Abstract
Dry-cured meat products are frequently colonised by toxigenic Penicillium nordicum and Penicillium verrucosum throughout their ripening that can produce ochratoxin A (OTA). The predictive nature of the molecular analyses can be used to determine the risk of toxigenic species. For this reason, the objective of the present work was to analyse the temporal changes in the expression of the otapks and otanps genes by P. nordicum and P. verrucosum in relation to OTA production on slices of dry-fermented sausage salchichón and dry-cured ham. Gene expression was higher in P. nordicum than in P. verrucosum in both meat matrices. The otapks gene was overexpressed by both Penicillium species, especially in salchichón. OTA was only detected in inoculated salchichón regardless of the ochratoxigenic species used. The high significant correlation found between the early relative expression of the otapks gene and OTA production in salchichón leads to propose the relative gene expression of the otapks gene as a good indicator to predict OTA accumulation throughout the ripening of this product.
Similar content being viewed by others
References
Andrade MJ, Thorsen L, Rodríguez A, Córdoba JJ, Jespersen L (2014) Inhibition of ochratoxigenic moulds by Debaryomyces hansenii strains for biopreservation of dry-cured meat products. Int J Food Microbiol 170:70–77. https://doi.org/10.1016/j.ijfoodmicro.2013.11.004
Andrés AI, Ventanas S, Ventanas J, Cava R, Ruíz J (2005) Physicochemical changes throughout the ripening of dry cured hams with different salt content and processing conditions. Eur Food Res Technol 221:30–35. https://doi.org/10.1007/s00217-004-1115-y
Bailly JD, Tabuc C, Quérin A, Guerre P (2005) Production and stability of patulin, ochratoxin A, citrinin, and cyclopiazonic acid on dry cured ham. J Food Prot 68:1516–1520. https://doi.org/10.4315/0362-028X-68.7.1516
Battilani P, Pietri A, Giorni P, Formenti S, Bertuzzi T, Toscani T, Virgili R, Kozakiewicz Z (2007) Penicillium populations in dry-cured ham manufacturing plants. J Food Prot 70:975–980. https://doi.org/10.4315/0362-028X-70.4.975
Bernáldez V, Rodríguez A, Rodríguez M, Sánchez-Montero L, Córdoba JJ (2017) Evaluation of different RNA extraction methods of filamentous fungi in various food matrices. LWT Food Sci Technol 78:47–53. https://doi.org/10.1016/j.lwt.2016.12.018
Comi G, Chiesa L, Panseri S, Orlic S, Iacumin L (2013) Evaluation of different methods to prevent Penicillium nordicum growth on and ochratoxin A production in country style sausages. World Mycotoxin J 6:411–418. https://doi.org/10.3920/WMJ2013.1548
Córdoba JJ, Antequera T, García C, Ventanas J, López-Bote C, Asensio MA (1994) Evolution of free amino acids and amines during ripening of Iberian cured ham. J Agric Food Chem 42:2296–2301. https://doi.org/10.1021/jf00046a040
EFSA (European Food Safety Authority) (2006) Opinion of the scientific panel on contaminants in the food chain on a request from the commission related to ochratoxin A in food. The EFSA J 365:1–56. https://doi.org/10.2903/j.efsa.2006.365
Fadda S, Oliver G, Vignolo G (2002) Protein degradation by Lactobacillus plantarum and Lactobacillus casei in a sausage model system. J Food Sci 67:1179–1183. https://doi.org/10.1111/j.1365-2621.2002.tb09473.x
Ferrara M, Magist D, Lippolis V, Cervellieri S, Susca A, Perrone G (2016) Effect of Penicillium nordicum contamination rates on ochratoxin A accumulation in dry-cured salami. Food Control 67:235–239. https://doi.org/10.1016/j.foodcont.2016.03.010
Geisen R (2004) Molecular monitoring of environmental conditions influencing the induction of ochratoxin A biosynthesis genes in Penicillium nordicum. Mol Nutr Food Res 48:532–540. https://doi.org/10.1002/mnfr.200400036
Iacumin L, Chiesa L, Boscolo D, Manzano M, Cantoni C, Orlic S, Comi G (2009) Moulds and ochratoxin A on surfaces of artisanal and industrial dry sausages. Food Microbiol 26:65–70. https://doi.org/10.1016/j.fm.2008.07.006
