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.     

Transformation ability of fungi isolated from cork and grape to produce 2,4,6-trichloroanisole from 2,4,6-trichlorophenol

The ability of eight fungal strains to transform 2,4,6-trichlorophenol (TCP) to 2,4,6-trichloroanisole (TCA) was studied. These fungi were isolated from cork, belonging to the genera Penicillium, Aspergillus, Trichoderma and Chrysonilia, and from grapes Botrytis cinerea. All, except Chrysonilia, produced TCA when grown directly on cork in the presence of TCP, Aspergillus and Botrytis cinerea being the ones with the highest level of production. It is the first time that Botrytis cinerea, a microorganism often present on grapes and in winery environments, has been shown to transform TCP into TCA. This result can partially explain the wine cork taint before being bottled.     

The mycobiota of three dry-cured meat products from Slovenia

The surface mycobiota of three types of Slovenian dry-cured meat products were isolated from a total of 75 items of product that were sampled periodically during the drying/ripening stage of processing. The predominant filamentous fungal genus isolated was Penicillium. Eurotium spp., Aspergillus versicolor and Cladosporium spp. were isolated from only two of the products. Eight Penicillium species were identified. Penicillium nordicum was recovered frequently. Penicillium nalgiovense was recovered less frequently, from one product only (a salami), while a yet-to-be described species Penicillium “milanense” was isolated from 21 items. The other penicillia were rarely isolated. Of the isolated and identified species, those that can produce mycotoxins are: A. versicolor, Penicillium brevicompactum, Penicillium chrysogenum, P. nordicum, and Penicillium polonicum. Their growth on dry-cured meat products is undesirable.     

Contamination with storage fungi of human food from Cameroon

In a mycological study, a total of 95 human food samples were investigated to evaluate the incidence of fungal contamination in Cameroon by conventional identification method and partly confirmed by DNA sequencing. The isolated fungal spp. were further studied to determine their toxigenic potentials. The investigation revealed the predominance of Aspergillus and Penicillium with 96% of samples contaminated with at least one species of these fungi, whereas the incidence of co-contamination of samples was 85%. Aspergillus flavus and Aspergillus parasiticus (Flavi section) were the most predominant species contaminating mainly maize and peanuts. In addition, P. crustosum and P. polonicum were the most common contaminants belonging to the genus Penicillium. On the other hand, A. ochraceus (Circumdati section) registered a low incidence rate of 5%, including other members of the Aspergillus group. Other members of the genera Rhizopus and Alternaria spp. were also registered in the study. A majority of fungal strains of A. ochraceus, A. parasiticus, P. crustosum and P. polonicum isolated were toxigenic, producing the mycotoxins tested for, while none was detected in cultures of A. fumigatus. The high incidence rate of fungi contamination coupled with their potentials in producing mycotoxins gives a strong indication that the samples tested may likely be contaminated with various mycotoxins. There is need for further study to assess the incidence of mycotoxins contamination in similar food samples.     

The challenge of Brettanomyces in wine

Brettanomyces spp. is an indigenous yeast that can grow during wine fermentation and impact its flavor. The characteristic flavor is often described as phenolic/medicinal, spices, cloves, earthy, barnyard, and horsy. The acceptability of Brettanoymces-induced flavors depends on the flavor intensity, personal preference, and consumer expectations. The yeast has been isolated from grapes, barrels, and winery equipment. It impacts wines from all the top producing regions in the world, with associated flavors most often found in red wines. Prevention of growth of this yeast in wine involves attention to fruit quality and winery sanitation, control of sulfite and oxygen levels, and the use uncontaminated barrels. Greater understanding of how Brettanomyces and its metabolites contribute to wine flavor is required to recognize its impact on consumer acceptability.     

