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Ultrasonics Sonochemistry
journal homepage: www.elsevier.com/locate/ultson

Ultrasound-assisted covalent reaction of myofibrillar protein: The

improvement of functional properties and its potential mechanism
Jiahui Chen a, 1, Xing Zhang b, 1, Mengying Fu c, Xing Chen d, Bassey Anthony Pius a,
Xinglian Xu a, *
a
Key Laboratory of Meat Processing, Ministry of Agriculture, Key Lab of Meat Processing and Quality Control, Ministry of Education, Jiangsu Collaborative Innovation
Center of Meat Production and Processing, College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, China
b
Department of Trauma and Reconstructive Surgery, RWTH Aachen University, Aachen 52074, Germany
c
School of Pharmaceutical Sciences, Xuzhou Medical University, Xuzhou 221002, China
d
State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu 214122, China

A R T I C L E I N F O A B S T R A C T

Keywords: The effects of the different combined manner of ultrasound and covalent reaction between polyphenol and
Myofibrillar protein (MP) myofibrillar protein (MP) from chicken were studied. More so, antioxidant activities, digestive properties, and
Ultrasound potential mechanism of ultrasound-assisted oxidation system of hydrophilic ((− )-Epicatechin gallate, ECG) and
Digestive properties
hydrophobic (Baicalein, BN) polyphenols was also analyzed in this study. Among all the combined treatments,
(− )-Epicatechin gallate (ECG)
Baicalein (BN)
surface hydrophobicity (SUH), active sulfhydryl contents (ASC), and specific surface area (SSA) of ultrasonic
assisted ECG oxidation group (T6) was relatively apparent, indicating that a more unfolding MP structure was
obtained. Furthermore, ultrasonic assisted ECG oxidation group showed the highest antioxidant activities
compared with other combined treatments on the basis of the results of DPPH free radical scavenging activities,
metal ion chelating activities, and hydroxyl radicals (OH⋅) scavenging activities. The results of simulated
digestion system and kinetic analysis also verified that ultrasonic assisted ECG oxidation had higher MP bio-
accessibility than the control group. In contrast, a lower digestibility was displayed in ultrasonic assisted BN
oxidation group. In summary, the ultrasound-assisted covalent reaction of MP and ECG might be a desirable
approach for industrial production of MP from chicken with better antioxidant activities and digestive properties.

1. Introduction in the transformation of structure and functional properties in protein

[8].
Myofibrillar proteins (MPs) have been widely studied in recent years Unlike other chemical groups mentioned above, phenolic com­
due to their unique essential amino acid contents and superior biological pounds have raised enormous interest because of the fact that these
digestibility [1–3]. Additionally, MPs have been considered as individ­ compounds have good antioxidant properties and healthy functions [4].
ualized building blocks in constructing gel or delivery systems that meet Since polyphenols could notably affect the structure and physicochem­
the requirements of health benefits and food quality for modern con­ ical properties of MPs, interactions of MP–polyphenol cannot be ignored
sumers [4,5]. In virtue of their flexible and sensitive structure, growing in the actual food production [9]. For example, the adjunction of poly­
interest has been drawn to utilizing meat proteins as ingredients in phenol can covalently react with active sites (e.g., sulfhydryl groups
functional foods. However, modern industrial applications of MPs are (-SH) and amino groups (–NH2)) in MP spontaneously, thus contributing
often compromised by their inherent imperfect nature, such as relative to the formation of soluble aggregates and enhancement of physical
aggregation state and poor functionality [6]. These shortcomings could stability [4]. Moreover, the covalent reaction of non-polar polyphenols
be alleviated by different structural modifications including physical and MPs could improve biological activities, surface hydrophobicity
and chemical ways [7]. Thereinto, chemical modification is the most (SUH), and active sulfhydryl contents (ASC) of proteins, thereby
conventional method to introduce functional groups into MPs, resulting enhancing the antioxidant activities and emulsifying properties of MPs

* Corresponding author.
E-mail address: [email protected] (X. Xu).
1
These authors contributed equally to this work.

https://doi.org/10.1016/j.ultsonch.2021.105652
Received 23 April 2021; Received in revised form 10 June 2021; Accepted 18 June 2021
J. Chen et al.

