- Email: [email protected]
Effect of halothane genotype on porcine meat quality and myoglobin autoxidation
Pll:
SO309-1740(97)00105-8
Meat Science, Vol. 49, No. I, 41-53, 1998 0 1998 Elsevier Science Ltd All rights reserved. Printed in Great Britain 0309-1740/98 $19.00+0.00
ELSEVIER
Effect of Halothane Genotype on Porcine Meat Quality and Myoglobin Autoxidationx L. G. Tarn,,, E. P. Berg,” D. E. Gerrard,b* E. B. Sheiss,” F. J. Taqd M. R. Okos’ & J. C. Forres@ aDepartment of Animal Science, Texas A&M University, College Station, TX 77843, USA bDepartment of Animal Sciences, Purdue University, West Lafayette, IN 47907, USA ‘Pig Improvement Company, Box 348, Franklin, KY 42135, USA dDepartment of Food Sciences, The Ohio State University, Columbus, OH 43210, USA PDepartment of Agricultural and Biological Engineering, Purdue University, West Lafayette, IN 47907, USA (Received
26 June 1997; revised version
received
3 September
1997; accepted
3 September
1997)
ABSTRACT The objective of this study was to determine effects of light (40-80 kg) or heavy (IOO130 kg) slaughter weight and halothane status (positive, nn; negative, NN; and heterozygous, Nn) on meat quality. Longissimus muscle (LM) pH at 45 min (pH4s) post-exsanguination was 6.25, 6,03, and 5.84 (different at p < 0.01) for NN, Nn, and nn genotype, respectively. At heavier weights (100-130 kg), genotype correlated (r = -0.71) with LM pH,,, 10th costae LM (TENLM) color score (r = -0.55)) TENLM Hunter L’value (r = 0.47), water holding capacity (r = 0.42) and TENLM subjectivejirmness-wetness score (r = 0.51). Rate constants for metmyoglobin accumulation and oxymyoglobin autoxidation, indicators for fresh meat color stability, increased ( p < 0.05) with decreasing pH. Color stability for NN muscle was more stable than nn muscle ( p ( 0.05). Electrofocusing of myoblobin revealed two bands (MW 17,1@) at pl 6.1 and 6.5 across genotypes. Because d$erences were not observed across genotypes, an observed increase (~~0.05) in 24 hr myoglobin autoxidation rate constant (associated with increased expression of the HAL gene) are presumed dependent upon post-mortem muscle changes. These data show that changes in halothane status affect fresh pork quality and that lowered meat quality results in further color destruction due to altered chemicaf reactions involving myoglobin oxidation. 0 1998 Elsevier Science Ltd. All rights reserved
INTRODUCTION Pigs
leaner
that are homozygous and possess greater
positive pigs (nn) for the halothane gene (HAL) are generally muscle than pigs lacking the gene (AW). However, these pigs
*To whom correspondence should be addressed. +Present address: l/181 Barkers Road, Kew 3101, Victoria, Australia. *Purdue University Agricultural Research Programs Journal Paper No. 15403.
41
L. G. Tam et al.
42
often yield carcasses exhibiting pale, soft and exudative (PSE) meat (MacLennan and Phillips, 1992; Louis, et al., 1993). The increase in lean yield provided by HAL status is negated by an increased pig mortality. Pigs that survive to market not only generate pork of low meat quality, but produce cuts with a shorter retail shelf-life due to reduced color stability. Breeding companies have developed hybrid lines that use heterozygous HAL (Nn) genotypes in an effort to prevent severe PSE associated with nn pigs while maintaining the desirable muscling attributes (Cannon et al., 1995). Although mortality rate is lowered in Nn pigs, conflicting results exist as to the quality of the meat produced from such animals. In addition, the severity of the PSE condition may be reduced if animals are delivered to market at a lower weight. The goal of this study, therefore, was to investigate the effect of the HAL gene and slaughter weight on porcine meat quality and color stability.
MATERIALS
AND
METHODS
Animals and slaughter data collection Eighty-four pigs (l&14 days of age) were secured from a commercial breeding company, DNA tested for the halothane gene (Fujii et al., 1991), and assigned a HAL status (NiV, Nn, nn). Five NN, 5 Nn and 4 nn pigs were allocated to six slaughter groups (40, 60, 80, 100, 115, 130 kg) and slaughtered according to normal (United States) industry procedures. Longissimus (LM; adjacent the last costae) and deep Semimembranosus (SM) muscle pH was measured using a portable pH’K21 probe (NWK Thien GmbH, West Germany) at exsanguination (pHu) and 45 min (pHa5) post mortem. Longissimus muscle was collected at the last costae immediately following exsanguination, frozen in liquid nitrogen, and stored at -40°C for use in myoglobin isoelectric focusing (IEF) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Dressed carcasses (head, peritoneal fat, and kidneys removed; feet left on) were weighed and sent to the chiller (4°C). Sample collection
at 24 hr post mortem
The LM was exposed at the 10th and 1 lth rib interface (TENLM) following a 24 hr chill (4°C) and subjectively evaluated by an experienced six member panel using National Pork Producers Council (NPPC, 1991) standards for fresh pork color, marbling, and firmnesswetness. Following subjective quality assessment, ultimate LM and SM muscle pH values were recorded using the pH*K21 probe and a three rib (8-10th rib) section of LM was removed for evaluation of objective quality parameters. Objective color measurements were obtained from 2.54 cm cross-sectional slices of LM (9th rib) and SM at two separate locations on the muscle surface using a HunterLab 45”joO D25-PC2A Colorimeter (Hunter Associates Laboratory Inc., Reston, VA). Water holding capacity was determined from triplicate 0.3 g samples of TENLM and SM by the filter paper press method (Hamm, 1986). Drip loss was estimated from 24 hr moisture loss of (duplicate) 2g TENLM core samples suspended from a hook in sealed centrifuge tubes at 4°C. In addition, LM and SM samples were collected, frozen in liquid nitrogen, and stored at -40°C for future analysis of 24 hr myoglobin autoxidation. Determination
of total meat pigment
Total pigment concentration (ST; light and dark portions)
was determined on 24 hr LM (10th rib) and Semitendinosus samples using the method of Garrido et al. (1994). Samples
Effect of halothane genotype on porcine meat quality and myoglobin autoxidation
43
were homogenised at 4°C in 40 mM potassium phosphate buffer, pH 6.5, and centrifuged at 1000 x g for 10 min at 4°C. Supernatants were removed and centrifuged at 10 000 x g for 20 min at 4°C. Filtered supernates were mixed with TX-l 14 (100 g 1-l) at 4°C heated at 37°C for 10 min, and centrifuged at 7500 x g for 5 min at 25°C. Sodium nitrite solution (65mM) was added to the upper pigment-rich phase. Absorbance was measured at 409hO.5 nm after 15 min using a Beckman DU6 UV-Visible spectrophotometer (Beckman Instruments, Inc., Irvine, California). Pigment amount was calculated as follows: Pigment MW=
(mgg-‘)=
((A,,,
17 142;dilutionfactor=83.51
Determination
of oxymyoglobin
x MW myoglobin mlg-‘;
x 1000)/&409) x dilution
molar absorbancecoefficient
factor
=79’6mM-‘cm-’
autoxidation rate constants
The method of Krzywicki (1982) was used to determine the relative concentration of myoglobin, oxymyoglobin, and metmyoglobin. Frozen TENLM, SM, and ST samples were powdered in liquid nitrogen for extraction at 4°C in 40mM potassium phosphate buffer, pH 5 and 6 (Warriss, 1979). Samples were diluted in buffer (1: 12.5), homogenised at 4°C and centrifuged at 1000 x g for 10 min at 4°C. Supernatants were removed, centrifuged, and filtered to minimise absorbance at 730 f 0.05 nm and remove lipid. The pH of each solution was recorded and samples were held for 10 hr at 25°C. Duplicate absorbance readings were made every hr at 572, 565, 545, 525 f 0.05 nm using a Beckman DU6 spectrophotometer. Absorbance at 730 * 0.05 nm was recorded as zero (Krzywicki, 1979). Rate constants for metmyoglobin accumulation and oxymyoglobin disappearance (autooxidation) were obtained by plotting In (C&J against time where C, was the fractional concentration of the myoglobin derivative at time t and C, was the initial fractional concentration of myoglobin derivative. The slope of the line of best fit was equal to the rate constant (k hr-‘). Isoelectric focusing Isoelectric focusing of myoglobin was carried out in PAGE tube gels (3 mm ODx 14 cm) using a modified method of hemoglobin separation developed by Antonini et al. (198 1). The gel mixture consisted of 23.7% (v/v) acrylamide (20%T, 2’7%Cbi,), 4.5% (v/v) ampholine (PI 5-8; Pharmacia LKB Biotechnology AB), 0.07% (v/v) TEMED, 0.49% (v/v) ammonium persulfate (20%), and 1.5% (v/v) Nonidet-10 (10% v/v). The cathode electrolyte was 0.01 M sodium hydroxide and the anode electrolyte was 0.01 M phosphoric acid. Tubes were cooled to 4°C. Gels were pre-run at 200 V for 15 min and 400 V for 30min on a vertical gel electrophoresis system (Model V-16-2, Bethesda Research Laboratories). Frozen meat samples were pulverized into a powder, diluted (1: 1.5) in potassium buffer pH 6.8) homogenised at 4°C. Samples were then packed in ice, gently agitated for 2 hr, centrifuged at 40 000 g for 1 hr at 4°C and passed through Whatman No. 1 filter paper to remove fat. Samples were mixed with glycerol (1:l) and stored at -20°C for no longer than five days. Ten ~1 of prepared sample was mixed with 15 ~1 sample buffer [lo% (w/v) glycerol; 6.75% (v/v) ampholine p1 5-81. Tube gels were loaded with 20 ~1 of sample and p1 standards of 5.9 to 7.2 (Sigma Chemical) and focused for 7.5 hr at a potential of 400 V. Tube gels were fixed for 1.5 hr with 3.5% (w/v) sulfosalicylic acid, 35% (v/v) methanol, and 13% (w/v) trichloroacetic acid. Proteins were stained for 30 min with Coomassie Blue and destained overnight. Tube gel duplicates were soaked in equilibration buffer pH 6.8 for
44
L. G. Tam et al.
30min, quick frozen in equilibration -70°C for future SDS-PAGE.
buffer
using
dry ice and ethanol,
and
stored
at
SDS-PAGE Tube gels from IEF were subjected to a 12% separating SDS-PAGE (3 mmx 15.2 cmx 16 cm). The gel mixture consisted of 40% (v/v) acrylamide (30%T, 2_7%Cbi,); 0.1% (W/V) SDS, 375 mM Tris-HCl pH 8.8; 0.05% (V/V) TEMED; 0.5% (V/V) ammonium persulfate (10%) (Laemmli, 1970). Tube gels were attached to the top of the slab separating gel with 1% (w/v) agarose and separating gel solution. Top and bottom electrophoresis reservoirs were filled with glycine buffer (25 mM Tris, 192 mM glycine, 0.1% (w/v) SDS). Molecular weight standards (MW = 14 00&70 000; Sigma Chemical) were embedded in agarose solution of the same diameter as the IEF tube gels and a 1 cm portion was attached to the top of the separating gel. Gels were run for 19 hr at 4°C at a constant current of 28 mA. After electrophoresis, slab gels were fixed and stained for 30 min with Coomassie Blue and destained overnight. Statistical analysis Statistical analysis was performed using the SAS general linear model procedure (SAS, 1985). A two factor (genotype, weight) fixed effect model with interaction was assumed for the analysis of meat quality and myoglobin autoxidation data. The statistical model was described as “ijk
=
/J- +
(ri)
+
(Bj) + (tb)q +
&ijk
where p is the overall mean effect, ti is the effect of the ith level of genotype, Bj is the effect of the jth level of weight and (tp)ij is the interaction between ti and pj and &ijk is the random error component.
