## 3 Results  
### Detection of Di- and Tripeptides and Cyclic Peptides Derived from CP after Yogurt Fermentation  
Peptide analysis using LC-MS revealed that the yogurt and the fermented mixture of milk and yogurt that were used for the preparation of the CP test foods contained negligible amounts of Hyp-containing peptides (data not shown). Therefore, we assumed that all Hyp-containing peptides were collagen-derived peptides. To evaluate the production of Hyp-containing di- and tripeptides and cyclic peptides from CP after yogurt fermentation, the three CP test foods were analyzed using LC-MS (once only) (Figure 2). The total concentration of Hyp-containing peptides in the CP test foods was relatively low compared to that of the CP that was previously prepared by extensive degradation with ginger protease [23]. The highest concentration of peptides detected in the CP test foods was 212.96 \(\upmu\)g/g CP for cyclo(Ser-Hyp) from \"CP in water,\" and the lowest concentration was 0.60 \(\upmu\)g/g CP for Pro-Hyp from \"CP in yogurt.\" Among the 13 linear Hyp-containing peptides tested, all the peptides, except Glu-Hyp-Gly, showed higher concentrations in \"yogurt-fermented CP\" compared to those in \"CP in yogurt\" and \"CP in water.\" In particular, Ala-Hyp, Leu-Hyp,  
Figure 1: Schematic representation of the study design. The supernatant samples of CP test foods were analyzed by liquid chromatography-mass spectrometry (LC-MS). Plasma samples were collected before and 1, 2, and 4 h after ingestion of CP test foods. Ethanol-soluble plasma samples were analyzed by LC-MS.  
Phe-Hyp, Ala-Hyp-Gly, and Leu-Hyp-Gly showed a significant (eight-fold or higher) increase in \"yogurt-fermented CP\" compared to their levels in \"CP in yogurt\" and \"CP in water.\" In addition, concentrations of Glu-Hyp-Gly in \"yogurt-fermented CP\" and \"CP in yogurt\" were five-fold higher than those in \"CP in water.\" Moreover, \"CP in yogurt\" showed a modest (non-significant) increase in the concentrations of Leu-Hyp, Ala-Hyp-Gly, Leu-Hyp-Gly, and Phe-Hyp-Gly compared to those in \"CP in water.\" There was no considerable change in the concentration of Hyp-containing cyclic peptides in any of the test foods. Detection of Food-Derived Hyp-Containing Peptides in Human Blood after Ingestion of CP with or without Yogurt Fermentation  
Figure 3 shows the average concentrations of Hyp and Hyp-containing peptides in the plasma of the four subjects after the ingestion of the test foods, \"yogurt-fermented CP,\" \"CP in yogurt,\" or \"CP  
Figure 2: The concentration (\(\upg\)/g CP) of Hyp-containing di- and tripeptides and cyclic peptides in the three CP test foods. Single measurement of the three CP test foods was conducted by LC-MS. The concentrations of Hyp-containing peptides are indicated as: “yogurt-fermented CP”, “CP in yogurt”, and “CP in water”. in water.\" Only negligible amounts of free Hyp and Hyp-containing peptides were observed in the plasma before the ingestion of the test foods. The concentrations of free Hyp and Hyp-containing peptides increased and reached their maximum levels 1-2 h after ingestion of each CP test food. The maximum concentrations (C\({}_{\text{max}}\)) of free Hyp were 68.15, 78.13, and 61.80 nmol/mL after ingestion of \"yogurt-fermented CP,\" \"CP in yogurt,\" and \"CP in water,\" respectively, and these levels were higher than those of Hyp-containing peptides (Table 2). Among the Hyp-containing peptides, the highest concentrations were observed for Pro-Hyp, which were 15.53, 20.86, and 16.36 nmol/mL after ingestion of \"yogurt-fermented CP,\" \"CP in yogurt,\" and \"CP in water,\" respectively, and there was no significant difference among their C\({}_{\text{max}}\) values. Compared to \"CP in water,\" \"yogurt-fermented CP\" significantly increased the plasma C\({}_{\text{max}}\) of Phe-Hyp, cyclo(Ala-Hyp), and cyclo(Pro-Hyp).