Agrobacterium-mediated insertional mutagenesis of the ochratoxigenic fungus Aspergillus westerdijkiae

2007 ◽  
Vol 53 (1) ◽  
pp. 148-151 ◽  
Author(s):  
Marcia M Mata ◽  
Marta H Taniwaki ◽  
Beatriz T Iamanaka ◽  
Daniele Sartori ◽  
André L.M Oliveira ◽  
...  
Keyword(s):  
Ochratoxin A ◽  
Insertional Mutagenesis ◽  
Aspergillus Ochraceus ◽  
Agrobacterium Mediated Transformation ◽  
Morphological Variations ◽  
Carcinogenic Potential ◽  
Coffee Beans ◽  
Aspergillus Westerdijkiae ◽  
Polymerase Chain ◽  
First Time

Aspergillus westerdijkiae is a potent ochratoxin A (OTA) producer that has been found in coffee beans. OTA is known to have nephrotoxic effects and carcinogenic potential in animal species. Here we report for the first time the Agrobacterium-mediated transformation for Aspergillus westerdijkiae and the generation of ochratoxin-defective mutants. Conidia were transformed to hygromycin B resistance using strain AGL-1 of Agrobacterium tumefaciens. The obtained transformation frequency was up to 47 transformants per 106 target conidia. Among 600 transformants, approximately 5% showed morphological variations. Eight transformants with consistently reduced OTA production were obtained. Two of these transformants did not produce OTA (detection limit: 0.1 µg/kg); the other six mutants produced lower amounts of OTA (1%–32%) compared with the wild-type strain. By using thermal asymmetric interlaced polymerase chain reaction, we successfully identified a putative flavin adenine dinucleotide monooxygenase gene.Key words: Aspergillus ochraceus, Aspergillus westerdijkiae, Agrobacterium-mediated transformation, Agrobacterium-mediated insertional mutagenesis, ochratoxin A.

Genetic relationships among Brazilian strains of Aspergillus ochraceus based on RAPD and ITS sequences

2004 ◽  
Vol 50 (11) ◽  
pp. 985-988 ◽  
Author(s):  
Maria Helena Pelegrinelli Fungaro ◽  
Marciane Magnani ◽  
Laurival Antônio Vilas-Boas ◽  
Patrícia Cristina Vissotto ◽  
Márcia Cristina Furlaneto ◽  
...  
Keyword(s):  
Ochratoxin A ◽  
Sequence Data ◽  
Genetic Relationships ◽  
Its Region ◽  
Aspergillus Ochraceus ◽  
Coffee Bean ◽  
Coffee Beans ◽  
Major Species ◽  
Group A ◽  
Group B

Ochratoxin A (OA) is a mycotoxin that has been found in coffee beans and coffee beverages. Its toxicological profile includes carcinogenicity, nephrotoxicity, and immunotoxicity. Aspergillus ochraceus is the major species responsible for OA production in Brazilian coffee beans. The genetic relationships among 25 A. ochraceus strains collected from Brazilian coffee-bean samples were determined based on RAPD and internal transcribed spacer (ITS) sequence data. The isolates were resolved into 2 distinct groups, one with 4 strains (group A) and the other with 21 strains (group B). Specific nucleotide variations characterizing group A and B were found for both ITS1 and ITS2 regions. Group B is a new group proposed here to accommodate the majority of the Brazilian isolates. Each group was found to contain both toxigenic and nontoxigenic strains, indicating that there is no association between molecular genotypes and the ability to produce OA.Key words: Aspergillus ochraceus, ochratoxin A, ITS region (ITS1–5.8S–ITS2), RAPD.

