Acrylamide inhibits autophagy, induces apoptosis and alters cellular metabolic profiles.

Acrylamide (ACR) is generated during thermal processing of carbohydrate-rich foods at high temperature and can directly enter the body through ingestion, inhalation and skin contact. The toxicity of ACR has been widely studied. The main results of these studies show that exposure to ACR can cause neurotoxicity in both animals and humans, and show reproductive toxicity and carcinogenicity in rodent animal models. However, the mechanism of toxicity of ACR has not been studied by metabolomics approaches, and the effect of ACR on autophagy remains unknown. Here, U2OS cell were treated with ACR 6 and 24 h and collected for further study. We have demonstrated that ACR inhibited autophagic flux, and increased ROS content. Accumulation of ROS resulted in increase of apoptosis rates and secretion of inflammatory factors. In addition, significant differences in metabolic profiles were observed between ACR treated and control cells according to multiple analysis models. A total of 73 key differential metabolites were identified. They were involved in multiple metabolic pathways. Among them, exposure to ACR caused glycolysis/gluconeogenesis attenuation by decreasing levels of glycolytic intermediates, reduced the rate of the TCA cycle, while elevating levels of several amino acid metabolites and lipid metabolites. In summary, our study provides useful evidence of cytotoxicity caused by ACR via metabolomics and multiple bioanalytic methods.

Combining metabolomics with bioanalysis methods to investigate the potential toxicity of dihexyl phthalate.

Dihexyl phthalate (DHP) is one of the most commonly used phthalate esters in various plastic and consumer products. Human are inevitably exposed to DHPs. Although several animal and human experiments have revealed that DHP can cause multiple toxicities, few studies have previously assessed the effects of DHP exposure by liquid chromatography mass spectrometry (LC-MS) analysis combine with molecular biology methods on human cells. Therefore, the purpose of our study was to investigate the effect of DHP on human cell metabolism by systems biology methods. In this study, U2 OS cancer cells were treated with 10 µM DHP for metabolomics analysis and apoptosis analysis at indicate time. Metabolomic study of the metabolic changes caused by DHP in U2 OS cells was performed for the first time using integrative liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (LC-Q-TOF-MS). To investigate the possible reason of fatty acids level altered by DHP, we measured some key fatty acid synthesis and oxidation-related enzyme expression levels by quantitative real-time PCR (Q-PCR). Apoptotic cells were analyzed by flow cytometry and apoptosis-related gene expressions were measured by Q-PCR. 2',7'-Dichlorofluorescein diacetate (DCFH-DA) staining was used to evaluate ROS content. Partial least squares-discriminate analysis (PLS-DA) clearly showed that significant differences in metabolic profiles were observed in U2 OS cells exposed to DHP compared with controls. A total of 58 putative metabolites in electrospray ionization source (ESI) + mode and 32 putative metabolites in ESI-mode were detected, the majority of the differential metabolites being lipids and lipid-like molecules. Among them, the altered fatty acids level corresponded to expression levels of genes encoding enzymes related to fatty acids synthesis and oxidation. Moreover, DHP induced reactive oxygen species (ROS) accumulation, promoted cell apoptosis and inflammation, and resulted in a significant increase in apoptosis and inflammation-related gene expression levels compared with controls. In summary, our results suggested that metabolomics combined with molecular bioanalysis methods could be an efficient tool to assess toxic effects, which contribute to explore the possible cytotoxicity mechanisms of DHP, and provide a basis for further research.

Chicken fillets subjected to UV-C and pulsed UV light: Reduction of pathogenic and spoilage bacteria, and changes in sensory quality.

