Mass spectrometric characterization and thermostability of turkey myoglobin

Pink color defect (PCD) is a major quality problem in the turkey industry leading to pink appearance of pre-cooked, uncured turkey. The present study determined the molecular mass of turkey myoglobin (Mb) using mass spectrometry and characterized the thermostability of turkey Mb, in comparison with beef Mb, to elucidate the molecular basis of PCD. Purified turkey and beef myoglobins were analyzed using Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry. The thermostability of turkey and beef oxymyoglobins was examined at pH and Mb concentrations (pH 6.2 and 0.04 mmol/L for turkey; pH 5.6 and 0.15 mmol/L for beef) reflecting inherent conditions in these meats. Turkey and beef oxymyoglobins were incubated at 71, 75, and 80 °C and percentage myoglobin denaturation (PMD) was determined. Molecular mass of turkey Mb (17,295 Da) was 346 Da greater than beef Mb (16,949 Da) and was approximately 300-350 Da greater than those of other red meat myoglobins, suggesting its unique primary structure. PMD was lower (P < 0.05) in turkey Mb than in beef Mb during incubation at 71, 75 and 80 °C, indicating that in-situ turkey Mb is less susceptible to heat-induced denaturation than beef Mb at typical meat cooking conditions. The observed greater thermostability of turkey Mb compared to beef Mb could be, partially, due to inherent greater pH in turkey than in beef. Possible unique primary structure of turkey Mb could have contributed to its greater thermostability, which is one of the reasons for PCD.     

Effects of malate,lactate, and pyruvate on myoglobin redox stability in homogenates of three bovine muscles

We investigated the effects of glycolytic and tricarboxylic acid cycle metabolic intermediates on myoglobin redox forms and meat colour stability. Eighteen combinations of malate (M), lactate (L), and pyruvate (P) were added to beef Longissimus lumborum, Psoas major, and Semitendinosus muscle homogenates to study their effect on metmyoglobin formation during incubation at 25 °C. Changes in surface colour at 0, 2, 4, 8, and 12 h were evaluated by using reflecto-spectrophotometry [both L*, a*, and b* and wavelengths specific for metmyoglobin (MMb)]. Addition of M, L, and P alone or in combinations stabilized (P < 0.05) L*, a*, and b* values and myoglobin redox forms in muscle homogenates; however, there was a trend for P to be least effective. At the 2% concentrations for the individual metabolites, L was most effective at retarding MMb formation in the Semitendinosus (M was intermediate and P was least effective), and M was most effective in the Psoas major and L. lumborum muscles (L was intermediate and P was least effective). Metmyoglobin was reduced most effectively with a combination of metabolites (M + L > M + P > L + P). Enhancing meat with these metabolites can effectively extend colour life of post-rigor meat, apparently by providing more reducing conditions for myoglobin, thus increasing myoglobin redox form stability.     

Effects of lactate/phosphate injection enhancement on oxidation stability and protein degradation in early postmortem beef cuts packaged in high oxygen modified atmosphere

The influence of lactate/phosphate enhancement on meat color and lipid oxidation stability, tenderness, protein degradation, and protein aggregation of early postmortem beef muscles packaged in a high oxygen modified atmosphere packaging (HiOx-MAP; 80% O₂, 20% CO₂) were studied. At 24 hr postmortem, three bovine muscles (longissimus, semimembranosus, and adductor; n = 10, respectively) were enhanced (10% injection rate) with either lactate (2.5%)/phosphate (0.3%) solution or water, packaged in HiOx-MAP, stored 9 days at 1 °C, and then displayed for 7 days at 1 °C. The lactate/phosphate injection significantly improved color stability (higher a* values) of all three bovine muscles throughout display period. Accumulation of lipid oxidation determined by 2-thiobarbituric acid-reactive substances values was also decreased (P < 0.05) in the lactate/phosphate injection compared to the water treatment during storage and display periods. The objective tenderness values of longissimus and semimembranosus were also improved (P < 0.05) by the lactate/phosphate enhancement treatment compared to the water treatment based on star probe measurement. There were no significant differences found in desmin and troponin-T degradation, or oxidative cross-linking of myosin between treatments. The results suggest that lactate/phosphate enhancement has beneficial effects on color and lipid oxidation stability, and tenderness development of beef cuts under HiOx-MAP conditions.     

