Effects of ultrasound-assisted tumbling on the quality and protein oxidative modification of spiced beef

Abstract

The aim of this study was to investigate the effects of ultrasound-assisted tumbling (UT) with different ultrasound powers (frequency 20 kHz, powers of 0, 300 W, 450 W and 600 W) on the quality of spiced beef as explained from the perspective of the changes of muscle fibers and myofibrillar proteins (MPs). The results showed that pH value, tenderness and yield rate of UT groups were all apparently improved compared with the single tumbling group (P < 0.05). Ultrasound-assisted tumbling treatment could loosen muscle fiber structure supported by scanning electron microscopy (SEM) result, and the increased myofibrillar fragmentation index (MFI) value (P < 0.05). Additionally, an upward trend was observed in protein oxidation degree with the rise of ultrasound power level (P < 0.05), while the difference between groups in MPs solubility was not significant (P > 0.05). Above all, ultrasound-assisted tumbling treatment could effectively improve the quality of spiced beef by exacerbating the modifications in muscle fiber structure and MPs.

1. Introduction

Spiced beef products are one of the traditional Chinese meat products [1]. After years of inheritance and development, they have been integrated with local tastes and formed various traditional types with unique characteristics. Owing to its special flavor and direct edible properties, spiced beef product has been widely appreciated by people for many years [2], [3]. Curing is the critical processing step for spiced beef production. During which, salt penetration allows beef to retain its basic saltiness and promote the diffusion of curing substances into the meat to enhance flavor [4], [5]. The conventional curing method of the spiced beef is static curing. However, it generally takes 1–2 days, negatively affecting the mass production efficiency [6]. To solve such problem, some industries apply tumbling-assisted technology, which can accelerate the penetration of sodium uniformly and shorten the processing time to some degree via external force [7], [8]. Nevertheless, Li et al. [9] and Chen et al. [10] both pointed out that the present single tumbling technologies applied in meat production were still time-consuming, and its impact on meat quality improvement was limited as well. Hence it is imperative to develop innovative techniques so as to further improve manufacturing efficiency and lower production expenses.

High-intensity ultrasound (20–100 kHz, >10 W/cm²) has been widely used in many fields attributed to its eco-friendly and non-thermal processing features [11]. Besides, many studies have pointed out its potential in boosting the curing efficiency and shortening the production cycle [12]. Kang et al. [13] also proved that ultrasound treatment could improve beef tenderness and water holding capacity without negatively affecting other quality indexes during the curing stage. In addition, the application of ultrasound did contribute to the enhancement of beef flavor during post-slaughter aging as well [14]. Thus from previous studies, the combination of ultrasound and tumbling technology for assisting meat processing has a certain degree of feasibility. Li et al. [9] also found that by means of the breathing ultrasonic tumbling technology, the marinade absorption capacity of chicken breast was enhanced, accompanied with the improved texture. On this basis, our laboratory has developed an ultrasound-assisted vacuum tumbling (UT) equipment, which has been verified to improve the pork curing efficiency and the flavor of spiced beef [15], [16]. While the effect of UT treatment on the eating quality index of final spiced beef product is still unknown. Therefore, the hypothesis of this study was to investigate whether the ultrasound-assisted vacuum tumbling technique had a significant effect on the tenderness and yield rate of spiced beef. Moreover, pH, microstructure, MFI, and protein solubility and oxidation degree were investigated to further elucidate the behind mechanism for the changed quality of spiced beef product.

2. Materials and methods

2.1. Materials

Beef knuckle muscles (Simmental × Charolais) were obtained from Nanjing Qilin Meat Co., Ltd. (Nanjing, China) after 48 h of slaughtering. The surface fat and the fascia were removed at the temperature of 4 °C. Then each piece of meat was divided into the size of 80 × 80 × 80 mm³, with the weight of around 500 g. After vacuum packaging (DC800-FB-E, CRYOVAC, USA), the meat was stored at −20 °C for further use. Before each use, the beef sample was thawed at 4 °C for 24 h, and then the blood on the surface was washed and removed.

2.2. Sample treatment

The composition of marinade and brine, intermittent tumbling mode and cooking procedure were all in accordance with Jiang et al. [15].

The equipment independently designed by our laboratory [17] was used for UT curing with four groups including one group of single tumbling (ST) as the control group, and three groups of UT treatment. All meat blocks were randomly injected with 4 °C pre-cooled curing solution, and the injection rate was 35 % (Dongwuan Zhier Electronics Co., Ltd., China).

