UHPLC as a suitable methodology for the analysis of carotenoids in food matrix

The purpose of this paper was to optimize an ultra-high performance liquid chromatography (UHPLC) method for the determination and quantification of carotenoid pigments in food products and compare it with a HPLC method in terms of resolution, selectivity, sensitivity, time and reagent saving. Less analysis times, solvent consumption and better peak resolution were achieved when the UHPLC methodology was used. Ultra-high performance liquid chromatography analysis was performed on Zorbax Eclipse Plus C18 column (2.1?×?50?mm; 1.8?μm) using a gradient solvent system and diode array detector. A total of seven carotenoids were separated and quantified in <8.5?min. For all components, the resolution was higher than 1.5 and LOD was below 1.0?μg?mL^?1. To determine the intraday and reproducibility, assays were conducted in 10 continuous days for method validation achieving recuperation rates ranging between 85 and 100?% and no significant differences among the results obtained in same and different days. The proposed methodology was applied for the determination of the carotenoid contents in honeybee pollen samples, pumpkin and nectarine flesh with satisfactory recovery.     

Genetic procedures for identification of enterotoxigenic strains of Staphylococcus aureus from three food poisoning outbreaks

Three food poisoning restaurant outbreaks due to Staphylococcus aureus, occurring during June-October 2002 in the Principality of Asturias (PA), Spain, provided the basis for investigating some aspects of the molecular epidemiology of this organism. The methods applied to identify strains and lineages included multiplex-polymerase chain reaction (PCR) to detect nine enterotoxin (se) genes, and three DNA fingerprinting procedures: pulsed-field gel electrophoresis (PFGE) with SmaI, randomly amplified polymorphic DNA (RAPD) with two selected primers, and plasmid restriction analysis with HindIII. Thirty-two isolates were differentiated into three non-se and 12 se strains, which were outbreak-specific, except for one that was represented in two of the outbreaks. In outbreak 1, the 16 food isolates analyzed had sec, seg and sei genes and generated a distinctive DNA fingerprint, being assigned to a single strain. This strain could be categorized as endemic in the PA and associated to manually handled dairy products and nasal carriers. In outbreak 2, the four food isolates analyzed fell into three strains, each one displaying a different se-gene profile (sea, sec and seg-seh-sei) and a distinctive DNA fingerprint. In outbreak 3, the five food isolates tested fell into four seg-sei strains generating identical RAPD but different PFGE and plasmid profiles, and one sea strain also collected from two nasal carriers. This last strain had also been found in manually handled vegetables in outbreak 2, and it belongs to a not very frequently found sea lineage in the PA. Multiplex-PCR to detect se genes together with the three applied DNA fingerprint typing procedures proved therefore to be useful tools in subclassifying S. aureus for epidemiological purposes.     

Analysis of Water-Soluble Vitamins in Honey by Isocratic RP-HPLC

An isocratic RP-HPLC method has been developed for the identification and quantification of water-soluble vitamins in honey. The mobile phase tested was an aqueous solution of sulphuric acid and the detection was carried out simultaneously by UV and fluorescence. The separation of vitamins C (l-ascorbic acid), B₁ (thiamine), B₃N (nicotinamide), B₃H (nicotinic acid), B₅ (d-pantothenic acid) and B₆ (pyridoxine) is achieved in these conditions in 15 min. The addition to the mobile phase of methanol 2 %?v/v reduced significantly the analysis time in the separation of these vitamins up to 10 min. Moreover, in presence of a cationic surfactant hexadecyltrimethylammonium bromide (CTAB) in the mobile phase, the separation of vitamin C, B₁, B₃N, B₃H, B₂ (riboflavin) and B₆ is possible in 6 min. The combination of both mobile phases, H₂SO₄/methanol and H₂SO₄/methanol/CTAB, has been applied to the analysis, in isocratic mode, of several monofloral honeys (rosemary, thyme, lavender, chestnut, echium) and a honeydew honey in a short time analysis.     

Specific detection and quantification of Aspergillus flavus and Aspergillus parasiticus in wheat flour by SYBR® Green quantitative PCR

Aflatoxins are important mycotoxins that represent a serious risk for human and animal health. These mycotoxins are mainly produced by Aspergillus flavus and Aspergillus parasiticus, two closely related species with different array of aflatoxins. In this work, two specific quantitative PCR (qPCR) assays were developed to detect and quantify both species in wheat flour using primers based on the multicopy ITS2 rDNA target sequence. The species specificity of the assays was tested in a wide range of strains of these species and others colonizing the same commodities. The sensitivity of the assay was estimated in 2.5 pg/reaction in both species. Discrimination capacity for detection and relative quantification of A. flavus and A. parasiticus DNA were analyzed using samples with DNA mixtures containing also other fungal species at different ratios. Both qPCR assays could detect spore concentrations equal or higher than 10⁶ spores/g in flour samples without prior incubation. These assays are valuable tools to improve diagnosis at an early stage and in all critical control points of food chain integrated in HACCP strategies.     

