Pre- and Postharvest Strategies to Minimize Mycotoxin Contamination in the Rice Food Chain.

Rice is part of many people's diet around the world, being the main energy source in some regions. Although fewer reports exist on the occurrence of mycotoxins in rice compared to other cereals, fungal contamination and the associated production of toxic metabolites, even at lower occurrence levels compared to other crops, are of concern because of the high consumption of rice in many countries. Due to the diversity of fungi that may contaminate the rice food chain, the co-occurrence of mycotoxins is frequent. Specific strategies to overcome these problems may be applied at the preharvest part of the crop chain, while assuring good practices at harvest and postharvest stages, since different fungi may find suitable conditions to grow at the various stages of the production chain. Therefore, the aim of this review is to present the state-of-the-art knowledge on such strategies in an integrated way, from the field to the final products, to reduce mycotoxin contamination in rice.

Group detection of DON and its modified forms by an ELISA kit.

Deoxynivalenol (DON) and its modified forms (3-, and 15-acetyl-DON, DON-3-glucoside) are commonly analysed by chromatographic methods. Indeed, coupled with proper extraction and clean-up, LC-MS represents the best approach for multi-mycotoxin measurements. On the other hand, immunochemistry-based methods are possibly able to detect a family of structurally related compounds, although the determination of single contributions is not possible so far. However, ELISA methods often lead to an apparent overestimation of the mycotoxins content because modified forms and matrix components can potentially cross-react with the antibodies (designed for the parent toxin). Several data about the possible cross-reactivity of commercial DON-detecting ELISA kit are reported in the literature so far. Data are commonly obtained in buffer solutions or in matrix-matched solutions, but comparison of a set of naturally incurred samples has never been reported. In the present work the accuracy of a commercial DON-detecting ELISA kit was evaluated on naturally incurred soft wheat (n = 15) and maize (n = 15), taking into account the matrix effect. Recovery was calculated considering the DON concentration found by LC-MS/MS and the total DON concentration, expressed as the sum of DON and its modified forms found by LC-MS/MS. The obtained data clearly show that, when 3-modified forms of DON occur in the sample, the ELISA kit does actually detect them, thus returning an apparent overestimation if only DON content is considered. When the ELISA recovery is calculated on the total DON content, the accuracy of the analysis increases and the variability decreases. According to our data, the ELISA kit seems to be a promising group detection tool for the accurate evaluation of DON and its modified forms, expressed as sum of DON, DON-3Glc and 3Ac-DON, for soft wheat and maize samples.

Oleoyl and linoleoyl esters of fumonisin B1 are differently produced by Fusarium verticillioides on maize and rice based media.

Fatty acid esters of fumonisins, namely oleoyl- and linoleoyl esters of fumonisin B1 (EFB1OA and EFB1LA, respectively), are modified forms of fumonisins whose formation and occurrence have been reported so far in naturally infected maize and in artificially inoculated rice. There is a lack of knowledge about the mechanism of formation, mainly in relation to the role played by the substrate. Therefore, in this work we studied the dynamics of accumulation of the toxin and its esters, together with their precursor, in maize and rice based media inoculated with different strains of F. verticillioides and incubated at 25 °C for 7-45 days. The production pattern of FB1 and its modified forms was significantly influenced by growth media, reaching a higher concentration in cornmeal compared to rice based medium. Similarly, cornmeal was more supportive for the conversion of FB1 by considering the esterification rate, with a prevalence of linoleoyl esters compared to oleoyl esters resembling the OA/LA rate in both media. The conversion of FB1 into fatty acid esters was also shown as strain-related. Results, thus, strongly support the hypothesis that fatty acid esters of FB1 are produced by the fungus itself at a late stage of growth, or at a certain point of FB1 accumulation in the medium, using fatty acids from the substrate.

A simple and reliable liquid chromatography-tandem mass spectrometry method for the determination of aflatoxin B1 in feed.

A chromatographic method is proposed for the analysis of aflatoxin B1 in cereal-based feed, particularly targeted to dairy animals. The method is based on a solid-liquid extraction followed by a Mycosep 226 clean-up. Accuracy and precision were established at the LOQ (1 µg kg⁻¹) with a spiked sample as well as with two other different naturally contaminated reference materials. The mean overall recovery (n = 18) was 100.8%, with a confidence interval of 2.7% and a CV% of 5.5%. The performance of the proposed method was compared with the AOAC method based on the use of immunoaffinity chromatography columns, proving that it could be considered a valid alternative. Moreover, the sample preparation is very simple and straightforward, potentially being applicable as a high-throughput method. On account of its simplicity and low cost, the method may be applied to the analysis of a large number of samples in the occasion of outbreaks of large-scale contamination.

