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We examined the absorption of balenine (Bal) in mouse blood after the administration of a high-purity Bal prepared from opah muscle. Using HPLC with phenyl isothiocyanate pre-column derivatization, we successfully isolated imidazole peptides and their constituents. We detected Bal and 3-methylhistidine (3-Me-His) in mouse blood 1 h after the administration of opah-derived Bal. The concentrations of Bal and 3-Me-His significantly increased to 128.27 and 69.09 nmol/mL in plasma, respectively, but were undetectable in control and carnosine (Car)-administrated mice. In contrast, β-alanine and histidine did not increase in mouse plasma 1 h after the administration of Car and opah-derived Bal. The present study is the first report on the absorption of food-derived Bal in mouse blood and serves as a pilot study for future clinical trials.

There are increasing reports demonstrating high bioavailability of 4-hydroxyproline (4Hyp)-containing oligopeptides after oral ingestion of collagen hydrolysate and their bioactivity. In contrast, no study investigates the fate of another collagen-specific but minor amino acid, 3Hyp. Here, we identified Gly-3Hyp-4Hyp tripeptide in human blood at high concentrations, comparable to other 4Hyp-containing oligopeptides, after ingesting porcine skin collagen hydrolysate. Additionally, Gly-3Hyp-4Hyp uniquely maintained the maximum concentration until 4 h after the ingestion due to its exceptionally high resistance to peptidase/protease demonstrated by incubation with mouse plasma. In mice, oral administration of collagen hydrolysate prepared from bovine tendon, which contains a higher amount of 3Hyp, further increased blood Gly-3Hyp-4Hyp levels compared to that from bovine skin. Furthermore, Gly-3Hyp-4Hyp showed chemotactic activity on skin fibroblasts and promoted osteoblast differentiation. These results highlight the specific nature of the Gly-3Hyp-4Hyp tripeptide and its potential for health promotion and disease treatment.

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.

This study showed that gelatin ingestion significantly increased prolyl-hydroxyproline (Pro-Hyp) levels in plasma of 9 subjects, with maximum concentrations of 15.5 ± 3.0 nmol/mL 2 h post-ingestion. Hydroxyprolyl-glycine (Hyp-Gly) concentrations were significantly increased and reached a maximal level of 2.3 ± 0.5 nmol/mL 1 h post-ingestion of gelatin. A low molecular weight gelatin hydrolysate (LMW-GH) significantly enhanced concentrations of both peptides, while gelatin hydrolysate ingestion did not significantly enhance the maximum concentration and area under the plasma concentration–time curve (AUC) of Hyp-Gly relative to gelatin. The absorption of free Hyp following gelatin ingestion (94.4 ± 16.4 nmol/mL) was significantly lower relative to GH (150.9 ± 15.3 nmol/mL) and LMW-GH (169.1 ± 32.5 nmol/mL). The present study is the first report demonstrating that Hyp-containing peptides are elevated to μM levels in human plasma after gelatin ingestion. These results suggested that gelatin is useful as a functional food as effectively as GH.

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.

Collagen-derived hydroxyproline (Hyp)-containing oligopeptides, known to have various physiological functions, are detected in blood at markedly higher concentrations after oral ingestion of collagen hydrolysate. Monitoring the absorption and metabolism of the bioactive peptides is essential to investigate the beneficial effects of collagen hydrolysate. We previously developed an internal standard mixture by sequential protease digestion of stable isotope-labeled collagen, which enabled highly accurate quantitation of collagen-derived oligopeptides by liquid chromatography-mass spectrometry (LC-MS). However, the use of proteases caused a profound imbalance in the generated peptides. Here, we employed partial acid hydrolysis to achieve more efficient and balanced peptide generation. Various stable isotope-labeled oligopeptides were detected after 0.5 h acid hydrolysis, and marked enhancement of peptide generation compared with the previous enzymatic method was observed, especially for Hyp-Gly (27.8 ± 0.6 ng/μg vs 0.231 ± 0.02 ng/μg). The acid hydrolysate was then heated to generate labeled cyclic dipeptides. Using the novel internal standard mixture in LC-MS, we were able to simultaneously quantitate 23 collagen-derived oligopeptides in human plasma and urine after oral administration of collagen hydrolysate.

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.

Levels of short linear hydroxyproline (Hyp)-containing peptides, such as prolyl-hydroxyproline (Pro-Hyp), increase in human blood after the ingestion of collagen hydrolysate, which has been associated with beneficial effects for human skin and joints. The present study demonstrates the presence of a novel food-derived collagen peptide, cyclic Pro-Hyp, in human blood after the ingestion of collagen hydrolysate. The cyclic Pro-Hyp levels in plasma samples were estimated by liquid chromatography mass spectrometry (LC-MS). Cyclic Pro-Hyp levels significantly increased in the plasma after ingestion of collagen hydrolysate, reaching a maximum level after 2 h and then decreasing. The maximum level of cyclic Pro-Hyp in plasma ranged from 0.1413 to 0.3443 nmol/mL, representing approximately 5% of linear Pro-Hyp in plasma after ingestion of collagen hydrolysate. Addition of cyclic Pro-Hyp in medium at 7 nmol/mL significantly enhanced the growth rate of mouse skin fibroblasts on collagen gel more extensively compared to linear Pro-Hyp.

