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Ultrason Sonochem
Ultrason Sonochem
Ultrasonics Sonochemistry
1350-4177
1873-2828
Elsevier

S1350-4177(24)00277-3
10.1016/j.ultsonch.2024.107029
107029
Original Research Article
Ultrasound-assisted improvement of thawing quality of Tibetan pork by inhibiting oxidation
Liu Junmei ab1
Li Xiefei a1
Geng Fang b
Li Xin a
Huang Yujie a
Wu Yingmei a
Luo Zhang c
Huang Qun huangqunlaoshi@126.com
abc⁎
Shang Peng nemoshpmh@126.com
c⁎
Liu Zhendong liu304418091@126.com
c⁎
a School of Public Health, Guizhou Province Engineering Research Center of Health Food Innovative Manufacturing, Guizhou Medical University, Guiyang 550025, China
b Institute for Egg Science and Technology, School of Food and Biological Engineering, Chengdu University, Chengdu 610106, China
c College of Food Science, Tibet Agriculture and Animal Husbandry University, Linzhi, Tibet Autonomous Region 860000, China
⁎ Corresponding authors at: School of Public Health, Guizhou Province Engineering Research Center of Health Food Innovative Manufacturing, Guizhou Medical University, Guiyang 550025, China (Q. Huang). huangqunlaoshi@126.comnemoshpmh@126.comliu304418091@126.com
1 Authors contributed equally to this work.

17 8 2024
11 2024
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110 10702921 6 2024
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© 2024 The Author(s)
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Highlights

• Ultrasound thawing (UT) significantly reduces thawing time with optimal tenderness.

• UT reduces MP oxidation and denaturation, and MP has the most stable structure.

• UT reduces amino acid metabolite production.

• UT reduces bitterness in the thawed meat and enhances the freshness of Tibetan pork.

The challenge of meat quality degradation due to transportation difficulties in high-altitude plateaus underscores the importance of an efficient thawing process for Tibetan pork to ensure its quality. This study compared four thawing methods ultrasound thawing (UT), refrigerator thawing (RT), hydrostatic thawing (HT), and microwave thawing (MT) to assess their impact on the quality of Tibetan pork, focusing on thawing loss, tenderness, color variation, and alterations in protein secondary structure and moisture content. Additionally, the study examined the impact of thawing on the metabolites of Tibetan pork using metabolomics techniques. The results indicated that UT yielded the highest quality samples. UT significantly accelerated the thawing rate and had minimal impact on tenderness compared to traditional thawing methods. Moreover, protein and lipid oxidation levels were reduced by UT treatment. Furthermore, it enhanced the binding capacity of protein and water molecules, reduced drip loss, and maintained meat color stability. What’s more, amino acid metabolites such as l-glutamic acid, l-proline, oxidized glutathione, and 1-methylhistidine played a significant role in thawing oxidation in Tibetan pork, exhibiting a positive correlation with protein oxidation. UT resulted in a notable decrease in the levels of hypoxanthine and 2-aminomethylpyrimidine, contributing to the reduction of bitterness in the thawed meat and consequently enhancing the freshness of Tibetan pork. This study offers novel insights into understanding the biological changes occurring during the thawing process, while also furnishing a theoretical framework and technical assistance to improve the quality of Tibetan pork and propel advancements in food processing technology.

Keywords

Tibetan pork
Ultrasonic thawing
Myofibrillar proteins
Oxidation
Metabolomics
Thawing methods
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pmc1 Introduction

Consumers highly favor the unique taste and high nutritional value of Tibetan pork, attributed to its distinctive growing environment. Freezing transportation is essential for extending the shelf life of fresh meat and maintaining its quality. Proper thawing processes are crucial before further processing of frozen meat. However, improper thawing techniques can lead to texture degradation, color changes, and nutrient loss. Additionally, protein degradation or aggregation can reduce meat quality and negatively impact consumer satisfaction. Therefore, an in-depth investigation of the thawing process of Tibetan pork and the exploration of appropriate thawing methods are essential.

Thawing methods significantly influence the quality of frozen meat products [1], [2], [3]. In recent years, novel thawing techniques such as microwave, radio frequency, ultrasound, vacuum, and high voltage electrostatic field thawing have been widely adopted [4]. These methods expedite the thawing process and reduce losses compared to conventional techniques. Researchers [5], [6], [7] have reported that ultrasound thawing is more effective than microwave, ambient temperature, and ohmic heating thawing. Ultrasound thawing not only accelerates the process but also prevents issues such as uneven thawing, thereby reducing losses. Wang et al. [3] discovered that high-temperature thawing significantly reduces the thermal stability of myofibrils compared to ultrasound thawing and severely damages protein structures. However, the use of high-voltage electrostatic field thawing, radio frequency thawing, and vacuum thawing in the food industry is limited due to practical operational challenges and cost implications. Ultrasound thawing technology has garnered increasing attention and application due to its advantages of rapid thawing speed, uniformity, and minimal loss of food components.

Ultrasound thawing technology has been widely used in meat processing as a non-thermal physical process [8]. During thawing, the microstreaming produced by ultrasound cavitation enhances both heat and mass transfer coefficients, thereby improving thawing efficiency. Multiple studies have shown that ultrasound thawing can mitigate the deterioration of thawing quality. Li et al. [9] found that ultrasound-assisted thawing effectively inhibits lipid and protein oxidation, reduces protein degradation and aggregation, and helps maintain the texture and taste of meat products compared to air thawing. Sun et al. [10] reported that ultrasound thawing accelerates thawing time and results in more compact and neatly arranged muscle fibers in common carp compared to other thawing treatments. Additionally, ultrasound thawing with appropriate power can reduce lipid oxidation and the production of flavor compounds [11]. Numerous studies have investigated the application of freeze–thaw technology in meat products. The oxidative degradation of unsaturated fatty acids generated unstable free radicals, accelerated the oxidation of fats and proteins, and altered the volatile flavor substances in meat products. Limited research has been reported on ultrasound thawing to reduce fatty acid changes in thawed Tibetan pork. Moreover, there is a scarcity of studies focusing on the use of ultrasound thawing to evaluate the meat quality characteristics of Tibetan pork from an oxidative perspective.

