Occurrence of Penicillium verrucosum in retail wheat flours from the Spanish market

In Spain, low ochratoxin A (OTA) levels have been detected in wheat and different wheat products but no information has been published about the fungi involved in this OTA contamination. Some species of the genera Penicillium and Aspergillus are known to form OTA but few of them are known to contaminate foods with this mycotoxin. Penicillium verrucosum, an important OTA producer typical of temperate and cold climates, is much more frequently found on cereals in countries where they occasionally have OTA problems as in North European countries compared with South Europe, where levels of OTA generally seem to be lower or is not detected. The aim of this study was to determine, identify and characterize the occurrence of potential OTA-producing Aspergillus spp. and Penicillium spp. from retail wheat flours purchased in the Spanish market and used for human consumption. A total of 105 Aspergillus isolates were analyzed in order to know whether they are able to produce OTA and/or citrinin (CIT). None of these isolates were able to produce these mycotoxins. However, 17 suspected P. verrucosum isolates were recovered and confirmed by RAPD analyses. Eleven isolates were OTA producers and 14 isolates produced CIT. Our results confirm the potential risk of OTA and CIT production in wheat flours if stored improperly and the occurrence of P. verrucosum in South European countries. This was the only species able to produce these mycotoxins.     

Ochratoxigenic species from Spanish wine grapes

The ochratoxigenic mycobiota of grapes belonging to representative wine regions located along the Mediterranean coast of Spain at different developmental stages was identified. During the development of the berries, the occurrence of Aspergillus spp. increased while the percentage of berries contaminated by non-ochratoxin A (OTA) producing species such as Alternaria spp. and Cladosporium spp. decreased. Penicillium verrucosum, the only confirmed Penicillium spp. that is able to produce OTA, was not isolated. The contamination by OTA-producing species comes from the surface of the berries and not from the inner fruit. Black aspergilli were predominant among the different Aspergillus spp. isolated. All the Aspergillus carbonarius isolates were able to produce OTA at different concentrations. None of the isolates belonging to Aspergillus niger aggregate and to Aspergillus japonicus var. aculeatus were able to produce OTA. These results are a strong evidence of the contribution of A. carbonarius in the OTA contamination in wine grapes, mainly at the last developmental stages of the berries.     

Ochratoxin A-producing strains of Penicillium spp. isolated from grapes used for the production of "passito" wines

The post-harvest mycobiota of dried grapes, used in Friuli Venezia Giulia (Northern-East Italy) for the production of "passito" dessert wines, was investigated in order to detect potential ochratoxin A (OTA)-producers. Five grape cultivars were analysed and only isolates belonging to the genera Aspergillus and Penicillium were evaluated. No Aspergillus spp. was found while 379 strains of Penicillium spp. were isolated. Four strains produced UV fluorescent metabolites on grape juice agar and synthetic liquid media as observed by thin-layer chromatography (TLC). Three of these resulted OTA producers when analyzed by high pressure liquid chromatography (HPLC), following immunoaffinity column purification. According to the results of morphological examinations and ribosomal DNA sequencing, the OTA producer strains did not belong to the species P. verrucosum or P. nordicum. The corresponding passito wines did not contain OTA.     

An overview of ochratoxin A in beer and wine

Ochratoxin A (OTA) is a mycotoxin produced mainly by several fungal species of the genera Aspergillus and Penicillium. This mycotoxin has been shown to be nephrotoxic, hepatotoxic, teratogenic and carcinogenic to animals and has been classified as a possible carcinogen to humans. OTA occurs in a variety of foods, including beer and wine. Reports on OTA occurrence in beer indicate that this is a worldwide problem due to the widespread consumption of this beverage. At present, the European Union (EU) has not set a maximum allowable limit (MAL) for this mycotoxin in beer, although there is a limit in barley and malt. Studies carried out in different countries agree in the high proportion of samples contaminated with OTA although levels are, usually, below 0.2 ng/ml. OTA occurrence has been related to the contamination of malt barley with ochratoxigenic species, particularly Penicillium verrucosum. OTA produced in grains is carried to wort and, although fermentation decreases the concentration, the toxin is not eliminated. Reducing the fungal contamination of malt barley is the most promising strategy for reducing OTA in beer. With regard to wine, surveys on the presence of OTA have been conducted worldwide. The proportion of wines in which OTA is detected is very high (above 50%) in some countries (especially in the Mediterranean basin) although only a few wines contained concentrations exceeding the MAL laid down by the EU (2.0 ng/ml). A gradient of concentration is usually recognized; OTA levels decrease in the order red, rose, and white wine but also with increasing latitude of the producing countries. OTA presence in wines is due to the black aspergilli, mainly A. carbonarius, which can grow on grapes in the vineyards and produce the toxin. At grape crushing, the juice can be contaminated with the toxin which is carried over into wine, where it persists due to its stability. Pre- and post-harvest treatments are being investigated to diminish contamination of wines as much as possible.     

