Whey protein (WP) fortification enhances sensory properties and physiological functions of fermented milk, yet the immunological and gut-microbiota impacts of altered WP-to-casein ratios remain unclear. In this study, fermented milks with varying WP-to-casein ratios were prepared using whey protein isolate (WPI), milk protein, or mixtures. Six-week gavage intervention in male Wistar rats revealed medium-low WPI doses (2.67 % and 1.33 %) increased beneficial gut bacteria including Peptostreptococcaceae, Clostridiaceae_1, Romboutsia, and Clostridium_sensu_stricto, while reducing harmful bacteria Erysipelotrichaceae. High-dose WPI (4.00 %) elevated fecal levels of short chain fatty acids, indole and its derivatives. Increasing WP-to-casein ratio to 10:8 enhanced Peptostreptococcaceae, Romboutsia, Intestinimonas (P < 0.05) while suppressing Erysipelotrichaceae. This ratio also elevated serum immunoglobulin A (IgA) and interleukin-2, colonic secretory IgA and recombinant mucin 2, improving gut barrier integrity. Thus, optimizing WP-to-casein ratio to 10:8 maximizes immunomodulatory and microbiota-regulating efficacy of protein-fortified fermented milk, providing basis for rational protein fortification strategies.
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