As an emerging technology in recent years, metabolomics has the advantages of high accuracy and sensitivity, so metabolomics has been widely used in muscle metabolism and meat quality research. The changes of various metabolites can be revealed through the comparative analysis of key metabolic markers and metabolic pathways. Li et al. [12] used metabolomic to reveal the meat quality characteristics of Lambs from different regions. Metabolomics techniques were used to predict potential biomarkers for determining the optimal aging time [13]. The dynamics of metabolic components and deterioration mechanisms of Tibetan pork during different thawing treatments are still unclear. Therefore, metabolomics technology is more suitable to be applied to the study of metabolites in Tibetan pork by different thawing treatments.

In this study, four thawing methods were compared to examine their impact on the quality of Tibetan pork. The methods, including ultrasound thawing, refrigerator thawing, hydrostatic thawing, and microwave thawing, were evaluated in terms of thawing loss, tenderness, and color variation. Additionally, the study analyzed alterations in protein oxidative denaturation, protein secondary structure, and moisture distribution in the meat products. The effects of thawing on the metabolites of Tibetan pork were also explored using widely targeted metabolome techniques. This study provides an in-depth understanding of the biochemical changes that occur during thawing, offering valuable insights for enhancing the quality of Tibetan pork. Moreover, it offers guidance for advancing food processing technology and meeting consumer expectations for high-quality food products.

2 Materials and methods

2.1 Sample processing

The Tibetan pork was obtained from Aba Bowen Agriculture and Animal Husbandry Technology Company Limited in Jiuzhaigou, China. Six 12-month-old male, castrated Tibetan pork, weighing approximately 25–30 kg, were slaughtered using standard commercial procedures. Fascia and fatty tissue were removed from the meat, which was then cut into 10 × 10 × 5 cm3 pieces and weighed. Each piece of meat was individually packed and immediately frozen in a refrigerator (571WDEMU, Haier Co., Ltd., China) at −20 °C for 24 h.

2.1.1 Thawing methods

In the HT group, frozen pork was thawed in polyethylene vacuum bags in thermostatic water (HH-S6, Beijing Great Wall Co., Ltd., China) at 25 ± 1 °C.

In the RT group, frozen pork was placed in beakers, sealed with cling film, and thawed in a refrigerator (571WDEMU, Haier Co., Ltd., China) at 4 °C.

In the MT group, frozen pork was removed from the refrigerator and placed in a microwave oven (M1-L213C, Guangdong Midea Kitchen Appliance Manufacturing Co., Ltd., China) at 300 W. Thawing was considered complete when the center temperature of the meat reached 4 °C.

In the UT group, frozen meat was placed in an ultrasonic cleaner (JY99-IIDN, Ningbo Xinzhi Co., Ltd., China) with ultrapure water as the immersion solution. The power was set at 300 W, with a frequency of 30 kHz, and operated in a 3-second on/3-s off cycle.

Thawing was considered complete when the center temperature of the meat reached 4 °C, and the extraction of myofibrillar protein was performed.

2.2 Determination of thawing quality indicators

2.2.1 Thawing curve

Changes in sample temperature during thawing were recorded using a thermometer (YET-610, Yuwen Co., Ltd., Shenzhen, Guangdong, China). The thermometer was connected to a 3.0 mm diameter type K thermocouple (NR-81539, Taishi Co., Ltd., Shenzhen, Guangdong, China) inserted into the geometric center of the frozen pork. The temperatures were recorded every minute.

2.2.2 Thawing loss

Frozen pork was weighed (M0 g) before thawing, and thawing continued until the center temperature reached 4 °C. After thawing, the surface water was absorbed with filter paper, and the pork was weighed again (M1 g). Thawing loss was calculated as follows:Thawingloss(%)=M0-M1M0×100%

2.2.3 Shear force

The shear force was measured following the method of Li et al [12]. Shear force was determined using a texture analyzer (TA.TOUCH, Shanghai Baosheng Co., Ltd., China) with a blade (HDP/BSW) oriented perpendicular to the muscle fibers. The parameters measured were: pre-test and post-test speeds of 2.0 mm/s, a contact force of 10.0 gf, and a contact point value of 20.0 gf.

2.2.4 Color measurement

The color of the samples was measured according to the method described by Huang et al [13]. The chromameter (XD-1058, Shenzhen 3nh Technology Co., Ltd., China) was calibrated using a whiteboard and a blackboard before the measurement. The L* (luminance), a* (redness), and b* (yellowness) values of the samples were then recorded. Measurements were taken at five random positions on the surface of each sample.

2.2.5 Total volatile basic nitrogen (TVB-N)

A 10 g sample was weighed, cut, and placed in a beaker with 100 mL of water. The mixture was shaken for 30 min at 25 °C and 200 rpm. 1 mL of the filtrate was pipetted into a 25 mL cuvette, and 2 mL of NaCl reagent (consisting of mercuric iodide, potassium iodide, and sodium hydroxide) was added. Water was added to bring the volume to 25 mL, and the solution was left for 10 min before measuring absorbance at 420 nm (ReadMax-1900, Shanghai Sembcorp Biotechnology Co., Ltd., China), with water serving as a reference.TVB-N=(C-C0)×10-3m×0.25×V25×100%

Note: C − TVB-N content in sample assay solution; C0 − TVB-N content in blank assay solution; m − sample mass; V − volume of sample solution for assay.

