Molecular and morphological identification of fungal species isolated from rice meju

The microflora of natural fermented Korean rice meju are assumed to be diverse, but has not yet been investigated. The fungi was isolated and identified from rice meju on the bases of morphology, culture characteristics, and molecular approaches of internal transcribed spacer and β-tubulin gene sequencing, and their enzyme activities were determined. Six species of fungi were primarily isolated from rice meju, specifically Aspergillus candidus, Aspergillus tritici, Cladosporium oxysporum, Penicillium commune, Penicillium griseofulvum, and Rhizomucor variabilis var. regularior. All isolates produced protease (5.17–15.78), and all strains except Mucor circinelloides PR06 expressed amylase activities (0.04–0.23). In comparison, lipase was produced by zygomycetous fungi isolates (1.70–0.25) and Aspergillus oryzae PR07 (0.68). The enzyme producing fungal strains might be involved in the rice meju fermentation. The identification of fungal diversity in meju is useful for understanding the fermentation processes and these strains might be used in the fermentation of foods.     

Effect of solute and matric potential on growth rate of fungal species isolated from cheese

The effect of water potential (Ψ_w) on the growth of 15 fungal species isolated from cheeses was analysed. The species, identified mainly by analysis of DNA sequences, belonged to genera Penicillium, Geotrichum, Mucor, Aspergillus, Microascus and Talaromyces. Particularly, the effect of matric potential (Ψ_m), and ionic (NaCl) and non-ionic (glycerol) solute potentials (Ψ_s) on growth rate was studied. The response of strains was highly dependent on the type of Ψ_w. For Ψ_s, clear profiles for optimal, permissive and marginal conditions for growth were obtained, and differences in growth rate were achieved comparing NaCl and glycerol for most of the species. Conversely, a sustained growth was obtained for Ψ_m in all the strains, with the exception of Aspergillus pseudoglaucus, whose growth increased proportionally to the level of water stress. Our results might help to understand the impact of environmental factors on the ecophysiology and dynamics of fungal populations associated to cheeses.     

The inhibitory effect of oregano extract on the growth of Aspergillus spp. and on sterigmatocystin biosynthesis

The aim of this study was to investigate the effect of oregano extract on the growth of Aspergillus species (Aspergillus niger, Aspergillus carbonarius, and Aspergillus wentii) isolated from food and biosynthesis of sterigmatocystin (STC) by Aspergillus versicolor. Antifungal determinations were conducted using the agar plate method. The effect of oregano extract on the biosynthesis of STC was determined in a Yeast Extract Sucrose (YES) broth. The STC content and mycelial growth of A. versicolor were determined on the 7th, 14th, and 21st day with all applied concentrations of oregano extract and control sample. The composition of oregano extract was determined by gas chromatography–mass spectrometry (GC–MS) and 21 different components were identified. The major components were carvacrol (34.20%) and carvone (18.05%). At 2.5 mL/100 mL concentration, the oregano extract completely inhibited the growth of A. wentii, while the growth of A. carbonarius and A. niger was reduced by 95.6% and 45.6%, respectively. Significant reductions in STC biosynthesis during 21 days of incubation were observed for extract concentrations higher than 0.06 mL/100 mL. The obtained results showed that the tested oregano extract could be used as a food preservative to prevent food-borne fungal infections and mycotoxin production.     

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.     

