Impact of pre-rigor ultrasonication on beef quality: A mechanistic perspective involving glycolytic and mitochondrial metabolism at the early postmortem

Graphical abstract

Highlights

• •Beef muscle of pre-rigor was innovatively subjected to power ultrasonication (US).
• •Pre-rigor US, particularly US600, accelerated early postmortem pH decline.
• •Both pre-rigor US improved beef quality during postmortem aging.
• •Both pre-rigor US induced mitochondrial dysfunction at the early postmortem.
• •Increased glycolytic enzymes activities and sarcoplasmic calcium levels were mainly observed in pre-rigor US600.

Abstract

This study investigated the impacts of pre-rigor ultrasonication (0, US300, and US600 W for 30 min with the frequency of 20 kHz) on pH variation and quality attributes of beef muscle, along with the underlying mechanisms related to glycolytic and mitochondrial metabolism at the early postmortem. Results demonstrated that both pre-rigor US treatments, particularly US600, accelerated early postmortem pH decline as well as improved meat color redness, water-holding capacity, and tenderness during subsequent aging. Mechanistically, US600 potentiated pH decline through dual pathways involving direct upregulation of sarcoplasmic calcium and glycolytic enzyme activities, coupled with the disruption of mitochondrial structure and function. However, US300 accelerated glycolytic flux and pH decline only through regulating mitochondrial metabolism without directly affecting glycolytic enzyme activities. Moreover, the reasons for the improved beef quality were integrated from both pH-dependent and independent pathways. This work elucidated how postmortem energy metabolism and the resulting beef quality responded to the variations in pre-rigor US, informing the rational design of potential US technology for consistent high-quality beef production.

1. Introduction

Abnormal changes in postmortem energy metabolism and thus pH decline directly determine beef quality characteristics. Specifically, insufficient or delayed pH drops are closely associated with cold shortening in beef and with the development of both typical and atypical dark, firm, and dry (DFD) beef [1], [2]. The primary shortcomings of such meat lie in its compromised color and tenderness, which severely limit consumer acceptability and the economic value of fresh beef. Traditionally, considerable attention has been paid to the link between energy metabolism, particularly glycolysis, and pH variation during the early postmortem [3], [4]. However, with the recent discovery that mitochondrial structure and function were maintained for several hours postmortem, their regulatory role in postmortem pH decline has gradually gained attention. As reviewed by Ma et al. [5], intact mitochondria could be involved in sustaining aerobic metabolism to supply energy, thereby delaying postmortem glycolysis and subsequent pH decline. Conversely, damaged mitochondria could release mitochondria-based proteins, which further increased glycolytic flux and caused a more rapid drop in postmortem pH. Thus, the development of targeted interventions to modulate early postmortem energy metabolism, mainly concerning glycolysis and mitochondria, represents a critical strategy for managing pH variation and thereby mitigating beef quality deterioration.

Ultrasound (US), a promising non-thermal technology, employs sound waves above the audible threshold of 20 kHz. Its application generates mechanical and cavitation effects, including micro-jets, shock waves, and shear forces, which can physically disrupt cellular structure and modulate enzyme activity [6], [7]. Recently, its mechanical and cavitation effects have been increasingly harnessed in meat processing to accelerate operations (e.g., tenderization and curing) and enhance product attributes (e.g., flavor and emulsification) [8], [9], [10], [11]. In terms of its action mechanism, ultrasound not only potentially disrupted cell morphology but also modulated enzyme conformation and their interactions with substrates [12], [13], [14]. It is worth noting that, to date, numerous investigations in meat science have mainly focused on ultrasound treatment of post-rigor meat (about 24 h postmortem) [9]. However, the application of ultrasound during the pre-rigor phase, a critical window of active muscle metabolism and preserved sub-organelles integrity, has received far less attention. Based on an in vitro energy metabolism model, sporadic studies, such as that by Kent et al. [15], recently reported that ultrasound exerted a limited effect on glycolytic pathway and the resulting pH change. Yet such simplified systems cannot fully replicate the complex microenvironment of intact muscle, which contains organelles, enzyme complexes, and intracellular signaling networks that collectively modulate metabolic flux [5]. Therefore, the comprehensive investigation of the effects of pre-rigor ultrasound on postmortem energy metabolism consisting of glycolysis and mitochondria within the native muscle context is warranted.

Based on the above, this study was designed to investigate the effects of pre-rigor ultrasound treatment (0.5 h postmortem) at different intensities on energy metabolism, pH alteration, and ultimately beef quality. To achieve this, we integrated analyses of meat quality, glycolytic flux, calcium homeostasis, mitochondrial stability, and targeted energy metabolomics of postmortem muscle following pre-rigor ultrasound exposure. Hopefully, this work can contribute to a theoretical basis for regulating postmortem energy metabolism and improving the consistent production of premium beef.

