Amedeo Pietri

Occurrence of ochratoxin A in Italian wines

A total of 96 red wines and 15 white dessert wines produced mostly in the years 1995–97 in 19 Italian regions were analysed for ochratoxin A (OTA). The amount of OTA ranged from < 1 to 3856 ng/l, the median (mean) was found to be 90 (419)ng/l for the red wines and 8 (736)ng/l for the white dessert wines. Our survey shows that the geographic region of origin has a strong influence on OTA contamination, both for red and for dessert wines: in fact, wines produced in southern Italy were markedly more contaminated. The overall median (mean) OTA concentration in the red wines produced in the four Italian areas (northwest, northeast, centre and south) was 2 (11), 90 (81), 134 (295) and 1264 (1233)ng/l. The same trend was observed for the white dessert wines: OTA concentrations of over 1000ng/l were found in four out of five samples from southern Italy (1185, 2454, 3477, 3856ng/l), while central and northern samples showed very low contamination. The contribution of wine to mean daily OTA intake can be considered negligible in the case of people drinking wine manufactured in northern and central Italy; this is not true if a medium drinker constantly consumes red wine produced in southern Italy: in this case wine alone could supply the diet with an amount of OTA equal to or even above the tolerable daily intake of 5ng/kg body weight recommended by the Scientific Committee on Food of the European Commission.

Ochratoxin A Production and Amplified Fragment Length Polymorphism Analysis of Aspergillus carbonarius, Aspergillus tubingensis, and Aspergillus niger Strains Isolated from Grapes in Italy

ABSTRACT Ochratoxin A is a potent nephrotoxin and a possible human carcinogen that can contaminate various agricultural products, including grapes and wine. The capabilities of species other than Aspergillus carbonarius within Aspergillus section Nigri to produce ochratoxin A from grapes are uncertain, since strain identification is based primarily on morphological traits. We used amplified fragment length polymorphisms (AFLPs) and genomic DNA sequences (rRNA, calmodulin, and β-tubulin genes) to identify 77 black aspergilli isolated from grape berries collected in a 2-year survey in 16 vineyards throughout Italy. Four main clusters were distinguished, and they shared an AFLP similarity of <25%. Twenty-two of 23 strains of A. carbonarius produced ochratoxin A (6 to 7,500 μg/liter), 5 of 20 strains of A. tubingensis produced ochratoxin A (4 to 130 μg/liter), 3 of 15 strains of A. niger produced ochratoxin A (250 to 360 μg/liter), and none of the 19 strains of Aspergillus “uniseriate” produced ochratoxin A above the level of detection (4 μg/liter). These findings indicate that A. tubingensis is able to produce ochratoxin and that, together with A. carbonarius and A. niger, it may be responsible for the ochratoxin contamination of wine in Italy.

Ochratoxin A in grapes and wine

The mycotoxin ochratoxin A is a potent nephrotoxin and a possible human carcinogen. It occurs in a variety of plant products, including wine, grape juice and dried vine fruits. Several surveys have shown that the range of ochratoxin A contents detected in wine produced in Europe varied between 0.01 and 3.4μgl−1. Both incidence and concentration of the toxin were higher in wines from southern regions and increased in the order white < rosè < red. In Italy, field trials were conducted in 1999 and 2000 to study fungi associated with grapes and their ability to produce ochratoxin. Aspergillus and/or Penicillium strains were present on grapes, starting from setting in a few vineyards. The highest level of grape colonisation was found at early veraison in 1999 and at ripening in 2000. In both years, 95% of strains belonged to the genus Aspergillus. Aspergillus niger aggregate was dominant, with about 50% of the ochratoxin-positive strains identified as A carbonarius. Other authors have confirmed the relevance of these fungi and underlined the contribution of A. carbonarius to the ochratoxin contamination of wine. This species is very invasive and colonises and penetrates berries, even without skin damage. It emerges that temperature, rain and relative humidity are the main factors that influence ochratoxin production in grapes.