Iacumin L, Manzano M, Andyanto D, Comi G (2017) Biocontrol of ochratoxigenic moulds (Aspergillus ochraceus and Penicillium nordicum) by Debaryomyces hansenii and Saccharomycopsis fibuligera during speck production. Food Microbiol 62:188–195. https://doi.org/10.1016/j.fm.2016.10.017
IARC, International Agency for Research on Cancer (1993) IARC monographs on the evaluation of the carcinogenic risk to humans 56: 449
Kamala A, Ortiz J, Kimanya M, Haesaert G, Donoso S, Tiisekwa B, De Meulenaer B (2015) Multiple mycotoxin co-occurrence in maize grown in three agro-ecological zones of Tanzania. Food Control 54:208–215. https://doi.org/10.1016/j.foodcont.2015.02.002
Karolewiez A, Bogs C, Geisen R (2005) Genetic background of ochratoxin A production in Penicillium. Mycotoxin Res 21:46–48. https://doi.org/10.1007/BF02954816
Karolewiez A, Geisen R (2005) Cloning a part of the ochratoxin A biosynthetic gene cluster of Penicillium nordicum and characterization of the ochratoxin polyketide synthase gene. Syst Appl Microbiol 28:588–595. https://doi.org/10.1016/j.syapm.2005.03.008
Larsen TO, Svendsen A, Smedsgaard J (2001) Biochemical characterization of ochratoxin A-producing strains of the genus Penicillium. Appl Environ Microbiol 67:3630–3635. https://doi.org/10.1128/AEM.67.8.3630-3635.2001
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using realtime quantitative PCR and the 2 (−ΔΔCT) method. Methods 25:402–408. https://doi.org/10.1006/meth.2001.1262
Long GL, Winefordner JD (1983) Limit of detection a closer look at the IUPAC definition. Anal Chem 55:712A–724A. https://doi.org/10.1021/ac00258a724
Lorenzo JM, Franco D (2012) Fat effect on physico-chemical, microbial and textural changes through the manufactured of dry-cured foal sausage lipolysis, proteolysis and sensory properties. Meat Sci 92:704–714. https://doi.org/10.1016/j.meatsci.2012.06.026
Lozano-Ojalvo D, Rodríguez A, Bernáldez V, Córdoba JJ, Rodríguez M (2013) Influence of temperature and substrate conditions on the omt-1 gene expression of Aspergillus parasiticus in relation to its aflatoxin production. Int J Food Microbiol 166:263–269. https://doi.org/10.1016/j.ijfoodmicro.2013.07.011
Lund F, Frisvad JC (2003) Penicillium verrucosum in wheat and barley indicates presence of ochratoxin A. J Appl Microbiol 95:1117–1123. https://doi.org/10.1046/j.1365-2672.2003.02076.x
Martín A, Córdoba JJ, Aranda E, Córdoba MG, Asensio MA (2006) Contribution of a selected fungal population to the volatile compounds on dry cured ham. Int J Food Microbiol 110:8–18. https://doi.org/10.1016/j.ijfoodmicro.2006.01.031
Martín L, Córdoba JJ, Antequera T, Timón ML, Ventanas J (1998) Effect of salt and temperature on proteolysis during ripening of Iberian ham. Meat Sci 49:145–153. https://doi.org/10.1016/S0309-1740(97)00129-0
Medina A, Schmidt-Heydt M, Rodríguez A, Parra R, Geisen R, Magan N (2015) Impacts of environmental stress on growth, secondary metabolite biosynthetic gene clusters and metabolite production of xerotolerant/xerophilic fungi. Curr Genet 61:325–334. https://doi.org/10.1007/s00294-014-0455-9
Ministero della Sanità (1999) Circolare 09.06.1999. In M. D. Sanità (Ed.), Gazzetta Ufficiale Repubblica Italiana 11.06.1999 (Vol. 135)
Núñez F, Rodríguez MM, Bermúdez E, Córdoba JJ, Asensio MA (1996) Composition and toxigenic potential of the mould population on dry-cured Iberian ham. Int J Food Microbiol 32:185–197. https://doi.org/10.1016/0168-1605(96)01126-9
Rao VK, Ramana MV, Girisham S, Reddy SM (2013) Culture media and factors influencing ochratoxin A production by two species of Penicillium isolated from poultry feeds. Natl Acad Sci Lett 36:101–110. https://doi.org/10.1007/s40009-012-0096-9
Rodríguez A, Rodríguez M, Luque MI, Justesen AF, Córdoba JJ (2011) Quantification of ochratoxin Aproducing molds in food products by SYBR Green and TaqMan real-time PCR methods. Int J Food Microbiol 149:226–235. https://doi.org/10.1016/j.ijfoodmicro.2011.06.019
