Physicochemical and microstructural attributes of marinated chicken breast influenced by breathing ultrasonic tumbling

Journal Pre-proofs Physicochemical and microstructural attributes of marinated chicken breast influenced by breathing ultrasonic tumbling Yan Li, Ting Feng, Jingxin Sun, Liping Guo, Baowei Wang, Ming Huang, Xinglian Xu, Jiying Yu, Harvey Ho PII: DOI: Reference:

S1350-4177(19)32021-8 https://doi.org/10.1016/j.ultsonch.2020.105022 ULTSON 105022

To appear in:

Ultrasonics Sonochemistry

Received Date: Revised Date: Accepted Date:

12 December 2019 9 February 2020 11 February 2020

Please cite this article as: Y. Li, T. Feng, J. Sun, L. Guo, B. Wang, M. Huang, X. Xu, J. Yu, H. Ho, Physicochemical and microstructural attributes of marinated chicken breast influenced by breathing ultrasonic tumbling, Ultrasonics Sonochemistry (2020), doi: https://doi.org/10.1016/j.ultsonch.2020.105022

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Physicochemical and microstructural attributes of marinated chicken breast influenced by breathing ultrasonic tumbling Yan Li1, Ting Feng1, Jingxin Sun1*, Liping Guo1, Baowei Wang1, Ming Huang2,3, Xinglian Xu4, Jiying Yu5, Harvey Ho6 1. College of Food Science & Engineering, Qingdao Agricultural University, Qingdao 266109, China 2. National Center of Meat Quality and Safety Control, Nanjing Agricultural University, Nanjing 210095, China 3. Nanjing Huangjiaoshou Food Sci. & Tech. Co., Ltd., Nanjing 211226, China 4. College of Food Science & Technology, Nanjing Agricultural University, Nanjing 210095, China 5. Hainan (Tanniu) Wenchang Chicken Co., Ltd., Haikou 571133, China 6. Auckland Bioengineering Institute, The University of Auckland, Auckland, New Zealand

* is the corresponding author Email: [email protected] Tel.: +86-532-58957771

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ABSTRACT: Currently, the conventional atmospheric pressure-based and vacuum-based tumbling processes have a limited improvement on the chicken characteristic attributes during the marination process. In view of this, through a breathing (pressure change) tumbling strategy, ultrasonication (40 kHz, 140 W) was applied to improve tenderness, taste, and microstructure of chicken by a redesigned tumbler. The results showed that the tumbling with the breathing action and ultrasonication significantly enhanced the marinating absorptivity, tenderness and taste, and accelerated the degradation of myosin light chain. Free peptides (from 1465.9 ± 34.6 to 4725.7 ± 43.2 μg/mL) and amino acids (from 1.503 ± 0.096 to 2.593 ± 0.109 mg/mL) rose evidently for the control and the breathing tumbling treatment assisted by ultrasound respectively. Raman analysis revealed that strength of disulfide bonds declined from 0.731 ± 0.006 to 0.607 ± 0.011 a.u. and the conversion from α-helix (decreased by 67.23%) into β-fold (increased by 1573%) conformation occurred. Low field NMR analysis indicated that the content of immobilized water increased from 77385 ± 14 to 137011 ± 106 au · ms by integral calculus. Scanning and transmission electron microscopies clearly showed a prospective rupture of myofibers, myofibrils, and lysosomes. Overall, as a potential alternative, the breathing ultrasonic tumbling means improved the marinating efficiency and characteristics of marinated chicken breast. KEYWORDS: ultrasound; breathing; tumbling; chicken breast; physicochemical property; microstructure

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1. Introduction The marinating process is a prevailing preservation strategy for meat products through addition of sodium chloride, sodium nitrate, phosphates, vinegars, sugars, spices, etc [1]. It is used to prevent rottenness, enhance water-holding property, stabilize color, and release substances of taste [2]. The tumbling marinating technology is a frequently used process that can not only improve the texture of meat products, but can also achieve tenderization under mechanical forces (e.g. friction, extrusion, and impact forces). Briefly, the tumbling actions can enlarge the intercellular space within tissues. It also reduces mechanical strength of myofibers, accelerates permeation and diffusion of saline ions, and even breaks myofilaments (cellular damage) [3]. These mechanical forces also help rearrange the distribution of moisture within muscular tissues. Generally, tumbling processing effects are affected by some considerable physical factors such as the tumbling time, rotation speed, temperature, pressure intensity, load capacity, etc. Low-speed, low-pressure (e.g. in a vacuum), and low-temperature tumbling modes are deemed to be an ideal processing method with such advantages as causing a low temperature fluctuation derived from rubbing action, allowing the uniform permeability of brine, and promoting the removal of bubbles [4]. Nowadays, the traditional processing methods for marinated raw meat products are being modified and improved in pretreatment (such as trimming, tenderization, and freeze-thawing) and marinating/salting (such as ultrasound, vacuum impregnation, pulsed pressure, pulsed electric fields, etc.) [5]. Among these methods, the traditional single vacuum-based tumbling approach is prevailing in some meat processing enterprises. However, there are nonnegligible factors in its applications such as marinating efficiency, temperature, oxidation, and vacuum efficiency. If these factors are not properly controlled, they may cause risks such as poor marinating efficiency, time-consumption, temperature build-up of meat, microbial reproduction, color deterioration, and excessive 3

moisture loss [6]. Deumier et al. [4] showed that pulsed vacuum brining led to a product that was more salted and less dehydrated than products brined under atmospheric brining conditions. A similar result was also reported, i.e. pulse vacuum brining could be used to improve the brining efficiency, promote the actomyosin dissociation and improve the water-holding capacity of lamb [7]. Due to its accessibility, the ultrasound technology is widely applied in the meat product industry for marinating, unfreezing, tenderization and sterilization [8]. A reduction of marinating time may be caused by ultrasound because of the increased cell membrane permeability and the promotion of penetration and diffusion of electrolyte ions [9]. Specially, ultrasonication could accelerate the thawing process due to the heat transfer within meat products [10]. Zou et al. [11] showed that myofibrils were presented with the largest interfibrillary spaces and the highest degree of actomyosin dissociation in the USB (ultrasound and sodium bicarbonate) group. However, thermal effects are a nonnegligible factor, demanding attention. At present, chicken products are marinated mostly by employing traditional atmospheric pressure-based tumbling or vacuum-based tumbling or ultrasonic-assisted treatment. From a production’s perspective, the tumbling process with a shortened marinating time is indeed favored. However, there are some shortcomings associated with traditional single forms and processing equipment, such as low marinating absorptivity, uneven penetration, uncontrolled heat production, and limited promotion in meat quality. Moreover, there is no report on the redesigned (refitted) tumbler with an ultrasonic module and an adjustable variable pressure module, synergistically applied to the study on the improvement of marinating effects and processed chicken quality. Hence, the main objective of the present research is to apply the practical redesigned tumbler to marinating processing of chicken-derived products to improve its attitudes (e.g. tenderness and/or gelation) and productivity. In this study, four tumbling modes are performed by controlling air pressure and ultrasound. Meanwhile, marinating absorptivity, molecular conformation, water distribution, and morphology are evaluated from a productive and structural point of view. 4

