Novel quadrupole-time of flight-based methodology for determination of multiple mycotoxins in human hair.

The potential of hair as matrix for assessing long-term exposure to mycotoxins remains scarcely explored. Therefore, this study aimed to develop and validate an analytical methodology for the simultaneous determination of aflatoxins, enniatins, beauvericin and T-2 toxin in human hair, based on a pretreatment stage prior to salt-assisted liquid-liquid extraction and followed by high performance liquid chromatography coupled to high resolution Q-TOF mass spectrometry for the first time. Washing with a non-ionic detergent was successfully applied, whereas enzymatic digestion with Pronase E was mandatory for releasing mycotoxins from the hair matrix. The methodology was validated according to Commission Decision 2002/657/EC, with limits of quantification ranging from 0.6 to 8.7 ng/g. The analysis of 10 samples showed at least one mycotoxin occurring in 67% of samples, including the carcinogenic aflatoxins. This is the first validated methodology for the quantification of multiple mycotoxins in human hair.

Extraction of Phenolic Compounds from Fresh Apple Pomace by Different Non-Conventional Techniques.

Red Delicious apple pomace was produced at laboratory scale with a domestic blender and different non-conventional extraction techniques were performed to isolate phenolic compounds, such as ultrasound-assisted extraction (UAE), ultraturrax extraction (UTE), accelerated solvent extraction (ASE) and pulsed electric field (PEF) extraction pre-treatment. Total phenolic content (TPC) was determined by Folin-Ciocalteu assay. Phloridzin, the main phenolic compound in apples, was determined by chromatographic analysis Q-TOF-LC/MS. The results obtained with these techniques were compared in order to identify the most efficient method to recover polyphenols. The highest value of TPC (1062.92 ± 59.80 µg GAE/g fresh apple pomace) was obtained when UAE was performed with EtOH:H2O (50:50, v/v), while ASE with EtOH:H2O (30:70, v/v) at 40 °C and 50% of flush was the most efficient technique in the recovery of phloridzin. The concentration of the main phenolic compounds ranged from 385.84 to 650.56 µg/g fresh apple pomace. The obtained results confirm that apple pomace represents an interesti-ng by-product, due to the presence of phenolic compounds. In particular, phloridzin could be considered a biomarker to determine the quality of numerous apple products. Therefore, this research could be a good starting point to develop a value-added product such as a functional food or nutraceutical.

Multi-mycotoxin contamination of green tea infusion and dietary exposure assessment in Moroccan population.

Green tea infusion is one of the most widely drunk beverages worldwide due to its health benefits associated with microelements, essential oils, and polyphenols, etc. Several studies have reported that green tea is subjected to contamination by various toxigenic fungi. Thus, this work aims to investigate the co-occurrence of 15 mycotoxins [four aflatoxins (AFB1, AFB2, AFG1, AFG2), ochratoxin A (OTA), beauvericin (BEA), four enniatins (ENA, ENA1, ENB, ENB1), zearalenone (ZEN), alternariol (AOH), tentoxin (TENT), T-2 and HT-2 toxins] in green tea samples available in Morocco by liquid chromatography tandem mass spectrometry method. Analytical and consumption data were then used to assess the dietary exposure for the population. Out of 111 total green tea samples, 62 (56%) were contaminated by at least one mycotoxin. The most found mycotoxins in samples were AOH (40%), ZEN (35%), AFG1 (2%), AFB2 (2%), ENB (2%) and TENT (1%). The highest level was found for ZEN with 45.8 ng/g. There is no sample that exceeded the recommended levels set by European Pharmacopoeia for certain mycotoxins in plant material. Although multi-mycotoxin co-occurred in samples (33%), the probable estimated daily intake values show that the intake of mycotoxins through the consumption of green tea does not represent a risk for the population.

Coffee Silverskin and Spent Coffee Suitable as Neuroprotectors against Cell Death by Beauvericin and α-Zearalenol: Evaluating Strategies of Treatment.

