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Aims: Present study was conducted to optimize the processing parameters for galactooligosaccharides (GOS) synthesis from whey powder followed by exploring its prebiotic efficiency., Main Methods: All factors (initial lactose concentration, pH, reaction time, temperature and enzyme to substrate ratio; E/S) were analyzed by single factor analysis and optimization for GOS yield was done following the orthogonal experimental design. For in vivo analysis, 60 mice were equally divided into four groups (normal control, NC; low, medium, and high dose of GOS, LG, MG and HG) and fed varying levels (0, 0.25, 0.5 and 1.0 g/kg bw per day) of GOS, for 30 days and sampling was done at the end of experiment regarding gut health, immunity, cecal microbiota and metabolites., Key Findings: Optimum yield of GOS (25.1%) was obtained at reaction time 25 min, temperature 50 °C, pH 4.5 and the enzyme to substrate ratio (E/S) of 20 U/g. In vivo experiment, shallower crypt and greater villus to crypt ratio (V/C) were found in the duodenum of LG treatment compared to NC mice (P < .05). The GOS promotes thymus development and improve immunity. Intervention with GOS increased the population of bifidobacterium and lactobacillus in MG and bifidobacterium in LG mice (P < .05), and was accompanied by decreased proliferation of desulfovibrio. Correlation analysis also supported the above findings., Significance: This study optimized the processing parameters for GOS preparation and provided data encouraging to suggest that GOS can be a potential option to improve the gut health and immunity., (Copyright © 2020 Elsevier Inc. All rights reserved.)