2. Materials and methods 2.1 Materials Fresh chicken breast (pectoralis major, 4 h postmortem) was purchased from a local supermarket in Qingdao, China. Sodium pyrophosphate (SPP), sodium tripolyphosphate (TPP), and sodium hexameta phosphate (HMP) were provided by Jianglai industrial Inc. (Shanghai, China). The composite sodium phosphate composed of SPP/TPP/HMP = 4/5/3 (w/w/w) was purchased from Macklin Biochemical Co., Ltd. (Shanghai, China). Other chemical reagents used in the marinating process, including extraction of myofibrillar proteins, measurement of amino acids and oligopeptides, electrophoresis, and electron microscopy are of analytical or guaranteed purity. 2.2 Marination and tumbling processes The fresh chicken breast was divided into three parts: muscles, tendons, and fats. The muscles were cut into small pieces (5 cm × 5 cm × 2 cm) and precooled to 4℃ at a small refrigerator house prior to processing, followed by uniformly marinating in a 7.0 wt% sodium chloride solution (brine) containing 1.2 wt% composite sodium phosphate (pH 6.2-6.3) at 4 ℃. The blending proportion of chicken breast and brine was set as 100/30 (w/v). Then, the four tumbling modes were carried out respectively at 4 ℃ at a stirring rate of 16 r/min in a redesigned tumbler (internal valid volume 50 liters) with a vacuum pump unit (an adjustable negative pressure module), an ultrasonic probe (Φ12 mm diameter) unit and an ultrasonic controller unit (Fig. 1). The two accessories were installed on the basis of a commercial tumbler (GR-20, Xindeli Food Machinery Co., Ltd., Zhucheng, China). An ultrasonic vibrator (JM1003, 40 kHz, 140 W) provided by the Rikangdachuang Ultrasound Inc., (Shenzhen, China) was placed at the bottom of the tumbler. The four tumbling modes were carried out respectively at initial 4 ℃ as follows: (1) constant pressure (0.101 MPa) tumbling for 40 min, namely PT; (2) tumbling in a vacuum (−0.08 MPa) for 40 min, namely VT; 5

(3) tumbling in a vacuum (−0.08 MPa) for 15 min, constant pressure (0.101 MPa) tumbling for 5 min, and cyclically tumbling in a total of 40 min, namely PV; (4) based on (3), synchronously tumbling through ultrasonication for 5 min in the constant pressure (0.101 MPa) stage, and cyclical treatment in a total of 40 min, namely UPV.

Fig. 1 Schematic diagram of a redesigned tumbler with a vacuum pump unit and an ultrasonic unit. 1. roller; 2. ultrasonic probe; 3. spiral baffle; 4. roller chain sprocket; 5. sprocket shaft; 6. motor; 7. coupling; 8. reduction wheel; 9. ultrasonic controller; 10. frame; 11. friction wheel; 12. safety valve; 13. sealed cap; 14. roller opening; 15. roller friction wheel; 16. adjustable variable pressure control device with a vacuum machine

Furthermore, the process without tumbling, pressure changes and ultrasonication but only marinating for 40 min in an atmospheric stainless steel container (10 L) was performed as the control, namely WT. The filling volume of chicken breast accounted for 2/3 of the volume capacity of the tumbler or the stainless steel container. Briefly, the size of the batch is 40 kg for tumbler, while 8 kg for WT control using the stainless steel container. 2.3 The marinating absorptivity 6

The marinating absorptivity (MA) was calculated using the following formula.

𝑀𝐴(%) =

𝑤2 - 𝑤1 × 100% 𝑤1

where w1 and w2 are the weight (wet weight) of chicken breast before and after marinating (unit: g). The marinated chicken breast is lightly drained to remove surface liquid before determination. 2.4 Tenderness evaluation The chicken breast treated by above processes was cut into five identical pieces in size (approximately 5 cm × 1 cm × 2 cm) along myofibers. The maximal shear force, acting as a tenderness index, was determined using a muscle tenderness meter (MODEL2000D, G-R Inc., US) and was expressed as Newton's strength (unit: N). The samples were penetrated by a cylindrical probe (P0.5, 15mm diameter). The shearing rate was set as 5.0 mm/s and the cutter edge was perpendicular to myofibers. A force-penetration time curve was obtained and the maximum value of the curve was recorded. 2.5 Extraction of myofibrillar proteins, SDS-PAGE analysis and myofibril fragmentation index Myofibrillar proteins were extracted based on the method of Doerscher et al. [12] with some modifications. The treated chicken breast (around 2 g) was cut into small pieces and homogenized at 6000 × g for 4 min in a 0.02 M phosphate buffer (pH 6.5) with a solid-liquid ratio of 1/10 (g/ml). The homogenate was centrifuged at 10,000 × g for 20 min at 4 ℃. The supernatant liquid was removed and the insoluble matter was repeatedly treated following the same procedures (homogenization, centrifugation, and supernatant removal) for three times. The resulted insoluble matter was reintroduced into a 0.1 M phosphate buffer (pH 6.5) with a solid-liquid ratio of 1/10 (g/ml). Then, potassium iodide and sodium azide were added and adjusted to 0.7 M and 0.02%, respectively. The mixture was homogenized at 7000 × g for 3 min and centrifuged at 10,000 × g for 20 min at 4 ℃. The resulting supernatant was collected as isolated myofibrillar protein solution. In addition, the determination method of protein

