Low-Cost Pocket Fluorometer and Chemometric Tools for Green and Rapid Screening of Deoxynivalenol in Durum Wheat Bran.

Cereal crops are frequently contaminated by deoxynivalenol (DON), a harmful type of mycotoxin produced by several Fusarium species fungi. The early detection of mycotoxin contamination is crucial for ensuring safety and quality of food and feed products, for preventing health risks and for avoiding economic losses because of product rejection or costly mycotoxin removal. A LED-based pocket-size fluorometer is presented that allows a rapid and low-cost screening of DON-contaminated durum wheat bran samples, without using chemicals or product handling. Forty-two samples with DON contamination in the 40-1650 µg/kg range were considered. A chemometric processing of spectroscopic data allowed distinguishing of samples based on their DON content using a cut-off level set at 400 µg/kg DON. Although much lower than the EU limit of 750 µg/kg for wheat bran, this cut-off limit was considered useful whether accepting the sample as safe or implying further inspection by means of more accurate but also more expensive standard analytical techniques. Chemometric data processing using Principal Component Analysis and Quadratic Discriminant Analysis demonstrated a classification rate of 79% in cross-validation. To the best of our knowledge, this is the first time that a pocket-size fluorometer was used for DON screening of wheat bran.

Fourier transform near-infrared and mid-infrared spectroscopy as efficient tools for rapid screening of deoxynivalenol contamination in wheat bran.

BACKGROUND: Deoxynivalenol (DON) is the most common Fusarium mycotoxin occurring in wheat and wheat-derived products, with several adverse and toxic effects in animals and humans. Although bran fractions produced by milling wheat have numerous health benefits, cereal bran is the part of the grain with the highest concentration of DON, thus representing a risk for consumers. Increased efforts have been made to develop analytical methods suitable for rapid DON screening. RESULTS: The applicability of Fourier transform near-infrared (FTNIR), or mid-infrared (FTMIR) spectroscopy, and their combination for rapid analysis of DON in wheat bran, was investigated for the classification of samples into compliant and non-compliant groups regarding the EU legal limit of 750 µg kg-1 . Partial least squares-discriminant analysis (PLS-DA) and principal component-linear discriminant analysis (PC-LDA) were employed as classification techniques using a cutoff value of 400 µg kg-1 DON to distinguish the two classes. Depending on the classification model, overall discrimination rates were from 87% to 91% for FTNIR and from 86% to 87% for the FTMIR spectral range. The FTNIR spectroscopy gave the highest overall classification rate of wheat bran samples, with no false compliant samples and 18% false noncompliant samples when the PC-LDA classification model was applied. The combination of the two spectral ranges did not provide a substantial improvement in classification results in comparison with FTNIR. CONCLUSIONS: Fourier transform near-infrared spectroscopy in combination with classification models was an efficient tool to screen many DON-contaminated wheat bran samples and assess their compliance with EU regulations. © 2018 Society of Chemical Industry.

Rapid prediction of deoxynivalenol contamination in wheat bran by MOS-based electronic nose and characterization of the relevant pattern of volatile compounds.

BACKGROUND: Deoxynivalenol (DON) is a mycotoxin, mainly produced by Fusarium sp., most frequently occurring in cereals and cereal-based products. Wheat bran refers to the outer layers of the kernel, which has a high risk of damage due to chemical hazards, including mycotoxins. Rapid methods for DON detection in wheat bran are required. RESULTS: A rapid screening method using an electronic nose (e-nose), based on metal oxide semiconductor sensors, has been developed to distinguish wheat bran samples with different levels of DON contamination. A total of 470 naturally contaminated wheat bran samples were analyzed by e-nose analysis. Wheat bran samples were divided in two contamination classes: class A ([DON] ≤ 400 µg kg-1 , 225 samples) and class B ([DON] > 400 µg kg-1 , 245 samples). Discriminant function analysis (DFA) classified wheat bran samples with good mean recognizability in terms of both calibration (92%) and validation (89%). A pattern of 17 volatile compounds of wheat bran samples that were associated (positively or negatively) with DON content was also characterized by HS-SPME/GC-MS. CONCLUSIONS: These results indicate that the e-nose method could be a useful tool for high-throughput screening of DON-contaminated wheat bran samples for their classification as acceptable / rejectable at contamination levels close to the EU maximum limit for DON, reducing the number of samples to be analyzed with a confirmatory method. © 2018 Society of Chemical Industry.