Colour and sarcoplasmic protein evaluation of pork following water bath and ohmic cooking

The objective of this study was to investigate the effects of ohmic (OH) and waterbath (WB) cooking on colour attributes and sarcoplasmic changes of porcine longissimus dorsi muscle at the same endpoint temperatures (EPTs; range 10 °C–80 °C). The OH treatment was carried out at 10 V cm^− 1, and the WB temperature at 85 °C. The colour parameters, deoxymyoglobin% (DeoMb) and metmyoglobin% (MetMb) of the OH-cooked meat were significantly lower (P < 0.05) than those obtained by WB-cooking at the same EPTs (range 60 °C–80 °C). SDS-PAGE analysis showed that the meat treated with WB-cooking had a lower sarcoplasmic protein solubility (5.97 mg/g vs.14.89 mg/g, P < 0.05) and fainter protein bands than that of OH-cooking thus, indicating paler colour, and lower water-holding capacity especially in WB-cooked meat at EPTs above 40 °C. Strong correlations among lightness, browness, metmyoglobin% and soluble proteins were observed in meat following OH-cooking.     

Mass spectrometric investigations on lactate adduction to equine myoglobin

Research focused on determining the fundamental mechanisms by which lactate influences color stability has not considered a direct effect of lactate on myoglobin. Thus, the objective of this study was to use Matrix Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry to examine lactate adduction to myoglobin. Equine oxymyoglobin and equine carboxymyoglobin (0.15 mM) were incubated with sodium lactate (200 mM) at 4 °C, pH 5.6 in 50 mM sodium citrate buffer or at 37 °C, pH 7.4 in 50 mM sodium phosphate buffer, simulating typical meat storage and physiological conditions, respectively. Controls consisted of myoglobin plus a volume of deionized water equivalent to that used to deliver the lactate treatments. No peaks corresponding to lactate-Mb adducts could be detected in the mass spectra of samples incubated up to 360 min at pH 7.4, 37 °C or 8 days at pH 5.6 and 4 °C. Our results suggest that lactate did not form covalent adducts with equine oxy- and carboxy-myoglobin.     

Gelling properties of white shrimp (Penaeus vannamei) meat as influenced by setting condition and microbial transglutaminase

The properties of white shrimp (Penaeus vannamei) gel added with different levels of microbial transglutaminase (MTGase) and subjected to setting at 25 °C for 2 h or 40 °C for 30 min, prior to heating at 90 °C for 20 min were studied. Breaking force of gels with and without setting increased with increasing MTGase amount added (P<0.05). However, no changes in deformation in all samples were noticeable (P>0.05). Directly heated gels showed the lower breaking force than those with prior setting at all MTGase levels added (P<0.05). Generally, gels prepared by setting at 25 °C exhibited the greater breaking force than those set at 40 °C, possibly associated with the appropriate protein structure for cross-linking at 25 °C and greater degradation at 40 °C as evidenced by a greater trichloroacetic acid soluble peptide content (P<0.05). Sodium dodecyl sulfate polyacrylamide gel electrophoretic study revealed that myosin heavy chain (MHC) underwent polymerization to a higher extent in the presence of MTGase, but the strengthening effect on gel was dependent on setting temperature. Regardless of setting condition, microstructure of gel added with MTGase was finer with a smaller void, compared with those of gel without MTGase. Therefore, setting temperature played an essential role in gel property of white shrimp meat added with MTGase.     

