## 3 Results and Discussion
### Amino Acid Composition  
Amino acid analysis was carried out using LCMS to determine changes in amino acid composition in the kefir-fermented coconut water. All the nine experimental sets of CWK (Table 1) were analysed at 0, 24, 48, 72, and 96 h. The PCA-2D score plot shown in Figure 3A described 75.9% and 7.7% of the total variances for PC1 and PC2, respectively. The scores for the different kefir-fermented coconut waters were clearly separated according to fermentation time (T = 0, 24, 48, 72, and 96 h). Samples at time T = 0 had negative scores along PC1. With increasing fermentation time (T= 24, 48, 72 and 96 h), the sample scores became increasingly positive. The PCA biplot in Figure 3B, generated for all the 18 identified amino acids showed both the scores and the loadings of CWK fermented at 0, 24, 48, 72, and 96 h. The 0 h (control) samples had high negative scores that corresponded to high negative loadings for tyrosine, valine, leucine, isoleucine, methionine, and phenylalanine. This finding was supported by the ANOVA results shown in Tables S7 and S8, which showed significantly higher values for tyrosine, valine, leucine, isoleucine, methionine, and phenylalanine in 0 h experimental sets.  
With increasing fermentation time, samples fermented for 24, 48, and 72 h had high positive scores, which corresponded to the high positive loadings of glutamic acid. All the samples fermented for 24, 48, 72, and 96 h had positive scores, which corresponded to high positive loadings of glutamic acid. These observations are supported by results summarised in the ANOVA table shown in Tables S7 and S8, which showed significantly higher values for glutamic acid, particularly in samples fermented for 24, 48, and 72 h. The presence of glutamic acid in these fermentations is desirable in the current study as this amino acid could potentially enhance the flavour profile of CWK beverage. The 95% confidence ellipse can be seen in Figure 3A. The ellipses clearly define statistically significant class separation. The ellipsoid for 96 h samples was wide vertically, with a variance that can be explained along PC2. The ellipses for samples fermented for T = 48 and 72 h overlapped tightly and corresponded to high positive glutamic acid loading along PC1, as seen in the PCA-biplot in Figure 3B. Glutamate dehydrogenase activity in LAB, which leads to the production of glutamic acid, is a very important criterion for selecting the strains utilised for producing fermented foods, such as cheese. The production of glutamate from \(\alpha\)-ketoglutarate by LAB produces flavour compounds which are associated with the formation of desirable aromas in fermented foods [63]. Glutamate, the anionic form of glutamic acid, can be further converted into glutamine or gamma-aminobutyric acid (GABA). GABA possesses several well-known physiological functions, such as anti-hypertension [64] and anti-diabetic [65]. Studies carried out on sourdough show that glutamine deamidation by LAB during wheat sourdough fermentation could account for the increase in glutamate levels that could positively impact the flavour of bread [66]. An increased production of glutamic acid with incubation time has also been reported for the fermentation of kefir grains in cow and buffalo milk [47].  
Changes in the amino acid profile can be related to the differences in the number of microorganisms that thrive in the different sugar mediums during CWK fermentation. A similar study was carried out on the water kefir microflora by [PERSON] and [PERSON] in 2002 [67], who reported that yeasts in kefir are responsible for the breakdown of sucrose into glucose and fructose units. [PERSON] et al. [39] further reported that the presence of amino acids in milk kefir may be due to the combined effects of the release of amino acids during fermentation, assimilation of peptides, and proteolytic activity.