The active peptides in Qingke baijiu fermented from Qingke (highland barley) are rarely reported. This work was designed to accurately identify peptides in Qingke baijiu and evaluate their angiotensin-converting enzyme inhibitory activities in vitro. Four novel peptides, Val-Val-Thr-Gly-Val-Gly-Gly-Gln (VVTGVGGQ), Leu-Pro-Val-Gly-Pro (LPVGP), Leu-Leu-Ser-Pro-Pro (LLSPP), and Phe-Pro-Leu-Gln-Pro-His-Gln-Pro (FPLQPHQP) were identified by Nano-UPLC-MS/MS. Molecular docking showed that LPVGP and FPLQPHQP had a high affinity with ACE (binding energy −8.78, −10.02 kcal mol⁻¹), which matched its in vitro ACE inhibitory activity (IC₅₀ 9.05, 5.03 µM). This might be related to their high hydrophobicity. Moreover, three peptides have C-terminal proline, which may contribute to their anti-digestive activity. The content of LPVGP was over 91.23% after digestion, while the content of VVTGVGGQ dropped to 55.47%. Finally, the four peptides have no obvious toxicity to Caco-2 cells. This study clarifies the ACE inhibitory activity and the structure–activity relationship of the four peptides identified in Qingke baijiu.

青稞（青稞）发酵而成的青稞白酒中的活性肽很少见报道。本工作旨在准确鉴定青客白酒中的多肽，并在体外评估其对血管紧张素转化酶的抑制活性。四种新型肽，Val-Val-Thr-Gly-Val-Gly-Gly-Gln (VVTGVGGQ)、Leu-Pro-Val-Gly-Pro (LPVGP)、Leu-Leu-Ser-Pro-Pro (LLSPP) 和通过 Nano-UPLC-MS/MS 鉴定 Phe-Pro-Leu-Gln-Pro-His-Gln-Pro (FPLQPHQP)。分子对接表明 LPVGP 和 FPLQPHQP 与 ACE 具有高亲和力（结合能 -8.78，-10.02 kcal mol ^-1），与其体外ACE 抑制活性相匹配（IC ₅₀9.05, 5.03 µM)。这可能与它们的高疏水性有关。此外，三种肽具有 C 末端脯氨酸，这可能有助于它们的抗消化活性。消化后LPVGP含量达91.23%以上，而VVTGVGGQ含量降至55.47%。最后，这四种肽对Caco-2细胞没有明显的毒性。本研究阐明了青客白酒中鉴定的四种肽的 ACE 抑制活性和构效关系。