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[Gas chromatographic determination of amino acids with nitrogen-selective detection].

Heptafluorobutyryl isobutyl ester derivatives of amino acids were determined, using the thermionic nitrogen-phosphorus detector. The linearity of the detector (more than 5 decades), allows determinations above 0.3 pMol and 110 nMol, without the need for further dilution. Due to the specificity of the detector, no fractionation or other pretreatment of the samples prior to hydrolysis were necessary. The separation of the amino acid derivatives ranges from fair to excellent when comparing separations on packed columns with those on capillary columns. On the capillary analyser, however, only a few runs could be performed, since it was not available for a long enough period of time. The reproducibility of the determination was high in the monobasic amino acids, with a coefficient of variation (c.v.) of less than 3, but somewhat worse in arginine, histidine, cystine and minor components. Histidine needs an on-column acylation by the co-injection of the hydrolysate with acetic anhydride. The amino acids tryptophan, cysteic acid and methionine sulphone have not been investigated until now. A disadvantage of the procedure compared with other analytical methods is the rather complicated derivatisation, which until now, has not been easily automated. Finally, the practicability of the method is demonstrated by the results of eightfold determinations in 13 heat-treated (canned) food samples.

Amino Acids↗

Lipid extraction from blood and biological samples and concentrations of dioxin-like compounds.

The extraction of lipid from human blood and other biological materials in the determination of dioxin-like and organochlorine compounds has been studies using different amounts of extracting solvent. When large volumes of ethanol-hexane are used to extract small blood samples, the total lipid extracted is incomplete as the polar phospholipids do not partition into the organic phase. This failure to extract all the blood lipid results in an inaccurately high concentration expressed on a lipid basis and, by corollary, a high biased body burden. This solvent dependence of lipid extraction does not appear to be a problem for most other biological and food samples since over 90% of the lipid is in the form of readily extractable neutral glycerides.

Adipose Tissue↗

Detection of pathogenic Yersinia enterocolitica in milk and pork using a DIG-labelled probe targeted against the yst gene.

A PCR-amplified, digoxigenin (DIG)-labelled yst probe was evaluated in naturally and artificially contaminated pork and milk samples for its ability to detect and differentiate between pathogenic and nonpathogenic strains of Yersinia enterocolitica following enrichment in irgasan-ticarcillin-chlorate (ITC) enrichment broth. A total of 44 raw pork samples were obtained from one meat-processing factory and two local retail stores. Y. enterocolitica serogroup O:3/biovar 4 was isolated from 6 of the 44 (14%) pork samples tested. The hybridization results were in good agreement when compared with those from standard biochemical and serological tests. The lowest concentration detectable by a colony hybridization technique in the milk samples inoculated with Y. enterocolitica O:8 PA-A corresponded to 10 cfu/ml in the original sample. No pathogenic serovars were detected in 6 raw milk and 3 pasteurized milks tested. The results of this investigation demonstrate that the DIG-labelled yst probe is a reliable tool for rapid detection of pathogenic Y. enterocolitica in naturally and artificially contaminated food samples.

Animals↗

Outbreak of acute gastroenteritis in an air force base in Western Greece.

BACKGROUND: On the 20th September 2005, soldiers and staff at the Air Force base in Western Greece experienced an outbreak of acute gastroenteritis. The purpose of this study was to identify the agent and the source of the outbreak in order to develop control measures and to avoid similar outbreaks in the future. METHODS: A case-control analytical approach was employed with 100 randomly selected cases and 66 controls. Patients completed standardized questionnaires, odds ratios were calculated and statistical significance was determined using chi2 test. In addition, to identify the source of the infection, we performed bacteriological examination of food samples (included raw beef, cooked minced meat, grated cheese and grated cheese in sealed package) collected from the cuisine of the military unit. RESULTS: More than 600 out of the 1,050 individuals who ate lunch that day, became ill. The overall attack rate, as the military doctor of the unit estimated it, was at least 60%. The overall odds ratio of gastroenteritis among those who had lunch was 370 (95% CI: 48-7700) as compared to those who didn't eat lunch. Among the symptoms the most prominent were watery diarrhoea (96%) and abdominal pain (73%). The mean incubation period was 9 h and the median duration of the symptoms was 21 h. In the bacteriological examination, Staphylococcus aureus was detected in a sample of raw beef (2,000 cfu per g) and in two samples of grated cheese; leftover cheese from lunch (7,800 cfu per g) and an unopened package purchased from the market (3,000 cfu per g). CONCLUSION: The findings of this study suggest that the aetiological agent of this outbreak was S. aureus. The food vehicle was the grated cheese, which was mixed with the beef and served for lunch in the military unit. This outbreak highlights the capacity of enterotoxin-producing bacteria to cause short term, moderately-severe illness in a young and healthy population. It underscores the need for proper food handling practices and reinforces the public health importance of timely notification of such outbreaks.

