Ultrasonicated brine: implications for polyphosphate functionality and beef quality

TL;DR

Ultrasonication of brine improves the effectiveness of polyphosphate in enhancing beef quality, potentially reducing the need for high phosphate levels.

Contribution

Ultrasonication of brine before adding polyphosphate enhances meat quality more effectively than traditional methods.

Findings

Ultrasonicated brine before polyphosphate addition improved water-holding capacity and reduced cooking loss.

Ultrasonication promoted protein unfolding and a denser protein network, enhancing thermal stability.

Lower phosphate levels achieved comparable functionality when brine was ultrasonicated prior to phosphate addition.

Abstract

This study evaluated whether ultrasonication of brining solutions can enhance the functional effectiveness of polyphosphate in beef systems, thereby providing a technological basis for reducing phosphate dependency. A 1.5% (w/v) NaCl brine was ultrasonicated using probe ultrasound (20 kHz, 300 W) for 0, 15, or 30 min either before polyphosphate addition (UBP) or after polyphosphate addition (UAP). Sodium tripolyphosphate was incorporated at 0, 0.1, or 0.2% (w/v), and brines were mixed with ground Longissimus lumborum at a 1:1 (w/v) ratio. Brine pH and redox potential were measured, and meat pH, water-holding capacity (WHC), cooking loss, emulsifying activity and stability, interfacial protein concentration, and color parameters were evaluated. Polyphosphate concentration was the dominant factor influencing meat pH, WHC, cooking loss, and emulsifying properties. Brine ultrasonication significantly modulated these effects, particularly through interactions with polyphosphate concentration. Pre-sonication of brine followed by polyphosphate addition (UBP) consistently resulted in higher WHC, lower cooking loss, and improved emulsifying activity compared with non-sonicated brine at the same phosphate levels. In contrast, ultrasonication of phosphate-containing brine (UAP) showed limited or inconsistent benefits at low phosphate concentrations. Structural and spectroscopic analyses (FESEM and FTIR) revealed that ultrasonicated brine promoted protein unfolding and formation of a denser, more cohesive protein network, consistent with improved water retention and thermal stability. Overall, the results demonstrate that ultrasonicated brine acts as a process-level enhancer that intensifies the functional performance of polyphosphate. While increasing phosphate concentration continuously improved meat quality, brine ultrasonication—particularly when applied prior to phosphate addition—amplified these effects, supporting the feasibility of achieving comparable functionality at lower phosphate inputs.

Figures

Interaction plots showing the effects of phosphate level and processing time on brine pH for UBP and UAP treatments. Data represent mean ± SD (n = 3). Different letters indicate significant differences among treatment combinations (P < 0.05).

Conceptual mechanistic diagram illustrating the differential pathways of UBP (phosphate added after sonication) and UAP (phosphate added before sonication). The diagram summarizes the proposed cavitation-mediated effects of brine ultrasonication on protein structural responses and the resulting changes in water-holding capacity and emulsifying properties. This schematic represents a conceptual framework and does not imply direct causality.

Two-panel interaction plots showing the effects of phosphate level and ultrasonic time on water-holding capacity (WHC, %) for (A) ultrasonicated NaCl before phosphate addition (UBP), and (B) ultrasonicated NaCl after phosphate addition (UAP). Data represent mean ± SD (n = 3). Different letters indicate significant differences among treatment combinations (P < 0.05).

Two-panel interaction plots showing the effects of phosphate level and ultrasonic time on cooking loss (%) for (A) ultrasonicated NaCl before phosphate addition (UBP), and (B) ultrasonicated NaCl after phosphate addition (UAP). Data represent mean ± SD (n = 3). Different letters indicate significant differences among treatment combinations (P < 0.05).

B](#f0025)). When polyphosphate was combined with prolonged brine ultrasonication, a reduction in ESI was observed. Because meat proteins were not directly exposed to ultrasound in this study, this effect is attributed to indirect changes induced by the ultrasonicated brine during subsequent emulsification. Prolonged ultrasonication of brine may increase protein unfolding and promote protein aggregation which reduce the flexibility of the interfacial protein film, increasing the likelihood of droplet association. In addition, protein-mediated droplet flocculation, arising from bridging interac

Interaction effects of polyphosphate concentration and brine ultrasonication time on color attributes of meat, including lightness (L*), redness (a*), yellowness (b*), and total color difference (ΔE). Results are shown separately for brine ultrasonicated before phosphate addition (UBP) and after phosphate addition (UAP). Data represent mean ± SD (n = 3). Different letters indicate significant differences among treatment combinations (P < 0.05).

Scanning electron microscope images of myofibrillar proteins from non-ultrasonicated brine without phosphate (UBP0), ultrasonicated brine before phosphate addition (UBP30), and ultrasonicated brine after addition of 0.2% phosphate for 30 min (UAP30).Fig. 8FTIR spectra of myofibrillar proteins from non-ultrasonicated brine without phosphate (UBP0), ultrasonicated brine before phosphate addition (UBP30), and ultrasonicated brine after addition of 0.2% phosphate for 30 min (UAP30).

Full text

Introduction

1

Growing concerns about the use of artificial additives in processed meats have limited their consumption over the past decade because of potential health risks associated with these compounds [1]. In the meat processing industry, sodium chloride (NaCl) and polyphosphates play an important role in enhancing flavor, preserving product quality, and extending shelf life. NaCl provides the characteristic salty taste and acts as a preservative; it also improves aroma and can suppress the bitterness of certain compounds [2]. Polyphosphates are commonly applied to improve oxidative and microbiological stability and to enhance texture and tenderness [3]. Together, these additives help maintain product moisture and water content, improving eating quality and storage stability. However, excessive polyphosphate intake is a serious concern for patients with kidney disease, as impaired excretion elevates serum polyphosphate, which may reduce calcium absorption and contribute to bone disorders [2]. Even in healthy individuals, long-term high polyphosphate intake has been linked to an increased risk of bone and metabolic disorders; therefore, limiting polyphosphate addition to meat products is increasingly important. To reduce polyphosphate levels in meat products while maintaining quality, various alternatives have been investigated, including modified starches [4], protein [5], edible fiber [6], carbonate salts [5], and alkaline compounds [7]. Although these strategies may mitigate health risks, they can also increase processing costs and influence product performance [5].

Recently, new technologies that are environmentally friendly, energy-efficient, and capable of shortening processing time while improving shelf life have gained considerable attention [8]. Among these, ultrasound is notable for its ability to modify food systems through acoustic cavitation. Cavitation generates shock waves and shear forces that alter protein functionality and can improve the water-holding capacity (WHC) of meat [9]. Microjets generated during bubble collapse have been proposed to interact with myofibrils, potentially inducing micro-scale disruptions within the protein matrix and enhancing additive diffusion, as discussed in recent reviews [10]. Ultrasound may also enhance electrolyte solubility, including NaCl and polyphosphates, which are essential for increasing juiciness, tenderness, and yield during immersion, injection, or other brining methods. Conventional brining improves marination but is often slow, costly, and susceptible to microbial growth; therefore, ultrasound-assisted brining has been explored to accelerate salt transport and improve product quality while reducing processing time and operational costs [11], [12], [13], [14], [15]. In many applications, however, the meat itself is directly exposed to ultrasonic waves).