Polyphasic approach for differentiating Penicillium nordicum from Penicillium verrucosum

The aim of this research was to use a polyphasic approach to differentiate Penicillium verrucosum from Penicillium nordicum, to compare different techniques, and to select the most suitable for industrial use. In particular, (1) a cultural technique with two substrates selective for these species; (2) a molecular diagnostic test recently set up and a RAPD procedure derived from this assay; (3) an RP-HPLC analysis to quantify ochratoxin A (OTA) production and (4) an automated system based on fungal carbon source utilisation (Biolog Microstation?) were used. Thirty strains isolated from meat products and originally identified as P. verrucosum by morphological methods were re-examined by newer cultural tests and by PCR methods. All were found to belong to P. nordicum. Their biochemical and chemical characterisation supported the results obtained by cultural and molecular techniques and showed the varied ability in P. verrucosum and P. nordicum to metabolise carbon-based sources and to produce OTA at different concentrations, respectively.     

Multiplex Real‐Time PCR Method for Detection and Quantification of Mycotoxigenic Fungi Belonging to Three Different Genera

Abstract: Cereal crop plants are colonized by many fungal species such as Aspergillus ochraceus and Penicillium verrucosum, which produce ochratoxins, and Fusarium graminearum, which produces trichothecene mycotoxins. A multiplex real‐time PCR method using TaqMan probes was developed to simultaneously detect and quantify these mycotoxigenic Fusarium, Penicillium and Aspergillus species in cereal grains. Primers and probes used in this method were designed targeting the trichothecene synthase (Tri5) gene in trichothecene‐producing Fusarium, rRNA gene in Penicillium verrucosum, and polyketide synthase gene (Pks) in Aspergillus ochraceus. The method was highly specific in detecting fungal species containing these genes and was sensitive, detecting up to 3 pg of genomic DNA. These PCR products were detectable over five orders of magnitude (3 pg to 30 ng of genomic DNA). The method was validated by evaluating sixteen barley culture samples for the presence of deoxynivalenol (DON) and ochratoxin A (OTA) producing fungi. Among the barley culture samples tested, 9 were positive for Fusarium spp, 5 tested positive for Penicillium spp, and 2 tested positive for Aspergillus spp. Results were confirmed by traditional microbiological methods. These results indicate that DON‐ and OTA‐producing fungi can be detected and quantified in a single reaction tube using this multiplex real‐time PCR method. Practical Application: This method would be helpful in detecting and quantifying the mycotoxin producing fungi such as Fusarium, Aspergillus, and Penicillium in cereal grains and cereal‐based foods.     

The mycobiota of speck, a traditional Tyrolean smoked and cured ham

Speck is a ham specialty product traditionally produced in South Tyrol (Italy) and North Tyrol (Austria) by farmers, butcheries, and meat industries. To date, nothing has been learned about fungi associated with this smoked and cured meat. Therefore, it was the main objective of this study to assess the typical mycobiota of Speck in relation to the different production types and the geographic provenance. A total of 121 Speck samples from North Tyrol and South Tyrol was analyzed. From 63 isolated fungal species, only a few can be regarded as typical colonizers: Eurotium rubrum and Penicillium solitum were the dominating species in all types and parts of Speck (crust, meat, and fat). Eight other Penicillium spp. were relatively frequent. The species diversity increased from industrially produced Speck to products from butcheries and farmers, and it was higher in all types of South Tyrolean products. Among the typical mycobiota, Penicillium verrucosum, Penicillium canescens, and Penicillium commune are known as potentially mycotoxigenic.     

谷物类食品中赭曲霉毒素A分析方法的研究进展

赭曲霉毒素A是由真菌产生的次级代谢产物,由于其具有极强的肾脏毒性、致癌性、致畸性,且对食品污染的范围广泛,许多国家对其制定了严格的限量标准。目前对谷物中赭曲霉毒素A的检测方法主要有薄层色谱分析法、高效液相色谱法、酶联免疫吸附法、毛细管电泳法和胶体金试纸条法等。随着检测水平的提高,对食品中的赭曲霉毒素A限量标准的要求也在不断地提高。建立安全、准确、灵敏度高、重现性好的检测体系将成为今后研究的重点。     

赭曲霉毒素A的研究进展

赭曲霉毒素A主要由赭曲霉和青霉属的某些菌株产生,广泛分布于谷物、其他植物性食品及相关产品和动物性食品,对动物和人体具有肾脏毒性、肝脏毒性,另外还有致畸、致突变和致癌作用,并有免疫抑制作用.随着对其研究的不断深入,现在愈来愈受到人们的关注,本文就赭曲霉毒素A对食品的污染、毒性、检测方法等方面进行综述.     