RESULTS
AND
DISCUSSION
Meat quality No significant quality differences were seen between 40, 60, or 80 kg pigs. Therefore, slaughter groups were pooled into two groups for statistical analysis; a lightweight (L-WT) group slaughtered at 40, 60, and 80 kg and a heavyweight (H-WT) group slaughtered at 100, 115 and 130. Color Hunter L’ a* b* values provide an objective means for comparison of meat color variation. Longissimus muscle (10th rib) subjective color scores from pigs possessing the stress gene (Nn and nn) were lower (p < 0.05) than the NN genotype, however, LM Hunter L* values of Nn muscle were not different from NN muscles (Table 1). Higher (p < 0.05) L’ values for H-WT nn pigs were observed supporting the subjective color scoring methodology. No differences in color scores were observed across genotypes of the L-WT group (Table 3). The SM of the Nn genotype was significantly darker (~~0.05) than NN (Table 1) and may be a reflection of the difficulty of assessing SM color due to inherent color variations within this muscle (Pommier and Houde, 1993; Klont and Lambooy, 1995).
EfSect of halothane genotype on porcine meat quality and myoglobin autoxidation
Meat Quality
TABLE 1 Characteristics (least square means and standard errors) for Pigs Differing thane Status (positive, nn; negative, NN, and Heterozygous, Nn) NN*
Characteristic
Semimembranosus
in Halo-
nn**
2.70(0.07) 2.92(0.08) 1.75(0.09) 4.54(0.30)
2,40(0.07)b 2.93(0.08) 1?32(0.09)” 3.1 l(0.30)b
L*-value a*-value b*-value
49.27(0.46)6 11+X5(0.22) 14.08(0.1 7)6
49. 16(0.46)b 12.10(0.22) 14.85(0.17)b
5 1.40(0.52)” 12.22(0.25) 15.43(0.19)
L*-value a*-value b*-value
47.90(0.62)” 12.91(0.30) 14.59(0.22)
45.55(0.62)b 13.86(0.30) 14.67(0.22)
47.37(0.69yb 12.77(0.34) 14.25(0.25)
1.33(0.04) 4.29(0.16)b 1’ 13(0.05)
1.31(0.04) 4.80(0.16) 1.44(0.05)
1.36(0.04) 5.04(0.18) 1.36(0.06)
6.63(0.03) 6.25(0.03) 5.51(0.02)
6.60(0.03) 6.03(0.03)b 5.51(0.02)
6.55(0.04) 5.84(0.03) 5.45(0.04)
6.51(0.04) 6.12(0.03) 5.44(0.02)b
6.52(0.04) 6.02(0.03)b 5.54(0.02)
6.45(0.04) 5g6(0.03)’ 5.48(0.02)b
Colord Firmness-wetnessd Marblingd Drip loss (%) Longissimus muscle
2.25(0.08)b 2.40(0.08)b 1.48(0. 10)b 5.06(0.33)
muscle
Total pigment (mg gg’ fresh muscle) Longissimus muscle Semitendinosus muscle Longissimus
45
Dark Light
muscle PHO’ ~H45
PH, Semimembranosus
muscle PHO ~H45
PH,
*n = 30. **n = 24. a,b,cMeans within a row with different superscripts differ (p < 0.05). dNPPC quality standards: 1= least/lightest and 5 = greatest/darkest. “pHo = pH at exsanguination; pH 45 = pH 45 min post-exsanguination; 24 hr chill.
pH, = ultimate
pH after a
A significant increase (p < 0.05) in LM Hunter b* value was observed for nn LM compared with NN LM for both weight groups (Tables 2 and 3), suggesting a shift from blue to yellow along the yellow to blue continuum of color. An increase (~~0.05) in LM Hunter b* value was observed for the nn genotype compared to NN and Nn genotypes (Table 1). Pommier and Houde (1993) reported greater b’ values for 3212pigs, while Klont and Lambooy (1995) reported no difference between genetic lines. Marbling andjrmnesslwetness Heavyweight pigs representing the NN genotype possessed a higher firmness-wetness and marbling score (p< O-OS) than NN L-WT pigs indicating that full expression of PSE muscle may not be manifested until heavier live weights are achieved. Less marbling (p < 0.05) was observed in the LM of nn pigs compared to NN and Nn genotypes (Table l), however, L-WT marbling scores (Table 3) did not differ (~~0.05). The average meat firmness and exudativeness of LM was lower (p > 0.05) for nn pigs compared to NN and Nn pigs for all weight endpoints (Table 1) as well as for L- and H-WT (Tables 2 and 3).
L. G. Tam et al.
46
TABLE 2
Meat Quality Characteristics (least square means and standard errors) for Heavyweight Pigs (lo&l30 kg) Differing in Halothane Status (positive, nn; negative, NN; and heterozygous, Nn) Characteristic
NN*
Colold Firmness-wetnes& Marblingd Drip loss (%) Longissimus muscle
2+33(0.09)” 3.03(0.08) 2.17(0.12) 5.83(0.44)”
-
Nn* 2.47(0.09)b 2.90(0.08) 1.83(0.12)” 3.92(044)b
-
nn** 2.25(0.10)b 2.33(0.09)b 1.46(0. 14)b 5.94(0.52)
L*-value a*-value b*-value
49.44(0.67)b 12.81(0.36) 14.70(0.26)b
49.63(0.67)b 13.03(0.36) 15.51(0.25)“b
52.68(0.75) 12.60(0.41) 15.70(0.29)
L*-value a*-value b*-value
5 1.38(0.94) 12.63(0.38) 14.88(0.33)
48.97(0.94) 14.18(0.38) 15.53(0.33)
49.56( 1.05) 13.25(0.42) 14.93(0.36)
Semimembranosus muscle
Total pigment (mg gg’ fresh muscle) Longissimus muscle Semitendinosus muscle
Dark Light
Water holding capacity Longissimus muscle PHO’ ~H45
PH”
Semitendinosus muscle
PHO ~H45
PH,
-
1.41(0.07)
2.21(0.06)b
5.94(0.30) 1.47(0.10) 2.38(0.06)
6.65(0.04) 6.22(0.03) 5.47(0.04)
6.63(0.05) 6.04(0.03)b 5.51(0.04)
6.54(0.05) 5.89(0.03) 5.42(0.04)
6.48(0.06) 6.06(0.04) 5.40(0.03)b
6.52(0.06) 6.00(0.04) 5.52(0.03)
6.42(0.07) 5.80(0.05)b 5.43(0.03)“b
*n= 15. **n= 12. a,b,cMeans within a row with different superscripts differ (p < 0.05). dNPPC quality standards: 1 = least/lightest and 5 = greatest/darkest. epHo = pH at exsanguination; pH45 = pH 45 min post-exsanguination;
Drip loss and water-holding
1.31(0.06) 5.32(0.27yb 1.60(0.09)
1.40(0.06) 4.89(0.27)b 1.46(0.09) 2. 14(0.06)b
pH, = ultimate pH after a 24 hr chill.
capacity
Drip loss for nn muscle was > 5%, placing the average muscle from this genotype between exudative and non exudative according to Kauffman et al. (1993). No differences were observed for drip loss between nn and NN LM, however, drip loss was lower (p < 0.05) for Nn loins compared to NN and Nn loins (Table 1). This trend was consistent across genotypes for both heavy and light weight groups (Tables 2 and 3). The small meat sample size used for drip loss may have produced increased free water loss during sampling and may explain differences between genotypes. Water holding capacity (WHC) was determined by the filter paper press method of Hamm (1986) where a* value > 2.4 indicated poor WHC. The WHC for all three genotypes was considered acceptable yet the Longissimus muscle WHC score for H-WT nn pigs was higher (p < 0.05) than NN and Nn genotypes (Table 2). Meat pigment Myoglobin content observed in muscle
of meat is responsible for color. pigment levels among all treatment
No differences (p > 0.05) were (Table 2). Klont and Lambooy
47
Effect of halothane genotype on porcine meat quality and myoglobin autoxidation
Meat Quality Characteristics (least (4&80 kg) Differing in Halothane
TABLE 3 square means and standard errors) for Lightweight Pigs Status (positive, nn; negative, NN; and heterozygous, Nn) NN*
Characteristic
2.57(0.11) 2.80(0.12)“b 1.33(0.14) 3.24(0.37yb
Colold Firmness-wetnes& Marblingd Drip loss (%) Longissimus muscle
Semimembranosus
nn** 2.25(0.12) 2.46(0. 14)b 1.50(0.15) 4.18(0.41)”
2.33(0.11) 2.97(0.12) 1.80(0.14) 2.30(0.37)b
L*-value a*-value b*-value
49.10(0.64) 10.96(0.26) 13.48(0.21)b
48.68(0.64) 11.18 (0.26) 14.21(0.21)b
50.11(0.71) 11.84(0.29) 15.14(0.24)
L*-value a*-value b*-value
44.30(0.80)“b 13.22(0.47) 14.31(0.30)
42.03(0.80)b 13.54(0.47) 13.76(0.30)
45.16(0.89) 12.27(0.53) 13.55(0.34)
1.27(0.05) 3.69(0.19) 1’ 15(0.04)
1.31(0.05) 4.28(0.19) 1.28(0.04)
1.30(0.05) 4.25(0.21) 1.25(0.05)
6.60(0.05) 6.27(0.04)” 555(0.03)
6.59(0.05) 6.02(0.04)b 5.51(0.03)
6.55(0.05) 5.79(0.05)’ 5.47(0.03)
S.SS(O.05) 6.18(0.04) 5.48(0.03)
6.51(0.05) 6.05(0.04) 5.57(0.03)
6.48(0.06) 5.92(0.05)b 5.52(0.03)
muscle
Total pigment (mg g-t fresh muscle) Longissimus muscle Semitendinosus muscle Longissimus
Nn*
Dark Light
muscle PHO’ ~H45
PH” Semitendinosus
muscle PHO ~H45
PH,
*n= 15. **n = 12. “,b,cMeans within a row with different superscripts differ (p > 0.05). “NPPC quality standards: 1= least/lightest and 5 = greatest/darkest. ‘pHo = pH at exsanguination; pH45 = pH 45 min post-exsanguination; 24 hr chill.
