Cattle transport and slaughter

Using specialized vehicles, the cattle were transported 110 km from the growing area to a Federal Inspection Type (Tipo Inspección Federal - TIF) slaughterhouse. Twelve animals per treatment (three per pen) were randomly selected for meat quality and oxidative stability measurements. Using a scale (Revuelta RGI model), the animals were weighed individually upon arriving at the slaughterhouse; average weight for males and females was 450.5 ± 30.5 kg. Transport and slaughter of animals complied with applicable federal regulations: NOM-051-ZOO-1995¹⁹, and NOM-033-ZOO-1995²⁰. The carcasses were electrically stimulated with alternating current (60 Hz, 50 volts x 2 min) immediately after slaughter, with electrodes placed on the Achilles tendon and the nose.

pH measurement and meat samples

The carcasses were cut longitudinally to produce two half carcasses and placed in cold storage (0.3 °C). Measurement of pH was done 45 min postmortem in the semimembranosus muscle using an electrode connected to a potentiometer (Hanna Instruments^®). A meat sample (approx. 15 cm long) was taken from the Longissimus thoracis muscle between the fifth and thirteenth intercostal spaces of the left half of each carcass for meat quality analysis. The carcasses remained in refrigeration for 24 ± 2 h.

Color and pH measurements in fresh meat

Color and pH measurements were done of meat samples from each carcass 24 h postmortem. After 30 min blooming, color was measured in triplicate with a spectrophotocolorimeter (MiniScan EZ HunterLab^®, USA. Iluminante D65/10°) and recorded as L* (luminosity), a* (red to green tones) and b* (yellow to blue tones). Measurement of pH was done with a previously calibrated portable digital potentiometer (pH-meter, Hanna Instruments^®, USA). All analyses were done in triplicate²¹. After the measurements were taken the samples were vacuum packed and frozen at -20 °C until analysis at the Meat Laboratory of the National Animal Physiology and Improvement Disciplinary Research Center (Centro Nacional de Investigación Disciplinaria en Fisiología y Mejoramiento Animal) of the INIFAP in Colón, Querétaro, México.

Sample preparation and analysis of aged meat

Using a bench saw (St-295-PE, Torrey^®, Mexico), each frozen sample was sectioned into five approximately 1-inch-thick cutlets from the region near the cranial area. The cutlets were weighed and numbered as cut, and placed in a vertical refrigerator (Torrey^®, Mexico) at 2 °C. Once defrosted they were weighed again to calculate water loss by subtracting defrosted weight from frozen (-20 °C) weight, which was expressed as the percentage weight lost compared to the initial weight. Cutlet No. 1 from each individual was used to evaluate pH²², color^23,24, water holding capacity²⁵, concentration of thiobarbituric acid-reactive substances (TBARS)²⁶, and activities of the enzymes glutathione peroxidase (GPX) and catalase (CAT)²⁷. Cutlet No. 3 was used to measure drip water loss²⁸. Cutlet No. 4 was prepared on an electric grill and then measurements were taken of shear force and water loss from cooking²⁹. Cutlets numbers 2 and 5 were placed on foam trays, covered with plastic wrap and aged for 8 days at 2 °C. After aging, measurements were taken of pH, color, water loss from cooking, shear force, lipid oxidation by TBARS, GPX and CAT activity. All measurements were done in triplicate.

Statistical analysis

All variables were processed with an ANOVA using a 2 x 2 factorial arrangement, and PROC MIXED in the SAS ver. 9.3 package.

Statistical model:

μ = effect of general mean;

α_i = effect of i-th treatment of mineral source;

β_j = effect of j-th treatment of vitamin E;

(αβ) _ij = effect of mineral source / vitamin E interaction;

E_ijk = random error of each observation.

Also using SAS, a multivariate analysis with Pearson correlations was run to evaluate the relationships between the studied variables.