Occurrence of pathogenic fungal species in Tunisian vineyards

Mycotoxins are secondary metabolites produced by filamentous fungi detected in food, such are grapes. OTA was evaluated in ten handle musts from different Tunisian vineyard. This mycotoxin was found at levels 1.1 mug/L to 4.3 mug/L. A survey was conducted to assess the contamination of the Tunisian vineyard with pathogenic fungal species, in particular those responsible of the OTA production. The results were evaluated for the first time in parcels cultivated in the North, in the Centre and in the South of the country. Italia Muscate and Superior Seedless varieties were concerned at three developmental stages of the berry, setting, veraison and maturity. Carigon variety was used as positive control for musts contaminating by OTA. The main fungal species isolated were Aspergillus spp. (33.32%), Botrytis cinerea (23.32%), Alternaria spp. (12.80%), Cladosporium spp. (10.59%) and Penicillium spp. (8.3%). The isolates of the Aspergillus genus were identified as Aspergillus niger aggregate (77%), Aspergillus carbonarius (15%) and Aspergillus flavus (8%). Their presence was characterized by a significant decrease in the Centre during the veraison and a slight increase in the North and the South during the maturity stage. Furthermore, when comparing Superior Seedless and Italia Muscate cultivated in the same area, the aspergilli were particularly less abundant at the setting stage in the case of Superior Seedless. There is no correlation between the OTA amount in musts and the contamination by Aspergillus species in different vineyards and for grape varieties studied.     

果品及其制品中赭曲霉毒素A污染的发生、控制和检测

赭曲霉毒素A（ochratoxin A,OTA）是由赭曲霉、炭黑曲霉和青霉属等真菌产生的聚酮类次生代谢产物,广泛存在于果品及其制品中,特别是葡萄及其制品中检出率较高。由于OTA具有毒性强、可致癌且结构非常稳定、难以去除等特点,世界各国都制定了果品及其制品中OTA的限量。本文就OTA的生物合成、果品及其制品中OTA检测技术、OTA污染状况和控制策略分别进行阐述,并对其快速检测、绿色安全防控等方面进行展望,以期为OTA污染的高效分析和有效控制提供理论依据和指导。     

Mycoflora and ochratoxin A producing strains of Aspergillus in Algerian wheat

Wheat is a basic staple food for very large segments of the population of Algeria. The aim of this study is to analyse ochratoxin A (OTA)-producing mould and OTA-contaminated wheat. To evaluate the mycoflora and the potential for OTA production by Aspergillus strains, a total of 85 samples of wheat destined for human consumption were collected from two regions in Algeria (Tizi Ouzou and Setif) during the following phases: preharvest, storage in silos, and after processing. The mean value counts of fungi ranged from 275 to 1277 CFU g(-1). The dominant genus was Aspergillus, predominantly A. flavus, A. niger and A. versicolor. The other isolated species were A. ochraceus, A. alliaceus, A. carbonarius, A. terreus, A. fumigatus, A. candidus and Aspergillus spp. The occurrence and the levels of the genus Penicillium, Fusarium, Alternaria and Mucor were substantially lower than those of Aspergillus. The storage in silos shows high levels of Aspergillus (66 to 84%), especially A. flavus, but A. niger and other fungi were isolated at relatively low percentages. Equal distribution of the fungal contamination into the bran, flour and semolina fractions was observed from Flour Mill and Semolina Mill. The genus Aspergillus remained present at high levels at several phases of the production process. In addition, the ability to produce OTA by 135 isolates belonging to eleven species of Aspergillus and 23 isolates of Penicillium spp. was analyzed using fluorescent detection-based HPLC. Thus, it was found that 51 isolates (32.3%) were ochratoxigenic. All isolated strains of A. ochraceus (12) and A. alliaceus (6) produced OTA at concentrations ranging from 0.23 to 11.50 microg g(-1). Most of the A. carbonarius strains (80%) were OTA producers (0.01 to 9.35 microg g(-1)), whereas A. terreus (50%), A. niger (28%), A. fumigatus (40%), A. versicolor (18%) and Penicillium spp. (21.7%) were low level producers (0.01 to 0.07 microg g(-1)). The concentration of OTA was determined in 30 samples of wheat. OTA was detected in 12 (40%) of the samples at levels ranging from 0.21 to 41.55 microg kg(-1).     