Coffee silverskin and spent coffee have been evaluated in a neuroblastoma cell line (SH-SY5Y cells) against beauvericin (BEA) and α-zearalenol (α-ZEL)-induced cytotoxicity with different strategies of treatment. First, the direct treatment of mycotoxins and coffee by-products extracts in SH-SY5Y cells was assayed. IC50 values for α-ZEL were 20.8 and 14.0 µM for 48 h and 72 h, respectively and, for BEA only at 72 h, it was 2.5 µM. Afterwards, the pre-treatment with spent coffee obtained by boiling water increased cell viability for α-ZEL at 24 h and 48 h from 10% to 16% and from 25% to 30%, respectively; while with silverskin coffee, a decrease was observed. Opposite effects were observed for BEA where an increase for silverskin coffee was observed at 24 h and 48 h, from 14% to 23% and from 25% to 44%, respectively; however, a decrease below 50% was observed for spent coffee. Finally, the simultaneous treatment strategy for the highest concentration assayed in SH-SY5Y cells provided higher cytoprotection for α-ZEL (from 44% to 56% for 24 h and 48 h, respectively) than BEA (30% for 24 h and 48 h). Considering the high viability of coffee silverskin extracts for SH-SY5Y cells, there is a forthcoming promising use of these unexploited residues in the near future against mycotoxins effects.

Reducing the effect of beauvericin on neuroblastoma SH-SY5Y cell line by natural products.

In the present work, different natural compounds from coffee by-product extracts (coffee silverskin and spent coffee) rich in polyphenols, was investigated against beauvericin (BEA) induced-cytotoxicity on SH-SY5Y cells. Spent coffee arise as waste products through the production of instant coffee and coffee brewing; while the silverskin is a tegument which is removed and eliminated with toasting coffee grains. First of all, polyphenol extraction methods, measurement of total polyphenols content and its identification were carried out. Afterwards evaluating in vitro effects with MTT assay on SH-SY5Y cells of coffee by-product extracts and mycotoxins at different concentrations and exposure times was performed. TPC in silverskin coffee by-product extracts was >10 times higher than in spent coffee by-product extracts. Chlorogenic acid was the majority polyphenol detected. Viability for BEA reached IC50 values at 72h (2.5 µM); boiling water silverskin coffee extract reached the highest viability also in pre-treatment BEA exposure and compared with MeOH and MeOH:H2O (v/v, 50:50) extracts. These results in SH-SY5Y cells highlight the use of such residues as supplements or bioactive compounds in the future.

Chemoprotective effect of carotenoids from Lycium barbarum L. on SH-SY5Y neuroblastoma cells treated with beauvericin.

Goji berry has recently been introduced in Mediterranean diet and its consumption is increasing. This study aims to determine cytoprotection of lutein (LUT), zeaxanthin (ZEAX) and goji berry extract (GBE) rich in carotenoids against Beauvericin (BEA)-induced cytotoxicity on SH-SY5Y neuroblastoma cells. Both carotenoids and GBE showed cytoprotective effects. Cytoprotection was evaluated by simultaneous combination of the two xanthophylls LUT and ZEAX with BEA, as well as using pre-treatment assays. The highest protective effect occurred in 16%, 24% and 12% respectively for LUT, ZEAX and LUT + ZEAX incubating simultaneously with BEA, while by pre-treatment assay LUT showed a cytoprotection effect over 30% and ZEAX alone or LUT + ZEAX promoted only a slight cytoprotection (<10%). Pre-treatment assays with GBE, showed a cytoprotection, between 3 and 20%, for BEA concentrations ranging from 0.1 to 6.25 µM, whereas no protective effect was observed when the cells were simultaneously incubated with GBE and BEA. Finally, by means of CI-isobologram method, the interaction between LUT, ZEAX and BEA were evaluated, and the results showed an synergism effect for almost all combinations tested. The data presented shows a option of using goji berries to potentially mitigate the toxicity of beauvericin eventually present in foods.

Impact of Ultrasound Extraction Parameters on the Antioxidant Properties of Moringa Oleifera Leaves.

