Determination of aflatoxins in walnut sujuk and Turkish delight by HPLC-FLD method

This study aims to assess the risk of aflatoxins (AFs) in traditional confectionery products (walnut sujuk and Turkish delight) of Turkey. A high performance liquid chromatography coupled with fluorescence detection (HPLC-FLD) method was used for the determination of AFs. Evaluation of the method showed good selectivity, linearity, recovery and precision. The limit of quantification (LOQ) ranged from 0.106 to 0.374 μg kg⁻¹. The expanded measurement uncertainty was less than 40% for all target analytes. The validated method was successfully applied to the determination of AFs in 112 traditional confectionery products containing nuts (hazelnuts and walnuts). AFs were detected in 43.8% of walnuts and 60.9% of hazelnuts used as ingredients in walnut sujuk and Turkish delight and at levels ranging from 0.58 to 15.2 μg kg⁻¹ and 0.43–63.4 μg kg⁻¹, respectively. This means that AFs levels in walnut sujuk and Turkish delight were up to levels of 6.1 and 9.5 μg kg⁻¹, respectively. Six walnut samples and twenty-one hazelnut samples were above the EU maximum limits (MLs) of 2 and 5 μg kg⁻¹ for aflatoxin B₁ (AFB₁), respectively.     

Aflatoxins and ochratoxin A in dried eggplant and green bell pepper

In this study, 50 dried eggplant and 50 dried green bell pepper samples were analyzed in terms of their aflatoxin and ochratoxin A (OTA) content. Aflatoxins G₂, G₁, B₂, and B₁, and OTA contents were analyzed using high-performance liquid chromatography with a flame ionization detector (HPLC–FID). Total aflatoxin and, as well as aflatoxin G₂, G₁, B₂, and B₁ content in dried eggplant samples were ranged between 0.82 and 2.58, 0.10–0.23, 0.32–1.35, 0.12–0.67, and 0.17–0.71 μg kg⁻¹, respectively. Total aflatoxin and, as well as aflatoxin G₂, G₁, B₂ and B₁ content in dried green bell pepper samples were 0.81–2.42, 0.11–0.22, 0.32–1.38, 0.13–0.66, and 0.18–0.91 μg kg⁻¹, respectively. OTA content was varied from 8.88 to 21.35 μg kg⁻¹ in eggplant samples, and from 15.38 to 24.70 μg kg⁻¹ in dried green bell pepper samples. Of the dried eggplant samples and dried green bell pepper samples, 36% and 24% of them, respectively, had aflatoxin B₁ values which were below the minimum limit of detection (LOD) of 0.05 μg kg⁻¹. None of the analyzed samples exceeded the legal limit values of 10 μg kg⁻¹ for total aflatoxin content, and 5 μg kg⁻¹ for aflatoxin B₁ content. However, 80% of the dried eggplant samples and 100% of the dried green bell pepper samples exceeded the legal limit value for OTA content (15 μg kg⁻¹). According to the results, it was concluded that dried vegetables should be examined in terms of their aflatoxins. It is essential to analyze OTA content more thoroughly, as it has the potential to pose a risk for public health, as well as for the economy.     

Isolation of recombinant antibody fragments (scFv) by phage display technology for detection of almond allergens in food products

Tree nut allergies are considered an important health issue in developed countries. To comply with the regulations on food labeling, reliable allergen detection methods are required. In this work we isolated almond-specific recombinant antibody fragments (scFv) from a commercial phage display library bypassing the use of live animals, hence being consistent with the latest policies on animal welfare. To this end an iterative selection procedure employing the Tomlinson I phage display library and a crude almond protein extract was carried out. Two different almond-specific scFv (named PD1F6 and PD2C9) were isolated after two rounds of biopanning, and an indirect phage ELISA was implemented to detect the presence of almond protein in foodstuffs. The isolated scFvs demonstrated to be highly specific and allowed detection of 40 ng mL⁻¹ and 100 ng mL⁻¹ of raw and roasted almond protein, respectively. The practical detection limit of the assay in almond spiked food products was 0.1 mg g⁻¹ (110–120 ppm). The developed indirect phage ELISA was validated by analysis of 92 commercial food products, showing good correlation with the results obtained by a previously developed real-time PCR method for the detection of almond in foodstuffs. The selected phage clones can be affinity maturated to improve their sensitivity and genetically engineered to be employed in different assay formats.     