2. Materials and methods

2.1. Sample treatment and collection

Simmental cattle, weighing 650 ± 30 kg with the age of 24 months, were slaughtered in accordance with the Chinese National Slaughtering Standard (GB/T 19477-2018) at Shandong Huasheng Halal Meat Co., Ltd (Binzhou, China). At 0.5 h postmortem, the fifteen longissimus thoracis (LT) muscles (pH 6.71 ± 0.01) were taken out from one side of fifteen individual carcasses. Each LT muscle was immediately sliced perpendicular to its long axis into three 3-cm-thick meat samples and then individually placed into a sterile sampling bag.

All samples were assigned to US and non-US control (CON) treatments, with samples from five carcasses being subjected to identical treatment (n = 5). The meat samples in CON group were directly transferred to a 4 °C environment. For US groups, the meat samples were submerged in a water-filled ultrasonic pot (Tianhua Ultrasonic Electronic Instrument, Jining, China), sonicated for 30 min at 20 kHz (300 or 600 W) with ice being added to maintain the bath at 4 °C, and subsequently stored at 4 °C. At 0.5, 4, and 24 h postmortem, the muscle pH was detected, and 30 g of muscle were rapidly frozen in liquid nitrogen and then transferred to a −80 °C freezer for biochemical analysis. The remaining portions were used for meat quality evaluation at 24, 120, and 240 h postmortem, respectively.

2.2. pH

The postmortem muscle pH was examined via a handheld pH meter (205-PH1, Testo Fisher, Germany).

2.3. Meat quality

Meat quality was measured according to the methods of Ma et al. [16] with slight modifications. For drip loss, the muscle sample was trimmed into 1 × 1 × 2 cm³ cubes, weighed as M1, and then suspended at 4 ℃ for 48 h. Then, the sample was reweighed as M2, with drip loss being calculated as: drip loss (%) = (M1 – M2)/M1 × 100%. For the cooking loss, the muscle samples (2 × 3 × 5 cm³) were weighed (M3), packaged in boiling bags, and cooked in a water bath at 72 °C to a core temperature of 71 °C. After cooling the samples to 25 °C, the meat samples were reweighed as M4, and cooking loss was calculated as: cooking loss (%) = (M3 – M4)/M3 × 100%. Shear force was assessed using the samples after cooking loss determination. These samples were further trimmed into 1 × 1 × 3 cm strips parallel to the myofibril direction. Shear force was then measured perpendicular to the fiber orientation using a tenderness meter (C-LM, Northeast Agricultural University, China) with a shear rate of 5 mm/s. In addition, the meat color, including L*, a*, and b*, was detected by applying a colorimeter (Konica Minolta, Inc., Tokyo, Japan) with light source D65 and 2° observer. Before the determination, the freshly cut muscle sample was laid on the chopping board, and the cross-section was exposed to air for 25 min. Three parallel measurements were conducted for each sample, and the data were averaged.

2.4. Glycogen and lactate levels

Glycogen and lactate contents were detected by applying commercial kits (Jiancheng Bioengineering, Nanjing, China). The detailed steps were presented in our previous work [17].

2.5. Glycolytic enzymes activities

The activities of glycolytic enzymes, including phosphofructokinase (PFK), pyruvate kinase (PK), and lactate dehydrogenase (LDH), were analyzed by commercial kits (Solarbio, Beijing, China).

2.6. Sarcoendoplasmic reticulum (SR) calcium ATPase (SERCA) activity

Following the procedure of Zhang et al. [18], SR was isolated from postmortem muscle at 4 °C. Briefly, 2.0 g of muscle were homogenized (8,000 rpm and 20 s) in 6 mL of buffer A (0.1 M KCl, 0.1 mM EDTA, and 20 mM 3-(N-Morpholino)propanesulfonic acid (MOPS), pH 7.4) and centrifuged (5,000 g and 20 min). The supernatant was collected, and the pellet was again extracted by homogenization and centrifugation. After combining the supernatants, KCl was added, and the mixture was left to react for 20 min and then centrifuged (23,500 g and 1 h). The resulting pellet was incubated in pre-cooled buffer B (20 mM MOPS and 3 M sucrose, pH 7.0) for 1 h and centrifuged again (40,000 g and 1 h). The final SR pellet was resuspended in buffer B for SERCA activity measurement by applying an ultra-trace Ca²⁺-ATPase kit (Jiancheng, Nanjing, China).