We have compared the efficacy of continuous ultraviolet (UV-C) (254 nm) and pulsed UV light in reducing the viability of Salmonella Enteritidis, Listeria monocytogenes, Staphylococcus aureus, enterohemorrhagic Escherichia coli, Pseudomonas spp., Brochothrix thermospacta, Carnobacterium divergens, and extended-spectrum ß-lactamase producing E. coli inoculated on chicken fillet surface. Fluences from 0.05 to 3.0 J/cm2 (10 mW/cm2, from 5 to 300 s) used for UV-C light resulted in average reductions from 1.1 to 2.8 log cfu/cm2. For pulsed UV light, fluences from 1.25 to 18.0 J/cm2 gave average reductions from 0.9 to 3.0 log cfu/cm2. A small change in the odor characterized as sunburnt and increased concentration of volatile compounds associated with burnt odor posed restrictions on the upper limit of UV treatment, however no sensory changes were observed after cooking the meat. Treatments under modified atmosphere conditions using a UV permeable top film gave similar or slightly lower bacterial reductions. PRACTICAL APPLICATIONS: Ultraviolet (UV) light may be used for decontaminating the surface of food products and reduce viability of pathogenic and spoilage bacteria. Exposure of raw chicken fillet surface to various doses of continuous UV-C or pulsed UV light proposed in the present work represent alternatives for microbiological improvement of this product. Chicken fillets can be treated in intact packages covered with UV permeable top film, thus avoiding recontamination of the meat. UV-C light treatment is a low cost strategy with low maintenance, whereas pulsed UV light involves more elaborate equipment, but treatment times are short and less space is required. Both methods can be helpful for producers to manage the safety and quality of chicken fillets.

Microbial Safety and Sensory Analyses of Cold-Smoked Salmon Produced with Sodium-Reduced Mineral Salts and Organic Acid Salts.

Cold-smoked (CS) salmon contains high levels of sodium salts, and excess dietary sodium intake is associated with an array of health complications. CS salmon may also represent a food safety risk due to possible presence and growth of the foodborne pathogen Listeria monocytogenes which may cause fatal human infections. Here we determine how reformulated CS salmon using commercial sodium-reduced salt replacers containing KCl (e.g., Nutek, Smart Salt, SOLO-LITE) and acetate-based preservative salts (Provian K, proviant NDV) affect sensory properties, quality, and microbial safety. Initial sensory screening of sodium-reduced CS salmon was followed by L. monocytogenes growth analyses in selected variants of reformulated CS salmon, and finally by analyses of CS salmon variants produced in an industrial smokehouse. Projective mapping indicated overall minor sensory changes in sodium-replaced samples compared with a conventional product with NaCl. Growth of L. monocytogenes was temperature-dependent (4 °C vs. 8 °C storage) with similar growth in sodium-reduced and conventional CS salmon. The addition of 0.9% of the preservative salts Provian K or Provian NDV gave up to 4 log lower L. monocytogenes counts in both sodium-reduced and conventional cold-smoked salmon after 29 days of chilled storage. No changes in pH (range 6.20−6.33), aw levels (range 0.960−0.973), or weight yield (96.8 ± 0.2%) were evident in CS salmon with salt replacers or Provian preservative salts. Analyses of CS salmon produced with selected mineral salt and preservative salt combinations in an industrial salmon smokery indicated marginal differences in sensory properties. Samples with the preservative salt Provian NDV provided L. monocytogenes growth inhibition and low-level total viable counts (<2.8 log/g) dominated by Photobacterium and Carnobacterium during storage. Production of sodium-reduced CS salmon with inhibiting salts provides a simple method to achieve a healthier food product with increased food safety.

Identifying Lipid Metabolites Influenced by Oleic Acid Administration Using High-Performance Liquid Chromatography-Mass Spectrometry-Based Lipidomics.

Oleic acid (OA), one of the most important monounsaturated fatty acids, possesses protective properties against chronic liver disease (CLD) development, but the underlying metabolic metabolism remains unknown. HPLC-MS-based lipidomics was utilized to identify and quantify the endogenously altered lipid metabolites when hepatocytes were exposed to OA administration. The identified lipids could be grouped into 22 lipid classes; of which, 10 classes were significantly influenced by the OA treatment: lysophosphatidylcholine (LPC), phosphatidylglycerol (PG), ceramides (Cer), hexosylceramides (Hex1Cer), dihexosylceramides (Hex2Cer), cholesterol ester (ChE), and coenzyme (Co) were decreased, while diglyceride (DG), triglyceride (TG), and acyl carnitine (AcCa) were increased. In addition, as the variable importance in projection (VIP) list (VIP > 1.0 and P < 0.05) showed, 478 lipid species showed significant difference with OA administration, and these molecules could be potential biomarkers in conjunction with OA administration. In summary, our results provided a novel perspective to understand the influences of OA administration by investigating endogenous altered levels of lipid metabolites via lipidomics.