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concentration was recommended by means of the biuret reaction (bovine serum albumin as a standard). The protein concentration was required to be diluted for further analysis. According to the method of Tomaszewska-Gras, et al. [13], SDS-PAGE analysis of protein samples was carried out to identify the composition of extracted myofibrillar proteins by an electrophoresis apparatus (DYY-6C, Liuyi Biotechnology Inc., Beijing, China). The extraction of myofibrils from chicken breast were suspended in the isolating buffer containing 100 mM KCl, 20 mM K2HPO4, 1 mM EDTA, and 1 mM NaN3 with a pH of 7.0. The myofibril fragmentation index (MFI) was measured in myofibril suspension using an ultraviolet spectrophotometer (UV-2802, UNICO Instruments Inc., Shanghai, China) at 540 nm. The MFI values were calculated using the formula MFI = A × 200, where A is the absorbance of myofibrillar protein solution [14]. 2.6 Free amino acid analysis Chicken breast treated by the above process was lyophilized and milled into powder. Approximately 2 grams of powder was added and dissolved into 50 mL ultrapure water. The chicken breast suspension liquid was introduced into an isovolumetric 10% (v/v) sulfosalicylic acid solution, incubated for 2 h, filtrated by a 0.22 μm filter membrane, and mixed with 2 mL, 1% (v/v) EDTA-Na2 and 2 mL, 0.06 M HCl solutions for 5 min, and centrifuged for 20 min at 15,000 × g at 4 ℃. The resulting supernatant was analyzed using an amino acid analyzer (8900, Hitachi Inc., Japan). 2.7 Free oligopeptide analysis Approximately 3 grams of chicken breast treated by the above process was introduced into 60 mL, 0.2 M phosphate buffer (pH 6.5), followed by homogenization for 3 min at 6000 × g. The homogenate was centrifuged for 20 min at 4 ℃ at 10,000 × g. The supernatant (1 mL) was added into 2.5 mL acetonitrile, followed by centrifugation for 20 min at 4 ℃ at 15,000 × g. The supernatant (2.5 mL) was lyophilized and the residues dissolved in 40 μL, 0.05% (v/v) trifluoroacetic acid. The filtration was performed via a 10 kDa molecular weight 8

membrane before injection into liquid chromatograph (LC) system (1260, Agilent technologies Inc., US). The LC conditions: BioBasic C18 Column (250 mm in length, 4.6 mm I.D., Thermo Fisher Scientific Inc., US), eluent A (0.05% trifluoroacetic acid), eluent B (acetonitrile: water: 0.05% trifluoroacetic acid = 60/40/0.04, v/v/v), gradient elution program (100% decreased to 1% for eluent A, 1% increased to 100% for eluent B), elution time (25 min), flow rate (0.9 mL/min), and wavelength (214 nm). The L-Carnosine was used as an internal standard substance. 2.8 Roman spectroscopy analysis The myofibrillar protein suspension was lyophilized and then analyzed at room temperature by a laser microscopic Raman spectroscope (Labram HR 800, Jobin-Yvon Inc., France). The detecting parameters were set as follows: power (100 mW), aperture (200 µm), raster (600 g/mm), scanning area (300-3500 cm-1), integral time (60 s), resolution (2 cm-1), and scanning speed (120 cm-1/min). The field-normalization operation was performed in the amide I and III regions according to the stretching vibration intensity of the side chain of phenylalanine as an internal standard at 1003 cm-1. The spectrograms were smoothed using the software Labspec (Jobin-Yvon Inc., France). The content of assigned protein structure (α-helix, β-fold, β-turn, and random coil) from the amide I and III bands was calculated using the method by Alix et al. [15]. 2.9 Low field-nuclear magnetic resonance (LF-NMR) The mobility and distribution of moisture were evaluated at 32 ℃ under a resonant frequency of 22.6 MHz and a scanning frequency of 32 for treated chicken breast using a LF-NMR apparatus (PQ001, Niumag Inc., Shanghai, China). Approximately 2 grams of chicken breast (coated with a polyethylene preservative film) was placed in a Φ15-mm cylindrical glass tube and inserted in the NMR probe. The transverse relaxation time (T2) was measured under a τ-value of 300 μs between pulses of 90° and 180°. The NMR T2 data were analyzed using the Multi-Exp Inv Analysis Software (Niumag Inc., Shanghai, China). The following parameters were presented that T21, T22a, T22b, and T23 were the relaxation time, corresponding to the highest point of peaks of water populations, and PT21, 9

PT22, and PT23 were the corresponding integral area fractions. 2.10 Scanning electron microscopy (SEM) The microscopic surface structure of treated chicken breast was studied using a scanning electron microscope (7500F, JEOL Inc., Japan). According to the method introduced by Li et al. [16], chicken breast was cut into cubes with approximately 0.075 cm3 (0.5 cm × 0.5 cm × 0.3 cm) in size. Images were taken using SEM at an accelerating voltage of 2 kV. The micrographs of myofibers and myofibrils were taken at ×1000 magnification. 2.11 Transmission electron microscopy (TEM) The microscopic cross-section structure of chicken breast was studied using a transmission electron microscope (HT7700, Hitachi High-Technologies Inc., Japan). Chicken breast was cut into cubes with approximately 0.008 cm3 (0.2 cm × 0.2 cm × 0.2 cm) in size along the vertical direction of myofibrils. The treatment method was recommended by Li et al. [17] with slight modifications. The ultramicrotome (UC7, Leica Inc., Germany) was used in the slicing process. The samples were deposited onto a carbon-coated copper grid (300 meshes) and then lyophilized for 6 h for further observations. Images were taken using TEM at an accelerating voltage of 80 kV. The micrographs of the samples were taken at ×1000, ×4000, and ×8000 magnifications. 2.12 Statistics Samples were measured in triplicate. The SPSS 19.0 software was used to analyze the variance by one-way ANOVA. Duncan multiple comparisons were used in the numerical analysis. All values are expressed as means ± standard deviation with a significant difference level P < 0.05. 3. Results and discussion 3.1 Marinating absorptivity Marinating absorptivity is used to assess the quality of chicken products during marinating processing. An excellent marinating action aims to prevent spoilage through reducing water activity, and to further provide a 10