Muscle pH values 45 min postmortem

At 45 min postmortem, no differences (P>0.05) in pH were observed due to mineral source, vitamin E or their interaction (Table 3). Values ranged from 6.1 to 6.3, which are similar to those reported in studies using electrical stimulation after bleeding in cattle³⁰.

pH values 24 h postmortem, and after 1 and 8 days’ aging in laboratory

After 24 h refrigeration meat pH differed by mineral source (P<0.05), but no effect was found for vitamin E or the mineral source / vitamin E interaction. In the chelated minerals treatment pH values were higher (5.67 ± 0.05) than in the inorganic minerals treatment (5.55 ± 0.03; Table 3). This same effect on pH has been reported in response to supplementation with Se from chelated and inorganic sources¹¹, although always within ranges considered normal (5.4 to 5.87)^31,32, that do not affect meat organoleptic characteristics. However, these small variations in pH may have affected the configuration of some proteins, which in turn may be related to the observed differences in meat water holding capacity after defrosting, since pH was more acidic in the inorganic source treatment than in the chelated source treatment. No differences in pH were observed between the one and eight days’ aging treatments.

Color, water holding capacity and oxidative stability after aging

Oxidation of polyunsaturated fats in beef rapidly causes rancidity, but also affects its color, quality and texture³⁴. In terms of color, luminosity (L*) was not affected (P>0.05) by mineral source, vitamin E, their interaction or aging period (Table 3 and 4). In contrast, a* and b* values were affected (P<0.05) by aging period in all treatments. Red and yellow tones decreased in the meat samples as aging period increased (Table 5), which is associated with the negative correlation between the a* value and TBARS³³, and the related red tones with oxidation processes.

The value of b * was higher in treatments with inorganic minerals (P<0.05) at one and eight days’ aging (Table 4). This difference was due to an increase in oxidation of oxymyoglobin to metmyoglobin³⁵, which produced a brown color³⁴. Greater TBARS activity at eight days’ aging in the inorganic mineral treatments was largely responsible for this phenomenon (Table 6).

Water loss from defrosting

Mineral source affected (P<0.05) water loss from defrosting (WLF), but vitamin E and the mineral source / vitamin E interaction did not. Water loss was greater (P<0.05) in cutlets in the chelated minerals treatment than in the inorganic minerals treatment. At 24 h postmortem cutlet pH was higher in the chelated than in the inorganic minerals treatment (P<0.05), which favored stability of myofibrillar proteins and therefore water retention. A portion of the water retained in the meat in the chelated minerals treatment may have become ice crystals when frozen; when defrosted these crystals would have caused greater water loss during aging. In addition, the higher CAT activity in this treatment may be related to this greater water loss.

Water loss from drip

Vitamin E in the diets affected water loss from drip (WLD) (P<0.05), although mineral source and the mineral source / vitamin E interaction did not (P>0.05, Table 4). Supplementation with vitamin E favored antioxidant activity at the cell membrane level³⁶ and allowed the cell to preserve its sarcoplasmic components during storage³⁷. This antioxidant action is also linked to the lower TBARS activity in the vitamin E-supplemented treatments (Table 5).

Water holding capacity

Water holding capacity (WHC) was higher (P<0.05) in the chelated mineral treatments than in the inorganic mineral treatments. Vitamin E and the mineral source / vitamin E interaction had no effect (P>0.05). One of the conditions that can alter the arrangement of myofibrillar proteins and the space between them is net charge. This can be modified by changes in the anion/cation balance, especially the replacement of bivalents with monovalents³⁸, as in the case of added saline solution. In contrast, carcasses and cutlets from the inorganic mineral source treatments exhibited less water loss, even when showed less retention to the added saline solution used for the water holding capacity test. At 24 h postmortem the pH values in the chelated minerals treatments (Table 3) favored WHC because the myofibrillar proteins were further from their isoelectric point (pH 5.4-5.5)³⁸, leading to protein stability and their binding to water molecules.

