Effects of additional electrical stimulation and pre-rigor conditioning temperature on the ageing potential of hot-boned bovine muscles

Objective The aim of this study is to characterize the impact of additional electrical stimulation (AES) and various pre-rigor holding temperatures (for 3 h) on the ageing-potential of hot boned bovine M. longissimus lumborum (LL). Methods Paired LL loins from 12 bulls were hot-boned within 40 min of slaughter, immediate AES applied and subjected to various holding temperatures (5°C, 15°C, 25°C, and 35°C) for 3 h. Results AES did not accelerate the rate of rigor attainment, but the 3 h pre-rigor holding temperature did. Shear force values decreased as the pre-rigor holding temperatures increased. AES and holding for 3 h (at 25°C) resulted in higher water-holding capacity. Conclusion Data confirmed that AES did not influence the various meat quality parameters in the present study, but pre-rigor holding temperature (25°C) alone or in combination with AES resulted in superior meat quality.


INTRODUCTION
The red meat industry identified that improving the eating quality of beef as essential in meeting the fastgrowing demands for high value beef products in discerning and highly competitive international markets [1]. It is particularly important that the industry reduce the inconsistent tenderness of beef being contributed by the socalled 'intermediate pHu' bull beef [2].
The ultimate pH of meat (pHu) has a considerable impact on meat quality [3]. Normal pHu samples are a bright red color that consumer' s associate with good quality and the meat tenderizes with ageing. High pHu meat results in tender but dark, firm and dry (DFD) pro duct. In contrast to high and low pHu meat, the tenderness of intermediate pHu meat is much more inconsistent and consequently it is of poorer quality. A significant proportion of bull beef can be categorized as intermediate pHu (18%) and seasonal variation may also add to the inconsistency [4]. The underlying biochemical mechanisms related to this in consistent tenderness issue are not fully understood. Therefore, improving the meat quality through novel meat processing systems for these classes of carcasses needs to be developed.
Varying pre-rigor environments generated by the application of electrical stimulation (ES) and/or pre-rigor chilling conditions influence the rate of glycolysis and subsequent pH decline in muscles post mortem. One of the major post mortem interventions implemented by the meat industry for enhancing meat quality attributes is carcass ES [5]. There are discrepancies among various reports on the influence of ES on meat quality. Some authors found positive effects due to ES [6,7], while others found no effects [8] and others reported negative effects [9]. Despite the dif ferences, ES has been shown to enhance tenderness due to reduction in muscle ATP [5]. The complex interaction of pH and temperature decline in pre-rigor muscle has a signifi cant role in meat tenderisation by influencing proteolytic enzyme activity, particularly μcalpain [6]. The meat industry typically refers to this as the "pH/temperature window" as hot or cold shortening can result from over stimulation or high chilling rate before the pH has declined sufficiently. Optimal tenderness was evident when glycolysis had pro ceeded at an intermediate rate resulting in 6.1 pH at 3 h post mortem [10].
Recently, Balan et al [6] demonstrated that various prerigor holding temperatures (especially 25°C or 35°C) along with accelerated pH decline rates by low voltage electric stim ulation (LVES) can have positive effects on bull M. longissimus lumborum. The treatments resulted in improved tenderness, reduced cook loss and increased sarcomere length. Further more, the treatments positively influenced the myofibrillar proteins and small heat shock proteins (sHSP) activities as sociated with μcalpain activity in bull beef samples. LVES combined with the 3 h pre-rigor holding temperature (for ES25 and ES35 samples) resulted in no cold shortening (or heat induced shortening) even though the samples were aged at 1°C. Based on the above findings [6], it can be implied that there is an optimal condition (i.e. combination of LVES with 3 h pre-rigor holding temperature at 25°C or 35°C) for maxi mizing the ageingpotential for bull beef. Moreover, the results discussed above [6] from bulls which were stunned by captive bolt (carried out to avoid any electrical effects before stimu lation), need to be verified (in all the three ranges of pHu) using current slaughter practices where the animals are elec trical stunned and immobilized preslaughter.
We hypothesize that the additional electric stimulation (AES), pre-rigor pH, and temperature decline will positively influence the ageingpotential of hot boned bull beef muscle, particularly from intermediate pHu carcasses. Therefore, the objective of this study is to characterize the impact of AES and various pre-rigor holding temperatures (for 3 h) on the ageing potential of hot boned bull beef muscle.

