Ming Du

Changes in collagenous tissue microstructures and distributions of cathepsin L in body wall of autolytic sea cucumber (Stichopus japonicus)

The autolysis of sea cucumber (Stichopus japonicus) was induced by ultraviolet (UV) irradiation, and the changes of microstructures of collagenous tissues and distributions of cathepsin L were investigated using histological and histochemical techniques. Intact collagen fibers in fresh S. japonicus dermis were disaggregated into collagen fibrils after UV stimuli. Cathepsin L was identified inside the surface of vacuoles in the fresh S. japonicus dermis cells. After the UV stimuli, the membranes of vacuoles and cells were fused together, and cathepsin L was released from cells and diffused into tissues. The density of cathepsin L was positively correlated with the speed and degree of autolysis in different layers of body wall. Our results revealed that lysosomal cathepsin L was released from cells in response to UV stimuli, which contacts and degrades the extracellular substrates such as collagen fibers, and thus participates in the autolysis of S. japonicus.

Thrombin inhibitory peptides derived from Mytilus edulis proteins: identification, molecular docking and in silico prediction of toxicity

Optimization of the thrombin inhibitory activities of different enzymatic hydrolysates was conducted, and then an optimal hydrolysis condition by trypsin (5000xa0u/g) was determined as follows, digested at 45xa0°C and pH 8.5 for 2xa0h with a protein concentration of 25xa0mg/mL. Thrombin inhibitory activity was proved to be 76.92xa0±xa04.66% under this condition. A total of 39 peptides were identified in the hydrolysate by UPLC-Q-TOF–MS/MS, and all the peptides were predicted to be nontoxic by in silico predictive approaches. Twenty-six peptides were predicted to be anticoagulant peptides by molecular docking method, and the peptide 26 (Lys-Asn-Ala-Glu-Asn-Glu-Leu-Gly-Glu-Val-Thr-Val-Arg) was predicted to be a better anticoagulant peptide through both structure–activity relationship and affinity activity to thrombin. The interactional positions between peptide and thrombin were also involved in the interaction site on the S1 pocket of thrombin and strongly promoted its thrombin inhibitory activity. The firmly non-bonded interactions made the bound of peptide and thrombin firmly. Eventually, the chemical identification and activity verification of synthetic peptide 26 were conducted, and the thrombin inhibitory activity was 89.96xa0±xa05.30% at the concentration of 9xa0mg/mL. This study optimized an enzymatic hydrolysis and a virtual screening method for predicting and verifying the anticoagulant peptide from Mytilus edulis, respectively, which provided a good theoretical basis and application method for the research and development of the anticoagulant peptides, especially from the seafood products.

Simultaneous determination of glyoxal, methylglyoxal and diacetyl in beverages using vortex-assisted liquid–liquid microextraction coupled with HPLC-DAD

The detection of α-dicarbonyl compounds including glyoxal (GO), methylglyoxal (MGO) and diacetyl (DA) levels in beverages could be effectively used to evaluate the degree of sugar degradation. In this study, a rapid and inexpensive clean-up method called vortex-assisted liquid–liquid microextraction (VALLME) instead of the expensive solid phase extraction method was successfully applied to purify beverages (tea drinks, soft drinks and carbonated drinks) containing α-dicarbonyl compounds after derivatization with o-phenylenediamine. The clean-up samples were analyzed by using a high-performance liquid chromatography-diode array detector (HPLC-DAD). The matrix effects were corrected by using an internal standard (3,4-hexanedione). The limits of quantitation (LOQs) ranging from 22.3 μg L−1 to 22.7 μg L−1 for GO, from 6.4 μg L−1 to 6.7 μg L−1 for MGO and from 5.4 to 6.2 μg L−1 for DA were observed for standards spiked into tea drinks, soft drinks or carbonated drinks. In addition, the developed method exhibited excellent linearity (R2 > 0.99) for GO (recovery rates, 85.0–108.0%), MGO (recovery rates, 90.8–113.0%) and DA (recovery rates, 93.8–109.3%). The contents of GO (ranging from 265.8 to 9899.8 μg L−1), MGO (<319.7 μg L−1), and DA (<298.6 μg L−1) were accurately quantified in fifteen beverage samples. VALLME as an inexpensive, rapid and valid technique improved the HPLC-DAD determination of GO, MGO and DA in drinks.