Acute Disease↗

Impact of wastewater reuse on cobalt status in Egyptian environment.

Cobalt is used in the manufacture of alloys, catalysts in the petroleum industry, catalytic converters, and paint pigments. Thus the potential for Co releases into the environment is highly increased. Use of waste sludges and sewage effluent to fertilize and irrigate soils has also increased soil Co concentrations. Total cobalt contents of alluvial delta soil of Egypt show considerable variation ranging from 13.1 to 64.7 ppm. The impact of either wastewater irrigation or industrial activities on soil total Co was obvious due to accumulation of organic matter and solid waste in the surface soil samples. Food crops and vegetables should not be grown on soil highly contaminated by Co. It is noteworthy that the delayed neutron activation analysis (DNAA) technique could be used successfully for total Co determination due to its high sensitivity. It is quit clearly that dust samples of Cairo City contains higher Co level, as compared to Suez Canal Region (Ismailia, Port Said an El-Sues cities). The high values in Cairo City may be due to the existence of industries around the city and the intensive traffic. To minimize Co environmental hazards, waste effluents should be treated on site. Thus, levels of potentially toxic Co needs to be continuously monitored and should be removed during several treatment processes before the disposal of these wastes.

Agriculture↗

Determination of selenium in Canadian food items by cyclic instrumental neutron activation analysis.

Instrumental neutron activation analysis (INAA) and pseudocyclic INAA (PCINAA) have been used to determine the selenium content of a variety of Canadian food items. Use of the 162-keV gamma ray of short-lived 77mSe in INAA allowed relatively simple and rapid determinations and was suitable for many of the foods. PCINAA was found to give lower detection limits and was used for the low-selenium food samples. Both internal and external quality assessments were used to evaluate and assure the accuracy and precision of the methods developed.

Food Analysis↗

Media for Salmonella.

The main categories of selective plating media are reviewed. The selective enrichment media are considered to be of critical importance in salmonella isolation. During the last 10 years Rappaport-Vassiliadis broth has emerged as a new reliable enrichment medium. Likewise motility enrichment is a very promising technique, but will no doubt have its limitations. However, since it is based on a different principle compared with traditional enrichment, it may complement traditional techniques in a useful way. Most important, however, is the insight that the selectivity of a selective procedure is not solely defined by the medium and by incubation conditions. The food sample and its microflora apparently influences the result considerably. This finding may open a new and fascinating field of scientific research; the ecology of selective media.

Culture Media↗

Analysis of heterocyclic amines in food products: interlaboratory studies.

A feasibility study and two interlaboratory exercises on the determination of selected heterocyclic amines (HAs) in beef extract, organised in the framework of a European project, are presented. The aim of these exercises was to improve the quality of the laboratories and to evaluate the performance of a standardised analytical method and also the methods currently used by each of the participants for the analysis of these compounds. Three lyophilised portions of a commercial beef material previously spiked with HAs at different concentration levels ranging from 10 to 75 ng g(-1) were used as laboratory reference materials (lot A, B and C). Firstly, a feasibility study was carried out using a test standard solution and the beef extract (lot A), which contained only five HAs. Then, two interlaboratory exercises were carried out using the laboratory reference materials lot B and lot C, containing 10 selected HAs at two different concentration levels, 75 and 10 ng/g, respectively. The results obtained by all participant laboratories using the proposed method showed satisfactory agreement and the CV(%) between-laboratories obtained were from 8.3 to 24.1% for lot B and from 8.7 to 44.5% for lot C. The standardised method evaluated in these collaborative studies is therefore proposed for the analysis of HAs in food material. Moreover, LC-MS is recommended as the most suitable technique for the analysis of a large number of HAs in food samples.