special taste after processing or during preservation. Generally, the taste substances mainly included some degraded protein fragments, such as oligopeptides and free amino acids, which endowed marinated chicken breast with a special taste (a compound of saltiness, bitterness, umami, sourness, etc.) [1,18]. As shown in Table 1, the breathing (alternating cycles of vacuum and atmospheric pressure) tumbling process assisted by ultrasound (UPV) revealed a maximal marinating absorptivity reaching 22.14 ± 1.22% (P < 0.05). In contrast to the traditional processing technologies WT, PT and VT, significant differences in marinating absorptivity may be attributed to regular pressure fluctuation and intermittent ultrasonication for PV and UPV in the airtight tumbler. The application of adjustable pressure (negative pressure) accelerated the permeation of brine and reduced the loss of moisture in meat products [4]. Actually, the breathing i.e. pressure fluctuation applied in this study led to a fast release of gases (e.g. CO2, O2) dissolving in tissues and a spontaneous release of proteins into tissue spaces. In the latter case, a portion of proteins (e.g. SSPs) susceptible to dissolution in brine indicated that a disassembly action of myofiber skeleton protein structure could result in a loose chicken structure and benefit tenderization [19]. Importantly, ultrasonication may shorten marinating time [8]. In this study, the ultrasonication with fixed frequency and intensity (40 kHz, 140 W) produced a strong molecular (or ionic) vibration within tissues or between tissues and brine. Moreover, an environmental differential shearing pressure (ultrasonic mechanical effects) caused myofiber disruption. It was predictable that myofiber disruption would be liable to release some endogenous proteolytic enzymes from lysosomes, and therefore disorganization of structural proteins and proteolysis reactions would occur. The anticipated looser structure of myofibers was in favor of capturing more brines. In any case, the permeation, diffusion, and retention of brine became easier and the potential tenderization could be more readily guaranteed by tumbling, breathing operation, and ultrasonication (PV and UPV) than the WT, PT and VT processes.

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Table 1 Physicochemical parameters of chicken breast via different tumbling processes Determinations

WT

PT

VT

PV

UPV

Marinating absorptivity, %

−

14.69 ± 0.93a

18.01 ± 0.92b

19.94 ± 1.69c

22.14 ± 1.22d

Maximal shear force, N

13.82 ± 0.51e

10.11 ± 0.43d

8.70 ± 0.25c

8.03 ± 0.28b

6.70 ± 0.29a

Total amino acids*, mg/mL

1.503 ± 0.096a

1.710 ± 0.102b

2.095 ± 0.106c

2.361 ± 0.122d

2.593 ± 0.109e

Essential amino acids*, mg/mL

0.633 ± 0.042a

0.674 ± 0.029a

0.873 ± 0.048b

0.894 ± 0.039b

1.004 ± 0.066c

Total oligopeptides#, μg/mL

1465.9 ± 34.6a

2871.0 ± 46.5b

3844.1 ± 52.1c

4169.0 ± 10.1d

4725.7 ± 43.2e

The sample number was 3 for each treatment group. The different lowercase letters a-e in the same row indicate significant differences (P < 0.05). PT: atmospheric pressure (0.101 MPa) tumbling, VT: vacuum-assisted (−0.08 MPa) tumbling, PV: combination of breathing tumbling, UPV: breathing tumbling assisted by ultrasound, WT: the process without tumbling, vacuum treatment, and intermittent ultrasonication. * means that total amino acids and essential amino acids were detected based on a free form. # means that the retention time by liquid chromatography was set within 18 min. – means not given.

3.2 Tenderness analysis Shearing force values are used to evaluate the tenderness of chicken. The smaller the shear force value is, the better the tenderness is, and vice versa. The tenderness of meat products is generally dependent on meat sources, slaughter methods, processing methods, texture features, etc. Specially, tenderness differences mainly result from disruption of muscle structures, including the tightness between proteins (e.g. actin and myosin), connective tissue

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structures, and intermuscular fats. In this study, compared to WT, a significant decline (P < 0.05) of maximal shear force value from 13.82 ± 0.51 N (WT) to 10.11 ± 0.43 N (PT), 8.70 ± 0.25 N (VT), 8.03 ± 0.28 N (PV), 6.70 ± 0.29 N (UPV) in Table 1 meant that the tenderness tended to improve, which may be ascribed to degradation of myofibril structure by mechanical tumbling and ultrasonic cavitation [20]. Indeed, in the data displayed in Table 1, all of these above actions were to contribute to an improvement of tenderness. Aktaş and Kaya [21] found that the content and concentration of organic acid-based or saline-based pickling liquid had a significant effect on the recovery of muscle tenderness during processing. Therefore, amongst four tumbling treatment groups, the significant decrease of shear force value during the marinating process may also be related to the content of absorbed sodium chloride in loose myofiber structures, compared to WT. Another study reported by Lyng et al. [22] confirmed that ultrasonication with a strong penetrating power promoted the damages of lysosomes, myofibrillar proteins, and other constituents, which would produce a differentiated characteristics with an improved tenderness. In this study, the breathing (variable pressure) tumbling PV and UPV were better than PT and VT in weakening shear force. This phenomenon may be due to the fact that pressure changes indirectly aggravated the structural instability (i.e. degradation) of myofiber skeleton proteins by the infiltration of marinating liquid. Apparently, ultrasonic treatment was more conductive to an improvement of tenderness than other processing means. Jayasooriya et al. [23] showed that as proteins were degraded, a significant downtrend of shear force meant an uptrend of tenderness value for fresh chicken after ultrasonic processing (25.9 kHz), which was consistent with our present results. Furthermore, tumbling actions and saline permeation had also an unfavorable impact upon connective tissue structure with a certain toughness. 3.3 Amino acid and oligopeptide analysis Tumbling actions generated multiple mechanical forces thereby acting fast on myofibrillar structure and myofibrillar proteins in myofibers. The influence may be reflected to a certain extent in the release of endogenous 13

digestive proteases and enzymatic cleavage of proteins, which endowed meat products with particular desirable tastes [24]. In fact, the substances causing the tastes generally covered amino acids, oligopeptides, fatty acids, esters, nucleotides, and mineral ions [25]. Amongst them, amino acids and oligopeptides account for the majority of degradation products of myofibers, influencing the tastes and gelling properties during processing. The enzymatic products acted as precursor substances for certain volatile substances during cooking. Specifically, these amino acids (e.g. Glu, Ser, Gly, Asp, Thr, etc.) and oligopeptides could directly influence meat tastes. In this study, a phenomenon was confirmed that detected total amino acids, essential amino acids and oligopeptides were increased during the four types of tumbling processes, compared to WT (Table 1). All the above physical processes may strengthen the release of small molecular substances inside/outside cells and achieve (or enhance) the tastes and juiciness of meat. The physical power, ultrasound and pressure (breathing action), enhanced the dissociation of myofibers and molecular digestion. The maximal amount of hydrolysis products for the desirable UPV treatment were mainly caused by significant myofiber disruption that would be liable to release more endogenous proteolytic enzymes from lysosomes. Consequently, proteolysis reactions and dissociation of more substances would occur under the conditions of ultrasound and pressure changes. Similarly, Pohlman et al. [26] confirmed that the process of disassembly of membrane structure in cytomembrane, cytoplasm, and lysosome liberated endogenous proteases in the intercellular matrix. Furthermore, it has been shown that ultrasonication at appropriate frequencies and intensity levels led to enhanced enzyme activity due to favorable conformational changes in protein molecules without altering its structural integrity [27]. 3.4 Myofibril fragmentation index (MFI) and SDS-PAGE analysis The tumbling marinating processes achieved dissociation, release and enzymatic degradation of myofibrillar proteins, which was depicted in the electrophoretograms in Fig. 2. The tumbling actions were a favorable behavior 14