Shear force after 1 and 8 days’ aging

After one day of aging shear force (SF) was unaffected (P>0.05) by mineral source, vitamin E or their interaction. However, at eight days SF had decreased (P<0.05) in response to mineral source, but not due to vitamin E or the mineral source / vitamin E interaction (Table 4). Shear force positively correlated to water loss from cooking (r= 0.53; P<0.001; Table 6) and negatively correlated to pH (r= -0.23, P<0.05). A scale developed by Shackelford et al³⁹ for SF in beef uses four categories: <3.2 kg= very soft meat; 3.2 to 3.89 kg= soft meat; 3.89 to 4.59 kg= intermediate; and >4.6 kg= hard. Based on this scale, after one day aging the cutlets in all four treatments would be considered “hard”. By day eight the cutlets in the chelated minerals treatment qualified as “soft”, those in the inorganic minerals only treatment were “intermediate”, and those in the inorganic minerals / vitamin E treatment were “hard”.

Values for pH equal to or greater than 5.8 are considered unacceptable in beef, and produce dark-colored cuts⁴⁰. These are characterized by having higher WHC and lower SF^41,42, as well as high pH values (>6.0) which can increase Z disk degradation⁴³. The cutlets from the chelated mineral treatments exhibited higher pH values, and thus also had greater WHC and lower SF values. Another factor that may have lowered SF is supplementation with chelated Se, which causes greater accumulation of seleno amino acids, and modifies muscle tissue structure in such a way that it lowers SF¹¹. Softening of meat is related to aging during which enzymes such as calpain and cathepsin exercise proteolytic action on muscle fiber structural proteins^38,44. After 8 days’ aging enzymatic action had noticeably softened the cutlets, which was particularly favored by higher pH values in the chelated minerals treatments (Table 3).

Water loss from cooking after 1 and 8 days’ aging

Water loss from cooking of cutlets after one or eight days’ aging was not affected by mineral source, vitamin E, or their interaction. Aging time did have an effect since water loss was higher after one day than at eight days (Table 4). All the cutlets subjected to cooking had been previously frozen. This can modify WHC⁴⁵ by causing immobilized water to become ice crystals⁴⁴, which, when defrosted, become free water that can be lost during the aging process.

TBARS in cuts after one and eight days’ aging

Thiobarbituric acid-reactive substances (TBARS) concentrations were unaffected by any of the treatments after one day of aging (P>0.05), although after eight days the mineral source / vitamin E interaction did have an effect (P<0.05; Table 5). Values for TBARS increased four- to six-fold in the chelated minerals treatments, and from eight- to over 14-fold in the inorganic minerals treatments. The difference in TBARS activity between one and eight days’ aging is due to fat peroxidation, which increased as the meat samples aged under refrigeration. At day eight, the difference between the inorganic only treatment (T1) and the chelated only treatment (T3) may have been caused by the difference in the concentration of cofactors (e.g. Se) stored in the tissue. For example, in one study muscle Se concentrations were higher in cattle fed chelated minerals’ supplements⁴⁶. Vitamin E also prevents oxidative deterioration by neutralizing the effect of free radicals^8,37; in the present results this effect can be seen in the lower TBARS activity in the treatments containing vitamin E.

Catalase

Activity of the CAT enzyme was higher in the chelated minerals treatment at both aging times (P<0.05; Table 5), but vitamin E and the mineral source / vitamin E interaction had no effect on this variable. Apparently the increased CAT activity was associated with the higher bioavailability of chelated minerals such as Cu. This element participates, via ceruloplasmin, in oxidation of Fe from the heme group, a CAT cofactor, in this enzyme’s first action stage on hydrogen peroxide¹⁷. The higher CAT activity in this treatment also helped to reduce fat oxidation.

Glutathione peroxidase

Neither mineral source, vitamin E nor their interaction affected GPX activity. In contrast, this activity was lower at one days’ aging than at eight days’ (P<0.05; Table 5). This is related to free radical activity which increases with time due to meat exposure to the environment and bacterial multiplication. A previous study also found no effect on GPX activity in response to mineral source (chelated or inorganic) and presence or absence of vitamin E in beef cattle, and concluded that feed Se concentrations were sufficient to meet the requirements in all treatments⁸.