###### Abstract  
In this study, thermo-tolerant strain of _Lactobacillus bulgaricus_ (_L. bulgaricus_) was developed using gradual increase in temperature to induce Adaptive Laboratory Evolution (ALE). Viable colony count of 1.87 \(\pm\) 0.98 log cfu/mL was achieved at 52 \({}^{\circ}\)C, using MRS agar supplemented with 2% lactose. Changes in bacteria morphology were discovered, from rod (control) to filament (52 \({}^{\circ}\)C) to cocci after frozen storage (\(-\)80 \({}^{\circ}\)C). When milk was inoculated with thermo-tolerant _L. bulgaricus_, lactic acid production was absent, leaving pH at 6.84 \(\pm\) 0.13. This has caused weakening of the protein network, resulting in high whey separation and lower water-holding capacity (37.1 \(\pm\) 0.35%) compared to the control (98.10 \(\pm\) 0.60%). Significantly higher proteolytic activity was observed through free amino acids analysis by LC-MS. Arginine and methionine (237.24 \(\pm\) 5.94 and 98.83 \(\pm\) 1.78 \(\upmu\)g/100 g, respectively) were found to be 115- and 275-fold higher than the control, contributing to changing the aroma similar to cheese. Further volatile analysis through SPME-GC-MS has confirmed significant increase in cheese-aroma volatiles compared to the control, with increase in diacetyl formation. Further work on DNA profiling, metabolomics and peptidomics will help to answer mechanisms behind the observed changes made in the study. Lactobacillus bulgaricus; adaptive laboratory evolution; volatiles; amino acids 2021102944. [[https://doi.org/10.3390/foods10122944](https://doi.org/10.3390/foods10122944)]([https://doi.org/10.3390/foods10122944](https://doi.org/10.3390/foods10122944))  
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of LAB fermentation, other flavour compounds such as diacetyl, acetoin, acetaldehyde, or acetic acid create specific flavour profiles for yoghurts produced from _Streptococcus thermophilus_ and _L. bulgaricus_. _L. bulgaricus_ is an aerobic to anaerobic homoformentative bacteria that uses the Embden-Meyerhof-Parnas glycolytic pathway (EMP) [6]. In lactic acid fermentation, _L. bulgaricus_ produces hydrogen peroxide through oxidation of reduced NADH to NAD\({}^{+}\), resulting from the conversion of pyruvate into lactate. As a homoformentative lactic acid bacteria, it produces lactic acid as the primary by-product, along with proteolytic and diacetyl as well as organic acids and aromatic compounds [7]. Diacetyl is extensively used as food and beverage flavouring. Diacetyl formation in yoghurt is one of the most essential flavour contributors due to the low threshold for consumers to detect its presence. Diacetyl can be recognised by its distinctive buttery aroma in amounts as small as 1 mg/kg, in commercial yoghurt products, with a range from 0.2 to 3 mg/kg of diacetyl for a balanced buttery flavour in yoghurt [8; 9]  
Recently, the use of different LAB has been trialed to accelerate the acidification, and to enhance the health benefits of yoghurt [10]. This approach has also resulted in favorable changes in nutritional and sensorial properties in yoghurt products to satisfy the demand over novel dairy products. Productivity and cost are the primary concern by the industry producing fermented foods. An increasing number of studies have demonstrated the selection of new LAB strains with different and specific functional properties, such as stress tolerance and production of molecules (enzymes, polysaccharides, aromatic compounds) [11; 12]. Stress tolerance generally includes different kinds of variations during fermentation: temperature, pH, osmotic stress, and others.