ARTICLE pubs.acs.org/JAFC
Morphological Examinations of Oxidatively Stressed Pork Muscle and Myofibrils upon Salt Marination and Cooking To Elucidate the Water-Binding Potential Zelong Liu,†,‡ Youling L. Xiong,*,‡ and Jie Chen† †
State Key Laboratory of Food Science and Technology and School of Food Science and Technology, Jiangnan University, Wuxi 214122, China ‡ Department of Animal and Food Sciences, University of Kentucky, Lexington, Kentucky 40546, United States ABSTRACT: Pork longissimus muscle samples were subjected to the following three marination conditions: (A) oxidation (40 min) in hydroxyl radical-generating solutions (HRGS; 10 μM FeCl3/100 μM ascorbate with 5 or 20 mM H2O2, pH 6.2) containing 0.1 M NaCl and then marination (40 min) in 0.6 M NaCl with 15 mM pyrophosphate (PP); (B) simultaneous oxidation/marination (40 min) in HRGS containing 0.6 M NaCl and 15 mM PP; or (C) the same as condition B except that PP was omitted. Protein oxidation, measured by the carbonyl and tryptophan fluorescence changes, enhanced hydration but increased cooking loss of meat. Light microscopy revealed a dense muscle structure characterized by swollen fibers and reduced intercellular spacing in intermediately oxidized muscle samples marinated with 0.6 M NaCl and 15 mM PP. However, oxidized fibers were more susceptible to transverse shrinkage upon cooking than nonoxidized fibers, which was supported by the dynamic ultrastructural changes in myofibrils observed using phase contrast microscopy. These findings provide a further understanding of the complex impact of oxidation on meat hydration and water-binding. KEYWORDS: muscle oxidation, marination, cooking, hydration properties, morphology
’ INTRODUCTION Juiciness is one of the most important quality attributes of cooked meat and meat products. Most processed meats are manufactured with added water along with water-binding ingredients to enhance the ability of muscle to retain endogenous moisture as well as hold extraneous water during processing. Salt (NaCl or KCl) and phosphates are common ingredients used to improve water retention in cooked meat. Higher concentrations of NaCl (up to 4%) and phosphates (e.g., sodium pyrophosphate and tripolyphosphate up to 0.5%) are used in processed meats through tumbling, massaging, or marination to enhance products’ juiciness, tenderness, and taste. The ability to improve water retention of meats by a brine (salt) with phosphate has long been recognized.13 Increases in water-binding and hydration in salted meat are achieved through the promotion of physical entrapment (immobilization) of water due to electrostatic repulsions between myofilaments and the formation of gel networks made of solubilized proteins.46 The presence of certain phosphates, most notably pyrophosphate (PP) and tripolyphosphate (TPP), has been shown to facilitate meat hydration due to myofibril expansion and, thus, transverse swelling.5,7,8 These phosphates are capable of dissociating actomyosin and deploymerization of myosin filaments, resulting in the weakening of interfilamental interactions and loosening of the myofibril structure, thereby allowing water molecules to diffuse more readily into the interfilamental spaces to increase the extent of hydration. Because PP acts on the myosin head to initiate actomyosin dissociation and myosin head conformation is also susceptible to oxidation,9 it is tempting to suggest that the efficacy of PP might be influenced by the oxidative status of myofibrillar proteins. Indeed, protein oxidation has been recognized as a significant r 2011 American Chemical Society
factor influencing water-holding in many fresh and processed muscle foods.10 Several studies have linked deteriorations in the quality of muscle foods processed under nonvacuum conditions to the oxidative modification of proteins by reactive oxygen species, such as hydroxyl radicals.11,12 The aggregation of myofibrillar proteins in whole-muscle tissue exposed to oxidants during post-mortem aging was thought to be a primary cause for the increase in cooking loss of pork.13 Furthermore, studies with isolated myofibrillar proteins strongly suggest that changes in the gelling potential of proteins subjected to oxidative stress may play a significant role in altering the water-binding capacity in processed muscle foods.14,15 In a previous study, we found that hydroxyl radical-induced cross-linking of myosin reduced the extent of transverse expansion (swelling) of myofibrils upon brine (>0.4 M NaCl) irrigations, indicating losses in the hydration capacity of myofibrils.16 However, when whole-muscle tissue was exposed to the same oxidizing system, the resultant oxidative modification of proteins led to improved hydration potential or moisture uptake in lowsalt (0.1 M NaCl) solution, whereas the water-holding capacity was slightly decreased.17 The improved hydration was explained by the observed extracellular space enlargement that appeared to facilitate water diffusion into the muscle tissue as evidenced by the migration of tracing dye. The objective of the present study was to understand how the hydration and water-binding properties of raw and cooked meat at a higher ionic strength (0.6 M NaCl) were influenced by protein oxidation. This was accomplished through Received: September 2, 2011 Revised: November 15, 2011 Accepted: November 15, 2011 Published: November 15, 2011 13026
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Figure 1. Flowchart of oxidation and marination treatments. All solutions were made with 15 mM piperazine-N,N0 -bis(2-ethanesulfonic acid) (PIPES) buffer at pH 6.2.
