CONCLUSIONS The purpose of the present investigation was to isolate and identify some - PowerPoint Presentation

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CONCLUSIONS

The purpose of the present investigation was to isolate and identify some Saccharomyces spp. by classical (morphology and biochemical method ) and molecular (PCR technique) methods. The PCR method allow the highly sensitive detection of specific yeast and will have a significant impact on the analysis of gut community structure emphasis the species-specific primers for Saccharomyces spp. which can decrease the workload and save identification time for the investigation of complex microbial populations. We found that the data obtained by sequencing the 18S-ITS region is useful for rapid identification and intraspecific phylogenetic studies of strains. Saccharomyces spp. was widely used as probiotics in efforts to reduce the numbers of pathogens residing in the intestinal tract and to maintain a balanced microbiota. An additional aim was to registering patent protection for Saccharomyces spp. strains locally isolated from Egyptian resources to increase the additive value of the Egyptian microbial wealth and in this study show that Saccharomyces boulardii is genetically very close or nearly identical to Saccharomyces cerevisiae and physiologically is identical except galactose fermentation and formation of ascospores.

Genetic and Metabolic Strategies for The Classification of Some Bakery-Yeasts

Shalaby, S.D., Helmy E. A.*, Abd El-Fadeel, R.H. Dairy Science Dept., Faculty of Agriculture, Al-Azhar University*Regional Center for Mycology and Biotechnology Al-Azhar University

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Regarding baking and brewing, Saccharomyces cerevisiae has got a great commercial value considered as a Qualified Presumption of Safety (QPS) status according to the European Food Safety Authority (EFSA). While, other yeast species as Saccharomyces boulardii showed potential benefits of the host yet used as biotherapeutic agents. Therefore, our study deals with morpho-physiological characterization and taxonomical classification of 52 isolates collected from Egyptian local markets utilizing several potential sources to obtain patent protection for Saccharomyces spp. isolates, and used it in food and dairy industry. Physiological characterization was performed by assessing the ability to use organic compounds as sole carbon source under semi-aerobic and/or aerobic conditions. Additionally, fermentation of sugars, assimilation of carbohydrates and nitrogen compounds as well as growth at high concentrations of D-glucose and sodium chloride were assessed too. The results obtained allowed us to identify 10 and 7 isolates as belonging to Saccharomyces cerevisiae and Saccharomyces boulardii, respectively, by Physiological characterization. Moreover, molecular approach was carried out to confirm the identification of two isolates. Finally, DNA sequencing analyses products of the two isolates were accepted for the submission in the GenBank (NCBI) under accession numbers of MF597761 and MG019392 for Saccharomyces cerevisiae AZ-EG Bouza 37 isolated from Bouza and Saccharomyces cerevisiae var. boulardii PI 65 isolated from the pickle, respectively.

Isolation of yeasts: Yeasts seldom occur in the absence of either molds or bacteria. Consequently, selective techniques used for recovery of yeasts, according to (Beech and Davenport. 1971) and (Davenport. 1980) which deal with the isolation of non-pathogenic yeasts.Preliminary identification on the basis of morphological and physiological properties:Morphological observation:Yeasts from fresh growing culture were cultivated on YPGA in Petri dishes, and the surface, color, margin and elevation of the colonies were observed. The yeasts were also inoculated in liquid YPG medium for determination of their cultural characteristics (pellicle, sediment or ring formation). The microscopic appearance of the cells grown in the same liquid media was also observed (Kurtezman and Fell. 1998)Physiological and biochemical characteristic :Fermentation of carbohydrates and assimilation of carbon compounds : Fermentation and assimilation tests were determined in liquid medium to Suh et al. (2007). Assimilation of nitrogen compound : Assimilation in liquid medium was carried out according to Wickerham (1951) .Molecular characteristic :DNA Extraction from Yeast Cells: A- DNA was extracted according to manufacture instructions using Zymo Research fungal/Bacterial DNA MiniPrep™ Kit (Catalog No. D6005- ZR corp, India). For optimal performance, beta-mercaptoethanol was added to the Fungal/ Bacterial DNA binding buffer to a final dilution of 0.5 % (v/v) i.e., 500 μl per 100 ml.B-Yeast genomic DNA purification protocol was achieved according to the manufacture instructions of (Thermo Fisher Scientific GeneJET Genomic DNA Purification Kit (K0721, USA).Primers selectivity Based on In silico studies:PCR amplification of 26S rRNA gene and 5.8S ITS region . PCR amplification of 26S rRNA gene and 5.8S ITS region: Amplification reactions were prepared in a total volume of 25 μl containing; 12.5 μl GoTaq® master mix (Promega, Corporations 2800 Woods Hollow Road. Medison, WI 53711-5399, USA), a pair of specific primers (A, B and C) at the concentration of 0.25μmol of each primer, 100 ng of template DNA and nuclease free water up to 25μl. PCR amplifications were carried out in eppendorf master cycler gradient apparatus (Medison, USA). Applying the following PCR temperature profile, as follows; denaturation cycle of 94°C for 5 min, followed by 35 cycles of 94°C for 30 s. Annealing temperature was tested as 50°C, 55°C, 58°C and 60°C for 1 min for obtaining optimum annealing temperature. DNA extension was adjusted to 72°C for 45 s and the final elongation cycle to 72°C for 4 min.Sequencing analysis:PCR product sequencing was achieved by GATC Biotech using ABI 3730xl DNA Sequencer (Konstanz, Germany). Applying forward and reverse primers as will be described by combining the traditional Sanger technology. Sequence similarity search was performed using the NCBI BLAST online tool (http://ncbi.nlm.nih.gov/BLAST/) against the nucleotide collection (nr/nt) database.Sequences Submission and Accession Number: Sequences of this study have been submitted to NCBI using BankIt tool (http://www.ncbi.nlm.nih .gov/BankIt/) with the published data in the NCBI database accession number.Phylogenetic Tree Construction:Phylogenetic tree was constructed based on the 18S rRNA intergenic spacers (ITS) sequence comparisons length polymorphism of the PCR-amplified and sequences from database using BLAST tree constructed in www.clcbio.com using ClC workbench 7.5 system based on Neighbor Joining method.

NCBI flat file for

Egyptian

Saccharomyces

cerevisiae

PCR amplification and visualization using

18S rRNA

ABSTRACT

Methods

Results

Physiological and biochemical characteristic