2.3 Myofibrillar protein extraction

Myofibrillar protein extraction was conducted with some modifications to a previously described method [14]. Four grams of meat sample were weighed and combined with ten times the volume of homogenization buffer (1.35 M NaCl, 47 mM KCl, 100 mM Na2HPO4, 20 mM NaH2PO4, pH 7.3 ± 0.1). Homogenization was carried out using a high-speed homogenizer (XHF-DY, Xinzhi Co., Ltd., Ningbo, China) at 10,000 rpm for 30 s. The resulting homogenate was then centrifuged at 3,819 × g for 10 min at 4 °C (H175, Xiang Yi Co., Ltd., Hunan, China), and the supernatant was discarded. Protein precipitates were thoroughly resuspended by adding five times the volume of homogenization buffer. The washing step was repeated once, and a layer of gauze was utilized to filter out insoluble connective tissue. The filtrate was centrifuged at 4 °C for 10 min, and the remaining supernatant was discarded. The residue underwent three additional washes by adding five times the volume of homogenizing buffer, followed by suspension in two times the volume of homogenizing buffer to obtain myofibrillar protein. Protein concentrations were determined using a BCA kit (P0012, Beyotime Biotechnology Co., Ltd., China).

2.4 Protein and lipid oxidation assays

2.4.1 Reactive sulfhydryl

Following the method of Dong et al. [15] with some modifications, 2 mL of 1 mol/L myofibrillar protein was combined with 16 mL of Tris-Gly-urea buffer (containing 6 mmol/L EDTA and 8 mol/L urea, pH 7.2 ± 0.1). The mixture was homogenized and then centrifuged at 10,610 × g for 15 min to remove the precipitate. Subsequently, 4.50 mL of supernatant was pipetted and mixed thoroughly with 0.50 mL of 0.01 mol/L Ellman's reagent using a vortex mixer. The reaction proceeded for 30 min away from light. The control group received no additional protein, and absorbance was measured at 412 nm.Reactivesulfhydryl(nmol/mg)=A412C×13600×106

Note: C (mol/L) protein concentration in the test sample.

2.4.2 Determination of protein carbonyl

Following the method of Cheng et al. [14] with some modifications, 0.50 mL of a 5 mg/mL myofibrillar protein solution was divided into two tubes. One tube contained 2 mL of 2 mol/L HCl, while the other tube contained 2 mL of 2 mol/L HCl with the addition of 0.20 % DNPH. The mixture was then incubated for 1 h at room temperature, shielded from light, with vortexing every 10 min. Subsequently, 2 mL of 20 % trichloroacetic acid was added separately, followed by centrifugation at 10,610 × g for 5 min (H175, Xiang Yi Co., Ltd., Hunan, China). The resulting precipitate was washed three times with 2 mL of ethyl ethanol: acetate (1:1). After washing away unbound DNPH from the precipitate, 6 M guanidine hydrochloride was added. The precipitate was then dissolved in a 37 °C water bath for 30 min and centrifuged at 10,610 × g for 5 min. The absorbance of the supernatant at 370 nm (ReadMax-1900, Shanghai Sembcorp Biotechnology Co., Ltd., China) was recorded as A.Carbonylcontent(nmol/mg)=A×3×109ε×2500

Note: ε [L/(mol·cm)] molar absorption coefficient.

2.4.3 Surface hydrophobicity

Three milliliters of a 1 mg/mL myofibrillar protein solution were combined with 600 μL of a 1 mg/mL bromophenol blue solution. The resulting mixture was thoroughly mixed for 10 min at room temperature and then centrifuged at 5,198 × g for 15 min. The supernatant was collected and diluted tenfold. The absorbance value was measured at 595 nm (ReadMax-1900, Shanghai Sembcorp Biotechnology Co., Ltd., China) and recorded as A1. For the blank control (A0), 3 mL of a 0.04 mol/mL sodium phosphate buffer saline solution containing 600 μL of bromophenol blue was used. Surface hydrophobicity was calculated according to the equation:Surfacehydrophobicityug=200×(A0-A1)A0

2.4.4 TBARS

TBARS values were determined using the method of Dong et al. [16] with slight modifications. One gram of pork was weighed, followed by the addition of 5 mL of TBA-TCA-HCl solution (0.375 % TBA, 15 % TCA, 0.25 mol/L HCl). The mixture was heated in boiling water for 10 min and then quickly cooled to room temperature. The supernatant was centrifuged at 4,000 × g for 10 min, and the absorbance was measured at 532 nm (ReadMax-1900, Shanghai Sembcorp Biotechnology Co., Ltd., China). TBARS values were calculated using the following formula:TBARS(mg/kg)=OD532×2.77

2.5 Protein structure

2.5.1 Fourier transform infrared spectroscopy (FTIR)

Fourier infrared spectroscopy (VERTEX70, Bruker Instruments GmbH Co., Ltd., Germany) was performed according to the reference method [17]. Two milligrams of freeze-dried myofibrillar protein were weighed and mixed with 0.10 g of potassium bromide, then crushed and pressed. The sample was analyzed by FTIR in the range of 500–4,000 cm−1 with a scan accumulation of 32 and a resolution of 4 cm−1. The air background spectrum was subtracted before measurement.

