Investigation of the peptides with calcium chelating capacity in hydrolysate derived from spent hen meat.

Calcium peptide chelates are developed as efficient supplements for preventing calcium deficiency. Spent hen meat (SHM) contains a high percentage of proteins but is generally wasted due to the disadvantages such as hard texture. We chose the underutilized SHM to produce peptides to bind calcium by proteolysis and aimed to investigate chelation between calcium and peptides in hydrolysate for a sustainable purpose. The optimized proteolysis conditions calculated from the result of response surface methodology for two-step hydrolysis were 0.30% (wenzyme/wmeat) for papain with a hydrolysis time of 3.5 h and 0.18% (wenzyme/wmeat) for flavourzyme with a hydrolysis time of 2.8 h. The enzymatic hydrolysate (EH) showed a binding capacity of 63.8 ± 1.8 mg calcium/g protein. Ethanol separation for EH improved the capacity up to a higher value of 68.6 ± 0.6 mg calcium/g protein with a high association constant of 420 M-1 (25°C) indicating high stability. The separated fraction with a higher amount of Glu, Asp, Lys, and Arg had higher calcium-binding capacity, which was related to the number of ─COOH and ─NH2 groups in peptide side chains according to the result from amino acid analysis and Fourier transform infrared spectroscopy. Two-step enzymatic hydrolysis and ethanol separation were an efficient combination to produce peptide mixtures derived from SHM with high calcium-binding capacity. The high percentage of hydrophilic amino acids in the separated fraction was concluded to increase calcium-binding capacity. This work provides foundations for increasing spent hen utilization and developing calcium peptide chelates based on underutilized meat.

ESR spin trapping for in situ detection of radicals involved in the early stages of lipid oxidation of dried microencapsulated oils.

Different studies have shown that detection of free radicals by ESR spin trapping provides useful information on the susceptibility to oxidation of bulk oils and accordingly on the oxidative stability of different samples for comparative purposes. With the same goal, ESR spin trapping was evaluated in this work for in situ detection of radicals in dried microencapsulated oils (DMOs). By testing different oils, encapsulation matrices and oxidation conditions, results showed that ESR spin trapping can be useful to evaluate the oxidative susceptibility of DMOs, but ESR data should be interpreted cautiously, as the great complexity of the reactions involved may lead to data misinterpretations. Conditions for oxygen availability can have important impacts on the rates of both spin trapping and spin-adduct quenching affecting the levels of radicals observed. The kinetics of oxidation, spin trapping and spin-adduct decay should be known first in bulk oils for correct data interpretation in DMOs.

Plant derived ingredients rich in nitrates or phenolics for protection of pork against protein oxidation.

A pork model system containg phenolic extracts (citrus, rosemary, and acerola), traditional Spanish food ingredients (paprika, garlic, and oregano), or natural nitrate sources (beet, lettuce, arugula, spinach, chard, celery, and watercress) were oxidized by an hydrophilic (OXHydro, 2,2'-azobis(2-amidinopropane)-dihydrochloride; AAPH) or lipophilic (OXLip, 2,2'-azobis(2,4-dimethylvaleronitrile; AMVN) radical initiator. Citrus as well as lettuce and spinach protected almost fully against protein thiol loss and showed efficient radical scavenging activity as determined by ESR spectroscopy in both oxidizing systems. Rosemary was an efficient radical scavenger in both systems, but behaved as a prooxidant on thiols in the OXHydro system. Acerola was also found to be prooxidative as determined by increased radical signal intensity especially in the OXLip system, assigned to high concentration of ascorbate in the extract. Natural nitrate sources, especially lettuce and spinach, are accordingly potential substitutes for synthetic phenolic antioxidants protecting against protein thiol oxidation and radical formation in pork.

Mate extract is superior to green tea extract in the protection against chicken meat protein thiol oxidation.