[10]. A body of studies also indicated that reaction of quinone with 2.3. Experimental design
active sites (e.g. -SH) in MP is conducive to the improvement of
bioavailability for protein [11,12]. Therefore, it is of great significance To investigate the functional properties before and after ultrasound
to optimize the chemical covalent modification effects of polyphenols treatment, MPs were subjected to 7 treatments: T1: CON, without
and MPs for further production of excellent performance products. polyphenol and ultrasound treatment; T2: without ultrasound treat­
Covalent modification of polyphenol on MPs could be prepared ment, but ECG was incorporated. T3: without ultrasound treatment, but
through different chemical methods (e.g. enzymatic, free radical graft­ BN was incorporated; T4: ECG was incorporated after ultrasound
ing, and alkaline reaction). Conversely, enzymatic method is relatively treatment. T5: BN was incorporated after ultrasound treatment. T6: ECG
effective although the cost of enzyme with high catalytic efficiency are was incorporated before ultrasound treatment. T7: BN was incorporated
generally expensive [13]. In addition, hydrogen peroxide is challenging before ultrasound treatment. Power density and processing time were
to be avoided in free radical grafting method. For this reason, the in­ 350 ± 20 W/L and 6 min, respectively. The probe is about 2.0 ± 0.5 cm
dustrial application of this reagent may be significantly restricted on away from the bottom of the beaker. Alkaline environment (pH 9.0) is
account of its strong oxidation abilities [7]. In comparison, the alkaline necessary to ensure covalent reaction of MPs and polyphenol (0.45
reaction may be a suitable option, but its oxidative effect is usually mmol/L) after incorporation of polyphenol, and then pH was adjusted
undesirable, even ineffective [4]. Hence, it is highly significant but back immediately after all the treatments [4]. In this regard, poly­
challenging to enhance oxidation effect under alkaline condition. phenols are prone to be oxidized and rearranged into quinones, and then
Recently, ultrasound has been considered a process intensification covalently cross-linked with protein side chain groups. And the latter
method for advanced oxidation processes [14–17]. During this process, two treatments (T6-T7) correspond to ultrasound-assisted oxidation
cavitation bubbles can form rapidly in an cycle, and then break violently system of hydrophilic and hydrophobic polyphenols, respectively. In
to release abundant energy [18]. Meanwhile, extra hydroxyl radicals this study, the structural changes of different treatments (T1-T7) were
(OH⋅) was produced by cavitation bubbles through cleaving additional evaluated through particle sizes, rheology measurement, circular di­
solvent molecules [19], giving the opportunity for alkaline reaction. We chroism spectrum, surface hydrophobicity, and sulfhydryl contents.
tentatively hypothesized that the combination of ultrasound and alka­ Moreover, antioxidant properties and potential mechanism of
line reaction could act in a synergistic way, whereby the actual effect ultrasound-assisted oxidation system of hydrophilic and hydrophobic
was generally more superior than only single alkaline oxidation process polyphenols was systematically studied. Lastly, the in vitro digestion
[20–22]. properties and chromaticity of emulsified MP gel were further investi­
Thus, the present work was conducted to explore the effects of the gated and compared.
different combined manner of ultrasound and alkaline oxidation process
on the structure of MPs. Importantly, both chemical and non-chemical 2.4. Particle sizes
factors were systematically considered for the first time to demon­
strate potential mechanism of ultrasound-assisted covalent reaction of Specific surface area (SSA) and particle size parameters (D[3,2], D
MPs. Additionally, the effects of the various combined approaches on [4,3]) were determined via the optical principle (Mastersizer 3000,
the antioxidant properties and in vitro digestive properties were Malvern Instruments Ltd., UK) [24,25]. After automatic calibration,
compared and characterized, paving the way for a deeper understanding different MPs solution (T1-T7) were dropped into a sample box filled
the nutritional value of meat MPs. with water. Then, the refractive index was automatically converted into
the particle size parameter by the instrument. The refractive and ab­
2. Materials and methods sorption coefficient were 1.46 and 0.01, respectively. D[4,3] corre­
sponds to the mean diameter in volume, and D[3,2] represents the mean
2.1. Materials and reagents diameter in surface, namely, ‘Sauter diameter’.

Chicken breast was purchased from Jiangsu sushi meat Co., Ltd. 2.5. SUH, ASC and circular dichroism
(Nanjing, Jiangsu). ECG and BN were provided by Beijing Solarbio
Technology Co., Ltd. (Beijing, China). ECG was isolated from Chinese SUH of MPs (4 mL) after different treatments (T1-T7) was assessed to
green tea, whereas BN was extracted from skullcap (Scutellaria baica­ bond the 8-anilino-1-naphthalenesulfonic acid (ANS, 20 μL). To reduce
lensis Georgi), and these polyphenols were purified by column chroma­ the experimental error caused by fluorescence quenching, MP samples
tography for further use. Furthermore, ECG and BN exert their biological were stored and avoided light exposure for 20 min. Excitation wave­
activities by scavenging free radicals and anti-allergic reaction, respec­ length and emission wavelength were 375 nm and 470 nm, respectively.
tively. According to our previous study [18], visible connective tissues in ASC of MPs (4 mL) after different treatments (T1-T7) was assessed to
selected chicken breasts were removed and cut into cubes for further use bond the Ellman’s reagent (5,5′ -Dithiobis-(2-nitrobenzoic acid)).
(4 ◦ C). The purity of ECG and BN was chromatographic grade (≥98%). Briefly, MP solution was diluted to final concentration (1 mg/mL) using
PBS (20 mM K2HPO4, KH2PO4, pH 6.25), and then homogenized for 30 s
2.2. Isolation of MPs (6000 rpm). ASC were conducted through the measurement of absor­
bance (412 nm). For circular dichroism, MP solution was placed in a
Standard salt solution (1000 mL, 0.1 mol/L KCl, 2 mmol/L EGTA, 1 quartz tube (0.1 cm). The instrument (Jasco, Tokyo, 178 Japan) was
mmol/L MgCl2, 20 mmol/L K2HPO4/KH2PO4, pH 7.0) was prepared as utilized to scan based on the set procedure (190 nm-260 nm) [26].
per previous study [9,23]. Meat slurry was then homogenized with a
homogenizer at 8000 rpm for 30 s (4 times). Meat sample was also 2.6. Rheology measurement
filtered through cheesecloth to remove unnecessary ingredient. Ob­
tained slurry (250 mL) were rinsed thrice by standard salt solution Rheology measurement contained two different parts, the static and
(1000 mL) and then subjected to a 5-min centrifugation (4000 rpm). dynamic measurements (Anton Paar, MCR 301, Austria). Before mea­
After collection, meat sample was rinsed thrice with NaCl solution (0.1 surement, MPs were hold on the plate for 2 min to remove residual flow
M, 1000 mL) using the same procedures. The final protein concentration histories. Static rheological measurement was conducted as Chen et al.
and storage temperature were adjusted to 40 mg/mL and 4 ◦ C, [27] described; shear rates were set from 0.1 to 100 s− 1. Other dynamic
respectively. parameters including strain (0.02%), frequency (0.01 Hz), and tem­
perature (25 ℃), remain unchanged at this time [28]. Apparent viscosity
and shear stress were monitored during different shear rates.
J. Chen et al.

Dynamic rheological responses at various scans were measured heating rate of 4 ℃/min. After the MP emulsified gel was completely
through strain (0.01–750%), frequency (0.1–100 rad/s) changes. Addi­ formed, the plastic tube was immediately cooled in cold water (0–4 ℃),
tionally, the creep and creep-recovery measurement were performed and then stored overnight (0–4 ℃) for further use.
with constant stress (3 Pa), which was determined via ramp shear sweep.
And fresh samples were required for each creep test. The parameter 2.10. Measurement of chromaticity
conversions were calculated according to following formula:
√̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅ Chromaticity of MP emulsion gel was monitored through the changes
G∗ = (G′ )2 + (G′′ )2 (1) of color parameters (CR-400, Minolta Camera Co., Osaka, Japan) [32],
including lightness (L), chroma (C), redness (a), and yellowness (b).
G∗ Then, whiteness (W*) and total color difference (△E) of gel were
η∗ = (2)
w evaluated as follows:
√̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅
where η* corresponds to complex viscosity, w corresponds to angular ΔE = (L* − L)2 + (a* − a)2 + (b* − b)2 (4)
frequency, G*, G’ and G’’ are complex modulus, storage modulus and
loss modulus, respectively. where L*, a* and b* are 88.64, − 0.84 and − 0.37, respectively.

