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. 2024 Mar 3;104:106842. doi: 10.1016/j.ultsonch.2024.106842

The impact of ultrasound treatment on glycolytic enzymes when applied to crude extracts from early post-mortem bovine muscle

Mary Ann Kent a,b, Anne Maria Mullen a, Eileen O'Neill b, Carlos Álvarez a,⁎
PMCID: PMC10940754  PMID: 38460472

Graphical abstract

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Keywords: Glycolysis; Ultimate pH; Metabolites, pH decline; Carcass intervention

Highlights

  • •

    US treatment of muscle homogenates did not alter in vitro glycogen degradation.

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    US power and duration had a significant interactive effect on in vitro glycolysis.

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    The effects were observed following the 100 % amp 30 min US treatment.

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    Significant differences were observed in pH, and glucose and lactic acid content.

Abstract

The rate of pH decline post – mortem and its interaction with temperature influences the final tenderness of meat, and therefore, the manipulation of the rate of pH decline is a strategy of interest in order to obtain consistent high quality meat. Ultrasound is a potential early post - mortem carcass intervention, which may alter the rate of glycolysis based on its ability to alter enzyme activity. In this study, homogenates (prepared from early post-mortem Longissimus thoracis et lumborum muscle) were subjected to different ultrasound intensities (0 %/60 %/100 % amp) and treatment durations (15/ 30 min). The effect of these treatments on the inherent activity of the glycolytic enzymes was investigated using an in vitro glycolytic buffer model system. It was found that ultrasound treatment intensity and duration had a significant interactive effect on the rate of pH decline, and on reducing sugars and lactic acid concentrations, specifically following the 100 % amp ultrasound for 30 min treatment and between 30 and 240 min incubation. No significant differences in pH or metabolites content were observed between treatments after 1440 min of incubation. No effect of ultrasound intensity or treatment duration was observed on the degradation of glycogen. Under the reported conditions of this trial, it can be concluded that the application of ultrasound has limited potential to have an impact on the glycolytic pathways in bovine muscle.

1. Introduction

Tenderness, along with other organoleptic qualities, such as colour, flavour and juiciness, plays an important role in the overall eating quality of meat [1]. The rate of early post-mortem glycolysis [2], [3], as well as the interaction between pH and temperature decline in muscle [4], [5], has been found to play an important role in determining meat tenderness. For optimum tenderness, it has been recommended by the Meat Standards Australia (MSA) that the muscle is between the temperatures of 15 and 35 °C when pH 6 is reached, since muscle is susceptible to hot- and cold-shortening outside of this pH – temperature window [6]. Factors that influence the rate of pH decline in muscle include the muscle type (oxidative or glycolytic), the nutritional and stress status of the animal pre-slaughter [7], as well as the age, gender and size of the animal [8]. A number of post-mortem carcass interventions have been implemented in the meat industry to manipulate the rate of pH and temperature decline, such as chilling and electrical stimulation [9], [10], aimed at avoiding the detrimental effects of heat and cold shortening and optimising tenderness.

Ultrasound (US) is a non-thermal technology that involves sound waves operating at frequencies of 20 kHz or greater. Ultrasound treatment results in continuous compression and rarefaction of bubbles within the medium being treated, with the eventual collapse of the bubbles causing acoustic or transient cavitation [11] which can affect the macromolecules of enzymes and their substrates [12]. Ultrasound has been investigated for its ability to alter enzymatic activity in food systems. It can affect enzymes directly, the substrates or the interaction between the enzyme and substrate [13]. Studies have been carried out to investigate the impact of ultrasound treatment on post-mortem [14], [15], [16], and pre-rigor bovine muscle [17], [18], [19], primarily focused on the impact of ultrasound on the quality traits of the muscle, such as tenderness, colour and water-holding capacity, with varying results.