Water soluble caffeic acid derivatives in aqueous extracts of mate (Ilex paraguariensis) showed a direct dose-dependent response, protecting protein thiols in a chicken breast meat model system when oxidation was initiated by a lipophilic radical initiator (2,2'-azobis(2,4-dimethylvaleronitrile; AMVN). In contrast, an inverse dose-dependent response was obtained when initiated by a hydrophilic radical initiator (2,2'-azobis(2-amidinopropane)dihydrochloride; AAPH), pointing toward a mechanism, where a specific concentration of water-soluble polyphenol yields optimum protection. Optimal concentrations of mate extract for the protection of thiols against radicals initiated in the lipid phase (AMVN) or in the aqueous phase (AAPH) were 0.1-0.5% and 0.01-0.1%, respectively. Green tea (Camellia sinensis) extract showed a pro-oxidative effect involving quinones from oxidation of pyrogallol-type catechins (epigallocatechin-3-gallate (EGCG), gallocatechin-3-gallate (GCG), and epicatechin-3-gallate (ECG)) and not the catechol-type catechins (catechin and epicatechin).

Antioxidant efficiency and mechanisms of green tea, rosemary or maté extracts in porcine Longissimus dorsi subjected to iron-induced oxidative stress.

Plant extracts from rosemary (RE), green tea (GTE), and maté (ME) were compared for the protection against iron-induced oxidation in porcine homogenates at total phenolic concentrations from 25 to 250 ppm. Lipid oxidation as indicated by TBARS was in all cases sufficiently suppressed, especially for RE. Hydrophobic RE retarded overall oxidation in the homogenates with an inverted dose-dependent response. Optimum delay of oxygen consumption was found at the lowest concentration applied, similar to protection against thiols and formation of protein radicals as measured by ESR, whereas the high concentration increased oxygen consumption and caused additionally thiol loss possibly due to thiol-quinone interactions, generating protein-phenol complexes. Hydrophilic ME or GTE increased the initial oxygen consumption rate as an indication of prooxidant activities at elevated concentrations. However, they were found to protect myoglobin and protein at those high concentrations with GTE being more efficient, possibly due to better chelation effect.

Naturally occurring nanotube with surface modification as biocompatible, target-specific nanocarrier for cancer phototherapy.

Phototherapy has drawn increasing attention including the use of nanocarriers with high drug loading capacity and delivery efficacy for target-specific therapy. We have made use of naturally-occurring halloysite nanotubes (HNTs) to build a biomimetic nanocarrier platform for target-specific delivery of phototherapeutic agents. The HNTs were decorated with poly(sodium-p-styrenesulfonate) (PSS) to enhance the biocompatibility, and were further functionalized by lumen loading the type-II photosensitizer indocyanine green (ICG). The HNT-PSS-ICG nanocarrier, without further tethering targeting groups, was shown to associate with the membrane of giant unilamellar vesicles (GUVs) via Pickering effects. Application of HNT-PSS-ICG nanocarrier to human breast cancer cells gave rise to a cell mortality as high as 95%. The HNT-PSS-ICG nanocarrier was further coated with MDA-MB-436 cell membranes to endow it with targeting therapy performance against breast cancer, which was confirmed by in vivo experiments using breast cancer tumors in mice. The membrane-coated and biocompatible nanocarrier preferentially concentrated in the tumor tissue, and efficiently decreased the tumor volume by a combination of photodynamic and photothermal effects upon near-infrared light exposure. Our results demonstrate that the HNT-based nanocarrier by virtue of facial preparation and high loading capacity can be a promising candidate for membrane-targeting nanocarriers.

Protein Oxidation and Sensory Quality of Brine-Injected Pork Loins Added Ascorbate or Extracts of Green Tea or Maté during Chill-Storage in High-Oxygen Modified Atmosphere.