2.7. Sarcoplasmic calcium concentration

Muscle tissue (2.0 g) was dissected into fine pieces and maintained on ice for 20 min. Afterwards, the minced sample was subjected to centrifugation at 40,000 g for 30 min (4 °C) and the supernatant was obtained. The concentration of sarcoplasmic calcium in the supernatant was assayed with a commercial calcium kit (Jiancheng, Nanjing, China) and calibrated using a standard curve covering 0 to 2,000 μM.

2.8. Mitochondria isolation

Mitochondria were isolated at 4 °C by differential centrifugation [19]. Briefly, 1.0 g of sample was homogenized in 5 mL of ice-cold buffer (0.25 M sucrose, 1 mM EDTA, and 10 mM Tris-HCl, pH 7.4) using a Dounce homogenizer with 30–40 gentle strokes. The homogenate was subjected to sequential centrifugation at 1,000 g for 15 min and then at 12,000 g for 25 min. The resulting mitochondrial pellet was suspended in buffer (0.25 M sucrose, 20 mM 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), 10 mM Tris-HCl, and 1 mM EDTA, pH 7.4). Mitochondrial protein concentration was detected through the Biuret method.

2.8.1. Mitochondrial membrane permeability

The protein concentration of mitochondria was diluted to 0.5 mg/mL with suspension buffer and then the absorbance was recorded at 540 nm.

2.8.2. Mitochondrial membrane swelling

Mitochondrial membrane swelling was determined by incubating 180 μL of mitochondrial suspension (0.5 mg/mL) with 25 μL each of 0.5 mM FeSO₄ and 0.5 mM ascorbic acid at 37 °C for 15 min, followed by absorbance measurement at 520 nm.

2.8.3. Mitochondrial reactive oxygen species (ROS) levels

ROS levels were evaluated using the DCFH-DA fluorescent probe (Jiancheng, Nanjing, China) [20]. Briefly, 1 mL of mitochondrial suspension was incubated with 2 mL of potassium phosphate buffer (50 mM, pH 7.4) containing 5 μM DCFH-DA at 37 °C for 30 min under darkness. After centrifugation at 2,500 g for 5 min, the fluorescence intensity of the supernatant was recorded at excitation/emission wavelengths of 488/525 nm.

2.9. Caspase 3 activity

Caspase 3 activity was quantified using a commercial kit (Beyotime, Shanghai, China), as previously described [21].

2.10. Transmission electron microscopy (TEM)

The mitochondrial ultrastructure was visualized using a TEM (H-7650, Hitachi, Japan) following the method of Zhang et al. [22]. Briefly, fresh muscle samples were initially cut into 0.5 cm³ cubes and fixed in 2.5% glutaraldehyde at 4 °C for 24 h. Then, the specimens were further trimmed into small cuboids (0.5 × 0.2 × 0.1 cm³) for subsequent dehydration, embedding, curing, and observation.

2.11. Targeted energy metabolomics analysis

Muscle tissue (50.0 mg) was homogenized (8,000 rpm for 5 min) with 200 μL of pre-chilled 80% methanol at −10 °C. Then, 800 μL of 80% methanol were added, vortexed, and sonicated at 100 W for 30 min (ice bath). After centrifugation (16,000 g, 20 min, 4 °C), the supernatant was obtained and vacuum-DRIED. Finally, the samples were redissolved with 100 μL of 50% methanol for mass spectrometry (MS) analysis. Specifically, the measure procedures of MS as well as the subsequent processing of the raw data were consistent with those described in our earlier publication [23].

2.12. Statistical analysis

All statistical analyses were conducted with SPSS version 26 (IBM Corporation, Armonk, NY) and the results were reported as mean ± standard error. A univariate general linear model was fitted to evaluate the influence of pre-rigor treatment, postmortem time, and their interaction, followed by Fisher's LSD test for multiple comparisons. Significance was defined as P < 0.05.

3. Results

3.1. Change in muscle pH at the early postmortem

The postmortem muscle pH is presented in Fig. 1. Pre-rigor treatment, postmortem time, and their interaction all notably affected pH (P < 0.05). All groups presented a decreasing trend in muscle pH from 0.5 to 24 h postmortem (P < 0.05). In addition, at 4 h postmortem, only US600 group was markedly lower than the CON group (P < 0.05). At 24 h postmortem, compared to CON group, both US300 and US600 groups were significantly lower, with US600 exhibiting a lower level than US300 (P < 0.05).

Fig. 1.

Changes in postmortem pH of beef LT muscle subjected to pre-rigor US. Different letters (A-C) indicate a significant difference among three groups at the same postmortem time (P < 0.05). Different letters (a-c) indicate a significant difference among postmortem time at the same group (P < 0.05).

