C. M. Franco

Application of the assay of aflatoxins by liquid chromatography with fluorescence detection in food analysis

HPLC using fluorescence detection has already become the most accepted method for the determination of aflatoxins due to its several advantages over other analytical methods. Both normal- and reversed-phase HPLC can be used. However the reversed-phase HPLC methods are more popular. Liquid chromatographic determination of aflatoxins using fluorescence detection and its application in food analysis is reviewed in this article.

Inhibitory effects of eugenol and thymol on Penicillium citrinum strains in culture media and cheese

In the present work we studied the antifungal effect of eugenol and thymol on the growth and production of citrinin from Penicillium citrinum (NRRL 2274 and NRRL 2269) in culture media and in different Spanish cheeses (Arzúa-Ulloa, Cebreiro and San Simón). The rate of growth was assessed by measuring colony diameters and the production of citrinin was measured using a rapid semi-quantitative fluorometric technique confirmed by RP-HPLC. A stronger inhibitory effect of eugenol than thymol was evident. 200 microg/ml of eugenol in solid culture medium increased the lag time of growth up to 9 days, and decreased the rate of colony growth. In liquid medium, a complete inhibition of fungal growth was observed. By contrast, thymol in the liquid culture medium only affected the growth rate. In Arzúa-Ulloa cheese, 200 microg/ml of eugenol fully inhibited fungal growth, while in Cebreiro cheese no effect was observed for this compound. Regarding the capacity to inhibit mycotoxin production 100 microg/ml eugenol delayed citrinin production until the sixth day, after which a limiting effect persisted. In Arzúa-Ulloa cheese, no citrinin was detected at a concentration of 150 microg/ml of eugenol, but citrinin was detected after 5 days in the case of thymol at the same concentration. In Cebreiro cheese, neither eugenol nor thymol prevented the production of citrinin at the concentrations applied.

Determination of quinolones in animal tissues and eggs by high-performance liquid chromatography with photodiode-array detection

A rapid, specific reversed-phase HPLC method is described, with solid-phase extraction, for assaying five quinolones (ciprofloxacin, difloxacin, enrofloxacin, norfloxacin and marbofloxacin) with confirmative diode-array detection in samples of bovine kidney, muscle and eggs. The least efficient extraction was marbofloxacin from kidney tissue (64%). The lower detection limit for each quinolone was: enrofloxacin and ciprofloxacin, 1 ng; norfloxacin and difloxacin, 2 ng; marbofloxacin, 4 ng injected. The intra-day relative standard deviations were lower than 7.9% and lower than 8.6% for inter-day assays. These results indicate that the developed method had an acceptable precision.

New Additive for Culture Media for Rapid Identification of Aflatoxin-Producing Aspergillus Strains

ABSTRACT A new reliable, fast, and simple method for the detection of aflatoxigenic Aspergillus strains, consisting of the addition of a cyclodextrin (a methylated β-cyclodextrin derivative) to common media used for testing mycotoxin production ability, was developed. We propose the use of this compound as an additive for fungal culture media to enhance the natural fluorescence of aflatoxins. The production of aflatoxins coincided with the presence of a bright blue or blue-green fluorescent area surrounding colonies when observed under long-wavelength (365-nm) UV light after 3 days of incubation at 28°C. The presence of aflatoxins was confirmed by extracting the medium with chloroform and examining the extracts by high-pressure liquid chromatography with fluorescence detection.

Simple and sensitive high-performance liquid chromatography-fluorescence method for the determination of citrinin application to the analysis of fungal cultures and cheese extracts☆

A new and highly sensitive method for the detection of the important mycotoxin, citrinin, has been developed. Spectroscopic studies demonstrate that the fluorescence of this metabolite is influenced by the pH of the environment. This fact was exploited in the chromatographic determination of citrinin with fluorescence detection. The proposed method, based on the addition of 1 M hydrochloric acid as an acidic post-column reagent, has a limit of detection of 0.9 center dot 10(-7) M. Analytical validation shows that linearity can be assumed from 2 center dot 10(-7) to 10(-4) M citrinin. The repeatability and reproducibility are satisfactory, with R.S.D. = 5.1% (n = 9, c = 10(-5) M) and R.S.D. = 7.2% (n = 9, c = 10(-5) M). The method was also applied to the determination of this mycotoxin produced by mould cultures isolated from soft cheese and also from soft cheese and also from cheese extracts spiked with citrinin. The specificity of the method is demonstrated and the necessity for post-column acidification is illustrated on real samples.

Egg and Egg-Derived Foods: Effects on Human Health and Use as Functional Foods.