Background: Ascorbate is often applied to enhance stability and robustness of brine-injected pork chops sold for retail, but may affect protein oxidation, while plant extracts are potential substitutes. Methods: Brine-injected pork chops (weight-gain ~12%, NaCl ~0.9%) prepared with ascorbate (225 ppm), green tea extract (25 ppm gallic acid equivalents (GAE)), or maté extract (25 ppm GAE) stored (5 °C, seven days) in high-oxygen atmosphere packaging (MAP: 80% O2 and 20% CO2) were analyzed for color changes, sensory quality, and protein oxidation compared to a control without antioxidant. Results: No significant differences were observed for green tea and maté extracts as compared to ascorbate when evaluated based on lipid oxidation derived off-flavors, except for stale flavor, which maté significantly reduced. All treatments increased the level of the protein oxidation product, α-aminoadipic semialdehyde as compared to the control, and ascorbate was further found to increase thiol loss and protein cross-linking, with a concomitant decrease in the sensory perceived tenderness. Conclusions: Green tea and maté were found to equally protect against lipid oxidation derived off-flavors, and maté showed less prooxidative activity towards proteins as compared to ascorbate, resulting in more tender meat. Maté is a valuable substitute for ascorbate in brine-injected pork chops.

Dose-Dependent Effects of Green Tea or Maté Extracts on Lipid and Protein Oxidation in Brine-Injected Retail-Packed Pork Chops.

Background: Phenolic plant extracts are added as antioxidants in meat to prevent lipid oxidation, but depending on the concentration applied, may affect proteins either through covalent interactions or by serving as a prooxidant. Methods: Brine-injected pork chops prepared with green tea extract (25-160 ppm gallic acid equivalents (GAE)), or maté extract (25-160 ppm GAE) and stored (5 °C, 7 days) in high-oxygen atmosphere packaging (MAP: 80% O2 and 20% CO2) were analyzed for color changes, lipid oxidation by thiobarbituric acid reactive substances (TBARS), and protein oxidation evaluated by thiol loss and protein radical formation by electron spin resonance (ESR) spectroscopy, and compared to a control without antioxidant. Results: Extract of maté and green tea showed significant and comparable antioxidative effects against formation of TBARS in brine-injected pork chops for all concentrations applied compared to the control. Protein radical formation decreased significantly by addition of 25 ppm maté extract, but increased significantly by addition of 80-160 ppm green tea extract, when monitored as formation of protein radicals. Meanwhile, protein thiol groups disappeared when applying the extracts by reactions assigned to addition reactions of oxidized phenols from the extracts to protein thiols. Conclusion: Maté is accordingly a good source of antioxidants for protection of both lipids and proteins in brine-injected pork chops chill-stored in high-oxygen atmosphere, though the dose must be carefully selected.

Zinc Bioavailability from Phytate-Rich Foods and Zinc Supplements. Modeling the Effects of Food Components with Oxygen, Nitrogen, and Sulfur Donor Ligands.

Aqueous solubility of zinc phytate (Ksp = (2.6 ± 0.2) × 10-47 mol7/L7), essential for zinc bioavailability from plant foods, was found to decrease with increasing temperature corresponding to ΔHdis of -301 ± 22 kJ/mol and ΔSdis of -1901 ± 72 J/(mol K). Binding of zinc to phytate was found to be exothermic for the stronger binding site and endothermic for the weaker binding site. The solubility of the slightly soluble zinc citrate and insoluble zinc phytate was found to be considerably enhanced by the food components with oxygen donor, nitrogen donor, and sulfur donor ligands. The driving force for the enhanced solubility is mainly due to the complex formation between zinc and the investigated food components rather than ligand exchange and ternary complex formation as revealed by quantum mechanical calculations and isothermal titration calorimetry. Histidine and citrate are promising ligands for improving zinc absorption from phytate-rich foods.

Mate extract as feed additive for improvement of beef quality.