The objective of this study was to evaluate whether ultrasonication of the brining solution, applied prior to meat contact, can enhance the functional effectiveness of polyphosphate in beef systems and improve meat quality attributes without direct ultrasonic treatment of the meat. Specifically, the study compared the effects of brine ultrasonication applied before versus after polyphosphate addition on physicochemical, functional, and structural properties of beef.

Materials and methods

2

Materials

2.1

Sodium chloride (NaCl), tripolyphosphate (Na_5_P_3_O_10_), hydrochloric acid (HCl), boric acid (H_3_BO_3_), sulfuric acid (H_2_SO_4_), potassium sodium tartrate tetrahydrate (C_4_H_4_KNaO_6_·4H_2_O), copper sulfate pentahydrate (CuSO_4_·5H_2_O), and sodium dodecyl sulfate (SDS, C_12_H_25_SO_4_Na) were purchased from Merck Co., Germany. All reagents were of analytical grade.

Preparation and maintenance of samples

2.2

Five Longissimus lumborum muscles of Holstein veal (3–5 years old) were purchased within 12 h post-mortem from a local supermarket. The pH of the muscles was measured, and three muscles with pH 5.5–5.8 were selected to avoid DFD characteristics. Visible fat and connective tissue were removed and the muscle surface was briefly rinsed with chilled sterile distilled water to remove residual blood and surface debris, followed by immediate draining. Samples were minced whitin 20 h post-mortem using a meat grinder (Pars Khazar, Iran) quipped with a 4-mm diameter grinding plate to obtain uniform particles, packed in polyethylene bags, and then freezed and stored at −18 °C still-air freezer until analysis.

Meat treatment

2.3

Frozen samples were thawed at 20 ± 2 °C prior to treatment. A 1.5% (w/v) NaCl brine solution was prepared. A 1.5% (w/v) sodium chloride solution was used, as this concentration is commonly applied in meat curing systems and is sufficient to promote myofibrillar protein solubilization and protein–water interactions under practical processing conditions. Then brine was ultrasonicated using a probe-type ultrasonic processor operating at a constant frequency of 20 kHz and a nominal power 300 W, corresponding to a nominal and actual intensity of 382 W/Cm^2^ and ∼270 W/Cm^2^, respectively. The ultrasound probe (1 cm in diameter) was centrally immersed in 250 mL of brine contained in a cylindrical vessel (127 mm height, 85 mm internal diameter), with the probe tip positioned approximately 20 mm above the bottom. All treatments were conducted in batch mode.

Ultrasonication was applied for 0, 15, or 30 min, generating brines named as UBP0, UBP15, and UBP30, respectively. Ultrasound was applied exclusively to the brining solution; meat samples were not directly exposed to ultrasonic waves. During ultrasonication, the bulk temperature of the brine was continuously monitored and maintained between 10 and 20 °C using an ice bath.

Following ultrasonication, sodium tripolyphosphate was added to each brine at concentrations of 0, 0.1, or 0.2% (w/v). The polyphosphate-containing brines were immediately mixed with thawed ground meat at a brine-to-meat ratio of 1:1 (w/v) and homogenized using an Ultra-Turrax homogenizer (T25 digital, IKA, Germany) at 10,000 rpm for 2 min.

In the second experimental set, a 1.5% (w/v) NaCl brine was first supplemented with sodium tripolyphosphate at 0, 0.1, or 0.2% (w/v) and then ultrasonicated at 300 W for 0, 15, or 30 min under temperature-controlled conditions (15 ± 5 °C) using an ice bath, producing UAP0, UAP15, and UAP30 brines, respectively. The treated brines were subsequently added to ground meat at a 1:1 (w/v) ratio and homogenized under the same conditions described above.

Proximate analysis

2.4

Proximate composition of meat samples was determined after 24 h postmortem according to AOAC methods. Moisture content was measured by oven-drying at 105 °C to constant weight (AOAC 950.46). Crude protein was determined using the Kjeldahl method with a nitrogen-to-protein conversion factor of 6.25 (AOAC 981.10). Crude fat content was measured by Soxhlet extraction (AOAC 960.39), and ash content was determined by incineration in a muffle furnace at 550 °C (AOAC 920.153) [16].

pH and redox potential (Eh) of brines

2.5

The pH and redox potential of all brine solutions was measured prior to mixing with meat. Redox potential (Eh) was obtained using a potentiometric titration apparatus (Nanbei company, China) based on mV. Brine pH was determined by direct immersion of the electrode using a pH meter (model 3520, Jenway, United Kingdom) [17].

Effect of ultrasonicated brine on meat characteristics

2.6

pH measurement

2.6.1

Ten grams of meat were homogenized with 90 mL of distilled water at 15,000 rpm for 20 s. The pH was measured at 25 °C using a pH meter (model 3520, Jenway, UK) [18].

Water holding capacity (WHC)

2.6.2

WHC was determined following Jiang, Li, Tu, Zhong, Zhang, Wang and Tao [19]. Briefly, 30 g of meat suspension were transferred into pre-weighed Falcon tubes and centrifuged at 10,000g for 10 min at room temperature. The supernatant was discarded, tubes were re-weighed, and WHC was calculated using Equation (1).

$$[eqn]$$

Cooking loss

2.6.3

Ground beef samples were sealed in heat-resistant polyethylene cooking bags, ensuring minimal headspace before cooking, and heated in a water bath at 85 °C for 20 min until the internal temperature reached 70 °C. Cooked samples were cooled to room temperature, exudate was removed, and the samples were re-weighed [20]. Cooking loss was calculated using Equation (2).

$$[eqn]$$

Emulsifying properties

2.6.4

Emulsifying properties were evaluated using a homogenized meat suspension, following the method of Yu, Li, Sun, Yan and Zou [21] with minor modifications. Briefly, a homogenized meat suspension adjusted to a concentration of 0.1% (30 mL) was homogenized at 10,000 rpm for 2 min using an Ultra-Turrax homogenizer (T25, IKA, Germany). The suspension was then mixed with 10 mL of oil and further homogenized at 15,000 rpm for 1 min. Immediately after emulsification, 50 μL of emulsion was collected from the bottom of the tube, diluted with 5 mL of 0.1% SDS, vortex-mixed, and the absorbance was measured at 500 nm.

For emulsion stability, the same procedure was repeated after 10 min. Again 50 μL suspension was pipetted out from the bottom of emulsion and mixed with SDS solution. The absorbance was read at 500 nm. The emulsifying activity index (EAI) and emulsion stability index (ESI) were calculated using Equations (3), (4).

$$[eqn]$$ $$[eqn]$$

where A is absorbance at 500 nm, D is the dilution factor, C is protein concentration (g mL^−1^) before emulsification, and ϕ is oil volume fraction (v/v). A_0_ and A_10_ represent absorbance at 0 and 10 min, respectively.

Interfacial protein concentration

2.6.5

Emulsions were centrifuged at 10,000 rpm for 30 min, and the supernatant was filtered through a 0.22 μm syringe filter. One milliliter of filtrate was mixed with 4 mL Biuret reagent and incubated in the dark for 20 min. Absorbance was measured at 540 nm, and protein concentration was determined using a bovine serum albumin standard curve [22].

