A significant frequency decrease was found due to the coagulation of milk when the Lactobacillus spp. was cultivated in the media. The SPQC sensor system established in this study demonstrated the specificity and selectivity for detection of Lactobacillus spp. in milk sample. The calibration curve of detection time against density of Lactobacillus spp. shows a linear correlation coefficient (R ² = 0.8453) over the range of 10²–2.4 × 10⁵ CFU ml⁻¹. The detection time was influenced by the addition of peptone and glucose. The sensor exhibited rapid (within 4 h) and enabled real time monitoring of Lactobacillus spp. growth. This system is potentially applicable to detect Lactobacillus spp. concentration at local farm when a suitable temperature control device is adapted.     

Thermal inactivation kinetics of quality-related enzymes in cauliflower (<Emphasis Type="Italic">Brassica oleracea</Emphasis> var. <Emphasis Type="Italic">botrytis</Emphasis><Emphasis Type="Bold">)</Emphasis>

Thermal inactivation of quality-related enzymes in both cauliflower crude enzyme extracts and fresh tissue samples was studied in temperature range 50–100 °C. For crude enzyme extracts, several parameters, reaction rate constants (k) and activation energy (E _a) as well as decimal reduction time (D) and (z) values, were used to characterize the thermal stability. The rates of inactivation were found to follow first-order inactivation kinetics. Activation energies varied between 101.18 and 208.42 kJ mol⁻¹ with z values of 10.59–24.09 °C. The examined kinetics indicated that lipoxygenase was the most heat resistant followed by peroxidase, polyphenol oxidase, pectin methyl esterase and ascorbic acid oxidase. Furthermore, the obtained results from the blanched fresh tissues indicated that inactivation of lipoxygenase secured disappearing of any other enzyme activities. Therefore, this study recommends using lipoxygenase as an indicator enzyme to optimize the thermal treatments of cauliflower products.     

Molecular diversity among natural populations of <Emphasis Type="Italic">Lactobacillus paracasei</Emphasis> and <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis>/<Emphasis Type="Italic">paraplantarum</Emphasis> strains isolated from autochthonous dairy products

The diversity of 87 Lactobacillus paracasei and Lactobacillus plantarum/paraplantarum strains, previously identified from different autochthonous dairy products, was investigated by phenotypic and genotypic approaches. The increased resolution obtained using phenotypic and genotypic characterization allowed the level of strain heterogeneity detection to be widened. Phenotypic diversity was evaluated by studying biochemical characteristics of technological interest, including antimicrobial and proteinase activities, resistance to nisin, aggregation ability, production of exopolysaccharides, acetoin and diacetyl, citrate utilization, and antibiotic susceptibility. Genotypic diversity was generally evaluated by PCR amplification of repetitive bacterial DNA element fingerprinting using the (GTG)₅ primer [(GTG)₅-PCR]. Moreover, in cases where strains were not discriminated by (GTG)₅-PCR combined with phenotypic analysis, pulsed-field gel electrophoresis (PFGE) analysis was performed. The results indicate that L. plantarum/paraplantarum and L. paracasei natural isolates from artisanal dairy products are a gold mine in terms of diversity of strains and could be potentially interesting to dairy companies for the formulation of functional starter cultures in the production of innovative foods.