Recently, much interest has been focused on Moringa oleifera L., a highly versatile and sustainable plant. In addition to its nutritional properties, numerous bioactive compounds have been identified in M. oleifera leaves, for which healthy properties have been reported. In the present research, the impact of ultrasound-assisted extraction (UAE) on the recovery of the bioactive compounds from leaves was investigated. Firstly, an experimental design approach has been used to highlight the influence of some extraction parameters (solvent, solvent/dry leaves ratio, temperature, time) on phenol compound recovery and antioxidant activity. Solvent composition was the most influential factor; in fact, the presence of water in the solvent (50:50, v/v) corresponded to an increase in the extraction performance. The liquid/solid ratio (L/S) also influenced the extraction process; in fact, the total phenol content reached 13.4 mg gallic acid equivalent (GAE)/g dry matter (DM) in the following UAE conditions: 50% water, 60:1 L/S ratio, 60 °C, 60 min. In order to quantify flavonols, hydroalcoholic extracts were analysed by HPLC-DAD (high performance liquid chromatography-diode array detector). In the flavonol class, the glycosidic forms of quercetin and kaempferol were mainly detected. Their content ranged from 216.4 µg/g DM of quercetin 3-O-rhamnoside to 293.9 µg/g DM of quercetin 3-O-(6″-O-malonyl)-ß-D-glucoside. In summary, the leaves of M. oleifera are a potential natural source of bioactive compounds, proving to be very promising for the development of health-promoting food supplements.

Beauvericin and ochratoxin A mycotoxins individually and combined in HepG2 cells alter lipid peroxidation, levels of reactive oxygen species and glutathione.

The co-presence of more than one mycotoxin in food is being evidenced in last food surveys as reported in the literature. Beauvericin (BEA) is a non-legislated emergent mycotoxin while Ochratoxin A (OTA) has been widely studied and legislated. Concentration range individually studied was from 2.5 to 0.3 µM for BEA and from 25 to 3.1 µM for OTA; binary mixture [BEA + OTA] comprised concentrations of 1:10 ratio from [2.5 + 25] to [3.1 + 0.3] µM. Potential of toxicity of BEA in HepG2 cells was the highest at all times assayed (24, 48 and 72h). LPO was performed through malondyaldehyde (MDA) detection denoting in the binary mixture for [1.25 + 12.5] µM and at 24 and 72h the highest disturbance values. ROS denoted differences respect to the control at different times specially for OTA, while in binary combination only for few point times was denoted. Effects detected for ROS and LPO were connected with alterations detected for glutathione levels of oxidized and reduced form. A real scenario of consumers chronically exposed to different mycotoxins and their mixtures is here presented highlighting the good methodology to assess the risk from exposure to combinations of chemicals in food.

Evaluating the impact of supercritical-CO2 pressure on the recovery and quality of oil from "horchata" by-products: Fatty acid profile, α-tocopherol, phenolic compounds, and lipid oxidation parameters.

The effect of supercritical carbon dioxide (SC-CO2) (10-40 MPa) and conventional extraction (CE) to recover oil from by-products obtained during "horchata" production was assessed. To evaluate both extraction techniques, the fatty acid composition, polyphenols, α-tocopherol, antioxidant capacity and lipid oxidation parameters of the extracts obtained were analysed. A linear relationship between extraction pressure and oil yield was observed. However, the highest oil yield was obtained under conventional extraction. The by-products from "horchata" presented a profile where monounsaturated fatty acids (MUFA) were the predominant, representing ≈ 70% of total fatty acids. The amount of saturated fatty acids (SFA) and polyunsaturated fatty acids (PUFA) was higher and MUFA lower at 10 MPa samples compared to the oils extracted using SC-CO2 at 20, 30 or 40 MPa, where no differences were detected. The content of α-tocopherol was significantly higher after SC-CO2 treatments compared to conventional extraction, independently of the applied treatment. On the other hand, the values of phenolic compounds and total antioxidant activity (TAC) increased as the pressure conditions of the SC-CO2 extraction increased, presenting a linear adjustment of the data. Regarding lipid oxidation, the lower oxidation indexes were obtained when the SC-CO2 pressure increased. Finally, our results confirmed that the application of SC-CO2 could be a potential alternative to conventional extraction in order to obtain oils from "horchata" by-products rich in high-added value compounds without the use of organic solvents which can be toxic.

