Effects of Early Post-Mortem Rate of pH fall and aging on Tenderness and Water Holding Capacity of Meat from Cull Dairy Holstein-Friesian Cows

Fast or slow muscle pH fall may give unacceptable purge losses or tough meat, depending much on concomitant evolution of muscle temperature early post-mortem, costing millions of euros to the meat industry. Tenderness and purge losses of Longissimus thoracis/lumborum (LTL) and Gluteus medius (Gm) sampled from cull dairy cows differing in production status (10 lactating vs. 22 dried off) and aging time, were evaluated regarding different rates of pH2 fall. Shear force related to pH2 was dependent on muscle and aging time. The intermediate glycolysis led to lower shear force in LTL, while the faster produced best quality in Gm. Purge was influenced by pH2 (P=0.0077), aging (P<0.0001) and muscle*pH2 interaction (P<0.0001). Aging affected thawing (P<0.0001), grilling (P=0.0004) and overall losses (P<0.0001). Under the ruled chilling regime, the fast pH fall in Gm and the slow pH fall in LTL approached out of the ideal pH6/temperature limits, being compatible with heat and cold shortening, respectively.


Introduction
Meeting consumer requirements is a major concern for meat producers and retailers.Retaining moisture and a high tenderness degree are considered important meat traits for consumer acceptability (Lonergan & Lonergan, 2005;Miller, Carr, Ramsey, Crockett, & Hoover, 2001).Purge losses reach as much as 1-3 % in fresh retail cuts developing a normal quality pattern (Offer and Knight, 1988) or rise up to about 10-15% in abnormal muscle quality condition, such as the extremely PSE pork products (Roseiro et al., 1994;Melody et al., 2004).The loss of water from cell compartments is associated to different mechanisms, which may occur at distinct storage phases (Lonergan & Lonergan, 2005).With the polarity inversion occurring in cell membranes at the early stages of the apoptosis process taking place in muscles after bleeding and O 2 depletion (Ouali et al., 2006), the acidic components formed by glycolysis are replaced by others of basic nature, promoting partial neutralization and/or slowing down acidification, giving rise to transient "plateaus" in the pH fall dynamics (Herrera-Mendez, Becila, Boudjellal & Ouali, 2006).Such discontinuity in pH fall can not be addressed to any transient reduction of glycolytic enzymes activities (phosphocreatine kinase and others) but to modifications of either the buffering capacity and/or charge distribution within the muscle cell (replacement of acidic phosphatidylserine by basic components, such as phosphatidylcholine and phosphatidylethanolamine) (Ouali et al., 2006).Partial denaturation of myosin head at low pH, namely when the muscle temperature is high, is also thought to be a mechanism involved in the shrinkage of the myofibrillar spacing and the subsequent purging development (Offer & Trinick, 1983;Offer, 1991).In the other hand, modifications occurring in costameres, as the ones affecting the lateral shrinkage of the myofibrils (Honikel, Kim, Hamm & Roncales, 1986;Kristensen & Purslow, 2001;Bertram, Purslow & Andersen, 2002), also contribute to sarcomeres shortening and cell volume shrinkage.Both events, occurring under a general apoptosis process (Ouali et al., 2006), would create channels between cells and cell bundles, facilitating the purging out from meat (Offer & Knight, 1988;Schafer, Rosenvold, Purslow, Andersen, & Henckel, 2002).Thus, different post-mortem pH decline rates would influence the meat quality standards (Kauffman & Marsh, 1987;Marsh, 1993), since it influences the proteinaceous linkages formed post-mortem (Dransfield, 1992;Geesink et al., 1995;Sentandreu, Coulis, & Ouali, 2002) as well as the proteolytic degradation of myofibrils (Salm et al., 1983) and collagen (Judge, Reeves, & Aberle, 1981).The conjunction of high muscle temperature and fast pH decline can also result in protein/enzymes denaturarion/autolysis, thus minimizing tenderness improvement as the post-mortem storage progresses (Dransfield, Etherington, & Taylor, 1992;Rees, Trout, & Warner, 2003).
Cull dairy cows represent near 50% of the overall cattle population slaughtered at S. Miguel (Azores), with a carcass weight amounting to 9,600 tones in 2013.Despite poor carcasses meat yield (70% scored as P; 30% as O), an important amount of lean meat is still produced from hindquarter noble cuts.In view of the multiple subjacent reasons for culling, dairy cows are considerably heterogeneous in age and production status at slaughter, making expectable different final meat quality patterns.
The present study was undertaken to evaluate the relationship between muscle early post-mortem rate of pH fall (up to 6 hours) and meat purge losses and Warner-Bratzler (WB) shear force of mature dairy culled cows, after aging for 2, 7, 14, 28 and 42 days.Thawing and grilling weight losses after aging were also examined.The validity of those quality trait values against the ideal pH/temperature window expectations was also evaluated.

