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Balenine (Bal) in opah muscle was extracted using hot water and purified by ion-exchange chromatography and recrystallization to provide 41 g of over 95% pure Bal from 1 kg of opah muscle. The structure of purified Bal was identical to that of an authentic Bal standard by NMR analysis. The antioxidant (ORAC and HORAC values) and Fe(II) ion-chelating abilities of purified Bal were examined by comparison with two major imidazole dipeptides, carnosine (Car) and anserine (Ans). Opah-derived Bal showed significantly higher ORAC and HORAC values and Fe(II) ion-chelating ability at 0.3 mM. In silico molecular simulation revealed that Bal and Car formed hydrogen bonds between the hydrogen atom of the imidazole imino group and the carboxyl carbonyl oxygen, whereas Ans did not. The proposed method for extracting and purifying Bal from opah muscle suggests that opah can be utilized as a functional food or Bal resource.

Collagen peptides (CP) have been used as functional foods for enhancing skin and joint health. Further degradation of CP results in peptide sizes small enough to enter the bloodstream following absorption in the small intestine. We examined the effects of food matrices on CP degradation into short chain peptides and absorption efficiency after ingestion. Changes to hydroxyproline (Hyp)-containing peptide levels in CP after yogurt fermentation and in human plasma by co-ingestion of CP and yogurt, with or without fermentation, were evaluated by liquid chromatography-mass spectrometry (LC-MS). The fermentation of CP with yogurt resulted in the significant degradation of CP into several Hyp-containing peptides such as Ala-Hyp, Leu-Hyp, Phe-Hyp, Ala-Hyp-Gly, and Leu-Hyp-Gly. CP ingestion after yogurt fermentation significantly increased the plasma concentrations of Phe-Hyp, cyclo(Ala-Hyp), and cyclo(Pro-Hyp) compared to water-based CP ingestion. The co-ingestion of CP and yogurt without fermentation significantly increased the plasma levels of Ala-Hyp, Phe-Hyp, Ala-Hyp-Gly, Leu-Hyp-Gly, Pro-Hyp-Gly, cyclo(Ala-Hyp), cyclo(Glu-Hyp), and cyclo(Pro-Hyp). Overall, the co-ingestion of CP and yogurt with or without fermentation significantly enhanced the absorption of CP-derived peptides, represented by the high Cmax and area under the curve per 1 h (AUC, nmol/h&middot, mL) of Hyp-containing peptides. These results suggest that, in addition to increasing short chain Hyp-containing peptide levels via fermentation, yogurt matrices containing milk-derived peptides and/or lactic acid bacteria-derived peptidases may influence the efficient absorption of CP-derived peptides into human blood.

Daily ingestion of collagen hydrolysate for a long period improves skin and joint conditions. It has been speculated that the beneficial effects are exerted by food-derived hydroxyproline (Hyp) peptides, which are detected in human blood after single ingestions. In the present study, to investigate the effect of long-term ingestion of collagen hydrolysate on Hyp peptides profile in blood, the concentrations of Hyp-peptides in human blood before and after daily ingestion for a long period were examined.; Results: Hyp-peptides increased to a maximum level at 1 h after ingestion and reverted to their initial levels within 24 h during experimental period. Pro-Gly and Hyp-peptides such as Pro-Hyp-Gly, Pro-Hyp, Ile-Hyp, Leu-Hyp, Hyp-Gly, Glu-Hyp and Ala-Hyp were identified in the blood after ingestion of collagen hydrolysate at 4.5 g day-1 for 4 weeks. For the whole period, Pro-Hyp was the leading compound. The compositional rate of Hyp-Gly showed a tendency to increase, while that of Pro-Hyp tended to decrease after daily ingestion.; Conclusion: The present results indicate that daily ingestion of collagen hydrolysate for a long period can change compositional rate of Hyp peptides in human blood. This fact suggests that long-term ingestion of collagen hydrolysate might change exo- or endo-type protease activity in the digestive tract, which may consequently promote beneficial effects. © 2017 Society of Chemical Industry.; © 2017 Society of Chemical Industry.

Prolyl-hydroxyproline (Pro-Hyp) levels increase in human blood on ingesting collagen hydrolysate and trigger proliferation of primary cultured fibroblasts attached on collagen. Such proliferative effects have been associated with the beneficial effects of collagen hydrolysate ingestion including enhancement of wound healing. However, some fibroblast cell lines do not respond to Pro-Hyp. In this study, we focused on the mesenchymal stem cell marker p75 neurotrophin receptor (p75NTR), because p75NTR-positive cells are increased at wound healing sites. Most fibroblasts that had migrated from the dissected mouse skin for 1–4 days expressed p75NTR. After prolonged cultivation, however, the ratio of p75NTR-positive fibroblasts dropped. The p75NTR-positive fibroblasts specifically incorporated fluorescein isothiocyanate (FITC)-labeled Pro-Hyp and Pro-Hyp significantly increased the number of p75NTR-positive fibroblasts grown on collagen gel. These results suggest that Pro-Hyp could enhance wound healing by stimulating p75NTR-positive fibroblasts at wound healing sites, while it has little effect on p75NTR-negative resident fibroblasts in normal tissues.