Rodríguez A, Rodríguez M, Martín A, Delgado J, Córdoba JJ (2012) Presence of ochratoxin A on the surface of dry-cured Iberian ham after initial fungal growth in the drying stage. Meat Sci 90:728–734. https://doi.org/10.1016/j.meatsci.2012.06.029
Rodríguez A, Medina A, Córdoba JJ, Magan N (2014) The influence of salt (NaCl) on ochratoxin A biosynthetic genes, growth and ochratoxin A production by three strains of Penicillium nordicum on a dry-cured ham-based medium. Int J Food Microbiol 178:113–119. https://doi.org/10.1016/j.ijfoodmicro.2014.03.007
Rodríguez A, Capela D, Medina A, Córdoba JJ, Magan N (2015a) Relationship between ecophysiological factors, growth and ochratoxin A contamination of dry-cured sausage based matrices. Int J Food Microbiol 194:71–77. https://doi.org/10.1016/j.ijfoodmicro.2014.11.014
Rodríguez A, Rodríguez M, Córdoba JJ, Andrade MJ (2015b) Design of primers and probes for quantitative real-time PCR methods. In: Basu C (ed) PCR primer design (methods in molecular biology). Humana, New York, pp 31–56
Schmidt-Heydt M, Graf E, Stoll D, Geisen R (2012) The biosynthesis of ochratoxin A by Penicillium as one mechanism for adaptation to NaCl rich foods. Food Microbiol 29:233–241. https://doi.org/10.1016/j.fm.2011.08.003
Schmidt-Heydt M, Graf E, Batzler J, Geisen R (2011) The application of transcriptomics to understand the ecological reason of ochratoxin A biosynthesis by Penicillium nordicum on sodium chloride rich dry cured foods. Trends Food Sci Technol 22:39–48. https://doi.org/10.1016/j.tifs.2011.02.010
Simoncini N, Virgili R, Spadola G, Battilani P (2014) Autochthonous yeasts as potential biocontrol agents in dry-cured meat products. Food Control 46:160–167. https://doi.org/10.1016/j.foodcont.2014.04.030
Virgili R, Simoncini N, Toscani T, Leggieri MC, Formenti S, Battilani P (2012) Biocontrol of Penicillium nordicum growth and ochratoxin A production by native yeasts of dry cured ham. Toxins 4:68–82. https://doi.org/10.3390/toxins4020068
Wang Y, Wang L, Liu F, Wang Q, Selvaraj JN, Xing F, Zhao Y, Liu Y (2016) Ochratoxin A producing fungi, biosynthetic pathway and regulatory mechanisms. Toxins 8:83. https://doi.org/10.3390/toxins8030083
Funding
The authors would like to thank to the Spanish Interministerial Committee on Science and Technology (grant numbers CSD2007-00016 and AGL 2013-45729-P) and the Government of Extremadura and FEDER (grant number GR15108) for their financial support. V. Bernáldez and Dr. A. Rodríguez were supported by a pre-doctoral fellowship FPU (AP 2012-5457) from the Spanish Ministry of Education, Science and Sport and by a Juan de la Cierva-Incorporación senior research fellowship (IJCI-2014-20666) from the Spanish Ministry of Economy and Competitiveness, respectively. Technical support was provided by the Facility of Innovation and Analysis in Animal Source Foodstuffs (SIPA) of SAIUEx (financed by UEX, Junta de Extremadura, MICINN, FEDER, and FSE).
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of Interest
Victoria Bernáldez declares that she has no conflict of interest. Alicia Rodríguez declares that she has no conflict of Interest. Josué Delgado declares that he has no conflict of Interest. Lourdes Sánchez-Montero declares that she has no conflict of Interest. Juan J. Córdoba declares that he has no conflict of interest.
Ethical Approval
This article does not contain any studies with human or animal subjects.
This article has not been published before, and it is not under consideration for publication anywhere else.
Informed Consent
Not applicable.
Rights and permissions
About this article
Cite this article
Bernáldez, V., Rodríguez, A., Delgado, J. et al. Gene Expression Analysis as a Method to Predict OTA Accumulation in Dry-Cured Meat Products. Food Anal. Methods 11, 2463–2471 (2018). https://doi.org/10.1007/s12161-018-1231-0
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12161-018-1231-0