Gut Microbiota and Metabolic Modulation by Slow-Release Protein Substitutes in Phenylketonuria: Findings from the PREMP Study

<b>Background/Objectives</b>: Phenylketonuria (PKU) is an inherited metabolic disorder requiring early and lifelong dietary management through a low-phenylalanine (Phe) diet supplemented with Phe-free protein substitutes (PS). Recently developed slow-release PS formulations aim to mimic natural protein absorption, enhancing metabolic stability and tolerability. The PREMP study (effect of Protein RElease on the Microbiota composition and function in Phenylketonuric subjects) assessed the effects of a slow-release, Phe-free PS on gut microbiota composition and metabolic parameters in patients with PKU. <b>Methods</b>: Patients aged ≥6 years with PKU were enrolled from two Italian centers (Milan and Naples). Participants replaced ≥50% of their usual protein equivalent (P.Eq.) intake from Phe-free PS with a slow-release PS for 4 months. Clinical, biochemical, and nutritional assessments were performed at baseline and post-intervention. Gut microbiota composition was analyzed by 16S rRNA gene sequencing, and fecal fatty acids were quantified by gas chromatography-mass spectrometry. <b>Results</b>: Thirteen patients (median age 17 years) completed the intervention, replacing on average 78% of their usual P.Eq. intake with the slow-release formulation. Plasma phenylalanine and tyrosine levels remained stable, while fasting insulin (<i>p</i> = 0.0185) and HOMA-IR (<i>p</i> = 0.0099) significantly decreased, indicating improved insulin sensitivity. Anthropometric and dietary parameters showed no significant changes. Gut microbiota diversity remained stable, with modest increases in microbial richness and beneficial genera such as <i>Bacteroides</i>, <i>Bifidobacterium</i>, and <i>Gemmiger</i>, while <i>Hafnia, Anaerostipes</i> and <i>Romboutsia</i> decreased. Fecal butyrate and other fatty acids showed slight, non-significant increases. <b>Conclusions</b>: The slow-release PS was safe, well-tolerated, and improved insulin sensitivity without affecting amino acid or nutritional status. Microbial changes suggest potential benefits for gut health, warranting confirmation in larger, long-term studies.