Color changes

2.6.6

The colour of ground beef samples was measured using a colourimeter (ZE6000, Nippon Denshuko, Japan) operating in the CIE L*, a*, b* colour space. The instrument was calibrated prior to measurements using a standard white calibration plate provided by the manufacturer. Measurements were performed using D65 illuminant and a 10° standard observer, with an aperture diameter of 10 mm. Ground beef samples were gently packed into Petri dishes to obtain a uniform surface. Colour parameters L* (lightness), a* (redness), and b* (yellowness) were recorded. Three readings were taken at different locations on each sample, and the average value was used for statistical analysis.

Microstructure

2.6.7

Myofibrillar proteins were extracted from meat treated with UAP0 and UAP30 brines. Minced meat was homogenized in 0.02 mMbuffer (pH 7.0) at a 1:10 (w/v) ratio using an Ultra-Turrax (T25, IKA, Germany) for 2 min. Sarcoplasmic proteins were removed by centrifugation at 8000 g for 20 min at 4 °C (Z36 HK, Hermle, Germany). The pellet was washed twice with polyphosphate buffer containing 0.6 M KCl and centrifuged under the same conditions. After the second wash, the supernatant was filtered through polyethylene strainer (18 mesh) to remove connective tissue and debris. The separated myofibrillar proteins was stored at −80 °C and freeze-dried [23]. Microstructures were observed using a field-emission scanning electron microscope (FE-SEM, TESCAN Mira3 XMU, USA Inc.) after sputter-coating dried powders with gold [24].

Fourier transform infrared spectroscopy (FTIR)

2.6.8

Fourier transform infrared (FTIR) spectra were obtained using an infrared spectrometer (Tensor 27, Germany). FTIR spectra of freeze-dried myofibrillar proteins were recorded at 25 °C over 4000–400 cm^−1^ [25].

Statistical analysis

2.7

All experiments were conducted using a factorial experimental design. Data were analyzed using a three-way analysis of variance (ANOVA) to evaluate the effects of ultrasound treatment sequence (UBP vs. UAP), ultrasonication time (0, 15, and 30 min), and polyphosphate concentration (0, 0.1, and 0.2%), as well as their interaction effects. When significant interaction effects were detected (P < 0.05), simple effects were examined using slice analysis, and mean comparisons were performed within relevant factor combinations. In the absence of significant interactions, main effects were interpreted. Mean comparisons were carried out using Tukey’s honestly significant difference (HSD) test at a significance level of P < 0.05. Results are presented as mean ± standard deviation (SD). All statistical analyses were performed using SAS software (version 9.4; SAS Institute Inc., Cary, NC, USA). All treatments were prepared in triplicate as independent experimental replicates.

Results and discussion

3

Chemical composition

3.1

The veal Longissimus lumborum muscle used in this study contained 74.00 ± 0.11% moisture, 2.83 ± 0.04% fat, 22.02 ± 0.20% protein, and 1.15 ± 0.01% ash. These values confirm that the raw material had a typical proximate composition [26], suitable for evaluating the effects of ultrasonicated brines.

Effect of ultrasound treatment on properties of brine

3.2

pH

3.2.1

The pH of the brine was significantly affected by polyphosphate concentration (P < 0.0001) and ultrasonication time (P < 0.01), with a significant ultrasonic time × polyphosphate interaction (P < 0.01). In contrast, the sequence of ultrasound application relative to polyphosphate addition (UBP vs. UAP) did not significantly influence brine pH (P > 0.05). These results indicate that changes in brine pH were governed primarily by polyphosphate chemistry and the duration of ultrasonic treatment rather than by the ultrasound sequence itself.

Addition of polyphosphate caused a pronounced increase in brine pH from mildly acidic values (≈5.4–5.9) to strongly alkaline levels (≈9.1–9.6) (Fig. 1). This sharp alkalization is consistent with the inherent buffering capacity and alkaline dissociation behavior of sodium polyphosphates in aqueous systems, which has been widely reported in food and meat-processing literature. The magnitude of this elevation depends on the type and concentration of polyphosphate as well as environmental conditions, including the presence of other ions [27].Fig. 1. Interaction plots showing the effects of phosphate level and processing time on brine pH for UBP and UAP treatments. Data represent mean ± SD (n = 3). Different letters indicate significant differences among treatment combinations (P < 0.05).

Importantly, the significant effect of ultrasonication duration on brine pH demonstrates that the duration of ultrasonic exposure modulated the expression of polyphosphate alkalinity. Increasing ultrasonication time resulted in a gradual elevation of brine pH at polyphosphate-containing levels, indicating that ultrasound promoted time-dependent physicochemical processes in the aqueous phase. Ultrasonic cavitation and acoustic streaming are known to enhance micro-mixing, mass transfer, and ion hydration in liquids, thereby accelerating polyphosphate dissociation, redistribution of ionic species, and establishment of acid–base equilibrium [28].

The presence of a significant sonication time × polyphosphate interaction suggests that the influence of ultrasonication time became more pronounced as polyphosphate concentration increased. In polyphosphate-free brines, ultrasonication time had little effect on pH, whereas in polyphosphate-containing brines prolonged ultrasonic treatment attenuated alkalization. This behavior indicates that ultrasound did not independently alter the brine pH but rather influence polyphosphate-driven chemical processes. The pH decline induced by ultrasound in UAP brines can be explained by cavitation. Cavitation involves cyclic bubble formation, growth, and collapse; rapid collapse causes adiabatic compression, producing localized hot spots with extremely high temperature and pressure. Under these conditions, water and dissolved gases dissociate and generate reactive radicals such as •OH and •OOH. Decomposition of •OOH yields O_2_^−^ and H^+^, and the release of H^+^ contributes to the observed pH decrease [29]. Ultrasound-induced micro-hotspots may promote conversion of PO_4_^3−^ into more protonated forms (HPO_4_^2−^ and/or H_2_PO_4_^−^) [30], thereby lowering pH. Prolonged sonication likely intensifies these conversions.

Despite these time-dependent changes, no significant differences in brine pH were detected between UBP and UAP treatments. This finding suggests that, under the applied conditions, the overall buffering action of polyphosphate dominated the system and minimized differences arising from the order of ultrasound application. Therefore, the role of ultrasound sequence appears to be secondary with respect to pH control, although it may still influence other functional properties of the brine, such as ion availability and interaction efficiency during subsequent meat processing.

Redox potential (Eh)

3.2.2

The redox potential of the brine (Eh, mV) was not significantly affected by ultrasound-treatment sequence (UBP vs. UAP), ultrasonic time (0–30 min), polyphosphate level (0–0.2%), or their interactions (P > 0.05). Across all experimental conditions, Eh values remained within a narrow range (Table 1), indicating that the overall redox environment of the brine was stable under the studied treatment intensities and formulation.Table 1. Changes in brine redox potential (Eh, mV) under different ultrasonication times and polyphosphate levels.TreatmentTime (min)Polyphosphate (%)Eh (mV)UAP0026.85 ± 0.21UAP00.126.90 ± 0.00UAP00.227.45 ± 0.07UAP15026.55 ± 0.07UAP150.126.25 ± 1.48UAP150.227.50 ± 0.71UAP30026.45 ± 0.07UAP300.126.65 ± 0.07UAP300.227.35 ± 0.35UBP0026.85 ± 0.21UBP00.126.90 ± 0.00UBP00.227.45 ± 0.07UBP15026.55 ± 0.07UBP150.126.95 ± 0.49UBP150.227.05 ± 0.07UBP30026.45 ± 0.07UBP300.126.60 ± 0.14UBP300.227.05 ± 0.07Data represent mean ± SD (n = 3). No significant differences were observed among treatments (factorial ANOVA, P > 0.05).

