Increase of Kokumi γ-Glutamyl Peptides in Porcine Hemoglobin Hydrolysate Using Bacterial γ-Glutamyltransferase.

The kokumi sensation of protein hydrolysates could be enhanced by γ-glutamylation through forming a series of γ-glutamyl di- and tri-peptides. In this study, porcine hemoglobin hydrolysate was γ-glutamylated using enzymes from Bacillus amyloliquefaciens (Ba) or Bacillus licheniformis (Bl), which are sold as glutaminases but identified as γ-glutamyltransferases (GGTs). To yield more γ-glutamyl peptides, reaction conditions were optimized in terms of GGT source (BaGGT and BlGGT), substrate concentration (10, 20, and 40%), reaction time (3, 6, 12, and 24 h), and glutamine supplementation (20, 40, and 80 mM). Results showed that both the GGTs had the highest transpeptidase activity at similar pH values but different temperatures. In addition, BaGGT had stronger catalytic ability to form γ-glutamyl dipeptides, while BlGGT was more capable to generate γ-Glu-Val-Gly. Adding glutamine was more efficient to obtain more target peptides than adjusting the hydrolysate concentration and reaction time. This study contributes to the valorization of animal side streams.

Novel antioxidant and anti-inflammatory peptides from the Siamese crocodile (Crocodylus siamensis) hemoglobin hydrolysate.

Novel antioxidant and anti-inflammatory peptides were isolated from hydrolysates of Siamese crocodile (Crocodylus siamensis) hemoglobin. C. siamensis hemoglobin hydrolysates (CHHs) were obtained by pepsin digestion at different incubation times (2, 4, 6, and 8 H) at 37 °C and subjected to antioxidant and anti-inflammatory activity assessment. CHH obtained by 2-H hydrolysis (2H-CHH) showed the highest anti-inflammatory activity with respect to decreasing nitric oxide (NO) production, whereas the strongest antioxidant activity was found for 6-H hydrolysis (6H-CHH) against nitric oxide radicals. To evaluate the anti-inflammatory and antioxidant activity of individual peptide components, 2H-CHH and 6H-CHH were purified by semipreparative HPLC. Peptide fraction P57 isolated from 6H-CHH was found to exhibit the highest nitric oxide radical inhibition activity (32.0%). Moreover, purification of 2H-CHH yielded peptide fraction P16, which displayed a high efficacy in decreasing NO production of macrophage RAW 264.7 cells (83.2%) and significantly reduced proinflammatory cytokines and inflammatory mediators interleukin-6 (IL-6), interleukin-1 beta (IL-1ß), and prostaglandin-E2 (PGE2 ) production to about 2.0, 0.3, and 1.9 ng/mL, respectively. Using LTQ orbitrap XL mass spectrometry, active peptide sequences were identified as antioxidant KIYFPHF (KF7), anti-inflammatory SAFNPHEKQ (SQ9), and IIHNEKVQAHGKKVL (IL15). Additionally, CHHs simulated gastric and intestinal in vitro digestion positively contributed to antioxidant and anti-inflammatory activity. Taken collectively, the results of this work demonstrate that CHHs contain several peptides with anti-inflammatory and antioxidant properties, which may prove valuable as treatment or supplement against diseases associated with inflammation and oxidative stress.

Antioxidant activity of hydrolysates and peptide fractions derived from porcine hemoglobin.

Porcine hemoglobin hydrolysate (PHH) was prepared with 6 different proteases (flavourzyme, papain, A.S.1398, alcalase, pepsin and trypsin). There was no correlation between extent of hydrolysis and antioxidant activity (p > 0.05). The peptic hydrolysate prepared at 60 min possessed the strongest antioxidant potential (67.0 ± 1.84%) among different hydrolysates, which was fractionated into 4 major types by ultrafiltration membranes with different molecular weight cut-off (MWCO), PHH-I (Mw > 10 kDa), PHH-II (Mw = 5-10 kDa), PHH-III (Mw = 3-5 kDa) and PHH-IV (Mw < 3 kDa). PHH-IV possessed higher inhibitory effects of lipid peroxidation and scavenging effects on superoxide radical compared with larger MW fractions. Four fractions possessed the scavenging effects on 1, 1-diphenyl-2-picrylhy-drazyl (DPPH) and hydroxyl radicals in the order PHH-IV > PHH-I > PHH-III > PHH-II. MW of the 2 major peptide fractions from PHH-IV was located at 2476 Da (F1) and 1042 Da (F2), respectively. PHH-IV could be utilized to develop physiologically functional foods or therapy drugs.