2.7. Antioxidant properties 2.11. Measurement of in vitro digestion properties

2.7.1. DPPH free radical scavenging activities Measurement of the in vitro digestion properties for MP emulsion gel
DPPH free radical scavenging activities of samples with different was performed as per previous studies [33–35] with some slight modi­
concentrations (0, 0.01, 0.1, 1, 3 and 5 mg/mL) were tested by the fications. Simulated gastric fluid (SGF) and simulated intestinal fluid
detection kit, which was purchased from Beijing Solarbio Science & (SIF) were prepared three days in advance (Table S1). Briefly, the
Technology Co., Ltd. (Beijing, China). Briefly, the mixture, containing mixture (pH 2.0, 70 mL) containing MP slurry (1.0 mL), SGF, and pepsin
MP solution (1.0 mL), DPPH ethanol solution (0.4 mL, 0.4 mM), and (2000 U/mL) was heated in the water bath with magnetic stirring (400
deionized water (2.0 mL) were stored at 25 ◦ C in darkness for 30 min rpm) at 37 ◦ C. Trichloroacetic acid (15%) was introduced to the mixture
[29]. Samples without DPPH ethanol were the background group. DPPH at different times (0, 5, 10, 15, 30, 60, 90 and 120 min). Hydrolysis of
free radical scavenging activities were expressed as changes in absor­ MP during pepsin digestion was expressed as the change in absorbance
bance at 517 nm after centrifugation for 10 min (6000 rpm). at 280 nm after centrifugation for 15 min (4 ◦ C, 4000 rpm). Lastly, the
pH was adjusted to 7.0 for further intestinal digestion.
2.7.2. Metal ion chelating activities For the second part, the mixture (pH 7.0, 70 mL) containing MP fluid
Metal ion chelating activities of MP solution with different concen­ after digestion of stomach (35 mL), SIF (35 mL), trypsin (1000 U/mL)
trations (0, 0.01, 0.1, 1, 3 and 5 mg/mL) were performed by the method and α-chymotrypsin (25 U/mL) was heated in the water bath with
of Xiao et al. [30]. MP solution (1.0 mL), deionized water (2.75 mL), iron magnetic stirring (400 rpm) at 37 ◦ C. Trichloroacetic acid (15%) was
zinc solution (0.2 mL, 5 mM), and ferrous chloride (FeCl2) solution then introduced to the mixture at different times (0, 5, 10, 15, 30, 60, 90
(0.05 mL, 2 mM) were mixed, and then stored at 25 ◦ C for 10 min. and 120 min) to inactivate the enzyme. Hydrolysis of MP during intes­
Deionized water were applied as the blank group. Metal ion chelating tinal digestion was expressed as the change in absorbance at 280 nm
activities were expressed as changes in absorbance at 562 nm after after centrifugation for 15 min (4 ◦ C, 4000 rpm).
centrifugation for 10 min (6000 rpm). Digestion kinetics were also analyzed according to the previous
studies [36,37]. The changes of absorbance for MP samples (T1-T7)
2.7.3. OH⋅ scavenging activities were fitted through Python, which was as follows:
OH⋅ scavenging activities of MP solution after different treatments
(T1-T7) were performed by the method of Xiao et al. [31]. Briefly, the OD = ODmax × exp(− B/t) (5)
mixture containing MP solution (1.0 mL), ethanol-salicylic acid (9 mM), where OD is the absorbance at time t, ODmax corresponds to the
ferrous sulfate (9 mM), and deionized water, was stored for 5 min. peptide concentration after infinite digestion, and B = half-life time of
Subsequently, H2O2 (0.01%, v/v) was added to the system at 37 ◦ C for MP samples was multiplied by ln2.
reaction (30 min). OH⋅ scavenging activities were expressed as changes
in absorbance at 510 nm after centrifugation for 10 min (6000 rpm). 2.12. Statistical analysis

2.8. Mass spectrum Difference of experimental data was showed as statistically signifi­
cant (p < 0.05). Fitting of described model was performed by Python.
Mass spectrometry experiments were conducted to analyze the po­
tential mechanism of ultrasound-assisted oxidation system for hydro­ 3. Results and discussion
philic and hydrophobic polyphenols. Briefly, MPs after treatments were
hydrolyzed using hydrochloric acid (6 M) for 24 h at a completely ni­ 3.1. Structural changes
trogen environment, and then re-dissolved by hydrochloric acid (0.02
M) [11]. Triple four stage rod mass liquid chromatography (Agilent 3.1.1. Particle sizes
1290-6460C, USA) was used to evaluate molecular weight. The mobile SSA and particle size parameters (D[3,2], D[4,3]) after treatments
phases A and B were water and methanol, respectively. Acq method: were shown in Table 1. Different treatments (T2-T7) resulted in a sig­
20200829-P. nificant increase in SSA of MPs, showing that the adsorption capacity of
protein was improved. Moreover, the SSA increased by 373.00% and
2.9. Preparation of MP emulsified gel 128.31%, respectively, at ultrasound-assisted oxidation system of ECG
and BN when comparing to the control group (T1). This may be attrib­
Fresh emulsion, containing MP solution after different treatments uted to the fact that ultrasound assisted oxidation promoted the
(T1-T7) and soybean oil (10%) were mixed, and then homogenized unfolding of protein structure significantly, resulting in more internal
(Ultra Turrax T-25 Basic, IKS company, Germany) for 1 min to ensure pores and larger SSA. Interestingly, SSA of the MP was higher with the
the uniformity. And then, MP emulsion was heated (25 ℃-85 ℃) at a ultrasound-assisted oxidation system of ECG, compared to BN. This
J. Chen et al.