histological and microscopic examinations of the muscle fiber and myofibril structures upon marination and thermal processing.
’ MATERIALS AND METHODS Preparation of Muscle Samples. Loins (Longissimus lumborum) were excised from 24 h post-mortem pork carcasses from 10 pigs processed at the University of Kentucky Meat Laboratory, a USDAapproved facility. The loins were individually packaged, vacuum sealed, and stored in a 30 °C freezer. On the day of use, one randomly selected frozen loin was used to prepare muscle samples for marination treatments as described in our previous paper.17 Round disks (2.54 cm diameter 1.27 cm thickness) were obtained from partially frozen meat with a sharpedged stainless steel corer and used for oxidation, marination, cooking, and structural analyses. Oxidation and Marination of Muscle. All freshly prepared muscle sample disks were equilibrated at 4 °C for 2 h to completely thaw and then gently blotted with a paper towel to remove any surface moisture. Muscle disks were randomly divided into three groups that were subjected to different oxidation and marination treatments (A, B, and C) as detailed in Figure 1. In treatment A, samples were immersed and incubated in hydroxyl radical-generating solutions (pH 6.2) containing 5 or 20 mM H2O2, 10 μM FeCl3, and 100 μM ascorbic acid with 0.1 M NaCl for 40 min and then placed on a stainless steel rack to drain for 5 min. The samples were subsequently marinated for 40 min in 0.6 M NaCl with 15 mM PP (pH 6.2). In treatment B, samples were immersed and incubated for 40 min in mixed solutions containing the above oxidants and marinades with 0.6 M NaCl and 15 mM PP. Treatment C was the same as treatment B except PP was not included in the marinade. A nonoxidized control for each treatment was created under the same conditions but without oxidants (FeCl3, ascorbic acid, and H2O2). All treatments were carried out at 4 °C with constant agitation, and the ratio of muscle sample to solution was 1:10 (w/v). After marination, samples were drained for 5 min on a stainless steel rack. Some of the marinated samples were subjected to cooking as described later. Raw and cooked muscle samples were immediately analyzed for protein oxidation and morphology. To determine the influence of oxidation on marinade migration into the muscle tissue, a separate oxidation and marination experiment identical to that outlined in Figure 1 was conducted except that all marination solutions also contained 1% (w/v, final concentration) FD&C Blue No. 1 dye. Raw and cooked muscle samples after being partially frozen were dissected.