2.5.2 Fluorescence spectra

The method was slightly modified according to Huang et al. [18]. Myofibrillar proteins, after various treatments, were dissolved in a phosphate buffer (1.35 M NaCl, 47 mM KCl, 100 mM Na2HPO4, 20 mM NaH2PO4, pH 7.3 ± 0.1), and the protein concentration was adjusted to 0.70 mol/mL. PBS buffer (pH 7.3 ± 0.1) was used as a blank solution. The fluorescence spectra were measured using an FL970 (FL970, Tianmei Co., Ltd., China) with an excitation wavelength of 280 nm, a scanning range of 300–500 nm, a slit width of 5.00 nm, and a scanning speed of 600 nm/min.

2.6 Low-field nuclear magnetic resonance (LF-NMR)

LF-NMR was determined using the reference method [15] with slight modifications. At room temperature, 1.50 g of meat was weighed and placed in a 25.00 mm diameter glass NMR tube (NMI20-060V-I, Suzhou Newmax Electronic Technology Co., Ltd., China). The parameters were set as follows: sampling frequency of 200 kHz, sampling parameter of 21 MHz, 90° pulse time of 6.40 μs, repeat sample wait time of 5,000 ms, cumulative number of 8.00, radio frequency delay of 0.02 ms, analog gain of 10.00 dB, and digital gain of 3.00 dB. The Multi-Exp Inv Analysis software was used to perform the inversion using the joint iterative reconstruction inversion algorithm.

2.7 LC-MS/MS analysis of metabolites

Metabolomic analysis of Tibetan pork was conducted using Ultra Performance Liquid Chromatography (UPLC) (ExionLC AD, Shanghai Abacus Analytical Instruments Trading Co., Ltd., China) and tandem mass spectrometry (MS/MS) (QTRAP 6500, Shanghai Abacus Analytical Instruments Trading Co., Ltd., China). The column used was a Waters ACQUITY UPLC HSS T3 C18 (1.8 μm, 2.1 mm × 100 mm). The mobile phases were ultrapure water with 0.1 % formic acid (phase A) and acetonitrile with 0.1 % formic acid (phase B), with a flow rate of 0.4 mL/min at 40 °C. The injection volume was 2 μL, and the gradient elution procedure was as follows: 5 % phase B at 0 min, 90 % phase B at 11.0–12 min, and 5 % phase B at 12.1–14 min.

The electrospray ionization (ESI) temperature was 500 °C, with a mass spectrometry voltage of 5,500 V (positive) and −4,500 V (negative). The ion source gas I (GS1) was set to 55 psi, gas II (GSII) to 60 psi, and the curtain gas (CUR) to 25 psi. The collision-activated dissociation (CAD) parameter was set to high. In the triple quadrupole (QTRAP), each ion pair was detected by scanning based on optimized declustering potential (DP) and collision energy (CE).

2.8 Statistical analysis

Three separate batches of different thawing treatment samples, three parallel for each sample. Data were analyzed using SPSS 27.0. Analysis of variance (ANOVA) was used to assess statistical differences in the results. All data results are presented as mean ± standard deviation (SD).

3 Results and discussion

3.1 Effect of different thawing methods on the quality of Tibetan pork

3.1.1 Thawing rate

The thawing rate is a crucial index for evaluating thawing effectiveness. As shown in Fig. 1A, different thawing methods significantly impacted the thawing time of Tibetan pork. Microwave thawing (MT) resulted in the shortest thawing time (13 min), while room temperature thawing (RT) had the longest thawing time (285 min). These differences are primarily attributed to varying rates of heat transfer through ultrasound, water, air convection, and thawing at 4 °C [19]. MT excites the molecules inside the pork, causing rapid movement and heat generation, which accelerates the thawing process. However, uneven heat distribution and depth attenuation of microwave energy can result in non-uniform thawing. HT has a shorter thawing time than RT due to the higher surface heat transfer coefficient of water compared to air. Compared to MT, UT better controls energy attenuation at the boundaries, improves the uniformity of heat and mass transfer, and significantly enhances the quality of defrosted meat. Therefore, UT is considered superior to other thawing methods.Fig. 1 Effect of different thawing methods on thawing curves (A), thawing loss rate (B), shear force (C), and TVB-N (D) of Tibetan pork. UT: Ultrasound thawing; HT: Hydrostatic thawing; RT: Refrigerator thawing; MT: Microwave thawing.

3.1.2 Thawing loss

Thawing loss is an important indicator of the quality of frozen meat [20]. As shown in Fig. 1B, the thawing loss of UT (2.52 %) was markedly lower than that of HT (5.24 %) and RT (4.10 %). UT's cavitation effect and microstreaming enhance heat transfer [21], and the rupture of cavitation bubbles converts acoustic energy into thermal energy. This process reorganizes the structure of non-covalent bonds between protein molecules and alters their functional properties [4]. Additionally, the relaxation of pores between muscle fibers increases water retention in the muscle, reducing thawing loss. In contrast, the MT samples showed the highest thawing loss of 8.37 %. This was due to the large temperature difference generated by the transient high temperature during MT, which destroyed the myofiber structure. Under these conditions, the ability of water to pass through the cell membrane was reduced, impeding water reabsorption and leading to water loss. The results indicate that UT can better preserve the internal moisture of the sample.