Eggs are sources of protein, fats and micronutrients that play an important role in basic nutrition. However, eggs are traditionally associated with adverse factors in human health, mainly due to their cholesterol content. Nowadays, however, it is known that the response of cholesterol in human serum levels to dietary cholesterol consumption depends on several factors, such as ethnicity, genetic makeup, hormonal factors and the nutritional status of the consumer. Additionally, in recent decades, there has been an increasing demand for functional foods, which is expected to continue to increase in the future, owing to their capacity to decrease the risks of some diseases and socio-demographic factors such as the increase in life expectancy. This work offers a brief overview of the advantages and disadvantages of egg consumption and the potential market of functional eggs, and it explores the possibilities of the development of functional eggs by technological methods.

Determination of the Principal Sources of Listeria spp. Contamination in Poultry Meat and a Poultry Processing Plant

The presence of Listeria spp. in chicken drumsticks, wings, breasts, and livers taken from a poultry processing plant was investigated. The poultry pieces found to be most contaminated were drumstick meat and skin, with 96% of samples shown to contain Listeria spp. Drumsticks contained the highest number of Listeria spp., with counts in many cases exceeding 3 log CFU/g for skin and muscle. Samples were also taken from various surfaces in the processing plant, where the most contaminated areas were found to be in the final stages of meat processing. Three samples were positive for Listeria spp. immediately after washing and disinfection had taken place. The results suggest that drumsticks are responsible for a large amount of the contamination of chicken carcasses, and that the surfaces that come into contact with these pieces of meat play an important role in spreading Listeria spp. Disinfection procedures used by this processing plant do not effectively control Listeria spp.

Molecular Typing by Pulsed-Field Gel Electrophoresis of Spanish Animal and Human Listeria monocytogenes Isolates

ABSTRACT A total of 153 strains of Listeria monocytogenesisolated from different sources (72 from sheep, 12 from cattle, 18 from feedstuffs, and 51 from humans) in Spain from 1989 to 2000 were characterized by pulsed-field gel electrophoresis. The strains ofL. monocytogenes displayed 55 pulsotypes. The 84 animal, 51 human, and 18 feedstuff strains displayed 31, 29, and 7 different pulsotypes, respectively, indicating a great genetic diversity among the Spanish L. monocytogenes isolates studied. L. monocytogenes isolates from clinical samples and feedstuffs consumed by the diseased animals were analyzed in 21 flocks. In most cases, clinical strains from different animals of the same flock had identical pulsotypes, confirming the existence of a listeriosis outbreak. L. monocytogenes strains with pulsotypes identical to those of clinical strains were isolated from silage, potatoes, and maize stalks. This is the first study wherein potatoes and maize stalks are epidemiologically linked with clinical listeriosis.

Simultaneous high-performance liquid chromatographic determination of ochratoxin A and citrinin in cheese by time-resolved luminescence using terbium

Abstract A simultaneous reversed-phase HPLC determination of two major mycotoxins, ochratoxin A and citrinin, in soft cheese is proposed. Both mycotoxins are eluted on a C18 RP support (25 × 4.6 mm I.D.) using an isocratic eluent consisting of methanol-water (70:30, v/v) containing tetrabutylammonium hydroxide (10−3 M), acidified to pH 5.5 with HCl, and pumped at a flow-rate of 0.8 ml/min. Prior to detection, a butanolic solution of 5·10−3 M terbium-5 · 10−4 M trioctylphosphine oxide (TOPO)-2.5 · 10−2 M triethylamine (TEA) was pumped in a postcolumn mode at a flow-rate of 0.2 ml/min to perform time-resolved luminescence (TRL) detection of the corresponding terbium chelates (λex = 331 nm/λem = 545 nm). The method is linear from 3.5·10−6 to 2·10−5 M for citrinin and from 1·10−5 to 5·10−5 M for ochratoxin A. The repeatability and reproducibility (R.S.D.) are 1.9 and 2.4% for citrinin (c = 3.5·10−6 M; n = 10), and 7.2 and 8.3% for ochratoxin A (c = 1.0·10−5 M; n = 10). The limits of detection, for a signal-to-background ratio of 3, are 2·10−6 and 3·10−6 M for citrinin and ochratoxin A, respectively. With the proposed method, ochratoxin A and citrin are easily determined in soft cheeses, with a significative increase in selectivity in comparison with direct fluorescence detection.

Quantitative LC-MS/MS method for the sensitive and simultaneous determination of natural hormones in bovine serum

We have developed a liquid chromatography tandem mass spectrometric (LC-MS/MS) method for the simultaneous quantitative analysis of several free forms of steroid hormones in bovine serum [pregnenolone (P(5)), progesterone (P(4)), 17hydroxyP(5), 17hydroxyP(4), testosterone (T), dehydroepiandrosterone (DHEA), androstenedione (A), estrone (E(1)), 2, 4 and 16 hydroxyE(1), 2 and 4 methoxyE(1)]. Deuterated analogs were used as internal standards. Serum proteins were eliminated with acetonitrile. Oxime derivatives of steroids were extracted with tert-butylmethylether and analyzed in positive MRM mode. Methodology was validated in accordance with the European Commission Decision 2002/657/EC. Performance characteristics permit the use of this methodology for steroid determination in animal serum samples.