Longitudinal slices were digitized and binarized, and the percent area of dye-covered area by pixels in the image was obtained as described in detail previously.17 Oxidation-Induced Chemical Changes. Both raw and cooked muscle samples were homogenized in a 10 times (w/v) amount of a rigor buffer containing 0.1 M KCl, 2 mM MgCl2, 1 mM EGTA, and 10 mM K2HPO4 (pH 7.0) and then centrifuged at 2000g for 10 min. The supernatant and pellet, which comprised crude sarcoplasmic proteins (SP) and myofibrillar proteins (MP), respectively, were collected and properly diluted for the following analyses. Tryptophan Fluorescence. The oxidation-induced fluorescence loss of tryptophan was examined using a FluoroMax-3 spectrofluorometer (Jobin Yvon Inc., Edison, NJ) according to the method of Gieauf et al.18 Before determination, both supernatant (SP fraction) and pellet (MP fraction) were diluted to 25 μg protein/mL with deionized water. The excitation and emission wavelengths were set to 283 and 338 nm, respectively, and the fluorescence intensity was recorded as photon counts per second (pc/s). Protein Carbonyls. The protein carbonyl content was determined using the procedure of Levine et al.19 The result was expressed as nanomoles of carbonyls per milligram of protein. Morphological Changes in Muscle Ultrastracture. Structural information on oxidized and nonoxidized raw and cooked meat and information on the morphology of myofibrils were obtained through histological and microscopic examinations. Histology and Light Microscopy of Muscle Fibers. Cylindrical samples (1.0 cm diameter 1.3 cm thickness) were carefully excised from raw and cooked muscle disks with a sharp-edged stainless steel corer. These cored samples were fixed with 4% paraformaldehyde in 10 mM phosphate-buffered saline at 4 °C overnight, dehydrated through a graded series of ethanol solutions, and processed for paraffin embedding.20 Samples were cut in a plane perpendicular to the muscle fiber direction into 8 μm thick sections using a microtome (HM325, MICROM, Waldorf, Germany), reshaped in water, then mounted on a glass slide and air-dried. After deparaffination and rehydration, the sections were stained with hematoxylin and eosin (H&E, Sigma, St. Louis, MO), which color the nuclei and cytoplasmic proteins blue and deep pink, respectively. To monitor fiber morphological changes as marinades penetrated into the muscle tissue, selected muscle samples after marination were dissected into outer, middle, and inner cross-section layers 1, 3, and 5 mm, respectively, from the end surface and then prepared for microscopic 13027
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Figure 2. Tryptophan fluorescence intensity of myofibrillar (a) and sarcoplasmic (b) protein fractions from nonoxidized (control) and oxidized, brinemarinated muscle samples before (raw) and after (cooked) cooking. Means (n = 3) across all samples without a common letter differ significantly (P < 0.05). Positive control, fresh muscle; negative control, marinated muscle samples without added oxidants. For detailed oxidation and marination treatment conditions (AC), see Figure 1. examination as described above. The stained specimens were observed by a light microscope (MICROPHOT-FXA Nikon photomicroscope, Nikon, Tokyo, Japan) via a 20 objective using bright field settings. Phase Contrast Microscopy of Myofibrils. To investigate how oxidation could influence the hydration of myofibrils during brine irrigation and thermal processing, an additional experiment was performed under the same treatment conditions as described above except that a shorter irrigation time was used. Briefly, myofibrils were isolated from fresh loin muscle (nonoxidized) as described by Xiong et al.,21 and the final pH of the myofibrillar pellet was adjusted to 6.2. Isolated myofibril samples were mounted to glass slides and then irrigated up to 3 min with the same solutions used for whole-muscle sample treatments (AC) (Figure 1) according to the procedure of Liu et al.16 Images of irrigated myofibrils were taken using the same microscope as previously mentioned on the phase contrast mode via a 100 oil immersion objective with a digital camera (Optronics, Goleta, CA). Glass slides containing irrigated myofibrils were removed from the microscope platform, placed on an equilibrated hot plate (45 °C), and heated at this temperature for 90 s. After cooling to room temperature, the myofibrils were again viewed under the microscope with the phase contrast as described above. Thereafter, the glass slides were removed, heated on a 76 °C hot plate for 90 s, and then subjected to phase contrast microscopy the same way. The exact sample temperature was validated with a thermocouple.
