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Enzymic determination of ammonia in food by flow injection.

Ammonia in food samples was determined by its reaction in an immobilised enzyme reactor containing glutamate dehydrogenase (GIDH) in a flow injection system, by measuring the decrease in the absorbance of ultraviolet radiation by reduced nicotinamide adenine dinucleotide (NADH). There was a linear relationship (r = 0.9995) between peak height and ammonia concentration over the range 0.05-0.6 mM. The detection limit was 0.005 mM for an injection volume of 19 microliters. Sampling frequency was 60 h-1 and the precision was better than 1.09% for 11 successive assays. The interference effect of urea and ascorbic acid at concentrations greater than 100 mg per 100 g of product should be taken into account. The interference caused by glycine, creatinine and amino acids is negligible. Only a 20% loss in the activity of the GIDH column was observed after 500 determinations during a 3-month period.

Ammonia↗

Molecular techniques to detect and identify protozoan parasites in the environment.

The environmental route of transmission is important for many protozoan and helminth parasites, with water, soil and food being particularly significant. Both the potential for producing large numbers of transmissive stages and their environmental robustness pose persistent threats to public and veterinary health. The introduction of molecular techniques, in particular those based on the amplification of nucleic acids, has provided researchers with highly sensitive and specific assays for the detection and identification of these pathogens. The application of these techniques to clinical, environmental, and food samples is instrumental for a thorough understanding of the epidemiology of the infection and for the implementation of control measures. Here, the advantages and drawbacks of some molecular techniques (Polymerase Chain Reaction--PCR; Reverse-Transcriptase PCR--RT-PCR; Real-time PCR--qPCR; Nucleic Acid Sequence-Based Amplification--NASBA) will be briefly reviewed. Some application of these techniques will be illustrated with reference to two important and widespread human parasites, the apicomplexan Cryptosporidium and the flagellate Giardia.

Animals↗

[Detection and characterization of verocytoxin-producing Escherichia coli types (VTEC) from different sources].

Hemolytic uremic syndrome (HUS) is caused by enterohemorrhagic E. coli (EHEC) belonging to a few serovars embracing strains of O26, O103, O111, O118, O145 and O157 serogroup, respectively. In own investigations 3.791 food specimen of animal origin were investigated by use of an enzyme-immuno-assay (EIA) and the polymerase chain reaction (PCR). All E. coli isolates (n = 459) of food as well as isolates from cattle feces (n = 440), from HUS patients (n = 50) and asymptomatic human carriers (n = 16) were investigated by means of the PCR using primer pairs for verocytotoxin genes (vtx1, vtx2, vtx2c, vtx2d, vtx2e), the E. coli attaching und effacing gene (eae), the enterohemolysin-gene (ehlyA) and the vt2 transporter protein-gene (ile X tRNA). Differences were found in respect to the eae- and the ile X tRNA genes, which could be detected in significantly higher ratios in the isolates from patients and human carriers. Furthermore vtx2d strains were exclusively analyzed to each 25% in food and cattle strains. In six food samples pathogenic strains of serovar O157 were detected whereas some of the cattle strains were estimated to belong to EHEC serovars O26:H11, O118:H- and O157:H7. The own data support the thesis that the risk for human beings is affiliated to a great extent by direct contact with ruminants, followed by person-to-person transmission. Regarding the epidemiological data the thesis that each VTEC strain ist potentially an EHEC strain can not longer be substantiated.

Animals↗

Quantitative determination of coenyzme Q10 by liquid chromatography and liquid chromatography/mass spectrometry in dairy products.

The dietary sources of CoQ10 and the evaluation of CoQ10 in dairy products were characterized. For quantitation of CoQ10 in food samples, 2 liquid chromatography (LC) methods with UV and mass spectrometry (MS) detections were developed. LC with UV detection was performed at 25 degrees C on a Hyperclone ODS 5 microm 150 x 4.6 mm column with mobile phase consisting of methanol-ethanol-2-propanol (70 + 15 + 15, v/v/v). Flow rate was 1.0 mL/min. Retention time of CoQ10 was 10.9 +/- 0.1 min. The method was sensitive [limit of detection (LOD) = 0.2 mg/kg], reproducible [relative standard deviation (RSD) = 3:0%), and linear up to 25 mg/kg (R > 0.999). LC/MS analysis was performed on a LUNA C18 3 microm, 150 x 4.6 mm column, using mobile phase consisting of ethanol-dioxane-acetic acid (9 + 1 + 0.01, v/v/v), flow rate was 0.6 mL/min, and the retention time of CoQ10 was 4.1 +/- 0.1 min. Identification and quantitation were performed with a Finnigan-LCQ mass detector in positive atmospheric pressure chemical ionization mode. Mass spectra were obtained in selected-ion monitoring mode; molecular mass (M+H)+ m/z 863.4 +/- 1 was used for quantitative determination. MS detection is more sensitive than UV detection (LOD = 0.1 mg/kg), less reproducible (RSD = 4.0%), and linear in selected range. Analytical recoveries are 75-90% and depend on the ratio between the amount of fat in the matrix and the concentration of CoQ10 in the sample. Some soybean milk products were analyzed together with different cow, goat, and sheep milk products. Concentrations obtained with LC and LC/MS were compared with a few accessible results available from the literature. Concentrations varied from 0 ppm in soybean milk to nearly 2 ppm in fresh milk from local farms.