Animals and Carcasses Handling
Thirty two culled dairy cows (lactating-10 cows; dried-off -22 cows) were used for the present study.They were reared at different farms and fed under a regime based on natural pasture or green silage and concentrate until drying (diet restriction to straw concomitantly with some water deprivation or, simultaneously by mating, personal communication).Cows were slaughtered conventionally at a commercial abattoir, in average 2 cows/day, depending on their availability in farms according to the variables under study (Table 1).Carcasses were electrically stimulated (90 V) for about 1 minute during the bleeding stage, about 5 minutes after stunning (captive bolt).After dressing, weighting and classification [conformation and fatness, according to EC Regulation Nº 103/2006], carcass sides were cooled down in a chiller at 0 ˚C -1 ˚C and 3-4 m/s ventilation rate for about 1 hour and then chilled at 2 ˚C and 0.5-1 m/s ventilation rate, for the next 48 hours.At this stage, the LTL (quarters separated at the 12 th rib level) and the whole Gm muscles were taken from the left hindquarter and sent under refrigeration to the laboratory for analysis.to exist.The final pH was measured 48 hours post-mortem and samples having a pH 48 higher than 6.0 were not used in this study.Muscles were grouped according to fall rate of pH measured at 2 h post-mortem (pH 2 ) as follows: slow (pH 2 > 6.4), intermediate (6.0 < pH 2 < 6.4) and fast (pH 2 < 6.0).During this period of time, muscle temperature was also continuously monitored throughout a probe connected to a Delta -T Logger (Delta -T devices, Burwell, Cambridge, UK).

Purge Loss and Shear Force Evaluation
Sub-samples of LTL and Gm muscles randomly assigned to each aging condition (vacuum packed and held at 2˚C for 2, 7, 14, 28 and 42 days) were used to evaluate related purge losses % = [fresh meat weight -(meat weight after each aging period)] x 100 / fresh meat weight.After aging, samples were dried with adsorbent paper, cut into two similar portions, vacuum packed, immediately frozen at -30ºC and held thereafter at -18˚C/-20˚C until grilling.Thawing and grilling losses (%) were calculated as follows: [aged samples weight -(thawed or grilled sample weight) x 100] / aged sample weight.Total meat losses (%) = [initial fresh meat weight -(aging losses + thawing losses + grilling losses)] x 100 / initial fresh meat weight.
For shear force evaluation, samples about 2.5-3.0 cm thick were cooked in a water bath at 85 ˚C until 70 ˚C in the critical point, cooled down in ice water for about 30 minutes and then stored under vacuum packaging in a refrigerator (0-2 °C).Before analysis, samples were kept at room temperature (15-18˚C) for equilibrium.Square sectioned cores (1x1x4/5 cm) for Warner-Blatzer (WB) shear tests (500 kg cell) were prepared parallel to the muscle fibers direction.Before analysis, they were consistently held at room temperature for about 1/2 hour (equalization) and then completely cut by the shear blade (triangular slot cutting edge, of 1 mm of thickness), perpendicular to the fibers, at a crosshead speed of 1 mm/s (Instron 4501 model, H3279, England).Each mean value was obtained from six to ten recordings and expressed as Newtons.

Statistical Analysis
To determine the effect of rate pH fall and aging time on variation of purge losses and shear force values, analysis of variance (ANOVA) was performed using JMP statistical software (Version 9.0.1,SAS Institute, Inc., Cary, NC, USA, 2010), following a linear mixed model.When significantly affected (P<0.05),least square means were compared using the Tukey HSD post hoc test.
The relationship between pH and temperature was examined in order to determine the risk of cold and heat-shortening occurrence in the two muscles (Gm and LTL) grouped according the different rates of pH 2 fall studied.Second-order polynomial regression was the best model for the fitting of data.