A survey on the occurrence of aflatoxin M1 in raw milk produced in Adana province of Turkey

During 2012, a total of 176 samples of raw milk obtained from dairy plants of Adana province of Turkey were analysed for the presence of aflatoxin M₁ (AFM₁). Aflatoxin M₁ analysis was carried out by centrifugation, liquid–liquid extraction, immunoaffinity column clean-up and high performance liquid chromatography with fluorescence detection (HPLC-FD). The limits of detection (LOD) and quantification (LOQ) of the analytical method were 0.021 μg kg⁻¹ and 0.025 μg kg⁻¹. Accuracy of the method obtained from bias ranged from 2.94 to 8.70. Aflatoxin M₁ was detected in 53 out of 176 samples analysed (30.1%). The ranges for positive samples were 0.042–0.552, 0.033–1.01, 0.047–0.150 and 0.025–0.102 μg kg⁻¹ in autumn, winter, spring and summer seasons, respectively. Thirty samples of raw milk (17%) were above the legal limits of Turkey and EU regulations.     

Development and validation of an LC–MS/MS/MS method for the quantification of fluoroquinolones in several matrices from treated turkeys

The study presents a sensitive and reliable confirmatory method for the extraction, identification, quantification of five fluoroquinolones (FQ) namely enrofloxacin, ciprofloxacin, difloxacin, sarafloxacin and flumequine, in plasma, liver, kidney, muscle, skin + fat, lung and intestinal content from turkeys.For the extraction and matrix clean-up of FQ residues from all biological matrices, the Quick Easy Cheap Effective Rugged Safe (QuEChERS) methodology was adopted; only for plasma samples acetonitrile was used.The analyses were performed by liquid chromatography with mass spectrometry detection (LC–MS). LC separation was performed on a C18 Kinetex column (100 × 2.1 mm, 2.6 μm, Phenomenex, CA, USA) with gradient elution using ammonium acetate solution (10 mM, pH 2.5) and methanol containing 0.1% formic acid. Mass spectrometric identification was done using an LTQ XL ion trap (Thermo Fisher Scientific, CA, USA), with a heated electrospray ionization probe, in positive ion mode.The method was validated according to the European Legislation (decision 2002/657/EC) and EMA guideline (EMA/CVMP/VICH/463202/2009); selectivity, linearity response, trueness (in terms of recovery), precision (within-day repeatability and within-laboratory reproducibility), limit of detection, limit of quantification, decision limits, detection capability, absolute recovery and robustness were evaluated using turkey blank matrices. All data were within the required limits established for confirmatory methods except for flumequine which presented a recovery value slightly higher than 110% in muscle and intestinal content. For all FQs, all the extraction rates were greater than 70% and limits of quantification ranged from 1.2 μg kg⁻¹ to 118.8 μg kg⁻¹.This fast and robust method was suitable for the identification and quantification of FQ residues in tissues, plasma and intestinal content as confirmed by data obtained from incurred samples of turkeys treated at farm for therapeutic purposes.     

A fast and simple LC-ESI-MS/MS method for detecting pyrrolizidine alkaloids in honey with full validation and measurement uncertainty

A fast and simple method was developed to determine pyrrolizidine alkaloids (PAs) in honey using liquid chromatography tandem mass spectrometry (LC- MS/MS) with electrospray ionization (ESI). An efficient extraction procedure was carried out by simply diluting with water, without the need of any additional clean-up steps. A full validation of the method was performed according to Commission Decision 2002/657/EC. The method was linear in the 050 μg kg⁻¹ range and presented satisfactory intra-day and inter-day precision with relative standard deviations of 1.45–10.2% and 1.60–1–0.2%, respectively. The measurement uncertainty, limit of detection (LOD) (0.1–1.0 μg kg⁻¹) and limit of quantification (LOQ) (0.2–1.5 μg kg⁻¹) were also calculated. The proposed method was applied to analyse eight PAs, namely, senecionine, senecionine-N-oxide, echimidine, intermedine, lycopsamine, retrorsine, monocrotaline and retrorsine-N-oxide, in 92 commercial honey samples from Brazil. At least three PAs were detected in 99.1% of the samples. PAs were not detectable (<LOD) in only one sample. Because PAs are natural toxins biosynthesized by plants, the importance of monitoring their concentration in honey is evident. For this purpose, a simple, low-cost extraction procedure was performed, and a high-throughput method was developed.     