Ochratoxins A and B, Xanthomegnin, Viomellein and Vioxanthin Production by Isolates of Aspergillus ochraceus from Green Coffee Beans

1983 ◽  
Vol 46 (11) ◽  
pp. 965-968 ◽  
Author(s):  
MICHAEL E. STACK ◽  
PHILIP B. MISLIVEC ◽  
TURGUT DENIZEL ◽  
REGINA GIBSON ◽  
ALBERT E. POHLAND
Keyword(s):  
High Performance Liquid Chromatography ◽  
Liquid Chromatography ◽  
Ochratoxin A ◽  
High Performance ◽  
Toxin Production ◽  
Aspergillus Ochraceus ◽  
Average Level ◽  
Green Coffee ◽  
Green Coffee Beans ◽  
Coffee Beans

Isolates from Aspergillus ochraceus obtained from green coffee beans were cultured on rice and water. After 20 d of growth the cultures were extracted with chloroform and the extracts were analyzed by high performance liquid chromatography for ochratoxin A (OA), ochratoxin B (OB), xanthomegnin (X), viomellein (V) and vioxanthin (VX). Forty-three percent of the isolates produced OA at an average level of 397 μg of toxin/g rice, 17% produced OB at an average level of 312 μg/g, and 84% produced X, V, and VX at an average level of 281, 417 and 386 μg/g, respectively. The highest levels of toxin production were OA, 2088 μg/g; OB, 3375 μg/g; X, 1562 μg/g; V, 2514 μg/g; and VX, 2054 μg/g. VX has not previously been reported as an A. ochraceus metabolite.

scholarly journals Impact of interacting climate change factors on growth and ochratoxin A production by Aspergillus section Circumdati and Nigri species on coffee

2016 ◽  
Vol 9 (5) ◽  
pp. 863-874 ◽  
Author(s):  
A. Akbar ◽  
A. Medina ◽  
N. Magan
Keyword(s):  
Climate Change ◽  
Ochratoxin A ◽  
Toxin Production ◽  
Aspergillus Ochraceus ◽  
Change Factors ◽  
Significant Stimulation ◽  
Coffee Beans ◽  
Higher Temperature ◽  
Stimulation Of ◽  
Lag Phases

The objectives of this study were to evaluate the effect of interacting climate change (CC) factors (water stress [water activity, aw; 0.99-0.90]); temperature [30, 35 °C]; and elevated CO2 [400 and 1000 ppm] on (1) lag phases prior to growth, (2) growth and (3) ochratoxin A (OTA) production by species of Aspergillus sections Circumdati and Nigri on coffee-based media and stored coffee beans. The lag phases, prior to growth, of all strains/species were slightly increased as aw, temperature and CO2 were modified. The interacting CC factors showed that most strains/species examined grew well at 30 °C and slightly less so at 35 °C except for Aspergillus niger (A 1911) which could tolerate the higher temperature. In addition, the interaction of elevated CO2 (1000 ppm) + temperature (35 °C) increased OTA production when compared with 30 °C but only for strains of Aspergillus westerdijkiae (B2), Aspergillus ochraceus (ITAL 14) and Aspergillus steynii (CBS 112814). Most of the strains had optimum growth at 0.95 aw at 35 °C, while at 30 °C the optimum was at 0.98 aw. On stored coffee beans there was only a significant stimulation of OTA production by A. westerdijkiae strains in elevated CO2 (1000) at 0.90 aw. These results suggest differential effects of CC factors on OTA production by species in the Sections Circumdati and Nigri in stored coffee and that for most species there is a reduction in toxin production.

Effect of roasting on ochratoxin A level in green coffee beans inoculated with Aspergillus ochraceus

1987 ◽  
Vol 97 (2) ◽  
pp. 111-115 ◽  
Author(s):  
Haruo Tsubouchi ◽  
Katsuhiko Yamamoto ◽  
Kazuo Hisada ◽  
Yoshio Sakabe ◽  
Shun- ichi Udagawa
Keyword(s):  
Ochratoxin A ◽  
Aspergillus Ochraceus ◽  
Green Coffee ◽  
Green Coffee Beans ◽  
Coffee Beans

scholarly journals Degrading Ochratoxin A and Zearalenone Mycotoxins Using a Multifunctional Recombinant Enzyme