A simple and reliable liquid chromatography-tandem mass spectrometry method for the determination of aflatoxin M1 in milk.

A new chromatographic method is proposed for the analysis of aflatoxin M(1) in milk. The method is based on liquid-liquid extraction followed by LC-MS/MS analysis. Liquid-liquid extraction (LLE) is performed on the defatted milk plus sodium chloride by using ethyl acetate as an extraction solvent. Accuracy and precision were evaluated at the LOQ (15 ng kg(-1)) spiked sample as well as with three other different naturally contaminated reference materials. The mean overall recovery (n = 24) was 95% with a confidence interval of 1.9% and a CV% of 4.5%. The performance of the proposed method was compared with that of the Official ISO Method based on the use of immunoaffinity chromatography columns (IAC): LLE protocol could be considered a valid alternative to the LC-IAC. In general it showed better accuracy with lower data dispersion. Moreover, the sample preparation is very simple and straightforward, potentially being applicable as a high-throughput method which, on account of its simplicity and low cost, may be applied to the analysis of a large number of samples in the occasion of outbreaks of large-scale contamination.

Volatile fingerprinting of chestnut flours from traditional Emilia Romagna (Italy) cultivars.

The volatile profile of nine monocultivar chestnut flours, obtained from fruits grown in Italy (Parma province), was characterised by a head-space solid-phase microextraction (HS-SPME) coupled with GC-MS technique. The volatile fraction was composed of 44 main compounds belonging to different classes, mainly aldehydes, ketones, alcohols, furans and terpenes. Aldehydes, in particular hexanal, are the most abundant components. In order to better understand the origin of the different volatile compounds during the drying and milling processes, samples of fresh fruit were also analysed by the same technique and the data obtained were statistically and critically compared in order to get a picture of the volatile evolution in chestnut from fresh fruit to flour. Finally, the nine monocultivar flours were chemometrically classified on the basis of the main odour descriptors associated with the volatile fingerprinting.

Development of a simple and high-throughput method for detecting aflatoxins production in culture media.

AIMS: To develop a simple, high-throughput and inexpensive procedure to detect and quantify aflatoxins into the culture media of growing mycelia. METHODS AND RESULTS: Fungal conidia (Aspergillus flavus) were inoculated into the wells of a microplate containing 200 µl of different formulations of coconut-derived liquid medium. Time-dependent production of aflatoxins in the culture media was evaluated by a procedure relying on the UV-induced fluorescence emission by the toxin, using a microplate reader. These data were validated by comparison with the outputs of a conventional HPLC-based procedure. Determinations of aflatoxin concentration, according to the fluorimetric procedure, were performed either by withdrawing samples from the plates or by direct 'in situ' readings, the latter method reinforcing the high-throughput feature of the procedure. Fluorescence enhancers (cyclodextrins) did not ameliorate the sensitivity of the procedure to low concentrations of the toxin into the medium. The efficacy of the procedure was also validated by testing the effect on toxin yield of adding an antioxidant agent (α-lipoic acid) to the medium. CONCLUSIONS: We give evidence that our improved procedure is reliable and suitable to analyse aflatoxin accumulation time course in coconut-derived culture medium. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that our procedure may profitably be used to give insights into the mechanisms of regulation of mycotoxin production and, consequently, to implement different strategies for the containment of aflatoxin contamination of food and feed commodities.

Hyphenated chromatographic techniques for structural characterization and determination of masked mycotoxins.

Mycotoxins are secondary metabolites produced by fungi that can contaminate a wide range of food and feed commodities and that are harmful to humans for their poisonous and toxic effects. An increasing amount of data have been accumulated in the last years, showing that mycotoxins may also occur in modified forms originating by plant, fungi or animal metabolism or by food processing. In particular, this modified forms may be produced via conjugation with sugars or other biological components (masked mycotoxins) or may occur as non extractable forms on account of strong interaction, association or binding with macromolecules in the food matrix (bound or hidden mycotoxins). Analytical methods have been set up in order to check for the occurrence of these forms and to evaluate their amount, in order to obtain reliable data for toxicity and exposure studies. In this paper hyphenated chromatographic methods for the determination and structural characterization of masked mycotoxins are reviewed, with a particular emphasis on liquid chromatography-(tandem) mass spectrometry as the most effective approach for their determination.

Brand-dependent volatile fingerprinting of Italian wines from Valpolicella.