Mate (Ilex paraguariensis A.St.-Hil.) is generally recognized as safe (GRAS status) and has a high content of alkaloids, saponins, and phenolic acids. Addition of mate extract to broilers feed has been shown to increase the oxidative stability of chicken meat, however, its effect on beef quality from animals supplemented with mate extract has not been investigated so far. Addition of extract of mate to a standard maize/soy feed at a level of 0.5, 1.0 or 1.5% w/w to the diet of feedlot for cattle resulted in increased levels of inosine monophosphate, creatine and carnosine in the fresh meat. The content of total conjugated linoleic acid increased in the meat as mate extract concentration was increased in the feed. The tendency to radical formation in meat slurries as quantified by EPR spin-trapping decreased as increasing mate extract addition to feed, especially after storage of the meat, indicating higher oxidative stability. Mate supplementation in the diet did not affect animal performance and carcass characteristics, but meat from these animals was more tender and consequently more accepted by consumers. Mate extract is shown to be a promising additive to feedlot diets for cattle to improve the oxidative stability, nutritive value and sensory quality of beef.

Regeneration of ß-Carotene from Radical Cation by Eugenol, Isoeugenol, and Clove Oil in the Marcus Theory Inverted Region for Electron Transfer.

The rate of regeneration of ß-carotene by eugenol from the ß-carotene radical cation, an initial bleaching product of ß-carotene, was found by laser flash photolysis and transient absorption spectroscopy to be close to the diffusion limit in chloroform/methanol (9:1, v/v), with a second-order rate constant (k2) of 4.3 × 109 L mol-1 s-1 at 23 °C. Isoeugenol, more reducing with a standard reduction potential of 100 mV lower than eugenol, was slower, with k2 = 7.2 × 108 L mol-1 s-1. Regeneration of ß-carotene following photobleaching was found 50% more efficient by eugenol, indicating that, for the more reducing isoeugenol, the driving force exceeds the reorganization energy for electron transfer significantly in the Marcus theory inverted region. For eugenol/isoeugenol mixtures and clove oil, kinetic control by the faster eugenol determines the regeneration, with a thermodynamic backup of reduction equivalent through eugenol regeneration by the more reducing isoeugenol for the mixture. Clove oil, accordingly, is a potential protector of provitamin A for use in red palm oils.

Antioxidative and prooxidative effects in food lipids and synergism with α-tocopherol of açaí seed extracts and grape rachis extracts.

Extracts of açaí seed and of grape rachis alone or in combination with α-tocopherol were evaluated as antioxidants in (i) bulk soybean oil, (ii) soybean oil liposomes and (iii) soybean-oil/water emulsions. The extracts made with 57% aqueous ethanol showed an antioxidant activity not dependent on concentration for grape rachis extracts and a concentration-dependent prooxidative activity for açaí seed extracts in bulk soybean oil. Both the extracts, however, protected liposome suspensions and oil/water emulsions against lipid oxidation. Synergism was demonstrated when extracts were combined with α-tocopherol, effects explained by the solubility of extract components in the water-phase and of α-tocopherol in the lipid-phase. Phenolic profiling of the extracts by U-HPLC-ESI-LTQ-MS was used to identify active antioxidants. Açaí seed and grape rachis extracts served as good sources of procyanidins and flavan-3-ols, imparted high antioxidant activity especially when combined with α-tocopherol and are suggested for protection of food oil/water emulsions.

Competitive kinetics as a tool to determine rate constants for reduction of ferrylmyoglobin by food components.

