Contamination And Characteristics Of Ochratoxigenic-Fungi In Grapes

Ochratoxin A (OTA) is a widespread mycotoxin. Grape and its products are oneof the main sources of food for OTA. Investigation on the distribution ofOTA-producing fungi in grapes can effectively control the root causes of growth andOTA yield by OTA-producing fungi and reducing the OTA content in grapes. Themain contents and results were listed as the followings.(1) Contamination of mold and OTA-producing fungi on the surface of severalmajor grape cultivars in Shaanxi province were investigated. The results showed thatthe fungal contamination and species were different among grape cultivars. The moldcontamination was higher in rotten berries than in intact ones. Thompson seedlessshowed the highest fungal contamination in the tested grape cultivars. Isolates wereobtained and identified as seven genera including Aspergillus, Penicillium, Mucor,Rhizopus, Alternaria, Cladosporium, and Fusarium.(2) Ultraviolet (UV) fluorescence method and colloidal gold immunoassay stripmethod were used to screen out OTA-producer from189strains isolated from grape.According to the high-performance liquid chromatography (HPLC) results, the UVfluorescence method yielded false-positive rate of85.71%, and false-negative rate of0%. The colloidal gold test strip method yields false-negative rate of47.4%andfalse-positive rate of0%. Finally,19strains showed OTA-produing ability.(3)According to the morphological and molecular biological methods,13of the19OTA-producing fungi belonged to Aspergillus sp..5belonged to Penicilliumsp.and one belong to Cladosporium sp..The final identification results were asfollows:090614,091314,090908and120209were indentified as Aspergilluscarbonariu;091718,090612,091327and091721were indentified as Aspergillus niger;120208was indentified as Aspergillus japonicus var.aculeatus;091902wasindentified as Aspergillus ochraceus;091506was indentified as Aspergillus melleus;091319was indentified as Aspergillus parasiticus;091709was indentified asAspergillus wentii;101119was indentified as Penicillium verruculosum;091325wasindentified as Penicillium polonicum;092506was indentified as Penicillium variabile;101104and101108were indentified as Penicillium chrysogenum;091306was indentified as Cladosporium cladosporioides. Among the19strains, Aspergilluscarbonarius090614produced the highest amount of OTA on CYA medium, whichwas13179.24ng/g.(4)Study on the growth and toxin production conditions of several OTA high-yieldstrains showed that the optimum temperatures for cell growth and OTA production byA. carbonarius090614were30℃and20℃, respectively, and the optimum pH forcell growth and OTA production were6.0and5.0respectively. As for A. ochraceus091902, the optimum temperatures for cell growth and OTA production were30℃and25℃, respectively, and the optimum pH for cell growth and OTA production waswere6.0and7.0respectively. For P. verruculosum101119, the optimumtemperatures for cell growth and OTA production were37℃and25℃, respectively,and the optimum pH for cell growth and OTA production was4.0. For C.cladosporioidas091306, the optimum temperatures for cell growth and OTAproduction was25℃, and the optimum pH for cell growth and OTA production was9.0. The optimum relative humidity for cell growth and OTA production was86%forall of the four strains.(5)Berry rot and OTA production were not observed for the undamagedinoculated grape berries by A.carbonarius090614at all temperatures. All damagedinoculated grape berries showed rotten damage and OTA production at the cultivatedtemperatures except at4℃. A higher proportion of rotten damage and OTAproduction were observed at30°C than at25°C. Comparatively, the extent of rottendamage and OTA accumulation were the most rapid in Thompson seedless, theslowest in Red Earth, and the intermediate in Kyoho grapes. Chemical propertieschange in the process of grape rot were tracked and analyzed. The results showed thatthe trends of reducing sugar content and soluble solids content correlated with asimilar first decrease followed by an increase. The total acid content increased, whilethe pH decreases gradually. A high initial value of reducing sugar content, solublesolids content, and low hardness of berry skin in grape berries had a significant linearrelationship (R2>0.98) with fast rotting and high OTA production at the end of theincubation.(6) Bacteria subtillis was used to inhibit the growth of OTA-producing fungi ongrapes. Results showed that liquid culture of strain B.subtillis CCTCC M207209withcells and the cell-free liquid culture could significantly inhibit the growth ofA.carbonarius090614in medium and grapes. The cell-free liquid culture had the highest inhibitng ability in grape berries, while the volatile products had no evidentability to inhibit the growth of A.carbonarius090614. Comparatively, B.subtillisshowed the highest antifungal activity on Red Earth grape, followed by Kyoho, andThompson seedless was the lowest one.