Susana Amézqueta

Occurrence of ochratoxin A in cocoa beans: Effect of shelling

The aim of this study was to investigate the influence of the shelling process on the presence of ochratoxin A (OTA) in cocoa samples. Twenty-two cocoa samples were analysed for the determination of OTA before (cocoa bean) and after undergoing manual shelling process (cocoa nib). In order to determine OTA contamination in cocoa samples, a validated high-performance liquid chromatography (HPLC) method with fluorescence detection was used for the quantitative analysis of ochratoxin A (OTA). In both types of samples, OTA was extracted with methanol–3% sodium hydrogen carbonate solution and then purified using immunoaffinity columns prior to HPLC analysis. Due to the fact that different recovery values were obtained for OTA from both types of samples, a revalidation of the method in the case of cocoa nibs was needed. Revalidation was based on the following criteria: Selectivity, limits of detection and quantification (0.03 and 0.1 µg kg−1, respectively), precision (within-day and between-day variability) and recovery 84.2% (RSD = 7.1%), and uncertainty (30%). Fourteen of the twenty-two cocoa bean samples (64%) suffered a loss of OTA of more than 95% due to shelling, six samples suffered a loss of OTA in the range 65–95%, and only one sample presented a reduction of less than 50%. The principal conclusion derived from this study is that OTA contamination in cocoa beans is concentrated in the shell; therefore, improvements of the industrial shelling process could prevent OTA occurrence in cocoa final products.

Validation of a high-performance liquid chromatography analytical method for ochratoxin A quantification in cocoa beans

A validated high-performance liquid chromatography (HPLC) method with fluorescence detection for the quantitative analysis of ochratoxin A (OTA) in cocoa beans is described. OTA was extracted with methanol–3% sodium hydrogen carbonate solution and then purified with immunoaffinity columns before its analysis by HPLC. The validation of the analytical method was based on the following criteria: selectivity, linearity, limit of detection and quantification, precision (within- and between-day variability) and recovery, robustness and uncertainty. Detection and quantification limits were 0.04 and 0.1 μg kg−1, respectively. Recovery was 88.9% (relative standard deviation = 4.0%). This method was successfully applied to the measurement of 46 cocoa bean samples of different origins. A total of 63% of cocoa bean samples was contaminated with a level greater than the limit of detection. The means and medians obtained for cocoa bean were 1.71 and 1.12 μg kg−1, respectively. Surveillance controls should be set up in both crops and factories involved in transformation processes to avoid this mycotoxin in final products.

Development and validation of a microemulsion electrokinetic chromatography method for patulin quantification in commercial apple juice

A microemulsion electrokinetic chromatography (MEECK) method for patulin (PAT) quantification in apple juice samples has been developed. The effects of several important factors such as co-surfactant type, concentration of surfactant, acetonitrile percentage in the microemulsion, and running voltage and temperature were investigated to determine the optimum conditions. They resulted to be: a background electrolyte (BGE) composed of 25mM of sodium tetraborate, SDS (2.16%w/w), ethanol (6.49%w/w), n-octanol (0.82%w/w) and 2%v/v acetonitrile; applied voltage of +15kV; and a capillary temperature of 35 degrees C. PAT was detected at 276nm. Quantification and detection limits (LOQ and LOD) in apple juice samples were 8.0microgL(-1) and 3.2microgL(-1), respectively. Patulin was extracted from apple juice using ethyl acetate with a mean recovery value of 75.3% (RSD=4.5). This method was applied to the measurement of patulin in twenty commercial apple juice samples obtained from different Danish supermarkets. The PAT apple juice mean and median levels obtained were 35.9 and 10.9microgL(-1), respectively. The comparison with a previously validated micellar electrokinetic chromatography (MEKC) method for PAT analysis showed the suitability of using MEEKC for this mycotoxin analysis. However, the expectations of obtaining a higher efficiency and thus lower limits of detection and quantitation when using MEEKC were not met.

Comparison between capillary electrophoresis and high performance liquid chromatography for the study of the occurrence of patulin in apple juice intended for infants.