Amines↗

Biosensor detection of botulinum toxoid A and staphylococcal enterotoxin B in food.

Immunoassays were developed for the simultaneous detection of staphylococcal enterotoxin B and botulinum toxoid A in buffer, with limits of detection of 0.1 ng/ml and 20 ng/ml, respectively. The toxins were also spiked and measured in a variety of food samples, including canned tomatoes, sweet corn, green beans, mushrooms, and tuna.

Animals↗

Comparison of colorimetric monoclonal enzyme immunoassay screening methods for detection of Salmonella in foods.

A colorimetric enzyme immunoassay (EIA) method for detection of Salmonella in foods has been compared to the AOAC colorimetric monoclonal EIA screening method (986.35, 15th ed.; 46.B21-46.B29, 14th ed.). The assays use the same monoclonal antibodies and have similar reactivity toward Salmonella. However, the new assay uses antibody-coated microtiter wells instead of coated magnetic beads to capture Salmonella antigens. Compared with the bead assay, the coated-well assay format requires significantly less time to complete, and was consistently able to detect lower levels of Salmonella in mixed culture. Compared to the standard AOAC culture method for food samples, the plate assay was as productive. No false negatives were obtained by the immunoassay; the false negative rate was 1.1% by the culture method. The rate of agreement between the 2 methods was 99.1%. The official final action bead assay method for Salmonella in foods, 986.35, and the same assay for use with low-moisture foods, 987.11, have been modified official first action to use antibody-coated microtiter strip-wells.

Antibodies, Monoclonal↗

Specific detection of cytopathogenic Listeria monocytogenes using a two-step method of immunoseparation and cytotoxicity analysis.

The development of rapid methods for detection of viable Listeria monocytogenes is crucial to prevent listeriosis and product recalls. While immunomagnetic separation has been used for isolating Listeria spp., lack of specificity and pathogenicity determination render this method unsatisfactory. A two-step method using Protein A agarose beads (Immunobeads) coated with a more specific antibody, monoclonal antibody (MAb)-C11E9 for L. monocytogenes was developed. Immunobeads were allowed to capture Listeria cells from a variety of samples and tested for cytopathogenic action on a murine hybridoma B-lymphocyte, Ped-2E9 cell line by Trypan blue staining, and by an alkaline phosphatase (AP)-based cytotoxicity assay. The two-step method was used to test uninoculated hotdogs, bologna, and raw beef, chicken, and pork samples, following selective enrichment in half-Fraser broth. Pure culture studies proved the assay to be specific for L. monocytogenes, while a similar assay with Dynal Anti-Listeria immunomagnetic beads was positive for L. monocytogenes, L. ivanovii, and L. seeligeri. Detection and confirmation of cytopathogenicity of Listeria cells from food samples after 24-h selective enrichment were completed in 2-4 h. Isolates were further analyzed by the CAMP test for hemolytic activity and RiboPrinter for genomic patterns. Using immunoseparation and cytotoxicity as a two-step rapid method, viable L. monocytogenes could be isolated, detected, and confirmed as cytopathogenic in 28 h or less.

Animals↗

Filtration-based tyramide amplification technique--a new simple approach for rapid detection of aflatoxin B1.

The catalyzed reporter deposition (CARD) method of signal amplification, also called "tyramide signal amplification", has been used in immunoassays not only to increase sensitivity but also to reduce assay time. The current approach to tyramide amplification in immunoassays involves slow incubation with agitation. In this paper we describe new filtration-based tyramide amplification and substrate visualization techniques. Compared with the standard method, this new approach greatly enhances spot intensities in membrane immunoassay and reduces biotinylated tyramide (B-T) and substrate consumption approximately fiftyfold, without loss of specificity. An improved test device and a cost-effective method for preparation of membranes for Super-CARD amplification have also been developed. The techniques have been used for rapid detection of aflatoxin B(1) (AFB(1)) in a variety of foodstuffs with a detection limit of 12.5 microg kg(-1). The assay procedure involves sequential addition of standards or sample, AFB(1)-horseradish peroxidase (HRP) conjugate, B-T, avidin-HRP, and substrate solution over anti-AFB(1) antibody-spotted zones of the membrane surface. The method saves time, improves reproducibility, eliminates many washing steps and avoids manipulation of the membranes between the different steps, while maintaining the sensitivity of the standard method. Average recoveries from different non-infected food samples spiked with AFB(1) at concentrations from 25 to 100 mg kg(-1) were between 95 and 105%. AFB(1) results obtained on different days for Aspergillus parasiticus infection of corn and groundnut samples correlated well with estimates obtained by HPLC.