Competitive kinetics were applied as a tool to determine apparent rate constants for the reduction of hypervalent haem pigment ferrylmyoglobin (MbFe(IV)O) by proteins and phenols in aqueous solution of pH 7.4 and I=1.0 at 25°C. Reduction of MbFe(IV)O by a myofibrillar protein isolate (MPI) from pork resulted in kMPI=2.2 ± 0.1 × 10(4)M(-1)s(-1). Blocking of the protein thiol groups on the MPI by N-ethylmaleimide (NEM) markedly reduced this rate constant to kMPI-NEM=1.3 ± 0.4 × 10(3)M(-1)s(-1) consistent with a key role for the Cys residues on MPI as targets for haem protein-mediated oxidation. This approach allows determination of apparent rate constants for the oxidation of proteins by haem proteins of relevance to food oxidation and should be applicable to other systems. A similar approach has provided approximate apparent rate constants for the reduction of MbFe(IV)O by catechin and green tea extracts, though possible confounding reactions need to be considered. These kinetic data suggest that small molar excesses of some plant extracts relative to the MPI thiol concentration should afford significant protection against MbFe(IV)O-mediated oxidation.

pH dependent antioxidant activity of lettuce (L. sativa) and synergism with added phenolic antioxidants.

Influence of pH on the antioxidant activities of combinations of lettuce extract (LE) with quercetin (QC), green tea extract (GTE) or grape seed extract (GSE) was investigated for both reduction of Fremy's salt in aqueous solution using direct electron spin resonance (ESR) spectroscopy and in L-α-phosphatidylcholine liposome peroxidation assay measured following formation of conjugated dienes. All examined phenolic antioxidants showed increasing radical scavenging effect with increasing pH values by using both methods. QC, GTE and GSE acted synergistically in combination with LE against oxidation of peroxidating liposomes and with QC showing the largest effect. The pH dependent increase of the antioxidant activity of the phenols is due to an increase of their electron-donating ability upon deprotonation and to their stabilization in alkaline solutions leading to polymerization reaction. Such polymerization reactions of polyphenolic antioxidants can form new oxidizable -OH moieties in their polymeric products resulting in a higher radical scavenging activity.

Inhibition of Cholesterol and Polyunsaturated Fatty Acids Oxidation through the Use of Annatto and Bixin in High-Pressure Processed Fish.

Annatto and bixin, the main carotenoid of annatto seeds, were both found to inhibit cholesterol oxidation in minced herring (Clupea harengus) and minced mackerel (Scomber scombrus) during high pressure processing (600 MPa for 10 min) and subsequent chilled storage for 2 wk, a treatment which otherwise increased the content of cholesterol oxidation products above a critical limit for human consumption. Annatto but not bixin reduced the loss of docosahexaenoic acid caused by high pressure processing of herring from 12% to 7%, an effect assigned to antioxidative effects of phenolic compounds in annatto, while bixin as a carotenoid binds to membranes protecting membrane cholesterol.

Synergism between soluble and dietary fiber bound antioxidants.

This study investigates the synergism between antioxidants bound to dietary fibers (DF) of grains and soluble antioxidants of highly consumed beverages or their pure antioxidants. The interaction between insoluble fractions of grains containing bound antioxidants and soluble antioxidants was investigated using (i) a liposome-based system by measuring the lag phase before the onset of oxidation and (ii) an ESR-based system by measuring the reduction percentage of Fremy's salt radical. In both procedures, antioxidant capacities of DF-bound and soluble antioxidants were measured as well as their combinations, which were prepared at different ratios. The simple addition effects of DF-bound and soluble antioxidants were compared with measured values. The results revealed a clear synergism for almost all combinations in both liposome- and ESR-based systems. The synergism observed in DF-bound-soluble antioxidant system paints a promising picture considering the role of fiber in human gastrointestinal (GI) tract health.

Competitive reduction of perferrylmyoglobin radicals by protein thiols and plant phenols.

Radical transfer from perferrylmyoglobin to other target species (myofibrillar proteins, MPI) and bovine serum albumin (BSA), extracts from green tea (GTE), maté (ME), and rosemary (RE), and three phenolic compounds, catechin, caffeic acid, and carnosic acid) was investigated by electron paramagnetic resonance (EPR) spectroscopy to determine the concentrations of plant extracts required to protect against protein oxidation. Blocking of MPI thiol groups by N-ethylmaleimide was found to reduce the rate of reaction of MPI with perferrylmyoglobin radicals, signifying the importance of protein thiols as radical scavengers. GTE had the highest phenolic content of the three extracts and was most effective as a radical scavenger. IC50 values indicated that the molar ratio between phenols in plant extract and MPI thiols needs to be >15 in order to obtain efficient protection against protein-to-protein radical transfer in meat. Caffeic acid was found most effective among the plant phenols.

