Semimembranosus muscles (Angus×Hereford, 51 steers, 18 months of age, ~2.5 kg/steer) were obtained from a local meat market. AMP and tris (hydroxymethyl) metyl aminomethane were purchased from Sigma Chemical Co. (St. Louis, MO, USA). All the chemicals and reagents in this study were of analytical grade.

The beef meats were vacuum packed and aged at 2°C±2°C for 6 days post mortem. The muscle was removed of visible fat, fibrillar and areolar connective tissue and cut into cubes with a mean mass of 50±2 g. The raw meat was randomly divided into five groups and stored at −18°C. Before treatment, the raw beef was thawed at 4°C for about 12 h. In this experiment, raw meat that had no treatment during 30 min of conditioning was used as Control (n = 6). The second group (DW, n = 6) was marinated with deionized water (pH 7.0, 250 mL) for 30 min. The third group (UDW, n = 6) was treated with ultrasound for 5 min (20 kHz, 200 W, 15.6 W/cm², ultrasound solution is deionized water) and then marinated in deionized water (pH 7.0, 250 mL) for 25 min. The fourth group (AMP, n = 6) was marinated with 32 mM AMP solution (pH 8.0, 250 mL) for 30 min, and the last group (UAMP, n = 6) was treated with ultrasound for 5 min (20 kHz, 200 W, 15.6 W/cm², ultrasound solution is deionized water) followed by AMP (32 mM, pH 8.0, 250 mL, 25 min). All samples were treated at 20°C. Duplicate measurements per sample were taken for all analysis. After these treatments, the samples were frozen by liquid nitrogen and then kept in a −40°C refrigerator for further use.

Myofibrillar fragmentation index (MFI) was determined based on the method of Wang et al [15] with slight modifications. The above uncooked beef (2.0 g) was homogenized in phosphate buffer (0.1 M KCl, 1 mM NaN₃, 7 mM KH₂PO₄, 18 mM K₂HPO₄, 1 mM MgCl₂, 1 mM ethylene diamine tetraacetic acid, pH 7.0, 20 mL, 4°C) in a falcon tube. The homogenate was filtered using mesh strainers to remove connective tissue and centrifuged at 4°C. The pellets of myofibrils were collected and centrifuged again with added phosphate buffer. The sediment was re-suspended in buffer (10 mL) and centrifuged again. This process was repeated, and the pellet was finally re-suspended in cold buffer solution. The concentration of protein was diluted to 0.5 mg/mL with the bicinchoninic acid (BCA kit, A045-3, Jiancheng Institute of Biotechnology, Nanjing, China) and measured at 540 nm in a UV spectrophotometry for 5 times. The mean reading of absorbance was multiplied with 200 as MFI.

Beef samples were cooked in plastic bags (48±2 g, width× length, 10 cm×15 cm) individually in a water bath kettle set at 95°C until the internal meat temperature reached 75°C. After cooking, the samples were cooled immediately in ice-water bath to the internal temperature of room temperature (25°C) and wiped with filter paper to remove excess water on the surface and weighted. Cooking loss was calculated as reported by Zou et al [1].

Meullenet-Owens razor shear force (MORS) method uses an extremely sharp blade with a certain size and connects it to physical property analyzer (texture analyzer) for cutting/shearing test. MORS text of the cooked samples was measured through a texture analyzer (TVT-300XP, TexVol Instruments, Viken, Sweden). A perpendicular strip (10×10×50 mm) of 1 cm² area was cut from each muscle sample. The test speed was set at 150 mm/min and the trigger force was 200 g, and 6 technical replicates per sample were tested [7].

Denaturation of proteins in the untreated and ultrasonic treated meat (uncooked sample) was analyzed by differential scanning calorimeter (DSC). DSC data were obtained by a NETZSCH DSC (STA-449C, NETZSCH Scientific Instruments Trading Ltd., Chemnitz, Germany). Analysis was done immediately after treatments, unless otherwise stated. A blank was a sealed empty pan. Uncooked beef samples (6.0 mg) were heated at a rate of 10°C/min from 30°C to 150°C, respectively. The integrated area above the endothermic peak from the thermal curve stands for the enthalpy (J/g). At least three runs average of the calorimetric results for samples were determined, respectively.

Cross and longitudinal sections were cut from each sample. Tissues (uncooked samples) were fixed with phosphate-buffered (10%, pH 6.9 to 7.1) formaldehyde for 1 day. Blocks of tissue (20 mm×10 mm×5 mm) were chipped from the surface of the treated meat samples. After the dehydration, paraffin embedding, sectioning, and dyeing treatment, the microstructure of the beef myofibrils were observed with an optical microscope (Eclipse E100, Nikon, Kyoto, Japan). Pictures were recorded with digital video camera (DS-U3, Nikon, Japan).

All determinations were carried out with at least triplicate samples. The results in this study are presented as the mean± standard deviation. Analysis of variance with p<0.05 by means of SPSS 19.0 was employed to assess the effect of different treatment with significant difference. The Duncan’s multiple range tests were used to locate differences. All analyses including figures and calculations were conducted with the Origin Pro 9.0.