The impact of ultrasound treatment on the rate of glycolysis in pre-rigor muscle has not been studied in depth, to the best of our knowledge. Our group carried out a recent study (not yet published) investigating the impact of ultrasound treatments (25/45 kHz for 15/30/45 min) of pre–rigor bovine muscle on the inherent activities of glycolytic enzymes using an in vitro glycolytic buffer system. This glycolytic buffer system had previously been used by other researchers to study post-mortem glycolysis in porcine, chicken and bovine muscle [20], [21], [22], [23], [24], [25]. In a previous trial (not published yet), we found that the ultrasound treatment of intact bovine Longissimus thoracis et lumborum did not affect the inherent activity of glycolytic enzymes, when examined in an in vitro glycolytic buffer, post - treatment. Lyng, Allen and McKenna [18], [26] reported that ultrasound (20 kHz for periods of 15 sec) had no significant impact on the proteolysis of pre- and post-rigor bovine and ovine muscle, and hypothesised that the structure of the muscle restricted the vibrations resulting from the ultrasound treatments, reducing the intensity of the ultrasound waves travelling through the muscle. Therefore, we have hypothesised that by removing the physical protection that the whole muscle matrix may exert, US may have the potential of affecting the kinetics of glycolytic enzymes. Thus, the aim of the current study is to investigate the effect of ultrasound treatment (20 kHz, 60 % amp (11.4 W/cm2) and 100 % amp (19 W/cm2) amplitude for 15 or 30 min) of muscle homogenates (muscle:water ratio of 1:5) on glycolysis, monitored in vitro using a glycolytic buffer system.

2. Materials and methods

2.1. Chemicals and reagents

ADP, creatine, disodium hydrogen phosphate, NAD+, potassium sodium tartrate, sodium acetate (Sigma Aldrich, Ireland), ATP (Cytiva), carnosine (Apollo Scientific, United Kingdom), 3,5-dinitrosalicyclic acid, glycogen, hydrochloric acid, sodium hydroxide (Acrós Organic-ThermoFisher, UK), L-Lactic Acid assay kit (Megazyme, Ireland), magnesium chloride, potassium chloride (VWR Chemicals, Ireland), perchloric acid (Alfa Aesar, United Kingdom), potassium hydroxide (Merck Emsure, Ireland), liquid nitrogen (AirProducts, Ireland). All chemicals were at least of reagent grade.

2.2. Animal and sample preparation

Bovine Longissimus thoracis et lumborum muscles were collected from 4 crossbred steers of similar carcass classification (conformation score O - to R +, aged 22 – 29 months, average side hot weight of 183.15 ± 28.11 kg) from a local commercial slaughterhouse (Kepak, Ireland). The muscle from the left hand side was hot-boned from the carcass and collected within 90 min of slaughter. Under refrigeration, the muscles were transported to Teagasc Research Centre, Ashtown, which took approximately 60 min. On arrival, the pH and temperature of the muscle were measured at three different locations, using a temperature/pH meter, with integrated temperature compensation (Hanna HI 98163 portable pH meter for meat and FC2323 meat pH electrode, Hanna Instruments Ltd., Leighton Buzzard, UK). Prior to measurement, the pH meter was calibrated using three pH buffers of 4.0, 7.0, and 10.0. Visible fat was trimmed from the muscles and each muscle was cut into 6 steaks of equal thickness (1.5 cm). The steaks were randomly assigned to a control group and treatment group, comprised of two ultrasound treatment durations (15, 30 min) and two ultrasound powers (100 % or 60 % amp (19 W/cm2 and 11.4 W/cm2, respectively)), giving a total of 4 different combinations. A control sample was assigned to each treatment duration (15, 30 min). The steaks were finely chopped and portions (5 g), then were placed in cryovials, which were then snap frozen in liquid nitrogen. These portions of frozen muscle were pooled and powdered using liquid nitrogen and a freezer mill (SPEX SamplePrep 6750 Freezer/Mill). The powdered samples were stored at −80 °C until the time of treatment. An additional steak from each animal was finely chopped and portions (5 g) placed in cryovials, snap frozen in liquid nitrogen and stored at – 80 °C for determination of initial metabolite concentrations in muscles.