Table 1
SSA, particle size (D [3,2] and D [4,3]) and chromaticity parameter (L, a, b, W*, △E, H and C) of MPs after different treatments (T1-T7). Different letters (a-g) indicated
significant differences within different MPs.
Treatments T1 T2 T3 T4 T5 T6 T7

SSA 124.025 ± 7.719e 414.789 ± 54.988c 369.956 ± 46.181c 500.100 ± 28.235b 502.567 ± 82.157b 586.644 ± 103.748a 283.160 ± 79.894d
D [3,2] 48.550 ± 3.069a 14.711 ± 1.953 cd 16.467 ± 2.234c 12.031 ± 0.679de 12.322 ± 2.733de 10.587 ± 2.317e 23.180 ± 7.662b
D [4,3] 118.000 ± 11.265bc 164.311 ± 51.419b 253.000 ± 180.586a 30.400 ± 12.586d 45.778 ± 16.452d 79.333 ± 89.524 cd 62.447 ± 30.578 cd
L 77.574 ± 0.439b 77.246 ± 0.757bc 76.664 ± 0.648c 80.610 ± 0.675a 77.640 ± 0.249b 77.206 ± 0.796bc 76.954 ± 0.576bc
a − 2.346 ± 0.059d − 1.730 ± 0.050b − 6.450 ± 0.065 g − 1.462 ± 0.084a − 5.556 ± 0.057e − 2.189 ± 0.068c − 6.255 ± 0.142f
b 0.296 ± 0.158f 0.911 ± 0.233e 18.210 ± 0.336a 1.429 ± 0.218d 14.600 ± 0.444b 0.812 ± 0.513e 11.155 ± 0.450c
W* 77.449 ± 0.438b 77.161 ± 0.749c 69.700 ± 0.466f 80.501 ± 0.660a 72.719 ± 0.090e 77.080 ± 0.778c 73.635 ± 0.355d
△E 11.189 ± 0.430d 11.504 ± 0.731d 22.815 ± 0.358a 8.260 ± 0.624e 19.172 ± 0.223b 11.589 ± 0.739d 17.298 ± 0.206c
C 2.369 ± 0.062d 1.968 ± 0.071f 19.319 ± 0.322a 2.054 ± 0.131e 15.622 ± 0.417b 2.387 ± 0.143d 12.791 ± 0.401c

clearly evidenced that ECG is prone to be oxidized since the dispersion of further application.
hydrophilic polyphenol is better at this time [7,11]. Besides, more However, the SUH was difficult to judge the side chain oxidation,
benzene rings and phenolic hydroxyl groups in ECG give MP more and the ASCs were also detected to determine the covalent modification
flexible structure through non-covalent force [9,38], leading to the in different treatments [44]. For ECG, ultrasound led to the expansion of
significant increase of SSA under cavitation. Previous study also indi­ protein structure and the increase of ASCs. Notably, ASC decreased from
cated that this change was closely correlated with the transformation of 126.7 ± 0.6 to 122.7 ± 1.7 (μmol/100 mg), corresponding to group T4
non-covalent bonds [39]. Amir et al. concluded that ultrasound could and T6. It was implied that more -SH groups were consumed by ultra­
open the protein chain, leading to the modification of 3-dimensional sound assisted oxidation group (T6) when comparing with ultrasonic
structure and an increase of the SSA [40]. In comparison, the change treatment before oxidation (T4) in hydrophilic phenols. Similar results
of SSA in T6 was significantly evident (from 124.025 ± 7.719 cm2/cm3 were also obtained in hydrophobic phenols (BN). Based on this reason,
to 586.644 ± 103.748 cm2/cm3). It was therefore concluded that MP we hypothesized that more –SH in MPs reacted with oxidized poly­
was easily damaged in ultrasound-assisted oxidation system of ECG. phenol intermediates (quinone) in ultrasound assisted oxidation group.
It was noteworthy that a similar trend was also observed in particle From the point of view of the flexibility for MP structure, the mechanical
size parameters. D [3,2] of MPs after T4-T7 were 75.22%, 74.62%, effect of ultrasound contributed to a sensitive state of myosin in MP
78.19%, and 52.26% less than that of their control counterparts, [23], which was conducive to the exposure and covalent reaction of
respectively, indicating that ultrasound effectively reduced MP aggre­ oxidation sites. In the current study, there were significant changes in
gation. More so, D [4,3] decreased significantly after ultrasound treat­ ASC, revealing the potential of ultrasound assisted oxidation in MP
ment, but no significant change was reported in T4-T7, which was unfolding and active groups exposure. Although no significant differ­
consistent with the findings found by Liu et al. [26]. It can be inferred ence was observed in T2 and T3, BN-treated MP showed a higher ASC
that the strong force provided by ultrasound destroyed the ordered value than that of ECG-treated after ultrasound (T4 and T5). This may be
structure of protein. Chen et al. also verified that [18] the enhancement related to the chemical structure of these two polyphenols. More ben­
of pH during ultrasound promoted the electrostatic repulsion and action zene rings and phenolic hydroxyl groups in ECG promote the non-
effect on MPs, thereby decreasing the D [4,3]. This change was also covalent interaction between MP and polyphenols and indirectly
related to the non-covalent interaction between damaged MPs [41]. restrict the expansion of protein structure [7,9]. Meanwhile, the sensi­
Chen et al. [23] further suggested that ultrasound damaged the elec­ tivity of MP after ultrasound was enhanced [19,45], thereby promoting
trostatic interaction and hydrogen bonds between MP molecules, the exposure of –SH under the action of nonpolar polyphenols. Circular
causing the decrease in the average particle size. D [4,3] increased dichroism measurement was also performed to assess the structural
significantly in T2 and T3, unlike T4-T7 where D [4,3] was smaller changes of MPs at this time (Fig. 1C). Two typical bands were exhibited
compared to the control. These acquired data validated that MP and in the spectrum of MP, corresponding to 207–209 and 221–228 nm,
polyphenols react covalently to promote protein aggregation, thereby respectively [46]. In comparison, the spectral bands noticeably shifted
increasing the particle size (T2 and T3) under the weak alkaline con­ upward, confirming that intra-molecular hydrogen bonds between the
dition. In contrast, the introduction of ultrasound decreases MP particle amino hydrogen (NH–) and carbonyl oxygen (C– – O) in MP chain were
size, regardless of the covalent reaction. Also, the effects of particle size destroyed after ultrasound assisted BN oxidation (T6) [47]. From the
variations through ultrasound assisted oxidation method on functional perspective of quorum sensing, a sensitive state of MP might be obtained
properties should be further analyzed. after ultrasonic treatment. The head of myosin, located in most hydro­
phobic parts, could not revert to its normal state; however, the repeated
3.1.2. SUH, ASC and circular dichroism cavitation during ultrasound promoted the formation of molten spher­
Actually, for control groups (T1), SUH decreased from 32208 ± 2041 oids and covalent binding with ECG and BN.
to 29948 ± 1136 and 29703 ± 1481, respectively, as oxidation occurred
in ECG and BN (Fig. 1A). Moreover, SUH values increased after the
3.2. Rheological changes
introduction of ultrasound. As expected, ultrasound assisted oxidation
group showed a higher SUH value, corresponding to 45132 ± 2579 and
3.2.1. Steady state changes
43562 ± 1564, respectively, in group T6-T7. These results were in
For a complex MP system, its rheological changes are vital to the
accordance with other studies [9,40]. Such a change could be ascribed
synthesis, storage, and final products. The rheograms of MP suspension
to the strong turbulence of cavitation bubbles produced by ultrasound,
after treatments (T1-T7) were shown in Fig. 2. All systems, except for
resulting in the exposure of hydrophobic groups and the increase of SUH
T4, showed newtonian flow at a relatively low shear rate, followed by a
[40]. Besides, repeated stretching of protein side chain during ultra­
markedly shear-thinning one when the shear rate increased to 0.03 1/s
sound contributed to the enhancement of SUH values [42]. Thereafter, a
and above. Viscosities of T2, T3, T4 and T5 at 100 1/s were 0.069, 0.086,
melting state of protein head and partial unfolding of MP tail formed
9.95 × 10− 4, and 0.099 Pa⋅s, respectively. In contrast, ultrasound
after ultrasound maintain the susceptibility of MP, promoting the SUH
assisted oxidation system of ECG and BN showed higher viscosities than
changes [18]. Zhou et al. [43] concluded that the unfolding of MP
those of other treatments, corresponding to 0.135 and 0.206 Pa⋅s,
molecules and the increase of SUH after ultrasound were conductive to
respectively. Predictably, the shape and shear resistance of MP
J. Chen et al.

