## 3 Result and Discussion
### Level of Fitness for Thermo-Tolerant L. bulgarcus and pH  
_Lactobacillus_ feed on lactose during fermentation and produce lactic acid, which is the major contributor to flavour and acidity of yoghurt. About 20 to 40% of lactose is transformed into lactic acid during fermentation, resulting in around 0.9% of the lactic acid [29]. In the current study, L-lactic acid and D-lactic acid produced by the thermo-tolerant strain of _L. bulgarcus_ were compared against the control (grown at 42 \({}^{\circ}\)C) to determine whether the mutation has caused a decline in its primary role of producing lactic acid or not. Measurements of lactic acid was taken from inoculated milk samples that were produced by inoculating with the two bacterial strains: thermo-tolerant and wild-type control. In the inoculated milk produced from the control strain, 7.62 \(\pm\) 0.23 log CFU/mL from LMRS was inoculated, and 7.41 \(\pm\) 0.36 log CFU/mL was observed post fermentation. However, with the thermo-tolerant mutant strain, a large drop in viable counts was observed between inoculated and post fermentation of 4.66 \(\pm\) 0.59 and 1.87 \(\pm\) 0.98 log CFU/mL, respectively. This result indicated that the weaker thermo-tolerant _L. bulgarcus_ mutant loses viability during the fermentation at higher temperatures.  
L-lactic acid is the predominant metabolite formed during fermentation of milk. D-lactic is an isomer, found in small amount in yoghurt that causes acidosis [44]. Both L- and D- lactic acid isomers were found in the control inoculated milk samples; 5.40 \(\pm\) 0.20 and 2.13 \(\pm\) 0.80 g/L, respectively, where neither of the lactic acid isomers were found from milk inoculated with the thermo-tolerant strain. The result may be explained by the lack of lactate dehydrogenase, which is the primary enzyme for lactic acid production. A decrease in interconversion of pyruvate and lactate using NADH and NAD\({}^{+}\) may have taken place, as proposed by [45]. Another possible explanation could be that by-product (ethanol or acetic acid) formation may have interfered with the lactic acid production [46].  
There was a significant (\(p\) < 0.05) difference in pH between the inoculated milk samples made from the two different strains. Samples made from the thermo-tolerant strain had average pH of 6.84 \(\pm\) 0.13, which was much higher than the average pH of yoghurt in general, 4.2 to 4.6, and compared to the control in the study, 4.55 \(\pm\) 0.04. Low pH in inoculated milk results from precipitation of casein and their coagulation, producing dense texture. With the absence of lactic acid in inoculated milk made with the thermo-tolerant strain, low level of casein cross-linking would have taken place to form the gel network. Although [PERSON] et al. (2001) [47] claims that the use of a high incubation temperature for yoghurt production increases the rate of acidification, the concept was not applicable to having bacteria grown in higher temperature as a starter culture. Despite the higher pH, the texture of the inoculated milk after fermentation remained firm, and this could be explained by the production of EPS from the bacteria. Further testing of the production of EPS will be required to verify this explanation.