Head-space solid-phase microextraction (HS-SPME) coupled to gas-chromatography-mass spectrometry was developed and applied to obtain the volatile aromatic fingerprints of three typical Italian wines, Valpolicella, Amarone and Recioto, all produced in the restricted geographical area of Valpolicella (Veneto, Italy) with the same grape cultivars within the regulations of a rigid disciplinary of production. Differences between the three typologies are mainly linked to the different withering times to which grapes are subjected before vinification, which strongly influences the concentration and the development of volatile aroma compounds. A total of 22 different wines (7 Valpolicella, 10 Amarone and 5 Recioto) were characterised in terms of aromatic volatile profile with the aim to distinguish the different products and to evaluate the possibility to differentiate the same product from different brands. For the chemometric evaluation of the data one-way analysis of variance (ANOVA), principal component analysis (PCA) and hierarchical cluster analysis (HCA) were tested. All the chemometric tools employed allow to differentiate between the three products. More intriguing is the ability of the chemometric approach to differentiate between the same product (Amarone, Recioto) from different winery, thus showing the potential of this approach to characterize the brand-dependent typicality of wines, which is usually related to subtle technological differences which nevertheless have strong influences on the organoleptic characteristics of the products.

Fate of Fusarium mycotoxins in the cereal product supply chain: the deoxynivalenol (DON) case within industrial bread-making technology.

Fusarium mycotoxins are a relevant problem in the cereal supply chain at a worldwide level, with wheat, maize and barley being the main contaminated crops. Mould growth can happen in the pre-harvest phase and also during transport and storage due to ineffective drying conditions. Among Fusarium toxins, deoxynivalenol (DON) is considered the most important contaminant in wheat due to its widespread occurrence. In the last years the European Food Safety Authority (EFSA) and the European Commission have frequently expressed opinions on Fusarium toxins, setting limits, regulations and guidelines in order to reduce their levels in raw materials and food commodities. In particular, European legislation (Reg. 1881/2006) sets the maximum limit for DON in flour and bread as 750 and 500 microg kg(-1) respectively. Relatively few studies have taken into account the loss of trichothecenes during processing, focusing on how processing factors may influence their degradation. In particular, the description of DON behaviour during bread-making is very difficult, since complex physico-chemical modifications occur during the transformation of the raw ingredients into the final product. In the present study, we studied how DON concentration may be influenced by modifying bread-making parameters, with a special emphasis on the fermentation and baking stages, starting from a naturally contaminated flour at both pilot and industrial scales. Exploiting the power of a Design of Experiments (DoE) approach to consider the great complexity of the studied system, the obtained model shows satisfying goodness-of-fit and prediction, suggesting that the baking step (time/temperature ranges) is crucial for minimizing native DON level in bread.

Occurrence of deoxynivalenol and its 3-beta-D-glucoside in wheat and maize.

Deoxynivalenol-3-beta-D-glucoside (D3G), a phase II plant metabolite of the mycotoxin deoxynivalenol (DON), occurs in naturally Fusarium-contaminated cereals. In order to investigate the frequency of occurrence as well as the relative and absolute concentrations of D3G in naturally infected cereals, 23 wheat samples originating from fields in Austria, Germany and Slovakia as well as 54 maize samples from Austrian fields were analysed for DON and D3G by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Both analytes were detected in all the 77 field samples. DON was found at levels from 42 to 4130 ng g(-1) (977 +/- 1000 ng g(-1) on average). The D3G concentrations in all cereal samples were in the range 10-1070 ng g(-1) (216 +/- 253 ng g(-1) on average), corresponding to about 5-46 mol% of their DON concentrations (15 +/- 8 mol% on average).

Analysis of GNAS1 and PRKAR1A gene mutations in human cardiac myxomas not associated with multiple endocrine disorders.

Cardiac myxomas are rare tumors that usually occur as sporadic lesions or,more rarely, in the familial form,mostly in the context of Carney complex (CNC). The molecular basis for the development of cardiac myxomas is unclear. However, somatic activating mutations in the GNAS1 gene (the gsp oncogene) are detected in the myocardium ofMcCune-Albright syndrome patients while germ-line mutations in the PRKAR1A gene are associated with CNC and familial myxomas. We investigated the presence of activating missense mutations in the GNAS1 gene as well as of inactivating mutations in PRKAR1A in 29 sporadically occurring cardiac myxomas. No gsp and no PRKAR1A mutations were found by direct sequencing of PCR products amplified from tumoral DNA. This is the first study including a large series of sporadic, isolated cardiac myxomas and showing that these cardiac neoplasms do not share the same mutations found in familial forms.

