Lu-juan Xing

Purification and identification of antioxidative peptides from dry-cured Xuanwei ham.

This study mainly focused on the purification and identification of antioxidative peptides generated in dry-cured Xuanwei ham. Based on scavenging effect on free radicals and ferrous ion, the antioxidant activity of crude peptides from Xuanwei ham was assessed. From the scavenging effects on 1,1-diphenyl-2-picrylhydrazyl (DPPH), hydroxyl (OH) radicals and superoxide anion (O2(-)), it was suggested that XHP generated during the ripening period had a strong antioxidant activity. By using size exclusion chromatography, anion exchange column and reversed-phase HPLC, fractions with a strong antioxidative activity were separated based on their molecular weight and polarity differences. The fraction with strong antioxidant effect was further characterized by LC-MS/MS. The results suggest that antioxidative peptides are produced during the long processing of Xuanwei ham among which the tetrapeptide Asp-Leu-Glu-Glu could be one of the main peptides that play key role in the antioxidant activity.

Effects of power ultrasound on oxidation and structure of beef proteins during curing processing

The aim of this study was to evaluate the effects of power ultrasound intensity (PUS, 2.39, 6.23, 11.32 and 20.96Wcm(-2)) and treatment time (30, 60, 90 and 120min) on the oxidation and structure of beef proteins during the brining procedure with 6% NaCl concentration. The investigation was conducted with an ultrasonic generator with the frequency of 20kHz and fresh beef at 48h after slaughter. Analysis of TBARS (Thiobarbituric acid reactive substances) contents showed that PUS treatment significantly increased the extent of lipid oxidation compared to static brining (P<0.05). As indicators of protein oxidation, the carbonyl contents were significantly affected by PUS (P<0.05). SDS-PAGE analysis showed that PUS treatment increased protein aggregation through disulfide cross-linking, indicated by the decreasing content of total sulfhydryl groups which would contribute to protein oxidation. In addition, changes in protein structure after PUS treatment are suggested by the increases in free sulfhydryl residues and protein surface hydrophobicity. Fourier transformed infrared spectroscopy (FTIR) provided further information about the changes in protein secondary structures with increases in β-sheet and decreases in α-helix contents after PUS processing. These results indicate that PUS leads to changes in structures and oxidation of beef proteins caused by mechanical effects of cavitation and the resultant generation of free radicals.

Inactivation of Escherichia coli O157:H7 and Bacillus cereus by power ultrasound during the curing processing in brining liquid and beef

The objective of this study was to evaluate the effects of ultrasound on the number of Escherichia coli O157:H7 and vegetative cells of Bacillus cereus in brining and beef during the curing processing. The mechanisms of the inactivation for the pathogens were preliminarily explored. The ultrasound intensity (2.39, 6.23, 11.32 and 20.96Wcm-2) and sonication time (30, 60, 90 and 120min) at 10°C were assessed in brine and meat. Plate count data indicated that 20.96Wcm-2 for 120min was the optimal treatment for bacterial reductions. E. coli O157:H7 was more susceptible to ultrasonic treatment in the first 30min than B. cereus in brine. However, a similar reduction percentage with around 40% in both cultures could be achieved when sonicated for 120min. Good fitness of Weibull model (R2>0.98; RMSE<0.12) was obtained with the distinct shape of the curves which predicted microbial inactivation by ultrasound in salt solution (6%). The production of hydrogen peroxide in brine by ultrasound confirmed the effects of ultrasound on the inactivation of microorganisms during the curing processing. The particle size distribution of bacterial and cell fluorescence staining analysis showed that ultrasound could result in the formation of cell fragments through destroying the integrality of the membrane of E. coli O157:H7 and B. cereus. However, the promoting effect of ultrasound on the number of bacteria in beef suggested that the sanitation conditions of equipment and raw meat must be paid attention when applying ultrasound for curing procedure.

Identification of Rosmarinic Acid-Adducted Sites in Meat Proteins in a Gel Model under Oxidative Stress by Triple TOF MS/MS

Triple TOF MS/MS was used to identify adducts between rosmarinic acid (RosA)-derived quinones and meat proteins in a gel model under oxidative stress. Seventy-five RosA-modified peptides responded to 67 proteins with adduction of RosA. RosA conjugated with different amino acids in proteins, and His, Arg, and Lys adducts with RosA were identified for the first time in meat. A total of 8 peptides containing Cys, 14 peptides containing His, 48 peptides containing Arg, 64 peptides containing Lys, and 5 peptides containing N-termini that which participated in adduction reaction with RosA were identified, respectively. Seventy-seven adduction sites were subdivided into all adducted proteins including 2 N-terminal adduction sites, 3 Cys adduction sites, 4 His adduction sites, 29 Arg adduction sites, and 39 Lys adduction sites. Site occupancy analyses showed that approximately 80.597% of the proteins carried a single RosA-modified site, 14.925% retained two sites, 1.492% contained three sites, and the rest 2.985% had four or more sites. Large-scale triple TOF MS/MS mapping of RosA-adducted sites reveals the adduction regulations of quinone and different amino acids as well as the adduction ratios, which clarify phenol-protein adductions and pave the way for industrial meat processing and preservation.

