## 3 Result and Discussion
### Physicochemical Properties
#### 3.3.4 Volatile Compounds  
A total of 56 volatile compounds were detected by SPME-GC-MS in both inoculated milk samples, which exceeded the findings of FAEs in other yoghurt studies reported in the literature. With the use of _Streptococcus thermophilus_ as a single starter strain in yoghurt fermentation, 53 volatile compounds have been reported [64], where 31 volatile compounds were attributed to the use of cow milk [65]. The 56 volatile compounds detected in this study were sub-classified into chemical groups as shown in Table 2, as 10 carboxylic acids, 8 aldehydes, 11 ketones, 17 alcohols and 10 esters. Although many volatile compounds were detected, not all of them contribute significantly to the overall flavour and aroma profile of yoghurt. According to the literature, the major volatile compounds found in yoghurt are acetaldehyde, ethanol, acetone, diacetyl, and 2-butanone, and these are known to have a significant impact on the flavour [29; 66; 67]. However, acetaldehyde was not detected from neither of the inoculated milk samples tested in this study. Similar finding was reported by [PERSON] et al. (1986), suggesting that acetaldehyde had not formed from pyruvate due to the absence of \(\alpha\)-carboxylate or aldehyde dehydrogenase which generate acetaldehyde in _L. bulgarcius_[68]. However, acetic acid was found in both samples. A large difference between the control and the thermo-tolerant strain inoculated milk was found in hexanoic acid, octanoic acid and benzoic acid, where a double or more of the concentration was found in the control. Except for oxalic acid and 2-methyl-propanoic acid, the other carboxylic acids detected in milk inoculated with the thermo-tolerant strain were much lower in concentration compared to the control. 3-methyl-butanoic acid was found in both of the samples, which must have been converted from 3-Methylbutanal through leucine catabolism, as shown in many dairy products, contributing to the pleasant fresh cheese aroma [69; 70]. A high amount of diacetyl (2,3-butanediol) production was evident in both samples made from the control and the thermo-tolerant strain, 313.62 \(\pm\) 0.20 and 844.79 \(\pm\) 0.13 \(\mathrm{\SIUnitSymbolMicro g}\)/L, respectively. This compound is known to carry buttery and cheese-like odour, which was found to be approximately 2.7 times higher than the control, shifting the overall volatile profile to be more on cheese-like side. This may be partly explained by the use of growth medium supplemented with lactose in the current study. Growth medium used for LAB has been reported to play a significant role in diacetyl production. According to [PERSON] et al. (2016) [71], MRS media enriched with glucose and citrate supplement increased more than 3 fold in diacetyl production compared to the minimal media. We have attempted to grow _L. bulgarcius_ in both MRS media only and MRS supplemented with lactose. From our preliminary experiments, survival rate of _L. bulgarcius_ was much higher with the increase in temperature through ALE with supplementation of lactose, as additional nutrient. Similar finding has been reported in _L. lactis_ grown in modified culture mediums (potassium phosphate buffer with 0.5% glucose; reconstituted skim milk with 1% glucose), which over-expressed NADH oxidase to re-route the metabolic flux away from lactate production against oxidised product in metabolic pathway of citrate, resulting in increased diacetyl production to 0.36 to 0.38 g/L [72; 73]. Among the 11 ketone compounds detected, the key contributors to the overall flavor profile were 2,3-butanedione (313.62 \(\pm\) 0.20 and 844.79 \(\pm\) 0.13 \(\mathrm{\SIUnitSymbolMicro g}\)/L), 2-heptanone (126.81 \(\pm\) 0.04 and 2045.73 \(\pm\) 0.35 \(\mathrm{\SIUnitSymbolMicro g}\)/L) and 2-nonanone (50.58 \(\pm\) 0.27 and 12.50 \(\pm\) 0.77 \(\mathrm{\SIUnitSymbolMicro g}\)/L), in milk inoculated with the control and the thermo-tolerant strain, respectively. Ester biosynthesis by lipase and LAB is favoured by alcoholysis in fermented dairy products [74].