Rendiconti Lincei. Scienze Fisiche e Naturali

, Volume 30, Issue 4, pp 767–773 | Cite as

Genotypic identification of ochratoxigenic Aspergilli that contaminated beef luncheon and their protease activity

  • Mohamed A. HusseinEmail author
  • Youssuf Gherbawy


Twenty-six fungal species belonging to 10 genera were collected from 40 beef luncheon samples marketed in the Qena city on Czapek–Dox agar at 28 °C. A. section Nigri and A. flavus were the most prevalent species among isolated fungi; they were contaminating 65% and 50% of the luncheon samples. According to the phylogenetic analysis using β-tubulin gene, the selected A. section Nigri was identified as A. niger and A. tubingensis. All selected isolates belonging to A. niger and A. tubingensis showed positive results for the presence of the PKS gene. Using VICAM fluorometer, the average level of ochratoxin A was found to range from 4.1 to 7.1 and 4.6 to 8.2 ppm for A. tubingensis and A. niger, respectively. All Aspergillus section Nigri isolates were protease producers except two isolates belonging to A. tubingensis. A significant difference was observed in protease production between A. tubingensis and A. niger (P = 0.005); the most active protease producers were AnL 12 and 19 (A. niger) and AtL 3 and 4 (A. tubingensis) were weak producers.


Luncheon Aspergillus section Nigri Phylogenetic analyses Ochratoxin A Protease 


Compliance with ethical standards

Conflict of interest

There is no conflict of interest.