Interaction between fish myoglobin and myosin in vitro

Interaction between tuna myoglobin and myosins from tuna and sardine was investigated in a model system at 4 °C for up to 24 h. Both sardine and tuna myosins bound progressively with tuna myoglobin as the storage time increased (P < 0.05). The soret absorption peak was noticeable in the myoglobin–myosin mixture. The oxidation of oxymyoglobin in the presence of myosin was generally greater than that found in the absence of myosin (P < 0.05). Oxymyoglobin underwent oxidation to a greater extent in the presence of tuna myosin than sardine myosin (P < 0.05). The interaction between fish myoglobin and myosin also caused changes in reactive sulfhydryl content and altered the tryptophan fluorescent intensity. The loss in Ca²⁺-ATPase activity of myosin varied with fish species and was governed by the myoglobin added. Thus, the interaction between fish myoglobin and myosin most likely occurred as a function of time and was species-specific.     

Oxidative stability and total lipids on thigh and breast meat of broilers fed diets with two fat sources and supplemented with conjugated linoleic acid

Two hundred broiler chickens of 21 days of age were distributed in a completely randomized factorial arrangement 2×5 (two oil sources, i.e. soybean or canola oil and five levels of CLA supplementation, i.e. 0.0%, 0.25%, 0.50%, 0.75% and 1.00%). The purpose of this study was to evaluate the dietary supplementation of broiler diets with CLA and oil sources on the lipid content and on the oxidative stability of chicken meat submitted to refrigeration or freezing storage temperatures. The use of canola oil and increasing CLA levels resulted in a linear reduction (P<0.05) on the total lipids in breast meat. These results can explain a linear reduction (P<0.05) observed in the malonaldehyde content of refrigerated and frozen meat of birds receiving canola oil. Birds receiving soybean oil and supplemented with CLA showed an abrupt reduction of total lipids on breast meat from 0.89 g/100 g at 0% CLA to 0.36 g/100 g at 0.5% CLA followed by a small increase at higher levels of CLA (P<0.05). These observations may help to explain the reduction (P<0.05) of oxidation in breast meat during frozen storage at 50 and 100 days as well as during cold storage at 5 °C.     

Effect of lamb age and retail packaging types on the quality of long-term chilled lamb loins

The objective of this study was to determine the impact of lamb age and packaging types on meat quality. Paired loins (M. longissimus dorsi) were obtained from 36 carcasses that included 4-month-old (New season; NS) and 11-month-old lambs (Old season; OS). The loins were vacuum-stored for eight weeks at − 1.5 °C. After storage, the loins were cut into subsamples and were randomly assigned to either high-oxygen modified atmosphere packaging (HiOx-MAP) or PVC-overwrapping for further display for 8 days. No differences between the age classes in shear force and drip loss were found (P > 0.05). OS loins had significantly higher myoglobin and redness compared with NS loins. HiOx-MAP initially induced a more intense red colour compared with PVC (P < 0.05), but resulted in severe discolouration at the end of display regardless of the age class. This observation suggests that the retail packaging type is the dominant factor over the age on lamb colour stability.     

Near-infrared oximetry of three post-rigor skeletal muscles for following myoglobin redox forms

We investigated the response of frequency-domain multidistance (FDMD) near-infrared (NIR) tissue oximetry for detecting absolute amounts of myoglobin (Mb) redox forms and their relationship to meat colour stability. Four packaging formats were used to create different blends of Mb redox forms and meat colours during display. Changes in surface colour and subsurface pigment forms during simulated display time (0, 2, 4, and 10 d at 2 °C) were evaluated using surface reflecto-spectrophotometry (both L∗a∗b∗ and specific wavelengths) and FDMD NIR tissue oximetry. Data for both methods of direct measurement of oxymyoglobin and deoxymyoglobin were strongly related and accounted for 86–94% of the display variation in meat colour. Indirect estimates of metmyoglobin ranged from r² = 59–85%. It appears that NIR tissue oximetry has potential as a noninvasive, rapid method for the assessment of meat colour traits and may help improve our understanding of meat colour chemistry in post-rigor skeletal muscle.     