Aflatoxin B1 production by Aspergillus parasiticus and strains of Aspergillus section Nigri in currants of Greek origin

Aflatoxin B₁ (AFB₁) mostly produced by Aspergillus flavus and Aspergillus parasiticus, is an extremely toxic and carcinogenic metabolite. Currants are used in the Mediterranean diet as a food with antioxidant properties. Four strains of Aspergillus section Nigri have been isolated from currants originated from Crete and Corinth. In this study AFB₁ production by A. parasiticus and the four strains of Aspergillus section Nigri in Cretan and Corinthian currants (Vitis vinifera L.) is investigated. AFB₁ determination was performed by HPLC–FID. Results revealed that the four strains Aspergillus section Nigri, as well as the aflatoxigenic strain A. parasiticus produced AFB₁ (0.0052–1.31 μg AFB₁ 15 g⁻¹, corresponding to 0.0003–0.087 μg AFB₁ g⁻¹) in both type of currants (Cretan and Corinthian) on the 12th day of observation. Moreover, AFB₁ production, by A. parasiticus in the synthetic Yeast Extract Sucrose (YES) medium was also studied. The ability of AFB₁ production has been affected by the special characteristics of each isolate and the currants substrate.     

Development of an aptasensor for the fast detection of Versicolorin A

Aflatoxins (AFs) are secondary metabolites produced by Aspergillus flavus and Aspergillus parasiticus. The molds may contribute to pre-harvest aflatoxin contamination of susceptible crops. For the customer and food producer, a predictive model for aflatoxin detection is very desirable. Versicolorin A (VerA), which is the first precursor in the pathway of aflatoxin B₁ (AFB₁) biosynthesis, shares similar toxic group with the furofuran structure in aflatoxin B₁. VerA exhibits a much lower teratogenic toxicity than AFB₁ and may be used as a predictive indicator for aflatoxin B₁ contamination of storage crops. Therefore, the development of a fast detection method for VerA is important. One of the randomly computer-generated aptamers of VerA was confirmed by isothermal titration calorimetry with K_d = 9.26 × 10⁻⁶ mol l⁻¹. In addition, a simple and sensitive label-free aptasensor was developed for the electrochemical detection of VerA. According to the results from differential pulse voltammetry (DPV), a linear relationship existed between the log conc. of VerA (ranged from 0.01 to 100 ng ml⁻¹) and the current (△I_p) with a limit of detection (LOD) of 10 pg ml⁻¹. The resulting aptasensor exhibited good reproducibility for detecting VerA and stability after storage for 15 days at 4 °C with acceptable anti-interference against ZEN, OTA, DON, and FB₁. When used in corn samples, the recoveries of VerA were determined to be in the range of 81.3%–104.4 %. Although with some intercross, result suggests that the obtained aptamer for VerA is potentially used as a sorbent for the preparation of solid-phase-extraction procedure to clean up food samples in conjunction with high-performance liquid chromatography analysis.     

Co-occurrence of aflatoxins and ochratoxin A in cereal flours commercialised in Turkey

In this study, we aim to determine co-occurrence of aflatoxins (AFs) and ochratoxin A (OTA) in cereal flours commercialised in Corum, Turkey. One hundred cereal flours were checked for target fungal metabolites between the years 2011 and 2013. The samples were analysed by high-performance liquid chromatography with fluorescence detection (HPLC-FLD) after immunoaffinity column (IAC) clean-up procedure. The method was successfully validated in accordance to European Union guidelines acceptance criteria for specificity, linearity, sensitivity, trueness and repeatability. All the results are well below the maximum limits specified in the EU legislation. AFs were detected neither wheat flour nor rice flour samples, while 66.7% of maize flours contained AFs with maximum concentration of 1.12 μg kg⁻¹. OTA was present in 26.7% of wheat flour, 41.7% of maize flour and 18.8% of rice flour samples, with mean levels of 0.247, 0.218 and 0.154 μg kg⁻¹, respectively. The co-occurence of AFs and OTA was found in 9 maize flour samples.     

