Rhamnogalacturonan–I (RG–I) pectin recovered from sugar beet pulp (SBP) and discarded red beetroot (DRB) were subjected to enzymatic hydrolysis to obtain pectooligosaccharides (POS). The hydrolysates with the highest amount of oligomer with a degree of polymerization from 2 to 6 were selected for a detailed characterization of the composition and the structure by MALDI-TOF MS and 1H NMR. The yields of pectin and POS products ranged from 32.7% (g DRB POS/g DRB) to 37.0% (g SBP Pectin/g SBP) with the POS content from 64.4% (DRB products) to 68.7% (SBP POS). A comparative study was conducted between SBP Pectin, SBP POS, DRB Pectin, DRB POS, and commercial prebiotics (inulin and fructooligosaccharides) for their antioxidant capacity and prebiotic potential. The results revealed the much higher antioxidant capacity of DRB products (DPPH: 176.8 μmol Trolox equivalent/g pectin and 162.5 μmol Trolox equivalent/g POS) over the other products. Both bacteria strains evaluated grew with all substrate products. DRB POS and inulin exhibited the highest maximum growth rate for Lactobacillus rhamnosus (0.25 and 0.26 h−1, respectively), whereas Bifidobacteria longum grew faster on DRB Pectin (0.89 h−1). The short chain fatty acids and lactate production were also measured. Highest concentrations arose using SBP products as substrates (125.3 mM for pectin and 115.7 mM for POS with Lactobacillus rhamnosus and 87.8 mM for pectin and 95.0 mM for POS with Bifidobacterium longum). The results demonstrated the potential applications of SBP and especially DRB products in the food industry due to their high antioxidant and prebiotic properties.
