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.     

Sodium lactate influences myoglobin redox stability in vitro

Injection-enhancement of beef with lactate improves color stability; however, the mechanism is unclear. Thus, our objectives were to assess the effects of sodium lactate on equine myoglobin redox stability in vitro. Oxymyoglobin at pH 5.6 (50 mM sodium citrate) and pH 7.4 (50 mM sodium phosphate) was incubated at 4 °C with lactate (0, 5, 10, 100, or 200 mM) and myoglobin redox form was determined using absorbance spectra. Metmyoglobin formation at pH 5.6 and 7.4 was significantly (P < 0.05) decreased by lactate at concentrations of 100 and 200 mM. In general, increasing lactate concentration from 100 to 200 mM increased (P < 0.05) oxymyoglobin redox stability. This effect of lactate on myoglobin redox stability could be partially responsible for the improved color stability associated with lactate injection-enhanced beef products. Further work should determine the effect of lactate on beef myoglobin.     

Mass spectrometric evidence for aldehyde adduction in carboxymyoglobin

Lipid oxidation generates secondary oxidation products, which compromise myoglobin (Mb) redox stability. Although lipid oxidation-induced discoloration in carboxymyoglobin (COMb) is documented, the molecular basis for interactions between COMb and lipid oxidation products has not been investigated. Our objective was to characterize the adduction of 4-hydroxy-2-nonenal (HNE), a reactive lipid oxidation product, with COMb, utilizing mass spectrometry. Equine COMb and equine OxyMb (0.15 mM) were incubated with HNE (1.0 mM) at pH 7.4, 37 °C (physiological condition) for 6 h, and at pH 5.6, 4 °C (typical meat storage condition) for 7 days. The samples were analyzed in Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS). MS spectra revealed that HNE formed mono-, di-, and tri-adducts with COMb at physiological conditions, whereas mono-, di-, tri-, and tetra-adducts were detected in OxyMb. This observation suggested a lower reactivity of COMb towards HNE at physiological conditions, compared to OxyMb. In contrast, at meat storage conditions, HNE formed mono- and di-adducts with both COMb and OxyMb, thus revealing a similar trend for aldehyde adduction in the cherry-red colored Mb redox forms. The present study is the first to report HNE adduction in COMb, and proteomic investigations are underway to determine the sites of HNE adduction in COMb.     

Characterization of bison (Bison bison) myoglobin

Bison is an alternate meat species gaining increased popularity in North America. Although previous investigations reported that bison meat discolors faster than beef, the molecular basis of this observation has not been investigated. Therefore, the objective of the present study was to determine the redox stability, thermostability, and primary structure of bison myoglobin (Mb), in comparison with beef Mb. Purified bison and beef myoglobins were analyzed for autoxidation, lipid oxidation-induced oxidation, and thermostability. Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry was utilized for determining the exact molecular mass of bison Mb, whereas Edman degradation was employed to determine the amino acid sequence. Bison and beef myoglobins behaved similarly in autoxidation, lipid oxidation-induced oxidation, and thermostability. The observed molecular mass of bison and beef myoglobins was 16,949 Da, and the primary structure of bison Mb shared 100% similarity with beef and yak myoglobins. Noticeably, the amino acid sequence of bison Mb was different from other ruminant myoglobins, such as water-buffalo, sheep, goat, and red-deer. The present study is the first to report the primary structure of bison Mb. Same primary structure and similar biochemical attributes of bison and beef myoglobins suggested that the observed rapid discoloration in bison meat could not be attributed to biochemistry of bison Mb.     

Temperature- and pH-dependent effect of lactate on in vitro redox stability of red meat myoglobins

Our objective was to evaluate the influence of lactate on in vitro redox stability and thermostability of beef, horse, pork, and sheep myoglobins. Lactate (200 mM) had no effect (P > 0.05) on redox stability at physiological (pH 7.4, 37 °C) and meat (pH 5.6, 4 °C) conditions. However, lactate increased (P < 0.05) metmyoglobin formation at a condition simulating stressed live skeletal muscle (pH 6.5, 37 °C). The redox stability of myoglobins at stressed live skeletal muscle and meat conditions was species–specific (P < 0.05). Myoglobin thermostability at 71 °C was lower (P < 0.05) in the presence of lactate compared with controls and was influenced (P < 0.05) by species. The results of the present study indicate that the effects of lactate on myoglobin are temperature and pH dependent. The observed lack of influence of lactate on myoglobin redox stability at meat condition suggests that the color stability of lactate-enhanced fresh meat is not due to direct interactions between the ingredient and the heme protein.     

