Real-Time Recombinase Polymerase Amplification Assay for the Detection of <Emphasis Type="Italic">Vibrio cholerae</Emphasis> in Seafood

Vibrio cholerae, the most hazardous Vibrio species, is threatening aquacultured animals and even human beings. In recent years, a few genetic markers that target V. cholerae have been reported, but their application was limited by techniques or themselves. The outer membrane lipoprotein gene lolB has been identified as a specific marker by using PCR and quantitative PCR methods. In this study, we developed a real-time recombinase polymerase amplification (RPA) assay targeting lolB gene in an attempt to provide a sensitive, rapid, and reliable detection method of V. cholerae. The sensitivity of RPA assay was determined by amplifying the standard plasmid dilutions. The detection limit down to five copies was achieved within 10 min, which was lower than that of qPCR (ten copies after 25 cycles within 1.5 h). The reproducibility of RPA assay was verified by eight independent experiments, presenting a high R ² value of the standard regression line (0.97). In addition, the specificity was confirmed with DNA extracted from other bacteria, shrimps, clams, and fishes using both methods. Finally, V. cholerae in shrimp samples were quantified using real-time RPA and qPCR with consistent outcomes, while no amplification was obtained from clam and fish samples using both methods. The applicability in the food industry was confirmed by quantitative detection of natural seafood.     

Rapid Detection of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> in Food Using a Recombinase Polymerase Amplification-Based Assay

Staphylococcus aureus (S. aureus) is of great importance and is a leading cause of food poisoning, which is a public health concern in terms of the frequency and seriousness of the disease. In the present study, RPA and real-time RPA assays were developed and validated to detect S. aureus with high sensitivity and specificity by targeting the nuc gene for the first time. The analytical sensitivity of real-time RPA was 10² copies/reaction, which was higher than the sensitivity of the real-time PCR method. The analysis time was reduced to 10 min, but this method was as reliable as real-time PCR. Furthermore, the potential use of RPA to detect S. aureus was validated with five different artificially contaminated foods. In conclusion, the RPA and real-time RPA assays developed here, similar to real-time PCR, are rapid and simple and exhibit with high sensitivity and specificity. These assays serve as efficient tools for the detection of S. aureus in less advanced laboratories and are suitable for point-of-care detection.     

Detection of Viable <Emphasis Type="Italic">Vibrio cholerae</Emphasis> Cells in Seafood Using a Real-Time Visual Loop-Mediated Isothermal Amplification Combined with Propidium Monoazide

Vibrio cholerae is an important foodborne pathogen causing severe intestinal infectious diseases that have high incidence and mortality. Almost all of rapid testing methods including immunological and molecular assays for V. cholerae are incapable of distinguishing live cells from dead ones, which may overestimate the number of bacteria and result in many false positive results. To address the problems, live cell-specific dye such as propidium monoazide (PMA) is employed. The loop-mediated isothermal amplification (LAMP) assay is a nucleic acid amplification method that is fast, specific, and sensitive. In this study, we developed a real-time visual LAMP assay using PMA dye to detect thyA gene, thereby identifying viable V. cholerae cells. The results showed that only V. cholarae strains could be detected, and there was no cross-reaction with non-V. cholarae strains. Besides, the sensitivity of the PMA-LAMP assay was 1.1 × 10² CFU/mL and the entire reaction could be accomplished within 1 h. The sensitivity was on par with that of the PMA-qPCR assay. The detection limit in different artificially inoculated samples was 5 CFU/25 g materials for the tested pathogens. In the practical test, the PMA-LAMP assay performed well in comparison with PMA-qPCR and the culture method. Hence, PMA-LAMP assay can provide a highly effective and rapid approach for detecting viable V. cholerae.     

Quantitative Detection of <Emphasis Type="Italic">Clostridium perfringens</Emphasis> by Real-Time PCR in Raw Milk