3.2. Change in meat quality during postmortem aging

Table 1 presents beef quality attributes during postmortem aging. L* was affected only by the postmortem time. The b* value and drip loss were notably affected by pre-rigor treatment, postmortem time, and their interaction (P < 0.05), while cooking loss and shear force were notably affected by pre-rigor treatment and postmortem time (P < 0.05). A significant effect with pre-rigor treatment was observed in a* values (P < 0.05). Additionally, as compared with CON group, the drip and cooking losses as well as shear force in both US groups were notably lower throughout the entire postmortem aging (P < 0.05). These suggest that beef subjected to pre-rigor US had enhanced water holding capacity (WHC) and tenderization process. Furthermore, the L* and a* values in US groups were notably increased during postmortem aging, while the b* only increased at 240 h postmortem, with the US600 group showing higher levels (P < 0.05).

Table 1.

Effect of pre-rigor US on beef quality during postmortem aging.

Index	US	Postmortem time (h)			P-value
		24	120	240	US	Time	US × Time
L*	CON	31.65 ± 1.09Ac	34.77 ± 0.87Bb	38.06 ± 0.96Ba	0.077	< 0.001	0.228
	US300	30.11 ± 0.87Ac	37.42 ± 0.77ABb	40.42 ± 1.34ABa
	US600	31.48 ± 1.31Ac	37.74 ± 0.87Ab	41.03 ± 0.84Aa
a*	CON	12.92 ± 0.91Bb	14.65 ± 0.67Ba	15.28 ± 0.45Aa	< 0.001	0.051	0.340
	US300	16.69 ± 0.35Aa	17.37 ± 0.59Aa	16.84 ± 0.40Aa
	US600	16.47 ± 0.28Aa	17.17 ± 0.54Aa	16.87 ± 0.58Aa
b*	CON	3.39 ± 0.04Aa	3.93 ± 0.16Aa	3.83 ± 0.21Ca	0.005	< 0.001	0.010
	US300	3.74 ± 0.27Ab	4.28 ± 0.26Aa	4.59 ± 0.27Ba
	US600	3.46 ± 0.28Ab	4.08 ± 0.54Aab	5.45 ± 0.58Aa
Drip loss (%)	CON	6.50 ± 0.36Aa	4.58 ± 0.42Ab	4.57 ± 0.16Ab	< 0.001	< 0.001	0.044
	US300	4.39 ± 0.50Ba	3.32 ± 0.43Ba	3.73 ± 0.54Aa
	US600	3.38 ± 0.22Ba	3.23 ± 0.30Ba	3.90 ± 0.30Aa
Cooking loss (%)	CON	27.37 ± 0.97Aa	25.19 ± 1.30Aa	23.38 ± 0.78Ab	< 0.001	< 0.001	0.952
	US300	24.35 ± 1.32Ba	21.48 ± 0.47Bb	19.60 ± 0.84Bb
	US600	23.03 ± 1.03Ba	20.32 ± 0.71Bb	19.56 ± 0.92Bb
Shear force (N)	CON	67.54 ± 1.26Aa	56.12 ± 1.16Ab	48.47 ± 0.86Ac	< 0.001	< 0.001	0.161
	US300	57.74 ± 2.24Ba	48.52 ± 1.17Bb	41.54 ± 1.08Bc
	US600	59.41 ± 1.66Ba	43.15 ± 1.49Cb	37.77 ± 1.57Bc

Notes: Different letters (A-C) indicate a significant difference among three groups at the same postmortem time (P < 0.05). Different letters (a-c) indicate a significant difference among postmortem time at the same group (P < 0.05).

3.3. Change in glycogenolysis/glycolysis of beef muscle at the early postmortem

Glycogenolysis/glycolysis rates were reflected by glycogen and lactate levels as well as the activities of critical glycolytic enzymes. As shown in Fig. 2A and B, pre-rigor treatment, postmortem time, and their interaction all notably affected glycogen and lactate levels as well as the activities of PFK and LDH (P < 0.05). Notable effects of pre-rigor treatment and its interaction with postmortem time were observed in the PK activity (P < 0.05). As postmortem time progressed, glycogen decreased and lactate increased significantly, and this metabolic pattern at 4 h postmortem was significantly stronger in US600 than CON groups (P < 0.05). By 24 h postmortem, notable variations in glycogen breakdown and lactate production were observed among the three groups, with US600 showing the highest levels, followed by US300 and CON groups (P < 0.05). In addition, relative to CON group, the activity of PFK (4 h), PK (24 h), and LDH (4 and 24 h) was notably higher in US600 group (P < 0.05). Meanwhile, US300 treatment did not markedly impact the activities of these enzymes within 24 h postmortem.

Fig. 2.