Easy and rapid method for the quantitative determination of pyrrolizidine alkaloids in honey by ultra performance liquid chromatography-mass spectrometry: An evaluation in commercial honey

Pyrrolizidine alkaloids (PAs) are toxins biosynthesised by plants and they are known to be present in approximately 6000 plant species, about 3% of all flowering plants. PAs are probably the most widely distributed natural toxins and represent a potential risk to human health, since poisoning caused by these toxins is associated with acute and chronic liver damage and may lead to death. One of the most common sources of PAs exposure in humans is honey consumption. We have developed a quick and easy method to quantify nine different PAs (echimidine, heliotrine, intermedine, lycopsamine, lasiocarpine, retrorsine, seneciphylline, senecionine, senkirkine) in honey based on QuEChERS sample extraction and ultra fast liquid chromatography coupled with mass spectrometry detection. We performed a validation study of the method and it resulted in good precision and accuracy, high recoveries, and good linear calibrations. The limit of detection ranged from 0.021 to 1.39 μg Kg⁻¹ and the limit of quantification from 0.081 to 4.35 μg Kg⁻¹. This new approach was applied to the quantification of PAs in retail honeys purchased in local supermarkets, classified by their country of origin: Italian honeys, blends of honey of European countries and blends of honey of European and non-European countries. The concentrations detected ranged from 1 to 169 μg PAs/kg⁻¹ with higher concentrations in blends of European and non-European honeys. This study reveals that many samples tested would exceed the tolerable daily intake suggested for these substances and they could be a hazard to human health.     

A reliable and sensitive time-resolved fluorescent immunochromatographic assay (TRFICA) for ochratoxin A in agro-products

Immunochromatographic assays (ICAs) are considered as a suitable diagnostic tool for the detection of mycotoxins. Mycotoxins and especially, ochratoxin A are analytes with more demanding sensitivity requirements. To enhance the sensitivity of current immunochromatographic assays for ochratoxin A (OTA), a novel sensitive ICA was developed in this study. In the assay, microspheres enclosing fluorescent europium (III) [Eu(III)] nanoparticles (EuNPs) were used as a label for OTA monoclonal antibody (OTA-mAb) conjugation. Accordingly, assay was called time-resolved fluorescent immunochromatographic assay (TRFICA). The test strip was composed of three parts: a sample pad, nitrocellulose membrane and an absorbent pad. As for detection, a proper concentration of conjugated microspheres was pipetted into the microtube and sample extract was added to it. Then the strip was inserted into the tube and the fluid flow along the strip. The TRFICA results were obtained in 8 min and read by a portable TRFICA strip reader. The established method allows quantitative determination of OTA with limit of detection as low as 1.0 μg kg⁻¹ in the samples. For validation, spiked samples including wheat, maize, soybean and rice were respectively assayed by TRFICA and a standard high performance liquid chromatography equipped with a fluorescence detector (HPLC-FLD), and good agreement of results was obtained between two methods.     

The efficacy of disinfectant misting in the lairage of a pig abattoir to reduce Salmonella and Enterobacteriaceae on pigs prior to slaughter