Isolation of Fungal Species from Fermentating Pearl Millet Gruel and Determination of their Antagonistic Activities against Indicator Bacterial Species

The mycological and physicochemical qualities (pH and titratable acidity) of fermenting pearl millet gruel were evaluated using routine methods. Several species of yeasts and moulds were isolated and the mould species identified based on their observable macroscopic and microscopic characteristics. The moulds identified included: Penicillium sp (FM1), Rhizopus spp (FM2, FM3 and FM6), Aspergillus flavus (FM7) and Aspergillus niger (FM8). These isolates were screened for production of antimicrobial compounds using agar well diffusion method. Escherichia coli, Staphylococcus aureus, Bacillus cereus, Bacillus lichieniformis, Salmonella spp., Pseudomonas fluorescens, Pseudomonas aeruginosa, Pseudomonas syringae, Proteus sp. and Serratia sp. were utilized as indicator organisms. Secondary metabolites were also extracted from the respective moulds and the antimicrobial properties of these metabolites were tested against pure cultures of E. coli, S. aureus, P. flourescens and B. lichieniformis. All the moulds exhibited antimicrobial activity against P. flourescens while the metabolic extract of Aspergillus flavus (FM7) displayed the highest zone of inhibition (24 mm) against an overnight culture of P. flourescens.     

Toxigenic molds in Tunisian and Egyptian sorghum for human consumption

The objective of this study was to characterize the mycoflora of sorghum grains commercialized in the Tunisian retail market and to identify aflatoxins (AFs), ochratoxin A (OTA) and zearalenone (ZEA) producing species. Sixty four samples of sorghum (37 samples of Tunisian sorghum and 27 samples of Egyptian sorghum) were analyzed. Dilution plating (CFU, colony forming units) was used for fungal enumeration. The isolation of mycobiota was carried out by plating of grains on PDA and malachite green medium. Aspergillus section Flavi and section Nigri and Fusarium isolates were sub-cultured in CYA to test their ability to produce AFs, OTA and ZEA, respectively. The selected Aspergillus section Flavi and section Nigri, Penicillium and Fusarium isolates were subjected to specific PCR assays using published species-specific primers. The results revealed the dominance of Fusarium (95.3%), followed by Aspergillus (87.2%) and Alternaria (81.2%) species. The fungal counts ranged from 100 to 1.3·10⁴ CFU/g for Tunisian sorghum and from 100 to 5.7·10³ CFU/g for Egyptian sorghum. Among Aspergillus section Flavi isolates identified by molecular biology, Aspergillus flavus was the most dominant (90.1%) while Aspergillus parasiticus represent 9.9% only. About Aspergillus section Nigri, results showed the dominance of Aspergillus niger aggregate species, including Aspergillus niger, Aspergillus tubingensis and other species. Among Fusarium species, Fusarium incarnatum was the most dominant in both Tunisian and Egyptian sorghum. Penicillium citrinum was the dominant Penicillium species in the studied samples. More than 890 isolates belonging to the genus Aspergillus and Fusarium were tested in order to test their capacity to produce AFs, OTA and ZEA. The percentage of mycotoxin producing isolates in Aspergillus section Flavi, A. section Nigri, and Fusarium was 30.0%, 4.6% and 11.1%, respectively.     

Identification and quantification of fungi and mycotoxins from Pu-erh tea

Pu-erh tea originates from the province of Yunnan in south-western China. As this tea is produced by so called Aspergillus post-fermentation the question arises which molds and mycotoxins may be found in this tea. In total 36 samples of Pu-erh tea were investigated for their content of filamentous fungi and the mycotoxins aflatoxins B₁, B₂, G₁, and G₂, fumonisins B₁, B₂, and B₃, and ochratoxin A. Fungi were isolated from all samples in a concentration of 1.0 × 10¹ to 2.6 × 10⁶ colony forming units (cfu)/g tea, all together 19 fungal genera and 31 species were identified. The most prevalent species were Aspergillus acidus and Aspergillus fumigatus, followed by Zygomycetes and Penicillium species. Aflatoxins and fumonisins were not found in the samples investigated, ochratoxin A was detected in 4 of 36 teas (11.1%).     