Efficient scavenging of ß-carotene radical cations by antiinflammatory salicylates.

The radical cation generated during photobleaching of ß-carotene is scavenged efficiently by the anion of methyl salicylate from wintergreen oil in a second-order reaction approaching the diffusion limit with k2 = 3.2 × 10(9) L mol(-1) s(-1) in 9 : 1 v/v chloroform-methanol at 23 °C, less efficiently by the anion of salicylic acid with 2.2 × 10(8) L mol(-1) s(-1), but still of possible importance for light-exposed tissue. Surprisingly, acetylsalicylate, the aspirin anion, reacts with an intermediate rate in a reaction assigned to the anion of the mixed acetic-salicylic acid anhydride formed through base induced rearrangements. The relative scavenging rate of the ß-carotene radical cation by the three salicylates is supported by DFT-calculations.

Oxidative stability and chemical safety of mayonnaise enriched with grape seed extract.

Grape seed extract (GSE), a by-product from the wine industry, was explored for its use as enrichment to mayonnaise, due to its potential health effects. Mayonnaises were enriched with 0 mg GSE per mL, 0.5 mg GSE per mL (~0.050%), 0.9 mg GSE per mL (~0.10%) and 1.4 mg GSE per mL (~0.15%) during preparation and stored in the dark at room temperature for 8 weeks. The antioxidative capacity of the mayonnaises was evaluated by their ability to scavenge the stable radical TEMPO by electron spin resonance (ESR) spectroscopy. The oxidative stability of the mayonnaises was determined by the content of lipid hydroperoxides (peroxide value, POV), the content of conjugated diene hydroperoxides and the content of thiobarbituric acid reactive substances (TBARS). The highest antioxidative capacity and the lowest content of lipid hydroperoxides and TBARS were found in the mayonnaise with the highest percentage of GSE (0.15%). Therefore, the oxidative stability of the mayonnaises enriched with GSE was slightly improved through storage. However, mayonnaise without GSE had the highest sensorial acceptability compared to mayonnaise enriched with GSE. In the Artemia salina assay, a fast screening method for overall toxicity, the death rate of brine shrimps larvae was found to increase for increasing percentage of GSE. A level of 0.05% GSE in mayonnaise is concluded not to constitute any toxicological risks, but to provide significant protection against oxidation during storage.

Palatability and chemical safety of apple juice fortified with pomegranate peel extract.

Pomegranate peel extract (PPE), a by-product of the pomegranate juice industry with potential health effects, was explored for use to fortify reconstituted apple juice in the concentration range 0.5 to 2.0% (w/w). Radical scavenging and antioxidative capacities of the fortified apple juices were evaluated using (i) electron spin resonance (ESR) to quantify their ability to scavenge the stable radical Fremy's salt and (ii) the Trolox equivalent antioxidant capacity (TEAC) assay and compared to apple juice without fortification as control. The highest antioxidative capacity was found in the apple juice fortified with the highest percentage of pomegranate peel extract, while the optimal sensory quality was found by addition of 0.5 g PPE per 100 mL. The Artemia salina assay was used as a fast screening method for evaluating overall toxicity, and showed little toxicity with up to 1.0 g per 100 mL addition of PPE, but increasing toxicity at higher concentrations. Accordingly, it is important to balance addition of PPE, when used for enrichment of apple juice in order to obtain a healthier product, without compromising the sensorial quality or toxicological safety of the apple juice. Concentrations between 0.5 and 1.0 g PPE per 100 mL seem to be acceptable.