Color-stabilizing effect of lactate on ground beef is packaging-dependent

Previous research on lactate-induced color stability in ground beef did not address the potential influence of packaging. The objective of the present study was to examine the effects of lactate on the color stability of ground beef patties stored in different modified atmosphere packaging (MAP) systems. Ground beef patties with either 2.5% potassium lactate or no lactate were packaged in vacuum (VP), high-oxygen MAP (HIOX; 80% O₂ + 20% CO₂), carbon monoxide MAP (CO; 0.4% CO + 19.6% CO₂ + 80% N₂), or aerobic packaging (PVC) and stored for 0, 2, or 4 days at 2 °C. Lactate-treated patties were darker (P < 0.05; lower L∗ values) than control patties. Surface redness (a∗ values) was greater (P < 0.05) for lactate patties than the controls when stored in PVC, HIOX, and VP. However, lactate’s effects on a∗ values were not evident when packaged in CO (P > 0.05). The color-stabilizing effect of CO could have masked lactate’s effect on surface redness. While lactate patties in PVC and VP demonstrated lower (P < 0.05) discoloration than controls, no differences (P > 0.05) existed between controls and lactate samples in CO and HIOX. Our results indicated that the effects of lactate on ground beef color are dependent on packaging.     

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.     

Primary structure of goat myoglobin

Color stability attributes of goat meat are different from those of sheep meat, possibly due to species-specific differences in myoglobin (Mb) biochemistry. An examination of post-genomic era protein databases revealed that the primary structure of goat Mb has not been determined. Therefore, our objective was to characterize the primary structure of goat Mb. Goat Mb was isolated from cardiac muscles employing ammonium sulfate precipitation and gel-filtration chromatography, and Edman degradation was utilized to determine the amino acid sequence. Sequence analyses of intact Mb as well as tryptic- and cyanogen bromide-peptides yielded the complete primary structure of goat Mb, which shared 98.7% similarity with sheep Mb. Similar to other livestock myoglobins goat Mb has 153 residues. Comparison of the sequences of goat and sheep myoglobins revealed two amino acid substitutions – THRgoat8GLNsheep and GLYgoat52GLUsheep. Goat Mb contains 12 histidine residues. As observed in other meat-producing livestock species, distal and proximal histidines, responsible for stabilizing the heme group and coordinating oxygen-binding, are conserved in goat Mb.     

Effects of pyruvate, succinate, and lactate enhancement on beef longissimus raw color

Beef strip loins (n=30) were divided into halves, and each half was assigned randomly to one of four injection enhancements: (1) non-enhanced control, (2) 3% pyruvate, (3) 3% succinate, and (4) 3% lactate. Steaks were cut and packaged in either vacuum, high oxygen (80% O(2)/20% CO(2)), or PVC. Color and lipid oxidation were measured on days 0, 5, and 13 of storage at 1°C. Enhancement had a significant effect on steak pH. On day 13 of storage, steaks enhanced with lactate, pyruvate, and succinate were less discolored (P<0.05) than control steaks in PVC and high oxygen. Enhancement darkened steaks (P<0.05) compared with control steaks. Succinate had the greatest and pyruvate had the least metmyoglobin-reducing activity (P<0.05). Lactate and pyruvate decreased the TBARS values of steaks packaged in PVC (P<0.05) whereas pyruvate was most effective for lowering lipid oxidation in high-oxygen packaging.     

Amino acid sequence of myoglobin from emu (Dromaius novaehollandiae) skeletal muscle

The objective of the present study was to characterize the primary structure of emu myoglobin (Mb). Emu Mb was isolated from Iliofibularis muscle employing gel-filtration chromatography. Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry was employed to determine the exact molecular mass of emu Mb in comparison with horse Mb, and Edman degradation was utilized to characterize the amino acid sequence. The molecular mass of emu Mb was 17,380 Da and was close to those reported for ratite and poultry myoglobins. Similar to myoglobins from meat-producing livestock and birds, emu Mb has 153 amino acids. Emu Mb contains 9 histidines. Proximal and distal histidines, responsible for coordinating oxygen-binding property of Mb, are conserved in emu. Emu Mb shared more than 90% homology with ratite and chicken myoglobins, whereas it demonstrated only less than 70% sequence similarity with ruminant myoglobins.     

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.     

Antioxidant status,colour stability and myoglobin resistance to oxidation of longissimus dorsi muscle from lambs fed a tannin-containing diet

Fourteen lambs were individually penned for 60 days. Seven lambs (C) were fed a barley-based concentrate, while 7 animals (T) received the same concentrate supplemented with 8.96% (dry matter basis) quebracho (Schinopsis lorentzii) extract. Tannin supplementation extended colour stability of fresh longissimus dorsi muscle (LM) over 7 days of aerobic storage with lower changes in hue angle over time in LM from T-fed animals compared to C-fed lambs (P = 0.032). Muscle from T-fed lambs had lower metmyoglobin percentages (MMb%) than that from C-fed animals after 3 days of storage (P = 0.05) and tended to have slower rates of change in (K/S)₅₇₂ ÷ (K/S)₅₂₅ values (P = 0.07). Compared to the C diet, tannin supplementation increased the concentration of total phenols in muscle (+31.29%, P = 0.001) and resulted in a higher muscle antioxidant capacity measured by FRAP (+16.81%, P = 0.026) and TEAC (+24.81%, P = 0.001) tests. Furthermore, feeding tannins resulted in lower MMb% after nitrite-induced oxidation measured both at the meat surface (−10%, P = 0.012) and in a meat extract (−8.22%, P = 0.004).     