pH, = ultimate
pH after
a
(1995) reported a significant increase in pigment concentration from NN to nn pigs. Pigment concentration found in this study were higher than Klont and Lambooy (1995) but similar to Garrido et al. (1994). At a low pH, the heme group of myoglobin becomes labile and will dissociate into heme and globin (Antonini and Brunori, 1971) providing less native myoglobin to bind oxygen, generating a more pale appearance. Low pH meat appeared paler, as supported by increased Hunter L’ values. Rapid post-mortem pH decline at an elevated carcass temperature contributes to development of PSE meat. The pH recorded at 45 min post exsanguination (pH4s) is often lower in pigs expressing the HAL genotype (Oliver et al., 1993; Klont and Lambooy, 1995; Kocwin-Podsiadla et al., 1995; Leach et al., 1995). A lower (~~0.05) pH4s was observed for the Nn and nn genotypes compared to NN (Table 1). Strong negative correlations between pH4s and halothane genotype were found. With increased halothane susceptibility, pH4s was lower (Table 5). Muscles with a higher pH4s tended to have darker, less exudative meat. No genotype differences were observed for initial (pHo) or ultimate PH (PH”).
muscle
muscle
PH”
~H45
PHO
PH”
~H45
PHO’
Dark Light
L*-value a*-value b*-value
L*-value a*-value b*-value
6.50(0.06) 6.07(0.04) 5.48(0.03)b
6.54(0.05) 6.00(0.04) 5.48(0.03)“b
1.31(0.06) 3.61(0.24) 1.20(0.07)
42.04(0.91)d 10.85(0.45)b 11.78(0.33)b
47. 17(0.68)b 10.77(0.33)d 13.1 9(0.25)b
2.34(0.10) 2.54(0.1 l)bc 1.46(0.14)“b 1.27(0.44)’
40 kg
80 kg
6.55(0.06) 5.96(0.04) 5.45(0.03)b
6.62(0.05) 6.09(0.04) 4.48(0.03yb
1.34(0.06) 4.85(0.24)b 1.34(0.07)6
49.87(0.91yb 13.25(0.45) 15.68(0,33)
50.42(0.68) 11.99(0.33)b’ 14.58(0.25)
pH after a 24 hr chill.
6.56(0.06) 6.08(0.04) 5.59(0.03)
6.66(0.05) 6.1 l(O.04) 5.57(0.03)
1.28(0.06) 4.62(0.24)bC 1.26(0.07)b
44.13(0.9l)“d 14.75(0.45) 14.81(0.33)
50.42(0.68) 1 l.87(0.33)bc 15.01(0.25)
2.53(0.10) 3.03(0.11) 1.93(0.14)“b 4.94(044)
100 kg ll5kg
6.47(0.06) 5.96(0.04) 5.48(0.03)b
6.59(0.05) 6.04(0.04) 5.50(0.03)“b
1.28(0.06) 5.1 1(0.24)b 1.49(0.07)“b
48.23(0.91)b 13.91(0.45) 14,47(0,33)
50.28(0.68) 12.82(0.33yb 15.40(0.25)
2.53(0.10) 2.88(0.1 l)ab 1.77(0.14)” 5.51(0.44)
errors) for Pigs 4&l 30 kg
2.53(0.10) 2.88(0.1 l)ab 1.82(0. 14)ub 3.85(0.44)
pH, = ultimate
6.48(0.06) 6.00(0.04) 5.50(0.03)b
6.53(0.05) 5.98(0.04) 5.48(0.03yb
1.29(0.06) 3.97(0.24)‘d 1.22(0.07)b
45.31(0.91) 13.37(0.45) 15.05(0.33)
50.27(0.68) 11.33(0.33)‘d 14.60(0.25)
2.28(0.10) 2.80(0.1 lyb 1.36(0.14)b 4.60(0.44)
60 kg
TABLE 4 (least square means and standard
ObcMeans within a row with different superscripts differ (p < 0.05). dNpPC quality standards: 1= least/lightest and 5 = greatest/darkest. ‘pHo = pH at exsanguination; pH4, = pH 45 m’in post-exsanguination;
Semitendinosus
Longissimus muscle
Total pigment (mg gg’ fresh muscle) Longissimus muscle Semitendinosus muscle
Semimembranosus
Colold Firmness-wetnes& Marblingd Drip loss (X) Longissimus muscle
Characteristic
Meat Quality Characteristics
6.47(0.06) 5.95(0.04) 5.41(0.03)b
6.61(0.05) 6.02(0.04) 5.41(0.03)b
1.50(0.06) 6.18(0.24)” 1.70(0.07)
51.82(0.91) 12.90(0.45)” 15.19(0.33)”
5 1.08(0.68) 13.53(0.33) 15.95(0.25)
2.49(0.10) 2.37(0.11) 1.77(0.14) 5.25(0.44yb
130 kg
;;I 3 4 g
p a
49
E#ect of halothane genotype on porcine meat quality and myoglobin autoxidation
Pearson’s
Correlation
Coefficients 2
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
TABLE 5 of Variables Affecting 4&l 30 kg 4
3
5
the Color of Longissimus 7
6
8
0.035 -0.419 0.280 0.128 0.355 -0.176 0.309 -0.343 0.318 0.018 -0.024 -0.402 0.152 -0.259 0.181 0.003 0.543 0.015 0.689 0.038 0.045
Genotype Pigment Color” L*-value a*-value b*-value pHob
Correlation
Coefficients 2
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
-0.738 0.090 0.504 -0.363 -0.037 -0.322 0.187
PH, %Drip loss Firmness-wetness0
Pearson’s
3
TABLE 6 of Variables Affecting the Color Heavyweight Pigs 100-130 kg 4
0.006- -0.550 0.471 0.289-0.434 -0442
Genotype Pigment Color” L*-value a*-value b*-value pHob ~H45
PH,
%Drip loss Firmness-wetness” WHC’
5
6
7
8
Autoxidation order kinetic
autoxidation
-0.186 0.070 -0.258 0.179 0.197 -0.005 -0.091 0.218 0.009 0.337 -0.068 0.334 0.013 -0.00 0.186 -0.204 -0.245
pH, = ultimate
of Longissimus 9
10
II -0.364 -0.212 0.441 -0.268 -0.061 -0.273 0.152 0.467 0.233 -0.166 1.000
pH after a
Muscle II
from 12
0.034 0.350-0~257-0~711-0.126-0~010-0~508 0.417 0.374 0.064-0.049 0.176-0.262 0.272-0.294-0.034 0.023-0.271 0.258 0.592-0.007-0.114 0.448-0.453 -0.301 0.227-0.151-0.487 0.01 l-0.142-0.323 0.392 0.756 0.069-0.027 0.107 0.078-0.312-0.139 -0.013-0.317 0~111-0~030-0~419 0.117 0.239-0.180-0.039 0.117-0.030 0.015 0.091 0.637-0.373 -0.296 0.226-0.010 -0.278 0.007 -0.277 1JJOO
“NPPC quality standards: 1 = least/lightest and 5 = greatest/darkest. bpHo = pH at exsanguination; pH 45 = pH 45 min post-exsanguination; 24 hr chill. ‘WHC = water holding capacity; filter paper press method.
Oxymyoglobin
10
9
pH45
“NPPC quality standards: 1 = least/lightest and 5 = greatest/darkest. bpH, = pH at exsanguination; pH 45 = pH 45 min post-exsanguination; 24 hr chill.
Muscle from Pigs
and metmyoglobin
accumulation
pH, = ultimate
pH after a
rate constants
of oxymyoglobin accompanied by the formation of metmyoglobin is a first reaction, d[Mb02]/dt = -k,,[MbOz] (George and Stratmann, 1952; Brown
and Mebine, 1969; Shikama and Sugawara, 1978; Bertelsen and Skibsted, 1987; Renerre and Gatellier, 1992). Rate constants increased (p < 0.05) for metmyoglobin accumulation and oxymyoglobin autoxidation when pH 5 buffer was used for myoglobin extraction because, at this pH, myoglobin is more susceptible to unfolding and cleavage. Rate constants (least square means and standard errors) for metmyoglobin accumulation were
50
L. G. Tam et al.
Pearson’s
Correlation
2 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
Genotype Pigment Color” L*-value a*-value b*-value pHob
TABLE 7 of Variables Affecting the Color Lightweight Pigs 4W30 kg
Coefficients 3
4
5
6
7
0.075 -0.300 0.130 0.299 0.404 -0.101 0.294 -0.029 0.009 -0.198 -0.029 -0.461 0.167 -0.405 0.088 0.138 0.799 0.134 0.502 -0.094 0.046
of Longissimus
8
9
-0.773 0.009 0.453 -0.304 -0.094 -0.384 0.151
-0.281 -0.198 0.555 -0.164 0.071 -0.196 0.287 0.391
PH~S
PH, %Drip loss Firmness-wetnessa
ONPPC quality standards: 1 = least/lightest and 5 = greatest/darkest. bpHo = pH at exsanguination; pH4s = pH 45 min post-exsanguination; 24 hr chill.
