ABSTRACT: The degree to which lipid and myoglobin (Mb) oxidation processes interact in meat can be species-specific. We investigated the effects of beef and pork sarcoplasmic extracts containing different Mb concentrations on lipid oxidation in a liposome system. Sarcoplasm was extracted from beef and pork longissimus dorsi and psoas major muscles. Beef sarcoplasm was diluted with 0.1 M phosphate buffer to obtain a Mb concentration equivalent to that in pork sarcoplasm. Conversely, equine heart Mb was added to pork sarcoplasm to match the myoglobin concentration of beef sarcoplasm. This resulted in beef and pork sarcoplasms, each with 2 different Mb concentrations for the longissimus (0.02 mM and 0.07 mM) and psoas (0.05 and 0.12 mM). Sarcoplasm (or phosphate buffer control) was incorporated within a phosphatidylcholine liposome preparation and incubated at 25°C. Thiobarbituric acid reactive substances (TBARS) were measured at 0, 30, 60, 90, and 120 min of incubation. Regardless of species, greater Mb concentration within the sarcoplasm increased lipid oxidation (P < 0.05). Across muscles, pork sarcoplasm had lower TBARS values than beef sarcoplasm (P < 0.05). Our results suggest that pork sarcoplasm has a lesser effect on lipid oxidation than beef sarcoplasm for a common Mb concentration. However, increased myoglobin concentration within sarcoplasm promotes lipid oxidation regardless of species.
Secondary products of lipid oxidation, including aldehydes and ketones, can decrease the redox stability of OxyMb (Chan and others 1997). Increased OxyMb oxidation concurrent with covalent binding of α, β unsaturated aldehydes to Mb from meat-producing species has been demonstrated in vitro (Alderton and others 2003; Suman and others 2007). However, Suman and others (2006, 2007) recently reported that porcine Mb is less susceptible than bovine Mb to alkylation by the α, β unsaturated aldehyde, 4-hydroxynonenal, because it contains fewer nucleophilic histidine residues. This has provided 1 possible explanation for the observed species-specific effect of α-tocopherol on myoglobin redox stability in pork and beef. That is, α-tocopherol inhibits lipid oxidation in both species, but bovine myoglobin's apparent greater susceptibility to alkylation intensifies its redox instability more than that of porcine Mb.
While the fundamental evidence for this mechanism is sound, it does not account for potential differences in antioxidant or prooxidant effects of sarcoplasmic constituents that could also influence the species-specific effect of α-tocopherol. Muscle sarcoplasm is a complex mixture of biological macromolecules and low molecular weight compounds (Kanner and others 1991). The antioxidant and prooxidant effects of specific sarcoplasmic constituents (for example, nonheme iron) have been investigated in meat systems (Rhee and Ziprin 1987; Gopalakrishnan and others 1999; Sista and others 2000; Monin and others 2003; Li and others 2005). A complicating factor to studies of this type is that 1 of the sarcoplasmic constituents that can impact lipid oxidation is Mb (Rhee and Ziprin 1987).
Fatty acids in pork are generally more unsaturated than those in beef (Enser and others 1996) and would be expected to oxidize more readily and generate more reactive secondary products of lipid oxidation. The latter should result in greater Mb redox instability in pork, not less as has been reported. Liposome systems provide the opportunity to control fatty acid profile when attempting to study species-specific effects of sarcoplasmic constituents (Yin and Faustman 1993). The objective of this study was to investigate the effects of sarcoplasmic extracts from porcine and bovine muscles, with and without added Mb, on lipid oxidation in a liposome model.
Materials and Methods
Sarcoplasm was extracted from beef and pork longissimus dorsi and psoas major muscles obtained from a local purveyor (n= 3 loins and tenderloins per species). Muscle samples, devoid of fat and connective tissues, were passed through a tissue grinder and 30 g of ground muscle was blended with 90 mL of 0.1 M phosphate buffer (pH 7.2) using a Waring table-top blender (Dynamics Corp. of America, New Hartford, Conn., U.S.A.) for 40 s. Homogenized muscle was centrifuged at 1500 ×g for 15 min at 4°C using a Sorvall refrigerated RCB 5 centrifuge (Thermo Fisher Scientific, Waltham, Mass., U.S.A.). The supernatant was passed through double-layered cheesecloth and the filtrate was again centrifuged at 36600 ×g for 20 min at 4°C. The remaining supernatant was sequentially filtered twice using 0.45 and 0.22 μm syringe filters (Fisher Scientific, Pittsburgh, Pa., U.S.A.). Myoglobin concentration was determined by measuring absorbance at 525 nm (A525nm= 7.6/mM/cm; Broumand and others 1958) using a Shimadzu UV-2102 PC spectrophotometer (Shimadzu Inc., Columbia, Md., U.S.A.).