that promoted denaturation, rearrangement, and aggregation of proteins in terms of forming a desirable thermally induced network structure. For instance, as a key component of myofibrillar proteins, the released actin was increased in its concentration via four tumbling marinating processes as compared to WT, whilst tropomyosin was decreased slightly (Fig. 2). Herein, a hypothesis was that the joint effects of release of actin and dissociation (from a complex state to a free state) of actomyosin determined an uptrend profile of actin content, while the dissolution and dilution effects may result in a downtrend towards tropomyosin content during marinating. The combination of low levels of actin with myosin could contribute to the excellent gel elasticity while an opposite side at higher levels [27]. The conceivable dissociation and degradation of actomyosin (a complex protein formed by actin and myosin), as a myofibrillar skeleton protein, could have a favorable impact on the tenderness, water-retaining property, and gelling property [28]. However, if the strong degradation and obvious content decrement of tropomyosin occur, the results would otherwise show a decrease in the hardness and the springiness of myosin and actomyosin gels, thus influence the chicken quality seriously [29]. Actually, we could not confirm that the tumbling processes induced the degradation of actin (a larger molecular weight protein) and tropomyosin (a smaller molecular weight protein) from an electrophoresis experiment only. The myosin light chain (MLC) protein content was decreased gradually as becoming shallow in some electrophoretic bands, especially for UPV. The reasons underlying the phenomenon were due to the obviously enzymatic degradation of a small number of MLC proteins, the potential dilution of brine, and the dissolution of substances. The content differences of these MLC proteins could influence the tenderization even though they have no advantage in quantitative terms [30]. Importantly, it should be underlined that intermittent ultrasonication indeed facilitated the degradation of smaller molecular weight MLC A and B subunits (Fig. 2), whilst pressure changes principally brought about extraction and release of fragments (e.g. subunits and peptides) from despiralized myosin.

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Fig. 2 SDS-PAGE patterns and myofibril fragmentation index (MFI) of myofibrillar proteins and myofibrils from chicken breast treated by four tumbling processes. PT: atmospheric pressure (0.101 MPa) tumbling, VT: vacuum-assisted (−0.08 MPa) tumbling, PV: combination of breathing tumbling, UPV: breathing tumbling assisted by ultrasound, WT: the process without tumbling, vacuum treatment, and intermittent ultrasonication.

MFI can reflect the structural integrity of myofibrils and their cytoskeleton proteins. Generally, the higher the MFI value is, the more myofibrillar proteins are decomposed. The improvement of tenderization was closely correlated with the increment of MFI value [31]. In this study, the myofibrils were cut into various segments of sarcomeres by the tumbler, suggesting that tumbling processes possessed the great potential in improving MFI value of chicken breast, especially significantly (P < 0.05) by UPV (Fig. 2). The above results were in accordance with those of shear force trial and amino acid analysis. Moreover, it should be emphasized that the usage of

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ultrasonication needs a relatively strong intensity and a long time to achieve its function in fragmentation of proteins [32]. Pressure changes were not liable to establish an osmotic equilibrium between intracellular and intercellular matter, but just maintained a continuous and exchanged state between osmosis and dissolution. 3.5 Raman spectroscopy analysis Table 2 Effects of tumbling treatment on disulfide bonds, aromatic side chains, and protein secondary structure of myofibrillar proteins from chicken breast through Raman spectra Determinations

WT

PT

VT

PV

UPV

Strength of disulfide bonds*(a.u.)

0.731 ± 0.006a (535 cm-1)

0.723 ± 0.002b (524 cm-1)

0.720 ± 0.005b (521 cm-1)

0.721 ± 0.004b (521 cm-1)

0.607 ± 0.011c (519 cm-1)

I760/I1003

0.761 ± 0.016a

0.781 ± 0.002b

0.819 ± 0.013c

0.847 ± 0.010d

0.811 ± 0.003c

I850/I830

1.001 ± 0.002a

1.035 ± 0.003c

1.023 ± 0.017bc

1.024 ± 0.006b

1.016 ± 0.008b

α-helix# (%)

76.97 ± 6.02a

55.71 ± 4.63b

39.72 ± 4.17c

33.99 ± 1.61d

25.22 ± 3.32e

β-fold# (%)

2.55 ± 0.23a

19.08 ± 1.32b

31.86 ± 2.03c

35.93 ± 2.02d

42.67 ± 1.87e

β-turn# (%)

11.56 ± 1.04a

14.95 ± 1.07b

17.32 ± 0.88c

18.53 ± 0.34c

20.31 ± 0.97c

Random coil# (%)

8.92 ± 0.53a

10.26 ± 0.82b

11.10 ± 0.08c

11.55 ± 0.51cd

11.80 ± 0.43d

The sample number was 3 for each treatment group. The different lowercase letters a-d in the same row indicate significant differences (P < 0.05); PT: atmospheric pressure (0.101 MPa) tumbling, VT: vacuum-assisted (−0.08 MPa) tumbling, PV: combination of breathing tumbling, UPV: breathing tumbling assisted by ultrasound, WT: the process without tumbling, vacuum treatment, and intermittent ultrasonication.* means normalized intensities for the vibration of disulfide bonds in Raman spectra, and the maximal peaking position of disulfide bonds of myofibrillar proteins is assigned to the corresponding bracket. # means the relative percentage composition of conformations in protein secondary structure.