Antibacterial Activity of Oregano (Origanum vulgare L.) Essential Oil Vapors against Microbial Contaminants of Food-Contact Surfaces.

The antimicrobial effect of eight essential oils' vapors against pathogens and spoilage bacteria was assayed. Oreganum vulgare L. essential oil (OVO) showed a broad antibacterial effect, with Minimum Inhibitory Concentration (MIC) values ranging from 94 to 754 µg cm-3 air, depending on the bacterial species. Then, gaseous OVO was used for the treatment of stainless steel, polypropylene, and glass surfaces contaminated with four bacterial pathogens at 6-7 log cfu coupon-1. No viable cells were found after OVO treatment on all food-contact surfaces contaminated with all pathogens, with the exception of Sta. aureus DSM 799 on the glass surface. The antimicrobial activity of OVO after the addition of beef extract as a soiling agent reduced the Sta. aureus DSM 799 viable cell count by more than 5 log cfu coupon-1 on polypropylene and glass, while no viable cells were found in the case of stainless steel. HS-GC-MS analysis of the headspace of the boxes used for the antibacterial assay revealed 14 different volatile compounds with α-Pinene (62-63%), and p-Cymene (21%) as the main terpenes. In conclusion, gaseous OVO could be used for the microbial decontamination of food-contact surfaces, although its efficacy needs to be evaluated since it depends on several parameters such as target microorganisms, food-contact material, temperature, time of contact, and relative humidity.

Does alteration of fumonisin production in Fusarium verticillioides lead to volatolome variation?

Fusarium verticillioides, a major fungal pathogen of maize, produces fumonisins, mycotoxins of global food safety concern. Control practices are needed to reduce the negative health and economic impacts of fumonisins. Therefore, we investigated volatile organic compounds (VOCs) emitted by fumonisin-producing (wild-type) and nonproducing (mutant) strains of F. verticillioides. VOC emissions were analyzed by gas chromatography-mass spectrometry following inoculation of maize kernels, and fumonisin accumulation was analyzed by high-performance liquid chromatography. Mutants emitted VOCs, including ethyl 3-methylbutanoate, that the wild type did not emit. In particular, ANOVA analysis showed significant differences between mutants and wild type for 4 VOCs which emission was correlated with absence of fumonisins. Exogenous ethyl 3-methylbutanoate reduced growth and fumonisin production in wild-type F. verticillioides, showing its potential in biocontrol. Together, our findings offer valuable insights into how mycotoxin production can impact VOC emissions from F. verticillioides and reveal a potential biocontrol strategy to reduce fumonisin contamination.

Comparison of slurry mixing and dry milling in laboratory sample preparation for determination of ochratoxin A and deoxynivalenol in wheat.

The significance of laboratory sample preparation for the determination of two important mycotoxins, ochratoxin A (OTA) and deoxynivalenol (DON), in wheat was investigated by comparing water-slurry mixing and dry-milling procedures. The distribution of OTA and DON in 10 kg samples of naturally contaminated wheat was established by analyzing one hundred 100 g subsamples of each sample. A normal distribution and a good repeatability of DON measurements was observed for both water-slurry mixing (mean 2290 microg/kg, CV 4.6%, median 2290 microg/kg) and dry milling (mean 2310 microg/kg, CV 6.4%, median 2290 microg/kg) procedures. For OTA determinations, reliable results could be obtained only by slurry mixing sample preparation (mean 2.62 microg/kg, CV 4.0%, median 2.62 microg/kg), whereas dry-milling comminution resulted in an inhomogeneous distribution with a high variability (mean 0.83 microg/kg, CV 75.2%, median 0.60 microg/kg) and a positive skewness (2.12). Ad hoc experiments were performed on different size portions of the same sample (10 kg) to assess accuracy and precision of the comminution/homogenization procedures (slurry mixing and dry milling). Very good results were obtained for DON determination with both procedures in terms of accuracy (>98.7% of the "weighted value") and precision (CV <3%). For OTA determination good results were only obtained by slurry mixing (99.4% of the "weighted value," CV 10%), whereas dry milling provided results with low accuracy (43.2% of the "weighted value") and high variability (CV 110%). This study clearly demonstrated that sample preparation by slurry mixing is strictly necessary to obtain reliable laboratory samples for OTA determination in wheat to minimize misclassification of acceptable/rejectable lots, mainly within official control.

