Michelangelo Pascale

The valorization of agri-food residues is crucial for advancing circular bioeconomy strategies and mitigating environmental impacts. Turnip greens (Brassica rapa subsp. sylvestris) are a traditional vegetable cultivated in southern Italy. While the edible portions include flower sprouts, buds, and young leaves, the more leathery leaves and stems are typically discarded. These wastes represent valuable sources of compounds with antioxidant and antimicrobial potential. This study aims to develop the extraction of phenolic compounds from turnip green residues using two techniques: silent maceration and ultrasound-assisted extraction (UAE). Ethanol was selected over methanol as a food-safe alternative solvent, with preliminary tests confirming equivalent efficiency. A Design of Experiments (DoE) approach was applied to both leaves and stems to assess the effects of solvent composition, solvent-to-matrix ratio, and extraction time on Total Phenolic Content and Trolox Equivalent Antioxidant Capacity. DoE results identified UAE as the most effective method for stems, while for leaves, the solvent-to-dry-mass ratio was the key parameter. HPLC-DAD analysis was performed to identify and quantify the phenolic acids in selected extracts. The antibacterial activity of these extracts against biofilms of six pathogenic strains was evaluated using crystal violet and MTT assays, confirming efficacy in both biofilm formation and mature stages.

There is an increasing demand to design user-friendly specific assays for the detection of analytes of interest for healthcare, environment, and agrifood. Modern biotechnology has approached this problem by using proteins, enzymes, or RNA/DNA fragments (aptamers) as biological recognition elements for biosensors/assays. The idea is to exploit the extremely wide range of selective affinities sculpted into the various proteins or aptamers by biological evolution. The number of compounds specifically recognized by different proteins and aptamers is very large and ranges from small molecules to macromolecules. The advantages of using proteins and aptamers as molecular recognition elements (MRE) for assays/biosensors are many, and involve relatively low costs in design and synthesis, water solubility, and finally high specificity. Many of the analytes of interest in the food control industry are relatively small. In this case, aptamers and antibodies are widely used as specific MREs in designing advanced biosensors. Aflatoxin B1 (AFB1) is the most frequently found aflatoxin in contaminated food samples, and is one of the most potent natural compounds in terms of genotoxicity and carcinogenicity. Aflatoxin M1 (AFM1) is the hydroxylated metabolite of AFB1 and is usually found in milk and milk products as a carry-over of AFB1 in animals that have ingested contaminated feed. AFM1 is also found in human milk, and has been shown to be hepatotoxic and carcinogenic. Here, we present recent advances in assays and biosensors based on the use of antibodies and aptamers as MREs that have been developed for monitoring the presence of AFM1 in milk and dairy products. The limitations and advantages of aptamer- and antibody-based assays/biosensors are discussed, as well as future research perspectives.

There is an increasing demand in designing user-friendly specific assays for the detec-tion of analytes of interest for healthcare, environment, and agrifood. Modern bio-technology has approached this problem by using proteins, enzymes, or RNA/DNA fragments (aptamers) as biological recognition element of biosensors/assays. The idea is to exploit the extremely wide range of selective affinities sculpted into the various proteins or aptamers by biological evolution. The number of compounds specifically recognized by different proteins and aptamers is very large and ranges from small molecules to macromolecules. The advantages of using proteins and aptamers as mo-lecular recognition element (MRE) of assays/biosensors are many and include relative-ly low costs in design and synthesis, water solubility, and finally, high specificity. Many of the analytes of interest in the food control industry are relatively small. In this case, the use of aptamers and antibodies as specific MREs in designing advanced bio-sensors is widely used. Aflatoxin B1 (AFB1) is the most frequently found aflatoxin in contaminated food samples and it is one of the most potent natural compounds in terms of genotoxicity and carcinogenicity. Aflatoxin M1 (AFM1) is the hydroxylated metabolite of AFB1 and is usually found in milk and milk products as carry-over of AFB1 in animals that have ingested contaminated feed. AFM1 is found also in human milk and has been shown to be hepatotoxic and carcinogenic. Here, we present recent advances in assays and biosensors based on the use of antibodies and aptamers as MREs that have been developed for monitoring the presence of AFM1 in milk and dairy products. Limitations and advantages of aptamer- and antibody-based as-says/biosensors are discussed, as well as future research perspectives.

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.

