Improved Sensitivity of Allergen Detection by Immunoaffinity LC-MS/MS Using Ovalbumin as a Case Study.

Food allergies are caused by severe hypersensitivity to specific food allergens such as the egg protein ovalbumin. It is therefore important to test food products for the presence of allergens to protect allergic people from accidental ingestion. For egg detection, ELISA is the only reasonable commercially available test format, although the recognition of target allergens can be affected by food processing, which may lead to false negative results. Current mass spectrometry-based detection methods may overcome this issue, but these approaches are often less sensitive. Here we combined the advantages of antibody-based and MS-based methods by developing an immunoaffinity LC-MS/MS technique to detect the common egg allergen Gal d 2. We investigated the principal functionality of this method with incurred cookie material containing whole egg powder. We found that the new method matched easily the sensitivity of egg specific ELISA tests. Further western blot experiments indicated that this strategy may be unaffected by food processing, providing an important alternative strategy for the detection and quantification of allergens in food.

Pulsed-Light Treatment of Dried Parsley: Reduction of Artificially Inoculated Salmonella and Impact in Given Quality Parameters.

ABSTRACT: Dried parsley is regularly contaminated with foodborne pathogens, especially Salmonella. Application of contaminated ingredients in ready-to-eat dishes without further thermal treatment represents a considerable health risk. This study examined the suitability of pulsed light as a novel decontamination method of Salmonella in dried parsley, along with the impact on selected quality parameters (chlorophyll content, phenolic compounds, color, and odor) and product characters (temperature and water activity value). Samples were inoculated with one of three Salmonella isolates (Salmonella Cerro or one of two isolates of Salmonella Agona) at two contamination levels of 103 or 107 CFU/g and treated under various experimental factors, including distance to the light source and exposure time, resulting in fluences in the range of 1.8 to 19.9 J/cm2. At selected parameter settings (9.8 and 13.3 J/cm2), the effect of prolonged storage time (48 h) of inoculated samples before treatment on the reduction of Salmonella Cerro was examined. Samples treated at the same fluences were also stored for 35 days at 22 to 25°C. The three Salmonella isolates were significantly reduced by pulsed light (P < 0.05). Reduction factors ranged between 0.3 and 5.2 log CFU with varying sensitivities of the isolates. In general, increasing fluences (depending on exposure time and distance to the light source) resulted in increasing reductions of Salmonella. However, on closer examination, exposure time and distance to the light source had a varying influence on the reduction of the different Salmonella isolates. Decreasing reduction factors were observed by increasing the contamination level and prolonging the storage time of inoculated samples before treatment. No undesirable changes in quality parameters and sensory analysis were detectable at fluences of 9.8 and 13.3 J/cm2, indicating that pulsed light may be a suitable alternative for the decontamination of dried parsley.

Postprandial Metabolic Response to Rapeseed Protein in Healthy Subjects.

Plant proteins have become increasingly important for ecological reasons. Rapeseed is a novel source of plant proteins with high biological value, but its metabolic impact in humans is largely unknown. A randomized, controlled intervention study including 20 healthy subjects was conducted in a crossover design. All participants received a test meal without additional protein or with 28 g of rapeseed protein isolate or soy protein isolate (control). Venous blood samples were collected over a 360-min period to analyze metabolites; satiety was assessed using a visual analog scale. Postprandial levels of lipids, urea, and amino acids increased following the intake of both protein isolates. The postprandial insulin response was lower after consumption of the rapeseed protein than after intake of the soy protein (p < 0.05), whereas the postmeal responses of glucose, lipids, interleukin-6, minerals, and urea were comparable between the two protein isolates. Interestingly, the rapeseed protein exerted stronger effects on postprandial satiety than the soy protein (p < 0.05). The postmeal metabolism following rapeseed protein intake is comparable with that of soy protein. The favorable effect of rapeseed protein on postprandial insulin and satiety makes it a valuable plant protein for human nutrition.

Prediction of flocculation ability of brewing yeast inoculates by flow cytometry, proteome analysis, and mRNA profiling.

