Effective Preparation Method of Phosphopeptides from Phosvitin and the Analysis of Peptide Profiles Using Tandem Mass Spectrometry.

The effect of high-temperature and mild-pressure (HTMP) pretreatment on the enzymatic hydrolysis of phosvitin and the structural characteristics of the phosphopeptides produced were analyzed using tandem mass spectrometry. The HTMP pretreatment hydrolyzed phosvitin at random sites and helped the subsequent enzyme hydrolysis of the peptides produced. With the HTMP pretreatment alone, 154 peptides were produced, while the use of trypsin, Protex 6L, and Multifect 14L in combination with the pretreatment produced 252, 280, and 164 peptides, respectively. The use of two enzyme combinations (trypsin + Protex 6L and trypsin + Multifect 14L) helped the hydrolysis further. The number of phosphopeptides produced increased when the modifications within the same amino acid sequences were considered. This study indicated that HTMP pretreatment was a breakthrough method to improve the enzymatic hydrolysis of phosvitin that enabled an easy production of phosvitin phosphopeptides for their subsequent functional characterizations.

Effect of Packaging and Antioxidant Combinations on Physicochemical Properties of Irradiated Restructured Chicken Rolls.

Effects of double packaging (combinational use of aerobic and vacuum conditions) and antioxidants on physicochemical properties in irradiated restructured chicken rolls were determined. Chicken breast treated with antioxidants (none, sesamol+a-tocopherol) was used to process restructured chicken breast rolls. The sliced rolls were vacuum, aerobic, or double packaged (vacuum for 7 d then aerobic for 3 d) and electron beam irradiated at 2.5 kGy. Color, 2-thiobarbituric acid reactive substances (TBARS), oxidation reduction potentials (ORP), and volatile profiles of the samples were determined at 0 and 10 d. Irradiation made restructured chicken rolls redder (p<0.05), and the increased redness was more distinct in irradiated vacuum-packaged than irradiated aerobic or double packaged meats. TBARS values of antioxidant-treated double packaged rolls were lower than even nonirradiated vacuum-packaged meat, and those were distinct at 10 d (p<0.05). ORP and lipid oxidation values were lower in irradiated vacuum and double packaged samples than those in irradiated aerobic packaged ones at 0 d (p<0.05). Irradiation of restructured chicken rolls increased the amount of total volatiles. Considerable amounts of off-odor volatiles were reduced or not detected by double packaging and antioxidant treatment at 10 d. Therefore, the combined use of antioxidants and double packaging would be useful to reduce redness and control the oxidative quality changes of irradiated restructured chicken rolls.

Induction of apoptosis by isoegomaketone from Perilla frutescens L. in B16 melanoma cells is mediated through ROS generation and mitochondrial-dependent, -independent pathway.

We have demonstrated for the first time the mechanism underlying ROS-mediated mitochondria-dependent apoptotic cell death triggered by isoegomaketone (IK) treatment in melanoma cells. We showed that IK induced apoptotic cell death and tumor growth inhibition using tissue culture and in vivo models of B16 melanoma. Furthermore, we observed that IK effectively induced apoptotic cell death, including sub-G1 contents up-regulation, nuclei condensation, DNA fragmentation, and caspase activation in B16 melanoma cells. Pretreatment with caspase inhibitor increased the survival rate of IK-treated B16 cells, implying that caspases play a role in IK-induced apoptosis. Furthermore, IK treatment generated ROS in melanoma cells. We also determined whether or not IK-induced cell death is due to ROS production in B16 cells. N-acetyl cysteine (NAC) inhibitedIK-induced Bcl-2 family-mediated apoptosis. This result indicates that IK-induced apoptosis involves ROS generation as well as up-regulation of Bax and Bcl-2 expression, leading to release of cytochrome c and AIF. Our data suggest that IK inhibits growth and induces apoptosis in melanoma cells via activation of ROS-mediated caspase-dependent and -independent pathways.

Protein oxidation: basic principles and implications for meat quality.