Aflatoxin B1↗

Occurrence of patulin in conventional and organic fruit products in Italy and subsequent exposure assessment.

The occurrence of patulin was investigated in 100 conventional and 69 organic fruity foodstuffs samples commercially available in Italy by using an HPLC method with a limit of quantification of 0.5 microg kg(-1). Patulin was detected in 26 (26%) conventional and 31 (45%) organic products with a significantly higher (p<0.01) mean concentration in the organic products (4.78 vs. 1.15 microg kg(-1)). Mean patulin concentrations in conventional apple juices, pear juices, other juices and fruits purees were 3.14, 0.22, 0.19, 0.11 microg kg(-1), respectively, and 7.11, 11.46, 2.10, 0.18 microg kg(-1) in the relevant organic products. Four samples of juices (one conventional and two organic apple, and one organic pear) contained patulin at concentrations above the limit of 50 microg kg(-1), four at concentrations between 10 and 25 microg kg(-1), and the remaining ones below 10 microg kg(-1). Patulin was detected (<1 microg kg(-1)) in only three of the 23 fruity baby food samples tested (homogenized fruits, 11 conventional and 12 organic). Based on the available data on Italian intakes of fruit juices, the estimated daily intakes of patulin, were 0.38 and 1.57 ng kg(-1) body weight (bw) from conventional and organic products, respectively. Estimated daily intakes of patulin for children were higher, 3.41 ng kg(-1) bw from conventional and 14.17 ng kg(-1) bw from organic products, but largely below the provisional maximum tolerable daily intake (PMTDI) of 400 ng kg(-1) bw. Patulin was also found in two samples of organic apple vinegar (<5 microg kg(-1)) and in fresh apples with rotten spots (12 out of 24 samples) with maximum levels at 16,402 and 44,572 microg kg(-1) for conventional and organic apples, respectively.

Beverages↗

Possible person-to-person transmission of Escherichia coli O111--associated hemolytic uremic syndrome.

Over a 3-month period, ten children (aged 1-13 years) from a 15-km radius in southern Picardy developed typical D+ hemolytic uremic syndrome (HUS). Polymerase chain reaction, using two pairs of verocytotoxin 1-(VT1) and VT2-specific oligonucleotide primers and an internal control was used to detect VT genes directly from stools samples. VT2 gene was detected in seven of nine patients' stools and in 5 of 14 contacts' stool samples. A VT2-producing Escherichia coli (VTEC) O111 was isolated from five of nine children's stools and in 3 adults' stools of the 14 tested. A retrospective case-control study was performed which showed a higher rate of absence in school A, where the first four cases were detected, compared with a control school. The odds ratio for the whole school was 2.77 (confidence interval 1.46-5.26), and 15 (confidence interval 2.54-115.6) if only the nursery classes were considered. A culture of all food samples from households was always negative for VTEC. A retrospective cohort study performed in 89% of children attending school A showed no linkage between food or drink and gastroenteritis. These findings emphasize the potential for person-to-person transmission of VT2-producing E. coli O111, since the only salient risk factor was close contact.

Adolescent↗

Antioxidant capacity of lycopene-containing foods.