For each muscle, half of the extracted beef sarcoplasm was diluted with 0.1 M phosphate buffer (pH 7.2) to achieve the desired concentration of Mb in pork sarcoplasm. The remaining beef sarcoplasm was not diluted. Half of the extracted pork sarcoplasm received an addition of equine heart Mb to achieve the Mb concentration in beef sarcoplasm (Sigma Chemical Co., St. Louis, Mo., U.S.A.). The remaining pork sarcoplasm was used without any added equine Mb. This resulted in 4 treatments for the longissimus muscle sarcoplasm: 1 = beef sarcoplasm with 0.02 mM Mb; 2 = beef sarcoplasm with 0.07 mM Mb; 3 = pork sarcoplasm with 0.02 mM Mb; and 4 = pork sarcoplasm with 0.07 mM Mb. The treatments for the psoas major sarcoplasm were: 1 = beef sarcoplasm with 0.05 mM Mb; 2 = beef sarcoplasm with 0.12 mM Mb; 3 = pork sarcoplasm with 0.05 mM Mb; and 4 = pork sarcoplasm with 0.12 mM Mb.
Phosphatidylcholine liposome preparation
Liposomes were prepared according to the procedure of New (1990) for multilamellar vesicles. Thirty milligrams of phosphotidyl choline, 12 mg cholesterol, and 3 mg dicetyl phosphate were dissolved in 5 mL of methylene chloride/methanol (2: 1 v/v) and placed in a 100-mL round-bottom flask. Solvent was removed and a lipid film was formed by rotary evaporation. Phosphatidylcholine, cholesterol, and dicetyl phosphate were obtained from Avanti Polar Lipids Inc. (Alabaster, Ala., U.S.A.). Reagent grade methylene chloride and methanol were obtained from Fisher Scientific (Pittsburgh, Pa., U.S.A.).
Ten milliliters sarcoplasm (or phosphate buffer control) and 15 glass beads (1 mm diameter) were mixed with phosphatidylcholine liposome preparations by mechanical shaking of the mixture for 30 min at 4°C on a table-top shaker and the contents were allowed to stand for 30 min at the same temperature.
Sarcoplasm–liposome preparations were incubated at 25°C in a water bath (Versa-bath, Fisher Scientific) and TBARS of samples were measured following 0, 30, 60, 90, and 120 min of incubation. One milliliter of sarcoplasm–liposome preparation was removed and combined with 2 mL of trichloroacetic acid (TCA) solution (20% w/v). The mixture was centrifuged at 1400 ×g for 10 min (Model 5414d, Eppendorf North America, Westbury, N.Y., U.S.A.). The supernatant (1 mL) was mixed with 1 mL aqueous thiobarbituric acid solution (20 mM). A blank was prepared by mixing 2 mL of 11% TCA and distilled water (2: 1 v/v) with 2 mL of 20 mM TBA solution. The solutions were incubated at 25°C for 20 h and absorbance was measured at 532 and 450 nm.
Data for longissimus dorsi and psoas major muscles were analyzed separately. The experimental design was a completely randomized design with repeated measures. Fixed effects included 5 treatments consisting of 2 different myoglobin concentrations per species (0.02 and 0.07 mM for the longissimus and 0.05 and 0.12 mM for the psoas) and a control with 0.1 M phosphate buffer. Repeated TBARS measurements were taken at 5 time periods (0, 30, 60, 90, and 120 min). Each experiment was replicated thrice (n= 3). Data were analyzed using the mixed procedure of SAS (SAS Institute, Inc., Cary, N.C., U.S.A.) with the repeated option to assess covariance resulting from the repeated measures. Least square means were generated for significant F-tests (P < 0.05) and separated using least significant differences.
Myoglobin concentrations in sarcoplasm extracted from pork longissimus and psoas were 0.02 and 0.05 mM, respectively, whereas beef contained 0.07 and 0.12 mM Mb in sarcoplasms extracted from longissimus and psoas muscles, respectively.
The effects of Mb concentration within beef and pork sarcoplasms extracted from longissimus and psoas muscles on TBARS values are presented in Figure 1 and 2, respectively. Pork sarcoplasm from the longissimus with 0.02 mM Mb had lower (P < 0.05) TBARS values than beef sarcoplasm with 0.02 mM Mb (Figure 1). In addition, pork sarcoplasm with 0.07 mM Mb had less lipid oxidation than beef sarcoplasm with 0.07 mM Mb. Sarcoplasmic extracts with greater Mb concentration within each muscle resulted in greater (P < 0.05) TBARS values regardless of species (Figure 1 and 2). Interestingly, pork sarcoplasm containing either 0.02 or 0.05 mM Mb showed lesser TBARS values than the control (P < 0.05). TBARS values for pork sarcoplasm with 0.02 mM Mb did not change during incubation. Thus, pork sarcoplasm demonstrated less lipid oxidation than beef sarcoplasm at a comparable Mb concentration within each muscle type.