All groups of meat samples were marinated in an intermittent tumbling mode for 12 h. And based on our previous study [15], three UT groups were treated under ultrasound conditions with the power of 300 W (UT300), 450 W (UT450) and 600 W (UT600) for total 2 h each (20 kHz, ultrasound for 30 min, rest 30 min), which was conducted simultaneously with the tumbling treatment. After the cooking procedure, the meat samples were finally taken out, placed in a 4 °C environment and cooled to 25 °C.

2.3. pH

A 2 g meat sample was taken from the same position in the center of each meat block and deionized water was added at a meat/liquid ratio of 1:9 (w/v). Then the samples were homogenized (PD500-TP, PRIMA, UK) at 10,000 rpm for 30 s (repeated twice with an interval of 10 s). After centrifugation (Avanti J-E, Beckman Coulter, USA) at 12,100 × g for 10 min at 4 ℃, the supernatant was remained and the pH was measured using calibrated meter probe (FE20, Mettler Toledo, CH) with being immersed 2–3 cm into the samples.

2.4. Warner-Bratzler shear force

Referring to Kang's method [13] with slight modifications, prepared meat blocks were cut into 1 × 1 × 5 cm³ columns along the muscle fiber direction. The column was sheared perpendicular to the direction of the muscle fibers using a digital muscle tenderizer (College of Engineering, Northeast Agricultural University, China), and the shear speed was 5 mm/s. The maximum shear force (N) during the cutting process was observed and recorded.

2.5. Yield rate

The method was modified from that of Zhang et al. [16]. The thawed beef product was washed with 4 ℃ deionized water and weighed after surface drying as W₀. Then the samples cooled to 25 ℃ was weighed as W₁. The yield rate (%) was expressed as W₁/W₀ × 100 %.

2.6. SEM

According to Zhang et al. [16], the beef slices (5 × 5 × 2 mm³) were cut along the direction of muscle fibers at the same part of each constituent meat block, which were fixed with 2.5 % glutaric aldehyde acid and placed in 4 °C cold storage (no more than 1 week). The observation parameters of the instrument (SU8010, Hitachi, Japan) were 5.0 kV and 5,000 × magnification.

2.7. Extraction of myofibrillar proteins

The separation method was similar with Zhang et al. [17]. And purified proteins after multiple extractions were used to determine protein concentration by the bis-urea method (Thermo, USA).

2.8. MFI

The method of Kang et al. [13] was referenced and slightly modified. The absorbance (Spectral Max M2e, MD, Germany) was measured at 540 nm (A₅₄₀) after diluting MP to 0.5 mg/mL with 20 mM phosphate buffer (20 mM NaH₂PO₄·2H₂O, 20 mM Na₂HPO₄·12H₂O, 4 °C, pH 7.0). The MFI value was expressed as A₅₄₀ × 200.

2.9. Mps solubility

MP was diluted to 2 mg/mL using 20 mM phosphate buffer. The 5 mL of the solution was centrifuged (Avanti J-E, Beckman Coulter, USA) at 8,000 g for 10 min at 4 °C and the supernatants were collected to determine the protein concentration by the bis-urea method. Protein solubility (%) was expressed as protein concentration in the supernatant after centrifugation / (2 mg/mL) × 100 %.

2.10. Protein oxidation

2.10.1 Carbonyl content.

The measurement method was adopted by Zhou et al. [18] with slight modification. The 0.1 mL MP was diluted by phosphate buffer, and mixed with 0.4 mL of 10 mmol/L 2,4-dinitrophenylhydrazine (DNPH) or 0.4 mL of 2 mol/L HCl solution (blank group). After being vortexed and mixed for 1 min, the samples reacted at 37 °C for 1 h. Subsequently, 0.5 mL of 20 % trichloroacetic acid (TCA) was added and vortexed for 1 min to stop the reaction, following by standing out of light for 5 min. After centrifugation (Avanti J-E, Beckman Coulter, USA) at 17,420 g for 15 min at 4 °C, the precipitates were washed by adding 1 mL of ethanol:acetate mixture (1:1, v/v) and centrifuged for 10 min under the same condition. The above washing and centrifugation process was then repeated 4 times until the precipitate became white. Then the precipitate was dissolved in 1.25 mL of 6 mol/L guanidine hydrochloride solution for 15 min at 37 ℃ and centrifuged (Avanti J-E, Beckman Coulter, USA) at 17,420 × g for 15 min at 4 °C, and the absorbance was measured at 370 nm in the supernatant.