Development and Validation of a LC-ESI-MS/MS Method for the Determination of Alternaria Toxins Alternariol, Alternariol Methyl-Ether and Tentoxin in Tomato and Tomato-Based Products.

Alternaria species are capable of producing several secondary toxic metabolites in infected plants and in agricultural commodities, which play important roles in food safety. Alternaria alternata turn out to be the most frequent fungal species invading tomatoes. Alternariol (AOH), alternariol monomethyl ether (AME), and tentoxin (TEN) are some of the main Alternaria mycotoxins that can be found as contaminants in food. In this work, an analytical method based on liquid chromatography (LC) tandem mass spectrometry (MS/MS) detection for the simultaneous quantification of AOH, AME, and TEN in tomato and tomato-based products was developed. Mycotoxin analysis was performed by dispersive liquid-liquid microextraction (DLLME) combined with LC-ESI-MS/MS. Careful optimization of the MS/MS parameters was performed with an LC/MS system with the ESI interface in the positive ion mode. Mycotoxins were efficiently extracted from sample extract into a droplet of chloroform (100 µL) by DLLME technique using acetonitrile as a disperser solvent. Method validation following the Commission Decision No. 2002/657/EC was carried out by using tomato juice as a blank matrix. Limits of detection and quantitation were, respectively, in the range 0.7 and 3.5 ng/g. Recovery rates were above 80%. Relative standard deviations of repeatability (RSDr) and intermediate reproducibility (RSDR) were ≤ 9% and ≤ 15%, respectively, at levels of 25 and 50 ng/g. Five out of 30 analyzed samples resulted positive to at least one Alternaria toxin investigated. AOH was the most common Alternaria toxin found, but at levels close to LOQ (average content: 3.75 ng/g).

In vitro bioaccessibility, transepithelial transport and antioxidant activity of Urtica dioica L. phenolic compounds in nettle based food products.

Nettle (Urtica dioica L.) is a well-known plant with a wide historical background use of stems, roots and leaves. Nettle leaves are an excellent source of phenolic compounds, principally 3-caffeoylquinic acid (3-CQA), caffeoylmalic acid (CMA) and rutin. The aim of this work was to evaluate the bioaccessibility (BAC), the bioavailability (BAV) and the antioxidant activity of nettle phenolic compounds present in foods and supplements. The BAC of nettle phenolics was evaluated with an in vitro dynamic digestion of real food matrices: the type of food matrix and chemical characteristic affected the kinetics of release and solubilization, with the highest BAC after duodenal digestion. A study of duodenal trans epithelial transport evidenced low bioavailability of native forms of 3-CQA, CMA and rutin. Simulation of colonic metabolism confirmed that phenolic compounds are fermented by gut microflora, confirming the need for further investigations on the impact of phenolic compounds at the large intestine level. Photochemiluminescence assay of the simulated digestion fluids demonstrated that ingestion of Urtica based foods contributes to create an antioxidant environment against superoxide anion radicals in the entire gastrointestinal tract (GIT).

Effect of the oriental and yellow mustard flours as natural preservative against aflatoxins B1, B2, G1 and G2 production in wheat tortillas.

Reduction of the AFs produced by Aspergillus parasiticus CECT 2681 in wheat tortillas by isothiocyanates (ITCs) from oriental and yellow mustard flours was evaluated in this study. Polyethylene plastic bags were introduced with wheat tortillas contaminated with A. parasiticus and treated with 0, 0.1, 0.5 or 0.1 g of either oriental or yellow mustard flour added with 2 ml of water. The wheat tortillas were stored at room temperature during 1 month. The quantification of the AFs produced was analyzed by liquid chromatography (LC) coupled to the mass spectrometry detection in tandem (MS/MS). Gaseous allyl isothiocyanate (AITC) from oriental mustard was more effective than p-hydroxybenzyl isothiocyanate (p-HBITC) from yellow mustard to inhibit the production of AFs. More importantly, 1 g of AITC was able to reduce >90 % of AFs B1, B2, G1 and G2. p-HBITC is less stable and volatile than AITC, leading to a much lower AFs (average of 17.7 to 45.2 %). Further studies should investigate the use of active packaging using oriental mustard flour and water to reduce the production of AFs by Aspergillus species in bakery goods.