This outcome is mechanistically plausible because Eh represents the equilibrium (or near-equilibrium) potential established at the interface between a noble metal electrode and an aqueous solution containing electroactive species, and it often reflects qualitative trends rather than a single well-defined equilibrium value. In saline/polyphosphate-containing systems, bulk redox potential may remain relatively constant unless ultrasound generates sufficiently large and persistent concentrations of redox-active products (or consumes dissolved oxygen) to shift the solution’s electrochemical balance.

Although ultrasonic cavitation can promote sonochemical reactions in water and generate reactive intermediates such as hydroxyl radicals and hydrogen peroxide [31], the lack of a measurable Eh shift suggests that, in the present brine matrix, any transient formation of oxidizing species was either too limited, rapidly quenched, or effectively buffered by the solution chemistry and dissolved constituents. In other words, ultrasonication modified the brine primarily in ways relevant to functionality rather than producing a sustained redox perturbation detectable by Eh under these conditions [32].

The impact of ultrasonicated brine on meat properties

3.3

A conceptual mechanistic diagram summarizing the proposed cavitation-mediated effects of brine ultrasonication and the differential pathways of UBP and UAP treatments is presented in Fig. 2.Fig. 2. Conceptual mechanistic diagram illustrating the differential pathways of UBP (phosphate added after sonication) and UAP (phosphate added before sonication). The diagram summarizes the proposed cavitation-mediated effects of brine ultrasonication on protein structural responses and the resulting changes in water-holding capacity and emulsifying properties. This schematic represents a conceptual framework and does not imply direct causality.

pH

3.3.1

Meat pH is a critical determinant of tenderness, WHC, color, juiciness, and shelf life. Lower pH typically reduces juiciness and increases hardness, whereas higher pH can enhance juiciness but may shorten shelf life [33]. Meat pH was significantly influenced by polyphosphate concentration (P < 0.001), whereas sonication time and the sequence of ultrasound application (UBP vs. UAP) had no significant effect (P > 0.05). No significant interaction effects among treatment, time, and polyphosphate level were observed, indicating that the effects of polyphosphate and time on meat pH were independent.

An increase in meat pH with increasing polyphosphate concentration was consistently observed across all treatments (Table 2). This behavior is well documented in meat systems and is primarily attributed to the buffering capacity of polyphosphate salts and their ability to shift the muscle environment toward a less acidic pH. Polyphosphates increase meat pH by dissociating into alkaline phosphate species and by modifying the ionic environment of myofibrillar proteins, thereby increasing net negative charge and electrostatic repulsion within the muscle matrix [34].Table 2. Main effects of order of ultrasonication, ultrasonication time, and polyphosphate concentration on meat pH.TreatmentMeat pHOrder of ultrasonicationUAP5.57 ± 0.07 ^a^UBP5.65 ± 0.08 ^a^Ultrasonication time (min)05.64 ± 0.09 ^a^155.66 ± 0.04 ^a^305.68 ± 0.07 ^a^Polyphosphate concentration (%)05.60 ± 0.04 ^a^0.15.64 ± 0.03 ^a^0.25.75 ± 0.04^b^Main effects were compared because interaction terms were not significant (P > 0.05). Different letters within each factor indicate significant differences (P < 0.05).

Meat pH was not significantly affected by the ultrasonication time of the brine (P > 0.05). This result indicates that extending the duration of ultrasound treatment applied to the brine did not lead to measurable changes in the acid–base balance of the meat. Since the meat samples were not directly exposed to ultrasonic waves, any potential effect of ultrasonication on meat pH could only occur indirectly through modifications in brine properties.

Although the sequence of ultrasound application did not significantly affect meat pH, insight into the underlying mechanisms can be gained by considering the corresponding pH behavior of the brine system. Brine pH measurements showed pronounced polyphosphate-induced alkalization, with pH values exceeding 9 depending on polyphosphate level and processing time. Despite this strong alkalinity in the brine, the resulting changes in meat pH were comparatively moderate. This attenuation reflects the inherent buffering capacity of muscle tissue, which limits extreme pH shifts and stabilizes the intracellular environment [35]. Thus, while ultrasonicated polyphosphate brines can substantially modify the pH of the surrounding solution, the muscle matrix dampens these effects, leading to controlled and gradual pH elevation in the meat.

The absence of a treatment effect on meat pH further suggests that ultrasound application sequence primarily influenced brine physicochemical properties. Ultrasound-related effects on meat quality therefore appear to be mediated more strongly through structural and hydration-related mechanisms, such as protein unfolding and enhanced water binding, rather than through pH modification. This interpretation is consistent with previous studies indicating that pH changes alone cannot fully explain improvements in WHC and functional properties of meat systems [36].

Water-holding capacity (WHC)

3.3.2

WHC of meat was significantly affected by polyphosphate concentration and ultrasonic time and showed a significant ultrasound treatment sequence × ultrasonic time × polyphosphate concentration interaction (P < 0.01).

The increase in WHC observed with increasing polyphosphate concentration (Fig. 3) can be partially attributed to the concomitant elevation in meat pH. Polyphosphate addition shifted the muscle system away from its isoelectric point, thereby increasing the net negative charge of myofibrillar proteins and enhancing electrostatic repulsion within the myofibrillar lattice. This process promotes myofibrillar swelling and improves water retention, a mechanism that is well established as a primary contributor to WHC in meat systems [7].Fig. 3. Two-panel interaction plots showing the effects of phosphate level and ultrasonic time on water-holding capacity (WHC, %) for (A) ultrasonicated NaCl before phosphate addition (UBP), and (B) ultrasonicated NaCl after phosphate addition (UAP). Data represent mean ± SD (n = 3). Different letters indicate significant differences among treatment combinations (P < 0.05).

