Advances in Food Mycology

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Evaluation of Molecular Methods for the Analysis of Yeasts in Foods 89 PCR-DGGE Phyllospheric yeast species and fungicide Gildemacher et al. (2004) treatments on russetting of Elstar apples 18S rDNA; nested PCR; NS1-NS8, NS1gc-NS2+10 PCR-DGGE Yeast species in raw milk Cocolin et al. (2002b) 26S rDNA; NL1gc-LS2 PCR-DGGE Yeast species involved in coffee fermentation Masoud et al. (2004) 26S rDNA; nested PCR; NL1-NL4, NL1gc-LS2; or NL1-LS2, NL1gc-LS2 PCR-DGGE Yeast populations associated with Ghanaian Nielsen et al. (2005) 26S rDNA; NL1gc-LS2 cocoa fermentations PCR-DGGE Differentiation of Saccharomyces sensu strictu strains; Manzano et al. (2004) S. cerevisiae, S. paradoxus and S. bayanus/S. pastorianus ITS2; Schafgc-Schar (Saccharomyces sensustricto specific) PCR-TGGE S. cerevisiae and S. paradoxus in wine fermentation Manzano et al. (2005) ITS2; Schafgc-Schar
90 Ai Lin Beh et al. (2003) have proposed the use of RT-PCR protocols that target RNA rather than DNA templates from the food or beverages. In several studies, culture methods have revealed the presence of yeast species not found by PCR-DGGE (Mills et al., 2002; Masoud et al., 2004; Prakitchaiwattana et al., 2004). Such yeasts were present at populations at less than 10 2 -10 3 CFU/ml or g of product and this is considered to be the lower limit of detection by PCR-DGGE. Nevertheless, there are reports where yeast species present at 10 4 -10 5 CFU/ml or g product have not been detected by PCR-DGGE. This can occur when there is a mixture of different yeast species in the sample, with one species being numerically present at populations 100- 1000 times more than other species. The DNA of the dominant species may bind primers more favorably, resulting in weaker amplification of the minority species. Also, when universal primers are used, there may be stronger hybridization of the primers with the DNA of some species over others (Mills et al., 2002; Prakitchaiwattana et al., 2004; Nielsen et al., 2005). While the relative mobility of DNA bands on DGGE/TGGE gels can discriminate between closely related species (e.g. those in Saccharomyces sensu stricto, Manzano et al., 2004), exceptions can occur. In some cases, different species have produced bands with similar mobilities (Hernán-Gómez et al., 2000; Gildemacher et al., 2004). There are several reports where multiple bands have been found for the one species (eg. two bands for Candida sp. (Mills et al., 2002), three bands for Pichia kluyveri (Masoud et al., 2004), and several bands for Metschnikowia pulcherrima (Prakitichaiwattana et al., 2004). The reasons for multiple banding within the one species are not well understood, but may reflect artefacts of PCR using primers with GC clamps and DNA denaturation kinetics during electrophoresis. Multiple bands could arise from nucleotide variations among multiple rDNA copies within a single strain, or could also indicate the presence of different strains within a species. To address these anomalies, it is good practice therefore, to isolate individual bands from DGGE/TGGE gels and confirm their identity by sequencing. Finally, food samples often contain large amounts of DNA from plants, animals and other microbial groups (eg. bacteria and filamentous fungi) that have the potential to interfere with the specific PCRamplification of yeast DNA and compromise the reliability and quality of the data obtained by DGGE/TGGE. For example, we have experienced particular difficulty in detecting yeasts in mould ripened cheeses with PCR-DGGE and PCR-TGGE because of the large
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Advances in Food Mycology
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Advances in Food Mycology Edited by
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FOREWORD This book represents the P
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CONTENTS Foreword .................