Effects of lactate on ground lamb colour stability and mitochondria-mediated metmyoglobin reduction

Previous research suggests that lactate’s colour stabilizing effect in beef is through NADH production and antioxidant activity. However, no research has assessed lactate’s role in lamb colour. Hence, our objectives were to evaluate the effects of lactate on lamb surface discolouration, oxygen consumption, and metmyoglobin reduction. In experiment 1, lactate (final meat concentration = 2.5% w/w) was added to ground lamb (n = 20 carcasses) and patties were stored for 3 days at 1 °C in PVC packaging. Surface colour (CIE L∗ and a∗) and metmyoglobin reducing activity of ground lamb patties were measured. Addition of lactate improved colour stability and metmyoglobin reducing activity (p < 0.05). In experiment 2, mitochondria were isolated from lamb longissimus muscle (n = 3). Addition of lactate–LDH–NAD to mitochondria resulted in significant oxygen consumption and metmyoglobin reduction compared with mitochondrial controls without lactate (p < 0.05). Lactate can improve the colour stability of lamb, possibly by increasing metmyoglobin reducing activity.     

Role of lactate dehydrogenase in metmyoglobin reduction and color stability of different bovine muscles

The role of lactate dehydrogenase (LDH) in metmyoglobin reducing activity (MRA) and color stability of different bovine muscles was studied in two consecutive experiments. In experiment 1, three different bovine muscles – M. longissimus lumborum (LL), M. semimembranosus (SM), and M. psoas major (PM) – were obtained (n = 7, respectively), cut into steaks, PVC packaged, and then displayed for 7 days at 1 °C. The LL was the most red over display time and had more (P < 0.05) LDH-B activity (catalyzing toward NADH generation), LDH1 isoform expression, NADH, and higher (P < 0.05) MRA than the other two muscles studied. The PM had the least color stability and lowest MRA. In experiment 2, LL steaks (n = 8) were cut in half, one side syringe-injected with oxamate, and the other injected with distilled water. Inclusion of oxamate decreased (P < 0.05) LDH-B activity, NADH, and a* values after 10 days display at 1 °C. These results suggest that variation in color stability of physiologically different muscles is regulated by different replenishment rates of NADH via different LDH isozymes.     

Effect of water activity and temperature on degradation of 5′-inosine monophosphate in a meat model system

The influence of water activity (a_w) (0.7,0.8 and 0.9) and temperature (80° and 120 °C) on the degradation of meat flavor precursor inosine monophosphate (IMP) was studied in a meat fiber model system. Breast and leg muscle from Indian domesticated layer chicken (Gallus gallus) were washed repeatedly with 0.1 mol/l phosphate buffer of pH 6 to obtain pigment free and with minimum content of natural IMP in muscle fiber. The freeze-dried breast and leg meat fiber had a protein content of 86.5±0.48% and 85.6±0.50%, respectively. The IMP contents (mg/100 g) of leg muscle fiber (7.3±0.60) was higher (P?0.05) than in the breast meat fiber (5.1±1.20). The degradation of IMP was temperature-dependent (P?0.05) in both types of meat fiber systems. In the samples of a_w 0.8, the IMP degradation in breast meat fiber system was lower (P?0.05) than in a_w at 0.7 and 0.9 samples, when treated at 80 °C, whereas, there was no significance difference (P>0.05) in the degradation of IMP at a_w 0.7 and 0.9 when heated at 120 °C. The degradation of IMP in leg meat fiber model system at higher a_w (0.9) was more (P?0.05) as compared to lower a_w (0.7 and 0.8) at 80 °C, while the samples treated at 120 °C, the degradation of IMP at a_w 0.8 and 0.9 was more (P?0.05) than at a_w 0.7.     