Generation of collagen dipeptides and deposition of orally administered prolylhydroxyproline (Pro-Hyp) in local inflammatory sites were examined in mice with hapten (2,4-dinitrofluorobenzene)-induced dermatitis in the ear. Pro-Hyp content in the hapten-treated ear was significantly higher in the chronic phase of contact dermatitis than the vehicle control. In contrast, hydroxyprolylglycine contents remained at lower levels in all cases compared to Pro-Hyp. Four hours after the ingestion of [13C5,15N]Pro and [13C5,15N]Pro-Hyp, labeled-Pro-Hyp and Pro, respectively, appeared only in the ear with dermatitis. Thus, Pro-Hyp is generated and degraded as part of the rapid synthesis and degradation of collagen in the ear with dermatitis. In addition to the endogenously generated Pro-Hyp, the orally administered Pro-Hyp was deposited in the ears.

The presence of hydroxyproline (Hyp)-containing peptides in human blood after collagen hydrolysate ingestion is believed to exert beneficial effects on human health. To estimate the effective beneficial dose of these peptides, we examined the relationship between ingested dose and food-derived Hyp levels in human plasma. Healthy volunteers (n = 4) ingested 30.8, 153.8 and 384.6 mg per kg body weight of collagen hydrolysate. The average plasma concentration of Hyp-containing peptides was dose-dependent, reaching maximum levels of 6.43, 20.17 and 32.84 nmol/ml following ingestion of 30.8, 153.8 and 384.6-mg doses of collagen hydrolysate, respectively. Ingesting over 153.8 mg of collagen hydrolysate significantly increased the average concentrations of the free and peptide forms of Hyp in plasma. The Hyp absorption limit was not reached with ingestion of as much as 384.6 mg of collagen hydrolysate. These finding suggest that ingestion of less than 30.8 mg of collagen hydrolysate is not effective for health benefits.

Collagen is defined as proteinous components with a triple helical structure in the extracellular matrix. Collagen forms a family consisting of closely related but different gene products and divided into two groups: fibrillar and nonfibrillar collagens (van der Rest and Garrone, 1991; Heino, 2007). In mammalian and avian tissues, the fibrillar collagen includes type I, II, III, V, and XI collagens (Sato et al., 1989; Zylberberg et al., 1992). Type I and II collagens are major constituents in skin, bone, tendon (type I), and cartilage (type II) in vertebrates, including fish, and type V collagen is codistributed with type I collagen as minor components in fish (Sato et al., 1989). In cartilage, type XI collagen is codistributed with type II collagen (Kelly et al., 1988). Thus, type I and II collagens are mainly used for collagen-related food ingredients. The collagens specifically consist of hydroxyproline (Hyp) and hydroxylysine (Hyl), posttranslational modified amino acids of proline (Pro) and lysine (Lys), respectively. The triple helical domain of the fibrillar collagen has a Gly-X-Y repeating motif, where X and Y are frequently occupied by Pro and Hyp, respectively (van der Rest and Garrone, 1991). The triple helical structure will be lost by heating the solution and converted into a globular structure. The denatured form of collagen is referred to as gelatin, which can be dissolved in hot water, yielding a viscous solution, and gel formation occurs by cooling the gelatin solution. Gelatin has long been used as a food ingredient as well as in folk medicine in Asia to improve blood circulation and arrest bleeding (Yao et al., 1989). In Western countries, gelatin consumption has been believed to improve joint condition by reducing pain (Moskowitz, 2000). Recently, partially hydrolyzed gelatin products have been prepared by enzymatic hydrolysis in order to increase their solubility in cold water. These are referred to as collagen peptides, and in some cases, simply as collagen in commercial products. Large amounts of collagen peptide preparations have been obtained from bone and skin of cattle, pig, chicken, etc. However, after the prevalence of bovine spongiform encephalopathy, much interest has been focused on production of collagen peptides from fish scales, skin, bone, etc. Episodes suggesting that ingestion of the collagen peptide would improve subjective skin conditions are widely prevalent in Japan and other countries. In Japan, annual sales of collagen peptide as a food supplement reached approximately US$300 million in 2007. Recent human CONTENTS

We examined the effect of prolyl-hydroxyproline (Pro-Hyp), which occurs in human peripheral blood after ingestion of collagen peptide, on the migration and growth of mouse skin fibroblasts. Mouse skin discs were cultured on a 24-well plastic plate in a fetal bovine serum (FBS)-free medium. Addition of Pro-Hyp (200 nmol/mL) significantly increased the number of fibroblasts migrating from the skin to the plate after incubation for 72 h. This effect of Pro-Hyp was abolished by the addition of mitomycin C. The fibroblasts that had migrated from the mouse skin were collected and cultured on collagen gel. The growth of fibroblasts on the collagen gel was suppressed even in the presence of FBS, while rapid fibroblast growth was observed on the plastic plate. Addition of Pro-Hyp (0-1000 nmol/mL) to the medium containing 10% FBS enhanced the growth of fibroblasts on the collagen gel in a dose-dependent manner. These results suggest that Pro-Hyp might stimulate the growth of fibroblasts in the skin and consequently increase the number of fibroblasts migrating from the skin.