lower viscosity [13,40]. It was indicated that ultrasound assisted cova­

lent reaction of MP, thereby promoting the rearrangement and stability
of MP through the chemical force.
Rheological changes were fitted with the Ostwald-de-waele model
and Carreau model [49]:

η = k × γn− 1
(6)

[ ]n−2 1
η = η0 1 + (λγ)2 (7)

where η corresponds to the apparent viscosity (Pa⋅s), η0 corresponds to

zero shear viscosity, k is the consistence index (Pa⋅sn), γ is the shear rate
(1/s), λ is relaxation parameter, and n is the flow characteristic
parameters.
Generally, consistence index (k) was related to the pseudoplastic
behavior and internal interaction of MPs. Higher k were observed in T6
(5.838 Pa⋅sn) and T7 (13.366 Pa⋅sn), whereas MPs treated with (T4)
showed the lowest values (0.023 Pa⋅sn). It was suggested that the
interaction between myosin in MPs increased after treatment T6 and T7,
thereby contributing to the formation of weak elastic network. This may
be due to the fact that more myosin participates in covalent reaction and
forming stable chemical structure.
Although it is easier to compare the shear resistance of MPs at
relatively high shear rates (100 1/s), the increase of shear rate during
the measurement limited the direct observation of the initial MPs state.
Furthermore, the change of shear force resulted in the breaking speed of
MP chains faster than the winding speed, thereby destroying the com­
plex cross-linking structure of protein. Zero shear viscosity (η0) was
calculated by Carreau model in this study to avoid the damage of protein
structure in the process of experimental shearing. As observed in
Table 2, T7 showed the highest η0 (423.114). It was showed that ultra­
sound assisted BN oxidation promoted friction in the protein chain,
resulting in an entanglement state and higher viscosity. Unlike previous
studies [18], the rod like molecules of MPs in this system tended to
overlap due to the enhanced oxidation effect. Chen et al. [50] proposed
that meat protein has a typical rod-shaped chain structure and can be
oriented under shearing. Wang et al. also demonstrated that the change
of viscosity was related to the overlapping and folding of protein rod
structure [42].
In general, relaxation modulus was connected with the instantaneous
elastic properties of MPs [51]. As shown in Fig. 2B, relaxation modulus
value (at 0.01 1/s) decreased in the order of T7 > T1 > T4 > T6 > T3 >
T2 > T5. It was note that the initial modulus of hydrophobic polyphenols
(BN, T7) was larger than that of other treatments. This indicated that the
interface deformation between myosin in MPs was greater after ultra­
sound assisted covalent reaction [52,53]. In comparison, relaxation
modulus varied at relatively high shear rates (at 100 1/s), which was
affected by both interface deformation and shear rate. Therefore, it
could be concluded that ultrasound assisted covalent reaction of MPs,
thereby promoting the unfolding and overlapping of myosin tail via
altering the protein structure. Additionally, myosin in the action area of
ultrasound may form a more stable state via “quorum sensing” [18],
leading to covalent modification with ECG and BN.

3.2.2. Dynamic rheological changes

Typical shear stress sweeps of MPs after different treatments (T1-T7)
Fig. 1. SUH (A), ASC (B) and circular dichroism curves (C), for the MPs with were displayed in Fig. 2C-D. Take ECG as an example, ultrasound
different treatments (T1-T7) obtained through optical tests. Different letters (a- assisted covalent reaction group (T6) presented the highest G’ under the
g) indicated significant differences within different treatments. same shear stress, followed by T2 and T4. During all shear stress tests, G’
was higher than G’’ for all MP solutions, implying that the solid-like
macromolecules increased after being treated with the combination of vicious behavior dominated the protein over liquid-like one. In addi­
ultrasound and oxidation. In addition, the viscosity changes were closely tion, the stress sensitivity of protein liquid was variant after different
related to the arrangement of treated MPs, especially after ultrasonic treatments. This were highly consistent with BN. Feng et al. findings
treatment [48]. Of note, the changes related to viscosity were connected [54], in which these changes were related to the flocculation state of
with the breakdown of chemical bonds [39], so MP strands were regu­ final conjugate. Additionally, the ordered structure of macromolecular
lated along the flow field and exhibited less resistance to flow, leading to substances was destroyed after treatment of high shear stress, and it was
J. Chen et al.