2.10.2. Sulfhydryl content

MPs were diluted with 20 mM phosphate buffer to a concentration of 1 mg/mL. Then 0.5 mL MP solution was added to 5 mL 20 mM Tris-HCl buffer (20 mM Tris-HCl, 8 M urea, 10 mM EDTA, 4 °C, pH 8.0) and 0.1 mL Ellman's reagent (pH 8.0). Notably, no urea was added to the 20 mM Tris-HCl buffer for reactive sulfhydryl determination.

The samples for the determination of total sulfhydryl groups were placed at the condition of 40 °C for 25 min, while the samples for the determination of reactive sulfhydryl groups reacted at 4 °C for 1 h under darkness. After the reaction, the solution was measured with the absorbance at 412 nm. The molar absorption coefficient was 136,000 mol^-1cm^-1.

2.11. Statistics and analyses of data

SPSS 25.0 software was used to analyze the data by one-way ANOVA. Data were analyzed for variance using Duncan's multiple test, where P < 0.05 was considered to be a significant difference among the groups. And results were expressed as mean ± standard error.

3. Results and Discussions

3.1. pH

Significant differences were observed for pH values between the ST and UT groups (P < 0.05), and the result showed an overall upward tendency as the pH reaching the peak at 450 W (Table S1). This demonstrates that the application of UT treatment could heighten the pH of spiced beef, which is in accordance with Wang et al. [19] who also reported increased pH of beef semitendinosus with varying ultrasound powers (0 W, 300 W, 600 W). The collapse of ultrasonic cavitation bubble could generate a localized high temperature and pressure, that not only damaged the protein chemical bonds and exposed more alkaline groups [20], but also migrated the ions outward into the cytoplasm via disrupting the cell membrane, thus altering the ionic functionality positions and increasing the pH value [1], [21]. Furthermore, Zhang et al. [1] found that owing to the sterilization effect of ultrasonic wave, the ultrasound-assisted cooked spiced beef remained a stable and much higher pH value throughout the 28 d storage period, while the pH of control group was constantly decreasing due to microbial spoilage.

3.2. Tenderness and MFI

In this study, Warner-Bratzler shear force was selected to reflect the tenderness of final spiced beef product (Fig. 1A). It was found that there was an apparent reduction in shear force value of UT groups in comparison with the ST group (P < 0.05). At the same time, among the UT groups, the shear force values of the UT450 and UT600 groups were obviously lower than those of the UT300 group (P < 0.05), which were respectively decreased by 12.84 % and 10.48 % (P < 0.05). These findings indicate that UT treatment facilitated the tenderization of spiced beef within the appropriate power range. Similar results are also found in the study of Kang et al. [13], under the 300 W ultrasound treatment (120 min), beef tenderness was significantly enhanced by 22.28 % than the untreated group. Notably, the difference between the UT450 and UT600 groups was not apparent according to the result (P > 0.05). This implied that there would be no further enhanced tenderness effects for the spiced beef when the ultrasonic power reached 450 W. Excessively high-intensity ultrasound treatments might induce significant changes in protein structure [22]. As reported by Zhang et al. [17], the 600 W ultrasound-assisted treatment (120 min) resulted in the denaturation and aggregation of proteins, which might have a negative effect on the meat product tenderness. Therefore, it is critical to select appropriate ultrasound parameters to achieve a balance between enhancing tenderness and minimizing protein aggregation.

Fig. 1.

Shear force value (A) and MFI (B) of spiced beef at different UT powers. a ∼ c different lowercase letters in each figure indicate significant differences between different groups at P < 0.05.

To further verify the effect of ultrasound-assisted tumbling treatment on the spiced beef tenderness, the MFI which indicated the myofibril’s fragmentation extent was also evaluated [23]. Fig. 1B shows that the UT groups had significantly higher MFI values than the ST group (P < 0.05). In consistent with our result, Zou et al. [24] also found that applying ultrasound (350 W) combined with sodium bicarbonate to chicken breast meat during curing process could significantly increase MFI and reduce shear force. The above results might be attributable to the destructive effects of ultrasound to meat product structure and thus the application of ultrasound could be beneficial to improve the tenderness of spiced beef.

