Lactic acid bacteria (LAB) has been studied excessively to create positive changes in nutritional and sensorial properties in yoghurt products. The aim of this approach is to meet market demand for novel dairy products and increase cost efficiency and productivity. Creating new LAB strains with distinct functional properties such as stress tolerance and molecule production would be one of the most efficient ways to meet the requirement motioned above. A mutant strain of Lactobacillus bulgaricus (L.bulgaricus) was developed by increasing temperature gradually with Adaptive Laboratory Evolution (ALE). A viable count of the control strain on De Man, Rogosa and Sharpe agar (MRS) agar with 2% lactose (LMRS) was 4.66 ± 0.59 log cfu/mL. The colony count of the mutant strain was 1.87 ± 0.98 log cfu/mL. The morphology of the mutant changed became more elongated when compared to the wild type strain as the incubation temperature increased to 52°C. This morphology change remained stable after storing the samples at -80°C for 3 weeks. When mutant L.bulgaricus was used to produce yoghurt, lactic acid was absent, resulting in a pH of 6.84± 0.13 compared to the control (pH 4.55 ± 0.04). The low acid production has caused a weakened protein network, and shrinkage of the milk gel structure, resulting with a significantly lower water holding capacity of 37.1 ± 0.35% compared to the control (98.10 ± 0.60). Free amino acids analysis by LC-MS showed high proteolytic activity. Arginine and methionine were found 115 and 275 folds higher than the control, (237.24 ± 5.94, 98.83 ± 1.78 µg/100g), which changed the aroma of the yoghurt to be more like cheese. VOCs analysis through SPME-GC-MS discovered both control (313.62 ± 0.20 µg/L) and mutant yoghurt samples (844.79 ±0.13 µg/L) showed high diacetyl production. In the mutant yoghurt samples, esters such as ethyl butanoate, ethyl butanoate, and ethyl decanoate were higher than the control strain, contributing to a more cheesy-like flavour. These suggest that applying ALE to probiotics produce new peptides and free amino acids to increase the nutritional value of dairy products.