Changes in glycogenolysis/glycolysis of beef LT muscle subjected to pre-rigor US, (A) glycogen content, (B) lactate concentration, and (C) the activity of critical glycolytic enzymes, including PFK, PK, and LDH. Different letters (A-C) indicate a significant difference among three groups at the same postmortem time (P < 0.05). Different letters (a-c) indicate a significant difference among postmortem time at the same group (P < 0.05).

3.4. Change in intracellular calcium homeostasis of beef muscle at the early postmortem

SERCA is primarily involved in reabsorbing calcium into the SR, and its changes can lead to the dysregulation of intracellular calcium. Thus, the SERCA activity and the sarcoplasmic calcium concentration were evaluated to reflect intracellular calcium homeostasis of beef muscle at the early postmortem. As presented in Fig. 3, pre-rigor treatment, postmortem time, and their interaction all markedly affected SERCA activity and sarcoplasmic calcium concentration (P < 0.05). Compared to US300 and CON groups, the US600 group showed significantly lower SERCA activity at 4 and 24 h postmortem, particularly exhibiting a reduction of more than 60% at 24 h postmortem (P < 0.05). Meanwhile, sarcoplasmic calcium rose significantly with postmortem time, and its levels in US600 group showed consistently higher compared to CON and US300 groups (P < 0.05). In addition, US300 group only became significantly higher at 24 h postmortem relative to CON group (P < 0.05).

Fig. 3.

Changes in (A) SERCA activity and (B) sarcoplasmic calcium concentration of beef LT muscle subjected to pre-rigor US. Different letters (A-C) indicate a significant difference among three groups at the same postmortem time (P < 0.05). Different letters (a-c) indicate a significant difference among postmortem time at the same group (P < 0.05).

3.5. Change in mitochondrial stability of beef muscle at the early postmortem

Mitochondrial stability was reflected by a combination of parameters, including mitochondrial swelling, membrane permeability, and ROS levels. As shown in Fig. 4A and B, mitochondrial swelling and membrane permeability, as indicated by lower absorbance values, were significantly influenced by pre-rigor treatment, postmortem time, and their interaction (P < 0.05). In addition, the extent of both exhibited a similar increasing trend over postmortem time, and this increase was significantly accelerated by US treatment, with US600 demonstrating a more pronounced effect (P < 0.05). For mitochondrial ROS levels, notable impacts of two main factors and their interaction were also observed (Fig. 4C, P < 0.05). As postmortem time progressed, mitochondrial ROS levels increased significantly, and their increase at 4 h postmortem was significantly stronger in US600 than CON groups (P < 0.05). By 24 h postmortem, significant variations in its levels were observed among the three groups, with US600 showing the highest levels, followed by US300 and CON groups (P < 0.05).

Fig. 4.

Changes in mitochondrial stability and ultrastructure of beef LT muscle subjected to pre-rigor US, (A) mitochondrial membrane permeability, (B) mitochondrial membrane swelling, (C) mitochondrial ROS levels, (D) caspase 3 activity, and (E) mitochondrial ultrastructure. Different letters (A-C) indicate a significant difference among three groups at the same postmortem time (P < 0.05). Different letters (a-c) indicate a significant difference among postmortem time at the same group (P < 0.05).

3.6. Change in caspase 3 activity of beef muscle at the early postmortem

Caspase 3 is primarily activated via the mitochondrial pathway, and its activity also serves as a reliable indicator of mitochondrial damage severity. It was observed that caspase 3 activity was affected by pre-rigor treatment, postmortem time, and their interaction (Fig. 4D, P < 0.05). In addition, a transient elevation in caspase 3 activity was observed across three groups from 0.5 to 4 h postmortem (P < 0.05), after which its levels remained stable until 24 h postmortem. Besides, although both US treatments notably enhanced caspase 3 activity within 24 h postmortem relative to CON group, the US600 group showed a stronger effect, particularly at 4 h postmortem (P < 0.05).

3.7. Change in mitochondrial ultrastructure of beef muscle at the early postmortem

TEM was employed to observe mitochondrial ultrastructure in beef muscle. As shown in Fig. 4E, mitochondrial ultrastructure gradually altered with the extension of postmortem time. Specifically, at 0.5 h postmortem, the outer membrane of the mitochondria appeared transparent and intact, and the cristae inside were long, uniform, and neatly arranged. Furthermore, in CON samples at 24 h postmortem, although a slight outer membrane disruption and swelling was observed, the mitochondrial cristae remained substantially intact. In contrast, both US treatments, particularly US600, caused more severe damage to mitochondrial structure, characterized by significant membrane and cristae disruption, vacuolization, and the collapse of mitochondria into numerous electron-dense black particles.

