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In Ethiopia spontaneous fermentation is the sole means of making fermented food, which is prone to contamination and under quality product. Hence, the present study aimed at isolating and evaluating starter function of lactic acid bacteria for milk fermentation. Twenty samples were aseptically collected from Holetta agricultural research center dairy farm, Biftu berga farmer’s union, Holland dairy farm, Genesis dairy farm and Adama residential households. A
total of 350 isolates were obtained on MRS agar media and repeatedly cultured till uniform colonies were obtained. The purified isolates were then tested for acidifying efficacy. From 350 isolates twenty isolates were able to produce sufficient acid for milk fermentation. Then the 20 isolates were selected and tested for acid tolerance ability at 3.5, 3.8, 4 and 4.5 pH being incubated at 37°C of for 0, 2 and 4 hours. Seven (37%) isolates out of twenty showed both tolerance and growth at 4hours incubation at all pH values and further tested for temperature stress at 15, 25, 45 and 55° for the same incubation period. Of the seven isolates four (57%) were able to grow at 45 and 55° C for 4 and 2hours of incubation, while three of them (AD11, G16 and BB13) were found to be growing at 45 and 55°C, and 3.8 and 4 pH as optimum growth parameter. Based on screening techniques conducted AD11, G16 and BB13 could possibly be potential candidates for starter culture development for dairy fermentation.