  1. Abd-Elghany SM, Sallam KI (2015) Rapid determination of total aflatoxins and ochratoxins A in meat products by immuno-affinity fluorimetry. Food Chem 179:253–256Google Scholar
  2. Abdel-Rahman AH, Saad SM (1989) Studies on the proteolytic activity of some prevalent mould species in relation meat hygiene. Zagazig Vet Med J 17:228–236Google Scholar
  3. Abdel-Sater MA, Al-Sharjabi FA, Al-Ashwal ES (2017) Mycological and enzymatic studies on fresh beef meat sold in Taiz City, Yemen. Eur J Biol Res 7(4):337–347Google Scholar
  4. Accensi F, Abarca ML, Cano J, Figuera L, Cabañes FJ (2001) Distribution of ochratoxin A-producing strains in the Aspergillus niger aggregate. Antonie Van Leeuwenhoek 79:365–370Google Scholar
  5. Ali FHM, Farghaly RM, Hammad AM (2005) Mycological investigations in beef and chicken luncheon. Beni-Suef Vet Med J 15(2):98–102Google Scholar
  6. Asefa DT, Kure CF, Gjerde RO, Omer MK, Langsrud S, Nesbakken T, Skaar I (2010) Fungal growth pattern, sources and factors of mould contamination in a dry-cured meat production facility. Int J Food Microbiol 140:131–135Google Scholar
  7. Ashie INA, Sorensen TL, Nielsen PM (2002) Effects of papain and a microbial enzyme on meat proteins and beef tenderness. J Food Sci 67(6):2138–2142Google Scholar
  8. Bahobail A (2016) Studies on mycobiota associated with wheat bran and soybean seeds in Jeddah city, Saudia Arabia. AJBAS 10(16):176–184Google Scholar
  9. Banwart GJ (1989) Basic food microbiology, 2nd edn. An Avi book published by Van Nostrand Reinhold, New YorkGoogle Scholar
  10. Barrett AJ, Rawlings ND, Woessner JF (2004) Handbook of proteolytic enzymes. Academic Press, LondonGoogle Scholar
  11. Bisbal F, Gil JV, Ramón D, Martínez-Culebras PV (2009) ITS-RFLP characterization of black Aspergillus isolates responsible for ochratoxin A contamination in cocoa beans. Eur Food Res Technol 229:751–755Google Scholar
  12. Chiotta ML, Susca A, Stea G, Mule G, Perrone G, Logrieco A, Chulze SN (2011) Phylogenetic characterization and ochratoxin A-fumonisin profile of black Aspergillus isolated from grapes in Argentina. Int J Food Microbiol 149:171–176Google Scholar
  13. Chulze SN, Magnoli CE, Dalcero AM (2006) Occurrence of ochratoxin A in wine and ochratoxigenic mycoflora in grapes and dried vine fruits in South America. Int J Food Microbiol 111:5–9Google Scholar
  14. El-Hamaky AM, Hassan AA, Abo El Yazeed H, Refai MK (2016) Prevalence and detection of toxigenic A. flavus, A. niger and A. ochraceus by traditional and molecular biology methods in feeds. Int J Curr Res 8(1):25621–25633Google Scholar
  15. Gabal MA, Hegazy SM, Nagwa YH (1994) Aflatoxin production by Aspergillus flavus field isolates. Vet Hum Toxicol 39:519–521Google Scholar
  16. Gherbawy YA, Shebany YA, Alharthy HF (2016) Molecular characterization of aflatoxigenic aspergilli contaminated poultry and animal feedstuff samples from the western region of Saudi Arabia. Ital J Food Sci 28:32–42Google Scholar
  17. Glass NL, Donaldson GC (1995) Development of primer sets designed for use with the PCR to amplify conserved genes from filamentous ascomycetes. Appl Environ Microbiol 61:1323–1330Google Scholar
  18. Ismail MA, Zaky ZM (1999) Evaluation of the mycological status of luncheon meat with special reference to aflatoxigenic moulds and aflatoxin residues. Mycopathologia 146:147–154Google Scholar
  19. Ismail SA, Shehata AA, El-diasty EM (2013) Microbiological quality of some meat products in local markets with special reference to mycotoxins. Glob Vet 10(5):577–584Google Scholar
  20. Jukes TH, Cantor CR (1969) Evolution of protein molecules. In: Munro HN (ed) Mammalian protein metabolism, vol 3. Academic Press, New York, pp 21–132Google Scholar
  21. Jurjevic Z, Peterson SW, Stea G, Solfrizzo M, Varga J, Hubka V, Perrone G (2012) Two novel species of Aspergillus section Nigri from indoor air. IMA Fungus 3:159–173Google Scholar
  22. Kusai NA, Azmi MMZ, Zulkifly S, Yusof MT, Zainudin NAIM (2016) Morphological and molecular characterization of Curvularia and related species associated with leaf spot disease of rice in Peninsular Malaysia. Rend Fis Acc Lincei 27:205–214Google Scholar
  23. Kusai NA, Ayob Z, Maidin MST, Safari S, Ali SRA (2018) Characterization of fungi from different ecosystems of tropical peat in Sarawak, Malaysia. Rend Fis Acc Lincei 29:469–482Google Scholar
  24. Kusters-van SMA, Samson RA, Visser J (1991) The use of RFLP analysis in classification of the black Aspergilli: reinterpretation of Aspergillus niger aggregate. Curr Genet 19:21–26Google Scholar
  25. Lahouar A, Marin S, Crespo-Sempere A, Saïd S, Sanchis V (2017) Influence of temperature, water activity and incubation time on fungal growth and production of ochratoxin A and zearalenone by toxigenic Aspergillus tubingensis and Fusarium incarnatum isolates in sorghum seeds. Int J Food Microbiol 242:53–60Google Scholar
  26. Lewis L, Onsongo M, Njapau H, Schurz-Rogers H, Luber G, Kieszak S, Nyamongo J, Backer L, Dahiye AM, Misore A, DeCock K, Rubin C, Kenya Aflatoxicosis Investigation Group (2005) Aflatoxin contamination of commercial maize products during an outbreak of acute aflatoxicosis in eastern and central Kenya. Environ Health Perspect 113(12):1763–1767Google Scholar
  27. Magnoli CE, Andrea L, Astoreca AL, Chiacchiera SM, Dalcero AM (2007) Occurrence of ochratoxin A and ochratoxigenic mycoflora in corn and corn-based foods and feeds in some South American countries. Mycopathologia 163:249–260Google Scholar
  28. Medina A, Mateo R, López-Ocaña L, Valle-Algarra FM, Jiménez M (2005) Study of Spanish grape mycobiota and ochratoxin A production by isolates of Aspergillus tubingensis and other members of Aspergillus section Nigri. Appl Environ Microbiol 71(8):4696–4702Google Scholar