With regard to natural protein folding, the disulfide bonds possess some differentiated molecular conformations in general, which is the structural basis of protein folding [33]. The appearance and fracture of disulfide bonds 17

influence the formation of heat-induced protein (e.g. myosin) gels to some extent. In this work, we found that the intensity and shift of the Raman peaks corresponding to disulfide bonds at around 510-540 cm-1 were varied after tumbling treatment [34]. The normalized intensity of disulfide bonds was 0.731 ± 0.006 for WT while its decrease was observed in PT, VT, PV, and UPV, suggesting a potential fracture to disulfide bonds (Table 2). In addition, the shift towards a low wavenumber direction was observed, compared to WT (at 535 cm-1), signifying changes in the conformation of disulfide bonds, namely changes of protein structure. As a consequence, changes of its spatial configuration may mean exposing more sulfydryl, peptide bonds, hydrophobic groups, and side chains of amino acids in a spatial level. Surely, these above groups were also more liable to reform a new disulfide linkage (disassembly and self-assembly of protein subunits) and a highly ordered protein gel network basis than existing external groups during heat treatment [35]. Due to the presence of benzene ring in side chains, the hydrophobicity of exposed hydrophobic amino acids inside proteins would alter some intermolecular stability and molecular gelation [36]. The I760/I1003 and I850/I830 values were used to indicate the normalized intensity of the side chain of aromatic amino acids (tryptophan and tyrosine residues, respectively) of myofibrillar proteins, which reflected the polarity of microenvironment near aromatic amino acid residues. If the tryptophan residue was situated into a polar solvent microenvironment instead of a hydrophobic microenvironment, the I760/I1003 value was declined [37]. Actually, an evident increment in I760/I1003 value was confirmed via tumbling processing in Table 2, especially PV (0.847 ± 0.012), which meant that part of surface tryptophan residues could be buried again due to rearrangement of new protein folding conformations or subunits. As an indicator applied to detect the microenvironment (e.g. interfacial polarity and hydrogen bond produced by phenolic hydroxyl group) around the tyrosine residues, the tyrosyl doublet ratio (I850/I830) would present a differentiated trend at Raman spectra and be proposed to determine whether the tyrosine residue is solvent exposed or buried [37]. If a polar solvent microenvironment wrapped tyrosine residues or 18

tyrosine residues were acted as a weak hydrogen bond donor on the interface of proteins, the I850/I830 value reached > 0.9; however if a hydrophobic microenvironment wrapped tyrosine residues, or tyrosine residues were acted as a strong hydrogen bond donor on the interface of proteins, the I850/I830 value came up to < 1.0 [37]. Actually, we found that I850/I830 values were all in the range of > 1.0 and increased via tumbling processing in contrast to WT, indicating that tyrosine residues were also buried or embedded into a hydrophobic microenvironment like tyrosine residues. Therefore, tumbling treatment assisted in rearrangement (rupture and reformation of disulfide bonds and conformations) of protein folding and re-embedding action of a portion of hydrophobic residues (tryptophan and tyrosine). Surely, a high concentration of hydrophobic groups on the interface of proteins were in favor of heat-induced gelation of proteins, however, it should be pointed out that the major forces involved in the gelation of soy protein is hydrogen bonding and van der Waals interactions; the contribution of hydrophobic and electrostatic interaction is negligible [38].

19

Fig. 3 Raman spectra and band assignment of myofibrillar proteins from chicken breast via different tumbling processes at (A) 400-2500 cm-1 and (B) 2500-3500 cm-1

The shift of Raman peak of amide bands similarly meant changes of group conformation or hydrophobic microstructure or molecular polarity. We found that the maximum peak of amide band Ⅰ shifted toward a high wavenumber via tumbling processing, while at 1655.5 cm-1 for WT (Fig. 3A). Moreover, the relative intensity of the peak was weakened. The maximum peak of amide band Ⅲ shifted toward an irregular wavenumber via tumbling processing, whilst it should be certain that a shift toward a high wavenumber by pressure changes (PV and UPV) was observed. The amide band III′ at around 932-1034 cm-1 was deemed to be an otherwise useful indicator of determining α-helix structure [39]. The relative intensity of peak at amide band III′ was likewise weakened, as potentially ascribed to a decrease of α-helix content and an increase of other conformation content. Overall, these shift and intensity profiles were mostly in consistence with the following conformation content profiles for amide bands I and III peaks (Table 2 and Fig. 3A). Generally, changes in protein conformation may affect texture and gel properties of foods [30]. The α-helix 20

content of the fresh chicken breast untreated by tumbling processing accounted for 77.97 ± 3.02% (WT) of entire conformations in protein secondary structure, while 2.02 ± 0.14% for β-fold conformation. In addition, the reduction of α-helix content and increases of other three conformations were confirmed. Importantly, α-helix conformation dominated myofibrillar proteins for WT group, and α-helix and β-fold conformations accounted for a major part. The UPV group produced the lowest content of α-helix conformation and the highest content of β-fold conformation. The reasons were that tumbling and cavitation effects destroyed the hydrogen bonds that maintained the structural stability of α-helix conformation, and made α-helix structure despiralized into a single or independent chain [40]. Afterwards, the chain would rearrange and form new conformations (e.g. β-fold) by intermolecular hydrophobic residues and self-assembly [41]. Indeed, the cavitation effect from continuous ultrasonication (UPV) may also denature and destroy proteins in stereochemistry to some extent, especially the long-chain secondary structure of proteins [41]. When the conformation was β-fold or random coil, the proteins generally possessed a better water-holding capacity, and if α-helix conformation was transformed into β-turn, the water-holding capacity was reduced [40]. Liu et al. [34] also verified that the appearance of β-fold conformation facilitated the formation of favorable protein gels. The C-H stretching vibration of aliphatic residues was observed and recognized at 2800-3050 cm-1 at Raman spectra [42]. The corresponding peaks of aliphatic residues shifted toward a higher wavenumber from 2929.77 cm-1 for WT to 2932.95 cm-1, 2931.36 cm-1, 2934.54 cm-1, and 2939.32 cm-1 for PT, VT, PV, and UPV respectively (Fig. 3B). Especially, peak shift extent of UPV group was marked, compared to WT. Xu et al. [43] reported that the shift towards a high wavenumber was attributed to the unfolding of proteins causing the exposure of methyl and methylene. Shao et al. [44] showed that exposure of more aliphatic residues enhanced hydrophobic interactions on the proteinic interfaces and further improved gelation properties. 3.6 Water distributions 21

Regarding the determination of water distribution through LF-NMR, changes of transverse T2 relaxation time were generally employed to distinguish populations of intracellular water and extracellular water [45]. Generally, the shorter the T2 relaxation time, the closer the combination of water and macromolecules, and vice versa. In this study, four peaks were observed (Fig. 4), and T21 (0-2 ms), T22a (4-10 ms), T22b (10-100 ms), and T23 (100-1000 ms) were assigned to the corresponding water populations. According to the results reported by Hinrichs et al. [46], T21 represented the water in combination with macromolecular proteins; T22a represented a component between bound water and immobilized water; T22b represented immobilized water between myofibrils and their perimysium, which dominated the water populations; T23 represented free water in the extracellular space. Specifically, T22a and T22b are considered as tightly-immobilized water and lightly-immobilized water, respectively [47].