Influence of the antimicrobial compound allyl isothiocyanate against the Aspergillus parasiticus growth and its aflatoxins production in pizza crust.

Aflatoxins (AFs) are secondary metabolites produced by different species of Aspergillus, such as Aspergillus flavus and Aspergillus parasiticus, which possess mutagenic, teratogenic and carcinogenic activities in humans. In this study, active packaging devices containing allyl isothiocyanate (AITC) or oriental mustard flour (OMF) + water were tested to inhibit the growth of A. parasiticus and AFs production in fresh pizza crust after 30 d. The antimicrobial and anti-aflatoxin activities were compared to a control group (no antimicrobial treatment) and to a group added with commercial preservatives (sorbic acid + sodium propionate). A. parasiticus growth was only inhibited after 30 d by AITC in filter paper at 5 µL/L and 10 µL/L, AITC sachet at 5 µL/L and 10 µL/L and OMF sachet at 850 mg + 850 µL of water. However, AFs production was inhibited by all antimicrobial treatments in a dose-dependent manner. More importantly, AITC in a filter paper at 10 µL/L, AITC sachet at 10 µL/L, OMF sachet at 850 mg + 850 µL of water and sorbic acid + sodium propionate at 0.5-2.0 g/Kg completely inhibited AFs formation. The use of AITC in active packaging devices could be a natural alternative to avoid the growth of mycotoxinogenic fungi in refrigerated bakery products in substitution of common commercial preservatives.

Determination of mycotoxins in bee pollen by gas chromatography-tandem mass spectrometry.

Bee pollen, promoted as a natural food supplement, is consumed increasingly by people to maintain a healthy diet. Depending on environmental conditions, pollen can also be an optimum medium for growth of molds such as Fusarium and Penicillium . A quick, easy, cheap, rapid, and safe (QuEChERS) extraction procedure followed by a gas chromatography-tandem mass spectrometry (GC-MS/MS) determination of eight selected Fusarium toxins in bee pollen was developed and optimized. Recovery studies at 20, 80, and 1000 µg/kg showed values between 73 and 95% with relative standard deviations (RSDs) of <15% for all studied mycotoxins. Limits of quantitation (LOQ) ranged from 1 to 4 µg/kg. The proposed method was applied to the analysis of 15 commercial samples. Two of 15 samples showed quantifiable values for neosolaniol and nivalenol.

Multi-mycotoxin analysis in wheat semolina using an acetonitrile-based extraction procedure and gas chromatography-tandem mass spectrometry.

A new analytical method for the rapid and simultaneous determination of ten mycotoxins including patulin, zearalenone and eight trichothecenes (nivalenol, fusarenon-X, diacetoxyscirpenol, 3-acetyl-deoxynivalenol, neosolaniol, deoxynivalenol, T-2 and HT-2) in wheat semolina has been developed and optimized. Sample extraction and purification were performed with a modified QuEChERS-based (acronym of Quick, Easy, Cheap, Effective, Rugged and Safe) procedure and determined by gas chromatography (GC) coupled to triple quadrupole instrument (QqQ). This is the first paper on the application of GC-QqQ-MS/MS to analysis of mycotoxins. Careful optimization of the gas chromatography-tandem mass spectrometry parameters was achieved in order to attain a fast separation with the best sensitivity allowing a total run time of 16 min. The validation was performed by analyzing recovery samples at three different spiked concentrations, 20, 40 and 80 µg kg(-1), with four replicates (n=4) at each concentration. Recoveries ranged from 74% to 124% and the intra-day precision and inter-day precision, expressed as relative standard deviation, were lower than 13% and 17%, respectively for all studied compounds, except for zearalenone. Limits of quantification (LOQ) were lower than 10 µg kg(-1) for all studied mycotoxins. Eight concentration levels were used for constructing the calibration curves which showed good linearity between LOQ and 100 times LOQ concentration levels (linear range). Matrix-matched calibration for applying the method in routine analysis is recommended for reliable quantitative results. The method validated was successfully applied to fifteen wheat semolina samples detecting occurrence of mycotoxins at concentrations below the maximum permissible level.