Apple juice samples intended for infants purchased in Navarra (Spain) have been analyzed for PAT occurrence. Two capillary electrophoresis methods, based on a MEKC and a CEC system, and an HPLC method were evaluated for the aforementioned study. The CEC system gave less satisfying separations and several practical problems, so samples have been analyzed by MEKC and HPLC. Both methods have been comparable in terms of recovery, precision, limits of detection, volume of organic solvents used and adequate selectivity with regard to PAT and HMF. The analysis time in HPLC has been slightly lower than in the MEKC methodology. The PAT levels obtained in apple juice by both validated methods showed a strong correlation (p<0.001). Therefore, both methodologies are useful for the accurate quantification of patulin in this matrix. The PAT levels obtained in the 20 infant apple juices samples were in a range between <LOD and 29.6 microg L(-1), with a mean concentration of 8.0 microg L(-1) which implies a dietary intake estimation of 104 ng kg(-1)b.w.day(-1) considering a body weight of 10 kg and an apple juice consumption of 130 mL day(-1), 26% of the PMTDI recommended by JECFA.

A Simple Chemical Method Reduces Ochratoxin A in Contaminated Cocoa Shells

Ochratoxin A (OTA) is a mycotoxin produced by Aspergillus and Penicillium species, which contaminates cocoa among other food commodities. It has been previously demonstrated that the toxin is concentrated in cocoa shells. The aim of this study was to assay a simple chemical method for ochratoxin A reduction from naturally contaminated cocoa shells. In order to determine the efficiency of the method, a high-performance liquid chromatography method with fluorescence detection was set up beforehand and validated. Ochratoxin A was extracted from cocoa shells with methanol-3% sodium bicarbonate solution and then purified with immunoaffinity columns. The recovery attained was 88.7% (relative standard deviation = 6.36%) and the limits of detection and quantification were 0.06 and 0.2 kg/kg, respectively. For decontamination experiments, the solvent extractor ASE 200 was used. First, aqueous solutions of 2% sodium bicarbonate and potassium carbonate were compared under the same conditions (1,500 lb/in2 at 40 degrees C for 10 min). Higher ochratoxin A reduction was obtained with potassium carbonate (83 versus 27%). Then, this salt was used under different conditions of pressure, temperature, and time. The greatest ochratoxin A reduction was achieved with an aqueous potassium carbonate solution (2%), at 1,000 lb/in2 at 90 degrees C for 10 min. This method could probably be applicable to the cocoa industry because it is fast and relatively economic. From the point of view of human health, the use of potassium carbonate, partially eliminated by rinsing the sample with water, does not likely represent a risk for human health.

Occurrence of Ochratoxin A in Southern Spanish Generous Wines under the Denomination of Origin “Jerez-Xérès-Sherry and ‘Manzanilla’ Sanlúcar de Barrameda”

The mycotoxin ochratoxin A (OTA) has toxic effects in animals; the most relevant of them is nephrotoxicity. OTA has also been classified as a possible carcinogen for humans (group 2B) by the International Agency for Research on Cancer (IARC). Therefore, exposure to OTA through contaminated food can represent health impairment to humans. The maximum permitted level for this mycotoxin in wine is 2.0 μg/L. The presence of OTA in Spanish wines produced using the traditional methods under the Denomination of Origin “Jerez-Xérès-Sherry and manzanilla Sanlúcar de Barrameda” was evaluated by a High performance Liquid Chromatography method with fluorescence detection and immunoaffinity column purification. A recovery of 95.4% and a limit of detection and quantification of 0.009 μg/L and 0.02 μg/L respectively, were achieved. In manzanilla, fino, amontillado and oloroso wine, the mean OTA values were 0.042, 0.044, 0.144, and 0.319 μg/L, respectively. These levels are not different from other data given in the reference literature on white wines, although fino and manzanilla wines have very low OTA levels.

OTA-producing fungi isolated from stored cocoa beans

Aims:  The aim of this study was to identify fungal populations in unroasted cocoa beans stored in Spain in order to evaluate the ochratoxin A (OTA)‐production ability of certain Aspergillus isolates.