西藏高原粮油作物曲霉菌污染及菌株产毒力研究

为探明西藏高原粮油作物曲霉菌污染状况及黄曲霉菌产毒能力,连续5年对西藏青稞、小麦、花生3种作物中曲霉菌污染情况进行分析,并对其分离到的黄曲霉菌株开展产毒力研究,结果表明,204份样品中,共分离出15种曲霉菌,曲霉菌污染率呈花生>青稞>小麦。青稞、小麦中曲霉属优势种均为黑曲霉(Aspergillus niger),真菌毒素主要为杂色曲霉毒素和赭曲霉毒素;花生优势种为黄曲霉(A.flavus);仅受黄曲霉毒素污染。来源于不同作物的黄曲霉菌,其产毒类型也有差异,麦类作物产毒菌株以产黄曲霉毒素B₁(AFB₁)、黄曲霉毒素B₂(AFB₂)为主;花生产毒菌株以产AFB₁、AFB₂、AFG₁、AFG₂为主。     

Incidence of lactic acid bacteria and Aspergillus flavus in brewer's grains and evaluation of potential antifungal activity of these bacteria

Feed destined for animal production as brewer's grains can be contaminated by Aspergillus section Flavi species. Lactic acid bacteria (LAB) play a defining role in the preservation and microbial safety of fermented foods. The objective of this study was to study the incidence of lactic acid bacteria, Aspergillus section Flavi and AFB₁ in brewer's grains and the preliminary antifungal activity of native LAB in vitro. LAB and aflatoxigenic Aspergillus were found in high counts in brewer's grains used as raw material for pig feedstuff. However, AFB₁ had low AFB₁ natural incidence in samples. In vitro antifungal activity of LAB isolated showed that all bacteria tested inhibited two Aspergillus flavus strains assayed. The high incidence of LAB could be inhibiting the AFB₁ production in by-products obtained from the beer industry. LAB strains with excellent antimicrobial activity were also found in this substrate.     

Antifungal activity of natural and enzymatically-modified flavonoids isolated from citrus species

The antifungal activity of isolated flavonoids from Citrus species, such as naringin, hesperidin and neohesperidin, and enzymatically-modified derivatives of these compounds, was studied on four fungi often found as food contaminants: Aspergillus parasiticus, Aspergillus flavus, Fusarium semitectum and Penicillium expansum. Although all the flavonoids showed antifungal activity, the intensity of this activity depended on the type of fungus and compound used. The hesperetin glucoside laurate strongly inhibited the mycelial growth of P. expansum, while prunin decanoate was the most inhibiting flavonoid for A. flavus, A. parasiticus, and F. semitectum.     

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     

Application of plant essential oils in prevention of fungal growth on Para rubber wood

Antifungal activities of essential oils (cinnamon, clove, eucalyptus, peppermint and lemongrass oils) against moulds (Penicillium sp., Aspergillus niger and Aspergillus versicolor) isolated from rubber wood surfaces were examined. Clove oil possessed consistent antifungal activity with the minimal inhibitory concentration (MIC) of 5 μl ml^?1 against all these fungi, while cinnamon oil had MICs from 2.5 to 10 μl ml^?1. However, only dip treatment with cinnamon oil inhibited the growth of A. niger on rubber wood for at least 25 weeks.     

Stored product pests as models for trapping entomopathogenic fungi from olive tree orchards in Western Greece

We used the following new range of insect species as baits for trapping entomopathogenic fungi: Plodia interpunctella, Rhyzopertha dominica, Tribolium confusum, Trogoderma granarium, Ephestia kuehniella and Tenebrio molitor, which constitute important stored product pests. Soil samples were collected from random points in olive tree orchards in Western Greece using Geographical Information Systems (GIS) and sample point coordinates were located with the Global Positioning System (GPS). Entomopathogenic fungi were identified by sequencing the ITS4 and ITS5 regions of the genomic DNA. Among the isolated fungal species, Beauveria bassiana and Metarhizium anisopliae showed the highest frequency, at 33.8% and 25.4% respectively. They also caused the most infestations of R. dominica and T. confusum whose mortality reached 33–97% and 87–100% respectively. We have also isolated various other fungal taxa such as Aspergillus alliaceus, Aspergillus insuetus, Aspergillus sp., Apophysomyces ossiformis, Chaetomium acropullum, Chaetomium sp., Chaetomium globosum, Chaetomium truncatulum, Trichoderma gamsii and Purpureocillium lilacinum. Our study confirms that T. confusum and R. dominica may be further utilized as baits for entomopathogenic fungi.     