3.8. Targeted energy metabolomics analysis of beef muscle at 4 h postmortem

As shown in Fig. 5A-C, the PCA score plot showed that the US600 group was clearly separated from both US300 and CON groups, whereas the distinction between US300 and CON groups was less evident. This suggests that US treatment, especially US600, markedly modified energy metabolite profiles in beef muscle at 4 h postmortem. To further visualize the differential energy metabolites among the three groups, a hierarchical clustering analysis was performed in the present study. As displayed in Fig. 5E-G, a total of 35 energy metabolites were discovered using ultra-performance liquid chromatography MS with targeted metabolic profiling. Also, the similarity of energy metabolite content within groups was high, yet it was poor across the groups, implying distinct response mechanisms of postmortem energy metabolism in beef muscle subjected to pre-rigor US. To be specific, compared to CON group, only three metabolites, including nicotinamide adenine dinucleotide (NADH), flavin mononucleotide (FMN), and acetyl-CoA, were found to be markedly up-regulated in US300 group (P < 0.05). However, in US600 group, it was identified that three metabolites (glucose 6-phosphate, glucose 1-phosphate, and 6-phosphogluconate) were significantly down-regulated, while seven metabolites (lactate, glutamate, succinate, pyruvate, adenosine monophosphate (AMP), guanosine diphosphate (GDP), and guanosine triphosphate (GTP)) were significantly up-regulated (P < 0.05). Meanwhile, the AMP/ATP ratio was significantly elevated in US600 compared to CON and US300 groups (Table 2).

Fig. 5.

Targeted energy metabolomics analysis of beef LT muscle at 4 h postmortem subjected to pre-rigor US, (A-C) principal component analysis (PCA) score plot and (D-F) hierarchical clustering results of energy metabolites. * indicates P < 0.05, ** indicates P < 0.01.

Table 2.

Changes in the nucleotide pool (ATP, ADP, and AMP) of beef LT muscle at 4 h postmortem subjected to pre-rigor US treatment.

	CON	US300	US600
ATP	0.23 ± 0.01A	0.23 ± 0.01A	0.25 ± 0.01A
ADP	0.19 ± 0.06A	0.17 ± 0.01A	0.28 ± 0.08A
AMP	0.02 ± 0.01B	0.02 ± 0.01B	0.10 ± 0.03A
AMP/ATP	0.08 ± 0.06B	0.07 ± 0.05B	0.42 ± 0.15A

Different letters (A-C) indicate a significant difference among three groups (P < 0.05).

Besides, to comprehensively profile metabolic alterations, differential abundance (DA) scores were calculated to evaluate pathway-level trends. As shown in Fig. 6, the energy metabolites were mainly enriched in glycolysis, mitochondrial redox, pyruvate metabolism, pentose phosphate pathway, and adenosine monophosphate-activated protein kinase (AMPK) signaling. Importantly, pre-rigor US treatment, especially US600, contributed to the marked down-regulation of metabolites in glycolysis and pentose phosphate pathways, whereas metabolites involved in mitochondrial redox, pyruvate metabolism, and AMPK signaling were predominantly up-regulated.

Fig. 6.

Differential abundance scores plot of energy metabolites of beef LT muscle at 4 h postmortem subjected to pre-rigor US. Notes: Each circle denotes a pathway, with size proportional to the number of annotated metabolites. Color from blue to red reflects the DA score from −1 to 1, and a score of −1 or 1 indicates that all metabolites in the pathway are decreased or increased, respectively.

4. Discussion

The rate and the extent of postmortem pH decline, governed by energy metabolism, are critical to beef quality. In particular, insufficient or delayed pH decline is primarily responsible for the occurrence of cold shortening in beef and for the development of both typical and atypical DFD beef [5], [24]. Therefore, it is of great significance to develop innovative technologies for regulating early postmortem energy metabolism and thus pH variation. In the current study, it was found that pre-rigor ultrasound treatment notably accelerated pH decline within 24 h postmortem, with US600 exhibiting the most pronounced effect. Also, the underlying mechanisms were further investigated from two core aspects of postmortem energy metabolism, consisting of glycolytic and mitochondrial metabolism.