The ability of brewing yeast to flocculate is an important feature for brewing of qualitatively good beer. Flocculation involves two main cell wall structures, which are the flocculation proteins (flocculins) and mannans, to which these flocculins bind. Unfortunately, in practice, the flocculation ability may get lost after several repitches. Flow cytometry was employed to analyze glucose and mannose structures of the cell surface by application of fluorescent lectins. Validation of the expression of the flocculin genes Lg-FLO1, FLO1, FLO5, and FLO9 was carried out using microarray techniques. SDS-PAGE, western blot, and ESI-MS/MS analyses served to isolate and determine yeast cell flocculins. Mannose and glucose labeling with fluorescent lectins allowed differentiating powdery and flocculent yeast cells under laboratory conditions. Using microarray techniques and proteomics, the four flocculation genes Lg-FLO1, FLO1, FLO5, FLO9, and the protein Lg-Flo1p were identified as factors of major importance for flocculation. The expression of the genes was several times higher in flocculent yeast cells than in powdery ones. Flow cytometry is a fast and simple method to quantify the proportions of powdery and flocculent yeast cells in suspensions under defined cultivation conditions. However, differentiation under industrial conditions will require mRNA and protein expression profiling.

Pulsed light treatment for the reduction of Salmonella Typhimurium and Yersinia enterocolitica on pork skin and pork loin.

The aim of the presented study was to investigate the impact of pulsed light on the reduction of Salmonella Typhimurium and Yersinia enterocolitica on pork skin and loin. Fluences of 0.52 to 19.11 J/cm2 were applied to the pathogen-inoculated products to perform microbiological studies, as well as analyses of color, temperature, lipid peroxidation and odor. Reductions on pork skin ranged from 1.73 to 3.16 log for Salmonella and from 1.48 to 4.37 log for Yersinia. Microbial reduction was significantly lower on pork loin, varying between a minimum of 0.4 and a maximum of 1.7 log for both pathogens. Treatments ≥7.36 J/cm2 modified the color parameters of pork skin and fluences ≥9.66 J/cm2 rendered pork loin samples less red. All studies with pulsed light resulted in odor changes, except for the experiment on pork skin at 0.52 J/cm2. Despite significant microbiological reduction on pork skin, further studies should be carried out to optimize this promising technology.

Fat perception in the human frontal operculum, insular and somatosensory cortex.

Here, we combined magnetic resonance imaging with lesion-symptom mapping in patients with chronic brain lesions to investigate brain representations of sugar and fat perception. Patients and healthy controls rated chocolate milkshakes that only differed in sugar or fat content. As compared to controls, patients showed an impaired fat, but not sugar perception. Impairments in fat perception overlapped with the anterior insula and frontal operculum, together assumed to underpin gustatory processing. We also identified the mid-dorsal insula as well as the primary and secondary somatosensory cortex - regions previously assumed to integrate oral-sensory inputs. These findings suggest that fat perception involves a specific set of brain regions that were previously reported to underpin gustatory processing and oral-sensory integration processes.

Atmospheric pressure plasma jet treatment of Salmonella Enteritidis inoculated eggshells.

Contamination of eggshells with Salmonella Enteritidis remains a food safety concern. In many cases human salmonellosis within the EU can be traced back to raw or undercooked eggs and egg products. Atmospheric pressure plasma is a novel decontamination method that can reduce a wide range of pathogens. The aim of this work was to evaluate the possibility of using an effective short time cold plasma treatment to inactivate Salmonella Enteritidis on the eggshell. Therefore, artificially contaminated eggshells were treated with an atmospheric pressure plasma jet under different experimental settings with various exposure times (15-300s), distances from the plasma jet nozzle to the eggshell surface (5, 8 or 12mm), feed gas compositions (Ar, Ar with 0.2, 0.5 or 1.0% O2), gas flow rates (5 and 7slm) and different inoculations of Salmonella Enteritidis (101-106CFU/cm2). Atmospheric pressure plasma could reduce Salmonella Enteritidis on eggshells significantly. Reduction factors ranged between 0.22 and 2.27 log CFU (colony-forming units). Exposure time and, particularly at 104CFU/cm2 inoculation, feed gas had a major impact on Salmonella reduction. Precisely, longer exposure times led to higher reductions and Ar as feed gas was more effective than ArO2 mixtures.

