Molecular characterization and spoilage potential of yeasts isolated from traditional Egyptian Karish cheese

Abstract

Traditional soft cheeses are susceptible to microbial contamination, particularly when unsterilized flavoring additives are used during production or retail handling. This study aimed to quantify and molecularly characterize the yeast flora associated with spiced Karish cheese, a traditional Egyptian fresh cheese supplemented with peppers and olives. Yeast counts ranged from 2.25 to 2.99 log10 CFU/g, while molds were detected in 60% of the samples, reaching 1.47 log CFU/g. These counts exceeded the maximum permissible limits specified by the Egyptian Standard (ES 1008-4/2005), indicating potential deficiencies in hygienic practices during production, handling, or storage. Morphological and physiological characterization grouped the 12 yeast isolates into four phenotypic profiles. Representative non-Saccharomyces isolates were further identified by sequencing the ITS1–5.8 S–ITS2 rDNA region, followed by BLASTN analysis and phylogenetic reconstruction using the Neighbor-Joining method with 1,000 bootstrap replicates. Two representative species were identified as Kodamaea ohmeri (AZ EG 3) and Pichia kudriavzevii (AZ EG 2). BLASTN analysis showed > 99% sequence identity with representative GenBank sequences, while phylogenetic analysis placed both isolates within well-supported monophyletic clades (bootstrap > 95%), with evolutionary distances of 0.009 and 0.020 substitutions/site, respectively. The representative sequences were deposited in GenBank under accession numbers PX406129 and PX503693. These findings provide baseline molecular data for the surveillance of spoilage yeasts in traditional Egyptian dairy products. Although the role of spice additives was not directly investigated, the results support the need for future studies to evaluate their contribution as potential sources of yeast contamination in artisanal cheese production.