Taxonomy of Aspergillus section Flavi and their production of aflatoxins, ochratoxins and other mycotoxins.

Aflatoxins and ochratoxins are among the most important mycotoxins of all and producers of both types of mycotoxins are present in Aspergillus section Flavi, albeit never in the same species. Some of the most efficient producers of aflatoxins and ochratoxins have not been described yet. Using a polyphasic approach combining phenotype, physiology, sequence and extrolite data, we describe here eight new species in section Flavi. Phylogenetically, section Flavi is split in eight clades and the section currently contains 33 species. Two species only produce aflatoxin B1 and B2 (A. pseudotamarii and A. togoensis), and 14 species are able to produce aflatoxin B1, B2, G1 and G2: three newly described species A. aflatoxiformans, A. austwickii and A. cerealis in addition to A. arachidicola, A. minisclerotigenes, A. mottae, A. luteovirescens (formerly A. bombycis), A. nomius, A. novoparasiticus, A. parasiticus, A. pseudocaelatus, A. pseudonomius, A. sergii and A. transmontanensis. It is generally accepted that A. flavus is unable to produce type G aflatoxins, but here we report on Korean strains that also produce aflatoxin G1 and G2. One strain of A. bertholletius can produce the immediate aflatoxin precursor 3-O-methylsterigmatocystin, and one strain of Aspergillus sojae and two strains of Aspergillus alliaceus produced versicolorins. Strains of the domesticated forms of A. flavus and A. parasiticus, A. oryzae and A. sojae, respectively, lost their ability to produce aflatoxins, and from the remaining phylogenetically closely related species (belonging to the A. flavus-, A. tamarii-, A. bertholletius- and A. nomius-clades), only A. caelatus, A. subflavus and A. tamarii are unable to produce aflatoxins. With exception of A. togoensis in the A. coremiiformis-clade, all species in the phylogenetically more distant clades (A. alliaceus-, A. coremiiformis-, A. leporis- and A. avenaceus-clade) are unable to produce aflatoxins. Three out of the four species in the A. alliaceus-clade can produce the mycotoxin ochratoxin A: A. alliaceus s. str. and two new species described here as A. neoalliaceus and A. vandermerwei. Eight species produced the mycotoxin tenuazonic acid: A. bertholletius, A. caelatus, A. luteovirescens, A. nomius, A. pseudocaelatus, A. pseudonomius, A. pseudotamarii and A. tamarii while the related mycotoxin cyclopiazonic acid was produced by 13 species: A. aflatoxiformans, A. austwickii, A. bertholletius, A. cerealis, A. flavus, A. minisclerotigenes, A. mottae, A. oryzae, A. pipericola, A. pseudocaelatus, A. pseudotamarii, A. sergii and A. tamarii. Furthermore, A. hancockii produced speradine A, a compound related to cyclopiazonic acid. Selected A. aflatoxiformans, A. austwickii, A. cerealis, A. flavus, A. minisclerotigenes, A. pipericola and A. sergii strains produced small sclerotia containing the mycotoxin aflatrem. Kojic acid has been found in all species in section Flavi, except A. avenaceus and A. coremiiformis. Only six species in the section did not produce any known mycotoxins: A. aspearensis, A. coremiiformis, A. lanosus, A. leporis, A. sojae and A. subflavus. An overview of other small molecule extrolites produced in Aspergillus section Flavi is given.

Fumonisin B2 production by Aspergillus niger in Thai coffee beans.

During 2006 and 2007, a total of 64 Thai dried coffee bean samples (Coffea arabica) from two growing sites in Chiangmai Province and 32 Thai dried coffee bean samples (Coffea canephora) from two growing sites in Chumporn Province, Thailand, were collected and assessed for fumonisin contamination by black Aspergilli. No Fusarium species known to produce fumonisin were detected, but black Aspergilli had high incidences on both Arabica and Robusta Thai coffee beans. Liquid chromatography (LC) with high-resolution mass spectrometric (HRMS) detection showed that 67% of Aspergillus niger isolates from coffee beans were capable of producing fumonisins B(2) (FB(2)) and B(4) when grown on Czapek Yeast Agar with 5% NaCl. Small amounts (1-9.7 ng g(-1)) of FB(2) were detected in seven of 12 selected coffee samples after ion-exchange purification and LC-MS/MS detection. Two samples also contained FB(4). This is the first record of freshly isolated A. niger strains producing fumonisins and the first report on the natural occurrence of FB(2) and FB(4) in coffee.