Acetic Acid↗

Health risks of dietary intake of environmental pollutants by elite sportsmen and sportswomen.

The dietary intake of arsenic (As), cadmium (Cd), mercury (Hg), lead (Pb), hexachlorobenzene (HCB), polychlorinated naphthalenes (PCNs), polycyclic aromatic hydrocarbons (PAHs), polychlorinated diphenyl ethers (PCDEs), polybrominated diphenyl ethers (PBDEs), polychlorinated biphenyls (PCBs), and polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) by elite sportsmen and sportswomen of Catalonia, Spain, was assessed. In 2000, food samples were randomly acquired in various cities of Catalonia. Analysis of the above pollutants were determined according to the appropriate analytical techniques (ICP-MS, HRGC/HRMS, HPLC). In general terms, elite sportsmen and sportswomen showed a higher intake of Cd, Hg, Pb, HCB, PCNs, PCDD/Fs and PAHs than the general population, while it was lower for PCDEs (both sexes), and PCBs and PBDEs (women). According to the FAO/WHO provisional tolerable weekly intake (PTWI) for metals, the WHO tolerable daily intake (TDI) for HCB, and the US EPA's reference dose (RfD) for PAHs, the dietary intakes of environmental pollutants should not mean a potential toxic hazard. However, the WHO-TDI for PCDD/Fs and "dioxin-like" PCBs is exceeded in sportsmen. The current results indicate that the consumption of those food groups showing the highest contribution to the intake of these pollutants should be diminished. In relation to this, the reduction of the consumption of dairy products and cereals would be important.

Arsenic↗

Isolation of verotoxigenic Escherichia coli from the Tasmanian environment.

Growing concerns on the emergence of verotoxin producing Escherichia coli (VTEC) in Australia have focused our attention on the possible sources of VTEC within the island state of Tasmania. An analysis of 156 food samples and 194 water samples obtained from various areas revealed evidence of eight possible sources. Six strains, with serotypes Ont:Hnt, O86:H-, O88:H-, O126:H21 and O134:H-, were isolated from water samples. Two VTEC of serotypes Ont:H8, 081:H- were isolated from raw meat samples. The waterborne isolates produced verocytotoxin. VT1, while both foodborne isolates were strong producers of VT2. Three VTEC isolates produced haemolysins, only one produced enterohaemolysin (EntHly) and the remaining were reported with alpha-haemolysin (alpha-Hly) activity. An important feature in the majority of isolates from water was their lack of ability to ferment lactose these isolates are routinely overlooked in public health laboratories.

Animals↗

[The occurrence of cadmium in some types of food].

An examination of 732 samples of foods of animal and vegetable origin revealed that 95% sea fish products, 94% samples of sea and fresh-water fish, 69.9% samples of foods of vegetable origin and 32.9% samples of foods of animal origin -- tinned beefand pork -- pâtés and spreads -- did not comply with the requirements of the standard on contaminants in foods for cadmium content. Together with the investigation of cadmium content in foods the applied method of atomic absorption spectrophotometry was tested, from the point of interferences during establishing the actual cadmium content; at the same time, this study concerned cadmium losses during the process of mineralization of the biologic material samples. It was concluded that the measured cadmium values were affected by the presence of a higher content of sodium chloride in the samples. The re-gained yield of cadmium by the used method of mineralization is 85%.

Animals↗

Low occurrence of pathogenic Yersinia enterocolitica in clinical, food, and environmental samples: a methodological problem.

While Yersinia enterocolitica is an important pathogen, which can cause yersiniosis in humans and animals, its epidemiology remains obscure. The pig is the major reservoir of pathogenic Y. enterocolitica of bioserotype 4/O:3, the most common type found in humans. Y. enterocolitica is thought to be a significant food-borne pathogen, although pathogenic isolates have seldom been recovered from foods. The low isolation rate of this pathogenic bacterium in natural samples, including clinical, food, and environmental samples, may be due to the limited sensitivity of culture methods. During the last decade, numerous DNA-based methods, such as PCR and colony hybridization assays, have been designed to detect pathogenic Y. enterocolitica in natural samples more rapidly and with better sensitivity than can be achieved by culture methods. In addition, the occurrence of pathogenic Y. enterocolitica in natural samples is clearly higher with PCR than with culture methods. The methods available for detection of pathogenic Y. enterocolitica in natural samples are reviewed in this article.

