## 4 Discussion
Studies have shown that the co-ingestion of foods affects the absorption of food components in the small intestine. [PERSON] et al. demonstrated that the co-ingestion of fermented milk and CP could increase the plasma concentration of collagen-specific amino acids, and they suggested that the absorption of CP-derived amino acids might be controlled by yogurt-derived components in the human intestine [17]. \(\beta\)-cascomorphin (BCM) is a representative bioactive peptide in milk products. BCM exhibits an opioid type effect on gastrointestinal motility [27] and the absorption of electrolytes [28] due to its interaction with the opioid receptors expressed in the intestine. [PERSON] et al. revealed that BCM promotes the intestinal absorption of nutrients via the opioid receptor or via specific binding sites in the brush border membrane [29]. Based on these studies, BCM in yogurt could promote the transport of amino acids and peptides derived from CP and consequently enhance the levels of amino acids as well as those of the eight Hyp-containing peptides in human blood (observed in the present study) (Table 2). However, the _Lactobacillus delbrueckii subsp. Bulgarius_ and _Streptococcus thermophiles_ used in present study may degrade BCM via PepX [25; 26] and consequently suppress the absorption of CP-derived short chain peptides. Although the mechanisms are still not completely clear, we suggest that PepX's presence in the yogurt and BCM degradation in the milk that was used for the preparation of the CP test foods might differentially affect the absorption of Hyp-containing peptides in our study. This may explain the unexpected observation that the ingestion of \"yogurt-fermented CP\" could not significantly enhance the intestinal absorption of Hyp-containing peptides such as Ala-Hyp, Ala-Hyp-Gly, Leu-Hyp-Gly, and cyclo(Glu-Hyp) compared to the ingestion of \"CP in yogurt.\" Moreover, milk contains dipeptidyl peptidase-IV (DPP-IV) inhibitors such as LKPTPEGDL, LYPYP, IPIQY, and IPI [30]. DPP-IV is expressed in the gastrointestinal tract and possibly enables the degradation of CP, as well as PepX. Thus, the short incubation time during the preparation of our CP test foods might have decreased the amount of these milk-derived DPP-IV inhibitory peptides, and consequently the activity of DPP-IV was retained in the \"CP in yogurt\" preparation. Therefore, the intestinal absorption of Ala-Hyp, Ala-Hyp-Gly, Leu-Hyp-Gly, and cyclo(Glu-Hyp) after the ingestion of \"yogurt-fermented CP\" was not more than that observed after ingestion of \"CP in yogurt.\"  
Among all the short chain Hyp-containing peptides that were analyzed in this study, Phe-Hyp was the only peptide whose levels were significantly increased in either \"yogurt-fermented CP\" and plasma. This result might be attributed to the production of over thirty-fold higher levels of Phe-Hyp in \"yogurt-fermented CP\" compared to that in \"CP in yogurt,\" a level that was potentially high enough to invalidate the suppression of peptide transport by PepX or DPP-IV. In order to clarify the effects of PepX, BCM, and DPP-IV inhibitors, as well as different lactic acid bacteria on peptide absorption, we plan to quantify them in yogurt in follow-up studies. Additionally, we also intend to increase our sample size (use a higher number of subjects) in order to accurately and undoubtedly disclose the mechanisms behind the effects of food matrices on peptide absorption in humans. [PERSON] et al. demonstrated that theaflavins inhibited the peptide transport system via the Pept1 that is expressed in human intestinal Caco-2 cells and that Pept1 expression in the small intestine was controlled by AMP-activated protein kinase [31]. [PERSON] et al. reported that theaflavins could enhance the intestinal barrier function through the expression of claudin-1, occludin, and ZO-1 via AMP-activated protein kinase [32].