Biodiversity of Aspergillus species in some important agricultural products.

The genus Aspergillus is one of the most important filamentous fungal genera. Aspergillus species are used in the fermentation industry, but they are also responsible of various plant and food secondary rot, with the consequence of possible accumulation of mycotoxins. The aflatoxin producing A. flavus and A. parasiticus, and ochratoxinogenic A. niger, A. ochraceus and A. carbonarius species are frequently encountered in agricultural products. Studies on the biodiversity of toxigenic Aspergillus species is useful to clarify molecular, ecological and biochemical characteristics of the different species in relation to their different adaptation to environmental and geographical conditions, and to their potential toxigenicity. Here we analyzed the biodiversity of ochratoxin producing species occurring on two important crops: grapes and coffee, and the genetic diversity of A. flavus populations occurring in agricultural fields. Altogether nine different black Aspergillus species can be found on grapes which are often difficult to identify with classical methods. The polyphasic approach used in our studies led to the identification of three new species occurring on grapes: A. brasiliensis, A. ibericus, and A. uvarum. Similar studies on the Aspergillus species occurring on coffee beans have evidenced in the last five years that A. carbonarius is an important source of ochratoxin A in coffee. Four new species within the black aspergilli were also identified in coffee beans: A. sclerotioniger, A. lacticoffeatus, A. sclerotiicarbonarius, and A. aculeatinus. The genetic diversity within A. flavus populations has been widely studied in relation to their potential aflatoxigenicity and morphological variants L- and S-strains. Within A. flavus and other Aspergillus species capable of aflatoxin production, considerable diversity is found. We summarise the main recent achievements in the diversity of the aflatoxin gene cluster in A. flavus populations, A. parasiticus and the non-toxigenic A. oryzae. Studies are needed in order to characterise the aflatoxin biosynthetic genes in the new related taxa A. minisclerotigenes and A. arachidicola.

Antimicrobial activity of ultrasound-assisted solvent-extracted spices.

AIMS: The objective of this research was to determine the antimicrobial activity of conventional and high-intensity ultrasound-assisted (HI-US) solvent-extracted Thai spices, including ginger (Zingiber officinale Rose), fingerroot (Bosenbergia pandurata Holtt) and turmeric (Curouma longa Linn). METHODS AND RESULTS: Extracts were obtained using hexane, isopropanol and a 7 : 3 isopropanol : hexane mixture as solvents with and without HI-US. The antimicrobial activity of the extracts was assayed against four strains each of Listeria monocytogenes and Salmonella Typhimurium DT 104 using an agar dilution assay. Application of HI-US did not alter antibacterial activity against S. Typhimurium, but antilisterial activity of some HI-US spice extracts decreased. Solvent type affected antimicrobial efficacy of extracts with hexane producing the least antimicrobial activity. Fingerroot extracted with isopropanol-hexane and without HI-US had the best antilisterial effect while HI-US-isopropanol fingerroot extract had the greatest antimicrobial efficacy against S. Typhimurium. CONCLUSIONS: Application of HI-US reduced time of extraction to 5 min, compared with the 24 h required for conventional extraction and maintained antimicrobial activity against Salmonella but slightly reduced activity against Listeria. SIGNIFICANCE AND IMPACT OF THE STUDY: HI-US in combination with proper solvent selection may offer a new tool to optimize extraction of spice essential oil for use as antimicrobial agents, and reduce processing time and costs.

Influence of temperature shifts on survival, growth, and toxin production by psychrotrophic and mesophilic strains of Bacillus cereus in potatoes and chicken gravy.