Hydration and Moisture Retention Properties of Muscle. To investigate the impact of oxidation on moisture absorption and retention, raw and cooked muscle samples were analyzed for hydration and cooking yield, respectively. Hydration of Muscle Samples. Freshly prepared muscle disks were weighed before (W0, ∼ 5 g) and after (W1, obtained after 5 min of draining) the oxidationmarination incubation. Hydration capacity (%) was defined using the following equation: hydration capacity ð%Þ ¼
W1 W0 100 W0
Cooking Loss and Product Yield. After the above hydration capacity measurement, samples were cooked to an internal temperature of 71 °C
on a George Foreman Grill (Salton Inc., Mt. Prospect, IL). After the samples had cooled to room temperature, the cooked weight (W2) was recorded. Cooking loss (%) and product yield (%) were calculated as cooking loss ð%Þ ¼
product yield ð%Þ ¼
W1 W2 100 W1 W2 100 W0
Statistical Analysis. Three independent trials (n = 3) conducted on different days with triplicate or quadruplicate sample analyses were performed. Each independent trial was treated as a replication in which all marination and oxidation experiments and sample analyses were repeated. Data were analyzed using the general linear model procedure of Statistix software 9.0 (Analytical Software, Tallahassee, FL). Analysis of variance (ANOVA) was conducted to determine the significance of major effects (e.g., oxidation, PP). Significant differences (P < 0.05) between individual means were identified by Tukey HSD all-pairwise multiple comparisons.
’ RESULTS Oxidation-Induced Chemical Changes in Muscle Proteins. Protein oxidation is generally recognized as a detrimental cause for reduced water binding in fresh or low-salt meat.10 Hence, tryptophan fluorescence and protein carbonyls were measured to identify how •OH produced from HRGS would affect both myofibrillar and sarcoplasmic protein fractions at a high ionic strength (0.6 M NaCl). As shown in Figure 2, oxidation caused significant losses of fluorescence in both myofibrillar (a) and sarcoplasmic (b) proteins in raw samples. The loss was remarkable in raw samples oxidized by 20 mM H2O2 (P < 0.05) irrespective of the oxidationmarination sequence. However, at the lower H2O2 level (5 mM), only treatment B samples were sensitive to oxidation. When cooked to 71 °C, the fluorescence of both protein fractions, especially SP, was significantly less (P < 0.05) than that of 13028
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Figure 3. Protein carbonyls in myofibrillar (a) and sarcoplasmic (b) protein fractions from nonoxidized (control) and oxidized, brine-marinated muscle samples before (raw) and after (cooked) cooking. Means (n = 3) across all samples without a common letter differ significantly (P < 0.05). Positive control, fresh muscle; negative control, marinated muscle samples without added oxidants. For detailed oxidation and marination treatment conditions (AC), see Figure 1.
Figure 4. Binarized images showing quantitative changes of the diffusion dye in nonoxidized (control) and oxidized, brine-marinated muscle samples before (raw) and after (cooked) cooking. The value below each sample image represents the mean percentage (( standard deviation) of the dark area (n = 3). For detailed oxidation and marination treatment conditions (AC), see Figure 1.
corresponding raw samples. No significant differences were seen between treatments (AC), and heating tended to diminish the oxidation effect. In addition to changes in fluorescence intensity, the fluorescence spectra (300400 nm, not shown) of MP and SP fractions showed a red shift in the emission maximum by approximately 510 nm as a result of oxidation. As expected, protein carbonyls were also generated in oxidatively stressed muscle tissue in all three marination treatments (Figure 3). The carbonyl production was consistent with the tryptophan loss in a H2O2 dose-dependent manner. Although PP, a metal ion chelator, is capable of slowing the Fenton reaction through stabilizing Fe(III) in the catalytic Fe(II)/Fe(III) cycle,22 • OH can still be generated in the presence of ascorbate and appropriate precursors.23 In fact, at 5 mM H2O2, treatment B (with PP) produced slightly more (P < 0.05) protein carbonyls in MP when compared with treatment C (without PP). The oxidation promotion rather than oxidation protection effect of PP might be due to its facilitation of oxidant/marinade diffusion and, therefore, more free radical production in the muscle tissue. Cooking caused substantial increases (P < 0.05) in the carbonyl content in
both MP (a) and SP (b), which was most remarkable in oxidized samples. Diffusion of Marinades in Muscle. The impact of chemical changes in proteins as presented above on the hydration properties of muscle tissue was first tested through the examination of marinade penetration (Figure 4). Because the tracing dye used (FD&C Blue No. 1) was completely soluble in aqueous solutions, it was assumed that the dye moved together with the marinade brine. Thus, the stained areas (dark) displayed in the figure, with the proportions indicated as percentage values, represent marinades that were distributed in raw and cooked muscles. For raw muscle samples within the same column (same treatment), the stained areas of oxidized muscles at both 5 and 20 mM H2O2 were larger than those of nonoxidized samples, namely, by a net of 7.1 and 5.3% for treatment A, 11.9 and 10.9% for treatment B, and 1.5 and 8.0% for treatment C. Except for samples oxidized at 5 mM H2O2 in treatment C, the percent increases of the stained areas in all oxidized muscles were significant (P < 0.05), indicating that oxidation enhanced the diffusion of the brine (0.6 M NaCl) into the muscle tissue. Comparison between treatments AC showed 13029
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Figure 5. Micrographs of H&E stained cross sections (layer 1) of nonoxidized (negative control) and oxidized, brine-marinated muscle samples before (a, raw) and after (b, cooked) cooking. For detailed oxidation and marination treatment conditions (AC), see Figure 1.