pH,
Muscle
from
10
11
0.160 0.225 -0.068 -0.132 -0.067 0.439 0.411 -0.236 0.254 0.210 0.449 -0.184 -0.053 0.182 -0.133 0.353 -0.088 0.266 -0.126 1.000
= ultimate
pH after a
0.0926 (0.0021) hr-’ and 0.0456 (o-0021) hr-’ when pH 5 and 6 buffer were used for myoglobin extraction, respectively. Rate constants for oxymyoglobin autoxidation were -0.0666 (0.0013) hr-’ and -0.0456 (o-0013) hr-’ when pH 5 and 6 were used for myoglobin extraction, respectively (Table 8). These results are similar to those reported by Bembers and Satterlee (1975) for a crude extract of porcine myoglobin from LM (0.025 hr-’ at pH 5.8-6.0 and 30°C). Autoxidation of oxymyoglobin theoretically utilizes one mole of oxymyoglobin to form one mole of metmyoglobin. When pH 5 buffer was used for myoglobin extraction the rate constant for autoxidation of oxymyoglobin was 1.39 times less than for metmyoglobin accumulation. The large increase in rate constant between pH 6 and 5 was confirmed by Shikama and Sugawara (1978). Rate constants calculated in this study were similar in magnitude to those reported by Shikama and Sugawara (1978). Differences in rate constants between genotypes reflect oxymyoglobin stability. Rate constants calculated for metmyoglobin accumulation (Table 8) of nn meat, 0.0797 (0.0029) hr-’ were larger (p < 0.05) than rate constants calculated for NIV, 0.0639 (0.0023)
Rate Constants
TABLE 8 (least square means and standard errors) for Metmyoglobin Oxymyoglobin Autoxidation for Pigs of Three Genotypes
Accumulation
and
Rate constant
Metmyoglobin
Oxymyoglobin
Genotype
Average
NN Nn nn
0.0639(0.0023) 0.0667(0.0024) 0.0797(0.0029)
accumulation
autoxidation NN Nn nn
-0.0612(0.0015) -0.0565(0.0015) -0.0500(0.0019)
Extracted
pHS
0.0926(0.0021) 0.0837(0.0033) 0.0902(0.0033) 0.1 lOO(O.0042) -0~0666(0~0013) -0.0697(0.0021) -0.0679(0.0021) -0.0621(0.0026)
Extracted
pH6
0.0456(0.0021) 0.0441(0.0033) 0.0433(0.0033) 0~0495(0~0041) -0.0456(0.0013) -0.0538(0.0020) -0.0451(0.0021) -0.0379(0.0026)
Effect of halothane genotype on porcine meat quality and myoglobin autoxidation
51
hr-‘, and Nn meat, 0.0667 (0.0024) hr-‘. In contrast the rate constant (Table 8) for autoxidation of oxymyoglobin for nn pigs [-0.0500 (0.0019] hr-‘) was smaller in magnitude (~~0.05) than rate constants for NN [-O-0612 (0.0015) hr-‘1 and Nn genotype [-O-O565 (0.0015) hrr ‘1. A comparison of rate constants for metmyoglobin accumulation and oxymyoglobin autoxidation of nn genotype showed the rate constant for metmyoglobin accumulation was 1.59 times larger than the rate constant reported for oxymyoglobin autoxidation. This result was in contrast to NN and Nn metmyoglobin accumulation and oxymyoglobin autoxidation rate constants where the rate constants for metmyoglobin accumulation were 1.04 and 1-18 times larger than oxymyoglobin autoxidation rate constants, respectively. Rate constants for metmyoglobin accumulation when pH 5 and 6 buffers were used for myoglobin extraction are reported in Table 8. Electrofocusing
of porcine muscle extract
Porcine muscle extract was successfully electrofocused in two dimensions to test for differences in porcine myoglobin banding patterns across halothane genotypes. Longissimus muscle from two pigs of each genotype were selected for electrofocusing. Two bands were identified on SDS-PAGE to be of an approximate molecular weight of 17 000 (Satterlee and Zachariah, 1972). The isoelectric point of the two bands identified in this study of approximate molecular weight 17 000 were calculated to be 6.1 for the major band, and 6.5 for the faint band. Identical banding patterns were maintained across genotypes similar to Wykle et al. (1978) who reported no differences between banding patterns of porcine heart and skeletal muscle myoglobin extracted from halothane positive, negative or seven cross bred groups of pigs. However, Wykle et al. (1978) identified eight bands of MW 17 000 that could be a result of their different staining methodology or myoglobin denaturation during electrophoreses conducted at room temperature. Sample preparation and electrofocusing were conducted in this study at temperatures no greater than 4°C to minimize protein denaturation and oxidation. Isoelectric points were similar to Bembers and Satterlee (1975) for purified myoglobin from normal muscle. The faint band identified in this study at p1 = 6.5 corresponded to that of metmyoglobin identified by Bembers and Satterlee (1975). The major band identified in this study at pI=6.1 was similar to the upper range reported by Bembers and Satterlee (1975) for oxymyoglobin from PSE muscle (PI= 6.09). No differences in banding patterns were observed for porcine skeletal muscle myoglobin extracted from pigs of different halothane sensitivity. Given no differences in myoglobin primary structure across genotypes, the observed increase (~(0.05) in myoglobin autoxidation rate constant with increased gene expression reported in this study was dependent upon post-mortem changes in the muscle and their effect on myoglobin conformation. Results of this study show that the average rate constant for metmyoglobin formation is 1.59 times higher than myoglobin autoxidation in LM from the nn. The affect of low pH has long been attributed to myoglobin denaturation and pale color of PSE pork. Low pH has been shown to reduce the stability constant for the heme-globin linkage and increase the autoxidation rate (Renerre et al., 1992). Renerre et al. (1992) stated that the accumulation of metmyoglobin depends on several contradictory mechanisms, such as the rate of oxygen diffusion and consumption, pigment autoxidation and enzymatic reduction of metmyoglobin. Many different factors could ultimately affect the action of these mechanisms on fresh meat color stability, breed and pre-slaughter handling most notably. Monin et al. (1987) described Hampshire sired pigs to possess higher muscle glycolytic potential and lower ultimate meat pH. The 1995 Terminal Sire Line National Genetic Evaluation Program (NPPC, 1995) reported that Hampshire sired pigs produced carcasses with a low ultimate pH, pale loin muscle, high
52
L. G. Tam et al.
drip loss and low water holding capacity. The Rendement Napole (RN) gene has been identified as the gene responsible for these poor quality characteristics (Monin et al., 1987). A high percentage of the pigs used in this study presented the typical phenotype of the Hampshire breed. The RN gene and its affect on post-mortem muscle metabolism are, as yet, not well understood. It is conceivable that the variation in myoglobin autoxidation and metmyoglobin accumulation could be a result of the RN mutation, where muscle from this genotype will possess a lower potential for myoglobin autoxidation and more rapid metmyoglobin accumulation.
ACKNOWLEDGEMENTS The authors would like to thank Pig Improvement Company, especially Andrej Sosnicki, for their support and identification of the genetic lines used in this study, which was supported in part by a grant from the National Pork Producers Council (DEG, JCF).
REFERENCES Antonini,
E. and Brunori, M. (1971) Frontiers of Biology. Vol. 21. Hemoglobin and Myoglobin in Their Reactions with Ligunds. North-Holland Publishing Company, Amsterdam. Antonini, E., Rossi-Bernardi, L. and Chiancone, (1981) Method in Enzymology. Vol. 76. Hemoglobins. Academic Press, New York. Bembers, M. and Satterlee, L. D. (1975) Physico-chemical characterization of normal and PSE (pale, soft and exudative) porcine muscle myoglobins. J. Food Sci. 40, 4&43. Bertelsen, G. and Skibsted, L. H. (1987) Photooxidation of oxymyoglobin-wavelength dependence of quantum yields in relation to light discoloration of meat. Meat Sci. 19, 243-251. Brown, W. D. and Mebine, L. B. (1969) Autoxidation of oxymyoglobins. J. Biol. Chem. 244,6696 6701. Cannon, J. E., Morgan, J. B., Heavner, J., McKeith, F. K., Smith, G. C. and Meeker, D. L. (1995) Pork quality audit: a review of the factors influencing pork quality. J. Muscle Foods 6, 369402. Fujii, J., Otsu, K., Zorzato, F., deleon, S., Khanna, V. K., Weiler, J. E., O’Brien, P. J. and MacLennan, D. H. (1991) Identification of a mutation in porcine ryanodine receptor associated with malignant hyperthermia. Science 253, 448451. Garrido, D., Perez, A. and Sanchez-Ferrer, A. (1994) Meat pigment determination by phase partitioning in Triton X-l 14 and oxidation with sodium nitrite. J. Sci. Food Agric. 64, 327329. George, P. and Stratmann, C. J. (1952) The oxidation of myoglobin to metmyoglobin by oxygen. 2. The relation between first-order rate constant and the partial pressure of oxygen. Biochem. J. 51, 418. Hamm, R. (1986) Properties of the myofibrillar system and their measurements. In Muscle as Food, ed. P. J. Bechtel. Academic Press, Orlando, FL. Harrison, G. G. (1979) Porcine malignant hyperthermia. Znt. Anesthesiol. Clin. 17, 25-61. Kauffman, R. G., Sybesma, W., Smulders, F. J. M, Eikelenboom, G., Engel, B., Laack, R. L. J. M. van, Hoving-Bolink, A. H., Sterrenberg, P., Nordheim, E. V., Walstra, P. and van der Wal, P. G. (1993) The effectiveness of examining early post-mortem musculature to predict ultimate pork quality. Meat Sci. 34, 283-300. Klont, R. E. and Lambooy, E. (1995) Effects of preslaughter muscle exercise on muscle metabolism and meat quality studied in anesthetized pigs of different halothane genotypes. J. Anim. Sci. 73, 108-l 17. Kocwin-Podsiadla, M., Przybylski, W., Kuryl, F., Talmant, A. and Monin, G. (1995) Muscle glycogen level and meat quality inpigs of diferent halothane genotypes. Meat Sci. 40, 121-125. Krzywicki, K. (1979) Assessment of relative content of myoglobin, oxymyoglobin and metmyoglobin at the surface of beef meat quality storage. Meat Sci. 3, l-10.
Effect
of halothane genotype on porcine meat quality and myoglobin autoxidation
53
Krzywicki, K. (1982) The determination of haem pigments in meat heme. Meat Sci. 7, 29-36. Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage. Nature 227, 68&685. Leach, L. M., Ellis, M., Sutton, D. S., McKeith, F. K. and Wilson, E. J. (1995). A within litter comparison of the growth performance, carcass characteristics, and meat quality of halothane carrier and negative pigs at three slaughter weights. Proceedings Vol. ZZ 41st Annual Znternational Congress of Meat Science and Technology, San Antonio, Texas. Louis, C. F., Rempel, W. E. and Mickelson, J. R. (1993) Porcine stress syndrome: Biochemical and genetic basis of this inherited syndrome of skeletal muscle. Proc. Recip. Meat Confr. 46, 89-99. MacLennan, D. H. and Phillips, M. S. (1992) Malignant hyperthermia. Science 256, 789-794. Monin, G., Mejenes-Quijano, A. and Talmant, A. (1987) Influence of breed and metabolic type on muscle glycolytic potential and meat pH in pigs. Meat Sci. 20, 149-158. National Pork Producers Council (1991) Procedures to Evaluate Market Hogs, 3rd edn. National Pork Producers Council, Des Moines, IA. National Pork Producers Council (1995) Terminal Sire Line National Genetic Evaluation Program. National Pork Producers Council, Des Moines, IA. Oliver, M. A., Gispert, M. and Diestre, A. (1993) The effects of breed and halothane sensitivity on pig meat quality. Meat Sci. 35, 105-l 18. Pommier, S. A. and Houde, A. (1993) Effect of genotype for malignant hyperthermia as determined by restriction endonuclease assay on the quality characteristics of commercial pork loins. J. Anim. Sci. 71, 42@425. Renerre, M., Anton, M. and Gatellier, P. (1992) Autoxidation of purified myoglobin from two bovine muscles. Meat Sci. 32, 331-342. Satterlee, L. D. and Zachariah, N. Y. (1972) Porcine and ovine myoglobin: isolation, purification, characterization and stability. J. Food Sci. 37, 909-912. Shikama, K. and Sugarwara, Y. (1978) Autoxidation of native oxymyoglobin. Kinetic analysis of the pH profile. Eur. .Z. Biochem. 91, 407413. Statistical Analysis System Institute Inc. (1985) SAS Users Guide to the Statistical Analysis System. NC State University, Raleigh, NC. van der Wal, P. G., Vries, A. G. and Eikelenboom, G. (1994) Predictive value of slaughterhouse measurements of ultimate pork quality in seven halothane negative Yorkshire populations. Meat Sci. 40, 183-191. Warriss, P. D. (1979) The extraction of haem pigments myoglobin and haemoglobin from fresh meat. J. Food TechnoI. 14, 75-80. Wykle, B., Gillett, T. A. and Addis, P. B. (1978) Myoglobin heterogeneity in pigs with PSE pale, soft andexudative and normal muscle by an improved isoelectric focusing technique. J. Anim. Sci. 47, 126&1264.
