PCR-DGGE analysis of fungal community in manufacturing process of a traditional Iranian cheese
AbstractBackground and Objectives: The microbial communities of traditional milk-based food are of great importance in its manufacturing process, especially when using raw milk with natural cultures. Liqvan (Lighvan or Levan) is a traditional Iranian buried cheese, which is made from raw ewe´s milk without a starter addition. The aim of this study was to explore the fungal active population during this cheese manufacturing process by comparing DNA and RNA based culture independent method Denaturing Gradient Gel Electrophoresis (DGGE).Materials and Methods: Four samples of each milk, curd and ripened cheese were collected from Liqvan village located in East Azerbaijan province of Iran. Total DNA and RNA of each sample were extracted and PCR amplicons of D1 region of 26S rRNA gene was targeted for DGGE analysis. This method applied at both DNA and RNA levels in order to examine which taxonomic groups of fungi are active at each step of ripening.Results: DGGE profiles of yeast amplicons showed different results between extracted DNA and RNA during ripening process. However, the main group that is present in all stages of ripening process belongs to the genus Candida although Kluyveromyces, Pichia, Galactomyces, Saccharomyces and Cryptococcus are most abundant fungi.Conclusion: As no starter culture added to Liqvan cheese it seems fungal diversity are mainly rely on the indigenous microbiota of milk. Furthermore, the percentage of the dominant fungal genera from the total sequences differed among DNA and cDNA libraries.
Bautista-Gallego J, Alessandria V, Fontana M, Bisotti S, Taricco S, Dolci P, et al. Diversity and functional characterization of Lactobacillus spp. isolated throughout the ripening of a hard cheese. Int J Food Microbiol 2014;181:60-66.
Cocolin L, Diez A, Urso R, Rantsiou K, Comi G, Bergmaier I, et al. Optimization of conditions for profiling bacterial populations in food by culture-independent methods. Int J Food Microbiol 2007;120:100-109.
Carraro L, Maifreni M, Bartolomeoli I, Martino ME, Novelli E, Frigo F, et al. Comparison of culture-dependent and-independent methods for bacterial community monitoring during Montasio cheese manufacturing. Res Microbiol 2011;162:231-239.
Cocolin L, Alessandria V, Dolci P, Gorra R, Rantsiou K. Culture independent methods to assess the diversity and dynamics of microbiota during food fermentation. Int J Food Microbiol 2013;167:29-43.
Ndoye B, Rasolofo EA, LaPointe G , Roy D. A review of the molecular approaches to investigate the diversity and activity of cheese microbiota. Dairy Sci Technol 2011;91:495.
Edalatian MR, Habibi Najafi MB, Mortazavi A, Mayo B. The biodiversity and evolution of lactic flora during ripening of the Iranian semisoft Lighvan cheese. Int J Dairy Technol 2012;65:81-89.
Kafili T, Razavi SH, EmamDjomeh Z, Naghavi MR, Álvarez-Martín P, Mayo B. Microbial characterization of Iranian traditional Lighvan cheese over manufacturing and ripening via culturing and PCR-DGGE analysis: identification and typing of dominant lactobacilli. Eur Food Res Technol 2009;229:83-92.
Navidghasemizad S, Hesari J, Saris P, Nahaei MR. Isolation of lactic acid bacteria from Lighvan cheese, a semihard cheese made from raw sheep milk in Iran. Int J Dairy Technol 2009;62:260-264.
Barouei J, Karbassi A, Ghoddusi HB, Mortazavi A. Lactic microflora present in Liqvan Ewes' milk cheese. Int J Food Prop 2008;11:407-414.
Cocolin L, Aggio D, Manzano M, Cantoni C, Comi G. An application of PCR-DGGE analysis to profile the yeast populations in raw milk. Int Dairy J 2002;12:407-411.
Rantsiou K, Urso R, Dolci P, Comi G, Cocolin L. Microflora of Feta cheese from four Greek manufacturers. Int J Food Microbiol 2008;126:36-42.
Masoud WMH, Takamiya MKW, Vogensen FK, Lillevang S, Abu Al-Soud, Waleed S, et al. Characterization of bacterial populations in Danish raw milk cheeses made with different starter cultures by denaturating gradient gel electrophoresis and pyrosequencing. Int Dairy J 2011;21:142-148.
Alegría A, Szczesny P, Mayo B, Bardowski J, Kowalczyk M. Biodiversity in Oscypek, a traditional Polish cheese, determined by culture-dependent and-independent approaches. Appl Environ Microbiol 2012;78:1890-1898.