that PP promoted water uptake, that is, hydration, regardless of oxidation. Yet, the efficacy of PP appeared reduced if the muscle was preoxidized (treatment A) when compared with samples exposed to oxidants and PP simultaneously (treatment B). When the marinated samples were cooked, the stained areas shrank substantially, suggesting that a large quantity of absorbed water was expelled from the muscle. The remaining dark areas in the oxidized muscle for all treatments retracted to the level of their respective controls (nonoxidized). The samples in treatments A and B, which contained PP, still showed a slightly higher amount of residual marinade than the sample without PP (treatment C) after cooking, that is, by an average 25%. Microstructural Changes in Oxidized Muscle. The microstructural features of the cross section of marinated muscle samples are presented in Figure 5. In the presence of 15 mM PP (treatments A and B) together with 0.6 M NaCl, fibers in control (nonoxidized) raw muscle exhibited a regular pattern (Figure 5a). When oxidized with 5 mM H2O2, muscle fibers after brine marination became more compact, and the extracellular space was visibly thinner when compared with controls, suggesting an increased hydration of the muscle cell (i.e., fiber swelling). At 20 mM H2O2, the extracellular space in both oxidation treatments was widened, likely due to transversal shrinkages of myofibrils and, thus, reduced water-binding potential. Detailed comparisons showed that treatment A (oxidation followed by marination) and treatment B (simultaneous oxidation and marination) produced somewhat different muscle morphologies, with the latter showing an overall more uniform, compact structure. In comparison with treatments A and B, treatment C (from which PP was absent) produced a slightly unpacked structure in oxidized samples (Figure 5a). Cooked muscle continued to exhibit distinct fiber structures in all oxidationmarination treatments; however, the arrangement of fibers appeared to become irregular and the extracellular space also lost its uniformity (Figure 5b). For muscle samples that were oxidized with 20 mM H2O2, pronounced reductions of fiber size (diameter) occurred, whereas the space between muscle fibers was decreased. Because the diffusion of marinades into the muscle tissue was affected by the extent of protein oxidation (Figure 2), a further experiment was performed to examine the fiber structure as the marinades penetrated into different layers of the muscle tissue. A representative micrograph, taken from treatment B, is used to highlight the oxidation impact (Figure 6). There were significant
Figure 6. Micrographs of H&E stained sections dissected from different layers (1, 3, and 5 mm below the surface) of nonoxidized and oxidized, brine-marinated muscle samples before (a, raw) and after (b, cooked) cooking. The results were from treatment B (simultaneous oxidation and marination) as an example to illustrate the oxidative effect.