Overview of Recent Liquid Chromatography Mass Spectrometry-Based Methods for Natural Toxins Detection in Food Products.

Natural toxins include a wide range of toxic metabolites also occurring in food and products, thus representing a risk for consumer health. In the last few decades, several robust and sensitive analytical methods able to determine their occurrence in food have been developed. Liquid chromatography mass spectrometry is the most powerful tool for the simultaneous detection of these toxins due to its advantages in terms of sensitivity and selectivity. A comprehensive review on the most relevant papers on methods based on liquid chromatography mass spectrometry for the analysis of mycotoxins, alkaloids, marine toxins, glycoalkaloids, cyanogenic glycosides and furocoumarins in food is reported herein. Specifically, a literature search from 2011 to 2021 was carried out, selecting a total of 96 papers. Different approaches to sample preparation, chromatographic separation and detection mode are discussed. Particular attention is given to the analytical performance characteristics obtained in the validation process and the relevant application to real samples.

Mass spectrometry-based electronic nose to authenticate 100% Italian durum wheat pasta and characterization of volatile compounds.

Headspace solid-phase microextraction (HS-SPME) coupled with mass spectrometry-based electronic nose (MS-eNose), in combination with multivariate statistical analysis was used as untargeted method for the rapid authentication of 100% Italian durum wheat pasta. Among the tested classification models, i.e. PCA-LDA, PLS-DA and SVMc, SVMc provided the highest accuracy results in both calibration (90%) and validation (92%) processes. Potential markers discriminating pasta samples were identified by HS-SPME/GC-MS analysis. Specifically, the content of a pattern of 8 out of 59 volatile organic compounds (VOCs) was significantly different between samples of 100% Italian durum wheat pasta and pasta produced with durum wheat of different origins, most of which were related to different lipidic oxidation in the two classes of pasta. The proposed MS-eNose method is a rapid and reliable tool to be used for authenticating Italian pasta useful to promote its typicity and preserving consumers from fraudulent practices.

Effect of Gaseous Citral on Table Grapes Contaminated by Rhizopus oryzae ITEM 18876.

Rhizopus oryzae is responsible for rapidly producing a deliquescent appearance in grape berries, generally favoured by cold chain interruptions. To counteract fruit spoilage and to meet consumer acceptance, innovative strategies based on the application of natural compounds are ongoing. Due to their biological activities, including antimicrobial ones, natural flavour compounds extend the shelf life and improve the nutritional value as well as the organoleptic properties of foods. Thus, in this work, the application of the antimicrobial citral, a flavor component of monoterpenes identified in plant and fruit essential oils, was developed and validated against one spoiler of R. oryzae. Citral, as pure compound, was first investigated in vitro against R. oryzae ITEM 18876; then, concentrations equal to the minimal inhibitory concentration (MIC) and 4-fold MIC (4MIC) value were applied on the table grape cv Italia infected with this strain and stored. The MIC value was equal to 0.0125 µL/cm3; both citral concentrations (0.0125 and 0.05 µL/cm3) were effective in counteracting the microbial decay of infected table grapes over the storage period. The HS-SPME/GC-MS method showed citral persistence in the head space of plastic trays with the infected samples; as expected, a higher content of citral isomers was found in the sample treated with 4MIC value. In conclusion, citral revealed its efficacy to counteract the onset of soft rot by R. oryzae ITEM 18876 under storage conditions. Thus, it could be successfully exploited to develop an active packaging or natural preservatives to extend table grape shelf life without affecting its quality and sensory characteristics, whilst also satisfying the consumer demand for natural preservative agents.

Industrial-Scale Cleaning Solutions for the Reduction of Fusarium Toxins in Maize.

