## 3 Results
### Acidification and Growth of Probiotics in Presence of Antioxidants and Food Matrices
More specifically, the following six experimental treatments were tested:  
* The-1: HC fecal media + FOS + inulin. * The-2: HC fecal media + FOS inulin + Pom.S.E. * The-3a: HC fecal media + FOS + inulin + tomato extract variety ResTom. * The-3b: HC fecal media + FOS + inulin + tom. ex. var. Indigo. * The-3c: HC fecal media + FOS + inulin + tom. ex. var. Bronze. * The-3d: HC fecal media + FOS + inulin + tom. ex. from WT tomato. In addition, HC fecal medium with only glucose added was used as the control. Each fecal medium was inoculated with the selected probiotics. After 24 h of incubation, most of the probiotics acidified the fecal media, showing a \(\Delta\)pH ranging from 0.2 to 0.4 (Figure 5A). _B. animalis_ BLC1 showed the highest \(\Delta\)pH (value: 0.78) when inoculated in The-1, whereas _B. breve_ 15A showed the highest \(\Delta\)pH (value: 0.60) in The-2. Probably due to the production of basic compounds (e.g., NH\({}_{3}\)), some strains showed a pH increase, specifically _Lc. casei_ LC4P1 grown The-1 and The-3d (\(-\)0.18 and \(-\)0.19, respectively) and _Lrp. plantarum_ ONI3 grown in The-2 (\(-\)0.15). Figure 5: (**A**) Heatmap of the fecal media acidification degree (\(\Delta\)pH) after 24 h of probiotic growth at 37 \({}^{\circ}\)C; values range  
from dark orange (lower \(\Delta\)pH values, \(<\)25 th percentile) to dark green (higher \(\Delta\)pH values, \(>\)75 th percentile), while light yellow values correspond to intermediate \(\Delta\)pH values (50 th percentile). All fecal media were obtained using pooled fecal extracts of healthy subjects (HC) adding a low content of glucose (2 g/L). Prebiotic supplements (FOS and inulin) were added at a concentration of 2.5 g/L, while antioxidant ones (Pom.S.E. and tomato extracts) were added at a concentration of 5 g/L. Experimental treatments: The-1, HC fecal media + FOS + inulin; The-2, HC fecal media + FOS + inulin + Pom.S.E. ; The-3a, HC fecal media + FOS + inulin + tomato extract var. ResTom; The-3b, HC fecal media + FOS + inulin + tomato extract var. Indigo; The-3c, HC fecal media + FOS + inulin + tomato extract var. Bronze; The-3d, HC fecal media + FOS + inulin + tomato extract from WT tomato. (**B**) Probiotics viable cell count (log CFU/mL) after 24 h of growth at 37 \({}^{\circ}\)C in fecal media obtained from pooled fecal extracts of healthy subjects (HC) with the addition of a low content of glucose (2 g/L). Control (C): fecal medium with pooled HC fecal extracts + glucose (2 g/L). The experimental treatments (The-1, The-2, The-3a, The-3b, The-3c, and The-3d) are reported above. Data are presented as the average of one biological triplicate \(\pm\) SD. All the probiotics showed an increase (about 1-2 logarithmic cycles) in cell density (Figure 5B). Compared with the control, almost all probiotics showed higher cell count when cultured in the presence of prebiotics alone (The-1) or in combination with pomergnate extract (The-2). The only exception was Synbio 100 in The-2. In presence of tomato extracts (The-3a, The-3b, The-3c, and The-3d), all the strains showed cell density values similar to or lower than that of the control. Therefore, probiotics growth was assessed in fecal media prepared starting from pooled feces from patients with chronic kidney disease (CKD). The experimental treatments, in which probiotics were singly added, were:  
* FM(CKD) + FOS + inu (The-1) = CKD fecal extract + glucose (2 g/L) + FOS (2.5 g/L) + inulin (2.5 g/L);
* FM(CKD) + FOS + inu + Pom.S.E. (The-2) = CKD fecal extract + glucose (2 g/L) + FOS (2.5 g/L) + inulin (2.5 g/L) + pomergnate seed extract (5 g/L). In addition, a CKD fecal medium, with the addition of glucose only, was used as the control, called FM(CKD). Except for _B. animalis_ 13A grown in the presence of both prebiotics and pomergnate, all the probiotics showed an increase of at least one logarithmic cycle. Compared with the control, lower viable cell counts were found for _B. animalis_ 13A and Synbio 100 when grown in the presence of prebiotics and pomergnate seed extract.