Clostridium perfringens causes a broad spectrum of diseases in both humans and animals and is an important cause of foodborne illness. We developed and tested a real-time (Q-)PCR assay for the species-specific detection of C. perfringens that targets the phospholipase C (plc) gene and includes an internal amplification control (IAC), making it possible to identify false-negative results, which are common due to the high level of PCR inhibition by food compounds. The CPplc-IAC real-time PCR (RTi-PCR) assay was 100% selective, as shows with 36 Clostridium strains and 85 non-Clostridium strains, with an analytical sensitivity of 1 genome equivalent (GE) in 23% of the reactions and 10 GE in 100% of the reactions. The quantification was linear (R ² = 0.9990) over a 7-log dynamic range, down to 10 GE, with a PCR efficiency E = 0.841. The applicability of this RTi-PCR assay was assessed in milk samples. The assay detected as few as 300 spores in 25 mL of artificially contaminated raw sheep milk with 78% probability and 30 spores in 25 mL with 50% probability. It also has accuracy of 83.03 to 151.18%, as shown by an evaluation of the correspondence between RTi-PCR assay results and the number of spores per milliliter determinated by standard plating. This RTi-PCR method was effective for the detection and quantification of C. perfringens in milk having an important applicability in the control of this pathogen in the dairy food industry.     

A Rapid and Visual Single Primer Isothermal Amplification-Based Method for the Detection of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> in Raw Pork Products

Staphylococcus aureus (S. aureus) is an important food-borne pathogen which poses a severe threat to public health worldwide. Rapid detection of S. aureus with high sensitivity is of particular importance for food safety. In this study, a novel single primer isothermal amplification (SPIA) method was established to detect S. aureus in food, targeting the accessory gene regulator (agr) gene with a DNA/RNA primer. The developed SPIA method has the advantages of visualization and avoiding tedious electrophoresis. In order to confirm the specificity of this method, 7 S. aureus strains and 26 non-S. aureus strains were detected with their pure cultures. The sensitivity and detection limit of S. aureus with artificially inoculated raw pork products by SPIA were evaluated through fluorescence and turbidity by naked eye and the amplification curve, which were 4.3?×?10⁰ CFU/mL and 5.6?×?10⁰ CFU/g, respectively. Compared with the conventional PCR method, the SPIA has 100-fold higher sensitivity and 100-fold lower detection limit. Therefore, the developed SPIA method is a potentially reliable tool for rapid and visual detection of S. aureus in food.     

Evaluation of <Emphasis Type="Italic">eryC</Emphasis> as a Molecular Marker for the Quantitative Detection of <Emphasis Type="Italic">Brucella</Emphasis> Spp. by Real-Time PCR in Food Samples

We evaluated the capacity of the Brucella sp. eryC gene as a diagnostic marker for brucellosis by quantitative real-time PCR. eryC gene encodes the enzyme d-erythrulose-1-phosphate dehydrogenase that plays an important role in the erythritol metabolism and is related with the Brucella survival in the intracellular environment of the macrophage. The assay includes an internal amplification control (IAC) in order to avoid false negative results. It was 100% specific, with an analytical sensitivity of 1 genome equivalent (GE) in 43% of the reactions, being the quantification highly linear (R ² > 0.9953) and efficient (PCR efficiency >0.8820) over a 6-log dynamic range, down to 10 GE. Finally, the applicability of this assay was evaluated with artificially contaminated biological matrices implicated in the transmission of this bacterium such as sheep raw milk and pig blood. The eryC-IAC real-time PCR assay allowed detection of as few as ten Brucella cells per 25 ml of sheep raw milk or per 1 ml of pig blood. In conclusion, we present an alternative for the detection of Brucella genus and therefore facilitate the establishment of preventive and prophylactic measures in food and farm environments.     

Development and characterization of reconstituted hydrogel from <Emphasis Type="Italic">Aloe vera</Emphasis> (<Emphasis Type="Italic">Aloe barbadensis</Emphasis> Miller) powder

Structural and rheological characterization of reconstituted hydrogels developed from A. vera non-fibrous alcohol insoluble residue (NFAIR) powder using different methods [viz., shaking (S), heating-shaking (HS), and heating (H)] and concentrations (viz., 0.2–1.6 %, w/v) was carried out. Functional group distribution by FTIR spectroscopy and Congo red (CR) method revealed the presence of acetylated acemannan in A. vera powder. Dynamic oscillation studies of A. vera (NFAIR) fluids at all concentrations of 0.2–1.6 %, w/v, showed gel strength in the order of H > HS > S method. However, in H method, increase in concentration from 0.2 to 1.6 %, w/v showed the conformational transition from semi-diluted solution to weak gel nature. Rheological models described the effect of heating temperatures (HT); 30–90 °C, and times (Ht); 15–60 min on viscoelastic behavior in reconstituted A. vera fluids. The reconstituted A. vera hydrogel prepared with a concentration of 1.6 %, w/v using 50 °C (HT) and 30 min (Ht) condition showed a good agreement with the Power law (storage modulus, G′) and Weak gel model (complex modulus, G*) fitted data (R² > 0.94) resulting higher viscoelastic moduli intercepts; G′₀ (71.5 Pa s ^n′), G″₀ (33.5 Pa s ^n″), lower slopes; n′ (0.22), n″ (0.06), higher network strength (A _F , 121.3 Pa s^1/z ) and number of network (z, 5.3) values. The obtained results suggested that heating at 50 °C/30 min can develop aqueous weak gel networks of A. vera with enhanced gel strength which may be utilized as a novel gelling agent for wide variety of targeted applications in food and pharmaceutical sectors.     