However, differences in WHC between UBP and UAP treatments cannot be explained solely by changes in meat pH, since no significant differences in pH were observed between the two treatments. This indicates that the enhanced WHC resulted from mechanisms other than direct pH effects. Importantly, in the present study, meat samples were not exposed to ultrasonic waves; instead, ultrasound was applied exclusively to the brine prior to or after polyphosphate incorporation. Therefore, the observed improvements in WHC must be attributed to ultrasound-induced modifications of the brine. Although the high pH of the ultrasonicated brine was attenuated once incorporated into the muscle, ultrasound-modified ionic environments likely facilitated more effective polyphosphate–protein interactions during brine diffusion into the meat, consistent with previous findings that ultrasonically treated brines alter hydration properties and brine component distribution, thereby influencing protein–water interactions in muscle tissue [37]. The stronger WHC response observed in the UBP treatment (Fig. 3) suggests that ultrasonication of the brine prior to polyphosphate addition produced a brine with enhanced functional efficiency, possibly through improved polyphosphate solubility, hydration, and availability for interaction with myofibrillar proteins. In contrast, when ultrasound was applied after polyphosphate incorporation (UAP), these effects were less pronounced or developed more slowly, resulting in a weaker or more time-dependent WHC response despite similar meat pH values. Previous study indicated that he structure of water itself contributes to WHC. Water molecules form unstable polymer-like assemblies through hydrogen bonding, whereas unstructured water that does not participate in these assemblies can more readily associate with other molecules, including proteins. Ultrasound generates intense pressure pulses that disrupt water’s molecular bonds and shift it into a thermodynamically non-equilibrium condition. In this condition, water has a higher hydration capacity. Ultrasound may also increase hydrogen peroxide formation and modify the ionic capacity of water-soluble constituents. Salts dissolved in water dissociate into ions that are either trapped within polar monomolecular water layers or interact with protein structures [38]. Dissociation of NaCl produces positively and negatively charged ions and can lead to formation of species such as NaOH, OCl^−^, and Cl^−^. These ions increase the net negative charge on myofibrillar proteins and thereby enhance WHC [39]. Tsirulnichenko, Potoroko, Krasulya and Gudina [40] likewise reported that acoustic vibrations generated by ultrasound increase the water-absorption capacity of myofibrillar proteins in poultry meat. Accordingly, ultrasonicated brine may improve the functional and processing properties of meat by ensuring adequate moisture retention in the final product and enabling incorporation of additional water.

Also, processing time exerted a significant effect on WHC, as evidenced by higher WHC values at longer ultrasonication times, particularly in polyphosphate-containing systems (Fig. 3). Although a definitive mechanism cannot be established based on the present data, this shift may be attributed to time-dependent changes in the functionality of polyphosphate-containing brines induced by ultrasonication. Prolonged sonication may have promoted more effective ion dispersion and polyphosphate–protein interactions, leading to improved water retention. These effects facilitate protein swelling and stabilization of immobilized water, resulting in enhanced WHC even in the absence of substantial changes in meat pH.

Cooking loss

3.3.3

Cooking loss was significantly affected by polyphosphate concentration (P < 0.0001) and brine ultrasonication time (P < 0.05), with a significant time × polyphosphate interaction (P < 0.01). In contrast, the ultrasound application sequence (UBP vs. UAP) had no significant effect on cooking loss, indicating that the duration of ultrasonic treatment applied to the brine was more influential than the order of ultrasound application relative to polyphosphate addition.

The cooking loss results indicate that the system response to the applied treatments was not linear and depended strongly on the presence of polyphosphate. In polyphosphate-free samples, increasing the brine ultrasonication time did not reduce cooking loss; instead, a slight increase was observed in some cases (Fig. 4). This behavior suggests that ultrasonication of the brine alone was insufficient to improve water retention during cooking and may even have increased the mobility of free water, facilitating moisture release during thermal processing.Fig. 4. Two-panel interaction plots showing the effects of phosphate level and ultrasonic time on cooking loss (%) for (A) ultrasonicated NaCl before phosphate addition (UBP), and (B) ultrasonicated NaCl after phosphate addition (UAP). Data represent mean ± SD (n = 3). Different letters indicate significant differences among treatment combinations (P < 0.05).

In contrast, the addition of polyphosphate fundamentally altered the cooking loss pattern. Polyphosphate addition also intensified electrostatic interactions among myosin molecules. Because myosin carries a net negative charge at postmortem pH, the inclusion of negatively charged polyphosphate ions is expected to increase the overall charge density on myosin, thereby strengthening electrostatic effects. Polyphosphate-supported dissociation of myofibrillar proteins promotes unfolding of protein molecules and exposure of hydrophobic groups. The exposed hydrophobic regions facilitate assembly of myosin molecules through hydrophobic interactions, which promotes development of a more cohesive myosin gel network. Formation of this gel network reduces water release during heating and thus lowers cooking loss during thermal processing [41].

In polyphosphate-containing systems, longer brine ultrasonication times resulted in a pronounced and consistent reduction in cooking loss, with the lowest values observed at higher polyphosphate levels combined with extended ultrasonication. This non-linear response is reflected in the significant time × polyphosphate interaction and indicates that ultrasound did not act as an independent factor, but rather enhanced the functional effectiveness of polyphosphate in the system.

Importantly, the observed differences in cooking loss cannot be attributed solely to changes in meat pH, as meat pH values showed only minor variations among treatments. Instead, brine pH data demonstrate that ultrasonication time influenced the alkaline behavior and buffering characteristics of polyphosphate in the aqueous phase. These ultrasound-induced modifications in the brine likely affected the manner in which polyphosphate interacted with myofibrillar proteins once incorporated into the meat, particularly under thermal conditions where protein stability governs moisture retention.

From a structural perspective, the reduction in cooking loss observed in polyphosphate-containing, ultrasonicated brine treatments can be interpreted as the formation of a protein network that was more resistant to heat-induced shrinkage and water expulsion. In this context, water retention during cooking was not governed simply by pH-related effects, but by improved organization and functionality of protein–polyphosphate interactions that stabilized water within the muscle matrix. The agreement between reduced cooking loss and increased WHC further supports the conclusion that the retained water in these samples was more structurally immobilized and less prone to release during heating.

Finally, the similarity in cooking loss trends between UBP and UAP treatments confirms that the sequence of ultrasound application relative to polyphosphate addition was not a determining factor for cooking loss. Rather, the duration of brine ultrasonication and the presence of polyphosphate were the critical parameters controlling thermal moisture retention.

Emulsifying properties

3.3.4

EAI quantifies a protein’s ability to adsorb at the oil–water interface during emulsion formation. A higher EAI indicates a greater capacity to form and stabilize emulsions, because proteins with elevated EAI are more efficiently positioned at the interface and thus reduce the likelihood of phase separation [42].

EAI was significantly affected by polyphosphate concentration (P < 0.0001) and brine ultrasonication time (P < 0.05), with a significant time × polyphosphate interaction (P < 0.01). The ultrasound application sequence (UBP vs. UAP) also exerted a significant effect on EAI (P < 0.05), indicating that not only the duration but also the timing of ultrasonication relative to polyphosphate addition influenced emulsification behavior.

The EAI response to the applied treatments was clearly non-linear and strongly dependent on polyphosphate presence. In polyphosphate-free systems, increasing the ultrasonication time of the brine resulted in only limited improvements in EAI, and in some cases no significant enhancement was observed (Fig. 5A). This indicates that ultrasonication of the brine alone was insufficient to markedly improve protein surface activity in the absence of polyphosphate.Fig. 5. Effect of polyphosphate concentration and ultrasonication time of brine on (A) emulsifying capacity (m^2^/g), (B) emulsion stability (%), of myofibrillar proteins, and (C) Emulsion surface protein concentration under UBP and UAP conditions. Data are presented as mean ± SD (n = 3). Different letters indicate significant differences among treatment combinations (P < 0.05).

