Novel Ca-Chelating Peptides from Protein Hydrolysate of Antarctic Krill (Euphausia superba): Preparation, Characterization, and Calcium Absorption Efficiency in Caco-2 Cell Monolayer Model.

Antarctic krill (Euphausia superba) is the world's largest resource of animal proteins and is thought to be a high-quality resource for future marine healthy foods and functional products. Therefore, Antarctic krill was degreased and separately hydrolyzed using flavourzyme, pepsin, papain, and alcalase. Protein hydrolysate (AKH) of Antarctic krill prepared by trypsin showed the highest Ca-chelating rate under the optimized chelating conditions: a pH of 8.0, reaction time of 50 min, temperature of 50 °C, and material/calcium ratio of 1:15. Subsequently, fourteen Ca-chelating peptides were isolated from APK by ultrafiltration and a series of chromatographic methods and identified as AK, EAR, AEA, VERG, VAS, GPK, SP, GPKG, APRGH, GVPG, LEPGP, LEKGA, FPPGR, and GEPG with molecular weights of 217.27, 374.40, 289.29, 459.50, 275.30, 300.36, 202.21, 357.41, 536.59, 328.37, 511.58, 516.60, 572.66, and 358.35 Da, respectively. Among fourteen Ca-chelating peptides, VERG presented the highest Ca-chelating ability. Ultraviolet spectrum (UV), Fourier Transform Infrared (FTIR), and scanning electron microscope (SEM) analysis indicated that the VERG-Ca chelate had a dense granular structure because the N-H, C=O and -COOH groups of VERG combined with Ca2+. Moreover, the VERG-Ca chelate is stable in gastrointestinal digestion and can significantly improve Ca transport in Caco-2 cell monolayer experiments, but phytate could significantly reduce the absorption of Ca derived from the VERG-Ca chelate. Therefore, Ca-chelating peptides from protein hydrolysate of Antarctic krill possess the potential to serve as a Ca supplement in developing healthy foods.

Purification and Characterization of Antioxidant Peptides Derived from Protein Hydrolysate of the Marine Bivalve Mollusk Tergillarca granosa.

In this report, protein hydrolysate (TGH) of blood cockle (Tegillarca granosa) was prepared using a two-enzyme system (Alcalase treatment for 1.5 h following Neutrase treatment for 1.5 h). Subsequently, six antioxidant peptides were isolated from TGH using ultrafiltration and chromatography methods, and their amino acid sequences were identified as EPLSD, WLDPDG, MDLFTE, WPPD, EPVV, and CYIE with molecular weights of 559.55, 701.69, 754.81, 513.50, 442.48, and 526.57 Da, respectively. In which, MDLFTE and WPPD exhibited strong scavenging activities on DPPH radical (EC50 values of 0.53 ± 0.02 and 0.36 ± 0.02 mg/mL, respectively), hydroxy radical (EC50 values of 0.47 ± 0.03 and 0.38 ± 0.04 mg/mL, respectively), superoxide anion radical (EC50 values of 0.75 ± 0.04 and 0.46 ± 0.05 mg/mL, respectively), and ABTS cation radical (EC50 values of 0.96 ± 0.08 and 0.54 ± 0.03 mg/mL, respectively). Moreover, MDLFTE and WPPD showed high inhibiting ability on lipid peroxidation. However, MDLFTE and WPPD were unstable and could not retain strong antioxidant activity at high temperatures (>80 °C for 0.5 h), basic pH conditions (pH > 9 for 2.5 h), or during simulated GI digestion. In addition, the effect of simulated gastrointestinal digestion on TGP4 was significantly weaker than that on MDLFTE. Therefore, MDLFTE and WPPD may be more suitable for serving as nutraceutical candidates in isolated forms than as food ingredient candidates in functional foods and products.

Anticancer Activity of a Hexapeptide from Skate (Raja porosa) Cartilage Protein Hydrolysate in HeLa Cells.

In this study, the hexapeptide Phe-Ile-Met-Gly-Pro-Tyr (FIMGPY), which has a molecular weight of 726.9 Da, was separated from skate (Raja porosa) cartilage protein hydrolysate using ultrafiltration and chromatographic methods, and its anticancer activity was evaluated in HeLa cells. Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay indicated that FIMGPY exhibited high, dose-dependent anti-proliferation activities in HeLa cells with an IC50 of 4.81 mg/mL. Acridine orange/ethidium bromide (AO/EB) fluorescence staining and flow cytometry methods confirmed that FIMGPY could inhibit HeLa cell proliferation by inducing apoptosis. Western blot assay revealed that the Bax/Bcl-2 ratio and relative intensity of caspase-3 in HeLa cells treated with 7-mg/mL FIMGPY were 2.63 and 1.83, respectively, significantly higher than those of the blank control (p < 0.01). Thus, FIMGPY could induce apoptosis by upregulating the Bax/Bcl-2 ratio and caspase-3 activation. Using a DNA ladder method further confirmed that the anti-proliferation activity of FIMGPY was attributable to its role in inducing apoptosis. These results suggest that FIMGPY from skate cartilage protein hydrolysate may have applications as functional foods and nutraceuticals for the treatment and prevention of cancer.