Bacteriological Techniques↗

The selenium content of infant food.

The selenium content of food exhibits great regional differences. Food samples of infants and young children from the North Rhine-Westphalia State in the Fed. Rep. of Germany were analysed by instrumental neutron activation analysis and showed that the average selenium content of local vegetable and fruit is below 5 ng/g wet weight. Only samples of potatoes and bananas exhibit higher selenium contents. The bananas, probably imported from different areas of the world, show an extremely high variance with values between 4 and 164 ng Se/g. Commercially available ready-for-use meals for infants or young children have a low Se content (median 24 ng/g) which is comparable to cow's milk. Besides eggs, cereals enriched with milk and some other cereal products contain more than 100 ng Se/g.

Food Analysis↗

Update of PCDD/PCDF-intake from food in Germany.

Between 1993 and 1996, 1414 food samples were collected in the south-western part of Germany and analysed for PCDD/PCDF contamination. The mean daily intake was calculated as 0.88 pg I-TEQ/kg b.w/day. The results show that the actual PCDD/PCDF intake is about half of the intake calculated for Germany between 1986 and 1991. Now, the mean daily intake is below the amount of 1 pg I-TEQ/kg b.w./day which should not be exceeded with respect to the value of precaution in Germany.

Benzofurans↗

Fine tuning a bile-enzymatic-gravimetric total dietary fiber method.

A recently proposed bile-enzymatic-gravimetric total dietary fiber (TDF) method was modified and the new procedure was compared with the original method, the traditional AOAC enzymatic-gravimetric determination (AOAC Official Method 985.29), and another simplified AOAC procedure by analyzing several diet composites, including National Institute of Standards and Technology 1548 total diet reference material. The original and modified bile-enzymatic-gravimetric procedures also were compared by analyzing 9 food samples from a collaborative study of the original method. The modified method consistently yielded values about 10% lower than the original method but closer to reference values and to values from AOAC Official Method 985.29, suggesting results that are more in line with accepted TDF standard methodology. Our modified method was used to analyze 180 fresh-frozen diet composites with TDF values ranging from 0.6 to 3.2 g/100 g wet weight. Samples were from 2 multicenter feeding studies sponsored by the National Heart, Lung and Blood Institute: DELTA (Dietary Effects on Lipoproteins and Thrombogenic Activity) and DASH (Dietary Approaches to Stop Hypertension). The mean relative standard deviation (RSD) for duplicate analyses was 1.1%. For 40 assays of a quality control diet composite over 9 months, the standard deviation was 0.1 g/100 g wet weight (4.9% RSD), indicating the method's excellent precision for routine use.

Diet↗

Production of type II heat-labile enterotoxin by Escherichia coli isolated from food and human feces.

Escherichia coli strains isolated in Sao Paulo, Brazil, from feces of patients with diarrhea and from food samples produced toxin(s) that was shown to be related both immunologically and genetically to the recently characterized type II heat-labile enterotoxin of E. coli. The new isolates of type II heat-labile enterotoxin-producing E. coli belonged to five different serotypes and did not represent a single clone.

Bacterial Toxins↗

Evaluation of a new chromogenic agar for the detection of Listeria in food.

AIM: Rapid identification of Listeria in food is important in protecting consumers from infection. The development of chromogenic media such as agar Listeria according to Ottaviani and Agosti (ALOA) has allowed more rapid detection of Listeria monocytogenes, with presumptive identification of this pathogenic species after only 24 h of incubation. The aim of this study was to evaluate Oxoid chromogenic Listeria agar (OCLA) in comparison with ALOA and a traditional, nonchromogenic medium, Oxford agar. METHODS AND RESULTS: Media were compared using pure cultures, spiked food samples and naturally contaminated samples. Whilst development of typical colony morphology took 48 h on Oxford agar, Listeria spp. were frequently detected after 24 h of incubation on OCLA and ALOA. There was no significant difference in recovery between the two chromogenic media. CONCLUSIONS: Results indicate that OCLA gives equivalent recovery of Listeria spp. compared with ALOA. Whilst L. monocytogenes was frequently detected after 24 h of incubation, a 48-h incubation time was necessary to ensure detection of both L. monocytogenes and other Listeria spp. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has shown that a commercially available chromogenic medium other than ALOA is appropriate for use in the international standard method. The commercial availability of more than one medium will facilitate the more widespread use of the method, thus increasing confidence in the ability to detect L. monocytogenes in food in the presence of other Listeria spp.

Agar↗

[Selenium concentration in food consumed in Brazil].