The presence of D-fagomine in the human diet from buckwheat-based foodstuffs

Buckwheat (Fagopyrum esculentum Moench) groats contain the iminosugar D-fagomine as a minor component that might contribute to the alleged health benefits of this pseudo-cereal. This study presents analysis of D-fagomine in buckwheat-based foodstuffs by liquid chromatography coupled to mass spectrometry and an estimation of its presence in the human diet based on a published population-based cross-sectional nutrition survey. D-fagomine is present in common buckwheat-based foodstuffs in amounts ranging from 1 to 25 mg/kg or mg/L, it is stable during boiling, baking, frying and fermentation, and it is biosynthesised upon sprouting. The estimated total intake of D-fagomine resulting from a diet that includes such foodstuffs would be between 3 and 17 mg per day (mean for both genders; range from P5 to P95). A diet rich in buckwheat products would provide a daily amount of D-fagomine that may in part explain the beneficial properties traditionally attributed to buckwheat consumption.

Mechanistically different effects of fat and sugar on insulin resistance, hypertension and gut microbiota in rats

Insulin resistance (IR) and impaired glucose tolerance (IGT) are the first manifestations of diet-induced metabolic alterations leading to Type 2 diabetes, while hypertension is the deadliest risk factor of cardiovascular disease. The roles of dietary fat and fructose in the development of IR, IGT, and hypertension are controversial. We tested the long-term effects of an excess of fat or sucrose (fructose/glucose) on healthy male Wistar-Kyoto (WKY) rats. Fat affects IR and IGT earlier than fructose through low-grade systemic inflammation evidenced by liver inflammatory infiltration, increased levels of plasma IL-6, PGE2, and reduced levels of protective short-chain fatty acids without triggering hypertension. Increased populations of gut Enterobacteriales and Escherichia coli may contribute to systemic inflammation through the generation of lipopolysaccharides. Unlike fat, fructose induces increased levels of diacylglycerols (lipid mediators of IR) in the liver, urine F2-isoprostanes (markers of systemic oxidative stress), and uric acid, and triggers hypertension. Elevated populations of Enterobacteriales and E. coli were only detected in rats given an excess of fructose at the end of the study. Dietary fat and fructose trigger IR and IGT in clearly differentiated ways in WKY rats: early low-grade inflammation and late direct lipid toxicity, respectively; gut microbiota plays a role mainly in fat-induced IR, and hypertension is independent of inflammation-mediated IR. The results provide evidence that suggests that the combination of fat and sugar is potentially more harmful than fat or sugar alone when taken in excess.

Tadpole toxicity prediction using chromatographic systems

Toxicity has been emulated in tadpole species through chromatographic systems. The parameter studied to evaluate the non-specific toxicity of a compound is the narcosis concentration (Cnar), which is defined as the concentration needed for the immobilization of the organism. Because experimental investigation with animals is lengthy, costly, technically difficult, and ethically questionable, there is a great interest in developing surrogate physicochemical systems able to emulate biological systems to obtain the same information in a faster, more economic, and easier manner. In order to see which chromatographic systems would be able to emulate tadpole narcosis, both, tadpole narcosis data and data in several chromatographic and electrophoretic systems, were fitted to a linear solvation energy relationship (LSER) model. Thus, by comparison of the models it was possible to see which of the chromatographic systems were more similar to the biological one. The physicochemical systems that best emulate tadpole narcosis were an HPLC system based on an immobilized artificial membrane (IAM) column, and two micellar electrokinetic chromatography (MEKC) systems based on sodium taurocholate (STC) and a mixture of sodium dodecylsulphate (SDS) and Brij 35 as surfactants. A system based on a RP18 HPLC column also was selected for comparison because it is a common column in most analytical laboratories. To establish the models, a set of compounds with known Cnar values were analyzed in the chromatographic, and electrophoretic selected systems and, then, the retention factor (k) was correlated to the concentration of narcosis. Statistics showed that the system based on STC micelles was the best to emulate toxicity in tadpoles. The robustness and predictive ability of the developed models were validated.