Kinetics of Quality Changes of Shrimp (<Emphasis Type="Italic">Litopenaeus setiferus</Emphasis>) During Pasteurization

Effect of cooking between 1 and 80 min at 60 to 100 °C on several quality attributes of whole peeled shrimp (Litopenaeus setiferus) (80–90 counts/kg) was studied using an isothermal heating method. Cook loss, area shrinkage, and hardness of shrimp increased with increasing heating time and temperature, following a fractional first-order kinetic model with activation energies (E _a ) of 71.0, 53.3, and 29.9 kJ/mol, respectively. Cook loss, area shrinkage, and hardness were positively correlated. The toughness of shrimp muscle increased in the initial period of heating, then decreased in the later period during the treatments. The overall color change (ΔE) increased with increasing treatment time and temperature, and followed a zero kinetic model with an E _a of 37.2 kJ/mol. The kinetic parameters obtained from this study can be applied toward understanding and predicting shrimp-quality changes during pasteurization treatments, and further provides insight into the pasteurization conditions required to achieve safe and high-quality shrimp products and potentially other crustacean shellfish and seafood products.     

Enterohemorrhagic <Emphasis Type="Italic">Escherichia coli</Emphasis> (EHEC) in water from karst springs: detection with real-time PCR and isolation of strains

Within 2 months, two water sources in a karst area in Switzerland were sampled 9 times each, and analyzed by real-time PCR for 6 EHEC O-types, Shiga-like-toxin (stx1 and stx2) and intimin (eae) genes. With the exception of O111, 5 O-types were recorded regularly and at high frequencies (O26: 33.3 %; O157: 33.3 %; O104: 66.6 %; O103: 72.2 %; O145: 94.4 %). Genes for Shiga-like-toxins and intimin were almost omnipresent (stx1: 77.8 %; stx2: 83.3 %; eae: 77.8 %). Strain isolation was undertaken for O-groups 26, 103, 104, 145 and 157. Sample selection for strain isolation was based on Cq-values for the O-groups and stx1, stx2 and eae. From selected samples, frozen enrichment cultures were cultivated on EHLY-agar and 50 typical colonies screened for the O-type and genes encoding for stx1, stx2 and eae. With this approach, only one virulent EHEC-strain could be isolated (Escherichia coli O103, stx1 +; stx2 ?; eae +). We carried out one extensive testing with 800 colonies of O-group O145, and no virulent strain was isolated. Our findings showed that PCR-results are not sufficient to formulate epidemiological conclusions and that the isolation of strains is necessary. However, as the detection procedure of EHEC in foods is cumbersome and expensive, the appropriateness of such an approach in official food control is a matter of debate.     

Characterization and evaluation of antimicrobial activity of actinonin against foodborne pathogens

This study revealed the antimicrobial properties of actinonin against major foodborne pathogens, Escherichia coli O157:H7, Listeria monocytogenes, Salmonella Typhimurium, Staphylococcus aureus, and Vibrio vulnificus. Among them, actinonin caused growth defect in S. Typhimurium and V. vulnificus. Minimal inhibitory concentration (MIC) values of actinonin were determined by broth microdilution methods. The MICs of actinonin were ≤0.768 μg/ml for S. Typhimurium and ≤0.192 μg/ml for V. vulnificus. Susceptibility to actinonin in both pathogens was measured by colony-forming ability and disc diffusion test. The results showed actinonin had antimicrobial activity against S. Typhimurium and V. vulnificus in a dose-dependent manner. The inhibitory effects on swarming motility were determined, and cytotoxicity of each pathogen against HeLa cells was decreased significantly by actinonin treatment. Furthermore, actinonin showed an antimicrobial efficacy in food models infected with these pathogens. These results demonstrate that actinonin is potentially an effective agent for food sanitization or preservation.     