2.3. Preparation of supernatants for determination of initial metabolite concentrations in muscles

Samples for the analysis of glucose and glycogen were prepared according to Hammelman et al. [27], with modifications. Briefly, for glucose analysis, 1.5 g of frozen muscle was homogenised in 10 ml of 0.5 M perchloric acid (PCA) using an Ultraturrax (13,500 rpm for 30 sec). Aliquots (2 ml) of homogenate were centrifuged, using Sigma 1–15 Benchtop Centrifuge (SIGMA Laborzentrifugen GmbH, Germany) at 10,000 g for 10 min. For glycogen analysis, frozen muscle (1.5 g) was homogenised in 10 ml of 0.5 M PCA using an Ultraturrax (13,500 rpm for 30 sec). Homogenate (1 ml) was added to an equal amount of 2.5 M HCl, hydrolysed at 90 °C for 2 hr, centrifuged for 10 min at 4,000 g and the supernatant was neutralised with 1.25 M KOH (2 ml). Sample preparation for lactic acid analysis was carried out according to the method outlined in the L-Lactic Acid Kit Assay kit (Megazyme, Ireland), where 5 g of frozen muscle was homogenised in 20 ml of 1 M PCA using an Ultraturrax, for 5 min (2 x 2.5 min with a 30 sec break in between) at 13,500 rpm. Approximately 40 ml of distilled water was added to the homogenate. The pH of the homogenate was adjusted to pH 10 using 2 M KOH, before being made to volume in a 100 ml volumetric flask. Aliquots (2 ml) of the homogenate were removed, centrifuged for 10 min at 10,000 g and subsequently analysed.

2.4. Ultrasound treatment

For ultrasound treatment, 20 g of powdered muscle (Section 2.2) was suspended in 100 ml of water using an Ultraturrax (6,000 rpm for 30 sec). The homogenates were sonicated using a 20 kHz ultrasound probe (UIP500hdT, Hielscher ultrasound technology, Germany), where the probe, with a diameter of 1.8 cm, was submerged into the homogenate at a depth of 1.5 cm. The homogenates were treated with ultrasound for 15 and 30 min at amplitude levels of 60 % or 100 % amp (11.4 W/cm2 and 19 W/cm2, respectively), in a jacketed flask, where the temperature of the treated homogenate was maintained at 32 °C ± 2.5 °C with a circulating water bath (LTD20G, Grant Instrument, UK). The mixtures were stirred constantly throughout the treatments. Controls were prepared where 20 g of powdered muscle was suspended in 100 ml of water using an Ultraturrax (6,000 rpm for 30 sec) and the homogenate was stirred constantly for 15 and 30 min in a jacketed flask, without the application of US. The temperature of the control was maintained at 32 °C with a circulating water bath (Lauda ECO RE415S (Lauda Dr. R. Wobser Gmbh & Co. KG, Germany)).

2.5. In vitro glycolytic buffer system model system

Experiments using the in vitro glycolytic buffer were carried out based on the method of England et al. [22]. Initially, the buffer was made up to contain 20 mM Na2HPO4, 10 mM MgCl2, 120 mM KCl, 10 mM ATP, 1 mM ADP, 1 mM NAD+, 60 mM glycogen, 50 mM carnosine, 60 mM creatine and 20 mM sodium acetate (pH 7.4) (this is a double strength buffer which is subsequently diluted (1:1) on addition of the homogenate). On completion of the individual ultrasound treatments, half of the control or treated homogenate (50 ml) was added to 50 ml of the buffer and immediately transferred into a jacketed reaction buffer vessel. This resulted in an overall homogenate containing 10 mM Na2HPO4, 5 mM MgCl2, 60 mM KCl, 5 mM ATP, 0.5 mM ADP, 0.5 mM NAD+, 30 mM glycogen, 25 mM carnosine, 30 mM creatine and 10 mM sodium acetate, with a 1:10 muscle (w/v) inclusion; resulting in the final concentrations as described by England et al [22]. The temperature of the homogenate was maintained at 25 °C for the duration of the experiment, using a recirculating water bath (Lauda ECO RE415S (Lauda Dr. R. Wobser Gmbh & Co. KG, Germany)), with constant stirring using a magnetic stirrer. Aliquots were removed at 0, 30, 90, 240 and 1440 min for subsequent metabolite analysis. The pH of the glycolytic buffer system was measured at each time point using a calibrated pH meter, with built in temperature compensation (Mettler-Toledo AG, Analytical, CH-8603 Schwerzenbach, Switzerland).