Raw materials and processing
A total of 12 cattle (around 24monthold bulls) were slaugh tered at a New Zealand meat plant over three slaughter days. Bulls in this study were electrically stunned for Halal kill (fre quency = 50 Hz, pulse width = 3.5 milliseconds, peak voltage = 583 volts) and the carcasses electrically immobilized follow ing exsanguination. Both loins (M. longissimus lumborum; LL) from the 12 beef carcasses were hotboned within 40 min post mortem. In this study, two main treatment effects i.e., AES, no additional electrical stimulation (NAES) and prerigor holding temperature (at 5°C, 15°C, 25°C, and 35°C for 3 h) and their interactions were tested giving a total of 8 dif ferent treatment combinations ( Figure 1).
Initial pH (pH 40min ) was recorded and the LL from either left or right (randomly selected) side of the carcass was im mediately subject to low voltage electrical stimulation (AES) for 30 S after boning (frequency = 13.3 Hz, pulse width = 5.4 milliseconds, peak voltage = 104 volts). After stimulation, pH was recorded once again. The LL from the other side of the carcass was not additionally electrically stimulated (NAES). Immediately after stimulation or nonstimulation, approxi mately 10 g of muscle was removed, snap frozen using liquid nitrogen and stored at -80°C for initial biochemical analyses. The loins were then divided into four different subsamples, placed in plastic bags and randomly submerged in either 5°C, 15°C, 25°C, or 35°C water baths for 3 h, which was the prerigor holding temperature. A temperature probe was inserted in to the geometric center of each loin section to monitor continuous drop in temperature ( Figure 2). After 3 h had elapsed, the meat/muscle samples were removed from the plastic bags; their pH measured, and further sampled for biochemical analysis. The loin samples were transferred to the AgResearch laboratory where they were vacuum packed and aged at 10°C for 48 h. After 48 h sampling and measure ments, loins were again vacuum packed and aged at 1°C until 14 days post mortem. pH pH of the LL samples was measured in duplicate by insert ing a calibrated pH probe (Hanna HI99163 pH meter with a FC232D combined pH/temperature probe, HANNA instru ments, Woonsocket, RI, USA) directly into the muscle at 40 min (before and after stimulation), 3 h, 6 h, 24 h, 48 h, and 14 days post mortem respectively.

Shear force
The LL samples were cooked in a water bath set at 99°C (controlled by a DigiSense scanning temperature logger [Eutech Instruments Pte Ltd., Singapore] with a thermocouple positioned into the center of each sample) to an internal temperature of 75°C. After cooking, the samples were trans ferred to icewater slurry for at least 10 min. Shear force was measured by determining the force required to shear through a 10 mm×10 mm cross section sample at right angles to the fiber axis using the MIRINZ tenderometer [11]. Ten repli cates were measured for each precooked sample. The results were expressed as shear force (kgF) and final values of peak  shear force were calculated as an average from the 10 repli cates.

Cooking loss
LL samples were weighed before and after cooking. The cook loss was calculated as weight before cooking minus weight after cooking and expressed as a percentage of the precooked weight [6].

Purge and drip loss (water holding capacity)
The LL sections were weighed prior to vacuumpackaging to obtain initial weight for the purge loss measurement. After the assigned storage time, the samples were removed from the vacuum bags, patted dry on paper towels and reweighed (final weight) to determine purge loss as the difference be tween initial weight and final weight expressed as percentage of initial weight.
Drip loss was measured after each assigned storage (48 h and 14 days) following the procedure of Balan et al [12]. A sample (about 50 g) of meat with any visible fat and connec tive tissue removed was weighed and then placed in plastic 'onion bag' netting and suspended by a hook within a closed container. After placing the container for 48 h at 4°C, the sam ple was blotted dry and then reweighed. The drip loss was calculated as weight lost expressed as a percentage of the original sample weight.