The involvement of oxidized proteins to the development of biological diseases has been studied for a few decades, but the effects and the mechanisms of protein oxidation in food systems are largely unknown. Protein oxidation is defined as the covalent modification of a protein induced either by the direct reactions with reactive oxygen species (ROS) or indirect reactions with secondary by-products of oxidative stress. ROS can cause oxidation in both amino acid side chains and protein backbones, resulting in protein fragmentation or protein-protein cross-linkages. Although all amino acids can be modified by ROS, cysteine, and methionine that are the most susceptible to oxidative changes due to high reaction susceptibility of the sulfur group in those amino acids. Oxidative modifications of proteins can change their physical and chemical properties, including conformation, structure, solubility, susceptibility to proteolysis, and enzyme activities. These modifications can be involved in the regulation of fresh meat quality and influence the processing properties of meat products. Oxidative stress occurs when the formation of oxidants exceeds the ability of antioxidant systems to remove the ROS in organisms. Increased levels of protein oxidation have been associated with various biological consequences, including diseases and aging, in humans and other animal species. The basic principles and products of protein oxidation and the implications of protein oxidation in food systems, especially in meat, are discussed in this review.

Effects of high-pressure processing and enzymatic dephosphorylation on phosvitin properties.

BACKGROUND: Egg phosvitin could be a good source of functional peptides. Enzymatic dephosphorylation and high-pressure processing combined with thermal treatment applied before proteolysis could produce phosvitin hydrolysates with different properties compared to its native form. RESULTS: Phosvitin structure was maintained overall during high-pressure treatment of 600 MPa applied at an initial temperature of 65 °C regardless of the pH and duration of treatment, confirming the high structural stability of this phosphoprotein. Treatment of phosvitin with phosphatase increased the degree of dephosphorylation from 24% to 63%, after 2 and 18 h, respectively. Moderate dephosphorylation of phosvitin prior to proteolytic digestion improved its hydrolysis, allowing formation of peptides with a molecular weight lower than 17,000 kDa as determined by size exclusion chromatography. Angiotensin-converting enzyme (ACE) inhibition and antioxidant activity of dephosphorylated and protease-treated phosvitin was increased by 52% and 39%, respectively, as compared to protease-digested native phosvitin. CONCLUSION: Enzymatic dephosphorylation before proteolysis mimicking in vivo gut conditions improved ACE inhibition and antioxidant activity of phosvitin hydrolysates.

Effect of cooking on radiation-induced chemical markers in beef and pork during storage.

UNLABELLED: Raw and cooked beef and pork loins were irradiated at 0 or 5 kGy. The radiation-induced marker compounds, such as hydrocarbons, 2-alkylcyclobutanones (2-ACBs), and sulfur volatiles, were determined after 0 and 6 mo of frozen storage. Two hydrocarbons (8-heptadecene [C(17:1)] and 6,9-heptadecadiene [C(17:2)]) and two 2-ACBs (2-dodecylcyclobutanone [2-DCB] and 2-tetradecylcyclobutanone [2-TCB]) were detected only in irradiated raw and cooked meats. Although precooked irradiated meats produced more hydrocarbons and 2-ACBs than the irradiated cooked ones, the amounts of individual hydrocarbons and 2-ACBs, such as 8-heptadecene, 6,9-heptadecadiene, 2-DCB, and 2-TCB, were sufficient enough to detect whether the meat was irradiated or not. Dimethyl disulfide and dimethyl trisulfide were also determined only in irradiated meats but dimethyl trisulfide disappeared after 6 mo of frozen storage under oxygen-permeable packaging conditions. The results indicated that 8-heptadecene, 6,9-heptadecadiene, 2-DCB, 2-TCB, and dimethyl disulfide, even though they were decreased with storage, could be used as marker compounds for the detection of irradiated beef and pork regardless of cooking under the frozen conditions for 6 mo. PRACTICAL APPLICATION: Radiation-induced chemical changes such as specific hydrocarbons, 2-ACBs, and sulfur volatiles may be used as potential identification markers by regulatory authorities to confirm irradiation history of frozen stored raw or cooked beef and pork.

Consumption of oxidized oil increases oxidative stress in broilers and affects the quality of breast meat.