Grain cleaning is the most effective non-destructive post-harvest mitigation strategy to reduce high levels of mycotoxins on account of the removal of mold-infected grains and grain fractions with high mycotoxin content. In this study, the reduction in the concentration of some co-occurring Fusarium toxins in maize, namely deoxynivalenol (DON), zearalenone (ZEA) and fumonisins B1 and B2 (FBs), was evaluated at an industrial-scale level by mechanical removal (sieving and density separation) of dust, coarse, small, broken, shriveled and low-density kernels and/or optical sorting of defected kernels. Samples were dynamically collected according to the Commission Regulation No. 401/2006 along the entire process line. Mycotoxin analyses of water-slurry aggregate samples were performed by validated LC methods. Depending on the contamination levels in raw incoming maize, the overall reduction rates ranged from 36 to 67% for DON, from 67 to 87% for ZEA and from 27 to 67% for FBs. High levels of DON, ZEA and FBs were found in all rejected fractions with values, respectively, up to 3030%, 1510% and 2680%, compared to their content in uncleaned maize. Results showed that grain cleaning equipment based on mechanical and or optical sorting technologies can provide a significant reduction in Fusarium toxin contamination in maize.

A rapid fluorescence polarization immunoassay for the determination of T-2 and HT-2 toxins in wheat.

A rapid fluorescence polarization (FP) immunoassay has been developed for the simultaneous determination of T-2 and HT-2 toxins in naturally contaminated wheat samples. Syntheses of four fluorescein-labelled T-2 or HT-2 toxin tracers were carried out and their binding response with seven monoclonal antibodies was evaluated. The most sensitive antibody-tracer combination was obtained by using an HT-2-specific antibody and a fluorescein-HT-2 tracer. The developed competitive FP immunoassay in solution showed high cross-reactivity for T-2 toxin (CR% = 100%) while a very low CR% for neosolaniol (0.12%) and no cross-reactivity with other mycotoxins frequently occurring in wheat. A rapid extraction procedure using 90% methanol was applied to wheat samples prior to FP immunoassay. The average recovery from spiked wheat samples (50 to 200 µg kg(-1)) was 96% with relative standard deviation generally lower than 8%. A limit of detection of 8 µg kg(-1) for the combined toxins was determined. Comparative analyses of 45 naturally contaminated and spiked wheat samples by both the FP immunoassay and high-performance liquid chromatography/immunoaffinity clean-up showed a good correlation (r = 0.964). These results, combined with the rapidity (10 min) and simplicity of the assay, show that this method is suitable for high throughput screening as well as for quantitative determination of T-2 and HT-2 toxins in wheat.

Rapid Authentication of 100% Italian Durum Wheat Pasta by FT-NIR Spectroscopy Combined with Chemometric Tools.

Italy is the country with the largest durum wheat pasta production and consumption. The mandatory labelling for pasta indicating the country of origin of wheat has made consumers more aware about the consumed pasta products and is influencing their choice towards 100% Italian wheat pasta. This aspect highlights the need to promote the use of domestic wheat as well as to develop rapid methodologies for the authentication of pasta. A rapid, inexpensive, and easy-to-use method based on infrared spectroscopy was developed and validated for authenticating pasta made with 100% Italian durum wheat. The study was conducted on pasta marketed in Italy and made with durum wheat cultivated in Italy (n = 176 samples) and on pasta made with mixtures of wheat cultivated in Italy and/or abroad (n = 185 samples). Pasta samples were analyzed by Fourier transform-near infrared (FT-NIR) spectroscopy coupled with supervised classification models. The good performance results of the validation set (sensitivity of 95%, specificity and accuracy of 94%) obtained using principal component-linear discriminant analysis (PC-LDA) clearly demonstrated the high prediction capability of this method and its suitability for authenticating 100% Italian durum wheat pasta. This output is of great interest for both producers of Italian pasta pointing toward authentication purposes of their products and consumer associations aimed to preserve and promote the typicity of Italian products.

A simple design for the validation of a FT-NIR screening method: Application to the detection of durum wheat pasta adulteration.

The demand for the development of fast, easy-to-use and low-cost analytical methods for food adulteration analysis has being increasing in the last years. Although infrared spectroscopic techniques offer these advantages, the validation of screening methods requiring the application of multivariate data treatment is less frequently described in literature thus limiting their use as routine tools in control laboratories for food fraud monitoring. In this paper, an EU-validation procedure for screening methods was successfully applied to a multivariate FT-NIR spectroscopic method for the screening of durum wheat pasta samples adulterated with common wheat at the screening target concentration of 3%. Good results in terms of the cut-off value (2.32% mass fraction of soft wheat) and false suspect rates (0.1% for blanks; 13% at 1% mass fraction) demonstrated that the present validation approach would be a proof-of-strategy to be used for multivariate infrared methods applied for screening purposes.

