ABSTRACT: Milk, renowned for its nutritional value, harbours bioactive compounds, including lactoferrin (Lf) [1], immunoglobulins, and functional proteins suggesting broader functionalities beyond nutrition. Lf, an iron glycoprotein, exhibits antimicrobial properties against bacteria, fungi, and viruses."In"this st"dy, Immune Powder (a functional dairy formulation) and Fractionated Milk Protein (FMP; a blend of bioactive proteins isolated from skim milk) containing Lf, zinc, and immunoglobulins produced by Ausnutria Australia Dairy Pty Ltd were evaluated for their broad spectrum pharmacological activity. In particular, the study aims to investigate the antibacterial (against pathogenic Escherichia coli), prebiotic (promoting Lactobacillus delbrueckii growth), anti-inflammatory (against RAW264.7 macrophages), and antiviral (against human coronavirus 229E, HCoV-229E cultured on MCR-5 cells) effects of the formulations. In addition, the impact of simulated gastric digestion on the efficacy of the formulations was explored. LCMS-based proteomics analysis was implemented to unveil cellular and molecular mechanisms underlying antiviral activity. The Immune Powder demonstrated antibacterial activity against E. coli (up to 99.74 ± 11.47% inhibition at 62.5 mg/mL), coupled with prebiotic action (10.84 ± 2.2 viability fold change at 125 mg/mL), albeit diminished post-digestion (p <0.01). The Immune Powder effectively mitigated inflammation by reducing NO production, with efficacy declining post-digestion (p < 0.0001). The potential antiviral activity of Immune Powder against HCoV-229E also shown an increase of 2.62 ± 0.42 in cell viability when treated at 500 µg/mL. Similarly, FMP exhibited antibacterial potency pre-digestion at high concentration (95.56 ± 1.23% inhibition at 125 mg/mL), and post-digestion at lower doses (61.82 ± 5.58% inhibition at 3906.25 µg/mL). FMP also shown enhanced prebiotic activity post-digestion (p < 0.0001), anti-inflammation pre-digestion, and significant antiviral activity against HCoV-229E (1.72 ± 0.52 cell viability fold change). The proteomics study suggested both formulas shared similar antiviral mechanism by inhibiting scavenger receptor binding and ECM interaction.
Muhammad Alsherbiny 