Fig. 2. Shear viscosities (A), relaxation modulus (B) for MP samples after different shear rate tests. Storage modulus (C) and loss modulus (D) for MP samples after
different shear stress tests. Dynamic modulus responses for MP samples (T1-T7) obtained through dynamic frequency and strain sweeps at various frequencies.

Table 2
Rheological change and model fitting of MPs after different treatments (T1-T7). Different letters (a-g) indicated significant differences within different MPs.
Treatments T1 T2 T3 T4 T5 T6 T7

k 12.088 ± 0.084b 2.998 ± 0.013e 3.796 ± 0.031d 0.023 ± 0.001 g 2.673 ± 0.011f 5.838 ± 0.019c 13.366 ± 0.074a
n 0.051 ± 0.011e 0.092 ± 0.007d 0.054 ± 0.013e 0.213 ± 0.014b 0.256 ± 0.005a 0.131 ± 0.004c 0.014 ± 0.009f
R2 0.999 1.000 1.000 0.998 1.000 1.000 1.000
η0 353.646 ± 51.385a 50.717 ± 3.995e 76.610 ± 6.109d 269.418 ± 1.820b 53.016 ± 5.472e 135.456 ± 17.362c 423.114 ± 72.514a
γcs 81.9 40.2 103 103 103 103 74.6
J. Chen et al.

highly difficult to reform the ordered structure again [55]. Notably, the
change in stress sensitivity also contributed to applying shear stress-
controlled release additive [56].
Internal interactions and stability of MPs can be easily monitored
through conventional angular frequency and strain sweeps. As illus­
trated in Fig. 2E, G’ occupied dominant position during the whole small
amplitude oscillatory shear measurement, suggesting that MPs solution
successful formed gel structure. This was also confirmed by the calcu­
lated tan δ, since this value was always < 1. Furthermore, all MP samples
were conventional weak gels (>0.1) rather than traditional elastic gels
(<0.1), according to the results of tan δ [57]. In addition, a dense
network structure was positively related to strong electrostatic in­
teractions in macromolecular substances, leading to a higher visco­
elastic moduli [58]. In this regard, it was expected that a higher amount
of MP might take part in covalent reaction during ultrasound assisted
treatments, contributing to the formation of a stronger intermolecular
MP/ECG or MP/BN network.
Typical strain sweeps of MPs after different treatments (T1-T7) was
given in Fig. 2G-H. G’ of treated MP suspensions was higher than G’’ in
the linear region, suggesting elastic response was dominant. Compared
with other oxidation treatment group, a marked increase in G’ of T6 or
T7 could be attributed to the enhancement of MP/ECG or MP/BN in­
teractions, which was also reported by Raei et al. [59]. Besides, most of
the oxidation groups increased the strain resistance of MPs in terms of
γcs values (Table 2). It was reported that disulfide bonds (S-S) and hy­
drophobic interactions were closely connected with the stability of
protein products [60]. As previously hypothesized herein, ultrasound
assisted oxidation treatment increased the oxidation effect and hydro­
phobic group exposure, thereby increasing protein interaction and
decreasing the MP gap.
Creep behaviors of MPs after different treatments (T1-T7) can be
seen in Fig. 3. Burger’s model was also applied to describe the visco­
elastic property of the MP/ECG or MP/BN composites, which was as
follows:
[ ( x) ] x
J(t) = J0 + J1 × 1 − exp − + (8)
c d

where J(t) is the creep compliance at time t, J0 represents the instan­

taneous elastic compliance, J1 is the retarded elastic compliance, c is the
retardation parameter, d is the viscosity parameter.
Generally, the changes of J0 and J1 were associated with stretching
energy of covalent bonds and transformation of non-covalent bonds
(hydrogen bonds and hydrophobic interactions), respectively [61].
Fig. 3. Strain responses (A) and creep compliance (B) of different MP samples
Notably, J0 decreased from 0.028 (T1) to − 84.462 (T6) and 0.017 (T7).
obtained through creep and creep-recovery sweeps (stress level: 3 Pa). Viscosity
More so, ultrasound assisted ECG oxidation treatment (− 1.458 × 106)
responses (C) of different MP samples through creep sweeps.
significantly reduced the J1 of initial protein sample (− 0.995), whereas
ultrasound assisted BN oxidation treatment resulted in a converse result
(141.492). Compared with other oxidation treatment group (T2-T5), the seven different treatment groups were performed in the concentration
transformation in creep compliance validated that ultrasound assisted ranging from 0.01 to 5 mg/mL. All the samples exhibited desirable
oxidation treatment promoted deformation resistance of MP solution. DPPH radical scavenging abilities with the characteristics of dose
This further indicated that such a treatment could improve the stability dependence. At a concentration of 5 mg/mL, the DPPH free radical
of chemical bonds in MPs. Results of viscosity responses during creep scavenging activities were 62.92%, 80.35%, 62.40%, 71.15%, 57.52%,
sweeps were in consistent with those of static shearing, verifying the 87.15% and 66.22% for T1-T7, respectively. This indicated that the
potential of ultrasound assisted oxidation treatment in viscosity prop­ increased SSA due to more ECG-MP or BN-MP structure formation
erties. Zhuang et al. also confirmed that values of J0 and J1 were posi­ facilitated the kinetics of their actions with DPPH radicals [65]. In
tively correlated with the cross-linking density of MPs [62].Besides, Fan addition, the improvement of DPPH radical scavenging ability may be
et al. attributed creep behaviors of MP to the hydrophobic interactions owing to the introduction of phenolic hydroxyl groups in ECG or BN
and disulfide covalent bonding (S-S) [63]. [54]. In our study, the incorporation of polyphenols after ultrasound (T4
and T5) produce lower DPPH free radical scavenging abilities compared
to treatments where ultrasound was applied first and then polyphenols
3.3. Antioxidant properties changes
were applied to the MP. It is well established that MP is in a special
sensitive state after ultrasound [18,40], and the synergistic effect on
Cells could be effortlessly damaged by excessive free radical oxida­
alkaline oxidation process can promote the grafting of polyphenols,
tion induced by metabolism, accelerating the body’s aging process [64].
thereby contributing to the enhancement of DPPH free radical scav­
Therefore, researchers have been dedicated to investigating various
enging abilities. In contrast, the weak oxidation after ultrasound is not
approaches to effectively reduce the damage of free radicals for a long
conducive to the covalent reaction of hydrophobic BN and MP since
time. As shown in Fig. 4, the DPPH free radical scavenging activities of
J. Chen et al.