SO309-1740(97)00105-8
Meat Science, Vol. 49, No. I, 41-53, 1998 0 1998 Elsevier Science Ltd All rights reserved. Printed in Great Britain 0309-1740/98 $19.00+0.00
ELSEVIER
Effect of Halothane Genotype on Porcine Meat Quality and Myoglobin Autoxidationx L. G. Tarn,,, E. P. Berg,” D. E. Gerrard,b* E. B. Sheiss,” F. J. Taqd M. R. Okos’ & J. C. Forres@ aDepartment of Animal Science, Texas A&M University, College Station, TX 77843, USA bDepartment of Animal Sciences, Purdue University, West Lafayette, IN 47907, USA ‘Pig Improvement Company, Box 348, Franklin, KY 42135, USA dDepartment of Food Sciences, The Ohio State University, Columbus, OH 43210, USA PDepartment of Agricultural and Biological Engineering, Purdue University, West Lafayette, IN 47907, USA (Received
26 June 1997; revised version
received
3 September
1997; accepted
3 September
1997)
ABSTRACT The objective of this study was to determine effects of light (40-80 kg) or heavy (IOO130 kg) slaughter weight and halothane status (positive, nn; negative, NN; and heterozygous, Nn) on meat quality. Longissimus muscle (LM) pH at 45 min (pH4s) post-exsanguination was 6.25, 6,03, and 5.84 (different at p < 0.01) for NN, Nn, and nn genotype, respectively. At heavier weights (100-130 kg), genotype correlated (r = -0.71) with LM pH,,, 10th costae LM (TENLM) color score (r = -0.55)) TENLM Hunter L’value (r = 0.47), water holding capacity (r = 0.42) and TENLM subjectivejirmness-wetness score (r = 0.51). Rate constants for metmyoglobin accumulation and oxymyoglobin autoxidation, indicators for fresh meat color stability, increased ( p < 0.05) with decreasing pH. Color stability for NN muscle was more stable than nn muscle ( p ( 0.05). Electrofocusing of myoblobin revealed two bands (MW 17,1@) at pl 6.1 and 6.5 across genotypes. Because d$erences were not observed across genotypes, an observed increase (~~0.05) in 24 hr myoglobin autoxidation rate constant (associated with increased expression of the HAL gene) are presumed dependent upon post-mortem muscle changes. These data show that changes in halothane status affect fresh pork quality and that lowered meat quality results in further color destruction due to altered chemicaf reactions involving myoglobin oxidation. 0 1998 Elsevier Science Ltd. All rights reserved
INTRODUCTION Pigs
leaner
that are homozygous and possess greater
positive pigs (nn) for the halothane gene (HAL) are generally muscle than pigs lacking the gene (AW). However, these pigs
*To whom correspondence should be addressed. +Present address: l/181 Barkers Road, Kew 3101, Victoria, Australia. *Purdue University Agricultural Research Programs Journal Paper No. 15403.
41
L. G. Tam et al.
42
often yield carcasses exhibiting pale, soft and exudative (PSE) meat (MacLennan and Phillips, 1992; Louis, et al., 1993). The increase in lean yield provided by HAL status is negated by an increased pig mortality. Pigs that survive to market not only generate pork of low meat quality, but produce cuts with a shorter retail shelf-life due to reduced color stability. Breeding companies have developed hybrid lines that use heterozygous HAL (Nn) genotypes in an effort to prevent severe PSE associated with nn pigs while maintaining the desirable muscling attributes (Cannon et al., 1995). Although mortality rate is lowered in Nn pigs, conflicting results exist as to the quality of the meat produced from such animals. In addition, the severity of the PSE condition may be reduced if animals are delivered to market at a lower weight. The goal of this study, therefore, was to investigate the effect of the HAL gene and slaughter weight on porcine meat quality and color stability.
MATERIALS
AND
METHODS
Animals and slaughter data collection Eighty-four pigs (l&14 days of age) were secured from a commercial breeding company, DNA tested for the halothane gene (Fujii et al., 1991), and assigned a HAL status (NiV, Nn, nn). Five NN, 5 Nn and 4 nn pigs were allocated to six slaughter groups (40, 60, 80, 100, 115, 130 kg) and slaughtered according to normal (United States) industry procedures. Longissimus (LM; adjacent the last costae) and deep Semimembranosus (SM) muscle pH was measured using a portable pH’K21 probe (NWK Thien GmbH, West Germany) at exsanguination (pHu) and 45 min (pHa5) post mortem. Longissimus muscle was collected at the last costae immediately following exsanguination, frozen in liquid nitrogen, and stored at -40°C for use in myoglobin isoelectric focusing (IEF) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Dressed carcasses (head, peritoneal fat, and kidneys removed; feet left on) were weighed and sent to the chiller (4°C). Sample collection
at 24 hr post mortem
The LM was exposed at the 10th and 1 lth rib interface (TENLM) following a 24 hr chill (4°C) and subjectively evaluated by an experienced six member panel using National Pork Producers Council (NPPC, 1991) standards for fresh pork color, marbling, and firmnesswetness. Following subjective quality assessment, ultimate LM and SM muscle pH values were recorded using the pH*K21 probe and a three rib (8-10th rib) section of LM was removed for evaluation of objective quality parameters. Objective color measurements were obtained from 2.54 cm cross-sectional slices of LM (9th rib) and SM at two separate locations on the muscle surface using a HunterLab 45”joO D25-PC2A Colorimeter (Hunter Associates Laboratory Inc., Reston, VA). Water holding capacity was determined from triplicate 0.3 g samples of TENLM and SM by the filter paper press method (Hamm, 1986). Drip loss was estimated from 24 hr moisture loss of (duplicate) 2g TENLM core samples suspended from a hook in sealed centrifuge tubes at 4°C. In addition, LM and SM samples were collected, frozen in liquid nitrogen, and stored at -40°C for future analysis of 24 hr myoglobin autoxidation. Determination
of total meat pigment
Total pigment concentration (ST; light and dark portions)
was determined on 24 hr LM (10th rib) and Semitendinosus samples using the method of Garrido et al. (1994). Samples
Effect of halothane genotype on porcine meat quality and myoglobin autoxidation
43
were homogenised at 4°C in 40 mM potassium phosphate buffer, pH 6.5, and centrifuged at 1000 x g for 10 min at 4°C. Supernatants were removed and centrifuged at 10 000 x g for 20 min at 4°C. Filtered supernates were mixed with TX-l 14 (100 g 1-l) at 4°C heated at 37°C for 10 min, and centrifuged at 7500 x g for 5 min at 25°C. Sodium nitrite solution (65mM) was added to the upper pigment-rich phase. Absorbance was measured at 409hO.5 nm after 15 min using a Beckman DU6 UV-Visible spectrophotometer (Beckman Instruments, Inc., Irvine, California). Pigment amount was calculated as follows: Pigment MW=
(mgg-‘)=
((A,,,
17 142;dilutionfactor=83.51
Determination
of oxymyoglobin
x MW myoglobin mlg-‘;
x 1000)/&409) x dilution
molar absorbancecoefficient
factor
=79’6mM-‘cm-’
autoxidation rate constants
The method of Krzywicki (1982) was used to determine the relative concentration of myoglobin, oxymyoglobin, and metmyoglobin. Frozen TENLM, SM, and ST samples were powdered in liquid nitrogen for extraction at 4°C in 40mM potassium phosphate buffer, pH 5 and 6 (Warriss, 1979). Samples were diluted in buffer (1: 12.5), homogenised at 4°C and centrifuged at 1000 x g for 10 min at 4°C. Supernatants were removed, centrifuged, and filtered to minimise absorbance at 730 f 0.05 nm and remove lipid. The pH of each solution was recorded and samples were held for 10 hr at 25°C. Duplicate absorbance readings were made every hr at 572, 565, 545, 525 f 0.05 nm using a Beckman DU6 spectrophotometer. Absorbance at 730 * 0.05 nm was recorded as zero (Krzywicki, 1979). Rate constants for metmyoglobin accumulation and oxymyoglobin disappearance (autooxidation) were obtained by plotting In (C&J against time where C, was the fractional concentration of the myoglobin derivative at time t and C, was the initial fractional concentration of myoglobin derivative. The slope of the line of best fit was equal to the rate constant (k hr-‘). Isoelectric focusing Isoelectric focusing of myoglobin was carried out in PAGE tube gels (3 mm ODx 14 cm) using a modified method of hemoglobin separation developed by Antonini et al. (198 1). The gel mixture consisted of 23.7% (v/v) acrylamide (20%T, 2’7%Cbi,), 4.5% (v/v) ampholine (PI 5-8; Pharmacia LKB Biotechnology AB), 0.07% (v/v) TEMED, 0.49% (v/v) ammonium persulfate (20%), and 1.5% (v/v) Nonidet-10 (10% v/v). The cathode electrolyte was 0.01 M sodium hydroxide and the anode electrolyte was 0.01 M phosphoric acid. Tubes were cooled to 4°C. Gels were pre-run at 200 V for 15 min and 400 V for 30min on a vertical gel electrophoresis system (Model V-16-2, Bethesda Research Laboratories). Frozen meat samples were pulverized into a powder, diluted (1: 1.5) in potassium buffer pH 6.8) homogenised at 4°C. Samples were then packed in ice, gently agitated for 2 hr, centrifuged at 40 000 g for 1 hr at 4°C and passed through Whatman No. 1 filter paper to remove fat. Samples were mixed with glycerol (1:l) and stored at -20°C for no longer than five days. Ten ~1 of prepared sample was mixed with 15 ~1 sample buffer [lo% (w/v) glycerol; 6.75% (v/v) ampholine p1 5-81. Tube gels were loaded with 20 ~1 of sample and p1 standards of 5.9 to 7.2 (Sigma Chemical) and focused for 7.5 hr at a potential of 400 V. Tube gels were fixed for 1.5 hr with 3.5% (w/v) sulfosalicylic acid, 35% (v/v) methanol, and 13% (w/v) trichloroacetic acid. Proteins were stained for 30 min with Coomassie Blue and destained overnight. Tube gel duplicates were soaked in equilibration buffer pH 6.8 for
44
L. G. Tam et al.
30min, quick frozen in equilibration -70°C for future SDS-PAGE.
buffer
using
dry ice and ethanol,
and
stored
at
SDS-PAGE Tube gels from IEF were subjected to a 12% separating SDS-PAGE (3 mmx 15.2 cmx 16 cm). The gel mixture consisted of 40% (v/v) acrylamide (30%T, 2_7%Cbi,); 0.1% (W/V) SDS, 375 mM Tris-HCl pH 8.8; 0.05% (V/V) TEMED; 0.5% (V/V) ammonium persulfate (10%) (Laemmli, 1970). Tube gels were attached to the top of the slab separating gel with 1% (w/v) agarose and separating gel solution. Top and bottom electrophoresis reservoirs were filled with glycine buffer (25 mM Tris, 192 mM glycine, 0.1% (w/v) SDS). Molecular weight standards (MW = 14 00&70 000; Sigma Chemical) were embedded in agarose solution of the same diameter as the IEF tube gels and a 1 cm portion was attached to the top of the separating gel. Gels were run for 19 hr at 4°C at a constant current of 28 mA. After electrophoresis, slab gels were fixed and stained for 30 min with Coomassie Blue and destained overnight. Statistical analysis Statistical analysis was performed using the SAS general linear model procedure (SAS, 1985). A two factor (genotype, weight) fixed effect model with interaction was assumed for the analysis of meat quality and myoglobin autoxidation data. The statistical model was described as “ijk
=
/J- +
(ri)
+
(Bj) + (tb)q +
&ijk
where p is the overall mean effect, ti is the effect of the ith level of genotype, Bj is the effect of the jth level of weight and (tp)ij is the interaction between ti and pj and &ijk is the random error component.