In contrast, polyphosphate addition fundamentally altered the EAI pattern. Increasing polyphosphate concentration from 0 to 0.2% resulted in a pronounced and statistically significant increase in EAI across all ultrasonication times. Polyphosphate increases the net negative charge of myofibrillar proteins, thereby enhancing electrostatic repulsion among protein molecules and promoting partial dissociation and unfolding. This unfolding exposes hydrophobic groups and increases protein flexibility, facilitating adsorption and rearrangement of proteins at the oil–water interface. These structural changes improve interfacial coverage and explain the substantially higher EAI values observed in polyphosphate-containing treatments [43]. Polyphosphates and salt also act synergistically to improve EAI, largely because salt increases myofibrillar protein solubility [44]. Cl^−^ ions from NaCl increase electrostatic repulsion between proteins, causing swelling of the myofibrillar [45]. Consequently, after polyphosphates facilitate actomyosin dissociation, myosin becomes more readily solubilized by salt, binds more water, and contributes to higher EAI.

Within polyphosphate-containing systems, longer brine ultrasonication times further enhanced EAI, particularly in the UBP treatments. This behavior is reflected in the significant sonication time × polyphosphate interaction and indicates that ultrasonication did not act as an independent factor, but rather amplified the functional effectiveness of polyphosphate. Ultrasonication applied prior to polyphosphate addition likely modified the physicochemical properties of the aqueous phase, such as hydration behavior and ionic organization, allowing polyphosphate to interact more efficiently with myofibrillar proteins upon incorporation. Vaz Leães, Pinton, de Aguiar Rosa, Robalo, Wagner, de Menezes, Barin, Campagnol and Cichoski [39] examined alkaline and supersaturated electrolyzed brine as a strategy to reduce NaCl in meat products and reported that Cl^−^ ions produced during electrolysis bind to protein chains, increasing electrostatic repulsion, expanding the filament network, and improving protein solubility, thereby enhancing emulsion stability. Similarly, as noted earlier, generating active brine produces NaCl-derived ions such as Cl^−^ and OCl^−^, which can increase the solubility of myofibrillar proteins and improve EAI by elevating pH and strengthening the net negative charge on these proteins.

A direct comparison between UBP and UAP revealed that UBP samples exhibited significantly higher EAI than UAP at identical polyphosphate concentrations and ultrasonication times (P < 0.05), especially at 0.1% and 0.2% polyphosphate. This confirms that the sequence of ultrasonication relative to polyphosphate addition influenced emulsifying capacity and that ultrasonication prior to polyphosphate addition generated a brine environment more favorable for enhancing protein interfacial activity.

Emulsion stability index (ESI) reflects the ability of an emulsion to resist destabilization processes such as phase separation during storage, with higher values indicating greater stability [46]. Emulsion stability was also significantly influenced by polyphosphate concentration (P < 0.001), whereas the effect of ultrasonication time was moderate but significant (P < 0.05) and strongly treatment-dependent. A significant time × polyphosphate interaction was detected for ESI (P < 0.05), indicating that stability responses depended on the combined effects of these factors. In polyphosphate-free samples, ultrasonication time had little effect on ESI, suggesting limited stabilization of the interfacial protein film in the absence of polyphosphate.

In polyphosphate-containing systems, ESI improved significantly; however, the pattern differed from that observed for EAI. While EAI reflects the ability of proteins to rapidly adsorb at the interface, ESI depends on the strength and integrity of the interfacial protein network formed after emulsification. In UBP samples, moderate ultrasonication times enhanced ESI, whereas prolonged ultrasonication did not provide further benefits, indicating that excessive modification of the brine environment may compromise optimal interfacial packing. In contrast, UAP treatments showed a stronger dependence of ESI on ultrasonication time, particularly at intermediate polyphosphate levels, resulting in a significant interaction effect (Fig. 5B). When polyphosphate was combined with prolonged brine ultrasonication, a reduction in ESI was observed. Because meat proteins were not directly exposed to ultrasound in this study, this effect is attributed to indirect changes induced by the ultrasonicated brine during subsequent emulsification. Prolonged ultrasonication of brine may increase protein unfolding and promote protein aggregation which reduce the flexibility of the interfacial protein film, increasing the likelihood of droplet association. In addition, protein-mediated droplet flocculation, arising from bridging interactions or depletion effects in the continuous phase, may further compromise emulsion stability, leading to lower ESI values despite improved emulsifying activity [47].

Despite these differences, no consistent overall superiority of UBP or UAP was observed for ESI across all treatment combinations (P > 0.05). This suggests that, unlike EAI, emulsion stability was less sensitive to the ultrasound application sequence and more dependent on the combined physicochemical conditions established in the brine after polyphosphate incorporation.

Overall, the results demonstrate that polyphosphate concentration was the primary determinant of both EAI and ESI, while brine ultrasonication time significantly modulated these properties through interaction effects. The distinct responses of EAI and ESI further indicate that ultrasonication of the brine selectively influenced different aspects of protein interfacial functionality. These findings confirm that ultrasonic modification of the brine can be used as an effective indirect strategy to tailor emulsion-related properties without directly exposing myofibrillar proteins to ultrasonic waves.

Interfacial protein concentration

3.3.5

Interfacial protein concentration is critical for reducing oil droplet size and maintaining emulsion stability. Proteins that adsorb at the oil–water interface form a protective layer around droplets and thereby enhance emulsion stability during storage [48]. Surface protein concentration exhibited response patterns that closely paralleled those observed for EAI and, to a lesser extent, emulsion stability (ESI), confirming its central role in governing interfacial functionality. As shown in Fig. 5C, polyphosphate concentration had a highly significant effect on surface protein concentration (P < 0.0001), which was directly reflected in the marked increase in EAI across all treatments. In contrast, the effects of brine ultrasonication time (P < 0.05) and the time × polyphosphate interaction (P < 0.01) modulated the magnitude and efficiency of this response rather than acting as independent drivers.

In polyphosphate-free systems, surface protein concentration remained low regardless of ultrasonication time, and correspondingly low EAI values were observed. This indicates that ultrasonication of the brine alone did not provide sufficient interfacial protein adsorption to enhance emulsification performance. The limited amount of protein adsorbed at the oil–water interface restricted the formation of a continuous interfacial layer [49], resulting in poor emulsifying capacity and only moderate emulsion stability.

The addition of polyphosphate fundamentally altered this behavior. At all ultrasonication times, increasing polyphosphate concentration led to a statistically significant increase in surface protein concentration, which was accompanied by a pronounced enhancement of EAI. This strong correspondence demonstrates that the improvement in emulsifying capacity was primarily driven by the increased availability of protein at the interface. Higher surface protein concentration facilitated more rapid and extensive coverage of newly formed oil droplets, thereby increasing the total interfacial area that could be stabilized [50].

With increasing brine ultrasonication time, the relationship between surface protein concentration and EAI became treatment-dependent. In UBP samples, moderate ultrasonication times promoted higher surface protein concentration and EAI, whereas prolonged ultrasonication led to a saturation behavior, particularly at higher polyphosphate levels. At 30 min, surface protein concentration at 0.1% and 0.2% polyphosphate became statistically indistinguishable, and a similar plateau was observed for EAI. This indicates that, beyond a certain point, additional polyphosphate or extended ultrasonication did not further enhance interfacial adsorption or emulsification efficiency.

In contrast, UAP samples showed a stronger time dependence, especially at intermediate polyphosphate levels. At 0.1% polyphosphate, prolonged ultrasonication resulted in a marked increase in surface protein concentration, which coincided with higher EAI values compared with shorter ultrasonication times. This suggests that ultrasonication applied after polyphosphate addition enhanced the mobility or accessibility of polyphosphate–protein complexes, thereby promoting protein adsorption at the interface and improving emulsifying performance.

