This study aimed to investigate if imidazole dipeptides (IDPs) undergo heat-induced denaturation and to identify denatured IDPs in food products. Herein, IDPs were heated at 120 °C for 4 h in both model systems and various foods for denaturation and subjected to mass spectrometry and nuclear magnetic resonance analyses. Notably, denatured carnosine, anserine, and balenine were identified as acryloyl-histidine, acryloyl-Nπ-methyl-histidine, and acryloyl-Nτ-methyl-histidine, respectively. These acryloyl-histidine-related components were not detected in raw meat samples, but their levels increased significantly after heat treatment. Additionally, they were found in commercially available processed meat products and IDP-containing supplements. Further, acrylamide was not detected post-heating at 120 °C, indicating that IDPs do not serve as a precursor for acrylamide under typical cooking conditions below 150 °C. Overall, these findings link the IDP denaturation pathway to the Maillard reaction and its influence on the bioavailability of acryloyl-histidine-related components and potential health effects, considering their regular consumption in daily diets.

Opah muscle is one of the abundant biological sources of balenine (Bal), which together with carnosine (Car) and anserine (Ans), is among the most widely studied of vertebrate imidazole dipeptides. The characteristics of imidazole dipeptides have been extensively investigated. We examined changes in the concentration of Bal and its constituent amino acids in human plasma following the ingestion of opah muscle extract and opah flesh. Bal plasma concentrations increased after ingestion, with maximum plasma concentrations (Cmax) of 89.87, 152.13, and 81.82 nmol/mL observed after ingestion of 5 and 10 g of opah muscle extract and 80 g of opah flesh, respectively. The constituent amino acids of Bal, 3-methyl-histidine (3-Me-His) and (3-alanine ((3-Ala), increased in plasma after ingestion. The Cmax of Bal was the highest of any food-derived peptide in human plasma reported to date. The findings showed that ingestion of opah muscle and muscle extract may be beneficial for human health.

Balenine is one of the endogenous imidazole dipeptides derived from marine products. It is composed of beta-alanine and 3-methyl-L-histidine, which exist mainly in the muscles of marine organisms. The physiological functions of dietary balenine are not well-known. In this study, we investigated whether the supplementation of dietary balenine was associated with muscle function in a cardiotoxin-indued muscle degeneration/regeneration model. Through morphological observation, we found that the supplementation of balenine-enriched extract promoted the regeneration stage. In addition, the expression of regeneration-related myogenic marker genes, such as paired box protein 7, MyoD1, myogenin, and Myh3, in a group of mice fed a balenine-enriched extract diet was higher than that in a group fed a normal diet. Moreover, the supplementation of balenine-enriched extract promoted the expression of anti-inflammatory cytokines as well as pro-inflammatory cytokines at the degeneration stage. Interestingly, phagocytic activity in the balenine group was significantly higher than that in the control group in vitro. These results suggest that balenine may promote the progress of muscle regeneration by increasing the phagocytic activity of macrophages.

Balenine is one of the endogenous imidazole dipeptides, mainly found in the muscle of marine animals. In this study we focused on the safety evaluation and physiological function of dietary balenine derived from opah Lampris guttatus in skeletal muscle of mice. There were no significant differences among different concentrations (0–1%) of balenine diet in the basic data included the weight of body weight, food intake, skeletal muscle and some organs, indicating that less than 1% dietary balenine showed no side effects. The cross-sectional area of myofibers in the group fed a balenine diet was similar to that observed in the group fed a normal diet. In physiological function, supplementation of balenine significantly induced the expression of peroxisome proliferator-activated receptor-γ coactivator-1α and pyruvate dehydrogenase kinase 4, which are related to mitochondrial biogenesis and lipid metabolism. Moreover, the activity of superoxide dismutase (SOD) in the skeletal muscle of the group fed a balenine diet was significantly increased, compared with that of the group fed a normal diet, whereas the supplementation of balenine did not affect the mRNA transcription of SODs in skeletal muscle. Our results suggest that dietary balenine contributes to the regulation of mitochondrial biogenesis and metabolism, and SOD activity in skeletal muscle of mice.

Balenine is one of the endogenous imidazole dipeptides. It is composed of beta-alanine and 3-methyl-l-histidine, which exist mainly in the muscles and the brain. The exact biological properties of balenine are still not well known, although the antioxidant activity of carnosine, another imidazole dipeptide, is known. In this study we investigated whether balenine exhibits antioxidant activity. It was found to decrease the superoxide anion (O2−) and increased hydrogen peroxide (H2O2) generation. We found that SOD activity increased in balenine-treated C2C12 myotubes, although balenine did not increase expression of SOD mRNA. On the other hand, there were no changes in other antioxidant enzymes, CAT and GPX activity, and mRNA levels. In an in vitro assay, the direct activation of SOD treated by balenine was significantly higher than with carnosine treatment. Moreover, balenine constituent amino acids did not have the ability to activate SOD. Our results suggest that balenine contributes to antioxidant effects through activation of SOD.

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.

We examined the method of analysis and quantitation of balenine (Bal) previously reported. In this method, Bal in muscle extract was hydrolyzed to 3-methylhistidine (3-Mehis) and beta-alanine in 6 M hydrochloric acid heated at 110 degrees C for 24 hours, analyzed and quantitated 3-Mehis by automatic amino acid analyzer. And it was found that 3-Mehis content was equal to Bal content before hydrolysis. However, in this study, the minimum variation of 3-Mehis content was achieved at 110 degrees C for 3 hours by six repetitions of hydrolysis and quantitation of 3-Mehis derived from Bal. In addition, a high linear correlation between 3-Mehis content after hydrolysis and Bal content before hydrolysis was observed at the Bal standard solution concentrations of 0.05 to 10 mu mol/mL. The ratio of 3-Mehis content after hydrolysis to Bal content before hydrolysis was not equal but 89%. Based on these considerations, the Bal content of opah Lampris guttatus muscle extract was quantitated and a remarkably high Bal content was found. The Bal content of opah was much higher than that of some species of baleen whales, such as mink whale Balaenoptera acutorostrata, Sei whale B. borealis and Bryde's whale B. brydei.