We conducted a study to examine aroma leakage from orange juice packed in gable-top paper containers for chilled distribution. Limonene, an aromatic component of orange juice, was considered as an index compound of aroma leakage, and its seepage on the surface of the container and concentration in the orange juice were measured by GC-MS for 12 commercial samples. After 3 days of storage, limonene was detected on the surface of 8 orange juice containers, and the concentration of limonene in the orange juice was found to have decreased. Thus, limonene leaked through the container within a few days, and the extent of leakage differed between containers, presumably depending upon their barrier properties. In addition, limonene was detected in green tea and milk that was stored together with the unopened orange juice containers at 4℃. The transference of orange aroma into milk was significant, because the contamination of the milk was confirmed by subjective sensory evaluation. This study suggests the possibility of transfer of aroma compounds through paper containers to other beverages.

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.

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.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 dayThe 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.

Elastin hydrolysate has apparent beneficial effects, and the food-derived peptide prolyl-glycine (Pro-Gly) is present in human blood after oral ingestion. Following ingestion of elastin hydrolysate (10 g/60 kg body weight) by healthy human volunteers, peripheral blood was used to prepare plasma samples from which peptides were extracted by solid phase extraction and fractionated by size-exclusion chromatography (SEC). Peptides in the SEC fractions were derivatized with phenyl isothiocyanate (PITC) and resolved by reversed phase (RP)-HPLC. Pro-Gly was the major food-derived elastin peptide, reaching a maximum (18 μM) at 30 min after ingestion, and decreasing to approximately 20% at 4 h after ingestion. Finally, in cell culture, levels of Pro-Gly in the medium above 0.1 μg/mL significantly enhanced elastin synthesis of normal human dermal fibroblasts (NHDF) without affecting the rate of cell proliferation.

In this paper, the detection of type I collagen degradation during the softening phenomenon of yellowtail muscle, was examined. Acid soluble collagen was isolated from dorsal ordinary muscle at death and after 24-h chilled storage. In the abundant ratio of subunit components, an increase in β12 chain (5.4 points) and a decrease in components with molecular weights larger than γ chain (7.0 points) after 24-h chilled storage, was found. Type I collagen was detected in the alkali-soluble fraction by SDS-PAGE. Its amount calculated from hydroxyproline contents in alkali-soluble fraction was increased from 0.097 mg/g muscle to 0.155 mg/g muscle during 24-h storage. The increased alkali-soluble collagen (0.058 mg/g muscle) was about 1.4% of whole collagen. These results suggest that a slight decomposition of type I collagen of yellowtail muscle may occur and subsequently becomes alkali-soluble corresponding to postmortem softening.

In order to resolve the relationship between type V collagen and post-mortem softening of fish meat during chilled storage, the breaking strength and type V collagen content of meat were measured in seven fish species. The meats of seven fish species were classified into three groups according to the type of textural change. Three types of meat showed earlier softening than others. Two types of meat showed gradual softening and the other two types showed no softening during 1 day of chilled storage. The relative contents of type V collagen were different among fish species. The content of striped jack meat was fivefold that of tiger puffer immediately after death. The ratio of decline of type V collagen content during 1 day of chilled storage showed a significant relationship with the softening ratio (%/24 h, r = 0.8914) and average softening rate (%/h, r = 0.9195). In other words, the meat that contained most type V collagen showed notable and early softening after the death of the fish. These results suggest a correlation between type V collagen and post-mortem softening of fish meat during chilled storage.

The present paper demonstrates that a nonstntggling slaughter method can delay degradation of type V collagen in meat of chub mackerel Scomber japonicus and softening of the meat during postharvest chilled storage. The fish were slaughtered by piercing a knife into nape (nonstruggling method) or by leaving on ground (struggling method) and then stored in an ice box. Sensory study revealed that the postharvest softening of the meat was moderated at 4 and 8 h by the non-struggling slaughter method in comparison with the struggling method. On the basis of the specific solubilization of type V collagen and reduced tyrosine content in it, a cleavage of the nonhelical regions (telopeptides) of the type V collagen occurred during the chilled storage in the fish slaughtered by the struggling method. The degradation of type V collagen was also slower in the meat of the fish slaughtered by the nonstruggling method, which can be directly linked to the moderation of the postharvest softening.

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

Peptides in the blood of subjects before and after collagen hydrolysate ingestion were fractionated by ion exchange and size-exclusion chromatographies and then derivatised with phenyl isothiocyanate. The derivatives were characterised by reserved phase (RP)-HPLC. Prolyl-hydroxyproline (Pro-Hyp), which has been identified in the previous studies, was detected as a major food-derived collagen peptide in the blood of all subjects (n=5). Another major peptide was identified as hydroxyprolyl-glycine (Hyp-Gly) in the blood of four subjects, which has not been detected in previous studies. The ratio of Hyp-Gly to Pro-Hyp depended on subjects and ranged from 0.00 to 5.04. Hyp-Gly was less susceptible to human serum peptidase than Pro-Hyp. Hyp-Gly enhanced the growth of mouse primary fibroblasts on collagen gels in a higher extent than Pro-Hyp. These findings suggest that Hyp-Gly plays a significant role in exerting the biological effects by ingestion of collagen hydrolysate.

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.