Growth Behavior of Listeria monocytogenes in a Traditional Norwegian Fermented Fish Product (Rakfisk), and Its Inhibition through Bacteriophage Addition.

Listeria monocytogenes may persist in food production environments and cause listeriosis. In Norway, a product of concern is the traditional and popular fermented fish product "rakfisk", which is made from freshwater salmonid fish by mild-salting and brine maturation at low temperatures for several months. It is eaten without any heat treatment, and L. monocytogenes, therefore, poses a potential hazard. We investigated the effect of salt and temperature on the growth of L. monocytogenes in rakfisk during the 91 days of maturation. The amounts of organic acids produced during fermentation were too low to inhibit growth of L. monocytogenes. Temperature was clearly the most important parameter for controlling L. monocytogenes. At 7 °C, approximately 2 log growth was observed during the first 14 days of fermentation, and the level of L. monocytogenes thereafter remained constant. At 4 °C, only a little growth potential of the pathogen was recorded. We also investigated the effect of the anti-Listeria bacteriophage P100 on rakfisk with added L. monocytogenes. The phage was introduced to the L. monocytogenes-inoculated fish before fermentation, and an average of 0.9 log reduction was observed throughout the fermentation period. This is the first study of L. monocytogenes behavior in rakfisk and points to possible measures for increasing the product safety.

Comparison of UV-C and Pulsed UV Light Treatments for Reduction of Salmonella, Listeria monocytogenes, and Enterohemorrhagic Escherichia coli on Eggs.

Ten percent of all strong-evidence foodborne outbreaks in the European Union are caused by Salmonella related to eggs and egg products. UV light may be used to decontaminate egg surfaces and reduce the risk of human salmonellosis infections. The efficiency of continuous UV-C (254 nm) and pulsed UV light for reducing the viability of Salmonella Enteritidis, Listeria monocytogenes, and enterohemorrhagic Escherichia coli on eggs was thoroughly compared. Bacterial cells were exposed to UV-C light at fluences from 0.05 to 3.0 J/cm2 (10 mW/cm2, for 5 to 300 s) and pulsed UV light at fluences from 1.25 to 18.0 J/cm2, resulting in reductions ranging from 1.6 to 3.8 log, depending on conditions used. Using UV-C light, it was possible to achieve higher reductions at lower fluences compared with pulsed UV light. When Salmonella was stacked on a small area or shielded in feces, the pulsed UV light seemed to have a higher penetration capacity and gave higher bacterial reductions. Microscopy imaging and attempts to contaminate the interior of the eggs with Salmonella through the eggshell demonstrated that the integrity of the eggshell was maintained after UV light treatments. Only minor sensory changes were reported by panelists when the highest UV doses were used. UV-C and pulsed UV light treatments appear to be useful decontamination technologies that can be implemented in continuous processing.

Survival of Five Strains of Shiga Toxigenic Escherichia coli in a Sausage Fermentation Model and Subsequent Sensitivity to Stress from Gastric Acid and Intestinal Fluid.

The ability of foodborne pathogens to exhibit adaptive responses to stressful conditions in foods may enhance their survival when passing through the gastrointestinal system. We aimed to determine whether Escherichia coli surviving stresses encountered during a model dry-fermented sausage (DFS) production process exhibit enhanced tolerance and survival in an in vitro gastrointestinal model. Salami sausage batters spiked with five E. coli isolates, including enterohaemorrhagic E. coli strains isolated from different DFS outbreaks, were fermented in a model DFS process (20°C, 21 days). Control batters spiked with the same strains were stored at 4°C for the same period. Samples from matured model sausages and controls were thereafter exposed to an in vitro digestion challenge. Gastric exposure (pH 3) resulted in considerably reduced survival of the E. coli strains that had undergone the model DFS process. This reduction continued after entering intestinal challenge (pH 8), but growth resumed after 120 min. When subjected to gastric challenge for 120 min, E. coli that had undergone the DFS process showed about 2.3 log10⁡ lower survival compared with those kept in sausage batter at 4°C. Our results indicated that E. coli strains surviving a model DFS process exhibited reduced tolerance to subsequent gastric challenge at low pH.