Antioxidant and ACE Inhibitory Activity of Enzymatic Hydrolysates from Ruditapes philippinarum

Ruditapes philippinarum proteins were hydrolyzed by trypsin, neutrase, and pepsin. The antioxidant activities and ACE inhibitory activity of hydrolysates were analyzed and the antioxidant activities were related to their molecular weight distribution and amino acid compositions. Results indicated the hydrolysis of proteins led to an increase in small peptides and free amino acids. The antioxidant activities of Ruditapes philippinarum hydrolysates against DPPH radical scavenging, inhibition on linoleic acid peroxidation, and reducing power showed that the neutrase hydrolysate exhibited the strongest antioxidant activity. In addition, an ACE inhibition assay revealed that the pepsin hydrolysate had the highest ACE inhibitory ability. Ruditapes philippinarum protein hydrolysates could be a promising source of natural antioxidant and ACE inhibitory.

Identification of a novel ACE-inhibitory peptide from casein and evaluation of the inhibitory mechanisms

Various bioactive peptides are continuously being identified from casein hydrolysates. In this work, a novel angiotensin I-converting enzyme (ACE)-inhibitory (ACEI) peptide, NMAINPSKENLCSTFCK, derived from the αs2-casein fragment residues 25-41, was screened and identified by UPLC-ESI-Q-TOF-MS/MS from tryptic casein hydrolysate. The IC50 value of the peptide, determined by an HPLC method, was 129.07u202fμM. The Lineweaver-Burk plot showed that this peptide acted as a mixed-type inhibitor against ACE, which might be attributed to the peptide being susceptible to degradation by ACE, indicating that the mixed-type inhibition could partly be a result of newly generated peptide fragments. The physicochemical characteristics and the secondary structure were evaluated by circular dichroism analysis and online prediction software (Expasy, PepDraw, and ProtParam) to identify the basic characteristics of this peptide. Moreover, molecular docking was simulated by Discovery Studio 2017 R2 software to provide the potential mechanisms underlying the ACEI activity of the peptides.

Effects of ball-milling treatment on mussel (Mytilus edulis) protein: structure, functional properties and in vitro digestibility

Summary nIn this study, the effects of ball-milling treatment on structural properties and functional properties of mussel protein were investigated. In vitro protein digestibility and free amino acid contents were also evaluated. Ball-milling treatment did not change the primary structure of mussel protein, but caused changes of secondary structure. The tertiary and quaternary structure also changed. After 20xa0min of ball-milling treatment, the whiteness and oil-binding capability of mussel protein significantly improved from 13.67 to 26.62 and 43.76% to 196.00%, while the protein solubility and water-holding capability of mussel protein significantly decreased from 74.89% to 53.10% and 215.67% to 90.91%. Ball-milling treatment increased the inxa0vitro digestibility significantly. These results provide theoretical basis for the utilisation of mussel protein in food industry.

Effects of high-pressure homogenization on functional properties and structure of mussel (Mytilus edulis) myofibrillar proteins