Mycobiota of cocoa: from farm to chocolate

The present work was carried out to study the mycobiota of cocoa beans from farm to chocolate. Four hundred and ninety-four samples were analyzed at various stages of cocoa processing: (i) primary stage at the farm (fermentation, drying, and storage), (ii) secondary stage at processing (testa, nibs, liquor, butter, cake and powder) and (iii) the final chocolate product (dark, milk, white and powdered) collected from retail outlets. Direct plating or dilution plating on Dichloran 18% Glycerol agar were used for cocoa beans and processed product analyses, respectively. Fungi were isolated and identified using different keys of identification. The largest numbers and diversity of fungi were observed in the samples collected at the farm, especially during drying and storage. The species with the highest occurrence among samples were: Absidia corymbifera, Aspergillus sp. nov., A. flavus, Penicillium paneum and yeasts. A total of 1132 potentially toxigenic fungi were isolated from the following species or species groups: A. flavus, Aspergillus parasiticus, Aspergillus nomius, Aspergillus niger group, Aspergillus carbonarius and Aspergillus ochraceus group. The highest percentage of toxigenic fungi was found at the drying and storage stages. The industrial processing reduced the fungal contamination in all fractions and no fungi were found in the final chocolate products. The knowledge of which fungi are dominant at each processing stage of cocoa provides important data about their ecology. This understanding leads to a reduction in fungal spoilage and mycotoxin production in this product.     

Specific detection and quantification of Aspergillus flavus and Aspergillus parasiticus in wheat flour by SYBR® Green quantitative PCR

Aflatoxins are important mycotoxins that represent a serious risk for human and animal health. These mycotoxins are mainly produced by Aspergillus flavus and Aspergillus parasiticus, two closely related species with different array of aflatoxins. In this work, two specific quantitative PCR (qPCR) assays were developed to detect and quantify both species in wheat flour using primers based on the multicopy ITS2 rDNA target sequence. The species specificity of the assays was tested in a wide range of strains of these species and others colonizing the same commodities. The sensitivity of the assay was estimated in 2.5 pg/reaction in both species. Discrimination capacity for detection and relative quantification of A. flavus and A. parasiticus DNA were analyzed using samples with DNA mixtures containing also other fungal species at different ratios. Both qPCR assays could detect spore concentrations equal or higher than 10⁶ spores/g in flour samples without prior incubation. These assays are valuable tools to improve diagnosis at an early stage and in all critical control points of food chain integrated in HACCP strategies.     

Fungi and natural incidence of selected mycotoxins in barley rootlets

The storage of barley rootlets is increasingly employed to provide raw material for pig feeding in Brazil. Barley rootlets represent an important feedstuff for animal production due to their high levels of protein and fiber, and low price. However, poor management of raw materials during storage can result in fungal growth, the loss of nutritive substances and contamination by mycotoxins. The aims of this work were (1) to identify fungi associated with barley rootlets used as pig feedstuff raw material, and (2) to identify and quantify selected mycotoxins naturally produced by isolated mycotoxin-producing species in this substrate over a year. Samples were examined for fungal counts and genera distribution. Fumonisin B₁ and aflatoxin B₁ contamination were determined using high pressure liquid chromatography (HPLC). Barley rootlet samples were of low hygienic quality. Although a broad survey was undertaken, low fungal diversity was found. Fusarium verticillioides was the most prevalent species followed by Aspergillus flavus. Despite Aspergillus clavatus being widely associated with high-moisture sprouted grains including brewers’ grains, and causing toxicity to livestock, it was not detected in this work. Although pre-harvest contamination of the barley crop, as in the maize, could occur, the barley might support F. verticillioides/Fusarium proliferatum growth when grain is remoistened during the germination and malting process and it might even continue during storage on pig farms. All samples were positive for fumonisin B₁ whereas aflatoxin B₁ contamination was not detected. It is important to point out the potential risk of fumonisin contamination in barley rootlets used as animal feed. Fusarium toxins are important not so much for their acute effects as for the chronic syndromes reported worldwide. The obtained results reveal the need for periodic monitoring of raw materials to avoid problems in animal production and hazards to animal and human health.     