Real-time closed tube single nucleotide polymorphism (SNP) quantification in pooled samples by quencher extension (QEXT).

Quencher extension (QEXT) is a novel single step closed tube real-time method to quantify SNPs using reporters and quenchers in combination with primer extension. A probe with a 5'-reporter dye is single base extended with a dideoxy nucleotide containing a quencher dye if the target SNP allele is present. The extension is recorded from the quenching (reduced fluorescence) of the reporter dye. This avoids the influence of the unincorporated dye-labeled nucleotides, resulting in high accuracy and a high signal-to-noise ratio. The relative amount of a specific SNP allele is determined from the nucleotide incorporation rate in a thermo-cycling reaction. We tested the QEXT assay using five SNPs in the Listeria monocytogenes inlA gene as a model system. The presence of the target SNP alleles was determined with high statistical confidence (P < 0.0005). The quantitative detection limits were between 0 and 5% for the targeted SNP alleles on a background of other SNP alleles (P < 0.05). The QEXT method is directly adaptable to current real-time PCR equipment and is thus suited for high throughput and a wide application range.

Effect of relevant environmental stresses on survival of enterohemorrhagic Escherichia coli in dry-fermented sausage.

Dry-fermented sausages (DFSs) have been linked to several serious foodborne outbreaks of enterohemorrhagic Escherichia coli (EHEC). The ability of pathogens to utilize adaptive responses to different stressful conditions intended to control their growth in foods, food preparation and production processes may enhance their survival. In certain cases, induced tolerance to one type of stress may lead to enhanced resistance to the applied stress as well as to other stresses. We exposed two EHEC strains, MF3582 of serotype O157:H- and MF5554 of serogroup O145, to different stresses commonly encountered during a production process. The two EHEC strains, previously shown to have different abilities to survive DFS production process conditions, were subjected to low temperatures (4°C and 12°C), 5% NaCl or 1% lactic acid for 6days prior to being added to sausage batters. Survival of EHEC was recorded in salami of two recipes, fermented at two temperatures (20°C and 30°C). The results showed that recipe type had the largest impact on EHEC reductions where Moderate recipe (MR) salami batters containing increased levels of NaCl, glucose and NaNO2 provided enhanced EHEC reductions in salami (2.6 log10) compared to Standard recipe (SR) salami (1.7 log10). Effects of pre-exposure stresses were dependent both on strain and recipe. While acid adaptation of MF5554 provided enhanced log10 reductions from 2.0 to 3.0 in MR sausages, adaptation to a combination of acid and salt stress showed the opposite effect in SR sausages with reductions of only 1.1 log10 as compared to the average of 1.8 log10 for the other SR sausages. Otherwise, the salt and acid adaptation single stresses had relatively small effects on EHEC survival through the DFS production process and subsequent storage and freeze/thaw treatments. Growing cells and cells frozen in batter survived poorly in MR sausages with an average reduction of 3.4 and 3.2 log10, respectively. The reductions of EHEC after storage of DFS increased with higher temperature and storage time. Up to 3.7 log10 additional reduction was obtained when MF3582 was stored for 2months at 20°C. In conclusion, adaptation of EHEC to acid, salt and low temperatures prior to being introduced in a DFS production process has limited, but strain dependent effects on EHEC reductions. Producers should avoid conditions leading to acid and salt adapted cells that can contaminate the sausage batter. Recipe parameters had the largest impact on EHEC reductions while storage at 20°C is effective for enhanced reductions in finished products.

Ethylhexylglycerin Impairs Membrane Integrity and Enhances the Lethal Effect of Phenoxyethanol.