Water misting/showers are used in abattoir lairages to improve meat quality, and to cool and calm pigs after transport and during hot weather. One novel approach, which has not been investigated to date, is to add a disinfectant to the misting water as a means of topically reducing Salmonella on pigs prior to slaughter, thereby potentially controlling this organism in the abattoir. The objective of this study was therefore to evaluate misting with water or with Virkon^® S (an approved disinfectant for use in the presence of animals), for their ability to topically reduce Salmonella on high seroprevalence pig herds before stunning and to reduce Enterobacteriaceae.Three experimental groups were investigated: control group (i.e., no misting); water group (misting with cold, 15–17 °C, water, herein referred to as water); and a disinfectant group (misting with 0.5% Virkon^® S). As pigs entered the abattoir, each animal was swabbed along its back before being allocated to its experimental group. Each group was randomly assigned to one of 3 lairage pens that were separated by non-trial pens. After 30 min of misting with water or disinfectant, pigs were moved to the stunning area, where each pig was again swabbed, as above. Swabs were analyzed for the presence of Salmonella and enumeration of Enterobacteriaceae.Before misting, Salmonella prevalence on the pigs was 79.0%, 72.1% and 83.6% for the control, water and disinfectant groups, respectively. After misting, Salmonella prevalence increased to 94.3% in the water group; whereas for the disinfectant group, the prevalence increased marginally to 85.9%. No change in Salmonella prevalence was detected for the control group. In line with the Salmonella results, no significant differences were observed in Enterobacteriaceae counts in the control group at either time point (4.37 and 5.01 log₁₀ CFU/cm², respectively) or in the disinfectant group before and after misting (4.02 and 4.26 log₁₀ CFU/cm², respectively). However, a 2.3 log₁₀ CFU/cm² increase in Enterobacteriaceae was recorded for the water group after misting as compared to before misting (p < 0.05).Since misting with water alone increased topical Salmonella contamination on pigs before slaughter, a risk assessment based on known Salmonella data, meat quality and welfare is recommended to determine whether its use is justifiable. On the other hand, the findings from this study suggest that misting with Virkon^® S at 0.5% could have a role in topical antisepsis of pigs contaminated with Salmonella prior to slaughter and as such this warrants further investigation.     

A structural modeling on food safety knowledge,attitude, and behaviour among Bum Bum Island community of Semporna,Sabah

The objective of this study was to evaluate the relationship among food safety knowledge, attitude and behavior in Bum Bum Island community, Semporna, Sabah. Proportional stratified sampling method was used in this survey. A total of 250 respondents were selected randomly from ten villages in Bum Bum Island. Face-to-face interview was conducted to complete the questionnaire. In general, respondents exhibited average food safety knowledge level especially in their awareness of personal hygiene and kitchenware hygiene. Food safety attitude of the community was found strongly affected their food safety behavior in positive way, which was proven by the highest standard β among variables tested (β₁ = 0.885, p < 0.05). However, food safety knowledge was negatively affected the food safety behavior of the respondents (β₁ = −0.128, p < 0.05). Our result confirmed that Structural Equation Modeling (SEM) was successfully used to model the relationship among food safety knowledge, attitude and behavior.     

Antibacterial mechanism of bifidocin A,a novel broad-spectrum bacteriocin produced by Bifidobacterium animalis BB04

Bifidocin A, a novel broad-spectrum bacteriocin produced by Bifidobacterium animalis BB04, was isolated from the feces of a healthy centenarian. To understand the mechanism of the antibacterial action of bifidocin A against gram-negative bacteria, its effects at a minimum inhibitory concentration on cell morphology, intracellular organization, membrane permeability, membrane integrity, and membrane proton motive force (PMF) of Escherichia coli 1.90 were investigated. Scanning and transmission electron microscopy analyses showed that bifidocin A induced alterations in the morphology and intracellular organization of E. coli cells. The intracellular organization was more susceptible to changes induced by bifidocin A than the morphology. Bifidocin A treatment caused the leakage of K⁺ and inorganic phosphate, the release of ATP and UV-absorbing materials, and a collapse of the transmembrane electrical potential and pH gradient in E. coli cells. Confocal laser scanning microscopy images showed that E. coli cells treated with bifidocin A took up propidium iodide. These results suggested that the mechanism of action bifidocin A against E. coli involved dissipation of the PMF of the cytoplasmic membrane, an increase in membrane permeability, pore formation in the cell membrane, a change in membrane integrity, and complete cell disintegration.     

Mycological quality and mycotoxin contamination of Sri Lankan peppers (Piper nigrum L.) and subsequent exposure assessment