The MFI is a helpful indicator to measure the degradation degree of myofibril and tenderness in meat. The increased MFI value may be correlated with the breaking down of the MPs into segments or loss of Z-disks which occurred during treatment. The MFI values of five groups are shown in Figure 1A. Compared to Control, the value of MFI in other groups significantly increased (p<0.05) except for that of DW. Furthermore, UAMP had the highest MFI values followed by UDW and AMP. The result indicated that UAMP could enhance the MFI value over ultrasound or AMP treatment alone. The MFI values of AMP were higher than those of Control and DW. Wang et al [18] reported that AMP marination exhibited a significant increased MFI over the untreated duck breast, which was consistent with our results. The reason UAMP has a remarkable ability to increase MFI might be due to the ultrasound causing mechanical and acoustic cavitation, and turbulence. This may have led to the exposure of the hydrophobic cores, which are buried inside the molecules of the protein and disrupted the interactions between the local sequences of amino acids and between the different parts of the protein molecule. The combined effect could cause biological transformations of the beef tissue, and accelerated AMP permeation and chemical reactions inside the meat, which agrees with the reports of Kang et al [19].

The moisture data of Control, DW, UDW, AMP, and UAMP beef was 72.25%, 73.06%, 72.72%, 72.54%, and 73.13%, respectively. There was no significant difference between these different treatments. The cooking loss of the meat samples with different treatments is shown in Figure 1B. The DW had the highest cooking loss among the treatments (p<0.05). There were no significant differences in cooking loss between AMP and Control, which was different from the study of Wang et al [20]. This phenomenon might be due to the short treatment time (30 min), while the curing time of AMP was 10 h in the study of Wang et al [20]. The cooking loss of UDW was significantly lower than that of Control and DW. Ultrasound treatment could not only destroy the structure of MP and release a large amount of salt-soluble protein, but also promote the enrichment of salt-soluble protein at the surface of muscle and enhance the ability of muscle surface to prevent water spillover. The cooking loss significantly decreased in UAMP over that in UDW (p<0.05). This indicated that ultrasound treatment could improve the cooking yield in AMP solution more than in DW. With the penetration of 32 mM AMP, phosphate or other charged particles bound to beef myofibrils could induce static shielding and increase the electrostatic repulsion between filaments, which caused myofibrils expanding and increase water retention [21].

Tenderness is one of the most indispensable meat qualitative attributes for the consumer of meat and meat products. The changes of shear force in five groups are shown in Figure 1C. Among five treatments, the shear force of DW and Control did not have significant differences (p>0.05). Shear force of UDW and AMP were significantly lower than that of those two treatments (p<0.05). Wang et al [20] found that duck breast meat became tender with AMP marination (40 mM, 10 h, 5°C), which induced the breakdown of cellular architecture and disruption of the integrity of myofibrillar structure. These findings are coincidental with the research findings of Siró et al [22] who observed lower shear force values than the control when meat was treated with the appropriate ultrasonic intensity. However, McDonnell et al [23] found that there was no change in hardness of beef when samples were treated by ultrasound-assisted curing, but Ozuna et al [9] demonstrated that the hardness of pork meat increased when ultrasound treatment was applied. This result of UDW could be attributed to the formation of localized hot spots upon collapse of bubbles, and to the generation of microjets, shear forces, shock waves and turbulence which were induced by the acoustic cavitation. The cavitation not only increased the water content in beef but also produced the physical disruption and degradation of meat protein by ultrasound treatment [6]. Additionally, the lowest shear force values for UAMP were observed in this study. These results seem to confirm the positive effect of ultrasound assisting the penetration of AMP solution inside meat pieces because of the increase of spaces between myofibrils.

The cross (a) and longitudinal (b) sectional microstructures of meats in different groups are presented in Figure 2. The samples exhibited an intact cell membrane with uniform shape and narrow intervals in Control and DW, while AMP showed a small cross-sectional area compared to that of control. The reason might be the short marination time (30 min) for AMP group. The myofibrillar spacing was smaller in AMP, UAMP, and UDW than Control. However, samples in UDW and UAMP had wider fragmented cross section. On the other hand, the loss of structural integrity, tissue damage and myofibrillar swelling was seen in a longitudinal sections of UDW and UAMP as compared to other treatment. Dolatowski [24] found that ultrasound pretreatment for horseflesh combined with curing led to destruction myofibrils and rupture of cellular structures. Siró et al [22] manifested that ultrasound with 20 kHz and 2 to 4 W/cm could breakdown the Z-line and increase the wide channels between muscle myofibrils, indicating that ultrasonication for 30 to 180 min may result in an irregular nebulin network. In this study, application of UAMP resulted in damage to histiocytes, which separated beef fibers with larger intracellular spaces. This result might be owing to more water in the muscle fibers interspaces in UAMP than single ultrasound or AMP treatments. Extensive changes in the microstructure of muscle fibers have induced improvement of beef tenderness and acceptability.

The peak temperature (T_d) usually reflects the stability of biological macromolecule structure, while enthalpy (ΔH) is the required energy to arouse a conformational transformation in DSC. The Control sample showed two endothermic peaks at 66.07°C and 79.30°C (Table 1), identified as myosin and actin, respectively in the samples. Shifts in the T_d of the proteins from their control to lower T_d and lower energy required denaturing signified by destabilisation of protein structure in UDW and UAMP groups. This might be due to the unfolding of protein molecule, due to the disruption of intra-molecular bonds of myosin. Furthermore, the denaturation temperature peak of myosin and actin reduced to 63.30°C and 76.25°C in UAMP, respectively. Owing to low ion concentrations, beef filament swelled in other studies [25]. This expansion of beef filaments might weaken the bond between the F-actin polymer and myosin molecule, thus lowering denaturation temperature of proteins in AMP treatments. The lowest T_d and ΔH were observed in the UAMP treatment. The result demonstrated that ultrasound treatment could unfold the protein molecule, disrupt intramolecular bonds (such as hydrophobic interactions) and cause some degree of protein degeneration. The decline in T_d and ΔH was due to molecular transformation in protein structure in the different treatments.