4.1. Pre-rigor ultrasound accelerated early postmortem glycogenolysis/glycolysis

Glycogenolysis/glycolysis involves the enzymatic conversion of glycogen into lactate, accompanied by a decrease in postmortem muscle pH [3]. Among the regulatory enzymes involved such as PFK, PK, and LDH, the PFK was generally considered the dominant rate-limiting enzyme, exerting greater control over glycolytic flux than the other catalytic enzymes [25]. In the current study, US600 induced varying degrees of increases in the activities of all three enzymes, coinciding with heightened glycogenolysis, reduced content of intermediate metabolites (e.g., glucose 6-phosphate), and ultimately elevated lactate accumulation within 24 h postmortem. These reveal that the accelerated pH decline in US600 samples could be partly attributable to enhanced glycogenolysis/glycolysis. Meanwhile, the reasons for the US-mediated enhancement of such processes could be explained by two aspects. First, the cavitation effect of US might directly alter the conformation of glycolytic enzymes, leading to increased exposure of their active sites and enzyme-substrate affinity [14]. Second, ultrasound likely up-regulated upstream signaling involved in the control of glycolysis, particularly AMPK signaling, which further improved enzyme activity and thus glycolytic flux. Specifically, AMPK activation is mediated by two kinases: liver kinase B1 (LKB1) which responds to an elevated AMP/ATP ratio, and calcium/calmodulin-dependent protein kinase kinase (CaMKK) which is activated by an increase in intracellular calcium [26]. In this study, targeted energy metabolomics revealed a significantly higher AMP/ATP ratio in US600 samples, further supporting the potential intensification of the LKB1/AMPK/glycolysis pathway. More importantly, US600 treatment markedly inhibited SERCA activity and elevated sarcoplasmic calcium levels within 24 h postmortem. This elevation might result from US-induced mechanical disruption and oxidative modification of the SR [9], which in turn promoted CaMKK-mediated AMPK phosphorylation and the resulting up-regulation of AMPK signaling. Collectively, these findings demonstrate that pre-rigor US600 treatment accelerated glycogenolysis/glycolysis, potentially through both direct modulation of enzyme activity and enhancement of AMPK signaling. Furthermore, unlike US600, US300 showed a relatively limited improvement on sarcoplasmic calcium levels, glycolytic flux, and pH decline, with notable changes confined to 24 h postmortem. Also, given that US300 did not enhance the activities of glycolytic enzymes and SERCA within 24 h postmortem, it is reasonable to infer that its influence on glycolysis and pH drop in intact muscle might be mediated by other pathways such as mitochondrial metabolism. This speculation is further supported by two previous studies [15], [27]. Specifically, it was found that when analyzed in intact muscle, US treatment (particularly the 30  min duration) had a significant impact on the rate of pH decline in postmortem bovine muscle. However, when tested in an in vitro glycolytic buffer system, the impact of US on glycolytic enzyme activity was limited, as evidenced by the lack of significant differences in the rates of pH decline and of reducing sugar, glycogen, and lactate conversion. Thus, the impact of pre-rigor US (especially at lower intensity) on intact muscle might be not due to permanent changes in glycolytic enzyme activity, but rather to US-induced modifications of muscle microenvironment.

4.2. Pre-rigor ultrasound impaired early postmortem mitochondrial stability

Different from glycolysis, the structure and the function of mitochondria were similarly influenced by both US applications throughout the early postmortem. These interventions consistently elevated mitochondrial swelling, membrane permeability, and ROS production as well as caspase 3 activity, accompanied by severe ultrastructural damage such as cristae disruption and membrane fragmentation. Such biochemical and morphological changes represented critical manifestations of compromised mitochondrial structure and function. Meanwhile, mitochondrial disruption could impede the progression of the tricarboxylic acid cycle and cause metabolite buildup, impairing aerobic metabolism and thus ATP generation [28]. As a consequence, the accumulation of tricarboxylic acid cycle-related metabolites (e.g., pyruvate, acetyl-CoA, and succinate) in US samples could further confirm the abnormalities in mitochondrial aerobic metabolism. The mechanism linking US to mitochondrial dysfunction could be ascribed to both direct mechanical effects and indirect calcium-mediated pathways. US-induced cavitation generated shear forces that physically damaged mitochondrial cristae and membranes. Concurrently, as sarcoplasmic calcium levels rose due to SR disruption and SERCA inhibition observed in US600 group, mitochondria might actively sequester this cation, leading to its internal calcium overload and consequently membrane disruption [29].

Research in recent years has indicated that mitochondria act as a dual role in postmortem energy metabolism. As reported by Ma et al. [5], intact mitochondria from livestock remained metabolically active for several hours postmortem, potentially delaying glycolysis by providing alternative ATP sources. However, upon structural damage, this capacity was severely impaired or even entirely reversed. To be specific, the disruption of mitochondrial cristae and membrane directly impaired electron transport chain integrity, causing the uncoupling of oxidative phosphorylation and reducing ATP generation efficiency. As a result, the glycogenolysis/glycolysis might in turn be increased to meet ATP demand, alongside a notable decline in postmortem pH. More importantly, as membrane integrity was compromised, mitochondrial-based proteins could be released into the cytoplasm and subsequently participated in the regulation of glycolysis [30]. This mechanism was supported by two in vitro studies showing that damaged mitochondria indeed accelerated glycolytic flux via enhancing ATP hydrolysis under acidic conditions, with mitochondrial F1-ATPase being identified as the primary causative agent [31], [32]. Consequently, this mitochondria-initiated metabolic pathway was likely another contributing mechanism for the accelerated decline in muscle pH following US, especially in US300 where the activities of glycolytic enzymes remained unaffected. Besides, intact mitochondria initially could enhance calcium uptake but reduce calpain-1 autolysis and thus proteolysis [33]. Therefore, US-induced disruption of mitochondrial integrity might further affect postmortem energy metabolism and pH decline by modifying proteolysis and muscle contraction processes.

