It is well documented that caffeic acid (3,4‐dihydroxycinnamic acid) (CA) interacts with and inhibits the oxidative reactions of myoglobin (Mb) and hemoglobin (Hb), and this interaction underlies its antioxidative action in meat. Sickle cell hemoglobin (HbS) is known for its tendency to oxidize more readily than normal HbA in the presence of hydrogen peroxide (H2O2), which leads to a more persistent and highly oxidizing ferryl Hb (HbFe4+). We have investigated the effects of CA on HbS oxidation intermediates, specifically on the ferric/ferryl forms. At a low concentration of H2O2 (0.5‐fold over heme), we observed a fivefold reduction in the amount of HbFe4+ accumulated in a mixture of ferric and H2O2 solution. Higher levels of H2O2 (onefold and twofold over heme) led to a lesser threefold and twofold reduction in the content of HbFe4+, respectively, possibly due to the saturation of the binding sites on the Hb molecule. The most intriguing finding was that when 5‐molar excess CA over heme was used, and a considerable increase in the delay time of HbS polymerization to approximately 200 s was observed. This delay in polymerization of HbS is theoretically sufficient to avoid microcapillary blockage and prevent vasoconstrictions in vivo. Mass spectrometry analysis indicated that CA was more extensively covalently bonded to βCys93 than to βCys112 and αCys104. The dual antioxidant and antisickling properties of CA may be explored further to maximize its therapeutic potential in SCD.

Rosemary extract (R) was added to pork sausage and compared with a novel antioxidant mixture of R and phospholipase A2 (R + P) as well as a treatment of synthetic antioxidants (Syn). Through 245 days of −20°C storage, lipid hydroperoxides (LOOH), redness, and hexanal were similar between R and R + P. However, earlier in storage, R + P decreased LOOH compared with R (p < .05); the neutral lipid hydroperoxides were decreased 3.3‐fold (p < .001), whereas free fatty acid hydroperoxides and polar lipid hydroperoxides were not significantly lowered. Through the long‐term storage, Syn had lower LOOH and hexanal than R + P yet redness was lower in Syn (p < .05). In unseasoned sausage, R + P demonstrated higher redness and lower hexanal values than R treatment (p < .05). These results suggest that R + P could offer an effective natural antioxidant in pork sausage and the antioxidant mechanism may be related to the suppression of neutral lipid hydroperoxides. Novelty impact statement This research demonstrates a novel antioxidant mixture consisting of low concentrations of rosemary extract and phospholipase A2 (PLA2) in pork sausage. The fractionation of total lipids into lipid classes suggested that the antioxidant action of the mixture was related to decreasing neutral lipid hydroperoxides. The antioxidant mixture stabilized color more effectively than synthetic antioxidants in seasoned sausage yet markers of lipid oxidation were lower in the synthetic treatment.

Conversion of the heme iron in myoglobin (Mb) and hemoglobin (Hb) from Fe2+ to Fe3+is a critical step that causes quality deterioration in muscle foods, such as discoloration and generation of oxidative species, including dissociated heme that oxidize lipids and proteins. Increased solvent access to the heme pocket has been proposed to cause oxidation of the heme iron and decrease heme affinity, although empirical results are lacking.&nbsp; This review introduces plasma induced modification of biomolecules (PLIMB) as an approach to modify amino acids of Mb and Hb and thereby assess solvent access to the heme pocket. After PLIMB, liquid chromatography tandem mass spectrometry (LC-MS/MS) peptide analysis and a user-friendly, software platform is utilized to quantify modified amino acid side chains of the heme proteins. Our current findings indicate that PLIMBàLC-MSMS provides a platform to measure solvent access to portions of the heme pocket environment. Evaluation of PLIMB at additional conditions (e.g.&nbsp;different pH values) is underway to better delineate the role of solvent access to the heme pocket relative to the ‘outer-sphere’ mechanism of heme protein oxidation and the ability of hydrogen bonding to stabilize heme within metHbs. Some aspects of heme protein-mediated lipid oxidation that occur at low O2&nbsp;partial pressures are discussed in relation to solvent access to the heme pocket. Other approaches to study mechanisms of discoloration and lipid oxidation related to Mb/Hb oxidation and heme loss from metHb are also discussed.

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ObjectivesThe objective of this study was to measure the loss of redness and onset of lipid oxidation in pre-rigor pork sausage containing synthetic antioxidants (Syn) compared to rosemary extract (R), and a combination of R with different concentrations of phospholipase A2 (R+P) over both light display and frozen storage.Materials and MethodsOur work examined pre-rigor spiced and unspiced pork sausage. Tissue from sows for both spiced and unspiced sausage was coarse ground and cooled to 1–3°C with dry ice within 1-h post-exsanguination. Water, treatments and seasonings were added, and the sausage stuffed within 2 h post-exsanguination for spiced sausages. Water and treatments were added 24 h post-exsanguination for the unspiced sausage. Sausages were stored in the dark at –20°C (to 110 and 245 d for unspiced and spiced, respectively) prior to light display. Sausages were sampled for color and lipid oxidation on approximately 40-d intervals of –20°C dark storage and 7–9 d of light display (5°C). In spiced sausage, R (type HT-P) was added at 200 ppm, PLA2 was added at 0.4 ppm. Butylated hydroxyanisole (BHA), propyl gallate (PG) and citric acid (CA) were each added at 0.01% of the estimated fat and collectively formed the Syn treatment. Spices consisted of sucrose, ginger, coriander, nutmeg, white pepper, and MSG. In unspiced sausage R was added at 200 ppm, PLA2 added at 0.4 ppm and 10 ppm, and BHA, CA and PG added at the same levels as in spiced sausage. Color stability was measured based on redness (a*). Peroxide values (PVs) were measured spectrophotometrically, headspace hexanal was measured via gas chromatography (GC) and α tocopherol depletion was measured with HPLC fluorescence detection as markers of lipid oxidation. Total lipids were fractionated to gravimetrically quantify neutral lipids, free fatty acids and polar lipids and to measure PVs in the aforesaid fractions. Unspiced sausages were only stored for 110 d because of rampant lipid oxidation and loss of color.ResultsIn spiced sausage, R and R+P displayed better color stability than both the control (no antioxidant, C) and Syn. Syn displayed the lowest hexanal values. R had the highest PVs and both Syn and R+P were significantly lower. Free fatty acids were the most heavily oxidized fraction on an oil basis, while neutral lipids were the most oxidized lipid on a wet weight basis. Alpha tocopherol did not deplete through 245 d in spiced sausage but was not detected in the unspiced sausage.In unspiced sausage, R+P was examined at two different levels (0.4 ppm and 10 ppm PLA2). R+P (10 ppm) exhibited lower headspace hexanal than R alone and R+P at both levels performed as well as Syn. In addition, R+P at both levels displayed significantly better color stability than R alone and was as good as Syn.ConclusionIn conclusion, R+P decreased lipid oxidation (compared to R) and enhanced color stability (compared to R) and offer an alternative to synthetic antioxidants in pre-rigor pork sausage. Furthermore, spiced pork sausage displayed mean redness values above 9 through 245 d, compared to only 75 d in unspiced sausage.