Preservatives are added to cosmetics to protect the consumers from infections and prevent product spoilage. The concentration of preservatives should be kept as low as possible and this can be achieved by adding potentiating agents. The aim of the study was to investigate the mechanisms behind potentiation of the bactericidal effect of a commonly used preservative, 2-phenoxyethanol (PE), by the potentiating agent ethylhexylglycerin (EHG). Sub-lethal concentrations of EHG (0.075%) and PE (0.675%) in combination led to rapid killing of E. coli (> 5 log reduction of cfu after 30 min), leakage of cellular constituents, disruption of the energy metabolism, morphological deformities of cells and condensation of DNA. Used alone, EHG disrupted the membrane integrity even at low concentrations. In conclusion, sub-lethal concentrations of EHG potentiate the effect of PE through damage of the cell membrane integrity. Thus, adding EHG to PE in a 1:9 ratio has a similar effect on membrane damage and bacterial viability as doubling the concentration of PE. This study provides insight about the mechanism of action of a strong potentiating agent, EHG, which is commonly used in cosmetics together with PE.

Prolonged shelf life and reduced drip loss of chicken filets by the use of carbon dioxide emitters and modified atmosphere packaging.

Modified atmosphere packaging containing CO2 is widely used for extending the shelf life of chicken meat. Active packaging by adding CO2 emitter sachets to packages of meat is an alternative to traditional modified atmosphere packaging. The purpose of the study was to investigate the shelf life of chicken filets under different CO2 concentrations at 4°C storage. The inhibition of microbial growth was proportional to the CO2 concentration. Storage in 100% CO2 both with and without a CO2 emitter sachet gave a microbiological shelf-life extension of 7 days compared with 60% CO2. Carnobacterium divergens, Carnobacterium sp., and Lactococcus sp. were the dominating species at the end of the storage period. During storage in pure CO2, the carbon dioxide dissolved in the meat and caused the collapse of the packages. The resulting squeeze of the meat lead to a severe increase in drip loss. The drip loss was reduced profoundly by using the CO2 emitting sachet in the packages. The addition of CO2 emitters can easily be implemented at industrial packaging lines without reduction in production efficiency.

Multiplex, quantitative, ligation-dependent probe amplification for determination of allergens in food.

Legislation requires labeling of foods containing allergenic ingredients. Here, we present a robust 10-plex quantitative and sensitive ligation-dependent probe amplification method, the allergen-multiplex ligation-dependent probe amplification (MLPA) method, for specific detection of eight allergens: sesame, soy, hazelnut, peanut, lupine, gluten, mustard, and celery. Ligated probes were amplified by polymerase chain reaction (PCR), and amplicons were detected using capillary electrophoresis. Quantitative results were obtained by comparing signals with an internal positive control. The limit of detection varied from approximately 5 to 400 gene copies, depending on the allergen. The method was tested using different foods spiked with mustard, celery, soy, or lupine flour in the 1-0.001% range. Depending on the allergen, sensitivities were similar or better than those obtained with qPCR. The allergen-MLPA method is modular and can be adapted by adding probe pairs for other allergens. The DNA-based allergen-MLPA method will constitute a complementary method to the traditional protein-based methods.

Reduction of verotoxigenic Escherichia coli in production of fermented sausages.

After a number of foodborne outbreaks of verotoxigenic Escherichia coli involving fermented sausages, some countries have imposed regulations on sausage production. For example, the US Food Safety and Inspection Service requires a 5 log(10) reduction of E. coli in fermented products. Such regulations have led to a number of studies on the inactivation of E. coli in fermented sausages by changing processing and post-processing conditions. Several factors influence the survival of E. coli such as pre-treatment of the meat, amount of NaCl, nitrite and lactic acid, water activity, pH, choice of starter cultures and addition of antimicrobial compounds. Also process variables like fermentation temperature and storage time play important roles. Though a large variety of different production processes of sausages exist, generally the reduction of E. coli caused by production is in the range 1-2 log(10). In many cases this may not be enough to ensure microbial food safety. By optimising ingredients and process parameters it is possible to increase E. coli reduction to some extent, but in some cases still other post process treatments may be required. Such treatments may be storage at ambient temperatures, specific heat treatments, high pressure processing or irradiation. HACCP analyses have identified the quality of the raw materials, low temperature in the batter when preparing the sausages and a rapid pH drop during fermentation as critical control points in sausage production. This review summarises the literature on the reduction verotoxigenic E. coli in production of fermented sausages.