Eating quality of beef from biotypes included in the PGI “Ternera Asturiana” showing distinct physicochemical characteristics and tenderization pattern

Different biotypes of the Protected Geographical Indication (PGI) “Ternera Asturiana” were studied to determine if their differences in physicochemical characteristics and tenderization pattern during maturation (3 to 21 days) had an effect on the consumer evaluation of beef palatability. Biotype affected significantly pH, water holding capacity, chemical composition (P < 0.001) and meat lightness (P < 0.05). Ageing time affected significantly (P < 0.05) colour, meat toughness and sensory attributes in a different way within each biotype. Multivariate analysis showed two different meat groups: 1) meat from mh-genotypes, characterized by high juice losses, lightness (L*), protein content and high sensory acceptability at intermediate (7 and 14 days) ageing times; 2) meat from rustic (AM) breed and biotypes free of myostatin mutation (AV (+/+) and AV × AM), showing higher intramuscular fat, myoglobin content, and instrumental toughness and requiring longer storage times (21 days). This should be taken into account for the proper post-mortem management and commercialization of each product to achieve its best sensory quality.     

Effects of lactate and modified atmospheric packaging on premature browning in cooked ground beef patties

Our objectives were to determine the effects of lactate and modified atmosphere packaging on raw surface color, lipid oxidation, and internal cooked color of ground beef patties. Eight chubs (85% lean) were divided in half and each half was either assigned to the control (no lactate) or mixed with 2.5% lactate (w/w). Following treatment, patties were prepared and packaged in either vacuum, PVC (atmospheric oxygen level), high-oxygen (80% O₂ + 20% CO₂), or 0.4% CO (30% CO₂ + 69.6% N₂) and stored for 0, 2, or 4 days at 2 °C. After storage, raw surface color and lipid oxidation were measured and patties were cooked to either 66 °C or 71 °C. Lactate improved (p < 0.05) color stability of PVC, high-oxygen, and vacuum packaged raw patties, but had no effect (p > 0.05) on the a∗ values and visual color scores of patties in 0.4% CO. Lactate decreased (p < 0.05) lipid oxidation in all packaging atmospheres. Nevertheless, high-oxygen and PVC-packaged patties had more (p < 0.05) lipid oxidation than patties in CO and vacuum. Lactate had no effect (p > 0.05) on premature browning, whereas patties packaged in high-oxygen demonstrated premature browning. Conversely, cooked patties in 0.4% CO and vacuum were more red (p < 0.05) than both high-oxygen and PVC-packaged patties. Although lactate improved raw color stability, it did not minimize premature browning in cooked ground beef patties.     

Efficacies of soy sauce and wine base marinades for controlling spoilage of raw beef

Fresh beef slices were marinated by immersion in marinades based on soy sauce without (SB) or with lactic acid (SBLA) or red wine base without (WB) or with 0.5% v/v oregano essential oil (WBO). For control samples (immersed in saline), a mean increase of 0.9log CFU/cm² in total viable counts (TVCs) occurred during the 24 h treatment. During marination with WB and SB, mean TVC decreased by 0.7 and 0.3log CFU/cm², respectively. The mean decrease in TVC for samples marinated in WBO or SBLA was 1.2log CFU/cm². Subsequent storage of beef resulted in a rapid increase of TVC in control samples, to ≥9.5log CFU/cm² after 8 days at 5 °C or 3 days at 15 °C. Significant (P < 0.05) microbial growth occurred in marinated samples stored at 5 °C. During storage at 15 °C TVC increased in only WB samples but the final numbers of 5.9log CFU/cm² were significantly lower (P < 0.05) than the numbers in the control. Results similar to those for TVC were observed for Pseudomonas spp. All marinades also gave meat with significant lower TBARS values than the controls. There were no significant differences (P > 0.05) in the toughness of the marinated samples compared to the control, except for SBLA samples which had significantly higher (P < 0.05) shear force values. Marination with soy sauce or red wine marinades can evidently control microbial spoilage and oxidation of meat.     

