Background <p>Lactic acid bacteria (LAB), extensively used in milk fermentation, enhance its functional properties by generating bioactive peptides, including Angiotensin-converting enzyme (ACE) inhibitory peptides that effectively lower hypertension. This study aimed to optimize ideal fermentation conditions for producing ACE-inhibitory peptides in milk from different cowbreeds using LABs.</p> Methods <p>The effect of temperature (25–45 °C), inoculum amount (1–9%), and incubation period (1–5 days) on the ACE inhibition capacity of fermented cow milk was explored using a single-factor experiment. Kohonen’s Self-Organizing Maps (KSOMs), which categorized samples based on similar characteristics, were used to examine the results. With Silhouette scores ranging from 0.98 to 0.99 and Davies–Bouldin values between 0.17 and 0.26, the analysis produced positive results, indicating that the sample groups were well-defined. Additionally, pH, proteolytic activity, and titratable acidity of the fermented samples were analyzed to further understand the biochemical changes occurring during fermentation.</p> Results <p>Results showed that fermenting Holstein Friesian and American cow milk with a combination ofLactobacillus plantarumandPediococcus acidilacticistrains at 37 °C by 1% inoculum led to high ACE inhibitory activity. Specifically, fermentation for 24 h in Holstein Friesian cow milk achieved 89.80% in hibition, while a longer fermentation period of 48 h inAmerican cow milk resulted in 89.59% inhibition.</p> Conclusion <p>Thus, findings demonstrate that using a specific combination of LAB strains under optimized fermentation conditions can produce fermented cow milk with ACE-inhibitory peptides, which could be incorporated into functional foods or supplements to promote health.</p> Graphical Abstract