In Vitro Prebiotic Potential of Purified Black Rice Oligosaccharides: Simulated Digestion Stability, Short-Chain Fatty Acid Production, and Probiotic Biofilm Formation

Black rice oligosaccharides (BO) were extracted with 80% aqueous ethanol (v/v) and purified by charcoal-celite chromatography followed by dialysis using a 500 Da molecular weight cut-off (MWCO) membrane, yielding an oligosaccharide fraction with a degree of polymerisation (DP) between three and eight (DP3-DP8; 2.87 {+/-} 0.29% w/w). MALDI-TOF MS showed sodium adduct ions from m/z 527 to 1330, and GC-MS analysis of hydrolysed samples identified glucose and galactose as the major monomers, while ketosyl residues were detected in the intact fraction by selective staining and are most plausibly attributed to fructosyl units based on cereal origin and DP distribution. BO showed high resistance to simulated salivary, gastric, and pancreatic digestion (only 2.08 {+/-} 0.51%, 0.34 {+/-} 0.03%, and 4.29 {+/-} 0.73% hydrolysis, respectively) with approximately 93% remaining carbohydrate available for fermentation. All Lactobacillus strains showed positive prebiotic activity scores, with the highest response observed for Lactobacillus rhamnosus (1.165 {+/-} 0.255) and Lactobacillus plantarum (0.980 {+/-} 0.163). Fermentation produced metabolically relevant short-chain fatty acids (SCFA), mainly acetate (34.82 {+/-} 2.08 mM), as well as strain-dependent propionate and butyrate levels. BO greatly promoted probiotic biofilm formation, with biomass reaching 391.33 {+/-} 26.08% and viable cell counts of 9.01 {+/-} 0.70 log CFU/mL relative to the control. Collectively, the results indicate that BO represents a digestion-resistant, hexose-based oligosaccharide series that is selectively utilised by probiotic lactobacilli, promotes SCFA production and enhances biofilm development. To our knowledge, this work is the first to combine structural profiling with in vitro functional evaluation of a purified, low-DP oligosaccharide fraction obtained from black rice. HighlightsO_LIPurified oligosaccharides (DP3-DP8) were obtained from black rice using charcoal-celite chromatography followed by dialysis. C_LIO_LIStructural analysis confirmed that the oligosaccharides were hexose-based and composed mainly of glucose and galactose. C_LIO_LIBlack rice oligosaccharides exhibited higher resistance to simulated gastric and intestinal digestion compared with starch. C_LIO_LIPositive prebiotic activity scores were observed due to selective utilisation by probiotic Lactobacillus strains. C_LIO_LIFermentation of black rice oligosaccharides significantly increased short-chain fatty acid production. C_LIO_LIPurified oligosaccharides enhanced probiotic biofilm formation, indicating improved colonisation potential. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=179 SRC="FIGDIR/small/705216v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@9a5915org.highwire.dtl.DTLVardef@14eac7aorg.highwire.dtl.DTLVardef@1db8baorg.highwire.dtl.DTLVardef@14ad50a_HPS_FORMAT_FIGEXP M_FIG C_FIG