3.1.3 Shear force

Shear force is an important indicator of the tenderness of Tibetan pork. The effect of different thawing treatments on shear force is shown in Fig. 1C. UT had the lowest shear force, followed by RT, HT, and MT, with tenderness decreasing as the shear force increased. The maximum shear force of MT may be attributed to the shortening of sarcomeres and the increased density of muscle fibers' surface area due to water loss during the microwave thawing process, leading to an increase in the force required to cut the fibers [22], [23]. In this study, HT shear force was significantly higher than UT (P < 0.05), and the reduction in tenderness was associated with fluid loss and muscle fiber shrinkage during thawing. UT contributes to lysosome release from the meat, which increases proteolysis [20], thereby increasing the meat's moisture content. Therefore, UT can promote the interaction between protein molecules, improve the structural properties of myosin, increase the muscle protein's water-holding capacity, and ultimately reduce shear force.

3.1.4 Color analysis

Meat color is an important sensory attribute that influences consumer purchasing decisions. As shown in Table 1, UT, HT, and MT had significantly higher L* values and lower a* values compared to RT (P < 0.05). MT exhibited the highest L* values, likely due to higher temperatures causing rapid melting of ice crystals, disruption of protein structure, and increased light reflection from free water on the surface. UT samples had higher L* values than HT samples because immersion in ultrapure water during thawing allowed some water to infiltrate the muscle, increasing light reflection and L* values [5]. Additionally, UT generates microstreaming during thawing, which enhances heat transfer, improves thawing efficiency, protects muscle structure, and increases L* values.Table 1 Effect of different thawing methods on the color of Tibetan pork.

Thawing Methods	L*	a*	b*	
UT	37.82 ± 1.49b	1.27 ± 0.39c	4.49 ± 0.25b	
HT	35.89 ± 0.70b	2.91 ± 0.52b	6.16 ± 0.83b	
RT	28.30 ± 1.03c	3.14 ± 0.41b	4.95 ± 0.60b	
MT	52.13 ± 0.59a	3.82 ± 0.40a	18.15 ± 0.76a	
Means with different lowercase letters (a–d) in the same column differ significantly (p < 0.05). UT: Ultrasound thawing; HT: Hydrostatic thawing; RT: Refrigerator thawing; MT: Microwave thawing.

As shown in Table 1, UT had significantly lower a* values compared to RT (P < 0.05). This decrease could be due to the destruction of myoglobin reductase during thawing and its exudation from the sarcoplasmic milieu, reducing the amount of myoglobin on the muscle surface. Furthermore, the production of free radicals promotes protein oxidation, destabilizes hemoglobin structure, and accelerates the loss of redness [24]. Muela et al. [25] found that a decrease in methemoglobin reductase activity correlates with reduced redness in frozen muscle. Loss of water-soluble myoglobin and increased free radicals from lipid oxidation lead to increased myoglobin oxidation and metmyoglobin formation, reducing a* values. The significant increase in b* values for MT compared to RT, as shown in Table 1, may be attributed to protein oxidation [26].

3.1.5 TVB-N

Total Volatile Basic Nitrogen (TVB-N) serves as a crucial indicator reflecting the extent of proteolysis during meat product processing and storage, as well as the freshness of meat. Illustrated in Fig. 1D, the TVB-N value of the MT treatment notably surpassed that of other thawing methods, suggesting that microwave thawing induced the most substantial protein damage, followed by RT and HT treatments. During the MT thawing process, prolonged thawing and uneven heat exposure promote proteolysis, resulting in the generation of nitrogenous compounds. Additionally, endogenous enzymatic activity may occur during thawing, catalyzing the degradation of proteins into smaller molecule compounds [12]. UT treatment significantly accelerates the thawing rate, mitigating high temperatures and uneven heating within the samples, thereby minimizing the destruction of tyrosine and methionine in the proteins [27]. This preservation of nutrients helps uphold the quality of Tibetan pork.

3.2 Protein and lipid oxidation analysis

3.2.1 Free sulfhydryl

Changes in the free sulfhydryl content under different thawing methods are shown in Fig. 2A. During the storage and processing of meat products, free sulfhydryl content can easily undergo oxidation to form disulfide bonds, leading to changes in protein structure [21]. The free sulfhydryl content in UT was significantly higher than in other thawing treatments (P < 0.05). It has been found [28] that protein oxidation and reduced free sulfhydryl content are associated with ice crystal damage to the cellular structure, which may result in proteins being affected by oxidation and cause a reduction in free sulfhydryl content during thawing. Cysteine residues are susceptible to oxidation during freezing, which promotes the formation of disulfide bonds by myofibrillar protein aggregation during thawing [29]. MT's lowest free sulfhydryl content is due to microwave heating producing transient high temperatures, leading to protein and lipid oxidation. The short thawing time and the cavitation effect of ultrasound may be responsible for the highest free sulfhydryl content in the UT group. Ultrasound can induce the migration of reactive sulfhydryl groups from the interior of the protein to the surface, which helps protect myofibrillar proteins [30].Fig. 2 Effect of different thawing treatments on the active sulfhydryl content (A), carbonyl content (B), surface hydrophobicity (C), and TBARS (D) of Tibetan pork. UT: Ultrasound thawing; HT: Hydrostatic thawing; RT: Refrigerator thawing; MT: Microwave thawing.

3.2.2 Carbonyl and surface hydrophobicity

The carbonyl content reflects the degree of protein oxidation [6]. As shown in Fig. 2B, there was a significant difference between thawing treatments (P < 0.05). The carbonyl content in UT was significantly lower than in HT and RT. UT reduces thawing time, during which ultrasound induces ice crystal vibrations and cavitation bubbles, reducing the exposure of sulfhydryl and hydrophobic groups, decreasing the production of hydroxyl radicals, and retaining water in the meat. The highest carbonyl content in the MT treatment may be due to changes in polar molecules under the influence of the alternating magnetic field, leading to the transformation of amino acid side-chain groups and accelerated carbonyl compound formation. This resulted in higher levels of protein oxidation, impairing the structure of Tibetan pork tissues and reducing water-holding capacity [9].