Occurrence of toxigenic fungi in ochratoxin A contaminated liquorice root

Fungi associated with ochratoxin A (OTA)-contaminated liquorice root and their capabilities for OTA production were investigated. Medicinal materials of mouldy liquorice root were collected from herbal markets located in Jiangxi, Zhejiang, Henan provinces and Beijing, China, respectively. Sixteen fungal species belonging to Penicillium, Aspergillus, Eurotium, Fusarium, Mucor and Scopulariopsis were isolated; the fungal composition was different in each liquorice root sample. Penicillium polonicum was predominant, comprising 54% of the total isolates in the liquorice root sample from Jiangxi province, which was contaminated with OTA at the highest level. In other samples with lower OTA contents, species of Aspergillus and Eurotium were predominant. OTA production of representative strains on rice media was detected by LC-MS/MS; all Penicillium polonicum isolates and a P. chrysogenum were ochratoxigenic; OTA concentrations ranged from 6.94 to 217.37?ng?g(-1). This is the first study to report P. polonicum as an OTA-producing fungus. OTA contamination of mouldy liquorice root constitutes a major health hazard in consumption. This situation demands urgent and undivided attention.     

A SURVEY OF OCHRATOXIN A IN WHEAT AND BARLEY IN INDIA

Ochratoxin A (OTA), a secondary metabolite produced by the species of Aspergillus and Penicillium has been found to be nephrotoxic, immunosuppressive and teratogenic in many species and is a possible human carcinogen. Its prevalence in cereals, namely wheat and barley, has been reported from many European countries. In this study, 126 samples of wheat and barley collected from different regions in India were analyzed by high‐performance liquid chromatography for the presence of OTA. The highest OTA content determined in one sample of wheat was 2.3 µg/kg. None of the 126 samples of wheat and barley contained more than 5 µg/kg. Wheat (82.6%) and barley (90%) samples contained less than 0.1 µg/kg, which is the limit of quantification of OTA in this study. The maximum limit of OTA for wheat, barley and rye that is being debated at Codex Alimentarius Commission meetings is 5 µg/kg. In an attempt to determine if there was any correlation between the ochratoxigenic mold mycoflora and the ochratoxin content in the samples analyzed, enumeration of the molds that belonged to Aspergillus and Penicillium species was determined using general mycological media. The aflatoxigenic and ochratoxigenic mycoflora was also enumerated using selective agar media. It was observed that more aflatoxigenic molds were recovered in both wheat and barley samples. None of the species of Penicillium detected from the samples was found to be ochratoxigenic. However, Aspergillus species belonging to section Nigri, Aspergillus niger group, also an ochratoxin producer, were recovered from the samples.     

Current importance of ochratoxin A-producing Aspergillus spp

Ochratoxin A (OA) is receiving attention worldwide because of the hazard it poses to human and animal health. OA contamination of commodities, such as cereals or pork and poultry meat, is well recognized. Nevertheless, there is an increasing number of articles reporting OA contamination in other food commodities, such as coffee, beer, wine, grape juice, and milk, in the last few years. This continuous and increasing exposure to OA that humans experience is reflected in the high incidence of OA in both human blood and milk in several countries. OA was believed to be produced only by Aspergillus ochraceus and closely related species of section Circumdati and by Penicillium verrucosum; however, in the genus Aspergillus, the production of OA has been recently reported by species outside the section Circumdati. Thus, it has been clearly established as a metabolite of different species of the section Nigri, such as Aspergillus niger and Aspergillus carbonarius. OA production ability by Aspergillus spp. is more widespread than previously thought; therefore, there is the possibility that unexpected species can be new sources of this mycotoxin in their natural substrates.     

Co‐occurrence of citrinin and ochratoxin A in rice in Asia and its implications for human health

Citrinin (CIT) and ochratoxin A (OTA) are nephrotoxic mycotoxins, produced by several Aspergillus and Penicillium species and their co‐occurrence in rice may cause health effects in humans. Rice is an important food crop worldwide and is a major staple food in Asia which may be invaded by CIT and OTA producing fungal spores in the field, during harvest and storage. Humans are exposed to these mycotoxins through ingestion of contaminated rice and other food commodities. Yet, data on the combined presence to these food contaminants are still insufficient to estimate human exposure in Asia. This review describes the prevalence of CIT and OTA in rice in Asia and its implications on human health, which may help in establishing and carrying out proper management strategies against mould development on rice. From the health point of view, combined exposition of CIT and OTA should be a public concern as both are nephrotoxic and long‐term exposure can pose detrimental health effects. Thus, it is necessary for local farmers and food factories to implement strict measures and to improve methods for rice preservation during the distribution to consumers, particularly in the markets. Moreover, regular surveys for CIT and OTA occurrence in rice and human biomonitoring are recommended to reduce the health effects in Asian population. © 2017 Society of Chemical Industry     