The relationship between surface protein concentration and ESI was more complex. While higher surface protein concentration generally supported improved stability, the correspondence was not strictly proportional. In several treatments, particularly at longer ultrasonication times, surface protein concentration reached a plateau whereas ESI continued to vary. This indicates that ESI was governed not only by the amount of protein adsorbed at the interface, but also by the structural organization, packing density, and mechanical strength of the interfacial protein film [51]. Consequently, treatments that produced similar surface protein concentrations could still differ in emulsion stability.

Meat color

3.3.6

Typically, bright red meat is associated with greater freshness and higher perceived quality, whereas pale, discolored, or darkened meat is commonly viewed as less fresh or closer to spoilage [52]. Lightness (L*) was significantly affected by polyphosphate concentration (P < 0.001) and brine ultrasonication time (P < 0.05). In contrast, the ultrasound application sequence (UBP vs. UAP) did not exert a significant main effect on L* (P > 0.05). L* was influenced by the combined effects of polyphosphate concentration and brine ultrasonication time, as evidenced by the non-parallel trends observed in the interaction plots (P < 0.01). In both UBP and UAP systems, L* increased noticeably at 0.1% polyphosphate when the brine was not ultrasonicated, whereas extended ultrasonication times altered this response. In UAP, particularly at 30 min, L* decreased at 0.1% polyphosphate, indicating that prolonged ultrasonication of polyphosphate-containing brine modified the way polyphosphate affected surface light scattering in the meat matrix (Fig. 6).Fig. 6. Interaction effects of polyphosphate concentration and brine ultrasonication time on color attributes of meat, including lightness (L*), redness (a*), yellowness (b*), and total color difference (ΔE). Results are shown separately for brine ultrasonicated before phosphate addition (UBP) and after phosphate addition (UAP). Data represent mean ± SD (n = 3). Different letters indicate significant differences among treatment combinations (P < 0.05).

Because meat was not subjected directly to ultrasound, these changes cannot be attributed to mechanical disruption of muscle structure. Instead, they likely reflect ultrasound-induced modifications in brine properties that altered protein hydration and ionic interactions after incorporation into the meat. Changes in protein–water distribution and surface reflectance may therefore explain the observed L* variations. In general, UBP brines and polyphosphates can increase WHC and thus enhance lightness. Nevertheless, during ultrasonication, reactive species are generated that may oxidize myoglobin to metmyoglobin [53], which can contribute to a reduction in L*.

Fig. 6 indicate change in the redness (a*) of meat after mixing with ultrasonicated brine. a* exhibited a pronounced interaction-type response, particularly in the UAP system. At 0.1% polyphosphate, a* values increased markedly at 15 min of brine ultrasonication, while a sharp decline was observed at 0.2% polyphosphate under the same ultrasonication time. In contrast, the UBP system showed a more gradual increase in a* with increasing polyphosphate concentration, especially at longer ultrasonication times. These findings suggest that the ultrasonication of brine containing polyphosphate (UAP) altered the redox and ionic environment of the aqueous phase, which in turn influenced pigment stability and light absorption once the brine interacted with the meat. Ultrasound-generated free radicals promote oxidation of heme pigments (myoglobin and hemoglobin), destabilizing meat color. In addition, further changing a* [54]. The non-linear behavior of a* indicates that the effectiveness of polyphosphate in maintaining or enhancing redness depended on both its concentration and the duration of brine ultrasonication. Polyphosphate addition changed a* values. This behavior can be explained by several physicochemical mechanisms that affect myoglobin stability and oxidative reactions. First, polyphosphates raise meat pH. A higher pH environment stabilizes myoglobin by slowing its conversion to metmyoglobin and favoring persistence of deoxymyoglobin and oxymyoglobin, which are responsible for purplish-red and bright red colors, respectively [55]. Second, polyphosphates act as mild antioxidants through strong chelation of pro-oxidant metal, delaing pigment oxidation [56].

Yellowness (b*) values were also affected by the interaction between polyphosphate concentration and brine ultrasonication time. In the UBP treatments, b* increased consistently with increasing polyphosphate concentration, particularly at longer ultrasonication times. In contrast, the UAP system displayed a more variable pattern, with higher b* values at 0.1% polyphosphate under extended ultrasonication. This behavior indicates that ultrasound-induced changes in the brine modified the way polyphosphate interacted with muscle proteins and pigments, thereby affecting color balance. The stronger response of b* to polyphosphate in ultrasonicated brine suggests enhanced protein–ion interactions that influenced light reflection in the yellow spectrum. The reduction in b* following exposure to ultrasonicated brine may be related to available chloride ions and reactive species that denature the globin portion of myoglobin; together with the reduced redness, this produces a paler appearance. Adding polyphosphate raises brine pH and may lessen this denaturation effect. At the same time, free radicals produced during ultrasonication can oxidize myoglobin to metmyoglobin and thereby increase b* [57].

Total color difference (ΔE) showed clear interaction-type trends, with the highest ΔE values generally observed at 0.1% polyphosphate, particularly in the UAP system at 30 min of brine ultrasonication. At higher polyphosphate levels (0.2%), ΔE values decreased in most cases, indicating greater overall color stability. These results suggest that moderate polyphosphate levels combined with extended brine ultrasonication amplified perceptible color changes, whereas higher polyphosphate concentrations contributed to color stabilization. The reduction in ΔE at 0.2% polyphosphate is consistent with improved protein–water and protein–ion interactions that limited pigment redistribution and surface color variability.

Microstructure

3.3.7

The microstructure of myofibrillar proteins extracted from control treatment (UBP0), UBP30 nad UAP30 containing 0.2% polyphosphate at magnification of × 2000 are presented in Fig. 7. In the UBP0, the fragmented and loosely connected protein matrix observed in FESEM corresponds well with the lower WHC and higher cooking loss values. The irregular and discontinuous network suggests limited protein unfolding and weak intermolecular interactions, which reduces the ability of the protein system to immobilize water. This structural weakness facilitates water expulsion during thermal processing, explaining the higher cooking loss recorded in polyphosphate-free, non-ultrasonicated brine treatments (Fig. 8).Fig. 7. Scanning electron microscope images of myofibrillar proteins from non-ultrasonicated brine without phosphate (UBP0), ultrasonicated brine before phosphate addition (UBP30), and ultrasonicated brine after addition of 0.2% phosphate for 30 min (UAP30).Fig. 8FTIR spectra of myofibrillar proteins from non-ultrasonicated brine without phosphate (UBP0), ultrasonicated brine before phosphate addition (UBP30), and ultrasonicated brine after addition of 0.2% phosphate for 30 min (UAP30).

In contrast, samples treated with ultrasonicated brine (UBP30 and UAP30) exhibited progressively more organized and cohesive protein networks, as evidenced by the formation of elongated strands and larger interconnected pores in FESEM micrographs. The structural features indicate partial unfolding and rearrangement of myofibrillar proteins, allowing the formation of a continuous three-dimensional network capable of physically entrapping water. These differences can be attributed to physicochemical modifications induced by ultrasonicated polyphosphate brine. Ultrasonication increased ionic activity and enhancing ion dispersion and effective pH within the brine. The higher pH facilitated unfolding and solubilization of myofibrillar proteins, improving water retention. This interpretation is directly supported by the significantly higher WHC values observed in these treatments, particularly in polyphosphate-containing systems.