As depicted in Fig. 2C, the surface hydrophobicity differed significantly (P < 0.05) among the various thawing treatments compared with the control RT. This discrepancy primarily stemmed from alterations in the hydrophobic groups of proteins induced by the thawing method. The surface hydrophobicity of HT, using the conventional thawing method, was significantly lower than that of RT, owing to its shorter thawing time resulting from the higher heat transfer efficiency of water. Furthermore, UT demonstrated the lowest surface hydrophobicity compared to the other thawing methods (RT, MT, and HT). Prolonged or uneven heating may induce changes in the local properties of proteins, thereby affecting protein function and structure [31]. Meanwhile, the uniform and rapid cavitation effect of ultrasound effectively preserves hydrophobic bonds and hydrogen bonds between hydrophobic molecules during thawing, minimizing the destruction of noncovalent forces and stabilizing the three-dimensional structure of proteins. This, in turn, reduces protein denaturation and aggregation [32].

3.2.3 Thiobarbituric acid reactive substances (TBARS)

As depicted in Fig. 2D, TBARS values were relatively lower in the UT group compared to RT, HT, and MT groups. This may be attributed to slower oxidation in a low oxygen environment and the uniform and rapid thawing induced by mechanical waves generated by ultrasound, thus reducing lipid oxidation. Studies by Li et al. [2], [33] and others have demonstrated that ultrasound disrupts noncovalent interactions and promotes protein-water interactions, fostering a homogeneous three-dimensional structure and diminishing protein and lipid oxidation. The TBARS value following MT treatment was 0.33, significantly higher than other thawing methods (P < 0.05). During freezing, ice crystal formation may damage muscle cells, leading to increased release of free fatty acids and intracellular oxidative precursors. There was no significant difference between HT and RT thawing treatments, likely due to the longer thawing time and sufficient exposure to oxygen, resulting in similar levels of lipid oxidation [34].

3.3 Protein structure analysis

3.3.1 Fourier transform infrared (FTIR) spectroscopy

FTIR spectroscopy is a valuable tool for assessing unfolding and intermolecular force changes in the secondary structure of proteins through the amide-I band [19]. As shown in Fig. 3B, HT and MT thawing treatments resulted in lower α-helix content and significantly higher β-turn and random coil content. During freezing, ice crystal growth disrupts the cellular structure, leading to the rapid breaking of hydrogen bonds and oxidative damage to the protein's secondary structure. The secondary structure changes observed in MT treatment align with experimental results showing a decrease in carbonyl groups and an increase in sulfhydryl groups.Fig. 3 Effect of different thawing treatments on Fourier infrared spectra (A), percentage of secondary structure (B), fluorescence spectra (C), and LF-NMR (D) of Tibetan pork. UT: Ultrasound thawing; HT: Hydrostatic thawing; RT: Refrigerator thawing; MT: Microwave thawing.

The high-speed jets and bursting of irregular bubbles generated by UT treatment enhanced heat transfer during thawing, accelerating the process. Consequently, UT treatment significantly increased α-helix content compared to other thawing methods. Chen et al. [35] showed that α-helix content reflects the stability of protein secondary structure, and UT maintained stability among hydrophobic residues, leading to a more stable secondary structure. Additionally, UT treatment reduced protein oxidation damage during thawing, resulting in increased tenderness, lower moisture loss, and decreased protein oxidation in Tibetan pork. Fig. 3B shows that the α-helix content of HT and RT treatments was significantly lower than that of UT treatment, while β-sheet content increased in varying proportions. This can be attributed to the oxidation of protein molecules during prolonged thawing, which weakens interactions between some protein residues and water molecules, disrupting hydrogen bonds accelerating protein oxidation and structural unfolding, and converting α-helices into β-sheets.

3.3.2 Fluorescence spectroscopy

Fluorescence intensity is used to assess tertiary structural changes in proteins, with the environmental polarity and position of tryptophan residues determining their fluorescence properties [36]. As shown in Fig. 3C, the fluorescence intensity of UT, HT, RT, and MT samples decreased, indicating alterations in the hydrophobic nuclei of myofibrillar proteins during thawing. UT treatment showed the highest fluorescence intensity among the thawing methods, likely due to the cavitation effect produced by ultrasound, which makes thawing more uniform and rapid, thereby reducing destruction and quality changes in myofibrillar proteins [37]. This result is consistent with FTIR and surface hydrophobicity data, indicating that UT treatment effectively inhibits oxidative denaturation of proteins. In UT-treated samples, proteins remain folded with tryptophan residues in hydrophobic environments, resulting in strong fluorescence intensity. MT accelerates the denaturation of the indole side chain in tryptophan residues, exposing tryptophan and increasing the polar climate. HT and RT thawing treatments caused accelerated oxidative damage due to temperature and prolonged thawing time, leading to protein structure unfolding and tryptophan exposure.

3.4 Low-field nuclear magnetic resonance (LF-NMR)

LF-NMR can characterize the water distribution and mobility in muscle samples, with the relaxation time (T2) and corresponding peak areas shown in Fig. 3D and Table 2. As shown in Fig. 3D, two peaks appeared between T21 (0–10 ms), suggesting that different thawing treatments affected the bound water status of Tibetan pork. These peaks represent strongly and weakly bound water to macromolecules [38]. Longer relaxation time (T22) implies increased water mobility and redistribution [39].Table 2 Effect of different thawing methods on the moisture distribution of Tibetan pork.