RESULTS
AND
DISCUSSION
Meat quality No significant quality differences were seen between 40, 60, or 80 kg pigs. Therefore, slaughter groups were pooled into two groups for statistical analysis; a lightweight (L-WT) group slaughtered at 40, 60, and 80 kg and a heavyweight (H-WT) group slaughtered at 100, 115 and 130. Color Hunter L’ a* b* values provide an objective means for comparison of meat color variation. Longissimus muscle (10th rib) subjective color scores from pigs possessing the stress gene (Nn and nn) were lower (p < 0.05) than the NN genotype, however, LM Hunter L* values of Nn muscle were not different from NN muscles (Table 1). Higher (p < 0.05) L’ values for H-WT nn pigs were observed supporting the subjective color scoring methodology. No differences in color scores were observed across genotypes of the L-WT group (Table 3). The SM of the Nn genotype was significantly darker (~~0.05) than NN (Table 1) and may be a reflection of the difficulty of assessing SM color due to inherent color variations within this muscle (Pommier and Houde, 1993; Klont and Lambooy, 1995).
EfSect of halothane genotype on porcine meat quality and myoglobin autoxidation
Meat Quality
TABLE 1 Characteristics (least square means and standard errors) for Pigs Differing thane Status (positive, nn; negative, NN, and Heterozygous, Nn) NN*
Characteristic
Semimembranosus
in Halo-
nn**
2.70(0.07) 2.92(0.08) 1.75(0.09) 4.54(0.30)
2,40(0.07)b 2.93(0.08) 1?32(0.09)” 3.1 l(0.30)b
L*-value a*-value b*-value
49.27(0.46)6 11+X5(0.22) 14.08(0.1 7)6
49. 16(0.46)b 12.10(0.22) 14.85(0.17)b
5 1.40(0.52)” 12.22(0.25) 15.43(0.19)
L*-value a*-value b*-value
47.90(0.62)” 12.91(0.30) 14.59(0.22)
45.55(0.62)b 13.86(0.30) 14.67(0.22)
47.37(0.69yb 12.77(0.34) 14.25(0.25)
1.33(0.04) 4.29(0.16)b 1’ 13(0.05)
1.31(0.04) 4.80(0.16) 1.44(0.05)
1.36(0.04) 5.04(0.18) 1.36(0.06)
6.63(0.03) 6.25(0.03) 5.51(0.02)
6.60(0.03) 6.03(0.03)b 5.51(0.02)
6.55(0.04) 5.84(0.03) 5.45(0.04)
6.51(0.04) 6.12(0.03) 5.44(0.02)b
6.52(0.04) 6.02(0.03)b 5.54(0.02)
6.45(0.04) 5g6(0.03)’ 5.48(0.02)b
Colord Firmness-wetnessd Marblingd Drip loss (%) Longissimus muscle
2.25(0.08)b 2.40(0.08)b 1.48(0. 10)b 5.06(0.33)
muscle
Total pigment (mg gg’ fresh muscle) Longissimus muscle Semitendinosus muscle Longissimus
45
Dark Light
muscle PHO’ ~H45
PH, Semimembranosus
muscle PHO ~H45
PH,
*n = 30. **n = 24. a,b,cMeans within a row with different superscripts differ (p < 0.05). dNPPC quality standards: 1= least/lightest and 5 = greatest/darkest. “pHo = pH at exsanguination; pH 45 = pH 45 min post-exsanguination; 24 hr chill.
pH, = ultimate
pH after a
A significant increase (p < 0.05) in LM Hunter b* value was observed for nn LM compared with NN LM for both weight groups (Tables 2 and 3), suggesting a shift from blue to yellow along the yellow to blue continuum of color. An increase (~~0.05) in LM Hunter b* value was observed for the nn genotype compared to NN and Nn genotypes (Table 1). Pommier and Houde (1993) reported greater b’ values for 3212pigs, while Klont and Lambooy (1995) reported no difference between genetic lines. Marbling andjrmnesslwetness Heavyweight pigs representing the NN genotype possessed a higher firmness-wetness and marbling score (p< O-OS) than NN L-WT pigs indicating that full expression of PSE muscle may not be manifested until heavier live weights are achieved. Less marbling (p < 0.05) was observed in the LM of nn pigs compared to NN and Nn genotypes (Table l), however, L-WT marbling scores (Table 3) did not differ (~~0.05). The average meat firmness and exudativeness of LM was lower (p > 0.05) for nn pigs compared to NN and Nn pigs for all weight endpoints (Table 1) as well as for L- and H-WT (Tables 2 and 3).
L. G. Tam et al.
46
TABLE 2
Meat Quality Characteristics (least square means and standard errors) for Heavyweight Pigs (lo&l30 kg) Differing in Halothane Status (positive, nn; negative, NN; and heterozygous, Nn) Characteristic
NN*
Colold Firmness-wetnes& Marblingd Drip loss (%) Longissimus muscle
2+33(0.09)” 3.03(0.08) 2.17(0.12) 5.83(0.44)”
-
Nn* 2.47(0.09)b 2.90(0.08) 1.83(0.12)” 3.92(044)b
-
nn** 2.25(0.10)b 2.33(0.09)b 1.46(0. 14)b 5.94(0.52)
L*-value a*-value b*-value
49.44(0.67)b 12.81(0.36) 14.70(0.26)b
49.63(0.67)b 13.03(0.36) 15.51(0.25)“b
52.68(0.75) 12.60(0.41) 15.70(0.29)
L*-value a*-value b*-value
5 1.38(0.94) 12.63(0.38) 14.88(0.33)
48.97(0.94) 14.18(0.38) 15.53(0.33)
49.56( 1.05) 13.25(0.42) 14.93(0.36)
Semimembranosus muscle
Total pigment (mg gg’ fresh muscle) Longissimus muscle Semitendinosus muscle
Dark Light
Water holding capacity Longissimus muscle PHO’ ~H45
PH”
Semitendinosus muscle
PHO ~H45
PH,
-
1.41(0.07)
2.21(0.06)b
5.94(0.30) 1.47(0.10) 2.38(0.06)
6.65(0.04) 6.22(0.03) 5.47(0.04)
6.63(0.05) 6.04(0.03)b 5.51(0.04)
6.54(0.05) 5.89(0.03) 5.42(0.04)
6.48(0.06) 6.06(0.04) 5.40(0.03)b
6.52(0.06) 6.00(0.04) 5.52(0.03)
6.42(0.07) 5.80(0.05)b 5.43(0.03)“b
*n= 15. **n= 12. a,b,cMeans within a row with different superscripts differ (p < 0.05). dNPPC quality standards: 1 = least/lightest and 5 = greatest/darkest. epHo = pH at exsanguination; pH45 = pH 45 min post-exsanguination;
Drip loss and water-holding
1.31(0.06) 5.32(0.27yb 1.60(0.09)
1.40(0.06) 4.89(0.27)b 1.46(0.09) 2. 14(0.06)b
pH, = ultimate pH after a 24 hr chill.
capacity
Drip loss for nn muscle was > 5%, placing the average muscle from this genotype between exudative and non exudative according to Kauffman et al. (1993). No differences were observed for drip loss between nn and NN LM, however, drip loss was lower (p < 0.05) for Nn loins compared to NN and Nn loins (Table 1). This trend was consistent across genotypes for both heavy and light weight groups (Tables 2 and 3). The small meat sample size used for drip loss may have produced increased free water loss during sampling and may explain differences between genotypes. Water holding capacity (WHC) was determined by the filter paper press method of Hamm (1986) where a* value > 2.4 indicated poor WHC. The WHC for all three genotypes was considered acceptable yet the Longissimus muscle WHC score for H-WT nn pigs was higher (p < 0.05) than NN and Nn genotypes (Table 2). Meat pigment Myoglobin content observed in muscle
of meat is responsible for color. pigment levels among all treatment
No differences (p > 0.05) were (Table 2). Klont and Lambooy
47
Effect of halothane genotype on porcine meat quality and myoglobin autoxidation
Meat Quality Characteristics (least (4&80 kg) Differing in Halothane
TABLE 3 square means and standard errors) for Lightweight Pigs Status (positive, nn; negative, NN; and heterozygous, Nn) NN*
Characteristic
2.57(0.11) 2.80(0.12)“b 1.33(0.14) 3.24(0.37yb
Colold Firmness-wetnes& Marblingd Drip loss (%) Longissimus muscle
Semimembranosus
nn** 2.25(0.12) 2.46(0. 14)b 1.50(0.15) 4.18(0.41)”
2.33(0.11) 2.97(0.12) 1.80(0.14) 2.30(0.37)b
L*-value a*-value b*-value
49.10(0.64) 10.96(0.26) 13.48(0.21)b
48.68(0.64) 11.18 (0.26) 14.21(0.21)b
50.11(0.71) 11.84(0.29) 15.14(0.24)
L*-value a*-value b*-value
44.30(0.80)“b 13.22(0.47) 14.31(0.30)
42.03(0.80)b 13.54(0.47) 13.76(0.30)
45.16(0.89) 12.27(0.53) 13.55(0.34)
1.27(0.05) 3.69(0.19) 1’ 15(0.04)
1.31(0.05) 4.28(0.19) 1.28(0.04)
1.30(0.05) 4.25(0.21) 1.25(0.05)
6.60(0.05) 6.27(0.04)” 555(0.03)
6.59(0.05) 6.02(0.04)b 5.51(0.03)
6.55(0.05) 5.79(0.05)’ 5.47(0.03)
S.SS(O.05) 6.18(0.04) 5.48(0.03)
6.51(0.05) 6.05(0.04) 5.57(0.03)
6.48(0.06) 5.92(0.05)b 5.52(0.03)
muscle
Total pigment (mg g-t fresh muscle) Longissimus muscle Semitendinosus muscle Longissimus
Nn*
Dark Light
muscle PHO’ ~H45
PH” Semitendinosus
muscle PHO ~H45
PH,
*n= 15. **n = 12. “,b,cMeans within a row with different superscripts differ (p > 0.05). “NPPC quality standards: 1= least/lightest and 5 = greatest/darkest. ‘pHo = pH at exsanguination; pH45 = pH 45 min post-exsanguination; 24 hr chill.