3.3. Yield rate

Yield rate is largely dependent on the water holding capacity of meat samples, and directly relates to the product economic value. As Table S1 shows, compared to the ST group, the UT groups had significant higher yields (P < 0.05). The yield values of UT300, UT450 and UT600 groups increased by 7.30 %, 9.91 % and 7.07 % respectively, while the differences between the UT groups were not apparent (P > 0.05). It can be seen that ultrasound-assisted tumbling could increase the yield rate and water holding capacity of spiced beef even at a low ultrasound power (150 W). Similarly, Zhang et al. [16] observed that, among the 500 W power range, the ultrasonic-treated samples consistently exhibited a markedly higher pork tumbling yield relative to the untreated one, irrespective of the duration of the treatment (60 or 120 min). These results were owing to the ultrasound-induced cavitation, which effectively disrupted the muscle fiber structure, thereby loosening the myogenic fiber arrangement and increasing the internal space available for water retention and marinade absorption [25]. Besides, the UT treatment could promote the penetration of sodium chloride, which further increased the sodium concentration and promoted the swelling of beef muscle fibers [16]. On the other hand, the continuous infiltration of the marinade accelerated the formation of the gel network structure between salt-soluble proteins, which reduced the water loss [26]. Conventional spiced beef is normally restricted with low yield rate because of the long curing time and inappropriate cooking condition, hence this ultrasound-assisted tumbling equipment shows considerable potential for improved yield. However some researchers also reported that ultrasound had no significant effects on yield and water retention of cooked ham [27]. This could be due to that their ultrasonic intensity did not reach the threshold of being sufficient to induce alterations in muscle structure along with varieties in muscle structure and composition among species.

3.4. Microstructure

As illustrated in Fig. 2, the muscle fiber structure of spiced beef in each group exhibited distinct characteristics. The ones in the ST group were neatly arranged, with small gaps and only slight fracture. In contrast, as the ultrasound power reinforced, the muscle fiber membrane structure became scattered, and the muscle fibers were further swollen and stretched outwards with obvious bending and fracture. The myofibril gaps were enlarged and the general structure of muscle fiber was more diffused as a result. Furthermore, according to Zou et al. [24], the arrangement of myofibrillar fibers also gradually became scattered with the increase of power level in cooked beef samples accompanied with the broken Z-lines and loss M−lines, resulting in the dissolution of myofibrillar integrity. When the ultrasound power level reached 1,000 W, there was an apparent fracture between the sarcomeres, and the boundary became blurred.

Fig. 2.

The SEM micrographs of spiced beef at different UT powers (magnification, 5000 × ). The yellow arrow indicates the muscle fiber gap. A: ST; B: UT300; C: UT450; D: UT600.

The cavitation bubbles and micro-jets generated by the cavitation effect of UT treatment physically damage the connective tissue and myofibrils, directly affecting the skeletal structure of muscle fibers and thus reducing the overall integrity [29]. Many studies have proved the direct positive correlation between the disruption of muscle fibers and the increase in meat tenderness [30]. And the loosening of the muscle fiber structure provided more water holding space, so the yield rate was improved in turn. Additionally, this physical effect can also trigger the destruction of sarcoplasmic reticulum, lysosomes and other organelles to regulate the activity of endogenous proteolytic enzymes such as calpains and cathepsins, which accelerate the degradation of muscle fiber structural proteins and junction proteins [19]. This gave the beef a much higher basic tenderness before cooking. Kang’s study [13] also found that compared with static curing, the degradation of crucial cytoskeletal proteins including desmin and troponin-T in beef were significantly improved under the assistance of ultrasound. Additionally, some scholars proposed that the improvement of meat quality index was caused by the synergistic effect of ultrasound and NaCl [31].

3.5. Mps oxidation

A series of protein oxidation and denaturation during meat processing can change physicochemical and functional properties of proteins, thereby affecting the macroscopic quality of the product [32]. In this study, the carbonyl and sulfhydryl contents were chosen to reflect the effect of UT treatment on the oxidation extent of myofibrillar proteins in spiced beef.

