engleski

Evaluation of DGGE method as a tool to study dynamic of microbial communities in Istrian cheese

To investigate in situ microbial communities’ structure, molecular methods based on nucleic acids analysis have become very popular. However, the results are strongly dependent on primers applied for investigation. Therefore we tested three different commonly used primer sets as well as denaturing gradient gel electrophoresis (DGGE) conditions in order to analyze microbial communities dynamic in cheese. To optimize DGGE 15 type strains commonly found in cheese were tested. Primers 338GCf/518r and 984GCf/1401r distinguish well among 15 LAB strains applied, with 338GCf/518r being more efficient such, opposite to 984GCf/1401r, discriminating between E. faecalis and E. faecium. No satisfied results were obtained by applying 41GCf/ 111r primers which generating more distinct bands from one species. Interesting, investigation on complex community DNA directly isolated from Istrian cheese revealed poor separation when amplified with 338GCf/518r primers. However, resolution of 984GCf/1401r was very high and finds to be the most suitable between three primers tested. 350 ng of loaded DNA gave the best results. There were no differences between results obtained after visualization of gels by silver staining or after staining with SybrGreen I. Our results clearly demonstrated demand on DGGE optimization for each cheese type separately. Primer sets selected and DGGE conditions depend on specific microbial communities’ properties and must be therefore carefully optimized according to particular communities’ demands.

Bacterial diversity; DGGE; sequencing; cheese ripening

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