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Contents ix Mycobiota, mycotoxigeni
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CONTRIBUTORS M. Lourdes Abarca, Dep
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Contributors xiii Dilek Heperkan, I
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Section 1. Understanding the fungi
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4 Jens C. Frisvad et al. Furthermor
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6 Jens C. Frisvad et al. Table 1. M
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8 Jens C. Frisvad et al. Major sour
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10 Jens C. Frisvad et al. Major sou
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12 Jens C. Frisvad et al. 3.4. Cycl
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14 Jens C. Frisvad et al. and poult
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16 Jens C. Frisvad et al. 3.9. Zear
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18 Jens C. Frisvad et al. 4.5. Myco
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20 Jens C. Frisvad et al. 4.9. Peni
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22 Jens C. Frisvad et al. Major sou
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24 Jens C. Frisvad et al. only occu
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26 Jens C. Frisvad et al. 1977, Myc
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28 Jens C. Frisvad et al. Kamyar, M
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30 Jens C. Frisvad et al. Pitt, J.
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RECOMMENDATIONS CONCERNING THE CHRO
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Recommendations Concerning the Chro
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Page 80 and 81: EVALUATION OF MOLECULAR METHODS FOR
Page 82 and 83: Evaluation of Molecular Methods for
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Page 120 and 121: Standardization of Methods for Dete
Page 122 and 123: Standardization of Methods for Dete
Page 124 and 125: ECOPHYSIOLOGY OF FUMONISIN PRODUCER
Page 126 and 127: Ecophysiology of Fumonisin Producer
Page 132 and 133: ECOPHYSIOLOGY OF FUSARIUM CULMORUM
Page 134 and 135: Ecophysiology of Fusarium culmorum
Page 146 and 147: FOOD-BORNE FUNGI IN FRUIT AND CEREA
Page 148 and 149: Fungi and Mycotoxins in Fruit and C
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Fungi and Mycotoxins in Fruit and C
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BLACK ASPERGILLUS SPECIES IN AUSTRA
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Black Aspergillus Species in Austra
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174 Marta Bau et al. sporadically i
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176 Marta Bau et al. Table 1. Occur
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178 Marta Bau et al. Table 2. Ochra
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FUNGI PRODUCING OCHRATOXIN IN DRIED
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Fungi Producing Ochratoxin in Dried
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AN UPDATE ON OCHRATOXIGENIC FUNGI A
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An Update on Ochratoxigenic Fungi a
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MYCOBIOTA, MYCOTOXIGENIC FUNGI, AND
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Mycobiota and Citrinin in Black Oli
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BYSSOCHLAMYS: SIGNIFICANCE OF HEAT
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Byssochlamys Heat Resistance and My
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EFFECT OF WATER ACTIVITY AND TEMPER
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Aflatoxin and CPA Production in Pea
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INACTIVATION OF FRUIT SPOILAGE YEAS
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High Pressure Inactivation of Fungi
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ACTIVATION OF ASCOSPORES BY NOVEL F
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Activation of Ascospores by Novel F
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MIXTURES OF NATURAL AND SYNTHETIC A
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Mixtures of Natural and Synthetic A
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PROBABILISTIC MODELLING OF ASPERGIL
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Probabilistic Modelling of Aspergil
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ANTIFUNGAL ACTIVITY OF SOURDOUGH BR
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Antifungal Activity of Sourdough Br
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PREVENTION OF OCHRATOXIN A IN CEREA
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Prevention of Ochratoxin A in Cerea
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RECOMMENDED METHODS FOR FOOD MYCOLO
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Recommended Methods for Food Mycolo
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Recommended Methods for Food Mycolo
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APPENDIX 1 - MEDIA All media are st
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Appendix 1 - Media 351 CYA (1): Cza
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Appendix 1 - Media 353 note that th
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Appendix 1 - Media 355 PCA: Potato
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Appendix 1 - Media 357 Pitt, J. I.,
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Appendix 2 - International Commissi
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362 Index Antifungal agents (Contin
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364 Index Cellulase, from F. culmor
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366 Index Fusarenon X, 15 Fusarin C
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368 Index Mycophenolic acid, 18, 21
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370 Index Phaffia, 74 Phoma tenuazo
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