Studies on the selection of a suitable packaging material for pre- and post-irradiation storage of cereal grains in Ghana

Twenty different species of fungi belonging to the genera Aspergillus, Cephalosporium, Dreschlera, Fusarium, Mucor, Neurospora, Rhizoctonia, Rhizopus, Penicillium and Trichoderma were isolated from jute and woven polypropylene sacks using the blotter test, solid medium test and the decimal serial dilution technique. There was a highly significant difference (Students' t-test, P < 0.05) between the larger number of fungal colonies associated with jute sacks than with woven polypropylene sacks. Correspondingly, more fungal species (16) were isolated from jute sacks than from woven polypropylene sacks (9). The blotter test showed that in the absence of an exogenous supply of nutrients, 88% of the sections of jute sacks supported in vivo growth of fungal spores whilst woven polypropylene sacks could not support the growth of contaminating spores. Evidence is presented that mould and yeast counts on sections of new-woven polypropylene sacks incubated at 80 and 90% relative humidity (R.H.) for four months increased by less than 1 log cycle, whilst in similar sections hung on a line under ambient conditions (75 ± 10% R.H. and 28 ± 3°C) viable counts of spores decreased. Sections of fresh jute sacks similarly treated supported 1 log cycle increase in mould and yeast counts at 80% R.H. and 2 log cycles increase at 90% R.H. after four months of storage. Gamma-ray irradiation (4.0 kGy) reduced the mould and yeast counts on new jute and new woven polypropylene sacks by 1 and 2 log cycles, respectively, but post-irradiation storage at 80% R.H. allowed moulds like Aspergillus flavus, A. niger, Fusarium nivale and Penicillium verrucosum var. cyclopium to commence growth on jute sacks. This presumably, may act as a springboard for infecting grain contents of jute sacks. Inert woven polypropylene sacks failed to support fungal growth.     

Biodiversity of Aspergillus section Nigri populations in Argentinian vineyards and ochratoxin A contamination

Aspergillus section Nigri are described as the main source of ochratoxin A (OTA) contamination in grapes and wine worldwide. The knowledge of the factors affecting grape contamination by species included in this section and OTA production is essential to be able to reduce their presence, not only to improve wine quality, but also to maintain their safety. Therefore, the aims of this study were to determine the incidence of Aspergillus section Nigri species harvested in different grape-growing regions from Argentina, their ability to produce OTA, to correlate with meteorological conditions and geographical coordinates with their prevalence and to evaluate the OTA natural occurrence in grapes and wines. The morphological identification showed that Aspergillus niger aggregate species were the most prevalent ones, followed by Aspergillus carbonarius and Aspergillus uniseriate. These populations were confirmed through using AFLP markers and sequencing and, Aspergillus tubingensis was separated from A. niger aggregate. Climatic factors, altitude, longitude and latitude have influenced on the distribution of species included in the section. A. carbonarius and A. niger were OTA producers but differed in their OTA producing ability. Temperature was the factor which influenced the most over the highest incidence of A. carbonarius in La Rioja and San Juan regions. The trellis system in vineyards and drip irrigation also influenced the species isolation. The OTA levels detected in grapes and wines were low, but grape variety was more important in susceptibility to fungal infection and OTA levels.