Fig. 4. Scavenging activities on DPPH radicals (A), and metal ion chelating activities (C) of MPs after different treatments (T1-T7). Fig. 5B and 5D correspond to the
scavenging capacity of DPPH radicals and metal ion for different samples at the concentration of 5 mg/mL. Different letters (a-f) indicated significant differences
within different treatments (T1-T7).

there is no synergistic effect at this time. Besides, the BN dispersion was 3.4. Proposed chemical structures and reaction pathway
not uniform [9], and the original properties of MP were also destroyed
after ultrasound [21,23]. Mass analysis was performed to confirm the proposed chemical
Besides, the metal ion chelating activities of MP samples after structures and reaction pathway. As shown in Fig. S1, a peak of 231 Da
different treatments (T1-T7) were shown in Fig. 4. All the samples dis­ was observed in second order mass spectra of ECG, whereas a peak of
played a decent metal ion chelating effect, with the concentration range 510.6 Da was shown in mass spectra of BN. Additionally, the multi-
from 0.01 to 5 mg/mL. At a concentration of 5 mg/mL, the metal ion cyclic structure of ECG was hydrolyzed due to the presence of high
chelating activities were 111.44%, 155.02%, 170.58%, 130.39%, concentration of hydrochloric acid. It was assumed that cysteine (121
148.58%, 310.58% and 209.50% for T1-T7, respectively. Notably, the Da) formed a complex with two hydroxyl groups in the benzene ring
chelating power of T6 was significantly higher than that of other groups (110 Da), and other fragment ion peaks were also consistent with these
at equivalent concentrations using the chromogenic method. This veri­ results. Similarly, BN had a molecular weight of 270 Da, and two
fied that ultrasound assisted ECG oxidation treatment remarkably cysteine (121 Da) molecules might bind one BN molecule to form Cys-
enhanced the metal ion chelating activities on account of the incorpo­ BN-Cys (510 Da).
ration of polyphenol. Moreover, tons of ECG-MP covalent complexes Above results indicated that ultrasound assisted oxidation of BN and
might be generated compared with other ECG incorporation groups. ECG were mainly via -SH addition pathway, which was consistent with
Additionally, the changes in chelating power may also be ascribed to the the previous results of ASC. As given in Fig. 5, ECG or BN quinone
side chain structure and unfolding degree of MP. Deng et al. have re­ structure was firstly formed by enhanced OH⋅ attack. Then, it continued
ported that the structure changes of MP was a dominant factor for to react with –SH in MP to engender ECG quinone-thiol adduct or BN
chelating activities variations [66]. quinone-thiol adduct. BN tended to bind another protein molecule
OH⋅ scavenging activities of MP samples after various treatments (cysteine) to produce MP-S-BN-S-MP polymer compared with ECG. Pan
(T1-T7) were illustrated in Fig. S2. At a concentration of 5 mg/mL, T6 et al. [11] applied ultrasound to decompose water molecules into
displayed the highest OH⋅ scavenging activities (67.56%), followed by reactive OH⋅ (H2O → H + OH ), which promoted formation of MP-S-GA-
• •

T3 (65.94%) and T2 (55.64%), implying that ultrasound assisted ECG S-MP polymer or MP-N-GA-N-MP polymer. This reaction pathway was
oxidation treatment elicited notable OH⋅ scavenging activities at a in agreement with the findings of Cao et al., [70] who reported that
relatively high concentration. It was well-acknowledged that OH⋅ treatment of MP with oxidized CA produced dimer. Notably, the
scavenging activities were related to the hydroxyl groups (–OH) sub­ unfolding of protein structure in the process of ultrasound assisted
stituents in a polyphenol ring [38,67]. Besides, some studies [68,69] oxidation was conducive to enhance the non-covalent interaction be­
confirmed that high intake of chicken protein may increase the risk of tween ECG/BN and MP, including hydrogen bonds and hydrophobic
chronic diseases, while ultrasound-assisted oxidative modification interaction [9]. Therefore, it can be concluded that ultrasound assisted
lowered the probability by scavenging free radicals in the body. Further oxidation contributed to the covalent and non-covalent interactions
investigations concerning the mechanism of gut-brain axis shall be between MP and ECG/BN. These interaction changes contributed to the
further studied in the near future. stability of finial products, since extra energy was required for
destroying the chemical bonds of MP-BN or MP-ECG.
J. Chen et al.

Fig. 5. Mechanism diagrams of oxidation for MP-ECG (A) and MP-BN (B). Proposed non-covalent interactions between MPs and different polyphenols (C and D
represent interactions between MPs and ECG, MPs and BN, respectively).