Effect of weight loss through laparoscopic gastric banding on blood pressure, plasma renin activity and aldosterone levels in morbid obesity.

BACKGROUND AND AIMS: Several mechanisms are probably involved in obesity-related hypertension. This study was aimed to investigate the effect of significant weight loss on blood pressure and plasma renin activity (PRA) and aldosterone levels, other then on metabolic profile, in normotensive and hypertensive obese subjects. METHODS AND RESULTS: Forty hypertensive and 55 normotensive obese subjects were studied under basal conditions and again 1 year after significant weight loss obtained through laparoscopic adjustable gastric banding (LAGB). Weight, waist circumference, blood glucose, insulin, electrolytes (Na and K), lipids and supine and upright PRA and aldosterone were evaluated. All parameters evaluated improved, except for total cholesterol, and electrolytes that did not change. Blood pressure decreased in hypertensive subjects, with a concordant decrease in PRA and supine aldosterone levels, not observed in normotensive patients. CONCLUSION: Weight loss is associated with reduction of blood pressure and of PRA and aldosterone levels in obese hypertensive subjects.

Echocardiographic alterations in patients with non-functioning adrenal incidentaloma.

OBJECTIVE: While left ventricular (LV) dysfunction has been described in patients with Cushing's syndrome (CS), data concerning morphologic and functional cardiac alterations in patients with incidentally discovered adrenal masses [adrenal "incidentaloma" (AI)], without overt hypercortisolism, are lacking. In this study the echocardiographic characteristics of patients with AI were evaluated and then compared with those of lean and obese normotensive subjects. SUBJECTS AND METHODS: Twenty-one patients with AI, without clinical or subclinical hypercortisolism, 18 normotensive obese subjects matched for gender and body mass index (BMI) and 20 normotensive lean subjects were studied. Echocardiography was performed in all subjects. In all patients plasma ACTH, serum cortisol, and DHEA-S levels were measured. RESULTS: Patients with AI showed greater impairment of several echocardiographic indices of LV hypertrophy and diastolic dysfunction compared to normotensive lean subjects (p<0.05), but did not differ from those in obese subjects. Hypertensive AI patients showed a greater alteration of echocardiographic parameters (p<0.05) and higher BMI (p<0.01) and cortisol values (p<0.05) than normotensive ones. Plasma ACTH and serum cortisol were similar in AI patients and in obese controls, while DHEA-S levels were lower in AI (p<0.05). No correlations between cortisol secretion and echocardiographic parameters were found. CONCLUSION: In patients with non-functioning AI there is an impairment of cardiac morphology and function. These data suggest that patients with AI should be carefully screened also by means of echocardiographic studies.

Complexation of the mycotoxin zearalenone with ß-cyclodextrin: Study of the interaction and first promising applications.

This work reports the study of the interactions between native and substituted ß-cyclodextrins and zearalenone and its derivatives α- and ß-zearelonol. The data obtained by fluorescence and NMR experiments suggested that zearalenone, α- and ß-zearalenol and cyclodextrins give rise to host-guest complexation, with the inclusion of the phenolic moiety inside the cyclodextrin cavity. The high stability of these complexes induces a high fluorescence enhancement upon complexation. These results have been successfully applied to the spectrofluorimetric determination of zearalenone in maize raw samples, without any chromatographic separation.

Usefulness of chemical-shift MRI in discriminating increased liver echogenicity in glycogenosis.

BACKGROUND: Glycogen storage diseases are inherited defects which cause accumulation of glycogen in the tissues. Hepatic steatosis is defined as accumulation of fat within hepatocytes. On sonography, liver shows increased echogenicity both in glycogen storage diseases and steatosis. Liver hyperechogenicity in glycogen storage diseases may depend on accumulation of glycogen and/or fat. Chemical-shift magnetic resonance imaging can discriminate tissues only containing water from those containing both fat and water. AIM: The primary aim of the present study was to evaluate the usefulness of liver chemical-shift magnetic resonance imaging for detecting liver steatosis in patients with metabolic impairment due to glycogen storage diseases. SUBJECTS: Twelve patients with type I (n=8) or type III (n=4) glycogen storage diseases were studied and compared to 12 obese-overweight subjects with known liver steatosis. As control group 12 lean normal voluntary subjects were recruited. METHODS: Liver was evaluated by sonography and chemical-shift magnetic resonance imaging to calculate hepatic fat fraction. RESULTS: A significant difference in echogenicity between patients with glycogen storage diseases and normal subjects was observed (p<0.05), while this difference was not present between overweight-obese and glycogen storage diseases patients. On the contrary, fat fraction was similar between glycogen storage diseases patients and normal subjects and different between glycogen storage diseases patients and overweight-obese (p<0.05). CONCLUSION: The present data suggest that chemical-shift magnetic resonance imaging may exclude fat deposition as a cause of liver hyperechogenicity in subjects with glycogen storage diseases.