A study was done to determine the influence of temperature on growth and toxin production characteristics of psychrotrophic and mesophilic strains of Bacillus cereus when inoculated into mashed potatoes and chicken gravy containing various concentrations of sodium chloride and held at temperatures different from those at which cells had been cultured. Logarithmic growth phase cells (10 h, 30 degrees C) of psychrotrophic (F3802A/84) and mesophilic (B4ac-1) strains of Bacillus cereus were inoculated into rehydrated commercially processed instant mashed potatoes and chicken gravy supplemented with 0, 2, or 4% sodium chloride. Growth, survival, and diarrheal toxin production in potatoes and gravy held at 30, 37, and 10 degrees C (strain F3802A/84) or 30, 40, and 10 degrees C (strain B4ac-1) were monitored. Both strains grew in both foods containing no added sodium chloride or 2% sodium chloride when held at 30, 37, or 40 degrees C for 2 days. Strain B4ac-1 grew better than strain F3802A/84 in foods containing 4% sodium chloride. Maximum amounts of enterotoxin (1024 ng/g) were produced by strain B4ac-1 in chicken gravy held at 30 and 40 degrees C. Strain F3802A/84 grew to populations of 7 log10 CFU/g in foods containing no added sodium chloride or 2% sodium chloride at 10 degrees C. Strain F3802A/84 produced the highest amount of enterotoxin (1024 ng/g) at 30 degrees C in chicken gravy containing 0.7 or 2% sodium chloride; however, little or low amounts of toxin (4-16 ng/g) were produced in chicken gravy at 10 degrees C. Compared to strain B4ac-1, cells of strain F3802A/84 subjected to a downward shift in incubation temperature (10 degrees C) grew more rapidly in chicken gravy. Strain B4ac-1 produced the highest amount of toxin (1024 ng/g) at 30 degrees C in gravy containing 4% sodium chloride and at 40 degrees C in gravy containing 0.7% sodium chloride. Toxin was not detected in inoculated mashed potatoes. Results of this study indicate that shifts in incubation temperature influence growth and toxin production by psychrotrophic and mesophilic strains of B. cereux differently. It is important to store pasteurized, ready-to-eat foods at a temperature low enough to prevent the growth of B. cereus.

Effect of shift in growth temperature on tolerance of psychrotrophic and mesophilic strains of Bacillus cereus to heat and sodium chloride.

A shift in growth temperature of a psychrotrophic (F3802A/84) strain and a mesophilic strain (B4ac-1) of Bacillus cereus grown at 30 degrees C for 10 h, then at 37 degrees C or 40 degrees C for 14 h, enhanced thermotolerance. Sodium chloride, at concentrations of 2.0 and 4.0% in brain heart infusion (BHI) broth, had no effect on thermotolerance of strain B4ac-1 heated at 50 degrees C, whereas the same concentrations of NaCl caused a decrease in thermotolerance of strain F3802A/84 heated at 48 degrees C. A downshift in growth temperature from 30 degrees C to 10 degrees C followed by incubation for 3 to 9 days increased thermotolerance of strain F3802A/84 but not strain B4ac-1 heated in BHI broth containing 2.0 or 4.0% NaCl compared to thermotolerance in BHI broth containing 0.5% NaCl. Protein analysis using one-dimensional gel electrophoresis revealed an increase in proteins with molecular weights of 54, 50, 44, and 42 kDa in cells of strain F3802A/84 and 83 and 69 kDa in cells of strain B4ac-1 subjected to an upshift in growth temperature from 30 degrees C to 37 degrees C or 40 degrees C, respectively. A downshift in growth temperature from 30 degrees C to 10 degrees C resulted in substantial amounts of proteins with molecular weights of 63, 40, and 29 kDa produced by strain F3802A/84 and 63 kDa to be produced by strain B4ac-1. Proteins produced in response to upshift or downshift in growth temperature are suspected to play an important role in heat resistance of the psychrotrophic and mesophilic strains of B. cereus examined in this study. Changes in resistance to heat or refrigeration temperatures, as well as tolerance to NaCl, as affected by previous exposure of cells to temperature shifts may influence the ability of B. cereus to grow in minimally processed foods during distribution and storage.