Natural Occurrence of Ochratoxin A in Blood and Milk Samples from Jennies and Their Foals after Delivery.

An assessment of the natural ochratoxin A (OTA) exposure of seven Martina Franca jennies was carried out by analyzing blood and milk samples collected close to and after delivery. A total of 41 and 34 blood samples were collected from jennies and foals, respectively, and analyzed by ELISA. A total of 33 milk samples were collected from jennies and analyzed by the HPLC/FLD method based on IAC clean-up. Furthermore, 53 feed samples were collected from January to September and analyzed by a reference method (AOAC Official Method No. 2000.03) for OTA content. Feed samples showed OTA levels up to 2.7 ng/g with an incidence of 32%, while the OTA incidence rate in jennies' blood samples was 73%, with a median value of 97 ng/L and concentrations ranging from

Quality evaluation of table grapes during storage by using 1H NMR, LC-HRMS, MS-eNose and multivariate statistical analysis.

Three non-targeted methods, i.e. 1H NMR, LC-HRMS, and HS-SPME/MS-eNose, combined with chemometrics, were used to classify two table grape cultivars (Italia and Victoria) based on five quality levels (5, 4, 3, 2, 1). Grapes at marketable quality levels (5, 4, 3) were also discriminated from non-marketable quality levels (2 and 1). PCA-LDA and PLS-DA were applied, and results showed that, the MS-eNose provided the best results. Specifically, with the Italia table grapes, mean prediction abilities ranging from 87% to 88% and from 98% to 99% were obtained for discrimination amongst the five quality levels and of marketability/non-marketability, respectively. For the cultivar Victoria, mean predictive abilities higher than 99% were achieved for both classifications. Good models were also obtained for both cultivars using NMR and HRMS data, but only for classification by marketability. Satisfying models were further validated by MCCV. Finally, the compounds that contributed the most to the discriminations were identified.

Geographical Origin Discrimination of Monofloral Honeys by Direct Analysis in Real Time Ionization-High Resolution Mass Spectrometry (DART-HRMS).

An untargeted method using direct analysis in real time and high resolution mass spectrometry (DART-HRMS) combined to multivariate statistical analysis was developed for the discrimination of two monofloral (chestnut and acacia) honeys for their geographical origins-i.e., Italy and Portugal for chestnut honey and Italy and China for acacia honey. Principal Component Analysis, used as an unsupervised approach, showed samples of clusterization for chestnut honey samples, while overlapping regions were observed for acacia honeys. Three supervised statistical approaches, such as Principal Components-Linear Discriminant Analysis, Partial Least Squares-Discriminant Analysis and k-nearest neighbors, were tested on the dataset gathered and relevant performances were compared. All tested statistical approaches provided comparable prediction abilities in cross-validation and external validation with mean values falling between 89.2-98.4% for chestnut and between 85.8-95.0% for acacia honey. The results obtained herein indicate the feasibility of the DART-HRMS approach in combination with chemometrics for the rapid authentication of honey's geographical origin.

Aflatoxin Reduction in Maize by Industrial-Scale Cleaning Solutions.

Different batches of biomass/feed quality maize contaminated by aflatoxins were processed at the industrial scale (a continuous process and separate discontinuous steps) to evaluate the effect of different cleaning solutions on toxin reduction. The investigated cleaning solutions included: (i) mechanical size separation of coarse, small and broken kernels, (ii) removal of dust/fine particles through an aspiration channel, (iii) separation of kernels based on gravity and (iv) optical sorting of spatial and spectral kernel defects. Depending on the sampled fraction, dynamic or static sampling was performed according to the Commission Regulation No. 401/2006 along the entire cleaning process lines. Aflatoxin analyses of the water-slurry aggregate samples were performed according to the AOAC Official Method No. 2005.008 based on high-performance liquid chromatography and immunoaffinity column cleanup of the extracts. A significant reduction in aflatoxin content in the cleaned products, ranging from 65% to 84% with respect to the uncleaned products, was observed when continuous cleaning lines were used. Additionally, an overall aflatoxin reduction from 55% to 94% was obtained by combining results from separate cleaning steps. High levels of aflatoxins (up to 490 µg/kg) were found in the rejected fractions, with the highest levels in dust and in the rejected fractions from the aspirator and optical sorting. This study shows that a cleaning line combining both mechanical and optical sorting technologies provides an efficient solution for reducing aflatoxin contamination in maize.

