## 3 Result and Discussion
### Physicochemical Properties
#### 3.3.3 Free Amino Acid Profile  
A total of 27 FAAs was identified in the inoculated milk samples, with total FAAs content of 225.25 \(\pm\) 37.45 and 1747.42 \(\pm\) 34.69 \(\upmu\)g/100 g, for the control and the thermo-tolerant strain, respectively. As shown in Table 1, most of the individual FAAs detected in the current study were significantly different between those made from the control and the thermo-tolerant strain (\(p\) < 0.001). Significantly higher proteolytic activity, measured from the release of FAAs [53], was found in inoculated milk produced from the thermo-tolerant strain in both total FAAs amount and also in individual FAAs. Approximately 7.76 times higher in total FAAs was found when compared to the use of control. There were number of FAAs which differed in great quantity between the two samples. Glutamic acid was found 4.8 times higher than the control sample; 265.88 \(\pm\) 5.08 and 55.27 \(\pm\) 9.67 \(\upmu\)g/100 g, respectively. Glutamate dehydrogenase (GDH) is known to use NADH or NADPH as coenzymes to produce glutamic acid from 2-oxoglutaric acid in the tricarboxylic acid cycle [54]. Mutation may have occurred through the generation of thermo-tolerant strain by ALE in the current study, and this may have caused higher activity of the GDH. As shown in Table 1 the concentration of the following amino acid found in milk inoculated with the thermo-tolerant strain increased greatly compared to the control sample: histidine (19.95 times), arginine (115.73 times), threonine (10.31 times), ornithine (55.64 times), methionine (274.52 times), valine (19.27 times), lysine (7.69 times), tyrosine (37.27 times), isoleucine (14.99 times), leucine (47.67 times), phenylalanine (22.56 times) and tryptophan (19.63 times). The observed increases in FAAs may have been caused by the release of the hydrolys polypeptide chain from storage proteins by exopeptidases [55]. It is likely that the exopeptidases have hydrolys peptide bonds in the inoculated milk and continued the hydrolysis to a higher degree compared to the control sample [56; 57]. Arginine and methionine have increased over 100 folds or more compared to the control. The increase in arginine may be explained by the high yield of glutamic acid [58] and the pH being high in the inoculated milk produced by the thermo-tolerant strain, which favours arginine production [59]. Arginine and methionine are classified as functional amino acids that helps with immune defense and as antioxidant [60]. One of the primary roles of _L. bulgarcius_ is to hydrolyse milk protein, casein, which degrade into peptides and FAAs. Some of these FAAs also contribute to odour as precursors for volatile compounds as shown in Tables 2 and 3[61; 62; 63]. With the mutation occurring in the thermo-tolerant strain, the proteolytic activity of _L. bulgarcius_ has improved. Further analysis using metabolomics and peptidomics may help to answer the change in metabolic pathways and the resultant amino acid profile. Figure 3: Shear stress against shear rate measured in milk inoculated with: (**a**) the control strain (42 \({}^{\circ}\)C) and (**b**) the thermo-tolerant strain (52 \({}^{\circ}\)C).