## 3 Results
### Effects of NatuREN G on Fecal Microbiota of HC and CKD Subjects  
To evaluate the in vitro effect(s) of NatuREN G on CKD, viable bacterial cell counts were determined after adding fresh feces from HC and CKD subjects to fecal media, with the aim of mimicking the composition of gut microbial community. Cultivable bacteria in HC and CKD subjects were estimated at baseline (T0) and after 24 h of incubation at 37 \({}^{\circ}\)C, with or without NatuREN G, under anaerobic conditions. The results of cell densities of the main gut cultivable bacterial groups were elaborated using principal components analysis (PCA). At T0, CKD samples were mainly characterized by high _Enterobacteriaceae_ and staphylococci and low lactic acid bacteria (LABs) and lactobacilli (Figure 9). The opposite was found in HC samples at T0. The addition of NatuREN G determined after 24 h showed a shift in CKD samples (CKD-NG-(24) toward the side of the PCA factor-plan wherein all the HC samples had fallen. This was mainly determined by the increase in the viable cell count of both LABs and lactobacilli. Differently, 24 h after adding NatuREN G to HC samples (HC-NG-(24), an increase in viable cell count of the groups _Bacteroides-Prevotella-Porphyromonas_, _Pseudomonas-Aeromonas_, and particularly of the bifidobacterial taxa, was found. This increase was related to decreases in total bacterial, total anaerobes, and _Enterococcus_. Few differences in the HC and CKD samples where NatuREN G had not been added were found compared with T0. Figure 9: Principal components analysis (PCA) of the main cultivable bacteria found in fecal batches fabricated using sterilized fecal media with the addition of feces from healthy (HC) or nephropathic (CKD) subjects. Data obtained from three independent analyses were collected from baseline samples (sterilized fecal media with fresh feces added, named t0), after 24 h of incubation at 37 \({}^{\circ}\)C (t24), and 24 h at 37 \({}^{\circ}\)C after adding the innovative symbiotic NatuREN G (NG-t24). The analysis of volatile organic compounds (VOCs) showed that NatuREN G produced significant variations in VOCs, particularly in fecal batches obtained from CKD patients (CKD + NGt24). Few differences (e.g., decreases in nonanoic acid and 1-pentanol) were found in batches obtained from HC fecal extracts (Figure 10). Decane increased in HC (HC + NGt24) and CKD (CKD + NGt24) fecal batches after 24 h of the addition of the innovative symbiotic. Similar to HC + NG, the nonanoic acid decreased in CKD at t24. Different from HC + NGt24, acetic acid was significantly increased in CKD + NGt24 batches. Interestingly, CKD + NGt24 contained a higher concentration of propanoic acid than HC + NGt24. Additionally, in CKD samples where NatuREN G was not added, an increase in dimethyl trisulfide was found. Conversely, dimethyl trisulfide was significantly decreased after 24 h of incubation of the CKD fecal batch with the addition of NatuREN G.  
An analysis of uremic toxins by nuclear magnetic resonance (NMR) was carried out with fresh fecal samples obtained from CKD and HC subjects to evaluate the effects of NatuREN G after 24 h of incubation. Parallel fecal samples, without the addition of NatuREN G, were used as controls. The uremic toxin detection was also evaluated immediately after the addition of fresh feces to fecal media (t0). No significant differences were found in HC samples, comparing concentrations of uremic toxins in both the incubated HC samples (with and without NatuREN G, HCt24 and HC + NGt24, respectively) with the baseline values (HCt0) (Figure 11A). Conversely, indole, \(p\)-cresol, and \(p\)-cresyl sulfate significantly increased after 24 h of incubation in CKD samples without the addition of NatuREN G (CKDt24). Furthermore, a significant decrease in indoxyl sulfate was detected comparing CKD + NGt24 to the relative baseline samples (CKDt0) (Figure 11A).