<p>Lactosucrose (4G-β-D-galactosylsucrose) is a functional trisaccharide that promotes gut microbiota growth; however, the high purification costs limit its industrial use. This study evaluated the growth-promoting potential of a minimally refined lactosucrose enzyme product (LSmix), containing residual sugars, on nine probiotic strains. Growth responses, acidification behavior, and lactosucrose utilization were systematically analyzed under controlled in vitro conditions. Principal component analysis (PCA) integrating OD₆₀₀, pH reduction, and lactosucrose utilization variables showed that the first three principal components (PC1–PC3) cumulatively explained 100% of the total variance, categorizing the strains into four distinct clusters. Cluster A (<i>Bifidobacterium bifidum</i>, <i>Bifidobacterium breve</i>, <i>Lactobacillus acidophilus</i>, and <i>Lactiplantibacillus plantarum</i>) showed high biomass formation, strong acidification, and efficient lactosucrose utilization. In contrast, <i>Enterococcus faecium</i> (Cluster C) showed limited metabolic activity toward lactosucrose. LSmix supported significantly higher growth than the residual-sugar control (LSminus). These results suggest that enzymatically produced, minimally refined lactosucrose mixture is a cost-effective alternative for functional food applications and probiotic fermentation.</p>