Use of ethidium monoazide and PCR in combination for quantification of viable and dead cells in complex samples.

The distinction between viable and dead cells is a major issue in many aspects of biological research. The current technologies for determining viable versus dead cells cannot readily be used for quantitative differentiation of specific cells in mixed populations. This is a serious limitation. We have solved this problem by developing a new concept with the viable/dead stain ethidium monoazide (EMA) in combination with real-time PCR (EMA-PCR). A dynamic range of approximately 4 log(10) was obtained for the EMA-PCR viable/dead assay. Viable/dead differentiation is obtained by covalent binding of EMA to DNA in dead cells by photoactivation. EMA penetrates only dead cells with compromised membrane/cell wall systems. DNA covalently bound to EMA cannot be PCR amplified. Thus, only DNA from viable cells can be detected. We evaluated EMA-PCR with the major food-borne bacterium Campylobacter jejuni as an example. Traditional diagnosis of this bacterium is very difficult due to its specific growth requirements and because it may enter a state where it is viable but not cultivable. The conditions analyzed included detection in mixed and natural samples, survival in food, and survival after disinfection or antibiotic treatment. We obtained reliable viable/dead quantifications for all conditions tested. Comparison with standard fluorescence-based viable/dead techniques showed that the EMA-PCR has a broader dynamic range and enables quantification in mixed and complex samples. In conclusion, EMA-PCR offers a novel real-time PCR method for quantitative distinction between viable and dead cells with potentially very wide application.

Increased speed and accuracy of single nucleotide polymorphism (SNP) quantification by quencher extension (QEXT).

Extract: Single nucleotide polymorphisms (SNPs) often represent allelic variants of genes. The differences between individuals within a species are mainly due to SNPs. SNPs are commonly used as genetic markers for genetic diseases and other phenotypic traits. Currently, throughput and accuracy are limiting factors in the widespread use of SNPs in diagnostics. A large number of both individuals and SNPs often have to be screened when searching for correlation between specific alleles and phenotypic traits. SNP genotyping analysis of one sample at a time is extremely costly and time consuming. Pooling DNA samples prior to quantitative SNP allele frequency determination in affected and control populations is an attractive way to increase throughput and reduce costs. Individual genotyping is only done when there is a significant discrepancy in allele frequencies between the affected and control populations. Despite extensive efforts, the technology to accurately quantify SNPs in sample pools is still lacking.

Biofilm formation and the presence of the intercellular adhesion locus ica among staphylococci from food and food processing environments.

In clinical staphylococci, the presence of the ica genes and biofilm formation are considered important for virulence. Biofilm formation may also be of importance for survival and virulence in food-related staphylococci. In the present work, staphylococci from the food industry were found to differ greatly in their abilities to form biofilms on polystyrene. A total of 7 and 21 of 144 food-related strains were found to be strong and weak biofilm formers, respectively. Glucose and sodium chloride stimulated biofilm formation. The biofilm-forming strains belonged to nine different coagulase-negative species of Staphylococcus. The icaA gene of the intercellular adhesion locus was detected by Southern blotting and hybridization in 38 of 67 food-related strains tested. The presence of icaA was positively correlated with strong biofilm formation. The icaA gene was partly sequenced for 22 food-related strains from nine different species of Staphylococcus, and their icaA genes were found to have DNA similarities to previously sequenced icaA genes of 69 to 100%. Northern blot analysis indicated that the expression of the ica genes was higher in strong biofilm formers than that seen with strains not forming biofilms. Biofilm formation on polystyrene was positively correlated with biofilm formation on stainless steel and with resistance to quaternary ammonium compounds, a group of disinfectants.