Saltatory Rolling Circle Amplification (SRCA): a Novel Nucleic Acid Isothermal Amplification Technique Applied for Rapid Detection of <Emphasis Type="Italic">Shigella</Emphasis> Spp. in Vegetable Salad

Shigella spp. are enteric pathogens that pose a serious threat to public health worldwide. A novel saltatory rolling circle amplification (SRCA) assay was developed to detect Shigella spp. in food targeting the ipaH gene. SRCA as an isothermal amplification method requires no expensive thermocycle instrument and could avoid electrophoresis as visualization results was successfully applied for SRCA. In order to confirm the specificity of this assay, 34 strains including 11 strains belonging to different Shigella species and 23 non-Shigella bacteria were detected with pure cultures. The sensitivity of Shigella flexneri by SRCA was evaluated using agarose gel electrophoresis, which was 7.3 × 10¹ fg/μL. In addition, the amplification results were also determined by adding the fluorochrome, SYBR Green I (1 μL of 1000×), allowing naked eye visualization of results, and the sensitivity was 7.3 × 10⁰ fg/μL. Moreover, the sensitivity of PCR was 7.3 × 10² fg/μL, showing that the sensitivity of SRCA by electrophoresis and SYBR Green I fluorescence were 10- and 100-fold higher than that of PCR, respectively. The detection limit of SRCA was also evaluated with artificially inoculated vegetable salad without enrichment, and it was 4.7 × 10² and 4.7 × 10¹ CFU/g by electrophoresis and fluorescence, respectively. The detection limit by PCR was 4.7 × 10³ CFU/g, which was 10- and 100-fold higher than that of SRCA. Therefore, SRCA is a potentially reliable tool for rapid and specific detection of Shigella in food and could be useful in underdeveloped countries with limited resources.     

Reversed-phase HPLC determination of mangiferin,isomangiferin and hesperidin in <Emphasis Type="Italic"> Cyclopia </Emphasis>and the effect of harvesting date on the phenolic composition of <Emphasis Type="Italic"> C. genistoides</Emphasis>

A reversed-phase HPLC method for separation of polyphenols in honeybush tea (Cyclopia spp.) is presented. Separation of eriodictyol, luteolin, medicagol, formononetin, mangiferin, isomangiferin, hesperetin and hesperidin was investigated. A C12 stationary phase was required to separate mangiferin and isomangiferin. The method was used to quantify the three major polyphenols (mangiferin, isomangiferin and hesperidin) in C. genistoides, C. intermedia, C. maculata and C. sessiliflora and to study the effect of harvesting date on these compounds in two types of C. genistoides. The highest levels of the xanthones, mangiferin (3.61 g/100 g) and isomangiferin (0.54 g/100 g), and the flavanone, hesperidin (1.74 g/100 g), were found for C. genistoides (both xanthones) and C. intermedia, respectively. Cyclopia sessiliflora contained the lowest levels of mangiferin (1.04 g/100 g) and hesperidin (0.29 g/100 g). The mangiferin content of both the Overberg and West Coast types decreased with harvesting date (P <0.05). The Overberg type contained more mangiferin, but hesperidin was more prominent in the West Coast type.     

Quantification and Discrimination of Viable and Dead <Emphasis Type="Italic">Escherichia coli</Emphasis> O157:H7 Cells from Chicken Without Enrichment by Ethidium Bromide Monoazide Real-time Loop-Mediated Isothermal Amplification

In this study, a rapid and sensitive method of real-time loop-mediated isothermal amplification (Rti-LAMP) assays was developed for quantification and discrimination of viable and heat-killed E. coli O157:H7 cells treated with low concentration of ethidium bromide monoazide (EMA). Four micrograms per milliliter of EMA was chosen as the optimal concentration which did not inhibit DNA amplification derived from viable cells, but significantly increased the Tt values of dead cells in Rti-LAMP assays. When the DNA from 2.0?×?10³ viable CFU of E. coli O157:H7 was subjected to EMA-Rti-LAMP, the resulting Tt value was 17.73 min. In contrast, the DNA from 2.0?×?10³?CFU completely heat destroyed CFU of E. coli O157:H7 did not yield a positive amplification which Tt value was regarded as 60 min. When the DNA from viable plus heat-killed CFU at a ratio of 5:2995 was subjected to EMA-Rti-LAMP, the resulting Tt value was 23.06 min, which was statistically identical (P?<?0.05) to the Tt value of 24.07 min obtained with the DNA from only 5 viable CFU. The results indicate that even though 3.0?×?10³ dead cells yielded a negative amplification setting the Tt value as 60 min, low numbers of viable cells in the presence of much higher numbers of dead cells still yielded a linear plot for enumerating viable CFU from Tt values. Detection of E. coli O157:H7 derived from contaminated chicken samples, the EMA-Rti-LAMP could notably distinguish viable and heat-killed cells from 5.0?×?10¹ to 1.0?×?10⁴?CFU/g without enrichment.     