Tracing the Geographical Origin of Durum Wheat by FT-NIR Spectroscopy.

Fourier transform near infrared (FT-NIR) spectroscopy, in combination with principal component-linear discriminant analysis (PC-LDA), was used for tracing the geographical origin of durum wheat samples. The classification model PC-LDA was applied to discriminate durum wheat samples originating from Northern, Central, and Southern Italy (n = 181), and to differentiate Italian durum wheat samples from those cultivated in other countries across the world (n = 134). Developed models were validated on a separated set of wheat samples. Different pre-treatments of spectral data and different spectral regions were selected and compared in terms of overall discrimination (OD) rates obtained in validation. The LDA models were able to correctly discriminate durum Italian wheat samples according to their geographical origin (i.e., North, Central, and South) with OD rates of up of 96.7%. Better results were obtained when LDA models were applied to the discrimination of Italian durum wheat samples from those originating from other countries across the world, having OD rates of up to 100%. The excellent results obtained herein clearly indicate the potential of FT-NIR spectroscopy to be used for the discrimination of durum wheat samples according to their geographical origin.

Performance Evaluation of LC-MS Methods for Multimycotoxin Determination.

The co-occurrence of regulated mycotoxins in foods and feeds, together with modified ("masked") and emerging mycotoxins, has been increasingly reported worldwide in recent years. Therefore, sensitive, accurate, and validated methods for the simultaneous determination of these hazardous contaminants in different matrices are highly demanded to fulfil regulatory requirements and to carry out reliable surveillance programs. In these last years, LC-MS methodologies for multimycotoxin screening and/or quantification are being routinely used in control laboratories. However, to date, only one European Standard for multimycotoxin determination is based on LC-MS (EN 16877:2016). The need for standardized LC-MS methods for multimycotoxin determination has been highlighted by regulatory authorities and scientific advisory bodies, including the U.S. Food and Drug Administration and the European Commission. The European Committee for Standardization (CEN) has issued calls for tender for the development of standardized LC-MS methods for mycotoxins in food and animal feeding stuffs. As deliverables, some LC-MS based methods for multimycotoxin determination are currently under approval as European Standards. In addition, the European Commission has recently established specific criteria with which screening methods for mycotoxins, including LC-MS methods, have to comply for use for regulatory purposes. Validation procedures by single-laboratory and collaborative trials have been defined. This paper provides insights and advances on guidelines and tools for performance evaluation of LC-MS methods intended for quantitative determination and for semiquantitative screening of multimycotoxins. In particular, performance criteria set in the European Union and the United States are critically overviewed, and expectations, needs, and future challenges relevant to LC-MS methods for multimycotoxin determination are also discussed.

Discrimination of geographical origin of oranges (Citrus sinensis L. Osbeck) by mass spectrometry-based electronic nose and characterization of volatile compounds.

An untargeted method using headspace solid-phase microextraction coupled to electronic nose based on mass spectrometry (HS-SPME/MS-eNose) in combination with chemometrics was developed for the discrimination of oranges of three geographical origins (Italy, South Africa and Spain). Three multivariate statistical models, i.e. PCA/LDA, SELECT/LDA and PLS-DA, were built and relevant performances were compared. Among the tested models, SELECT/LDA provided the highest prediction abilities in cross-validation and external validation with mean values of 97.8% and 95.7%, respectively. Moreover, HS-SPME/GC-MS analysis was used to identify potential markers to distinguish the geographical origin of oranges. Although 28 out of 65 identified VOCs showed a different content in samples belonging to different classes, a pattern of analytes able to discriminate simultaneously samples of three origins was not found. These results indicate that the proposed MS-eNose method in combination with multivariate statistical analysis provided an effective and rapid tool for authentication of the orange's geographical origin.