differences in the appearance of the outer layers between oxidized and control samples, with or without cooking. For example, the extracellular space in oxidized muscle was visibly diminished, and the muscle cells appeared to be more hydrated. However, the structural patterns of the middle and inner layers progressively became similar, suggesting that muscle fiber and connective tissue were resistant to reducing concentrations of oxidants caused by diffusion limitations. The different oxidant diffusivities could also be a straightforward reason for the differences in physicochemical changes, including tryptophan fluorescence (Figure 2) and protein carbonyls (Figure 3) observed between oxidized muscle samples. Phase Contrast Microscopy of Myofibrils. Because myofibrils make up the bulk of muscle fiber and are largely responsible for water-binding in fresh meat,3 their interaction with marinades was examined to help explain the hydration properties of oxidatively stressed muscle. A typical structural feature of myofibrils during simultaneous oxidationmarination (treatment B) followed by heating, in comparison with nonoxidized controls, is provided in a series of captured images (Figure 7). The results are 13030
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Figure 7. Phase contrast images of typical control and oxidized myofibrils subjected to brine marination followed by heating at 45 °C and then 76 °C. Schematic sarcomere changes are drawn on both sides. The results for oxidized samples were from treatment B (simultaneous oxidation and marination) as an example to illustrate the oxidative effect.
Figure 8. Changes in the relative diameter, sarcomere length, and A-bandwidth of myofibrils after exposures to oxidationmarination treatment conditions similar to those for whole-muscle samples (see Figure 4 caption) and then heated at different temperatures. For detailed oxidation and marination treatment conditions (AC), see Figure 1.
schematized on both sides of the images to highlight processinduced changes and the oxidative effect. Fresh control myofibrils showed a typical sarcomere structural pattern with regular repeating A-bands being the most prominent feature. Upon brine marination, there was significant transverse expansion of the A-band. In comparison, concurrently oxidized and marinated myofibrils displayed less swelling. During cooking, the swollen A-band in marinated control samples progressively shrunk to a cohesive rigid block, apparently due
to retraction resulting from myosin denaturation, along with a somewhat lost continuity of myofibrils at the I-bands. On the other hand, the A-band in oxidized myofibrils underwent more lateral shrinkage (diameter) but less longitudinal shortening (band width) than control myofibrils. The M-line was visible at 45 °C but obscured when samples were heated to 76 °C. The above morphological examinations of oxidation-dependent myofibril changes upon brine irrigation and heating were extended to treatment conditions A and C, and the myofibril 13031
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Journal of Agricultural and Food Chemistry diameter, sarcomere length, and A-band width of all samples were recorded. The results, summarized in Figure 8, showed that through the process of irrigation and then heating, myofibrils underwent dramatic structural changes in the presence of PP (treatments A and B). The changes for oxidized myofibrils were less than those for the controls, but the differences diminished in the absence of PP (treatment C). More specifically, treatment A at 20 mM H2O2 caused a significant shrinkage of myofibril diameter and restricted lateral myofibril expansion during subsequent brine irrigation. Comparison of oxidized myofibrils in treatments A and B showed the latter had more extensive swelling than the former, which may be attributed to structural hindrances of myosin. There was no apparent alteration of the A-band width and sarcomere length for all myofibril samples after oxidation and brine irrigation (Figure 8). As the heating temperature was raised, the diameter of myofibrils progressively decreased, and the length of the A-band became shortened, yet the sarcomere length appeared to be largely unchanged. Hydration and Moisture Retention Properties of Muscle. The above morphological examinations offered solid evidence that oxidation-induced chemical changes affected brine penetration and distribution in muscle tissues. To more accurately quantify the hydration and water-holding capacities, raw and cooked muscle samples were analyzed. As summarized in Table 1, oxidatively stressed muscle samples had a greater hydration capacity than control samples in each oxidation treatment. The weight gain after marination was approximately 5060% more for oxidation treatments with PP (A and B) and 30% more for treatment without PP (C) when compared with nonoxidized controls. Hence, oxidized muscles were able to absorb more extraneous water from marinades. These results were in accordance with those from dye tracing (Figure 4) and, more directly, the microstructural features of marinated samples (Figures 5 and 6). The degree of hydration of treatment A was slightly higher than that of treatment B, contrary to the result of dye tracing (Figure 4). The discrepancy can be explained by the fact that in hydration measurement, marinated samples were drained for 5 min, whereas in dye tracing, marinated samples were immediately frozen, dissected, and analyzed. Despite the increase in hydration, marinated oxidized muscle samples demonstrated a higher overall cooking loss when compared with nonoxidized samples. Although PP-added treatments (A and B) showed a markedly less weight loss than the non-PP treatment (C), oxidation seemed to offset the positive effect of PP. The increased cooking loss of oxidatively stressed muscle samples was in accordance with the more notable thermal liberation of initially imbedded dye (Figure 4), the more pronounced size reduction of muscle cells (Figures 5 and 6), and the greater lateral shrinkage of myofibrils (Figures 7 and 8). Of all oxidized samples, the treatment with 5 mM H2O2 plus PP resulted in the least moisture loss.