The aim of the study was to characterize the toxigenic moulds and to screen different mycotoxins in peppers (Piper nigrum L.) of Sri Lankan origin. Aspergillus flavus, Aspergillus parasiticus, Aspergillus niger and Penicillium spp. were found to be the most dominant fungi. Characterization of the moulds was carried out in A. flavus and parasiticus agar (AFPA) and malt extract agar (MEA) in 77 black pepper (BP) and 11 white pepper (WP) samples. In total, 73% of the BP and 64% of the WP samples were contaminated with A. flavus and/or A. parasiticus (AfAp). A BP sample with water activity (a_w) 0.70 recorded the highest count of AfAp (4.3*10⁴ CFU/g). Moreover, 75% of the BP samples exceeded the safe a_w limit (0.65) set by the European Spice Association (ESA). The frequency of occurrence of A. niger in BP was 62% with counts up to 1.3*10³ CFU/g. Penicillium spp. were found in 61% and 55% of the BP and WP samples, respectively. In BP 94% of the samples had a Penicillium contamination below 10³ CFU/g. Other Aspergillus spp, found in peppers included, Aspergillus terrus, Aspergillus tamarii, Aspergillus candidus, Aspergillus penicilloides, Aspergillus sydowii and Aspergillus fumigatus. Mould counts in BP (10²–10⁴ CFU/g) were significantly higher than that of WP (<10² CFU/g). Apart from the occurrence of “classical mycotoxins” of spices, aflatoxins (<LOQ-18 μg/kg) and ochratoxin A (<LOQ-79 μg/kg), other toxins including fumonisin B1 (<LOQ-135 μg/kg), sterigmatocystin (<LOQ-49 μg/kg) and citrinin (<LOQ-112 μg/kg) were detected in peppers. In total, 63% of the BP samples were contaminated with at least one mycotoxin. Mycotoxin contamination in WP was significantly less compared to BP. The exposure to aflatoxins and ochratoxin A by consuming pepper remains harmless considering the existing pepper dietary intake data of the Sri Lankan population.     

Prevalence and antimicrobial resistance of Salmonella isolated from an integrated broiler chicken supply chain in Qingdao,China

The present study analyzed the prevalence and antimicrobial resistance of Salmonella along an integrated broiler chicken supply chain. A total of 172 Salmonella isolates were recovered from 1148 samples collected from four sample sources (breeder farms, broiler farms, abattoir, and retail markets), representing nine production stages. These Salmonella isolates were examined for antimicrobial susceptibility to 12 different antimicrobial agents using a disk diffusion assay. Among them, 168 were identified as six different serotypes of Salmonella enterica. The predominant serotype was S. Enteritidis (n = 116), followed by S. Infantis (n = 18), S. Gueuletapee (n = 16), S. Derby (n = 12), S. Meleagridis (n = 4), and S. London (n = 2). The remaining four isolates were serogroup-untypeable. A majority of the 172 isolates (96.51%) was resistant to one or more antibiotics and 61.05% of the Salmonella isolates showed a multidrug resistance phenotype. Statistical analysis indicated the one risk product stage for Salmonella contamination occurred in the sample source at the abattoir, specifically the stage of Carcasses after chilling. The majority of S. Enteritidis isolates shared the same pulsed-field gel electrophoresis (PFGE) cluster, suggesting that the S. Enteritidis strain might spread along the broiler chicken supply chain. The prevalence and antimicrobial resistance of Salmonella in different production stages suggest the importance of controlling Salmonella in the broiler chicken supply chain for public health, underlying the need for improved measures of reducing carcass contamination in abattoirs and the appropriate use of antimicrobials in broiler flocks.     

Geobacillus stearothermophilus ATCC 7953 spore chemical germination mechanisms in model systems

Bacterial endospores through their strong resistance to both chemical and physical hurdles constitute a risk for food industry. Inactivation strategies are based on thermal and/or chemical treatments but rely on incomplete knowledge of the mechanisms of inactivation. Alternative strategies were suggested to achieve food safety while improving product quality. One of them relies on the successive germination and inactivation by pasteurization of bacterial spores. However, to date, a gap of knowledge on bacterial spore germination remains and hinders such an application for food sterilization. Geobacillus stearothermophilus ATCC 7953 spore germination mechanisms were investigated by in situ fluorometry combined with plate counts. G. stearothermophilus spores' inner membrane was stained with Laurdan fluorescent dye. While nutrient pathways showed no strong germination with the combinations tested, successful germination up to 3 log₁₀ could be achieved using 60 mmol l⁻¹ calcium-dipicolinic acid (CaDPA) at 55 °C for 2 h. A model for the CaDPA germination mechanism in two phases could be derived which suggested a potential key role of cortex fragments in the germination path, before completion of the cortex degradation. Additionally, it was confirmed that the germination potential of CaDPA, which does not rely on nutrient receptors, is a widespread germination trigger across spore formers. Understanding germination mechanisms and the limitations of different germination paths is important for the development of multi-hurdle approaches to achieve commercial sterility with reduced thermal load.     