2.6. Preparation of supernatants for metabolite analysis of the in vitro glycolytic buffer model system

For glycogen analysis, aliquots (1 ml) of homogenate or glycolytic buffer mixture were added to an equal amount of 2.5 M HCl, where the mixture was heated for 2 hr at 90 °C in a water bath, centrifuged for 10 min at 4,000 g and neutralised with 1.25 M KOH (2 ml). The neutralised homogenate was subsequently diluted with an equal amount of Milli – Q water prior to analysis. For glucose (and other reducing sugars) and lactic acid analysis, aliquots (2 ml) of homogenate were removed and added to an equal amount of ice-cold 1 M PCA, before being centrifuged at 4,000 g for 10 min and the supernatants were neutralised using 1 ml of 2 M KOH prior to analysis. For lactic acid quantification, a 50/50 dilution of the neutralised PCA extract was prepared with Milli – Q water to be within the detection limits of the kit.

2.7. Determination of concentration of metabolites in supernatants

The concentration of glucose (and other reducing sugars such as fructose and its phosphorylated conjugates) and glycogen (as glucose equivalents) in the supernatants was measured spectrophotometrically using the DNS method [28], [29]. Lactic acid concentration in the supernatants was determined using an enzymatic kit (L-Lactic Acid (L-Lactate) Assay Kit, Megazyme, Wicklow, Ireland).

2.8. Statistical analysis

Data in this paper is presented as average ± SEM. To determine the number of replicates required for this study, a power analysis was performed, based on preliminary studies, where the resolutions of the analytical methods and variance of glucose (and other reducing sugars), glycogen and lactic acid were evaluated. To obtain a pH difference of 0.2, based on a power of 0.8 and alpha value of 0.05, a sample size of 2 was required. From this, a sample size of 4 was selected. Analysis of all variables from the in vitro buffer was carried out using MIXED procedure of the SAS, version 9.4 (SAS Institute Inc., Cary, NC, USA) with 4 replicates and 3 factors: intensity (control, 100 % or 0 %), treatment duration (15 or 30 min) and the repeated measure factor, sample time (0, 30, 90, 240 and 1440 min). Animals were treated as a random effect. Treatment means and interactions were compared using Tukey’s test. In all cases, a P – value < 0.05 was considered as significant.

3. Results

3.1. Biochemical status of muscle prior to ultrasound treatment

The average pH value of the muscles, at 2.5 hr post-mortem, was pH 6.42 (±0.07) and the average temperature was 29.83 °C (±0.60). The pH values obtained in this study were similar to those found by Onopiuk et al. [30] and Byrne et al. [31] in bovine Longissimus dorsi, at a similar time post-mortem.

The glycogen concentration was 1.28 ± 0.13 g/100 g, which is within the range of that reported by Coombes et al. [32] and Onopiuk et al.[30], who reported quantities of between 1.36 g/100 g and 0.62 g/100 g glycogen, measured at 1 and 4 h post-mortem, respectively, in bovine Longissimus muscle. The lactic acid content of muscle used in this study, 0.28 ± 0.03 g/100 g, was within the range reported in literature for bovine Longissimus and Semitendinosus muscle between 2 and 4 h post-mortem, where quantities of between 0.22 and 0.38 g/100 g were found [30], [33]. The content of reducing sugars (mainly glucose at this stage) in the muscle used in this study was 0.068 ± 0.008 g/100 g, which was within the range found in the literature, where levels of 0.036 g/100 g [34] to 0.169 g/100 g [35] were reported in bovine Longissimus lumborum within the first 3 h post-mortem. The results show that muscles employed for this study were a faithful representation of an average bovine Longissimus muscle at 2 or 3 h post-mortem.

3.2. pH decline

When muscle homogenates were added to the glycolytic buffer system (as sources of glycolytic enzymes) the pH declined over the 1440 min duration of incubation at 25 °C, with the pH decreasing, for controls and treatments, from an initial pH of 7.42 ± 0.01, prior to the addition of muscle, to an average of pH 5.50 ± 0.04, except for buffers containing muscle treated with 100 % amp for 30 min, where the average pH after 1440 min was pH 5.78 ± 0.15 (Fig. 1). While the average pH value of the 100 % amp, 30 min treatment containing buffer system at 1440 min was numerically higher than the combined average pH of the control and other treatment containing buffers, no significant differences were observed between treatments at 1440 min. The majority of the decline in pH took place within the first 240 min. The rate of pH decline was significantly affected by ultrasound treatment duration (P < 0.05) and intensity (P < 0.05) and there was a significant interaction between these two parameters (P < 0.05); where the treatment carried out at 100 % amp for 30 min resulted in a slower rate of pH decline when compared to the controls and other treatments. The slower rate of pH decline was observed for the first 240 min in particular, with the pH of the buffer system containing the 100 %, 30 min treatment being significantly higher (P < 0.05) when compared to control and other treatment containing buffers between 30 and 240 min.