Influence of wood origin in the polyphenolic composition of a Spanish red wine aging in bottle, after storage in barrels of Spanish, French and American oak wood

In this paper, the changes in the composition of a red wine obtained from Tempranillo grapes (Vitis vinifera L.), stored from 12 to 24 months in bottles were studied. The wine was previously aging for 21 months in barrels made of Spanish oak wood (Quercus spp.). The changes of chromatic data, global polyphenolic families assessment (polyphenols, catechins, proanthocyanidins and anthocyanins) and individually polyphenols by HPLC during their storage time in bottles were studied and compared with these of the same wine aged in barrels made of French oak and American oak and stored in bottle for 24 months. Samples of wines obtained after 12 months in bottle were also compared with those after 24 months. The stepwise discriminant analysis with data of chromatic parameters and global polyphenolic families indicates that the wines aged in Spanish and French oak wood barrels, after 24 months stored in bottle, have similar characteristics, but they are significantly different to those of wines aged in barrels made of American oak wood. Regarding the analysis of individually non-anthocyanic polyphenols, discriminant analysis shows that wines stored for 24 months in bottle, aged in barrels made of Spanish, French and American oak woods, show overlapping results, while those from wines after 12 months in bottle are more dispersed. The discriminant analysis carried out on anthocyanidin concentrations of wines stored 24 months in bottles has shown three groups according the kind of wood used, indicating that wines aged in Spanish and French oak barrels are much more similar in anthocyanidin concentrations than those aged in the American barrels.     

Influence of wine turbidity on the accumulation of biogenic amines during aging

In this work the concentration of biogenic amines in filtered and unfiltered wine during aging in oak barrels was studied. Red wine from Merlot grapes was used. The wine remained for 243 days in barrels of American oak (Quercus alba) and French oak (Quercus sessilis) from the Allier and Nevers regions. Results showed that the different degree of wine turbidity did not have any influence on the accumulation of biogenic amines during the aging period. Histamine and tyramine were degraded at the end of aging; however, the concentration of histamine in both matured wines was higher than 8 mg l^?1, which could be a toxic danger to consumers. Putrescine and cadaverine were formed during aging and were not degraded. Phenylethylamine + spermidine were formed, above all, towards the end of the aging period, and the concentration of spermine did not show any appreciable variation. Copyright © 2004 Society of Chemical Industry     

Use of microfungi in the treatment of oak chips: possible effects on wine

BACKGROUND: Oak barrels are commonly used in the aging of wines and spirits because of their positive effects on the product. In recent years the addition of oak chips has been used to introduce desirable wood aromas and flavours into wines. In this study, oak chips in saline solution or laboratory medium were inoculated with Penicillium purpurogenum, Aureobasidium pullulans, Phialemonium obovatum, Phanerochaete chrysosporium and a combination of Ph. chrysosporium and A. pullulans. After 12 weeks of incubation, oak chips (2 g L^?1) were macerated in a red wine for 17 days. Gas chromatography/mass spectrometry and high‐performance liquid chromatography were used to evaluate 14 compounds, namely furfural, furfuryl alcohol, guaiacol, syringol, cis‐β‐methyl‐γ‐octalactone, 2‐phenylethanol, 4‐vinylguaiacol, benzyl alcohol, 2,3‐butanediol, γ‐butyrolactone, benzaldehyde, 4‐ethylguaiacol, gallic acid and ellagic acid. RESULTS: The microfungal treatments increased the concentration of some components. In particular, P. purpurogenum resulted in a significant improvement in the levels of guaiacol, furfural, syringol, furfuryl alcohol and 2‐phenylethanol. CONCLUSION: Penicillium purpurogenum and Ph. chrysosporium showed a constant trend (enrichment of furfural and benzaldehyde) independent to some extent of the medium used for chip treatment. Copyright © 2010 Society of Chemical Industry     

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.     

Bacterial and Fungal Communities in Pu'er Tea Samples of Different Ages

Pu'er is a major kind of postfermented tea and is made with a “large leaf” variety of Camellia sinensis (C. sinensis assamica), whose distribution is limited to the mountains of southern Yunnan, China. The quality of Pu'er tea is believed to increase with storage (aging, maturing) because of postfermentation by microbes. The effect of storage period (from < 1 to 192 mo) on the bacteria and fungi in Pu'er tea was investigated by a culture‐dependent and a PCR‐DGGE method. The individual numbers of fungi and bacteria decreased with increasing storage time and were significantly greater in ripened tea than in raw Pu'er tea. Both methods indicated that yeast, Aspergillus spp., and Penicillium spp. were the dominant fungi in almost all the samples. However, the common bacteria detected by the culture‐dependent method were species of Pseudomonas, Achromobacter, Alcaligenes, Sporosarcina, and Bacillus, whereas those detected by PCR‐DGGE were species of Staphylococcus, Arthrobacter, and Streptomyces. According to ordination analysis, bacterial community structure differed between ripened and raw Pu'er tea. Bacterial diversity was positively correlated with aging time, while fungal diversity in both raw and ripened tea increased during the first 60 mo of aging and then decreased. Changes in polyphenol content were correlated with the changes in fungal diversity. These results suggest that the relationship between storage time and the quality of Pu'er tea is complex and involves changes in polyphenol content and microbial abundance and diversity.     

