## 2 Sources and Composition of Human Saliva: Host and Microbial Contributions
Figure 1: Partial \({}^{1}\)H NMR profiles of **a** WMS supernatant sample collected from a healthy human participant, stored and prepared for analysis according to our protocols described in Section 6. (**a**) and (**b**,**c**) show the 0.80-2.50, 2.55-4.20, and 5.30–8.60 ppm regions of spectra acquired. (**d**) 5.30–8.60 ppm region of the spectral profile acquired on a WMS supernatant collected from  
a second healthy participant. Abbreviations: Assignment codes correspond to those listed in Table 1. Tentative assignments only are also indicated in Table 1. Single-pulse \({}^{1}\)H NMR spectra were recorded on a Bruker Avance 600 spectrometer operating at frequency of 600.13 MHz, with a probe operating temperature of 20 \({}^{\circ}\)C. Spectra were acquired with suppression of the large H\({}_{2}\)O/HOD resonance (\(\delta\) = 4.80 ppm) using radiofrequency pulse presaturation. Pulsing conditions were sweep width 8389 Hz; 5 us pulses; 1.0 s pulse repetition rate; 32,768 (subsequently zero-filled to 65,536) datapoints; 128 transients. An exponential line-broadening function of 0.30 Hz was applied to FIDs before Fourier transformation. Typical spectra are shown. Although previous \({}^{1}\)H NMR-based metabolomics investigations of PS and SM/SL saliva samples have been very restricted in the context of poor spectral resolution, limited numbers of participant donors, and gland inflammation complications for the latter [22, 23], [PERSON] et al. [7] compared the \({}^{1}\)H NMR profiles of PS and SM/SL saliva samples collected from the same donors and concluded that there were no major differences between them. However, although understandable because of collection problems, unlike those experienced with WMS, the relatively limited sample size involved (n = 10) may be insufficient to draw robust conclusions from this, and therefore the authors recommend that a full \({}^{1}\)H NMR-based metabolomics investigation should be performed in order to seek, and possibly confirm and verify, any metabolic differences detectable, if indeed justified. Particularly noteworthy is the phenomenon that, as noted by [PERSON] et al. [7], the overall physiological significance of salivary metabolites such as short-chain organic acid anions is predominantly unknown. However, in the human colon, such bacterial catabolites, e.g., \(n\)-butyrate, propionate, lactate, and succinate have been ascribed principal actions in relation to human health. Indeed, these catabolites exert important roles in the maintenance of gut epithelial integrity, especially for \(n\)-butyrate, which is known to stimulate the tight junction expression and synchronizes colonic epithelial cell proliferation; these processes occur through the inhibition of histone deacetylase inhibition, an upregulation of the expression of apoptosis-supporting-genes [24, 25], and the ability to exert actions regarding metabolic and immune regulation functions [24, 26]. Interestingly, they also play roles in the regulating the satiety and appetite of the host [27]. In 2017, [PERSON] and [PERSON] [28] provided an overview of the generation of propionate and \(n\)-butyrate by human colonic mucosa, and they note that both these microbial catabolites exert or promote a series of favorable health-friendly actions. Such organic acid anions may arise from the bacterial degradation of dietary carbohydrates, and from amino acids liberated from the actions of proteolytic bacteria on proteins. The critical role of cross-feeding of intermediary metabolites such as lactate, succinate, and 1,2-propanediol between different species of gut bacteria was outlined, as was the influence of environmental elements such as host dietary changes, on the growth and preponderance of propionate- and \(n\)-butyrate-generating bacteria; all these metabolites are routinely detectable in the \({}^{1}\)H NMR profiles of human saliva [3].