Food allergies are an exceptional response of the immune system caused by the ingestion of specific foods. The main foods responsible for allergic reactions are milk, eggs, seafood, soy, peanuts, tree nuts, wheat, and their derived products. Chicken egg ovalbumin (OVA), a common allergen molecule, is often used for the clarification process of wine. Traces of OVA remain in the wine during the fining process, and they can cause significant allergic reactions in sensitive consumers. Consequently, the European Food Safety Authority (EFSA) and the American Food and Drug Administration (FDA) have shown the risks for allergic people to assume allergenic foods and food ingredients, including eggs. Commonly, OVA detection requires sophisticated and time-consuming analytical techniques. Intending to develop a faster assay, we designed a proof-of-concept non-Faradaic impedimetric immunosensor for monitoring the presence of OVA in wine. Polyclonal antibodies anti-OVA were covalently immobilised onto an 11-mercaptoundecanoic-acid (11-MUA)-modified gold surface. The developed immunosensor was able to detect OVA in diluted white wine without the need for an external probe or any pre-treatment step with a sensitivity of 0.20 µg/mL, complying with the limit established by the resolution OIV/COMEX 502–2012 for the quantification of allergens in wine.

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.

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.

Aptamers are single-stranded oligonucleotides selected by SELEX (Systematic Evolution of Ligands by EXponential Enrichment) able to discriminate target molecules with high affinity and specificity, even in the case of very closely related structures. Aptamers have been produced for several targets including small molecules like mycotoxins; however, the high affinity for their respective target molecules is a critical requirement. In the last decade, the screening through computational methods of aptamers for their affinity against specific targets has greatly increased and is becoming a commonly used procedure due to its convenience and low costs. This paper describes an in-silico approach for rapid screening of ten ssDNA aptamer sequences against fumonisin B1 (FB1, n = 3), aflatoxin B1 (AFB1, n = 2) and ochratoxin A (OTA, n = 5). Theoretical results were compared with those obtained by testing the same aptamers by fluorescent microscale thermophoresis and by magnetic beads assay for their binding affinity (K_D) revealing a good agreement.

An analytical method for the simultaneous determination of trichothecenes—namely, nivalenol (NIV), deoxynivalenol (DON) and its acetylated derivatives (3- and 15-acetyl-DON), T-2 and HT-2 toxins—and zearalenone (ZEN) in wheat, wheat flour, and wheat crackers was validated through a collaborative study involving 15 participants from 10 countries. The validation study, performed within the M/520 standardization mandate of the European Commission, was carried out according to the IUPAC (International Union of Pure and Applied Chemistry) International Harmonized Protocol. The method was based on mycotoxin extraction from the homogenized sample material with a mixture of acetonitrile-water followed by purification and concentration on a solid phase extraction column. High-performance liquid chromatography coupled with tandem mass spectrometry was used for mycotoxin detection, using isotopically labelled mycotoxins as internal standards. The tested contamination ranges were from 27.7 to 378 μg/kg for NIV, from 234 to 2420 μg/kg for DON, from 18.5 to 137 μg/kg for 3-acetyl-DON, from 11.4 to 142 μg/kg for 15-acetyl-DON, from 2.1 to 37.6 μg/kg for T-2 toxin, from 6.6 to 134 μg/kg for HT-2 toxin, and from 31.6 to 230 μg/kg for ZEN. Recoveries were in the range 71–97% with the lowest values for NIV, the most polar mycotoxin. The relative standard deviation for repeatability (RSD_r) was in the range of 2.2–34%, while the relative standard deviation for reproducibility (RSD_R) was between 6.4% and 45%. The HorRat values ranged from 0.4 to 2.0. The results of the collaborative study showed that the candidate method is fit for the purpose of enforcing the legislative limits of the major Fusarium toxins in wheat and wheat-based products.

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 <LOD to 6000 ng/L. A seasonal effect on OTA levels in positive blood samples was observed, with increases in the 53% of the positive ones from April to June. Concerning foals, the incidence rate of blood samples was 50%, with a median value of 52 ng/L, and concentrations ranged from <LOD to 4034 ng/L. The incidence of milk samples was 36%, with levels ranging from <LOD to 82 ng/L. In conclusion, the results showed a natural exposure of jennies and foals to OTA, and its presence in jenny milk could pose a risk for human newborns, considering its well-known nutritional and health properties.