4.3. Pre-rigor ultrasound affected beef quality during postmortem aging

Taking the above into account, an intensity-dependent mechanism was proposed to reveal pre-rigor ultrasound-induced modifications of postmortem energy metabolism and pH decline. Moderate-intensity treatment (US300) accelerated postmortem glycolysis and contributed to a lower ultimate pH primarily through disrupting the structural and functional integrity of mitochondria. Distinctly, high-intensity treatment (US600) exerted its effects through dual pathways. In addition to mitochondrial-mediated mechanisms similar to US300, US600 could increase glycolytic flux via improving enzyme activity and upstream signaling of glycolysis, particularly AMPK signaling and PFK. These further amplified the alteration of postmortem energy metabolism in US600 samples and thereby maximized the rate and extent of pH decline. Such intensity-dependent response highlighted a threshold effect, where higher intensities recruited more comprehensive regulatory networks to modify postmortem energy metabolism. However, despite distinct regulatory mechanisms governing postmortem energy metabolism and the differential effects on pH decline, both US treatments similarly improved water-holding capacity, tenderness, and redness (a*) during subsequent aging. The only exception was yellowness (b*), which was negatively affected by both US treatments at the later stage of postmortem aging, likely due to enhanced lipid oxidation, as reported by Fang et al. [34] and Zhang et al. [35].

Besides, the improvements in WHC and tenderness could be associated with both pH-dependent and −independent mechanisms. Regarding the pH-dependent pathway, pre-rigor US reduced the ultimate pH of beef muscle to below 5.65, which favored the activity of acid-preferring proteolytic enzymes such as caspases and cathepsins [5]. Their enhanced proteolysis contributed to increased WHC while averting the development of atypical DFD beef, a defect typically characterized by poor tenderness. Additionally, the accelerated pH declines could enable postmortem beef muscle to enter the appropriate pH-temperature window (pH 6.0 at 12–35 °C), potentially preventing the occurrence of cold shortening [36]. The pH-independent mechanism, meanwhile, could be involved in the ultrasound-induced direct disruption of myofibrils, the activation of endogenous proteolytic enzyme system, and the exacerbation of mitochondrial-derived apoptosis. Such mechanisms have been thoroughly validated in other studies that were achieved by applying ultrasound to post-rigor meat [37], [38], [39], [40]. Regarding redness in meat color, two plausible explanations emerged. First, ultrasound-mediated mitochondrial dysfunction and rapid pH decline suppressed mitochondrial oxidative metabolism, reducing mitochondrial competition for oxygen and thereby promoting the formation of oxymyoglobin [5], [41]. Second, the elevated lactate levels in US-treated samples enhanced NADH regeneration and thus metmyoglobin reduction ability, contributing to the increased oxymyoglobin and thereby a desirable bright red meat color [42].

5. Conclusion

This study demonstrated that pre-rigor ultrasound treatment, especially US600, notably altered postmortem energy metabolism, strengthened the rate and extent of pH decline, and improved beef quality. Specifically, for high-intensity treatment (US600), the underlying mechanisms involved sarcoplasmic calcium overload, increased glycolytic enzyme activities, and disruption of mitochondrial structure and function. However, moderate-intensity treatment (US300) primarily lowered postmortem ultimate pH through mitochondria-mediated metabolism without directly affecting glycolytic enzyme activity. These findings provided a theoretical foundation for optimizing ultrasound technology to achieve desired postmortem metabolic outcomes and the resulting beef quality. Future research should explore the intensity-response relationships across muscle types and consider combining ultrasound with other interventions targeting mitochondrial stability to further enhance beef quality.

CRediT authorship contribution statement

Chao Ma: Writing – review & editing, Writing – original draft, Visualization, Validation, Software, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Binyu Zhang: Writing – review & editing. Jie Li: Writing – review & editing. Lujuan Xing: Writing – review & editing. Xiangli Chen: Investigation. Wangang Zhang: Writing – review & editing, Supervision, Project administration, Funding acquisition.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.