Effect of heat-treated <Emphasis Type="Italic">Nuruk</Emphasis> on the quality characteristics of aged <Emphasis Type="Italic">Yakju</Emphasis>

Long-term aging of Yakju, a traditional Korean liquor made of rice and Nuruk (a fermentation agent), causes browning and odor and flavor development. This study investigated the effects of heat-treated Nuruk (50–80 °C, 30 min) on Yakju quality. The saccharogenic powers and glucoamylase, α-amylase, and carboxypeptidase activities were similar in non-heat-treated Nuruk and that treated at 50 °C. However, acidic protease and alcohol dehydrogenase decreased above 50 °C. The content of nitrogen-containing compounds was inversely proportional to the heat-treatment temperature. Compounds that cause off-flavors decreased at 50–60 °C, but increased at 70–80 °C, whereas compounds that provide fragrance increased at 50–60 °C. Sensory evaluation indicated that bad taste attributes were higher in Yakju produced using non-heat-treated Nuruk. Therefore, heat treatment of Nuruk at 50 °C can be adopted as a method for improving Yakju quality, as enzymatic activities that affect color, aroma, and taste are regulated.     

Steady- and Unsteady-State Determination of the Water Vapor Permeance (<Emphasis Type="Italic">WVP</Emphasis>) of Polyethylene Film to Estimate the Moisture Gain of Packed Dry Mango

The water vapor permeance (WVP; g m^?2 d^?1 Pa^?1) of packaging films quantifying the water vapor transfer rate between foods and its surroundings is usually determined in units operating under steady-state conditions that do not necessarily reflect food handling scenarios. This study evaluated the determination of the WVP of a polyethylene (PE) film by steady-state method ASTM F1249-06 using a permeability cell and unsteady-state method ASTM E96/E96M in which 102 vacuum-sealed PE bags containing silica gel were stored (37.8 °C, 75% relative humidity) and weighed over 25 days. Average steady-state WVP (2.935 ± 0.365 × 10^?3, n = 4) fell within the 95% quantiles of unsteady-state WVP values (1.818–3.183 × 10^?3, n = 2142). Moisture uptake of dehydrated mango stored at 37.8 °C and 75% relative humidity was predicted with WVP values obtained by both methods. Predictions were validated by monitoring over 25 days the weight gain of 100 PE bags with dry mango. Experimental moisture averages during storage fell within one standard deviation of predictions using the unsteady-state WVP (R ² = 0.974). The same was observed only until day 15 for predictions obtained with the steady-state WVP. Calculations for days 20–25 overestimated the moisture uptake by 6.0–7.2%, resulting in registered R ² = 0.924. The unsteady-state WVP determination is low-cost, uses large numbers of film samples, and allowed more accurate predictions of dry mango moisture uptake. Knowledge of the moisture uptake controlled by the film WVP is essential when predicting the safety and quality changes limiting the shelf-life of foods.     

Validation of a Loop-Mediated Amplification/ISO 6579-Based Method for Analysing Soya Meal for the Presence of <Emphasis Type="Italic">Salmonella enterica</Emphasis>

An alternative method for detection of Salmonella spp. in animal feed, based on the use of loop-mediated amplification (LAMP) in conjunction with a standard culturing procedure, was compared with the standard ISO 6579 as reference method, using soya meal as the test matrix. In the method comparison study, the sensitivities for both the alternative and reference methods were 100 %. The relative level of detection was 1.000. Tested against 100 Salmonella and 30 non-Salmonella strains, the LAMP-based method was 99 % inclusive and 100 % exclusive. The interlaboratory trial involved ten laboratories from eight European countries, testing eight samples at three contamination levels: 0 cfu/100 g, 1–5 cfu/100 g and 14–68 cfu/100 g. The trial specificity, or percentage correct identification of uncontaminated samples, was 96.3 % for both the reference methods and the LAMP/ISO 6579 alternative method, thus demonstrating its suitability for adoption as a procedure for rapid identification of Salmonella uncontaminated soya meal.     