Variation in the Rate of Muscles pH Fall
Within the wide range of glycolytic rates found in both muscles among carcasses at 2 hours post-mortem (Table 2), no particular pH evolution pattern (slow, intermediate or fast pH fall) by age and production status of cows at slaughter was found.Since no additive effect in the glycolytic variation was objectively induced by cooling rate (same chilling regime applied) and considering the scored EUROP carcass classification (Table 1), which indicates that most of them would cool down at similar rates, the results show that the electrical stimulation response differed strongly among tested animals with normal muscle pH 48 (Gm -between 5.76 and 5.38; LTLbetween 5.92 and 5.41) (Table 2).The slope of pH decline between 2 and 6 hours post-mortem was more pronounced in the Gm muscle, namely with intermediate (0.66 pH units) and slow (0.95 pH units) pH 2 evolutions when compared to LTL muscle (0.31 and 0.69 pH units, respectively).Concerning the faster pH 2 group, differences between muscles were not so great (Gm -0.43 pH units vs. LTL -0.38 pH units).
No evident transient pH stability pattern was found within the pH 2 groups for each muscle type, probably due to the interference of electrical stimulation applied to carcasses.Corroborating the results reported by Ouali et al. (2006), our muscles also showed, most frequently, one transient stage (55%), followed by those having two (36%) and none (9%).In terms of post-mortem timing, plateaus mostly appeared after the first 2 hours following slaughter but one Gm muscle exhibited a short event during the first hour post-mortem.Only one case with 2 concomitant plateaus in both muscles of the same carcass was found, with this transient frequency profile being slightly higher in LTL muscle.

Early Post-Mortem Muscle pH Condition and Meat Shear Force
The tendency of meat from muscles with two pH transient steps to be tougher than that having only one (Ouali et al., 2006), was not confirmed in our study.The results from LTL muscle (Table 3) show the former samples type with significantly improved overall mean tenderness (45.49N vs. 58.89N), with such difference consistently registered up to the 14 days aging period.The tenderization process within samples with 2 pH transient steps was apparently faster than in the other groups, reaching a mean shear force level at day 2 of aging equivalent to those obtained after 28 (1 transient step) and 14 days (no transient step).The evolution of meat tenderness with the extension of aging also differed among the 3 groups, varying those samples with 2 transient stages, comparatively, between more narrowed limits (49.83 N at day 2 and 41.35 N at day 14).The other two groups (0 and 1 transient step) behaved similarly, showing a more extended but progressive decreasing of values.It must be yet underlined that the mean shear force in samples with 1 transient step tended to stabilize around 58.8 N somewhere between day 7 and day 14 and those having no transient steps showed the best mean tenderness degree after 28 days of aging (39.72 N).This last result must be taken under reserve due to the exiguous number of tested samples (Table 3) and the enormous difference in values found between them.The shear force discrimination between samples with two and one pH transient steps can not be dissociated from the results obtained with 3 samples within the later group, which shear force mean values were seemingly above 100 N along the entire aging duration.Among such cases, only one had a pH 2 > 6.4, which according to the data represented in Figure 1, could be cold-shortened, while the other two had intermediate pH 2 evolutions, which make the cold-shortening occurrence almost unlikely (van de Ven, Pearce & Hopkins, 2013).The exact meaning of pH transient steps and their expected effects in the multiple enzyme systems activity involved in meat tenderization calls for new investigations, to clarify their subjacent complex mechanisms and the key interacting factors determining their functionality..31N at day 2), while the fast group extends the tenderization process, reaching even a lower mean shear force value after 28 days of aging (Table 5).Such different behaviors were also reported by other authors, who underlined that they were possibly due to distinct pH sensitivity patterns of aging enzymes in muscles with distinct anatomical locations, thus promoting the inhibition or activation of caspases and calpains (Kim, Lonergan & Huff-Lonergan, 2010;Kim, Stuart, Nygaard & Rosenvold, 2012), enzymes involved in the cleavage of cellular structures early post-mortem (Herrera-Mendez et al., 2006, referring Fischer et al., 2003).
Despite the greater effective probability of heat shortening development in Gm muscles within pH 2 <6.0 group, the resulting overall mean shear force value (56.98N) did not significantly differ from the other groups with intermediate (54.45N) and slow (48.38 N) rates of pH 2 fall.The lower effect related to heat shortening on sarcomere contraction rate at rigor on set and consequently on meat tenderness, may explain the lower differences among Gm samples.Nevertheless, the shear force value obtained at day 2 was considerably higher in pH < 6.0 (68.19 N) than in the other groups (60.7 N and 61.15 N in intermediate and slow groups, respectively).Also the difficulty, frequently mentioned (Devine et al., 1999;Dransfield et al., 1992) in heat shortened samples to pursuit post-mortem tenderization only appeared slightly attenuated in Gm samples grouped in fast and intermediate rates of pH 2 decline (28 % and 23 % reduction in shear force from day 2 up to day 28) in relation to that within the slow group (35 % reduction).The reported most tender beef obtained when the temperature/pH 6.0 is around 29-30 ºC (Hwang & Tompson, 2001) was not totally confirmed in our study for the Gm muscle.