A total of 120 4-week-old broiler chickens were allotted to 12 pens and fed one of three diets including control, oxidized diet (5% oxidized oil), or antioxidant-added diet (500 IU vitamin E) for 2 weeks. Blood samples were collected at the end of feeding trial, and breast muscles were sampled immediately after slaughter. Breast meats were also collected 24 h after slaughter and used for meat quality measurements. Oxidative stress in blood, lipid and protein oxidation, and sarcoplasmic reticulum Ca²(+)-ATPase (SERCA) activity of breast muscle were determined. The oxidized diet increased oxidative stress in blood and increased carbonyl content in breast meat compared with the other two dietary treatments (P < 0.05). Lipid oxidation of breast muscles with the antioxidant-supplemented diet was lower than that with the oxidized and control diet groups (P < 0.05). Meat from birds fed the oxidized diet showed higher drip loss after 1 and 3 days of storage and greater 0-1 h post-mortem pH decline (P < 0.05). Significant differences in specific SERCA activity in breast muscles from birds fed control and oxidized diets (P < 0.05) were detected. This suggested that dietary oxidized oil induced oxidative stress in live birds and increased lipid and protein oxidation in breast muscle. Decrease in SERCA activity in breast muscles due to oxidative stress in live animals accelerated post-mortem glycolysis, which sped the pH drop after slaughter and increased drip loss, indicating that oxidation of diet can cause PSE-like (pale, soft, and exudative) conditions in broiler breast muscles.

Far-infrared radiation increases the antioxidant properties of rice hull extract in cooked turkey meat.

To determine the antioxidant effects of rice hull extract exposed to far-infrared radiation, the added extracts were compared with sesamol in cooked turkey breast. Rice hull extract showed antioxidant properties in cooked turkey breast by reducing lipid oxidation and volatile aldehydes. Far-infrared radiation increased significantly the antioxidant activities of rice hull extracts. Rice hull extract irradiated by far-infrared (FRH) had lower TBARS values and fewer volatile aldehydes (hexanal, pentanal, and propanal) than a non-irradiated extract (IRH) during the 3 days of aerobic storage. Addition of FRH at 0.2% (w/w) in turkey meat could reduce the amounts of volatile hexanal to 18-47% of the control during the storage. However, the antioxidant activities of rice hull extracts did not last as long as those of pure sesamol due to the relatively low concentration of phenolics, and the extracts had some peculiar odor. Addition of rice hull extracts also increased both a and b values of the samples due to its brown intensity.

Nutritional regulation of porcine bacterial-induced colitis by conjugated linoleic acid.

Excessive intake of saturated fatty acids and/or linoleic acid favors the induction of an array of lipid mediators and cytokines enhancing inflammatory responses. Conversely, dietary supplementation with (n-3) fatty acids or vitamin D ameliorates inflammation and autoimmune diseases. Although it was well accepted that conjugated linoleic acid (CLA) prevented diseases with a common inflammatory pathogenesis (i.e., cancer and atherosclerosis), no studies were available on the roles of CLA in mucosal inflammation. The present study was designed to investigate the anti-inflammatory actions and molecular mechanisms underlying the regulation of colonic health by CLA. We hypothesized that colonic inflammation can be ameliorated by dietary CLA supplementation. To test this hypothesis, inflammation of the colonic mucosa was triggered by challenging pigs fed either soybean oil-supplemented or CLA-supplemented diets with an enteric bacterial pathogen (i.e., Brachyspira hyodysenteriae). Immunoregulatory cytokines and peroxisome proliferator-activated receptor-gamma (PPAR-gamma) mRNA expression were assayed in colonic lymph nodes and colon of pigs. Colonic mucosal lesions and lymphocyte subset distribution were evaluated by histology and immunohistochemistry. Supplementation of CLA in the diet before the induction of colitis decreased mucosal damage; maintained cytokine profiles (i.e., interferon-gamma and interleukin-10) and lymphocyte subset distributions (i.e., CD4+ and CD8+), resembling those of noninfected pigs; enhanced colonic expression of PPAR-gamma; and attenuated growth failure. Therefore, CLA fed preventively before the onset of enteric disease attenuated inflammatory lesion development and growth failure.