Microbial quality and presence of moulds in Kuflu cheese

The chemical and microbial qualities, including fungal flora, of 30 samples of Kuflu cheese randomly purchased from different markets in Turkey were investigated. The gross composition of the cheese samples ranged between 37.65-53.65% moisture, 6.21-40.09% fat-in-dry matter, 4.70-10.07% salt-in-moisture and 26.18-44.85% protein. The mean pH value of the cheeses was 6.29+/-0.28 and pH values ranged from 5.52 to 7.22. Variations between the samples in terms of their gross composition suggested a lack of quality standards in cheesemilk, cheesemaking procedure and ripening conditions. The levels of main microbial groups including total mesophilic and coliform bacteria, yeasts and moulds and the presence of some potentially pathogenic microorganisms (E. coli, Salmonella spp. and Staphylococcus aureus) were determined. The high numbers of all microbial groups and presence of potentially pathogenic organisms in the cheese samples suggested that the production and maturation of Kuflu cheese should be improved by better hygiene. Moulds at the cheese surface were isolated and identified. A total of 24 different mould species were detected and the genus most frequently isolated was Penicillium spp. which represented 70.25% of total isolates. Penicillium commune, P. roqueforti and P. verrucosum were the most abundant species in the cheeses sampled. The other dominant fungal groups were Geotrichum candidum, Penicillium expansum and P. chrysogenum. Other genera isolated from the cheese were Acremonium, Alternaria, Aspergillus, Cladosporium, Geotrichum, Mucor, Rhizopus and Trichoderma. The potentially toxigenic species, including some Penicillum spp. and Aspergillus flavus, were also detected.     

赭曲霉毒素A分析方法进展

赭曲霉毒素A(OchratoxinA,OTA)是曲霉属和青霉属的某些菌种产生的一种具有致癌、致畸、免疫抑制和肝肾毒性的有毒代谢产物,世界上许多国家已制定了食品中OTA的相关法规和标准,而准确、可靠、灵敏的分析OTA方法是法规标准实施的重要依据。有机溶剂与酸(或碳酸氢钠)的混合溶液是提取食品中OTA的常用溶剂系统;含有OTA提取液的净化手段包括液液分配、固相柱萃取和免疫亲和层析等;反相液相色谱配荧光检测器、酶联免疫吸附法和液相色谱质谱联机是分析OTA的常用方法,本文就OTA的检测方法进行综述。     

Fungal mycoflora and mycotoxins in Korean polished rice destined for humans

Rice samples collected from the Republic of Korea were analyzed for fungal mycoflora and mycotoxins: fumonisins, ochratoxin A, trichothecenes, and zearalenone. The potential of the fungi to produce each mycotoxin was also examined, so that the fungal isolates associated with mycotoxins occurring in rice could be verified. Penicillium citrinum and Aspergillus candidus were the most prevalent species infecting the samples, while Fusarium proliferatum was found as the dominant Fusarium species. Ochratoxin A was the most commonly detected mycotoxin analyzed in the present study; moreover, its level in some samples was above the EU tolerable limit (3 ng/g). According to rice culture experiments, it was revealed that in Korea, fumonisins detected in rice were due to F. proliferatum infection, whereas the occurrence of ochratoxin A was caused by Penicillium verrucosum, though there were no symptoms of disease in rice found in any sample. Furthermore, there appears to be an uneven geographical distribution of P. verrucosum as well as ochratoxin A in that most of them are found in the rice samples produced in the northern region of Korea.     

葡萄赭曲霉毒素污染及其产毒素菌株的筛选方法研究进展

赭曲霉毒素A(ochratoxin,OTA)是由曲霉属和青霉属等真菌产生的一类真菌毒素,其毒性很强,分布广泛,对人类和动植物的健康有着巨大的影响。葡萄及其制品是食品中OTA的主要来源之一,从病害的葡萄表面筛选分离产生赭曲霉毒素的菌株是最常用的研究产毒素菌株的方法。由于产毒素菌株主要分布在葡萄果实的表面,葡萄组织受损后会极大提高赭曲霉素的污染程度,研究产毒菌株分布及产毒素能力对控制赭曲霉素污染及寻找一种有效地生物防治方法提供参考。本文综述了葡萄赭曲霉毒素的污染情况及其产毒素菌株的筛选方法,为控制葡萄及其产品中的OTA污染提供依据。