Determination of methenamine residues in edible animal tissues by HPLC-MS/MS using a modified QuEChERS method: Validation and pilot survey in actual samples

A modified QuEChERS (quick, easy, cheap, effective, rugged, and safe) method was developed for the determination of methenamine in edible animal tissues by high performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS). Samples were extracted with acetonitrile, and cleaned with anhydrous sodium sulfate and primary secondary amine (PSA) sorbent. An isotope dilution mass spectrometry technique was applied to compensate for matrix effect. The separation was performed on a HILIC column, and the mobile phase composed of acetonitrile-0.1% of formic acid and 5.0 mmol/L of ammonium acetate in water. The method showed a linear relationship in the range of 1.0–20.0 μg/L for methenamine, and the determination coefficients (R²) ranged from 0.9939 to 0.9995. The limit of detection (LOD) and quantification (LOQ) for methenamine in animal tissues sample was 1.5 μg/kg and 5.0 μg/kg, respectively. The average recoveries were 86.7–109.5% at spiked levels of 5.0, 25.0, 100.0 μg/kg. The intra-day precision ranged from 2.6 to 7.0%, and the inter-day precision ranged from 4.9 to 11.3%. The validated method was successfully applied to determination of methenamine in swinish muscle, kidney and liver.     

Antibacterial activity and mechanism of bifidocin A against Listeria monocytogenes

Bifidocin A, produced by Bifidobacterium animalis BB04, is a novel bacteriocin with antimicrobial activity against a wide range of gram-positive and gram-negative foodborne bacteria. The objective of this study was to investigate the antibacterial activity and mechanism of action of bifidocin A against Listeria monocytogenes, one of the most susceptible bacteria to this bacteriocin. The minimum inhibitory concentration (MIC) of bifidocin A for L. monocytogenes 35152 was 0.029 mg/mL. Time-kill assays showed that bifidocin A effectively inhibited the growth of L. monocytogenes in a time-and concentration-dependent manner. The mechanism of action of bifidocin A was studied by analyzing its effects at a MIC on the cell morphology, intracellular organization, membrane permeability, membrane integrity, and membrane proton motive force (PMF) of L. monocytogenes. Scanning and transmission electron microscopy analyses showed that bifidocin A induced alterations in the morphology and intracellular organization of L. monocytogenes cells. Confocal laser scanning microscopy images showed that L. monocytogenes cells treated with bifidocin A took up propidium iodide. Bifidocin A treatment also induced the leakage of K⁺ and inorganic phosphate, the hydrolysis and release of ATP, and a collapse of the transmembrane electrical potential and pH gradient in L. monocytogenes cells. These results suggested that bifidocin A exerted its anti-Listeria monocytogenes effect through the dissipation of the cytoplasmic membrane PMF, increased membrane permeability, cell membrane pore formation, destruction of membrane integrity, and ultimately complete disintegration of the cells.     

Species determination of pine nuts in commercial samples causing pine nut syndrome

Consumption of pine nuts from the species of Pinus armandii has been reported to cause dysgeusia, commonly known as pine mouth, or pine nut syndrome (PNS). However, the number of reports on pine nut consumptions of the different species and PNS is limited. This leaves open the possibility that other pine species than P. armandii could be involved in PNS as well. This study investigated 18 samples involved in PNS and received at the Danish Veterinary and Food Administration in 2011 through 2012. Samples were subjected to gas chromatographic analysis of fatty acids. The content of 11 individual fatty acids was used together with the diagnostic index and the sum of Δ5-fatty acids as diagnostic parameters. Diagnostic parameters from samples were then compared to reference material and literature data to determine the species. In a limited number of samples, the diagnostic parameters matched neither our reference materials nor literature data. However, the morphology, the fatty acid analysis, and externally obtained DNA sequencing data suggest a P. armandii subspecies or a variety. With these possible P. armandii subspecies, P. armandii was identified in all analyzed samples. The application of principal component analysis (PCA) to the data set showed a satisfactory separation of the majority of the 13 pine species included in the study.