Tawing Methods	T2 (ms)	P2 (%)	
T21	T22	T23	P21	P22	P23	
RT	2.17 ± 0.55b	45.29 ± 0.38a	612.05 ± 1.58a	8.98 ± 0.36a	90.41 ± 0.40a	0.61 ± 0.04b	
UT	2.62 ± 0.30a	43.65 ± 0.69b	432.19 ± 5.77c	9.20 ± 0.33a	90.71 ± 0.41a	0.09 ± 0.05c	
HT	1.56 ± 0.13c	45.34 ± 0.57a	555.29 ± 6.80b	9.11 ± 0.41a	90.43 ± 0.69a	0.46 ± 0.04b	
MT	1.67 ± 0.34c	46.20 ± 0.58a	614.83 ± 6.45a	8.64 ± 0.29b	87.14 ± 0.31b	1.42 ± 0.51a	
UT: Ultrasound thawing; HT: Hydrostatic thawing; RT: Refrigerator thawing; MT: Microwave thawing.

As shown in Fig. 3D, the UT treatment resulted in the highest immobilized water and the lowest free water among the different thawing treatments. As shown in Table 2, P22 was the largest and P23 was the smallest for UT treatment. The cavitation effect induced by UT treatment in the structure of myogenic fibers helps protect intracellular water. Less free water in the cell reduces damage and protein oxidation during thawing [12], [40]. This also corresponds to our results for thawing losses and carbonyls. MT treatment showed a significant decrease in P22 and a significant increase in P23 compared to UT and HT. Hydrophobic-hydrophilic linkage breakage of leucine, valine, and alanine during MT treatment increases muscle fiber dehydration, reducing tenderness and water-holding capacity [41]. RT and HT treatments, T22 was significantly increased compared to UT. Prolonged thawing resulted in lipid and protein oxidation, weakening water-protein interactions, leading to water redistribution and conversion of immobilized water to free water, which flowed out of the meat.

3.5 Metabolomics analysis

3.5.1 Metabolite characterization

To better analyze the effects of different thawing methods on the nutrients of Tibetan pork, multivariate data analysis was performed using chemometric methods such as principal component analysis (PCA) and orthogonal partial least squares-discriminant analysis (OPLS-DA). These methods elucidated detailed information on the differences between hydrostatic and ultrasound thawing. The predicted results of PCA and OPLS-DA are shown in Fig. 4A and B, indicating that samples of each group were well reproduced and significant differences were observed between groups. In PCA, PC1 accounted for 73.96 % and PC2 accounted for 5.96 % of the variance, with a cumulative variance contribution of 79.92 %. In OPLS-DA, PC1 accounted for 75.1 % and PC2 for 14.5 %, with a cumulative variance contribution of 89.6 %. These results show that the predictive effect of the OPLS-DA model is significantly better than that of the PCA model. Therefore, the variable importance in projection (VIP) in the OPLS-DA model was used to determine the differential metabolites in samples with different thawing methods. In this study, VIP > 1 and P < 0.05 were used as criteria to identify differentially abundant metabolites, resulting in the identification of 64 differentially abundant metabolites (Appendix A). Fig. 4C shows the classification of differential metabolites in different thawing methods, including 16 amino acids and their metabolites, 14 nucleotides and their metabolites, 11 organic acids and their derivatives, 8 carbohydrates and their metabolites, 4 alcohols and amines, 3 fatty amides, 2 coenzymes and vitamins, and 6 other compounds.Fig. 4 Metabolite changes in Tibetan pork after different thawing treatments. PCA (A), OPLS-DA (B), classification of differential metabolites (C), clustering heat map analysis (D). UT: Ultrasound thawing; HT: Hydrostatic thawing.

3.5.2 Analysis of metabolite differences

To visually analyze the significant differences in metabolites between HT and UT, a clustered heat map analysis was performed using the relative values of metabolites under different experimental conditions. The colors on the heat map represent the relative levels of metabolites, with differences visualized by color gradients, as shown in Fig. 4D.

Different thawing methods can impact the flavor of meat products. The flavor of meat products depends mainly on the metabolic levels of flavor substances such as unsaturated fatty acids, free amino acids, inosinic acid, inorganic salts, ribose, peptides, and organic acids during the preparation process. The presence and content of these substances directly affect the taste and flavor of the meat.

In this study, we found that the amino acid and metabolite contents were significantly higher in the HT group compared to the UT group, as shown in Fig. 4D. This increase may be due to the enzymatic breakdown of muscle proteins, lipids, and other macromolecules during the thawing process, resulting in the release of metabolites. Different thawing methods may lead to varying degrees of enzyme activity and metabolite release, which in turn affects the taste of meat products. In the HT treatment, the levels of l-glutamic acid, l-lysine, and l-proline were significantly upregulated compared to the UT treatment. This may be attributed to the slower thawing rate in the HT treatment, where prolonged thawing disrupts the muscle fiber structure, leading to the release of proteases that activate protein hydrolysis [42]. It has been found [6] l-glutamic acid biosynthesis is closely related to arginine biosynthesis and d-glutamine and aspartic acid metabolism, and that l-glutamic acid can be used as a marker to distinguish between UT and HT processes. Additionally, l-glutamic acid metabolites were found to be positively correlated with the L* value [15], [43].