Toxins ◽  
2019 ◽  
Vol 11 (5) ◽  
pp. 301 ◽  
Author(s):  
Md Shofiul Azam ◽  
Dianzhen Yu ◽  
Na Liu ◽  
Aibo Wu
Keyword(s):  
Ochratoxin A ◽  
Previous Investigation ◽  
Cell Cytotoxicity ◽  
Chain Reaction ◽  
Fusarium Mycotoxin ◽  
Fusion Enzyme ◽  
Toxic Product ◽  
Optimum Ph ◽  
Polymerase Chain ◽  
First Time

Zearalenone (ZEA) is an estrogenic and ochratoxin A (OTA) is a hepatotoxic Fusarium mycotoxin commonly seen in cereals and fruits products. No previous investigation has studied on a single platform for the multi degradation mycotoxin. The current study aimed to investigate the bifunctional activity of a novel fusion recombinant. We have generated a recombinant fusion enzyme (ZHDCP) by combining two single genes named zearalenone hydrolase (ZHD) and carboxypeptidase (CP) in frame deletion by crossover polymerase chain reaction (PCR). We identified enzymatic properties and cell cytotoxicity assay of ZHDCP enzyme. Our findings have demonstrated that ZEA was completely degraded to the non-toxic product in 2 h by ZHDCP enzyme at an optimum pH of 7 and a temperature of 35 °C. For the first time, it was found out that ZEA 60% was degraded by CP degrades in 48 h. Fusion ZHDCP and CP enzyme were able to degrade 100% OTA in 30 min at pH 7 and temperature 30 °C. ZEA- and OTA-induced cell death and increased cell apoptosis rate and regulated mRNA expression of Sirt1, Bax, Bcl2, Caspase3, TNFα, and IL6 genes. Our novel findings demonstrated that the fusion enzyme ZHDCP possess bifunctional activity (degrade OTA and ZEA), and it could be used to degrade more mycotoxins.

scholarly journals Effect of a Debaryomyces hansenii and Lactobacillus buchneri Starter Culture on Aspergillus westerdijkiae Ochratoxin A Production and Growth during the Manufacture of Short Seasoned Dry-Cured Ham

2020 ◽  
Vol 8 (10) ◽  
pp. 1623
Author(s):  
Lucilla Iacumin ◽  
Martina Arnoldi ◽  
Giuseppe Comi
Keyword(s):  
Ochratoxin A ◽  
Starter Culture ◽  
Debaryomyces Hansenii ◽  
Aspergillus Ochraceus ◽  
In Situ Tests ◽  
Potential Health ◽  
Lactobacillus Buchneri ◽  
Dry Cured Ham ◽  
Aspergillus Westerdijkiae

Recently, specific dry-cured hams have started to be produced in San Daniele and Parma areas. The ingredients are similar to protected denomination of origin (PDO) produced in San Daniele or Parma areas, and include pork leg, coming from pigs bred in the Italian peninsula, salt and spices. However, these specific new products cannot be marked as a PDO, either San Daniele or Parma dry cured ham, because they are seasoned for 6 months, and the mark PDO is given only to products seasoned over 13 months. Consequently, these products are called short-seasoned dry-cured ham (SSDCH) and are not branded PDO. During their seasoning period, particularly from the first drying until the end of the seasoning period, many molds, including Eurotium spp. and Penicillium spp., can grow on the surface and work together with other molds and tissue enzymes to produce a unique aroma. Both of these strains typically predominate over other molds. However, molds producing ochratoxins, such as Aspergillus ochraceus and Penicillium nordicum, can simultaneously grow and produce ochratoxin A (OTA). Consequently, these dry-cured hams may represent a potential health risk for consumers. Recently, Aspergillus westerdijkiae has been isolated from SSDCHs, which could represent a potential problem for consumers. Therefore, the aim of this study was to inhibit A. westerdijkiae using Debaryomyces hansenii or Lactobacillus buchneri or a mix of both microorganisms. Six D. hansenii and six L. buchneri strains were tested in vitro for their ability to inhibit A. westerdijkiae. The strains D. hansenii (DIAL)1 and L. buchneri (Lb)4 demonstrated the highest inhibitory activity and were selected for in situ tests. The strains were inoculated or co-inoculated on fresh pork legs for SSDCH production with OTA-producing A. westerdijkiae prior to the first drying and seasoning. At the end of seasoning (six months), OTA was not detected in the SSDCH treated with both microorganisms and their combination. Because both strains did not adversely affect the SSDCH odor or flavor, the combination of these strains are proposed for use as starters to inhibit OTA-producing A. westerdijkiae.