The antioxidant activity and transcellular pathway of Asp-Leu-Glu-Glu in a Caco-2 cell monolayer

Asp-Leu-Glu-Glu (DLEE) is one of the antioxidant peptides purified from Chinese dry-cured Xuanwei ham in our previous study. In the current work, the stability in a simulated digestion system, the transportation pathway and the antioxidant ability of DLEE were further investigated in a Caco-2 cell monolayer. In the simulated gastrointestinal digestion system, no oligopeptides were generated. In the transport trial, the inhibitors cytochalasin D increased the transport of DLEE across the Caco-2 cell monolayer, with Papp values of 3.22 9 10 6 cm s . A decreased expression occludin was observed with the DLEE incubation in the cell monolayer, and the antioxidant activity showed to be increased gradually in basolateral side. This study indicates that the absorption of DLEE could mainly occur via paracellular transport and provides information about its antioxidant activity after being absorbed across a cell monolayer.

The proteomics homology of antioxidant peptides extracted from dry-cured Xuanwei and Jinhua ham

The objective of this study was to investigate the antioxidant activity and the homology of peptides extracted from dry-cured Xuanwei and Jinhua ham. The antioxidant activity of crude peptides was assessed by ORAC and ABTS assays and the scavenging effects on DPPH and O2- free radicals. LC-ESI-Q-TOF-MS/MS was used to analyze the peptide composition. Based on the identified peptides, homologous proteins were matched by the Peaks software. Overall, 243 and 213 peptides were identified with their parent proteins in Xuanwei and Jinhua dry-cured hams. Based on the ORAC and TEAC values, XHP showed higher antioxidant ability than JHP (P < 0.05). After further purification by G-15 chromatography, the results indicate that the oligopeptides with less than 1000 Da had greater antioxidant activity than the other two fractions. Homology-based proteomics showed that the majority of peptides originated from myosin which accounted for 26% in XHP and 32% in JHP respectively.

Caco-2 cell-based electrochemical biosensor for evaluating the antioxidant capacity of Asp-Leu-Glu-Glu isolated from dry-cured Xuanwei ham

A cell-based electrochemical biosensor was developed to determine the antioxidant activity of Asp-Leu-Glu-Glu (DLEE) isolated from dry-cured Chinese Xuanwei ham. A platinized gold electrode (Pt NPs/GE) covered with silver nanowires (Ag NWs) was fabricated to detect H2O2 using redox signaling via cyclic voltammetry (CV), differential pulse voltammetry (DPV), and electrochemical impedance spectroscopy (EIS). Under optimal condition, the detection limit of the modified electrode was 0.12μM with a linear relationship from 0.2 to 2μM, which showed relatively outstanding catalytic effects towards the reduction of H2O2. Furthermore, the generation of reactive oxygen species (ROS) in the cell can be used to indirectly assess changes in intercellular oxidative stress by detecting variations in electrochemical signals. A 3D cell culture of alginate/graphene oxide (NaAlg/GO) was used to encapsulate and immobilize Caco-2 cells. Based on ROS generation and electrochemical results, we found that DLEE could effectively reduce oxidative stress level in Caco-2 cells under external stimulation. DLEE showed high antioxidant activity with a relative antioxidant capacity (RAC) rate of 88.17% at 1.5mg/mL. Finally, an efficient electrochemical biosensor was established using the active 3D Caco-2 cell platform. This system is sensitive and simple to operate with the property to evaluate the antioxidant activity of peptides by the detection of H2O2 in cell membrane. In summary, this work describes a new method for assessing antioxidant capacity of peptide DLEE using cell-based electrochemical signaling with a rapid screening pattern.

Effect of Flavourzyme on proteolysis, antioxidant activity and sensory qualities of Cantonese bacon

The aim of this study was to investigate the influence of Flavourzyme on protein degradation, antioxidant activity and sensory qualities of Cantonese bacon made at 60°C for 28h. Flavourzyme was used at doses of 8, 16, 24, 32 and 40LAUP/kg of raw meat. Results indicated that Flavourzyme addition in Cantonese bacon promoted proteolysis, which was reflected by the increase of various free amino acids and SDS-PAGE-detected changes in the myogen and myofibrillar proteins. Sensory qualities were improved while TPA (texture profile analysis) was decreased significantly (P<0.05). The best sensory scores were obtained at between 24 and 32LAPU/kg. Besides, Flavourzyme addition improved antioxidant activity, and decreased water activity and protein carbonyl content of Cantonese bacon. This study indicated that the addition of Flavourzyme improved eating attributes and storage stability of Cantonese bacon.