Purification And Structural Identification Of Umami Peptide From Air Dried And Fermented Megalobrama Amblycephala

The umami peptide extracted from air dried and fermented Megalobrama amblycephala were purified, and also analyzed its structure and formation mechanism of umami flavor in this study. The nutritional components including ash, oil, protein, polypeptides, free amino acid, free fatty acid composition and the volatile compounds were investigated to evaluate the nutrition value and characteristic flavor of air dried and fermented Megalobrama amblycephala. The extraction conditions for water-soluble protein from air dried and fermented Megalobrama amblycephala were optimized through the single factor experiment, and the umami peptide with single purity was separated and purified by using ultrafiltration, RP-HPLC, and electronic tongue from the fish peptides. Moreover, molecular mass, amino acid composition, primary and secondary structure of the purified umami peptide were determined using the matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS), automatic amino acid analyzer, automatic protein/peptide sequence analyzer, and fourier transform infrared spectroscopy (FTIR).(1)The contents of protein, oligopeptide, fat, and ash were 32.72%, 4.26%,9.40% and 9.38% in air dried and fermented Megalobrama amblycephala, respectively. The air dried and fermented Megalobrama amblycephala had all essential amino acids, and the contents of Lysine and Methionine were 3.31% and 1.26%. Moreover, the contents of flavor amino acids (Aspartic, Glutamic, Glycine, Alanine, Serine, Proline) and umami amino acids (Aspartic, Glutamic, Glycine, Alanine) were 58.19% and 51.07%, respectively. The unsaturated fatty acids accounted for 68.80% in the total free fatty acids, and also the C18:1 content was the highest. The contents of polyunsaturated fatty acids were 21.98%, and also the contents of C20:5(EPA) and C22:6(DHA) were 0.40% and 0.89%, respectively. In addition, odd-carbon fatty acids (pentadecanoic acid and heptadecanoic acid) were also determined in air dried and fermented Megalobrama amblycephala. The volatile compounds were rich, and the contents of aldehydes and alcohols were respectively 16.65% and 10.91%. The characteristic volatile components such as 3-Octenol,2-pentyl-furan, nonanal contributed to the air dried and fermented Megalobrama amblycephala’s characteristic flavor.(2)The extraction conditions for water-soluble protein from air dried and fermented Megalobrama amblycephala were optimized, and the results showed that the optimized parameters including 1:10 ratio of fish meat to deionized water,40℃ of extraction temperature,120 mins of extraction time were confirmed, and also 29.05% of extraction rate of water-soluble protein gained at the optimized conditions. Two fractions including molecular weight above 3 kDa (P1) and below 3 kDa (P2) were separated from the extracted polypeptides using ultrafiltration, and also fraction P2a isolated from P2 with better umami taste using nanofiltration desalination. In addition, four fractions including P2a-1, P2a-2, P2a-3, and P2a-4 were purified from P2a using RP-HPLC with linear gradient elution. The flavor of four fractions were evaluated by electronic tongue, the result showed that fraction P2a-2 had the highest umami flavor. The RP-HPLC analysis indicated that P2a-2 was a single polypeptide.(3)MALDI-TOF-MS was used to determine the molecular weight of P2a-2, and the results showed that the molecular weight of P2a-2 with a single purity was 1305.59Da. The contents of umami amino acids were 53.29% and 42.41% in fraction P2a and P2a-2, respectively. The content of glutamic (21.97%) in P2a-2 decreased 1.3% than that in P2a. However, the content of histidine (27.54%) increased 9.91% than P2a. The secondary structure of P2a and P2a-2 were mainly β-sheet and β-turn, respectively. The high content of histidine, glutamic in P2a-2 and higher content of proline than P2a were benefit to the formation of β-turn. Polar amino acids presented umami flavor such as glutamic, aspartic, asparagine, glycine were usually apper in β-turn. They were all umami amino acids, which could make contribution to the formation of umami flavor. Therefore, higher content of umami amino acids and the secondary structure (β-turn) may make P2a-2 present better umami flavor than P2a.