Characterization of acid-and pepsin-soluble collagens from spines and skulls of skipjack tuna (Katsuwonus pelamis).

Acid-soluble collagen (ASC) and pepsin-soluble collagen (PSC) from the spine (ASC-SP and PSC-SP) and skull (ASC-SK and PSC-SK) of the skipjack tuna, Katsuwonus pelamis, were successfully isolated and characterized. The yields of ASC-SP, PSC-SP, ASC-SK and PSC-SK were (2.47 ± 0.39)%, (5.62 ± 0.82)%, (3.57 ± 0.40)%, and (6.71 ± 0.81)%, respectively, on the basis of dry weight. The four collagens contained Gly (330.2-339.1 residues/1 000 residues) as the major amino acid, and their imino acid contents were between 168.8 and 178.2 residues/1 000 residues. Amino acid composition, SDS-PAGE, and FTIR investigations confirmed that ASC-SP and ASC-SK were mainly composed of type I collagen, and had higher contents of high-molecular weight cross-links than those of PSC-SK and PSC-SP. The FTIR investigation also certified all the collagens had triple helical structure. The denaturation temperatures of ASC-SK, PSC-SK, ASC-SP, and PSC-SP were 17.8, 16.6, 17.6, and 16.5 °C, respectively. All isolated collagens were soluble at acidic pH (1-5) and lost their solubilities when the NaCl concentration was above 2% (W/V). The isolated collagens from the spines and skulls of skipjack tuna could serve as an alternative source of collagens for further application in food, cosmetic, biomedical, and pharmaceutical industries.

Identification and analysis of an intracellular Cu/Zn superoxide dismutase from Sepiella maindroni under stress of Vibrio harveyi and Cd2+.

Superoxide dismutases (SODs) are ubiquitous family of metalloenzymes involved in protecting organisms from excess reactive oxygen species damage. In this paper, a novel intracellular Cu/ZnSOD from Sepiella maindroni (designated as SmSOD) was identified and characterized. The full-length cDNA sequence of SmSOD (GenBank accession No. KF908850) was 709 bp containing an open reading frame (ORF) of 459 bp, encoding 153 amino acid residues peptide with predicted pI/MW (6.02/15.75 kDa), a 131 bp-5'- and 116 bp-3'- untranslated region (UTR). BLASTn analysis and phylogenetic relationship strongly suggested that the sequence shared high similarity with known Cu/Zn SODs. Several highly conserved motifs, including two typical Cu/Zn SOD family domains, two conserved Cu-/Zn-binding sites (H-47, H-49, H-64, H-120 for Cu binding, and H-64, H-72, H-81, D-84 for Zn binding) and intracellular disulfide bond (C-58 and C-146), were also identified in SmSOD. Time-dependent mRNA expression of SmSOD in hepatopancreas was recorded by quantitative real-time RT-PCR after Vibrio harveyi injection and Cd(2+) exposure. The results indicated that SmSOD was an acute-phase protein involved in the immune responses against pathogens and biological indicator for metal contaminants in aquatic environment.

[Determination of 8 trace elements in mantle muscle and cuttlebone of Sepiella maindroni by ICP-MS using microwave digestion for sample preparation].

An inductively coupled plasma mass spectrometry (ICP-MS) for determination of the contents of 8 trace elements in mantle muscle and cuttlebone of Sepiella maindroni after microwave digestion of the sample has been developed. Satisfactory linearity of working curves for the 8 elements was obtained, giving all their correlation coefficients over 0.997 3. The precision of measurement ranges from 2.4% to 8.7% in terms of relative standard deviation. The recoveries and the limits of detection are in the range of 96.5%-106.3% and 0.002-0.032 microg x L(-1), respectively. It was indicated that the proposed method had the advantages of simplicity, speediness and sensitivity. The results showed that the mantle muscle and cuttlebone of Sepiella maindroni contained rich trace elements Zn and Cu, but the contents of Cd and As are higher than the limits of Chinese Pharmacopoeia, Green Trade Standard for Importing and Exporting Medicinal Plant and Preparation and U.S. Food and Drug Standard. Furthermore, our study provides new scientific foundation for the quality control, culture, general application, resource utilization and exporting of Sepiella maindroni.