OBJECTIVE: To determine the selenium content of foods consumed in Brazil. METHODS: The project was carried out between 1993 and 1999. The food samples were collected in stores in several Brazilian states. The levels of selenium were determined by hydride generation atomic absorption spectrophotometry and by wet oxidation. RESULTS: The highest levels of selenium were found in animal products, especially fish, and in wheat products. We found that plant products generally had selenium levels of less than 5.0 micrograms/100 g. CONCLUSIONS: In Brazil a diet rich in animal products, especially fish, is important in ensuring consumption of the recommended levels of selenium.

Brazil↗

A synthetic oligonucleotide probe and a cloned polynucleotide probe based on the yopA gene for detection and enumeration of virulent Yersinia enterocolitica.

We compared a synthetically produced 19-mer oligonucleotide probe with a polynucleotide probe consisting of a cloned fragment of the virulence gene yopA for their relative efficiencies in identification and enumeration of virulent Yersinia enterocolitica. The probes were used in DNA-DNA colony hybridization assays to differentiate 70 Yersinia strains with known plasmid profiles. All 19 strains harboring the 40- to 50-megadalton virulence plasmid were positive in the hybridization assay, whereas their isogenic derivatives lacking this plasmid were negative. Both probes correctly identified plasmid-bearing variants of Y. enterocolitica serogroups O:3, O:5,27, O:8, O:9, O:13, and O:21 from three continents. In contrast, none of the probes hybridized with DNA from 32 environmental yersiniae belonging to 26 serogroups not associated with disease. Colony hybridization was used to detect and enumerate virulent Y. enterocolitica in three artificially contaminated food samples. Despite a large background of indigenous bacteria (3 x 10(4) CFU), the efficiency of enumeration ranged from 33 to 82%. The use of nylon filters did not impair the growth of virulent yersiniae. Both probes showed a perfect concordance in their specific differentiation and enumeration of virulent Y. enterocolitica. DNA colony hybridization with these two probes permitted rapid and reliable identification of all common pathogenic serogroups without the need for enrichment or esoteric identification protocols.

Animals↗

Report of a collaborative trial to investigate the performance of the R5 enzyme linked immunoassay to determine gliadin in gluten-free food.

OBJECTIVE: Analytical methods for measurements of gluten in the low level range of 20-200 ppm of gluten, as required for gluten-free food, have never been endorsed by the Codex Alimentarius. With the aim of investigating standardized and reliable methods for the detection of gliadin in food with detection limits lower than 200 ppm, as proposed by the Codex Alimentarius, the Working Group on Prolamin Analysis and Toxicity (WGPAT) coordinated a large collaborative study to validate a monoclonal antibody based ELISA, which uses an antibody (R5) against rye secalins. METHODS: Twelve food samples in which gliadin was present in the range of 0-168 ppm were analysed with two different commercially available R5 ELISA tests and a special extraction solvent, based on a reducing and a dissociating agent designed to extract heat denatured proteins. Twenty laboratories participated in this study. RESULTS: Recovery values ranged from 65 to 110% in general. The repeatability (RSDr) and reproducibility (RSDR) figures ranged between 13 and 25, resp. 23 and 47 for one test, and between 11 and 22, resp. 25 and 33 for the other. CONCLUSION: Both assays are comparable and robust. The repeatability and reproducibility data are in a range that is acceptable for ELISAs. Kits from both suppliers fulfilled performance criteria of regular ELISA methods, and it is shown that both ELISA kits guarantee a sensitivity of 1.5 ppm gliadin for gluten-free food.

Antibodies, Monoclonal↗

Immunomagnetic separation and conventional culture procedure for detection of naturally occurring Salmonella in raw pork sausages and chicken meat.

The aim of the study was to compare immunomagnetic separation (IMS) and conventional selective enrichment procedures using selenite cystine broth (SC) and Rappaport-Vassiliadis broth (RV) in 137 naturally contaminated food samples (69 raw pork sausages and 68 chicken meat). The utilization of SC or IMS appeared to be the most appropriate enrichment procedure: 15 out of 18 Salmonella-positive samples (83.3%) were detected by SC and 12 (66.7%) by IMS; RV yielded only seven positive isolations (38.9%). However, RV yielded the highest count of Salmonella colonies per plate and the lowest interference by competing organisms. IMS could become a reliable alternative to standard enrichment procedures and a combined IMS and selective enrichment broth could increase the chance of Salmonella recovery.

Animals↗

Isolation and characterization of Listeria monocytogenes isolates from ready-to-eat foods in Florida.

Of 3,063 ready-to-eat food samples tested, 91 (2.97%) were positive for Listeria monocytogenes, and lineage 1 strains outnumbered lineage 2 strains 57 to 34. Seventy-one isolates (78%) exhibited multiple antibiotic resistance, and an L. monocytogenes-specific bacteriophage cocktail lysed 65 of 91 (71%) isolates. Determining phage, acid, and antibiotic susceptibility phenotypes enabled us to identify differences among strains which were otherwise indistinguishable by conventional methods.

Animals↗