As shown in Table S2, increasing ultrasound power level was found to cause an upturn of the carbonyl content and a downtrend of the total sulfhydryl content in spiced beef (P < 0.05). This implies that the MPs oxidation degree was greatly improved due to the ultrasound application. Many reactive oxygen radicals generated by the release of ultrasonic mechanical waves could directly oxidize the protein side-chain residues, leading to the formation of carbonyl groups and disulphide bonds [18]. On the other hand, according to our previous studies, it was found that the degree of lipid oxidation was increased after ultrasound-assisted treatment [15]. It is well known that lipid secondary oxidation products (such as MDA ect.) also promoted the protein oxidation process and led to an increase in intermolecular covalent bonds [32]. Sun et al. [33] found a gradual shift from alpha-helix to beta-folding of the protein secondary structure and the spatial structure of the protein became sparser as the level of oxidation increased. Meanwhile, disulfide bond formation due to protein oxidation also has been shown to promote the formation of protein networks in muscle tissue. This process was observed to increase the percentage of immobilised water, which promoted the water holding capacity in turn [28]. Nevertheless, excessive protein oxidation may adversely affect the final product quality because of the increased protein dimers and cross-linking structures. It not only expands the particle size of protein aggregates, but also exacerbates the muscle structural destruction, resulting in a reduction of water-holding capacity and poor product quality [21]. Besides, excessive oxidation of proteins also causes loss of amino acids and reduces the protein digestibility, which may have a negative effect on the quality and nutritional value of meat. Hence, it is significant to select the appropriate parameters of ultrasonic treatment for meat products to improve their eating quality.

Apart from these results, it can also be seen that the reactive sulfhydryl contents of UT groups were significantly increased compared with the ST group (P < 0.05, Table S2), and the UT600 group had the highest reactive sulfhydryl content. This may be due to that the UT treatment could unfold the myofibrillar protein structure and then expose more hydrophobic sulfhydryl groups to the protein surface, resulting in the rise of pH and improved meat product quality [34]. Similar conclusions are also drawn by Zhang et al. [17] who observed 33.21 % increase of reactive sulfhydryl groups in pork (300 W, 120 min). In addition, the energy released by the ultrasound cavitation cleaved the water molecules to form numerous free radicals that penetrated into the interior of myofibrillar proteins, affecting hydrophobic interactions and thereby altering the protein conformation. However, some researchers proposed ultrasonic treatment did not apparently affect or even decreased the reactive sulfhydryl content of MPs [35], which may be owing to the variations in ultrasound intensities and system media.

3.6. Mps solubility

Protein solubility is associated with the water holding capacity which can reflect the structural changes and aggregation of myofibrillar proteins. According to Fig. 3, the protein solubility of spiced beef showed no significant difference between groups in our study (P > 0.05). Long tumbling time might be the explanation of the altered MPs’ structures and improved protein solubility for all treatments. Kim et al. [36] reported that the 3 h tumbling treatment resulted in a significant increase in protein solubility of marinated of pork loins. However the effects of ultrasound treatment on muscle protein solubility are not consistent across different studies. For example, Ma et al. [37] and Amir et al. (fresh beef, powers of 100 W, 300 W) [38] both discovered ultrasound-assisted treatment could lead to an increase in protein solubility. While according to Zhang et al. [17], the protein solubility of high ultrasound power group (500 W) even decreased compared with that of the non-treated group in pork. It was attributed to that different ultrasound parameters led to the various aggregation and refolding extent of proteins and then different protein particle size and solubility.

Fig. 3.

Solubility of beef myofibrillar proteins at different UT powers. a ∼ c different lowercase letters significant differences between different groups at P < 0.05.

3. Conclusion

In this study, it was found that ultrasound-assisted tumbling treatment of spiced beef showed better tenderness and yield rate with a higher pH value, compared with the single tumbling group. The analysis of MFI values and microstructural images showed that UT treatment did help to increase the degree of destruction of muscle fibers and loosen the structure of myofibers, which led to the improved product quality. Besides, the spiced beef from UT groups presented a higher protein oxidation level as proved by increased carbonyl content, and decreased total sulfhydryl content. However the myofibrillar protein solubility did not change significantly between groups possibly due to the effect of long tumbling time. To summarize, ultrasound-assisted vacuum tumbling treatment has great prospects for being applied in meat industry to improve the eating quality and yield rate of spiced beef through regulating muscle fiber structure and protein properties.

CRediT authorship contribution statement

Wenxuan Wang: Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Software, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. Feiyan Jiang: Validation, Data curation, Conceptualization. Lujuan Xing: Validation, Conceptualization. Yan Huang: Validation, Conceptualization. Wangang Zhang: Supervision, Resources, 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.

We would like to submit the enclosed manuscript entitled “Effects of ultrasound-assisted tumbling on the quality and protein oxidative modification of spiced beef”, which we wish to be considered for publication in “Ultrasonics Sonochemistry”. No conflict of interest exits in the submission of this manuscript, and manuscript is approved by all authors for publication. I would like to declare on behalf of my co-authors that the work described was original research that has not been published previously, and not under consideration for publication elsewhere, in whole or in part. All the authors have approved the manuscript that is enclosed.

Appendix A. Supplementary data

The following are the Supplementary data to this article: