Abstract: The consumption of collagen hydrolysate (CH) has been reported to promote the healing of pressure ulcers and alleviate symptoms of osteoarthritis. The -Gly-Pro-Hyp-Gly- motif is abundantly present in collagen. The ingestion of CH has been demonstrated to significantly increase Pro-Hyp and Hyp-Gly levels in human peripheral blood. However, the increase in Gly-Pro levels is negligible. To investigate the underlying mechanisms affecting the bioavailability of CH-derived peptides, we conducted both in vivo and in vitro digestion studies. After oral administration of CH (800 mg/kg body weight) to rats, Pro-Hyp levels significantly increased in the intestinal lumen, small intestinal tissue, and blood from the abdominal vein. In contrast, Gly-Pro was detected only in negligible amounts. In vitro digestion of CH using digestive exopeptidases, including aminopeptidase N, leucine aminopeptidase, and carboxypeptidase A, predominantly generated Gly-Pro. Interestingly, when CH was digested using a crude extract of small intestinal mucosa, Pro-Hyp was predominantly generated, while Gly-Pro levels were negligible. Synthetic Gly-Pro was rapidly degraded by the crude intestinal mucosa extract, whereas Pro-Hyp exhibited resistance to degradation. Exopeptidases in the mucosa extract were fractionated by Size Exclusion Chromatography and Ion Exchange Chromatography followed by analyses using SDS-PAGE-in gel digestion and LC-MS/MS. Prolidase and aminopeptidase N were identified, which cleaved X-Pro and Pro-X dipeptides, respectively. Furthermore, pyroglutamyl peptides, in rice protein hydrolysate, were inhibited the prolidase activity and increase of bioavailability of Pro-Hyp in rat model. These findings suggest that the presence of two exopeptidases with different substrate specificity in the intestinal mucosa, namely prolidase and aminopeptidase N, plays a critical role in determining the bioavailability of food-derived peptides, forming lumen-blood barrier to most peptides.
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