Animal performance and fatty acid composition of lambs fed with different vegetable oils

Twenty-seven lambs were used to investigate the effects of the inclusion of 4% hydrogenated palm oil (HPO) or sunflower oil (SFO) in the concentrate on animal performance, carcass and meat quality and fat characteristics and fatty acid composition. Animals (16.2 ± 0.27 kg initial weight) were fed concentrate (Control, HPO or SFO) and barley straw ad libitum and slaughtered at 25 kg. SFO lambs tended to eat less concentrate than HPO animals (P < 0.10). Neither HPO nor SFO affected any of the carcass characteristics studied, meat pH and meat and fat colour (P > 0.05). SFO decreased proportions of C16:0, C18:1 cis-11 and C18:3 (P < 0.05) and increased C18:1 trans (P < 0.001) and C18:2/C18:3 ratio (P < 0.05). Atherogenicity index was lower (P < 0.05) when SFO was included in the concentrate. HPO did not affected and SFO improved fatty acid composition of fattening lambs without affecting animal performance.     

Detection of 4-hydroxy-2-nonenal adducts of turkey and chicken myoglobins using mass spectrometry

4-Hydroxy-2-nonenal (HNE), an unsaturated aldehyde generated by peroxidation of polyunsaturated fatty acids, is highly reactive and destabilizes myoglobin (Mb) redox state, affecting meat colour. Our objective was to characterise the adduction of HNE to turkey and chicken Mbs using tandem mass spectrometry (MS/MS). Turkey and chicken oxymyoglobins (OxyMbs) were incubated with HNE at 25 °C, pH 5.8 or 7.4. MetMb formation was greater in the presence of HNE than controls (p < 0.05). Electrospray ionisation-Q-TOF mass spectrometry of HNE-reacted Mbs revealed covalent adduction of HNE to both turkey and chicken Mbs via Michael addition. LC–ESI-MS/MS of chicken Mb reacted with HNE identified covalent adduction of histidine (His) residues 64 and 93 at pH 7.4, whereas at pH 5.8 only His 64 was adducted. These results suggest that HNE accelerates chicken OxyMb oxidation in vitro by covalent modification at histidine residues.     

Effects of 4-hydroxy-2-nonenal on beef heart mitochondrial ultrastructure, oxygen consumption, and metmyoglobin reduction

The effects of 4-hydroxy-2-nonenal (HNE) on mitochondria isolated from bovine hearts (n = 5) were assessed using ultrastructure, oxygen consumption, membrane permeability, HNE binding, and metmyoglobin reduction in vitro. Pre-incubation (pH 5.6 and 7.4 at 25 °C) of mitochondria with HNE decreased oxygen consumption compared with samples without HNE (P < 0.05). Electron microscopy revealed that HNE-treated mitochondria were swollen and had increased membrane permeability at pH 7.4, compared with ethanol controls. Conversely, mitochondria incubated with HNE at pH 5.6 had decreased volume and permeability. Fluorescence studies indicate that HNE binds to the membrane of mitochondria isolated from bovine cardiac muscle (at pH 5.6 and 7.4). HNE-treated mitochondria at both pH 5.6 and 7.4 had lower metmyoglobin reduction and NADH dependent metmyoglobin reductase activity compared with control mitochondria without HNE (P < 0.05). In addition to covalent binding with myoglobin, HNE may influence beef color stability by interacting with mitochondria.     

Temperature induced denaturation of myosin: Evidence of structural alterations of myosin subfragment-1

Denaturation of myofibrillar proteins in porcine longissimus thoracis et lumborum muscle was investigated after pre-rigor temperature incubation at 20, 30 and 40 °C. At 24 h myofibrils were isolated and myosin was further cleaved by chymotrypsin. High temperature pre-rigor induced release of myosin S1 (subfragment-1), less (P < 0.05) Ca²⁺-ATPase activity and structural alterations of the region of the myosin molecule that harbors S1. Surface hydrophobicity of myofibrils from the 40 °C group increased (P < 0.001), suggesting a temperature-induced structural rearrangement exposing hydrophobic groups on the surface of myofibrils which in turn may explain the reduced water-holding of PSE meat.