A multiplex RT-PCR approach to detect aflatoxigenic strains of Aspergillus flavus.

AIMS: To develop a multiplex reverse transciption-polymerase chain reaction (RT-PCR) protocol to discriminate aflatoxin-producing from aflatoxin-nonproducing strains of Aspergillus flavus. METHODS AND RESULTS: The protocol was first optimized on a set of strains obtained from laboratory collections and then validated on A. flavus strains isolated from corn grains collected in the fields of the Po Valley (Italy). Five genes of the aflatoxin gene cluster of A. flavus, two regulatory (aflR and aflS) and three structural (aflD, aflO and aflQ), were targeted with specific primers to highlight their expression in mycelia cultivated under inducing conditions for aflatoxins production. 48-h-old cultures expressed the complete set of the genes analysed here whereas 24-h-old ones did not. Genomic PCR (quadruplex PCR) was also performed in parallel using chromosomal DNA extracted from the same set of strains to correlate the integrity of the genes with their expression. CONCLUSIONS: We show that a good correlation exists between gene expression of the aflatoxin genes, here analysed by multipex RT-PCR, and aflatoxin production, except for one strain that apparently transcribed all the relevant genes but did not produce aflatoxin in the medium. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first example of the application of a combination of multiplex PCR and RT-PCR approaches to screen a population of A. flavus for the presence of aflatoxigenic and nonaflatoxigenic strains. The proposed protocol will be helpful in evaluating the risk posed by A. flavus in natural environments and might also be a useful tool to monitor its presence during the processing steps of food and feed commodities.

A new validated HPLC-FLD method for detecting ochratoxin A in dry-cured meat and in blue cheese.

In the present study, a fast and sensitive method for the quantification of ochratoxin A in two lipidicproteic food matrices has been developed. In particular, the sample preparation procedure has been optimized for dry-cured meat products and blue cheeses and tested for several validation parameters (LOD, LOQ, recovery, repeatability and within-laboratory precision). The procedure has been then applied to several dry-cured meat products and blue cheeses from the market.Ochratoxin A has been occasionally found in dry-cured and smoked ham from the market and the contamination occurred both in the outer and in the inner part of the products. Concerning the blue cheese, the occurrence of ochratoxin A is reported for the first time: OTA was occasionally found at low levels (0.1-3 µg/kg) in commercial samples of Roquefort from France and Gorgonzola from Italy, opening a new issue for risk assessment and quality control.

A primary adrenal non-Hodgkin's lymphoma presenting as an incidental adrenal mass.

The primary adrenal localization of a non-Hodgkin's lymphoma (NHL) is a rare event. We report the case of a 70-yr-old woman, who was admitted at our Institute for a hormonal evaluation after the incidental discovery of a right adrenal mass during ultrasonography (US) performed for cardiovascular disease. At the physical examination, no sign of adrenal hyperfunction was present. She showed only an androgenetic alopecia and her blood pressure was 180/70 mm Hg, with an arrhythmic heart rate of 100 beats/min. No alterations in hormonal and biochemical data were observed. US studies showed a right adrenal mass (major diameter 16 mm), and an abdominal computed tomography (CT) scan confirmed this solid lesion (major diameter 15 mm) with a high density. [75Se] methylnorcholesterol adrenal scintigraphy exhibited a normal symmetrical radiotracer uptake. After 8 months of follow-up, an abdominal CT scan demonstrated a significant increase of the right adrenal mass (major diameter: 40 mm), with a solid tissue density and enhancement after i.v. contrast. [75Se] methylnorcholesterol adrenal scintigraphy showed an absent uptake on the right side versus the contralateral side. The hematological, hormonal and radiological evaluation did not reveal any sign of malignancy. Owing to the mass enlargement and the modification of scintigraphic pattern, the patient underwent unilateral adrenalectomy. Histological examination revealed a primary diffuse large B-cell NHL (REAL classification) of the adrenal gland. After surgery, she underwent a combined polychemotherapy (cyclophospamide, adriamycin, vincristine and prednisone) and subsequently one cycle of radiotherapy. At present, the patient is in good conditions and there are no signs or symptoms of recurrent disease.