To study the effects of sarcoplasmic constituents other than Mb on lipid oxidation, the ideal system would be to produce sarcoplasm without Mb. One way to achieve this is with the use of knockout mice that lack Mb in their skeletal muscles (Grange and others 2001). However, it would be very difficult to obtain mouse skeletal muscles in quantities sufficient for studies of relevance to meat science. Another approach would be to selectively remove Mb from muscle sarcoplasmic preparations (for example, affinity columns containing Mb antibodies) and then add Mb back in desired concentrations. However, we have been unsuccessful at accomplishing this and are unaware of any related attempts published in the literature. Thus, our approach included the addition of equine heart myoglobin to pork sarcoplasm and dilution of beef sarcoplasm to achieve common Mb concentrations between these 2 species. Equine myoglobin was selected as the candidate protein because it is easily obtained through commercial sources and because it shares 88.2% and 90.8% homology with bovine and porcine myoglobins, respectively (http://www.expasy.org). Admittedly, the dilution of beef sarcoplasm with buffer to achieve a Mb concentration relevant to pork also dilutes all other sarcoplasmic constituents. However, our results demonstrated that despite such dilution, beef sarcoplasm consistently enhanced lipid oxidation to a greater extent than that of pork.
Regardless of species, greater Mb concentrations within the sarcoplasm promoted more lipid oxidation than lesser concentrations. Beef sarcoplasm extracted from the psoas tended to result in more lipid oxidation than sarcoplasm extracted from the longissimus. Jensen and others (1997) also reported increased lipid oxidation in the psoas compared with the longissimus. Anton and others (1993) suggested that this may be due to production of free radicals being more pronounced in aerobic muscles than in anaerobic muscles. In addition, bovine longissimus contained more glutathione peroxidase activity and lower tissue and soluble selenium concentrations than the psoas (Daun and others 2001). This may help to explain lower TBARS values in the longissimus; however, we did not measure these constituents in the present study and cannot confirm that this was responsible for our observation.
Previous reports have concluded that beef is more susceptible to lipid oxidation than pork (Rhee and Ziprin 1987; Rhee and others 1996; Kim and others 2002; Min and others 2008). Our results are in agreement and demonstrated that beef sarcoplasm from the longissimus and psoas muscles caused greater lipid oxidation than pork sarcoplasm in vitro. Min and others (2008) reported that ferrylmyoglobin and/or hematin, rather than free ionic iron (which binds with hemosiderin), were primarily responsible for greater TBARS values in raw beef loin than in pork during storage. In the present study, and at comparable Mb concentrations, beef sarcoplasm resulted in greater TBARS values than pork. The lower TBARS values in pork may be due to increased catalase activity (Rhee and others 1996) than that in beef. Increased catalase activity minimizes H2O2 production, resulting in less available H2O2 to react with Mb, and consequently, less ferrylmyoglobin formation (Kanner and Harel 1985).
Min and others (2008) suggested that increased TBARS values in beef may also be due to greater lipoxygenase-like activities and lower ferric ion reducing capacities (FRC) than in pork. FRC represents the total reducing potential contributed by ascorbic acid, glutathione, and NADH present in muscle (Benzie and Strain 1999). Lipoxygenase requires nonheme iron in the active site (Theerakulkait and Barrett 1995) and nonheme iron content in beef increased during storage due to its release from myoglobin by the of action H2O2 (Min and Ahn 2005). Thus, relatively high lipoxygenase-like activity in beef can be attributed to a high concentration of myoglobin. However, in the present study, beef sarcoplasm resulted in more lipid oxidation at a similar Mb concentration, both for longissimus and psoas extracts, suggesting a role for molecules other than Mb for increasing TBARS values. Min and others (2008) further suggested that free radical scavenging activities of both beef and pork were similar during storage. At the same time, TBARS values increased for beef during storage, suggesting that the amount of antioxidants in raw pork loin were sufficient to minimize lipid oxidation. This may provide partial explanation for the observation that pork sarcoplasm extract, containing either 0.02 or 0.05 mM Mb, resulted in lesser TBARS values than controls (P < 0.05).
The present study revealed that beef sarcoplasmic extracts from longissimus and psoas resulted in greater TBARS values than the same pork muscles with comparable Mb concentrations, and that pork sarcoplasm from longissimus and psoas resulted in lower TBARS values than the control. This suggests that the balance between antioxidant–prooxidant potential in sarcoplasmic extracts is species-specific.