’ DISCUSSION Chemical modifications of proteins occur ubiquitously in fresh and processed muscle foods when exposed to radicals. In the present study, the oxidation of myofibrillar and sarcoplasmic proteins was indicated by the loss of intrinsic fluorescence (tryptophan) and the formation of protein carbonyls. Fluorescence can be quenched by nearby polar amino acid side chains, such as glutamine, asparagine, and histidine,24 and disulfide bonds in proximity to the tryptophan residue,25 therefore, can
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Table 1. Hydration, Cooking Loss, and Product Yield of Nonoxidized (Control) and Oxidized Pork Muscle Samples a sample
hydration (%)
cooking loss (%)
product yield (%)
Treatment A control
23.6 ( 2.8 b
32.4 ( 2.6 cd
83.5 ( 1.1 abc
H2O2 5 mM H2O2 20 mM
35.2 ( 3.7 a 34.9 ( 3.8 a
39.4 ( 2.6 a 40.6 ( 1.7 a
82.0 ( 0.2 bcd 80.1 ( 1.5 cd
Treatment B control
22.5 ( 2.9 b
28.4 ( 1.5 d
86.8 ( 1.9 a
H2O2 5 mM
34.3 ( 4.2 a
37.4 ( 1.7 ab
84.1 ( 0.7 ab
H2O2 20 mM
33.1 ( 3.1 a
38.8 ( 2.9 abc
81.4 ( 1.3 bcd
control
19.7 ( 2.3 b
34.3 ( 1.8 bc
78.7 ( 1.6 d
H2O2 5 mM
25.9 ( 3.3 b
36.2 ( 2.9 abc
80.2 ( 0.3 bcd
H2O2 20 mM
25.0 ( 3.5 b
36.9 ( 3.3 abc
78.8 ( 2.1 d
Treatment C
Data represent the mean ( standard deviation of three replications (n = 3). Means within the same column that lack a common letter differ significantly (P < 0.05). a
be used to assess the nature of the tryptophan microenvironment.26 The oxidation-induced red shift of fluorescence spectra (by approximately 510 nm) can be explained by the exposure of tryptophan residues to an aqueous environment, that is, moving away from previous hydrophobic interior implying protein denaturation.27 On the other hand, exposed amino acid side chain groups, such as those of arginine, lysine, and proline residues, can be readily modified by •OH to produce carbonyls.28 Cooking aggravated protein oxidation and denaturation because of the enhanced •OH formation and increased exposures of susceptible side-chain groups.29 Furthermore, the liberation of heme and iron from heat-denatured myoglobin would also promote protein oxidation.30,31 These oxidation-induced chemical modifications are known to alter the distribution of surface charges and hydrogen bonds involved in the molecular interaction and water-binding potential of MP.32,33 Moreover, oxidatively denatured sarcoplasmic proteins can precipitate on myofibrils, hindering water binding and thereby lowering the water-holding capacity of myofibrils.34,35 As reported previously, in low-salt solution (0.1 M NaCl), protein oxidation promoted the association of myofilaments through hydrophobic and covalent (disulfide) interactions, resulting in the shrinkage of individual muscle fibers and the enlargement of extracellular spaces conducive to water penetration.17 This phenomenon was true also under the high-salt condition (0.6 M NaCl) as observed in the present study except that fibers in oxidized muscle tissue at 0.6 M NaCl appeared to be more hydrated when compared to fibers at 0.1 M NaCl. The extra space created between muscle fibers by oxidation had a poor capillary effect; the entrapped marinade was loosely held and readily expelled upon heating, irrespective of ionic strength. Similar in situ findings have been reported with ground pork stored under a high-oxygen-atmosphere packaging condition.36 Although PP was capable of dissociating the myosinactin cross-bridges and thereby promoting myofibril swelling or hydration in the presence of 0.6 M NaCl, its function was significantly weakened when muscle was oxidatively stressed. This was because disulfide linkages16 and possibly also carbonylamine cross-links31 formed between thick filaments in oxidized samples 13032