Color
The cuts (48 h post mortem) for color measurements were placed in Cryovac food grade trays (Cryovac TQD0900; 22.5 cm×17.3 cm×4.1 cm; CRYOVAC, Sealed Air Corpo ration, Duncan, SC, USA) with the cross sectional side up and then sealed with oxygen barrier film using Cryovac LID 1050 (CRYOVAC, Sealed Air Corporation, USA) into HiOxMAP. A highoxygen modified atmosphere (80% O 2 / 20% CO 2 , certified standard within ±5%, BOC GASES; Hamil ton, New Zealand) was accomplished using a ROSS Inpack Junior A10 Packaging Machine (Ross industries packaging division, Midland, VA, USA) by applying vacuum, then flush ing the package with the gas mixture and sealing. The gas mixture composition inside each pack was checked before opening with a PBI Dansensor, CheckPoint handheld gas analyzer (Ringsted, Denmark) by piercing the top layer and reading the oxygen and carbon dioxide levels. The packaged cuts were displayed for 7 days at 3°C±1°C under continuous fluorescent natural white light (2,800 lx, color rendering index = 82, color temperature = 4,000 K; Osram, Auckland, New Zealand).
On days 1, 4, and 7 of simulated retail display under light, the cut cross sectional meat surface was measured using a Minolta Color Meter (Illuminant D65, 1 cm diameter aper ture, 10° standard observer; CR300; Konica Minolta Photo Imaging Inc., Tokyo, Japan) for color using the CIE L* a* b* color space. Calibration was performed by using a standard white tile prior to the color measurement. L* (lightness), a* (redness), and b* (yellowness) values were used to calculate chroma ([a* 2 +b* 2 ] 1/2 ) and hue angle ([b*/a*] tan1 ) [13]. The surface meat color was scanned with the color meter covered with the same film after removing the plastic film on the top of the HiOxMAP tray.

Statistical analysis
All statistical analysis was performed using Genstat 16th edi tion [14]. The pH fall was analyzed using analysis of variance (ANOVA) where side within animal was the blocking vari able and the treatment variables were temperature, electric stimulation, time and all possible 2 and 3way interactions. Firstly, data was analyzed excluding pHu using ANOVA. The shear forces, cook loss, drip and purge loss (for 48 h and 14 days) data were the dependent variables. For the above men tioned analysis, side within animal was included as a blocking variable and the treatment variables were temperature (5°C, 15°C, 25°C, and 35°C) and additional low voltage electrical stimulation and their interaction. Then these variables were reanalyzed including pHu as a treatment variable, where pHu was split into three levels: low (5.4 to 5.79); intermediate (5.8 to 6.19) and high (>6.2). The initial pH for the animal was also included in the treatment structure. The color vari ables were analyzed using repeated measure ANOVA across 1, 4, and 7 days. Side within animal was included as a block ing variable and the treatments were temperature, AES, time and their 2 and 3way interactions. Then this analysis was rerun including pHu (low, intermediate, and high) as a treat ment and all 2, 3, and 4way interactions. Least squares means for each attribute were separated using least significant differences (F test, p<0.05).

Ultimate pH of meat samples
In this study, out of the 12 bull carcasses used, six had high (>6.2), three intermediate (5.8 to 6.2) and three low pHu (5.4 to 5.79) ( Table 1).

Temperature and pH decline
AES had no effect on pH decline (p>0.05). AES did not re sult in an immediate fall in pH as expected (Table 1) when compared to our previous findings [6,7]. This could be due to the electrical inputs into the carcasses from headonly elec trical stunning and immobilization that masked any effect of AES. Pre-rigor holding temperature (for 3 h) and timepoint significantly influenced pH fall (p<0.001). Generally, the highest pH decline was at 6 h post mortem except for 35°C samples where the highest pH fall was at 3 h post mortem. It is well documented that high pre-rigor temperature accelerates post mortem pH decline [6,7,15]. In comparison to our pre vious trial in which no electrical stunning or immobilization was used [6,7], in the present study, AES35 samples muscle pH (in low pHu samples, Table 1) declined only by ΔpH 0.17 pH units and 0.75 soon after ES and 3 h of holding when com pared to 0.43 pH units and 1.37 in the previous trial [6]. This loss of AES effect was due to the headonly electrical stunning and immobilization procedure carried out before AES and continued to be low when compared to rest of the samples (Table 1).

Shear force
The AES treatment applied to the hotboned loin samples did not result in significant (p>0.05) changes in shear force values (Table 2). At 48 h post mortem, shear force values were   [16]). However, after 14 days of ageing this observed effect was lost as all the samples (intermediate pHu) were tender (less than 7.5 kgF) except the ES35 samples which was ~8.8 kgF. This observa tion supports previous findings, that the increased toughness of meat with a ultimate pH between 5.8 to 6.19 is due to slow rate of tenderization [4,17].
Earlier findings have suggested that pre-rigor temperature had the most dominant effect on the shear force values [18]. It was found that samples held at 38°C had significantly higher shear force values when compared to 15°C samples. However, in this study the effect was evident only in the intermediate pHu beef samples (Figure 3a, 3b). The difference in the find ings of the two studies may be due to the long storage at high temperature (38°C) by Kim et al [18] that would have caused the protein to denature. However, in the present study the samples were held at 35°C only for 3 h in pre-rigor compared to the 24 h at 38°C in the former study.