Development of a Simple,Rapid Multiplex PCR Method Using the 16S rRNA Gene to Determine the Country of Origin of Brackish Water Bivalve <Emphasis Type="Italic">Corbicula japonica</Emphasis>

In this study, a multiplex PCR detection method was developed to identify the country of origin of Corbicula japonica (clams), a commercially important bivalve in Asia. Specific primer sets that have a single nucleotide mismatch at the 3′ terminus were designed after sequencing the mitochondrial 16S rRNA gene of clams identified as C. japonica originating from Korea, China, and Japan. Using this method, each origin was clearly identified based on the PCR products: three bands for Korean C. japonica (100, 283, and 384 bp), one band for Chinese C. japonica (384 bp), and two bands for Japanese C. japonica (384 and 100 bp). These results indicate that the 16S rRNA gene, which is usually used to identify species, can distinguish the country of origin within C. japonica. Our multiplex PCR assay should be a useful tool for the fair trade of the species.     

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.     

Determination of Seven <Emphasis Type="Italic">N</Emphasis>-nitrosamines in Agricultural Food Matrices Using GC-PCI-MS/MS

The quantitative analytical methods for seven N-nitrosamines including N-nitrosodimethylamine (NDMA), N-nitrosomethylethylamine (NMEA), N-nitrosodiethylamine (NDEA), N-nitrosodibutylamine (NDBA), N-nitrosopiperidine (NPIP), N-nitrosopyrrolidine (NPYR), and N-nitrosomorpholine (NMOR) were established for agricultural food matrices. Four food matrices were used for the method development: rice soup as a fatless solid matrix, apple juice as a fatless liquid matrix, corn oil as a fat-rich liquid matrix, and 20 % alcohol as an alcohol matrix. A combination of solid-supported liquid-liquid extraction (SLLE) using Extrelut NT and a solid phase extraction (SPE) using Florisil was employed for fatless matrices. For an alcohol matrix, only SLLE was used without SPE, and liquid-liquid extraction (LLE) was established for a fat-rich matrix. The extract was analyzed by gas chromatography-positive chemical ionization-tandem mass spectrometry (GC-PCI-MS/MS) using ammonia gas as an ion source. Linearity, recovery, repeatability, inter-day precision, reproducibility, and uncertainty were evaluated for method validation using four matrices. Method detection limits for all of the investigated N-nitrosamines were ranged from 0.10 to 0.18 μg/kg for the rice soup, from 0.10 to 0.19 μg/kg for the apple juice, 0.10 μg/kg for the corn oil, and from 0.10 to 0.25 μg/kg for 20 % alcohol, depending on N-nitrosamines. Established methods were applied to determine seven N-nitrosamines in some agricultural food products.     

Effect of Tannin Extracts on Biofilms and Attachment of <Emphasis Type="Italic">Escherichia coli</Emphasis> on Lettuce Leaves

Lettuce is often involved in foodborne outbreaks caused by pathogenic Escherichia coli. Current control strategies have often proved ineffective to ensure safe food production. For that reason, the present study compared the efficacy of tannin extracts and chlorine treatments on the reduction of E. coli ATCC 25922 adhered to lettuce leaves. E. coli was inoculated artificially on leaf surfaces of fresh crisp lettuce. Effectiveness of water, chlorine (200 mg/L), and three commercial available tannin extracts from Acacia mearnsii De Wild. (tannin AQ (2 %, w/v), tannin SG (1 %, v/v) and tannin SM (1 %, v/v)) treatments was evaluated using the viable plate count method and scanning electron microscopy (SEM). SEM results revealed that bacterial cells are attached as individual cells and in clusters to the leaf surface after 2 h of incubation. Biofilm formation was observed after 24 h of incubation. The tannin SM treatment was able to reduce counts in approximately 2 log CFU/cm² on leaf segments. However, treatment was less effective in the reduction of E. coli counts after 24 h of incubation when compared to 2 h incubation of the same extract. The results suggest that the tannin SM extract diminishes E. coli counts adhered to and under biofilm formation on lettuce leaves and its effect is similar to the use of chlorine solutions.