scholarly journals Influence of physiological factors on growth, sporulation and ochratoxin A/B production of the new Aspergillus ochraceus grouping

2009 ◽  
Vol 2 (4) ◽  
pp. 429-434 ◽  
Author(s):  
A. Abdel-Hadi ◽  
N. Magan
Keyword(s):  
Ochratoxin A ◽  
Maximum Growth ◽  
Spore Production ◽  
Aspergillus Ochraceus ◽  
Optimum Temperature ◽  
Physiological Factors ◽  
Large Numbers ◽  
Asexual Spore ◽  
The Impact ◽  
First Time

Recently, new species within the Aspergillus section Circumdati group responsible for ochratoxin production were reported. This study has examined the impact of interactions between water activity (aw, 0.99-0.90), temperature (20-35 °C) on growth, asexual spore production and ochratoxin A and B (OTA and OTB) on strains of each of the three species from this new grouping (A. ochraceus, A. steynii, and A. westerdijkiae) for the first time. The maximum growth occurred at 0.95 aw and 30 °C for both A. ochraceus and A. westerdijkiae, while it was at 0.99 aw and 30 °C for A. steynii. No conidial spore production occurred at 0.99 aw in cultures of A. ochraceus and A. steynii but large numbers of spores (2.3×107/cm2) were produced by A. westerdijkiae. Optimum temperature for spore production was 0.95 aw and 30 °C for A. westerdijkiae and A. ochraceus, and 0.95 aw and 35 °C for A. steynii. Quantification of OTA showed that optimum was produced at 0.99 aw, by A. steynii at 30 °C, for A. westerdijkiae at 25 °C and for A. ochraceus at 20 °C. As water stress was imposed (0.95 aw), the temperature for maximum OTA production changed. For example, for A. steynii and A. westerdijkiae this was at 35 °C, for A. ochraceus, 25 °C. Much less OTB was produced relative to OTA, but the production followed the same pattern at all aw levels and temperatures. This is the first detailed study to examine the similarities and differences in ecology of these related species in this important mycotoxigenic group.

scholarly journals Resilience of Aspergillus westerdijkiae Strains to Interacting Climate-Related Abiotic Factors: Effects on Growth and Ochratoxin A Production on Coffee-Based Medium and in Stored Coffee

2020 ◽  
Vol 8 (9) ◽  
pp. 1268 ◽  
Author(s):  
Asya Akbar ◽  
Angel Medina ◽  
Naresh Magan
Keyword(s):  
Water Stress ◽  
Ochratoxin A ◽  
Elevated Co2 ◽  
Abiotic Factors ◽  
Green Coffee ◽  
Roasted Coffee ◽  
Green Coffee Beans ◽  
Coffee Beans ◽  
Aspergillus Westerdijkiae ◽  
Stimulation Of