Cook loss
At the sampling points of 48 h and 14 days post mortem, there was a significant interaction (p<0.01) effect of AES and prerigor holding temperature on cook loss for the AES and NAES (Table 2). Previous findings have shown that cooking loss was not affected by ES [6,7]. Among the AES samples, the cook loss was greatest for AES5 and lowest for AES25. The cook loss was significantly higher for AES5 when com pared to at all the other pre-rigor holding temperatures (p< 0.05) [6,7]. Contrary to our results, earlier findings have shown that higher pre-rigor temperature results in higher cook loss [19]. The difference between the findings of the two studies may be due to the long storage at different tem perature (15°C or 35°C) until rigor. Generally cooking loss was lowest for the high pHu groups at both time points (p = 0.008 and 0.055 for 48 h and 14 days post mortem respec tively) (Figure 4a, 4b).

Purge loss
The influence of AES on purge loss was dependent on pre rigor holding temperature (Table 2; p = 0.03). Previous findings have shown that ES had no effect on purge loss [18]. Among, the AES samples, AES25 had significantly lower purge than AES5. However, in NAES samples there was no significant (p> 0.05) difference in the purge loss for the different tem peratures. Purge loss was significantly influenced by pHu ( Figure 5; p<0.05). Generally, the purge loss was greater for the low pHu group than for the high pHu group. Interest ingly, for the low pHu group (among the AES samples) the purge loss was least for 25°C than 5°C and 15°C and among NAES samples, purge loss was highest for 35°C than for 5°C and there was no difference for the intermediate and high pHu groups [18].

Drip loss
AES had no effect on drip loss at 48 h post mortem (Table 2; p>0.05), but the 3 h pre-rigor holding temperature did. For both AES and NAES samples, drip loss was higher for 5°C and 35°C and lower for 15°C and 25°C. Our findings are somewhat similar to the earlier findings [18], which reported that drip loss was higher for the meat held at 38°C pre-rigor when compared to the 15°C beef samples. After 14 days of ageing, (Table 2) drip loss was significantly influenced by AES, pre-rigor holding temperature and their interaction ef fect (p = 0.029). These results are contradictory to the earlier finding [18], which reported that additional stimulation did not influence the drip loss values. The difference between the current findings and the previous findings may be due to different pre-rigor conditions. For NAES samples there was no significant difference in drip loss between the various pre-rigor holding temperatures. Among AES samples, the samples held at 25°C for 3 h had the lowest drip loss compared to the others (p<0.05). At rigor and during the early post rigor period, the drip loss value of AES15 samples was lower compared to AES35 samples. However, as the ageing (14 days post mortem) progressed the drip loss values of the AES15 samples increased to greater than AES35 [20].
Drip loss was significantly influenced by pHu (Figure 6a, 6b; p<0.001). In the intermediate and high pHu groups there was no significant difference in the drip loss for the various pre-rigor holding temperatures. Whereas for the low pHu sam ples, the drip loss for 15°C and 25°C is lower than for 5°C and 35°C. It is well documented that the formation of drip is thought to be as a result of shrinkage of the myofibrils due to the pH fall post mortem and the denaturation of protein such as myosin, which occurs when a low pH and a high pre-rigor temperature environment is created [21]. In this study, based on the pH (Table 1) and temperature (Figure 2) data, AES35 reached low value of pH (<5.9), whereas the sample tem perature was maintained at 35°C for 3 h. The pH decline after death was much slower in AES25 and AES15 sam ples. The observed pH and pre-rigor holding temperature conditions in the AES35 are severe enough to cause the excess drip loss. Interestingly, after 14 days of ageing, drip loss was significantly influenced by AES, pHu, pre-rigor holding temperature and their interaction effect (p<0.05)  Figure 6b). However, for AES samples, there was signifi cantly higher drip loss observed at 5°C than at 25°C in the low pHu group only. As expected, drip loss was greatest in low pHu group when compared to other pHu groups. How ever, the AES25 treatment of the low pHu group had almost similar drip loss values to other samples from high and in termediate pHu groups. This observation requires further investigation to validate the finding in order to establish whether it is a feasible method of reducing drip loss in low pHu beef. It was proposed that there was a clear relation ship between drip loss and shear force values (r 2 = 0.70) which was observed in this study [20].