Early Post-Mortem Muscle pH Condition and Meat Moisture Weight Losses
Least square mean purge, thawing and grilling losses of Gm and LTL muscles, as affected by aging time, and muscle rate of pH 2 decline are shown in Tables 4 and 5.The aging time, as expected, affected very significantly purge losses in both muscles (Tables 4 and 5).Differently, the significant effect from the rate of pH 2 fall was only for purge losses recorded after aging, in both muscles, but to a lower extent in LTL (P=0.04)than in Gm muscle (P<0.001).
Regarding the earliest post-mortem stage evaluated in the current study, corresponding to the samples kept in carcasses for 2 days and immediately frozen at -30ºC after excision and preparation for analysis (held thereafter at -18˚C/-20˚C), the least thawing loss situation was found in the fast rate of pH 2 fall in both muscles, reaching 6.63 % and 6.26 % in Gm and LTL muscles, respectively (Tables 4 and 5).Regarding the other pH 2 groups, the respective weight losses appeared quite leveled between them in both muscles as well, but with that formed in Gm at a higher level (8.11 % -slow vs. 8.74 % -intermediate) than in LTL (6.85 % -slow vs. 6.95 %intermediate).Such quantitative early post-mortem discrimination among the pH 2 fall rates assayed, once discounted the increased thawing loss level added by the blast air freezing to the samples (Moore & Young, 1991;Sacks, Casey, Boshof & van Zyl, 1993), lack a coherent explanation in view of the results expressed in Figure 1, namely for the Gm muscle (best response while submitted to highly probable heat shortening effect).
However, such trend was not confirmed when samples aged under vacuum packaging at 0 ºC -2 ºC were analyzed.Under these processing conditions, the worse purge formation in LTL muscle appeared associated to the slow group, with an overall mean value significantly higher than the fast group (7.78 % vs. 5.76 %) but not differing from that achieved in the intermediate group (5.94 %).If the purging behavior in the slow group may be explained by the higher probability of cold shortening occurrence (Figure 1), the higher overall mean purge level from the intermediate in relation to the fast group (P > 0.05) is, apparently, questionable, because these last samples will necessarily reach similar pH values at higher muscle temperatures (30.25˚C vs. 17.95˚C).These conditions would increase the associated protein denaturation and implicate a reduction in the respective water holding capacity.However, the higher muscle temperature/pH 6 could also speed up the aging enzymes activity and thus the subsequent hydrolysis of the costameres structure, preventing the myofibrils lateral shrinkage accomplishment and promoting a lower purging intensity (Wang & Ramirez-Mitchell, 1983;Kristensen & Purslow, 2001;Melody et al., 2004).Yet, the marginal incidence of cold shortened samples within the intermediate group, as well as their faster pH decline rate between the 2 nd and the 6 th hour post-mortem, referred before, could also be responsible for those different values.Concerning the Gm muscle, the worse purge quality status came from the intermediate pH 2 fall rate (8.19 % -overall mean value) significantly higher than the level achieved from the slow group (5.95 %) but not differing from that obtained within the faster group (7.20 %).
Here again, the relative purge overall mean discrimination between fast and intermediate groups seems to be somewhat distorted, attending to their temperature at pH 6.0 crossing point within the ideal window of quality, showed in Figure 1.Apparently, under the Gm physico-chemical post-mortem environment, the balance resulting from the factors affecting the water holding capacity is slightly more negative in the intermediate than in the fast group (Kim et al., 2012).Hopkins et al., (2014) also reported no significant differences in the eating quality of two beef cuts obtained from stimulated and non stimulated carcasses aged for 1 and 14 days, which had temperature/pH 6.0 mean values of 40.9 ºC and 33.3 ºC, respectively.The main reason referred by the authors for this result was the fact that the model used to define the beef quality standards did not include the pH decline rate as a predictive trait.Differences between the most and the least purging pH 2 groups after aging for 7, 14 and 28 days were greater in Gm than in LTL muscle (7.11 % vs 6.49 %) (Tables 4 and 5).Comparing LTL samples with pH 2 > 6.4 and pH 2 < 6.0, the former condition showed 38.9 %, 33.2 % and 32.0 % higher mean purge levels after 7, 14 and 28 days of aging, respectively.These gaps in Gm muscles were considerably lower, namely those found for the longer aging periods (+33.4% -7 days; 16.9% -14 days and 12.9% -28 days).Such apparent lower relative impact in purge formation of samples passing by a heat-shortening situation is an unexpected result.Under this condition, the amount of free water in and out of the myofibrillar compartments is referred to increase considerably, due to the protein denaturation and the concomitant decrease in the sarcomere length at rigor, which should facilitate its flow into the extracellular space (Guinot, Vignon, & Monin, 1993).Regarding the different groups of pH 2 fall rates aged for 42 days, the differences in purge formation appeared much more attenuated in LTL (5.1%) comparatively to Gm muscle (14.6%).The slow decrease in purging after 42 days of aging or even its lower mean level in relation to that found after 28 days aging period (LTL slow group), could be due to some reabsorption by the muscle protein network or to the sponge effect suggested by Farouk, Mustafa, Wu, & Krsinic (2012).Irrespective of muscle pH 2 fall rate, purging production decreased with the aging time in both muscles.This corroborates the findings of Farouk et al. (2012), who stressed that, apart from the increasing values of drip after the first week of aging, the water holding capacity of beef improves thereafter, whatever the temperature that rigor sets in.Other studies also confirm this behavior in different meat animal species (Farouk et al., 2007;Farouk, Wiklund, Stuart & Dobbie, 2009;Zhang, Lonergan, Gardner, & Huff-Lonergan, 2006).
The thawing losses decreased with the meat aging time.According to Bertram et al. (2002), moisture weight losses from meat develop as an ongoing process, involving the water transfer from the myofibrils microstructure to the extra myofibrillar and then to the extracellular space.Thawing weight loss from Gm was, in average, higher than that of LTL muscle (Tables 4 and 5) for samples having similar temperature/pH 6.0 values (eg.Gm intermediate group vs. LTL fast group).
Under the heat pressure of grilling treatment, the distinct rates of pH 2 fall did not produce significantly different losses in both muscles.The effect attributed to the aging time (P=0.0009and P=0.013 for LTL and Gm muscles, respectively) reflected a somewhat erratic behavior within each pH 2 group.Nevertheless, a trend for slightly higher purge values in samples aged for longer periods was verified for both muscles, which is opposite to that reported in Devine et al. (2002) and Wheeler, Savell, Cross, Lunt & Smith (1990) for cooking processed samples.Under grilling, our results mostly seem to express differences in sample geometry (shape/dimension) and volume relationship, which may provide distinct heating intensities, affecting conjunctive tissue structure and determining weight losses.At grilling the results varied between a minimum of 22 % in both muscles and a maximum of about 27 % in LTL and 29% in Gm.If samples aged for different periods were pooled together, then the average losses under grilling within the different pH 2 groups were very close in both muscles (less than 1 %).
Based on total purge losses obtained from both muscles, no coherent relationship among distinct pH 2 conditions and samples aging periods assayed can be formulated.Overall, the difference among mean values reached around 1 % in LTL and less than 3 % in Gm.In the former muscle, samples with pH 2 >6.4 presented the lower level whereas in Gm this performance was related to the faster condition (Tables 4 and 5).