pH, = ultimate
pH after
a
(1995) reported a significant increase in pigment concentration from NN to nn pigs. Pigment concentration found in this study were higher than Klont and Lambooy (1995) but similar to Garrido et al. (1994). At a low pH, the heme group of myoglobin becomes labile and will dissociate into heme and globin (Antonini and Brunori, 1971) providing less native myoglobin to bind oxygen, generating a more pale appearance. Low pH meat appeared paler, as supported by increased Hunter L’ values. Rapid post-mortem pH decline at an elevated carcass temperature contributes to development of PSE meat. The pH recorded at 45 min post exsanguination (pH4s) is often lower in pigs expressing the HAL genotype (Oliver et al., 1993; Klont and Lambooy, 1995; Kocwin-Podsiadla et al., 1995; Leach et al., 1995). A lower (~~0.05) pH4s was observed for the Nn and nn genotypes compared to NN (Table 1). Strong negative correlations between pH4s and halothane genotype were found. With increased halothane susceptibility, pH4s was lower (Table 5). Muscles with a higher pH4s tended to have darker, less exudative meat. No genotype differences were observed for initial (pHo) or ultimate PH (PH”).
muscle
muscle
PH”
~H45
PHO
PH”
~H45
PHO’
Dark Light
L*-value a*-value b*-value
L*-value a*-value b*-value
6.50(0.06) 6.07(0.04) 5.48(0.03)b
6.54(0.05) 6.00(0.04) 5.48(0.03)“b
1.31(0.06) 3.61(0.24) 1.20(0.07)
42.04(0.91)d 10.85(0.45)b 11.78(0.33)b
47. 17(0.68)b 10.77(0.33)d 13.1 9(0.25)b
2.34(0.10) 2.54(0.1 l)bc 1.46(0.14)“b 1.27(0.44)’
40 kg
80 kg
6.55(0.06) 5.96(0.04) 5.45(0.03)b
6.62(0.05) 6.09(0.04) 4.48(0.03yb
1.34(0.06) 4.85(0.24)b 1.34(0.07)6
49.87(0.91yb 13.25(0.45) 15.68(0,33)
50.42(0.68) 11.99(0.33)b’ 14.58(0.25)
pH after a 24 hr chill.
6.56(0.06) 6.08(0.04) 5.59(0.03)
6.66(0.05) 6.1 l(O.04) 5.57(0.03)
1.28(0.06) 4.62(0.24)bC 1.26(0.07)b
44.13(0.9l)“d 14.75(0.45) 14.81(0.33)
50.42(0.68) 1 l.87(0.33)bc 15.01(0.25)
2.53(0.10) 3.03(0.11) 1.93(0.14)“b 4.94(044)
100 kg ll5kg
6.47(0.06) 5.96(0.04) 5.48(0.03)b
6.59(0.05) 6.04(0.04) 5.50(0.03)“b
1.28(0.06) 5.1 1(0.24)b 1.49(0.07)“b
48.23(0.91)b 13.91(0.45) 14,47(0,33)
50.28(0.68) 12.82(0.33yb 15.40(0.25)
2.53(0.10) 2.88(0.1 l)ab 1.77(0.14)” 5.51(0.44)
errors) for Pigs 4&l 30 kg
2.53(0.10) 2.88(0.1 l)ab 1.82(0. 14)ub 3.85(0.44)
pH, = ultimate
6.48(0.06) 6.00(0.04) 5.50(0.03)b
6.53(0.05) 5.98(0.04) 5.48(0.03yb
1.29(0.06) 3.97(0.24)‘d 1.22(0.07)b
45.31(0.91) 13.37(0.45) 15.05(0.33)
50.27(0.68) 11.33(0.33)‘d 14.60(0.25)
2.28(0.10) 2.80(0.1 lyb 1.36(0.14)b 4.60(0.44)
60 kg
TABLE 4 (least square means and standard
ObcMeans within a row with different superscripts differ (p < 0.05). dNpPC quality standards: 1= least/lightest and 5 = greatest/darkest. ‘pHo = pH at exsanguination; pH4, = pH 45 m’in post-exsanguination;
Semitendinosus
Longissimus muscle
Total pigment (mg gg’ fresh muscle) Longissimus muscle Semitendinosus muscle
Semimembranosus
Colold Firmness-wetnes& Marblingd Drip loss (X) Longissimus muscle
Characteristic
Meat Quality Characteristics
6.47(0.06) 5.95(0.04) 5.41(0.03)b
6.61(0.05) 6.02(0.04) 5.41(0.03)b
1.50(0.06) 6.18(0.24)” 1.70(0.07)
51.82(0.91) 12.90(0.45)” 15.19(0.33)”
5 1.08(0.68) 13.53(0.33) 15.95(0.25)
2.49(0.10) 2.37(0.11) 1.77(0.14) 5.25(0.44yb
130 kg
;;I 3 4 g
p a
49
E#ect of halothane genotype on porcine meat quality and myoglobin autoxidation
Pearson’s
Correlation
Coefficients 2
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
TABLE 5 of Variables Affecting 4&l 30 kg 4
3
5
the Color of Longissimus 7
6
8
0.035 -0.419 0.280 0.128 0.355 -0.176 0.309 -0.343 0.318 0.018 -0.024 -0.402 0.152 -0.259 0.181 0.003 0.543 0.015 0.689 0.038 0.045
Genotype Pigment Color” L*-value a*-value b*-value pHob
Correlation
Coefficients 2
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
-0.738 0.090 0.504 -0.363 -0.037 -0.322 0.187
PH, %Drip loss Firmness-wetness0
Pearson’s
3
TABLE 6 of Variables Affecting the Color Heavyweight Pigs 100-130 kg 4
0.006- -0.550 0.471 0.289-0.434 -0442
Genotype Pigment Color” L*-value a*-value b*-value pHob ~H45
PH,
%Drip loss Firmness-wetness” WHC’
5
6
7
8
Autoxidation order kinetic
autoxidation
-0.186 0.070 -0.258 0.179 0.197 -0.005 -0.091 0.218 0.009 0.337 -0.068 0.334 0.013 -0.00 0.186 -0.204 -0.245
pH, = ultimate
of Longissimus 9
10
II -0.364 -0.212 0.441 -0.268 -0.061 -0.273 0.152 0.467 0.233 -0.166 1.000
pH after a
Muscle II
from 12
0.034 0.350-0~257-0~711-0.126-0~010-0~508 0.417 0.374 0.064-0.049 0.176-0.262 0.272-0.294-0.034 0.023-0.271 0.258 0.592-0.007-0.114 0.448-0.453 -0.301 0.227-0.151-0.487 0.01 l-0.142-0.323 0.392 0.756 0.069-0.027 0.107 0.078-0.312-0.139 -0.013-0.317 0~111-0~030-0~419 0.117 0.239-0.180-0.039 0.117-0.030 0.015 0.091 0.637-0.373 -0.296 0.226-0.010 -0.278 0.007 -0.277 1JJOO
“NPPC quality standards: 1 = least/lightest and 5 = greatest/darkest. bpHo = pH at exsanguination; pH 45 = pH 45 min post-exsanguination; 24 hr chill. ‘WHC = water holding capacity; filter paper press method.
Oxymyoglobin
10
9
pH45
“NPPC quality standards: 1 = least/lightest and 5 = greatest/darkest. bpH, = pH at exsanguination; pH 45 = pH 45 min post-exsanguination; 24 hr chill.
Muscle from Pigs
and metmyoglobin
accumulation
pH, = ultimate
pH after a
rate constants
of oxymyoglobin accompanied by the formation of metmyoglobin is a first reaction, d[Mb02]/dt = -k,,[MbOz] (George and Stratmann, 1952; Brown
and Mebine, 1969; Shikama and Sugawara, 1978; Bertelsen and Skibsted, 1987; Renerre and Gatellier, 1992). Rate constants increased (p < 0.05) for metmyoglobin accumulation and oxymyoglobin autoxidation when pH 5 buffer was used for myoglobin extraction because, at this pH, myoglobin is more susceptible to unfolding and cleavage. Rate constants (least square means and standard errors) for metmyoglobin accumulation were
50
L. G. Tam et al.
Pearson’s
Correlation
2 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
Genotype Pigment Color” L*-value a*-value b*-value pHob
TABLE 7 of Variables Affecting the Color Lightweight Pigs 4W30 kg
Coefficients 3
4
5
6
7
0.075 -0.300 0.130 0.299 0.404 -0.101 0.294 -0.029 0.009 -0.198 -0.029 -0.461 0.167 -0.405 0.088 0.138 0.799 0.134 0.502 -0.094 0.046
of Longissimus
8
9
-0.773 0.009 0.453 -0.304 -0.094 -0.384 0.151
-0.281 -0.198 0.555 -0.164 0.071 -0.196 0.287 0.391
PH~S
PH, %Drip loss Firmness-wetnessa
ONPPC quality standards: 1 = least/lightest and 5 = greatest/darkest. bpHo = pH at exsanguination; pH4s = pH 45 min post-exsanguination; 24 hr chill.