A preliminary study of presence of resveratrol in skins and pulps of European and Japanese plum cultivars.

BACKGROUND: Resveratrol is a polyphenol with health properties being mainly present in the skins of several foods. However, any study has been carried out to analyze the presence of this stilbene in the plum fruit from the genus Prunus in European and Japanese cultivars. RESULTS: The analysis of resveratrol from the skin in different cultivars of plums from Spanish markets with liquid chromatography coupled to ultraviolet (LC-UV) detector with subsequent confirmation by LC-MS/MS has been demonstrated that contents of this compound in plums ranged from 0.1 to 6.2 µg/g. CONCLUSIONS: Values of resveratrol in European plum cultivars is higher than in Japanese cultivars.

Analysis of staphylococcal enterotoxin A in milk by matrix-assisted laser desorption/ionization-time of flight mass spectrometry.

Staphylococcal enterotoxin A (SEA) is an exotoxin excreted mainly by Staphylococcus aureus and nowadays is the most prevalent compound in staphylococcal food poisoning worldwide. SEA is highly heat-resistant, and usual cooking times and temperatures are unlikely to completely inactivate it. A procedure for extraction of this toxin based on protein precipitation with a mixture of dichloromethane and acidified water was used before SDS-PAGE separation of soluble proteins. Finally, bands of interest were excised from the gel and in-gel enzymatic digestion was done. SEA from pasteurized milk was detected with matrix-assisted laser-desorption/ionization-time of flight (MALDI-TOF) mass spectrometry. Nineteen peptides (range 800-2400 Da) were identified as products of trypsin cleavage of the SEA standard with a score of 204 and 73% coverage of the protein sequence, whereas thirteen peptides were revealed for SEA extracted from milk with a score of 148 and 58% sequence coverage obtained. This procedure has been applied successfully for identification of SEA in milk.

Apple-products phytochemicals and processing: a review.

Epidemiological studies have linked the consumption of apples with reduced risk of some cancers, cardiovascular disease, asthma, and diabetes. Extensive research exists on apples and the health benefits of their beverages and phytochemicals. The purpose of this paper is to review the most recent literature in this area focusing on phytochemicals, phytochemical bioavailability and antioxidant behavior.

Review on the toxicity, occurrence, metabolism, detoxification, regulations and intake of zearalenone: an oestrogenic mycotoxin.

Zearalenone (ZEA) is a mycotoxin produced mainly by fungi belonging to the genus Fusarium in foods and feeds. It is frequently implicated in reproductive disorders of farm animals and occasionally in hyperoestrogenic syndromes in humans. There is evidence that ZEA and its metabolites possess oestrogenic activity in pigs, cattle and sheep. However, ZEA is of a relatively low acute toxicity after oral or interperitoneal administration in mice, rat and pig. The biotransformation for ZEA in animals involves the formation of two metabolites alpha-zearalenol (alpha-ZEA) and beta-zearalenol (beta-ZEA) which are subsequently conjugated with glucuronic acid. Moreover, ZEA has also been shown to be hepatotoxic, haematotoxic, immunotoxic and genotoxic. The exact mechanism of ZEA toxicity is not completely established. This paper gives an overview about the acute, subacute and chronic toxicity, reproductive and developmental toxicity, carcinogenicity, genotoxicity and immunotoxicity of ZEA and its metabolites. ZEA is commonly found on several foods and feeds in the temperate regions of Europe, Africa, Asia, America and Oceania. Recent data about the worldwide contamination of foods and feeds by ZEA are considered in this review. Due to economic losses engendered by ZEA and its impact on human and animal health, several strategies for detoxifying contaminated foods and feeds have been described in the literature including physical, chemical and biological process. Dietary intakes of ZEA were reported from few countries from the world. The mean dietary intakes for ZEA have been estimated at 20 ng/kgb.w./day for Canada, Denmark and Norway and at 30 ng/kgb.w./day for the USA. The Joint FAO/WHO Expert Committee on Food Additives (JECFA) established a provisional maximum tolerable daily intake (PMTDI) for ZEA of 0.5 microg/kg of body weight.