A reliable screening of mycotoxins and pesticide residues in paprika using ultra-high performance liquid chromatography coupled to high resolution Orbitrap mass spectrometry

In this study, an ultra-high performance liquid chromatography (UHPLC) coupled to a high resolution Orbitrap mass spectrometry (Orbitrap-HRMS) was demonstrated as a promising technique in high-throughput method development for the routine analysis and contamination control of mycotoxins and pesticide residues in spices. The method was applied for the analysis of fifty ground paprika samples containing blends of sweet and hot paprika harvested in Brazil and China. The efficiency and detection sensitivity of the used UHPLC-Orbitrap-HRMS technique were compared to the results obtained using a triple quadrupole tandem mass spectrometric detector (UHPLC-QqQ-MS/MS). The values of recovery (75–120%) and repeatability (8–15%) for both methods, calculated as the average (n = 5) from the results of spiked (10–500 μg kg⁻¹), paprika samples, were in good conformity to the relevant EU guidelines. The high resolution of the used Orbitrap-HRMS technique provided a better sensitivity for quantitative determination of several pesticide contaminants in paprika, compared to the results obtained by the QqQ-MS/MS method and were comparable in case of mycotoxins. The results of analysis demonstrated the ubiquitous presence of three mycotoxins (fumonisin B₁, ochratoxin A, and sterigmatocystin) and twelve pesticide residues in paprika. The concentrations of determined contaminants were below the MRLs set by the Regulations of the European Union with exception of iprovalicarb, which violated the EU MRL in two samples of hot paprika. In addition, a notable difference in the concentration of fumonisin B₁ was determined depending on the harvest period (2009–2013), reaching the maximum concentrations of 33 μg kg⁻¹ in sweet paprika and 140 μg kg⁻¹ in hot paprika. There was no significant correlation found between the determined mycotoxin contamination levels and the pesticide residues, with the sole exception of decreased fumonisin B₁ content in samples with an elevated concentration of metalaxyl fungicide.     

Volatile compounds on the surface and within Iberian dry-cured loin

The profile of volatile compounds from external and internal areas of Iberian dry-cured loin was studied by headspace solid-phase microextraction. Higher levels of 16 volatile compounds (hexane, decane, 3-methylbutanal, several sulphur compounds and some aromatic hydrocarbons) were detected in the outer part of the loins. These differences could be owing to several factors, such as greater exposure to oxygen and dehydration conditions in the surface of the product, which favour oxidation reactions and Strecker degradation of amino acids. Moreover, the addition of spices on the surface and the proximity of a mould layer growing on the surface of the product could also contribute to the higher levels of these compounds on the external layer. However, other sulphur compounds coming from spices showed similar levels on the surface and within the loins. This different behaviour could be a consequence of different diffusion rates depending on the features of the compound and the matrix.     

A novel approach for color degradation kinetics of paprika as a function of water activity

The influence of temperature and water activity on color degradation in paprika powders was investigated. The Hunter color parameters (L, a, b), hue angle (h), and total color difference (TCD) were used to estimate the color changes of paprika during thermal treatment at different temperatures (60, 80 and 99 °C) and water activities (0.459, 0.582 and 0.703). The changes in L, a, b and TCD values were significantly influenced by the processing time, temperature and water activity. The color changes during processing and storage were described by a first order kinetic model. The temperature dependence of the degradation followed the Arrhenius relation. Samples with a high water activity exhibited a high rate constant of the color degradation. The effect of water activity on activation energy was not significant. A linear relationship between the water activity and rate constant of the color parameters was found. By incorporating the linear relationship k_refa_w into the Arrhenius equation, a modified Arrhenius equation was proposed which was used to predict paprika color changes as influenced by temperature and water activity.     

Aflatoxins and ochratoxin A in Brazilian paprika

Seventy paprika samples collected in the city of São Paulo, Brazil, from January to April 2006 were analysed for aflatoxins and ochratoxin A (OTA) using an immunoaffinity column clean-up and HPLC-FLD. For aflatoxins, the limit of quantification (LOQ) were 0.23, 0.23, 0.45 and 0.45 μg/kg for AFB₁, AFB₂, AFG₁ and AFG₂, respectively. For OTA the LOQ was 0.80 μg/kg. Aflatoxins were found in 82.9% of samples and AFB₁ was detected in 61.4% at levels ranging from 0.5 to 7.3 μg/kg with mean concentration of 3.4 μg/kg. OTA was found in 85.7% at levels ranging from 0.24 to 97.2 μg/kg with mean concentration of 7.0 μg/kg.     