pH,
Muscle
from
10
11
0.160 0.225 -0.068 -0.132 -0.067 0.439 0.411 -0.236 0.254 0.210 0.449 -0.184 -0.053 0.182 -0.133 0.353 -0.088 0.266 -0.126 1.000
= ultimate
pH after a
0.0926 (0.0021) hr-’ and 0.0456 (o-0021) hr-’ when pH 5 and 6 buffer were used for myoglobin extraction, respectively. Rate constants for oxymyoglobin autoxidation were -0.0666 (0.0013) hr-’ and -0.0456 (o-0013) hr-’ when pH 5 and 6 were used for myoglobin extraction, respectively (Table 8). These results are similar to those reported by Bembers and Satterlee (1975) for a crude extract of porcine myoglobin from LM (0.025 hr-’ at pH 5.8-6.0 and 30°C). Autoxidation of oxymyoglobin theoretically utilizes one mole of oxymyoglobin to form one mole of metmyoglobin. When pH 5 buffer was used for myoglobin extraction the rate constant for autoxidation of oxymyoglobin was 1.39 times less than for metmyoglobin accumulation. The large increase in rate constant between pH 6 and 5 was confirmed by Shikama and Sugawara (1978). Rate constants calculated in this study were similar in magnitude to those reported by Shikama and Sugawara (1978). Differences in rate constants between genotypes reflect oxymyoglobin stability. Rate constants calculated for metmyoglobin accumulation (Table 8) of nn meat, 0.0797 (0.0029) hr-’ were larger (p < 0.05) than rate constants calculated for NIV, 0.0639 (0.0023)
Rate Constants
TABLE 8 (least square means and standard errors) for Metmyoglobin Oxymyoglobin Autoxidation for Pigs of Three Genotypes
Accumulation
and
Rate constant
Metmyoglobin
Oxymyoglobin
Genotype
Average
NN Nn nn
0.0639(0.0023) 0.0667(0.0024) 0.0797(0.0029)
accumulation
autoxidation NN Nn nn
-0.0612(0.0015) -0.0565(0.0015) -0.0500(0.0019)
Extracted
pHS
0.0926(0.0021) 0.0837(0.0033) 0.0902(0.0033) 0.1 lOO(O.0042) -0~0666(0~0013) -0.0697(0.0021) -0.0679(0.0021) -0.0621(0.0026)
Extracted
pH6
0.0456(0.0021) 0.0441(0.0033) 0.0433(0.0033) 0~0495(0~0041) -0.0456(0.0013) -0.0538(0.0020) -0.0451(0.0021) -0.0379(0.0026)
Effect of halothane genotype on porcine meat quality and myoglobin autoxidation
51
hr-‘, and Nn meat, 0.0667 (0.0024) hr-‘. In contrast the rate constant (Table 8) for autoxidation of oxymyoglobin for nn pigs [-0.0500 (0.0019] hr-‘) was smaller in magnitude (~~0.05) than rate constants for NN [-O-0612 (0.0015) hr-‘1 and Nn genotype [-O-O565 (0.0015) hrr ‘1. A comparison of rate constants for metmyoglobin accumulation and oxymyoglobin autoxidation of nn genotype showed the rate constant for metmyoglobin accumulation was 1.59 times larger than the rate constant reported for oxymyoglobin autoxidation. This result was in contrast to NN and Nn metmyoglobin accumulation and oxymyoglobin autoxidation rate constants where the rate constants for metmyoglobin accumulation were 1.04 and 1-18 times larger than oxymyoglobin autoxidation rate constants, respectively. Rate constants for metmyoglobin accumulation when pH 5 and 6 buffers were used for myoglobin extraction are reported in Table 8. Electrofocusing
of porcine muscle extract
Porcine muscle extract was successfully electrofocused in two dimensions to test for differences in porcine myoglobin banding patterns across halothane genotypes. Longissimus muscle from two pigs of each genotype were selected for electrofocusing. Two bands were identified on SDS-PAGE to be of an approximate molecular weight of 17 000 (Satterlee and Zachariah, 1972). The isoelectric point of the two bands identified in this study of approximate molecular weight 17 000 were calculated to be 6.1 for the major band, and 6.5 for the faint band. Identical banding patterns were maintained across genotypes similar to Wykle et al. (1978) who reported no differences between banding patterns of porcine heart and skeletal muscle myoglobin extracted from halothane positive, negative or seven cross bred groups of pigs. However, Wykle et al. (1978) identified eight bands of MW 17 000 that could be a result of their different staining methodology or myoglobin denaturation during electrophoreses conducted at room temperature. Sample preparation and electrofocusing were conducted in this study at temperatures no greater than 4°C to minimize protein denaturation and oxidation. Isoelectric points were similar to Bembers and Satterlee (1975) for purified myoglobin from normal muscle. The faint band identified in this study at p1 = 6.5 corresponded to that of metmyoglobin identified by Bembers and Satterlee (1975). The major band identified in this study at pI=6.1 was similar to the upper range reported by Bembers and Satterlee (1975) for oxymyoglobin from PSE muscle (PI= 6.09). No differences in banding patterns were observed for porcine skeletal muscle myoglobin extracted from pigs of different halothane sensitivity. Given no differences in myoglobin primary structure across genotypes, the observed increase (~(0.05) in myoglobin autoxidation rate constant with increased gene expression reported in this study was dependent upon post-mortem changes in the muscle and their effect on myoglobin conformation. Results of this study show that the average rate constant for metmyoglobin formation is 1.59 times higher than myoglobin autoxidation in LM from the nn. The affect of low pH has long been attributed to myoglobin denaturation and pale color of PSE pork. Low pH has been shown to reduce the stability constant for the heme-globin linkage and increase the autoxidation rate (Renerre et al., 1992). Renerre et al. (1992) stated that the accumulation of metmyoglobin depends on several contradictory mechanisms, such as the rate of oxygen diffusion and consumption, pigment autoxidation and enzymatic reduction of metmyoglobin. Many different factors could ultimately affect the action of these mechanisms on fresh meat color stability, breed and pre-slaughter handling most notably. Monin et al. (1987) described Hampshire sired pigs to possess higher muscle glycolytic potential and lower ultimate meat pH. The 1995 Terminal Sire Line National Genetic Evaluation Program (NPPC, 1995) reported that Hampshire sired pigs produced carcasses with a low ultimate pH, pale loin muscle, high
52
L. G. Tam et al.
drip loss and low water holding capacity. The Rendement Napole (RN) gene has been identified as the gene responsible for these poor quality characteristics (Monin et al., 1987). A high percentage of the pigs used in this study presented the typical phenotype of the Hampshire breed. The RN gene and its affect on post-mortem muscle metabolism are, as yet, not well understood. It is conceivable that the variation in myoglobin autoxidation and metmyoglobin accumulation could be a result of the RN mutation, where muscle from this genotype will possess a lower potential for myoglobin autoxidation and more rapid metmyoglobin accumulation.
ACKNOWLEDGEMENTS The authors would like to thank Pig Improvement Company, especially Andrej Sosnicki, for their support and identification of the genetic lines used in this study, which was supported in part by a grant from the National Pork Producers Council (DEG, JCF).
REFERENCES Antonini,
E. and Brunori, M. (1971) Frontiers of Biology. Vol. 21. Hemoglobin and Myoglobin in Their Reactions with Ligunds. North-Holland Publishing Company, Amsterdam. Antonini, E., Rossi-Bernardi, L. and Chiancone, (1981) Method in Enzymology. Vol. 76. Hemoglobins. Academic Press, New York. Bembers, M. and Satterlee, L. D. (1975) Physico-chemical characterization of normal and PSE (pale, soft and exudative) porcine muscle myoglobins. J. Food Sci. 40, 4&43. Bertelsen, G. and Skibsted, L. H. (1987) Photooxidation of oxymyoglobin-wavelength dependence of quantum yields in relation to light discoloration of meat. Meat Sci. 19, 243-251. Brown, W. D. and Mebine, L. B. (1969) Autoxidation of oxymyoglobins. J. Biol. Chem. 244,6696 6701. Cannon, J. E., Morgan, J. B., Heavner, J., McKeith, F. K., Smith, G. C. and Meeker, D. L. (1995) Pork quality audit: a review of the factors influencing pork quality. J. Muscle Foods 6, 369402. Fujii, J., Otsu, K., Zorzato, F., deleon, S., Khanna, V. K., Weiler, J. E., O’Brien, P. J. and MacLennan, D. H. (1991) Identification of a mutation in porcine ryanodine receptor associated with malignant hyperthermia. Science 253, 448451. Garrido, D., Perez, A. and Sanchez-Ferrer, A. (1994) Meat pigment determination by phase partitioning in Triton X-l 14 and oxidation with sodium nitrite. J. Sci. Food Agric. 64, 327329. George, P. and Stratmann, C. J. (1952) The oxidation of myoglobin to metmyoglobin by oxygen. 2. The relation between first-order rate constant and the partial pressure of oxygen. Biochem. J. 51, 418. Hamm, R. (1986) Properties of the myofibrillar system and their measurements. In Muscle as Food, ed. P. J. Bechtel. Academic Press, Orlando, FL. Harrison, G. G. (1979) Porcine malignant hyperthermia. Znt. Anesthesiol. Clin. 17, 25-61. Kauffman, R. G., Sybesma, W., Smulders, F. J. M, Eikelenboom, G., Engel, B., Laack, R. L. J. M. van, Hoving-Bolink, A. H., Sterrenberg, P., Nordheim, E. V., Walstra, P. and van der Wal, P. G. (1993) The effectiveness of examining early post-mortem musculature to predict ultimate pork quality. Meat Sci. 34, 283-300. Klont, R. E. and Lambooy, E. (1995) Effects of preslaughter muscle exercise on muscle metabolism and meat quality studied in anesthetized pigs of different halothane genotypes. J. Anim. Sci. 73, 108-l 17. Kocwin-Podsiadla, M., Przybylski, W., Kuryl, F., Talmant, A. and Monin, G. (1995) Muscle glycogen level and meat quality inpigs of diferent halothane genotypes. Meat Sci. 40, 121-125. Krzywicki, K. (1979) Assessment of relative content of myoglobin, oxymyoglobin and metmyoglobin at the surface of beef meat quality storage. Meat Sci. 3, l-10.
Effect
of halothane genotype on porcine meat quality and myoglobin autoxidation
53
Krzywicki, K. (1982) The determination of haem pigments in meat heme. Meat Sci. 7, 29-36. Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage. Nature 227, 68&685. Leach, L. M., Ellis, M., Sutton, D. S., McKeith, F. K. and Wilson, E. J. (1995). A within litter comparison of the growth performance, carcass characteristics, and meat quality of halothane carrier and negative pigs at three slaughter weights. Proceedings Vol. ZZ 41st Annual Znternational Congress of Meat Science and Technology, San Antonio, Texas. Louis, C. F., Rempel, W. E. and Mickelson, J. R. (1993) Porcine stress syndrome: Biochemical and genetic basis of this inherited syndrome of skeletal muscle. Proc. Recip. Meat Confr. 46, 89-99. MacLennan, D. H. and Phillips, M. S. (1992) Malignant hyperthermia. Science 256, 789-794. Monin, G., Mejenes-Quijano, A. and Talmant, A. (1987) Influence of breed and metabolic type on muscle glycolytic potential and meat pH in pigs. Meat Sci. 20, 149-158. National Pork Producers Council (1991) Procedures to Evaluate Market Hogs, 3rd edn. National Pork Producers Council, Des Moines, IA. National Pork Producers Council (1995) Terminal Sire Line National Genetic Evaluation Program. National Pork Producers Council, Des Moines, IA. Oliver, M. A., Gispert, M. and Diestre, A. (1993) The effects of breed and halothane sensitivity on pig meat quality. Meat Sci. 35, 105-l 18. Pommier, S. A. and Houde, A. (1993) Effect of genotype for malignant hyperthermia as determined by restriction endonuclease assay on the quality characteristics of commercial pork loins. J. Anim. Sci. 71, 42@425. Renerre, M., Anton, M. and Gatellier, P. (1992) Autoxidation of purified myoglobin from two bovine muscles. Meat Sci. 32, 331-342. Satterlee, L. D. and Zachariah, N. Y. (1972) Porcine and ovine myoglobin: isolation, purification, characterization and stability. J. Food Sci. 37, 909-912. Shikama, K. and Sugarwara, Y. (1978) Autoxidation of native oxymyoglobin. Kinetic analysis of the pH profile. Eur. .Z. Biochem. 91, 407413. Statistical Analysis System Institute Inc. (1985) SAS Users Guide to the Statistical Analysis System. NC State University, Raleigh, NC. van der Wal, P. G., Vries, A. G. and Eikelenboom, G. (1994) Predictive value of slaughterhouse measurements of ultimate pork quality in seven halothane negative Yorkshire populations. Meat Sci. 40, 183-191. Warriss, P. D. (1979) The extraction of haem pigments myoglobin and haemoglobin from fresh meat. J. Food TechnoI. 14, 75-80. Wykle, B., Gillett, T. A. and Addis, P. B. (1978) Myoglobin heterogeneity in pigs with PSE pale, soft andexudative and normal muscle by an improved isoelectric focusing technique. J. Anim. Sci. 47, 126&1264.