Conclusions
The post-mortem rate of pH decline significantly affected purging and tenderness of meat from culled dairy cows after aging.Meat from LTL with intermediate and fast pH 2 fall rates showed the best tenderness and water holding capacity, respectively, while the slow group presented the poorest quality standard due to its temperature/pH 6.0 relationship compatible with cold shortening development.Differently, the most tender and less purging meat from Gm muscle was obtained from samples having slow rate of pH 2 fall, with the other two pH 2 fall groups showing the poorest condition for both traits, due to temperature/pH 6.0 relationship passing the ideal quality interval at the hot end, being more exposed to heat shortening events.
Additional processing treatments before consumption (thawing, grilling) tend to level the existing differences in moisture losses among pH 2 groups.In order to optimize culled dairy cows meat quality, their carcasses processing, namely the chilling regime management, has to attend to the large range of variation in animals body condition and to the intrinsic biochemical characteristics of distinct muscles.

Table 1 .
Live culled cows characteristics and respective carcasses quality attributes used in the experimental design

Table 2 .
pH 2 means, minimum and maximum values within each pH 2 group in Gm and LTL muscles

Table 3 .
Shear force (N) mean values (±SE) in LTL muscle aged for 7, 14, 28 and 42 days of culled dairy cows as affected by the frequency of pH transient steps ocurred up to 6 hours post-mortem Overall means with different superscript letters are significantly different, P<0.05 (Tukey HSD post hoc test).

Table 4 .
Purge, thawing and grilling losses and Warner-Bratzler shear force (WBsf) least square means of samples from Gm muscle of cull dairy cows in relation to the rate of pH 2 fall and aging time

Table 5 .
Purge, thawing and grilling losses and Warner-Bratzler shear force (WBsf) least square means of samples from LTL muscle of cull dairy cows in relation to the rate of pH 2 fall and aging time