Ochratoxin A in red paprika: relationship with the origin of the raw material

The occurrence of ochratoxin A (OA) in paprika elaborated from peppers grown in several countries (Peru, Brazil, Zimbabwe and Spain) was studied, using an immunoaffinity clean-up column coupled to liquid chromatography and fluorescence detection. The preparation of the methyl ester (OA-Me) and liquid chromatography-electrospray-ion trap-mass spectrometry was used both to confirm the identity of the chromatographic peak that correspond to OA and to quantify it at low levels or in dirty fractions. A total of 115 strains of moulds were isolated; 85 of the fungal strains were obtained from OA contaminated paprika samples and identified as belonging to the Aspergillus Section Circumdati group (A. ochraceus) and Section Nigri group (A. niger, A. carbonarius). Among the latter ones, 31% of the A. ochraceus isolates and one A. niger were OA producers in vitro. None of the mould strains isolated from paprika samples with undetectable levels of OA or concentrations below 1 microg kg(-1) were toxin producers. Great differences in OA content in paprika samples were found, and a relationship with the climatic conditions of the geographic origin of the samples, and with cultural and technical practices in pepper manipulation is suggested.     

Potential of high-throughput sequencing for broad-range detection of pathogenic bacteria in spices and herbs

A broad-range culture-independent method was developed and evaluated regarding its sensitivity of detection of pathogenic bacteria in spices and herbs, with focus on paprika powder. The method involved DNA extraction using cetyltrimethylammonium bromide (CTAB), 16S rDNA amplification using universal bacterial polymerase chain reaction, and high-throughput sequencing on Illumina MiSeq platform. The sensitivity of the method was evaluated with series of model samples contaminated at different levels with Salmonella enterica and Escherichia coli (as representatives of Gram-negative bacteria) and Staphylococcus aureus (as a representative of Gram-positive bacteria). For spices (paprika, black pepper), the method had a screening-level sensitivity with limits of detection in the range of 10⁴–10⁵ CFU/g, and a semi-quantitative response. Low sensitivity (LOD ≥10⁷ CFU/g) was observed with herbs (oregano, parsley). The developed method demonstrated a good potential for microbiological screening of spices, with a prospect of further improvement of sensitivity based on progress in high-throughput sequencing technology.     

Differentiation of Spanish paprika from Protected Designation of Origin based on color measurements and pattern recognition

A method based on the measurement of color is proposed to differentiate paprika samples from different geographical origin. ASTA scale and coordinates in the CIELAB color space were obtained from UV–Vis spectra of acetone extracts of samples. Samples of sweet, hot/sweet and hot paprika from the two recognized Spanish Protected Designation of Origin, Murcia and Extremadura, were analyzed. Two strategies were considered with the aim of building classification models. The first used the computed color parameters as input data, whilst the second used the scores of the samples obtained after applying principal component analysis to reduce the dimensionality of the data matrix from the absorbance spectrum. The developed pattern recognition models were based on linear discriminant analysis, support vector machines and multilayer perceptron artificial neural networks. In both considered strategies, the best results were obtained in the case of artificial neural networks, with classification efficiencies ranging from 92% to 95% for the different varieties of paprika.     

NIR-based Sudan I to IV and Para-Red food adulterants screening

ABSTRACT

Spices are added in order to enhance the organoleptic characteristics of food and culinary dishes, making them more attractive for consumers. The use of illicit cheap colourants might be profitable along the food supply chain, posing undue risks to human health. This work evaluates the feasibility of NIR spectroscopy with chemometrics as a rapid, simple, non-destructive and affordable screening tool to determine the presence of Sudan I, II, III, IV and Para-red dyes in paprika. The dataset comprised unadulterated and adulterated samples with the five studied dyes at different concentration levels. Several multivariate classification models were built with Linear Discriminant Analysis (LDA) and different machine learning techniques. Preliminary results show that a classifier based on only six wavenumbers is able to determine the presence of some of these dyes in food samples in levels that may represent risk to human health. Sensitivities and specificities above 90% were obtained in almost all cases. These results show the feasibility of inexpensive and portable devices that can be useful for screening out adulterated stock along the food chain supply.     

Investigation of regional differences of the dominant microflora of spice paprika by molecular methods

As microbial contamination of spices comes primarily from the soil, and soil microbiota are dependent on climate, geography, agriculture, etc a qualitative focus on the dominant microflora of spices instead of the quantitative determination of commonly studied spoilage and pathogenic microflora may give a better insight into the geological origin of the sample. The aim of the present study was to identify using molecular microbiological methods the dominant bacteria of spice paprika produced at different countries and to look for species characteristic of spices from the given regions. According to our data, no differentiation could be made among spice paprika samples of different geographical origin using the total bacterial count or extent of mould contamination. However, when the dominant microflora is examined, bacterial species could be identified in the spice paprika samples characteristic of a particular climate. According to our study, the presence of Bacillus mycoides and Bacillus licheniformis was characteristic of the microflora of spice paprika grown in Central Europe; Bacillus safensis could be detected in all four paprika samples examined from the tropical monsoon climate; the species common to all three samples of the tropical climate group were Bacillus amyloliquefaciens subsp. plantarum and subsp. amyloliquefaciens, while Bacillus mojavensis was detected as being characteristic of Spanish origin for paprika. No common species were found in the paprika samples originating from China.