We examined the resilience of strains of Aspergillus westerdijkiae in terms of growth and ochratoxin A (OTA) production in relation to: (a) two-way interacting climate-related abiotic factors of water activity (aw, 0.99–0.90) × temperature (25–37 °C) on green coffee and roasted coffee-based media; (b) three-way climate-related abiotic factors (temperature, 30 vs. 35 °C; water stress, 0.98–0.90 aw; CO2, 400 vs. 1000 ppm) on growth and OTA production on a 6% green coffee extract-based matrix; and (c) the effect of three-way climate-related abiotic factors on OTA production in stored green coffee beans. Four strains of A. westerdijkiae grew equally well on green or roasted coffee-based media with optimum 0.98 aw and 25–30 °C. Growth was significantly slower on roasted than green coffee-based media at 35 °C, regardless of aw level. Interestingly, on green coffee-based media OTA production was optimum at 0.98–0.95 aw and 30 °C. However, on roasted coffee-based media very little OTA was produced. Three-way climate-related abiotic factors were examined on two of these strains. These interacting factors significantly reduced growth of the A. westerdijkiae strains, especially at 35 °C × 1000 ppm CO2 and all aw levels when compared to 30 °C. At 35 °C × 1000 ppm CO2 there was some stimulation of OTA production by the two A. westerdijkiae strains, especially under water stress. In stored green coffee beans optimum OTA was produced at 0.95–0.97 aw/30 °C. In elevated CO2 and 35 °C, OTA production was stimulated at 0.95–0.90 aw.

Study of the Occurrence of Ochratoxin A in Green Coffee Beans

1974 ◽  
Vol 57 (4) ◽  
pp. 866-870 ◽  
Author(s):  
Colette P Levi ◽  
Hugh L Trenk ◽  
Herman K Mohr
Keyword(s):  
Heat Treatment ◽  
Ochratoxin A ◽  
Room Temperature ◽  
Aspergillus Ochraceus ◽  
Green Coffee ◽  
Optimal Conditions ◽  
Green Coffee Beans ◽  
Coffee Beans ◽  
Almost All

Abstract Moldy green coffee beans were found to contain Aspergillus ochraceus and detectable levels of ochratoxin A. The official first action method for the detection of ochratoxins in barley, 26.C15–26.C22, was modified for analysis of coffee beans and a number of survey samples of coffee beans were analyzed. A. ochraceus was found to be present in almost all samples but ochratoxin A was infrequently observed. Ochratoxin A production in sterile green coffee beans inoculated with A. ochraceus under optimal conditions was maximal at 13 days at room temperature but the total amount was low (450 μg/kg). Considerable destruction (approximately 80%) of ochratoxin A occurred during a heat treatment which simulated the roasting of coffee beans.

Effect of Water Activity and Temperature on Mycelial Growth and Ochratoxin A Production by Isolates of Aspergillus ochraceus on Irradiated Green Coffee Beans

2005 ◽  
Vol 68 (1) ◽  
pp. 133-138 ◽  
Author(s):  
E. PARDO ◽  
S. MARÍN ◽  
A. J. RAMOS ◽  
V. SANCHIS
Keyword(s):  
Ochratoxin A ◽  
Water Activity ◽  
Mycelial Growth ◽  
Marked Decrease ◽  
Limit Of Detection ◽  
Aspergillus Ochraceus ◽  
Primary Data ◽  
Green Coffee ◽  
Green Coffee Beans ◽  
Coffee Beans

Aspergillus ochraceus as a fungal contaminant and ochratoxin A (OTA) producer plays an important role in coffee quality. Temperature and water activity (aw) significantly influence mycelial growth and OTA production by isolates of A. ochraceus on green coffee beans. Maximum mycelial growth was found at 30°C and 0.95 to 0.99 aw. A marked decrease in growth rate was observed when temperature and aw were reduced. At 0.80 aw, mycelial growth occurred only at 30 and 20°C for one isolate. Maximum OTA production was found at 20°C and 0.99 aw. At 10°C, OTA was not produced, regardless of aw. Similarly, no OTA was detected at 0.80 aw. OTA production ranged from the limit of detection (40 ng g−1 of green coffee) to 17,000 ng g−1 of green coffee. Significant intraspecific differences in mycelial growth and OTA production were found. Primary data for lag phases prior to mycelial growth under the influence of temperature and aw were modelled by multiple linear regression, and the response surface plots were obtained.

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