  29. Medina A, Mateo EM, Valle-Algarra MV, Mateo F, Mateo R, Jiménez M (2008) Influence of nitrogen and carbon sources on the production of ochratoxin A by ochratoxigenic strains of Aspergillus spp. isolated from grapes. Int J Food Microbiol 122:93–99Google Scholar
  30. Misra N (1983) New Record of fungi from the bark of cinnamon in storage. Sci Cult 49(5):133–135Google Scholar
  31. Mizakova A, Pipova M, Turek P (2002) The occurrence of moulds in fermented raw meat products. Czech J Food Sci 20:89–94Google Scholar
  32. Nasser LA (2015) Molecular identification of isolated fungi, microbial and heavy metal contamination of canned meat products sold in Riyadh, Saudi Arabia. Saudi J Biol Sci 22:513–520Google Scholar
  33. Noonim P, Mahakarnchanakul W, Varga J, Frisvad JC, Samson RA (2008) Two novel species of Aspergillus section Nigri from Thai coffee beans. Int J Syst Evol Microbiol 58:1727–1734Google Scholar
  34. O’Callaghan J, Caddick MX, Dobson ADW (2003) A polyketide synthase gene required for ochratoxin A biosynthesis in Aspergillus ochraceus. Microbiology 149:3485–3491Google Scholar
  35. Palumbo JD, O’Keeffe TL, Vasquez SJ, Mahoney NE (2011) Isolation and identification of ochratoxin A-producing Aspergillus section Nigri strains from California raisins. Lett Appl Microbiol 52:330–336Google Scholar
  36. Patino B, Gonzalez-Salgado A, Gonzalez-Jaen MT, Vazquez C (2005) PCR detection assays for the ochratoxin-producing Aspergillus carbonarius and Aspergillus ochraceus species. Int J Food Microbiol 104:207–214Google Scholar
  37. Perrone G, Mulè G, Susca A, Battilani P, Pietri A, Logrieco A (2006) Ochratoxin A production and amplified fragment length polymorphism analysis of Aspergillus carbonarius, Aspergillus tubingensis, and Aspergillus niger strains isolated from grapes in Italy. Appl Environ Microbiol 72(1):680–685Google Scholar
  38. Perrone G, Varga J, Susca A, Frisvad JC, Stea G, Kocsube S, Toth B, Kozakiewicz Z, Samson RA (2008) Aspergillus uvarum sp nov., a uniseriate black Aspergillus species isolated from grapes in Europe. Int J Syst Evol Microbiol 58:1032–1039Google Scholar
  39. Perrone G, Stea G, Epifani F, Varga J, Frisvad JC, Samson RA (2011) Aspergillus niger contains the cryptic phylogenetic species A. awamori. Fungal Biol 115:1138–1150Google Scholar
  40. Petzinger E, Weidenbach A (2002) Mycotoxins in the food chain: the role of ochratoxins. Livest Sci 76:245–250Google Scholar
  41. Reddy KV, Naveen K, Reddy IB (2013) Incidence and molecular detection of ochratoxigenic fungi from Indian cereal grains. Int J Pharm Bio Sci 4(3):31–40Google Scholar
  42. Rehbar S, Batool R (2017) Protease production by Brevundimonas sp. and Trabulsiella guamensis isolated from contaminated soil. Rend Fis Acc Lincei 28:529–534Google Scholar
  43. Rodriguez MS, Crettaz-Minaglia MC, Gianello D, Piaggio M (2019) Microbiological water quality of a temperate third-order stream. Rend Fis Acc Lincei 30:417–426Google Scholar
  44. Saleem A (2008) Effect of some food preservatives on the lipolytic activity of beef luncheon fungi. Mycobiology 36(3):167–172Google Scholar
  45. Saleem A, El-Said AH (2009) Proteolytic activity of beef luncheon fungi as affected by incorporation of some food preservatives. Acta Microbiol Immunol Hung 56(4):417–426Google Scholar
  46. Salem Y, EL-Mossalami H, Mousa M (2016) Microbiological criteria of local manufactured beef luncheon, Alexandria. J Vet Sci 51(2):381–385Google Scholar
  47. Serra R, Cabanes FJ, Perrone G, Castella G, Venancio A, Mule G, Kozakiewicz Z (2006) Aspergillus ibericus: a new species of section Nigri isolated from grapes. Mycologia 98:295–306Google Scholar
  48. Sonjak S, Licen M, Frisvad JC, Gunde-Cimerman N (2011) The mycobiota of three dry-cured meat products from Slovenia. Food Microbiol 28:373–376Google Scholar
  49. Susca A, Proctor RH, Morelli M, Haidukowski M, Gallo A, Logrieco AF, Moretti A (2016) Variation in fumonisin and ochratoxin production associated with differences in biosynthetic gene content in Aspergillus niger and A. welwitschiae isolates from multiple crop and geographic origins. Front Microbiol 7:1–15Google Scholar
  50. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The Clustal X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25:4876–4882Google Scholar
  51. Van De Peer Y, De Wachter R (1994) Treecon software package for the construction and drawing of evolutionary trees. Comput Appl Biosci 9:177–182Google Scholar
  52. Varga J, Kevei F, Vriesema A, Debets F, Kozakiewicz Z, Croft JH (1994) Mitochondrial DNA restriction fragment length polymorphisms in field isolates of the Aspergillus niger aggregate. Can J Microbiol 40:612–621Google Scholar
  53. Varga J, Kocsube S, Toth B, Frisvad JC, Perrone G, Susca A, Meijer M, Samson RA (2007) Aspergillus brasiliensis sp. nov., a biseriate black Aspergillus species with world-wide distribution. Int J Syst Evol Microbiol 57:1925–1932Google Scholar
  54. Yee TL, Tajuddin R, Nor NMIM, Mohd MH, Zakaria L (2016) Filamentous ascomycete and basidiomycete fungi from beach sand. Rend Fis Acc Lincei 27:603–607Google Scholar
  55. Youssef M, Saleem A (2017) Proteolytic activity of recent foraminiferal fungi isolated from different coastal lagoons red sea coast, Egypt. Wulfenia J 24(9):258–270Google Scholar
  56. Zohri AA, Moharram AM, Refaie RRS (2014) Mycobiota contaminating beef burger and sausage with reference to their toxins and enzymes. J Basic Appl Mycol Egypt 5:61–73Google Scholar

Copyright information

© Accademia Nazionale dei Lincei 2019

Authors and Affiliations

  1. 1.Botany and Microbiology Department, Faculty of ScienceSouth Valley UniversityQenaEgypt

Personalised recommendations