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Journal of Agricultural and Food Chemistry not only hindered the liberation of myosin but also restricted the lateral movement of myofilaments. Oxidation-generated intraand interfilamental cross-linking37 can also explain why the longitudinal retraction (A-band shortening) in heated myofibrils was obstructed when samples were marinated either concurrently with oxidation (treatment B) or after oxidation (treatment A). In both cases, oxidation prevented myofilaments from sliding past each other. The generation of these swelling-restriction forces was obviously oxidant dose-dependent, that is, the higher the H2O2 concentration, the stronger the restriction forces produced. Because myofibrils have a loose or disrupted structure in 0.6 M NaCl due to electrostatic repulsions, individual proteins, including myosin, would be more susceptible to diffusive hydroxyl radicals. The cross-links generated would result in a reduced PP efficacy in treatment B as discussed above. Conversely, in 0.1 M NaCl, the intact structure of myofibrils would less likely predispose myosin to oxidative modification, in agreement with the protein carbonyl analysis (Figure 3). The foreseeable fewer cross-links produced would allow myofibrils in treatment A to attract and retain more moisture upon subsequent brine marination than samples in treatment B. However, despite the numerical trend suggesting this being the case (Table 1), the difference between treatments A and B was mostly nonsignificant, indicating that the exact mechanism underlying the influence of oxidation on the function of PP and NaCl is more complicated. It is worth noting that the use of myofibrils, the primary waterbinding apparatus in the cell, as a model to investigate the morphology, hydration, and water-binding properties of oxidatively stressed muscle was of practical significance because it would avoid interferences from other materials such as endomysial collagen.3841 In fact, the remarkable shrinkage of some muscle fibers or widening of the intercellular gaps at 20 mM H2O2 (treatments B and C) but only moderate to negligible changes in myofibrils (e.g., A-band width) at the same H2O2 concentration served to suggest that hydration (marinade uptake), not marinade retention, in oxidized muscle involved greater contraction of the collagen network than the aggregation of myofibrils. These differential structural changes may also account for the aggravating effect of oxidizing radicals during cooking.42 In conclusion, hydroxyl radical-initiated oxidative stress at either 0.1 or 0.6 M NaCl promoted hydration of pork muscle when marinated in a 0.6 M NaCl brine with or without PP. However, upon cooking, the normal thermally induced moisture loss was aggravated by oxidation. Combined morphological evidence from muscle and myofibril samples led to the conclusion that the hydration enhancement was due to two action modes depending on the extent of oxidation, that is, most extraneous water was either tightly held in cells (for moderately oxidized muscle) or loosely held in the enlarged extracellular spaces (for severely oxidized muscle). The intensified transverse shrinkage of myofibrils and muscle fiber was responsible for increased cooking loss in oxidized muscle. These findings provided further understanding of the mechanism by which fresh meat marinated at high ionic strength salt solutions binds water when processed under oxidative conditions.
’ AUTHOR INFORMATION Corresponding Author
*Phone: (859) 257-3822. Fax: (859) 257-5318. E-mail: ylxiong@ uky.edu.
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Funding Sources
This research was supported by NRI/CSREES/USDA, USA (Grant 2008-35503-18790), the Natural Science Foundation, China (Grant 30972290), the Ministry of Science and Technology, China (Grant SKLF-MB-200803), and an Oversea Study Fellowship from the China Scholarship Council (to Z.L.). Approved for publication as journal article 11-07-075 by the Director of the Kentucky Agricultural Experiment Station.
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