Whole wheat and white wheat flour— the mycobiota and potential mycotoxins

Mold growth has detrimental effects on the quality of flour and may result in mycotoxin contamination. The search for potential mycotoxins—almost 400 are known—is time-consuming and expensive. However, detailed knowledge about the mycobiota and especially the toxin producing fungi enables the effective search for these toxic fungal metabolites. Therefore, a whole wheat flour and a white wheat flour (type 405) were investigated for their total qualitative as well as quantitative mycobiota. Overall, 51 species belonging to 14 different genera could be isolated. Total fungal counts of the whole wheat flour amounted to 1833 molds while the white wheat flour contained 1730 cfu 2 g⁻¹. The mycobiota of both flours was dominated by Aspergillus spp. accounting for 84% and 77·3% of the isolations, respectively. Fungi of the genus Penicillium spp. occurred only to a minor degree: 8% of the isolations in whole wheat flour and 15% in white wheat flour. Aspergillus candidus was the most frequently encountered mold.Penicillium aurantiogriseum , Cladosporium cladosporioides, A. flavus, Eurotium herbariorum,P. griseofulvum , P. brevicompactum and P. viridicatum were isolated to a lesser degree. From the 3563 identified fungi 93·3% (32 species) belong to the group of toxigenic molds.     

Application of Lactobacillus amylovorus as an antifungal adjunct to extend the shelf-life of Cheddar cheese

Lactobacillus amylovorus DSM 19280 is an antifungal strain that is inhibitory to a range of fungi including Penicillium expansum, Penicillium roqueforti, Aspergillus niger, Aspergillus fumigatus and Fusarium culmorum. In this study, the strain was used as an adjunct culture in a Cheddar cheese model system. During the ripening period, P. expansum spores were applied to the cheese surface to mimic fungal contamination. The presence of the antifungal L. amylovorus adjunct resulted in a four-day delay in appearance of Penicillium growth on the cheese in comparison to the adjunct-free control. When cheeses were exposed to natural airborne fungi, the presence of the adjunct resulted in a six-day delay in the appearance of mycelia on the cheese surface. Significantly, its presence had no detectable negative impact on cheese quality. The results indicate that the strain could have an application for extending the shelf-life of cheeses which are prone to fungal spoilage.     

Artificial aging of Uva di Troia and Primitivo wines using oak chips inoculated with Penicillium purpurogenum

BACKGROUND: Two red wines (Primitivo and Uva di Troia) treated with oak chips inoculated with Penicillium purpurogenum were analysed in order to assess their contents of furfural, cis‐β‐methyl‐γ‐octalactone, syringol, eugenol, vanillin and 4‐vinylguaiacol. Two different sizes of oak chips (small and big, of length 2 and 8 mm respectively) and two different degrees of toasting (low and high) were used in the study. Aroma compounds were analysed by gas chromatography/mass spectrometry to determine differences among samples after 15 days of chip contact time. RESULTS: Big oak chips inoculated with P. purpurogenum increased the level of 4‐vinylguaiacol, while small oak chips inoculated with P. purpurogenum, in some conditions, increased the level of eugenol. Chip size and degree of toasting also played an important role in the content of eugenol. CONCLUSION: The use of oak chips inoculated with mould might be a promising alternative to barrel aging. Moreover, different fungal inocula could contribute to the enrichment of wine with specific compounds (e.g. 4‐vinylguaiacol and eugenol). Copyright © 2011 Society of Chemical Industry     

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.     