Glutathione is an important peptide in animal tissues that reduces oxidative stress and acts as a strong antioxidant. In our study, oxidized glutathione content was upregulated, while reduced glutathione content was significantly downregulated in the HT group compared to the UT group. Reduced glutathione can bind to free radicals, preventing the destruction of sulfhydryl groups by antioxidants. Among the two thawing treatments, the HT treatment had significantly higher oxidized glutathione content than the UT treatment, which corresponds with our sulfhydryl content results. The determination of free sulfhydryl content can indicate the degree of protein structure disruption, and the higher free sulfhydryl content in the UT group suggests a stronger antioxidant capacity. Increased oxidized glutathione levels suggest that prolonged thawing in still water exposes the sample to oxygen, accelerating oxidative reactions. This may lead to lipid oxidation in the food, adversely affecting energy metabolism. It has been found [44] that glutathione can act as an antioxidant and prevent lipid oxidation. These results suggest that UT treatment is more effective in inhibiting protein oxidation, thereby maintaining the original flavor and freshness of Tibetan pork.

It is evident that the content of nearly all nucleotides and their metabolites increased significantly after HT treatment compared to the UT group (Appendix A). Hypoxanthine is generated through the breakdown of ribonucleotides via phosphorylation in several biochemical reactions. This phosphorylation can occur with the involvement of various enzymes in the ribonucleotide metabolic pathway, converting ribonucleotides into products like hypoxanthine. Hypoxanthine, being bitter, can impart a bitter flavor to Tibetan pork. 2-Aminomethylpyrimidine, a pyrimidine derivative containing an amino group and a methyl group, is commonly found in the ribonucleic acid metabolic pathway. It is primarily formed through the breakdown of ribonucleotides following methylation in specific biochemical reactions. 2-Aminomethylpyrimidine interacts with bitter taste receptors, thereby intensifying the transmission signals of bitter substances and enhancing the bitter taste.

Reports indicate that carbohydrates play a pivotal role in predicting final pH as they are major metabolites. Glycolytic pathways are stimulated in the presence of high carbohydrate stores [45]. The abundance of carbohydrate metabolites was higher in the HT treatment compared to the UT group, primarily consisting of d-fructose-6-phosphate disodium salt, d-glucose, UDP glucose, etc. d-fructose 6-phosphate disodium salt serves as a sugar intermediate in the glycolysis pathway, resulting from the isomerization of 6-phosphate glucose by phosphoglucose isomerase. Elevated levels of it signify increased activity of the glucose phosphate isomerization reaction in the glycolytic pathway. Moreover, an increase in its content may lead to a reduction in antioxidant activity, potentially affecting the reduced activity of metmyoglobin and consequently impacting the redness of HT-treated meat.

We also observed significantly higher levels of l-lysine, l-proline, 5-oxoproline, and 1-methylhistidine in the HT group compared to the UT group. These amino acids are highly prone to protein carbonylation. Carbonyl compounds serve as crucial indicators of protein oxidation in meat and meat products. The results revealed fewer carbonyl derivatives in the UT group and more in the HT group. This suggests that UT treatment can decrease the production of oxidation products in meat, thereby reducing the extent of protein oxidation. It also validates that UT treatment enhances the oxidative stability of meat.

4 Conclusions

This study demonstrates that UT not only accelerates the thawing rate but also enhances the tenderness of Tibetan pork. Protein oxidation in UT-treated pork was significantly reduced compared to other thawing methods. Additionally, carbonyl content, surface hydrophobicity, TVB-N value, and TBARS value were significantly lower in UT-treated samples compared to other thawing methods, whereas the content of α-helices and fluorescence intensity were notably higher. This suggests that protein denaturation of Tibetan pork after UT thawing was minimized, and the binding capacity of protein and water molecules was more stable, resulting in more tenderness. Furthermore, the levels of hypoxanthine and 2-aminomethylpyrimidine decreased significantly after UT treatment, contributing to the reduction of meat bitterness and enhancing the freshness of Tibetan pork. l-glutamic acid has been proposed as a marker for distinguishing between UT and HT treatments, providing a robust theoretical framework for the advancement and industrial application of ultrasound technology. Moreover, UT offers a theoretical foundation and technical support for enhancing the quality of Tibetan pork and promoting the development of Tibetan pork processing technology. The specific effects of protein degradation and oxidation products on Tibetan pork after thawing nutritional attributes need to be further focused on infuture studies.

CRediT authorship contribution statement

Junmei Liu: Writing – original draft, Investigation, Funding acquisition, Formal analysis, Data curation. Xiefei Li: Writing – original draft, Investigation, Funding acquisition, Formal analysis, Data curation. Fang Geng: Methodology, Formal analysis, Data curation. Xin Li: Formal analysis. Yujie Huang: Data curation. Yingmei Wu: Writing – review & editing, Writing – original draft. Zhang Luo: Writing – review & editing, Visualization. Qun Huang: Resources, Funding acquisition, Formal analysis. Peng Shang: Writing – review & editing, Supervision, Conceptualization. Zhendong Liu: Writing – review & editing, Funding acquisition, 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.

Appendix A Supplementary data

The following are the Supplementary data to this article:Supplementary Data 1

Acknowledgments

This study was financially supported through grants from the 10.13039/501100012166 National Key Research and Development Program of China (2022YFD1600905 ), the Major Science and Technology Projects of Tibet Autonomous Region (XZ202101ZD0005N ), Joint Project of 10.13039/501100007548 Northwest A & F University -Xizang Agriculture and Animal Hus bandry College (XNLH2022-03 ), and the 10.13039/501100001809 National Natural Science Foundation of China (32160773 ).

Appendix A Supplementary data to this article can be found online at https://doi.org/10.1016/j.ultsonch.2024.107029.
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