Fig. 4 Water distributions in chicken breast treated by different tumbling processes. PT: atmospheric pressure (0.101 MPa) tumbling, VT: vacuum-assisted (−0.08 MPa) tumbling, PV: combination of breathing tumbling, UPV: breathing 22

tumbling assisted by ultrasound, WT: the process without tumbling, vacuum treatment, and intermittent ultrasonication.

The free water for UPV was undetected. Different shift profiles of moisture were based on their top peaks, as displayed in the left bottom of the figure.

Table 3 The normalized integral area of water population peaks by LF-NMR analysis for different tumbling processes Treatment

AT21

AT22a

AT22b

AT23

WT

57.0 ± 0.0b

170.7 ± 3.5d

77385 ± 14e

948 ± 20a

PT

48.0 ± 1.3d

111.3 ± 4.6e

116695 ± 95c

684 ± 17c

VT

54.0 ± 3.0c

375.7 ± 5.8b

101109 ± 47d

464 ± 25d

PV

43.7 ± 1.5e

360.7 ± 0.6c

128870 ± 25b

817 ± 60b

UPV

70.1 ± 1.0a

781.0 ± 3.6a

137011 ± 106a

—

The sample number was 3 for each treatment group. PT: atmospheric pressure (0.101 MPa) tumbling, VT: vacuum-assisted (−0.08 MPa) tumbling, PV: combination of breathing tumbling, UPV: breathing tumbling assisted by ultrasound, WT: the process without tumbling, vacuum treatment, and intermittent ultrasonication. The different lowercase letters a-e in the same column indicate significant differences (P < 0.05). — means undetected. The unit of AT21, AT22a, AT22b, and AT23 was au·ms.

As depicted in Fig. 4, the shift and relative composition of water populations were evaluated. T21, T22a, and T22b shifted towards a high relaxation time, whilst T23 shifted towards a low relaxation time after tumbling treatment. Such a variance was also reflected in the relative composition content determined by integral calculus. We found that the relative composition content was altered according to the normalized integral area in Table 3, displaying on one hand an evident increase for UPV at T22a while PV took the second place, and on the other hand a significant increase for PV at T22b while UPV took the second place. The phenomenon revealed that the content of 23

lightly-immobilized water and tightly-immobilized water was increased under ultrasonication and breathing pressure-transform treatment, even though some evidence in Fig. 4 showed a weakened affinity (shift of moisture) or combination between proteins and lightly-immobilized water (main water population) as considered in this study [48]. The profile seemed not to be regularly varied in the relative composition content for the peaks at T21 while a decreased profile was observed for T23. We even found that the peak at T23 disappeared for UPV, an evacuation phenomenon of free water (Fig. 4 and Table 3). Bertram et al. [49] similarly showed that the relative water content for T22a and T22b peaks was increased after adding brine into meat but decreased for T23 peak. Zhu et al. [50] showed that tumbling actions helped increase the water content of meat products and especially changed water distributions. When tumbling, the mechanical friction between myofibers or between tumblers and myofibers destroyed spatial structure of myofibrillar proteins (e.g. exposure of polar hydrophilic groups within proteins), made them easily released, and finally helped hold more moisture (e.g. immobilized water). Meanwhile, the permeation of brine helped boost exudation and exchange of intracellular or extracellular water from myofibers by adjusting osmotic pressure. In any case, the better water-holding effects of PV and UPV could not do without the presence of breathing pressure changes and/or ultrasonication. Briefly, the intermittent vacuum environment during breathing action promoted the release of air from intercellular space, allowing more marinating liquids to enter intercellular space and holding more immobilized water. The cavitation effect of ultrasonication could also penetrate and destroy the tissue structure indirectly influencing water distributions. Overall, the synergistic effect of breathing pressure changes and ultrasonication on water distributions within myofibers was noticeable during the tumbling treatment, especially influencing the tissue structure and intramembranous and intermembranous permeation of substances, allowing for more immobilized water and bound water [23]. In combination of above results, protein degradation promoted the tenderization of chicken breast whilst the increase of immobilized water improved the water retention. 24

3.7 SEM and TEM Myofibers coated with perimysium and endomysium connective tissues are slender cylindrical cells, and each myofiber is composed of perimysium, myoplasm, cell nucleus, and a large number of myofibrils [51]. Generally, tumbling marinating process influenced myofiber structure in morphology and tenderness of fabricated meat products. When subjected to ultrasonic processing as a supplementary means, the myofibrillar proteins would be further fragmentized, perimysium was torn apart, and Z line ruptured, which could directly improve tenderness [8]. Bhat et al. [52] showed that the release of lysosomal enzymes accelerated the decomposition of myofibrillar proteins and thus shortened the tenderization time.

Fig. 5 Scanning electron microscopic images of myofibers and myofibrils in chicken breast treated by different tumbling processes. The scale bar is 10 μm and the magnification is ×1000 times. PT: atmospheric pressure (0.101 MPa) tumbling, VT: vacuum-assisted (−0.08 MPa) tumbling, PV: combination of breathing tumbling, UPV: breathing tumbling assisted by ultrasound, WT: the process without tumbling, vacuum treatment, and intermittent ultrasonication.

The images named the capitals (top left corner) from A to E belong to WT, PT, VT, PV, and UPV treatment respectively. The orange, green, and blue arrows were separately assigned to perimysium, myofiber, and myofibril. 25