Bench-scale study of the effect of phosphate on an aerobic iron oxidation plant for mine water treatment.

At the opencast pit Nochten acidic iron- and sulfate-rich mine waters are treated biotechnologically in a mine-water treatment plant by microbial iron oxidation. Due to the low phosphate concentration in such waters the treatment plant was simulated in bench-scale to investigate the influence of addition of potassium dihydrogen phosphate on chemical and biological parameters of the mine-water treatment. As a result of the phosphate addition the number of cells increased, which resulted in an increase of the iron oxidation rate in the reactor with phosphate addition by a factor of 1.7 compared to a reference approach without phosphate addition. Terminal restriction fragment length polymorphism (T-RFLP) analysis during the cultivation revealed a shift of the microbial community depending on the phosphate addition. While almost exclusively iron-oxidizing bacteria related to "Ferrovum" sp. were detected with phosphate addition, the microbial community was more diverse without phosphate addition. In the latter case, iron-oxidizing bacteria ("Ferrovum" sp., Acidithiobacillus spp.) as well as non-iron-oxidizing bacteria (Acidiphilium sp.) were identified.

New cultivation medium for "Ferrovum" and Gallionella-related strains.

Since the first isolation of the well-known iron oxidizer Acidithiobacillus ferrooxidans various media and techniques have been developed to isolate new species of acidophilic iron-oxidizing bacteria. A successful strategy in many cases was the use of iFeo medium in double-layer plates with a heterotrophic strain in the underlayer. However, even with samples which had been shown by molecular techniques to be dominated by "Ferrovum myxofaciens" and Gallionella-related bacteria, these bacteria were isolated considerably less frequently than Acidithiobacillus spp. on iFeo. Therefore, a new medium was designed which corresponded largely to the chemical composition of the mine water in a treatment plant dominated by the bacterial groups mentioned and was called artificial pilot-plant water (APPW). The analyses of approximately 500 colonies obtained from mine waters of two different sampling sites by PCR with primers specific for Acidithiobacillus spp., "Ferrovum" spp., Gallionella relatives, and Acidiphilium spp. revealed higher abundances of "Ferrovum" spp. and Gallionella relatives on the newly designed APPW medium than on iFeo which favored Acidithiobacillus spp. Molecular analysis of the colonies obtained indicated the occurrence of at least two species of iron-oxidizing bacteria and/or the heterotrophic Acidiphilium spp. in most of the colonies. Furthermore, the influence on the isolation of the concentrations of iron, phosphate, and ammonium of APPW, in levels of the iFeo medium previously described was studied.

A cytomic approach reveals population heterogeneity of Cupriavidus necator in response to harmful phenol concentrations.

The understanding of functions of cells within microbial populations or communities is certainly needed for existing and novel cytomic approaches which grip the individual scale. Population behaviour results from single cell performances and is caused by the individual genetic pool, history, life cycle states and microenvironmental surroundings. Mimicking natural impaired environments, the paper shows that the Gram-negative Betaproteobacterium Cupriavidus necator dramatically altered its population heterogeneity in response to harmful phenol concentrations. Multiparametric flow cytometry was used to follow variations in structural cellular parameters like chromosome contents and storage materials. The functioning of these different cell types was resolved by ensuing proteomics after the cells' spatial separation by cell sorting, finding 11 proteins changed in their expression profile, among them elongation factor Tu and the trigger factor. At least one third of the individuals clearly underwent starving states; however, simultaneously these cells prepared themselves for entering the life cycle again. Using cytomics to recognise individual structure and function on the microbial scale represents an innovative technical design to describe the complexity of such systems, overcoming the disadvantage of small cell volumes and, thus, to resolve bacterial strategies to survive harmful environments by altering population heterogeneity.