**Background:** Citrus canning generates large volumes of wastewater rich in pectic polysaccharides, which are currently discarded, causing environmental problems. Pectin is a complex heteropolysaccharide composed of homogalacturonan (HG), rhamnogalacturonan-I (RG-I), and rhamnogalacturonan-II (RG-II) domains. RG-I, the 'hairy region,' consists of a backbone of repeating GalA and rhamnose with neutral sugar side chains (arabinose, galactose). Pectin is considered a prebiotic candidate due to its indigestibility and fermentability by gut microbiota, and slowly fermentable carbohydrates may be more beneficial than rapidly fermentable ones because they can reach the distal colon. However, the relationship between pectin structure—particularly the RG-I domain—and fermentation characteristics remained unclear. This study aimed to recover RG-I pectic polysaccharides from citrus canning wastewater and evaluate how the proportion of the RG-I domain influences in vitro fermentation characteristics.
**Methods:** Three pectic polysaccharides were extracted from citrus segment membranes using an acid–alkali sequential extraction method. RG-46 was from satsuma mandarin (0.4% citric acid/0.1% HCl for 40 min, then 0.2% NaOH/0.1% KOH for 10 min). RG-67 was from sweet orange under similar conditions (50 min acid treatment). RG-56 was obtained by repeating the RG-67 extraction three times. The RG-I domain content was calculated as RG-I (%) ≈ 2Rha(mol%) + Ara(mol%) + Gal(mol%). Monosaccharide composition was analyzed by HPLC after PMP derivatization. Molecular weight was measured by HPSEC-MALLS-RI. Degrees of methyl esterification (DM) and acetylation (DA) were determined by HPLC. In vitro fermentation used fresh feces from six healthy donors in an anaerobic chamber at 37°C for 24 h. Samples were collected at 0, 4, 8, 12, and 24 h. SCFAs were quantified by GC-FID. Gut microbiota was analyzed by 16S rDNA V4 region sequencing on the Illumina Miseq platform. Statistical analysis used one-way ANOVA (p < 0.05).
**Key Results:** The three pectic polysaccharides differed primarily in RG-I domain proportion: RG-46 (46%), RG-56 (56%), and RG-67 (67%). GalA, Ara, and Gal were the main monosaccharides (>30%, >20%, and >10%, respectively). Molecular weights were similar (RG-67 highest, RG-46 lowest), and DM was low (~10%) with negligible DA. During fermentation, pH decreased in all groups; RG-67 showed the greatest pH decline. Total SCFA concentrations at 24 h ranked: RG-67 > RG-56 > RG-46 > FOS > blank. Acetate was the dominant SCFA, followed by propionate and butyrate. RG-67 produced the most acetate, propionate, and butyrate. HPSEC showed that 0–4 h was the main degradation period, with nearly half of substrates utilized. RG-67 degraded more slowly but was nearly exhausted by 24 h. Neutral sugars (arabinose, xylose, galactose, mannose) were utilized more readily than acidic sugars (GalA, rhamnose). Alpha diversity indices (Sobs, Chao, Ace, Shannon, Simpson, Heip) showed that all three RG-I pectins outperformed FOS in promoting microbiota richness and diversity. At the phylum level, Bacteroidota and Actinobacteriota increased in all treatment groups. At the genus level, RG-I pectins enhanced Bacteroides, Phascolarctobacterium, Bifidobacterium, and Prevotella while reducing Escherichia-Shigella and Klebsiella. At the species level, Bacteroides_vulgatus, Phascolarctobacterium_faecium, Bifidobacterium_pseudocatenulatum, and Bacteroides_thetaiotaomicron were enriched. Eubacterium_eligens_group and Monoglobus showed particular preference for RG-I pectins, with abundances positively correlated with RG-I domain proportion. LEfSe analysis identified Phascolarctobacterium, Sutterella, and Lachnospira as biomarkers for RG-46; Bacteroides, Collinsella, and Eubacterium_eligens_group for RG-56; and TM7x, Monoglobus, Granulicatella, and Lachnospiraceae_NK4A136_group for RG-67. PICRUSt analysis showed RG-67 had the highest functional abundance in carbohydrate transport and metabolism, cell wall/membrane biogenesis, and energy production. Pearson correlation analysis revealed that Ara was highly positively correlated with Bacteroides, Bifidobacterium, Monoglobus, and SCFA accumulation. Gal was negatively correlated with Escherichia-Shigella (p < 0.01). GalA was positively correlated with Bifidobacterium (p < 0.05).
**Clinical Implications:** This study demonstrates that RG-I-rich pectic polysaccharides recovered from citrus canning wastewater have strong prebiotic potential, exceeding that of FOS in SCFA production and beneficial gut microbiota modulation. The slow fermentability of high-RG-I pectins (especially RG-67) may be particularly advantageous for delivering fermentable carbohydrates to the distal colon, potentially reducing harmful protein fermentation and its associated metabolites. The selective promotion of butyrate-producing bacteria such as Eubacterium_eligens_group and Collinsella, along with the suppression of pathogenic genera like Escherichia-Shigella and Klebsiella, suggests potential benefits for intestinal health, including anti-inflammatory and anti-tumor effects. These findings support a strategy for food processing industries to recover functional ingredients from wastewater, enabling both value addition and environmental sustainability. However, the authors note that the structure–function relationship and health effects need further confirmation through in vivo studies.