Mussel myofibrillar proteins (MMP) suspensions (10.6%u202f±u202f0.5%, w/v) were treated by high-pressure homogenization (HPH) at 0 (control), 20, 40, 60, 80 or 100u202fMPa for 3u202fcycles. Particle size distribution, zeta potential, solubility, water and oil holding capacity, emulsifying, foaming properties, secondary structure, free sulfhydryl and surface hydrophobicity of the obtained suspensions were analyzed. The results showed that functional properties of MMP significantly (Pu202f<u202f0.05) improved after HPH treatment. Absolute zeta potential, emulsifying activity index, emulsion stability index, foaming ability and foaming stability increased by 23.64u202fmV, 14.99u202fm2/g, 4.3u202fmin, 17.3% and 29.7% at 80u202fMPa, protein solubility and oil holding capacity increased by 7.4% and 1300% at 100u202fMPa. However, HPH treatment significantly (Pu202f<u202f0.05) decreased particle size and water holding capacity. HPH treatment altered secondary structure, tertiary and quaternary structure. Functionality improvements mainly resulted from changes in structure and decrease in particle size. The results showed that HPH has potential for improving functional properties of MMP, thus expand its application in food industry.

Isotope dilution determination for the trace level of 4(5)-methylimidazole in beverages using dispersive liquid-liquid microextraction coupled with ESI-HPLC–MS/MS

In this study, a rapid and reliable pretreatment method called dispersive liquid-liquid microextraction (DLLME) was developed for the determination of 4(5)-methylimidazole (4-MeI) in beverages. The clean-up samples were analyzed by a high performance liquid chromatography coupled with triple quadrupole mass spectrometer (HPLC-MS/MS) using a reversed-phase C18 column (250u202fmmu202f×u202f4.6u202fmm, 5u202fμm). The matrix effects and the fault of DLLME operation were corrected by the isotope dilution technique. The developed method exhibited the limit of quantification (0.3u202fμgu202fL-1) and excellent linearity (R2u202f=u202f0.999). In addition, the recovery values of 4-MeI in carbonated beverage were ranging from 102.60% to 113.22%, in soft drinks were ranging from 103.24% to 108.85%. The content 4-MeI was detected in thirteen beverage samples treated with DLLME and solid phase extraction method (SPE) (ranging from 6.4 to 165.5u202fμgu202fL-1). The consistent result of 4-MeI content detected by DLLME and SPE method concluded that the established method on the basis of DLLME was rapid, robust and accurate for the trace 4-MeI determination in beverages.

Structure, Function, and Nutrition of Lactoferrin

Lactoferrin (Lf) is a polyfunctional protein from varied secretions of organisms. To date, about 20 different physiological roles of Lf have been reported. More and more studies on the structure and functions of Lf as well as the relationship of them have been intensively reported. In order to understand the Lf bioactivities and the mechanisms of different Lf functions, especially on the relationship between structure and functions, the research advancements are described in this study. In particular, the structure, bioactive site, thermal stability, several kinds of bioactive mechanisms such as antimicrobial, osteogenic, immunomodulatory, antitumor, antioxidant, and enzymic activities, and so forth are involved. Altogether, these are expected to provide some new ideas for the interesting topics about Lf, not only helpful for scientific research, but also for practical application in the medical and food industries, respectively.

Identification and mechanism evaluation of a novel osteogenesis promoting peptide from Tubulin Alpha-1C chain in Crassostrea gigas

Marine shellfish provides a series of biofunctionality account of its high-protein level. In this study, the osteogenic effect of a novel peptide, YRGDVVPK, from Crassostrea gigas protein hydrolysates on preosteoblast MC3T3-E1 proliferation was examined. Synthetic peptide with 100u202fnM significantly promoted the proliferation of MC3T3-E1 cells for a treatment of 72u202fh assayed by MTT method, and which was confirmed by the increase of alkaline phosphatase (ALP) activity. The peptide, YRGDVVPK, was docked with integrin α5β1 (PDB ID: 3VI4), which is a surface receptor of MC3T3-E1. The interaction of the peptide with integrin α5β1 (PDB ID: 3VI4) was analyzed by the molecular modeling algorithm of CDOCKER, which showed a more stable combination than the original ligand. The results suggested the novel peptide could promote the preosteoblast MC3T3-E1 proliferation probably by activating the signaling pathway of MAPK, which is induced through binding with peptide YRGDVVPK.