Water holding capacity
The water holding capacity (WHC % loss) data presented in this study is defined as the sum of the purge and the drip loss values, where lower values denote higher WHC and vice versa. The interaction between AES and pre-rigor holding     (Table 2; p = 0.009) as was previously reported [18]. A significant increase in WHC was observed for AES25 when compared to the NAES25 treatment. There was no significant difference in WHC between NAES samples due to the various pre-rigor holding temperatures, whereas there was significant difference between AES25 compared to AES5. WHC was significantly influenced by pHu (p< 0.05). Among the three pHu groups, the low pHu treatment had the least WHC for each pre-rigor holding temperature (Figure 7). By contrast, in the AES treatments, there was significantly less WHC for AES5 and AES15 for the low pHu treatment when compared to high and intermediate pHu treatments. As expected, the low pHu samples (AES 25 and AES35) had significantly lower WHC when compared to high pHu samples at the same temperatures.
Among the NAES samples, there was no significant dif ference between the various pre-rigor holding temperatures for each of the pHu samples. However, AES (in high and low but not in intermediate pHu) significantly increased the WHC for AES25 when compared to AES5 treatments. These find ings contradicted earlier findings [22], which reported low pH and high temperature condition in post mortem muscle reduced WHC due to the denaturation of muscle proteins such as myosin. In the current study, AES and suitable prerigor holding temperature such as AES25 based can override the negative effects of ES [20].

Color stability
There are inconsistent reports in the literature on the effects of ES on color stability. Some studies found no color differ ence due to ES treatment [23,24]. In general, it is documented that ES significantly improves lean color appearance, possibly by ensuring more complete post mortem glycolysis within 24 h [25,26]. Overall, the AES applied to the hotboned loin samples did not influence (p>0.05) color stability (Table 3; p>0.05).
The various pre-rigor holding temperatures and time (display days) did influence (p<0.05) color stability but no significant difference was found due to AES (p>0.05). When compared to AES5, AES35 samples had significantly lower L* values (indicator of lightness) regardless of time (display days) or stimulation and AES35 samples had the least L* values at day 1 and 4. Farouk and Swan [27], found 24 h post mortem samples held at 35°C had significantly higher L* values when compared to 5°C samples and it was suggested that high rigor temperature caused protein denaturation. In this study, sam ples were exposed to high pre-rigor temperature only for 3 h. The values of red (a* values), yellow (b*) color, hue angle (in dication of discoloration) and chroma (indication of saturation of color or vividness) values decreased as display days in creased. The values were significantly lower on day 7 when  compared to day 1. Our results are contradictory to the re cent findings of Kim et al [15], who reported that hotboned beef loins within 30 min post mortem kept in a 38°C water bath during the pre-rigor seemed to have greater L*, a*, hue and chroma angle values at 1 day post mortem compared with the loins held at 15°C, regardless of ES of the beef carcass. The discrepancy between the two studies may have been due to the long storage at high temperature (at 38°C) until rigor in their studies which had resulted in the protein denaturation and myofibrillar lattice shrinkage [24,28]. For a* and b* values, there was a significant interaction effect between display day and pHu group (Table 4). For a* there was also a significant interaction effect between 3 h prerigor holding temperature and pHu group. Among the low and intermediate pHu groups, day 1 a* values were the highest when compared to days 4 and 7. In the high pHu group, there was no significant difference due to the duration of display, except for the observation that samples held at 35°C for 3 h pre-rigor had a lower a* values for day 4 and 7 when com pared to day 1. Similarly, day 4 samples that were held at high pre-rigor temperature (i.e. 35°C) had lower a* values when compared to other pre-rigor temperature treatments. There was a significant interaction effect between display day and pHu interaction on Hue angle. For chroma angle, the dis play day and pre-rigor temperatures are influenced by the pHu group (Table 4).

CONCLUSION
The results from the current study demonstrate that AES applied, after electrical stunning and Halal slaughter did not influence the various meat quality parameters of bull beef M. longissimus lumborum except the drip loss (only at 14 days of ageing). However, the pre-rigor holding temperature (i.e. 25°C) alone or in combination with AES resulted in more tender meat, less cook loss, decreased purge and drip loss and increased WHC in bull beef samples.

CONFLICT OF INTEREST
We certify that there is no conflict of interest with any financial organization regarding the material discussed in the manu script. Farouk MM, Staincliffe M, Stuart AD, Kemp R, Craigie C are employees of AgResearch Ltd.

ACKNOWLEDGMENTS
This study was supported by the AGMARDT Postdoctoral fellowship fund (A19076). The authors would like to acknowl edge to Pete Dobbie, Sorivan ChemKieth, Kevin Taukiri, and Carolijn van der Stok (Food Assurance and Meat Quality team at AgResearch) and Suchitra Prabhu for assistance in sample and data collection.