Epidemiology of toxin-producing fungi and ochratoxin A occurrence in grape

Fungi responsible for ochratoxin A (OTA) production have been studied especially on cereals, where Penicillium verrucosum and Aspergillus ochraceus are to be considered the main producers. Until 1998, these fungi were also believed to be responsible for the production of the toxin in grape, but OTA-producing A. carbonarius and A. niger were identified in dried vine fruits in 1999. Further studies pointed out that mycoflora potentially responsible for the presence of OTA in grapes are present in the field. Aspergilli are dominant to Penicillia, and among these Aspergilli section Nigri. A. carbonarius probably plays an important role because of the high percentage of positive strains and the amount of OTA produced. Aspergilli section Nigri are present on grape bunches early in the season and their frequency increases during later growth stages. At early veraison and ripening, the incidence of colonised berries is more related to the year than to the growth stage, but not to visible symptoms, since it is normal to isolate fungi from intact berries. Differences in ochratoxin content of berries have been detected between years, when the same vineyards, managed in the same way, showed high levels (1999) or the absence (2000) of the toxin. The results suggest that meteorological differences between years and grape-growing areas are responsible for differences in OTA levels, but the data are at present insufficient to draw firm conclusions.

Occurrence of mycotoxins and ergosterol in maize harvested over 5 years in Northern Italy

Maize samples collected from storage bins and feed mills in Northern Italy between 1995 and 1999 were surveyed for the occurrence of aflatoxin B1 (AFB1), zearalenone (ZEA), deoxynivalenol (DON) and fumonisin (FB1); further, ergosterol was analysed as a fungal growth marker. The incidence and mean content of AFB1 were generally low; nevertheless, a remarkable contamination was found in two samples (109 and 158 μg kg−1), while five others exceeded 20 μg kg−1. DON and ZEA mean levels were significantly higher in 1996 (2716 and 453 μg kg−1) with respect to the other years, when mean contents ranged from 7 to 30% and from 3 to 17%, respectively, expressed in per cent of 1996 contents. FB1 was present in all samples and was by far the most remarkable mycotoxin in Northern Italian maize, with the exception of samples from 1996. The average level was 3064 μg kg−1, 69.6% of samples resulted over 1000 μg kg−1 and 16.9% over 5000 μg kg−1. Significant correlations were found between ergosterol and the major mycotoxin(s) in each year (FB1 in 1995 and 1997–99; ZEA + DON in 1996). Consequently, ergosterol seems to be a good index of the toxicological quality of maize. Climatic conditions influenced the growth of different fungal species. In 1996, the first 20 days of October were extremely rainy; these weather conditions delayed the harvest until the first week of November and favoured the growth of DON and ZEA producing fungi and the synthesis of mycotoxins. On the contrary, the temperate and dry climate of the other years supported the growth of FB1-producing fungi.

DETOXIFICATION METHODS OF AFLATOXINS. A REVIEW.

Detoxification of aflatoxin contaminated foods and feeds is a current problem, as aflatoxins are highly carcinogenic and capable of passing unaltered through metabolic processes and accumulating in the tissues (seriously jeopardizing human and animal health). Although numerous detoxification methods have been tested, none seems able to fulfill the efficacy, safety, safeguarding of nutritional elements and costs requisites of a detoxification process. This paper critically reviews the main chemical detoxification methods and the latest approach to the problem using added sorbents capable of adsorbing aflatoxins.

Penicillium populations in dry-cured ham manufacturing plants

Seven ham manufacturing plants were sampled for 1 year to assess the mycoflora present in the air and on hams, with special attention given to potential mycotoxin producers. Temperature and relative humidity were recorded in the ripening rooms. Maturing rooms held hams from 2 to 3 through 6 to 7 ripening months, and aging rooms held hams for the following 6 to 7 months, until the 14-month ripening point, when they were ready for the market. Mean temperatures and relative humidities registered during the study were 14.9 degrees C and 62.4%, respectively, in maturing rooms and 16.3 degrees C and 57.6% in aging rooms. Aspergilli and penicillia, potential mycotoxin producers, were isolated in all the plants from the air and the ham. Aspergilli represented 5% of the isolates, while penicillia were largely dominant, with Penicillium nalgiovense being the most represented species (around 60% of the penicillia), followed by Penicillium nordicum, with 10 and 26% of the penicillia isolated, respectively, from the air or the ham. Ochratoxin A production ability, checked in vitro at 250C, was observed in 50% of the P. nordicum isolates obtained both from the air and the ham. Air and ham surface contamination by penicillia was greater in the ripening rooms, where higher temperatures were registered. A certain correlation was also observed between air and ham surface contamination. On the basis of this study, P. nordicum, the ochratoxin A producer that is notable on proteinaceous substrates, is normally present in ham manufacturing plants in Italy, even though not a dominant species. Further studies are necessary to clarify and ensure if dry-curing conditions minimize the potential risk of ochratoxin A formation in the product.