Phage types, virulence genes and PFGE profiles of Shiga toxin-producing Escherichia coli O157:H7 isolated from raw beef, soft cheese and vegetables in Lima (Peru)

The present study was conducted in Lima Metropolitana to evaluate the prevalence of Shiga toxin-producing Escherichia coli (STEC) O157:H7 in raw beef, raw ground beef, soft cheese and fresh vegetables, sampled at different markets in the city. Between October 2000 and February 2001, 407 food samples were collected from different markets in the 42 districts of Lima Metropolitana. Samples were assayed for E. coli O157 by selective enrichment in modified Tryptic Soy Broth containing novobiocin, followed by immunomagnetic separation (IMS) and plating onto sorbitol MacConkey agar supplemented with cefixime and potassium tellurite. Fifty (12.3%) of 407 food samples resulted positive for E. coli O157 isolation (23 of 102 ground beef; 15 of 102 beef meat; eight of 102 soft cheese and four of 101 fresh vegetables). Thirty-five E. coli O157 isolates were further analysed for the presence of virulence genes. All 35 were positive by PCR for O157 rfbE, fliCh7, eae-gamma1 and ehxA genes. In addition, genes encoding Shiga toxins were detected in 33 of 35 isolates, five isolates (14%) encoded stx(1), stx(2), and 28 (80%) stx2 only. The isolates were of seven different phage types (PT4, PT8, PT14, PT21, PT34, PT54, and PT87) with three phage types accounting for 80% of isolates: PT4 (15 isolates), PT14 (8 isolates), and PT21 (5 isolates). Interestingly, the majority (31 of 35; 89%) of E. coli O157:H7 isolates characterized in this study belonged mainly to the phage types previously found in STEC O157:H7 strains associated with severe human disease in Europe and Canada. Pulsed-field gel electrophoresis (PFGE) of 32 isolates revealed 14 XbaI-PFGE groups (I to XIV) of similarity >85%, with 23 (72%) isolates grouped in five clusters. Some isolates from different districts presented a high clonal relatedness. Thus, PFGE group VIII clustered eleven strains from nine different districts. The broad range of PFGE subtypes found in this study demonstrates the natural occurrence of many genetic variants among STEC O157:H7 spread in Lima.     

Dietary intake of ochratoxin A from conventional and organic bread

Ochratoxin A (OTA) was extracted from 100 bread samples by using accelerated solvent extraction (ASE) and analyzed with liquid chromatography coupled with fluorescence detection. The presence of OTA was confirmed by methyl-ester derivatization. Bread samples were bought from different bakeries and supermarkets, 74 of non-organic and 26 of organic bread. The incidence of OTA varied between 20.3% and 23.0% for non-organic and organic bread, respectively. The highest values were obtained with non-organic versus organic products, five samples exceeded the European maximum permitted limit of OTA (3 ng/g) for this product. Estimated daily intake of OTA in this study was 1.6 ng/kg b.w./day. This value represents 32% and 10% of the tolerable daily intake (TDI) according to the Scientific Committee on Food of the European Commission and the FAO/WHO Committee of Experts on Food Additives, respectively. The daily intake estimated from this study reflects the necessity to take a vigilant attitude to guarantee food safety.     

Modification of the optical and structural properties of ZnO nanowires by low-energy Ar+ ion sputtering

The effects of low-energy (≤2 kV) Ar⁺ irradiation on the optical and structural properties of zinc oxide (ZnO) nanowires (NWs) grown by a simple and cost-effective low-temperature technique were investigated. Both photoluminescence spectra from ZnO NW-coated films and cathodoluminescence analysis of individual ZnO NWs demonstrated obvious evidences of ultraviolet/visible luminescent enhancement with respect to irradiation fluence. Annihilation of the thinner ZnO NWs after the ion bombardment was appreciated by means of high-resolution scanning electron microscopy and transmission electron microscopy (TEM), which results in an increasing NW mean diameter for increasing irradiation fluences. Corresponding structural analysis by TEM pointed out not only significant changes in the morphology but also in the microstructure of these NWs, revealing certain radiation-sensitive behavior. The possible mechanisms accounting for the decrease of the deep-level emissions in the NWs with the increasing irradiation fluences are discussed according to their structural modifications.     

Discriminating power of the hydrocarbon content from virgin olive oil of extremadura cultivars

Samples of virgin olive oils (105) from seven Extremaduran olive varieties (Cacereña, Carrasqueña, Cornezuelo, Corniche, Morisca, Picual, and Verdial de Badajoz) in three stage of maturity (green, semi-ripe, and ripe) were collected and the alkane, alkene, and sesquiterpene contents determined. There were significant differences at the 0.01 probability level in most of the hydrocarbons, both by variety and by state of maturity. Discriminant analysis applied to 70 samples explained 72.3% of the variance between the different groups of varieties and allowed 90% of the samples to be classified according to their variety. The acceptability of the model was verified against the remaining 35 samples, giving a mean level of correct classification of 94%.