3.5. Chromaticity changes BN increased it. This may be due to the light waves’ selective ability by
chromophores in hydrophilic (ECG) and non-hydrophilic (BN) poly­
The values of L, C, a, b, W* and ΔE represent the lightness, chroma, phenols. Notably, ultrasound assisted ECG or BN oxidation significantly
redness, yellowness, whiteness, and total color difference, respectively, dimished the whiteness (w*) of gels, accompanied by the improved
as summarized in Table 1. Relatively huge changes of b* values were antioxidant properties. This was in agreement with some studies that
observed in the BN-treated group, whereas relatively small changes indicated that polyphenols were served as copigments for color and
were obtained in the ECG-treated group. The transformation of b values stability enhancement of protein [71]. The potential effect of chemical
for ECG and BN indicated that the color of MP emulsion gel changed structure on chromaticity was considered by Fan et al. [72], while the
from white to a faint yellow. Moreover, b* reached its minimum value hydrophilicity analysis of polyphenols was no referred. Take together,
(ECG: 0.812, BN: 11.155) after ultrasound assisted oxidation, implying our previous investigations also vadalited that BN could be applied as
that more ECG/BN were involved in covalent modification after treat­ the natural pigment [18].
ment T6 and T7. As reported, the acceptance and preference of con­
sumers were closely related to individual sensitivity to color. For 3.6. Digestive properties changes
instance, consumers with relatively small grades tend to choose protein
colloids with a faint yellow. Generally, ΔE values below control group Previous observations indicated that ultrasound assisted ECG or BN
(T1) suggested that oxidation treatment made an positive effect on oxidation significantly increased antioxidant properties of MPs,
improving the color stability of various MPs. Moreover, T7 treatment including DPPH free radical scavenging activities, metal ion chelating
allowed the most desirable result with the smallest ΔE value (54.598%, activities and OH⋅ scavenging activities. However, the digestive prop­
17.298) and higher remaining percentage of BN after T5 treatment erties of MPs under this situation remained unknown as human beings in
(71.347%, 19.172), followed by T3 treatment (103.90%, 22.815). modern society attached enormous attention to the absorption of pro­
Additionally, the ECG incorporation effectively decreased C value, while tein. The protein bio-accessibility of MP gels using the simulation
J. Chen et al.

Fig. 6. Measured (A) and fitted curves (B) of digestion in gastric digestion stage; measured (C) and fitted curves (D) of digestion in intestinal digestion stage; the E-F
represent initial slope, and average rate of digestion of MP samples after various oxidation treatments; the G-H represent B and ODmax of fitted curves, respectively.
Labels are as follows: T1 (control group), T6 (ultrasound assisted ECG oxidation group), T7 (ultrasound assisted BN oxidation group).

digestion system was analyzed, and corresponding results were shown in highest ODmax, whereas BN under same treatment showed a lower level
Fig. 6. For gastric digestion stage, particularly in the initial stage of MP during the gastric digestion stage. It was shown that the enhanced effect
digestion, the peptide release rate increased significantly after ultra­ of hydrophilic polyphenols caused by ultrasound assisted oxidation may
sound assisted ECG oxidation treatment. Continuous digestion contribute to the improvement of protein bioavailability. Another study
decreased the number of MP reaction sites, lowering digestive rate of also concluded that the absorption of protein nutrients was closely
follow-up. A similar result was reported by Zhao et al. [36]. As the OD correlated with the covalent modification effect of protein [74].
(280 nm) did not reach its maximal value at the end of digestion, ODmax For intestinal digestion stage, the trend of hydrolysis was consider­
was applied in the model to reflect the level of MP hydrolysis [73]. As ably similar to that of gastric digestion. We found that the initial rate and
observed, ultrasound assisted ECG oxidation treatment exhibited the the average rate was highest in ultrasound-assisted hydrophilic ECG
J. Chen et al.

oxidation group (T6). Besides, the stimulated intestinal stage exhibited a CRediT authorship contribution statement
higher hydrolysis rate than that of the gastric stage, which was
confirmed by larger ODmax. In comparison, ultrasound assisted oxida­ Jiahui Chen: Conceptualization, Methodology, Data Curation,
tion of BN decreased protein bioavailability. Substantive studies have Experiment, Writing - original draft. Xing Zhang: Writing - original
suggested that the excessive covalent modification and protein aggre­ draft, Investigation, Conceptualization, Data Curation. Mengying Fu:
gation could decrease the digestion rates. Of note, this was also related Formal analysis, Conceptualization, Software. Xing Chen: Methodol­
to the protein types and processing method [35]. Chen et al. also re­ ogy, Conceptualization. Bassey Anthony Pius: Conceptualization.
ported that the covalent modification of MP and (− )-epigallocatechin Xinglian Xu: Writing - review & editing, Supervision, Funding
was beneficial for the gel properties [12]. Although ultrasound has been acquisition.
viewed as a green method for improving proteins bioavailability [75],
the protein digestibility of ultrasound-assisted oxidation for polyphenols
under alkaline conditions was still controversial [76]. In this study, our Declaration of Competing Interest
findings showed that ultrasound-assisted hydrophilic polyphenol (ECG)
oxidation hold tremendous potential for future application. The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
3.7. Mechanism of this system with or without ultrasound the work reported in this paper.

Advanced oxidation processes usually involved a method for pro­ Acknowledgments

ducing ⋅OH that unselectively oxidize macromolecular substances in
medium solution, protein in particular. In this study, it was speculated This work has been supported by the National Natural Science
that ultrasound in combination with alkaline oxidation processes actu­ Foundation of China (No. 31972097), China Agriculture Research Sys­
ally act in a synergistic way, although the effect of covalent modification tem of MOF and MARA (CARS-41), National Key Research Program of
was greater than that of single process only. Enhanced oxidation process China (2018YFD0401203), and a project funded by the Priority Aca­
facilitated the unfolding of MP, thereby contributing to the covalent and demic Program Development of Jiangsu Higher Education Institution
non-covalent interactions (e.g. hydrophobic interaction and hydrogen (PAPD). We thank Qingdao Sci-tech Innovation Quality Testing Co. Ltd.
bondings) between MP and ECG/BN. In other words, ECG or BN quinone for technical assistances.
structure was formed by enhanced OH⋅ attack during ultrasound-
assisted covalent reaction, after which they then reacted with -SH in
Appendix A. Supplementary data
MP to produce ECG or BN quinone-thiol adduct. During this progress,
the enhancement of covalent and non-covalent interactions contributed
Supplementary data to this article can be found online at https://doi.
to the transformation in antioxidant and rheological properties.
org/10.1016/j.ultsonch.2021.105652.
Besides chemical reaction, “quorum sensing” was considered based
on our previous studies [18]. The sensitive state of myosin in MP during
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