Logistic regression modeling of cropping systems to predict fumonisin contamination in maize.

The aims of this research were to monitor the presence of fumonisins in maize crops in northern Italy over a 6 year period, to study the role of the cropping system on fumonisin levels, and to contribute to the development of a predictive system for fumonisin contamination. In the 6 year period from 2002 to 2007, 438 maize samples were collected in five regions, supported by agronomic data, and analyzed for fumonisin content. Fumonisin was detected in almost all of the grain samples, but 2007 was less and 2005 more contaminated compared to the other years. Preceding crop, maturity class of hybrids, nitrogen fertilization, sowing and harvest week, and grain moisture significantly affected the level of contamination. The logistic regression developed explained around 60% of variability with major roles for longitude, maturity class, and growing weeks. The function can be used to quantify the effect of these factors in a predictive system.

Reduction of carryover of aflatoxin from cow feed to milk by addition of activated carbons

According to a double-reversal experimental design on 12 late-lactation Friesian cows the effect of two activated carbons (ACs) (CAC1 and CAC2) and a hydrated sodium calcium aluminosilicate (HSCAS) on carryover of aflatoxin B1 (AFB1) from feed to aflatoxin M1 (AFM1) in milk was determined. Cows were fed a basal diet containing AFB1 naturally contaminated corn meal and copra, During week 1 cows were fed diets containing AFB1 alone (11.28 μg of AFB1/kg of feed); in week 2 the diets contained AFB1 plus 2.0% sorbent; and in week 3 the diets again contained AFB1 alone (13.43 μg of AFB1/kg of feed). ACs reduced the analytical content of AFB1 in the pelleted feed by from 40.6% to 73.6%, whereas reduction by HSCAS was 59.2%, The AFM1 concentrations in milk in weeks 1 and 3 were higher than that in week 2, Decreases in the AFM1 excreted in the milk by addition to feed of 2% of the sorbents ranged from 22% to 45%. CAC1 and HSCAS were significantly different from each other in reducing the AFM1concentration in milk (45.3% versus 32.5%); these reductions were significantly higher than that of CAC2 (22.0%). Carryover reduction by addition of CAC1 (50%) was significantly higher than that of HSCAS (36%). Addition of 2% CAC2 did not allow pelleting of feed because of the caking action of this carbon, The lower performance of CAC2 could be related to the unsuccessful pelleting. The addition of ACs did not influence feed intake, milk production, milk composition, or body weight. Our results suggest that ACs, high-affinity sorbents for AFB1 in vitro, are efficacious in reducing AFB1 carryover from cow feed to milk. Further in vivo investigations should establish lower amounts of ACs which can be efficacious.

Maternal dietary habits and mycotoxin occurrence in human mature milk

During 2006, 82 samples of human mature milk were collected at Italian hospitals and checked for aflatoxin M1 (AFM1) and ochratoxin A (OTA) by immunoaffinity column extraction and HPLC. AFM1 was detected in four (5%) of milk samples (ranging from < 7 ng/L to 140 ng/L; mean level: 55.35 ng/L); OTA was detected in 61 (74%) of milk samples (ranging from < 5 ng/L to 405 ng/L; mean level: 30.43 ng/L. OTA levels were significantly higher (p less, not double equals 0.05) in milk of habitual consumers of bread, bakery products and cured pork meat. No other statistically significant differences were observed although habitual consumers of pasta (p = 0.059), cookies (p = 0.061) and juices (p = 0.063) had mean contamination values of OTA higher than the moderate consumer. The very few AFB1 positive samples did not allow statistical comparisons. The present study confirms that the occurrence of OTA in human milk is related to maternal dietary habits. The findings support the possibility of dietary recommendations to woman, during pregnancy and lactation, aimed to tentatively reduce the OTA contamination of human milk.