The FESEM observations are further corroborated by FTIR results, which showed shifts in characteristic bands associated with protein secondary structure and hydrogen bonding (e.g., amide II and O–H/N–H stretching regions). The downshifts in amide II bands and changes in the polyphosphate-related P–O stretching region indicate strengthened hydrogen bonding and enhanced protein–polyphosphate interactions. These molecular-level modifications explain the macroscopic structural reorganization seen in FESEM, where ultrasonicated polyphosphate brine promoted a thicker and more cohesive protein framework.

The reduced cooking loss observed in polyphosphate-containing, ultrasonicated brine treatments is therefore not solely a consequence of pH changes, but rather a result of structural stabilization of the protein matrix. The sponge-like architecture seen in the UAP30 sample, characterized by larger and more uniform pores with reinforced protein walls, suggests that water was more effectively immobilized within the protein network. This structural resistance to heat-induced shrinkage directly accounts for the pronounced reduction in cooking loss during thermal processing.

FTIR spectroscopy

3.3.8

FTIR analysis demonstrated distinct spectral modifications in myofibrillar proteins subjected to ultrasonicated brine treatments, reflecting treatment-dependent molecular rearrangements. The broad band centered at 3314 cm^−1^ in UBP0, corresponding to O–H and N–H stretching vibrations, shifted to 3311 cm^−1^ in UAP30 and to 3317 cm^−1^ in UBP30. The shift toward a lower wavenumber in UAP30 suggests reinforcement of hydrogen bonding interactions, likely arising from enhanced protein–water and protein–polyphosphate associations when polyphosphate was present during ultrasonication. Conversely, the slight upshift observed in UBP30 implies comparatively weaker hydrogen bonding, indicating that the sequence of polyphosphate addition influenced the resulting interaction network.

Ultrasound treatment also affected aliphatic side-chain vibrations. The C–H stretching band at 2925 cm^−1^ in UBP0 shifted to 2929 cm^−1^ in both UAP30 and UBP30, while the symmetric C–H stretching band moved from 2855 to 2874 cm^−1^ following ultrasound exposure. These upward shifts indicate increased molecular mobility and reduced packing constraints of protein side chains, suggesting conformational loosening and partial unfolding of myofibrillar proteins induced by ultrasonicated brine [58].

Further structural evidence was observed in the amide II region, where the band at 1543 cm^−1^ in UBP0 shifted to 1541–1542 cm^−1^ after treatment with UAP30 and UBP30 containing 0.2% polyphosphate. This red shift reflects decreased vibrational energy of N–H bending and C–N stretching modes, commonly associated with strengthened hydrogen bonding and rearrangement of protein secondary structure [59]. Such changes imply partial unfolding of myofibrillar proteins and increased availability of hydrogen-bonding sites, facilitating interactions with water molecules and polyphosphate ions. The redistribution of secondary-structure elements, potentially involving a transition from α-helical regions toward more disordered conformations, is consistent with the enhanced water-holding capacity observed in the corresponding meat samples.

Additionally, the deformation band at 1457 cm^−1^, attributed to aliphatic C–H bending vibrations, shifted to lower wavenumbers in UAP30 and UBP30, indicating changes in the local environment of aliphatic side chains and increased intermolecular constraints within the protein matrix [58].

Pronounced effects of polyphosphate incorporation were evident in the 1000–1200 cm^−1^ region. The absorption band at 1083 cm^−1^ in UBP0 shifted to 1076 cm^−1^ in the ultrasonicated brine samples, reflecting stronger interactions between polyphosphate groups and myofibrillar proteins. Previous FTIR studies have shown that P–O stretching vibrations are highly sensitive to changes in hydration and coordination environment, with polyphosphate binding typically causing a shift toward lower wavenumbers [60]. Ultrasonication likely promotes interactions between polyphosphate groups and amide or hydroxyl functionalities of myofibrillar proteins, reducing P–O bond mobility and resulting in the observed spectral displacement.

In addition to the amide-related regions, a distinct absorption band at 1744 cm^−1^ was detected exclusively in the UBP0 sample. This band is generally attributed to non-amide carbonyl stretching vibrations and, in protein systems, may reflect contributions from protonated carboxylic groups or other non-amide C=O environments. The absence of this band in the treated samples indicates a modification in the local carbonyl environment following treatment. Considering that the pH of the UBP30 andUAP30 was higher, the disappearance of the 1744 cm^−1^ band suggests a reduced contribution of protonated carbonyl species and/or altered hydrogen-bonding conditions around these groups. However, as no clear concomitant changes were observed in the typical carboxylate regions, this effect is interpreted cautiously as a change in carbonyl microenvironment rather than a definitive shift in carboxylate formation [61].

Taken together, the disappearance of the 1744 cm^−1^ band and the subtle shifts in the ∼1450 cm^−1^ region indicate treatment-induced adjustments in the local protein environment and possible conformational reorganization, without requiring dramatic changes in backbone secondary structure. These interpretations align with recent discussions in the FTIR literature that highlight the complexity of spectral contributions and the sensitivity of protein FTIR spectra to hydrogen bonding and environmental changes [62].

These structural observations are consistent with the functional results (WHC and cooking loss) and provide microstructural and spectroscopic support for the superior performance of ultrasonically treated brine applied.

The observed differences between UBP and UAP treatments highlight the critical role of ultrasound timing in cavitation-mediated brine modification. These results underscore that the presence of polyphosphate during ultrasonication alters the physicochemical environment of cavitation, thereby influencing subsequent protein functionality.

Conclusion

4

This study demonstrated that ultrasonication applied exclusively to the brining solution modified the functional performance of polyphosphate-containing curing systems without direct ultrasonic exposure of meat. The results showed that both polyphosphate concentration and brine ultrasonication time significantly influenced meat quality attributes, including water-holding capacity, cooking loss, emulsifying properties, and microstructural characteristics.

Brines sonicated prior to polyphosphate addition (UBP) generally resulted in more favorable functional outcomes compared with brines sonicated after polyphosphate addition (UAP), particularly at moderate sonication times and higher polyphosphate levels. These differences were consistently reflected across physicochemical measurements, protein functionality indices, FTIR spectral changes, and FESEM observations. Overall, the findings indicate that controlled ultrasonication of curing solutions can be used as a process modification tool to modulate polyphosphate-related functionality in beef products, providing a basis for future studies aimed at optimizing polyphosphate usage levels through quantitative reduction strategies.

Declaration of Generative AI and AI-assisted technologies in the writing process

During the preparation of this work, the authors used Chat GPT in order to improve the language and readability of the manuscript. After using this tool/service, the author(s) reviewed and edited the content as needed and take(s) full responsibility for the content of the publication.

CRediT authorship contribution statement

Fatemeh Maleki-dashti: Investigation, Formal analysis. Nafiseh Soltanizadeh: Writing – review & editing, Supervision, Project administration, Methodology, Conceptualization. Mohsen Ebrahimi Hemmati Kaykha: Writing – original draft, Software.

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.