Increased consumption of tomatoes and tomato products has been associated with decreased cancer risks. One fat-soluble compound identified in tomatoes which may be responsible for this association is lycopene. There may, however, be other antioxidants present in tomato-based foods, and total antioxidant capacity may be another way to rate the health benefits of these foods. In this work, we examined the Trolox-equivalent antioxidant capacity (TEAC) of aqueous and organic extracts of lycopene-containing foods: ketchup, fresh tomatoes, tomato paste, tomato sauce, tomato soup, tomato juice, vegetable juice, canned tomatoes and watermelon. Antioxidant activity in these food extracts was greater in the aqueous versus organic fractions, except for watermelon and tomato sauce where the levels were similar in the two fractions. Lycopene levels in the food samples tested, however, were relatively greater in the organic fractions, with the exception of the two juices, which had similar levels in the two fractions, and two highly concentrated tomato products, tomato paste and ketchup, which had relatively higher lycopene levels in the aqueous fractions. The foods with the highest antioxidant capacity per serving overall (tomato soup was highest) did not have the highest lycopene levels. This indicates that it may be important to consume a variety of tomato-containing products in order to obtain the largest variety of dietary antioxidants possible.

Analysis of Variance↗

Clostridium perfringens food poisoning: use of serotyping in an outbreak setting.

An outbreak of Clostridium perfringens food poisoning occurred among attendees of a firehouse luncheon. The predominant symptoms of diarrhea (100%) and abdominal pain (81%) among case-patients, the mean incubation period (13.4 h), and the mean duration of illness (21.2 h) were all characteristic of C. perfringens enteritis. Roast beef, although not epidemiologically implicated, was the most likely vehicle of transmission. Fecal specimens from case-patients contained a median C. perfringens spore count of greater than 10(6) and yielded isolates that were heat sensitive and predominantly nonhemolytic, produced C. perfringens enterotoxin A, and, in the majority of specimens (four of five), were identical in serotype. Food samples were negative. This outbreak demonstrates that following enumeration of C. perfringens from a suitable number of fecal specimens from case-patients, serotyping of the isolates may be helpful in implicating C. perfringens as the cause of foodborne illness. This is especially true when implicated food items test negative or are no longer available for testing.

Adolescent↗

[Determination of deoxynivalenol and nivalenol in cereals at the microgram/kilogram range].

A method is described for the determination of Deoxynivalenol (DON) and Nivalenol (NV). A liquid chromatographic clean-up step is followed by silylation of the extract and the trimethylsilylether are determined by gas-chromatography-mass-spectrometry (GC-MS), monitoring the characteristic ions in the negative chemical ionisation mode. 71% of 42 food-samples were contaminated with DON and 38% with NV. The highest contamination detected was 2000 micrograms/kg DON and 120 micrograms/kg NV in a wheat bran sample. Deoxynivalenol and Nivalenol in concentrations of 10 micrograms/kg can be detected easily by this method.

Chromatography, Thin Layer↗

Modification of the bile salts-Irgasan-brilliant green agar for enumeration of Aeromonas species from food.

The present study evaluated the productivity of BIBG medium for the isolation of Aeromonas spp. from food and describes a modification of the BIBG medium (mBIBG) (increased pH (8.7), replacement of xylose by soluble starch as a carbon source, decreased concentration of bile salts) to increase its selectivity and electivity. Using the mBIBG medium, growth of the majority of the Enterobacteriaceae (9/10) was suppressed except for Citrobacter freundii. The mBIBG medium supported growth of Pseudomonas species but a clear distinction between Aeromonas and Pseudomonas colonies could be made. Interpretation of the mBIBG medium should be performed after 24 h of incubation. It was noted that three of the 27 Aeromonas strains tested did not develop on the mBIBG medium. The ability or inability to grow on a selective medium is strain-dependent. Enumeration of Aeromonas species (A. hydrophila LMG 3771, A. caviae LMG 3775, A. veronii biovar veronii LMG 9075, A. veronii biovar sobria LMG 13071) from artificially contaminated foods (shrimp, minced meat (beef/pork), precut leek, and shredded carrots) confirmed that the mBIBG medium is suitable for quantitative recovery of aeromonads (ca. 10(2)-10(7) cfu/g) in the presence of a high background flora (10(5)-10(6) cfu/g). Screening of naturally contaminated foods (vegetables, seafood, meat) for the presence of Aeromonas resulted in three out of 14 food samples showing presumptive Aeromonas colonies on mBIBG.

Aeromonas↗