الفهرس 
AcknowledgementOnly 14 pages are availabe for public view
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Abstract
The present investigation was done to study the mycological evaluation of some fermented dairy products, 180 random samples of yoghurt, laban rayeb, labenah, Domiati cheese, Cheddar cheese, and keshk (30 samples each), that were collected from different supermarkets, groceries, dairy shops in Assiut city, Egypt; during the period from July 2019 to March 2020. Some samples were selected for AFM1 screening using HPLC.
Identification of genera and species was performed using the morphological and microscopical characteristics in addition to the molecular in case of yeasts. Molecular techniques [Internal transcribed spacer (ITS) sequences of nuclear ribosomal DNA were amplified using primers ITS1, ITS4] were employed for all yeast isolates. All ITS gene sequences of the yeast were deposited at the National Center for Biotechnological Information (NCBI) website and Assiut University Moubasher Centre for Mycological science Culture Collection (AUMMC) and accession numbers are given for them. Phylogenetic trees were also constructed for those sequenced strains. Last but not least, Natamycin, potassium sorbate and thyme oil were applied to examine their inhibitory effect on the growth of some yeast strains during manufacturing and storage of Domiati cheese.
The obtained results revealed that a total of 76 species related to 37 genera were recovered from the six fermented dairy products investigated. Twenty-eight species assigned to 18 genera of yeasts and 48 species belong to 18 genera of filamentous fungi on dichloran rose bengal chloramphenicol agar (DRBC).
The current study proved that, keshk samples was heavily contaminated with fungi constituting 1.44 x 104 CFUs/g, followed by Cheddar cheese which registered 2.1 x 103 CFUs/g, while large scale yoghurt and labenah were the lowest representing 0.56 x 10 and 0.12 x 102 CFUs/g, respectively. Labenah (15 genera and 33 species) and Domiati cheese (19 & 32) were gained the highest number of total genera and species while the lowest was from large scale rayeb (9 & 15). The highest percentage counts of yeasts were recovered from balady rayeb (99.70 %) and the lowest from large scale yoghurt (1.18 %), while the reverse occurred with the filamentous fungi. from yeasts, 16 genera represented by 25 species were assigned to Ascomycota, while 2 genera and 3 species were Basidiomycota.
The obtained data comparing with the maximum permissible limits (MPL) of the local Egyptian Organization for Standardization and Quality control (EQSOC, 2005) showed that, all large scale yoghurt samples have acceptable quality for yeast and the filamentous fungi count. However, all labenah samples were compatible with the Egyptian standards for yeast counts, 73.3 % of samples were complied with the standards for filamentous fungi counts. Large scale rayeb and Domiati cheese samples were more compatible than the others, representing 93.3% and 90 % of samples acceptable quality for yeast counts and 86.7% and 83.3% for filamentous fungi, respectively, except for balady rayeb since 93.3 % of their samples were compatible for filamentous fungi counts. Two thirds of balady yoghurt samples and Cheddar cheese samples (66.7 % each) were of acceptable quality according to the Egyptian Standards for filamentous fungi and yeast counts, respectively. Keshk and Cheddar cheese samples were heavily contaminated with fungi, since 80 % and 76.7 % of their samples were not acceptable according to the Egyptian Standards for filamentous fungi counts, while 60 % of keshk and 53.3 % of balady yoghurt samples were incompatible with the Egyptian Standards for yeast counts.
Regarding to AFM1 screening, six samples were selected randomly from the investigated Cheddar cheese samples and were analyzed for contamination with AFM1. Data indicated that AFM1 was detected in five out of the six samples in a percentage of 83.3%, with a contamination level ranging from 2.35 to 8.94 µg /kg, all positive samples were exceeding the Egyptian Standards (0.05 μg/Kg), also all of them were exceeding The European Commission regulation (0.25 μg/Kg).
The results of the experimental part showed that, the minimum inhibitory concentrations (MIC) of the most powerful treatments; Natamycin, Natamycin with potassium sorbate, Natamycin and potassium sorbate with thyme oil on the most susceptible five yeast species which are: Debaryomyces fabryi, D. hansenii, Pichia membranifaciens, Meyerozyma guilliermondii, and Candida parapsilosis were tested to evaluate their effectiveness in controlling cheese spoilage and pathogenic yeasts.
The MICs of the antifungal Natamycin on the five yeast strains ranged from 15.6 - 31.2 μg/ml when used separately or in a mixture with potassium sorbate and the MICs of the potassium sorbate was 156 - 312 μg/ml, while the MICs of thyme oil was 0.65 - 10.31 μl/ml. The addition of thyme oil to the mixture of Natamycin and potassium sorbate increased their antimicrobial efficiency and their MICs ranged from 0.6 - 9.4 and 6.5 - 105 μg/ml, respectively.
Pichia membranifaciens was the most susceptible yeast affected by the antifungal Natamycin when used separately or in the mixtures gaining the lowest MICs. Candida parapsilosis and Meyerozyma guilliermondii were the most susceptible yeasts to Natamycin with potassium sorbate while Debaryomyces fabryi and Meyerozyma guilliermondii to the mixture of Natamycin, potassium sorbate with thyme oil.
The 3rd treatment that contained a mixture of Natamycin and potassium sorbate with thyme oil was the most effective one which sharply reduced the total yeast counts until completely removed yeasts at the 29th day of cheese ripening. The addition of thyme oil to Natamycin increases its efficiency in getting rid of contaminated yeast that cause spoilage of cheese during storage, as well as beneficial for reducing consumption of the added quantity from 20 mg/ L to 6.6 mg/L of Natamycin. So, there is an agreement between the in vitro MIC results and the in vivo results. Using the natural affordable thyme oil a synergistic antifungal effect has been found.
The public health hazards of fungi and the steps, which should be taken to control them in the dairy industry as well as the recommended sanitary measures, were also discussed.