The fresh chicken breast myofilaments without tumbling treatment have a parallel, tight myofiber structure with a clear, intact perimysium in Fig. 5. Normally, the destruction of perimysium played a crucial role in improving meat tenderness during processing. When performing PT treatment, myofilaments similarly displayed a parallel, tight tissue structure whereas perimysium structure started being damaged. After VT treatment, myofibers would rupture along the vertical myofibrils, followed by the distinct damage towards perimysium. In this case, myofibril structure would be exposed and very likely be eroded by external forces. The perimysium almost completely disappeared via PV and UPV treatment and myofibrils largely exposed as well, displaying a loose state. The structural integrity of the myofibers was obviously damaged, and myofibrils were completely exposed. Most of the myofibrils were fractured and fragmented significantly as reflected in the poor, disordered cell edge. Besides pressure influencing the processing properties of chicken breast for PT, VT, and PV, ultrasonic cavitation made chicken breast subjected to rapid molecular vibration, myofiber structure fracture and separation [53]. An uniform maceration of brine and a release of cellular endogenous proteases were successfully achieved by cavitation prior to tenderization [54]. As a result, the complete tissue structure was fast decomposed, and myofibrillar fragmentation was strengthened. For untreated WT, myofibrillar structure was complete and closely arranged in parallel. We found different tumbling was liable to loosen myofibrils and expose more intercellular spaces (Fig. 5 and 6A). The evidently fragmented myofibrils were observed through the treatment in a vacuum and an adjustable variable pressure, such as VT, PV, and UPV, directly confirming MFI results. The cell organelles such as lysosomes tended to reduce as tumbling process became more complicated, especially for UPV. Another phenomenon was that the bubbles inside interspace of myofilaments almost disappeared via tumbling processes, whereas air bubbles were enriched in WT group without tumbling (Fig. 6A). This indicated that gas (O2 or CO2) was readily released onto the surface of 26

chicken breast or exchanged with brine by physical forces (tumbling actions, ultrasonication, and pressure changes), which produced the maceration in depth.

Fig. 6 Transmission electron microscopic images of myofibril structure (A), myofibrillar fragmentation (B), and lysosome structure (C) in chicken breast treated by different tumbling processes. PT: atmospheric pressure (0.101 MPa) tumbling, VT: vacuum-assisted (−0.08 MPa) tumbling, PV: combination of breathing tumbling, UPV: breathing 27

tumbling assisted by ultrasound, WT: the process without tumbling, vacuum treatment, and intermittent ultrasonication.

The blue, white, and green arrows were assigned to interspace between myofibrils, primary lysosomes, and secondary lysosomes. The scale bar from image A-C is 10 μm, 2 μm, and 1 μm, and the magnifications are taken as 1000, 4000, and 8000 times respectively.

The light and shade horizontal stripes of each myofibril were arranged correspondingly on the same horizontal plane, presenting regularly alternating structure, namely parazone (band I) and diazone (band A). After staining, a dark line appearing at the center of band I is called Z line, whilst a dark line at the center of band A is called M line. The myofibrils between the adjacent Z lines were called sarcomere, and a sarcomere containing 1/2 band I, band A, and 1/2 band I, was the structural and functional basis of myofibrils (Fig. 6B). We found myofibril structures were arranged tightly with relatively unbroken bands and lines for WT. For PT and VT, Z lines were misplaced, myofibril structures were fractured at the Z lines, and degradation occurred. The space between myofibrils increased and organelles decreased and dispersed. For PV, most of the Z lines were misaligned and degraded, the number of broken sarcomere increased, and the space between myofibrils became optically larger. For UPV, most myofibrils were broken, displaying a state of bending and deformation and the structure of myofibrils was completely destroyed. In short, part of the sarcomere was broken, Z line proteins and perimysium proteins were degraded, and the space between myofibrils was increased [55]. Based on above results in Fig. 6B, we found that the thin myofilament proteins (e.g. actin and tropomyosin) near the Z line of band I and the thick myofilament proteins (e.g. myosin) near M line of band A had an obvious degradation behavior in contrast to other parts, which was consistent with the SDS-PAGE results. Generally, the myofibril gap within fresh myofibers is filled with organelles released via lysis of cell and metastasis, such as lysosomes, most of which are a primary form with a large amount but with a small volume. The lysosome contains more than 60 kinds of acidic hydrolases: proteases, lipases, glycosidases, and nucleases. The 28

lysosome also includes primary lysosomes (smaller, inactive state, induced activation, and containing only lyases) and secondary lysosomes (larger, active state, digestion in progress, and containing lyases and substances). In Fig. 6C, the volume of lysosomes was enlarged and the number was declined via tumbling processing. Meanwhile, the membrane of secondary lysosomes tended to rupture and then lyases were released, which mainly happened near a broken sarcomere or a broken Z line structure from myofibril. The compact secondary lysosomes were significantly decreased and ruptured even though the compact primary lysosomes were broke at different levels. A possible reason was that ultrasonication and tumbling treatment had a more direct and important impact on the damage of secondary lysosomes than primary lysosomes, which was caused by unstable volume for secondary lysosomes via phagocytosis. In addition, we hypothesized that uncracked primary lysosomes concentrated on a broken sarcomere, continued to digest zymolytes by phagocytosis, and formed ongoing secondary lysosomes. In brief, four types of tumbling treatment, especially UPV, could sufficiently guarantee the rupture of lysosome membranes, myofibrils, and myofibers in the morphological aspect. The release and functionating of hydrolytic enzymes are a crucial cause or precondition for the destruction of integrity of myofiber microstructure and the improvement of tenderness of meat products. 4. Conclusions The redesigned tumbler with a vacuum generator unit and an ultrasonic unit improved the marinating absorptivity, texture, taste, water-holding property, and morphological property of chicken breast during marinating processing. In addition, we stressed that the refit of a traditional tumbler was proposed to boost traditional equipment’s performance or modify technological parameters. Importantly, some efforts will be further made on characteristics of heat-induced gels of marinated chicken breast by this tumbler. This work would provide an experimental and theoretical basis for the development of innovative tumblers equipped with ultrasonic and adjustable variable pressure units in the processing of poultry products. 29

Conflict of interest The authors claimed that there was no conflict of interest. Acknowledgements This work was financially supported by the Shandong Modern Agricultural Technology and Industry System (SDAIT-11-11), China Agricultural Research System (CARS-42-5 and CARS-41-Z06), and High-Level Research Programme of Qingdao Agricultural University (663/1118006). The authors gave sincere thanks for the constructive assistance from Dr. Li-Juan Chai, Jiangnan University, China, in experimental methodology and paper writing.

30

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Author contribution statement Yan Li and Jingxin Sun: Conceptualization, Methodology, Data curation, Writing-Original draft preparation Ting Feng and Liping Guo Investigation, Formal analysis Baowei Wang: Supervision Ming Huang and Xinglian Xu: Project administration Jiying Yu and Harvey Ho: Writing-Review & Editing [56]

Conflict of interest The authors claimed that there was no conflict of interest.

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[57]

Highlights 

A controlled breathing ultrasonic tumbler for marinated chicken breast was designed



The marinating absorptivity, tenderness and taste were improved



The conversion from α-helix to β-fold conformation was confirmed by Raman analysis



LF-NMR analysis showed that water-holding property was enhanced



The occurrence of rupture in sarcomeres was easy at Z lines and M lines

[58] [59]

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