Fig. 1.

Fig. 1

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pH decline of the in vitro glycolytic reaction buffer over 1440 min (24 hr) at 25 °C following the addition of control or ultrasound treated muscle homogenates (Control for 15 min (C 15), Control for 30 min (C 30), 60 % amp for 15 min (US60 15), 60 % amp for 30 min (US60 30), 100 % amp for 15 min (US100 15), 100 % amp for 30 min (US100 30). N = 4. Data is mean ± SE.

The rate of pH decline in the glycolytic buffer system, illustrated in Fig. 1, for controls and all treatments except for that containing muscle homogenates treated at 100 % amp ultrasound for 30 min, was similar to that reported by Beline et al. [25], England et al [20], [21], [22] and Matarneh et al. [23], who used bovine, porcine and chicken muscle, respectively, as a source of glycolytic enzymes in a similar glycolytic buffer model system.

3.3. Concentration of metabolites in the in vitro glycolytic buffer model system

An in vitro glycolytic buffer system was employed in this study to assess the potential impact of the various ultrasound treatments on the inherent activities of glycolytic enzymes present in the muscle homogenates, and to monitor changes in glycogen, glucose (and other reducing sugars) and lactic acid content over a 24 h period of incubation. Overall, it was found that treatment time duration and intensity had a significant effect (P < 0.05) on the concentration of these metabolites, specifically following the 30 min, 100 % amp (19 W/cm2) treatment.

3.3.1. Glycogen

Glycogen was included in the in vitro glycolytic buffer at a concentration of 30 mM. During the incubation period, the concentration of glycogen decreased, as a result of the activity of glycolytic enzymes provided by the muscle homogenates. The majority of the glycogen degradation took place within the first 240 min, and this degradation pattern was in agreement with the findings of other studies using a similar buffer system [21], [22], [36], [37] (Fig. 2). No significant effect (P > 0.05) on glycogen degradation was observed for ultrasound intensity (Control, 60 % amp, 100 % amp) or treatment duration (15 min, 30 min) at any given sampling point (Fig. 2). These results indicate that ultrasound did not have an impact on the utilisation of glycogen, neither promoting its degradation nor impeding its depolymerisation.

Fig. 2.

Fig. 2

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Glycogen concentration (mM) of the in vitro glycolytic reaction buffer over 1440 min (24 hr) at 25 °C following the addition of control or ultrasound treated muscle homogenates (Control for 15 min (C 15), Control for 30 min (C 30), 60 % amp for 15 min (US60 15), 60 % amp for 30 min (US60 30), 100 % amp for 15 min (US100 15), 100 % amp for 30 min (US100 30). N = 4. Data is mean ± SE.

3.3.2. Glucose and other reducing sugars

In general, there was a rapid increase in the content of glucose and other reducing sugars within the first 240 min of the reaction buffer. The content of these monosaccharides continued to increase in the in vitro glycolytic buffer between 240 and 1440 min, but at a slower pace (Fig. 3). Ultrasound intensity (control, 60 % amp, 100 % amp) and treatment duration (15 min, 30 min) had a significant impact on the levels of glucose (and other reducing sugars) (P < 0.05), while a significant interaction (P < 0.05) was also observed between ultrasound intensity and treatment duration. The 100 % amp, 30 min ultrasound treatment resulted in a rapid increase in the levels of free sugars, with the content of glucose (and other reducing sugars) being significantly higher than in the control and other treatment containing buffer systems at sampling points of 30 min, 90 min and 240 min (P < 0.05). Their content in this buffer system peaked at 240 min before declining to levels similar to those of the control and other treatment containing buffer systems at 1440 min. At 1440 min, no significant differences (P > 0.05) in glucose content was observed between any of the buffer systems containing muscle of the various treatments (Fig. 3).

Fig. 3.

Fig. 3

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Glucose (and other reducing sugars) concentration (mM) of the in vitro glycolytic reaction buffer over 1440 min (24 hr) at 25 °C following the addition of control or ultrasound treated muscle homogenates (Control for 15 min (C 15), Control for 30 min (C 30), 60 % amp for 15 min (US60 15), 60 % amp for 30 min (US60 30), 100 % amp for 15 min (US100 15), 100 % amp for 30 min (US100 30). N = 4. Data is mean ± SE.

3.3.3. Lactic acid

Lactic acid content in the glycolytic buffer systems, including all treatments and controls except for the 100 % amp and 30 min treatment, increased rapidly in the first 90 min and then plateaued after 240 min (Fig. 4); which is in agreement with the pattern of pH decline. Ultrasound intensity and duration had a significant impact (P < 0.05) on lactic acid in the buffers, and a significant interaction (P < 0.05) between the two treatment parameters was also observed. The 100 % amp ultrasound treatment carried out for 30 min resulted in a significant delay in the production of lactic acid from between 30 and 240 min, where the lactic acid content of this buffer system was significantly lower (P < 0.05) to that of the control and other treatment containing buffers at the same time points (Fig. 4). No significant differences (P > 0.05) were observed in the content of lactic acid at 1440 min in any of the buffer systems.

Fig. 4.

Fig. 4

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Lactic acid concentration (mM) of the in vitro glycolytic reaction buffer over 1440 min (24 hr) at 25 °C following the addition of control or ultrasound treated muscle homogenates (Control for 15 min (C 15), Control for 30 min (C 30), 60 % amp for 15 min (US60 15), 60 % amp for 30 min (US60 30), 100 % amp for 15 min (US100 15), 100 % amp for 30 min (US100 30). N = 4. Data is mean ± SE.

4. Discussion

The initial step of glycolysis is the release of glucose from glycogen via the process of glycogenolysis and the enzyme responsible for this process is glycogen phosphorylase (GP). GP, together with glycogen debranching enzymes, release free glucose and glucose-1-phosphate to be utilised by the glycolytic pathway to produce ATP [38]. The decline in the rate of glycogen degradation following 240 min reaction time (Fig. 2) reflects a general decline in GP activity observed over time as described by England et al. [20]. The results of this study indicate that the ultrasound treatments of the muscle homogenates did not have a significant effect (P > 0.05) on the degradation or consumption of glycogen. This indicates that glycogen phosphorylase, nor the glycogen debranching enzymes, were not affected by subjecting muscle homogenates to any of the ultrasound treatments used.

The significant change in the concentration of glucose (and other reducing sugars) following the most aggressive ultrasound treatment used in this study (100 % amp for 30 min), could potentially be due to ultrasound having an impact on the activity of hexokinase, an enzyme responsible for the conversion of glucose to glucose-6-phosphate [37], [39]. Potential mechanisms that ultrasound could have an effect on the activity and structure of hexokinase includes the disruption of the enzyme’s molecular structure, as well as having an impact on the hexokinase’s active site, reducing its ability to form an enzyme-substrate complex [40]. It is interesting to note that the optimum pH for hexokinase activity is pH 7.5 and activity reduces below pH 6.5 [41]. The pH of the in vitro buffer system containing homogenate treated with 100 % amp, 30 min US, reduced from pH 7.42 ± 0.01 to pH 6.48 ± 0.20 over the first 240 min (Fig. 1) of the reaction. The pH of this buffer system remains closer to the optimum pH for hexokinase for longer and hence, if no other factor was coming into play, it could be expected that the enzyme would be more active for this treatment than controls or other treatments. However, the possible negative effect of the 100 % amp, 30 min ultrasound treatment on hexokinase activity could explain the higher reducing sugars (mainly glucose) concentrations for the 100 % amp, 30 min ultrasound treated group (compared to controls and other treatment groups) when pH is greater than pH 6.0 during the first 240 min (S Fig. 1); indicating that the inhibition originated in hexokinase activity by the 100 % amp, 30 min ultrasound treatment is more relevant than the inhibition observed after a decrease in pH, as observed in control and other treatment groups.

While hexokinase is a possible candidate for modification by the most powerful ultrasound treatment used in this study, in theory the activity of any enzyme involved in glycolytic reactions subsequent to glucose formation and the step after fructose-1,6-bisphosphatase is degraded into glyceraldehyde-3-phosphate, could be affected and hence be responsible for the accumulation of glucose (and other reducing sugars) and the delay in lactic acid production observed. Such enzymes include phosphoglucose isomerase, already mentioned PFK and aldolase. As indicated by Matarneh et al. [23], the loss of phosphofructokinase activity results in the prevention of further lactic acid production and the concomitant pH decline, as well as the accumulation of glucose and other reducing sugars such as glucose-6-phosphate and fructose-6-phosphate in post – mortem muscle. Interestingly, for the 100 % amp 30 min ultrasound treatment, it was observed that the concentration of glucose (and other reducing sugars) concentration decreased between 240 and 1440 min, allowing the in vitro buffer system to achieve a similar pHu and lactic acid content to the buffer systems containing homogenates following the control and other ultrasound treatments. Based on our experimental setup, it is not possible to determine if the inhibiting effect of ultrasound was partially reversible.

While there are few studies investigating the effects of ultrasound on enzymes linked to glycolysis, Braginskaya et al., [40] reported that hexokinase, extracted from yeast, exhibits partial loss of catalytic activity, following exposure to ultrasound, with significantly greater effects observed following 1.5 W/cm2 treatment compared to treatment of 1 W/cm2. The authors of the paper attributed the loss of activity to the mechanical damage of acoustic streaming and cavitation bubbles, resulting in a reduced enzyme-substrate affinity of hexokinase [40]. This could potentially explain the accumulation of glucose and other reducing sugars, and delay in lactic acid production within the first 30 to 240 min in the glycolytic buffer containing muscle treated with 100 % amp, 30 min US.

5. Conclusion

In this study it was observed that the ultrasound treatment of muscle homogenates using 100 % amplitude for 30 min had a significant impact on the inherent activities of glycolytic enzymes measured in vitro using a glycolytic buffer system. These effects were manifested as in changes in the rate of change of pH, and the profile of concentration of glucose (and other reducing sugars) and lactic acid during the incubation period, while the rate of glycogen consumption remained unchanged. Ultrasound power and duration had a significant and interactive effect. The rate of pH decline, as well as glucose and lactic acid concentrations appeared to differ significantly between 30 min and 240 min of incubation in the in vitro buffer model system containing muscle homogenates treated with 100 % amplitude (19 W/cm2) for 30 min, when compared to the control and other treatment containing buffers. However, no differences were found after 1440 min of incubation. Thus it appears that the strongest ultrasound treatment had no significant effect on the subsequent activity of glycogen phosphorylase but appeared to adversely affect the activity of other glycolytic enzymes. Further investigation is warranted to identify the specific enzyme(s) that are impacted by ultrasound through further experimental work.

While a change in pH decline was observed in the current study following ultrasound treatment, it was only found after applying the most intensive treatment. Based on the results of the current study on the application of ultrasound on homogenates containing powdered muscle, the potential of using ultrasound as a tool commercially to manipulate the rate of glycolysis as a mechanism to optimise tenderness is limited.

CRediT authorship contribution statement

Mary Ann Kent: Writing – original draft, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Anne Maria Mullen: Writing – review & editing, Supervision, Project administration, Conceptualization. Eileen O'Neill: Writing – review & editing, Supervision, Project administration, Methodology, Funding acquisition, Conceptualization. Carlos Álvarez: Writing – review & editing, Supervision, Resources, Project administration, Methodology, Funding acquisition, Data curation, Conceptualization.

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.

Acknowledgments

This work was funded as part of the Walsh Scholarship programme supported by Teagasc - The Agriculture and Food Development Authority of Ireland, project number 0802.

The authors would like to acknowledge Paula Reid for her statistical support.

Footnotes

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.ultsonch.2024.106842.

Appendix A. Supplementary data

The following are the Supplementary data to this article:

Supplementary data 1
mmc1.docx (21.9KB, docx)

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