The effect of high pressure and residual oxygen on the color stability of minced cured restructured ham at different levels of drying,pH, and NaCl

Color stability of minced cured restructured ham was studied by considering the effects of high pressure (HP) (600 MPa, 13 °C, 5 min), raw meat pH₂₄ (low, normal, high), salt content (15, 30 g/kg), drying (20%, 50% weight loss), and residual oxygen level (0.02%–0.30%). Raw hams were selected by pH₂₄ in Semimembranosus, mixed with additives, frozen, sliced, and dried by the Quick-Dry-Slice® (QDS) process followed by HP treatment or not (control). Packaging and storage simulated industrial packaging: modified atmosphere containing 80% N₂, 20% CO₂, and residual O₂ in one of three intervals: < 0.1%, 0.1%–0.2%, or 0.2%–0.3%, and retail storage conditions: chill storage, 12 h light, 12 h darkness. HP improved the stability of the redness of 20% QDS hams, while the stabilizing effect on 50% QDS hams was smaller, concluding that water has the dominating role. Raw meat pH₂₄, salt content, and residual oxygen level had varying effects on the stability of the red color.     

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.     

电感耦合等离子体质谱法测定糖果中的重金属元素

用电感耦合等离子体质谱法测定糖果中的微量元素Cu、Pb、Cd含量。采用内标法,能有效地克服基体效应、接口效应和质谱干扰所造成的影响,检测限、加标回收率、相对标准偏差均能很好地满足测定的要求。     

Application of Selected Ion Flow Tube Mass Spectrometry Coupled with Chemometrics to Study the Effect of Location and Botanical Origin on Volatile Profile of Unifloral American Honeys

Abstract: Ten Ohio and Indiana honey samples from star thistle (Centaurea Americana), blueberry (Vaccinium spp.), clover (Trifolium spp.), cranberry (Vaccinium spp.), wildflower, and an unknown source were collected. The headspace of these honeys was analyzed by selected ion flow tube mass spectrometry and soft independent modeling of class analogy (SIMCA). SIMCA was utilized to statistically differentiate between honeys based on their composition. Ohio honeys from star thistle, blueberry, and clover were similar to each other in volatile composition, while Ohio wildflower honey was different. Indiana honeys from star thistle, blueberry, and wildflower were different from each other in volatile composition, while clover and cranberry honeys were similar. Honeys from Ohio and Indiana with the same floral origins were different in volatile composition. Furfural, 1‐octen‐3‐ol, butanoic, and pentanoic acids were the volatiles with the highest discriminating power between types of floral honey. Methanol and ethanol followed by acetic acid were at the highest levels in most honeys, though furfural was at the highest concentration in Indiana blueberry honey, while 1‐octen‐3‐ol was at the highest concentration in Indiana wildflower honey. The highest concentration of volatile compounds was in Indiana wildflower honey followed by Ohio wildflower honey, while the lowest concentration of volatile compounds was observed in Ohio clover honey followed by Indiana clover honey. Practical Application: Using chemometrics, concentrations of volatile compounds in different honeys can be used to determine the influence of botanical and geographical origins on aroma, which is important for the quality of honey. Characterization of volatile compounds can also be a useful tool for assessing honey quality.     

Rapid primary structure determination of myoglobins by a complementary approach based on mass spectrometry and Edman degradation

The primary structure of two myoglobins (Mbs), isolated from the heart and muscle of Equus asinus L. and Struthio camelus L., respectively, was determined using a combined approach based on Edman degradation and mass spectrometry. The strategy allowed the determination of donkey Mb sequence, which was found to be identical to the horse Mb, as also confirmed by ESI/Q-TOF mass spectrometry. Indeed, donkey Mb accurate molecular mass (16951.50 Da) was in good agreement with the molecular mass of horse Mb (16951.48 Da). A similar strategy was also applied for revisiting the primary structure of ostrich Mb, revealing the presence of two amino acid substitutions (i.e. Asp53Glu and Asp60Glu), with respect to the previously reported sequence ( Enoki, Ohga, Ishidate, & Morimoto, 2008). The proposed approach represents a rapid and reliable tool for determining/revisiting the primary structures of the highly conserved myoglobins.