A combined morphologic and molecular approach for characterizing fungal microflora from a traditional Italian cheese (Fossa cheese)

Phenotypic and genotypic methods were used to identify filamentous fungi that characterize traditional Italian Fossa cheese and its ripening environment. After ageing for 60 days at a dairy, it was ripened for an additional three months in a pit. In the fully ripened cheese, moulds ranged from 3 to 3.4 log cfu g^?1 and Penicillium was the prevalent species. Pit environmental fungi ranged from 530 to 750 cfu m^?3 (air) and from 130 to 340 cfu cm^?2 (surfaces). The dominant pit strains were Alternaria spp., Aspergillus spp., Cladosporium spp. and Penicillium spp. Phylogenetic analyses of 18S rRNA gene and ITS1-5.8S-ITS2 regions highlighted Penicillium camemberti, Aspergillus nidulans and Aspergillus versicolor as traceable species occurring in both the cheese and pit environment, suggesting their involvement in the development of typical Fossa cheese characteristics. This approach may be used for the identification of microflora on other cheese varieties to better understand the fungal contribution in cheese ripening.     

Effect of Oak Chips on Evolution of Phenolic Compounds and Color Attributes of Bog Bilberry Syrup Wine During Bottle‐Aging

This study investigated the evolution of phenolic compounds of bog bilberry syrup wine during a bottle‐aging process, and further estimated the oak chip treatment on the wine color alteration. The wine was macerated with oak chips (2 or 5 g/L under light or medium toasting level) for 20 d and then bottle‐aged for 6 mo. Results showed that the oak chip treatment significantly increased the content of phenolic compounds and enhanced the copigmented anthocyanin level before aging. It also resulted in an increase on a^* and C^* but a decrease on L^*, b^*, and H^* of the wine. During aging process, a content decrease of total phenol and antioxidant capacity of the wine was observed. Phenolic acids, flavonol glycosides, and anthocyanins reduced the content, whereas flavonol increased the content. Free and copigmented anthocyanin levels decreased, whereas polymerized anthocyanins level increased. This process caused an increase on L^*, b^*, and H^*, but a decrease on a^* and C^*. The oak chip treatment delayed the wine color change and its effect was mainly depended on the addition amount. Partial least square regression revealed that flavonol glycosides, phenolic acids, and anthocyanins displayed a positive correlation with L^*, b^*, and H^*, but a negative correlation with a^* and C^*. Quercetin‐3‐O‐glucuronide, myricetin‐3‐O‐galactoside, chlorogenic acid, and quercetin exerted a more important effect on the color alteration in wine.     

橡木桶陈酿对红葡萄酒的作用与影响的研究

阐述了木桶陈酿对红葡萄酒的微氧熟化、增强理化稳定性、改善风味特征的重要作用,同时比较了不同植物来源橡木桶呈香组份的特性,说明了木桶容积、新旧程度对陈酿效果的影响,并且提出了如何根据葡萄酒的性质选择橡木桶的建议。     

橡木桶在高档陈酿型干红葡萄酒生产中的优化应用

橡木桶陈酿过程是酿造高品质葡萄酒最基本、最重要的环节。在生产高档陈酿型干红葡萄酒过程中,结合原料、发酵状况及陈酿过程中的酒体变化,分析研究陈酿过程中橡木桶的选择与陈酿管理等实际问题。由此制定出椽木桶优化应用方案,开发高档陈酿型干红葡萄酒系列产品。     

A comparative study on the fungal communities of wheat Qu for Qingshuang‐type Chinese rice wine

A traditional culture‐dependent method and ribosomal intergenic spacer analysis (RISA) were employed to investigate the fungal communities in two representative samples of wheat Qu (S1, S2). A total of seven species were isolated on media of potato dextrose agar, Czapek and wheat Qu extraction. Owing to the difference in the culture media, a high variability of predominant species was observed. RISA profiles of total DNA exhibited distinguishable bands corresponding to nine fungal species. Similar RISA profiles of two starter cultures were obtained. Sequencing of the internal transcribed spacer fragments from fungal isolates and RISA fingerprint profiles revealed the presence of nine species of fungi in wheat Qu S1: Lichtheimia ramosa, Absidia corymbifera, Aspergillus sydowii, Aspergillus oryzae, Rhizomucor pusillus, Eurotium amstelodami, Issatchenkia orientalis, Emericella nidulans and Cladosporium cladosporioides. In comparison with S1, S2 had a richer fungal diversity. The fungal species derived from S1 were all present in S2 coupled with another two species, namely, Aspergillus niger and Clavispora lusitaniae. Among all species isolated, Aspergillus sydowii was the dominant species in Qingshuang‐type Chinese rice wine wheat Qu. Copyright © 2012 The Institute of Brewing & Distilling